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Sample records for fluorescent labelled sulfonamides

  1. Electron Spin Resonance Studies of Carbonic Anhydrase: Transition Metal Ions and Spin-Labeled Sulfonamides*

    Science.gov (United States)

    Taylor, June S.; Mushak, Paul; Coleman, Joseph E.

    1970-01-01

    Electron spin resonance (esr) spectra of Cu(II) and Co(II) carbonic anhydrase, and a spin-labeled sulfonamide complex of the Zn(II) enzyme, are reported. The coordination geometry of Cu(II) bound in the enzyme appears to have approximately axial symmetry. Esr spectra of enzyme complexes with metal-binding anions also show axial symmetry and greater covalency, in the order ethoxzolamide cyanide complex suggests the presence of two, and probably three, equivalent nitrogen ligands from the protein. Esr spectra of the Co(II) enzyme and its complexes show two types of Co(II) environment, one typical of the native enzyme and the 1:1 CN- complex, and one typical of a 2:1 CN- complex. Co(II) in the 2:1 complex appears to be low-spin and probably has a coordination number of 5. Binding of a spin-labeled sulfonamide to the active center immobilizes the free radical. The similarity of the esr spectra of spin-labeled Zn(II) and Co(II) carbonic anhydrases suggests that the conformation at the active center is similar in the two metal derivatives. PMID:4320976

  2. Rapid trace level determination of sulfonamide residues in honey with online extraction using short C-18 column by high-performance liquid chromatography with fluorescence detection.

    Science.gov (United States)

    Sajid, Muhammad; Na, Na; Safdar, Muhammad; Lu, Xin; Ma, Lin; He, Lan; Ouyang, Jin

    2013-11-01

    A sensitive and inexpensive quantification method with online extraction using a short C-18 column for sulfonamide residues in honey by high performance liquid chromatography with fluorescence detector was developed and validated. In sample preparation, acid hydrolysis was used to break the N-glycoside bond between the honey sugar and sulfonamide drugs and derivatization of sulfonamide residues with fluorescamine was conducted at pH 3.5 using a citrate buffer (0.5M) in the honey matrix. The chromatography was carried out on Zorbax Extended C-18 (250mm×4.6mm; 5μm) column, using a mixture of acetonitrile and an acetate buffer (pH 4.50, 20mM) as a mobile phase. A Zorbax Extended C-18 (12mm×4.6mm; 5μm) column was used for online extraction of fifteen sulfonamide residues from honey sample with the help of a two position valve. The limit of quantification of sulfonamide residues in honey was less than 3ngg(-1), and the percentage recovery of study compounds in spiked honey sample was from 80% for sulfacetamide to 100% of sulfachloropyridazine. The developed method has excellent linearity for all studied sulfonamides with a correlation coefficient 0.993. Copyright © 2013 Elsevier B.V. All rights reserved.

  3. Electron Detachment Dissociation (EDD) of Fluorescently Labeled Sialylated Oligosaccharides

    Science.gov (United States)

    Zhou, Wen; Håkansson, Kristina

    2012-01-01

    We explored the application of electron detachment dissociation (EDD) and infrared multiphoton dissociation (IRMPD) tandem mass spectrometry to fluorescently labeled sialylated oligosaccharides. Standard sialylated oligosaccharides and a sialylated N-linked glycan released from human transferrin were investigated. EDD yielded extensive glycosidic cleavages and cross-ring cleavages in all cases studied, consistently providing complementary structural information compared to IRMPD. Neutral losses and satellite ions such as C – 2H ions were also observed following EDD. In addition, we examined the influence of different fluorescent labels. The acidic label 2-aminobenzoic acid (2-AA) enhanced signal abundance in negative-ion mode. However, few cross-ring fragments were observed for 2-AA labeled oligosaccharides. The neutral label 2-aminobenzamide (2-AB) resulted in more cross-ring cleavages compared to 2-AA labeled species, but not as extensive fragmentation as for native oligosaccharides, likely resulting from altered negative charge locations from introduction of the fluorescent tag. PMID:22120881

  4. Bioorthogonal fluorescent labeling of functional G-protein-coupled receptors

    DEFF Research Database (Denmark)

    Tian, He; Naganathan, Saranga; Kazmi, Manija A

    2014-01-01

    Novel methods are required for site-specific, quantitative fluorescent labeling of G-protein-coupled receptors (GPCRs) and other difficult-to-express membrane proteins. Ideally, fluorescent probes should perturb the native structure and function as little as possible. We evaluated bioorthogonal...

  5. Carbon "Quantum" Dots for Fluorescence Labeling of Cells.

    Science.gov (United States)

    Liu, Jia-Hui; Cao, Li; LeCroy, Gregory E; Wang, Ping; Meziani, Mohammed J; Dong, Yiyang; Liu, Yuanfang; Luo, Pengju G; Sun, Ya-Ping

    2015-09-02

    The specifically synthesized and selected carbon dots of relatively high fluorescence quantum yields were evaluated in their fluorescence labeling of cells. For the cancer cell lines, the cellular uptake of the carbon dots was generally efficient, resulting in the labeling of the cells with bright fluorescence emissions for both one- and two-photon excitations from predominantly the cell membrane and cytoplasm. In the exploration on labeling the live stem cells, the cellular uptake of the carbon dots was relatively less efficient, though fluorescence emissions could still be adequately detected in the labeled cells, with the emissions again predominantly from the cell membrane and cytoplasm. This combined with the observed more efficient internalization of the same carbon dots by the fixed stem cells might suggest some significant selectivity of the stem cells toward surface functionalities of the carbon dots. The needs and possible strategies for more systematic and comparative studies on the fluorescence labeling of different cells, including especially live stem cells, by carbon dots as a new class of brightly fluorescent probes are discussed.

  6. Fluorescent-labeled ligands for the benzodiazepine receptor - Part 1 : Synthesis and characterization of fluorescent-labeled benzodiazepines

    NARCIS (Netherlands)

    Janssen, M.J; Hulst, A.J R L; Kellogg, R.M; Hendriks, M.M W B; Ensing, K; de Zeeuw, R.A

    Because radioactive labeled ligands in receptor assays have several disadvantages, we synthesized a number of fluorescent-labeled benzodiazepines. Several fluorophores were attached at different positions of 1,4-benzodiazepine molecules in order to assess the impact of the fluorophores and their

  7. Electron detachment dissociation of fluorescently labeled sialylated oligosaccharides.

    Science.gov (United States)

    Zhou, Wen; Håkansson, Kristina

    2011-12-01

    We explored the application of electron detachment dissociation (EDD) and infrared multiphoton dissociation (IRMPD) tandem mass spectrometry to fluorescently labeled sialylated oligosaccharides. Standard sialylated oligosaccharides and a sialylated N-linked glycan released from human transferrin were investigated. EDD yielded extensive glycosidic cleavages and cross-ring cleavages in all cases studied, consistently providing complementary structural information compared with infrared multiphoton dissociation. Neutral losses and satellite ions such as C-2H ions were also observed following EDD. In addition, we examined the influence of different fluorescent labels. The acidic label 2-aminobenzoic acid (2-AA) enhanced signal abundance in negative-ion mode. However, few cross-ring fragments were observed for 2-AA-labeled oligosaccharides. The neutral label 2-aminobenzamide (2-AB) resulted in more cross-ring cleavages compared with 2-AA-labeled species, but not as extensive fragmentation as for native oligosaccharides, likely resulting from altered negative charge locations from introduction of the fluorescent tag. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. In Situ Live-Cell Nucleus Fluorescence Labeling with Bioinspired Fluorescent Probes.

    Science.gov (United States)

    Ding, Pan; Wang, Houyu; Song, Bin; Ji, Xiaoyuan; Su, Yuanyuan; He, Yao

    2017-08-01

    Fluorescent imaging techniques for visualization of nuclear structure and function in live cells are fundamentally important for exploring major cellular events. The ideal cellular labeling method is capable of realizing label-free, in situ, real-time, and long-term nucleus labeling in live cells, which can fully obtain the nucleus-relative information and effectively alleviate negative effects of alien probes on cellular metabolism. However, current established fluorescent probes-based strategies (e.g., fluorescent proteins-, organic dyes-, fluorescent organic/inorganic nanoparticles-based imaging techniques) are unable to simultaneously realize label-free, in situ, long-term, and real-time nucleus labeling, resulting in inevitable difficulties in fully visualizing nuclear structure and function in live cells. To this end, we present a type of bioinspired fluorescent probes, which are highly efficacious for in situ and label-free tracking of nucleus in long-term and real-time manners. Typically, the bioinspired polydopamine (PDA) nanoparticles, served as fluorescent probes, can be readily synthesized in situ within live cell nucleus without any further modifications under physiological conditions (37 °C, pH ∼7.4). Compared with other conventional nuclear dyes (e.g., propidium iodide (PI), Hoechst), superior spectroscopic properties (e.g., quantum yield of ∼35.8% and high photostability) and low cytotoxicity of PDA-based probes enable long-term (e.g., 3 h) fluorescence tracking of nucleus. We also demonstrate the generality of this type of bioinspired fluorescent probes in different cell lines and complex biological samples.

  9. High-level fluorescence labeling of gram-positive pathogens.

    Directory of Open Access Journals (Sweden)

    Simone Aymanns

    Full Text Available Fluorescence labeling of bacterial pathogens has a broad range of interesting applications including the observation of living bacteria within host cells. We constructed a novel vector based on the E. coli streptococcal shuttle plasmid pAT28 that can propagate in numerous bacterial species from different genera. The plasmid harbors a promoterless copy of the green fluorescent variant gene egfp under the control of the CAMP-factor gene (cfb promoter of Streptococcus agalactiae and was designated pBSU101. Upon transfer of the plasmid into streptococci, the bacteria show a distinct and easily detectable fluorescence using a standard fluorescence microscope and quantification by FACS-analysis demonstrated values that were 10-50 times increased over the respective controls. To assess the suitability of the construct for high efficiency fluorescence labeling in different gram-positive pathogens, numerous species were transformed. We successfully labeled Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus dysgalactiae subsp. equisimilis, Enterococcus faecalis, Enterococcus faecium, Streptococcus mutans, Streptococcus anginosus and Staphylococcus aureus strains utilizing the EGFP reporter plasmid pBSU101. In all of these species the presence of the cfb promoter construct resulted in high-level EGFP expression that could be further increased by growing the streptococcal and enterococcal cultures under high oxygen conditions through continuous aeration.

  10. Diffuse fluorescence tomography of exo- and endogenously labeled tumors

    Science.gov (United States)

    Balalaeva, Irina V.; Turchin, Ilya V.; Orlova, Anna G.; Plekhanov, Vladimir I.; Shirmanova, Marina V.; Kleshnin, Michail S.; Fiks, Ilya I.; Zagainova, Elena V.; Kamensky, Vladislav A.

    2007-06-01

    Strong light scattering and absorption limit observation of the internal structure of biological tissue. Only special tools for turbid media imaging, such as optical diffuse tomography, enable noninvasive investigation of the internal biological tissues, including visualization and intravital monitoring of deep tumors. In this work the preliminary results of diffuse fluorescence tomography (DFT) of small animals are presented. Usage of exogenous fluorophores, targeted specifically at tumor cells, and fluorescent proteins expressed endogenously can significantly increase the contrast of obtained images. Fluorescent compounds of different nature, such as sulphonated aluminium phthalocyanine (Photosens), red fluorescing proteins and CdTe/CdSe-core/shell nanocrystals (quantum dots) were applied. We tested diffuse fluorescence tomography method at model media, in post mortem and in vivo experiments. The animal was scanned in transilluminative configuration by low-frequency modulated light (1 kHz) from Nd:YAG laser with second harmonic generation at wavelength of 532 nm or semiconductor laser at wavelength of 655 nm. Quantum dots or protein DsRed2 in glass capsules (inner diameter 2-3 mm) were placed post mortem inside the esophagus of 7-day-old hairless rats to simulate marked tumors. Photosens was injected intravenously to linear mice with metastazing Lewis lung carcinoma. The reconstruction algorithm, based on Algebraic Reconstruction Technique, was created and tested numerically in model experiments. High contrast images of tumor simulating capsules with DsRed2 concentrations about 10 -6 M and quantum dots about 5x10 -11 M have been obtained. Organ distribution of Photosens and its accumulation in tumors and surrounding tissues of animals has been examined. We have conducted the monitoring of tumors, exogenously labeled by photosensitizer. This work demonstrates potential capabilities of DFT method for intravital detection and monitoring of deep fluorescent-labeled

  11. Hydroxylamine as an oxygen nucleophile: substitution of sulfonamide by a hydroxyl group in benzothiazole-2-sulfonamides

    NARCIS (Netherlands)

    Kamps, J.A.G.; Belle, R.; Mecinovic, J.

    2013-01-01

    Benzothiazole-2-sulfonamides react with an excess of hydroxylamine in aqueous solutions to form 2-hydroxybenzothiazole, sulfur dioxide, and the corresponding amine. Mechanistic studies that employ a combination of structure-reactivity relationships, oxygen labeling experiments, and (in)direct

  12. Analysis of fluorescently labeled glycosphingolipid-derived oligosaccharides following ceramide glycanase digestion and anthranilic acid labeling.

    Science.gov (United States)

    Neville, David C A; Coquard, Virginie; Priestman, David A; te Vruchte, Danielle J M; Sillence, Daniel J; Dwek, Raymond A; Platt, Frances M; Butters, Terry D

    2004-08-15

    Interest in cellular glycosphingolipid (GSL) function has necessitated the development of a rapid and sensitive method to both analyze and characterize the full complement of structures present in various cells and tissues. An optimized method to characterize oligosaccharides released from glycosphingolipids following ceramide glycanase digestion has been developed. The procedure uses the fluorescent compound anthranilic acid (2-aminobenzoic acid; 2-AA) to label oligosaccharides prior to analysis using normal-phase high-performance liquid chromatography. The labeling procedure is rapid, selective, and easy to perform and is based on the published method of Anumula and Dhume [Glycobiology 8 (1998) 685], originally used to analyze N-linked oligosaccharides. It is less time consuming than a previously published 2-aminobenzamide labeling method [Anal. Biochem. 298 (2001) 207] for analyzing GSL-derived oligosaccharides, as the fluorescent labeling is performed on the enzyme reaction mixture. The purification of 2-AA-labeled products has been improved to ensure recovery of oligosaccharides containing one to four monosaccharide units, which was not previously possible using the Anumula and Dhume post-derivatization purification procedure. This new approach may also be used to analyze both N- and O-linked oligosaccharides.

  13. A label-free, fluorescence based assay for microarray

    Science.gov (United States)

    Niu, Sanjun

    DNA chip technology has drawn tremendous attention since it emerged in the mid 90's as a method that expedites gene sequencing by over 100-fold. DNA chip, also called DNA microarray, is a combinatorial technology in which different single-stranded DNA (ssDNA) molecules of known sequences are immobilized at specific spots. The immobilized ssDNA strands are called probes. In application, the chip is exposed to a solution containing ssDNA of unknown sequence, called targets, which are labeled with fluorescent dyes. Due to specific molecular recognition among the base pairs in the DNA, the binding or hybridization occurs only when the probe and target sequences are complementary. The nucleotide sequence of the target is determined by imaging the fluorescence from the spots. The uncertainty of background in signal detection and statistical error in data analysis, primarily due to the error in the DNA amplification process and statistical distribution of the tags in the target DNA, have become the fundamental barriers in bringing the technology into application for clinical diagnostics. Furthermore, the dye and tagging process are expensive, making the cost of DNA chips inhibitive for clinical testing. These limitations and challenges make it difficult to implement DNA chip methods as a diagnostic tool in a pathology laboratory. The objective of this dissertation research is to provide an alternative approach that will address the above challenges. In this research, a label-free assay is designed and studied. Polystyrene (PS), a commonly used polymeric material, serves as the fluorescence agent. Probe ssDNA is covalently immobilized on polystyrene thin film that is supported by a reflecting substrate. When this chip is exposed to excitation light, fluorescence light intensity from PS is detected as the signal. Since the optical constants and conformations of ssDNA and dsDNA (double stranded DNA) are different, the measured fluorescence from PS changes for the same

  14. Fluorescently labeled bevacizumab in human breast cancer: defining the classification threshold

    Science.gov (United States)

    Koch, Maximilian; de Jong, Johannes S.; Glatz, Jürgen; Symvoulidis, Panagiotis; Lamberts, Laetitia E.; Adams, Arthur L. L.; Kranendonk, Mariëtte E. G.; Terwisscha van Scheltinga, Anton G. T.; Aichler, Michaela; Jansen, Liesbeth; de Vries, Jakob; Lub-de Hooge, Marjolijn N.; Schröder, Carolien P.; Jorritsma-Smit, Annelies; Linssen, Matthijs D.; de Boer, Esther; van der Vegt, Bert; Nagengast, Wouter B.; Elias, Sjoerd G.; Oliveira, Sabrina; Witkamp, Arjen J.; Mali, Willem P. Th. M.; Van der Wall, Elsken; Garcia-Allende, P. Beatriz; van Diest, Paul J.; de Vries, Elisabeth G. E.; Walch, Axel; van Dam, Gooitzen M.; Ntziachristos, Vasilis

    2017-07-01

    In-vivo fluorescently labelled drug (bevacizumab) breast cancer specimen where obtained from patients. We propose a new structured method to determine the optimal classification threshold in targeted fluorescence intra-operative imaging.

  15. Threshold Analysis and Biodistribution of Fluorescently Labeled Bevacizumab in Human Breast Cancer

    NARCIS (Netherlands)

    Koch, Maximilian; de Jong, Johannes S.; Glatz, Juergen; Symvoulidis, Panagiotis; Lamberts, Laetitia E.; Adams, Arthur L. L.; Kranendonk, Mariette E. G.; Terwisscha Van Scheltinga, Anton; Aichler, Michaela; Jansen, Liesbeth; de Vries, Jakob; Lub-de Hooge, Marjolijn N.; Schroder, Carolien P.; Jorritsma-Smit, Annelies; Linssen, Matthijs D.; de Boer, Esther; van der Vegt, Bert; Nagengast, Wouter B.; Elias, Sjoerd G.; Oliveira, Sabrina; Witkamp, Arjen J.; Mali, Willem P. T. M.; Van der Wall, Elsken; Garcia-Allende, P. Beatriz; van Diest, Paul J.; de Vries, Elisabeth G. E.; Walch, Axel; van Dam, Gooitzen M.; Ntziachristos, Vasilis

    2017-01-01

    In vivo tumor labeling with fluorescent agents may assist endoscopic and surgical guidance for cancer therapy as well as create opportunities to directly observe cancer biology in patients. However, malignant andnonmalignant tissues are usually distinguished on fluorescence images by applying

  16. Acylhydrazone bond dynamic covalent polymer gel monolithic column online coupling to high-performance liquid chromatography for analysis of sulfonamides and fluorescent whitening agents in food.

    Science.gov (United States)

    Zhang, Chengjiang; Luo, Xialin; Wei, Tianfu; Hu, Yufei; Li, Gongke; Zhang, Zhuomin

    2017-10-13

    A new dynamic covalent polymer (DCP) gel was well designed and constructed based on imine chemistry. Polycondensation of 4,4'-biphenyldicarboxaldehyde and 1,3,5-benzenetricarbohydrazide via Schiff-base reaction resulted in an acylhydrazone bond gel (AB-gel) DCP. AB-gel DCP had three-dimensional network of interconnected nanoparticles with hierarchically porous structure. AB-gel DCP was successfully fabricated as a monolithic column by an in-situ chemical bonding method for online enrichment and separation purpose with excellent permeability. AB-gel DCP based monolithic column showed remarkable adsorption affinity towards target analytes including sulfonamides (SAs) and fluorescent whitening agents (FWAs) due to its strong π-π affinity, hydrophobic effect and hydrogen bonding interaction. Then, AB-gel DCP based monolithic column was applied for online separation and analysis of trace SAs and FWAs in food samples coupled with high-performance liquid chromatography (HPLC). Sulfathiazole (ST) and sulfadimidine (SM2) in one positive weever sample were actually found and determined with concentrations of 273.8 and 286.3μg/kg, respectively. 2,5-Bis(5-tert-butyl-2-benzoxazolyl) thiophene (FWA184) was actually quantified in one tea infusion sample with the concentration of 268.5ng/L. The spiked experiments suggested the good recoveries in range of 74.5-110% for SAs in weever and shrimp samples with relative standard deviations (RSDs) less than 9.7% and in range of 74.0-113% for FWAs in milk and tea infusion samples with RSDs less than 9.0%. AB-gel DCP monolithic column was proved to be a promising sample preparation medium for online separation and analysis of trace analytes in food samples with complex matrices. Copyright © 2017 Elsevier B.V. All rights reserved.

  17. Novel biosensor system model based on fluorescence quenching by a fluorescent streptavidin and carbazole-labeled biotin.

    Science.gov (United States)

    Zhu, Xianwei; Shinohara, Hiroaki; Miyatake, Ryuta; Hohsaka, Takahiro

    2016-10-01

    In the present study, a novel molecular biosensor system model was designed by using a couple of the fluorescent unnatural mutant streptavidin and the carbazole-labeled biotin. BODIPY-FL-aminophenylalanine (BFLAF), a fluorescent unnatural amino acid was position-specifically incorporated into Trp120 position of streptavidin by four-base codon method. On the other hand, carbazole-labeled biotin was synthesized as a quencher for the fluorescent Trp120BFLAF mutant streptavidin. The fluorescence of fluorescent Trp120BFLAF mutant streptavidin was decreased as we expected when carbazole-labeled biotin was added into the mutant streptavidin solution. Furthermore, the fluorescence decrease of Trp120BFLAF mutant streptavidin with carbazole-labeled biotin (100 nM) was recovered by the competitive addition of natural biotin. This result demonstrated that by measuring the fluorescence quenching and recovery, a couple of the fluorescent Trp120BFLAF mutant streptavidin and the carbazole-labeled biotin were successfully applicable for quantification of free biotin as a molecular biosensor system. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

  18. Synthesis, characterization and bioimaging of fluorescent labeled polyoxometalates.

    Science.gov (United States)

    Geisberger, Georg; Gyenge, Emina Besic; Hinger, Doris; Bösiger, Peter; Maake, Caroline; Patzke, Greta R

    2013-07-21

    A fluorescent labeled Wells-Dawson type POM ({P2W17O61Fluo}) was newly synthesized and characterized by a wide range of analytical methods. {P2W17O61Fluo} was functionalized with fluorescein amine through a stable amide bond, and its long time stability was verified by UV/vis spectroscopic techniques at physiologically relevant pH values. No significant impact on the cell viability or morphology of HeLa cells was observed for POM concentrations up to 100 μg mL(-1). Cellular uptake of fluorescent {P2W17O61Fluo} was monitored by confocal laser scanning microscopy. POM uptake occurs mainly after prolonged incubation times of 24 h resulting in different intracellular patterns, i.e. randomly distributed over the entire cytoplasm, or aggregated in larger clusters. This direct monitoring strategy for the interaction of POMs with cells opens up new pathways for elucidating their unknown mode of action on the way to POM-based drug development.

  19. The internalization of fluorescence-labeled PLA nanoparticles by macrophages.

    Science.gov (United States)

    Li, Fengjuan; Zhu, Aiping; Song, Xiaoli; Ji, Lijun; Wang, Juan

    2013-09-10

    Rhodamine B (RhB)-labeled PLA nanoparticles were prepared through surface grafting copolymerization of glycidyl methacrylate (GMA) onto PLA nanoparticles during the emulsion/evaporation process. RhB firstly interacts with sodium dodecyl sulfate (SDS) through electrostatic interaction to form hydrophobic complex (SDS-RhB). Due to the high-affinity of SDS-RhB with GMA, hydrophilic RhB can be successfully combined into PLA nanoparticles. The internalization of RhB-labeled PLA nanoparticles by macrophages was investigated with fluorescence microscope technology. The effects of the PLA nanoparticle surface nature and size on the internalization were investigated. The results indicate that the PLA particles smaller than 200 nm can avoid the uptake of phagocytosis. The bigger PLA particles (300 nm) with polyethylene glycol (PEG) surface showed less internalization by macrophage compared with those with poly(ethylene oxide-propylene oxide) copolymer (F127) or poly(vinyl alcohol) (PVA) surface. The "stealth" function of PEG on the PLA nanoparticles from internalization of macrophages due to the low protein adsorption is revealed by electrochemical impedance technology. Copyright © 2013 Elsevier B.V. All rights reserved.

  20. The LB Films of Dansyl Chloride Labeled Octadecylamine and Its Fluorescence Lifetime

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Octadecylamine was derivatized with dansyl chloride (5-dimethylaminonaphthalene-1-sulfonyl chloride) in order to simplify and understand the LB films of fluorescent probe labeling proteins.Its monolayer and multilayers in the absence and presence of stearic acid were deposited by LB technique.Fluorescence spectra and lifetimes of the fluorescent products were studied to elucidate the microenvironment of molecules in the LB films.

  1. Investigation of antigen-antibody interactions of sulfonamides with a monoclonal antibody in a fluorescence polarization immunoassay using 3D-QSAR models

    Science.gov (United States)

    A three-dimensional quantitative structure-activity relationship (3D-QSAR) model of sulfonamide analogs binding a monoclonal antibody (MAbSMR) produced against sulfamerazine was carried out by Distance Comparison (DISCOtech), comparative molecular field analysis (CoMFA), and comparative molecular si...

  2. Cu2+-labeled dansyl compounds as fluorescent and PET probes for imaging apoptosis.

    Science.gov (United States)

    Han, Junyan; Wang, Xukui; Yu, MeiXiang

    2016-11-15

    Compound DNSTT-Cu 2+ , a novel chelate of Cu 2+ with DOTA conjugated to a fluorescent dansyl fragment, is developed for imaging cell apoptosis. Apoptotic U-87MG cells could be selectively visualized by the fluorescence of DNSTT-Cu 2+ from cytoplasm of cells, confirmed by the fluorescence of apoptosis cells co-labeled with Alexa Fluor 568-labeled annexin V, a conventional probe for selectively labeling membranes of apoptosis cells. A radioactive 64 Cu 2 + analog, DNSTT- 64 Cu 2+ , was easily synthesized, providing a potential PET probe for imaging apoptosis in vivo. Copyright © 2016 Elsevier Ltd. All rights reserved.

  3. Synthesis and Properties of the p-Sulfonamide Analogue of the Green Fluorescent Protein (GFP) Chromophore: The Mimic of GFP Chromophore with Very Strong N-H Photoacid Strength.

    Science.gov (United States)

    Chen, Yi-Hui; Sung, Robert; Sung, Kuangsen

    2018-04-06

    The para-sulfonamide analogue ( p-TsABDI) of a green fluorescent protein (GFP) chromophore was synthesized to mimic the GFP chromophore. Its S 1 excited-state p K a * value in dimethylsulfoxide (DMSO) is -1.5, which is strong enough to partially protonate dipolar aprotic solvents and causes excited-state proton transfer (ESPT), so it can partially mimic the GFP chromophore to further study the ESPT-related photophysics and the blinking phenomenon of GFP. In comparison with 8-hydroxypyrene-1,3,6-trisulfonate (HPTS) (p K a = 7.4, p K a * = 1.3 in water), p-TsABDI (p K a = 6.7, p K a * = -1.5 in DMSO) is a better photoacid for pH-jump studies.

  4. Threshold analysis and biodistribution of fluorescently labeled bevacizumab in human breast cancer

    NARCIS (Netherlands)

    Koch, Maximillian; de Jong, Johannes S; Glatz, Jürgen; Symvoulidis, Panagiotis; Lamberts, Laetitia E; Adams, Arthur; Kranendonk, Mariëtte E G; Terwisscha van Scheltinga, Anton G T; Aichler, Michaela; Jansen, Liesbeth; de Vries, Jakob; Lub-de Hooge, Marjolijn N; Schröder, Carolina P; Jorritsma-Smit, Annelies; Linssen, Matthijs D; de Boer, Esther; van der Vegt, Bert; Nagengast, Wouter B; Elias, Sjoerd G; Oliveira, Sabrina; Witkamp, Arjen; Mali, Willem P Th M; van der Wall, Elsken; Garcia-Allende, Beatriz P; Van Diest, Paul J; de Vries, Elisabeth G; Walch, Axel; van Dam, Gooitzen M; Ntziachristos, Vasilis

    2017-01-01

    In vivo tumor labeling with fluorescent agents may assist endoscopic and surgical guidance for cancer therapy as well as create opportunities to directly observe cancer biology in patients. However, malignant and non-malignant tissues are usually distinguished on fluorescence images by applying

  5. Threshold analysis and biodistribution of fluorescently labeled bevacizumab in human breast cancer

    NARCIS (Netherlands)

    Koch, Maximillian; de Jong, Johannes S; Glatz, Jürgen; Symvoulidis, Panagiotis; Lamberts, Laetitia E; Adams, Arthur; Kranendonk, Mariëtte E G; Terwisscha van Scheltinga, Anton G T; Aichler, Michaela; Jansen, Liesbeth; de Vries, Jakob; Lub-de Hooge, Marjolijn N; Schröder, Carolina P; Jorritsma-Smit, Annelies; Linssen, Matthijs D; de Boer, Esther; van der Vegt, Bert; Nagengast, Wouter B; Elias, Sjoerd G; Oliveira, Sabrina|info:eu-repo/dai/nl/304841455; Witkamp, Arjen; Mali, Willem P Th M; van der Wall, Elsken; Garcia-Allende, Beatriz P; Van Diest, Paul J; de Vries, Elisabeth G; Walch, Axel; van Dam, Gooitzen M; Ntziachristos, Vasilis

    2016-01-01

    In vivo tumor labeling with fluorescent agents may assist endoscopic and surgical guidance for cancer therapy as well as create opportunities to directly observe cancer biology in patients. However, malignant and non-malignant tissues are usually distinguished on fluorescence images by applying

  6. Nanodiamonds with silicon vacancy defects for nontoxic photostable fluorescent labeling of neural precursor cells.

    Science.gov (United States)

    Merson, Tobias D; Castelletto, Stefania; Aharonovich, Igor; Turbic, Alisa; Kilpatrick, Trevor J; Turnley, Ann M

    2013-10-15

    Nanodiamonds (NDs) containing silicon vacancy (SiV) defects were evaluated as a potential biomarker for the labeling and fluorescent imaging of neural precursor cells (NPCs). SiV-containing NDs were synthesized using chemical vapor deposition and silicon ion implantation. Spectrally, SiV-containing NDs exhibited extremely stable fluorescence and narrow bandwidth emission with an excellent signal to noise ratio exceeding that of NDs containing nitrogen-vacancy centers. NPCs labeled with NDs exhibited normal cell viability and proliferative properties consistent with biocompatibility. We conclude that SiV-containing NDs are a promising biomedical research tool for cellular labeling and optical imaging in stem cell research.

  7. Post-PCR detection of nucleic acids using metalloporphyrin labels and time-resolved fluorescence

    International Nuclear Information System (INIS)

    O'Shea, Desmond J.; O'Sullivan, Paul J.; Ponomarev, Gelii V.; Papkovsky, Dmitri B.

    2005-01-01

    Phosphorescent platinum(II)-coproporphyrin label (PtCP) was evaluated in post-PCR detection of nucleic acids by time-resolved fluorescence (TR-F) using three common formats. PtCP-labelled oligonucleotide primers and PtCP-dUTP were incorporated in a PCR to produce labelled amplified target -173 or 305 bp DNA. Alternatively, aminoallyl-dUTP was incorporated in a PCR and the product was subsequently labelled with PtCP. The resulting PCR mixtures containing labelled dsDNA were separated on 1.5% agarose gels and then analysed by ethidium bromide staining and by direct detection of PtCP label on a commercial TR-F plate reader Victor 2 (Perkin Elmer Life Sciences) used in scanning mode. In all cases label incorporation and high yields of amplified DNA were observed. Direct TR-F detection of PtCP-labelled DNA from a gel provided high sensitivity and signal to noise ratio, with limits of detection in the range of 9-22 pg for all three formats. The sensitivity achieved with PtCP label was considerably better than that achieved with ethidium bromide staining (∼1 ng of dsDNA) or with conventional fluorescent label FITC. Neither the FITC label nor ethidium bromide staining interfered with PtCP detection, thus allowing multiplexed detection

  8. Synthesis and Preliminary Biological Evaluations of Fluorescent or 149Promethium Labeled Trastuzumab-Polyethylenimine

    Directory of Open Access Journals (Sweden)

    Jonathan Fitzsimmons

    2015-12-01

    Full Text Available Background: Radioimmunotherapy utilize a targeting antibody coupled to a therapeutic isotope to target and treat a tumor or disease. In this study we examine the synthesis and cell binding of a polymer scaffold containing a radiotherapeutic isotope and a targeting antibody. Methods: The multistep synthesis of a fluorescent or 149Promethium-labeled Trastuzumab-polyethyleneimine (PEI, Trastuzumab, or PEI is described. In vitro uptake, internalization and/or the binding affinity to the Her2/neu expressing human breast adenocarcinoma SKBr3 cells was investigated with the labeled compounds. Results: Fluorescent-labeled Trastuzumab-PEI was internalized more into cells at 2 and 18 h than fluorescent-labeled Trastuzumab or PEI. The fluorescent-labeled Trastuzumab was concentrated on the cell surface at 2 and 18 h and the labeled PEI had minimal uptake. DOTA-PEI was prepared and contained an average of 16 chelates per PEI; the compound was radio-labeled with 149Promethium and conjugated to Trastuzumab. The purified 149Pm-DOTA-PEI-Trastuzumab had a radiochemical purity of 96.7% and a specific activity of 0.118 TBq/g. The compound demonstrated a dissociation constant for the Her2/neu receptor of 20.30 ± 6.91 nM. Conclusion: The results indicate the DOTA-PEI-Trastuzumab compound has potential as a targeted therapeutic carrier, and future in vivo studies should be performed.

  9. Fluorescence labelling as tool for zeolite particle tracking in nanoremediation approaches

    International Nuclear Information System (INIS)

    Gillies, Glenn; Mackenzie, Katrin; Kopinke, Frank-Dieter; Georgi, Anett

    2016-01-01

    Colloidal Fe-zeolites such as Fe-BEA-35 are currently under study as new adsorbent and catalyst materials for in-situ chemical oxidation with H_2O_2. As for nanoremediation in general, the availability of suitable particle detection methods is a requirement for successful process development and particle tracing. Detection and distinguishing between natural colloids and introduced particles with a similar composition are a challenge. By means of fluorescence labelling, a highly specific detection option for Fe-BEA-35 was developed. ‘Ship-in-a-bottle’ synthesis of fluorescein within the zeolite pores, which was applied for the first time for a BEA type zeolite, provides a product with stable and non-extractable fluorescence. When the fluorescent labelled zeolite is added at a concentration of 1 wt.% referring to the total zeolite mass, a very low detection limit of 1 mg/L of total zeolite is obtained. Compared to commonly applied turbidity measurements, detection via fluorescence labelling is much more specific and sensitive. Fluorescence is only marginally affected by carboxymethyl cellulose, which is frequently applied as stabilizer in application suspensions but will be depleted upon contact with H_2O_2. Transport properties of fluorescent labelled and non-labelled Fe-zeolite particles are in agreement as determined in a column study with quartz sand and synthetic groundwater (classified as very hard). - Highlights: • Fluorescent BEA zeolite was prepared for first time by ‘ship-in-a-bottle’ synthesis. • Fluorescein synthesized inside zeolite channels is stable and non-extractable. • Detection limit of Fe-zeolite particles in suspension with 1 wt.% fluorescent zeolite is 1 mg/L. • Transport properties of fluorescent and Fe-loaded BEA particles are identical.

  10. Aptamer-mediated indirect quantum dot labeling and fluorescent imaging of target proteins in living cells

    International Nuclear Information System (INIS)

    Liu, Jianbo; Zhang, Pengfei; Yang, Xiaohai; Wang, Kemin; Guo, Qiuping; Huang, Jin; Li, Wei

    2014-01-01

    Protein labeling for dynamic living cell imaging plays a significant role in basic biological research, as well as in clinical diagnostics and therapeutics. We have developed a novel strategy in which the dynamic visualization of proteins within living cells is achieved by using aptamers as mediators for indirect protein labeling of quantum dots (QDs). With this strategy, the target protein angiogenin was successfully labeled with fluorescent QDs in a minor intactness model, which was mediated by the aptamer AL6-B. Subsequent living cell imaging analyses indicated that the QDs nanoprobes were selectively bound to human umbilical vein endothelial cells, gradually internalized into the cytoplasm, and mostly localized in the lysosome organelle, indicating that the labeled protein retained high activity. Compared with traditional direct protein labeling methods, the proposed aptamer-mediated strategy is simple, inexpensive, and provides a highly selective, stable, and intact labeling platform that has shown great promise for future biomedical labeling and intracellular protein dynamic analyses. (paper)

  11. Site-Specific Bioorthogonal Labeling for Fluorescence Imaging of Intracellular Proteins in Living Cells.

    Science.gov (United States)

    Peng, Tao; Hang, Howard C

    2016-11-02

    Over the past years, fluorescent proteins (e.g., green fluorescent proteins) have been widely utilized to visualize recombinant protein expression and localization in live cells. Although powerful, fluorescent protein tags are limited by their relatively large sizes and potential perturbation to protein function. Alternatively, site-specific labeling of proteins with small-molecule organic fluorophores using bioorthogonal chemistry may provide a more precise and less perturbing method. This approach involves site-specific incorporation of unnatural amino acids (UAAs) into proteins via genetic code expansion, followed by bioorthogonal chemical labeling with small organic fluorophores in living cells. While this approach has been used to label extracellular proteins for live cell imaging studies, site-specific bioorthogonal labeling and fluorescence imaging of intracellular proteins in live cells is still challenging. Herein, we systematically evaluate site-specific incorporation of diastereomerically pure bioorthogonal UAAs bearing stained alkynes or alkenes into intracellular proteins for inverse-electron-demand Diels-Alder cycloaddition reactions with tetrazine-functionalized fluorophores for live cell labeling and imaging in mammalian cells. Our studies show that site-specific incorporation of axial diastereomer of trans-cyclooct-2-ene-lysine robustly affords highly efficient and specific bioorthogonal labeling with monosubstituted tetrazine fluorophores in live mammalian cells, which enabled us to image the intracellular localization and real-time dynamic trafficking of IFITM3, a small membrane-associated protein with only 137 amino acids, for the first time. Our optimized UAA incorporation and bioorthogonal labeling conditions also enabled efficient site-specific fluorescence labeling of other intracellular proteins for live cell imaging studies in mammalian cells.

  12. Direct fluorescence anisotropy assay for cocaine using tetramethylrhodamine-labeled aptamer.

    Science.gov (United States)

    Liu, Yingxiong; Zhao, Qiang

    2017-06-01

    Development of simple, sensitive, and rapid method for cocaine detection is important in medicine and drug abuse monitoring. Taking advantage of fluorescence anisotropy and aptamer, this study reports a direct fluorescence anisotropy (FA) assay for cocaine by employing an aptamer probe with tetramethylrhodamine (TMR) labeled on a specific position. The binding of cocaine and the aptamer causes a structure change of the TMR-labeled aptamer, leading to changes of the interaction between labeled TMR and adjacent G bases in aptamer sequence, so FA of TMR varies with increasing of cocaine. After screening different labeling positions of the aptamer, including thymine (T) bases and terminals of the aptamer, we obtained a favorable aptamer probe with TMR labeled on the 25th base T in the sequence, which exhibited sensitive and significant FA-decreasing responses upon cocaine. Under optimized assay conditions, this TMR-labeled aptamer allowed for direct FA detection of cocaine as low as 5 μM. The maximum FA change reached about 0.086. This FA method also enabled the detection of cocaine spiked in diluted serum and urine samples, showing potential for applications. Graphical Abstract The binding of cocaine to the TMR-labeled aptamer causes conformation change and alteration of the intramolecular interaction between TMR and bases of aptamer, leading to variance of fluorescence anisotropy (FA) of TMR, so direct FA analyis of cocaine is achieved.

  13. Cryo-imaging of fluorescently labeled single cells in a mouse

    Science.gov (United States)

    Steyer, Grant J.; Roy, Debashish; Salvado, Olivier; Stone, Meredith E.; Wilson, David L.

    2009-02-01

    We developed a cryo-imaging system to provide single-cell detection of fluorescently labeled cells in mouse, with particular applicability to stem cells and metastatic cancer. The Case cryoimaging system consists of a fluorescence microscope, robotic imaging positioner, customized cryostat, PC-based control system, and visualization/analysis software. The system alternates between sectioning (10-40 μm) and imaging, collecting color brightfield and fluorescent blockface image volumes >60GB. In mouse experiments, we imaged quantum-dot labeled stem cells, GFP-labeled cancer and stem cells, and cell-size fluorescent microspheres. To remove subsurface fluorescence, we used a simplified model of light-tissue interaction whereby the next image was scaled, blurred, and subtracted from the current image. We estimated scaling and blurring parameters by minimizing entropy of subtracted images. Tissue specific attenuation parameters were found [uT : heart (267 +/- 47.6 μm), liver (218 +/- 27.1 μm), brain (161 +/- 27.4 μm)] to be within the range of estimates in the literature. "Next image" processing removed subsurface fluorescence equally well across multiple tissues (brain, kidney, liver, adipose tissue, etc.), and analysis of 200 microsphere images in the brain gave 97+/-2% reduction of subsurface fluorescence. Fluorescent signals were determined to arise from single cells based upon geometric and integrated intensity measurements. Next image processing greatly improved axial resolution, enabled high quality 3D volume renderings, and improved enumeration of single cells with connected component analysis by up to 24%. Analysis of image volumes identified metastatic cancer sites, found homing of stem cells to injury sites, and showed microsphere distribution correlated with blood flow patterns. We developed and evaluated cryo-imaging to provide single-cell detection of fluorescently labeled cells in mouse. Our cryo-imaging system provides extreme (>60GB), micron

  14. In situ hybridization of phytoplankton using fluorescently labeled rRNA probes

    OpenAIRE

    Groben, R.; Medlin, Linda

    2005-01-01

    Fluorescently-labelled molecular probes were used to identify and characterise phytoplankton species using in situ hybridisation coupled with fluorescence microscopy and flow cytometry. The application of this technique is sometimes problematic, because of the many different species with which this method is to be used. Problems that may occur are: probe penetration versus maintanance of cell stability, strong autofluorescence and/or cell lost during the sample processing. Here we present a m...

  15. Synthesis and evaluation of radioactive and fluorescent residualizing labels for identifying sites of plasma protein catabolism

    International Nuclear Information System (INIS)

    Maxwell, J.L.; Baynes, J.W.; Thorpe, S.R.

    1986-01-01

    Inulin and lactose were each coupled to tyramine by reductive amination with NaBH 3 CN and the tyramine then labeled with 125 I. Dilactitol- 125 I-tyramine (DLT) and inulin- 125 I-tyramine (InTn) were coupled by reductive amination and cyanuric chloride, respectively, to asialofetuin (ASF), fetuin and rat serum albumin (RSA). Attachment of either label had no effect on the circulating half-lives of the proteins. Radioactivity from labeled ASF was recovered in rat liver (> 90%) by 1 h post-injection and remained in liver with half-lives of 2 and 6 days, respectively, for the DLT and InTn labels. Whole body recoveries of radioactivity from DLT- and InTn labels. Whole body recoveries of radioactivity from DLT- and InTn-labeled RSA were 5 and 6.5 days, respectively, again indicating that the larger glycoconjugate label residualized more efficiently in cells following protein degradation. (Lactitol) 2 -N-CH 2 -CH 2 -NH-fluroescein (DLF) was also coupled to ASF by reductive amination and recovered quantitatively in liver at 1 h post-injection. Native ASF was an effective competitor for clearance of DLF-ASF from the circulation. Fluorescent degradation products were retained in liver with a half-life of 1.2 days. Residualizing fluorescent labels should be useful for identification and sorting of cells active in the degradation of plasma proteins

  16. Reliable Assessment and Quantification of the Fluorescence-Labeled Antisense Oligonucleotides In Vivo

    Directory of Open Access Journals (Sweden)

    Maria Chiara Munisso

    2014-01-01

    Full Text Available The availability of fluorescent dyes and the advances in the optical systems for in vivo imaging have stimulated an increasing interest in developing new methodologies to study and quantify the biodistribution of labeled agents. However, despite these great achievements, we are facing significant challenges in determining if the observed fluorescence does correspond to the quantity of the dye in the tissues. In fact, although the far-red and near-infrared lights can propagate through several centimetres of tissue, they diffuse within a few millimetres as consequence of the elastic scattering of photons. In addition, when dye-labeled oligonucleotides form stable complex with cationic carriers, a large change in the fluorescence intensity of the dye is observed. Therefore, the measured fluorescence intensity is altered by the tissue heterogeneity and by the fluctuation of dye intensity. Hence, in this study a quantification strategy for fluorescence-labeled oligonucleotides was developed to solve these disadvantageous effects. Our results proved that upon efficient homogenization and dilution with chaotropic agents, such as guanidinium thiocyanate, it is possible to achieve a complete fluorescence intensity recovery. Furthermore, we demonstrated that this method has the advantage of good sensitivity and reproducibility, as well as easy handling of the tissue samples.

  17. Hydrogen sulfide deactivates common nitrobenzofurazan-based fluorescent thiol labeling reagents.

    Science.gov (United States)

    Montoya, Leticia A; Pluth, Michael D

    2014-06-17

    Sulfhydryl-containing compounds, including thiols and hydrogen sulfide (H2S), play important but differential roles in biological structure and function. One major challenge in separating the biological roles of thiols and H2S is developing tools to effectively separate the reactivity of these sulfhydryl-containing compounds. To address this challenge, we report the differential responses of common electrophilic fluorescent thiol labeling reagents, including nitrobenzofurazan-based scaffolds, maleimides, alkylating agents, and electrophilic aldehydes, toward cysteine and H2S. Although H2S reacted with all of the investigated scaffolds, the photophysical response to each scaffold was significantly different. Maleimide-based, alkylating, and aldehydic thiol labeling reagents provided a diminished fluorescence response when treated with H2S. By contrast, nitrobenzofurazan-based labeling reagents were deactivated by H2S addition. Furthermore, the addition of H2S to thiol-activated nitrobenzofurazan-based reagents reduced the fluorescence signal, thus establishing the incompatibility of nitrobenzofurazan-based thiol labeling reagents in the presence of H2S. Taken together, these studies highlight the differential reactivity of thiols and H2S toward common thiol-labeling reagents and suggest that sufficient care must be taken when labeling or measuring thiols in cellular environments that produce H2S due to the potential for both false-positive and eroded responses.

  18. Algorithms for Cytoplasm Segmentation of Fluorescence Labelled Cells

    OpenAIRE

    Carolina Wählby; Joakim Lindblad; Mikael Vondrus; Ewert Bengtsson; Lennart Björkesten

    2002-01-01

    Automatic cell segmentation has various applications in cytometry, and while the nucleus is often very distinct and easy to identify, the cytoplasm provides a lot more challenge. A new combination of image analysis algorithms for segmentation of cells imaged by fluorescence microscopy is presented. The algorithm consists of an image pre?processing step, a general segmentation and merging step followed by a segmentation quality measurement. The quality measurement consists of a statistical ana...

  19. Fluorescently labeled bionanotransporters of nucleic acid based on carbon nanotubes

    International Nuclear Information System (INIS)

    Novopashina, D.S.; Apartsin, E.K.; Venyaminova, A.G.

    2012-01-01

    We propose an approach to the design of a new type of hybrids of oligonucleotides with fluorescein-functionalized single-walled carbon nanotubes. The approach is based on stacking interactions of functionalized nanotubes with pyrene residues in conjugates of oligonucleotides. The amino- and fluorescein-modified single walled carbon nanotubes are obtained, and their physico-chemical properties are investigated. The effect of the functionalization type of carbon nanotubes on the efficacy of the sorption of pyrene conjugates of oligonucleotides was examined. The proposed noncovalent hybrids of fluorescein-labeled carbon nanotubes with oligonucleotides may be used for the intracellular transport of functional nucleic acids.

  20. Synthesis of a Fluorescently Labeled 68Ga-DOTA-TOC Analog for Somatostatin Receptor Targeting.

    Science.gov (United States)

    Ghosh, Sukhen C; Hernandez Vargas, Servando; Rodriguez, Melissa; Kossatz, Susanne; Voss, Julie; Carmon, Kendra S; Reiner, Thomas; Schonbrunn, Agnes; Azhdarinia, Ali

    2017-07-13

    Fluorescently labeled imaging agents can identify surgical margins in real-time to help achieve complete resections and minimize the likelihood of local recurrence. However, photon attenuation limits fluorescence-based imaging to superficial lesions or lesions that are a few millimeters beneath the tissue surface. Contrast agents that are dual-labeled with a radionuclide and fluorescent dye can overcome this limitation and combine quantitative, whole-body nuclear imaging with intraoperative fluorescence imaging. Using a multimodality chelation (MMC) scaffold, IRDye 800CW was conjugated to the clinically used somatostatin analog, 68 Ga-DOTA-TOC, to produce the dual-labeled analog, 68 Ga-MMC(IRDye 800CW)-TOC, with high yield and specific activity. In vitro pharmacological assays demonstrated retention of receptor-targeting properties for the dual-labeled compound with robust internalization that was somatostatin receptor (SSTR) 2-mediated. Biodistribution studies in mice identified the kidneys as the primary excretion route for 68 Ga-MMC(IRDye 800CW)-TOC, along with clearance via the reticuloendothelial system. Higher uptake was observed in most tissues compared to 68 Ga-DOTA-TOC but decreased as a function of time. The combination of excellent specificity for SSTR2-expressing cells and suitable biodistribution indicate potential application of 68 Ga-MMC(IRDye 800CW)-TOC for intraoperative detection of SSTR2-expressing tumors.

  1. Fluorescently Labeled Branched Polymers and Thermal Responsive Nanoparticles for Live Cell Imaging

    NARCIS (Netherlands)

    Zhou, D.; Ma, Y.; Poot, Andreas A.; Dijkstra, Pieter J.; Feijen, Jan

    2012-01-01

    Branched poly(methoxy-PEG acrylate) and thermally responsive poly(methoxy-PEG acrylate)-block-poly(N-isopropylacrylamide) are synthesized by RAFT polymerization. After reduction, these polymers are fluorescently labeled by reacting the free thiol groups with N-(5-fluoresceinyl)maleimide. As shown by

  2. Breast cancer cells synchronous labeling and separation based on aptamer and fluorescence-magnetic silica nanoparticles

    Science.gov (United States)

    Wang, Qiu-Yue; Huang, Wei; Jiang, Xing-Lin; Kang, Yan-Jun

    2018-01-01

    In this work, an efficient method based on biotin-labeled aptamer and streptavidin-conjugated fluorescence-magnetic silica nanoprobes (FITC@Fe3O4@SiNPs-SA) has been established for human breast carcinoma MCF-7 cells synchronous labeling and separation. Carboxyl-modified fluorescence-magnetic silica nanoparticles (FITC@Fe3O4@SiNPs-COOH) were first synthesized using the Stöber method. Streptavidin (SA) was then conjugated to the surface of FITC@Fe3O4@SiNPs-COOH. The MCF-7 cell suspension was incubated with biotin-labeled MUC-1 aptamer. After centrifugation and washing, the cells were then treated with FITC@Fe3O4@SiNPs-SA. Afterwards, the mixtures were separated by a magnet. The cell-probe conjugates were then imaged using fluorescent microscopy. The results show that the MUC-1 aptamer could recognize and bind to the targeted cells with high affinity and specificity, indicating the prepared FITC@Fe3O4@SiNPs-SA with great photostability and superparamagnetism could be applied effectively in labeling and separation for MCF-7 cell in suspension synchronously. In addition, the feasibility of MCF-7 cells detection in peripheral blood was assessed. The results indicate that the method above is also applicable for cancer cells synchronous labeling and separation in complex biological system.

  3. Single-Labeled Oligonucleotides Showing Fluorescence Changes upon Hybridization with Target Nucleic Acids

    Directory of Open Access Journals (Sweden)

    Gil Tae Hwang

    2018-01-01

    Full Text Available Sequence-specific detection of nucleic acids has been intensively studied in the field of molecular diagnostics. In particular, the detection and analysis of single-nucleotide polymorphisms (SNPs is crucial for the identification of disease-causing genes and diagnosis of diseases. Sequence-specific hybridization probes, such as molecular beacons bearing the fluorophore and quencher at both ends of the stem, have been developed to enable DNA mutation detection. Interestingly, DNA mutations can be detected using fluorescently labeled oligonucleotide probes with only one fluorophore. This review summarizes recent research on single-labeled oligonucleotide probes that exhibit fluorescence changes after encountering target nucleic acids, such as guanine-quenching probes, cyanine-containing probes, probes containing a fluorophore-labeled base, and microenvironment-sensitive probes.

  4. Selective Labeling of Proteins on Living Cell Membranes Using Fluorescent Nanodiamond Probes

    Directory of Open Access Journals (Sweden)

    Shingo Sotoma

    2016-03-01

    Full Text Available The impeccable photostability of fluorescent nanodiamonds (FNDs is an ideal property for use in fluorescence imaging of proteins in living cells. However, such an application requires highly specific labeling of the target proteins with FNDs. Furthermore, the surface of unmodified FNDs tends to adsorb biomolecules nonspecifically, which hinders the reliable targeting of proteins with FNDs. Here, we combined hyperbranched polyglycerol modification of FNDs with the β-lactamase-tag system to develop a strategy for selective imaging of the protein of interest in cells. The combination of these techniques enabled site-specific labeling of Interleukin-18 receptor alpha chain, a membrane receptor, with FNDs, which eventually enabled tracking of the diffusion trajectory of FND-labeled proteins on the membrane surface.

  5. Analysis of fluorescently labeled substance P analogs: binding, imaging and receptor activation

    Directory of Open Access Journals (Sweden)

    Simmons Mark A

    2001-06-01

    Full Text Available Abstract Background Substance P (SP is a peptide neurotransmitter found in central and peripheral nerves. SP is involved in the control of smooth muscle, inflammation and nociception. The amino acid sequence of SP is Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2. Five different forms of fluorescently labeled SP have recently been synthesized, in which Alexa 488, BODIPY Fl, fluorescein, Oregon Green 488 or tetramethylrhodamine has been covalently linked to SP at Lys3. Here, these novel analogs are characterized as to their ligand binding, receptor activation and fluorescence labeling properties. Results Competition binding studies, using radiolabeled [125I] SP, revealed that all of the labeled forms of SP, except for Alexa 488-SP, effectively competed with radiolabeled SP for binding at the rat SP receptor. With the exception of Alexa 488-SP, all of the SP analogs produced Ca++ elevations and fluorescence labeling of the SP receptor expressed in Chinese hamster ovary cells. In SP-responsive neurons, BODIPY Fl-SP and Oregon Green 488-SP were as effective as unlabeled SP in producing a reduction of the M-type K+ current. Fluorescein-SP produced variable results, while tetramethylrhodamine-SP was less potent and Alexa 488-SP was less effective on intact neurons. Conclusions The above results show that fluorescent labeling of SP altered the biological activity and the binding properties of the parent peptide. Oregon Green 488 and BODIPY FL-SP are the most useful fluorophores for labeling SP without affecting its biological activity. Given these results, these probes can now be utilized in further investigations of the mechanisms of SPR function, including receptor localization, internalization and recycling.

  6. Selective labeling of a single organelle by using two-photon conversion of a photoconvertible fluorescent protein

    Science.gov (United States)

    Watanabe, Wataru; Shimada, Tomoko; Matsunaga, Sachihiro; Kurihara, Daisuke; Arimura, Shin-ichi; Tsutsumi, Nobuhiro; Fukui, Kiichi; Itoh, Kazuyoshi

    2008-02-01

    We present space-selective labeling of organelles by using two-photon conversion of a photoconvertible fluorescent protein with near-infrared femtosecond laser pulses. Two-photon excitation of photoconvertible fluorescent-protein, Kaede, enables space-selective labeling of organelles. We alter the fluorescence of target mitochondria in a tobacco BY-2 cell from green to red by focusing femtosecond laser pulses with a wavelength of 750 nm.

  7. A SIMPLE FLUORESCENT LABELING METHOD FOR STUDIES OF PROTEIN OXIDATION, PROTEIN MODIFICATION, AND PROTEOLYSIS

    Science.gov (United States)

    Pickering, Andrew. M.; Davies, Kelvin. J. A.

    2014-01-01

    Proteins are sensitive to oxidation, and oxidized proteins are excellent substrates for degradation by proteolytic enzymes such as the Proteasome and the mitochondrial Lon protease. Protein labeling is required for studies of protein turnover. Unfortunately, most labeling techniques involve 3H or 14C methylation which is expensive, exposes researchers to radioactivity, generates large amounts of radioactive waste, and allows only single-point assays because samples require acid-precipitation. Alternative labeling methods, have largely proven unsuitable, either because the probe itself is modified by the oxidant(s) being studied, or because the alternative labeling techniques are too complex or too costly for routine use. What is needed is a simple, quick, and cheap labeling technique that uses a non-radioactive marker, that binds strongly to proteins, is resistant to oxidative modification, and emits a strong signal. We have devised a new reductive method for labeling free carboxyl groups of proteins with the small fluorophore 7-amino-4-methycoumarin (AMC). When bound to target proteins, AMC fluoresces very weakly but when AMC is released by proteinases, proteases, or peptidases, it fluoresces strongly. Thus, without acid-precipitation, the proteolysis of any target protein can be studied continuously, in multiwell plates. In direct comparisons, 3H-labeled proteins and AMC-labeled proteins exhibited essentially identical degradation patterns during incubation with trypsin, cell extracts, and purified proteasome. AMC-labeled proteins are well-suited to study increased proteolytic susceptibility following protein modification, since the AMC-protein bond is resistant to oxidizing agents such as hydrogen peroxide and peroxynitrite, and is stable over time and to extremes of pH, temperature (even boiling), freeze-thawing, mercaptoethanol, and methanol. PMID:21988844

  8. Algorithms for Cytoplasm Segmentation of Fluorescence Labelled Cells

    Directory of Open Access Journals (Sweden)

    Carolina Wählby

    2002-01-01

    Full Text Available Automatic cell segmentation has various applications in cytometry, and while the nucleus is often very distinct and easy to identify, the cytoplasm provides a lot more challenge. A new combination of image analysis algorithms for segmentation of cells imaged by fluorescence microscopy is presented. The algorithm consists of an image pre‐processing step, a general segmentation and merging step followed by a segmentation quality measurement. The quality measurement consists of a statistical analysis of a number of shape descriptive features. Objects that have features that differ to that of correctly segmented single cells can be further processed by a splitting step. By statistical analysis we therefore get a feedback system for separation of clustered cells. After the segmentation is completed, the quality of the final segmentation is evaluated. By training the algorithm on a representative set of training images, the algorithm is made fully automatic for subsequent images created under similar conditions. Automatic cytoplasm segmentation was tested on CHO‐cells stained with calcein. The fully automatic method showed between 89% and 97% correct segmentation as compared to manual segmentation.

  9. Sensitive determination of pipecolic acid in serum by high-performance liquid chromatography using 4-(5,6-dimethoxy-2-phthalimidinyl)-2-methoxyphenylsulfonyl chloride as a fluorescent labelling reagent

    International Nuclear Information System (INIS)

    Inoue, Hirofumi; Sakata, Yasuhiko; Fukunaga, Keiko; Nishio, Hiroaki; Tsuruta, Yasuto

    2004-01-01

    A simple and highly sensitive high-performance liquid chromatography (HPLC) for the determination of pipecolic acid (PA) in serum was developed. Pipecolic acid and nipecotic acid (internal standard (IS)) were derivatised with 4-(5,6-dimethoxy-2-phthalimidinyl)-2-methoxyphenylsulfonyl chloride (DMS-Cl) to produce fluorescent sulfonamides. The labelling reaction was carried out at 70 degree sign C for 15 min at pH 9.0. The fluorescent derivatives were separated on a reversed-phase column (45 degree sign C) with a stepwise elution using methanol/acetonitrile/10 mmol l -1 acetic acid (42:5:53) and methanol at a flow rate of 1.0 ml/min and detected at excitation and emission wavelengths of 316 and 403 nm, respectively. The labelling yield was 100.8%. The detection limit of pipecolic acid was 4 fmol at signal-to-noise ratio of 3. The within-day and day-to-day relative standard deviations were 3.3-8.1 and 1.4-6.4%, respectively. The concentration of pipecolic acid in normal human serum was 1.09±0.37 μmol l -1

  10. Fluorescence correlation spectroscopy analysis for accurate determination of proportion of doubly labeled DNA in fluorescent DNA pool for quantitative biochemical assays.

    Science.gov (United States)

    Hou, Sen; Sun, Lili; Wieczorek, Stefan A; Kalwarczyk, Tomasz; Kaminski, Tomasz S; Holyst, Robert

    2014-01-15

    Fluorescent double-stranded DNA (dsDNA) molecules labeled at both ends are commonly produced by annealing of complementary single-stranded DNA (ssDNA) molecules, labeled with fluorescent dyes at the same (3' or 5') end. Because the labeling efficiency of ssDNA is smaller than 100%, the resulting dsDNA have two, one or are without a dye. Existing methods are insufficient to measure the percentage of the doubly-labeled dsDNA component in the fluorescent DNA sample and it is even difficult to distinguish the doubly-labeled DNA component from the singly-labeled component. Accurate measurement of the percentage of such doubly labeled dsDNA component is a critical prerequisite for quantitative biochemical measurements, which has puzzled scientists for decades. We established a fluorescence correlation spectroscopy (FCS) system to measure the percentage of doubly labeled dsDNA (PDL) in the total fluorescent dsDNA pool. The method is based on comparative analysis of the given sample and a reference dsDNA sample prepared by adding certain amount of unlabeled ssDNA into the original ssDNA solution. From FCS autocorrelation functions, we obtain the number of fluorescent dsDNA molecules in the focal volume of the confocal microscope and PDL. We also calculate the labeling efficiency of ssDNA. The method requires minimal amount of material. The samples have the concentration of DNA in the nano-molar/L range and the volume of tens of microliters. We verify our method by using restriction enzyme Hind III to cleave the fluorescent dsDNA. The kinetics of the reaction depends strongly on PDL, a critical parameter for quantitative biochemical measurements. Copyright © 2013 Elsevier B.V. All rights reserved.

  11. Label free selective detection of estriol using graphene oxide-based fluorescence sensor

    Science.gov (United States)

    Kushwaha, H. S.; Sao, Reshma; Vaish, Rahul

    2014-07-01

    Water-soluble and fluorescent Graphene oxide (GO) is biocompatible, easy, and economical to synthesize. Interestingly, GO is also capable of quenching fluorescence. On the basis of its fluorescence and quenching abilities, GO has been reported to serve as an energy acceptor in a fluorescence resonance energy transfer (FRET) sensor. GO-based FRET biosensors have been widely reported for sensing of proteins, nucleic acid, ATP (Adenosine triphosphate), etc. GO complexes with fluorescent dyes and enzymes have been used to sense metal ions. Graphene derivatives have been used for sensing endocrine-disrupting chemicals like bisphenols and chlorophenols with high sensitivity and good reproducibility. On this basis, a novel GO based fluorescent sensor has been successfully designed to detect estriol with remarkable selectivity and sensitivity. Estriol is one of the three estrogens in women and is considered to be medically important. Estriol content of maternal urine or plasma acts as an important screening marker for estimating foetal growth and development. In addition, estriol is also used as diagnostic marker for diseases like breast cancer, osteoporosis, neurodegenerative and cardiovascular diseases, insulin resistance, lupus erythematosus, endometriosis, etc. In this present study, we report for the first time a rapid, sensitive with detection limit of 1.3 nM, selective and highly biocompatible method for label free detection of estriol under physiological conditions using fluorescence assay.

  12. Graphene oxide based photoinduced charge transfer label-free near-infrared fluorescent biosensor for dopamine.

    Science.gov (United States)

    Chen, Jin-Long; Yan, Xiu-Ping; Meng, Kang; Wang, Shu-Feng

    2011-11-15

    While the super fluorescence quenching capacity of graphene and graphene oxide (GO) has been extensively employed to develop fluorescent sensors, their own unique fluorescence and its potential for chemo-/biosensing have seldom been explored. Here we report a GO-based photoinduced charge transfer (PCT) label-free near-infrared (near-IR) fluorescent biosensor for dopamine (DA). The multiple noncovalent interactions between GO and DA and the ultrafast decay at the picosecond range of the near-IR fluorescence of GO resulted in effective self-assembly of DA molecules on the surface of GO, and significant fluorescence quenching, allowing development of a PCT-based biosensor with direct readout of the near-IR fluorescence of GO for selective and sensitive detection of DA. The developed method gave a detection limit of 94 nM and a relative standard deviation of 2.0% for 11 replicate detections of 2.0 μM DA and was successfully applied to the determination of DA in biological fluids with quantitative recovery (98-115%).

  13. In vivo tomographic imaging with fluorescence and MRI using tumor-targeted dual-labeled nanoparticles

    Directory of Open Access Journals (Sweden)

    Zhang Y

    2013-12-01

    Full Text Available Yue Zhang,1 Bin Zhang,1 Fei Liu,1,2 Jianwen Luo,1,3 Jing Bai1 1Department of Biomedical Engineering, School of Medicine, 2Tsinghua-Peking Center for Life Sciences, 3Center for Biomedical Imaging Research, Tsinghua University, Beijing, People's Republic of China Abstract: Dual-modality imaging combines the complementary advantages of different modalities, and offers the prospect of improved preclinical research. The combination of fluorescence imaging and magnetic resonance imaging (MRI provides cross-validated information and direct comparison between these modalities. Here, we report on the application of a novel tumor-targeted, dual-labeled nanoparticle (NP, utilizing iron oxide as the MRI contrast agent and near infrared (NIR dye Cy5.5 as the fluorescent agent. Results of in vitro experiments verified the specificity of the NP to tumor cells. In vivo tumor targeting and uptake of the NPs in a mouse model were visualized by fluorescence and MR imaging collected at different time points. Quantitative analysis was carried out to evaluate the efficacy of MRI contrast enhancement. Furthermore, tomographic images were also acquired using both imaging modalities and cross-validated information of tumor location and size between these two modalities was revealed. The results demonstrate that the use of dual-labeled NPs can facilitate the dual-modal detection of tumors, information cross-validation, and direct comparison by combing fluorescence molecular tomography (FMT and MRI. Keywords: dual-modality, fluorescence molecular tomography (FMT, magnetic resonance imaging (MRI, nanoparticle

  14. Magnetic targeting of iron-oxide-labeled fluorescent hepatoma cells to the liver

    Energy Technology Data Exchange (ETDEWEB)

    Luciani, Alain [Universite Rene Descartes, Hopital Europeen Georges Pompidou, Laboratoire de Recherche en Imagerie, EA 4062, Paris (France); Imagerie Medicale, Faculte de Medecine Paris XII, CHU Henri Mondor, Creteil cedex (France); Wilhelm, Claire; Gazeau, Florence [Universite Paris Diderot, Batiment Condorcet, Laboratoire Matiere et Systemes Complexes, CNRS-UMR 7057, Paris Cedex (France); Bruneval, Patrick [Anatomopathologie, Hopital Europeen Georges Pompidou, Paris (France); Cunin, Patrick [Unite de Recherche Clinique, Faculte de Medecine Paris XII, CHU Henri Mondor, Creteil cedex (France); Autret, Gwennhael; Clement, Olivier [Universite Rene Descartes, Hopital Europeen Georges Pompidou, Laboratoire de Recherche en Imagerie, EA 4062, Paris (France); Rahmouni, Alain [Imagerie Medicale, Faculte de Medecine Paris XII, CHU Henri Mondor, Creteil cedex (France)

    2009-05-15

    The purpose of this study was to determine whether an external magnet field can induce preferential trafficking of magnetically labeled Huh7 hepatoma cells to the liver following liver cell transplantation. Huh7 hepatoma cells were labeled with anionic magnetic nanoparticles (AMNP) and tagged with a fluorescent membrane marker (PKH67). Iron-uptake was measured by magnetophoresis. Twenty C57Bl6 mice received an intrasplenic injection of 2 x 10{sup 6} labeled cells. An external magnet (0.29 T; 25 T/m) was placed over the liver of 13 randomly selected animals (magnet group), while the remaining 7 animals served as controls. MRI (1.5 T) and confocal fluorescence microscopy (CFM) were performed 10 days post-transplantation. The presence and location of labeled cells within the livers were compared in the magnet group and controls, and confronted with histological analysis representing the standard of reference. Mean iron content per cell was 6 pg. Based on histology, labeled cells were more frequently present within recipient livers in the magnet group (p < 0.01) where their distribution was preferentially peri-vascular (p<0.05). MRI and CFM gave similar results for the overall detection of transplanted cells (kappa=0.828) and for the identification of peri-vascular cells (kappa=0.78). Application of an external magnet can modify the trafficking of transplanted cells, especially by promoting the formation of perivascular aggregates. (orig.)

  15. Magnetic targeting of iron-oxide-labeled fluorescent hepatoma cells to the liver

    International Nuclear Information System (INIS)

    Luciani, Alain; Wilhelm, Claire; Gazeau, Florence; Bruneval, Patrick; Cunin, Patrick; Autret, Gwennhael; Clement, Olivier; Rahmouni, Alain

    2009-01-01

    The purpose of this study was to determine whether an external magnet field can induce preferential trafficking of magnetically labeled Huh7 hepatoma cells to the liver following liver cell transplantation. Huh7 hepatoma cells were labeled with anionic magnetic nanoparticles (AMNP) and tagged with a fluorescent membrane marker (PKH67). Iron-uptake was measured by magnetophoresis. Twenty C57Bl6 mice received an intrasplenic injection of 2 x 10 6 labeled cells. An external magnet (0.29 T; 25 T/m) was placed over the liver of 13 randomly selected animals (magnet group), while the remaining 7 animals served as controls. MRI (1.5 T) and confocal fluorescence microscopy (CFM) were performed 10 days post-transplantation. The presence and location of labeled cells within the livers were compared in the magnet group and controls, and confronted with histological analysis representing the standard of reference. Mean iron content per cell was 6 pg. Based on histology, labeled cells were more frequently present within recipient livers in the magnet group (p < 0.01) where their distribution was preferentially peri-vascular (p<0.05). MRI and CFM gave similar results for the overall detection of transplanted cells (kappa=0.828) and for the identification of peri-vascular cells (kappa=0.78). Application of an external magnet can modify the trafficking of transplanted cells, especially by promoting the formation of perivascular aggregates. (orig.)

  16. Radio- and fluorescence-labelling of thapsigargin, a selective inhibitor of microsomal calcium-ATPase

    Energy Technology Data Exchange (ETDEWEB)

    Andersen, A.; Lauridsen, A.; Christensen, S.B. (Copenhagen Univ. (Denmark). Royal Danish School of Pharmacy)

    1992-03-01

    Debutanoylthapsigargin (2) labelled in the skeleton was prepared by stereoselective reduction of the ketone obtained by oxidation of debutanoylthapsigargin. Butanoylation of 2 yielded thapsigargin. The use of sodium boro({sup 3}H)hydride as a reducing agent afforded labelled debutanoyl thapsigargin with a specific activity of 22 Ci/mmol. A fluorescent analogue of thapsigargin (4a) was prepared by allowing 2 to react with N-dansyl-{beta}-alanine. Acetylation of 4a afforded a trisacetate the missing bioactivity of which allows it to be used as a negative control. (Author).

  17. Radio- and fluorescence-labelling of thapsigargin, a selective inhibitor of microsomal calcium-ATPase

    International Nuclear Information System (INIS)

    Andersen, A.; Lauridsen, A.; Christensen, S.B.

    1992-01-01

    Debutanoylthapsigargin (2) labelled in the skeleton was prepared by stereoselective reduction of the ketone obtained by oxidation of debutanoylthapsigargin. Butanoylation of 2 yielded thapsigargin. The use of sodium boro[ 3 H]hydride as a reducing agent afforded labelled debutanoyl thapsigargin with a specific activity of 22 Ci/mmol. A fluorescent analogue of thapsigargin (4a) was prepared by allowing 2 to react with N-dansyl-β-alanine. Acetylation of 4a afforded a trisacetate the missing bioactivity of which allows it to be used as a negative control. (Author)

  18. Production and testing of 244Cm-labeled fluorescent polystyrene latex microspheres

    International Nuclear Information System (INIS)

    Guilmette, R.A.; Mueller, H.L.; Brodbeck, R.D.

    1988-01-01

    To provide a useful tool for studying the mechanisms of retention and translocation of respirable-sized, alpha-emitting particles, we have developed a method of incorporating 244 Cm into fluorescent polystyrene latex microspheres. The resultant particles contain alpha radioactivity comparable to μm-size (AMAD) 239 Pu0 2 particles, but are easily visible by fluorescent light microscopy. Preliminary testing of the particles with dog macrophages in vitro has shown that the initial uptake of these Cm-labeled particles is more rapid than uptake of unlabeled particles of similar size. We are continuing to develop procedures for achieving better particle yields, smaller dispersity of particle size distributions and improved retention of the Cm label by the particles. (author)

  19. Production and testing of {sup 244}Cm-labeled fluorescent polystyrene latex microspheres

    Energy Technology Data Exchange (ETDEWEB)

    Guilmette, R A; Mueller, H L; Brodbeck, R D

    1988-12-01

    To provide a useful tool for studying the mechanisms of retention and translocation of respirable-sized, alpha-emitting particles, we have developed a method of incorporating {sup 244}Cm into fluorescent polystyrene latex microspheres. The resultant particles contain alpha radioactivity comparable to {mu}m-size (AMAD) {sup 239}Pu0{sub 2} particles, but are easily visible by fluorescent light microscopy. Preliminary testing of the particles with dog macrophages in vitro has shown that the initial uptake of these Cm-labeled particles is more rapid than uptake of unlabeled particles of similar size. We are continuing to develop procedures for achieving better particle yields, smaller dispersity of particle size distributions and improved retention of the Cm label by the particles. (author)

  20. Intrinsically Labeled Fluorescent Oligonucleotide Probes on Quantum Dots for Transduction of Nucleic Acid Hybridization.

    Science.gov (United States)

    Shahmuradyan, Anna; Krull, Ulrich J

    2016-03-15

    Quantum dots (QDs) have been widely used in chemical and biosensing due to their unique photoelectrical properties and are well suited as donors in fluorescence resonance energy transfer (FRET). Selective hybridization interactions of oligonucleotides on QDs have been determined by FRET. Typically, the QD-FRET constructs have made use of labeled targets or have implemented labeled sandwich format assays to introduce dyes in proximity to the QDs for the FRET process. The intention of this new work is to explore a method to incorporate the acceptor dye into the probe molecule. Thiazole orange (TO) derivatives are fluorescent intercalating dyes that have been used for detection of double-stranded nucleic acids. One such dye system has been reported in which single-stranded oligonucleotide probes were doubly labeled with adjacent thiazole orange derivatives. In the absence of the fully complementary (FC) oligonucleotide target, the dyes form an H-aggregate, which results in quenching of fluorescence emission due to excitonic interactions between the dyes. The hybridization of the FC target to the probe provides for dissociation of the aggregate as the dyes intercalate into the double stranded duplex, resulting in increased fluorescence. This work reports investigation of the dependence of the ratiometric signal on the type of linkage used to conjugate the dyes to the probe, the location of the dye along the length of the probe, and the distance between adjacent dye molecules. The limit of detection for 34mer and 90mer targets was found to be identical and was 10 nM (2 pmol), similar to analogous QD-FRET using labeled oligonucleotide target. The detection system could discriminate a one base pair mismatch (1BPM) target and was functional without substantial compromise of the signal in 75% serum. The 1BPM was found to reduce background signal, indicating that the structure of the mismatch affected the environment of the intercalating dyes.

  1. Alexa Fluor-labeled Fluorescent Cellulose Nanocrystals for Bioimaging Solid Cellulose in Spatially Structured Microenvironments

    Energy Technology Data Exchange (ETDEWEB)

    Grate, Jay W.; Mo, Kai-For; Shin, Yongsoon; Vasdekis, Andreas; Warner, Marvin G.; Kelly, Ryan T.; Orr, Galya; Hu, Dehong; Dehoff, Karl J.; Brockman, Fred J.; Wilkins, Michael J.

    2015-03-18

    Cellulose nanocrystal materials have been labeled with modern Alexa Fluor dyes in a process that first links the dye to a cyanuric chloride molecule. Subsequent reaction with cellulose nanocrystals provides dyed solid microcrystalline cellulose material that can be used for bioimaging and suitable for deposition in films and spatially structured microenvironments. It is demonstrated with single molecular fluorescence microscopy that these films are subject to hydrolysis by cellulose enzymes.

  2. Conformational detection of prion protein with biarsenical labeling and FlAsH fluorescence

    International Nuclear Information System (INIS)

    Coleman, Bradley M.; Nisbet, Rebecca M.; Han, Sen; Cappai, Roberto; Hatters, Danny M.; Hill, Andrew F.

    2009-01-01

    Prion diseases are associated with the misfolding of the host-encoded cellular prion protein (PrP C ) into a disease associated form (PrP Sc ). Recombinant PrP can be refolded into either an α-helical rich conformation (α-PrP) resembling PrP C or a β-sheet rich, protease resistant form similar to PrP Sc . Here, we generated tetracysteine tagged recombinant PrP, folded this into α- or β-PrP and determined the levels of FlAsH fluorescence. Insertion of the tetracysteine tag at three different sites within the 91-111 epitope readily distinguished β-PrP from α-PrP upon FlAsH labeling. Labelling of tetracysteine tagged PrP in the α-helical form showed minimal fluorescence, whereas labeling of tagged PrP in the β-sheet form showed high fluorescence indicating that this region is exposed upon conversion. This highlights a region of PrP that can be implicated in the development of diagnostics and is a novel, protease free mechanism for distinguishing PrP Sc from PrP C . This technique may also be applied to any protein that undergoes conformational change and/or misfolding such as those involved in other neurodegenerative disorders including Alzheimer's, Huntington's and Parkinson's diseases.

  3. Fluorescently labelled multiplex lateral flow immunoassay based on cadmium-free quantum dots.

    Science.gov (United States)

    Beloglazova, Natalia V; Sobolev, Aleksander M; Tessier, Mickael D; Hens, Zeger; Goryacheva, Irina Yu; De Saeger, Sarah

    2017-03-01

    A sensitive tool for simultaneous qualitative detection of two mycotoxins based on use of non-cadmium quantum dots (QDs) is presented for the first time. QDs have proven themselves as promising fluorescent labels for biolabeling and chemical analysis. With an increasing global tendency to regulate and limit the use of hazardous elements, indium phosphide (InP) QDs are highlighted as environmentally-friendly alternatives to the highly efficient and well-studied, but potentially toxic Cd- and Pb-based QDs. Here, we developed water-soluble InP QDs-based fluorescent nanostructures. They consisted of core/shell InP/ZnS QDs enrobed in a silica shell that allowed the water solubility (QD@SiO 2 ). Then we applied the QD@SiO 2 as novel, silica shell-encapsulated fluorescent labels in immunoassays for rapid multiplexed screening. Two mycotoxins, zearalenone and deoxynivalenol, were simultaneously detected in maize and wheat, since the two QD@SiO 2 labelled conjugates emit at two different, individually detectable wavelengths. The cutoff values for the simultaneous determination were 50 and 500μgkg -1 for zearalenone and deoxynivalenol, respectively, in both maize and wheat. Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) was used to confirm the result. Copyright © 2017 Elsevier Inc. All rights reserved.

  4. Fluorescent Staining of Tea Pathogenic Fungi in Tea Leaves Using Fluorescein-labeled Lectin

    Science.gov (United States)

    Yamada, Kengo; Yoshida, Katsuyuki; Sonoda, Ryoichi

    Fluorochrome-labeled lectin, fluorescein conjugated wheat germ agglutinin (F-WGA) was applied to stain tea pathogenic fungi in tea leaf tissue. Infected leaves were fixed and decolorized with a mixture of ethanol and acetic acid, and cleared with 10% KOH for whole mount before staining with F-WGA. Hyphae of Pestalotiopsis longiseta, Pseudocercospora ocellata, Botrytis cinerea and Colletotrichum theae-sinensis fluoresced brightly in whole mount and sectioned samples of infected leaf tissue. In browned tissue, hyphae did not fluoresce frequently in whole mount sample. Autofluorescence of leaf tissue was strong in browned tissue of sections, it was removed by 10% KOH treatment before staining. Penetration hyphae of C. theae-sinensis in cell wall of trichome and hyphae in basal part of trichome did not fluoresced frequently. In whole mount samples of tea leaf infected with Exobasidium vexans and E. reticulatum, hymenia appeared on leaf surface fluoresced, but hyphae in leaf tissue did not fluoresce. In sectioned samples, hyphae fluoresced brightly when sections were treated with 10% KOH before staining.

  5. Labeling RNAs in Live Cells Using Malachite Green Aptamer Scaffolds as Fluorescent Probes.

    Science.gov (United States)

    Yerramilli, V Siddartha; Kim, Kyung Hyuk

    2018-03-16

    RNAs mediate many different processes that are central to cellular function. The ability to quantify or image RNAs in live cells is very useful in elucidating such functions of RNA. RNA aptamer-fluorogen systems have been increasingly used in labeling RNAs in live cells. Here, we use the malachite green aptamer (MGA), an RNA aptamer that can specifically bind to malachite green (MG) dye and induces it to emit far-red fluorescence signals. Previous studies on MGA showed a potential for the use of MGA for genetically tagging other RNA molecules in live cells. However, these studies also exhibited low fluorescence signals and high background noise. Here we constructed and tested RNA scaffolds containing multiple tandem repeats of MGA as a strategy to increase the brightness of the MGA aptamer-fluorogen system as well as to make the system fluoresce when tagging various RNA molecules, in live cells. We demonstrate that our MGA scaffolds can induce fluorescence signals by up to ∼20-fold compared to the basal level as a genetic tag for other RNA molecules. We also show that our scaffolds function reliably as genetically encoded fluorescent tags for mRNAs of fluorescent proteins and other RNA aptamers.

  6. Fluorescent Lipids: Functional Parts of Fusogenic Liposomes and Tools for Cell Membrane Labeling and Visualization

    Directory of Open Access Journals (Sweden)

    Christian Kleusch

    2012-01-01

    Full Text Available In this paper a rapid and highly efficient method for controlled incorporation of fluorescent lipids into living mammalian cells is introduced. Here, the fluorescent molecules have two consecutive functions: First, they trigger rapid membrane fusion between cellular plasma membranes and the lipid bilayers of their carrier particles, so called fusogenic liposomes, and second, after insertion into cellular membranes these molecules enable fluorescence imaging of cell membranes and membrane traffic processes. We tested the fluorescent derivatives of the following essential membrane lipids for membrane fusion: Ceramide, sphingomyelin, phosphocholine, phosphatidylinositol-bisphosphate, ganglioside, cholesterol, and cholesteryl ester. Our results show that all probed lipids could more efficiently be incorporated into the plasma membrane of living cells than by using other methods. Moreover, labeling occurred in a gentle manner under classical cell culture conditions reducing cellular stress responses. Staining procedures were monitored by fluorescence microscopy and it was observed that sphingolipids and cholesterol containing free hydroxyl groups exhibit a decreased distribution velocity as well as a longer persistence in the plasma membrane compared to lipids without hydroxyl groups like phospholipids or other artificial lipid analogs. After membrane staining, the fluorescent molecules were sorted into membranes of cell organelles according to their chemical properties and biological functions without any influence of the delivery system.

  7. Fluorescent Labeling and Biodistribution of Latex Nanoparticles Formed by Surfactant-Free RAFT Emulsion Polymerization.

    Science.gov (United States)

    Poon, Cheuk Ka; Tang, Owen; Chen, Xin-Ming; Kim, Byung; Hartlieb, Matthias; Pollock, Carol A; Hawkett, Brian S; Perrier, Sébastien

    2017-10-01

    The authors report the preparation of a novel range of functional polyacrylamide stabilized polystyrene nanoparticles, obtained by surfactant-free reversible addition-fragmentation chain transfer (RAFT) emulsion polymerization, their fluorescent tagging, cellular uptake, and biodistribution. The authors show the versatility of the RAFT emulsion process for the design of functional nanoparticles of well-defined size that can be used as drug delivery vectors. Functionalization with a fluorescent tag offers a useful visualization tool for tracing, localization, and clearance studies of these carriers in biological models. The studies are carried out by labeling the sterically stabilized latex particles chemically with rhodamine B. The fluorescent particles are incubated in a healthy human renal proximal tubular cell line model, and intravenously injected into a mouse model. Cellular localization and biodistribution of these particles on the biological models are explored. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. Tomographic sensing and localization of fluorescently labeled circulating cells in mice in vivo

    International Nuclear Information System (INIS)

    Zettergren, Eric; Swamy, Tushar; Niedre, Mark; Runnels, Judith; Lin, Charles P

    2012-01-01

    Sensing and enumeration of specific types of circulating cells in small animals is an important problem in many areas of biomedical research. Microscopy-based fluorescence in vivo flow cytometry methods have been developed previously, but these are typically limited to sampling of very small blood volumes, so that very rare circulating cells may escape detection. Recently, we described the development of a ‘diffuse fluorescence flow cytometer’ (DFFC) that allows sampling of much larger blood vessels and therefore circulating blood volumes in the hindlimb, forelimb or tail of a mouse. In this work, we extend this concept by developing and validating a method to tomographically localize circulating fluorescently labeled cells in the cross section of a tissue simulating optical flow phantom and mouse limb. This was achieved using two modulated light sources and an array of six fiber-coupled detectors that allowed rapid, high-sensitivity acquisition of full tomographic data sets at 10 Hz. These were reconstructed into two-dimensional cross-sectional images using Monte Carlo models of light propagation and the randomized algebraic reconstruction technique. We were able to obtain continuous images of moving cells in the sample cross section with 0.5 mm accuracy or better. We first demonstrated this concept in limb-mimicking optical flow photons with up to four flow channels, and then in the tails of mice with fluorescently labeled multiple myeloma cells. This approach increases the overall diagnostic utility of our DFFC instrument. (paper)

  9. Fluorescent Photo-conversion: A second chance to label unique cells.

    Science.gov (United States)

    Mellott, Adam J; Shinogle, Heather E; Moore, David S; Detamore, Michael S

    2015-03-01

    Not all cells behave uniformly after treatment in tissue engineering studies. In fact, some treated cells display no signs of treatment or show unique characteristics not consistent with other treated cells. What if the "unique" cells could be isolated from a treated population, and further studied? Photo-convertible reporter proteins, such as Dendra2 , allow for the ability to selectively identify unique cells with a secondary label within a primary labeled treated population. In the current study, select cells were identified and labeled through photo-conversion of Dendra2 -transfected human Wharton's Jelly cells (hWJCs) for the first time. Robust photo-conversion of green-to-red fluorescence was achieved consistently in arbitrarily selected cells, allowing for precise cell identification of select hWJCs. The current study demonstrates a method that offers investigators the opportunity to selectively label and identify unique cells within a treated population for further study or isolation from the treatment population. Photo-convertible reporter proteins, such as Dendra2 , offer the ability over non-photo-convertible reporter proteins, such as green fluorescent protein, to analyze unique individual cells within a treated population, which allows investigators to gain more meaningful information on how a treatment affects all cells within a target population.

  10. In Vitro Osteogenic Potential of Green Fluorescent Protein Labelled Human Embryonic Stem Cell-Derived Osteoprogenitors

    Directory of Open Access Journals (Sweden)

    Intekhab Islam

    2016-01-01

    Full Text Available Cellular therapy using stem cells in bone regeneration has gained increasing interest. Various studies suggest the clinical utility of osteoprogenitors-like mesenchymal stem cells in bone regeneration. However, limited availability of mesenchymal stem cells and conflicting evidence on their therapeutic efficacy limit their clinical application. Human embryonic stem cells (hESCs are potentially an unlimited source of healthy and functional osteoprogenitors (OPs that could be utilized for bone regenerative applications. However, limited ability to track hESC-derived progenies in vivo greatly hinders translational studies. Hence, in this study, we aimed to establish hESC-derived OPs (hESC-OPs expressing green fluorescent protein (GFP and to investigate their osteogenic differentiation potential in vitro. We fluorescently labelled H9-hESCs using a plasmid vector encoding GFP. The GFP-expressing hESCs were differentiated into hESC-OPs. The hESC-OPsGFP+ stably expressed high levels of GFP, CD73, CD90, and CD105. They possessed osteogenic differentiation potential in vitro as demonstrated by increased expression of COL1A1, RUNX2, OSTERIX, and OPG transcripts and mineralized nodules positive for Alizarin Red and immunocytochemical expression of osteocalcin, alkaline phosphatase, and collagen-I. In conclusion, we have demonstrated that fluorescently labelled hESC-OPs can maintain their GFP expression for the long term and their potential for osteogenic differentiation in vitro. In future, these fluorescently labelled hESC-OPs could be used for noninvasive assessment of bone regeneration, safety, and therapeutic efficacy.

  11. Influence of bone affinity on the skeletal distribution of fluorescently labeled bisphosphonates in vivo.

    Science.gov (United States)

    Roelofs, Anke J; Stewart, Charlotte A; Sun, Shuting; Błażewska, Katarzyna M; Kashemirov, Boris A; McKenna, Charles E; Russell, R Graham G; Rogers, Michael J; Lundy, Mark W; Ebetino, Frank H; Coxon, Fraser P

    2012-04-01

    Bisphosphonates are widely used antiresorptive drugs that bind to calcium. It has become evident that these drugs have differing affinities for bone mineral; however, it is unclear whether such differences affect their distribution on mineral surfaces. In this study, fluorescent conjugates of risedronate, and its lower-affinity analogues deoxy-risedronate and 3-PEHPC, were used to compare the localization of compounds with differing mineral affinities in vivo. Binding to dentine in vitro confirmed differences in mineral binding between compounds, which was influenced predominantly by the characteristics of the parent compound but also by the choice of fluorescent tag. In growing rats, all compounds preferentially bound to forming endocortical as opposed to resorbing periosteal surfaces in cortical bone, 1 day after administration. At resorbing surfaces, lower-affinity compounds showed preferential binding to resorption lacunae, whereas the highest-affinity compound showed more uniform labeling. At forming surfaces, penetration into the mineralizing osteoid was found to inversely correlate with mineral affinity. These differences in distribution at resorbing and forming surfaces were not observed at quiescent surfaces. Lower-affinity compounds also showed a relatively higher degree of labeling of osteocyte lacunar walls and labeled lacunae deeper within cortical bone, indicating increased penetration of the osteocyte canalicular network. Similar differences in mineralizing surface and osteocyte network penetration between high- and low-affinity compounds were evident 7 days after administration, with fluorescent conjugates at forming surfaces buried under a new layer of bone. Fluorescent compounds were incorporated into these areas of newly formed bone, indicating that "recycling" had occurred, albeit at very low levels. Taken together, these findings indicate that the bone mineral affinity of bisphosphonates is likely to influence their distribution within the

  12. Site-directed fluorescence labeling of a membrane protein with BADAN: probing protein topology and local environment

    NARCIS (Netherlands)

    Koehorst, R.B.M.; Spruijt, R.B.; Hemminga, M.A.

    2008-01-01

    We present a new and simple method based on site-directed fluorescence labeling using the BADAN label that allows to examine protein-lipid interactions in great detail. We apply this approach to a membrane-embedded mainly -helical reference protein, the M13 major coat protein, of which in a

  13. The Non-Specific Binding of Fluorescent-Labeled MiRNAs on Cell Surface by Hydrophobic Interaction.

    Science.gov (United States)

    Lu, Ting; Lin, Zongwei; Ren, Jianwei; Yao, Peng; Wang, Xiaowei; Wang, Zhe; Zhang, Qunye

    2016-01-01

    MicroRNAs are small noncoding RNAs about 22 nt long that play key roles in almost all biological processes and diseases. The fluorescent labeling and lipofection are two common methods for changing the levels and locating the position of cellular miRNAs. Despite many studies about the mechanism of DNA/RNA lipofection, little is known about the characteristics, mechanisms and specificity of lipofection of fluorescent-labeled miRNAs. Therefore, miRNAs labeled with different fluorescent dyes were transfected into adherent and suspension cells using lipofection reagent. Then, the non-specific binding and its mechanism were investigated by flow cytometer and laser confocal microscopy. The results showed that miRNAs labeled with Cy5 (cyanine fluorescent dye) could firmly bind to the surface of adherent cells (Hela) and suspended cells (K562) even without lipofection reagent. The binding of miRNAs labeled with FAM (carboxyl fluorescein) to K562 cells was obvious, but it was not significant in Hela cells. After lipofectamine reagent was added, most of the fluorescently labeled miRNAs binding to the surface of Hela cells were transfected into intra-cell because of the high transfection efficiency, however, most of them were still binding to the surface of K562 cells. Moreover, the high-salt buffer which could destroy the electrostatic interactions did not affect the above-mentioned non-specific binding, but the organic solvent which could destroy the hydrophobic interactions eliminated it. These results implied that the fluorescent-labeled miRNAs could non-specifically bind to the cell surface by hydrophobic interaction. It would lead to significant errors in the estimation of transfection efficiency only according to the cellular fluorescence intensity. Therefore, other methods to evaluate the transfection efficiency and more appropriate fluorescent dyes should be used according to the cell types for the accuracy of results.

  14. The Non-Specific Binding of Fluorescent-Labeled MiRNAs on Cell Surface by Hydrophobic Interaction.

    Directory of Open Access Journals (Sweden)

    Ting Lu

    Full Text Available MicroRNAs are small noncoding RNAs about 22 nt long that play key roles in almost all biological processes and diseases. The fluorescent labeling and lipofection are two common methods for changing the levels and locating the position of cellular miRNAs. Despite many studies about the mechanism of DNA/RNA lipofection, little is known about the characteristics, mechanisms and specificity of lipofection of fluorescent-labeled miRNAs.Therefore, miRNAs labeled with different fluorescent dyes were transfected into adherent and suspension cells using lipofection reagent. Then, the non-specific binding and its mechanism were investigated by flow cytometer and laser confocal microscopy. The results showed that miRNAs labeled with Cy5 (cyanine fluorescent dye could firmly bind to the surface of adherent cells (Hela and suspended cells (K562 even without lipofection reagent. The binding of miRNAs labeled with FAM (carboxyl fluorescein to K562 cells was obvious, but it was not significant in Hela cells. After lipofectamine reagent was added, most of the fluorescently labeled miRNAs binding to the surface of Hela cells were transfected into intra-cell because of the high transfection efficiency, however, most of them were still binding to the surface of K562 cells. Moreover, the high-salt buffer which could destroy the electrostatic interactions did not affect the above-mentioned non-specific binding, but the organic solvent which could destroy the hydrophobic interactions eliminated it.These results implied that the fluorescent-labeled miRNAs could non-specifically bind to the cell surface by hydrophobic interaction. It would lead to significant errors in the estimation of transfection efficiency only according to the cellular fluorescence intensity. Therefore, other methods to evaluate the transfection efficiency and more appropriate fluorescent dyes should be used according to the cell types for the accuracy of results.

  15. Compact 3D printed module for fluorescence and label-free imaging using evanescent excitation

    Science.gov (United States)

    Pandey, Vikas; Gupta, Shalini; Elangovan, Ravikrishnan

    2018-01-01

    Total internal reflection fluorescence (TIRF) microscopy is widely used for selective excitation and high-resolution imaging of fluorophores, and more recently label-free nanosized objects, with high vertical confinement near a liquid-solid interface. Traditionally, high numerical aperture objectives (>1.4) are used to simultaneously generate evanescent waves and collect fluorescence emission signals which limits their use to small area imaging (filters to prevent specular reflection within the objective lenses. We have developed a compact 3D module called cTIRF that can generate evanescent waves in microscope glass slides via a planar waveguide illumination. The module can be attached as a fixture to any existing optical microscope, converting it into a TIRF and enabling high signal-to-noise ratio (SNR) fluorescence imaging using any magnification objective. As the incidence optics is perpendicular to the detector, label-free evanescent scattering-based imaging of submicron objects can also be performed without using emission filters. SNR is significantly enhanced in this case as compared to cTIRF alone, as seen through our model experiments performed on latex beads and mammalian cells. Extreme flexibility and the low cost of our approach makes it scalable for limited resource settings.

  16. C-Terminal Fluorescent Labeling Impairs Functionality of DNA Mismatch Repair Proteins

    Science.gov (United States)

    Brieger, Angela; Plotz, Guido; Hinrichsen, Inga; Passmann, Sandra; Adam, Ronja; Zeuzem, Stefan

    2012-01-01

    The human DNA mismatch repair (MMR) process is crucial to maintain the integrity of the genome and requires many different proteins which interact perfectly and coordinated. Germline mutations in MMR genes are responsible for the development of the hereditary form of colorectal cancer called Lynch syndrome. Various mutations mainly in two MMR proteins, MLH1 and MSH2, have been identified so far, whereas 55% are detected within MLH1, the essential component of the heterodimer MutLα (MLH1 and PMS2). Most of those MLH1 variants are pathogenic but the relevance of missense mutations often remains unclear. Many different recombinant systems are applied to filter out disease-associated proteins whereby fluorescent tagged proteins are frequently used. However, dye labeling might have deleterious effects on MutLα's functionality. Therefore, we analyzed the consequences of N- and C-terminal fluorescent labeling on expression level, cellular localization and MMR activity of MutLα. Besides significant influence of GFP- or Red-fusion on protein expression we detected incorrect shuttling of single expressed C-terminal GFP-tagged PMS2 into the nucleus and found that C-terminal dye labeling impaired MMR function of MutLα. In contrast, N-terminal tagged MutLαs retained correct functionality and can be recommended both for the analysis of cellular localization and MMR efficiency. PMID:22348133

  17. C-terminal fluorescent labeling impairs functionality of DNA mismatch repair proteins.

    Directory of Open Access Journals (Sweden)

    Angela Brieger

    Full Text Available The human DNA mismatch repair (MMR process is crucial to maintain the integrity of the genome and requires many different proteins which interact perfectly and coordinated. Germline mutations in MMR genes are responsible for the development of the hereditary form of colorectal cancer called Lynch syndrome. Various mutations mainly in two MMR proteins, MLH1 and MSH2, have been identified so far, whereas 55% are detected within MLH1, the essential component of the heterodimer MutLα (MLH1 and PMS2. Most of those MLH1 variants are pathogenic but the relevance of missense mutations often remains unclear. Many different recombinant systems are applied to filter out disease-associated proteins whereby fluorescent tagged proteins are frequently used. However, dye labeling might have deleterious effects on MutLα's functionality. Therefore, we analyzed the consequences of N- and C-terminal fluorescent labeling on expression level, cellular localization and MMR activity of MutLα. Besides significant influence of GFP- or Red-fusion on protein expression we detected incorrect shuttling of single expressed C-terminal GFP-tagged PMS2 into the nucleus and found that C-terminal dye labeling impaired MMR function of MutLα. In contrast, N-terminal tagged MutLαs retained correct functionality and can be recommended both for the analysis of cellular localization and MMR efficiency.

  18. [Tetracyclines, sulfonamides and metronidazole].

    Science.gov (United States)

    Pérez-Trallero, Emilio; Iglesias, Luis

    2003-11-01

    Tetracyclines form a group of natural and semisynthetic products that acts inhibiting the bacterial protein synthesis. They are bacteriostatic agents, exhibiting activity against a wide range of organisms, but they are at the present of limited use because of their acquired resistance. Doxycycline is currently the most frequently used tetracycline in human medicine and it is included in the List of Essential Medicines of the World Health Organization. Sulfonamides are synthetic, broad-spectrum bacteriostatic antibiotics. They were the first effective systemic antimicrobial agents. Their mode of action is based on the inhibition of DNA synthesis. Due to their toxicity and high adquired resistance their use is currently very low. Metronidazole is the main compound of 5-nitroimidazole family. It is a very active bactericidal antibiotic against anaerobic and some microaerophilic bacteria and it is still very useful in the treatment of bacterian and parasitic infections.

  19. Site-specific fluorescent labeling of nascent proteins on the translating ribosome.

    Science.gov (United States)

    Saraogi, Ishu; Zhang, Dawei; Chandrasekaran, Sandhya; Shan, Shu-ou

    2011-09-28

    As newly synthesized proteins emerge from the ribosome, they interact with a variety of cotranslational cellular machineries that facilitate their proper folding, maturation, and localization. These interactions are essential for proper function of the cell, and the ability to study these events is crucial to understanding cellular protein biogenesis. To this end, we have developed a highly efficient method to generate ribosome-nascent chain complexes (RNCs) site-specifically labeled with a fluorescent dye on the nascent polypeptide. The fluorescent RNC provides real-time, quantitative information on its cotranslational interaction with the signal recognition particle and will be a valuable tool in elucidating the role of the translating ribosome in numerous biochemical pathways.

  20. Synthesis of strongly fluorescent molybdenum disulfide nanosheets for cell-targeted labeling.

    Science.gov (United States)

    Wang, Nan; Wei, Fang; Qi, Yuhang; Li, Hongxiang; Lu, Xin; Zhao, Guoqiang; Xu, Qun

    2014-11-26

    MoS2 nanosheets with polydispersity of the lateral dimensions from natural mineral molybdenite have been prepared in the emulsions microenvironment built by the water/surfactant/CO2 system. The size, thickness, and atomic structure are characterized by transmission electron microscopy (TEM), atomic force microscopy (AFM), and laser-scattering particle size analysis. Meanwhile, by the analysis of photoluminescence spectroscopy and microscope, the MoS2 nanosheets with smaller lateral dimensions exhibit extraordinary photoluminescence properties different from those with relatively larger lateral dimensions. The discovery of the excitation dependent photoluminescence for MoS2 nanosheets makes them potentially of interests for the applications in optoelectronics and biology. Moreover, we demonstrate that the fabricated MoS2 nanosheets can be a nontoxic fluorescent label for cell-targeted labeling application.

  1. Real-time label-free quantitative fluorescence microscopy-based detection of ATP using a tunable fluorescent nano-aptasensor platform.

    Science.gov (United States)

    Shrivastava, Sajal; Sohn, Il-Yung; Son, Young-Min; Lee, Won-Il; Lee, Nae-Eung

    2015-12-14

    Although real-time label-free fluorescent aptasensors based on nanomaterials are increasingly recognized as a useful strategy for the detection of target biomolecules with high fidelity, the lack of an imaging-based quantitative measurement platform limits their implementation with biological samples. Here we introduce an ensemble strategy for a real-time label-free fluorescent graphene (Gr) aptasensor platform. This platform employs aptamer length-dependent tunability, thus enabling the reagentless quantitative detection of biomolecules through computational processing coupled with real-time fluorescence imaging data. We demonstrate that this strategy effectively delivers dose-dependent quantitative readouts of adenosine triphosphate (ATP) concentration on chemical vapor deposited (CVD) Gr and reduced graphene oxide (rGO) surfaces, thereby providing cytotoxicity assessment. Compared with conventional fluorescence spectrometry methods, our highly efficient, universally applicable, and rational approach will facilitate broader implementation of imaging-based biosensing platforms for the quantitative evaluation of a range of target molecules.

  2. NIR-to-visible upconversion nanoparticles for fluorescent labeling and targeted delivery of siRNA

    International Nuclear Information System (INIS)

    Jiang Shan; Zhang Yong; Lim, Kian Meng; Sim, Eugene K W; Ye Lei

    2009-01-01

    Near-infrared (NIR)-to-visible upconversion fluorescent nanoparticles were synthesized and used for imaging and targeted delivery of small interfering RNA (siRNA) to cancer cells. Silica-coated NaYF 4 upconversion nanoparticles (UCNs) co-doped with lanthanide ions (Yb/Er) were synthesized. Folic acid and anti-Her2 antibody conjugated UCNs were used to fluorescently label the folate receptors of HT-29 cells and Her2 receptors of SK-BR-3 cells, respectively. The intracellular uptake of the folic acid and antibody conjugated UCNs was visualized using a confocal fluorescence microscope equipped with an NIR laser. siRNA was attached to anti-Her2 antibody conjugated UCNs and the delivery of these nanoparticles to SK-BR-3 cells was studied. Meanwhile, a luciferase assay was established to confirm the gene silencing effect of siRNA. Upconversion nanoparticles can serve as a fluorescent probe and delivery system for simultaneous imaging and delivery of biological molecules.

  3. NIR-to-visible upconversion nanoparticles for fluorescent labeling and targeted delivery of siRNA

    Science.gov (United States)

    Jiang, Shan; Zhang, Yong; Lim, Kian Meng; Sim, Eugene K. W.; Ye, Lei

    2009-04-01

    Near-infrared (NIR)-to-visible upconversion fluorescent nanoparticles were synthesized and used for imaging and targeted delivery of small interfering RNA (siRNA) to cancer cells. Silica-coated NaYF4 upconversion nanoparticles (UCNs) co-doped with lanthanide ions (Yb/Er) were synthesized. Folic acid and anti-Her2 antibody conjugated UCNs were used to fluorescently label the folate receptors of HT-29 cells and Her2 receptors of SK-BR-3 cells, respectively. The intracellular uptake of the folic acid and antibody conjugated UCNs was visualized using a confocal fluorescence microscope equipped with an NIR laser. siRNA was attached to anti-Her2 antibody conjugated UCNs and the delivery of these nanoparticles to SK-BR-3 cells was studied. Meanwhile, a luciferase assay was established to confirm the gene silencing effect of siRNA. Upconversion nanoparticles can serve as a fluorescent probe and delivery system for simultaneous imaging and delivery of biological molecules.

  4. A flexible fluorescence correlation spectroscopy based method for quantification of the DNA double labeling efficiency with precision control

    International Nuclear Information System (INIS)

    Hou, Sen; Tabaka, Marcin; Sun, Lili; Trochimczyk, Piotr; Kaminski, Tomasz S; Kalwarczyk, Tomasz; Zhang, Xuzhu; Holyst, Robert

    2014-01-01

    We developed a laser-based method to quantify the double labeling efficiency of double-stranded DNA (dsDNA) in a fluorescent dsDNA pool with fluorescence correlation spectroscopy (FCS). Though, for quantitative biochemistry, accurate measurement of this parameter is of critical importance, before our work it was almost impossible to quantify what percentage of DNA is doubly labeled with the same dye. The dsDNA is produced by annealing complementary single-stranded DNA (ssDNA) labeled with the same dye at 5′ end. Due to imperfect ssDNA labeling, the resulting dsDNA is a mixture of doubly labeled dsDNA, singly labeled dsDNA and unlabeled dsDNA. Our method allows the percentage of doubly labeled dsDNA in the total fluorescent dsDNA pool to be measured. In this method, we excite the imperfectly labeled dsDNA sample in a focal volume of <1 fL with a laser beam and correlate the fluctuations of the fluorescence signal to get the FCS autocorrelation curves; we express the amplitudes of the autocorrelation function as a function of the DNA labeling efficiency; we perform a comparative analysis of a dsDNA sample and a reference dsDNA sample, which is prepared by increasing the total dsDNA concentration c (c > 1) times by adding unlabeled ssDNA during the annealing process. The method is flexible in that it allows for the selection of the reference sample and the c value can be adjusted as needed for a specific study. We express the precision of the method as a function of the ssDNA labeling efficiency or the dsDNA double labeling efficiency. The measurement precision can be controlled by changing the c value. (letter)

  5. Imaging of injured and atherosclerotic arteries in mice using fluorescence-labeled glycoprotein VI-Fc

    Energy Technology Data Exchange (ETDEWEB)

    Bigalke, Boris, E-mail: boris.bigalke@med.uni-tuebingen.de [Medizinische Klinik III, Kardiologie und Kreislauferkrankungen, Eberhard-Karls-Universitaet Tuebingen (Germany); Division of Imaging Sciences, St Thomas' Hospital, King' s College London (United Kingdom); Pohlmeyer, Ilka; Schoenberger, Tanja [Medizinische Klinik III, Kardiologie und Kreislauferkrankungen, Eberhard-Karls-Universitaet Tuebingen (Germany); Griessinger, Christoph M. [Labor fuer Praeklinische Bildgebung und Bildgebungstechnologie der Werner-Siemens-Stiftung, Radiologische Klinik, Eberhard-Karls-Universitaet Tuebingen (Germany); Ungerer, Martin [Corimmun GmbH, Martinsried (Germany); Botnar, Rene M. [Division of Imaging Sciences, St Thomas' Hospital, King' s College London (United Kingdom); Pichler, Bernd J. [Labor fuer Praeklinische Bildgebung und Bildgebungstechnologie der Werner-Siemens-Stiftung, Radiologische Klinik, Eberhard-Karls-Universitaet Tuebingen (Germany); Gawaz, Meinrad [Medizinische Klinik III, Kardiologie und Kreislauferkrankungen, Eberhard-Karls-Universitaet Tuebingen (Germany)

    2011-08-15

    Purpose: To assess endothelial injury and repair using fluorescence-labeled glycoprotein VI (GPVI)-Fc in a murine model. Materials and methods: Three 4-week-old male ApoE-deficient (ApoE{sup -/-})-mice were fed with a 1.25% cholesterol diet over 16 weeks and compared to three wild type (WT) C57BL/6J-mice in a wire-induced vascular injury model. Another group of WT mice (n = 10) were mechanically injured by carotid ligation. Fluorescence-labeled GPVI-Fc (150 {mu}g/mouse) was administered and assessed by optical imaging 24 h after injury and compared to another group (n = 3) which was injected two days after injury and sacrificed another day later. Results: After denudation, all injured carotids of WT mice showed a higher mean fluorescence signal than the corresponding intact carotids of the same animals (48.4 {+-} 18.9 vs. 10.4 {+-} 1.0; P = 0.028). Injection of unlabeled GPVI-Fc 20 h and 3 h before injecting GPVI-Fc-FITC significantly reduced the fluorescence signal in injured carotids to 14.6 {+-} 4.6, while intact carotids showed a signal of 9.2 {+-} 1.1; P = 0.046. Ligation injury resulted with an increased GPVI-Fc-binding to injured carotids compared to intact carotids (31.53 {+-} 6.18 vs. 16.48 {+-} 5.15; P = 0.039). Three days after injury and 24 h after GPVI-Fc-FITC injection, differences between intact and injured carotids have vanished (12.51 {+-} 2.76 vs. 14.76 {+-} 1.59; P = 0.519). Conclusions: A GPVI-based plaque imaging system could help to identify vascular lesions and to take a precautionary measure as necessary.

  6. Imaging of injured and atherosclerotic arteries in mice using fluorescence-labeled glycoprotein VI-Fc

    International Nuclear Information System (INIS)

    Bigalke, Boris; Pohlmeyer, Ilka; Schoenberger, Tanja; Griessinger, Christoph M.; Ungerer, Martin; Botnar, Rene M.; Pichler, Bernd J.; Gawaz, Meinrad

    2011-01-01

    Purpose: To assess endothelial injury and repair using fluorescence-labeled glycoprotein VI (GPVI)-Fc in a murine model. Materials and methods: Three 4-week-old male ApoE-deficient (ApoE -/- )-mice were fed with a 1.25% cholesterol diet over 16 weeks and compared to three wild type (WT) C57BL/6J-mice in a wire-induced vascular injury model. Another group of WT mice (n = 10) were mechanically injured by carotid ligation. Fluorescence-labeled GPVI-Fc (150 μg/mouse) was administered and assessed by optical imaging 24 h after injury and compared to another group (n = 3) which was injected two days after injury and sacrificed another day later. Results: After denudation, all injured carotids of WT mice showed a higher mean fluorescence signal than the corresponding intact carotids of the same animals (48.4 ± 18.9 vs. 10.4 ± 1.0; P = 0.028). Injection of unlabeled GPVI-Fc 20 h and 3 h before injecting GPVI-Fc-FITC significantly reduced the fluorescence signal in injured carotids to 14.6 ± 4.6, while intact carotids showed a signal of 9.2 ± 1.1; P = 0.046. Ligation injury resulted with an increased GPVI-Fc-binding to injured carotids compared to intact carotids (31.53 ± 6.18 vs. 16.48 ± 5.15; P = 0.039). Three days after injury and 24 h after GPVI-Fc-FITC injection, differences between intact and injured carotids have vanished (12.51 ± 2.76 vs. 14.76 ± 1.59; P = 0.519). Conclusions: A GPVI-based plaque imaging system could help to identify vascular lesions and to take a precautionary measure as necessary.

  7. Real-time label-free quantitative fluorescence microscopy-based detection of ATP using a tunable fluorescent nano-aptasensor platform

    Science.gov (United States)

    Shrivastava, Sajal; Sohn, Il-Yung; Son, Young-Min; Lee, Won-Il; Lee, Nae-Eung

    2015-11-01

    Although real-time label-free fluorescent aptasensors based on nanomaterials are increasingly recognized as a useful strategy for the detection of target biomolecules with high fidelity, the lack of an imaging-based quantitative measurement platform limits their implementation with biological samples. Here we introduce an ensemble strategy for a real-time label-free fluorescent graphene (Gr) aptasensor platform. This platform employs aptamer length-dependent tunability, thus enabling the reagentless quantitative detection of biomolecules through computational processing coupled with real-time fluorescence imaging data. We demonstrate that this strategy effectively delivers dose-dependent quantitative readouts of adenosine triphosphate (ATP) concentration on chemical vapor deposited (CVD) Gr and reduced graphene oxide (rGO) surfaces, thereby providing cytotoxicity assessment. Compared with conventional fluorescence spectrometry methods, our highly efficient, universally applicable, and rational approach will facilitate broader implementation of imaging-based biosensing platforms for the quantitative evaluation of a range of target molecules.Although real-time label-free fluorescent aptasensors based on nanomaterials are increasingly recognized as a useful strategy for the detection of target biomolecules with high fidelity, the lack of an imaging-based quantitative measurement platform limits their implementation with biological samples. Here we introduce an ensemble strategy for a real-time label-free fluorescent graphene (Gr) aptasensor platform. This platform employs aptamer length-dependent tunability, thus enabling the reagentless quantitative detection of biomolecules through computational processing coupled with real-time fluorescence imaging data. We demonstrate that this strategy effectively delivers dose-dependent quantitative readouts of adenosine triphosphate (ATP) concentration on chemical vapor deposited (CVD) Gr and reduced graphene oxide (r

  8. Comparison of three fluorescence labeling and tracking methods of endothelial progenitor cells in laser-injured retina

    Directory of Open Access Journals (Sweden)

    Hui Shi

    2018-04-01

    Full Text Available AIM: To compare three kinds of fluorescent probes for in vitro labeling and in vivo tracking of endothelial progenitor cells (EPCs in a mouse model of laser-induced retinal injury. METHODS: EPCs were isolated from human umbilical cord blood mononuclear cells and labeled with three different fluorescent probes: 5-(and-6-carboxyfluorescein diacetate succinimidyl ester (CFSE, 1,1′-dilinoleyl-3,3,3′,3′-tetramethylindo-carbocyanine perchlorate linked acetylated low-density lipoprotein (DiI-AcLDL, and green fluorescent protein (GFP. The fluorescent intensity of EPCs was examined by confocal microscopy. Survival rate of labeled EPCs was calculated with trypan blue staining, and their adhesive capability was assessed. A mouse model of retinal injury was induced by laser, and EPCs were injected into the vitreous cavity. Frozen section and fluorescein angiography on flat-mounted retinal samples was employed to track the labeled EPCs in vivo. RESULTS: EPCs labeled with CFSE and DiI-AcLDL exhibited an intense green and red fluorescence at the beginning; the fluorescence intensity decreased gradually to 20.23% and 49.99% respectively, after 28d. On the contrary, the florescent intensity of GFP-labeled EPCs increased in a time-dependent manner. All labeled EPCs showed normal morphology and no significant change in survival and adhesive capability. In the mouse model, transplantation of EPCs showed a protective effect against retinal injury. EPCs labeled with CFSE and DiI-AcLDL were successfully tracked in mice during the development of retinal injury and repair; however, GFP-labeled EPCs were not detected in the laser-injured mouse retina. CONCLUSION: The three fluorescent markers used in this study have their own set of advantages and disadvantages. CFSE and DiI-AcLDL are suitable for short-term EPC-labeling, while GFP should be used for long-term labeling. The choice of fluorescent markers should be guided by the purpose of the study.

  9. Dual fluorescence labeling of surface-exposed and internal proteins in erythrocytes infected with the malaria parasite Plasmodium falciparum

    DEFF Research Database (Denmark)

    Bengtsson, Dominique C; Sowa, Kordai M P; Arnot, David E

    2008-01-01

    There is a need for improved methods for in situ localization of surface proteins on Plasmodium falciparum-infected erythrocytes to help understand how these antigens are trafficked to, and positioned within, the host cell membrane. This protocol for confocal immunofluorescence microscopy combines...... and permeabilization; indirect labeling of the internal antigen using a secondary antibody tagged with a spectrally distinct fluorescent dye; and detection of the differentially labeled antigens using a laser scanning confocal microscope. The protocol can be completed in approximately 7 h. Although the protocol...... surface antigen labeling on live cells with subsequent fixation and permeabilization, which enables antibodies to penetrate the cell and label internal antigens. The key steps of the protocol are as follows: indirect labeling of the surface antigen using a fluorescently tagged secondary antibody; fixation...

  10. Labeling of neuronal differentiation and neuron cells with biocompatible fluorescent nanodiamonds.

    Science.gov (United States)

    Hsu, Tzu-Chia; Liu, Kuang-Kai; Chang, Huan-Cheng; Hwang, Eric; Chao, Jui-I

    2014-05-16

    Nanodiamond is a promising carbon nanomaterial developed for biomedical applications. Here, we show fluorescent nanodiamond (FND) with the biocompatible properties that can be used for the labeling and tracking of neuronal differentiation and neuron cells derived from embryonal carcinoma stem (ECS) cells. The fluorescence intensities of FNDs were increased by treatment with FNDs in both the mouse P19 and human NT2/D1 ECS cells. FNDs were taken into ECS cells; however, FNDs did not alter the cellular morphology and growth ability. Moreover, FNDs did not change the protein expression of stem cell marker SSEA-1 of ECS cells. The neuronal differentiation of ECS cells could be induced by retinoic acid (RA). Interestingly, FNDs did not affect on the morphological alteration, cytotoxicity and apoptosis during the neuronal differentiation. Besides, FNDs did not alter the cell viability and the expression of neuron-specific marker β-III-tubulin in these differentiated neuron cells. The existence of FNDs in the neuron cells can be identified by confocal microscopy and flow cytometry. Together, FND is a biocompatible and readily detectable nanomaterial for the labeling and tracking of neuronal differentiation process and neuron cells from stem cells.

  11. Micro-Droplet Detection Method for Measuring the Concentration of Alkaline Phosphatase-Labeled Nanoparticles in Fluorescence Microscopy

    Directory of Open Access Journals (Sweden)

    Rufeng Li

    2017-11-01

    Full Text Available This paper developed and evaluated a quantitative image analysis method to measure the concentration of the nanoparticles on which alkaline phosphatase (AP was immobilized. These AP-labeled nanoparticles are widely used as signal markers for tagging biomolecules at nanometer and sub-nanometer scales. The AP-labeled nanoparticle concentration measurement can then be directly used to quantitatively analyze the biomolecular concentration. Micro-droplets are mono-dispersed micro-reactors that can be used to encapsulate and detect AP-labeled nanoparticles. Micro-droplets include both empty micro-droplets and fluorescent micro-droplets, while fluorescent micro-droplets are generated from the fluorescence reaction between the APs adhering to a single nanoparticle and corresponding fluorogenic substrates within droplets. By detecting micro-droplets and calculating the proportion of fluorescent micro-droplets to the overall micro-droplets, we can calculate the AP-labeled nanoparticle concentration. The proposed micro-droplet detection method includes the following steps: (1 Gaussian filtering to remove the noise of overall fluorescent targets, (2 a contrast-limited, adaptive histogram equalization processing to enhance the contrast of weakly luminescent micro-droplets, (3 an red maximizing inter-class variance thresholding method (OTSU to segment the enhanced image for getting the binary map of the overall micro-droplets, (4 a circular Hough transform (CHT method to detect overall micro-droplets and (5 an intensity-mean-based thresholding segmentation method to extract the fluorescent micro-droplets. The experimental results of fluorescent micro-droplet images show that the average accuracy of our micro-droplet detection method is 0.9586; the average true positive rate is 0.9502; and the average false positive rate is 0.0073. The detection method can be successfully applied to measure AP-labeled nanoparticle concentration in fluorescence microscopy.

  12. Micro-Droplet Detection Method for Measuring the Concentration of Alkaline Phosphatase-Labeled Nanoparticles in Fluorescence Microscopy.

    Science.gov (United States)

    Li, Rufeng; Wang, Yibei; Xu, Hong; Fei, Baowei; Qin, Binjie

    2017-11-21

    This paper developed and evaluated a quantitative image analysis method to measure the concentration of the nanoparticles on which alkaline phosphatase (AP) was immobilized. These AP-labeled nanoparticles are widely used as signal markers for tagging biomolecules at nanometer and sub-nanometer scales. The AP-labeled nanoparticle concentration measurement can then be directly used to quantitatively analyze the biomolecular concentration. Micro-droplets are mono-dispersed micro-reactors that can be used to encapsulate and detect AP-labeled nanoparticles. Micro-droplets include both empty micro-droplets and fluorescent micro-droplets, while fluorescent micro-droplets are generated from the fluorescence reaction between the APs adhering to a single nanoparticle and corresponding fluorogenic substrates within droplets. By detecting micro-droplets and calculating the proportion of fluorescent micro-droplets to the overall micro-droplets, we can calculate the AP-labeled nanoparticle concentration. The proposed micro-droplet detection method includes the following steps: (1) Gaussian filtering to remove the noise of overall fluorescent targets, (2) a contrast-limited, adaptive histogram equalization processing to enhance the contrast of weakly luminescent micro-droplets, (3) an red maximizing inter-class variance thresholding method (OTSU) to segment the enhanced image for getting the binary map of the overall micro-droplets, (4) a circular Hough transform (CHT) method to detect overall micro-droplets and (5) an intensity-mean-based thresholding segmentation method to extract the fluorescent micro-droplets. The experimental results of fluorescent micro-droplet images show that the average accuracy of our micro-droplet detection method is 0.9586; the average true positive rate is 0.9502; and the average false positive rate is 0.0073. The detection method can be successfully applied to measure AP-labeled nanoparticle concentration in fluorescence microscopy.

  13. Development of flow-through and dip-stick immunoassays for screening of sulfonamide residues.

    Science.gov (United States)

    Zhang, Hongyan; Zhang, Yan; Wang, Shuo

    2008-08-20

    Two formats of membrane-based competitive enzyme immunoassays (flow-through and dip-stick) have been developed for the screening of sulfonamide residues in pig muscle and milk. Membrane was coated with anti-sulfonamide antibody and a sulfonamide hapten D2-horseradish peroxidase (HRP) conjugant was used as the labeled antigen for competitive assay of sulfonamides. Visual detection limits of the flow-through or dip-stick assay were 1-5 microg L(-1) or 1-10 microg L(-1) in buffer for seven sulfonamides, respectively. Assay validation was performed using samples spiked with single sulfonamide, spiked samples were tested using the developed strip assays and results were compared with those obtained by a validated high-performance liquid chromatograph (HPLC) method. Results showed that the two strip assays were correlated well with HPLC, respectively. With assay times of 5 min (flow-through) and 15 min (dip-stick), these rapid tests could offer simple, rapid and cost-effective on-site screening tools to detect sulfonamides in pig muscle (flow-through or dip-stick) or milk (only dip-stick).

  14. Quinoline-2-sulfonamide

    Directory of Open Access Journals (Sweden)

    Joachim Kusz

    2013-08-01

    Full Text Available In the title compound, C9H8N2O2S, the sulfamoyl –NH2 group is involved in intermolecular hydrogen bonding with the sulfonamide O and quinoline N atoms. In the crystal, molecules are linked into dimers via pairs of N—H...N hydrogen bonds, forming an R22(10 motif. The dimers are further assembled into chains parallel to the b axis through N—H...O hydrogen bonds, generating a C(4 motif. The crystal packing is additionally stabilized by intermolecular C—H...O interactions. The crystal studied was a non-merohedral twin with a domain ratio of 0.938 (2:0.062 (2. Density functional theory (DFT calculations, at the B3LYP/6–31 G(d,p level of theory, were used to optimize the molecular structure and to determine interaction energies for the title compound. The resulting interaction energy is ∼4.4 kcal mol−1 per bridge for the C(4 chain and ∼5.9 kcal mol−1 per bridge for the R22(10 motif.

  15. Fluorescent Affibody Molecule Administered In Vivo at a Microdose Level Labels EGFR Expressing Glioma Tumor Regions.

    Science.gov (United States)

    de Souza, Ana Luiza Ribeiro; Marra, Kayla; Gunn, Jason; Samkoe, Kimberley S; Hoopes, P Jack; Feldwisch, Joachim; Paulsen, Keith D; Pogue, Brian W

    2017-02-01

    Fluorescence guidance in surgical oncology provides the potential to realize enhanced molecular tumor contrast with dedicated targeted tracers, potentially with a microdose injection level. For most glioma tumors, the blood brain barrier is compromised allowing some exogenous drug/molecule delivery and accumulation for imaging. The aberrant overexpression and/or activation of epidermal growth factor receptor (EGFR) is associated with many types of cancers, including glioblastoma, and so the use of a near-infrared (NIR) fluorescent molecule targeted to the EGFR receptor provides the potential for improving tumor contrast during surgery. Fluorescently labeled affibody molecule (ABY-029) has high EGFR affinity and high potential specificity with reasonably fast plasma clearance. In this study, ABY-29 was evaluated in glioma versus normal brain uptake from intravenous injection at a range of doses, down to a microdose injection level. Nude rats were inoculated with the U251 human glioma cell line in the brain. Tumors were allowed to grow for 3-4 weeks. ABY-029 fluorescence ex vivo imaging of brain slices was acquired at different time points (1-48 h) and varying injection doses from 25 to 122 μg/kg (from human protein microdose equivalent to five times microdose levels). The tumor was most clearly visualized at 1-h post-injection with 8- to 16-fold average contrast relative to normal brain. However, the tumor still could be identified after 48 h. In all cases, the ABY-029 fluorescence appeared to localize preferentially in EGFR-positive regions. Increasing the injected dose from a microdose level to five times, a microdose level increased the signal by 10-fold, and the contrast was from 8 to 16, showing that there was value in doses slightly higher than the microdose restriction. Normal tissue uptake was found to be affected by the tumor size, indicating that edema was a likely factor affecting the expected tumor to normal tissue contrast. These results suggest

  16. Simultaneous cathodoluminescence and electron microscopy cytometry of cellular vesicles labeled with fluorescent nanodiamonds.

    Science.gov (United States)

    Nagarajan, Sounderya; Pioche-Durieu, Catherine; Tizei, Luiz H G; Fang, Chia-Yi; Bertrand, Jean-Rémi; Le Cam, Eric; Chang, Huan-Cheng; Treussart, François; Kociak, Mathieu

    2016-06-02

    Light and Transmission Electron Microscopies (LM and TEM) hold potential in bioimaging owing to the advantages of fast imaging of multiple cells with LM and ultrastructure resolution offered by TEM. Integrated or correlated LM and TEM are the current approaches to combine the advantages of both techniques. Here we propose an alternative in which the electron beam of a scanning TEM (STEM) is used to excite concomitantly the luminescence of nanoparticle labels (a process known as cathodoluminescence, CL), and image the cell ultrastructure. This CL-STEM imaging allows obtaining luminescence spectra and imaging ultrastructure simultaneously. We present a proof of principle experiment, showing the potential of this technique in image cytometry of cell vesicular components. To label the vesicles we used fluorescent diamond nanocrystals (nanodiamonds, NDs) of size ≈150 nm coated with different cationic polymers, known to trigger different internalization pathways. Each polymer was associated with a type of ND with a different emission spectrum. With CL-STEM, for each individual vesicle, we were able to measure (i) their size with nanometric resolution, (ii) their content in different ND labels, and realize intracellular component cytometry. In contrast to the recently reported organelle flow cytometry technique that requires cell sonication, CL-STEM-based image cytometry preserves the cell integrity and provides a much higher resolution in size. Although this novel approach is still limited by a low throughput, the automatization of data acquisition and image analysis, combined with improved intracellular targeting, should facilitate applications in cell biology at the subcellular level.

  17. Fluorescent labelling of DNA on superparamagnetic nanoparticles by a perylene bisimide derivative for cell imaging

    Energy Technology Data Exchange (ETDEWEB)

    Maltas, Esra, E-mail: maltasesra@gmail.com [Selcuk University, Faculty of Science, Department of Chemistry, 42075 Konya (Turkey); Malkondu, Sait [Selcuk University, Faculty of Science, Department of Chemistry, 42075 Konya (Turkey); Uyar, Pembegul [Selcuk University, Faculty of Science, Department of Biology, 42075 Konya (Turkey); Selcuk University, Advanced Technology Research and Application Center, Konya (Turkey); Ozmen, Mustafa [Selcuk University, Faculty of Science, Department of Chemistry, 42075 Konya (Turkey)

    2015-03-01

    N,N′-Bis[tris-(2-aminoethyl) amine]-3,4,9,10-perylenetetracarboxylic diimide (PBI-TRIS), nonfluorescent dye was used to fluorescent labelling of DNA. For this aim, (3-aminopropyl) triethoxysilane (APTS) modified superparamagnetic iron oxide nanoparticles (SPIONs) were synthesized to provide a suitable surface for binding of DNA. Amine functionalized nanoparticles showed a high immobilization capacity (82.70%) at 25 mg of nanoparticle concentration for Calf thymus DNA. Binding capacity of PBI-TRIS to DNA-SPION was also found as 1.93 μM on 25 mg of nanoparticles by using UV–vis spectroscopy. Binding of PBI-TRIS to DNA onto nanoparticles was also characterized by scanning electron microscopy and infrared spectroscopy. The confocal images of PBI-TRIS labelled DNA-SPION and breast cells were taken at 488 and 561.7 nm of excitation wavelengths. Cell image was also compared with a commercial dye, DAPI at 403.7 nm of excitation wavelength. Results showed that PBI-TRIS can be used for cell staining. - Highlights: • Functionalized SPIONs were synthesized and treated with DNA. • The binding of PBI-TRIS with DNA was studied. • The image of PBI-TRIS labelled DNA-SPION was detected by a confocal microscope.

  18. Fluorescent labelling of DNA on superparamagnetic nanoparticles by a perylene bisimide derivative for cell imaging

    International Nuclear Information System (INIS)

    Maltas, Esra; Malkondu, Sait; Uyar, Pembegul; Ozmen, Mustafa

    2015-01-01

    N,N′-Bis[tris-(2-aminoethyl) amine]-3,4,9,10-perylenetetracarboxylic diimide (PBI-TRIS), nonfluorescent dye was used to fluorescent labelling of DNA. For this aim, (3-aminopropyl) triethoxysilane (APTS) modified superparamagnetic iron oxide nanoparticles (SPIONs) were synthesized to provide a suitable surface for binding of DNA. Amine functionalized nanoparticles showed a high immobilization capacity (82.70%) at 25 mg of nanoparticle concentration for Calf thymus DNA. Binding capacity of PBI-TRIS to DNA-SPION was also found as 1.93 μM on 25 mg of nanoparticles by using UV–vis spectroscopy. Binding of PBI-TRIS to DNA onto nanoparticles was also characterized by scanning electron microscopy and infrared spectroscopy. The confocal images of PBI-TRIS labelled DNA-SPION and breast cells were taken at 488 and 561.7 nm of excitation wavelengths. Cell image was also compared with a commercial dye, DAPI at 403.7 nm of excitation wavelength. Results showed that PBI-TRIS can be used for cell staining. - Highlights: • Functionalized SPIONs were synthesized and treated with DNA. • The binding of PBI-TRIS with DNA was studied. • The image of PBI-TRIS labelled DNA-SPION was detected by a confocal microscope

  19. Separation-oriented derivatization of native fluorescent compounds through fluorous labeling followed by liquid chromatography with fluorous-phase.

    Science.gov (United States)

    Sakaguchi, Yohei; Yoshida, Hideyuki; Todoroki, Kenichiro; Nohta, Hitoshi; Yamaguchi, Masatoshi

    2009-06-15

    We have developed a new and simple method based on "fluorous derivatization" for LC of native fluorescent compounds. This method involves the use of a column with a fluorous stationary phase. Native fluorescent analytes with target functional groups are precolumn derivatized with a nonfluorescent fluorous tag, and the fluorous-labeled analytes are retained in the column, whereas underivatized substances are not. Only the retained fluorescent analytes are detected fluorometrically at appropriate retention times, and retained substrates without fluorophores are not detected. In this study, biologically important carboxylic acids (homovanillic acid, vanillylmandelic acid, and 5-hydroxyindoleacetic acid) and drugs (naproxen, felbinac, flurbiprofen, and etodolac) were used as model native fluorescent compounds. Experimental results indicate that the fluorous-phase column can selectively retain fluorous compounds including fluorous-labeled analytes on the basis of fluorous separation. We believe that separation-oriented derivatization presented here is the first step toward the introduction of fluorous derivatization in quantitative LC analysis.

  20. Synchrotron X-ray fluorescence studies of a bromine-labelled cyclic RGD peptide interacting with individual tumor cells

    International Nuclear Information System (INIS)

    Sheridan, Erin J.; Austin, Christopher J. D.; Aitken, Jade B.; Vogt, Stefan; Jolliffe, Katrina A.; Harris, Hugh H.; Rendina, Louis M.

    2013-01-01

    The first example of synchrotron X-ray fluorescence imaging of cultured mammalian cells in cyclic peptide research is reported. The study reports the first quantitative analysis of the incorporation of a bromine-labelled cyclic RGD peptide and its effects on the biodistribution of endogenous elements (for example, K and Cl) within individual tumor cells. The first example of synchrotron X-ray fluorescence imaging of cultured mammalian cells in cyclic peptide research is reported. The study reports the first quantitative analysis of the incorporation of a bromine-labelled cyclic RGD peptide and its effects on the biodistribution of endogenous elements (for example, K and Cl) within individual tumor cells

  1. Automated sorting of polymer flakes: fluorescence labeling and development of a measurement system prototype.

    Science.gov (United States)

    Brunner, S; Fomin, P; Kargel, Ch

    2015-04-01

    The extensive demand and use of plastics in modern life is associated with a significant economical impact and a serious ecological footprint. The production of plastics involves a high energy consumption and CO2 emission as well as the large need for (limited) fossil resources. Due to the high durability of plastics, large amounts of plastic garbage is mounting in overflowing landfills (plus 9.6 million tons in Europe in the year 2012) and plastic debris is floating in the world oceans or waste-to-energy combustion releases even more CO2 plus toxic substances (dioxins, heavy metals) to the atmosphere. The recycling of plastic products after their life cycle can obviously contribute a great deal to the reduction of the environmental and economical impacts. In order to produce high-quality recycling products, mono-fractional compositions of waste polymers are required. However, existing measurement technologies such as near infrared spectroscopy show limitations in the sorting of complex mixtures and different grades of polymers, especially when black plastics are involved. More recently invented technologies based on mid-infrared, Raman spectroscopy or laser-aided spectroscopy are still under development and expected to be rather expensive. A promising approach to put high sorting purities into practice is to label plastic resins with unique combinations of fluorescence markers (tracers). These are incorporated into virgin resins during the manufacturing process at the ppm (or sub ppm) concentration level, just large enough that the fluorescence emissions can be detected with sensitive instrumentation but neither affect the visual appearance nor the mechanical properties of the polymers. In this paper we present the prototype of a measurement and classification system that identifies polymer flakes (mill material of a few millimeters size) located on a conveyor belt in real time based on the emitted fluorescence of incorporated markers. Classification performance

  2. Single-label kinase and phosphatase assays for tyrosine phosphorylation using nanosecond time-resolved fluorescence detection.

    Science.gov (United States)

    Sahoo, Harekrushna; Hennig, Andreas; Florea, Mara; Roth, Doris; Enderle, Thilo; Nau, Werner M

    2007-12-26

    The collision-induced fluorescence quenching of a 2,3-diazabicyclo[2.2.2]oct-2-ene-labeled asparagine (Dbo) by hydrogen atom abstraction from the tyrosine residue in peptide substrates was introduced as a single-labeling strategy to assay the activity of tyrosine kinases and phosphatases. The assays were tested for 12 different combinations of Dbo-labeled substrates and with the enzymes p60c-Src Src kinase, EGFR kinase, YOP protein tyrosine phosphatase, as well as acid and alkaline phosphatases, thereby demonstrating a broad application potential. The steady-state fluorescence changed by a factor of up to 7 in the course of the enzymatic reaction, which allowed for a sufficient sensitivity of continuous monitoring in steady-state experiments. The fluorescence lifetimes (and intensities) were found to be rather constant for the phosphotyrosine peptides (ca. 300 ns in aerated water), while those of the unphosphorylated peptides were as short as 40 ns (at pH 7) and 7 ns (at pH 13) as a result of intramolecular quenching. Owing to the exceptionally long fluorescence lifetime of Dbo, the assays were alternatively performed by using nanosecond time-resolved fluorescence (Nano-TRF) detection, which leads to an improved discrimination of background fluorescence and an increased sensitivity. The potential for inhibitor screening was demonstrated through the inhibition of acid and alkaline phosphatases by molybdate.

  3. Preparation of fluorescent-dye-labeled cDNA from RNA for microarray hybridization.

    Science.gov (United States)

    Ares, Manuel

    2014-01-01

    This protocol describes how to prepare fluorescently labeled cDNA for hybridization to microarrays. It consists of two steps: first, a mixture of anchored oligo(dT) and random hexamers is used to prime amine-modified cDNA synthesis by reverse transcriptase using a modified deoxynucleotide with a reactive amine group (aminoallyl-dUTP) and an RNA sample as a template. Second, the cDNA is purified and exchanged into bicarbonate buffer so that the amine groups in the cDNA react with the dye N-hydroxysuccinimide (NHS) esters, covalently joining the dye to the cDNA. The dye-coupled cDNA is purified again, and the amount of dye incorporated per microgram of cDNA is determined.

  4. Bio-orthogonal Fluorescent Labelling of Biopolymers through Inverse-Electron-Demand Diels-Alder Reactions.

    Science.gov (United States)

    Kozma, Eszter; Demeter, Orsolya; Kele, Péter

    2017-03-16

    Bio-orthogonal labelling schemes based on inverse-electron-demand Diels-Alder (IEDDA) cycloaddition have attracted much attention in chemical biology recently. The appealing features of this reaction, such as the fast reaction kinetics, fully bio-orthogonal nature and high selectivity, have helped chemical biologists gain deeper understanding of biochemical processes at the molecular level. Listing the components and discussing the possibilities and limitations of these reagents, we provide a recent snapshot of the field of IEDDA-based biomolecular manipulation with special focus on fluorescent modulation approaches through the use of bio-orthogonalized building blocks. At the end, we discuss challenges that need to be addressed for further developments in order to overcome recent limitations and to enable researchers to answer biomolecular questions in more detail. © 2017 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.

  5. Automated characterization of nerve fibers labeled fluorescently: determination of size, class and spatial distribution.

    Science.gov (United States)

    Prodanov, Dimiter; Feirabend, Hans K P

    2008-10-03

    Morphological classification of nerve fibers could help interpret the assessment of neural regeneration and the understanding of selectivity of nerve stimulation. Specific populations of myelinated nerve fibers can be investigated by retrograde tracing from a muscle followed by microscopic measurements of the labeled fibers at different anatomical levels. Gastrocnemius muscles of adult rats were injected with the retrograde tracer Fluoro-Gold. After a survival period of 3 days, cross-sections of spinal cords, ventral roots, sciatic, and tibial nerves were collected and imaged on a fluorescence microscope. Nerve fibers were classified using a variation-based criterion acting on the distribution of their equivalent diameters. The same criterion was used to classify the labeled axons using the size of the fluorescent marker. Measurements of the axons were paired to those of the entire fibers (axons+myelin sheaths) in order to establish the correspondence between so-established axonal and fiber classifications. It was found that nerve fibers in L6 ventral roots could be classified into four populations comprising two classes of Aalpha (denoted Aalpha1 and Aalpha2), Agamma, and an additional class of Agammaalpha fibers. Cut-off borders between Agamma and Agammaalpha fiber classes were estimated to be 5.00+/-0.09 microm (SEM); between Agammaalpha and Aalpha1 fiber classes to be 6.86+/-0.11 microm (SEM); and between Aalpha1 and Aalpha2 fiber classes to be 8.66+/-0.16 microm (SEM). Topographical maps of the nerve fibers that innervate the gastrocnemius muscles were constructed per fiber class for the spinal root L6. The major advantage of the presented approach consists of the combined indirect classification of nerve fiber types and the construction of topographical maps of so-identified fiber classes.

  6. Non-Covalent Fluorescent Labeling of Hairpin DNA Probe Coupled with Hybridization Chain Reaction for Sensitive DNA Detection.

    Science.gov (United States)

    Song, Luna; Zhang, Yonghua; Li, Junling; Gao, Qiang; Qi, Honglan; Zhang, Chengxiao

    2016-04-01

    An enzyme-free signal amplification-based assay for DNA detection was developed using fluorescent hairpin DNA probes coupled with hybridization chain reaction (HCR). The hairpin DNAs were designed to contain abasic sites in the stem moiety. Non-covalent labeling of the hairpin DNAs was achieved when a fluorescent ligand was bound to the abasic sites through hydrogen bonding with the orphan cytosine present on the complementary strand, accompanied by quench of ligand fluorescence. As a result, the resultant probes, the complex formed between the hairpin DNA and ligand, showed almost no fluorescence. Upon hybridization with target DNA, the probe underwent a dehybridization of the stem moiety containing an abasic site. The release of ligand from the abasic site to the solution resulted in an effective fluorescent enhancement, which can be used as a signal. Compared with a sensing system without HCR, a 20-fold increase in the sensitivity was achieved using the sensing system with HCR. The fluorescent intensity of the sensing system increased with the increase in target DNA concentration from 0.5 nM to 100 nM. A single mismatched target ss-DNA could be effectively discriminated from complementary target DNA. Genotyping of a G/C single-nucleotide polymorphism of polymerase chain reaction (PCR) products was successfully demonstrated with the sensing system. Therefore, integrating HCR strategy with non-covalent labeling of fluorescent hairpin DNA probes provides a sensitive and cost-effective DNA assay. © The Author(s) 2016.

  7. Construction of a multiple fluorescence labeling system for use in co-invasion studies of Listeria monocytogenes

    DEFF Research Database (Denmark)

    Andersen, Jens Bo; Roldgaard, Bent; Lindner, A. B.

    2006-01-01

    strains at the single cell level by use of fluorescence microscopy. More than 90% of the L. monocytogenes host cells maintained the fluorescence tags for 40 generations. The fluorescence tags did not alter the invasive capacity of the L. monocytogenes cells in a traditional Caco-2 cell invasion assay......, and visual discrimination between invaded bacteria carrying different fluorescent labels inside the cells was possible. Conclusion The constructed fluorescent marker system is stable, easy to use, does not affect the virulence of L. monocytogenes in Caco-2 cell assays, and allows discrimination between...... deviations in the observed capacity for infection when animal models are used. One way to circumvent this problem is to carry out virulence studies as competition assays between 2 or more strains. This, however, requires invasion-neutral markers that enable easy discrimination between the different strains...

  8. Molecular imaging needles: dual-modality optical coherence tomography and fluorescence imaging of labeled antibodies deep in tissue

    Science.gov (United States)

    Scolaro, Loretta; Lorenser, Dirk; Madore, Wendy-Julie; Kirk, Rodney W.; Kramer, Anne S.; Yeoh, George C.; Godbout, Nicolas; Sampson, David D.; Boudoux, Caroline; McLaughlin, Robert A.

    2015-01-01

    Molecular imaging using optical techniques provides insight into disease at the cellular level. In this paper, we report on a novel dual-modality probe capable of performing molecular imaging by combining simultaneous three-dimensional optical coherence tomography (OCT) and two-dimensional fluorescence imaging in a hypodermic needle. The probe, referred to as a molecular imaging (MI) needle, may be inserted tens of millimeters into tissue. The MI needle utilizes double-clad fiber to carry both imaging modalities, and is interfaced to a 1310-nm OCT system and a fluorescence imaging subsystem using an asymmetrical double-clad fiber coupler customized to achieve high fluorescence collection efficiency. We present, to the best of our knowledge, the first dual-modality OCT and fluorescence needle probe with sufficient sensitivity to image fluorescently labeled antibodies. Such probes enable high-resolution molecular imaging deep within tissue. PMID:26137379

  9. Fluorescent labeling of NASBA amplified tmRNA molecules for microarray applications

    Directory of Open Access Journals (Sweden)

    Kaplinski Lauris

    2009-05-01

    Full Text Available Abstract Background Here we present a novel promising microbial diagnostic method that combines the sensitivity of Nucleic Acid Sequence Based Amplification (NASBA with the high information content of microarray technology for the detection of bacterial tmRNA molecules. The NASBA protocol was modified to include aminoallyl-UTP (aaUTP molecules that were incorporated into nascent RNA during the NASBA reaction. Post-amplification labeling with fluorescent dye was carried out subsequently and tmRNA hybridization signal intensities were measured using microarray technology. Significant optimization of the labeled NASBA protocol was required to maintain the required sensitivity of the reactions. Results Two different aaUTP salts were evaluated and optimum final concentrations were identified for both. The final 2 mM concentration of aaUTP Li-salt in NASBA reaction resulted in highest microarray signals overall, being twice as high as the strongest signals with 1 mM aaUTP Na-salt. Conclusion We have successfully demonstrated efficient combination of NASBA amplification technology with microarray based hybridization detection. The method is applicative for many different areas of microbial diagnostics including environmental monitoring, bio threat detection, industrial process monitoring and clinical microbiology.

  10. Multiplex fluorescence melting curve analysis for mutation detection with dual-labeled, self-quenched probes.

    Directory of Open Access Journals (Sweden)

    Qiuying Huang

    2011-04-01

    Full Text Available Probe-based fluorescence melting curve analysis (FMCA is a powerful tool for mutation detection based on melting temperature generated by thermal denaturation of the probe-target hybrid. Nevertheless, the color multiplexing, probe design, and cross-platform compatibility remain to be limited by using existing probe chemistries. We hereby explored two dual-labeled, self-quenched probes, TaqMan and shared-stem molecular beacons, in their ability to conduct FMCA. Both probes could be directly used for FMCA and readily integrated with closed-tube amplicon hybridization under asymmetric PCR conditions. Improved flexibility of FMCA by using these probes was illustrated in three representative applications of FMCA: mutation scanning, mutation identification and mutation genotyping, all of which achieved improved color-multiplexing with easy probe design and versatile probe combination and all were validated with a large number of real clinical samples. The universal cross-platform compatibility of these probes-based FMCA was also demonstrated by a 4-color mutation genotyping assay performed on five different real-time PCR instruments. The dual-labeled, self-quenched probes offered unprecedented combined advantage of enhanced multiplexing, improved flexibility in probe design, and expanded cross-platform compatibility, which would substantially improve FMCA in mutation detection of various applications.

  11. Simultaneous cathodoluminescence and electron microscopy cytometry of cellular vesicles labeled with fluorescent nanodiamonds

    Science.gov (United States)

    Nagarajan, Sounderya; Pioche-Durieu, Catherine; Tizei, Luiz H. G.; Fang, Chia-Yi; Bertrand, Jean-Rémi; Le Cam, Eric; Chang, Huan-Cheng; Treussart, François; Kociak, Mathieu

    2016-06-01

    Light and Transmission Electron Microscopies (LM and TEM) hold potential in bioimaging owing to the advantages of fast imaging of multiple cells with LM and ultrastructure resolution offered by TEM. Integrated or correlated LM and TEM are the current approaches to combine the advantages of both techniques. Here we propose an alternative in which the electron beam of a scanning TEM (STEM) is used to excite concomitantly the luminescence of nanoparticle labels (a process known as cathodoluminescence, CL), and image the cell ultrastructure. This CL-STEM imaging allows obtaining luminescence spectra and imaging ultrastructure simultaneously. We present a proof of principle experiment, showing the potential of this technique in image cytometry of cell vesicular components. To label the vesicles we used fluorescent diamond nanocrystals (nanodiamonds, NDs) of size ~150 nm coated with different cationic polymers, known to trigger different internalization pathways. Each polymer was associated with a type of ND with a different emission spectrum. With CL-STEM, for each individual vesicle, we were able to measure (i) their size with nanometric resolution, (ii) their content in different ND labels, and realize intracellular component cytometry. In contrast to the recently reported organelle flow cytometry technique that requires cell sonication, CL-STEM-based image cytometry preserves the cell integrity and provides a much higher resolution in size. Although this novel approach is still limited by a low throughput, the automatization of data acquisition and image analysis, combined with improved intracellular targeting, should facilitate applications in cell biology at the subcellular level.Light and Transmission Electron Microscopies (LM and TEM) hold potential in bioimaging owing to the advantages of fast imaging of multiple cells with LM and ultrastructure resolution offered by TEM. Integrated or correlated LM and TEM are the current approaches to combine the advantages of

  12. pH-responsive diblock copolymers with two different fluorescent labels for simultaneous monitoring of micellar self-assembly and degree of protonation

    DEFF Research Database (Denmark)

    Madsen, Jeppe; Madden, George; Themistou, Efrosyni

    2018-01-01

    methacrylate [PyMA] is statistically copolymerized with glycerolmonomethacrylate (GMA) to introduce a suitable fluorescent label. The chain-ends of the PDPA block are labelled with cresylviolet perchlorate [CV] by exploiting the spin trap properties of this dye molecule. Below pH 6, fluorescence from both...

  13. Fluorescent Oligonucleotides Containing a 2-Ethynylfluorene-or 2-Ethynylfluorenone-labeled 2'-Deoxyguanosine Unit: Fluorescence Changes upon Duplex Formation

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Min Ji; Hwang, Gil Tae [Kyungpook National University, Daegu (Korea, Republic of)

    2016-08-15

    Two new DNA probes bearing a fluorescent deoxyguanosine unit labeled with 2-ethynylfluorene (G{sup FL} )or 2-ethynylfluorenone (G{sup FO}) were synthesized and examined for their efficiency as quencher-free linear beacon probes. Oligodeoxynucleotides (ODNs) containing a G{sup FL} or G{sup FO} unit exhibit low thermal selectivity and few distinctive fluorescence changes upon duplex formation due to the syn conformation about the glycosidic bond. An exciplex emission was observed when the G{sup FL} unit of ODNs bearing adenine flanking bases was positioned opposite to the adenine nucleobases.

  14. Rapid genomic fingerprinting of Lactococcus lactis strains by arbitrarily primed polymerase chain reaction with 32P and fluorescent labels.

    OpenAIRE

    Cancilla, M R; Powell, I B; Hillier, A J; Davidson, B E

    1992-01-01

    Arbitrarily primed polymerase chain reaction, with incorporation of either radioactive or fluorescent labels, was used as a rapid and sensitive method for obtaining genomic fingerprints of strains of Lactococcus lactis. Closely related strains produced almost identical fingerprints. Fingerprints of other strains showed only some similarities.

  15. Development of a facile and sensitive HPLC-FLD method via fluorescence labeling for triterpenic acid bioavailability investigation.

    Science.gov (United States)

    You, Jinmao; Wu, Di; Zhao, Mei; Li, Guoliang; Gong, Peiwei; Wu, Yueyue; Guo, Yu; Chen, Guang; Zhao, Xianen; Sun, Zhiwei; Xia, Lian; Wu, Yongning

    2017-06-01

    Triterpenic acids are widely distributed in many fruits and are known for their medicinal benefits. The study of bioavailability has been an important task for a better understanding of the triterpenic acids. Although many methods based on fluorescence labeling for triterpenic acid determination have been established, these reported methods needed anhydrous conditions, which are not suitable for the convenient study of triterpenic acid bioavailability. Inspired by that, a versatile method, which overcomes the difficulty of the reported methods, has been first developed in this study. The novel method using 2-[12-benzo[b]acridin-5- (12H)-yl]-acetohydrazide (BAAH) as the fluorescence labeling reagent coupled with high-performance liquid chromatography with fluorescence detection was first developed for the study of triterpenic acid bioavailability. Furthermore, the labeling conditions have been optimized in order to achieve the best fluorescence labeling yield. Under the optimal conditions, the quantitative linear range of analytes was 2-1000 ng mL -1 , and the correlation coefficients were >0.9998. The detection limits for all triterpenic acid derivatives were achieved within the range of 0.28-0.29 ng mL -1 . The proposed method was successfully applied to the study of triterpenic acid bioavailability with excellent applicability and good reproducibility. Copyright © 2016 John Wiley & Sons, Ltd.

  16. Development of a fluorescent label tool based on lanthanide nanophosphors for viral biomedical application

    International Nuclear Information System (INIS)

    Le, Quoc Minh; Tran, Thu Huong; Nguyen, Thanh Huong; Hoang, Thi Khuyen; Nguyen, Thanh Binh; Do, Khanh Tung; Tran, Kim Anh; Nguyen, Dang Hien; Le, Thi Luan; Nguyen, Thi Quy; Dang, Mai Dung; Thu Nguyen, Nu Anh; Nguyen, Van Man

    2012-01-01

    We report for the first time the preparation of luminescent lanthanide nanomaterial (LLN) linked bioconjugates and their application as a label tool for recognizing virus in the processing line of vaccine industrial fabrication. Several LLNs with the nanostructure forms of particles or rods/wires with europium (III) and terbium (III) ions in lattices of vanadate, phosphate and metal organic complex were prepared to develop novel fluorescent conjugates able to be applied as labels in fluorescence immunoassay analysis of virus/vaccine. With regard to the LLNs, we have successfully synthesized nanoparticles around 10 nm of YVO 4 :Eu(III), with high emission in the red spectral region, nanorod and nanowire of TbPO 4 ·H 2 O and Eu 1-x Tb x PO 4 ·H 2 O, width 5–7 nm and length 300 nm, showing very bright luminescence in green, and core/shell nanosized Eu(III) and Tb(III)/Eu(III) complexes with naphthoyl trifluoroacetone and tri-n-octylphosphineoxide (Eu.NTA.TOPO-PVP, Eu X Tb 1-X .NTA.TOPO). The appropriated core/shell structures can play a double role, one for enhancing luminescence efficiency and another for providing nanophosphors with better stability in water media for facilitating the penetration of nanophosphor core into a biomedical environment. The organic functionalizations of the obtained LLNs were done through their surface encapsulation with a functional polysiloxane including active groups such as amine (NH 2 ), thiocyanate (SCN) or mecarpto (SH). The properties of functional sol-gel matrix have great influence on the luminescence properties, especially luminescence intensity of YVO 4 :Eu(III), Eu.NTA.TOPO-PVP, TbPO 4 ·H 2 O and Eu x Tb 1-x PO 4 ·H 2 O. Bioconjugation processes of the functionalized LLNs have been studied with some bioactive molecules such as biotin, protein immunoglobulin G (IgG) or bovine serum albumin (BSA). The results of LLN-bioconjugate linking with IgG for recognizing virus (vaccine) will be presented in brief. It is consistent

  17. Label-Free Fluorescent Detection of Trypsin Activity Based on DNA-Stabilized Silver Nanocluster-Peptide Conjugates

    Directory of Open Access Journals (Sweden)

    Cai-Xia Zhuo

    2016-11-01

    Full Text Available Trypsin is important during the regulation of pancreatic exocrine function. The detection of trypsin activity is currently limited because of the need for the substrate to be labeled with a fluorescent tag. A label-free fluorescent method has been developed to monitor trypsin activity. The designed peptide probe consists of six arginine molecules and a cysteine terminus and can be conjugated to DNA-stabilized silver nanoclusters (DNA-AgNCs by Ag-S bonding to enhance fluorescence. The peptide probe can also be adsorbed to the surface of graphene oxide (GO, thus resulting in the fluorescence quenching of DNA-AgNCs-peptide conjugate because of Förster resonance energy transfer. Once trypsin had degraded the peptide probe into amino acid residues, the DNA-AgNCs were released from the surface of GO, and the enhanced fluorescence of DNA-AgNCs was restored. Trypsin can be determined with a linear range of 0.0–50.0 ng/mL with a concentration as low as 1 ng/mL. This label-free method is simple and sensitive and has been successfully used for the determination of trypsin in serum. The method can also be modified to detect other proteases.

  18. Live Imaging of Cellular Internalization of Single Colloidal Particle by Combined Label-Free and Fluorescence Total Internal Reflection Microscopy.

    Science.gov (United States)

    Byrne, Gerard D; Vllasaliu, Driton; Falcone, Franco H; Somekh, Michael G; Stolnik, Snjezana

    2015-11-02

    In this work we utilize the combination of label-free total internal reflection microscopy and total internal reflectance fluorescence (TIRM/TIRF) microscopy to achieve a simultaneous, live imaging of single, label-free colloidal particle endocytosis by individual cells. The TIRM arm of the microscope enables label free imaging of the colloid and cell membrane features, while the TIRF arm images the dynamics of fluorescent-labeled clathrin (protein involved in endocytosis via clathrin pathway), expressed in transfected 3T3 fibroblasts cells. Using a model polymeric colloid and cells with a fluorescently tagged clathrin endocytosis pathway, we demonstrate that wide field TIRM/TIRF coimaging enables live visualization of the process of colloidal particle interaction with the labeled cell structure, which is valuable for discerning the membrane events and route of colloid internalization by the cell. We further show that 500 nm in diameter model polystyrene colloid associates with clathrin, prior to and during its cellular internalization. This association is not apparent with larger, 1 μm in diameter colloids, indicating an upper particle size limit for clathrin-mediated endocytosis.

  19. Rapid labeling of amino acid neurotransmitters with a fluorescent thiol in the presence of o-phthalaldehyde.

    Science.gov (United States)

    Maddukuri, Naveen; Zhang, Qiyang; Zhang, Ning; Gong, Maojun

    2017-02-01

    LIF detection often requires labeling of analytes with fluorophores; and fast fluorescent derivatization is valuable for high-throughput analysis with flow-gated CE. Here, we report a fast fluorescein-labeling scheme for amino acid neurotransmitters, which were then rapidly separated and detected in flow-gated CE. This scheme was based on the reaction between primary amines and o-phthalaldehyde in the presence of a fluorescent thiol, 2-((5-fluoresceinyl)aminocarbonyl)ethyl mercaptan (FACE-SH). The short reaction time (neurotransmitters by coupling in vitro microdialysis with online derivatization and flow-gated CE. It is also anticipated that this fluorophore tagging scheme would be valuable for on-chip labeling of proteins retained on support in SPE. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  20. Bioimaging of Fluorescence-Labeled Mitochondria in Subcutaneously Grafted Murine Melanoma Cells by the “In Vivo Cryotechnique”

    Science.gov (United States)

    Lei, Ting; Huang, Zheng; Ohno, Nobuhiko; Wu, Bao; Sakoh, Takashi; Saitoh, Yurika; Saiki, Ikuo

    2014-01-01

    The microenvironments of organs with blood flow affect the metabolic profiles of cancer cells, which are influenced by mitochondrial functions. However, histopathological analyses of these aspects have been hampered by technical artifacts of conventional fixation and dehydration, including ischemia/anoxia. The purpose of this study was to combine the in vivo cryotechnique (IVCT) with fluorescent protein expression, and examine fluorescently labeled mitochondria in grafted melanoma tumors. The intensity of fluorescent proteins was maintained well in cultured B16-BL6 cells after cryotechniques followed by freeze-substitution (FS). In the subcutaneous tumors of mitochondria-targeted DsRed2 (mitoDsRed)-expressing cells, a higher number of cancer cells were found surrounding the widely opened blood vessels that contained numerous erythrocytes. Such blood vessels were immunostained positively for immunoglobulin M and ensheathed by basement membranes. MitoDsRed fluorescence was detected in scattering melanoma cells using the IVCT-FS method, and the total mitoDsRed volume in individual cancer cells was significantly decreased with the expression of markers of hypoxia. MitoDsRed was frequently distributed throughout the cytoplasm and in processes extending along basement membranes. IVCT combined with fluorescent protein expression is a useful tool to examine the behavior of fluorescently labeled cells and organelles. We propose that the mitochondrial volume is dynamically regulated in the hypoxic microenvironment and that mitochondrial distribution is modulated by cancer cell interactions with basement membranes. PMID:24394469

  1. Detection of NT-pro BNP using fluorescent protein modified by streptavidin as a label in immunochromatographic assay

    Directory of Open Access Journals (Sweden)

    Haixia Li

    2016-12-01

    Full Text Available A novel fluorescent immunochromatographic assay for the detection of NT-proBNP in human serum has been developed. Based on a sandwich-type immunoassay format, analytes in samples were captured by one monoclonal antibody labeled with fluorescent protein and “sandwiched” by another monoclonal antibody immobilized on the nitrocellulose membrane, the fluorescence and concentration of analytes were measured and then calculated by fluoroanalyzer. The fluorescent protein is a fusion protein and was prepared through the application of Streptavidin gene SA, β subunit cpcB of Phycocyanin, lyase alr0617, and phycoerythrobilin synthetase gene ho1, pebA, pebB for covalent binding. It is characterized with higher stability, good solubility in water and it is not easy to quench fluorescence. Take the advantages of fluorescent protein, the immunochromatographic assay exhibited a wide linear range for NT-proBNP from 200 pg ml−1 to 26,000 pg ml−1, with a detection limit of 47 pg ml−1 under optimal conditions. Compared with chemiluminescence immunoassay (CLIA, 131 human serum samples were analyzed and the correlation coefficient of the developed immunoassay was 0.978. These results demonstrated that fluorescent immunochromatographic assay is a more rapid, sensitive, specific method and could be developed into a platform for more biomarkers determination in clinical practice. Keywords: NT-pro BNP, Fluorescent protein, Immunochromatographic assay

  2. Green fluorescent protein labeling of Listeria, Salmonella, and Escherichia coli O157:H7 for safety-related studies.

    Directory of Open Access Journals (Sweden)

    Li Ma

    Full Text Available Many food safety-related studies require tracking of introduced foodborne pathogens to monitor their fate in complex environments. The green fluorescent protein (GFP gene (gfp provides an easily detectable phenotype so has been used to label many microorganisms for ecological studies. The objectives of this study were to label major foodborne pathogens and related bacteria, including Listeria monocytogenes, Listeria innocua, Salmonella, and Escherichia coli O157:H7 strains, with GFP and characterize the labeled strains for stability of the GFP plasmid and the plasmid's effect on bacterial growth. GFP plasmids were introduced into these strains by a CaCl(2 procedure, conjugation or electroporation. Stability of the label was determined through sequential propagation of labeled strains in the absence of selective pressure, and rates of plasmid-loss were calculated. Stability of the GFP plasmid varied among the labeled species and strains, with the most stable GFP label observed in E. coli O157:H7. When grown in nonselective media for two consecutive subcultures (ca. 20 generations, the rates of plasmid loss among labeled E. coli O157:H7, Salmonella and Listeria strains ranged from 0%-30%, 15.8%-99.9% and 8.1%-93.4%, respectively. Complete loss (>99.99% of the plasmid occurred in some labeled strains after five consecutive subcultures in the absence of selective pressure, whereas it remained stable in others. The GFP plasmid had an insignificant effect on growth of most labeled strains. E. coli O157:H7, Salmonella and Listeria strains can be effectively labeled with the GFP plasmid which can be stable in some isolates for many generations without adversely affecting growth rates.

  3. Tumorigenicity and Validity of Fluorescence Labelled Mesenchymal and Epithelial Human Oral Cancer Cell Lines in Nude Mice

    Directory of Open Access Journals (Sweden)

    Wei Xin Cai

    2016-01-01

    Full Text Available Tumorigenicity and metastatic activity can be visually monitored in cancer cells that were labelled with stable fluorescence. The aim was to establish and validate local and distant spread of subcutaneously previously injected fluorescence transduced human tongue cancer cell lines of epithelial and mesenchymal phenotype in nude mice. A total of 32 four-week-old male athymic Balb/c nude mice were randomly allocated into 4 groups (n=8. A single dose of 0.3 mL PBS containing 1 × 107 of four different cancer cell-lines (UM1, UM1-GFP, UM2, and UM2-RFP was injected subcutaneously into the right side of their posterolateral back. Validity assessment of the labelled cancer cells’ tumorigenicity was assessed by physical examination, imaging, and histology four weeks after the injection. The tumor take rate of cancer cells was similar in animals injected with either parental or transduced cancer cells. Transduced cancer cells in mice were easily detectable in vivo and after cryosection using fluorescent imaging. UM1 cells showed increased tumor take rate and mean tumor volume, presenting with disorganized histopathological patterns. Fluorescence labelled epithelial and mesenchymal human tongue cancer cell lines do not change in tumorigenicity or cell phenotype after injection in vivo.

  4. A fluorescent and chemiluminescent difunctional mesoporous silica nanoparticle as a label for the ultrasensitive detection of cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Tao Liang [Key Laboratory of Analytical Chemistry for Life Science of Shaanxi Province, School of Chemistry and Chemical Engineering, Shaanxi Normal University, Xi' an 710062 (China); Song Chaojun; Sun Yuanjie [Department of Immunology, The Fourth Military Medical University, Xi' an 710032 (China); Li Xiaohua; Li Yunyun [Key Laboratory of Analytical Chemistry for Life Science of Shaanxi Province, School of Chemistry and Chemical Engineering, Shaanxi Normal University, Xi' an 710062 (China); Jin Boquan [Department of Immunology, The Fourth Military Medical University, Xi' an 710032 (China); Zhang Zhujun, E-mail: zhangzj@snnu.edu.cn [Key Laboratory of Analytical Chemistry for Life Science of Shaanxi Province, School of Chemistry and Chemical Engineering, Shaanxi Normal University, Xi' an 710062 (China); Yang Kun, E-mail: yangkunkun@fmmu.edu.cn [Department of Immunology, The Fourth Military Medical University, Xi' an 710032 (China)

    2013-01-25

    Highlights: Black-Right-Pointing-Pointer Difunctional amino mesoporous silica nanoparticles (FCMSN) were synthesized. Black-Right-Pointing-Pointer The fluorescence and chemiluminescence properties of the FCMSN were studied. Black-Right-Pointing-Pointer The NaIO{sub 4} oxidation method was used for modification of the FCMSN. Black-Right-Pointing-Pointer Liver cancer 7721 cell was detected. Black-Right-Pointing-Pointer The specificity affected by FCMSN's amino groups was studied. - Abstract: A new kind of ultrabright fluorescent and chemiluminescent difunctional mesoporous silica nanoparticle (FCMSN) is reported. A luminescent dye, Rhodamine 6G or tris(2,2 Prime -bipyridyl)dichlororuthenium(II) hexahydrate (Rubpy), is doped inside nanochannels of a silica matrix. The hydrophobic groups in the silica matrix avoid the leakage of dye from open channels. The amines groups on the surface of the FCMSN improve the modification performance of the nanoparticle. Because the nanochannels are isolated by a network skeleton of silica, fluorescence quenching based on the inner filter effect of the fluorescent dyes immobilized in nanochannels is weakened effectively. The Quantum Yield of obtained 90 nm silica particles was about 61%. Compared with the fluorescent core-shell nanoparticle, the chemiluminescence reagents can freely enter the nanoparticles to react with fluorescent dyes to create chemiluminescence. The results show that the FCMSN are both fluorescent labels and chemiluminescent labels. In biological applications, the NaIO{sub 4} oxidation method was proven to be superior to the glutaraldehyde method. The amount of amino could affect the specificity of the FCMSN. The fluorescence microscopy imaging demonstrated that the FCMSN is viable for biological applications.

  5. Multiplex competitive microbead-based flow cytometric immunoassay using quantum dot fluorescent labels

    International Nuclear Information System (INIS)

    Yu, Hye-Weon; Kim, In S.; Niessner, Reinhard; Knopp, Dietmar

    2012-01-01

    Highlights: ► First time, duplex competitive bead-based flow cytometric immunoassay was developed using ODs. ► Antibody-coated QD detection probes and antigen-immobilized microspheres were synthesized. ► The two model target analytes were low molecular weight compounds of microbial and chemical origin. ► The determination of different water types was possible after simple filtration of samples. - Abstract: In answer to the ever-increasing need to perform the simultaneous analysis of environmental hazards, microcarrier-based multiplex technologies show great promise. Further integration with biofunctionalized quantum dots (QDs) creates new opportunities to extend the capabilities of multicolor flow cytometry with their unique fluorescence properties. Here, we have developed a competitive microbead-based flow cytometric immunoassay using QDs fluorescent labels for simultaneous detection of two analytes, bringing the benefits of sensitive, rapid and easy-of-manipulation analytical tool for environmental contaminants. As model target compounds, the cyanobacterial toxin microcystin-LR and the polycyclic aromatic hydrocarbon compound benzo[a]pyrene were selected. The assay was carried out in two steps: the competitive immunological reaction of multiple targets using their exclusive sensing elements of QD/antibody detection probes and antigen-coated microsphere, and the subsequent flow cytometric analysis. The fluorescence of the QD-encoded microsphere was thus found to be inversely proportional to target analyte concentration. Under optimized conditions, the proposed assay performed well within 30 min for the identification and quantitative analysis of the two environmental contaminants. For microcystin-LR and benzo[a]pyrene, dose–response curves with IC 50 values of 5 μg L −1 and 1.1 μg L −1 and dynamic ranges of 0.52–30 μg L −1 and 0.13–10 μg L −1 were obtained, respectively. Recovery was 92.6–106.5% for 5 types of water samples like bottled

  6. Volume labeling with Alexa Fluor dyes and surface functionalization of highly sensitive fluorescent silica (SiO2) nanoparticles

    Science.gov (United States)

    Wang, Wei; Nallathamby, Prakash D.; Foster, Carmen M.; Morrell-Falvey, Jennifer L.; Mortensen, Ninell P.; Doktycz, Mitchel J.; Gu, Baohua; Retterer, Scott T.

    2013-10-01

    A new synthesis approach is described that allows the direct incorporation of fluorescent labels into the volume or body of SiO2 nanoparticles. In this process, fluorescent Alexa Fluor dyes with different emission wavelengths were covalently incorporated into the SiO2 nanoparticles during their formation by the hydrolysis of tetraethoxysilane. The dye molecules were homogeneously distributed throughout the SiO2 nanoparticles. The quantum yields of the Alexa Fluor volume-labeled SiO2 nanoparticles were much higher than nanoparticles labeled using conventional organic dyes. The size of the resulting nanoparticles was controlled using microemulsion reaction media with sizes in the range of 20-100 nm and a polydispersity of cultured macrophages. Differences in particle agglomeration and cell association were clearly associated with differences in observed nanoparticle toxicity. The capacity to maintain particle fluorescence while making significant changes to surface chemistry makes these particles extremely versatile and useful for studies of particle agglomeration, uptake, and transport in environmental and biological systems.A new synthesis approach is described that allows the direct incorporation of fluorescent labels into the volume or body of SiO2 nanoparticles. In this process, fluorescent Alexa Fluor dyes with different emission wavelengths were covalently incorporated into the SiO2 nanoparticles during their formation by the hydrolysis of tetraethoxysilane. The dye molecules were homogeneously distributed throughout the SiO2 nanoparticles. The quantum yields of the Alexa Fluor volume-labeled SiO2 nanoparticles were much higher than nanoparticles labeled using conventional organic dyes. The size of the resulting nanoparticles was controlled using microemulsion reaction media with sizes in the range of 20-100 nm and a polydispersity of cultured macrophages. Differences in particle agglomeration and cell association were clearly associated with differences in

  7. Label-free fluorescence strategy for sensitive microRNA detection based on isothermal exponential amplification and graphene oxide.

    Science.gov (United States)

    Li, Wei; Hou, Ting; Wu, Min; Li, Feng

    2016-01-01

    MicroRNAs (miRNAs) play an important role in many biological processes, and have been regarded as potential targets and biomarkers in cancer diagnosis and therapy. Also, to meet the big challenge imposed by the characteristics of miRNAs, such as small size and vulnerability to enzymatic digestion, it is of great importance to develop accurate, sensitive and simple miRNA assays. Herein, we developed a label-free fluorescence strategy for sensitive miRNA detection by combining isothermal exponential amplification and the unique features of SYBR Green I (SG) and graphene oxide (GO), in which SG gives significantly enhanced fluorescence upon intercalation into double-stranded DNAs (dsDNAs), and GO selectively adsorbs miRNA, single-stranded DNA and SG, to protect miRNA from enzymatic digestion, and to quench the fluorescence of the adsorbed SG. In the presence of the target miRNA, the ingeniously designed hairpin probe (HP) is unfolded and the subsequent polymerization and strand displacement reaction takes place to initiate the target recycling process. The newly formed dsDNAs are then recognized and cleaved by the nicking enzyme, generating new DNA triggers with the same sequence as the target miRNA, which hybridize with intact HPs to initiate new extension reactions. As a result, the circular exponential amplification for target miRNA is achieved and large amount of dsDNAs are formed to generate significantly enhanced fluorescence upon the intercalation of SG. Thus sensitive and selective fluorescence miRNA detection is realized, and the detection limit of 3 fM is obtained. Besides, this method exhibits additional advantages of simplicity and low cost, since expensive and tedious labeling process is avoided. Therefore, the as-proposed label-free fluorescence strategy has great potential in the applications in miRNA-related clinical practices and biochemical researches. Copyright © 2015 Elsevier B.V. All rights reserved.

  8. Synthesis of water-soluble, ring-substituted squaraine dyes and their evaluation as fluorescent probes and labels

    Energy Technology Data Exchange (ETDEWEB)

    Tatarets, Anatoliy L. [SSI ' Institute for Single Crystals' of the National Academy of Sciences of Ukraine, 60 Lenin Ave., Kharkov 61001 (Ukraine); Fedyunyayeva, Irina A. [SSI ' Institute for Single Crystals' of the National Academy of Sciences of Ukraine, 60 Lenin Ave., Kharkov 61001 (Ukraine); Dyubko, Tatyana S. [SSI ' Institute for Single Crystals' of the National Academy of Sciences of Ukraine, 60 Lenin Ave., Kharkov 61001 (Ukraine); Povrozin, Yevgeniy A. [SSI ' Institute for Single Crystals' of the National Academy of Sciences of Ukraine, 60 Lenin Ave., Kharkov 61001 (Ukraine); Doroshenko, Andrey O. [Institute of Chemistry, V.N. Karazin National University, 4 Svobody Sq., Kharkov 61077 (Ukraine); Terpetschnig, Ewald A. [SETA BioMedicals, LLC, 2014 Silver Ct East, Urbana, IL 61801 (United States) and ISS, Inc., 1602 Newton Drive, Champaign, IL 61822 (United States)]. E-mail: ewaldte@juno.com; Patsenker, Leonid D. [SSI ' Institute for Single Crystals' of the National Academy of Sciences of Ukraine, 60 Lenin Ave., Kharkov 61001 (Ukraine); SETA BioMedicals, LLC, 2014 Silver Ct East, Urbana, IL 61801 (United States)

    2006-06-16

    A series of ring-substituted squaraines absorbing and emitting in the red and NIR spectral region was synthesized and their spectral and photophysical properties (quantum yields, fluorescence lifetimes) and photostabilities were measured and compared to Cy5, a commonly used fluorescent label. The absorption maxima in aqueous media were found to be between 628 and 667 nm and the emission maxima are between 642 and 685 nm. Squaraine dyes exhibit high extinction coefficients (163,000-265,000 M{sup -1} cm{sup -1}) and lower quantum yields (2-7%) in aqueous buffer but high quantum yields (up to 45%) and long fluorescence lifetimes (up to 3.3 ns) in presence of BSA. Dicyanomethylene- and thio-substituted squaraines exhibit an additional absorption around 400 nm with extinction coefficients between 21,500 and 44,500 M{sup -1} cm{sup -1}. These dyes are excitable not only with red but also with blue diode lasers or light emitting diodes. Due to the favourable spectral and photophysical properties these dyes can be used as fluorescent probes and labels for intensity- and fluorescence lifetime-based biomedical applications.

  9. Synthesis of water-soluble, ring-substituted squaraine dyes and their evaluation as fluorescent probes and labels

    International Nuclear Information System (INIS)

    Tatarets, Anatoliy L.; Fedyunyayeva, Irina A.; Dyubko, Tatyana S.; Povrozin, Yevgeniy A.; Doroshenko, Andrey O.; Terpetschnig, Ewald A.; Patsenker, Leonid D.

    2006-01-01

    A series of ring-substituted squaraines absorbing and emitting in the red and NIR spectral region was synthesized and their spectral and photophysical properties (quantum yields, fluorescence lifetimes) and photostabilities were measured and compared to Cy5, a commonly used fluorescent label. The absorption maxima in aqueous media were found to be between 628 and 667 nm and the emission maxima are between 642 and 685 nm. Squaraine dyes exhibit high extinction coefficients (163,000-265,000 M -1 cm -1 ) and lower quantum yields (2-7%) in aqueous buffer but high quantum yields (up to 45%) and long fluorescence lifetimes (up to 3.3 ns) in presence of BSA. Dicyanomethylene- and thio-substituted squaraines exhibit an additional absorption around 400 nm with extinction coefficients between 21,500 and 44,500 M -1 cm -1 . These dyes are excitable not only with red but also with blue diode lasers or light emitting diodes. Due to the favourable spectral and photophysical properties these dyes can be used as fluorescent probes and labels for intensity- and fluorescence lifetime-based biomedical applications

  10. Automated microfluidic devices integrating solid-phase extraction, fluorescent labeling, and microchip electrophoresis for preterm birth biomarker analysis.

    Science.gov (United States)

    Sahore, Vishal; Sonker, Mukul; Nielsen, Anna V; Knob, Radim; Kumar, Suresh; Woolley, Adam T

    2018-01-01

    We have developed multichannel integrated microfluidic devices for automated preconcentration, labeling, purification, and separation of preterm birth (PTB) biomarkers. We fabricated multilayer poly(dimethylsiloxane)-cyclic olefin copolymer (PDMS-COC) devices that perform solid-phase extraction (SPE) and microchip electrophoresis (μCE) for automated PTB biomarker analysis. The PDMS control layer had a peristaltic pump and pneumatic valves for flow control, while the PDMS fluidic layer had five input reservoirs connected to microchannels and a μCE system. The COC layers had a reversed-phase octyl methacrylate porous polymer monolith for SPE and fluorescent labeling of PTB biomarkers. We determined μCE conditions for two PTB biomarkers, ferritin (Fer) and corticotropin-releasing factor (CRF). We used these integrated microfluidic devices to preconcentrate and purify off-chip-labeled Fer and CRF in an automated fashion. Finally, we performed a fully automated on-chip analysis of unlabeled PTB biomarkers, involving SPE, labeling, and μCE separation with 1 h total analysis time. These integrated systems have strong potential to be combined with upstream immunoaffinity extraction, offering a compact sample-to-answer biomarker analysis platform. Graphical abstract Pressure-actuated integrated microfluidic devices have been developed for automated solid-phase extraction, fluorescent labeling, and microchip electrophoresis of preterm birth biomarkers.

  11. Affinity fluorescence-labeled peptides for the early detection of cancer in Barrett's esophagus

    Science.gov (United States)

    Li, Meng; Lu, Shaoying; Piraka, Cyrus; Appelman, Henry; Kwon, Rich; Soetikno, Roy; Kaltenbach, Tonya; Wang, Thomas D.

    2009-02-01

    Fluorescence-labeled peptides that affinity bind to neoplastic mucsosa are promising for use as a specific contrast agent in the detection of pre-malignant tissue in the esophagus. This method is can be used to identify expression of biological markers associated with dysplasia on endoscopic imaging as a guide for biopsy and represents a novel method for the early detection and prevention of cancer. We demonstrate the use of phage display to select affinity peptides and identify the sequence "ASYNYDA" that binds with high target-to-background ratio to dysplastic esophageal mucosa compared to that of intestinal metaplasia. Validation of preferential binding is demonstrated for neoplasia in the setting of Barrett's esophagus. An optimal tradeoff between sensitivity and specificity of 82% and 85% was found at the relative threshold of 0.60 with a target-to-background ratio of 1.81 and an area under the ROC curve of 0.87. Peptides are a novel class of ligand for targeted detection of pre-malignant mucosa for purposes of screening and surveillance.

  12. Synthesis and biological investigation of PIM mimics carrying biotin or a fluorescent label for cellular imaging.

    Science.gov (United States)

    Front, Sophie; Bourigault, Marie-Laure; Rose, Stéphanie; Noria, Ségueni; Quesniaux, Valérie F J; Martin, Olivier R

    2013-01-16

    Phosphatidyl inositol mannosides (PIMs) are constituents of the mycobacterial cell wall; these glycolipids are known to exhibit potent inhibitory activity toward the LPS-induced production of cytokines by macrophages, and therefore have potential as anti-inflammatory agents. Recently, heterocyclic analogues of PIMs in which the inositol is replaced by a piperidine (aza-PIM mimics) or a tetrahydropyran moiety (oxa-PIM mimics) have been prepared by short synthetic sequences and shown to retain the biological activity of the parent PIM structures. In this investigation, the aza-PIM analogue was used as a convenient scaffold to link biotin or a fluorescent label (tetramethyl-rhodamine) by way of an aminocaproyl spacer, with the goal of using these conjugates for intracellular localization and for the study of the mechanism of their antiinflammatory action. The synthesis of these compounds is reported, as well as the evaluation of their activities as inhibitors of LPS-induced cytokine production by macrophages (TNFα, IL12p40); preliminary investigations by FACS and confocal microscopy indicated that PIM-biotin conjugate binds to macrophage membranes with rapid kinetics.

  13. Single Molecule 3D Orientation in Time and Space: A 6D Dynamic Study on Fluorescently Labeled Lipid Membranes

    DEFF Research Database (Denmark)

    Börner, Richard; Ehrlich, Nicky; Hohlbein, Johannes

    2016-01-01

    Interactions between single molecules profoundly depend on their mutual three-dimensional orientation. Recently, we demonstrated a technique that allows for orientation determination of single dipole emitters using a polarization-resolved distribution of fluorescence into several detection channels...... interesting in non-isotropic environments such as lipid membranes, which are of great importance in biology. We used giant unilamellar vesicles (GUVs) labeled with fluorescent dyes down to a single molecule concentration as a model system for both, assessing the robustness of the orientation determination...

  14. Double-labeled donor probe can enhance the signal of fluorescence resonance energy transfer (FRET) in detection of nucleic acid hybridization

    Science.gov (United States)

    Okamura, Yukio; Kondo, Satoshi; Sase, Ichiro; Suga, Takayuki; Mise, Kazuyuki; Furusawa, Iwao; Kawakami, Shigeki; Watanabe, Yuichiro

    2000-01-01

    A set of fluorescently-labeled DNA probes that hybridize with the target RNA and produce fluorescence resonance energy transfer (FRET) signals can be utilized for the detection of specific RNA. We have developed probe sets to detect and discriminate single-strand RNA molecules of plant viral genome, and sought a method to improve the FRET signals to handle in vivo applications. Consequently, we found that a double-labeled donor probe labeled with Bodipy dye yielded a remarkable increase in fluorescence intensity compared to a single-labeled donor probe used in an ordinary FRET. This double-labeled donor system can be easily applied to improve various FRET probes since the dependence upon sequence and label position in enhancement is not as strict. Furthermore this method could be applied to other nucleic acid substances, such as oligo RNA and phosphorothioate oligonucleotides (S-oligos) to enhance FRET signal. Although the double-labeled donor probes labeled with a variety of fluorophores had unexpected properties (strange UV-visible absorption spectra, decrease of intensity and decay of donor fluorescence) compared with single-labeled ones, they had no relation to FRET enhancement. This signal amplification mechanism cannot be explained simply based on our current results and knowledge of FRET. Yet it is possible to utilize this double-labeled donor system in various applications of FRET as a simple signal-enhancement method. PMID:11121494

  15. Multisegment one-step RT-PCR fluorescent labeling of influenza A virus genome for use in diagnostic microarray applications

    Energy Technology Data Exchange (ETDEWEB)

    Vasin, A V; Plotnikova, M A; Klotchenko, S A; Elpaeva, E A; Komissarov, A B; Egorov, V V; Kiselev, O I [Research Institute of Influenza of the Ministry of Health and Social Development of the Russian Federation, 15/17 Prof. Popova St., St. Petersburg (Russian Federation); Sandybaev, N T; Chervyakova, O V; Strochkov, V M; Taylakova, E T; Koshemetov, J K; Mamadaliev, S M, E-mail: vasin@influenza.spb.ru [Research Institute for Biological Safety Problems of the RK NBC/SC ME and S RK, Gvardeiskiy (Kazakhstan)

    2011-04-01

    Microarray technology is one of the most challenging methods of influenza A virus subtyping, which is based on the antigenic properties of viral surface glycoproteins - hemagglutinin and neuraminidase. On the example of biochip for detection of influenza A/H5N1 virus we showed the possibility of using multisegment RTPCR method for amplification of fluorescently labeled cDNA of all possible influenza A virus subtypes with a single pair of primers in influenza diagnostic microarrays.

  16. Fluorescence resonance energy transfer imaging of CFP/YFP labeled NDH in cyanobacterium cell

    International Nuclear Information System (INIS)

    Ji Dongmei; Lv Wei; Huang Zhengxi; Xia Andong; Xu Min; Ma Weimin; Mi Hualing; Ogawa Teruo

    2007-01-01

    The laser confocal scanning microscopy combined with time-correlated single photon counting imaging technique to obtain fluorescence intensity and fluorescence lifetime images for fluorescence resonance energy transfer measurement is reported. Both the fluorescence lifetime imaging microscopy (FLIM) and intensity images show inhomogeneous cyan fluorescent protein and yellow fluorescent protein (CFP /YFP) expression or inhomogeneous energy transfer between CFP and YFP over whole cell. The results presented in this work show that FLIM could be a potential method to reveal the structure-function behavior of NAD(P)H dehydrogenase complexes in living cell

  17. Fluorescence single-molecule counting assays for protein quantification using epi-fluorescence microscopy with quantum dots labeling

    International Nuclear Information System (INIS)

    Jiang Dafeng; Liu Chunxia; Wang Lei; Jiang Wei

    2010-01-01

    A single-molecule counting approach for quantifying the antibody affixed to a surface using quantum dots and epi-fluorescence microscopy is presented. Modifying the glass substrates with carboxyl groups provides a hydrophilic surface that reacts with amine groups of an antibody to allow covalent immobilization of the antibody. Nonspecific adsorption of single molecules on the modified surfaces was first investigated. Then, quantum dots were employed to form complexes with surface-immobilized antibody molecules and used as fluorescent probes for single-molecule imaging. Epi-fluorescence microscopy was chosen as the tool for single-molecule fluorescence detection here. The generated fluorescence signals were taken by an electron multiplying charge-coupled device and were found to be proportional to the sample concentrations. Under optimal conditions, a linear response range of 5.0 x 10 -14 -3.0 x 10 -12 mol L -1 was obtained between the number of single molecules and sample concentration via a single-molecule counting approach.

  18. Topology of OxlT, the oxalate transporter of Oxalobacter formigenes, determined by site-directed fluorescence labeling.

    Science.gov (United States)

    Ye, L; Jia, Z; Jung, T; Maloney, P C

    2001-04-01

    The topology of OxlT, the oxalate:formate exchange protein of Oxalobacter formigenes, was established by site-directed fluorescence labeling, a simple strategy that generates topological information in the context of the intact protein. Accessibility of cysteine to the fluorescent thiol-directed probe Oregon green maleimide (OGM) was examined for a panel of 34 single-cysteine variants, each generated in a His(9)-tagged cysteine-less host. The reaction with OGM was readily scored by examining the fluorescence profile after sodium dodecyl sulfate-polyacrylamide gel electrophoresis of material purified by Ni2+ linked affinity chromatography. A position was assigned an external location if its single-cysteine derivative reacted with OGM added to intact cells; a position was designated internal if OGM labeling required cell lysis. We also showed that labeling of external, but not internal, positions was blocked by prior exposure of cells to the impermeable and nonfluorescent thiol-specific agent ethyltrimethylammonium methanethiosulfonate. Of the 34 positions examined in this way, 29 were assigned unambiguously to either an internal or external location; 5 positions could not be assigned, since the target cysteine failed to react with OGM. There was no evidence of false-positive assignment. Our findings document a simple and rapid method for establishing the topology of a membrane protein and show that OxlT has 12 transmembrane segments, confirming inferences from hydropathy analysis.

  19. Synthesis and characterization of novel sulfonamides derivatives

    African Journals Online (AJOL)

    3Department of Chemistry, Comsat Institute of Information & Technology Riawind road. Lahore ... Newer sulfonamides and their derivatives has obtained great attention in pharmaceutical field in ... MS data was recorded on Finnigan MAT 112 mass ..... potent and orally active sulfonamide ETB selective antagonists. Bioorg.

  20. Gadolinium and fluorescent bi-functionally labeling and in vitro MRI of rat bone marrow mesenchymal stem cells

    International Nuclear Information System (INIS)

    Shen Jun; Zhou Cuiping; Cheng Li'na; Duan Xiaohui; Liang Biling; Fu Yue; Bi Xiaobin; Liu Yu; Deng Yubin

    2008-01-01

    Objective: To determine the feasibility of magnetically labeling and tracking mesenchymal stem cells (MSCs) in vitro by using a gadolinium and fluorescent bi-functionally transfection agent of polyethylenimine. Methods: A gadolinium bifunctional transfection reagent complex was obtained after the linear polyethylenimine derivative (JetPEI-FluoR) was incubated with Gd-DTPA. Mesenchymal stem cells isolated from the bone marrows of SD rats were cultured and expanded. The mesenchymal stem cells were incubated with the bi-functional labeling agents. After labeling, the MSCs were examined with fluoroscope and electron microscope and the biological characters were detected including trypan blue exclusion test, MTT, and apoptosis detection. On a 1.5 T MR system, the labeled MSCs were examined with spin echo T 1 WI and T 2 WI and T 1 measurement with mixed sequence. After labeling, the cells were cultured and undergone routine passage. Prior MR examinations were repeated for each passage of labeled cells. All data was statistically prolessed with SPSS for Windows. Results: Of 5 x 10 5 MSCs incubated with the bi-functional agents, 4.25 x 10 5 MSCs were successfully labeled, the percentage of labeled MSCs was 85% fluoroscopically. The high density electron particles of gadolinium observed electron microscopically existed around cellular apparatuses, especially around Golgi apparatus. In trypan blue exclusion test, the exclusion rate of labeled MSCs with incubation duration of 3,6,12,24 h was (96.55±2.90)%, (94.17± 2.56)%, (97.16±3.12)% and (94.23±2.67)%, respectively. The corresponding exclusion rate of unlabeled MSCs was (95.86±2.67)%, (92.04±2.21)%, (93.38±3.64)% and (92.12±2.53)%, respectively. There was no statistical difference of trypan blue exclusion rate between labeled cells and control unlabeled cells within 24 hours of incubation (F=4.523, P>0.05). In the proliferation test, the optical absorption value of labeled MSC with 2.5, 5.0, 10.0, 20.0, 30.0 and 40

  1. One-pot synthesis of fluorescent nitrogen-doped carbon dots with good biocompatibility for cell labeling.

    Science.gov (United States)

    Zhang, Zhengwei; Yan, Kun; Yang, Qiulian; Liu, Yanhua; Yan, Zhengyu; Chen, Jianqiu

    2017-12-01

    Here we report an easy and economical hydrothermal carbonization approach to synthesize the fluorescent nitrogen-doped carbon dots (N-CDs) that was developed using citric acid and triethanolamine as the precursors. The synthesis conditions were optimized to obtain the N-CDs with superior fluorescence performances. The as-prepared N-CDs are monodispersed sphere nanoparticles with good water solubility, and exhibited strong fluorescence, favourable photostability and excitation wavelength-dependent behavior. Furthermore, the in vitro cytotoxicity and cellular labeling of N-CDs were investigated using the rat glomerular mesangial cells. The results showed the N-CDs have more inconspicuous cytotoxicity and better biosafety in comparison with ZnSe quantum dots, although both targeted the cells successfully. Considering their admirable photostability, low toxicity and good compatibility, the as-obtained N-CDs could have potential applications in biosensors, cellular imaging, and other fields. Copyright © 2017 John Wiley & Sons, Ltd.

  2. A Dual Reporter Iodinated Labeling Reagent for Cancer Positron Emission Tomography Imaging and Fluorescence-Guided Surgery

    Science.gov (United States)

    2018-01-01

    The combination of early diagnosis and complete surgical resection offers the greatest prospect of curative cancer treatment. An iodine-124/fluorescein-based dual-modality labeling reagent, 124I-Green, constitutes a generic tool for one-step installation of a positron emission tomography (PET) and a fluorescent reporter to any cancer-specific antibody. The resulting antibody conjugate would allow both cancer PET imaging and intraoperative fluorescence-guided surgery. 124I-Green was synthesized in excellent radiochemical yields of 92 ± 5% (n = 4) determined by HPLC with an improved one-pot three-component radioiodination reaction. The A5B7 carcinoembryonic antigen (CEA)-specific antibody was conjugated to 124I-Green. High tumor uptake of the dual-labeled A5B7 of 20.21 ± 2.70, 13.31 ± 0.73, and 10.64 ± 1.86%ID/g was observed in CEA-expressing SW1222 xenograft mouse model (n = 3) at 24, 48, and 72 h post intravenous injection, respectively. The xenografts were clearly visualized by both PET/CT and ex vivo fluorescence imaging. These encouraging results warrant the further translational development of 124I-Green for cancer PET imaging and fluorescence-guided surgery. PMID:29388770

  3. Determination of sulfonamides in meat by liquid chromatography coupled with atmospheric pressure chemical ionization mass spectrometry

    International Nuclear Information System (INIS)

    Kim, Dal Ho; Choi, Jong Oh; Kim, Jin Seog; Lee, Dai Woon

    2002-01-01

    Liquid chromatography/atmospheric pressure chemical ionization-mass spectrometry (LC-APCI-MS) has been used for the determination of sulfonamides in meat. Five typical sulfonamides were selected as target compounds, and beef meat was selected as a matrix sample. As internal standards, sulfapyridine and isotope labeled sulfamethazine ( 13 C 6 -SMZ) were used. Compared to the results of recent reports, our results have shown improved precision to a RSD of 1.8% for the determination of sulfamethazine spiked with 75 ng/g level in meat

  4. Organic liquids-responsive β-cyclodextrin-functionalized graphene-based fluorescence probe: label-free selective detection of tetrahydrofuran.

    Science.gov (United States)

    Hu, Huawen; Xin, John H; Hu, Hong; Wang, Xiaowen; Lu, Xinkun

    2014-06-06

    In this study, a label-free graphene-based fluorescence probe used for detection of volatile organic liquids was fabricated by a simple, efficient and low-cost method. To fabricate the probe, a bio-based β-cyclodextrin (β-CD) was firstly grafted on reduced graphene surfaces effectively and uniformly, as evidenced by various characterization techniques such as Ultraviolet/Visible spectroscopy, Fourier transform infrared spectroscopy, X-ray diffraction, thermogravimetric analysis, scanning electron microscopy and transmission electron microscopy. The subsequent inclusion of Rhodamine B (RhB) into the inner cavities of the β-CD grafted on the graphene surfaces was achieved easily by a solution mixing method, which yielded the graphene-based fluorescent switch-on probe. In addition, the gradual and controllable quenching of RhB by Fluorescence Resonance Energy Transfer from RhB to graphene during the process of stepwise accommodation of the RhB molecules into the β-CD-functionalized graphene was investigated in depth. A wide range of organic solvents was examined using the as-fabricated fluorescence probe, which revealed the highest sensitivity to tetrahydrofuran with the detection limit of about 1.7 μg/mL. Some insight into the mechanism of the different responsive behaviors of the fluorescence sensor to the examined targets was also described.

  5. Detection of Thrombin Based on Fluorescence Energy Transfer between Semiconducting Polymer Dots and BHQ-Labelled Aptamers

    Directory of Open Access Journals (Sweden)

    Yizhang Liu

    2018-02-01

    Full Text Available Carboxyl-functionalized semiconducting polymer dots (Pdots were synthesized as an energy donor by the nanoprecipitation method. A black hole quenching dye (BHQ-labelled thrombin aptamers was used as the energy acceptor, and fluorescence resonance energy transfer between the aptamers and Pdots was used for fluorescence quenching of the Pdots. The addition of thrombin restored the fluorescence intensity. Under the optimized experimental conditions, the fluorescence of the system was restored to the maximum when the concentration of thrombin reached 130 nM, with a linear range of 0–50 nM (R2 = 0.990 and a detection limit of 0.33 nM. This sensor was less disturbed by impurities, showing good specificity and signal response to thrombin, with good application in actual samples. The detection of human serum showed good linearity in the range of 0–30 nM (R2 = 0.997, with a detection limit of 0.56 nM and a recovery rate of 96.2–104.1%, indicating that this fluorescence sensor can be used for the detection of thrombin content in human serum.

  6. Label-free fluorescence strategy for sensitive detection of adenosine triphosphate using a loop DNA probe with low background noise.

    Science.gov (United States)

    Lin, Chunshui; Cai, Zhixiong; Wang, Yiru; Zhu, Zhi; Yang, Chaoyong James; Chen, Xi

    2014-07-15

    A simple, rapid, label-free, and ultrasensitive fluorescence strategy for adenosine triphosphate (ATP) detection was developed using a loop DNA probe with low background noise. In this strategy, a loop DNA probe, which is the substrate for both ligation and digestion enzyme reaction, was designed. SYBR green I (SG I), a double-stranded specific dye, was applied for the readout fluorescence signal. Exonuclease I (Exo I) and exonuclease III (Exo III), sequence-independent nucleases, were selected to digest the loop DNA probe in order to minimize the background fluorescence signal. As a result, in the absence of ATP, the loop DNA was completely digested by Exo I and Exo III, leading to low background fluorescence owing to the weak electrostatic interaction between SG I and mononucleotides. On the other hand, ATP induced the ligation of the nicking site, and the sealed loop DNA resisted the digestion of Exo I and ExoIII, resulting in a remarkable increase of fluorescence response. Upon background noise reduction, the sensitivity of the ATP determination was improved significantly, and the detection limitation was found to be 1.2 pM, which is much lower than that in almost all the previously reported methods. This strategy has promise for wide application in the determination of ATP.

  7. Organic Liquids-Responsive β-Cyclodextrin-Functionalized Graphene-Based Fluorescence Probe: Label-Free Selective Detection of Tetrahydrofuran

    Directory of Open Access Journals (Sweden)

    Huawen Hu

    2014-06-01

    Full Text Available In this study, a label-free graphene-based fluorescence probe used for detection of volatile organic liquids was fabricated by a simple, efficient and low-cost method. To fabricate the probe, a bio-based β-cyclodextrin (β-CD was firstly grafted on reduced graphene surfaces effectively and uniformly, as evidenced by various characterization techniques such as Ultraviolet/Visible spectroscopy, Fourier transform infrared spectroscopy, X-ray diffraction, thermogravimetric analysis, scanning electron microscopy and transmission electron microscopy. The subsequent inclusion of Rhodamine B (RhB into the inner cavities of the β-CD grafted on the graphene surfaces was achieved easily by a solution mixing method, which yielded the graphene-based fluorescent switch-on probe. In addition, the gradual and controllable quenching of RhB by Fluorescence Resonance Energy Transfer from RhB to graphene during the process of stepwise accommodation of the RhB molecules into the β-CD-functionalized graphene was investigated in depth. A wide range of organic solvents was examined using the as-fabricated fluorescence probe, which revealed the highest sensitivity to tetrahydrofuran with the detection limit of about 1.7 μg/mL. Some insight into the mechanism of the different responsive behaviors of the fluorescence sensor to the examined targets was also described.

  8. Label-free detection of kanamycin based on a G-quadruplex DNA aptamer-based fluorescent intercalator displacement assay

    Science.gov (United States)

    Xing, Yun-Peng; Liu, Chun; Zhou, Xiao-Hong; Shi, Han-Chang

    2015-01-01

    This work was the first to report that the kanamycin-binding DNA aptamer (5'-TGG GGG TTG AGG CTA AGC CGA-3') can form stable parallel G-quadruplex DNA (G4-DNA) structures by themselves and that this phenomenon can be verified by nondenaturing polyacrylamide gel electrophoresis and circular dichroism spectroscopy. Based on these findings, we developed a novel label-free strategy for kanamycin detection based on the G4-DNA aptamer-based fluorescent intercalator displacement assay with thiazole orange (TO) as the fluorescence probe. In the proposed strategy, TO became strongly fluorescent upon binding to kanamycin-binding G4-DNA. However, the addition of kanamycin caused the displacement of TO from the G4-DNA-TO conjugate, thereby resulting in decreased fluorescent signal, which was inversely related to the kanamycin concentration. The detection limit of the proposed assay decreased to 59 nM with a linear working range of 0.1 μM to 20 μM for kanamycin. The cross-reactivity against six other antibiotics was negligible compared with the response to kanamycin. A satisfactory recovery of kanamycin in milk samples ranged from 80.1% to 98.0%, confirming the potential of this bioassay in the measurement of kanamycin in various applications. Our results also served as a good reference for developing similar fluorescent G4-DNA-based bioassays in the future.

  9. Fluorescent humanized anti-CEA antibody specifically labels metastatic pancreatic cancer in a patient-derived orthotopic xenograft (PDOX) mouse model

    Science.gov (United States)

    Lwin, Thinzar M.; Miyake, Kentaro; Murakami, Takashi; DeLong, Jonathan C.; Yazaki, Paul J.; Shivley, John E.; Clary, Bryan; Hoffman, Robert M.; Bouvet, Michael

    2018-03-01

    Specific tumor targeting can result in selective labeling of cancer in vivo for surgical navigation. In the present study, we show that the use of an anti-CEA antibody conjugated to the near-infrared (NIR) fluorescent dye, IRDye800CW, can selectively target and label pancreatic cancer and its metastases in a clinically relevant patient derived xenograft mouse model.

  10. Monitoring the diffusion behavior of Na,K-ATPase by fluorescence correlation spectroscopy (FCS) upon fluorescence labelling with eGFP or Dreiklang

    Science.gov (United States)

    Junghans, Cornelia; Schmitt, Franz-Josef; Vukojević, Vladana; Friedrich, Thomas

    2016-02-01

    Measurement of lateral mobility of membraneembedded proteins in living cells with high spatial and temporal precision is a challenging task of optofluidics. Biological membranes are complex structures, whose physico-chemical properties depend on the local lipid composition, cholesterol content and the presence of integral or peripheral membrane proteins, which may be involved in supramolecular complexes or are linked to cellular matrix proteins or the cytoskeleton. The high proteinto- lipid ratios in biomembranes indicate that membrane proteins are particularly subject to molecular crowding, making it difficult to follow the track of individual molecules carrying a fluorescence label. Novel switchable fluorescence proteins such as Dreiklang [1], are, in principle, promising tools to study the diffusion behavior of individual molecules in situations of molecular crowding due to excellent spectral control of the ON- and OFF-switching process. In this work, we expressed an integral membrane transport protein, the Na,K-ATPase comprising the human α2-subunit carrying an N-terminal eGFP or Dreiklang tag and human β1-subunit, in HEK293T cells and measured autocorrelation curves by fluorescence correlation spectroscopy (FCS). Furthermore,we measured diffusion times and diffusion constants of eGFP and Dreiklang by FCS, first, in aqueous solution after purification of the proteins upon expression in E. coli, and, second, upon expression as soluble proteins in the cytoplasm of HEK293T cells. Our data show that the diffusion behavior of the purified eGFP and Dreiklang in solution as well as the properties of the proteins expressed in the cytoplasm are very similar. However, the autocorrelation curves of eGFP- and Dreiklanglabeled Na,K-ATPase measured in the plasma membrane exhibit marked differences, with the Dreiklang-labeled construct showing shorter diffusion times. This may be related to an additional, as yet unrecognized quenching process that occurs on the same time

  11. Label-Free Carbon-Dots-Based Ratiometric Fluorescence pH Nanoprobes for Intracellular pH Sensing.

    Science.gov (United States)

    Shangguan, Jingfang; He, Dinggeng; He, Xiaoxiao; Wang, Kemin; Xu, Fengzhou; Liu, Jinquan; Tang, Jinlu; Yang, Xue; Huang, Jin

    2016-08-02

    Measuring pH in living cells is of great importance for better understanding cellular functions as well as providing pivotal assistance for early diagnosis of diseases. In this work, we report the first use of a novel kind of label-free carbon dots for intracellular ratiometric fluorescence pH sensing. By simple one-pot hydrothermal treatment of citric acid and basic fuchsin, the carbon dots showing dual emission bands at 475 and 545 nm under single-wavelength excitation were synthesized. It is demonstrated that the fluorescence intensities of the as-synthesized carbon dots at the two emissions are pH-sensitive simultaneously. The intensity ratio (I475 nm/I545 nm) is linear against pH values from 5.2 to 8.8 in buffer solution, affording the capability as ratiometric probes for intracellular pH sensing. It also displays that the carbon dots show excellent reversibility and photostability in pH measurements. With this nanoprobe, quantitative fluorescence imaging using the ratio of two emissions (I475 nm/I545 nm) for the detection of intracellular pH were successfully applied in HeLa cells. In contrast to most of the reported nanomaterials-based ratiometric pH sensors which rely on the attachment of additional dyes, these carbon-dots-based ratiometric probes are low in toxicity, easy to synthesize, and free from labels.

  12. Synthesis and characterization of fluorescence-labelled silica core-shell and noble metal-decorated ceria nanoparticles

    Directory of Open Access Journals (Sweden)

    Rudolf Herrmann

    2014-12-01

    Full Text Available The present review article covers work done in the cluster NPBIOMEM in the DFG priority programme SPP 1313 and focuses on synthesis and characterization of fluorescent silica and ceria nanoparticles. Synthetic methods for labelling of silica and polyorganosiloxane/silica core–shell nanoparticles with perylenediimide derivatives are described, as well as the modification of the shell with thiol groups. Photometric methods for the determination of the number of thiol groups and an estimate for the number of fluorescent molecules per nanoparticles, including a scattering correction, have been developed. Ceria nanoparticles decorated with noble metals (Pt, Pd, Rh are models for the decomposition products of automobile catalytic converters which appear in the exhaust gases and finally interact with biological systems including humans. The control of the degree of agglomeration of small ceria nanoparticles is the basis for their synthesis. Almost monodisperse agglomerates (40 ± 4–260 ± 40 nm diameter can be prepared and decorated with noble metal nanoparticles (2–5 nm diameter. Fluorescence labelling with ATTO 647N gave the model particles which are now under biophysical investigation.

  13. Application of fluorescently labelled lectins for the study of polysaccharides in biofilms with a focus on biofouling of nanofiltration membranes

    Directory of Open Access Journals (Sweden)

    Patrick Di Martino

    2016-07-01

    Full Text Available The biofilm state is the dominant microbial lifestyle in nature. A biofilm can be defined as cells organised as microcolonies embedded in an organic polymer matrix of microbial origin living at an interface between two different liquids, air and liquid, or solid and liquid. The biofilm matrix is made of extracellular polymeric substances, polysaccharides being considered as the major structural components of the matrix. Fluorescently labelled lectins have been widely used to stain microbial extracellular glycoconjugates in natural and artificial environments, and to study specific bacterial species or highly complex environments. Biofilm development at the membrane surface conducting to biofouling is one of the major problems encountered during drinking water production by filtration. Biofouling affects the durability and effectiveness of filtration membranes. Biofouling can be reduced by pretreatments in order to control two key parameters of water, the bioavailable organic matter concentration and the concentration of live bacteria. Nanofiltration (NF is a high technology process particularly suited to the treatment of surface waters to produce drinking water that is highly sensitive to biofouling. The development of strategies for fouling prevention and control requires characterizing the fouling material composition and organisation before and after NF membrane cleaning. The aim of this review is to present basics of biofilm analyses after staining with fluorescently labelled lectins and to focus on the use of fluorescent lectins and confocal laser scanning microscopy to analyse NF membrane biofouling.

  14. DNA-length-dependent quenching of fluorescently labeled iron oxide nanoparticles with gold, graphene oxide and MoS2 nanostructures.

    Science.gov (United States)

    Balcioglu, Mustafa; Rana, Muhit; Robertson, Neil; Yigit, Mehmet V

    2014-08-13

    We controlled the fluorescence emission of a fluorescently labeled iron oxide nanoparticle using three different nanomaterials with ultraefficient quenching capabilities. The control over the fluorescence emission was investigated via spacing introduced by the surface-functionalized single-stranded DNA molecules. DNA molecules were conjugated on different templates, either on the surface of the fluorescently labeled iron oxide nanoparticles or gold and nanographene oxide. The efficiency of the quenching was determined and compared with various fluorescently labeled iron oxide nanoparticle and nanoquencher combinations using DNA molecules with three different lengths. We have found that the template for DNA conjugation plays significant role on quenching the fluorescence emission of the fluorescently labeled iron oxide nanoparticles. We have observed that the size of the DNA controls the quenching efficiency when conjugated only on the fluorescently labeled iron oxide nanoparticles by setting a spacer between the surfaces and resulting change in the hydrodynamic size. The quenching efficiency with 12mer, 23mer and 36mer oligonucleotides decreased to 56%, 54% and 53% with gold nanoparticles, 58%, 38% and 32% with nanographene oxide, 46%, 38% and 35% with MoS2, respectively. On the other hand, the presence, not the size, of the DNA molecules on the other surfaces quenched the fluorescence significantly with different degrees. To understand the effect of the mobility of the DNA molecules on the nanoparticle surface, DNA molecules were attached to the surface with two different approaches. Covalently immobilized oligonucleotides decreased the quenching efficiency of nanographene oxide and gold nanoparticles to ∼22% and ∼21%, respectively, whereas noncovalently adsorbed oligonucleotides decreased it to ∼25% and ∼55%, respectively. As a result, we have found that each nanoquencher has a powerful quenching capability against a fluorescent nanoparticle, which can be

  15. Preparation and application of new fluorescein-labeled fumonisins B1 in fluorescence polarization analysis technique

    Science.gov (United States)

    Objective: To prepare a new fluorescent tracer against common mycotoxins such as fumonisin B1 in order to replace 6-(4,6-Dichlorotriazinyl) aminofluorescein (6-DTAF), an expensive marker, and to develop a technique for quick detection of fumonisin B1 based on the principle of fluorescence polarizati...

  16. In-stem labelling allows visualization of DNA strand displacements by distinct fluorescent colour change.

    Science.gov (United States)

    Barrois, Sebastian; Wagenknecht, Hans-Achim

    2013-05-21

    The combination of thiazole orange (TO) and thiazole red (TR) as an internal pair of fluorescent DNA base surrogates ("DNA traffic lights") allows us to follow at least two consecutive DNA strand displacements in real time through a distinct fluorescence colour change from green to red and vice versa.

  17. Zinc Phthalocyanine Labelled Polyethylene Glycol: Preparation, Characterization, Interaction with Bovine Serum Albumin and Near Infrared Fluorescence Imaging in Vivo

    Directory of Open Access Journals (Sweden)

    Tianjun Liu

    2012-05-01

    Full Text Available Zinc phthalocyanine labelled polyethylene glycol was prepared to track and monitor the in vivo fate of polyethylene glycol. The chemical structures were characterized by nuclear magnetic resonance and infrared spectroscopy. Their light stability and fluorescence quantum yield were evaluated by UV-Visible and fluorescence spectroscopy methods. The interaction of zinc phthalocyanine labelled polyethylene glycol with bovine serum albumin was evaluated by fluorescence titration and isothermal titration calorimetry methods. Optical imaging in vivo, organ aggregation as well as distribution of fluorescence experiments for tracking polyethylene glycol were performed with zinc phthalocyanine labelled polyethylene glycol as fluorescent agent. Results show that zinc phthalocyanine labelled polyethylene glycol has good optical stability and high emission ability in the near infrared region. Imaging results demonstrate that zinc phthalocyanine labelled polyethylene glycol can track and monitor the in vivo process by near infrared fluorescence imaging, which implies its potential in biomaterials evaluation in vivo by a real-time noninvasive method.

  18. A Rapid Label-Free Fluorescent Aptasensor PicoGreen-Based Strategy for Aflatoxin B₁ Detection in Traditional Chinese Medicines.

    Science.gov (United States)

    Zhang, Cheng; Dou, Xiaowen; Zhang, Lei; Sun, Meifeng; Zhao, Ming; OuYang, Zhen; Kong, Dandan; Antonio, F Logrieco; Yang, Meihua

    2018-02-28

    Aflatoxin B₁ (AFB₁) is a very hazardous carcinogen, readily contaminating foodstuffs and traditional Chinese medicines (TCMs) that has inspired increasing health concerns due to dietary exposure. Colloidal nanocrystals have been proposed as optical labels for aptasensor assembly, but these typically require tedious multistep conjugation and suffer from unsatisfactory robustness when used for complex matrices. In the present study, we report a rapid and sensitive method for screening for trace AFB₁ levels in TCMs using a label-free fluorescent aptasensor PicoGreen dye-based strategy. Using PicoGreen to selectively measure complementary double-stranded DNA, fluorescence enhancement due to dsDNA is 'turned off' in the presence of AFB₁ due binding of aptamer target over complementary sequence. Self-assembly of a label-free fluorescent aptasensor based on AFB₁ aptamer and PicoGreen dye was performed. Due to competition between the complementary sequence and AFB₁ target, this rapid method was capable of highly sensitive and selective screening for AFB₁ in five types of TCMs. This proposed approach had a limit of detection as low as 0.1 μg·L -1 and good linearity with a range of 0.1-10 μg·L -1 (0.1-10 ppb). Among the 20 samples tested, 6 batches were found to be contaminated with AFB₁ using this method, which was confirmed using sophisticated liquid chromatography-electrospray ionization-tandem mass spectrometry/mass spectrometry analysis. The results of this study indicate the developed method has the potential to be a simple, quick, and sensitive tool for detecting AFB₁ in TCMs.

  19. "Molecular beacon"-hosted thioflavin T: Applications for label-free fluorescent detection of iodide and logic operations.

    Science.gov (United States)

    Li, Yan-Yun; Jiang, Xiao-Qin; Lu, Ling-Fei; Zhang, Min; Shi, Guoyue

    2016-04-01

    In this work, we presented a simple, label-free and rapid-responsive fluorescence assay for iodide (I(-)) detection based on "molecular beacon (MB)"-hosted thioflavin T (ThT), achieving a limit of detection as low as 158 nM. The proposed method exhibited very good selectivity to I(-) ions over other anions interference due to the strong binding force between I(-) ions with Hg(2+). Upon the addition of I(-) ions, it would capture Hg(2+) from a T-Hg(2+)-T complex belonging to the MB-like DNA hairpin structure, which eventually quenched the initial fluorescence as output. In addition, it was successfully applied for operation of an integrated DNA logic gate system and to the determination of I(-) in real samples such as human urine. Copyright © 2016 Elsevier B.V. All rights reserved.

  20. Dual-Mode SERS-Fluorescence Immunoassay Using Graphene Quantum Dot Labeling on One-Dimensional Aligned Magnetoplasmonic Nanoparticles.

    Science.gov (United States)

    Zou, Fengming; Zhou, Hongjian; Tan, Tran Van; Kim, Jeonghyo; Koh, Kwangnak; Lee, Jaebeom

    2015-06-10

    A novel dual-mode immunoassay based on surface-enhanced Raman scattering (SERS) and fluorescence was designed using graphene quantum dot (GQD) labels to detect a tuberculosis (TB) antigen, CFP-10, via a newly developed sensing platform of linearly aligned magnetoplasmonic (MagPlas) nanoparticles (NPs). The GQDs were excellent bilabeling materials for simultaneous Raman scattering and photoluminescence (PL). The one-dimensional (1D) alignment of MagPlas NPs simplified the immunoassay process and enabled fast, enhanced signal transduction. With a sandwich-type immunoassay using dual-mode nanoprobes, both SERS signals and fluorescence images were recognized in a highly sensitive and selective manner with a detection limit of 0.0511 pg mL(-1).

  1. Diaminobenzidine photoconversion is a suitable tool for tracking the intracellular location of fluorescently labelled nanoparticles at transmission electron microscopy

    Directory of Open Access Journals (Sweden)

    M. Malatesta

    2012-04-01

    Full Text Available Chitosan-based nanoparticles (NPs deserve particular attention as suitable drug carriers in the field of pharmaceutics, since they are able to protect the encapsulated drugs and/or improve their efficacy by making them able to cross biological barriers (such as the blood-brain barrier and reach their intracellular target sites. Understanding the intracellular location of NPs is crucial for designing drug delivery strategies. In this study, fluorescently-labelled chitosan NPs were administered in vitro to a neuronal cell line, and diaminobenzidine (DAB photoconversion was applied to correlate fluorescence and transmission electron microscopy to precisely describe the NPs intracellular fate. This technique allowed to demonstrate that chitosan NPs easily enter neuronal cells, predominantly by endocytosis; they were found both inside membrane-bounded vesicles and free in the cytosol, and were observed to accumulate around the cell nucleus.

  2. A rhodamine-labeled citalopram analogue as a high-affinity fluorescent probe for the serotonin transporter

    DEFF Research Database (Denmark)

    Zhang, Peng; Jørgensen, Trine Nygaard; Løland, Claus Juul

    2013-01-01

    A novel fluorescent ligand was synthesized as a high-affinity, high specificity probe for visualizing the serotonin transporter (SERT). The rhodamine fluorophore was extended from an aniline substitution on the 5-position of the dihydroisobenzofuran ring of citalopram (2, 1-(3-(dimethylamino......)propyl)-1-(4-fluorophenyl)-1,3-dihydroisobenzofuran-5-carbonitrile), using an ethylamino linker. The resulting rhodamine-labeled ligand 8 inhibited [3H]5-HT uptake in COS-7 cells (Ki = 225 nM) with similar potency to the tropane-based JHC 1-064 (1), but with higher specificity towards the SERT relative...

  3. 3D Restoration Microscopy Improves Quantification of Enzyme-Labeled Fluorescence-Based Single-Cell Phosphatase Activity in Plankton

    OpenAIRE

    Diaz-de-Quijano, Daniel; Palacios, Pilar; Hornák, Karel; Felip, Marisol

    2014-01-01

    The ELF or fluorescence-labeled enzyme activity (FLEA) technique is a culture-independent single-cell tool for assessing plankton enzyme activity in close-to-in situ conditions. We demonstrate that single-cell FLEA quantifications based on two-dimensional (2D) image analysis were biased by up to one order of magnitude relative to deconvolved 3D. This was basically attributed to out-of-focus light, and partially to object size. Nevertheless, if sufficient cells were measured (25-40 cells), bia...

  4. Normal development of hamster and rabbit eggs fertilized by spermatozoa labelled with the fluorescent thiol alkylating agent, monobromobimane

    International Nuclear Information System (INIS)

    Fleming, A.D.; Cummins, J.M.; Kuehl, T.J.; Seidel, G.E. Jr.; Yanagimachi, R.

    1986-01-01

    Cauda epididymal spermatozoa of the golden hamster were labelled with the thiol-alkylating reagent, monobromobimane (MB). Female hamsters underwent uterine insemination with labelled spermatozoa at laparotomy under metofane anesthesia. All 12 females examined between 5 and 54 h postinsemination yielded a total of 83/100 (83%) eggs in the process of fertilization or embryos. Under ultraviolet (UV) exposure all exhibited a fluorescent tail which, in the 4- and 8-cell embryos, could be seen to be fraying or disintegrating. As cleavage progressed, labelled tail components came to be restricted among the blastomeres such that at the 4- and 8-cell stage the tail could be seen in only one to three blastomeres. To study complete development and pregnancy another 12 females received uterine insemination. After recovery from anesthesia (approximately equal to 4 h) these females were mated with a vasectomized male bearing a dominant genetic marker (black eyes) to allow unequivocal determination of paternity in the fetuses and young produced. Seven became pregnant with one female losing her pregnancy about Day 7 of gestation. Two females sacrificed on Day 13 produced 17 normal fetuses and one resorption. Four females delivered 16 young, all normal at birth and in subsequent growth and fertility. In addition, insemination of female rabbits with MB-labelled spermatozoa yielded normal embryos (50/52 96%) from 3 does on Day 2 and (38/64 60%) from 4 does on Days 4 or 5. Two normal litters (9 bunnies) have delivered from 3 does allowed to carry to term

  5. Human induced pluripotent stem cells labeled with fluorescent magnetic nanoparticles for targeted imaging and hyperthermia therapy for gastric cancer

    International Nuclear Information System (INIS)

    Li, Chao; Ruan, Jing; Yang, Meng; Pan, Fei; Gao, Guo; Qu, Su; Shen, You-Lan; Dang, Yong-Jun; Wang, Kan; Jin, Wei-Lin; Cui, Da-Xiang

    2015-01-01

    Human induced pluripotent stem (iPS) cells exhibit great potential for generating functional human cells for medical therapies. In this paper, we report for use of human iPS cells labeled with fluorescent magnetic nanoparticles (FMNPs) for targeted imaging and synergistic therapy of gastric cancer cells in vivo. Human iPS cells were prepared and cultured for 72 h. The culture medium was collected, and then was co-incubated with MGC803 cells. Cell viability was analyzed by the MTT method. FMNP-labeled human iPS cells were prepared and injected into gastric cancer-bearing nude mice. The mouse model was observed using a small-animal imaging system. The nude mice were irradiated under an external alternating magnetic field and evaluated using an infrared thermal mapping instrument. Tumor sizes were measured weekly. iPS cells and the collected culture medium inhibited the growth of MGC803 cells. FMNP-labeled human iPS cells targeted and imaged gastric cancer cells in vivo, as well as inhibited cancer growth in vivo through the external magnetic field. FMNP-labeled human iPS cells exhibit considerable potential in applications such as targeted dual-mode imaging and synergistic therapy for early gastric cancer

  6. Fluorescently labeled dengue viruses as probes to identify antigen-specific memory B cells by multiparametric flow cytometry.

    Science.gov (United States)

    Woda, Marcia; Mathew, Anuja

    2015-01-01

    Low frequencies of memory B cells in the peripheral blood make it challenging to measure the functional and phenotypic characteristics of this antigen experienced subset of B cells without in vitro culture. To date, reagents are lacking to measure ex vivo frequencies of dengue virus (DENV)-specific memory B cells. We wanted to explore the possibility of using fluorescently labeled DENV as probes to detect antigen-specific memory B cells in the peripheral blood of DENV immune individuals. Alexa Fluor dye-labeled DENV yielded viable virus that could be stored at -80°C for long periods of time. Using a careful gating strategy and methods to decrease non-specific binding, we were able to identify a small frequency of B cells from dengue immune individuals that bound labeled DENV. Sorted DENV(+) B cells from immune, but not naïve donors secreted antibodies that bound DENV after in vitro stimulation. Overall, Alexa Fluor dye-labeled DENVs are useful reagents to enable the detection and characterization of memory B cells in DENV immune individuals. Copyright © 2014 Elsevier B.V. All rights reserved.

  7. Infrared fluorescent protein 1.4 genetic labeling tracks engrafted cardiac progenitor cells in mouse ischemic hearts.

    Directory of Open Access Journals (Sweden)

    Lijuan Chen

    Full Text Available Stem cell therapy has a potential for regenerating damaged myocardium. However, a key obstacle to cell therapy's success is the loss of engrafted cells due to apoptosis or necrosis in the ischemic myocardium. While many strategies have been developed to improve engrafted cell survival, tools to evaluate cell efficacy within the body are limited. Traditional genetic labeling tools, such as GFP-like fluorescent proteins (eGFP, DsRed, mCherry, have limited penetration depths in vivo due to tissue scattering and absorption. To circumvent these limitations, a near-infrared fluorescent mutant of the DrBphP bacteriophytochrome from Deinococcus radiodurans, IFP1.4, was developed for in vivo imaging, but it has yet to be used for in vivo stem/progenitor cell tracking. In this study, we incorporated IFP1.4 into mouse cardiac progenitor cells (CPCs by a lentiviral vector. Live IFP1.4-labeled CPCs were imaged by their near-infrared fluorescence (NIRF using an Odyssey scanner following overnight incubation with biliverdin. A significant linear correlation was observed between the amount of cells and NIRF signal intensity in in vitro studies. Lentiviral mediated IFP1.4 gene labeling is stable, and does not impact the apoptosis and cardiac differentiation of CPC. To assess efficacy of our model for engrafted cells in vivo, IFP1.4-labeled CPCs were intramyocardially injected into infarcted hearts. NIRF signals were collected at 1-day, 7-days, and 14-days post-injection using the Kodak in vivo multispectral imaging system. Strong NIRF signals from engrafted cells were imaged 1 day after injection. At 1 week after injection, 70% of the NIRF signal was lost when compared to the intensity of the day 1 signal. The data collected 2 weeks following transplantation showed an 88% decrease when compared to day 1. Our studies have shown that IFP1.4 gene labeling can be used to track the viability of transplanted cells in vivo.

  8. Automated Slide Scanning and Segmentation in Fluorescently-labeled Tissues Using a Widefield High-content Analysis System.

    Science.gov (United States)

    Poon, Candice C; Ebacher, Vincent; Liu, Katherine; Yong, Voon Wee; Kelly, John James Patrick

    2018-05-03

    Automated slide scanning and segmentation of fluorescently-labeled tissues is the most efficient way to analyze whole slides or large tissue sections. Unfortunately, many researchers spend large amounts of time and resources developing and optimizing workflows that are only relevant to their own experiments. In this article, we describe a protocol that can be used by those with access to a widefield high-content analysis system (WHCAS) to image any slide-mounted tissue, with options for customization within pre-built modules found in the associated software. Not originally intended for slide scanning, the steps detailed in this article make it possible to acquire slide scanning images in the WHCAS which can be imported into the associated software. In this example, the automated segmentation of brain tumor slides is demonstrated, but the automated segmentation of any fluorescently-labeled nuclear or cytoplasmic marker is possible. Furthermore, there are a variety of other quantitative software modules including assays for protein localization/translocation, cellular proliferation/viability/apoptosis, and angiogenesis that can be run. This technique will save researchers time and effort and create an automated protocol for slide analysis.

  9. Metal plasmon-coupled fluorescence imaging and label free coenzyme detection in cells

    International Nuclear Information System (INIS)

    Zhang, Jian; Fu, Yi; Li, Ge; Zhao, Richard Y.

    2012-01-01

    Highlights: ► Metal nanoparticle for fluorescence cell imaging. ► Non-invasive emission detection of coenzyme in cell on time-resolved confocal microscope. ► Near-field interaction of flavin adenine dinucleotide with silver substrate. ► Isolation of emissions by coenzymes from cellular autofluorescence on fluorescence cell imaging. -- Abstract: Flavin adenine dinucleotide (FAD) is a key metabolite in cellular energy conversion. Flavin can also bind with some enzymes in the metabolic pathway and the binding sites may be changed due to the disease progression. Thus, there is interest on studying its expression level, distribution, and redox state within the cells. FAD is naturally fluorescent, but it has a modest extinction coefficient and quantum yield. Hence the intrinsic emission from FAD is generally too weak to be isolated distinctly from the cellular backgrounds in fluorescence cell imaging. In this article, the metal nanostructures on the glass coverslips were used as substrates to measure FAD in cells. Particulate silver films were fabricated with an optical resonance near the absorption and the emission wavelengths of FAD which can lead to efficient coupling interactions. As a result, the emission intensity and quantum yield by FAD were greatly increased and the lifetime was dramatically shortened resulting in less interference from the longer lived cellular background. This feature may overcome the technical limits that hinder the direct observation of intrinsically fluorescent coenzymes in the cells by fluorescence microscopy. Fluorescence cell imaging on the metallic particle substrates may provide a non-invasive strategy for collecting the information of coenzymes in cells.

  10. Metal plasmon-coupled fluorescence imaging and label free coenzyme detection in cells

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Jian, E-mail: jian@cfs.bioment.umaryland.edu [Center for Fluorescence Spectroscopy, University of Maryland School of Medicine, Department of Biochemistry and Molecular Biology, 725 West Lombard Street, Baltimore, MD 21201 (United States); Fu, Yi [Center for Fluorescence Spectroscopy, University of Maryland School of Medicine, Department of Biochemistry and Molecular Biology, 725 West Lombard Street, Baltimore, MD 21201 (United States); Li, Ge [Division of Molecular Pathology, Department of Pathology, University of Maryland School of Medicine, 10 South Pine Street, Baltimore, MD 21201 (United States); Zhao, Richard Y. [Division of Molecular Pathology, Department of Pathology, University of Maryland School of Medicine, 10 South Pine Street, Baltimore, MD 21201 (United States); Department of Microbiology-Immunology, University of Maryland School of Medicine, 10 South Pine Street, Baltimore, MD 21201 (United States); Institute of Human Virology, University of Maryland School of Medicine, 10 South Pine Street, Baltimore, MD 21201 (United States)

    2012-08-31

    Highlights: Black-Right-Pointing-Pointer Metal nanoparticle for fluorescence cell imaging. Black-Right-Pointing-Pointer Non-invasive emission detection of coenzyme in cell on time-resolved confocal microscope. Black-Right-Pointing-Pointer Near-field interaction of flavin adenine dinucleotide with silver substrate. Black-Right-Pointing-Pointer Isolation of emissions by coenzymes from cellular autofluorescence on fluorescence cell imaging. -- Abstract: Flavin adenine dinucleotide (FAD) is a key metabolite in cellular energy conversion. Flavin can also bind with some enzymes in the metabolic pathway and the binding sites may be changed due to the disease progression. Thus, there is interest on studying its expression level, distribution, and redox state within the cells. FAD is naturally fluorescent, but it has a modest extinction coefficient and quantum yield. Hence the intrinsic emission from FAD is generally too weak to be isolated distinctly from the cellular backgrounds in fluorescence cell imaging. In this article, the metal nanostructures on the glass coverslips were used as substrates to measure FAD in cells. Particulate silver films were fabricated with an optical resonance near the absorption and the emission wavelengths of FAD which can lead to efficient coupling interactions. As a result, the emission intensity and quantum yield by FAD were greatly increased and the lifetime was dramatically shortened resulting in less interference from the longer lived cellular background. This feature may overcome the technical limits that hinder the direct observation of intrinsically fluorescent coenzymes in the cells by fluorescence microscopy. Fluorescence cell imaging on the metallic particle substrates may provide a non-invasive strategy for collecting the information of coenzymes in cells.

  11. Fluorescent probes as a tool for labelling and tracking the amphibian chytrid fungus Batrachochytrium dendrobatidis.

    Science.gov (United States)

    Herbert, Sarah M; Leung, Tommy L F; Bishop, Phillip J

    2011-09-09

    The dissemination of the virulent pathogen Batrachochytrium dendrobatidis (Bd) has contributed to the decline and extinction of many amphibian species worldwide. Several different strains have been identified, some of which are sympatric. Interactions between co-infecting strains of a pathogen can have significant influences on disease epidemiology and evolution; therefore the dynamics of multi-strain infections is an important area of research. We stained Bd cells with 2 fluorescent BODIPY fatty acid probes to determine whether these can potentially be used to distinguish and track Bd cell lines in multi-strain experiments. Bd cells in broth culture were stained with 5 concentrations of green-fluorescent BODIPY FL and red-fluorescent BODIPY 558/568 and visualised under an epifluorescent microscope for up to 16 d post-dye. Dyed strains were also assessed for growth inhibition. The most effective concentration for both dyes was 10 pM. This concentration of dye produced strong fluorescence for 12 to 16 d in Bd cultures held at 23 degrees C (3 to 4 generations), and did not inhibit Bd growth. Cells dyed with BODIPY FL and BODIPY 558/568 can be distinguished from each other on the basis of their fluorescence characteristics. Therefore, it is likely that this technique will be useful for research into multi-strain dynamics of Bd infections.

  12. Label-free silicon nanodots featured ratiometric fluorescent aptasensor for lysosomal imaging and pH measurement.

    Science.gov (United States)

    Zhang, Yanan; Guo, Shan; Cheng, Shibo; Ji, Xinghu; He, Zhike

    2017-08-15

    The homeostasis of lysosomal pH is crucial in cell physiology. Developing small fluorescent nanosensors for lysosome imaging and ratiometric measurement of pH is highly demanded yet challenging. Herein, a pH-sensitive fluorescein tagged aptamer AS1411 has been utilized to covalently modify the label-free fluorescent silicon nanodots via a crosslinker for construction of a ratiometric pH biosensor. The established aptasensor exhibits the advantages of ultrasmall size, hypotoxicity, excellent pH reversibility and good photostability, which favors its application in an intracellular environment. Using human breast MCF-7 cancer cells and MCF-10A normal cells as the model, this aptasensor shows cell specificity for cancer cells and displays a wide pH response range of 4.5-8.0 in living cells. The results demonstrate that the pH of MCF-7 cells is 5.1, which is the expected value for acidic organelles. Lysosome imaging and accurate measurement of pH in MCF-7 cells have been successfully conducted based on this nanosensor via fluorescent microscopy and flow cytometry. Copyright © 2017 Elsevier B.V. All rights reserved.

  13. Pulmonary permeability assessed by fluorescent-labeled dextran instilled intranasally into mice with LPS-induced acute lung injury.

    Directory of Open Access Journals (Sweden)

    Honglei Chen

    Full Text Available Several different methods have been used to assess pulmonary permeability in response to acute lung injury (ALI. However, these methods often involve complicated procedures and algorithms that are difficult to precisely control. The purpose of the current study is to establish a feasible method to evaluate alterations in lung permeability by instilling fluorescently labeled dextran (FITC-Dextran intranasally.For the mouse model of direct ALI, lipopolysaccharide (LPS was administered intranasally. FITC-Dextran was instilled intranasally one hour before the mice were euthanized. Plasma fluorescence intensities from the LPS group were significantly higher than in the control group. To determine the reliability and reproducibility of the procedure, we also measured the lung wet-to-dry weight ratio, the protein concentration of the bronchoalveolar lavage fluid, tight and adherens junction markers and pathological changes. Consistent results were observed when the LPS group was compared with the control group. Simultaneously, we found that the concentration of plasma FITC-Dextran was LPS dose-dependent. The concentration of plasma FITC-Dextran also increased with initial intranasal FITC-Dextran doses. Furthermore, increased fluorescence intensity of plasma FITC-Dextran was found in the intraperitoneally LPS-induced ALI model.In conclusion, the measurement of FITC-Dextran in plasma after intranasal instillation is a simple, reliable, and reproducible method to evaluate lung permeability alterations in vivo. The concentration of FITC-Dextran in the plasma may be useful as a potential peripheral biomarker of ALI in experimental clinical studies.

  14. Pulmonary permeability assessed by fluorescent-labeled dextran instilled intranasally into mice with LPS-induced acute lung injury.

    Science.gov (United States)

    Chen, Honglei; Wu, Shaoping; Lu, Rong; Zhang, Yong-guo; Zheng, Yuanyuan; Sun, Jun

    2014-01-01

    Several different methods have been used to assess pulmonary permeability in response to acute lung injury (ALI). However, these methods often involve complicated procedures and algorithms that are difficult to precisely control. The purpose of the current study is to establish a feasible method to evaluate alterations in lung permeability by instilling fluorescently labeled dextran (FITC-Dextran) intranasally. For the mouse model of direct ALI, lipopolysaccharide (LPS) was administered intranasally. FITC-Dextran was instilled intranasally one hour before the mice were euthanized. Plasma fluorescence intensities from the LPS group were significantly higher than in the control group. To determine the reliability and reproducibility of the procedure, we also measured the lung wet-to-dry weight ratio, the protein concentration of the bronchoalveolar lavage fluid, tight and adherens junction markers and pathological changes. Consistent results were observed when the LPS group was compared with the control group. Simultaneously, we found that the concentration of plasma FITC-Dextran was LPS dose-dependent. The concentration of plasma FITC-Dextran also increased with initial intranasal FITC-Dextran doses. Furthermore, increased fluorescence intensity of plasma FITC-Dextran was found in the intraperitoneally LPS-induced ALI model. In conclusion, the measurement of FITC-Dextran in plasma after intranasal instillation is a simple, reliable, and reproducible method to evaluate lung permeability alterations in vivo. The concentration of FITC-Dextran in the plasma may be useful as a potential peripheral biomarker of ALI in experimental clinical studies.

  15. Mechanism-based fluorescent labeling of beta-galactosidases. An efficient method in proteomics for glycoside hydrolases.

    Science.gov (United States)

    Kurogochi, Masaki; Nishimura, Shin-Ichiro; Lee, Yuan Chuan

    2004-10-22

    (4-N-5-Dimethylaminonaphthalene-1-sulfonyl-2-difluoromethylphenyl)-beta-d-galactopyranoside was synthesized and successfully tested on beta-galactosidases from Xanthomonas manihotis (Wong-Madden, S. T., and Landry, D. Glycobiology (1995) 5, 19-28 and Taron, C. H., Benner, J. S., Hornstra, L. J., and Guthrie, E. P. (1995) Glycobiology 5, 603-610), Escherichia coli (Jacobson, R. H., Zhang, X. J., DuBose, R. F., and Matthews, B. W. (1994) Nature 369, 761-766), and Bacillus circulans (Fujimoto, H., Miyasato, M., Ito, Y., Sasaki, T., and Ajisaka, K. (1988) Glycoconj. J. 15, 155-160) for the rapid identification of the catalytic site. Reaction of the irreversible inhibitor with enzymes proceeded to afford a fluorescence-labeled protein suitable for further high throughput characterization by using antidansyl antibody and matrix-assisted laser desorption ionization time-of-flight/time-of-flight (MALDI-TOF/TOF). Specific probing by a fluorescent aglycon greatly facilitated identification of the labeled peptide fragments from beta-galactosidases. It was demonstrated by using X. manihotis beta-galactosidase that the Arg-58 residue, which is located within a sequence of 56IPRAYWKD63, was labeled by nucleophilic attack of the guanidinyl group. This sequence including Arg-58 (Leu-46 to Tyr-194) was similar to that (Met-1 to Tyr-151) of Thermus thermophilus A4, which is the first known structure of glycoside hydrolases family 42 (Hidaka, M., Fushinobu, S., Ohtsu, N., Motoshima, H., Matsuzawa, H., Shoun, H., and Wakagi, T. (2002) J. Mol. Biol. 322, 79-91). A catalytic glutamic acid (Glu-537) of E. coli beta-galactosidase was proved to be labeled by the same procedure, suggesting that the modification site with this irreversible substrate might depend both on the nucleophilicity of the amino acids and their spatial arrangement in the individual catalytic cavity. Similarly, a Glu-259 in 257TLEE260 was selectively labeled using B. circulans beta-galactosidase, indicating that Glu

  16. A Fluorescent Thermometer Based on a Pyrene-Labeled Thermoresponsive Polymer

    Directory of Open Access Journals (Sweden)

    Ulrich S. Schubert

    2010-08-01

    Full Text Available Thermoresponsive polymers that undergo a solubility transition by variation of the temperature are important materials for the development of ‘smart’ materials. In this contribution we exploit the solubility phase transition of poly(methoxy diethylene glycol methacrylate, which is accompanied by a transition from hydrophilic to hydrophobic, for the development of a fluorescent thermometer. To translate the polymer phase transition into a fluorescent response, the polymer was functionalized with pyrene resulting in a change of the emission based on the microenvironment. This approach led to a soluble polymeric fluorescent thermometer with a temperature range from 11 °C to 21 °C. The polymer phase transition that occurs during sensing is studied in detail by dynamic light scattering.

  17. An aggregated perylene-based broad-spectrum, efficient and label-free quencher for multiplexed fluorescent bioassays.

    Science.gov (United States)

    Liu, Tao; Hu, Rong; Lv, Yi-Fan; Wu, Yuan; Liang, Hao; Huan, Shuang-Yan; Zhang, Xiao-Bing; Tan, Weihong; Yu, Ru-Qin

    2014-08-15

    Fluorescent sensing systems based on the quenching of fluorophores have found wide applications in bioassays. An efficient quencher will endow the sensing system a high sensitivity. The frequently used quenchers are based on organic molecules or nanomaterials, which usually need tedious synthesizing and modifying steps, and exhibit different quenching efficiencies to different fluorophores. In this work, we for the first time report that aggregated perylene derivative can serve as a broad-spectrum and label-free quencher that is able to efficiently quench a variety of fluorophores, such as green, red and far red dyes labeled on DNA. By choosing nucleases as model biomolecules, such a broad-spectrum quencher was then employed to construct a multiplexed bioassay platform through a label-free manner. Due to the high quenching efficiency of the aggregated perylene, the proposed platform could detect nuclease with high sensitivity, with a detection limit of 0.03U/mL for EcoRV, and 0.05U/mL for EcoRI. The perylene quencher does not affect the activity of nuclease, which makes it possible to design post-addition type bioassay platform. Moreover, the proposed platform allows simultaneous and multicolor analysis of nucleases in homogeneous solution, demonstrating its value of potential application in rapid screening of multiple bio-targets. Copyright © 2014 Elsevier B.V. All rights reserved.

  18. Why sulfonamides are contraindicated in Rocky Mountain spotted fever

    OpenAIRE

    Ren, Vicky; Hsu, Sylvia

    2014-01-01

    Sulfonamide antibiotics are not effective for the treatment of Rocky Mountain spotted fever (RMSF). Patients suspected of having RMSF based on history and physical exam should be treated with doxycycline and not a sulfonamide to avoid increased morbidity and mortality.

  19. Why sulfonamides are contraindicated in Rocky Mountain spotted fever.

    Science.gov (United States)

    Ren, Vicky; Hsu, Sylvia

    2014-02-18

    Sulfonamide antibiotics are not effective for the treatment of Rocky Mountain spotted fever (RMSF). Patients suspected of having RMSF based on history and physical exam should be treated with doxycycline and not a sulfonamide to avoid increased morbidity and mortality.

  20. A fluorescent probe which allows highly specific thiol labeling at low pH

    DEFF Research Database (Denmark)

    Nielsen, Jonas W.; Jensen, Kristine Steen; Hansen, Rosa E.

    2012-01-01

    and properties of a thiol-specific reagent, fluorescent cyclic activated disulfide (FCAD), which includes the fluorescein moiety as fluorophore and utilizes a variation of thiol-disulfide exchange chemistry. The leaving-group character of FCAD makes it reactive at pH 3, allowing modification at low pH, limiting...

  1. Localization of fluorescently labeled structures in frozen-hydrated samples using integrated light electron microscopy

    NARCIS (Netherlands)

    Faas, F.G.A.; Bárcena, M.A.; Agronskaia, A.V.; Gerritsen, H.C.; Moscicka, K.B.; Diebolder, C.A.; Driel, L.F.; Limpens, R.W.A.L.; Bos, E.; Ravelli, R.B.G.; Koning, R.I.; Koster, A.J.

    2013-01-01

    Correlative light and electron microscopy is an increasingly popular technique to study complex biological systems at various levels of resolution. Fluorescence microscopy can be employed to scan large areas to localize regions of interest which are then analyzed by electron microscopy to obtain

  2. Gene Replacement and Fluorescent Labeling to Study the Functional Role of Exopolysaccharides in Bifidobacterium animalis subsp. lactis

    Directory of Open Access Journals (Sweden)

    Nuria Castro-Bravo

    2017-07-01

    Full Text Available An extracellular layer of exopolysaccharides (EPS covers the surface of some Bifidobacterium animalis subsp. lactis strains, which could be of relevance for its probiotic performance. In order to understand the functional characteristics of B. animalis subsp. lactis, two isogenic strains that differ in their EPS-producing phenotype, due to a single mutation in the gene Balat_1410, were studied. By means of a double crossover recombination strategy, successfully used for the first time in bifidobacteria, Balat_1410 in the type strain B. animalis subsp. lactis DSM10140 was replaced by a mutated gene containing a non-synonymous mutation previously associated with the appearance of a mucoid-ropy phenotype. Nuclear magnetic resonance and SEC-MALS analyses showed that the novel strain harboring the mutation acquired a ropy phenotype, due to the production of a high molecular weight (HMW-EPS that is not produced in the wild-type strain. Fluorescence labeling of both strains with two fluorescent proteins, m-Cherry and Green Fluorescent Protein, was achieved by expressing the corresponding genes under the control of a native selected promoter (the elongation factor Tu promoter. Remarkably, qualitative and quantitative fluorescence analyses demonstrated that the ropy strain displays a lower capability to adhere to human intestinal epithelial cells. In addition, the presence of the HMW-EPS reduced the capability of the producing strain to form biofilms upon three different abiotic surfaces. This work also highlights the fact that different EPS confer variable functional characteristics to the bifidobacterial surface, which may be relevant for the performance of B. animalis subsp. lactis as a probiotic. The construction of molecular tools allowing the functional characterization of surface structures in next generation probiotics is still a challenging issue that deserves further attention, given the relevant role that such molecules must play in the

  3. Fast neuronal labeling in live tissue using a biocytin conjugated fluorescent probe

    DEFF Research Database (Denmark)

    Harsløf, Mads; Müller, Christoph Felix; Rohrberg, Julie

    2015-01-01

    of local synapses within 10min. TMR biocytin is fixable, stable during methyl salicylate clearing, and can be visualized deep in nervous tissue. COMPARISON WITH EXISTING METHODS: Retrograde labeling with TMR biocytin enables long-range neuronal visualization and concurrent calcium imaging after only a few...

  4. Comparative microscopic and biochemical study of the uptake of fluorescent and 125I-labeled lipoproteins by skin fibroblasts, smooth muscle cells, and peritoneal macrophages in culture

    International Nuclear Information System (INIS)

    Reynolds, G.D.; St Clair, R.W.

    1985-01-01

    Uptake of low density lipoprotein (LDL) and of acetyl LDL was compared in skin fibroblasts, smooth muscle cells, and peritoneal macrophages with the use of lipoproteins labeled with either 125 I or the fluorescent probe 3,3'-dioctadecylindocarbocyanine (DiI). The uptake of DiI-labeled lipoproteins was assessed by quantitative spectrofluorometry and by fluorescence microscopy. The DiI was quantitatively retained by the cells, while the 125 I-LDL was degraded and 125 I-labeled degradation products were excreted from the cells. In smooth muscle cells and fibroblasts the uptake of LDL was virtually the same whether measured with the use of the DiI or 125 I-label. The labeling of acetyl LDL with DiI enhanced its uptake in peritoneal macrophages by an average of 18%. With the DiI label, lipoprotein uptake could be determined after as little as 10 minutes of incubation at 37 C. The pattern of uptake of the DiI-labeled lipoproteins was consistent with binding to specific receptors, because no DiI could be detected in mutant cells without LDL receptors, and uptake was competitively inhibited by addition of excess unlabeled lipoprotein. When the DiI-labeled lipoproteins were removed from the medium, there was a 5-15% loss of DiI from all cell types studied over the first 24 hours

  5. Label-free identification of macrophage phenotype by fluorescence lifetime imaging microscopy

    Science.gov (United States)

    Alfonso-García, Alba; Smith, Tim D.; Datta, Rupsa; Luu, Thuy U.; Gratton, Enrico; Potma, Eric O.; Liu, Wendy F.

    2016-04-01

    Macrophages adopt a variety of phenotypes that are a reflection of the many functions they perform as part of the immune system. In particular, metabolism is a phenotypic trait that differs between classically activated, proinflammatory macrophages, and alternatively activated, prohealing macrophages. Inflammatory macrophages have a metabolism based on glycolysis while alternatively activated macrophages generally rely on oxidative phosphorylation to generate chemical energy. We employ this shift in metabolism as an endogenous marker to identify the phenotype of individual macrophages via live-cell fluorescence lifetime imaging microscopy (FLIM). We demonstrate that polarized macrophages can be readily discriminated with the aid of a phasor approach to FLIM, which provides a fast and model-free method for analyzing fluorescence lifetime images.

  6. Data for analysis of mannose-6-phosphate glycans labeled with fluorescent tags.

    Science.gov (United States)

    Kang, Ji-Yeon; Kwon, Ohsuk; Gil, Jin Young; Oh, Doo-Byoung

    2016-06-01

    Mannose-6-phosphate (M-6-P) glycan plays an important role in lysosomal targeting of most therapeutic enzymes for treatment of lysosomal storage diseases. This article provides data for the analysis of M-6-P glycans by high-performance liquid chromatography (HPLC) and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. The identities of M-6-P glycan peaks in HPLC profile were confirmed by measuring the masses of the collected peak eluates. The performances of three fluorescent tags (2-aminobenzoic acid [2-AA], 2-aminobenzamide [2-AB], and 3-(acetyl-amino)-6-aminoacridine [AA-Ac]) were compared focusing on the analysis of bi-phosphorylated glycan (containing two M-6-Ps). The bi-phosphorylated glycan analysis is highly affected by the attached fluorescent tag and the hydrophilicity of elution solvent used in HPLC. The data in this article is associated with the research article published in "Comparison of fluorescent tags for analysis of mannose-6-phosphate glycans" (Kang et al., 2016 [1]).

  7. Data for analysis of mannose-6-phosphate glycans labeled with fluorescent tags

    Directory of Open Access Journals (Sweden)

    Ji-Yeon Kang

    2016-06-01

    Full Text Available Mannose-6-phosphate (M-6-P glycan plays an important role in lysosomal targeting of most therapeutic enzymes for treatment of lysosomal storage diseases. This article provides data for the analysis of M-6-P glycans by high-performance liquid chromatography (HPLC and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF mass spectrometry. The identities of M-6-P glycan peaks in HPLC profile were confirmed by measuring the masses of the collected peak eluates. The performances of three fluorescent tags (2-aminobenzoic acid [2-AA], 2-aminobenzamide [2-AB], and 3-(acetyl-amino-6-aminoacridine [AA-Ac] were compared focusing on the analysis of bi-phosphorylated glycan (containing two M-6-Ps. The bi-phosphorylated glycan analysis is highly affected by the attached fluorescent tag and the hydrophilicity of elution solvent used in HPLC. The data in this article is associated with the research article published in “Comparison of fluorescent tags for analysis of mannose-6-phosphate glycans” (Kang et al., 2016 [1].

  8. A label-free fluorescent adenosine triphosphate biosensor via overhanging aptamer-triggered enzyme protection and target recycling amplification.

    Science.gov (United States)

    Wang, Zhaoyin; Zhao, Jian; Dai, Zhihui

    2016-06-20

    Herein, a label-free fluorescent adenosine triphosphate (ATP) aptasensor is fabricated with a DNA hairpin and an overhanging aptamer. In the presence of ATP, the overhanging sequences of the aptamer may form preferred substrates of exo III, and thus trigger the enzyme-assisted amplification, which results in the release of G-rich sequences. Free G-rich sequences subsequently generate an enhanced flourescent signal by binding with thioflavin T. However, if ATP is absent, the overhanging sequence can induce steric hindrance and protect the DNA hairpin against the digestion of exo III, significantly reducing the noise of this biosensor. Accordingly, the signal-to-noise ratio of the sensing system is greatly improved, which ensures the desirable analytical performance of the proposed aptasensor both in pure samples and real samples.

  9. Introducing Ratiometric Fluorescence to MnO2 Nanosheet-Based Biosensing: A Simple, Label-Free Ratiometric Fluorescent Sensor Programmed by Cascade Logic Circuit for Ultrasensitive GSH Detection.

    Science.gov (United States)

    Fan, Daoqing; Shang, Changshuai; Gu, Wenling; Wang, Erkang; Dong, Shaojun

    2017-08-09

    Glutathione (GSH) plays crucial roles in various biological functions, the level alterations of which have been linked to varieties of diseases. Herein, we for the first time expanded the application of oxidase-like property of MnO 2 nanosheet (MnO 2 NS) to fluorescent substrates of peroxidase. Different from previously reported fluorescent quenching phenomena, we found that MnO 2 NS could not only largely quench the fluorescence of highly fluorescent Scopoletin (SC) but also surprisingly enhance that of nonfluorescent Amplex Red (AR) via oxidation reaction. If MnO 2 NS is premixed with GSH, it will be reduced to Mn 2+ and lose the oxidase-like property, accompanied by subsequent increase in SC's fluorescence and decrease in AR's. On the basis of the above mechanism, we construct the first MnO 2 NS-based ratiometric fluorescent sensor for ultrasensitive and selective detection of GSH. Notably, this ratiometric sensor is programmed by the cascade logic circuit (an INHIBIT gate cascade with a 1 to 2 decoder). And a linear relationship between ratiometric fluorescent intensities of the two substrates and logarithmic values of GSH's concentrations is obtained. The detection limit of GSH is as low as 6.7 nM, which is much lower than previous ratiometric fluorescent sensors, and the lowest MnO 2 NS-based fluorescent GSH sensor reported so far. Furthermore, this sensor is simple, label-free, and low-cost; it also presents excellent applicability in human serum samples.

  10. Selection of specific aptamer against enrofloxacin and fabrication of graphene oxide based label-free fluorescent assay.

    Science.gov (United States)

    Dolati, Somayeh; Ramezani, Mohammad; Nabavinia, Maryam Sadat; Soheili, Vahid; Abnous, Khalil; Taghdisi, Seyed Mohammad

    2018-05-15

    Specific ssDNA aptamers for the antibiotic enrofloxacin (ENR) were isolated from an enriched nucleotide library by SELEX (Systematic Evolution of Ligands by EXponential enrichment) method with high binding affinity. After seven rounds, five aptamers were selected and identified. Apt58 with highest affinity and sensitivity (K d  = 14.19 nM) was employed to develop a label-free fluorescent biosensing approach based on aptamer, graphene oxide (GO) and native fluorescence of ENR for determination of ENR residue in raw milk samples. Under optimized experimental conditions, the linear range was from 5 nM to 250 nM and LOD was calculated to be 3.7 nM, and the recovery rate was between 94.1% and 108.5%. The integration of aptamer and GO in this bioassay provides a promising way for rapid, sensitive and cost-effective detection of ENR in real samples like raw milk. Copyright © 2018 Elsevier Inc. All rights reserved.

  11. Ratiometric fluorescent sensing of pH values in living cells by dual-fluorophore-labeled i-motif nanoprobes.

    Science.gov (United States)

    Huang, Jin; Ying, Le; Yang, Xiaohai; Yang, Yanjing; Quan, Ke; Wang, He; Xie, Nuli; Ou, Min; Zhou, Qifeng; Wang, Kemin

    2015-09-01

    We designed a new ratiometric fluorescent nanoprobe for sensing pH values in living cells. Briefly, the nanoprobe consists of a gold nanoparticle (AuNP), short single-stranded oligonucleotides, and dual-fluorophore-labeled i-motif sequences. The short oligonucleotides are designed to bind with the i-motif sequences and immobilized on the AuNP surface via Au-S bond. At neutral pH, the dual fluorophores are separated, resulting in very low fluorescence resonance energy transfer (FRET) efficiency. At acidic pH, the i-motif strands fold into a quadruplex structure and leave the AuNP, bringing the dual fluorophores into close proximity, resulting in high FRET efficiency, which could be used as a signal for pH sensing. The nanoprobe possesses abilities of cellular transfection, enzymatic protection, fast response and quantitative pH detection. The in vitro and intracellular applications of the nanoprobe were demonstrated, which showed excellent response in the physiological pH range. Furthermore, our experimental results suggested that the nanoprobe showed excellent spatial and temporal resolution in living cells. We think that the ratiometric sensing strategy could potentially be applied to create a variety of new multicolor sensors for intracellular detection.

  12. Use of direct fluorescence labeling and confocal microscopy to determine the biodistribution of two protein therapeutics, Cerezyme and Ceredase.

    Science.gov (United States)

    Piepenhagen, Peter A; Vanpatten, Scott; Hughes, Heather; Waire, James; Murray, James; Andrews, Laura; Edmunds, Tim; O'Callaghan, Michael; Thurberg, Beth L

    2010-07-01

    Efficient targeting of therapeutic reagents to tissues and cell types of interest is critical to achieving therapeutic efficacy and avoiding unwanted side effects due to offtarget uptake. To increase assay efficiency and reduce the number of animals used per experiment during preclinical development, we used a combination of direct fluorescence labeling and confocal microscopy to simultaneously examine the biodistribution of two therapeutic proteins, Cerezyme and Ceredase, in the same animals. We show that the fluorescent tags do not interfere with protein uptake and localization. We are able to detect Cerezyme and Ceredase in intact cells and organs and demonstrate colocalization within target cells using confocal microscopy. In addition, the relative amount of protein internalized by different cell types can be quantified using cell type-specific markers and morphometric analysis. This approach provides an easy and straightforward means of assessing the tissue and cell type-specific biodistribution of multiple protein therapeutics in target organs using a minimal number of animals. (c) 2009 Wiley-Liss, Inc.

  13. Alzheimer's disease evaluation using label-free, stainless, fluorescence to measure tryptophan metabolism along the kynurenine pathway

    Science.gov (United States)

    Sordillo, Laura A.; Zhang, Lin; Shi, Lingyan; Sriramoju, Vidyasagar; Sordillo, Peter P.; Alfano, Robert R.

    2018-02-01

    Under stress conditions, pro-inflammatory cytokines, such as tumor necrosis factor-alpha, interleukin-1 beta, interleukin 6 and interferon gamma are released. It is known that these cytokines stimulate indoleamine 2,3-dioxygenase (IDO) and tryptophan 2,3-dioxygenase (TDO), which increase tryptophan metabolism through the kynurenine pathway, and that this can cause increased production of neurotoxic compounds. Brain tissues from Alzheimer's disease patients and agematched controls were investigated using label-free fluorescence spectroscopy. Tryptophan (exc. 280/ em. 340 nm) and its metabolites (N-formyl-L-kynurenine (exc. 325/em. 434 nm), kynurenine (exc. 365/em. 480 nm) and kynurenic acid (exc. 330/em. 390 nm)) have distinct spectral profiles. Preliminary results show a difference in the optical signatures in three important areas of the brain (hippocampus, BA 9, BA 17) between patients with Alzheimer's disease and agedmatched controls (normal), and a marked relative increase in tryptophan in the Alzheimer's patients. Thus determinations of tryptophan to tryptophan metabolite ratios could potentially be used to measure IDO and TDO activity and the degree of inflammation in the brain. This label-free optical technique may be useful in the study of Alzheimer's and other neurodegenerative diseases.

  14. Rhodamine-labeled 2beta-carbomethoxy-3beta-(3,4-dichlorophenyl)tropane analogues as high-affinity fluorescent probes for the dopamine transporter

    DEFF Research Database (Denmark)

    Cha, Joo Hwan; Zou, Mu-Fa; Adkins, Erika M

    2005-01-01

    linker. The resulting 2-substituted (5) and N-substituted (9) rhodamine-labeled ligands provided the highest DAT binding affinities expressed in COS-7 cells (Ki= 27 and 18 nM, respectively) in the series. Visualization of the DAT with 5 and 9 was demonstrated by confocal fluorescence laser scanning...

  15. Fluorescently labeled chimeric anti-CEA antibody improves detection and resection of human colon cancer in a patient-derived orthotopic xenograft (PDOX) nude mouse model.

    Science.gov (United States)

    Metildi, Cristina A; Kaushal, Sharmeela; Luiken, George A; Talamini, Mark A; Hoffman, Robert M; Bouvet, Michael

    2014-04-01

    The aim of this study was to evaluate a new fluorescently labeled chimeric anti-CEA antibody for improved detection and resection of colon cancer. Frozen tumor and normal human tissue samples were stained with chimeric and mouse antibody-fluorophore conjugates for comparison. Mice with patient-derived orthotopic xenografts (PDOX) of colon cancer underwent fluorescence-guided surgery (FGS) or bright-light surgery (BLS) 24 hr after tail vein injection of fluorophore-conjugated chimeric anti-CEA antibody. Resection completeness was assessed using postoperative images. Mice were followed for 6 months for recurrence. The fluorophore conjugation efficiency (dye/mole ratio) improved from 3-4 to >5.5 with the chimeric CEA antibody compared to mouse anti-CEA antibody. CEA-expressing tumors labeled with chimeric CEA antibody provided a brighter fluorescence signal on frozen human tumor tissues (P = 0.046) and demonstrated consistently lower fluorescence signals in normal human tissues compared to mouse antibody. Chimeric CEA antibody accurately labeled PDOX colon cancer in nude mice, enabling improved detection of tumor margins for more effective FGS. The R0 resection rate increased from 86% to 96% with FGS compared to BLS. Improved conjugating efficiency and labeling with chimeric fluorophore-conjugated antibody resulted in better detection and resection of human colon cancer in an orthotopic mouse model. © 2013 Wiley Periodicals, Inc.

  16. Genetic diversity analysis of sugarcane germplasm based on fluorescence-labeled simple sequence repeat markers and a capillary electrophoresis-based genotyping platform

    Science.gov (United States)

    Genetic diversity analysis, which refers to the elaboration of total extent of genetic characteristics in the genetic makeup of a certain species, constitutes a classical strategy for the study of diversity, population genetic structure, and breeding practices. In this study, fluorescence-labeled se...

  17. Localization of fluorescently labeled structures in frozen-hydrated samples using integrated light electron microscopy.

    Science.gov (United States)

    Faas, F G A; Bárcena, M; Agronskaia, A V; Gerritsen, H C; Moscicka, K B; Diebolder, C A; van Driel, L F; Limpens, R W A L; Bos, E; Ravelli, R B G; Koning, R I; Koster, A J

    2013-03-01

    Correlative light and electron microscopy is an increasingly popular technique to study complex biological systems at various levels of resolution. Fluorescence microscopy can be employed to scan large areas to localize regions of interest which are then analyzed by electron microscopy to obtain morphological and structural information from a selected field of view at nm-scale resolution. Previously, an integrated approach to room temperature correlative microscopy was described. Combined use of light and electron microscopy within one instrument greatly simplifies sample handling, avoids cumbersome experimental overheads, simplifies navigation between the two modalities, and improves the success rate of image correlation. Here, an integrated approach for correlative microscopy under cryogenic conditions is presented. Its advantages over the room temperature approach include safeguarding the native hydrated state of the biological specimen, preservation of the fluorescence signal without risk of quenching due to heavy atom stains, and reduced photo bleaching. The potential of cryo integrated light and electron microscopy is demonstrated for the detection of viable bacteria, the study of in vitro polymerized microtubules, the localization of mitochondria in mouse embryonic fibroblasts, and for a search into virus-induced intracellular membrane modifications within mammalian cells. Copyright © 2012 Elsevier Inc. All rights reserved.

  18. Near-infrared emitting fluorescent nanocrystals-labeled natural killer cells as a platform technology for the optical imaging of immunotherapeutic cells-based cancer therapy

    International Nuclear Information System (INIS)

    Lim, Yong Taik; Cho, Mi Young; Noh, Young-Woock; Chung, Bong Hyun; Chung, Jin Woong

    2009-01-01

    This study describes the development of near-infrared optical imaging technology for the monitoring of immunotherapeutic cell-based cancer therapy using natural killer (NK) cells labeled with fluorescent nanocrystals. Although NK cell-based immunotherapeutic strategies have drawn interest as potent preclinical or clinical methods of cancer therapy, there are few reports documenting the molecular imaging of NK cell-based cancer therapy, primarily due to the difficulty of labeling of NK cells with imaging probes. Human natural killer cells (NK92MI) were labeled with anti-human CD56 antibody-coated quantum dots (QD705) for fluorescence imaging. FACS analysis showed that the NK92MI cells labeled with anti-human CD56 antibody-coated QD705 have no effect on the cell viability. The effect of anti-human CD56 antibody-coated QD705 labeling on the NK92MI cell function was investigated by measuring interferon gamma (IFN- γ) production and cytolytic activity. Finally, the NK92MI cells labeled with anti-human CD56 antibody-coated QD705 showed a therapeutic effect similar to that of unlabeled NK92MI cells. Images of intratumorally injected NK92MI cells labeled with anti-human CD56 antibody-coated could be acquired using near-infrared optical imaging both in vivo and in vitro. This result demonstrates that the immunotherapeutic cells labeled with fluorescent nanocrystals can be a versatile platform for the effective tracking of injected therapeutic cells using optical imaging technology, which is very important in cell-based cancer therapies.

  19. Fluorescence-labeled methylation-sensitive amplified fragment length polymorphism (FL-MS-AFLP) analysis for quantitative determination of DNA methylation and demethylation status.

    Science.gov (United States)

    Kageyama, Shinji; Shinmura, Kazuya; Yamamoto, Hiroko; Goto, Masanori; Suzuki, Koichi; Tanioka, Fumihiko; Tsuneyoshi, Toshihiro; Sugimura, Haruhiko

    2008-04-01

    The PCR-based DNA fingerprinting method called the methylation-sensitive amplified fragment length polymorphism (MS-AFLP) analysis is used for genome-wide scanning of methylation status. In this study, we developed a method of fluorescence-labeled MS-AFLP (FL-MS-AFLP) analysis by applying a fluorescence-labeled primer and fluorescence-detecting electrophoresis apparatus to the existing method of MS-AFLP analysis. The FL-MS-AFLP analysis enables quantitative evaluation of more than 350 random CpG loci per run. It was shown to allow evaluation of the differences in methylation level of blood DNA of gastric cancer patients and evaluation of hypermethylation and hypomethylation in DNA from gastric cancer tissue in comparison with adjacent non-cancerous tissue.

  20. Automated parasite faecal egg counting using fluorescence labelling, smartphone image capture and computational image analysis.

    Science.gov (United States)

    Slusarewicz, Paul; Pagano, Stefanie; Mills, Christopher; Popa, Gabriel; Chow, K Martin; Mendenhall, Michael; Rodgers, David W; Nielsen, Martin K

    2016-07-01

    Intestinal parasites are a concern in veterinary medicine worldwide and for human health in the developing world. Infections are identified by microscopic visualisation of parasite eggs in faeces, which is time-consuming, requires technical expertise and is impractical for use on-site. For these reasons, recommendations for parasite surveillance are not widely adopted and parasite control is based on administration of rote prophylactic treatments with anthelmintic drugs. This approach is known to promote anthelmintic resistance, so there is a pronounced need for a convenient egg counting assay to promote good clinical practice. Using a fluorescent chitin-binding protein, we show that this structural carbohydrate is present and accessible in shells of ova of strongyle, ascarid, trichurid and coccidian parasites. Furthermore, we show that a cellular smartphone can be used as an inexpensive device to image fluorescent eggs and, by harnessing the computational power of the phone, to perform image analysis to count the eggs. Strongyle egg counts generated by the smartphone system had a significant linear correlation with manual McMaster counts (R(2)=0.98), but with a significantly lower coefficient of variation (P=0.0177). Furthermore, the system was capable of differentiating equine strongyle and ascarid eggs similar to the McMaster method, but with significantly lower coefficients of variation (P<0.0001). This demonstrates the feasibility of a simple, automated on-site test to detect and/or enumerate parasite eggs in mammalian faeces without the need for a laboratory microscope, and highlights the potential of smartphones as relatively sophisticated, inexpensive and portable medical diagnostic devices. Copyright © 2016 Australian Society for Parasitology. Published by Elsevier Ltd. All rights reserved.

  1. Multiway study of hybridization in nanoscale semiconductor labeled DNA based on fluorescence resonance energy transfer

    DEFF Research Database (Denmark)

    Gholami, Somayeh; Kompany Zare, Mohsen

    2013-01-01

    donor-QD acceptor) upon hybridization with a label free target was monitored by two-dimensional photoluminescence excitation spectroscopy (2D-PLE). Detection of a target oligonucleotide strand, using sandwiched nanoassembly in a separation-free format, was performed with the appearance of a new feature...... and model based analysis of 2D-PLE data was implemented by means of PAR-AFAC and hard trilinear decomposition (HTD), allowing to fit a proper model for FRET-based sandwich DNA hybridization systems. This study is the first successful application of a multiway chemometric technique to consider FRET based DNA...... hybridization in sandwiched nanoassemblies. A multi-equilibria model was properly fitted to the data and confirmed there is a competition between ternary and binary complex formation. Equilibrium constants of DNA hybridization in sandwiched nanoassemblies were estimated for the first time. Equilibrium constants...

  2. Part-per-trillion level detection of estradiol by competitive fluorescence immunoassay using DNA/dye conjugate as antibody multiple labels.

    Science.gov (United States)

    Zhu, Shengchao; Zhang, Qin; Guo, Liang-Hong

    2008-08-22

    Fluorescent organic dyes are currently the standard signal-generating labels used in microarray quantification. However, new labeling strategies are needed to meet the demand for high sensitivity in the detection of low-abundance proteins and small molecules. In this report, a long-chain DNA/dye conjugate was used to attach multiple fluorescence labels on antibodies to improve signal intensity and immunoassay sensitivity. Compared with the 30 base-pair (bp) oligonucleotide used in our previous work [Q. Zhang, L.-H. Guo, Bioconjugate Chem. 18 (2007) 1668-1672], conjugation of a 219 bp DNA in solution with a fluorescent DNA binder SYBR Green I resulted in more than sixfold increase in signal intensity, consistent with the increase in bp number. In a direct immunoassay for the detection of goat anti-mouse IgG in a mouse IgG-coated 96-well plate, the long DNA conjugate label also produced higher fluorescence than the short one, accompanied by about 15-fold improvement in the detection limit. To demonstrate its advantage in real applications, the DNA/dye conjugate was employed in the competitive immunoassay of 17beta-estradiol, a clinically and environmentally important analyte. The biotin-terminated DNA was attached to biotinylated anti-estradiol antibody through the biotin/streptavidin/biotin bridge after the immuno-reaction was completed, followed by conjugation with SYBR Green I. The limit of detection for 17beta-estradiol is 1.9 pg mL(-1), which is 200-fold lower than the assay using fluorescein-labeled antibodies. The new multiple labeling strategy uses readily available reagents, and is also compatible with current biochip platform. It has great potential in the sensitive detection of protein and antibody microarrays.

  3. Using fluorescence measurement of zinc ions liberated from ZnS nanoparticle labels in bioassay for Escherichia coli O157:H7

    International Nuclear Information System (INIS)

    Cowles, Chad L.; Zhu Xiaoshan; Pai, Chi-Yun

    2011-01-01

    In this study, an alternative approach using ZnS nanoparticle biolabels as fluorescence signal transducers is reported for the immunoassay of E. coli O157:H7 in tap water samples. Instead of measuring the fluorescence of ZnS nanoparticles in the assay, the fluorescence signal is generated through the binding of zinc ions released from nanoparticle labels with zinc-ion sensitive fluorescence indicator Fluozin-3. In the assay, ZnS nanoparticles around 50 nm in diameter were synthesized, bioconjugated, and applied for the detection of E. coli O157:H7. The assay shows a detection range over two orders of magnitude and a detection limit around 1000 colony-forming units (cfu) of E. coli O157:H7.

  4. Labeling the oily core of nanocapsules and lipid-core nanocapsules with a triglyceride conjugated to a fluorescent dye as a strategy to particle tracking in biological studies

    Science.gov (United States)

    Fiel, Luana Almeida; Contri, Renata Vidor; Bica, Juliane Freitas; Figueiró, Fabrício; Battastini, Ana Maria Oliveira; Guterres, Sílvia Stanisçuaski; Pohlmann, Adriana Raffin

    2014-05-01

    The synthesis of novel fluorescent materials represents a very important step to obtain labeled nanoformulations in order to evaluate their biological behavior. The strategy of conjugating a fluorescent dye with triacylglycerol allows that either particles differing regarding supramolecular structure, i.e., nanoemulsions, nanocapsules, lipid-core nanocapsules, or surface charge, i.e., cationic nanocapsules and anionic nanocapsules, can be tracked using the same labeled material. In this way, a rhodamine B-conjugated triglyceride was obtained to prepare fluorescent polymeric nanocapsules. Different formulations were obtained, nanocapsules (NC) or lipid-core nanocapsules (LNC), using the labeled oil and Eudragit RS100, Eudragit S100, or poly(caprolactone) (PCL), respectively. The rhodamine B was coupled with the ricinolein by activating the carboxylic function using a carbodiimide derivative. Thin layer chromatography, proton nuclear magnetic resonance (1H-NMR), Fourier transform infrared spectroscopy (FTIR), UV-vis, and fluorescence spectroscopy were used to identify the new product. Fluorescent nanocapsule aqueous suspensions were prepared by the solvent displacement method. Their pH values were 4.6 (NC-RS100), 3.5 (NC-S100), and 5.0 (LNC-PCL). The volume-weighted mean diameter ( D 4.3) and polydispersity values were 150 nm and 1.05 (NC-RS100), 350 nm and 2.28 (NC-S100), and 270 nm and 1.67 (LNC-PCL). The mean diameters determined by photon correlation spectroscopy (PCS) ( z-average) were around 200 nm. The zeta potential values were +5.85 mV (NC-RS100), -21.12 mV (NC-S100), and -19.25 mV (LNC-PCL). The wavelengths of maximum fluorescence emission were 567 nm (NC-RS100 and LNC-PCL) and 574 nm (NC-S100). Fluorescence microscopy was used to evaluate the cell uptake (human macrophage cell line) of the fluorescent nanocapsules in order to show the applicability of the approach. When the cells were treated with the fluorescent nanocapsules, red emission was detected

  5. Identification of squid species by melting temperature shifts on fluorescence melting curve analysis (FMCA) using single dual-labeled probe

    Science.gov (United States)

    Koh, Eunjung; Song, Ha Jeong; Kwon, Na Young; Kim, Gi Won; Lee, Kwang Ho; Jo, Soyeon; Park, Sujin; Park, Jihyun; Park, Eun Kyeong; Hwang, Seung Yong

    2017-06-01

    Real time PCR is a standard method for identification of species. One of limitations of the qPCR is that there would be false-positive result due to mismatched hybridization between target sequence and probe depending on the annealing temperature in the PCR condition. As an alternative, fluorescence melting curve analysis (FMCA) could be applied for species identification. FMCA is based on a dual-labeled probe. Even with subtle difference of target sequence, there are visible melting temperature (Tm) shift. One of FMCA applications is distinguishing organisms distributed and consumed globally as popular food ingredients. Their prices are set by species or country of origin. However, counterfeiting or distributing them without any verification procedure are becoming social problems and threatening food safety. Besides distinguishing them in naked eye is very difficult and almost impossible in any processed form. Therefore, it is necessary to identify species in molecular level. In this research three species of squids which have 1-2 base pair differences each are selected as samples since they have the same issue. We designed a probe which perfectly matches with one species and the others mismatches 2 and 1 base pair respectively and labeled with fluorophore and quencher. In an experiment with a single probe, we successfully distinguished them by Tm shift depending on the difference of base pair. By combining FMCA and qPCR chip, smaller-scale assay with higher sensitivity and resolution could be possible, andc furthermore, enabling results analysis with smart phone would realize point-of-care testing (POCT).

  6. Ultra-sensitive DNA assay based on single-molecule detection coupled with fluorescent quantum dot-labeling and its application to determination of messenger RNA

    International Nuclear Information System (INIS)

    Li Li; Li Xincang; Li Lu; Wang Jinxing; Jin Wenrui

    2011-01-01

    An ultra-sensitive single-molecule detection (SMD) method for quantification of DNA using total internal reflection fluorescence microscopy (TIRFM) coupled with fluorescent quantum dot (QD)-labeling was developed. In this method, the target DNA (tDNA) was captured by the capture DNA immobilized on the silanized coverslip blocked with ethanolamine and bovine serum albumin. Then, the QD-labeled probe DNA was hybridized to the tDNA. Ten fluorescent images of the QD-labeled sandwich DNA hybrids on the coverslip were taken by a high-sensitive CCD. The tDNA was quantified by counting the bright spots on the images using a calibration curve. The LOD of the method was 1 x 10 -14 mol L -1 . Several key factors, including image acquirement, fluorescence probe, substrate preparation, noise elimination from solutions and glass coverslips, and nonspecific adsorption and binding of solution-phase detection probes were discussed in detail. The method could be applied to quantify messenger RNA (mRNA) in cells. In order to determine mRNA, double-stranded RNA-DNA hybrids consisting of mRNA and corresponding cDNA were synthesized from the cellular mRNA template using reverse transcription in the presence of reverse transcriptase. After removing the mRNA in the double-stranded hybrids using ribonuclease, cDNA was quantified using the SMD-based TIRFM. Osteopontin mRNA in decidual stromal cells was chosen as the model analyte.

  7. Ultra-sensitive DNA assay based on single-molecule detection coupled with fluorescent quantum dot-labeling and its application to determination of messenger RNA

    Energy Technology Data Exchange (ETDEWEB)

    Li Li [School of Chemistry and Chemical Engineering, Shandong University, Jinan 250100 (China); Li Xincang [School of Life Sciences, Shandong University, Jinan 250100 (China); Li Lu [School of Chemistry and Chemical Engineering, Shandong University, Jinan 250100 (China); Wang Jinxing [School of Life Sciences, Shandong University, Jinan 250100 (China); Jin Wenrui, E-mail: jwr@sdu.edu.cn [School of Chemistry and Chemical Engineering, Shandong University, Jinan 250100 (China)

    2011-01-24

    An ultra-sensitive single-molecule detection (SMD) method for quantification of DNA using total internal reflection fluorescence microscopy (TIRFM) coupled with fluorescent quantum dot (QD)-labeling was developed. In this method, the target DNA (tDNA) was captured by the capture DNA immobilized on the silanized coverslip blocked with ethanolamine and bovine serum albumin. Then, the QD-labeled probe DNA was hybridized to the tDNA. Ten fluorescent images of the QD-labeled sandwich DNA hybrids on the coverslip were taken by a high-sensitive CCD. The tDNA was quantified by counting the bright spots on the images using a calibration curve. The LOD of the method was 1 x 10{sup -14} mol L{sup -1}. Several key factors, including image acquirement, fluorescence probe, substrate preparation, noise elimination from solutions and glass coverslips, and nonspecific adsorption and binding of solution-phase detection probes were discussed in detail. The method could be applied to quantify messenger RNA (mRNA) in cells. In order to determine mRNA, double-stranded RNA-DNA hybrids consisting of mRNA and corresponding cDNA were synthesized from the cellular mRNA template using reverse transcription in the presence of reverse transcriptase. After removing the mRNA in the double-stranded hybrids using ribonuclease, cDNA was quantified using the SMD-based TIRFM. Osteopontin mRNA in decidual stromal cells was chosen as the model analyte.

  8. Measurement of apoptosis by SCAN©, a system for counting and analysis of fluorescently labelled nuclei

    Directory of Open Access Journals (Sweden)

    Neta Shlezinger

    2014-11-01

    Full Text Available Apoptosis-like programmed cell death (A-PCD is a universal process common to all types of eukaryotic organisms. Because A-PCD-associated processes are conserved, it is possible to define A-PCD by a standard set of markers. Many of the popular methods to measure A-PCD make use of fluorescent ligands that change in intensity or cellular localization during A-PCD. In single cell organisms, it is possible to quantify levels of A-PCD by scoring the number of apoptotic cells using flow cytometry instruments. In a multicellular organism, quantification of A-PCD is more problematic due to the complex nature of the tissue. The situation is further complicated in filamentous fungi, in which nuclei are divided between compartments, each containing a number of nuclei, which can also migrate between the compartments. We developed SCAN©, a System for Counting and Analysis of Nuclei, and used it to measure A-PCD according to two markers – chromatin condensation and DNA strand breaks. The package includes three modules designed for counting the number of nuclei in multi-nucleated domains, scoring the relative number of nuclei with condensed chromatin, and calculating the relative number of nuclei with DNA strand breaks. The method provides equal or better results compared with manual counting, the analysis is fast and can be applied on large data sets. While we demonstrated the utility of the software for measurement of A-PCD in fungi, the method is readily adopted for measurement of A-PCD in other types of multicellular specimens.

  9. Volumetric label-free imaging and 3D reconstruction of mammalian cochlea based on two-photon excitation fluorescence microscopy

    International Nuclear Information System (INIS)

    Zhang, Xianzeng; Zhan, Zhenlin; Xie, Shusen; Geng, Yang; Ye, Qing

    2013-01-01

    The visualization of the delicate structure and spatial relationship of intracochlear sensory cells has relied on the laborious procedures of tissue excision, fixation, sectioning and staining for light and electron microscopy. Confocal microscopy is advantageous for its high resolution and deep penetration depth, yet disadvantageous due to the necessity of exogenous labeling. In this study, we present the volumetric imaging of rat cochlea without exogenous dyes using a near-infrared femtosecond laser as the excitation mechanism and endogenous two-photon excitation fluorescence (TPEF) as the contrast mechanism. We find that TPEF exhibits strong contrast, allowing cellular and even subcellular resolution imaging of the cochlea, differentiating cell types, visualizing delicate structures and the radial nerve fiber. Our results further demonstrate that 3D reconstruction rendered with z-stacks of optical sections enables better revealment of fine structures and spatial relationships, and easily performed morphometric analysis. The TPEF-based optical biopsy technique provides great potential for new and sensitive diagnostic tools for hearing loss or hearing disorders, especially when combined with fiber-based microendoscopy. (paper)

  10. On optical detection of densely labeled synapses in neuropil and mapping connectivity with combinatorially multiplexed fluorescent synaptic markers.

    Directory of Open Access Journals (Sweden)

    Yuriy Mishchenko

    Full Text Available We propose a new method for mapping neural connectivity optically, by utilizing Cre/Lox system Brainbow to tag synapses of different neurons with random mixtures of different fluorophores, such as GFP, YFP, etc., and then detecting patterns of fluorophores at different synapses using light microscopy (LM. Such patterns will immediately report the pre- and post-synaptic cells at each synaptic connection, without tracing neural projections from individual synapses to corresponding cell bodies. We simulate fluorescence from a population of densely labeled synapses in a block of hippocampal neuropil, completely reconstructed from electron microscopy data, and show that high-end LM is able to detect such patterns with over 95% accuracy. We conclude, therefore, that with the described approach neural connectivity in macroscopically large neural circuits can be mapped with great accuracy, in scalable manner, using fast optical tools, and straightforward image processing. Relying on an electron microscopy dataset, we also derive and explicitly enumerate the conditions that should be met to allow synaptic connectivity studies with high-resolution optical tools.

  11. Fluorescently labeled methyl-beta-cyclodextrin enters intestinal epithelial Caco-2 cells by fluid-phase endocytosis.

    Directory of Open Access Journals (Sweden)

    Ferenc Fenyvesi

    Full Text Available Cyclodextrins are widely used excipients for increasing the bioavailability of poorly water-soluble drugs. Their effect on drug absorption in the gastrointestinal tract is explained by their solubility- and permeability-enhancement. The aims of this study were to investigate penetration properties of fluorescently labeled randomly methylated-beta-cyclodextrin (FITC-RAMEB on Caco-2 cell layer and examine the cellular entry of cyclodextrins on intestinal cells. The permeability of FITC-RAMEB through Caco-2 monolayers was very limited. Using this compound in 0.05 mM concentration the permeability coefficient was 3.35±1.29×10(-8 cm/s and its permeability did not change in the presence of 5 mM randomly methylated-beta-cyclodextrin. Despite of the low permeability, cellular accumulation of FITC-RAMEB in cytoplasmic vesicles was significant and showed strong time and concentration dependence, similar to the characteristics of the macropinocytosis marker Lucifer Yellow. The internalization process was fully inhibited at 0°C and it was drastically reduced at 37°C applying rottlerin, an inhibitor of macropinocytosis. Notably, FITC-RAMEB colocalized with the early endosome organizer Rab5a. These results have revealed that FITC-RAMEB is able to enter intestinal epithelial cells by fluid-phase endocytosis from the apical side. This mechanism can be an additional process which helps to overcome the intestinal barrier and contributes to the bioavailability enhancement of cyclodextrins.

  12. Simultaneous detection of Staphylococcus aureus and Salmonella typhimurium using multicolor time-resolved fluorescence nanoparticles as labels.

    Science.gov (United States)

    Wang, Xiaole; Huang, Yukun; Wu, Shijia; Duan, Nuo; Xu, Baocai; Wang, Zhouping

    2016-11-21

    Foodborne illnesses caused by Staphylococcus aureus and Salmonella typhimurium are common public health issues worldwide, affecting both developing and developed countries. In this study, aptamers labeled with multicolor lanthanide-doped time-resolved fluorescence (TRFL) nanoparticles were used as signal probes, and immobilized by Fe 3 O 4 magnetic nanoparticles were used as the capture probes. The signal probes were bonded onto the captured bacteria by the recognition of aptamer to form the sandwich-type complex. Under the optimal conditions, TRFL intensity at 544nm was used to quantify S. typhimurium (y=10,213×-12,208.92, R 2 =0.9922) and TRFL intensity at 615nm for S. aureus (y=4803.20×-1933.87, R 2 =0.9982) in the range of 10 2 -10 5 CFU/ml. Due to the magnetic separation and concentration of Fe 3 O 4 nanoparticles, detection limits of the developed method were found to be 15, 20CFU/ml for S. typhimurium and S. aureus, respectively. The application of this bioassay in milk was also investigated, and results were consistent with those of plate-counting method. Therefore, this simple and rapid method owns a great potential in the application for the multiplex analysis in food safety. Copyright © 2016 Elsevier B.V. All rights reserved.

  13. Endogenous Two-Photon Excited Fluorescence Provides Label-Free Visualization of the Inflammatory Response in the Rodent Spinal Cord

    Directory of Open Access Journals (Sweden)

    Ortrud Uckermann

    2015-01-01

    Full Text Available Activation of CNS resident microglia and invasion of external macrophages plays a central role in spinal cord injuries and diseases. Multiphoton microscopy based on intrinsic tissue properties offers the possibility of label-free imaging and has the potential to be applied in vivo. In this work, we analyzed cellular structures displaying endogenous two-photon excited fluorescence (TPEF in the pathologic spinal cord. It was compared qualitatively and quantitatively to Iba1 and CD68 immunohistochemical staining in two models: rat spinal cord injury and mouse encephalomyelitis. The extent of tissue damage was retrieved by coherent anti-Stokes Raman scattering (CARS and second harmonic generation imaging. The pattern of CD68-positive cells representing postinjury activated microglia/macrophages was colocalized to the TPEF signal. Iba1-positive microglia were found in areas lacking any TPEF signal. In peripheral areas of inflammation, we found similar numbers of CD68-positive microglia/macrophages and TPEF-positive structures while the number of Iba1-positive cells was significantly higher. Therefore, we conclude that multiphoton imaging of unstained spinal cord tissue enables retrieving the extent of microglia activation by acquisition of endogenous TPEF. Future application of this technique in vivo will enable monitoring inflammatory responses of the nervous system allowing new insights into degenerative and regenerative processes.

  14. Endogenous Two-Photon Excited Fluorescence Provides Label-Free Visualization of the Inflammatory Response in the Rodent Spinal Cord

    Science.gov (United States)

    Uckermann, Ortrud; Galli, Roberta; Beiermeister, Rudolf; Sitoci-Ficici, Kerim-Hakan; Later, Robert; Leipnitz, Elke; Chavakis, Triantafyllos; Koch, Edmund; Schackert, Gabriele; Steiner, Gerald; Kirsch, Matthias

    2015-01-01

    Activation of CNS resident microglia and invasion of external macrophages plays a central role in spinal cord injuries and diseases. Multiphoton microscopy based on intrinsic tissue properties offers the possibility of label-free imaging and has the potential to be applied in vivo. In this work, we analyzed cellular structures displaying endogenous two-photon excited fluorescence (TPEF) in the pathologic spinal cord. It was compared qualitatively and quantitatively to Iba1 and CD68 immunohistochemical staining in two models: rat spinal cord injury and mouse encephalomyelitis. The extent of tissue damage was retrieved by coherent anti-Stokes Raman scattering (CARS) and second harmonic generation imaging. The pattern of CD68-positive cells representing postinjury activated microglia/macrophages was colocalized to the TPEF signal. Iba1-positive microglia were found in areas lacking any TPEF signal. In peripheral areas of inflammation, we found similar numbers of CD68-positive microglia/macrophages and TPEF-positive structures while the number of Iba1-positive cells was significantly higher. Therefore, we conclude that multiphoton imaging of unstained spinal cord tissue enables retrieving the extent of microglia activation by acquisition of endogenous TPEF. Future application of this technique in vivo will enable monitoring inflammatory responses of the nervous system allowing new insights into degenerative and regenerative processes. PMID:26355949

  15. A combination of positive dielectrophoresis driven on-line enrichment and aptamer-fluorescent silica nanoparticle label for rapid and sensitive detection of Staphylococcus aureus.

    Science.gov (United States)

    Shangguan, Jingfang; Li, Yuhong; He, Dinggeng; He, Xiaoxiao; Wang, Kemin; Zou, Zhen; Shi, Hui

    2015-07-07

    Staphylococcus aureus (S. aureus) is an important human pathogen that causes several diseases ranging from superficial skin infections to life-threatening diseases. Here, a method combining positive dielectrophoresis (pDEP) driven on-line enrichment and aptamer-fluorescent silica nanoparticle label has been developed for the rapid and sensitive detection of S. aureus in microfluidic channels. An aptamer, having high affinity to S. aureus, is used as the molecular recognition tool and immobilized onto chloropropyl functionalized fluorescent silica nanoparticles through a click chemistry approach to obtain S. aureus aptamer-nanoparticle bioconjugates (Apt(S.aureus)/FNPs). The pDEP driven on-line enrichment technology was used for accumulating the Apt(S.aureus)/FNP labeled S. aureus. After incubating with S. aureus, the mixture of Apt(S.aureus)/FNP labeled S. aureus and Apt(S.aureus)/FNPs was directly introduced into the pDEP-based microfluidic system. By applying an AC voltage in a pDEP frequency region, the Apt(S.aureus)/FNP labelled S. aureus moved to the electrodes and accumulated in the electrode gap, while the free Apt(S.aureus)/FNPs flowed away. The signal that came from the Apt(S.aureus)/FNP labelled S. aureus in the focused detection areas was then detected. Profiting from the specificity of aptamer, signal amplification of FNP label and pDEP on-line enrichment, this assay can detect as low as 93 and 270 cfu mL(-1)S. aureus in deionized water and spiked water samples, respectively, with higher sensitivities than our previously reported Apt(S.aureus)/FNP based flow cytometry. Moreover, without the need for separation and washing steps usually required for FNP label involved bioassays, the total assay time including sample pretreatment was within 2 h.

  16. Fluorescent dye labeled influenza virus mainly infects innate immune cells and activated lymphocytes and can be used in cell-mediated immune response assay

    OpenAIRE

    Xie, Dongxu

    2009-01-01

    Early results have recognized that influenza virus infects the innate and adaptive immune cells. The data presented in this paper demonstrated that influenza virus labeled with fluorescent dye not only retained the ability to infect and replicate in host cells, but also stimulated a similar human immune response as did unlabeled virus. Influenza virus largely infected the innate and activated adaptive immune cells. Influenza B type virus was different from that of A type virus. B type virus w...

  17. CdSe/ZnS Quantum Dots-Labeled Mesenchymal Stem Cells for Targeted Fluorescence Imaging of Pancreas Tissues and Therapy of Type 1 Diabetic Rats.

    Science.gov (United States)

    Liu, Haoqi; Tang, Wei; Li, Chao; Lv, Pinlei; Wang, Zheng; Liu, Yanlei; Zhang, Cunlei; Bao, Yi; Chen, Haiyan; Meng, Xiangying; Song, Yan; Xia, Xiaoling; Pan, Fei; Cui, Daxiang; Shi, Yongquan

    2015-12-01

    Mesenchymal stem cells (MSCs) have been used for therapy of type 1 diabetes mellitus. However, the in vivo distribution and therapeutic effects of transplanted MSCs are not clarified well. Herein, we reported that CdSe/ZnS quantum dots-labeled MSCs were prepared for targeted fluorescence imaging and therapy of pancreas tissues in rat models with type 1 diabetes. CdSe/ZnS quantum dots were synthesized, their biocompatibility was evaluated, and then, the appropriate concentration of quantum dots was selected to label MSCs. CdSe/ZnS quantum dots-labeled MSCs were injected into mouse models with type 1 diabetes via tail vessel and then were observed by using the Bruker In-Vivo F PRO system, and the blood glucose levels were monitored for 8 weeks. Results showed that prepared CdSe/ZnS quantum dots owned good biocompatibility. Significant differences existed in distribution of quantum dots-labeled MSCs between normal control rats and diabetic rats (p quantum dots-labeled MSC injection. Statistical differences existed between the blood glucose levels of the diabetic rat control group and MSC-injected diabetic rat group (p < 0.01), and the MSC-injected diabetic rat group displayed lower blood glucose levels. In conclusion, CdSe/ZnS-labeled MSCs can target in vivo pancreas tissues in diabetic rats, and significantly reduce the blood glucose levels in diabetic rats, and own potential application in therapy of diabetic patients in the near future.

  18. A label-free fluorescence biosensor for highly sensitive detection of lectin based on carboxymethyl chitosan-quantum dots and gold nanoparticles

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Ziping; Liu, Hua; Wang, Lei; Su, Xingguang, E-mail: suxg@jlu.edu.cn

    2016-08-17

    In this work, we report a novel label-free fluorescence “turn off-on” biosensor for lectin detection. The highly sensitive and selective sensing system is based on the integration of carboxymethyl chitosan (CM-CHIT), CuInS{sub 2} quantum dots (QDs) and Au nanoparticles (NPs). Firstly, CuInS{sub 2} QDs featuring carboxyl groups were directly synthesized via a hydrothermal synthesis method. Then, the carboxyl groups on the CuInS{sub 2} QDs surface were interacted with the amino groups (−NH{sub 2}), carboxyl groups (−COOH) and hydroxyl groups (−OH) within CM-CHIT polymeric chains via electrostatic interactions and hydrogen bonding to form CM-CHIT-QDs assemblies. Introduction of Au NPs could quench the fluorescence of CM-CHIT-QDs through electron and energy transfer. In the presence of lectin, lectin could bind exclusively with CM-CHIT-QDs by means of specific multivalent carbohydrate-protein interaction. Thus, the electron and energy transfer process between CM-CHIT-QDs and Au NPs was inhibited, and as a result, the fluorescence of CM-CHIT-QDs was effectively “turned on”. Under the optimum conditions, there was a good linear relationship between the fluorescence intensity ratio I/I{sub 0} (I and I{sub 0} were the fluorescence intensity of CM-CHIT-QDs-Au NPs in the presence and absence of lectin, respectively) and lectin concentration in the range of 0.2–192.5 nmol L{sup −1}, And the detection limit could be down to 0.08 nmol L{sup −1}. Furthermore, the proposed biosensor was employed for the determination of lectin in fetal bovine serum samples with satisfactory results. - Graphical abstract: A label-free fluorescence biosensor for highly sensitive detection of lectin based on the integration of carboxymethyl chitosan, CuInS{sub 2} quantum dots and gold nanoparticles. - Highlights: • A label-free near-infrared fluorescence “turn off-on” biosensor for detection of lectin was established. • The highly sensitive biosensor was based on the

  19. A label-free fluorescence biosensor for highly sensitive detection of lectin based on carboxymethyl chitosan-quantum dots and gold nanoparticles

    International Nuclear Information System (INIS)

    Liu, Ziping; Liu, Hua; Wang, Lei; Su, Xingguang

    2016-01-01

    In this work, we report a novel label-free fluorescence “turn off-on” biosensor for lectin detection. The highly sensitive and selective sensing system is based on the integration of carboxymethyl chitosan (CM-CHIT), CuInS_2 quantum dots (QDs) and Au nanoparticles (NPs). Firstly, CuInS_2 QDs featuring carboxyl groups were directly synthesized via a hydrothermal synthesis method. Then, the carboxyl groups on the CuInS_2 QDs surface were interacted with the amino groups (−NH_2), carboxyl groups (−COOH) and hydroxyl groups (−OH) within CM-CHIT polymeric chains via electrostatic interactions and hydrogen bonding to form CM-CHIT-QDs assemblies. Introduction of Au NPs could quench the fluorescence of CM-CHIT-QDs through electron and energy transfer. In the presence of lectin, lectin could bind exclusively with CM-CHIT-QDs by means of specific multivalent carbohydrate-protein interaction. Thus, the electron and energy transfer process between CM-CHIT-QDs and Au NPs was inhibited, and as a result, the fluorescence of CM-CHIT-QDs was effectively “turned on”. Under the optimum conditions, there was a good linear relationship between the fluorescence intensity ratio I/I_0 (I and I_0 were the fluorescence intensity of CM-CHIT-QDs-Au NPs in the presence and absence of lectin, respectively) and lectin concentration in the range of 0.2–192.5 nmol L"−"1, And the detection limit could be down to 0.08 nmol L"−"1. Furthermore, the proposed biosensor was employed for the determination of lectin in fetal bovine serum samples with satisfactory results. - Graphical abstract: A label-free fluorescence biosensor for highly sensitive detection of lectin based on the integration of carboxymethyl chitosan, CuInS_2 quantum dots and gold nanoparticles. - Highlights: • A label-free near-infrared fluorescence “turn off-on” biosensor for detection of lectin was established. • The highly sensitive biosensor was based on the inner filter effect of Au NPs on CM

  20. Detection of radiation-induced brain necrosis in live rats using label-free time-resolved fluorescence spectroscopy (TRFS) (Conference Presentation)

    Science.gov (United States)

    Hartl, Brad A.; Ma, Htet S. W.; Sridharan, Shamira; Hansen, Katherine; Klich, Melanie; Perks, Julian; Kent, Michael; Kim, Kyoungmi; Fragoso, Ruben; Marcu, Laura

    2017-02-01

    Differentiating radiation-induced necrosis from recurrent tumor in the brain remains a significant challenge to the neurosurgeon. Clinical imaging modalities are not able to reliably discriminate the two tissue types, making biopsy location selection and surgical management difficult. Label-free fluorescence lifetime techniques have previously been shown to be able to delineate human brain tumor from healthy tissues. Thus, fluorescence lifetime techniques represent a potential means to discriminate the two tissues in real-time during surgery. This study aims to characterize the endogenous fluorescence lifetime signatures from radiation induced brain necrosis in a tumor-free rat model. Fischer rats received a single fraction of 60 Gy of radiation to the right hemisphere using a linear accelerator. Animals underwent a terminal live surgery after gross necrosis had developed, as verified with MRI. During surgery, healthy and necrotic brain tissue was measured with a fiber optic needle connected to a multispectral fluorescence lifetime system. Measurements of the necrotic tissue showed a 48% decrease in intensity and 20% increase in lifetimes relative to healthy tissue. Using a support vector machine classifier and leave-one-out validation technique, the necrotic tissue was correctly classified with 94% sensitivity and 97% specificity. Spectral contribution analysis also confirmed that the primary source of fluorescence contrast lies within the redox and bound-unbound population shifts of nicotinamide adenine dinucleotide. A clinical trial is presently underway to measure these tissue types in humans. These results show for the first time that radiation-induced necrotic tissue in the brain contains significantly different metabolic signatures that are detectable with label-free fluorescence lifetime techniques.

  1. Assessing a relationship between bone microstructure and growth rate: a fluorescent labelling study in the king penguin chick (Aptenodytes patagonicus).

    Science.gov (United States)

    de Margerie, E; Robin, J-P; Verrier, D; Cubo, J; Groscolas, R; Castanet, J

    2004-02-01

    Microstructure-function relationships remain poorly understood in primary bone tissues. The relationship between bone growth rate and bone tissue type, although documented in some species by previous works, remains somewhat unclear and controversial. We assessed this relationship in a species with extreme adaptations, the king penguin (Aptenodytes patagonicus). These birds have a peculiar growth, interrupted 3 months after hatching by the austral winter. Before this interruption, chicks undergo extremely rapid statural and ponderal growth. We recorded experimentally (by means of fluorescent labelling) the growth rate of bone tissue in four long bones (humerus, radius, femur and tibiotarsus) of four king penguin chicks during their fastest phase of growth (3-5 weeks after hatching) and identified the associated bone tissue types ('laminar', 'longitudinal', 'reticular' or 'radial' fibro-lamellar bone tissue). We found the highest bone tissue growth rate known to date, up to 171 microm day(-1) (mean 55 microm day(-1)). There was a highly significant relationship between bone tissue type and growth rate (P<10(-6)). Highest rates were obtained with the radial microarchitecture of fibro-lamellar bone, where cavities in the woven network are aligned radially. This result supports the heuristic value of a relationship between growth rate and bone primary microstructure. However, we also found that growth rates of bone tissue types vary according to the long bone considered (P<10(-5)) (e.g. growth rates were 38% lower in the radius than in the other long bones), a result that puts some restriction on the applicability of absolute growth rate values (e.g. to fossil species). The biomechanical disadvantages of accelerated bone growth are discussed in relation to the locomotor behaviour of the chicks during their first month of life.

  2. Topology of AspT, the aspartate:alanine antiporter of Tetragenococcus halophilus, determined by site-directed fluorescence labeling.

    Science.gov (United States)

    Nanatani, Kei; Fujiki, Takashi; Kanou, Kazuhiko; Takeda-Shitaka, Mayuko; Umeyama, Hideaki; Ye, Liwen; Wang, Xicheng; Nakajima, Tasuku; Uchida, Takafumi; Maloney, Peter C; Abe, Keietsu

    2007-10-01

    The gram-positive lactic acid bacterium Tetragenococcus halophilus catalyzes the decarboxylation of L-aspartate (Asp) with release of L-alanine (Ala) and CO(2). The decarboxylation reaction consists of two steps: electrogenic exchange of Asp for Ala catalyzed by an aspartate:alanine antiporter (AspT) and intracellular decarboxylation of the transported Asp catalyzed by an L-aspartate-beta-decarboxylase (AspD). AspT belongs to the newly classified aspartate:alanine exchanger family (transporter classification no. 2.A.81) of transporters. In this study, we were interested in the relationship between the structure and function of AspT and thus analyzed the topology by means of the substituted-cysteine accessibility method using the impermeant, fluorescent, thiol-specific probe Oregon Green 488 maleimide (OGM) and the impermeant, nonfluorescent, thiol-specific probe [2-(trimethylammonium)ethyl]methanethiosulfonate bromide. We generated 23 single-cysteine variants from a six-histidine-tagged cysteineless AspT template. A cysteine position was assigned an external location if the corresponding single-cysteine variant reacted with OGM added to intact cells, and a position was assigned an internal location if OGM labeling required cell lysis. The topology analyses revealed that AspT has a unique topology; the protein has 10 transmembrane helices (TMs), a large hydrophilic cytoplasmic loop (about 180 amino acids) between TM5 and TM6, N and C termini that face the periplasm, and a positively charged residue (arginine 76) within TM3. Moreover, the three-dimensional structure constructed by means of the full automatic modeling system indicates that the large hydrophilic cytoplasmic loop of AspT possesses a TrkA_C domain and a TrkA_C-like domain and that the three-dimensional structures of these domains are similar to each other even though their amino acid sequences show low similarity.

  3. Early kinetic intermediate in the folding of acyl-CoA binding protein detected by fluorescence labeling and ultrarapid mixing

    DEFF Research Database (Denmark)

    Teilum, Kaare; Maki, Kosuke; Kragelund, Birthe B

    2002-01-01

    showed a major increase in tryptophan-dansyl fluorescence energy transfer, indicating formation of a partially collapsed ensemble of states on the 100-micros time scale. A subsequent decrease in dansyl fluorescence is attributed to intramolecular quenching of donor fluorescence on formation of the native...

  4. Synthesis of highly fluorescent and thio-linkers stabilize gold quantum dots and nano clusters in DMF for bio-labeling

    Energy Technology Data Exchange (ETDEWEB)

    Rastogi, Shiva K., E-mail: srastogi@uidaho.edu [University of Idaho, Department of Chemistry (United States); Denn, Benjamin D.; Branen, A. Larry [University of Idaho, Coeur D' Alene, Biosensors and Nanotechnology Application Laboratory (BNAL) (United States)

    2012-01-15

    This study demonstrates a one versus two-step synthesis of fluorescent gold quantum dots (F-AuQDs) and nano clusters (F-AuNCs) functionalized with thiolated organic linkers using reduction of gold precursor in N,N Prime -dimethylformamide in 1 h of reaction. The F-AuQDs and F-AuNCs show fluorescence emission at 425 {+-} 5 nm upon excitation at 345 {+-} 5 nm of wavelength, with good water solubility and stability. Five different thiolated organic binary linkers consisting of various functional groups including: carboxylic acid, hydroxyl, and aromatic amine, were conjugated with the F-AuQDs and F-AuNCs. The formation mechanism and functionalization of the F-AuQDs and F-AuNCs was characterized using UV-vis absorption spectra, UV-vis light, fluorescent emission spectra, pH, TEM, and FTIR. The fluorescence emission of the F-AuQDs and F-AuNCs is greatly dependent on the thio-linker. This novel one-step approach provides facile and fast synthesis of F-AuQDs and F-AuNCs over the two-step method, with less than 5 h of reaction and workup compared to more than 28 h of reaction for the two-step approach. These thio-linker functionalized F-AuQDs and F-AuNCs have a wide application in fluorescent labeling of biomolecules, optical devices, imaging, energy transfer, and biosensing.

  5. Labelling of living mammalian spermatozoa with the fluorescent thiol alkylating agent, monobromobimane (MB): immobilization upon exposure to ultraviolet light and analysis of acrosomal status

    International Nuclear Information System (INIS)

    Cummins, J.M.; Fleming, A.D.; Crozet, N.; Kuehl, T.J.; Kosower, N.S.; Yanagimachi, R.

    1986-01-01

    Living spermatozoa of seven mammalian species were treated with the thiol-alkylating fluorescent labelling compound, monobromobimane (MBBR). MB-labelling alone had no effect on sperm motility, nor on the time course or ability of golden hamster spermatozoa to undergo the acrosome reaction when capacitated in vitro. Exposure of MB-labelled spermatozoa to ultraviolet (UV) light and excitation of the MB fluorochrome resulted in virtually immediate immobilization of the spermatozoa without affecting acrosomal status. UV exposure of unlabelled spermatozoa for up to 30 sec had no effect upon motility. Immobilization of MB-labelled spermatozoa depended on the midpiece being irradiated, as irradiation of the head alone, or of the more distal parts of the principal piece, had little or no effect upon motility. Labelling with MB followed by immobilization of individually selected spermatozoa was most useful for detailing the course and site of occurrence of the acrosome reaction during penetration of the cumulus oophorus by golden hamster spermatozoa in vitro. In these often hyperactivated spermatozoa, precise determination of the acrosomal status could not often otherwise be made due to the difficulty in visualizing the acrosomal region of a vigorously thrashing, hyperactivated spermatozoon. This technique should prove valuable in a variety of studies on sperm motility, capacitation and fertilization, and could also be extended to other cell systems

  6. CdSe/ZnS Quantum Dots-Labeled Mesenchymal Stem Cells for Targeted Fluorescence Imaging of Pancreas Tissues and Therapy of Type 1 Diabetic Rats

    Science.gov (United States)

    Liu, Haoqi; Tang, Wei; Li, Chao; Lv, Pinlei; Wang, Zheng; Liu, Yanlei; Zhang, Cunlei; Bao, Yi; Chen, Haiyan; Meng, Xiangying; Song, Yan; Xia, Xiaoling; Pan, Fei; Cui, Daxiang; Shi, Yongquan

    2015-06-01

    Mesenchymal stem cells (MSCs) have been used for therapy of type 1 diabetes mellitus. However, the in vivo distribution and therapeutic effects of transplanted MSCs are not clarified well. Herein, we reported that CdSe/ZnS quantum dots-labeled MSCs were prepared for targeted fluorescence imaging and therapy of pancreas tissues in rat models with type 1 diabetes. CdSe/ZnS quantum dots were synthesized, their biocompatibility was evaluated, and then, the appropriate concentration of quantum dots was selected to label MSCs. CdSe/ZnS quantum dots-labeled MSCs were injected into mouse models with type 1 diabetes via tail vessel and then were observed by using the Bruker In-Vivo F PRO system, and the blood glucose levels were monitored for 8 weeks. Results showed that prepared CdSe/ZnS quantum dots owned good biocompatibility. Significant differences existed in distribution of quantum dots-labeled MSCs between normal control rats and diabetic rats ( p pancreas of rats in the diabetes group, and was about 32 %, while that in the normal control group rats was about 18 %. The blood glucose levels were also monitored for 8 weeks after quantum dots-labeled MSC injection. Statistical differences existed between the blood glucose levels of the diabetic rat control group and MSC-injected diabetic rat group ( p pancreas tissues in diabetic rats, and significantly reduce the blood glucose levels in diabetic rats, and own potential application in therapy of diabetic patients in the near future.

  7. Preclinical evaluation of carbon-11 and fluorine-18 sulfonamide derivatives for in vivo radiolabeling of erythrocytes

    OpenAIRE

    Gheysens, Olivier; Akurathi, Vamsidhar; Chekol, Rufael; Dresselaers, Tom; Celen, Sofie; Koole, Michel; Dauwe, Dieter; Cleynhens, Bernard J; Claus, Piet; Janssens, Stefan; Verbruggen, Alfons M; Nuyts, Johan; Himmelreich, Uwe; Bormans, Guy M

    2013-01-01

    Background To date, few PET tracers for in vivo labeling of red blood cells (RBCs) are available. In this study, we report the radiosynthesis and in vitro and in vivo evaluation of 11C and 18F sulfonamide derivatives targeting carbonic anhydrase II (CA II), a metallo-enzyme expressed in RBCs, as potential blood pool tracers. A proof-of-concept in vivo imaging study was performed to demonstrate the feasibility to assess cardiac function and volumes using electrocardiogram (ECG)-gated positron ...

  8. Label-Free Detection of Sequence-Specific DNA Based on Fluorescent Silver Nanoclusters-Assisted Surface Plasmon-Enhanced Energy Transfer.

    Science.gov (United States)

    Ma, Jin-Liang; Yin, Bin-Cheng; Le, Huynh-Nhu; Ye, Bang-Ce

    2015-06-17

    We have developed a label-free method for sequence-specific DNA detection based on surface plasmon enhanced energy transfer (SPEET) process between fluorescent DNA/AgNC string and gold nanoparticles (AuNPs). DNA/AgNC string, prepared by a single-stranded DNA template encoded two emitter-nucleation sequences at its termini and an oligo spacer in the middle, was rationally designed to produce bright fluorescence emission. The proposed method takes advantage of two strategies. The first one is the difference in binding properties of single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) toward AuNPs. The second one is SPEET process between fluorescent DNA/AgNC string and AuNPs, in which fluorescent DNA/AgNC string can be spontaneously adsorbed onto the surface of AuNPs and correspondingly AuNPs serve as "nanoquencher" to quench the fluorescence of DNA/AgNC string. In the presence of target DNA, the sensing probe hybridized with target DNA to form duplex DNA, leading to a salt-induced AuNP aggregation and subsequently weakened SPEET process between fluorescent DNA/AgNC string and AuNPs. A red-to-blue color change of AuNPs and a concomitant fluorescence increase were clearly observed in the sensing system, which had a concentration dependent manner with specific DNA. The proposed method achieved a detection limit of ∼2.5 nM, offering the following merits of simple design, convenient operation, and low experimental cost because of no chemical modification, organic dye, enzymatic reaction, or separation procedure involved.

  9. Label-free tungsten disulfide quantum dots as a fluorescent sensing platform for highly efficient detection of copper (II) ions

    International Nuclear Information System (INIS)

    Zhao Xuan; He Da-Wei; Wang Yong-Sheng; Hu Yin; Fu Chen; Li Xue

    2017-01-01

    A fluorescent probe for the sensitive and selective determination of copper ion (Cu 2+ ) is presented. It is based on the use of tungsten disulfide quantum dots (WS 2 QDs) which is independent of the pH of solution and emits strong blue fluorescence. Copper ions could cause aggregation of the WS 2 QDs and lead to fluorescence quenching of WS 2 QDs. The change of fluorescence intensity is proportional to the concentration of Cu 2+ , and the limit of detection is 0.4 μM. The fluorescent probe is highly selective for Cu 2+ over some potentially interfering ions. These results indicate that WS 2 QDs, as a fluorescent sensing platform, can meet the selective requirements for biomedical and environmental application. (paper)

  10. Toehold strand displacement-driven assembly of G-quadruplex DNA for enzyme-free and non-label sensitive fluorescent detection of thrombin.

    Science.gov (United States)

    Xu, Yunying; Zhou, Wenjiao; Zhou, Ming; Xiang, Yun; Yuan, Ruo; Chai, Yaqin

    2015-02-15

    Based on a new signal amplification strategy by the toehold strand displacement-driven cyclic assembly of G-quadruplex DNA, the development of an enzyme-free and non-label aptamer sensing approach for sensitive fluorescent detection of thrombin is described. The target thrombin associates with the corresponding aptamer of the partial dsDNA probes and liberates single stranded initiation sequences, which trigger the toehold strand displacement assembly of two G-quadruplex containing hairpin DNAs. This toehold strand displacement reaction leads to the cyclic reuse of the initiation sequences and the production of DNA assemblies with numerous G-quadruplex structures. The fluorescent dye, N-Methyl mesoporphyrin IX, binds to these G-quadruplex structures and generates significantly amplified fluorescent signals to achieve highly sensitive detection of thrombin down to 5 pM. Besides, this method shows high selectivity towards the target thrombin against other control proteins. The developed thrombin sensing method herein avoids the modification of the probes and the involvement of any enzyme or nanomaterial labels for signal amplification. With the successful demonstration for thrombin detection, our approach can be easily adopted to monitor other target molecules in a simple, low-cost, sensitive and selective way by choosing appropriate aptamer/ligand pairs. Copyright © 2014 Elsevier B.V. All rights reserved.

  11. Labelling of algae and inorganic sediments with neutron-activable indicator elements and fluorescence dyes. Markierung von Algen und anorganischen Sedimenten mit neutronenaktivierbaren Indikatorelementen und mit Fluoreszenzfarbstoffen

    Energy Technology Data Exchange (ETDEWEB)

    Schulze, E.

    1986-04-15

    For an investigation of algae sinking characteristics in natural bodies of water, three different species (Chlorella fusca, Scenedesmus quadricauda, Nostoc variabilis) were labelled with neutron-activable elements. The rare earths Dy, Er, and Eu were preferred for their rare natural occurrence and favourable activation analysis characteristics. Growth and enrichment were monitored using a method of chlorophyll fluorescence measurement which enables measurements of the chlorophyll concentration and photosynthetic activity. After sampling, the suspensions were filtered and activated with thermal neutrons in a research reactor. The indicator masses were determined by a quantitative evaluation of the ..gamma.. spectra. In parallel to these investigation, labelling with fluorescent dyes was investigated. The transport characteristics of phosphate-carrying aggregates of the coarse clay fraction, glasses containing Dy, Eu and Er were prepared and fractionated in a centrifugal ball mill to obtain an earth with near-natural grain size distribution. The applicability of the labelling and detection methods developed for the dissertation was tested in a natural environment. Limits of application and costs were assessed.

  12. Radionuclide and Fluorescence Imaging of Clear Cell Renal Cell Carcinoma Using Dual Labeled Anti-Carbonic Anhydrase IX Antibody G250.

    Science.gov (United States)

    Muselaers, Constantijn H J; Rijpkema, Mark; Bos, Desirée L; Langenhuijsen, Johan F; Oyen, Wim J G; Mulders, Peter F A; Oosterwijk, Egbert; Boerman, Otto C

    2015-08-01

    Tumor targeted optical imaging using antibodies labeled with near infrared fluorophores is a sensitive imaging modality that might be used during surgery to assure complete removal of malignant tissue. We evaluated the feasibility of dual modality imaging and image guided surgery with the dual labeled anti-carbonic anhydrase IX antibody preparation (111)In-DTPA-G250-IRDye800CW in mice with intraperitoneal clear cell renal cell carcinoma. BALB/c nu/nu mice with intraperitoneal SK-RC-52 lesions received 10 μg DTPA-G250-IRDye800CW labeled with 15 MBq (111)In or 10 μg of the dual labeled irrelevant control antibody NUH-82 (20 mice each). To evaluate when tumors could be detected, 4 mice per group were imaged weekly during 5 weeks with single photon emission computerized tomography/computerized tomography and the fluorescence imaging followed by ex vivo biodistribution studies. As early as 1 week after tumor cell inoculation single photon emission computerized tomography and fluorescence images showed clear delineation of intraperitoneal clear cell renal cell carcinoma with good concordance between single photon emission computerized tomography/computerized tomography and fluorescence images. The high and specific accumulation of the dual labeled antibody conjugate in tumors was confirmed in the biodistribution studies. Maximum tumor uptake was observed 1 week after inoculation (mean ± SD 58.5% ± 18.7% vs 5.6% ± 2.3% injected dose per gm for DTPA-G250-IRDye800CW vs NUH-82, respectively). High tumor uptake was also observed at other time points. This study demonstrates the feasibility of dual modality imaging with dual labeled antibody (111)In-DTPA-G250-IRDye800CW in a clear cell renal cell carcinoma model. Results indicate that preoperative and intraoperative detection of carbonic anhydrase IX expressing tumors, positive resection margins and metastasis might be feasible with this approach. Copyright © 2015 American Urological Association Education and Research

  13. Continuous recording of long-chain acyl-coenzyme A synthetase activity using fluorescently labeled bovine serum albumin

    DEFF Research Database (Denmark)

    Demant, Erland J.F.; Nystrøm, Birthe T.

    2001-01-01

    acyl-Coenzyme A, synthetase, activity assay, fluorescence recording, fatty acid probe, serum albumin, hydroxycoumarin, detergent, micelles, Pseudomonas fragi, rat liver microsomes......acyl-Coenzyme A, synthetase, activity assay, fluorescence recording, fatty acid probe, serum albumin, hydroxycoumarin, detergent, micelles, Pseudomonas fragi, rat liver microsomes...

  14. One-pot synthesis of strongly fluorescent DNA-CuInS2 quantum dots for label-free and ultrasensitive detection of anthrax lethal factor DNA

    International Nuclear Information System (INIS)

    Liu, Ziping; Su, Xingguang

    2016-01-01

    Herein, high quality DNA-CuInS 2 QDs are facilely synthesized through a one-pot hydrothermal method with fluorescence quantum yield as high as 23.4%, and the strongly fluorescent DNA-CuInS 2 QDs have been utilized as a novel fluorescent biosensor for label-free and ultrasensitive detection of anthrax lethal factor DNA. L-Cysteine (L-Cys) and a specific-sequence DNA are used as co-ligands to stabilize the CuInS 2 QDs. The specific-sequence DNA consists of two domains: phosphorothiolates domain (sulfur-containing variants of the usual phosphodiester backbone) controls the nanocrystal passivation and serves as a ligand, and the functional domain (non-phosphorothioates) controls the biorecognition. The as-prepared DNA-CuInS 2 QDs have high stability, good water-solubility and low toxicity. Under the optimized conditions, a linear correlation was established between the fluorescence intensity ratio I/I 0 (I 0 is the original fluorescence intensity of DNA-CuInS 2 QDs, and I is the fluorescence intensity of DNA-CuInS 2 QDs/GO with the addition of various concentrations of anthrax lethal factor DNA) and the concentration of anthrax lethal factor DNA in the range of 0.029–0.733 nmol L −1 with a detection limit of 0.013 nmol L −1 . The proposed method has been successfully applied to the determination of anthrax lethal factor DNA sequence in human serum samples with satisfactory results. Because of low toxicity and fine biocompatibility, DNA-CuInS 2 QDs also hold potential applications in bioimaging. - Highlights: • Strongly fluorescent DNA-QDs were successfully prepared by a one-pot hydrothermal method with quantum yield up to 23.4%. • A biosensor for label-free detection of anthrax lethal factor DNA was established based on the as-prepared DNA-QDs. • The DNA sensor took advantage of the feature that ssDNA binds to GO with significantly higher affinity than dsDNA. • Good sensitivity and selectivity were obtained. • This method was utilized to detect

  15. One-pot synthesis of strongly fluorescent DNA-CuInS{sub 2} quantum dots for label-free and ultrasensitive detection of anthrax lethal factor DNA

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Ziping; Su, Xingguang, E-mail: suxg@jlu.edu.cn

    2016-10-26

    Herein, high quality DNA-CuInS{sub 2} QDs are facilely synthesized through a one-pot hydrothermal method with fluorescence quantum yield as high as 23.4%, and the strongly fluorescent DNA-CuInS{sub 2} QDs have been utilized as a novel fluorescent biosensor for label-free and ultrasensitive detection of anthrax lethal factor DNA. L-Cysteine (L-Cys) and a specific-sequence DNA are used as co-ligands to stabilize the CuInS{sub 2} QDs. The specific-sequence DNA consists of two domains: phosphorothiolates domain (sulfur-containing variants of the usual phosphodiester backbone) controls the nanocrystal passivation and serves as a ligand, and the functional domain (non-phosphorothioates) controls the biorecognition. The as-prepared DNA-CuInS{sub 2} QDs have high stability, good water-solubility and low toxicity. Under the optimized conditions, a linear correlation was established between the fluorescence intensity ratio I/I{sub 0} (I{sub 0} is the original fluorescence intensity of DNA-CuInS{sub 2} QDs, and I is the fluorescence intensity of DNA-CuInS{sub 2} QDs/GO with the addition of various concentrations of anthrax lethal factor DNA) and the concentration of anthrax lethal factor DNA in the range of 0.029–0.733 nmol L{sup −1} with a detection limit of 0.013 nmol L{sup −1}. The proposed method has been successfully applied to the determination of anthrax lethal factor DNA sequence in human serum samples with satisfactory results. Because of low toxicity and fine biocompatibility, DNA-CuInS{sub 2} QDs also hold potential applications in bioimaging. - Highlights: • Strongly fluorescent DNA-QDs were successfully prepared by a one-pot hydrothermal method with quantum yield up to 23.4%. • A biosensor for label-free detection of anthrax lethal factor DNA was established based on the as-prepared DNA-QDs. • The DNA sensor took advantage of the feature that ssDNA binds to GO with significantly higher affinity than dsDNA. • Good sensitivity and selectivity were

  16. A Label-Free and Sensitive Fluorescent Qualitative Assay for Bisphenol A Based on Rolling Circle Amplification/Exonuclease III-Combined Cascade Amplification.

    Science.gov (United States)

    Li, Xia; Song, Juan; Xue, Qing-Wang; You, Fu-Heng; Lu, Xia; Kong, Yan-Cong; Ma, Shu-Yi; Jiang, Wei; Li, Chen-Zhong

    2016-10-21

    Bisphenol A (BPA) detection in drinking water and food packaging materials has attracted much attention since the discovery that BPA can interfere with normal physiological processes and cause adverse health effects. Here, we constructed a label-free aptamer fluorescent assay for selective and sensitive detection of BPA based on the rolling circle amplification (RCA)/Exonuclease III (Exo III)-combined cascade amplification strategy. First, the duplex DNA probe (RP) with anti-BPA aptamer and trigger sequence was designed for BPA recognition and signal amplification. Next, under the action of BPA, the trigger probe was liberated from RP to initiate RCA reaction as primary amplification. Subsequently, the RCA products were used to trigger Exo III assisted secondary amplification with the help of hairpin probes, producing plenty of "G-quadruplex" in lantern-like structures. Finally, the continuously enriched "G-quadruplex lanterns" were lightened by zinc(II)-protoporphyrin IX (ZnPPIX) generating enhanced fluorescence signals. By integrating the primary RCA and secondary Exo III mediated cascade amplification strategy, this method displayed an excellent sensitivity with the detection limits of 5.4 × 10 -17 M. In addition, the anti-BPA aptamer exhibits high recognition ability with BPA, guaranteeing the specificity of detection. The reporter signal probe (G-quadruplex with ZnPPIX) provides a label-free fluorescence signals readout without complicated labeling procedures, making the method simple in design and cost-effective in operation. Moreover, environmental samples analysis was also performed, suggesting that our strategy was reliable and had a great potential application in environmental monitoring.

  17. A Label-Free and Sensitive Fluorescent Qualitative Assay for Bisphenol A Based on Rolling Circle Amplification/Exonuclease III-Combined Cascade Amplification

    Directory of Open Access Journals (Sweden)

    Xia Li

    2016-10-01

    Full Text Available Bisphenol A (BPA detection in drinking water and food packaging materials has attracted much attention since the discovery that BPA can interfere with normal physiological processes and cause adverse health effects. Here, we constructed a label-free aptamer fluorescent assay for selective and sensitive detection of BPA based on the rolling circle amplification (RCA/Exonuclease III (Exo III-combined cascade amplification strategy. First, the duplex DNA probe (RP with anti-BPA aptamer and trigger sequence was designed for BPA recognition and signal amplification. Next, under the action of BPA, the trigger probe was liberated from RP to initiate RCA reaction as primary amplification. Subsequently, the RCA products were used to trigger Exo III assisted secondary amplification with the help of hairpin probes, producing plenty of “G-quadruplex” in lantern-like structures. Finally, the continuously enriched “G-quadruplex lanterns” were lightened by zinc(II-protoporphyrin IX (ZnPPIX generating enhanced fluorescence signals. By integrating the primary RCA and secondary Exo III mediated cascade amplification strategy, this method displayed an excellent sensitivity with the detection limits of 5.4 × 10−17 M. In addition, the anti-BPA aptamer exhibits high recognition ability with BPA, guaranteeing the specificity of detection. The reporter signal probe (G-quadruplex with ZnPPIX provides a label-free fluorescence signals readout without complicated labeling procedures, making the method simple in design and cost-effective in operation. Moreover, environmental samples analysis was also performed, suggesting that our strategy was reliable and had a great potential application in environmental monitoring.

  18. Biomimetic synthesis of needle-like fluorescent calcium phosphate/carbon dot hybrid composites for cell labeling and copper ion detection.

    Science.gov (United States)

    Guo, Shanshan; Lu, Shousi; Xu, Pingxiang; Ma, Yi; Zhao, Liang; Zhao, Yuming; Gu, Wei; Xue, Ming

    2016-05-04

    Herein, we report a biomimetic method to synthesize needle-like calcium phosphate (CaP) with dimensions of ∼130 nm length and ∼30 nm width using carbon dots (CDs) and sodium carboxymethylcellulose as dual templates. In addition to acting as the template, the CDs enable the CaP/CDs hybrid composites to emit blue fluorescence under UV excitation. Moreover, the prepared CaP/CDs exhibited a negligible cytotoxicity towards HeLa cells. The potential of these CaP/CDs as a fluorescent probe for cell labeling was tested. In addition, it was demonstrated that the CaP/CDs were capable of selective detection of copper ions in drinking water.

  19. Effect of humic acid (HA) on sulfonamide sorption by biochars

    International Nuclear Information System (INIS)

    Lian, Fei; Sun, Binbin; Chen, Xi; Zhu, Lingyan; Liu, Zhongqi; Xing, Baoshan

    2015-01-01

    Effect of quantity and fractionation of loaded humic acid (HA) on biochar sorption for sulfonamides was investigated. The HA was applied in two different modes, i.e. pre-coating and co-introduction with sorbate. In pre-coating mode, the polar fractions of HA tended to interact with low-temperature biochars via H-bonding, while the hydrophobic fractions were likely to be adsorbed by high-temperature biochars through hydrophobic and π-π interactions, leading to different composition and structure of the HA adlayers. The influences of HA fractionation on biochar sorption for sulfonamides varied significantly, depending on the nature of interaction between HA fraction and sorbate. Meanwhile, co-introduction of HA with sulfonamides revealed that the effect of HA on sulfonamide sorption was also dependent on HA concentration. These findings suggest that the amount and fractionation of adsorbed HA are tailored by the surface properties of underlying biochars, which differently affect the sorption for organic contaminants. - Highlights: • Effect of quantity and fractionation of coated HA on sorption of sulfonamides by BC was studied. • Fractionation of coated HA is tailored by surface properties of BC. • Roles of HA in BC sorption depend on interaction between HA adlayer and sorbate. • Roles of HA in sulfonamide sorption by BC also depend on HA aqueous concentration. - The quantity and fractionation of adsorbed HA play a major role in sulfonamide sorption by biochars

  20. Transport of sulfonamide antibiotics in crop fields during monsoon season.

    Science.gov (United States)

    Park, Jong Yol; Ruidisch, Marianne; Huwe, Bernd

    2016-11-01

    Previous studies have documented the occurrence of veterinary sulfonamide antibiotics in groundwater and rivers located far from pollution sources, although their transport and fate is relatively unknown. In mountainous agricultural fields, the transport behaviour can be influenced by climate, slope and physico-chemical properties of the sulfonamides. The objective of this research is to describe the transport behaviour of three sulfonamide antibiotics (sulfamethoxazole, sulfadimethoxine and sulfamethazine) in sloped agricultural fields located in the Haean catchment, South Korea. During dry and monsoon seasons, a solute transport experiment was conducted in two typical sandy loam agricultural fields after application of antibiotics and potassium bromide as conservative tracers. Field measurement and modelling revealed that frequency and amount of runoff generation indicate a relation between slope and rain intensity during monsoon season. Since the steepness of slope influenced partitioning of precipitation between runoff and subsurface flow, higher loss of sulfonamide antibiotics and bromide by runoff was observed at the steeper sloped field. Bromide on topsoil rapidly infiltrated at high infiltration rates. On the contrary, the sulfonamides were relatively retarded in the upper soil layer due to adsorption onto soil particles. Presence of furrows and ridges affected the distribution of sulfonamide antibiotics in the subsurface due to gradient from wetter furrows to drier ridges induced by topography. Modelling results with HydroGeoSphere matched with background studies that describe physico-chemical properties of the sulfonamides interaction between soil and the antibiotic group, solute transport through vadose zone and runoff generation by storm events.

  1. A review of reagents for fluorescence microscopy of cellular compartments and structures, part I: BacMam labeling and reagents for vesicular structures.

    Science.gov (United States)

    Dolman, Nick J; Kilgore, Jason A; Davidson, Michael W

    2013-07-01

    Fluorescent labeling of vesicular structures in cultured cells, particularly for live cells, can be challenging for a number of reasons. The first challenge is to identify a reagent that will be specific enough where some structures have a number of potential reagents and others very few options. The emergence of BacMam constructs has allowed more easy-to-use choices. Presented here is a discussion of BacMam constructs as well as a review of commercially-available reagents for labeling vesicular structures in cells, including endosomes, peroxisomes, lysosomes, and autophagosomes, complete with a featured reagent for each structure, recommended protocol, troubleshooting guide, and example image. © 2013 by John Wiley & Sons, Inc.

  2. Iatrogenic, sulfonamide-induced hypothyroid crisis in a Labrador Retriever.

    Science.gov (United States)

    Brenner, K; Harkin, K; Schermerhorn, T

    2009-12-01

    This case report describes a sulfonamide-induced hypothyroid crisis in a 4-year-old Labrador Retriever bitch. Empirical therapy with high-dose trimethoprim-sulfamethoxazole for 10 days produced signs of weakness, ataxia and mental depression and the clinicopathological results supported hypothyroid-induced central nervous system depression. Short-term levothyroxine sodium therapy led to complete resolution of all clinical signs and follow-up thyroid hormone assays ruled out underlying thyroid pathology. This case report is the first to highlight this potentially life-threatening manifestation of sulfonamide-induced hypothyroidism. Sulfonamide combinations are widely used antimicrobials in veterinary medicine and early recognition of this syndrome is critical.

  3. Sulfonamidation of Aryl and Heteroaryl Halides through Photosensitized Nickel Catalysis.

    Science.gov (United States)

    Kim, Taehoon; McCarver, Stefan J; Lee, Chulbom; MacMillan, David W C

    2018-03-19

    Herein we report a highly efficient method for nickel-catalyzed C-N bond formation between sulfonamides and aryl electrophiles. This technology provides generic access to a broad range of N-aryl and N-heteroaryl sulfonamide motifs, which are widely represented in drug discovery. Initial mechanistic studies suggest an energy-transfer mechanism wherein C-N bond reductive elimination occurs from a triplet excited Ni II complex. Late-stage sulfonamidation in the synthesis of a pharmacologically relevant structure is also demonstrated. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. Surface sulfonamide modification of poly(N-isopropylacrylamide)-based block copolymer micelles to alter pH and temperature responsive properties for controlled intracellular uptake.

    Science.gov (United States)

    Cyphert, Erika L; von Recum, Horst A; Yamato, Masayuki; Nakayama, Masamichi

    2018-06-01

    Two different surface sulfonamide-functionalized poly(N-isopropylacrylamide)-based polymeric micelles were designed as pH-/temperature-responsive vehicles. Both sulfadimethoxine- and sulfamethazine-surface functionalized micelles were characterized to determine physicochemical properties, hydrodynamic diameters, zeta potentials, temperature-dependent size changes, and lower critical solution temperatures (LCST) in both pH 7.4 and 6.8 solutions (simulating both physiological and mild low pH conditions), and tested in the incorporation of a proof-of-concept hydrophobic antiproliferative drug, paclitaxel. Cellular uptake studies were conducted using bovine carotid endothelial cells and fluorescently labeled micelles to evaluate if there was enhanced cellular uptake of the micelles in a low pH environment. Both variations of micelles showed enhanced intracellular uptake under mildly acidic (pH 6.8) conditions at temperatures slightly above their LCST and minimal uptake at physiological (pH 7.4) conditions. Due to the less negative zeta potential of the sulfamethazine-surface micelles compared to sulfadimethoxine-surface micelles, and the proximity of their LCST to physiological temperature (37°C), the sulfamethazine variation was deemed more amenable for clinically relevant temperature and pH-stimulated applications. Nevertheless, we believe both polymeric micelle variations have the capacity to be implemented as an intracellular drug or gene delivery system in response to mildly acidic conditions. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 1552-1560, 2018. © 2018 Wiley Periodicals, Inc.

  5. Soil bacterial consortia and previous exposure enhance the biodegradation of sulfonamides from pig manure.

    Science.gov (United States)

    Islas-Espinoza, Marina; Reid, Brian J; Wexler, Margaret; Bond, Philip L

    2012-07-01

    Persistence or degradation of synthetic antibiotics in soil is crucial in assessing their environmental risks. Microbial catabolic activity in a sandy loamy soil with pig manure using 12C- and 14C-labelled sulfamethazine (SMZ) respirometry showed that SMZ was not readily degradable. But after 100 days, degradation in sulfadiazine-exposed manure was 9.2%, far greater than soil and organic manure (0.5% and 0.11%, respectively, p library from the treatment with highest degradation showed that most bacteria belonged to α, β and γ classes of Proteobacteria, Firmicutes, Bacteroidetes and Acidobacteria. Proteobacteria (α, β and γ), Firmicutes and Bacteroidetes which were the most abundant classes on day 1 also decreased most following prolonged exposure. From the matrix showing the highest degradation rate, 17 SMZ-resistant isolates biodegraded low levels of 14C-labelled SMZ when each species was incubated separately (0.2-1.5%) but biodegradation was enhanced when the four isolates with the highest biodegradation were incubated in a consortium (Bacillus licheniformis, Pseudomonas putida, Alcaligenes sp. and Aquamicrobium defluvium as per 16S rRNA gene sequencing), removing up to 7.8% of SMZ after 20 days. One of these species (B. licheniformis) was a known livestock and occasional human pathogen. Despite an environmental role of these species in sulfonamide bioremediation, the possibility of horizontal transfer of pathogenicity and resistance genes should caution against an indiscriminate use of these species as sulfonamide degraders.

  6. Label-free and enzyme-free detection of transcription factors with graphene oxide fluorescence switch-based multifunctional G-quadruplex-hairpin probe.

    Science.gov (United States)

    Zhu, Desong; Wang, Lei; Xu, Xiaowen; Jiang, Wei

    2016-01-15

    Transcription factors (TFs) play pivotal roles in the regulation of a variety of essential cellular processes and some of them have been recognized as potential diagnostic markers and therapeutic targets of some diseases. Sensitive and accurate detection of TFs is of great importance to better understanding their roles in gene regulation and evaluation of disease state. Here, we developed a simple, label-free and enzyme-free new fluorescent strategy for the detection of TFs by graphene oxide (GO) fluorescence switch-based multifunctional G-quadruplex-hairpin probe (MGHP). The MGHP possessed of three functions simultaneously, adsorbing onto GO with the loop part, binding to target with the stem part and serving as signal carrier with the terminal G-quadruplex. First, the MGHP was adsorbed quickly to GO. Next, the TF bound to the stem part of MGHP to form a huge target-MGHP complex, which led to desorption of the complex from GO. Finally, NMM was inserted into G-quadruplex in the complex to yield an enhanced fluorescence response. The GO used here, as a fluorescence switch, could quickly and efficiently quench the fluorescence of NMM inserted into the MGHP absorbed on the GO, guaranteeing a high signal-to-noise ratio. Sensitive detection of purified NF-κB p50 and HeLa cell nuclear extracts were achieved with detection limits of 0.2nM and 7.8ng/µL, respectively. Moreover, this proposed strategy could be used to screen inhibitors of NF-κB p50 activity. The strategy proposed here might offer a new potential approach for reliable quantification of TFs in clinical diagnostics and treatment research of some diseases. Copyright © 2015 Elsevier B.V. All rights reserved.

  7. A label-free fluorescence biosensor for highly sensitive detection of lectin based on carboxymethyl chitosan-quantum dots and gold nanoparticles.

    Science.gov (United States)

    Liu, Ziping; Liu, Hua; Wang, Lei; Su, Xingguang

    2016-08-17

    In this work, we report a novel label-free fluorescence "turn off-on" biosensor for lectin detection. The highly sensitive and selective sensing system is based on the integration of carboxymethyl chitosan (CM-CHIT), CuInS2 quantum dots (QDs) and Au nanoparticles (NPs). Firstly, CuInS2 QDs featuring carboxyl groups were directly synthesized via a hydrothermal synthesis method. Then, the carboxyl groups on the CuInS2 QDs surface were interacted with the amino groups (NH2), carboxyl groups (COOH) and hydroxyl groups (OH) within CM-CHIT polymeric chains via electrostatic interactions and hydrogen bonding to form CM-CHIT-QDs assemblies. Introduction of Au NPs could quench the fluorescence of CM-CHIT-QDs through electron and energy transfer. In the presence of lectin, lectin could bind exclusively with CM-CHIT-QDs by means of specific multivalent carbohydrate-protein interaction. Thus, the electron and energy transfer process between CM-CHIT-QDs and Au NPs was inhibited, and as a result, the fluorescence of CM-CHIT-QDs was effectively "turned on". Under the optimum conditions, there was a good linear relationship between the fluorescence intensity ratio I/I0 (I and I0 were the fluorescence intensity of CM-CHIT-QDs-Au NPs in the presence and absence of lectin, respectively) and lectin concentration in the range of 0.2-192.5 nmol L(-1), And the detection limit could be down to 0.08 nmol L(-1). Furthermore, the proposed biosensor was employed for the determination of lectin in fetal bovine serum samples with satisfactory results. Copyright © 2016 Elsevier B.V. All rights reserved.

  8. Fluorescent nanodiamond and lanthanide labelled in situ hybridization for the identification of RNA transcripts in fixed and CLARITY-cleared central nervous system tissues (Conference Presentation)

    Science.gov (United States)

    Parker, Lindsay M.; Staikopoulos, Vicky; Cordina, Nicole M.; Sayyadi, Nima; Hutchinson, Mark R.; Packer, Nicolle H.

    2016-03-01

    Despite significant advancement in the methodology used to conjugate, incorporate and visualize fluorescent molecules at the cellular and tissue levels, biomedical imaging predominantly relies on the limitations of established fluorescent molecules such as fluorescein, cyanine and AlexaFluor dyes or genetic incorporation of fluorescent proteins by viral or other means. These fluorescent dyes and conjugates are highly susceptible to photobleaching and compete with cellular autofluorescence, making biomedical imaging unreliable, difficult and time consuming in many cases. In addition, some proteins have low copy numbers and/or poor antibody recognition, further making detection and imaging difficult. We are developing better methods for imaging central nervous system neuroinflammatory markers using targeted mRNA transcripts labelled with fluorescent nanodiamonds or lanthanide chelates. These tags have increased signal and photostability and can also discriminate against tissue/cell autofluorescence. Brains and spinal cords from BALB/c mice with a chronic constriction model of neuropathic pain (neuroinflammation group) or that have undergone sham surgeries (control group) were collected. A subset of brains and spinal cords were perfused and fixed with paraformaldehyde (n=3 sham and n=3 pain groups) prior to sectioning and in situ hybridization using nanodiamond or lanthanide chelate conjugated complementary RNA probes. Another subset of brains and spinal cords from the same cohort of animals were perfused and processed for CLARITY hydrogel based clearing prior to in situ hybridization with the same probes. We will present our findings on the photostability, sensitivity and discrimination from background tissue autofluorescence of our novel RNA probes, compared to traditional fluorophore tags.

  9. Cytotoxicity Test Based on Human Cells Labeled with Fluorescent Proteins: Fluorimetry, Photography, and Scanning for High-Throughput Assay.

    Science.gov (United States)

    Kalinina, Marina A; Skvortsov, Dmitry A; Rubtsova, Maria P; Komarova, Ekaterina S; Dontsova, Olga A

    2018-06-01

    High- and medium-throughput assays are now routine methods for drug screening and toxicology investigations on mammalian cells. However, a simple and cost-effective analysis of cytotoxicity that can be carried out with commonly used laboratory equipment is still required. The developed cytotoxicity assays are based on human cell lines stably expressing eGFP, tdTomato, mCherry, or Katushka2S fluorescent proteins. Red fluorescent proteins exhibit a higher signal-to-noise ratio, due to less interference by medium autofluorescence, in comparison to green fluorescent protein. Measurements have been performed on a fluorescence scanner, a plate fluorimeter, and a camera photodocumentation system. For a 96-well plate assay, the sensitivity per well and the measurement duration were 250 cells and 15 min for the scanner, 500 cells and 2 min for the plate fluorimeter, and 1000 cells and less than 1 min for the camera detection. These sensitivities are similar to commonly used MTT (tetrazolium dye) assays. The used scanner and the camera had not been previously applied for cytotoxicity evaluation. An image processing scheme for the high-resolution scanner is proposed that significantly diminishes the number of control wells, even for a library containing fluorescent substances. The suggested cytotoxicity assay has been verified by measurements of the cytotoxicity of several well-known cytotoxic drugs and further applied to test a set of novel bacteriotoxic compounds in a medium-throughput format. The fluorescent signal of living cells is detected without disturbing them and adding any reagents, thus allowing to investigate time-dependent cytotoxicity effects on the same sample of cells. A fast, simple and cost-effective assay is suggested for cytotoxicity evaluation based on mammalian cells expressing fluorescent proteins and commonly used laboratory equipment.

  10. Fluorescent carbon dots nanosensor for label-free determination of vitamin B12 based on inner filter effect

    Science.gov (United States)

    Ding, Longhua; Yang, Hongmei; Ge, Shenguang; Yu, Jinghua

    2018-03-01

    A simple and effective fluorescent assay for the determination of vitamin B12 was developed. In this study, carbon dots (CDs) were prepared by one-pot hydrothermal method and directly used as a fluorophore in the inner filter effect (IFE). Both of the maximum absorption peak of vitamin B12 and excitation maxima of CDs are located at 360 nm, hence, the excited light of CDs can be absorbed by vitamin B12, resulting in the fluorescence reduction of CDs. And the fluorescence intensity of CDs decreases with the increasing concentration of vitamin B12. This IFE-based sensing strategy shows a good linear relationship between the normalized fluorescence intensity and the concentration of vitamin B12 ranging from 0 to 60 μM, with a limit of detection (LOD) of 0.1 μM at a signal-to-noise ratio of 3. Furthermore, this proposed approach was successfully applied to vitamin B12 sensing in injections. This IFE sensing platform based on various fluorescent nanomaterials has a high promise for the detection of other biomolecules due to its inherent convenience.

  11. Label-free imaging of brain and brain tumor specimens with combined two-photon excited fluorescence and second harmonic generation microscopy

    Science.gov (United States)

    Jiang, Liwei; Wang, Xingfu; Wu, Zanyi; Du, Huiping; Wang, Shu; Li, Lianhuang; Fang, Na; Lin, Peihua; Chen, Jianxin; Kang, Dezhi; Zhuo, Shuangmu

    2017-10-01

    Label-free imaging techniques are gaining acceptance within the medical imaging field, including brain imaging, because they have the potential to be applied to intraoperative in situ identifications of pathological conditions. In this paper, we describe the use of two-photon excited fluorescence (TPEF) and second harmonic generation (SHG) microscopy in combination for the label-free detection of brain and brain tumor specimens; gliomas. Two independently detecting channels were chosen to subsequently collect TPEF/SHG signals from the specimen to increase TPEF/SHG image contrasts. Our results indicate that the combined TPEF/SHG microscopic techniques can provide similar rat brain structural information and produce a similar resolution like conventional H&E staining in neuropathology; including meninges, cerebral cortex, white-matter structure corpus callosum, choroid plexus, hippocampus, striatum, and cerebellar cortex. It can simultaneously detect infiltrating human brain tumor cells, the extracellular matrix collagen fiber of connective stroma within brain vessels and collagen depostion in tumor microenvironments. The nuclear-to-cytoplasmic ratio and collagen content can be extracted as quantitative indicators for differentiating brain gliomas from healthy brain tissues. With the development of two-photon fiberscopes and microendoscope probes and their clinical applications, the combined TPEF and SHG microcopy may become an important multimodal, nonlinear optical imaging approach for real-time intraoperative histological diagnostics of residual brain tumors. These occur in various brain regions during ongoing surgeries through the method of simultaneously identifying tumor cells, and the change of tumor microenvironments, without the need for the removal biopsies and without the need for tissue labelling or fluorescent markers.

  12. Uptake pathways of fluorescent indicators by pea seed and seedlings and their potential as anti-counterfeiting labeling for plant seeds

    International Nuclear Information System (INIS)

    Lin, C.; Guan, Y. J.; Fu, H.; Hu, J.; Tian, Y. X.

    2015-01-01

    The present study investigates the effects of seed soaking in varying concentration of rhodamine B (RB) or safranine T (ST) solutions on germination and seedling growth of pea seeds. The fluorescence in pea seedling at different developmental stages was observed. The results indicate that there were no adverse effects of seed soaking in RB (0.1mg/ml) and ST (0.5, 0.3, 0.1mg/ml) solutions on germination, seedling growth, antioxidant enzyme activities, malondialdehyde (MDA) and chlorophyll contents. The seeds treated with RB showed bright red and orange fluorescence under green (546 nm) and blue (495 nm) light excitation, respectively while no red or orange color was observed in the control seeds. In addition, the vascular bundles of stem, seedling roots and aerial parts of seedlings treated with RB all emitted brilliant fluorescence for a longer time as compared with that treated with ST. It can be concluded that pea seed labeled with RB by seed soaking at appropriate concentration could be used as a potential anti-counterfeiting technique in pea seeds. (author)

  13. Multimodal Imaging of Integrin Receptor-Positive Tumors by Bioluminescence, Fluorescence, Gamma Scintigraphy, and Single-Photon Emission Computed Tomography Using a Cyclic RGD Peptide Labeled with a Near-Infrared Fluorescent Dye and a Radionuclide

    Directory of Open Access Journals (Sweden)

    W. Barry Edwards

    2009-03-01

    Full Text Available Integrins, particularly the αvβ3 heterodimers, play important roles in tumor-induced angiogenesis and invasiveness. To image the expression pattern of the αvβ3 integrin in tumors through a multimodality imaging paradigm, we prepared a cyclic RGDyK peptide analogue (LS308 bearing a tetraazamacrocycle 1,4,7,10-tetraazacyclododecane-N, N′, N″, N‴-tetraacetic acid (DOTA and a lipophilic near-infrared (NIR fluorescent dye cypate. The αvβ3 integrin binding affinity and the internalization properties of LS308 mediated by the αvβ3 integrin in 4t1luc cells were investigated by receptor binding assay and fluorescence microscopy, respectively. The in vivo distribution of 111In-labeled LS308 in a 4t1luc tumor-bearing mouse model was studied by fluorescence, bioluminescence, planar gamma, and single-photon emission computed tomography (SPECT. The results show that LS308 has high affinity for αvβ3 integrin and internalized preferentially via the αvβ3 integrin-mediated endocytosis in 4t1luc cells. We also found that LS308 selectively accumulated in αvβ3-positve tumors in a receptor-specific manner and was visualized by the four imaging methods. Whereas the endogenous bioluminescence imaging identified the ensemble of the tumor tissue, the fluorescence and SPECT methods with the exogenous contrast agent LS308 reported the local expression of αvβ3 integrin. Thus, the multimodal imaging approach could provide important complementary diagnostic information for monitoring the efficacy of new antiangiogenic drugs.

  14. Fluorescence of nitrobenzoxadiazole (NBD) labeled lipids in model membranes is connected not to lipid mobility, but to probe location

    Czech Academy of Sciences Publication Activity Database

    Amaro, Mariana; Filipe, H. A.; Ramalho, J. P. P.; Hof, Martin; Loura, L. M. S.

    2016-01-01

    Roč. 18, č. 10 (2016), s. 7042-7054 ISSN 1463-9076 R&D Projects: GA ČR GBP208/12/G016 Institutional support: RVO:61388955 Keywords : fluorescence * nitrobenzoxadiazole Subject RIV: CF - Physical ; Theoretical Chemistry Impact factor: 4.123, year: 2016

  15. A quencher-free molecular beacon design based on pyrene excimer fluorescence using pyrene-labeled UNA (unlocked nucleic acid)

    DEFF Research Database (Denmark)

    Karlsen, Kasper Kannegård; Okholm, Anders Hauge; Kjems, Jørgen

    2013-01-01

    A quencher-free molecular beacon capable of generating pyrene excimer fluorescence has been constructed using strategically positioned pyrene-UNA monomers. Hybridization of a fully complementary RNA target was accompanied by a pyrene excimer emission increase of more than 900%, and detection of RNA...

  16. Comparative analysis of monoclonal antibody N-glycosylation using stable isotope labelling and UPLC-fluorescence-MS.

    Science.gov (United States)

    Millán Martín, Silvia; Delporte, Cédric; Farrell, Amy; Navas Iglesias, Natalia; McLoughlin, Niaobh; Bones, Jonathan

    2015-03-07

    A twoplex method using (12)C6 and (13)C6 stable isotope analogues (Δmass = 6 Da) of 2-aminobenzoic acid (2-AA) is described for quantitative analysis of N-glycans present on monoclonal antibodies and other glycoproteins using ultra performance liquid chromatography with sequential fluorescence and accurate mass tandem quadrupole time of flight (QToF) mass spectrometric detection.

  17. A novel graphene-based label-free fluorescence 'turn-on' nanosensor for selective and sensitive detection of phosphorylated species in biological samples and living cells.

    Science.gov (United States)

    Ke, Yaotang; Garg, Bhaskar; Ling, Yong-Chien

    2016-02-28

    A novel label-free fluorescence 'turn-on' nanosensor has been developed for highly selective and sensitive detection of phosphorylated species (Ps) in biological samples and living cells. The design strategy relies on the use of Ti(4+)-immobilized polydopamine (PDA) coated reduced graphene oxide (rGO@PDA-Ti(4+)) that serves as an attractive platform to bind riboflavin 5'-monophosphate molecules (FMNs) through ion-pair interactions between phosphate groups and Ti(4+). The as-prepared rGO@PDA-Ti(4+)-FMNs (nanosensor), fluoresce only weakly due to the ineffective Förster resonance energy transfer between the FMNs and rGO@PDA-Ti(4+). The experimental findings revealed that the microwave-assisted interaction of the nanosensor with α-, β-casein, ovalbumin, human serum, non-fat milk, egg white, and living cells (all containing Ps) releases FMNs (due to the high formation constant between phosphate groups and Ti(4+)), leading to an excellent fluorescence 'turn-on' response. The fluorescence spectroscopy, confocal microscopy, and MALDI-TOF MS spectrometry were used to detect Ps both qualitatively and quantitatively. Under the optimized conditions, the nanosensor showed a detection limit of ca. 118.5, 28.9, and 54.8 nM for the tryptic digests of α-, β-casein and ovalbumin, respectively. Furthermore, the standard addition method was used as a bench-mark proof for phosphopeptide quantification in egg white samples. We postulate that the present quantitative assay for Ps holds tremendous potential and may pave the way to disease diagnostics in the near future.

  18. Europium-decorated graphene quantum dots as a fluorescent probe for label-free, rapid and sensitive detection of Cu(2+) and L-cysteine.

    Science.gov (United States)

    Lin, Liping; Song, Xinhong; Chen, Yiying; Rong, Mingcong; Wang, Yiru; Zhao, Li; Zhao, Tingting; Chen, Xi

    2015-09-03

    In this work, europium-decorated graphene quantum dots (Eu-GQDs) were prepared by treating three-dimensional Eu-decorated graphene (3D Eu-graphene) via a strong acid treatment. Various characterizations revealed that Eu atoms were successfully complexed with the oxygen functional groups on the surface of graphene quantum dots (GQDs) with the atomic ratio of 2.54%. Compared with Eu free GQDs, the introduction of Eu atoms enhanced the electron density and improved the surface chemical activities of Eu-GQDs. Therefore, the obtained Eu-GQDs were used as a novel "off-on" fluorescent probe for the label-free determination of Cu(2+) and l-cysteine (L-Cys) with high sensitivity and selectivity. The fluorescence intensity of Eu-GQDs was quenched in the presence of Cu(2+) owing to the coordination reaction between Cu(2+) and carboxyl groups on the surface of the Eu-GQDs. The fluorescence intensity of Eu-GQDs recovered with the subsequent addition of L-Cys because of the strong affinity of Cu(2+) to L-Cys via the Cu-S bond. The experimental results showed that the fluorescence variation of the proposed approach had a good linear relationship in the range of 0.1-10 μM for Cu(2+) and 0.5-50 μM for L-Cys with corresponding detection limits of 0.056 μM for Cu(2+) and 0.31 μM for L-Cys. The current approach also displayed a special response to Cu(2+) and L-Cys over the other co-existing metal ions and amino acids, and the results obtained from buffer-diluted serum samples suggested its applicability in biological samples. Copyright © 2015 Elsevier B.V. All rights reserved.

  19. Synthesis and evaluation of [{sup 14}C]-Labelled and fluorescent-Tagged paclitaxel derivatives as new biological probes

    Energy Technology Data Exchange (ETDEWEB)

    Rao, C.S.; Chu, J.-J.; Lai, Y.-K. [Department of Life Science, National Tsing Hua University, Hsinchu, Taiwan (China); Liu, R.-S. [Department of Chemistry, National Tsing Hua University, Hsinchu, Taiwan (China)

    1998-11-01

    Our present report deals with the preparation of hitherto unreported 7-([carbonyl-{sup 14}C]-acetyl)paclitaxel 4 and two new bioactive 7-substituted fluorescent taxoids (FITC 9 and rhodamine 11), as well as evaluation towards their applications as biological probes. The results in this report demonstrate that (a) the new paclitaxel derivatives 4, 9, 11 could be prepared with good yields starting from paclitaxel; (b) the [{sup 14}C]acetylation step was found to be better by using [{sup 14}C]acetic anhydride rather than [{sup 14}C]sodium acetate; (c) the radiochemical purity of 4 was 96% and its specific activity was 48 mCi/mmol; (d) the cytotoxicity of 4 was close to that of paclitaxel whereas 9, 11 were far less active than paclitaxel, but these cytotoxic levels were good enough for their biological applications; (e) the drug-quantitation by flow cytometric analysis using 9 and 11 was proved to be equally efficient with respect to the radioactivity-based determination employing 4; (f) the intracellular fluorescence mapping by 9 and 11 was found to be effective and the microtubule network pattern was visible in both the cases; (g) the overall fluorescence imaging efficiency was better with 11 while the intensity of fluorescence was higher with 9; (h) staining of nucleolus was observed in fluorescence studies of both 9 and 11. Based on these results, the newly prepared paclitaxel derivatives can be considered as efficient biological probes and should find further use in relevant applications. (Copyright (c) 1998 Elsevier Science B.V., Amsterdam. All rights reserved.)

  20. Construction of a viral T2A-peptide based knock-in mouse model for enhanced Cre recombinase activity and fluorescent labeling of podocytes.

    Science.gov (United States)

    Koehler, Sybille; Brähler, Sebastian; Braun, Fabian; Hagmann, Henning; Rinschen, Markus M; Späth, Martin R; Höhne, Martin; Wunderlich, F Thomas; Schermer, Bernhard; Benzing, Thomas; Brinkkoetter, Paul T

    2017-06-01

    Podocyte injury is a key event in glomerular disease leading to proteinuria and opening the path toward glomerular scarring. As a consequence, glomerular research strives to discover molecular mechanisms and signaling pathways affecting podocyte health. The hNphs2.Cre mouse model has been a valuable tool to manipulate podocyte-specific genes and to label podocytes for lineage tracing and purification. Here we designed a novel podocyte-specific tricistronic Cre mouse model combining codon improved Cre expression and fluorescent cell labeling with mTomato under the control of the endogenous Nphs2 promoter using viral T2A-peptides. Independent expression of endogenous podocin, codon improved Cre, and mTomato was confirmed by immunofluorescence, fluorescent activated cell sorting and protein analyses. Nphs2 pod.T2A.ciCre.T2A.mTomato/wild-type mice developed normally and did not show any signs of glomerular disease or off-target effects under basal conditions and in states of disease. Nphs2 pod.T2A.ciCre.T2A.mTomato/wild-type -mediated gene recombination was superior to conventional hNphs2.Cre mice-mediated gene recombination. Last, we compared Cre efficiency in a disease model by mating Nphs2 pod.T2A.ciCre.T2A.mTomato/wild-type and hNphs2.Cre mice to Phb2 fl/fl mice. The podocyte-specific Phb2 knockout by Nphs2 pod.T2A.ciCre.T2A.mTomato/wild-type mice resulted in an aggravated glomerular injury as compared to a podocyte-specific Phb2 gene deletion triggered by hNphs2.Cre. Thus, we generated the first tricistronic podocyte mouse model combining enhanced Cre recombinase efficiency and fluorescent labeling in podocytes without the need for additional matings with conventional reporter mouse lines. Copyright © 2016 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.

  1. A blue fluorescent labeling technique utilizing micro- and nanoparticles for tracking in LIVE/DEAD® stained pathogenic biofilms of Staphylococcus aureus and Burkholderia cepacia

    Directory of Open Access Journals (Sweden)

    Klinger-Strobel M

    2016-02-01

    Full Text Available Mareike Klinger-Strobel,1,2,* Julia Ernst,3,* Christian Lautenschläger,4 Mathias W Pletz,1,2 Dagmar Fischer,3,5 Oliwia Makarewicz1,2 1Center for Infectious Diseases and Infection’s Control, 2Center for Sepsis Control and Care, Jena University Hospital, 3Department of Pharmaceutical Technology, Friedrich Schiller University Jena, 4Department of Internal Medicine IV, Jena University Hospital, 5Jena Center for Soft Matter (JCSM, Friedrich Schiller University Jena, Jena, Germany*These authors contributed equally to this work Abstract: Strategies that target and treat biofilms are widely applied to bacterial cultures using popular live/dead staining techniques with mostly red or green fluorescent markers (eg, with SYTO® 9, propidium iodide, fluorescein. Therefore, visualizing drugs or micro- and nanoparticulate delivery systems to analyze their distribution and effects in biofilms requires a third fluorescent dye that does not interfere with the properties of the live/dead markers. The present study establishes and evaluates a model for tracking polymeric particles in fluorescently stained biological material. To this end, poly(D,L-lactide-co-glycolide (PLGA-based micro- and nanoparticles were used as well-established model systems, which, because of their favorable safety profiles, are expected to play important future roles with regard to drug delivery via inhalation. PLGA was covalently and stably labeled with 7-amino-4-methyl-3-coumarinylacetic acid (AMCA, after which blue fluorescent poly(ethylene glycol-block-PLGA (PEG-PLGA particles were prepared using a mixture of fluorescent AMCA-PLGA and PEG-PLGA. Because chitosan is known to reduce negative surface charge, blue fluorescent PEG-PLGA-particles with chitosan were also prepared. These micro- and nanoparticles were physicochemically characterized and could be clearly distinguished from live/dead stained bacteria in biofilms using confocal laser scanning microscopy. Keywords: 7-amino-4

  2. Synthesis of labeled compounds

    International Nuclear Information System (INIS)

    Whaley, T.W.

    1977-01-01

    Intermediate compounds labeled with 13 C included methane, sodium cyanide, methanol, ethanol, and acetonitrile. A new method for synthesizing 15 N-labeled 4-ethylsulfonyl-1-naphthalene-sulfonamide was developed. Studies were conducted on pathways to oleic-1- 13 C acid and a second pathway investigated was based on carbonation of 8-heptadecynylmagnesium bromide with CO 2 to prepare sterolic acid. Biosynthetic preparations included glucose- 13 C from starch isolated from tobacco leaves following photosynthetic incubation with 13 CO 2 and galactose- 13 C from galactosylglycerol- 13 C from kelp. Research on growth of organisms emphasized photosynthetic growth of algae in which all cellular carbon is labeled. Preliminary experiments were performed to optimize the growth of Escherichia coli on sodium acetate- 13 C

  3. Fluorescent quenching-based quantitative detection of specific DNA/RNA using a BODIPY® FL-labeled probe or primer

    Science.gov (United States)

    Kurata, Shinya; Kanagawa, Takahiro; Yamada, Kazutaka; Torimura, Masaki; Yokomaku, Toyokazu; Kamagata, Yoichi; Kurane, Ryuichiro

    2001-01-01

    We have developed a simple method for the quantitative detection of specific DNA or RNA molecules based on the finding that BODIPY® FL fluorescence was quenched by its interaction with a uniquely positioned guanine. This approach makes use of an oligonucleotide probe or primer containing a BODIPY® FL-modified cytosine at its 5′-end. When such a probe was hybridized with a target DNA, its fluorescence was quenched by the guanine in the target, complementary to the modified cytosine, and the quench rate was proportional to the amount of target DNA. This widely applicable technique will be used directly with larger samples or in conjunction with the polymerase chain reaction to quantify small DNA samples. PMID:11239011

  4. 25-gauge fibered-needle for label-free fluorescence analysis of breast masses: a first in vivo study

    Science.gov (United States)

    Toullec, Alexis; Mathieu, Marie-Christine; Benoit, Charlotte; Farcy, René; Tourasse, Christophe; Boisserie-Lacroix, Martine; Fontaine-Aupart, Marie-Pierre; Delaloge, Suzette; Balleyguier, Corinne

    2018-02-01

    Breast lesions diagnosis and characterization need additional cost-effective techniques to avoid unnecessary invasive procedures, such as core needle biopsies, in the case of benign tumors. Endogenous fluorescence is an effective method to highlight in situ metabolic and/or structural changes between cancerous and non-cancerous lesions. In this context, we developed an original set-up, consisting of a 405 nm laser diode transmitting light through a 25 Gauge (0.45 mm x 50mm) 14° sharp fibered needle to excite intranodular fluorophores around the needle tip and providing real-time labelfree fluorescence spectral analysis of lesions from 450 nm to 650 nm. The objective was to help radiologists to classify suspicious masses in vivo and in real-time within the lesion. We reported the results of spectral differences between 14 invasive lobular carcinomas and 6 intraductal papilloma enrolled in a clinical study.

  5. Fluorescent magnetic nanoparticles for cell labeling: flux synthesis of manganite particles and novel functionalization of silica shell

    Czech Academy of Sciences Publication Activity Database

    Kačenka, Michal; Kaman, Ondřej; Kikerlová, S.; Pavlů, B.; Jirák, Zdeněk; Jirák, D.; Herynek, Vít; Černý, J.; Chaput, F.; Laurent, S.; Lukeš, I.

    2015-01-01

    Roč. 47, Jun (2015), s. 97-106 ISSN 0021-9797 R&D Projects: GA ČR(CZ) GAP108/11/0807; GA MŠk(CZ) ED1.1.00/02.0109 Institutional support: RVO:68378271 ; RVO:68378041 Keywords : manganites * magnetic nanoparticles * molten salt synthesis * silica coating * dual probes * MRI * cell labeling Subject RIV: BM - Solid Matter Physics ; Magnetism Impact factor: 3.782, year: 2015

  6. Homogeneous immunoassay for the cancer marker alpha-fetoprotein using single wavelength excitation fluorescence cross-correlation spectroscopy and CdSe/ZnS quantum dots and fluorescent dyes as labels

    International Nuclear Information System (INIS)

    Wang, Jinjie; Liu, Heng; Huang, Xiangyi; Ren, Jicun

    2016-01-01

    The article describes sensitive and selective homogeneous immunoassays for the liver cancer biomarker alpha-fetoprotein (AFP) in human serum by using single wavelength excitation fluorescence cross-correlation spectroscopy (SW-FCCS). Both competitive and sandwich immunoassay modes were applied, and AFP served as a model analyte. Fluorescent CdSe/ZnS quantum dots (with a 655 nm emission peak) and the fluorophore Alexa Fluor 488 (520 nm emission) were chosen to label the antibodies in the sandwich mode, and the antibody and the antigen in the competitive mode. Under optimized conditions, the sandwich assay has a linear dynamic range that covers the 20 pM to 5.0 nM concentration range. The competitive assay, in turn, extends from 180 pM to 15.0 nM. The respective detection limits are 20 pM and 180 pM. The method was successfully applied to directly determine AFP in (spiked) clinical samples, and results were in good agreement with data obtained via ELISAs. (author)

  7. Europium-decorated graphene quantum dots as a fluorescent probe for label-free, rapid and sensitive detection of Cu{sup 2+} and L-cysteine

    Energy Technology Data Exchange (ETDEWEB)

    Lin, Liping [College of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou, 350002 (China); Song, Xinhong; Chen, Yiying; Rong, Mingcong; Wang, Yiru [Department of Chemistry and the MOE Key Laboratory of Spectrochemical Analysis & Instrumentation, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen, 361005 (China); Zhao, Li; Zhao, Tingting [Xiamen Huaxia College, Xiamen, 361024 (China); Chen, Xi, E-mail: xichen@xmu.edu.cn [Department of Chemistry and the MOE Key Laboratory of Spectrochemical Analysis & Instrumentation, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen, 361005 (China); State Key Laboratory of Marine Environmental Science, Xiamen University, Xiamen, 361005 (China)

    2015-09-03

    In this work, europium-decorated graphene quantum dots (Eu-GQDs) were prepared by treating three-dimensional Eu-decorated graphene (3D Eu-graphene) via a strong acid treatment. Various characterizations revealed that Eu atoms were successfully complexed with the oxygen functional groups on the surface of graphene quantum dots (GQDs) with the atomic ratio of 2.54%. Compared with Eu free GQDs, the introduction of Eu atoms enhanced the electron density and improved the surface chemical activities of Eu-GQDs. Therefore, the obtained Eu-GQDs were used as a novel “off-on” fluorescent probe for the label-free determination of Cu{sup 2+} and L-cysteine (L-Cys) with high sensitivity and selectivity. The fluorescence intensity of Eu-GQDs was quenched in the presence of Cu{sup 2+} owing to the coordination reaction between Cu{sup 2+} and carboxyl groups on the surface of the Eu-GQDs. The fluorescence intensity of Eu-GQDs recovered with the subsequent addition of L-Cys because of the strong affinity of Cu{sup 2+} to L-Cys via the Cu–S bond. The experimental results showed that the fluorescence variation of the proposed approach had a good linear relationship in the range of 0.1–10 μM for Cu{sup 2+} and 0.5–50 μM for L-Cys with corresponding detection limits of 0.056 μM for Cu{sup 2+} and 0.31 μM for L-Cys. The current approach also displayed a special response to Cu{sup 2+} and L-Cys over the other co-existing metal ions and amino acids, and the results obtained from buffer-diluted serum samples suggested its applicability in biological samples. - Highlights: • The europium-decorated graphene quantum dots (Eu-GQDs) have been successfully prepared. • Various characterizations results proved that Eu atoms were successfully introduced into graphene quantum dots. • The introduced Eu atoms changed the electron density and surface chemical activities of Eu-GQDs. • Eu-GQDs were used as an “off-on” fluorescent probe for Cu{sup 2+} and L-cysteine detection

  8. Target-induced structure switching of hairpin aptamers for label-free and sensitive fluorescent detection of ATP via exonuclease-catalyzed target recycling amplification.

    Science.gov (United States)

    Xu, Yunying; Xu, Jin; Xiang, Yun; Yuan, Ruo; Chai, Yaqin

    2014-01-15

    In this work, we described the development of a new label-free, simple and sensitive fluorescent ATP sensing platform based on exonuclease III (Exo III)-catalyzed target recycling (ECTR) amplification and SYBR Green I indicator. The hairpin aptamer probes underwent conformational structure switching and re-configuration in the presence of ATP, which led to catalytic cleavage of the re-configured aptamers by Exo III to release ATP and to initiate the ECTR process. Such ECTR process resulted in the digestion of a significant number of the hairpin aptamer probes, leading to much less intercalation of SYBR Green I to the hairpin stems and drastic suppression of the fluorescence emission for sensitive ATP detection down to the low nanomolar level. Due to the highly specific affinity bindings between aptamers and ATP, the developed method exhibited excellent selectivity toward ATP against other analogous molecules. Besides, our ATP sensing approach used un-modified aptamer probes and could be performed in a "mix-and-detect" fashion in homogenous solutions. All these distinct advantages of the developed method thus made it hold great potential for the development of simple and robust sensing strategies for the detection of other small molecules. © 2013 Elsevier B.V. All rights reserved.

  9. Phytoplankton IF-FISH: Species-specific labeling of cellular proteins by immunofluorescence (IF) with simultaneous species identification by fluorescence immunohybridization (FISH).

    Science.gov (United States)

    Meek, Megan E; Van Dolah, Frances M

    2016-05-01

    Phytoplankton rarely occur as unialgal populations. Therefore, to study species-specific protein expression, indicative of physiological status in natural populations, methods are needed that will both assay for a protein of interest and identify the species expressing it. Here we describe a protocol for IF-FISH, a dual labeling procedure using immunofluorescence (IF) labeling of a protein of interest followed by fluorescence in situ hybridization (FISH) to identify the species expressing that protein. The protocol was developed to monitor expression of the cell cycle marker proliferating cell nuclear antigen (PCNA) in the red tide dinoflagellate, Karenia brevis, using a large subunit (LSU) rRNA probe to identify K. brevis in a mixed population of morphologically similar Karenia species. We present this protocol as proof of concept that IF-FISH can be successfully applied to phytoplankton cells. This method is widely applicable for the analysis of single-cell protein expression of any protein of interest within phytoplankton communities. Copyright © 2016 Elsevier B.V. All rights reserved.

  10. A Real-Time Near-Infrared Fluorescence Imaging Method for the Detection of Oral Cancers in Mice Using an Indocyanine Green-Labeled Podoplanin Antibody.

    Science.gov (United States)

    Ito, Akihiro; Ohta, Mitsuhiko; Kato, Yukinari; Inada, Shunko; Kato, Toshio; Nakata, Susumu; Yatabe, Yasushi; Goto, Mitsuo; Kaneda, Norio; Kurita, Kenichi; Nakanishi, Hayao; Yoshida, Kenji

    2018-01-01

    Podoplanin is distinctively overexpressed in oral squamous cell carcinoma than oral benign neoplasms and plays a crucial role in the pathogenesis and metastasis of oral squamous cell carcinoma but its diagnostic application is quite limited. Here, we report a new near-infrared fluorescence imaging method using an indocyanine green (ICG)-labeled anti-podoplanin antibody and a desktop/a handheld ICG detection device for the visualization of oral squamous cell carcinoma-xenografted tumors in nude mice. Both near-infrared imaging methods using a desktop (in vivo imaging system: IVIS) and a handheld device (photodynamic eye: PDE) successfully detected oral squamous cell carcinoma tumors in nude mice in a podoplanin expression-dependent manner with comparable sensitivity. Of these 2 devices, only near-infrared imaging methods using a handheld device visualized oral squamous cell carcinoma xenografts in mice in real time. Furthermore, near-infrared imaging methods using the handheld device (PDE) could detect smaller podoplanin-positive oral squamous cell carcinoma tumors than a non-near-infrared, autofluorescence-based imaging method. Based on these results, a near-infrared imaging method using an ICG-labeled anti-podoplanin antibody and a handheld detection device (PDE) allows the sensitive, semiquantitative, and real-time imaging of oral squamous cell carcinoma tumors and therefore represents a useful tool for the detection and subsequent monitoring of malignant oral neoplasms in both preclinical and some clinical settings.

  11. Direct fluorescence in situ hybridization on human metaphase chromosomes using quantum dot-platinum labeled DNA probes

    Energy Technology Data Exchange (ETDEWEB)

    Hwang, Gyoyeon [Chemical Kinomics Research Center, Future Convergence Research Division, Korea Institute of Science and Technology, Hwarangno 14-gil 5, Seongbuk-gu, Seoul 136-791 (Korea, Republic of); Biological Chemistry, Korea University of Science and Technology, 217, Gajeong-ro, Yuseong-gu, Deajeon (Korea, Republic of); Lee, Hansol [Chemical Kinomics Research Center, Future Convergence Research Division, Korea Institute of Science and Technology, Hwarangno 14-gil 5, Seongbuk-gu, Seoul 136-791 (Korea, Republic of); Lee, Jiyeon, E-mail: jylee@kist.re.kr [Chemical Kinomics Research Center, Future Convergence Research Division, Korea Institute of Science and Technology, Hwarangno 14-gil 5, Seongbuk-gu, Seoul 136-791 (Korea, Republic of); Biological Chemistry, Korea University of Science and Technology, 217, Gajeong-ro, Yuseong-gu, Deajeon (Korea, Republic of)

    2015-11-13

    The telomere shortening in chromosomes implies the senescence, apoptosis, or oncogenic transformation of cells. Since detecting telomeres in aging and diseases like cancer, is important, the direct detection of telomeres has been a very useful biomarker. We propose a telomere detection method using a newly synthesized quantum dot (QD) based probe with oligonucleotide conjugation and direct fluorescence in situ hybridization (FISH). QD-oligonucleotides were prepared with metal coordination bonding based on platinum-guanine binding reported in our previous work. The QD-oligonucleotide conjugation method has an advantage where any sequence containing guanine at the end can be easily bound to the starting QD-Pt conjugate. A synthesized telomeric oligonucleotide was bound to the QD-Pt conjugate successfully and this probe hybridized specifically on the telomere of fabricated MV-4-11 and MOLT-4 chromosomes. Additionally, the QD-telomeric oligonucleotide probe successfully detected the telomeres on the CGH metaphase slide. Due to the excellent photostability and high quantum yield of QDs, the QD-oligonucleotide probe has high fluorescence intensity when compared to the organic dye-oligonucleotide probe. Our QD-oligonucleotide probe, conjugation method of this QD probe, and hybridization protocol with the chromosomes can be a useful tool for chromosome painting and FISH. - Highlights: • We prepared a probe linked between QD and telomeric oligonucleotide with platinum-guanine bonding. • Telomeres were detected by our new telomere probes successfully in three different human metaphase chromosomes. • QDPt-DNA probe has high fluorescence intensity in comparison with organic dye-DNA probe.

  12. Assessment of differential expression of oncogenes in adenocarcinoma of stomach with fluorescent labeling and simultaneous amplification of gene transcripts

    International Nuclear Information System (INIS)

    Rajcevic, U.; Hudler, P.; Komel, R.; Mijovski, G.; Gorjanc, G.; Kovac, M.; Hoelzl, G.; Repse, S.; Juvan, R.; Huber, C.G.

    2007-01-01

    Background. Gastric cancer is one of the leading malignancies with a poor prognosis and low survival rates. Although the mechanisms underlying its development are still unknown, there is a consensus that genetic instability, inactivation of tumor suppressor genes and over-expression of oncogenes are involved in the early and late stages of gastric carcinogenesis. In the present study we wanted to display differential expression of seven oncogenes, namely CCNE1, EGF, ERBB3, FGF4, HRG1, HGFR and TDGF1. Patients and methods. We employed a method based on the multiplex reverse transcription polymerase chain (RT-PCR) method with a fluorescence detection. Results. More than half of patients (74.3%) out of total 74 with gastric adenocarcinoma had over-expressed at least one oncogene, with the exception of FGF4, which was expressed in tumor tissue of less than one third of patients. 56.8% of the patients patients showed over-expression of two or more oncogenes. Conclusions. Patients with precancerous lesions had elevated levels of TDGF1 or cripto-1 (64.9%) and CCNE1 (57.1%), suggesting that they could be used as markers for an early detection of malignant changes in stomach. Finally, the fluorescent multiplex RT-PCR method could be of value for rapid assessment of oncogene mRNA levels in small samples of tumor or precancerous biopsies. (author)

  13. A Label-Free Microfluidic Biosensor for Activity Detection of Single Microalgae Cells Based on Chlorophyll Fluorescence

    Directory of Open Access Journals (Sweden)

    Junsheng Wang

    2013-11-01

    Full Text Available Detection of living microalgae cells is very important for ballast water treatment and analysis. Chlorophyll fluorescence is an indicator of photosynthetic activity and hence the living status of plant cells. In this paper, we developed a novel microfluidic biosensor system that can quickly and accurately detect the viability of single microalgae cells based on chlorophyll fluorescence. The system is composed of a laser diode as an excitation light source, a photodiode detector, a signal analysis circuit, and a microfluidic chip as a microalgae cell transportation platform. To demonstrate the utility of this system, six different living and dead algae samples (Karenia mikimotoi Hansen, Chlorella vulgaris, Nitzschia closterium, Platymonas subcordiformis, Pyramidomonas delicatula and Dunaliella salina were tested. The developed biosensor can distinguish clearly between the living microalgae cells and the dead microalgae cells. The smallest microalgae cells that can be detected by using this biosensor are 3 μm ones. Even smaller microalgae cells could be detected by increasing the excitation light power. The developed microfluidic biosensor has great potential for in situ ballast water analysis.

  14. A Label-Free Microfluidic Biosensor for Activity Detection of Single Microalgae Cells Based on Chlorophyll Fluorescence

    Science.gov (United States)

    Wang, Junsheng; Sun, Jinyang; Song, Yongxin; Xu, Yongyi; Pan, Xinxiang; Sun, Yeqing; Li, Dongqing

    2013-01-01

    Detection of living microalgae cells is very important for ballast water treatment and analysis. Chlorophyll fluorescence is an indicator of photosynthetic activity and hence the living status of plant cells. In this paper, we developed a novel microfluidic biosensor system that can quickly and accurately detect the viability of single microalgae cells based on chlorophyll fluorescence. The system is composed of a laser diode as an excitation light source, a photodiode detector, a signal analysis circuit, and a microfluidic chip as a microalgae cell transportation platform. To demonstrate the utility of this system, six different living and dead algae samples (Karenia mikimotoi Hansen, Chlorella vulgaris, Nitzschia closterium, Platymonas subcordiformis, Pyramidomonas delicatula and Dunaliella salina) were tested. The developed biosensor can distinguish clearly between the living microalgae cells and the dead microalgae cells. The smallest microalgae cells that can be detected by using this biosensor are 3 μm ones. Even smaller microalgae cells could be detected by increasing the excitation light power. The developed microfluidic biosensor has great potential for in situ ballast water analysis. PMID:24287532

  15. A 2D/3D image analysis system to track fluorescently labeled structures in rod-shaped cells: application to measure spindle pole asymmetry during mitosis.

    Science.gov (United States)

    Schmitter, Daniel; Wachowicz, Paulina; Sage, Daniel; Chasapi, Anastasia; Xenarios, Ioannis; Simanis; Unser, Michael

    2013-01-01

    The yeast Schizosaccharomyces pombe is frequently used as a model for studying the cell cycle. The cells are rod-shaped and divide by medial fission. The process of cell division, or cytokinesis, is controlled by a network of signaling proteins called the Septation Initiation Network (SIN); SIN proteins associate with the SPBs during nuclear division (mitosis). Some SIN proteins associate with both SPBs early in mitosis, and then display strongly asymmetric signal intensity at the SPBs in late mitosis, just before cytokinesis. This asymmetry is thought to be important for correct regulation of SIN signaling, and coordination of cytokinesis and mitosis. In order to study the dynamics of organelles or large protein complexes such as the spindle pole body (SPB), which have been labeled with a fluorescent protein tag in living cells, a number of the image analysis problems must be solved; the cell outline must be detected automatically, and the position and signal intensity associated with the structures of interest within the cell must be determined. We present a new 2D and 3D image analysis system that permits versatile and robust analysis of motile, fluorescently labeled structures in rod-shaped cells. We have designed an image analysis system that we have implemented as a user-friendly software package allowing the fast and robust image-analysis of large numbers of rod-shaped cells. We have developed new robust algorithms, which we combined with existing methodologies to facilitate fast and accurate analysis. Our software permits the detection and segmentation of rod-shaped cells in either static or dynamic (i.e. time lapse) multi-channel images. It enables tracking of two structures (for example SPBs) in two different image channels. For 2D or 3D static images, the locations of the structures are identified, and then intensity values are extracted together with several quantitative parameters, such as length, width, cell orientation, background fluorescence and

  16. Antimalarial activity of compounds comprising a primary benzene sulfonamide fragment.

    Science.gov (United States)

    Andrews, Katherine T; Fisher, Gillian M; Sumanadasa, Subathdrage D M; Skinner-Adams, Tina; Moeker, Janina; Lopez, Marie; Poulsen, Sally-Ann

    2013-11-15

    Despite the urgent need for effective antimalarial drugs with novel modes of action no new chemical class of antimalarial drug has been approved for use since 1996. To address this, we have used a rational approach to investigate compounds comprising the primary benzene sulfonamide fragment as a potential new antimalarial chemotype. We report the in vitro activity against Plasmodium falciparum drug sensitive (3D7) and resistant (Dd2) parasites for a panel of fourteen primary benzene sulfonamide compounds. Our findings provide a platform to support the further evaluation of primary benzene sulfonamides as a new antimalarial chemotype, including the identification of the target of these compounds in the parasite. Copyright © 2013 Elsevier Ltd. All rights reserved.

  17. Improved Resection and Outcome of Colon-Cancer Liver Metastasis with Fluorescence-Guided Surgery Using In Situ GFP Labeling with a Telomerase-Dependent Adenovirus in an Orthotopic Mouse Model.

    Directory of Open Access Journals (Sweden)

    Shuya Yano

    Full Text Available Fluorescence-guided surgery (FGS of cancer is an area of intense development. In the present report, we demonstrate that the telomerase-dependent green fluorescent protein (GFP-containing adenovirus OBP-401 could label colon-cancer liver metastasis in situ in an orthotopic mouse model enabling successful FGS. OBP-401-GFP-labeled liver metastasis resulted in complete resection with FGS, in contrast, conventional bright-light surgery (BLS did not result in complete resection of the metastasis. OBP-401-FGS reduced the recurrence rate and prolonged over-all survival compared with BLS. In conclusion, adenovirus OBP-401 is a powerful tool to label liver metastasis in situ with GFP which enables its complete resection, not possible with conventional BLS.

  18. Uptake and localization of fluorescent labelled gold nanoparticles in living zebrafish (Danio rerio) using Light Sheet Microscopy

    DEFF Research Database (Denmark)

    Skjolding, Lars Michael; Asmonaite, G.; Jolk, R.

    2015-01-01

    Despite nanoparticles being used in many different products and applications, the effects and fate in the environment are still not well understood. Uptake of nanoparticles into cells has been shown in vitro and in vivo. However, it is challenging to find suitable methods to identify uptake...... and determine localization on a whole organism level. Furthermore, methods used to identify nanoparticle uptake have been associated with artefacts induced by sample preparation including staining methods for electron microscopy.  This study used Fluorescent Light Sheet Microscopy (FLSM) to determine uptake...... to the particles through the diet or the water phase in a series of separate experiments. In the dietary exposure experiments Artemia salina were exposed to 1 mg Au/L for 24h before being fed to D. rerio. For exposure through the water phase 1 mg Au/L was added directly to aquaria holding the fish and non...

  19. Origin and characterization of retrograde labeled neurons supplying the rat urethra using fiberoptic confocal fluorescent microscopy in vivo and immunohistochemistry.

    Science.gov (United States)

    Lee, Keon-Cheol; Sharma, Seema; Tuttle, Jeremy B; Steers, William D

    2010-10-01

    Autonomic innervation of urethral smooth muscle may influence urinary continence after prostatectomy. It is unclear whether the cavernous nerves carry fibers that influence continence. Using a retrograde axonal tracer combined with real-time in vivo imaging and ex vivo immunohistochemistry we determined the course and type of neurons supplying urethral smooth muscle distal to the prostate in the rat. We injected the retrograde axonal tracers cholera toxin B fragment-Alexa Fluor 488 and Fast Blue in the distal urethral smooth muscle in 10 rats each. Five days later the cavernous nerves and pelvic ganglion were imaged using fiberoptic confocal fluorescence microscopy (cholera toxin B fragment-Alexa Fluor 488) or harvested for immunohistochemistry (Fast Blue). Dual immunofluorescence of Fast Blue neurons with tyrosine hydroxylase or neuronal nitric oxide synthase was done to characterize neurons as noradrenergic or nitrergic. To ascertain whether the cavernous nerves contain fibers to the urethra that originate in the pelvic ganglia we cut the cavernous nerves with their ancillary branches in 3 rats and imaged them for Fast Blue. Fluorescent neurons and axons were detected in cavernous nerves and the pelvic ganglion. Few neurons were seen in rats with cavernous nerve section. Of urethral neurons 53.1% showed neuronal nitric oxide synthase positivity while 40.6% were immunoreactive for tyrosine hydroxylase. About 6.2% of urethral neurons failed to show tyrosine hydroxylase or neuronal nitric oxide synthase immunoreactivity. Most of the autonomic innervation to the urethra beyond the prostatic apex travels in the cavernous nerves. Many nerves may be parasympathetic based on neuronal nitric oxide synthase immunoreactivity. Nerves supplying the urethra outside the cavernous nerves may course posterior to the prostate. Along with afferent fibers, tyrosine hydroxylase immunoreactivity expressing neuron fibers, ie noradrenergic nerves, traveling in the cavernous nerves may

  20. Discovery of tertiary sulfonamides as potent liver X receptor antagonists.

    Science.gov (United States)

    Zuercher, William J; Buckholz, Richard G; Campobasso, Nino; Collins, Jon L; Galardi, Cristin M; Gampe, Robert T; Hyatt, Stephen M; Merrihew, Susan L; Moore, John T; Oplinger, Jeffrey A; Reid, Paul R; Spearing, Paul K; Stanley, Thomas B; Stewart, Eugene L; Willson, Timothy M

    2010-04-22

    Tertiary sulfonamides were identified in a HTS as dual liver X receptor (LXR, NR1H2, and NR1H3) ligands, and the binding affinity of the series was increased through iterative analogue synthesis. A ligand-bound cocrystal structure was determined which elucidated key interactions for high binding affinity. Further characterization of the tertiary sulfonamide series led to the identification of high affinity LXR antagonists. GSK2033 (17) is the first potent cell-active LXR antagonist described to date. 17 may be a useful chemical probe to explore the cell biology of this orphan nuclear receptor.

  1. Analytik, Vorkommen und Verhalten aromatischer Sulfonamide in der aquatischen Umwelt

    OpenAIRE

    Hartig, Claudia

    2000-01-01

    Subject of this PhD-thesis are the analysis and behaviour of aromatic sulfonamides as organic micropollutants in the aquatic environment. On the grounds of their widespread application, bacteriostatics as well as benzene- and toluenesulfonamides (BTS) are investigated. Analysis of the sulfonamides consists of solid-phase extraction (LiChrolut EN) and HPLC-MS/MS. Limits of detection vary from 0.1-20 ng/L. The MS/MS detection is carried out in SRM mode. Matrix effects of the investigated second...

  2. Assessing Collagen and Elastin Pressure-Dependent Microarchitectures in Live, Human Resistance Arteries by Label-Free Fluorescence Microscopy

    DEFF Research Database (Denmark)

    Bloksgaard, Maria; Thorsted, Bjarne; Brewer, Jonathan R.

    2017-01-01

    The pathogenic contribution of resistance artery remodeling is documented in essential hypertension, diabetes and the metabolic syndrome. Investigations and development of microstructurally motivated mathematical models for understanding the mechanical properties of human resistance arteries...... in health and disease have the potential to aid understanding how disease and medical treatments affect the human microcirculation. To develop these mathematical models, it is essential to decipher the relationship between the mechanical and microarchitectural properties of the microvascular wall....... In this work, we describe an ex vivo method for passive mechanical testing and simultaneous label-free three-dimensional imaging of the microarchitecture of elastin and collagen in the arterial wall of isolated human resistance arteries. The imaging protocol can be applied to resistance arteries of any species...

  3. Functionalized graphene oxide/Fe3O4 hybrids for cellular magnetic resonance imaging and fluorescence labeling.

    Science.gov (United States)

    Zhou, Chaohui; Wu, Hui; Wang, Mingliang; Huang, Chusen; Yang, Dapeng; Jia, Nengqin

    2017-09-01

    In this work, we developed a T 2 -weighted contrast agent based on graphene oxide (GO)/Fe 3 O 4 hybrids for efficient cellular magnetic resonance imaging (MRI). The GO/Fe 3 O 4 hybrids were obtained by combining with co-precipitation method and pyrolysis method. The structural, surface and magnetic characteristics of the hybrids were systematically characterized by transmission electron microscopy (TEM), vibrating sample magnetometer (VSM), AFM, Raman, FT-IR and XRD. The GO/Fe 3 O 4 hybrids were functionalized by modifying with anionic and cationic polyelectrolyte through layer-by-layer assembling. The fluorescence probe fluorescein isothiocyanate (FITC) was further loaded on the surface of functionalized GO/Fe 3 O 4 hybrids to trace the location of GO/Fe 3 O 4 hybrids in cells. Functionalized GO/Fe 3 O 4 hybrids possess good hydrophilicity, less cytotoxicity, high MRI enhancement with the relaxivity (r 2 ) of 493mM -1 s -1 as well as cellular MRI contrast effect. These obtained results indicated that the functionalized GO/Fe 3 O 4 hybrids could have great potential to be utilized as cellular MRI contrast agents for tumor early diagnosis and monitoring. Copyright © 2017 Elsevier B.V. All rights reserved.

  4. Prediction of gradient retention data for hydrophilic interaction liquid chromatographic separation of native and fluorescently labeled oligosaccharides.

    Science.gov (United States)

    Vaňková, Nikola; Česla, Petr

    2017-02-17

    In this work, we have investigated the predictive properties of mixed-mode retention model and oligomeric mixed-mode model, taking into account the contribution of monomeric units to the retention, in hydrophilic interaction liquid chromatography. The gradient retention times of native maltooligosaccharides and their fluorescent derivatives were predicted in the oligomeric series with number of monomeric glucose units in the range from two to seven. The maltooligosaccharides were separated on a packed column with carbamoyl-bonded silica stationary phase and 15 gradient profiles with different initial and final mobile phase composition were used with the gradient times 5; 7.5 and 10min. The predicted gradient retention times were compared for calculations based on isocratic retention data and gradient retention data, which provided better accuracy of the results. By comparing two different mobile phase additives, the more accurate retention times were predicted in mobile phases containing ammonium acetate. The acidic derivatives, prepared by reaction of an oligosaccharide with 2-aminobenzoic acid or 8-aminonaphthalene-1,3,6-trisulfonic acid, provided more accurate predictions of the retention data in comparison to native oligosaccharides or their neutral derivatives. The oligomeric mixed-mode model allowed prediction of gradient retention times using only one gradient profile, which significantly speeded-up the method development. Copyright © 2017 Elsevier B.V. All rights reserved.

  5. Comparison of isocratic retention models for hydrophilic interaction liquid chromatographic separation of native and fluorescently labeled oligosaccharides.

    Science.gov (United States)

    Česla, Petr; Vaňková, Nikola; Křenková, Jana; Fischer, Jan

    2016-03-18

    In this work, we have investigated retention of maltooligosaccharides and their fluorescent derivatives in hydrophilic interaction liquid chromatography using four different stationary phases. The non-derivatized maltooligosaccharides (maltose to maltoheptaose) and their derivatives with 2-aminobenzoic acid, 2-aminobenzamide, 2-aminopyridine and 8-aminonaphthalene-1,3,6-trisulfonic acid were analyzed on silica gel, aminopropyl silica, amide (carbamoyl-bonded silica) and ZIC-HILIC zwitterionic sulfobetain bonded phase. The partitioning of the analytes between the bulk mobile phase and adsorbed water-rich layer, polar and ionic interactions of analytes with stationary phase have been evaluated and compared. The effects of the mobile phase additives (0.1% (v/v) of acetic acid and ammonium acetate in concentration range 5-30 mmol L(-1)) on retention were described. The suitability of different models for prediction of retention was tested including linear solvent strength model, quadratic model, mixed-mode model, and empirical Neue-Kuss model. The mixed-mode model was extended to the parameter describing the contribution of monomeric glucose unit to the retention of non-derivatized and derivatized maltooligosaccharides, which was used for evaluation of contribution of both, oligosaccharide backbone and end-group to retention. Copyright © 2016 Elsevier B.V. All rights reserved.

  6. Improved fluorescent labeling of chitin oligomers: Chitinolytic properties of acidic mammalian chitinase under somatic tissue pH conditions.

    Science.gov (United States)

    Wakita, Satoshi; Kimura, Masahiro; Kato, Naoki; Kashimura, Akinori; Kobayashi, Shunsuke; Kanayama, Naoto; Ohno, Misa; Honda, Shotaro; Sakaguchi, Masayoshi; Sugahara, Yasusato; Bauer, Peter O; Oyama, Fumitaka

    2017-05-15

    Acidic mammalian chitinase (AMCase) has been implicated in various pathophysiological conditions including asthma, allergic inflammation and food processing. AMCase is most active at pH 2.0, and its activity gradually decreases to up to pH 8. Here we analyzed chitin degradation by AMCase in weak acidic to neutral conditions by fluorophore-assisted carbohydrate electrophoresis established originally for oligosaccharides analysis. We found that specific fragments with slower-than-expected mobility as defined by chitin oligosaccharide markers were generated at pH 5.0∼8.0 as by-products of the reaction. We established an improved method for chitin oligosaccharides suppressing this side reaction by pre-acidification of the fluorophore-labeling reaction mixture. Our improved method specifically detects chitin oligosaccharides and warrants quantification of up to 50nmol of the material. Using this strategy, we found that AMCase produced dimer of N-acetyl-d-glucosamine (GlcNAc) at strong acidic to neutral condition. Moreover, we found that AMCase generates (GlcNAc) 2 as well as (GlcNAc) 3 under physiological conditions. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

  7. Fluorescence-Activated Cell Sorting of EGFP-Labeled Neural Crest Cells From Murine Embryonic Craniofacial Tissue

    Directory of Open Access Journals (Sweden)

    Saurabh Singh

    2005-01-01

    Full Text Available During the early stages of embryogenesis, pluripotent neural crest cells (NCC are known to migrate from the neural folds to populate multiple target sites in the embryo where they differentiate into various derivatives, including cartilage, bone, connective tissue, melanocytes, glia, and neurons of the peripheral nervous system. The ability to obtain pure NCC populations is essential to enable molecular analyses of neural crest induction, migration, and/or differentiation. Crossing Wnt1-Cre and Z/EG transgenic mouse lines resulted in offspring in which the Wnt1-Cre transgene activated permanent EGFP expression only in NCC. The present report demonstrates a flow cytometric method to sort and isolate populations of EGFP-labeled NCC. The identity of the sorted neural crest cells was confirmed by assaying expression of known marker genes by TaqMan Quantitative Real-Time Polymerase Chain Reaction (QRT-PCR. The molecular strategy described in this report provides a means to extract intact RNA from a pure population of NCC thus enabling analysis of gene expression in a defined population of embryonic precursor cells critical to development.

  8. Synthesis and evaluation of Tc-99m and fluorescence-labeled elastin-derived peptide, VAPG for multimodal tumor imaging in murine tumor model.

    Science.gov (United States)

    Kim, Myoung Hyoun; Kim, Chang Guhn; Kim, Seul-Gi; Kim, Dae-Weung

    2017-12-01

    We developed a Tc-99m and fluorescence-labeled peptide, Tc-99m TAMRA-GHEG-ECG-VAPG to target tumor cells and evaluated the diagnostic performance as a dual-modality imaging agent for tumor in a murine model. TAMRA-GHEG-ECG-VAPG was synthesized by using Fmoc solid-phase peptide synthesis. Radiolabeling of TAMRA-GHEG-ECG-VAPG with Tc-99m was done by using ligand exchange via tartrate. Binding affinity and in vitro cellular uptake studies were performed. Gamma camera imaging, biodistribution, and ex vivo imaging studies were performed in murine models with SW620 tumors. Tumor tissue slides were prepared and analyzed with immunohistochemistry by using confocal microscopy. After radiolabeling procedures with Tc-99m, Tc-99m TAMRA-GHEG-ECG-VAPG complexes were prepared in high yield (>96%). The K d of Tc-99m TAMRA-GHEG-ECG-VAPG determined by saturation binding was 16.8 ± 3.6 nM. Confocal microscopy images of SW620 cells incubated with TAMRA-GHEG-ECG-VAPG showed strong fluorescence in the cytoplasm. Gamma camera imaging revealed substantial uptake of Tc-99m TAMRA-GHEG-ECG-VAPG in tumors. Tumor uptake was effectively blocked by the coinjection of an excess concentration of VAPG. Specific uptake of Tc-99m TAMRA-GHEG-ECG-VAPG was confirmed by biodistribution, ex vivo imaging, and immunohistochemistry stain studies. In vivo and in vitro studies revealed substantial uptake of Tc-99m TAMRA-GHEG-ECG-VAPG in tumor cells. Tc-99m TAMRA-GHEG-ECG-VAPG has potential as a dual-modality tumor imaging agent. Copyright © 2017 John Wiley & Sons, Ltd.

  9. Detection of a fluorescent-labeled avidin-nucleic acid nanoassembly by confocal laser endomicroscopy in the microvasculature of chronically inflamed intestinal mucosa

    Directory of Open Access Journals (Sweden)

    Buda A

    2015-01-01

    Full Text Available Andrea Buda,1,* Sonia Facchin,1,* Elisa Dassie,2 Elisabetta Casarin,3 Mark A Jepson,4 Helmut Neumann,5 Giorgia Hatem,1 Stefano Realdon,6 Renata D’Incà,1 Giacomo Carlo Sturniolo,1 Margherita Morpurgo3 1Department of Surgical, Oncological, and Gastroenterological Sciences, University of Padova, 2Department of Molecular Medicine, University of Padova, Padova, Italy; 3Department of Pharmaceutical and Pharmacological Sciences, University of Padova, Padova, Italy; 4School of Biochemistry and Wolfson Bioimaging Facility, University of Bristol, Bristol, UK; 5Ludwig Demlig Endoscopic Center of Excellence, ESGE Endoscopy Training Center, University of Erlangen-Nuremberg, Erlangen, Germany; 6Veneto Institute of Oncology IOV-IRCCS, Padova, Italy *These authors contributed equally to this work Abstract: Inflammatory bowel diseases are chronic gastrointestinal pathologies causing great discomfort in both children and adults. The pathogenesis of inflammatory bowel diseases is not yet fully understood and their diagnosis and treatment are often challenging. Nanoparticle-based strategies have been tested in local drug delivery to the inflamed colon. Here, we have investigated the use of the novel avidin-nucleic acid nanoassembly (ANANAS platform as a potential diagnostic carrier in an experimental model of inflammatory bowel diseases. Fluorescent-labeled ANANAS nanoparticles were administered to mice with chemically induced chronic inflammation of the large intestine. Localization of mucosal nanoparticles was assessed in vivo by dual-band confocal laser endomicroscopy. This technique enables characterization of the mucosal microvasculature and crypt architecture at subcellular resolution. Intravascular nanoparticle distribution was observed in the inflamed mucosa but not in healthy controls, demonstrating the utility of the combination of ANANAS and confocal laser endomicroscopy for highlighting intestinal inflammatory conditions. The specific localization of

  10. Fluorescent molecularly imprinted polymers as plastic antibodies for selective labeling and imaging of hyaluronan and sialic acid on fixed and living cells.

    Science.gov (United States)

    Panagiotopoulou, Maria; Kunath, Stephanie; Medina-Rangel, Paulina Ximena; Haupt, Karsten; Tse Sum Bui, Bernadette

    2017-02-15

    Altered glycosylation levels or distribution of sialic acids (SA) or hyaluronan in animal cells are indicators of pathological conditions like infection or malignancy. We applied fluorescently-labeled molecularly imprinted polymer (MIP) particles for bioimaging of fixed and living human keratinocytes, to localize hyaluronan and sialylation sites. MIPs were prepared with the templates D-glucuronic acid (GlcA), a substructure of hyaluronan, and N-acetylneuraminic acid (NANA), the most common member of SA. Both MIPs were found to be highly selective towards their target monosaccharides, as no cross-reactivity was observed with other sugars like N-acetyl-D-glucosamine, N-acetyl-D-galactosamine, D-glucose and D-galactose, present on the cell surface. The dye rhodamine and two InP/ZnS quantum dots (QDs) emitting in the green and in the red regions were used as fluorescent probes. Rhodamine-MIPGlcA and rhodamine-MIPNANA were synthesized as monodispersed 400nm sized particles and were found to bind selectively their targets located in the extracellular region, as imaged by epifluorescence and confocal microscopy. In contrast, when MIP-GlcA and MIP-NANA particles with a smaller size (125nm) were used, the MIPs being synthesized as thin shells around green and red emitting QDs respectively, it was possible to stain the intracellular and pericellular regions as well. In addition, simultaneous dual-color imaging with the two different colored QDs-MIPs was demonstrated. Importantly, the MIPs were not cytotoxic and did not affect cell viability; neither was the cells morphology affected as demonstrated by live cell imaging. These synthetic receptors could offer a new and promising imaging tool to monitor disease progression. Copyright © 2016 Elsevier B.V. All rights reserved.

  11. Ultrasensitive Single Fluorescence-Labeled Probe-Mediated Single Universal Primer-Multiplex-Droplet Digital Polymerase Chain Reaction for High-Throughput Genetically Modified Organism Screening.

    Science.gov (United States)

    Niu, Chenqi; Xu, Yuancong; Zhang, Chao; Zhu, Pengyu; Huang, Kunlun; Luo, Yunbo; Xu, Wentao

    2018-05-01

    As genetically modified (GM) technology develops and genetically modified organisms (GMOs) become more available, GMOs face increasing regulations and pressure to adhere to strict labeling guidelines. A singleplex detection method cannot perform the high-throughput analysis necessary for optimal GMO detection. Combining the advantages of multiplex detection and droplet digital polymerase chain reaction (ddPCR), a single universal primer-multiplex-ddPCR (SUP-M-ddPCR) strategy was proposed for accurate broad-spectrum screening and quantification. The SUP increases efficiency of the primers in PCR and plays an important role in establishing a high-throughput, multiplex detection method. Emerging ddPCR technology has been used for accurate quantification of nucleic acid molecules without a standard curve. Using maize as a reference point, four heterologous sequences ( 35S, NOS, NPTII, and PAT) were selected to evaluate the feasibility and applicability of this strategy. Surprisingly, these four genes cover more than 93% of the transgenic maize lines and serve as preliminary screening sequences. All screening probes were labeled with FAM fluorescence, which allows the signals from the samples with GMO content and those without to be easily differentiated. This fiveplex screening method is a new development in GMO screening. Utilizing an optimal amplification assay, the specificity, limit of detection (LOD), and limit of quantitation (LOQ) were validated. The LOD and LOQ of this GMO screening method were 0.1% and 0.01%, respectively, with a relative standard deviation (RSD) < 25%. This method could serve as an important tool for the detection of GM maize from different processed, commercially available products. Further, this screening method could be applied to other fields that require reliable and sensitive detection of DNA targets.

  12. Bithiophene sulfonamide-based molecular and polymeric semiconductors

    Science.gov (United States)

    Facchetti, Antonio; Marks, Tobin J.; Xia, Yu; Drees, Martin; Melkonyan, Ferdinand; Zhao, Wei

    2017-05-30

    The present invention relates to new semiconducting compounds having at least one optionally substituted bithiophene sulfonamide moiety. The compounds disclosed herein can exhibit high carrier mobility and/or efficient light absorption/emission characteristics, and can possess certain processing advantages such as solution-processability and/or good stability at ambient conditions.

  13. Sorption of fluoroquinolones and sulfonamides in 13 Brazilian soils.

    Science.gov (United States)

    Leal, Rafael Marques Pereira; Alleoni, Luis Reynaldo Ferracciú; Tornisielo, Valdemar Luiz; Regitano, Jussara Borges

    2013-08-01

    Animal production is a leading economic activity in Brazil and antibiotics are widely used. However, the occurrence, behavior, and impacts of antibiotics in Brazilian soils are still poorly known. We evaluated the sorption behavior of four fluoroquinolones (norfloxacin, ciprofloxacin, danofloxacin, and enrofloxacin) and five sulfonamides (sulfadiazine, sulfachloropyridazine, sulfamethoxazole, sulfadimidine, and sulfathiazole) in 13 Brazilian soils with contrasting physical, chemical, and mineralogical properties. Fluoroquinolone sorption was very high (Kd≥544 L kg(-1)) whereas sulfonamide sorption ranged from low to high (Kd=0.7-70.1 L kg(-1)), consistent with previous reports in the literature. Soil texture and cation exchange capacity were the soil attributes that most affected sorption. Cation exchange was the most important sorption mechanism for the fluoroquinolones in highly weathered tropical soils, although cation bridging and ion pairing could not be ruled out. Hydrophobic partition played an important role in the sorption of the sulfonamides, but sorption was also affected by non-hydrophobic interactions with organic and/or mineral surfaces. Sorption for both compound classes tended to be higher in soils with high Al and Fe oxihydroxide contents, but they were not correlated with Kd values. No direct effect of soil pH was seen. The fluoroquinolones are not expected to leach even in worst-case scenarios (soils rich in sand and poor in organic carbon), whereas soil attributes dictate leaching potential for the sulfonamides. Copyright © 2013 Elsevier Ltd. All rights reserved.

  14. Fluorescent labelling of intestinal epithelial cells reveals independent long-lived intestinal stem cells in a crypt

    International Nuclear Information System (INIS)

    Horita, Nobukatsu; Tsuchiya, Kiichiro; Hayashi, Ryohei; Fukushima, Keita; Hibiya, Shuji; Fukuda, Masayoshi; Kano, Yoshihito; Mizutani, Tomohiro; Nemoto, Yasuhiro; Yui, Shiro; Okamoto, Ryuichi; Nakamura, Tetsuya; Watanabe, Mamoru

    2014-01-01

    Highlights: • Lentivirus mixed with Matrigel enables direct infection of intestinal organoids. • Our original approach allows the marking of a single stem cell in a crypt. • Time-lapse imaging shows the dynamics of a single stem cell. • Our lentivirus transgene system demonstrates plural long-lived stem cells in a crypt. - Abstract: Background and aims: The dynamics of intestinal stem cells are crucial for regulation of intestinal function and maintenance. Although crypt stem cells have been identified in the intestine by genetic marking methods, identification of plural crypt stem cells has not yet been achieved as they are visualised in the same colour. Methods: Intestinal organoids were transferred into Matrigel® mixed with lentivirus encoding mCherry. The dynamics of mCherry-positive cells was analysed using time-lapse imaging, and the localisation of mCherry-positive cells was analysed using 3D immunofluorescence. Results: We established an original method for the introduction of a transgene into an organoid generated from mouse small intestine that resulted in continuous fluorescence of the mCherry protein in a portion of organoid cells. Three-dimensional analysis using confocal microscopy showed a single mCherry-positive cell in an organoid crypt that had been cultured for >1 year, which suggested the presence of long-lived mCherry-positive and -negative stem cells in the same crypt. Moreover, a single mCherry-positive stem cell in a crypt gave rise to both crypt base columnar cells and transit amplifying cells. Each mCherry-positive and -negative cell contributed to the generation of organoids. Conclusions: The use of our original lentiviral transgene system to mark individual organoid crypt stem cells showed that long-lived plural crypt stem cells might independently serve as intestinal epithelial cells, resulting in the formation of a completely functional villus

  15. Fluorescent labelling of intestinal epithelial cells reveals independent long-lived intestinal stem cells in a crypt

    Energy Technology Data Exchange (ETDEWEB)

    Horita, Nobukatsu [Department of Gastroenterology and Hepatology, Graduate School, Tokyo Medical and Dental University (Japan); Tsuchiya, Kiichiro, E-mail: kii.gast@tmd.ac.jp [Department of Advanced Therapeutics for Gastrointestinal Diseases, Graduate School, Tokyo Medical and Dental University (Japan); Hayashi, Ryohei [Department of Gastroenterology and Hepatology, Graduate School, Tokyo Medical and Dental University (Japan); Department of Gastroenterology and Metabolism, Hiroshima University (Japan); Fukushima, Keita; Hibiya, Shuji; Fukuda, Masayoshi; Kano, Yoshihito; Mizutani, Tomohiro; Nemoto, Yasuhiro; Yui, Shiro [Department of Gastroenterology and Hepatology, Graduate School, Tokyo Medical and Dental University (Japan); Okamoto, Ryuichi; Nakamura, Tetsuya [Department of Advanced Therapeutics for Gastrointestinal Diseases, Graduate School, Tokyo Medical and Dental University (Japan); Watanabe, Mamoru [Department of Gastroenterology and Hepatology, Graduate School, Tokyo Medical and Dental University (Japan)

    2014-11-28

    Highlights: • Lentivirus mixed with Matrigel enables direct infection of intestinal organoids. • Our original approach allows the marking of a single stem cell in a crypt. • Time-lapse imaging shows the dynamics of a single stem cell. • Our lentivirus transgene system demonstrates plural long-lived stem cells in a crypt. - Abstract: Background and aims: The dynamics of intestinal stem cells are crucial for regulation of intestinal function and maintenance. Although crypt stem cells have been identified in the intestine by genetic marking methods, identification of plural crypt stem cells has not yet been achieved as they are visualised in the same colour. Methods: Intestinal organoids were transferred into Matrigel® mixed with lentivirus encoding mCherry. The dynamics of mCherry-positive cells was analysed using time-lapse imaging, and the localisation of mCherry-positive cells was analysed using 3D immunofluorescence. Results: We established an original method for the introduction of a transgene into an organoid generated from mouse small intestine that resulted in continuous fluorescence of the mCherry protein in a portion of organoid cells. Three-dimensional analysis using confocal microscopy showed a single mCherry-positive cell in an organoid crypt that had been cultured for >1 year, which suggested the presence of long-lived mCherry-positive and -negative stem cells in the same crypt. Moreover, a single mCherry-positive stem cell in a crypt gave rise to both crypt base columnar cells and transit amplifying cells. Each mCherry-positive and -negative cell contributed to the generation of organoids. Conclusions: The use of our original lentiviral transgene system to mark individual organoid crypt stem cells showed that long-lived plural crypt stem cells might independently serve as intestinal epithelial cells, resulting in the formation of a completely functional villus.

  16. Detection of immunomagnetically captured 4',6-diamidino-2-phenyl-indole (DAPI)-labeled Escherichia coli 0157:H7 by fluorescent microscopic imaging

    Science.gov (United States)

    Tu, Shu-I.; Uknalis, Joseph; Patterson, Deidre; Gehring, Andrew G.

    1999-01-01

    Live cells of E. coliO157:H7 were captured by goat anti-E. coliO157 serum coated on the surface of polystyrene based immunomagnetic beads (IMB). The captured bacteria were labeled by 4',6-diamidino-2-phenylindole (DAPI), a nucleic acid stain, for observation by epifluorescent microscopy. The beads with captured bacteria were then concentrated by magnetic separators. The efficiency of this magnetic concentration step was less than that of using high speed centrifugation. The antibody-captured and IMB-immobilized bacteria were then applied on HF-treated, bovine serum albumin (BSA)-coated microscope slides mounted on an automated stage, and magnetically aligned before fluorescence distribution was measured by a cooled CCD attached to an inverted microscope. Since the beads were concentrated and linearly aligned along the edge of the magnetic field, image capture along the edge for a few field widths was sufficient to account for most of captured bacteria. We applied this approach to determine the bacterial counts in spiked beef hamburger patties. The results showed that after a 6-hour enrichment, sufficient number of the bacteria could be detected from the samples spiked with 1 CFU of E. coliO157:H7 per gram of the hamburger.

  17. DNA methylation levels analysis in four tissues of sea cucumber Apostichopus japonicus based on fluorescence-labeled methylation-sensitive amplified polymorphism (F-MSAP) during aestivation.

    Science.gov (United States)

    Zhao, Ye; Chen, Muyan; Storey, Kenneth B; Sun, Lina; Yang, Hongsheng

    2015-03-01

    DNA methylation plays an important role in regulating transcriptional change in response to environmental stimuli. In the present study, DNA methylation levels of tissues of the sea cucumber Apostichopus japonicus were analyzed by the fluorescence-labeled methylation-sensitive amplified polymorphism (F-MSAP) technique over three stages of the aestivation cycle. Overall, a total of 26,963 fragments were amplified including 9112 methylated fragments among four sea cucumber tissues using 18 pairs of selective primers. Results indicated an average DNA methylation level of 33.79% for A. japonicus. The incidence of DNA methylation was different across tissue types in the non-aestivation stage: intestine (30.16%), respiratory tree (27.61%), muscle (27.94%) and body wall (56.25%). Our results show that hypermethylation accompanied deep-aestivation in A. japonicus, which suggests that DNA methylation may have an important role in regulating global transcriptional suppression during aestivation. Further analysis indicated that the main DNA modification sites were focused on intestine and respiratory tree tissues and that full-methylation but not hemi-methylation levels exhibited significant increases in the deep-aestivation stage. Copyright © 2014 Elsevier Inc. All rights reserved.

  18. Facile and sensitive determination of N-nitrosamines in food samples by high-performance liquid chromatography via combining fluorescent labeling with dispersive liquid-liquid microextraction.

    Science.gov (United States)

    Lu, Shuaimin; Wu, Di; Li, Guoliang; Lv, Zhengxian; Gong, Peiwei; Xia, Lian; Sun, Zhiwei; Chen, Guang; Chen, Xuefeng; You, Jinmao; Wu, Yongning

    2017-11-01

    The intake of N-nitrosamines (NAs) from foodstuffs is considered to be an important influence factor for several cancers. But the rapid and sensitive screening of NAs remains a challenge in the field of food safety. Inspired by that, a sensitive and rapid method was demonstrated for determination of five NAs (Nitrosopyrrolidine, Nitrosodimethylamine, Nitrosodiethylamine, Nitrosodipropylamine and Nitrosodibutylamine) using dispersive liquid-liquid microextraction (DLLME) followed by high-performance liquid chromatography with fluorescence detection (HPLC-FLD). The NAs were firstly denitrosated and labeled by 2-(11H-benzo[a]carbazol-11-yl) ethyl carbonochloridate (BCEC-Cl) and finally enriched by DLLME. Furthermore, the main DLLME conditions were optimized systematically. Under the optimal conditions, satisfactory limits of detection (LODs) were obtained with a range of 0.01-0.07ngg -1 , which were significantly lower than the reported methods. The developed method showed many merits including rapidity, simplicity, high sensitivity and excellent selectivity, which shows a broad prospect in food safety analysis. Copyright © 2017 Elsevier Ltd. All rights reserved.

  19. Comparing Avocado, Swamp Bay, and Camphortree as Hosts of Raffaelea lauricola Using a Green Fluorescent Protein (GFP)-Labeled Strain of the Pathogen.

    Science.gov (United States)

    Campbell, A S; Ploetz, R C; Rollins, J A

    2017-01-01

    Raffaelea lauricola, a fungal symbiont of the ambrosia beetle Xyleborus glabratus, causes laurel wilt in members of the Lauraceae plant family. North American species in the family, such as avocado (Persea americana) and swamp bay (P. palustris), are particularly susceptible to laurel wilt, whereas the Asian camphortree (Cinnamomum camphora) is relatively tolerant. To determine whether susceptibility is related to pathogen colonization, a green fluorescent protein-labeled strain of R. lauricola was generated and used to inoculate avocado, swamp bay, and camphortree. Trees were harvested 3, 10, and 30 days after inoculation (DAI), and disease severity was rated on a 1-to-10 scale. By 30 DAI, avocado and swamp bay developed significantly more severe disease than camphortree (mean severities of 6.8 and 5.5 versus 1.6, P < 0.003). The extent of xylem colonization was recorded as the percentage of lumena that were colonized by the pathogen. More xylem was colonized in avocado than camphortree (0.9% versus 0.1%, P < 0.03) but colonization in swamp bay (0.4%) did not differ significantly from either host. Although there were significant correlations between xylem colonization and laurel wilt severity in avocado (r = 0.74), swamp bay (r = 0.82), and camphortree (r = 0.87), even severely affected trees of all species were scarcely colonized by the pathogen.

  20. Studying the interaction between three synthesized heterocyclic sulfonamide compounds with hemoglobin by spectroscopy and molecular modeling techniques.

    Science.gov (United States)

    Naeeminejad, Samane; Assaran Darban, Reza; Beigoli, Sima; Saberi, Mohammad Reza; Chamani, Jamshidkhan

    2017-11-01

    The interaction between synthesized heterocyclic benzene sulfonamide compounds, N-(7-benzyl-56-biphenyl-2m-tolyl-7H-pyrrolo[23-d]pyrimidine-4-yl)-benzene sulfonamide (HBS 1 ), N-(7-benzyl-56-biphenyl-2-m-tolyl-7H-pyrrolo[23-d] pyrimidine-4-yl)-4-methyl- benzene sulfonamide (HBS 2 ), and N-(7-benzyl-56-biphenyl-2-m-tolyl-7H-pyrrolo[23-d]pyrimidine-4-yl)-4-chloro-benzene sulfonamide (HBS 3 ) with Hb was studied by fluorescence quenching, zeta potentional, circular dichroism, and molecular modeling techniques. The fluorescence spectroscopy experiments were performed in order to study the conformational changes, possibly due to a discrete reorganization of Trp residues during binding between HBS derivatives and Hb. The variation of the K SV value suggested that hydrophobic and electrostatic interactions were the predominant intermolecular forces stabilizing the complex. The K SV1 ans K SV2 values of HBS derivatives with Hb are .6 × 10 13 and 3 × 10 13  M -1 for Hb-HBS 1 , 1 × 10 13 and 4 × 10 13  M -1 for Hb-HBS 2 , .9 × 10 13 , and 6 × 10 13  M -1 for Hb-HBS 3 , respectively. The molecular distances between Hb and HBS derivatives in binary and ternary systems were estimated according to Förster's theory of dipole-dipole non-radiation energy transfer. The quantitative analysis data of circular dichroism spectra demonstrated that the binding of the three HBS derivatives to Hb induced conformational changes in Hb. Changes in the zeta potential of the Hb-HBS derivatives complexes demonstrated a hydrophobic adsorption of the anionic ligand onto the surface of Hb as well as both electrostatic and hydrophobic adsorption in the case of the complex. The modeling data thus confirmed the experimental results. This study is expected to provide important insight into the interaction of Hb with three HBS derivatives to use in various toxicological and therapeutic processes.

  1. Fluorescent multiplex cell flow systems and methods

    KAUST Repository

    Merzaban, Jasmeen; Abuelela, Ayman F.; Mohammad, Amal Jehad

    2017-01-01

    scanning system emits multiple electromagnetic wavelengths simultaneously it cause multiple fluorescent labels having different excitation wavelength maximums to fluoresce. The system can simultaneously capture real-time fluorescence images from at least

  2. Design of a single-step immunoassay principle based on the combination of an enzyme-labeled antibody release coating and a hydrogel copolymerized with a fluorescent enzyme substrate in a microfluidic capillary device.

    Science.gov (United States)

    Wakayama, Hideki; Henares, Terence G; Jigawa, Kaede; Funano, Shun-ichi; Sueyoshi, Kenji; Endo, Tatsuro; Hisamoto, Hideaki

    2013-11-21

    A combination of an enzyme-labeled antibody release coating and a novel fluorescent enzyme substrate-copolymerized hydrogel in a microchannel for a single-step, no-wash microfluidic immunoassay is demonstrated. This hydrogel discriminates the free enzyme-conjugated antibody from an antigen-enzyme-conjugated antibody immunocomplex based on the difference in molecular size. A selective and sensitive immunoassay, with 10-1000 ng mL(-1) linear range, is reported.

  3. In vivo fluorescence imaging of an orthotopic rat bladder tumor model indicates differential uptake of intravesically instilled near-infrared labeled 2-deoxyglucose analog by neoplastic urinary bladder tissues

    Science.gov (United States)

    Piao, Daqing; Davis, Carole A.; Hurst, Robert E.; Slaton, Joel W.

    2017-02-01

    Bladder cancer is one of the most expensive cancers to manage due to frequent recurrences requiring life-long surveillance and treatment. A near-infrared labeled 2-deoxy-d-glucose probe IRDye800CW-DG targeting glucose metabolism pathway has shown to enhance the sensitivity of diagnosing several types of cancers as tested on tumor models not including bladder tumor. This pilot study has explored differential uptake of intravesically administered IRDye800CW-DG in an orthotopic rat bladder tumor model. Twenty-five female Fischer rats were randomly grouped to four conditions: no-tumor-control (n=3), no-tumor-control intravesically instilled with IRDye800CWDG (n=6), rats bearing GFP-labeled AY-27 rat bladder urothelial cell carcinoma cells and washed with saline (n=5), and rats bearing AY-27 tumors and intravesically instilled with IRDye800CW-DG (n=11). Near-infrared fluorescence was measured from the opened bladder wall of anesthetized rat at an excitation wavelength of 750nm and an emission wavelength of 776nm, by using an in-house fluorescence imaging system. There is no statistically significant difference of the peak fluorescence intensity among the no-tumor-control bladders (n=3), the no-tumorcontrol bladders instilled with IRDye800CW-DG (n=6), and the GFP-labeled AY-27 treated bladders washed by saline (n=5). When compared to that of the no-tumor-control bladders instilled with IRDye800CW-DG (n=6), the fluorescence intensity of GFP-labeled AY-27 treated bladders instilled with IRDye800CW-DG and with histology confirmed neoplastic bladder tissue (n=11) was remarkably more intense (3.34 fold of over the former) and was also statistically significant (pbladder tissues suggests the potential for cystoscopy-adaptation to enhance diagnosis and guiding surgical management of flat urinary bladder cancer.

  4. Synthesis, Pharmacological Profile and Docking Studies of New Sulfonamides Designed as Phosphodiesterase-4 Inhibitors.

    Directory of Open Access Journals (Sweden)

    Isabelle Karine da Costa Nunes

    Full Text Available Prior investigations showed that increased levels of cyclic AMP down-regulate lung inflammatory changes, stimulating the interest in phosphodiesterase (PDE4 as therapeutic target. Here, we described the synthesis, pharmacological profile and docking properties of a novel sulfonamide series (5 and 6a-k designed as PDE4 inhibitors. Compounds were screened for their selectivity against the four isoforms of human PDE4 using an IMAP fluorescence polarized protocol. The effect on allergen- or LPS-induced lung inflammation and airway hyper-reactivity (AHR was studied in A/J mice, while the xylazine/ketamine-induced anesthesia test was employed as a behavioral correlate of emesis in rodents. As compared to rolipram, the most promising screened compound, 6a (LASSBio-448 presented a better inhibitory index concerning PDE4D/PDE4A or PDE4D/PDE4B. Accordingly, docking analyses of the putative interactions of LASSBio-448 revealed similar poses in the active site of PDE4A and PDE4C, but slight unlike orientations in PDE4B and PDE4D. LASSBio-448 (100 mg/kg, oral, 1 h before provocation, inhibited allergen-induced eosinophil accumulation in BAL fluid and lung tissue samples. Under an interventional approach, LASSBio-448 reversed ongoing lung eosinophilic infiltration, mucus exacerbation, peribronchiolar fibrosis and AHR by allergen provocation, in a mechanism clearly associated with blockade of pro-inflammatory mediators such as IL-4, IL-5, IL-13 and eotaxin-2. LASSBio-448 (2.5 and 10 mg/kg also prevented inflammation and AHR induced by LPS. Finally, the sulfonamide derivative was shown to be less pro-emetic than rolipram and cilomilast in the assay employed. These findings suggest that LASSBio-448 is a new PDE4 inhibitor with marked potential to prevent and reverse pivotal pathological features of diseases characterized by lung inflammation, such as asthma.

  5. Site-directed fluorescence labeling reveals a revised N-terminal membrane topology and functional periplasmic residues in the Escherichia coli cell division protein FtsK.

    Science.gov (United States)

    Berezuk, Alison M; Goodyear, Mara; Khursigara, Cezar M

    2014-08-22

    In Escherichia coli, FtsK is a large integral membrane protein that coordinates chromosome segregation and cell division. The N-terminal domain of FtsK (FtsKN) is essential for division, and the C terminus (FtsKC) is a well characterized DNA translocase. Although the function of FtsKN is unknown, it is suggested that FtsK acts as a checkpoint to ensure DNA is properly segregated before septation. This may occur through modulation of protein interactions between FtsKN and other division proteins in both the periplasm and cytoplasm; thus, a clear understanding of how FtsKN is positioned in the membrane is required to characterize these interactions. The membrane topology of FtsKN was initially determined using site-directed reporter fusions; however, questions regarding this topology persist. Here, we report a revised membrane topology generated by site-directed fluorescence labeling. The revised topology confirms the presence of four transmembrane segments and reveals a newly identified periplasmic loop between the third and fourth transmembrane domains. Within this loop, four residues were identified that, when mutated, resulted in the appearance of cellular voids. High resolution transmission electron microscopy of these voids showed asymmetric division of the cytoplasm in the absence of outer membrane invagination or visible cell wall ingrowth. This uncoupling reveals a novel role for FtsK in linking cell envelope septation events and yields further evidence for FtsK as a critical checkpoint of cell division. The revised topology of FtsKN also provides an important platform for future studies on essential interactions required for this process. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  6. A novel Tc-99m and fluorescence-labeled arginine-arginine-leucine-containing peptide as a multimodal tumor imaging agent in a murine tumor model.

    Science.gov (United States)

    Kim, Myoung Hyoun; Kim, Seul-Gi; Kim, Dae-Weung

    2018-06-15

    We developed a Tc-99m and TAMRA-labeled peptide, Tc-99m arginine-arginine-leucine (RRL) peptide (TAMRA-GHEG-ECG-RRL), to target tumor cells and evaluated the diagnostic performance of Tc-99m TAMRA-GHEG-ECG-RRL as a dual-modality imaging agent for tumor in a murine model. TAMRA-GHEG-ECG-RRL was synthesized using Fmoc solid-phase peptide synthesis. Binding affinity and in vitro cellular uptake studies were performed. Gamma camera imaging, biodistribution, and ex vivo imaging studies were performed in murine models with PC-3 tumors. Tumor tissue slides were prepared and analyzed with immunohistochemistry using confocal microscopy. After radiolabeling procedures with Tc-99m, Tc-99m TAMRA-GHEG-ECG-RRL complexes were prepared in high yield (>96%). The K d of Tc-99m TAMRA-GHEG-ECG-RRL determined by saturation binding was 41.7 ± 7.8 nM. Confocal microscopy images of PC-3 cells incubated with TAMRA-GHEG-ECG-RRL showed strong fluorescence in the cytoplasm. Gamma camera imaging revealed substantial uptake of Tc-99m TAMRA-GHEG-ECG-RRL in tumors. Tumor uptake was effectively blocked by the coinjection of an excess concentration of RRL. Specific uptake of Tc-99m TAMRA-GHEG-ECG-RRL was confirmed by biodistribution, ex vivo imaging, and immunohistochemistry stain studies. In conclusion, in vivo and in vitro studies revealed substantial uptake of Tc-99m TAMRA-GHEG-ECG-RRL in tumors. Tc-99m TAMRA-GHEG-ECG-RRL has potential as a dual-modality tumor imaging agent. Copyright © 2018 John Wiley & Sons, Ltd.

  7. In situ observation of the growth of biofouling layer in osmotic membrane bioreactors by multiple fluorescence labeling and confocal laser scanning microscopy.

    Science.gov (United States)

    Yuan, Bo; Wang, Xinhua; Tang, Chuyang; Li, Xiufen; Yu, Guanghui

    2015-05-15

    Since the concept of the osmotic membrane bioreactor (OMBR) was introduced in 2008, it has attracted growing interests for its potential applications in wastewater treatment and reclamation; however, the fouling mechanisms of forward osmosis (FO) membrane especially the development of biofouling layer in the OMBR are not yet clear. Here, the fouled FO membranes were obtained from the OMBRs on days 3, 8 and 25 in sequence, and then the structure and growing rule of the biofouling layer formed on the FO membrane samples were in-situ characterized by multiple fluorescence labeling and confocal laser scanning microscopy (CLSM). CLSM images indicated that the variations in abundance and distribution of polysaccharides, proteins and microorganisms in the biofouling layer during the operation of OMBRs were significantly different. Before the 8th day, their biovolume dramatically increased. Subsequently, the biovolumes of β-d-glucopyranose polysaccharides and proteins continued increasing and leveled off after 8 days, respectively, while the biovolumes of α-d-glucopyranose polysaccharides and microorganisms decreased. Extracellular polymeric substances (EPS) played a significant role in the formation and growth of biofouling layer, while the microorganisms were seldom detected on the upper fouling layer after 3 days. Based on the results obtained in this study, the growth of biofouling layer on the FO membrane surface in the OMBR could be divided into three stages. Initially, EPS was firstly deposited on the FO membrane surface, and then microorganisms associated with EPS located in the initial depositing layer to form clusters. After that, the dramatic increase of the clusters of EPS and microorganisms resulted in the quick growth of biofouling layer during the flux decline of the OMBR. However, when the water flux became stable in the OMBR, some microorganisms and EPS would be detached from the FO membrane surface. Copyright © 2015 Elsevier Ltd. All rights reserved.

  8. Review on enzyme-linked immunosorbent assays for sulfonamide residues in edible animal products.

    Science.gov (United States)

    Zhang, Hongyan; Wang, Shuo

    2009-10-31

    The current status of enzyme-linked immunosorbent assays (ELISAs) for sulfonamides in edible animal products is reviewed. The attention was focused on the design and synthesis of haptens, conjugation to carrier protein, production of antibody, application of homologous and heterologous systems, as well as the molecular modeling of the haptens and sulfonamides. Researches have shown that sulfonamides seem to be particularly resistant to attempts to produce broad specificity antibodies. By summarizing the available research on sulfonamide ELISAs, it is hoped that it can be considered as a basis for further investigation aimed at developing the most efficient approaches for detection.

  9. Biosolid-borne tetracyclines and sulfonamides in plants.

    Science.gov (United States)

    Mathews, Shiny; Reinhold, Dawn

    2013-07-01

    Tetracyclines and sulfonamides used in human and animal medicine are released to terrestrial ecosystems from wastewater treatment plants or by direct manure application. The interactions between plants and these antibiotics are numerous and complex, including uptake and accumulation, phytometabolism, toxicity responses, and degradation in the rhizosphere. Uptake and accumulation of antibiotics have been studied in plants such as wheat, maize, potato, vegetables, and ornamentals. Once accumulated in plant tissue, organic contaminants can be metabolized through a sequential process of transformation, conjugation through glycosylation and glutathione pathways, and ultimately sequestration into plant tissue. While studies have yet to fully elucidate the phytometabolism of tetracyclines and sulfonamides, an in-depth review of plant and mammalian studies suggest multiple potential transformation and conjugation pathways for tetracyclines and sulfonamides. The presence of contaminants in the vicinity or within the plants can elicit stress responses and defense mechanisms that can help tolerate the negative effects of contaminants. Antibiotics can change microbial communities and enzyme activity in the rhizosphere, potentially inducing microbial antibiotic resistance. On the other hand, the interaction of microbes and root exudates on pharmaceuticals in the rhizosphere can result in degradation of the parent molecule to less toxic compounds. To fully characterize the environmental impacts of increased antibiotic use in human medicine and animal production, further research is essential to understand the effects of different antibiotics on plant physiology and productivity, uptake, translocation, and phytometabolism of antibiotics, and the role of antibiotics in the rhizosphere.

  10. Fluorescent Metal-Organic Framework (MOF) as a Highly Sensitive and Quickly Responsive Chemical Sensor for the Detection of Antibiotics in Simulated Wastewater.

    Science.gov (United States)

    Zhu, Xian-Dong; Zhang, Kun; Wang, Yu; Long, Wei-Wei; Sa, Rong-Jian; Liu, Tian-Fu; Lü, Jian

    2018-02-05

    A Zn(II)-based fluorescent metal-organic framework (MOF) was synthesized and applied as a highly sensitive and quickly responsive chemical sensor for antibiotic detection in simulated wastewater. The fluorescent chemical sensor, denoted FCS-1, exhibited enhanced fluorescence derived from its highly ordered, 3D MOF structure as well as excellent water stability in the practical pH range of simulated antibiotic wastewater (pH = 3.0-9.0). Remarkably, FCS-1 was able to effectively detect a series of sulfonamide antibiotics via photoinduced electron transfer that caused detectable fluorescence quenching, with fairly low detection limits. Two influences impacting measurements related to wastewater treatment and water quality monitoring, the presence of heavy-metal ions and the pH of solutions, were studied in terms of fluorescence quenching, which was nearly unaffected in sulfonamide-antibiotic detection. Additionally, the effective detection of sulfonamide antibiotics was rationalized by the theoretical computation of the energy bands of sulfonamide antibiotics, which revealed a good match between the energy bands of FCS-1 and sulfonamide antibiotics, in connection with fluorescence quenching in this system.

  11. Fluorescence technique application of X-ray in labeling with Mn, Sr and Cu, of the parasitoid and host: Muscidifuax uniraptor Kogan and Legner, 1970 (Hymenoptera: Pteromalidae) and Musca domestica L., 1758 (Diptera: Muscidae)

    International Nuclear Information System (INIS)

    Itepan, Natanael Marcio

    2003-01-01

    The objective of this work was to develop the methodology of the labeling adult of Musca domestica and Muscidifurax uniraptor using the elements Mn, Sr and Cu with the use of x-ray fluorescence. This work was carried out in the Laboratory of Biological Control of House Flies, 'Eduardo Hiroshi Mizumoto', of the 'Entomology, Phytopatology and Zoology Department of the College of Agriculture 'Luiz de Queiroz' ESALQ/USP), and the Division of Methods the Development and Nuclear Analytics Techniques, of CENA/USP, Piracicaba, Sao Paulo, Brazil. The larvae was removed to the labeled diet with increasing level of the elements Mn, Sr and Cu. The levels tested for all element were: 0 (control); 0,25; 0,50; 1,00; 2,00; 4,00; 8,00; 16,00; 32,00 and 64,00 mg/gr of diet. Labeled pupae with 1,00 to 4,00 (Mn) and 1,00 (Sr and Cu) mg/gr of diet were tested for the pupal parasitoid M. uniraptor. The concentration quantity of the chemical elements was realized by the Analytical Technique denominated (EDXRF) energy dispersive X-ray fluorescence. Concentrations of 2,00 (Mn) and 1,00 (Sr) supplemented to the diet of M. domestica were sufficient for the adult insect labeled, however, not affecting its life expectancy. Pupae originated from the larvae of M. domestica treated with dose of 2,00 (Mn) and 1,00 (Sr and Cu) mg supplemented to the diet, and used as hosts of the parasitoid M. uniraptor, affected the viability of the immature phase and did not label the adults. (author)

  12. A novel single fluorophore-labeled double-stranded oligonucleotide probe for fluorescence-enhanced nucleic acid detection based on the inherent quenching ability of deoxyguanosine bases and competitive strand-displacement reaction.

    Science.gov (United States)

    Zhang, Yingwei; Tian, Jingqi; Li, Hailong; Wang, Lei; Sun, Xuping

    2012-01-01

    We develop a novel single fluorophore-labeled double-stranded oligonucleotide (OND) probe for rapid, nanostructure-free, fluorescence-enhanced nucleic acid detection for the first time. We further demonstrate such probe is able to well discriminate single-base mutation in nucleic acid. The design takes advantage of an inherent quenching ability of guanine bases. The short strand of the probe is designed with an end-labeled fluorophore that is placed adjacent to two guanines as the quencher located on the long opposite strand, resulting in great quenching of dye fluorescence. In the presence of a target complementary to the long strand of the probe, a competitive strand-displacement reaction occurs and the long strand forms a more stable duplex with the target, resulting in the two strands of the probe being separated from each other. As a consequence of this displacement, the fluorophore and the quencher are no longer in close proximity and dye fluorescence increases, signaling the presence of target.

  13. Hazard of Sulfonamides and Detection Technology Research Progress

    Science.gov (United States)

    Jiang, Jiahui; Wang, Guangyu

    2017-12-01

    As a kind of widely used antibiotic with typical characteristics, sulfonamides have been greatly applied in clinical medicine for long time. It can’t be effectively treated by pollutant disposal system during pharmaceutical process and utilization and will be discharged into natural environment to be one of the antibiotics with great effect. This kind of substance is difficult to be biodegraded and will be easy to accumulate in the environment, generating huge eco-toxicological effect with significant mutagenicity and teratogenic effect. It is the severe threat for ecological balance, human health and drinking water safety. Its environmental behavior and detection technology attract extensive attention home and abroad.

  14. [Interaction Between Sulfonamide Antibiotics Fates and Chicken Manure Composting].

    Science.gov (United States)

    Lin, Hui; Wang, Jian-mei; Sun, Wan-chun; Fu, Jian-rong; Chen, Hong-jin; Ma, Jun-wei

    2016-05-15

    Based on aerobic manure composting with or without the addition of a mixture of sulfadimethoxine SM2 and sulfamonomethoxine SMM (1:1, m/m), changes in the physic-chemical properties of manure compost, the microbial community physiological profiles, the antibiotics concentration and the abundances of five antibiotic resistance genes (ARGs) during the composting were tracked. The results indicated that the introduction of sulfonamide antibiotics led to inhibition on the basal respiration of manure compost during the early composting period, delayed the formation of thermophilic temperature and reduced the conversion of nutrients such as organic matter, ammonia nitrogen and nitrate nitrogen. Meanwhile, the introduction of sulfonamide antibiotics dramatically affected the physiological profile of microbial community in manure in the middle stage of composting. HPLC-MS/MS results showed that both SMM and SM2 in manure were completely degraded within 14 days, while the degradation rate of SMM was faster than that of SM2. For both composting treatments with or without addition of exogenous antibiotics, the relative abundance of sull and sul2 showed an initial decline in the first 14 or 21 days and a slight increase thereafter. The addition of exogenous antibiotics showed insignificant enhancement on increasing the relative abundance of sul1 and IntI1 in manure, but resulted in an apparent increase in sul2 relative abundance. Although the fates of tetQ and tetW during composting were different from that of sulfonamide ARGs, the introduction of sulfonamide antibiotics into manure increased the relative abundance of tetracycline ARGs. Redundancy analysis indicated that composting temperature correlated negatively with sul1, sul2 and IntI1 relative abundance in manure but had no obvious relationship with tetQ and tetW relative abundance. All the ARGs detected in this work correlated negatively with C/N ratio and the nitrate nitrogen concentration of manure compost but

  15. Multiresidue determination of fluoroquinolone, sulfonamide, trimethoprim, and chloramphenicol antibiotics in urban waters in China.

    Science.gov (United States)

    Peng, Xianzhi; Tan, Jianhua; Tang, Caiming; Yu, Yiyi; Wang, Zhendi

    2008-01-01

    A feasible method has been optimized to simultaneously determine multiclass antibiotic residues, including sulfonamides, fluoroquinolones, trimethoprim, and chloramphenicol in urban riverine water and wastewater by off-line solid phase extraction and high-performance liquid chromatography coupled with a diode-array ultraviolet detector and a fluorescence detector. Internal standard and standard addition methods were used in combination to identify and quantify these antibiotics to compensate for the matrix interference. The method quantification limits (MQLs) were determined to be 0.035 to 0.100 microg/L and 0.100 to 0.300 microg/L for the riverine water and wastewater, respectively. Recoveries of the investigated antibiotics ranged from 63 to 126%. Sulfamethoxazole was the most frequently detected antibiotic residue in Guangzhou section of the Major Pearl River, South China, with a maximum level of 0.510 microg/L. Fluoroquinolone antibiotics were relatively less detected with a maximum level of 0.459 microg/L. The maximum concentration of sulfamethoxazole reached 5.597 microg/L in the raw wastewater from a large-scale sewage treatment plant in Guangzhou city. Around 30% of sulfamethoxazole might survive the primary clarification and biotreatment processes in the sewage treatment plant. None of the investigated antibiotics have been found above MQLs in the final effluent after chlorine disinfection.

  16. 3D restoration microscopy improves quantification of enzyme-labeled fluorescence-based single-cell phosphatase activity in plankton. Cytometry Part A, 85A: 841–853

    Czech Academy of Sciences Publication Activity Database

    Diaz-de-Quijano, D.; Palacios, P.; Horňák, Karel; Felip, M.

    85A, č. 10 (2014), s. 841-853 ISSN 1552-4922 Institutional support: RVO:60077344 Keywords : 3D fluorescence microscopy * deconvolution * ELF phosphate * phosphatase activity * phytoplankton Subject RIV: EE - Microbiology, Virology Impact factor: 2.928, year: 2014

  17. A potential fluorescent probe: Maillard reaction product from glutathione and ascorbic acid for rapid and label-free dual detection of Hg(2+) and biothiols.

    Science.gov (United States)

    Dong, Jiang Xue; Song, Xiao Fang; Shi, Yan; Gao, Zhong Feng; Li, Bang Lin; Li, Nian Bing; Luo, Hong Qun

    2016-07-15

    Maillard reactions and their fluorescent products have drawn much attention in the fields of food and life science, however, the application of fluorescent products separated from the reaction as an indicator for detection of certain substances in sensor field has not been mentioned. In this article, we report on an easy-to-synthesize and water-soluble fluorescent probe separated from the typical Maillard reaction products of glutathione and ascorbic acid, with excellent stability and high quantum yield (18.2%). The further application of the probe has been explored for dual detection of Hg(2+) and biothiols including cysteine, homocysteine, and glutathione, which is based on Hg(2+)-induced fluorescence quenching of the Maillard reaction fluorescent products (MRFPs) and the fluorescence recovery as the introduction of biothiols. This sensing system exhibits a good selectivity and sensitivity, and the linear ranges for Hg(2+), cysteine, homocysteine, and glutathione are 0.05-12, 0.5-10, 0.3-20, and 0.3-20μM, respectively. The detection limits for Hg(2+), cysteine, homocysteine, and glutathione are 22, 47, 96, and 30nM at a signal-to-noise ratio of 3, respectively. Furthermore, the practical applications of this sensor for Hg(2+) and biothiols determination in water samples and human plasma sample have been demonstrated with satisfactory results. Copyright © 2016 Elsevier B.V. All rights reserved.

  18. Enzymatic Transformation and Bonding of Sulfonamide Antibiotics to Model Humic Substances

    Directory of Open Access Journals (Sweden)

    J. Schwarz

    2015-01-01

    Full Text Available Sulfonamides are consumed as pharmaceutical antibiotics and reach agricultural soils with excreta used as fertilizer. Subsequently, nonextractable residues rapidly form in soil, which has been researched in a couple of studies. To further elucidate conditions, strength, and mechanisms of the fixation to soil humic substances, three selected sulfonamides were investigated using the biochemical oligomerization of substituted phenols as a model for the humification process. Catechol, guaiacol, and vanillin were enzymatically reacted using laccase from Trametes versicolor. In the presence of the substituted phenols alone, the concentration of sulfonamides decreased. This decrease was even more pronounced when additional laccase was present. Upon the enzymatic oligomerization of the substituted phenols to a humic-like structure the sulfonamides were sorbed, transformed, sequestered, and nonextractable bound. Sulfonamides were transformed depending on their molecular properties. Fractions of different bonding strength were determined using a sequential extraction procedure. Isolated nonextractable products were analyzed by chromatographic, spectroscopic, and calorimetric methods to identify coupling and bonding mechanisms of the sulfonamides. Differential scanning calorimetry measurements suggested cross-linking of such incorporated sulfonamides in humic oligomers. Nuclear magnetic resonance spectroscopy measurements showed clear differences between the vanillin-sulfapyridine oligomer and the parent sulfapyridine indicating bound residue formation through covalent binding.

  19. Molecular dynamics-assisted pharmacophore modeling of caspase-3-isatin sulfonamide complex: Recognizing essential intermolecular contacts and features of sulfonamide inhibitor class for caspase-3 binding.

    Science.gov (United States)

    Kumar, Sivakumar Prasanth; Patel, Chirag N; Jha, Prakash C; Pandya, Himanshu A

    2017-12-01

    The identification of isatin sulfonamide as a potent small molecule inhibitor of caspase-3 had fuelled the synthesis and characterization of the numerous sulfonamide class of inhibitors to optimize for potency. Recent works that relied on the ligand-based approaches have successfully shown the regions of optimizations for sulfonamide scaffold. We present here molecular dynamics-based pharmacophore modeling of caspase-3-isatin sulfonamide crystal structure, to elucidate the essential non-covalent contacts and its associated pharmacophore features necessary to ensure caspase-3 optimal binding. We performed 20ns long dynamics of this crystal structure to extract global conformation states and converted into structure-based pharmacophore hypotheses which were rigorously validated using an exclusive focussed library of experimental actives and inactives of sulfonamide class by Receiver Operating Characteristic (ROC) statistic. Eighteen structure-based pharmacophore hypotheses with better sensitivity and specificity measures (>0.6) were chosen which collectively showed the role of pocket residues viz. Cys163 (S 1 sub-site; required for covalent and H bonding with Michael acceptor of inhibitors), His121 (S 1 ; π stack with bicyclic isatin moiety), Gly122 (S 1 ; H bond with carbonyl oxygen) and Tyr204 (S 2 ; π stack with phenyl group of the isatin sulfonamide molecule) as stringent binding entities for enabling caspase-3 optimal binding. The introduction of spatial pharmacophore site points obtained from dynamics-based pharmacophore models in a virtual screening strategy will be helpful to screen and optimize molecules belonging to sulfonamide class of caspase-3 inhibitors. Copyright © 2017 Elsevier Ltd. All rights reserved.

  20. Ultrafast fluorescence of photosynthetic crystals and light-harvesting complexes

    NARCIS (Netherlands)

    Oort, van B.F.

    2008-01-01

    This thesis focuses on the study of photosynthetic pigment protein complexes using time resolved fluorescence techniques. Fluorescence spectroscopy often requires attaching fluorescent labels to the proteins under investigation. With photosynthetic proteins this is not necessary, because these

  1. In situ fluorescence activation of DNA-silver nanoclusters as a label-free and general strategy for cell nucleus imaging.

    Science.gov (United States)

    Li, Duo; Qiao, Zhenzhen; Yu, Yanru; Tang, Jinlu; He, Xiaoxiao; Shi, Hui; Ye, Xiaosheng; Lei, Yanli; Wang, Kemin

    2018-01-25

    A facile, general and turn-on nucleus imaging strategy was first developed based on in situ fluorescence activation of C-rich dark silver nanoclusters by G-rich telomeres. After a simple incubation without washing, nanoclusters could selectively stain the nucleus with intense red luminescence, which was confirmed using fixed/living cells and several cell lines.

  2. Conjugated polymer with carboxylate groups-Hg2 + system as a turn-on fluorescence probe for label-free detection of cysteine-containing compounds

    Science.gov (United States)

    Mi, Hongyu; Guan, Mingming; Liu, Jilin; Shan, Hongyan; Fei, Qiang; Huan, Yanfu; Feng, Guodong

    2017-04-01

    In this work, a turn on fluorescent sensor, based on Hg2 + coordination conjugated polymer, was developed to detect cysteine-containing compounds. The fluorescence of conjugated polymer (poly(2,5-bis (sodium 4-oxybutyrate) -1,4 - phenylethynylene-alt-1,4-phenyleneethynylene; PPE-OBS) would be quenched by Hg2 + because of the coordination-induced aggregation and electron transfers of PPE-OBS toward Hg2 +. When there were some cysteine-containing compounds in PPE-OBS-Hg2 + system, the fluorescence of PPE-OBS would be recovered. It indicated that the PPE-OBS-Hg2 + system could be used to detect cysteine-containing compounds. Under the optimized conditions, the experiment results showed that there were particularly linear range, high sensitivity and selectivity over other amino acids. The limit of detection (LOD) of cysteine (Cys), homocysteine (Hcy) and glutathione (GSH) were 0.725 μmol L- 1, 0.982 μmol L- 1 and 1.21 μmol L- 1 by using this sensor. In addition, Cys standard recovery in several green tea drink and honey samples was also demonstrated. The recovery of Cys was range from 96.3 to 105.0% and RSD was less than 3.25%. The satisfactory results demonstrated that the proposed method could be as a potential fluorescent method for determining cysteine-containing compounds in real samples.

  3. Umbilical cord mesenchymal stem cells labeled with multimodal iron oxide nanoparticles with fluorescent and magnetic properties: application for in vivo cell tracking

    Science.gov (United States)

    Sibov, Tatiana T; Pavon, Lorena F; Miyaki, Liza A; Mamani, Javier B; Nucci, Leopoldo P; Alvarim, Larissa T; Silveira, Paulo H; Marti, Luciana C; Gamarra, LF

    2014-01-01

    Here we describe multimodal iron oxide nanoparticles conjugated to Rhodamine-B (MION-Rh), their stability in culture medium, and subsequent validation of an in vitro protocol to label mesenchymal stem cells from umbilical cord blood (UC-MSC) with MION-Rh. These cells showed robust labeling in vitro without impairment of their functional properties, the viability of which were evaluated by proliferation kinetic and ultrastructural analyzes. Thus, labeled cells were infused into striatum of adult male rats of animal model that mimic late onset of Parkinson’s disease and, after 15 days, it was observed that cells migrated along the medial forebrain bundle to the substantia nigra as hypointense spots in T2 magnetic resonance imaging. These data were supported by short-term magnetic resonance imaging. Studies were performed in vivo, which showed that about 5 × 105 cells could be efficiently detected in the short term following infusion. Our results indicate that these labeled cells can be efficiently tracked in a neurodegenerative disease model. PMID:24531365

  4. Umbilical cord mesenchymal stem cells labeled with multimodal iron oxide nanoparticles with fluorescent and magnetic properties: application for in vivo cell tracking.

    Science.gov (United States)

    Sibov, Tatiana T; Pavon, Lorena F; Miyaki, Liza A; Mamani, Javier B; Nucci, Leopoldo P; Alvarim, Larissa T; Silveira, Paulo H; Marti, Luciana C; Gamarra, Lf

    2014-01-01

    Here we describe multimodal iron oxide nanoparticles conjugated to Rhodamine-B (MION-Rh), their stability in culture medium, and subsequent validation of an in vitro protocol to label mesenchymal stem cells from umbilical cord blood (UC-MSC) with MION-Rh. These cells showed robust labeling in vitro without impairment of their functional properties, the viability of which were evaluated by proliferation kinetic and ultrastructural analyzes. Thus, labeled cells were infused into striatum of adult male rats of animal model that mimic late onset of Parkinson's disease and, after 15 days, it was observed that cells migrated along the medial forebrain bundle to the substantia nigra as hypointense spots in T2 magnetic resonance imaging. These data were supported by short-term magnetic resonance imaging. Studies were performed in vivo, which showed that about 5 × 10(5) cells could be efficiently detected in the short term following infusion. Our results indicate that these labeled cells can be efficiently tracked in a neurodegenerative disease model.

  5. Cerebellar projections to the red nucleus and inferior olive originate from separate populations of neurons in the rat: A non-fluorescent double labeling study

    NARCIS (Netherlands)

    T.M. Teune (Thea); J. van der Burg (Johannes); T.J.H. Ruigrok (Tom)

    1995-01-01

    textabstractIn the rat, the extent of collateralization of projections from the cerebellar nuclei to the red nucleus and inferior olive was investigated using a retrograde double labeling technique. The combination of tracers selected, cholera toxin-β-subunit and WGA-BSA-gold, not only enabled the

  6. Modularity and three-dimensional isostructurality of novel synthons in sulfonamide-lactam cocrystals.

    Science.gov (United States)

    Bolla, Geetha; Mittapalli, Sudhir; Nangia, Ashwini

    2015-07-01

    The design of novel supramolecular synthons for functional groups relevant to drugs is an essential prerequisite for applying crystal engineering in the development of novel pharmaceutical cocrystals. It has been convincingly shown over the past decade that molecular level control and modulation can influence the physicochemical properties of drug cocrystals. Whereas considerable advances have been reported on the design of cocrystals for carboxylic acids and carboxamide functional groups, the sulfonamide group, which is a cornerstone of sulfa drugs, is relatively unexplored for reproducible heterosynthon-directed crystal engineering. The occurrence of synthons and isostructurality in sulfonamide-lactam cocrystals (SO2NH2⋯CONH hydrogen bonding) is analyzed to define a strategy for amide-type GRAS (generally recognized as safe) coformers with sulfonamides. Three types of supramolecular synthons are identified for the N-H donor of sulfonamide hydrogen bonding to the C=O acceptor of amide. Synthon 1: catemer synthon C 2 (1)(4) chain motif, synthon 2: dimer-cyclic ring synthon R 2 (2)(8)R 4 (2)(8) motifs, and synthon 3: dimer-catemer synthon of R 2 (2)(8)C 1 (1)(4)D notation. These heterosynthons of the cocrystals observed in this study are compared with the N-H⋯O dimer R 2 (2)(8) ring and C(4) chain motifs of the individual sulfonamide structures. The X-ray crystal structures of sulfonamide-lactam cocrystals exhibit interesting isostructurality trends with the same synthon being present. One-dimensional, two-dimensional and three-dimensional isostructurality in crystal structures is associated with isosynthons and due to their recurrence, novel heterosynthons for sulfonamide cocrystals are added to the crystal engineer's toolkit. With the predominance of sulfa drugs in medicine, these new synthons provide rational strategies for the design of binary and potentially ternary cocrystals of sulfonamides.

  7. Selective Access to Heterocyclic Sulfonamides and Sulfonyl Fluorides via a Parallel Medicinal Chemistry Enabled Method.

    Science.gov (United States)

    Tucker, Joseph W; Chenard, Lois; Young, Joseph M

    2015-11-09

    A sulfur-functionalized aminoacrolein derivative is used for the efficient and selective synthesis of heterocyclic sulfonyl chlorides, sulfonyl fluorides, and sulfonamides. The development of a 3-step parallel medicinal chemistry (PMC) protocol for the synthesis of pyrazole-4-sulfonamides effectively demonstrates the utility of this reagent. This reactivity was expanded to provide rapid access to other heterocyclic sulfonyl fluorides, including pyrimidines and pyridines, whose corresponding sulfonyl chlorides lack suitable chemical stability.

  8. Label-free turn-on fluorescent detection of melamine based on the anti-quenching ability of Hg 2+ to gold nanoclusters.

    Science.gov (United States)

    Dai, Haichao; Shi, Yan; Wang, Yilin; Sun, Yujing; Hu, Jingting; Ni, Pengjuan; Li, Zhuang

    2014-03-15

    In this work, we proposed a facile, environmentally friendly and cost-effective assay for melamine with BSA-stabilized gold nanoclusters (AuNCs) as a fluorescence reader. Melamine, which has a multi-nitrogen heterocyclic ring, is prone to coordinate with Hg(2+). This property causes the anti-quenching ability of Hg(2+) to AuNCs through decreasing the metallophilic interaction between Hg(2+) and Au(+). By this method, detection limit down to 0.15 µM is obtained, which is approximately 130 times lower than that of the US food and Drug Administration estimated melamine safety limit of 20 µM. Furthermore, several real samples spiked with melamine, including raw milk and milk powder, are analyzed using the sensing system with excellent recoveries. This gold-nanocluster-based fluorescent method could find applications in highly sensitive detection of melamine in real samples. © 2013 Elsevier B.V. All rights reserved.

  9. Label-Free Platform for MicroRNA Detection Based on the Fluorescence Quenching of Positively Charged Gold Nanoparticles to Silver Nanoclusters.

    Science.gov (United States)

    Miao, Xiangmin; Cheng, Zhiyuan; Ma, Haiyan; Li, Zongbing; Xue, Ning; Wang, Po

    2018-01-16

    A novel strategy was developed for microRNA-155 (miRNA-155) detection based on the fluorescence quenching of positively charged gold nanoparticles [(+)AuNPs] to Ag nanoclusters (AgNCs). In the designed system, DNA-stabilized Ag nanoclusters (DNA/AgNCs) were introduced as fluorescent probes, and DNA-RNA heteroduplexes were formed upon the addition of target miRNA-155. Meanwhile, the (+)AuNPs could be electrostatically adsorbed on the negatively charged single-stranded DNA (ssDNA) or DNA-RNA heteroduplexes to quench the fluorescence signal. In the presence of duplex-specific nuclease (DSN), DNA-RNA heteroduplexes became a substrate for the enzymatic hydrolysis of the DNA strand to yield a fluorescence signal due to the diffusion of AgNCs away from (+)AuNPs. Under the optimal conditions, (+)AuNPs displayed very high quenching efficiency to AgNCs, which paved the way for ultrasensitive detection with a low detection limit of 33.4 fM. In particular, the present strategy demonstrated excellent specificity and selectivity toward the detection of target miRNA against control miRNAs, including mutated miRNA-155, miRNA-21, miRNA-141, let-7a, and miRNA-182. Moreover, the practical application value of the system was confirmed by the evaluation of the expression levels of miRNA-155 in clinical serum samples with satisfactory results, suggesting that the proposed sensing platform is promising for applications in disease diagnosis as well as the fundamental research of biochemistry.

  10. Umbilical cord mesenchymal stem cells labeled with multimodal iron oxide nanoparticles with fluorescent and magnetic properties: application for in vivo cell tracking

    Directory of Open Access Journals (Sweden)

    Sibov TT

    2014-01-01

    Full Text Available Tatiana T Sibov,1,2 Lorena F Pavon,1 Liza A Miyaki,1 Javier B Mamani,1 Leopoldo P Nucci,1,2 Larissa T Alvarim,1,3 Paulo H Silveira,1 Luciana C Marti,1 LF Gamarra1–31Hospital Israelita Albert Einstein, São Paulo, Brazil; 2Departamento de Neurologia e Neurociências, Universidade Federal de São Paulo, São Paulo, Brazil; 3Faculdade de Ciências Médicas da Santa Casa de São Paulo, São Paulo, BrazilAbstract: Here we describe multimodal iron oxide nanoparticles conjugated to Rhodamine-B (MION-Rh, their stability in culture medium, and subsequent validation of an in vitro protocol to label mesenchymal stem cells from umbilical cord blood (UC-MSC with MION-Rh. These cells showed robust labeling in vitro without impairment of their functional properties, the viability of which were evaluated by proliferation kinetic and ultrastructural analyzes. Thus, labeled cells were infused into striatum of adult male rats of animal model that mimic late onset of Parkinson's disease and, after 15 days, it was observed that cells migrated along the medial forebrain bundle to the substantia nigra as hypointense spots in T2 magnetic resonance imaging. These data were supported by short-term magnetic resonance imaging. Studies were performed in vivo, which showed that about 5 × 105 cells could be efficiently detected in the short term following infusion. Our results indicate that these labeled cells can be efficiently tracked in a neurodegenerative disease model.Keywords: mesenchymal stem cells, multimodal iron oxide nanoparticles, Rhodamine, magnetic resonance imaging, Parkinson's disease

  11. Multicapillary SDS-gel electrophoresis for the analysis of fluorescently labeled mAb preparations: a high throughput quality control process for the production of QuantiPlasma and PlasmaScan mAb libraries.

    Science.gov (United States)

    Székely, Andrea; Szekrényes, Akos; Kerékgyártó, Márta; Balogh, Attila; Kádas, János; Lázár, József; Guttman, András; Kurucz, István; Takács, László

    2014-08-01

    Molecular heterogeneity of mAb preparations is the result of various co- and post-translational modifications and to contaminants related to the production process. Changes in molecular composition results in alterations of functional performance, therefore quality control and validation of therapeutic or diagnostic protein products is essential. A special case is the consistent production of mAb libraries (QuantiPlasma™ and PlasmaScan™) for proteome profiling, quality control of which represents a challenge because of high number of mAbs (>1000). Here, we devise a generally applicable multicapillary SDS-gel electrophoresis process for the analysis of fluorescently labeled mAb preparations for the high throughput quality control of mAbs of the QuantiPlasma™ and PlasmaScan™ libraries. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  12. Electronically tuned sulfonamide-based probes with ultra-sensitivity for Ga"3"+ or Al"3"+ detection in aqueous solution

    International Nuclear Information System (INIS)

    Kumar, Ashwani; Chae, Pil Seok

    2017-01-01

    Three electronically tuned fluorescent probes (1–3) were synthesized by conjugating a fluorescent unit to N,N-bis-(hydroxyethyl)ethylenediamine. Probe 1 bearing an electron-deficient naphthalenedimide unit did not give a fluorescence response to the presence of various metal ions including monovalent metal ions (Na"+, K"+, and Ag"+), divalent metal ions (Ca"2"+, Cd"2"+, Co"2"+, Ni"2"+, Cu"2"+, Hg"2"+, Pb"2"+, and Zn"2"+) and trivalent metal ions (Al"3"+, Ga"3"+, Fe"3"+, and Cr"3"+) in an aqueous solution. By contrast, probes 2 and 3 possessing 1,8-naphthalimide and pyrene fluorophores, respectively, exhibited selective fluorescent “OFF-ON” behaviors as a result of Ga"3"+/Al"3"+ binding among the diverse metal ions, suggesting the importance of fluorophore electronic character with regard to metal ion sensing. The ethylenediamine analog of probe 3, corresponding to probe 4, was unable to yield a significant change in fluorescence intensity in the presence of any metal ions tested here, revealing the essential role of two hydroxyl groups for metal ion binding. A high association constant of K_a = 2.99 × 10"5 M"−"1 was obtained for probe 3 with Ga"3"+, with a limit of detection (LOD) of 10 nM. This LOD is the lowest value known for Ga"3"+ detection using chemical sensors. Along with an increase in aggregate sizes, PET suppression of probes upon metal ion binding was the primary contributor to the enhancement in fluorescence emission necessary for the sensitive detection of the target ions. The probe-metal ion complexes were fully characterized via TEM, FE-SEM, "1H NMR, fluorescence spectroscopy techniques and DFT calculations. - Highlights: • Three electronically tuned sulfonamide-based probes (probes 1, 2, and 3) were developed for metal ion-sensing. • Probes 2 and 3 exhibited AIE behavior with increasing water-content. • Probes 2 and 3 displayed a selective fluorescence “OFF-ON“ behavior for Ga"3"+ detection with the LOD of 10 nM. • PET

  13. Cryopreservation of green fluorescent protein (GFP)-labeled primordial germ cells with GFP fused to the 3' untranslated region of the nanos gene by vitrification of Japanese eel (Anguilla japonica) somite stage embryos.

    Science.gov (United States)

    Kawakami, Y; Ishihara, M; Saito, T; Fujimoto, T; Adachi, S; Arai, K; Yamaha, E

    2012-12-01

    Primordial germ cells (PGC) are the only cell type in developing embryos with the potential to transmit genetic information to the next generation. In this study, PGC of Japanese eel (Anguilla japonica) were visualized by injection of mRNA synthesized from a construct carrying the green fluorescent protein (GFP) gene fused to the 3' untranslated region of the Japanese eel nanos gene. We investigated the feasibility of cryopreserving Japanese eel PGC by vitrification of dechorionated whole somite stage embryos. The GFP-labeled PGC were rapidly cooled using liquid nitrogen after exposure to a pretreatment solution containing 1.5 M cryoprotectant (methanol, dimethyl sulfoxide, and glycerol for 10 min and ethylene glycol for 10, 20, and 30 min) and a vitrification solution containing 3 M cryoprotectant and 0.5 M sucrose for 1, 5, and 10 min. Ethylene glycerol is an effective cryoprotectant for embryonic cells and shows no evidence of ice formation after thawing. Vitrified and thawed PGC were transplanted into blastula stage embryos from zebrafish (Danio rerio). The GFP-labeled PGC migrated toward the host gonadal ridge, suggesting maintenance of their normal migration motility. These techniques may assist in achieving inter- and intraspecies germ-line chimers using donor Japanese eel PGC.

  14. A label-free ultrasensitive fluorescence detection of viable Salmonella enteritidis using enzyme-induced cascade two-stage toehold strand-displacement-driven assembly of G-quadruplex DNA.

    Science.gov (United States)

    Zhang, Peng; Liu, Hui; Ma, Suzhen; Men, Shuai; Li, Qingzhou; Yang, Xin; Wang, Hongning; Zhang, Anyun

    2016-06-15

    The harm of Salmonella enteritidis (S. enteritidis ) to public health mainly by contaminating fresh food and water emphasizes the urgent need for rapid detection techniques to help control the spread of the pathogen. In this assay, an newly designed capture probe complex that contained specific S. enteritidis-aptamer and hybridized signal target sequence was used for viable S. enteritidis recognition directly. In the presence of the target S. enteritidis, single-stranded target sequences were liberated and initiated the replication-cleavage reaction, producing numerous G-quadruplex structures with a linker on the 3'-end. And then, the sensing system took innovative advantage of quadratic linker-induced strand-displacement for the first time to release target sequence in succession, leading to the cyclic reuse of the target sequences and cascade signal amplification, thereby achieving the successive production of G-quadruplex structures. The fluorescent dye, N-Methyl mesoporphyrin IX, binded to these G-quadruplex structures and generated significantly enhanced fluorescent signals to achieve highly sensitive detection of S. enteritidis down to 60 CFU/mL with a linear range from 10(2) to 10(7)CFU/mL. By coupling the cascade two-stage target sequences-recyclable toehold strand-displacement with aptamer-based target recognition successfully, it is the first report on a novel non-label, modification-free and DNA extraction-free ultrasensitive fluorescence biosensor for detecting viable S. enteritidis directly, which can discriminate from dead S. enteritidis. Copyright © 2016 Elsevier B.V. All rights reserved.

  15. Binding of fluorescently labeled cholera toxin subunit B to glycolipids in the human submandibular gland and inhibition of binding by periodate oxidation and by galactose

    DEFF Research Database (Denmark)

    Kirkeby, S

    2016-01-01

    FITC-labeled cholera toxin subunit B (CTB) stained the surfaces of cells of mucous acini in the submandibular gland. CTB, also called choleragenoid, binds to the GM1 glycolipid in the cell membrane. The binding in most acini was inhibited by periodic acid oxidation of the sections, while some acini...... to the internal galactose residue linked to GalNAc, as in the GM1 glycolipid. Inhibition of the GM1 receptor binding to cholera toxin has potential for protection of humans against cholera. Galactose and agents that modify sialic acid inhibit the accessibility of the toxin to the GM1 carbohydrate receptor. Human...

  16. Nitrotriazole- and imidazole-based amides and sulfonamides as antitubercular agents.

    Science.gov (United States)

    Papadopoulou, Maria V; Bloomer, William D; Rosenzweig, Howard S; Arena, Alexander; Arrieta, Francisco; Rebolledo, Joseph C J; Smith, Diane K

    2014-11-01

    Twenty-three 3-nitrotriazole-based and 2-nitroimidazole-based amides and sulfonamides were screened for antitubercular (anti-TB) activity in aerobic Mycobacterium tuberculosis H37Rv by using the BacTiter-Glo (BTG) microbial cell viability assay. In general, 3-nitrotriazole-based sulfonamides demonstrated anti-TB activity, whereas 3-nitrotriazole-based amides and 2-nitroimidazole-based amides and sulfonamides were inactive. Three 3-nitrotriazole-based sulfonamides (compounds 4, 2, and 7) demonstrated 50% inhibitory concentration (IC50), IC90, and MIC values of 0.38, 0.43, and 1.56 μM (compound 4), 0.57, 0.98, and 3.13 μM (compound 2), and 0.79, 0.87, and 3.13 μM (compound 7), respectively. For 3-nitrotriazole-based sulfonamides, anti-TB activity increased with lipophilicity, whereas the one-electron reduction potential (E1/2) did not play a role. 2-Nitroimidazole-based analogs, which were inactive in the BTG assay, were significantly more active in the low-oxygen assay and more active than the 3-nitrotriazoles. All active nitrotriazoles in the BTG assay were similarly active or more potent (lower MIC values) against resistant strains, with the exception of compounds 2, 3, 4, and 8, which demonstrated greater MIC values against isoniazid-resistant strains. Five 3-nitrotriazole-based sulfonamides demonstrated activity in infected murine J774 macrophages, causing log reductions similar to those seen with rifampin. However, some compounds caused toxicity in uninfected macrophages. In conclusion, the classes of 3-nitrotriazole-based amides and sulfonamides merit further investigation as potential antitubercular agents. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  17. Crystal structures of 4-meth-oxy-N-(4-methyl-phenyl)benzene-sulfonamide and N-(4-fluoro-phenyl)-4-meth-oxy-benzene-sulfonamide.

    Science.gov (United States)

    Rodrigues, Vinola Z; Preema, C P; Naveen, S; Lokanath, N K; Suchetan, P A

    2015-11-01

    Crystal structures of two N-(ar-yl)aryl-sulfonamides, namely, 4-meth-oxy-N-(4-methyl-phen-yl)benzene-sulfonamide, C14H15NO3S, (I), and N-(4-fluoro-phen-yl)-4-meth-oxy-benzene-sulfonamide, C13H12FNO3S, (II), were determined and analyzed. In (I), the benzene-sulfonamide ring is disordered over two orientations, in a 0.516 (7):0.484 (7) ratio, which are inclined to each other at 28.0 (1)°. In (I), the major component of the sulfonyl benzene ring and the aniline ring form a dihedral angle of 63.36 (19)°, while in (II), the planes of the two benzene rings form a dihedral angle of 44.26 (13)°. In the crystal structure of (I), N-H⋯O hydrogen bonds form infinite C(4) chains extended in [010], and inter-molecular C-H⋯πar-yl inter-actions link these chains into layers parallel to the ab plane. The crystal structure of (II) features N-H⋯O hydrogen bonds forming infinite one dimensional C(4) chains along [001]. Further, a pair of C-H⋯O inter-molecular inter-actions consolidate the crystal packing of (II) into a three-dimensional supra-molecular architecture.

  18. Fluorescence detection of DNA, adenosine-5'-triphosphate (ATP), and telomerase activity by zinc(II)-protoporphyrin IX/G-quadruplex labels.

    Science.gov (United States)

    Zhang, Zhanxia; Sharon, Etery; Freeman, Ronit; Liu, Xiaoqing; Willner, Itamar

    2012-06-05

    The zinc(II)-protoporphyrin IX (ZnPPIX) fluorophore binds to G-quadruplexes, and this results in the enhanced fluorescence of the fluorophore. This property enabled the development of DNA sensors, aptasensors, and a sensor following telomerase activity. The DNA sensor is based on the design of a hairpin structure that includes a "caged" inactive G-quadruplex sequence. Upon opening the hairpin by the analyte DNA, the resulting fluorescence of the ZnPPIX/G-quadruplex provides the readout signal for the sensing event (detection limit 5 nM). Addition of Exonuclease III to the system allows the recycling of the analyte and its amplified analysis (detection limit, 200 pM). The association of the ZnPPIX to G-quadruplex aptamer-substrate complexes allowed the detection of adenosine-5'-triphosphate (ATP, detection limit 10 μM). Finally, the association of ZnPPIX to the G-quadruplex repeat units of telomers allowed the detection of telomerase activity originating from 380 ± 20 cancer 293T cell extract.

  19. Biological and Docking Studies of Sulfonamide Derivatives of 4-Aminophenazone

    International Nuclear Information System (INIS)

    Akhtar, M.S.; Ismail, A.; Murtaza, S.; Shamim, S.; Tahir, M.N.; Usman Ali Rana, U.A.

    2016-01-01

    Sulfonamide derivatives of 4-aminophenazone (4APZ) were synthesized and accordingly characterized by spectroscopic techniques. These newly synthesized compounds were examined for their biological activities such as enzyme inhibition, analgesic, antibacterial, antioxidant and DNA interaction. A direct correlation between enzyme inhibition activity and concentration of the compounds was observed both by experimental and molecular docking studies. Analgesic activity of the compounds was investigated by formalin-induced paw licking (FIPL), acetic acid-induced writhing (AIW) and heat conduction methods in mice. Membrane stabilization effect was determined by hypotonicity-induced hemolysis. Bacterial strains, S. aureus, S. epidermidis, B. subtilis, E. coli, P. aeruginosa, S. mutans and A. odontolyticus were used for investigating the antibacterial potential of the compounds. Antioxidant potential was investigated by Ferric Reducing Antioxidant Power assay (FRAP) and DPPH free radical scavenging method. DNA interaction studies of the synthesized compounds showed weak interaction. Hyperchromic effect was observed along the series and large positive K values were obtained for most of the compounds. (author)

  20. Two-photon excitation in chip electrophoresis enabling label-free fluorescence detection in non-UV transparent full-body polymer chips.

    Science.gov (United States)

    Geissler, David; Belder, Detlev

    2015-12-01

    One of the most commonly employed detection methods in microfluidic research is fluorescence detection, due to its ease of integration and excellent sensitivity. Many analytes though do not show luminescence when excited in the visible light spectrum, require suitable dyes. Deep-ultraviolet (UV) excitation (electrophoresis of small aromatic compounds. Various polymers, such as poly(methyl methacrylate), cyclic olefin polymer, and copolymer as well as poly(dimethylsiloxane) were investigated and compared with respect to achievable LOD and ruggedness against photodamage. To demonstrate the applicability of the technique, the method was also applied to the determination of serotonin and tryptamine in fruit samples. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  1. Food Labels

    Science.gov (United States)

    ... Staying Safe Videos for Educators Search English Español Food Labels KidsHealth / For Teens / Food Labels What's in ... to have at least 95% organic ingredients. Making Food Labels Work for You The first step in ...

  2. Synthesis of 1-(4-methylsulfone-phenyl)-5-(4-fluoro-phenyl)-5-[14C]-1,2,3- triazole and 1-(4-sulfonamide-phenyl)-5-(4-fluoro-phenyl)-5-[14C]-1,2,3- triazole as novel carbon-14 anticonvulsant

    International Nuclear Information System (INIS)

    Saemian, N.; Shirvani, G.; Matloubi, H.

    2006-01-01

    Two 1,2,3-triazole anticonvulsants, 1-(4-methylsulfone-phenyl)-5-(4-fluoro-phenyl)-5-[ 14 C]-1,2,3-triazole and 1-(4-sulfonamide-phenyl)-5-(4- fluoro-phenyl)-5-[ 14 C]-1,2,3-triazole, both labeled with carbon-14 in the 5-position were prepared from para-fluoro-benzonitrile-[cyano- 14 C]. (author)

  3. Stability Indicating HPLC Method for the Determination of Chiral Purity of R-(-)-5-[2-aminopropyl]-2-methoxybenzene Sulfonamide.

    Science.gov (United States)

    Kasawar, G B; Farooqui, M N

    2009-09-01

    A chiral reverse phase liquid chromatographic method was developed for the enantiomeric resolution of racemic mixture of (-)-5-[2-aminopropyl]-2-methoxybenzene sulfonamide in bulk drug. The enantiomeric separation of sulfonamide was resolved on a Crownpak CR (+) column using perchloric acid buffer of pH 1.0 as mobile phase and with UV detection at 226 nm. The method is validated and proved to be robust. The limit of detection and quantification of S (-)-(5)-[2-aminopropyl]-2-methoxybenzene sulfonamide] was found to be 0.084 and 0.159 mug/ml, respectively for 20 mul injection volume. The percentage recovery of S (-)-(5)-[2-aminopropyl]-2-methoxybenzene sulfonamide] ranged from 99.57 to 101.88 in bulk drug samples of R (-)-(5)-[2- aminopropyl]-2-methoxybenzene sulfonamide].

  4. Biomolecule-to-fluorescent-color encoder: modulation of fluorescence emission via DNA structural changes

    Science.gov (United States)

    Nishimura, Takahiro; Ogura, Yusuke; Yamada, Kenji; Ohno, Yuko; Tanida, Jun

    2014-01-01

    A biomolecule-to-fluorescent-color (B/F) encoder for optical readout of biomolecular information is proposed. In the B/F encoder, a set of fluorescence wavelengths and their intensity levels are used for coding of a biomolecular signal. A hybridization chain reaction of hairpin DNAs labeled with fluorescent reporters was performed to generate the fluorescence color codes. The fluorescence is modulated via fluorescence resonance energy transfer, which is controlled by DNA structural changes. The results demonstrate that fluorescent color codes can be configured based on two wavelengths and five intensities using the B/F encoder, and the assigned codes can be retrieved via fluorescence measurements. PMID:25071950

  5. Removal and factors influencing removal of sulfonamides and trimethoprim from domestic sewage in constructed wetlands.

    Science.gov (United States)

    Dan A; Yang, Yang; Dai, Yu-Nv; Chen, Chun-Xing; Wang, Su-Yu; Tao, Ran

    2013-10-01

    Twelve pilot-scale constructed wetlands with different configurations were set up in the field to evaluate the removal and factors that influence removal of sulfonamides (sulfadiazine, sulfapyridine, sulfacetamide, sulfamethazine and sulfamethoxazole) and trimethoprim from domestic sewage. The treatments included four flow types, three substrates, two plants and three hydraulic loading rates across two seasons (summer and winter). Most target antibiotics were efficiently removed by specific constructed wetlands; in particular, all types of constructed wetlands performed well for the degradation of sulfapyridine. Flow types were the most important influencing factor in this study, and the best removal of sulfonamides was achieved in vertical subsurface-flow constructed wetlands; however, the opposite phenomenon was found with trimethoprim. Significant relationships were observed between antibiotic degradation and higher temperature and redox potential, which indicated that microbiological pathways were the most probable degradation route for sulfonamides and trimethoprim in constructed wetlands. Copyright © 2013 Elsevier Ltd. All rights reserved.

  6. Detection of norovirus virus-like particles using a surface plasmon resonance-assisted fluoroimmunosensor optimized for quantum dot fluorescent labels.

    Science.gov (United States)

    Ashiba, Hiroki; Sugiyama, Yuki; Wang, Xiaomin; Shirato, Haruko; Higo-Moriguchi, Kyoko; Taniguchi, Koki; Ohki, Yoshimichi; Fujimaki, Makoto

    2017-07-15

    A highly sensitive biosensor to detect norovirus in environment is desired to prevent the spread of infection. In this study, we investigated a design of surface plasmon resonance (SPR)-assisted fluoroimmunosensor to increase its sensitivity and performed detection of norovirus virus-like particles (VLPs). A quantum dot fluorescent dye was employed because of its large Stokes shift. The sensor design was optimized for the CdSe-ZnS-based quantum dots. The optimal design was applied to a simple SPR-assisted fluoroimmunosensor that uses a sensor chip equipped with a V-shaped trench. Excitation efficiency of the quantum dots, degree of electric field enhancement by SPR, and intensity of autofluorescence of a substrate of the sensor chip were theoretically and experimentally evaluated to maximize the signal-to-noise ratio. As the result, an excitation wavelength of 390nm was selected to excite SPR on an Al film of the sensor chip. The sandwich assay of norovirus VLPs was performed using the designed sensor. Minimum detectable concentration of 0.01ng/mL, which corresponds to 100 virus-like particles included in the detection region of the V-trench, was demonstrated. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

  7. Slow aggregation of lysozyme in alkaline pH monitored in real time employing the fluorescence anisotropy of covalently labelled dansyl probe.

    Science.gov (United States)

    Homchaudhuri, Lopamudra; Kumar, Satish; Swaminathan, Rajaram

    2006-04-03

    The onset of hen egg white lysozyme aggregation on exposure to alkaline pH of 12.2 and subsequent slow growth of soluble lysozyme aggregates (at 298 K) was directly monitored by steady-state and time-resolved fluorescence anisotropy of covalently attached dansyl probe over a period of 24 h. The rotational correlation time accounting for tumbling of lysozyme in solution (40 microM) increased from approximately 3.6 ns (in pH 7) to approximately 40ns on exposure to pH 12.2 over a period of 6 h and remained stable thereafter. The growth of aggregates was strongly concentration dependent, irreversible after 60 min and inhibited by the presence of 0.9 M l-arginine in the medium. The day old aggregates were resistant to denaturation by 6 M guanidine.HCl. Our results reveal slow segmental motion of the dansyl probe in day old aggregates in the absence of L-arginine (0.9 M), but a much faster motion in its presence, when growth of aggregates is halted.

  8. Novel synthetic approach to asymmetric monocationic trimethine cyanine dyes derived from N-ethyl quinolinum moiety. Combined fluorescent and ICD probes for AT-DNA labelling

    Energy Technology Data Exchange (ETDEWEB)

    Kurutos, Atanas [Department of Pharmaceutical and Applied Organic Chemistry, Faculty of Chemistry, University of Sofia, 1164 Sofia (Bulgaria); Crnolatac, Ivo; Orehovec, Iva [Laboratory for Study of Interactions of Biomacromolecules, Division of Organic Chemistry and Biochemistry, Ruđer Bošković Institute, Bijenička c. 54, 10000 Zagreb (Croatia); Gadjev, Nikolai [Department of Pharmaceutical and Applied Organic Chemistry, Faculty of Chemistry, University of Sofia, 1164 Sofia (Bulgaria); Piantanida, Ivo, E-mail: pianta@irb.hr [Laboratory for Study of Interactions of Biomacromolecules, Division of Organic Chemistry and Biochemistry, Ruđer Bošković Institute, Bijenička c. 54, 10000 Zagreb (Croatia); Deligeorgiev, Todor, E-mail: ohtak@chem.uni-sofia.bg [Department of Pharmaceutical and Applied Organic Chemistry, Faculty of Chemistry, University of Sofia, 1164 Sofia (Bulgaria)

    2016-06-15

    Two asymmetric monocationic trimethine cyanine dyes were obtained via condensation reaction between 2-methylbenzothialolium salts containing various (aliphatic and benzyl) substituents on the nitrogen atom of the benzothiazolic chromophore, and 1-ethyl−4-(2-(phenylamine)vinyl)quinolin−1-ium iodide, by a novel improved method at room temperature under mild conditions. Both compounds bind non-covalently to double stranded DNA and RNA by micromolar affinity, but give highly selective fluorescent response>650 nm for only AT-DNA sequences at excess of DNA over dye, combined with equally AT-DNA selective ICD response at dye/DNA crowded conditions (r{sub [dye]/[AT-DNA]}>0.2)-namely ICD bands (attributed to dye-dimer formation) allow determination of AT-DNA at submicromolar concentrations. Selectivity was attributed to particular steric properties of AT-DNA minor groove in respect to other studied ds-DNA/RNA. Comparison of aliphatic- and benzyl- dye showed that only aliphatic- derivative revealed ICD band upon binding to AU-RNA major groove and short AT-sequences in mixed sequence (ct-)DNA.

  9. Identification and dynamic modeling of biomarkers for bacterial uptake and effect of sulfonamide antimicrobials

    International Nuclear Information System (INIS)

    Richter, Merle K.; Focks, Andreas; Siegfried, Barbara; Rentsch, Daniel; Krauss, Martin; Schwarzenbach, René P.; Hollender, Juliane

    2013-01-01

    The effects of sulfathiazole (STA) on Escherichia coli with glucose as a growth substrate was investigated to elucidate the effect-based reaction of sulfonamides in bacteria and to identify biomarkers for bacterial uptake and effect. The predominant metabolite was identified as pterine-sulfathiazole by LC-high resolution mass spectrometry. The formation of pterine-sulfathiazole per cell was constant and independent of the extracellular STA concentrations, as they exceeded the modeled half-saturation concentration K M S of 0.011 μmol L −1 . The concentration of the dihydrofolic acid precursor para-aminobenzoic acid (pABA) increased with growth and with concentrations of the competitor STA. This increase was counteracted for higher STA concentrations by growth inhibition as verified by model simulation of pABA dynamics. The EC value for the inhibition of pABA increase was 6.9 ± 0.7 μmol L −1 STA, which is similar to that calculated from optical density dynamics indicating that pABA is a direct biomarker for the SA effect. - Highlights: ► Elucidation of the effect-based reaction of sulfonamides in bacteria. ► Identification of a biomarker for uptake and effect-based reaction of sulfonamides. ► Investigation of a biomarker for the bacterial growth inhibition by sulfonamides. ► Quantitative mechanistic modeling of biomarker dynamics using enzyme kinetics. ► Mechanistic quantitative linking of sulfonamide concentrations and effects. - Identification of specific biomarkers for the uptake and effect-based reaction of sulfonamides in bacteria and resulting growth inhibition.

  10. Fish proteins as targets of ferrous-catalyzed oxidation: identification of protein carbonyls by fluorescent labeling on two-dimensional gels and MALDI-TOF/TOF mass spectrometry.

    Science.gov (United States)

    Pazos, Manuel; da Rocha, Angela Pereira; Roepstorff, Peter; Rogowska-Wrzesinska, Adelina

    2011-07-27

    Protein oxidation in fish meat is considered to affect negatively the muscle texture. An important source of free radicals taking part in this process is Fenton's reaction dependent on ferrous ions present in the tissue. The aim of this study was to investigate the susceptibility of cod muscle proteins in sarcoplasmic and myofibril fractions to in vitro metal-catalyzed oxidation and to point out protein candidates that might play a major role in the deterioration of fish quality. Extracted control proteins and proteins subjected to free radicals generated by Fe(II)/ascorbate mixture were labeled with fluorescein-5-thiosemicarbazide (FTSC) to tag carbonyl groups and separated by two-dimensional gel electrophoresis. Consecutive visualization of protein carbonyl levels by capturing the FTSC signal and total protein levels by capturing the SyproRuby staining signal allowed us to quantify the relative change in protein carbonyl levels corrected for changes in protein content. Proteins were identified using MALDI-TOF/TOF mass spectrometry and homology-based searches. The results show that freshly extracted cod muscle proteins exhibit a detectable carbonylation background and that the incubation with Fe(II)/ascorbate triggers a further oxidation of both sarcoplasmic and myofibril proteins. Different proteins exhibited various degrees of sensitivity to oxidation processes. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), nucleoside diphosphate kinase B (NDK), triosephosphate isomerase, phosphoglycerate mutase, lactate dehydrogenase, creatine kinase, and enolase were the sarcoplasmic proteins most vulnerable to ferrous-catalyzed oxidation. Moreover, NDK, phosphoglycerate mutase, and GAPDH were identified in several spots differing by their pI, and those forms showed different susceptibilities to metal-catalyzed oxidation, indicating that post-translational modifications may change the resistance of proteins to oxidative damage. The Fe(II)/ascorbate treatment significantly

  11. Hydroxylamine-O-sulfonamide is a versatile lead compound for the development of carbonic anhydrase inhibitors.

    Science.gov (United States)

    Di Fiore, Anna; Vergara, Alessandro; Caterino, Marco; Alterio, Vincenzo; Monti, Simona M; Ombouma, Joanna; Dumy, Pascal; Vullo, Daniela; Supuran, Claudiu T; Winum, Jean-Yves; De Simone, Giuseppina

    2015-07-21

    Hydroxylamine-O-sulfonamide, a molecule incorporating two zinc-binding groups (ZBGs), has been investigated as a carbonic anhydrase inhibitor (CAI) by means of kinetic, crystallographic and Raman spectroscopy studies, highlighting interesting results on its mechanism of action. These data can be exploited to design new, effective and selective CAIs.

  12. Deciphering the bacterial microbiome in huanglongbing-affected citrus treated with thermotherapy and sulfonamide antibiotics

    Science.gov (United States)

    Huanglongbing (HLB) is a serious citrus disease that threatens the citrus industry. In previous studies, sulfonamide antibiotics and heat treatment suppressed ‘Candidatus Liberibacter asiaticus’ (Las), but did not completely eliminate the Las. Furthermore, there are few reports studying the bacteria...

  13. Multicomponent ternary cocrystals of the sulfonamide group with pyridine-amides and lactams.

    Science.gov (United States)

    Bolla, Geetha; Nangia, Ashwini

    2015-11-04

    SMBA was selected as a bifunctional sulfa drug to design ternary cocrystals with pyridine amides and lactam coformers. Supramolecular assembly of five ternary cocrystals of p-sulfonamide benzoic acid with nicotinamide and 2-pyridone is demonstrated and reproducible heterosynthons are identified for crystal engineering.

  14. Lysine sulfonamides as novel HIV-protease inhibitors: Nepsilon-acyl aromatic alpha-amino acids.

    Science.gov (United States)

    Stranix, Brent R; Lavallée, Jean-François; Sévigny, Guy; Yelle, Jocelyn; Perron, Valérie; LeBerre, Nicholas; Herbart, Dominik; Wu, Jinzi J

    2006-07-01

    A series of lysine sulfonamide analogues bearing Nepsilon-acyl aromatic amino acids were synthesized using an efficient synthetic route. Evaluation of these novel protease inhibitors revealed compounds with high potency against wild-type and multiple-protease inhibitor-resistant HIV viruses.

  15. Preparation and application of epitope magnetic molecularly imprinted polymers for enrichment of sulfonamide antibiotics in water.

    Science.gov (United States)

    Hu, Yufeng; Wang, Cheng; Li, Xiangdao; Liu, Lifen

    2017-10-01

    Sulfonamides, which are widely used synthetic antibiotics, are hydrophilic and stable. They can easily migrate into the environment and aquatic animals, and increase the risk of cancer, drug resistance, and allergic symptoms if consumed by humans. Here, we developed an epitope magnetic imprinting approach to enrich multiple sulfonamide antibiotics from a water sample. Epitope magnetic molecularly imprinted polymers (EMMIPs) were prepared by free-radical polymerization using vinyl-functioned Fe 3 O 4 as a core, sulfanilamide (SA) as a dummy template, methacrylic acid as a functional monomer, and ethylene glycol dimethacrylate as a cross-linker. The performance of the EMMIPs was first evaluated by rebinding SA, and then an adsorption experiment was conducted to assess the extraction of multiple sulfonamide antibiotics containing the SA group. The binding experiments showed that the EMMIPs reached adsorption equilibrium in only 5 min with adsorption of SA at 2040 μg/g, compared with just 462 μg/g for the epitope magnetic non-imprinted polymers. EMMIPs were combined with HPLC for the detection of six sulfonamide antibiotics in surface water samples. The recoveries ranged from 79.3 to 92.4% and the relative standard deviations from 0.9 to 7.3%. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  16. Interaction of nitrogen dioxide with sulfonamide-substituted phthalocyanines: Towards NO2 gas sensor

    Czech Academy of Sciences Publication Activity Database

    Pochekailov, Sergii; Nožár, Juraj; Nešpůrek, Stanislav; Rakušan, J.; Karásková, M.

    2012-01-01

    Roč. 169, 5 July (2012), s. 1-9 ISSN 0925-4005 R&D Projects: GA AV ČR KAN400720701; GA MPO FR-TI1/144 Institutional research plan: CEZ:AV0Z40500505 Keywords : phthalocyanine * sulfonamide * nitrogen dioxide Subject RIV: CG - Electrochemistry Impact factor: 3.535, year: 2012

  17. Synthesis of New Thiazole Derivatives Bearing A Sulfonamide Moiety Of Expected Anticancer And Radiosensitizing Activities

    International Nuclear Information System (INIS)

    Mohamed, S.Sh.I.

    2012-01-01

    In a search for new cytotoxic agents with improved antitumor activity and selectivity, some new pyrano thiazole and thiazolopyranopyrimidine derivatives bearing sulfonamide moiety were synthesized. The newly synthesized compounds were evaluated for their antitumor activity alone and in combination with γ-irradiation. These new compounds were docked inside the active site of carbonic anhydrase II to predict their mechanism of action.

  18. Fate of sulfonamide antibiotics in contact with activated sludge--sorption and biodegradation.

    Science.gov (United States)

    Yang, Sheng-Fu; Lin, Cheng-Fang; Wu, Chien-Ju; Ng, Kok-Kwang; Lin, Angela Yu-Chen; Hong, Pui-Kwan Andy

    2012-03-15

    The sorption and biodegradation of three sulfonamide antibiotics, namely sulfamethoxazole (SMX), sulfadimethoxine (SDM), and sulfamonomethoxine (SMM), in an activated sludge system were investigated. Experiments were carried out by contacting 100 μg/L of each sulfonamide compound individually with 2.56 g/L of MLSS at 25±0.5 °C, pH 7.0, and dissolved oxygen of 3.0±0.1 mg/L in a batch reactor over different periods of 2 d and 14 d. All sulfonamides were removed completely over 11-13 d. Sorptive equilibrium was established well within the first few hours, followed by a lag period of 1-3 days before biodegradation was to deplete the antibiotic compounds linearly in the ensuing 10 days. Apparent zeroth-order rate constants were obtained by regression analysis of measured aqueous concentration vs. time profiles to a kinetic model accounting for sorption and biodegradation; they were 8.1, 7.9, and 7.7 μg/L/d for SDM, SMX, and SMM, respectively, at activated sludge concentration of 2.56 g/L. The measured kinetics implied that with typical hydraulic retention time (e.g. 6 h) provided by WWTP the removal of sulfonamide compounds from the wastewater during the activated sludge process would approximate 2 μg/L. Copyright © 2011 Elsevier Ltd. All rights reserved.

  19. Accumulation of sulfonamide resistance genes in arable soils due to repeated application of manure containing sulfadiazine.

    Science.gov (United States)

    Heuer, Holger; Solehati, Qodiah; Zimmerling, Ute; Kleineidam, Kristina; Schloter, Michael; Müller, Tanja; Focks, Andreas; Thiele-Bruhn, Sören; Smalla, Kornelia

    2011-04-01

    Two soils were amended three times with pig manure. The abundance of sulfonamide resistance genes was determined by quantitative PCR 2 months after each application. In both soils treated with sulfadiazine-containing manure, the numbers of copies of sul1 and sul2 significantly increased compared to numbers after treatments with antibiotic-free manure or a control and accumulated with repeated applications.

  20. Enhanced removal of sulfonamide antibiotics by KOH-activated anthracite coal: Batch and fixed-bed studies

    International Nuclear Information System (INIS)

    Zuo, Linzi; Ai, Jing; Fu, Heyun; Chen, Wei; Zheng, Shourong; Xu, Zhaoyi; Zhu, Dongqiang

    2016-01-01

    The presence of sulfonamide antibiotics in aquatic environments poses potential risks to human health and ecosystems. In the present study, a highly porous activated carbon was prepared by KOH activation of an anthracite coal (Anth-KOH), and its adsorption properties toward two sulfonamides (sulfamethoxazole and sulfapyridine) and three smaller-sized monoaromatics (phenol, 4-nitrophenol and 1,3-dinitrobenzene) were examined in both batch and fixed-bed adsorption experiments to probe the interplay between adsorbate molecular size and adsorbent pore structure. A commercial powder microporous activated carbon (PAC) and a commercial mesoporous carbon (CMK-3) possessing distinct pore properties were included as comparative adsorbents. Among the three adsorbents Anth-KOH exhibited the largest adsorption capacities for all test adsorbates (especially the two sulfonamides) in both batch mode and fixed-bed mode. After being normalized by the adsorbent surface area, the batch adsorption isotherms of sulfonamides on PAC and Anth-KOH were displaced upward relative to the isotherms on CMK-3, likely due to the micropore-filling effect facilitated by the microporosity of adsorbents. In the fixed-bed mode, the surface area-normalized adsorption capacities of Anth-KOH for sulfonamides were close to that of CMK-3, and higher than that of PAC. The irregular, closed micropores of PAC might impede the diffusion of the relatively large-sized sulfonamide molecules and in turn led to lowered fixed-bed adsorption capacities. The overall superior adsorption of sulfonamides on Anth-KOH can be attributed to its large specific surface area (2514 m"2/g), high pore volume (1.23 cm"3/g) and large micropore sizes (centered at 2.0 nm). These findings imply that KOH-activated anthracite coal is a promising adsorbent for the removal of sulfonamide antibiotics from aqueous solution. - Highlights: • A high efficiency adsorbent for sulfonamide removal is prepared from anthracite. • Effects of

  1. Using Fluorescent Viruses for Detecting Bacteria in Water

    Science.gov (United States)

    Tabacco, Mary Beth; Qian, Xiaohua; Russo, Jaimie A.

    2009-01-01

    A method of detecting water-borne pathogenic bacteria is based partly on established molecular-recognition and fluorescent-labeling concepts, according to which bacteria of a species of interest are labeled with fluorescent reporter molecules and the bacteria can then be detected by fluorescence spectroscopy. The novelty of the present method lies in the use of bacteriophages (viruses that infect bacteria) to deliver the fluorescent reporter molecules to the bacteria of the species of interest.

  2. A fluorescence scanning electron microscope

    International Nuclear Information System (INIS)

    Kanemaru, Takaaki; Hirata, Kazuho; Takasu, Shin-ichi; Isobe, Shin-ichiro; Mizuki, Keiji; Mataka, Shuntaro; Nakamura, Kei-ichiro

    2009-01-01

    Fluorescence techniques are widely used in biological research to examine molecular localization, while electron microscopy can provide unique ultrastructural information. To date, correlative images from both fluorescence and electron microscopy have been obtained separately using two different instruments, i.e. a fluorescence microscope (FM) and an electron microscope (EM). In the current study, a scanning electron microscope (SEM) (JEOL JXA8600 M) was combined with a fluorescence digital camera microscope unit and this hybrid instrument was named a fluorescence SEM (FL-SEM). In the labeling of FL-SEM samples, both Fluolid, which is an organic EL dye, and Alexa Fluor, were employed. We successfully demonstrated that the FL-SEM is a simple and practical tool for correlative fluorescence and electron microscopy.

  3. Multiple tag labeling method for DNA sequencing

    Science.gov (United States)

    Mathies, R.A.; Huang, X.C.; Quesada, M.A.

    1995-07-25

    A DNA sequencing method is described which uses single lane or channel electrophoresis. Sequencing fragments are separated in the lane and detected using a laser-excited, confocal fluorescence scanner. Each set of DNA sequencing fragments is separated in the same lane and then distinguished using a binary coding scheme employing only two different fluorescent labels. Also described is a method of using radioisotope labels. 5 figs.

  4. Enhanced removal of sulfonamide antibiotics by KOH-activated anthracite coal: Batch and fixed-bed studies.

    Science.gov (United States)

    Zuo, Linzi; Ai, Jing; Fu, Heyun; Chen, Wei; Zheng, Shourong; Xu, Zhaoyi; Zhu, Dongqiang

    2016-04-01

    The presence of sulfonamide antibiotics in aquatic environments poses potential risks to human health and ecosystems. In the present study, a highly porous activated carbon was prepared by KOH activation of an anthracite coal (Anth-KOH), and its adsorption properties toward two sulfonamides (sulfamethoxazole and sulfapyridine) and three smaller-sized monoaromatics (phenol, 4-nitrophenol and 1,3-dinitrobenzene) were examined in both batch and fixed-bed adsorption experiments to probe the interplay between adsorbate molecular size and adsorbent pore structure. A commercial powder microporous activated carbon (PAC) and a commercial mesoporous carbon (CMK-3) possessing distinct pore properties were included as comparative adsorbents. Among the three adsorbents Anth-KOH exhibited the largest adsorption capacities for all test adsorbates (especially the two sulfonamides) in both batch mode and fixed-bed mode. After being normalized by the adsorbent surface area, the batch adsorption isotherms of sulfonamides on PAC and Anth-KOH were displaced upward relative to the isotherms on CMK-3, likely due to the micropore-filling effect facilitated by the microporosity of adsorbents. In the fixed-bed mode, the surface area-normalized adsorption capacities of Anth-KOH for sulfonamides were close to that of CMK-3, and higher than that of PAC. The irregular, closed micropores of PAC might impede the diffusion of the relatively large-sized sulfonamide molecules and in turn led to lowered fixed-bed adsorption capacities. The overall superior adsorption of sulfonamides on Anth-KOH can be attributed to its large specific surface area (2514 m(2)/g), high pore volume (1.23 cm(3)/g) and large micropore sizes (centered at 2.0 nm). These findings imply that KOH-activated anthracite coal is a promising adsorbent for the removal of sulfonamide antibiotics from aqueous solution. Copyright © 2016 Elsevier Ltd. All rights reserved.

  5. Stability Indicating HPLC Method for the Determination of Chiral Purity of R-(-)-5-[2-aminopropyl]-2-methoxybenzene Sulfonamide

    OpenAIRE

    Kasawar, G. B.; Farooqui, M. N.

    2009-01-01

    A chiral reverse phase liquid chromatographic method was developed for the enantiomeric resolution of racemic mixture of (-)-5-[2-aminopropyl]-2-methoxybenzene sulfonamide in bulk drug. The enantiomeric separation of sulfonamide was resolved on a Crownpak CR (+) column using perchloric acid buffer of pH 1.0 as mobile phase and with UV detection at 226 nm. The method is validated and proved to be robust. The limit of detection and quantification of S (-)-(5)-[2-aminopropyl]-2-methoxyben...

  6. L-Threonine-derived novel bifunctional phosphine-sulfonamide catalyst-promoted enantioselective aza-morita-Baylis-Hillman reaction

    KAUST Repository

    Zhong, Fangrui

    2011-03-18

    A series of novel bifunctional phosphine-sulfonamide organic catalysts were designed and readily prepared from natural amino acids, and they were utilized to promote enantioselective aza-Morita-Baylis-Hillman (MBH) reactions. l-Threonine-derived phosphine-sulfonamide 9b was found to be the most efficient catalyst, affording the desired aza-MBH adducts in high yields and with excellent enantioselectivities. © 2011 American Chemical Society.

  7. Prevalence and characterization of plasmids carrying sulfonamide resistance genes among Escherichia coli from pigs, pig carcasses and human

    OpenAIRE

    Wu, Shuyu; Dalsgaard, Anders; Hammerum, Anette M; Porsbo, Lone J; Jensen, Lars B

    2010-01-01

    Abstract Background Sulfonamide resistance is very common in Escherichia coli. The aim of this study was to characterize plasmids carrying sulfonamide resistance genes (sul1, sul2 and sul3) in E. coli isolated from pigs and humans with a specific objective to assess the genetic diversity of plasmids involved in the mobility of sul genes. Methods A total of 501 E. coli isolates from pig feces, pig carcasses and human stools were tested for their susceptibility to selected antimicrobial. Multip...

  8. Fluorescence spectroscopy

    DEFF Research Database (Denmark)

    Bagatolli, Luis

    2016-01-01

    Fluorescence spectroscopy is a powerful experimental tool used by scientists from many disciplines. During the last decades there have been important developments on distinct fluorescence methods, particularly those related to the study of biological phenomena. This chapter discusses the foundati......Fluorescence spectroscopy is a powerful experimental tool used by scientists from many disciplines. During the last decades there have been important developments on distinct fluorescence methods, particularly those related to the study of biological phenomena. This chapter discusses...

  9. Fluorescent Labelled Thiourea-Bridged Glycodendrons

    Czech Academy of Sciences Publication Activity Database

    Krist, Pavel; Vannucci, Luca; Kuzma, Marek; Man, Petr; Sadalapure, K.; Patel, A.; Bezouška, Karel; Pospíšil, Milan; Petruš, L.; Lindhorst, T. K.; Křen, Vladimír

    2004-01-01

    Roč. 5, - (2004), s. 445-452 ISSN 1439-4227 R&D Projects: GA ČR GA203/01/1018; GA AV ČR IAA7020006; GA ČR GV312/98/K034; GA MŠk(CZ) 1P04OCD13.50 Institutional research plan: CEZ:AV0Z5020903 Keywords : nkr-p1a * nk * glcnac-coated Subject RIV: EE - Microbiology, Virology Impact factor: 3.474, year: 2004

  10. Nutrition Labeling

    DEFF Research Database (Denmark)

    Grunert, Klaus G

    2013-01-01

    because consumers will avoid products that the label shows to be nutritionally deficient, but also because food producers will try to avoid marketing products that appear, according to the label, as nutritionally problematic, for example, because of a high content of saturated fat or salt. Nutrition......Nutrition labeling refers to the provision of information on a food product’s nutritional content on the package label. It can serve both public health and commercial purposes. From a public health perspective, the aim of nutrition labeling is to provide information that can enable consumers...... to make healthier choices when choosing food products. Nutrition labeling is thus closely linked to the notion of the informed consumer, that chooses products according to their aims, on the basis of the information at their disposal. Because many consumers are assumed to be interested in making healthy...

  11. Private Labels

    OpenAIRE

    Kolmačková, Zuzana

    2013-01-01

    This Bachelor Thesis titled Private labels deals with distribution strategy based on the introduction of private labels especially in retail chains. At the beginning it is focused on the general concept of private label offered by retailers, where is mentioned basic characteristics, history and structuring of distribution brands. Subsequently this thesis informs readers about the introduction of new special distribution brands, which focus primarily on the new consumption habits of customers....

  12. Rationalization of activity cliffs of a sulfonamide inhibitor of DNA methyltransferases with induced-fit docking.

    Science.gov (United States)

    Medina-Franco, José L; Méndez-Lucio, Oscar; Yoo, Jakyung

    2014-02-21

    Inhibitors of human DNA methyltransferases (DNMT) are of increasing interest to develop novel epi-drugs for the treatment of cancer and other diseases. As the number of compounds with reported DNMT inhibition is increasing, molecular docking is shedding light to elucidate their mechanism of action and further interpret structure-activity relationships. Herein, we present a structure-based rationalization of the activity of SW155246, a distinct sulfonamide compound recently reported as an inhibitor of human DNMT1 obtained from high-throughput screening. We used flexible and induce-fit docking to develop a binding model of SW155246 with a crystallographic structure of human DNMT1. Results were in excellent agreement with experimental information providing a three-dimensional structural interpretation of 'activity cliffs', e.g., analogues of SW155246 with a high structural similarity to the sulfonamide compound, but with no activity in the enzymatic assay.

  13. Distribution of quinolones, sulfonamides, tetracyclines in aquatic environment and antibiotic resistance in Indochina

    Directory of Open Access Journals (Sweden)

    Satoru eSuzuki

    2012-02-01

    Full Text Available Southeast Asia has become the center of rapid industrial development and economic growth. However, this growth has far outpaced investment in public infrastructure, leading to the unregulated release of many pollutants, including wastewater-related contaminants such as antibiotics. Antibiotics are of major concern because they can easily be released into the environment from numerous sources, and can subsequently induce development of antibiotic-resistant bacteria. Recent studies have shown that for some categories of drugs this source-to-environment antibiotic resistance relationship is more complex. This review summarizes current understanding regarding the presence of quinolones, sulfonamides, and tetracyclines in aquatic environments of Indochina and the prevalence of bacteria resistant to them. Several noteworthy findings are discussed: 1 quinolone contamination and the occurrence of quinolone resistance are not correlated; 2 occurrence of the sul sulfonamide resistance gene varies geographically; and 3 microbial diversity might be related to the rate of oxytetracycline resistance.

  14. Synthesis and antimalarial activity of new 4-amino-7-chloroquinolyl amides, sulfonamides, ureas and thioureas.

    Science.gov (United States)

    Ekoue-Kovi, Kekeli; Yearick, Kimberly; Iwaniuk, Daniel P; Natarajan, Jayakumar K; Alumasa, John; de Dios, Angel C; Roepe, Paul D; Wolf, Christian

    2009-01-01

    We report the synthesis and in vitro antimalarial activities of more than 50 7-chloro-4-aminoquinolyl-derived sulfonamides 3-8 and 11-26, ureas 19-22, thioureas 23-26, and amides 27-54. Many of the CQ analogues prepared for this study showed submicromolar antimalarial activity versus HB3 (chloroquine sensitive) and Dd2 (chloroquine resistant strains of Plasmodium falciparum) and low resistance indices were obtained in most cases. Systematic variation of the side chain length and introduction of fluorinated aliphatic and aromatic termini revealed promising leads that overcome CQ resistance. In particular, sulfonamide 3 exhibiting a short side chain with a terminal dansyl moiety combined high antiplasmodial potency with a low resistance index and showed IC(50)s of 17.5 and 22.7 nM against HB3 and Dd2 parasites.

  15. Sulfonamide and carbamate derivatives of 6-chloropurine: synthesis, characterization and antimicrobial activity evaluation

    Directory of Open Access Journals (Sweden)

    K. Venkata Narayana

    2016-07-01

    Full Text Available A series of new sulfonamide derivatives, 9-(substitutedbenzenesulfonyl-6-chloro-9H-purines 7(a-e and carbamate derivatives, 6-chloro-purine-9-carboxylic acid substituted alkyl/arylester 9(a-d, have been synthesized through an intermediate, sodium salt of 6-chloro-9(H-purine (6 which was prepared by the treatment of 6-chloro-9(H-purine (4 with sodium hydride. Structures of the newly synthesized compounds were elucidated by IR, NMR ( 1H and 13C, mass spectra and elemental analysis. Antimicrobial activity against three bacterial strains and three fungal strains at two different concentrations, 100 and 200 µg/mL including MIC values was investigated. Bio-screening data disclosed that most of the sulfonamide derivatives, 7a, 7c and 7d, and one carbamate derivative 9a showed promising antimicrobial activity having MIC values in the range of 18.0-25.0 µg/mL.

  16. Rationalization of Activity Cliffs of a Sulfonamide Inhibitor of DNA Methyltransferases with Induced-Fit Docking

    Directory of Open Access Journals (Sweden)

    José L. Medina-Franco

    2014-02-01

    Full Text Available Inhibitors of human DNA methyltransferases (DNMT are of increasing interest to develop novel epi-drugs for the treatment of cancer and other diseases. As the number of compounds with reported DNMT inhibition is increasing, molecular docking is shedding light to elucidate their mechanism of action and further interpret structure–activity relationships. Herein, we present a structure-based rationalization of the activity of SW155246, a distinct sulfonamide compound recently reported as an inhibitor of human DNMT1 obtained from high-throughput screening. We used flexible and induce-fit docking to develop a binding model of SW155246 with a crystallographic structure of human DNMT1. Results were in excellent agreement with experimental information providing a three-dimensional structural interpretation of ‘activity cliffs’, e.g., analogues of SW155246 with a high structural similarity to the sulfonamide compound, but with no activity in the enzymatic assay.

  17. Piperazine sulfonamide BACE1 inhibitors: Design, synthesis, and in vivo characterization

    Energy Technology Data Exchange (ETDEWEB)

    Cumming, Jared; Babu, Suresh; Huang, Ying; Carrol, Carolyn; Chen, Xia; Favreau, Leonard; Greenlee, William; Guo, Tao; Kennedy, Matthew; Kuvelkar, Reshma; Le, Thuy; Li, Guoqing; McHugh, Nansie; Orth, Peter; Ozgur, Lynne; Parker, Eric; Saionz, Kurt; Stamford, Andrew; Strickland, Corey; Tadesse, Dawit; Voigta, Johannes; Zhang, Lili; Zhang, Qi (Ligand); (Merck)

    2010-08-17

    With collaboration between chemistry, X-ray crystallography, and molecular modeling, we designed and synthesized a series of novel piperazine sulfonamide BACE1 inhibitors. Iterative exploration of the non-prime side and S2{prime} sub-pocket of the enzyme culminated in identification of an analog that potently lowers peripheral A{beta}{sub 40} in transgenic mice with a single subcutaneous dose.

  18. Deciphering the Bacterial Microbiome in Huanglongbing-Affected Citrus Treated with Thermotherapy and Sulfonamide Antibiotics.

    Directory of Open Access Journals (Sweden)

    Chuanyu Yang

    Full Text Available Huanglongbing (HLB is a serious citrus disease that threatens the citrus industry. In previous studies, sulfonamide antibiotics and heat treatment suppressed 'Candidatus Liberibacter asiaticus' (Las, but did not completely eliminate the Las. Furthermore, there are few reports studying the bacterial microbiome of HLB-affected citrus treated by heat and sulfonamide antibiotics. In this study, combinations of heat (45°C or 40°C and sulfonamide treatment (sulfathiazole sodium-STZ, or sulfadimethoxine sodium-SDX were applied to HLB-affected citrus. The bacterial microbiome of HLB-affected citrus following thermotherapy and/or chemotherapy was characterized by PhyloChipTMG3-based metagenomics. Our results showed that the combination of thermotherapy at 45°C and chemotherapy with STZ and SDX was more effective against HLB than thermotherapy alone, chemotherapy alone, or a combination of thermotherapy at 40°C and chemotherapy. The PhyloChipTMG3-based results indicated that 311 empirical Operational Taxonomic Units (eOTUs were detected in 26 phyla. Cyanobacteria (18.01% were dominant after thermo-chemotherapy. Thermotherapy at 45°C decreased eOTUs (64.43% in leaf samples, compared with thermotherapy at 40°C (73.96% or without thermotherapy (90.68% and it also reduced bacterial family biodiversity. The eOTU in phylum Proteobacteria was reduced significantly and eOTU_28, representing "Candidatus Liberibacter," was not detected following thermotherapy at 45°C. Following antibiotic treatment with SDX and STZ, there was enhanced abundance of specific eOTUs belonging to the families Streptomycetaceae, Desulfobacteraceae, Chitinophagaceae, and Xanthomonadaceae, which may be implicated in increased resistance to plant pathogens. Our study further develops an integrated strategy for combating HLB, and also provides new insight into the bacterial microbiome of HLB-affected citrus treated by heat and sulfonamide antibiotics.

  19. Synthesis of Morpholine Containing Sulfonamides: Introduction of Morpholine Moiety on Amine Functional Group

    Directory of Open Access Journals (Sweden)

    D. Singh

    2004-01-01

    Full Text Available Sulfonamides have been the center of drug structures as this group is quite stable & well tolerated in human beings. The synthesis of these structures was started in search of new pharmacological active reagents. These compounds are being tested for the desired activity (ICAM-1/LFA-1 Interaction inhibitors as anti-adhesion therapeutic agents, the biological activity & structure activity relationship will be published elsewhere. Synthesis of morpholine moiety from amino group is done by using reagent 2-chloroethanol.

  20. Surface runoff and transport of sulfonamide antibiotics and tracers on manured grassland.

    Science.gov (United States)

    Burkhardt, Michael; Stamm, Christian; Waul, Christopher; Singer, Heinz; Müller, Stephan

    2005-01-01

    Despite their common use in animal production the environmental fate of the veterinary sulfonamide antibiotics after excretion is only poorly understood. We performed irrigation experiments to investigate the transport of these substances with surface runoff on grassland. Liquid manure from pigs treated with sulfadimidine was spiked with sulfadiazine, sulfathiazole, the herbicide atrazine (2-chloro-4-ethylamino-6-isopropylamino-1,3,5-triazine), and the conservative tracer bromide and spread onto eight plots. Four plots received the same amounts of the spiked substances in aqueous solution (controls). Apart from the application matrix we varied the time between application and irrigation. Manure increased the runoff volume up to six times compared with the controls. It seemed that manure enhanced the runoff by sealing the soil surface. On manured plots the relative antibiotic concentrations in runoff were higher than on the controls, reaching an average of 0.3% (sulfadiazine), 0.8% (sulfathiazole), and 1.4% (sulfadimidine) of the input concentrations after a 1-d contact time. The corresponding values on the controls were 0.16% for sulfadiazine and 0.08% for sulfathiazole. After 3 d, the maximum values on the manured plots were even higher, whereas they had fallen below the limit of quantification on the controls. As a consequence, the sulfonamide losses were 10 to 40 times larger on the manured plots. The relative mobility of the sulfonamides on the control plots followed the trend expected from their chromatographic separation but the opposite was found on the manured plots. Hence it is important to consider explicitly the physical and chemical effects of manure when assessing the environmental fate of sulfonamides.

  1. Ultrafast fluorescence of photosynthetic crystals and light-harvesting complexes

    OpenAIRE

    Oort, van, B.F.

    2008-01-01

    This thesis focuses on the study of photosynthetic pigment protein complexes using time resolved fluorescence techniques. Fluorescence spectroscopy often requires attaching fluorescent labels to the proteins under investigation. With photosynthetic proteins this is not necessary, because these proteins contain fluorescent pigments. Each pigment’s fluorescence is influenced by its environment, and thereby may provide information on structure and dynamics of pigment protein complexes in vitro a...

  2. Electronically tuned sulfonamide-based probes with ultra-sensitivity for Ga{sup 3+} or Al{sup 3+} detection in aqueous solution

    Energy Technology Data Exchange (ETDEWEB)

    Kumar, Ashwani, E-mail: ashwanirubal@gmail.com; Chae, Pil Seok, E-mail: pchae@hanyang.ac.kr

    2017-03-15

    Three electronically tuned fluorescent probes (1–3) were synthesized by conjugating a fluorescent unit to N,N-bis-(hydroxyethyl)ethylenediamine. Probe 1 bearing an electron-deficient naphthalenedimide unit did not give a fluorescence response to the presence of various metal ions including monovalent metal ions (Na{sup +}, K{sup +}, and Ag{sup +}), divalent metal ions (Ca{sup 2+}, Cd{sup 2+}, Co{sup 2+}, Ni{sup 2+}, Cu{sup 2+}, Hg{sup 2+}, Pb{sup 2+}, and Zn{sup 2+}) and trivalent metal ions (Al{sup 3+}, Ga{sup 3+}, Fe{sup 3+}, and Cr{sup 3+}) in an aqueous solution. By contrast, probes 2 and 3 possessing 1,8-naphthalimide and pyrene fluorophores, respectively, exhibited selective fluorescent “OFF-ON” behaviors as a result of Ga{sup 3+}/Al{sup 3+} binding among the diverse metal ions, suggesting the importance of fluorophore electronic character with regard to metal ion sensing. The ethylenediamine analog of probe 3, corresponding to probe 4, was unable to yield a significant change in fluorescence intensity in the presence of any metal ions tested here, revealing the essential role of two hydroxyl groups for metal ion binding. A high association constant of K{sub a} = 2.99 × 10{sup 5} M{sup −1} was obtained for probe 3 with Ga{sup 3+}, with a limit of detection (LOD) of 10 nM. This LOD is the lowest value known for Ga{sup 3+} detection using chemical sensors. Along with an increase in aggregate sizes, PET suppression of probes upon metal ion binding was the primary contributor to the enhancement in fluorescence emission necessary for the sensitive detection of the target ions. The probe-metal ion complexes were fully characterized via TEM, FE-SEM, {sup 1}H NMR, fluorescence spectroscopy techniques and DFT calculations. - Highlights: • Three electronically tuned sulfonamide-based probes (probes 1, 2, and 3) were developed for metal ion-sensing. • Probes 2 and 3 exhibited AIE behavior with increasing water-content. • Probes 2 and 3 displayed a

  3. Electrochemically modified carbon fiber bundles as selective sorbent for online solid-phase microextraction of sulfonamides

    International Nuclear Information System (INIS)

    Ling, Xu; Zhang, Wenpeng; Chen, Zilin

    2016-01-01

    The authors show that carbon fiber bundles electrochemically modified with the conducting polymer poly(3,4-ethylenedioxythiophene) (PEDOT) is a viable sorbent for online solid-phase microextraction (SPME) of the sulfonamides (sulfadiazine, sulfadimidine and sulfamethoxazole) prior to their determination by HPLC. The fibers were packed in a tube loop made from polyether ether ketone (PEEK) that was coupled to the HPLC system for online SPME. Preconcentration factors can reach values of up to 300, and the limit of detection (at an S/N ration of 3) can be as low as 0.05 ng⋅mL −1 . The method was applied to the analysis of the sulfonamides in spiked rat plasma with intra-day and inter-day RSDs of <3.33 and <4.57 %, and with recoveries in the range from 91.7 to 97.8 % in spiked plasma. The in-tube SPME was also applied to the determination of the 3 sulfonamides in rat plasma after oral administration (tablet powder) with high sensitivity. In addition to its efficient extraction, the PEEK tube based SPME has chemical and mechanical stability under even harsh conditions. (author)

  4. Effect of pH and soil structure on transport of sulfonamide antibiotics in agricultural soils.

    Science.gov (United States)

    Park, Jong Yol; Huwe, Bernd

    2016-06-01

    We investigated the effect of solution pH and soil structure on transport of sulfonamide antibiotics (sulfamethoxazole, sulfadimethoxine and sulfamethazine) in combination with batch sorption tests and column experiments. Sorption isotherms properly conformed to Freundlich model, and sorption potential of the antibiotics is as follows; sulfadimethoxine > sulfamethoxazole > sulfamethazine. Decreasing pH values led to increased sorption potential of the antibiotics on soil material in pH range of 4.0-8.0. This likely resulted from abundance of neutral and positive-charged sulfonamides species at low pH, which electrostatically bind to sorption sites on soil surface. Due to destruction of macropore channels, lower hydraulic conductivities of mobile zone were estimated in the disturbed soil columns than in the undisturbed soil columns, and eventually led to lower mobility of the antibiotics in disturbed column. The results suggest that knowledge of soil structure and solution condition is required to predict fate and distribution of sulfonamide antibiotics in environmental matrix. Copyright © 2016 Elsevier Ltd. All rights reserved.

  5. Structural Basis for the Inhibition of Helicobacter pylori α-Carbonic Anhydrase by Sulfonamides.

    Directory of Open Access Journals (Sweden)

    Joyanta K Modak

    Full Text Available Periplasmic α-carbonic anhydrase of Helicobacter pylori (HpαCA, an oncogenic bacterium in the human stomach, is essential for its acclimation to low pH. It catalyses the conversion of carbon dioxide to bicarbonate using Zn(II as the cofactor. In H. pylori, Neisseria spp., Brucella suis and Streptococcus pneumoniae this enzyme is the target for sulfonamide antibacterial agents. We present structural analysis correlated with inhibition data, on the complexes of HpαCA with two pharmacological inhibitors of human carbonic anhydrases, acetazolamide and methazolamide. This analysis reveals that two sulfonamide oxygen atoms of the inhibitors are positioned proximal to the putative location of the oxygens of the CO2 substrate in the Michaelis complex, whilst the zinc-coordinating sulfonamide nitrogen occupies the position of the catalytic water molecule. The structures are consistent with acetazolamide acting as site-directed, nanomolar inhibitors of the enzyme by mimicking its reaction transition state. Additionally, inhibitor binding provides insights into the channel for substrate entry and product exit. This analysis has implications for the structure-based design of inhibitors of bacterial carbonic anhydrases.

  6. Simultaneous occurrence of nitrates and sulfonamide antibiotics in two ground water bodies of Catalonia (Spain)

    Science.gov (United States)

    García-Galán, M. a. Jesús; Garrido, Teresa; Fraile, Josep; Ginebreda, Antoni; Díaz-Cruz, M. Silvia; Barceló, Damià

    2010-03-01

    SummaryIn the present work the occurrence of 19 selected sulfonamides, including one acetylated metabolite, was investigated in ground water samples taken from two ground water bodies in Catalonia (Plana de Vic and La Selva). Both include areas designated as nitrate vulnerable zones, according to Directive 91/676/EEC. A fully automated analytical methodology based on on-line solid-phase extraction-liquid chromatography-tandem mass spectrometry (on-line SPE-LC-MS/MS) was developed for this purpose. The high selectivity and sensitivity achieved (limits of detection between 0.005 and 0.8 ng/L) permitted to demonstrate the ubiquity of these antibiotics in both ground water bodies. Results showed a wide range of concentrations, from 0.01 ng/L up to 3460.57 ng/L. Since sulfonamides are related to livestock veterinary practices, they can be used as a specific indicator of manure contamination. However, the presence of sulfonamides appeared not to be directly related to the concentration of nitrates, as it is reflected on the low correlation coefficients found.

  7. Effects of pig slurry on the sorption of sulfonamide antibiotics in soil.

    Science.gov (United States)

    Thiele-Bruhn, S; Aust, M O

    2004-07-01

    Sorption of p-aminobenzoic acid (pABA) and five sulfonamide antibiotics to loess Chernozem topsoil amended with varied additions of pig slurry was investigated in batch trials. In unfertilized soil, partition coefficients (Kd) of sulfonamides ranged from 0.3 to 2.0. Strong sorption nonlinearity (1/n = 0.5 to 0.8) was best fitted by the Freundlich isotherm (R2 = 0.7 to 1.0) and was indicative for specific sorption mechanisms. Adsorption to pig slurry was much stronger, and nondesorbable portions were increased compared with soil. However, in a mixture of soil and slurry (50:1 w/w), sorption of the antibiotics was significantly decreased at a lower concentration range of pABA and the sulfonamides. This was attributed to competitive adsorption of dissolved organic matter (DOM) constituents from manure. An increase in pig slurry amendment resulted in increased total organic matter, DOM concentration, and ionic strength, but pH decreased. As a result, the nonadsorbed portions of pABA, sulfanilamide, and sulfadiazine (logD(ow) 0.1)--remained nearly constant in the presence of increased manure input. The pH changes caused by manure amendment strongly affected ionisation status of the latter compounds, thus resulting in increased adsorption, which compensated the mobilizing effect of DOM. It is suggested that the effect of manure be considered in test methods to determine the soil retention of pharmaceutical substances.

  8. Sustainability Labeling

    NARCIS (Netherlands)

    Dam, van Y.K.

    2017-01-01

    Sustainability labeling originated from a need to protect the identity of alternative systems of food production and to increase market transparency. From the 1980s onwards sustainability labeling has changed into a policy instrument replacing direct government regulation of the food market, and a

  9. Polymorphism in R-tamsulosin (an alpha blocker): The unexpected manifestation of a sulfonamide⋯o-diethoxybenzene heterosynthon

    Science.gov (United States)

    Nanubolu, Jagadeesh Babu; Sridhar, Balasubramanian; Ravikumar, Krishnan

    2014-12-01

    A two point Nsbnd H⋯O dimer or an infinite catemer are the most preferred motifs/synthons for sulfonamide structures. Such synthons are known to be so robust that they are only disrupted in the presence of highly activated O acceptors such as pyridine-N-oxide and sulfoxide. We demonstrate in this article that a multi-point synthon offered by much weaker ethoxy O and amine N acceptors can however strongly compete and disrupt the robust sulfonamide homosynthons. This has been illustrated with the synthon analysis in three polymorphic crystal structures of R-tamsulosin, an active drug used in the treatment of Benign Prostatic Hyperplasia (BPH) and its hydrochloride salt. These crystalline solids are characterized by Single crystal X-ray diffraction (SC-XRD), powder X-ray diffraction (PXRD), Fourier Transform Infrared (FT-IR) and Raman spectroscopy. Forms I, II of the free base and hydrochloride salt crystallize in the monoclinic P21, C2, and P21 space groups respectively with two molecules in the asymmetric unit (Z‧ = 2), whereas, form III of freebase crystallize in the orthorhombic P212121 space group with Z‧ = 1. Remarkably, all four crystal structures contain a totally unexpected sulfonamide⋯o-diethoxybenzene heterosynthon. The multi-point motifs observed in polymorphs are relatively stronger than those in the hydrochloride salt because of the gauche conformation of the tamsulosin linker chain which renders an additional hydrogen bond interaction with amine N acceptor, and resemble the crown ether sulfonamide recognition pattern. Observation of this new heterosynthon offers potential scope in the design of pharmaceutical cocrystals for sulfonamide bearing drug molecules. The present study also presents a detailed hydrogen bond motif analysis in 310 primary sulfonamide structures culled from the latest version of Cambridge Structural Database (CSD). The role of various competing groups is discussed in the context of understanding the most recurring

  10. [Occurrence of quinolone and sulfonamide antibiotics in swine and cattle manures from large-scale feeding operations of Guangdong Province].

    Science.gov (United States)

    Tai, Yi-Ping; Luo, Xiao-Dong; Mo, Ce-Hui; Li, Yan-Wen; Wu, Xiao-Lian; Liu, Xing-Yue

    2011-04-01

    The occurrence and distribution of four quinolones and four sulfonamides in swine and cattle feces sampled from twenty large-scale feeding operations in different areas of Guangdong province were detected using solid phase extraction (SPE) and high performance liquid chromatography (HPLC). Quinolone and sulfonamide compounds were observed in all pig dung samples. Their total concentrations ranged from 24.5 microg/kg to 1516.2 microg/kg (F. W.) with an average of 581.0 microg/kg and ranged from 1925.9-13399.5 microg/kg with an average of 4403.9 microg/kg respectively. The dominant compounds in pig feces were ciprofloxacin and enrofloxacin for quinolones and sulfamerazine and sulfamethoxazole for sulfonamides. Quinolone compounds which dominated with norfloxacin and ciprofloxacin were also observed in all cattle dung samples, its total concentrations ranged from 73.2 microg/kg to 1328.0 microg/kg which averaged 572.9 microg/kg. While the positive rates of sulfonamide compounds detected in cattle dung samples were above 90%, predominated by sulfamethoxazole and sulfamerazine. Concentration and distribution of both quinolone and sulfonamide compounds in swine and cattle dungs of different feeding operations varied greatly. Relatively high concentrations of the two kinds of antibiotics were found in both swine and cattle dungs from Guangzhou area, while sulfameter and sulfamethazine in cattle dungs from Foshan and Shenzhen areas were below the limit of detection.

  11. The lipid dependence of melittin action investigated by dual-color fluorescence burst analysis

    NARCIS (Netherlands)

    Bogaart, Geert van den; Mika, Jacek T.; Krasnikov, Viktor; Poolman, Bert

    Dual-color fluorescence-burst analysis was used to study melittin-induced leakage of macromolecules from liposomes of various lipid compositions. To perform dual-color fluorescence-burst analysis, fluorescently labeled size-marker molecules were encapsulated into liposomes, labeled with a second

  12. Pesticide Labels

    Science.gov (United States)

    Pesticide labels translate results of our extensive evaluations of pesticide products into conditions, directions and precautions that define parameters for use of a pesticide with the goal of ensuring protection of human health and the environment.

  13. Labelling patients

    International Nuclear Information System (INIS)

    Strudwick, R.M.

    2016-01-01

    This article looks at how diagnostic radiographers label their patients. An ethnographic study of the workplace culture in one diagnostic imaging department was undertaken using participant observation for four months and semi-structured interviews with ten key informants. One of the key themes; the way in which radiographers label their patients, is explored in this article. It was found from the study that within the department studied the diagnostic radiographers labelled or categorised their patients based on the information that they had. This information is used to form judgements and these judgements were used to assist the radiographers in dealing with the many different people that they encountered in their work. This categorisation and labelling of the patient appears to assist the radiographer in their decision-making processes about the examination to be carried out and the patient they are to image. This is an important aspect of the role of the diagnostic radiographer. - Highlights: • I have studied the culture in one imaging department. • Radiographers label or categorise their patients. • These labels/categories are used to manage the patient. • This is an important aspect of the way in which radiographers work.

  14. Nine New Fluorescent Probes

    Science.gov (United States)

    Lin, Tsung-I.; Jovanovic, Misa V.; Dowben, Robert M.

    1989-06-01

    Absorption and fluorescence spectroscopic studies are reported here for nine new fluorescent probes recently synthesized in our laboratories: four pyrene derivatives with substituents of (i) 1,3-diacetoxy-6,8-dichlorosulfonyl, (ii) 1,3-dihydroxy-6,8-disodiumsulfonate, (iii) 1,3-disodiumsulfonate, and (iv) l-ethoxy-3,6,8-trisodiumsulfonate groups, and five [7-julolidino] coumarin derivatives with substituents of (v) 3-carboxylate-4-methyl, (vi) 3- methylcarboxylate, (vii) 3-acetate-4-methyl, (viii) 3-propionate-4-methyl, and (ix) 3-sulfonate-4-methyl groups. Pyrene compounds i and ii and coumarin compounds v and vi exhibit interesting absorbance and fluorescence properties: their absorption maxima are red shifted compared to the parent compound to the blue-green region, and the band width broadens considerably. All four blue-absorbing dyes fluoresce intensely in the green region, and the two pyrene compounds emit at such long wavelengths without formation of excimers. The fluorescence properties of these compounds are quite environment-sensitive: considerable spectral shifts and fluorescence intensity changes have been observed in the pH range from 3 to 10 and in a wide variety of polar and hydrophobic solvents with vastly different dielectric constants. The high extinction and fluorescence quantum yield of these probes make them ideal fluorescent labeling reagents for proteins, antibodies, nucleic acids, and cellular organelles. The pH and hydrophobicity-dependent fluorescence changes can be utilized as optical pH and/or hydrophobicity indicators for mapping environmental difference in various cellular components in a single cell. Since all nine probes absorb in the UV, but emit at different wavelengths in the visible, these two groups of compounds offer an advantage of utilizing a single monochromatic light source (e.g., a nitrogen laser) to achieve multi-wavelength detection for flow cytometry application. As a first step to explore potential application in

  15. Vibrational Spectra And Potential Energy Distributions of Normal Modes of N,N'-Etilenbis(P-Toluen sulfonamide)

    International Nuclear Information System (INIS)

    Alyar, S.

    2008-01-01

    N-substituted sulfonamides are well known for their diuretic, antidiabetic, antibacterial and antifungal, anticancer e.g., and are widely used in the therapy of patients. These important bioactive properties are strongly affected by the special features of -CH 2 -SO 2 -NR-linker and intramolecular motion Thus, the studies of energetic and spatial properties on N-substituted sulfonamides are of great importance to improve our understanding of their biological activities and enhance abilities to predict new drugs. Density Functional Theory B3LYP /6-31G(d,p) level has been applied to obtain the vibrational force field for the most stable conformation of N,N'-etilenbis(p-toluensulfonamit)(ptsen)having sulfonamide moiety. The results of these calculation have been compared with spectroscopic data to verify accuracy of calculation and applicability of the DFT approach to ptsen. Additionally, complete normal coordinate analyses with quantum mechanical scaling (SQM) were performed to derive the potential energy distributions (PE)

  16. Solid-State Examination of Conformationally Diverse Sulfonamide Receptors Based on Bis(2-anilinoethynyl)pyridine, -Bipyridine, and -Thiophene.

    Science.gov (United States)

    Berryman, Orion B; Johnson, Charles A; Vonnegut, Chris L; Fajardo, Kevin A; Zakharov, Lev N; Johnson, Darren W; Haley, Michael M

    2015-03-04

    Utilizing an induced-fit model and taking advantage of rotatable acetylenic C(sp)-C(sp 2 ) bonds, we disclose the synthesis and solid-state structures of a series of conformationally diverse bis-sulfonamide arylethynyl receptors using either pyridine, 2,2'-bipyridine, or thiophene as the core aryl group. Whereas the bipyridine and thiophene structures do not appear to bind guests in the solid state, the pyridine receptors form 2 + 2 dimers with water molecules, two halides, or one of each, depending on the protonation state of the pyridine nitrogen atom. Isolation of a related bis-sulfonimide derivative demonstrates the importance of the sulfonamide N-H hydrogen bonds in dimer formation. The pyridine receptors form monomeric structures with larger guests such as BF 4 - or HSO 4 - , where the sulfonamide arms rotate to the side opposite the pyridine N atom.

  17. Solid-State Examination of Conformationally Diverse Sulfonamide Receptors Based on Bis(2-anilinoethynyl)pyridine, -Bipyridine, and -Thiophene

    Science.gov (United States)

    Berryman, Orion B.; Johnson, Charles A.; Vonnegut, Chris L.; Fajardo, Kevin A.; Zakharov, Lev N.; Johnson, Darren W.; Haley, Michael M.

    2015-01-01

    Utilizing an induced-fit model and taking advantage of rotatable acetylenic C(sp)–C(sp2) bonds, we disclose the synthesis and solid-state structures of a series of conformationally diverse bis-sulfonamide arylethynyl receptors using either pyridine, 2,2′-bipyridine, or thiophene as the core aryl group. Whereas the bipyridine and thiophene structures do not appear to bind guests in the solid state, the pyridine receptors form 2 + 2 dimers with water molecules, two halides, or one of each, depending on the protonation state of the pyridine nitrogen atom. Isolation of a related bis-sulfonimide derivative demonstrates the importance of the sulfonamide N–H hydrogen bonds in dimer formation. The pyridine receptors form monomeric structures with larger guests such as BF4− or HSO4−, where the sulfonamide arms rotate to the side opposite the pyridine N atom. PMID:26405435

  18. Efficacy of sulfonamides and Baycox(®) against Isospora suis in experimental infections of suckling piglets.

    Science.gov (United States)

    Joachim, Anja; Mundt, Hans-Christian

    2011-12-01

    Sulfonamide treatment of piglets against neonatal coccidiosis has frequently been suggested in the literature. In order to evaluate the efficacy of sulfonamides against experimental Isospora suis infections in suckling piglets (oral infection with 1,500 sporulated oocysts of I. suis per piglet on the fourth day of life), two trials were conducted. In trial I, oral sulfadimidine (group Sulfa-Oral) was applied in doses of 100 mg/kg of body weight (BW) 1 day before infection and 75 mg/kg BW daily for the following 5 days, and sulfamethoxypyrimidine (SMP) was applied parenterally in daily doses of 75 mg/kg BW for the same time period. In trial II, SMP was applied parenterally in doses of 75 mg/kg BW (a) from the day of infection daily for 7 days (SMP-Standard), (b) for 2 days starting on the day of infection (SMP-Early), (c) for 3 days starting 2 days post-infection (d.p.i.; SMP-Middle), (d) for 2 days starting 5 d.p.i. (SMP-Late), and (e) every other day from the day of infection until 6 d.p.i. (SMP-Alternating), as well as (f) orally in doses of 75 mg/kg BW from the day of infection for 7 days (SMP-Oral). The sulfonamide-treated groups were compared to a toltrazuril-treated group (single oral treatment with Baycox® 5% suspension, 20 mg/kg BW 2 d.p.i.) and to a water-treated Control group. Each group consisted of seven to nine piglets. The parameters evaluated were oocyst excretion and fecal consistency/diarrhea from 4 to 15 d.p.i. Sulfa-Oral, SMP-Early, and SMP-Late had no significant effect in reduction of oocyst excretion and diarrhea, whereas treatment for 3-7 days with SMP reduced both parasite shedding and diarrhea significantly. Oral treatment with SMP was comparable to parenteral application. Baycox® in a single application had the most pronounced effect and completely suppressed oocyst excretion and diarrhea during the examination period. It could be shown that repeated application of sulfonamides, provided that the appropriate time period after infection

  19. Sensitive turn-on fluorescent detection of tartrazine based on fluorescence resonance energy transfer.

    Science.gov (United States)

    Huang, Sheng Tian; Shi, Yan; Li, Nian Bing; Luo, Hong Qun

    2012-01-18

    We introduce a sensitive, rapid, label-free and general fluorescent method for the determination of tartrazine by competitive binding to reduced graphene oxide (rGO) against fluorescein, and the fluorescence recovery upon fluorescein desorption from rGO provides a quantitative readout for tartrazine, giving a detection limit of 0.53 ng mL(-1).

  20. Adsorption of sulfonamides to demineralized pine wood biochars prepared under different thermochemical conditions

    International Nuclear Information System (INIS)

    Xie, Mengxing; Chen, Wei; Xu, Zhaoyi; Zheng, Shourong; Zhu, Dongqiang

    2014-01-01

    The main objective of this study was to understand the key factors and mechanisms controlling adsorption of sulfonamides to biochars. Batch adsorption experiments were performed for sulfamethoxazole and sulfapyridine to three pine-wood biochars prepared under different thermochemical conditions: pyrolysis at 400 °C (C400) and 500 °C (C500), and pyrolysis at 500 °C followed with hydrogenation (C500-H). For both sulfonamides, the adsorbent surface area-normalized adsorption was stronger to C500 than to C400. This is attributable to the enhanced π–π electron-donor–acceptor interaction with the carbon surface of C500 due to the higher degree of graphitization. Despite the relatively large difference in surface O-functionality content between C500 (12.2%) and C500-H (6.6%), the two biochars exhibited nearly identical adsorbent surface area-normalized adsorption, indicating negligible role of surface O-functionalities in the adsorption to these adsorbents. Effects of solution chemistry conditions (pH, Cu 2+ , and dissolved soil humic acid) on adsorption were examined. -- Highlights: • Adsorption to biochars is dominated by π–π electron-donor–acceptor (EDA) interaction. • Graphitic surfaces of biochars are predominant adsorption sites. • Surface O-functionalities of biochars play minor roles in adsorption. • Adsorption affinities are markedly affected by Cu ions and humic acids. -- Adsorption of sulfonamides to biochars is dominated by π–π electron-donor–acceptor (EDA) interaction with the graphitic surface

  1. Malaria parasite carbonic anhydrase: inhibition of aromatic/heterocyclic sulfonamides and its therapeutic potential

    Science.gov (United States)

    Krungkrai, Sudaratana R; Krungkrai, Jerapan

    2011-01-01

    Plasmodium falciparum (P. falciparum) is responsible for the majority of life-threatening cases of human malaria, causing 1.5-2.7 million annual deaths. The global emergence of drug-resistant malaria parasites necessitates identification and characterization of novel drug targets and their potential inhibitors. We identified the carbonic anhydrase (CA) genes in P. falciparum. The pfCA gene encodes anα-carbonic anhydrase, a Zn2+-metalloenzme, possessing catalytic properties distinct from that of the human host CA enzyme. The amino acid sequence of the pfCA enzyme is different from the analogous protozoan and human enzymes. A library of aromatic/heterocyclic sulfonamides possessing a large diversity of scaffolds were found to be very good inhibitors for the malarial enzyme at moderate-low micromolar and submicromolar inhibitions. The structure of the groups substituting the aromatic-ureido- or aromatic-azomethine fragment of the molecule and the length of the parent sulfonamide were critical parameters for the inhibitory properties of the sulfonamides. One derivative, that is, 4- (3, 4-dichlorophenylureido)thioureido-benzenesulfonamide (compound 10) was the most effective in vitro Plasmodium falciparum CA inhibitor, and was also the most effective antimalarial compound on the in vitro P. falciparum growth inhibition. The compound 10 was also effective in vivo antimalarial agent in mice infected with Plasmodium berghei, an animal model of drug testing for human malaria infection. It is therefore concluded that the sulphonamide inhibitors targeting the parasite CA may have potential for the development of novel therapies against human malaria. PMID:23569766

  2. Thermodynamic aspects of solubility and partitioning processes of some sulfonamides in the solvents modeling biological media

    International Nuclear Information System (INIS)

    Perlovich, German L.; Ryzhakov, Alex M.; Strakhova, Nadezda N.; Kazachenko, Vladimir P.; Schaper, Klaus-Jürgen; Raevsky, Oleg A.

    2014-01-01

    Highlights: • Solubility processes of some sulfonamide isomers in water and 1-octanol were investigated. • Transfer processes from water to 1-octanol were evaluated by analysis of enthalpic and entropic terms. • Impact of various substituents in phenyl rings on solubility and transfer processes was studied. -- Abstract: The thermodynamic aspects of solubility processes of sulfonamides (SAs) with the general structures 4-NH 2 –C 6 H 4 –SO 2 NH–C 6 H 2 (R 1 )(R 2 )-R 3 (R 1 = 2-CH 3 , 2-Cl; R 2 = 4-CH 3 , 4-Cl; R 3 =5-H, 5-Cl), 4-NH 2 -2-Cl–C 6 H 3 –SO 2 NH–C 6 H 3 (R 1 )-R 2 (R 1 = 2-H, 2-Cl; R 2 = 4-H, 4-Cl) and 4-NH 2 -2-CH 3 –C 6 H 3 –SO 2 NH–C 6 H 3 (R 1 )-R 2 (R 1 = 2-H, 2-Cl, 2-NO 2 ; R 2 = 4-H, 4-Cl) in water and 1-octanol (as phases modeling various drug delivery pathways) were studied using the isothermal saturation method. For the sulfonamides with various substituents in phenyl rings the processes of transfer from water to 1-octanol were studied by a diagram method combined with analysis of enthalpic and entropic terms. Distinguishing between enthalpy and entropy, as is possible through the present approach, leads to the insight that the contribution of these terms is different for different molecules (entropy- or enthalpy-determined). Thus, in contrast to the interpretation of only the Gibbs energy of transfer (extensively used for pharmaceuticals in the form of the partition coefficient, logP), the analysis of thermodynamic functions of the transfer process provides additional mechanistic information. This may be important for further evaluation of the physiological distribution of drug molecules and may provide a better understanding of biopharmaceutical properties of drugs

  3. Novel 1,4-naphthoquinone-based sulfonamides: Synthesis, QSAR, anticancer and antimalarial studies.

    Science.gov (United States)

    Pingaew, Ratchanok; Prachayasittikul, Veda; Worachartcheewan, Apilak; Nantasenamat, Chanin; Prachayasittikul, Supaluk; Ruchirawat, Somsak; Prachayasittikul, Virapong

    2015-10-20

    A novel series of 1,4-naphthoquinones (33-44) tethered by open and closed chain sulfonamide moieties were designed, synthesized and evaluated for their cytotoxic and antimalarial activities. All quinone-sulfonamide derivatives displayed a broad spectrum of cytotoxic activities against all of the tested cancer cell lines including HuCCA-1, HepG2, A549 and MOLT-3. Most quinones (33-36 and 38-43) exerted higher anticancer activity against HepG2 cell than that of the etoposide. The open chain analogs 36 and 42 were shown to be the most potent compounds. Notably, the restricted sulfonamide analog 38 with 6,7-dimethoxy groups exhibited the most potent antimalarial activity (IC₅₀ = 2.8 μM). Quantitative structure-activity relationships (QSAR) study was performed to reveal important chemical features governing the biological activities. Five constructed QSAR models provided acceptable predictive performance (Rcv 0.5647-0.9317 and RMSEcv 0.1231-0.2825). Four additional sets of structurally modified compounds were generated in silico (34a-34d, 36a-36k, 40a-40d and 42a-42k) in which their activities were predicted using the constructed QSAR models. A comprehensive discussion of the structure-activity relationships was made and a set of promising compounds (i.e., 33, 36, 38, 42, 36d, 36f, 42e, 42g and 42f) was suggested for further development as anticancer and antimalarial agents. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

  4. Design, synthesis, characterization and computational docking studies of novel sulfonamide derivatives.

    Science.gov (United States)

    Saleem, Hira; Maryam, Arooma; Bokhari, Saleem Ahmed; Ashiq, Ayesha; Rauf, Sadaf Abdul; Khalid, Rana Rehan; Qureshi, Fahim Ashraf; Siddiqi, Abdul Rauf

    2018-01-01

    This study reports three novel sulfonamide derivatives 4-Chloro-N-[(4-methylphenyl) sulphonyl]-N-propyl benzamide ( 1A ), N-(2-hydroxyphenyl)-4-methyl benzene sulfonamide ( 1B ) and 4-methyl-N-(2-nitrophenyl) benzene sulfonamide ( 1C ). The compounds were synthesised from starting material 4-methylbenzenesulfonyl chloride and their structure was studied through 1 H-NMR and 13 C-NMR spectra. Computational docking was performed to estimate their binding energy against bacterial p -amino benzoic acid (PABA) receptor, the dihydropteroate synthase (DHPS). The derivatives were tested in vitro for their antimicrobial activity against Gram+ and Gram- bacteria including E. coli, B. subtilis, B. licheniformis and B. linen. 1A was found active only against B. linen ; 1B was effective against E. coli, B. subtilis and B. linen whereas 1C showed activity against E. coli, B. licheniformis and B. linen . 1C showed maximum activity with minimum inhibitory concentration (MIC) of 50, 100 and 150 µg/mL against E. coli, B. licheniformis and B. linen respectively. 1C exhibited maximum affinity to DHPS with binding free energy of -8.1 kcal/mol. It enriched in the top 0.5 % of a library of 7663 compounds, ranked in order of their binding affinity against DHPS. 1C was followed by 1B which showed a moderate to low level MIC of 100, 250 and 150 µg/mL against E. coli, B. subtilis and B. linen respectively, whereas 1A showed a moderate level MIC of 100 µg/mL but only against B. linen . These derivatives may thus serve as potential anti-bacterial alternatives against resistant pathogens.

  5. Antimicrobial activity of some sulfonamide derivatives on clinical isolates of Staphylococus aureus

    Directory of Open Access Journals (Sweden)

    Bekdemir Yunus

    2008-08-01

    Full Text Available Abstract Background Staphylococcus aureus is a non-motile, gram positive, non-sporforming, facultative anaerobic microorganism. It is one of the important bacteria as a potential pathogen specifically for nosocomial infections. The sulfonamide derivative medicines are preferred to cure infection caused by S. aureus due to methicillin resistance. Methods Antimicrobial activity of four sulfonamide derivatives have been investigated against 50 clinical isolates of S. aureus and tested by using MIC and disc diffusion methods. 50 clinical isolate which collected from specimens of patients who are given medical treatment in Ondokuz Mayis University Medical School Hospital. A control strain of S. aureus ATCC 29213 was also tested. Results The strongest inhibition was observed in the cases of I [N-(2-hydroxy-4-nitro-phenyl-4-methyl-benzensulfonamid], and II [N-(2-hydroxy-5-nitro-phenyl-4-methyl-benzensulfonamid] against S. aureus. Compound I [N-(2-hydroxy-4-nitro-phenyl-4-methyl-benzensulfonamid] showed higher effect on 21 S. aureus MRSAisolates than oxacillin antibiotic. Introducing an electron withdrawing on the ring increased the antimicrobial activity remarkably. Conclusion This study may help to suggest an alternative possible leading compound for development of new antimicrobial agents against MRSA and MSSA resistant S. aureus. It was also shown here that that clinical isolates of 50 S. aureus have various resistance patterns against to four sulfonamide derivatives. It may also be emphasized here that in vitro antimicrobial susceptibility testing results for S. aureus need standardization with further studies and it should also have a correlation with in vivo therapeutic response experiments.

  6. Exciton-controlled fluorescence: application to hybridization-sensitive fluorescent DNA probe.

    Science.gov (United States)

    Okamoto, Akimitsu; Ikeda, Shuji; Kubota, Takeshi; Yuki, Mizue; Yanagisawa, Hiroyuki

    2009-01-01

    A hybridization-sensitive fluorescent probe has been designed for nucleic acid detection, using the concept of fluorescence quenching caused by the intramolecular excitonic interaction of fluorescence dyes. We synthesized a doubly thiazole orange-labeled nucleotide showing high fluorescence intensity for a hybrid with the target nucleic acid and effective quenching for the single-stranded state. This exciton-controlled fluorescent probe was applied to living HeLa cells using microinjection to visualize intracellular mRNA localization. Immediately after injection of the probe into the cell, fluorescence was observed from the probe hybridizing with the target RNA. This fluorescence rapidly decreased upon addition of a competitor DNA. Multicoloring of this probe resulted in the simple simultaneous detection of plural target nucleic acid sequences. This probe realized a large, rapid, reversible change in fluorescence intensity in sensitive response to the amount of target nucleic acid, and facilitated spatiotemporal monitoring of the behavior of intracellular RNA.

  7. Synthesis and characterization of curcumin-sulfonamide hybrids: Biological evaluation and molecular docking studies

    Science.gov (United States)

    Banuppriya, Govindharasu; Sribalan, Rajendran; Padmini, Vediappen

    2018-03-01

    Curcumin-sulfonamide hybrids (4a-e) were synthesized and their in vitro antioxidant, anti-inflammatory and anticancer activities were studied. The synthesized compounds showed a very good potent activity towards antioxidant and anti-inflammatory studies rather than its parent as well as standard. These compounds have exhibited an excellent toxicity effect to the cancer cell lines such as A549 and AGS. The compounds 4a and 4c have showed good anticancer activity than curcumin. The molecular docking studies were also performed against various Epidermal Growth Factor Receptor (EGFR) enzymes. The DFT calculations were also done in order to support the docking results.

  8. Determination of sulfonamides and trimethoprim using high temperature HPLC with simultaneous temperature and solvent gradient.

    Science.gov (United States)

    Giegold, Sascha; Teutenberg, Thorsten; Tuerk, Jochen; Kiffmeyer, Thekla; Wenclawiak, Bernd

    2008-10-01

    A fast HPLC method for the analysis of eight selected sulfonamides (SA) and trimethoprim has been developed with the use of high temperature HPLC. The separation could be achieved in less than 1.5 min on a 50 mm sub 2 microm column with simultaneous solvent and temperature gradient programming. Due to the lower viscosity of the mobile phase and the increased mass transfer at higher temperatures, the separation could be performed on a conventional HPLC system obtaining peak widths at half height between 0.6 and 1.3 s.

  9. 4-Methyl-N-(1-methyl-1H-indazol-5-yl)benzene­sulfonamide

    Science.gov (United States)

    Chicha, Hakima; Oulemda, Bassou; Rakib, El Mostapha; Saadi, Mohamed; El Ammari, Lahcen

    2013-01-01

    In the title compound, C15H15N3O2S, the fused ring system is close to planar, the largest deviation from the mean plane being 0.030 (2) Å, and makes a dihedral angle of 48.84 (9)° with the benzene ring belonging to the methyl­benzene­sulfonamide moiety. In the crystal, mol­ecules are ­connected through N—H⋯N hydrogen bonds and weak C—H⋯O contacts, forming a two-dimensional network parallel to (001). PMID:24427093

  10. Development of an analytical method for determination of sulfonamide residues in eggs

    International Nuclear Information System (INIS)

    Ben Azzeddine, Chams

    2009-01-01

    For the determination of sulfonamide residues in eggs, Premitest is selected for screening, providing a qualitative biological approach, it is inexpensive, fast, multi-elements and easy to implement. The H. P.L.C. / UV is the quantitative method of choice for confirmation and determination of these contaminants. During my internship, I had the opportunity to participate in the development of this method. It is recognized slower and more expensive but more specific and more sensitive. In this report, I present this optimized method and some criteria checked during my internship. Other criteria are to be completed to validate the method that will be subsequently used for routine analysis.

  11. Efficient synthesis of sulfonamide derivatives on solid supports catalyzed using solvent-free and microwave-assisted methods

    Energy Technology Data Exchange (ETDEWEB)

    Camargo-Ordonez, Argelia; Moreno-Reyes, Christian; Olazaran-Santibanez, Fabian; Martinez-Hernandez, Sheila; Bocanegra-Garcia, Virgilio; Rivera, Gildardo [Universidad Autonoma de Tamaulipas, Reynosa (Mexico). Dep. de Farmacia y Quimica Medicinal

    2011-07-01

    In this work we report the synthesis of sulfonamide derivatives using a conventional procedure and with solid supports, such as silica gel, florisil, alumina, 4A molecular sieves, montmorillonite KSF, and montmorillonite K10 using solvent-free and microwave-assisted methods. Our results show that solid supports have a catalytic activity in the formation of sulfonamide derivatives. We found that florisil, montmorillonite KSF, and K10 could be used as inexpensive alternative catalysts that are easily separated from the reaction media. Additionally, solvent-free and microwave-assisted methods were more efficient in reducing reaction time and in increasing yield. (author)

  12. Efficient synthesis of sulfonamide derivatives on solid supports catalyzed using solvent-free and microwave-assisted methods

    International Nuclear Information System (INIS)

    Camargo-Ordonez, Argelia; Moreno-Reyes, Christian; Olazaran-Santibanez, Fabian; Martinez-Hernandez, Sheila; Bocanegra-Garcia, Virgilio; Rivera, Gildardo

    2011-01-01

    In this work we report the synthesis of sulfonamide derivatives using a conventional procedure and with solid supports, such as silica gel, florisil, alumina, 4A molecular sieves, montmorillonite KSF, and montmorillonite K10 using solvent-free and microwave-assisted methods. Our results show that solid supports have a catalytic activity in the formation of sulfonamide derivatives. We found that florisil, montmorillonite KSF, and K10 could be used as inexpensive alternative catalysts that are easily separated from the reaction media. Additionally, solvent-free and microwave-assisted methods were more efficient in reducing reaction time and in increasing yield. (author)

  13. The role of the acidity of N-heteroaryl sulfonamides as inhibitors of bcl-2 family protein-protein interactions.

    Science.gov (United States)

    Touré, B Barry; Miller-Moslin, Karen; Yusuff, Naeem; Perez, Lawrence; Doré, Michael; Joud, Carol; Michael, Walter; DiPietro, Lucian; van der Plas, Simon; McEwan, Michael; Lenoir, Francois; Hoe, Madelene; Karki, Rajesh; Springer, Clayton; Sullivan, John; Levine, Kymberly; Fiorilla, Catherine; Xie, Xiaoling; Kulathila, Raviraj; Herlihy, Kara; Porter, Dale; Visser, Michael

    2013-02-14

    Overexpression of the antiapoptotic members of the Bcl-2 family of proteins is commonly associated with cancer cell survival and resistance to chemotherapeutics. Here, we describe the structure-based optimization of a series of N-heteroaryl sulfonamides that demonstrate potent mechanism-based cell death. The role of the acidic nature of the sulfonamide moiety as it relates to potency, solubility, and clearance is examined. This has led to the discovery of novel heterocyclic replacements for the acylsulfonamide core of ABT-737 and ABT-263.

  14. Molecular recognition of DNA-protein complexes: A straightforward method combining scanning force and fluorescence microscopy

    NARCIS (Netherlands)

    H. Sanchez (Humberto); R. Kanaar (Roland); C. Wyman (Claire)

    2010-01-01

    textabstractCombining scanning force and fluorescent microscopy allows simultaneous identification of labeled biomolecules and analysis of their nanometer level architectural arrangement. Fluorescent polystyrene nano-spheres were used as reliable objects for alignment of optical and topographic

  15. Removal of tetracyclines, sulfonamides, and quinolones by industrial-scale composting and anaerobic digestion processes.

    Science.gov (United States)

    Liu, Hang; Pu, Chengjun; Yu, Xiaolu; Sun, Ying; Chen, Junhao

    2018-02-15

    This study evaluated and compared the removal of antibiotics by industrial-scale composting and anaerobic digestion at different seasons. Twenty compounds belonged to three classes of widely used veterinary antibiotics (i.e., tetracyclines, sulfonamides, and quinolones) were investigated. Results show that of the three groups of antibiotics, tetracyclines were dominant in swine feces and poorly removed by anaerobic digestion with significant accumulation in biosolids, particularly in winter. Compared to that in winter, a much more effective removal (> 97%) by anaerobic digestion was observed for sulfonamides in summer. By contrast, quinolones were the least abundant antibiotics in swine feces and exhibited a higher removal by anaerobic digestion in winter than in summer. The overall removal of antibiotics by aerobic composting could be more than 90% in either winter or summer. Nevertheless, compost products from livestock farms in Beijing contained much higher antibiotics than commercial organic fertilizers. Thus, industrial composting standards should be strictly applied to livestock farms to further remove antibiotics and produce high quality organic fertilizer.

  16. Development and Characterization of Novel Films Based on Sulfonamide-Chitosan Derivatives for Potential Wound Dressing

    Directory of Open Access Journals (Sweden)

    Oana Maria Dragostin

    2015-12-01

    Full Text Available The objective of this study was to develop new films based on chitosan functionalized with sulfonamide drugs (sulfametoxydiazine, sulfadiazine, sulfadimetho-xine, sulfamethoxazol, sulfamerazine, sulfizoxazol in order to enhance the biological effects of chitosan. The morphology and physical properties of functionalized chitosan films as well the antioxidant effects of sulfonamide-chitosan derivatives were investigated. The chitosan-derivative films showed a rough surface and hydrophilic properties, which are very important features for their use as a wound dressing. The film based on chitosan-sulfisoxazol (CS-S6 showed the highest swelling ratio (197% and the highest biodegradation rate (63.04% in comparison to chitosan film for which the swelling ratio was 190% and biodegradation rate was only 10%. Referring to the antioxidant effects the most active was chitosan-sulfamerazine (CS-S5 which was 8.3 times more active than chitosan related to DPPH (1,1-diphenyl-2-picrylhydrazyl radical scavenging ability. This compound showed also a good ferric reducing power and improved total antioxidant capacity.

  17. Rapid startup of thermophilic anaerobic digester to remove tetracycline and sulfonamides resistance genes from sewage sludge.

    Science.gov (United States)

    Xu, Rui; Yang, Zhao-Hui; Wang, Qing-Peng; Bai, Yang; Liu, Jian-Bo; Zheng, Yue; Zhang, Yan-Ru; Xiong, Wei-Ping; Ahmad, Kito; Fan, Chang-Zheng

    2018-01-15

    Spread of antibiotic resistance genes (ARGs) originating from sewage sludge is highlighted as an eminent health threat. This study established a thermophilic anaerobic digester using one-step startup strategy to quickly remove tetracycline and sulfonamides resistance genes from sewage sludge. At least 20days were saved in the startup period from mesophilic to thermophilic condition. Based on the results of 16S rDNA amplicons sequencing and predicted metagenomic method, the successful startup largely relied on the fast colonization of core thermophilic microbial population (e.g. Firmicutes, Proteobacteria, Actinobacteria). Microbial metabolic gene pathways for substrate degradation and methane production was also increased by one-step mode. In addition, real-time quantitative PCR approach revealed that most targeted tetracycline and sulfonamides resistance genes ARGs (sulI, tetA, tetO, tetX) were substantially removed during thermophilic digestion (removal efficiency>80%). Network analysis showed that the elimination of ARGs was attributed to the decline of their horizontal (intI1 item) and vertical (potential hosts) transfer-related elements under high-temperature. This research demonstrated that rapid startup thermophilic anaerobic digestion of wastewater solids would be a suitable technology for reducing quantities of various ARGs. Copyright © 2017 Elsevier B.V. All rights reserved.

  18. Preliminary investigation on the occurrence of several sulfonamide antibiotics in the Haihe River Basin of China

    Science.gov (United States)

    Zhang, S. L.; Zhang, J.; Wang, Z. G.; Wang, Y. Z.; Liang, S. T.; Liu, C.; Wang, Z.

    2017-08-01

    Several samples collected from lakes, rivers and reservoirs in Haihe river basin of China were analyzed for 8 sulfonamide antibiotics by using solid-phase extraction and liquid chromatography with tandem mass spectrometry (HPLC-MS/MS). All water samples were enriched with HLB extraction cartridges. The antibiotics were separated by gradient elution with methanol as the mobile phase adding 0.1% formic acid. The eluate was then analyzed by the mode of multiple reaction monitoring (MRM). The limits of detection (LOD) and quantification (LOQ) were 0.4-1.0 ng/L and 1.0-3.0 ng/L respectively. The method was used for the analysis of 13 samples from Haihe river basin in China. The results showed that sulfamethoxazole was present in all water samples with maximum concentration of 107.59 ng/L. Sulfadiazine was also frequently detected, concentrations ranging from 2.81 ng/L to 85.35 ng/L. Other sulfonamide antibiotics were not detected in most water samples, especially for those samples from drinking water resources.

  19. Spectroscopic characterization, antimicrobial activity, DFT computation and docking studies of sulfonamide Schiff bases

    Science.gov (United States)

    Mondal, Sudipa; Mandal, Santi M.; Mondal, Tapan Kumar; Sinha, Chittaranjan

    2017-01-01

    Schiff bases synthesised from the condensation of 2-(hydroxy)naphthaldehyde and sulfonamides (sufathiazole (STZ), sulfapyridine (SPY), sulfadiazine (SDZ), sulfamerazine (SMZ) and sulfaguanidine (SGN)) are characterized by different spectroscopic data (FTIR, UV-Vis, Mass, NMR) and two of them, (E)-4-(((2-hydroxynaphthalen-1-yl)methylene)amino)-N-(thiazol-2-yl)benzenesulfonamide (1a) and (E)-N-(diaminomethylene)-4-(((2-hydroxynaphthalen-1-yl)methylene)amino)benzenesulfonamide (1e) have been confirmed by single crystal X-ray structure determination. Antimicrobial activities of the Schiff bases have been evaluated against certified and resistant Gram positive (Staphylococcus aureus, Enterococcus facelis) and Gram negative (Streptococcus pyogenes, Salmonella typhi, Shigella dysenteriae, Shigella flexneri, Klebsiella pneumonia) pathogens. Performance of Schiff base against the resistant pathogens are better than standard stain and MIC data lie 32-128 μg/ml while parent sulfonamides are effectively inactive (MIC >512 μg/ml). The DFT optimized structures of the Schiff bases have been used to accomplish molecular docking studies with DHPS (dihydropteroate synthase) protein structure (downloaded from Protein Data Bank) to establish the most preferred mode of interaction. ADMET filtration, Cytotoxicity (MTT assay) and haemolysis assay have been examined for evaluation of druglike character.

  20. Elimination patterns of worldwide used sulfonamides and tetracyclines during anaerobic fermentation.

    Science.gov (United States)

    Spielmeyer, Astrid; Breier, Bettina; Groißmeier, Kathrin; Hamscher, Gerd

    2015-10-01

    Antibiotics such as sulfonamides and tetracyclines are frequently used in veterinary medicine. Due to incomplete absorption in the animal gut and/or unmetabolized excretion, the substances can enter the environment by using manure as soil fertilizer. The anaerobic fermentation process of biogas plants is discussed as potential sink for antibiotic compounds. However, negative impacts of antibiotics on the fermentation process are suspected. The elimination of sulfadiazine, sulfamethazine, tetracycline and chlortetracycline in semi-continuous lab-scale fermenters was investigated. Both biogas production and methane yield were not negatively affected by concentrations up to 38 mg per kg for sulfonamides and 7 mg per kg for tetracyclines. All substances were partly eliminated with elimination rates between 14% and 89%. Both matrix and structure of the target molecule influenced the elimination rate. Chlortetracycline was mainly transformed into iso-chlortetracycline. In all other cases, the elimination pathways remained undiscovered; however, sorption processes seem to have a negligible impact. Copyright © 2015 Elsevier Ltd. All rights reserved.

  1. Impact of manure-related DOM on sulfonamide transport in arable soils

    Science.gov (United States)

    Zhou, Dan; Thiele-Bruhn, Sören; Arenz-Leufen, Martina Gesine; Jacques, Diederik; Lichtner, Peter; Engelhardt, Irina

    2016-09-01

    Field application of livestock manure introduces colloids and veterinary antibiotics, e.g. sulfonamides (SAs), into farmland. The presence of manure colloids may potentially intensify the SAs-pollution to soils and groundwater by colloid-facilitated transport. Transport of three SAs, sulfadiazine (SDZ), sulfamethoxypyridazine (SMPD), and sulfamoxole (SMOX), was investigated in saturated soil columns with and without manure colloids from sows and farrows, weaners, and fattening pigs. Experimental results showed that colloid-facilitated transport of SMOX was significant in the presence of manure colloids from fattening pigs with low C/N ratio, high SUVA280 nm and protein C, while manure colloids from sows and farrows and weaners had little effect on SMOX transport. In contrast, only retardation was observed for SDZ and SMPD when manure colloids were present. Breakthrough curves (BTCs) of colloids and SAs were replicated well by a newly developed numerical model that considers colloid-filtration theory, competitive kinetic sorption, and co-transport processes. Model results demonstrate that mobile colloids act as carriers for SMOX, while immobile colloids block SMOX from sorbing onto the soil. The low affinity of SMOX to sorb on immobile colloids prevents aggregation and also promotes SMOX's colloid-facilitated transport. Conversely, the high affinity of SDZ and SMPD to sorb on all types of immobile colloids retarded their transport. Thus, manure properties play a fundamental role in increasing the leaching risk of hydrophobic sulfonamides.

  2. Thermodynamic aspects of solubility, solvation and partitioning processes of some sulfonamides

    Energy Technology Data Exchange (ETDEWEB)

    Perlovich, German L., E-mail: glp@isc-ras.r [Department of Computer-Aided Molecular Design, Institute of Physiologically Active Compounds, Russian Academy of Sciences, 142432 Chernogolovka (Russian Federation); Institute of Solution Chemistry, Russian Academy of Sciences, 153045 Ivanovo (Russian Federation); Ryzhakov, Alex M. [Institute of Solution Chemistry, Russian Academy of Sciences, 153045 Ivanovo (Russian Federation); Strakhova, Nadezda N.; Kazachenko, Vladimir P. [Department of Computer-Aided Molecular Design, Institute of Physiologically Active Compounds, Russian Academy of Sciences, 142432 Chernogolovka (Russian Federation); Schaper, Klaus-Juergen [Research Center Borstel, Leibniz Center for Medicine and Biosciences, D-23845 Borstel (Germany); Raevsky, Oleg A. [Department of Computer-Aided Molecular Design, Institute of Physiologically Active Compounds, Russian Academy of Sciences, 142432 Chernogolovka (Russian Federation)

    2011-05-15

    Research highlights: {yields} The thermodynamic aspects of sublimation processes of some sulfonamides were studied by investigating the temperature dependence of vapor pressure using the transpiration method. {yields} Solubility processes of the compounds in water, phosphate buffer with pH 7.4 and n-octanol were investigated and corresponding thermodynamic functions were calculated as well. {yields} Thermodynamic characteristics of the sulfonamides solvation were evaluated. - Abstract: The thermodynamic aspects of sublimation processes of three sulfonamides with the general structures C{sub 6}H{sub 5}-SO{sub 2}NH-C{sub 6}H{sub 4}-R (R = 4-NO{sub 2}) and 4-NH{sub 2}-C{sub 6}H{sub 4}-SO{sub 2}NH-C{sub 6}H{sub 4}-R (R = 4-NO{sub 2}; 4-CN) were studied by investigating the temperature dependence of vapor pressure using the transpiration method. These data together with those obtained earlier for C{sub 6}H{sub 5}-SO{sub 2}NH-C{sub 6}H{sub 4}-R (R = 4-Cl) and 4-NH{sub 2}-C{sub 6}H{sub 4}-SO{sub 2}NH-C{sub 6}H{sub 4}-R (R = 4-Cl; 4-OMe; 4-C{sub 2}H{sub 5}) were analyzed and compared. A correlation was derived between sublimation Gibbs free energies and the sum of H-bond acceptor factors of the molecules. Solubility processes of the compounds in water, phosphate buffer with pH 7.4 and n-octanol (as phases modeling various drug delivery pathways) were investigated and corresponding thermodynamic functions were calculated as well. Thermodynamic characteristics of the sulfonamides solvation were evaluated. Also in this case a correlation between solubility/solvation Gibbs free energy values and the sum of H-bond acceptor factors was observed. For the sulfonamides with various substituents at para-position the processes of transfer from one solvent (water or buffer) to n-octanol were studied by a diagram method combined with analysis of enthalpic and entropic terms. Distinguishing between enthalpy and entropy, as is possible through the present approach, leads to the insight

  3. Sulfonamide inhibition studies of two β-carbonic anhydrases from the ascomycete fungus Sordaria macrospora, CAS1 and CAS2.

    Science.gov (United States)

    Vullo, Daniela; Lehneck, Ronny; Pöggeler, Stefanie; Supuran, Claudiu T

    2018-12-01

    The two β-carbonic anhydrases (CAs, EC 4.2.1.1) recently cloned and purified from the ascomycete fungus Sordaria macrospora, CAS1 and CAS2, were investigated for their inhibition with a panel of 39 aromatic, heterocyclic, and aliphatic sulfonamides and one sulfamate, many of which are clinically used agents. CAS1 was efficiently inhibited by tosylamide, 3-fluorosulfanilamide, and 3-chlorosulfanilamide (K I s in the range of 43.2-79.6 nM), whereas acetazolamide, methazolamide, topiramate, ethoxzolamide, dorzolamide, and brinzolamide were medium potency inhibitors (K I s in the range of 360-445 nM). CAS2 was less sensitive to sulfonamide inhibitors. The best CAS2 inhibitors were 5-amino-1,3,4-thiadiazole-2-sulfonamide (the deacetylated acetazolamide precursor) and 4-hydroxymethyl-benzenesulfonamide, with K I s in the range of 48.1-92.5 nM. Acetazolamide, dorzolamide, ethoxzolamide, topiramate, sulpiride, indisulam, celecoxib, and sulthiame were medium potency CAS2 inhibitors (K I s of 143-857 nM). Many other sulfonamides showed affinities in the high micromolar range or were ineffective as CAS1/2 inhibitors. Small changes in the structure of the inhibitor led to important differences of the activity. As these enzymes may show applications for the removal of anthropically generated polluting gases, finding modulators of their activity may be crucial for designing environmental-friendly CO 2 capture processes.

  4. Rapid method for quantification of nine sulfonamides in bovine milk using HPLC/MS/MS and without using SPE.

    Science.gov (United States)

    Nebot, Carolina; Regal, Patricia; Miranda, Jose Manuel; Fente, Cristina; Cepeda, Alberto

    2013-12-01

    Sulfonamides are antimicrobial agents widely employed in animal production and their residues in food could be an important risk to human health. In the dairy industry, large quantities of milk are monitored daily for the presence of sulfonamides. A simple and low-cost extraction protocol followed by a liquid chromatographic-tandem mass spectrometry method was developed for the simultaneous detection of nine sulfonamides in whole milk. The method was validated at the maximum residue limits established by European legislation. The limits of quantification obtained for most sulfonamides were between 12.5 and 25 μg kg(-1), detection capabilities ranged from 116 to 145 μg kg(-1), and recoveries, at 100 μg kg(-1), were greater than 89±12.5%. The method was employed to analyse 100 raw whole bovine milk samples collected from dairy farms in the northwest region of Spain. All of the samples were found to be compliant, but two were positive; one for sulfadiazine and the other for sulfamethoxipyridazine. Copyright © 2013 Elsevier Ltd. All rights reserved.

  5. Accumulation of Sulfonamide Resistance Genes in Arable Soils Due to Repeated Application of Manure Containing Sulfadiazine ▿

    OpenAIRE

    Heuer, Holger; Solehati, Qodiah; Zimmerling, Ute; Kleineidam, Kristina; Schloter, Michael; Müller, Tanja; Focks, Andreas; Thiele-Bruhn, Sören; Smalla, Kornelia

    2011-01-01

    Two soils were amended three times with pig manure. The abundance of sulfonamide resistance genes was determined by quantitative PCR 2 months after each application. In both soils treated with sulfadiazine-containing manure, the numbers of copies of sul1 and sul2 significantly increased compared to numbers after treatments with antibiotic-free manure or a control and accumulated with repeated applications.

  6. Synthesis and Photophysical Properties of Biaryl-Substituted Nucleos(t)ides. Polymerase Synthesis of DNA Probes Bearing Solvatochromic and pH Sensitive Dual Fluorescent and 19F NMR Labels

    Czech Academy of Sciences Publication Activity Database

    Riedl, Jan; Pohl, Radek; Rulíšek, Lubomír; Hocek, Michal

    2012-01-01

    Roč. 77, č. 2 (2012), s. 1026-1044 ISSN 0022-3263 R&D Projects: GA ČR GA203/09/0317 Institutional research plan: CEZ:AV0Z40550506 Keywords : nucleotides * biaryls * fluorescence * DNA Subject RIV: CC - Organic Chemistry Impact factor: 4.564, year: 2012

  7. Fluorescein-labeled stable neurotensin derivatives.

    Science.gov (United States)

    Maes, Veronique; Hultsch, Christina; Kohl, Suzann; Bergmann, Ralf; Hanke, Thomas; Tourwé, Dirk

    2006-08-01

    Neurotensin(8-13) analogs containing a glycine or 5-aminovaleroyl spacer were labeled with fluorescein through formation of an N-terminal thiourea function. The receptor binding was measured in HT-29 cell cultures and showed a substantial decrease in affinity, especially for the metabolically stabilized [MeArg(9), Tle(11)] analog. Using fluorescence microscopy, the internalization of the fluorescent neurotensin analogs into HT-29 cells was observed. Copyright (c) 2006 European Peptide Society and John Wiley & Sons, Ltd.

  8. Development of a general methodology for labelling peptide-morpholino oligonucleotide conjugates using alkyne-azide click chemistry.

    Science.gov (United States)

    Shabanpoor, Fazel; Gait, Michael J

    2013-11-11

    We describe a general methodology for fluorescent labelling of peptide conjugates of phosphorodiamidate morpholino oligonucleotides (PMOs) by alkyne functionalization of peptides, subsequent conjugation to PMOs and labelling with a fluorescent compound (Cy5-azide). Two peptide-PMO (PPMO) examples are shown. No detrimental effect of such labelled PMOs was seen in a biological assay.

  9. Fluorescence microscopy.

    Science.gov (United States)

    Sanderson, Michael J; Smith, Ian; Parker, Ian; Bootman, Martin D

    2014-10-01

    Fluorescence microscopy is a major tool with which to monitor cell physiology. Although the concepts of fluorescence and its optical separation using filters remain similar, microscope design varies with the aim of increasing image contrast and spatial resolution. The basics of wide-field microscopy are outlined to emphasize the selection, advantages, and correct use of laser scanning confocal microscopy, two-photon microscopy, scanning disk confocal microscopy, total internal reflection, and super-resolution microscopy. In addition, the principles of how these microscopes form images are reviewed to appreciate their capabilities, limitations, and constraints for operation. © 2014 Cold Spring Harbor Laboratory Press.

  10. Food labels

    DEFF Research Database (Denmark)

    Selsøe Sørensen, Henrik; Clement, Jesper; Gabrielsen, Gorm

    2012-01-01

    evidence for dividing consumers into two profiles: one relying on general food knowledge and another using knowledge related to signpost labels. In a combined eyetracking and questionnaire survey we analyse the influence of background knowledge and identify different patterns of visual attention......The food industry develops tasty and healthy food but fails to deliver the message to all consumers. The consumers’ background knowledge is essential for how they find and decode relevant elements in the cocktail of signs which fight for attention on food labels. In this exploratory study, we find...... for the two consumer profiles. This underlines the complexity in choosing and designing the ‘right’ elements for a food package that consumers actually look at and are able to make rational use of. In spite of any regulation of food information provided by authorities, consumers will still be confronted...

  11. Sorption and biodegradation of sulfonamide antibiotics by activated sludge: experimental assessment using batch data obtained under aerobic conditions.

    Science.gov (United States)

    Yang, Sheng-Fu; Lin, Cheng-Fang; Lin, Angela Yu-Chen; Hong, Pui-Kwan Andy

    2011-05-01

    This study investigated the adsorption, desorption, and biodegradation characteristics of sulfonamide antibiotics in the presence of activated sludge with and without being subjected to NaN(3) biocide. Batch experiments were conducted and the relative contributions of adsorption and biodegradation to the observed removal of sulfonamide antibiotics were determined. Three sulfonamide antibiotics including sulfamethoxazole (SMX), sulfadimethoxine (SDM), and sulfamonomethoxine (SMM), which had been detected in the influent and the activated sludge of wastewater treatment plants (WWTP) in Taiwan, were selected for this study. Experimental results showed that the antibiotic compounds were removed via sorption and biodegradation by the activated sludge, though biodegradation was inhibited in the first 12 h possibly due to competitive inhibition of xenobiotic oxidation by readily biodegradable substances. The affinity of sulfonamides to sterilized sludge was in the order of SDM > SMM > SMX. The sulfonamides existed predominantly as anions at the study pH of 6.8, which resulted in a low level of adsorption to the activated sludge. The adsorption/desorption isotherms were of a linear form, as well described by the Freundlich isotherm with the n value approximating unity. The linear distribution coefficients (K(d)) were determined from batch equilibrium experiments with values of 28.6 ± 1.9, 55.7 ± 2.2, and 110.0 ± 4.6 mL/g for SMX, SMM, and SDM, respectively. SMX, SMM, and SDM desorb reversibly from the activated sludge leaving behind on the solids 0.9%, 1.6%, and 5.2% of the original sorption dose of 100 μg/L. The sorbed antibiotics can be introduced into the environment if no further treatments were employed to remove them from the biomass. Copyright © 2011 Elsevier Ltd. All rights reserved.

  12. Analysis of Cholesterol Trafficking with Fluorescent Probes

    DEFF Research Database (Denmark)

    Maxfield, Frederick R.; Wustner, Daniel

    2012-01-01

    Cholesterol plays an important role in determining the biophysical properties of biological membranes, and its concentration is tightly controlled by homeostatic processes. The intracellular transport of cholesterol among organelles is a key part of the homeostatic mechanism, but sterol transport...... that can bind to cholesterol to reveal its distribution in cells. We also discuss the use of intrinsically fluorescent sterols that closely mimic cholesterol, as well as some minimally modified fluorophore-labeled sterols. Methods for imaging these sterols by conventional fluorescence microscopy...... and by multiphoton microscopy are described. Some label-free methods for imaging cholesterol itself are also discussed briefly....

  13. Application of fluorescent-and radioactive tracers in Sedimentalogy

    International Nuclear Information System (INIS)

    Alencar, L.M.L. de.

    1981-01-01

    The development of techniques of sediment labelling, creating the possibility of using fluorescent and radioactive tracers not yet applied in Brazil, in the area of sedimentology, is studied. (A.R.H.) [pt

  14. Multispectral open-air intraoperative fluorescence imaging.

    Science.gov (United States)

    Behrooz, Ali; Waterman, Peter; Vasquez, Kristine O; Meganck, Jeff; Peterson, Jeffrey D; Faqir, Ilias; Kempner, Joshua

    2017-08-01

    Intraoperative fluorescence imaging informs decisions regarding surgical margins by detecting and localizing signals from fluorescent reporters, labeling targets such as malignant tissues. This guidance reduces the likelihood of undetected malignant tissue remaining after resection, eliminating the need for additional treatment or surgery. The primary challenges in performing open-air intraoperative fluorescence imaging come from the weak intensity of the fluorescence signal in the presence of strong surgical and ambient illumination, and the auto-fluorescence of non-target components, such as tissue, especially in the visible spectral window (400-650 nm). In this work, a multispectral open-air fluorescence imaging system is presented for translational image-guided intraoperative applications, which overcomes these challenges. The system is capable of imaging weak fluorescence signals with nanomolar sensitivity in the presence of surgical illumination. This is done using synchronized fluorescence excitation and image acquisition with real-time background subtraction. Additionally, the system uses a liquid crystal tunable filter for acquisition of multispectral images that are used to spectrally unmix target fluorescence from non-target auto-fluorescence. Results are validated by preclinical studies on murine models and translational canine oncology models.

  15. Role of the sulfonamide moiety of Ru(II) half-sandwich complexes in the asymmetric transfer hydrogenation of 3,4-dihydroisoquinolines

    Czech Academy of Sciences Publication Activity Database

    Matuška, O.; Zápal, J.; Hrdličková, R.; Mikoška, M.; Pecháček, J.; Vilhanová, B.; Václavík, Jiří; Kuzma, M.; Kačer, P.

    2016-01-01

    Roč. 118, č. 1 (2016), s. 215-222 ISSN 1878-5190 Institutional support: RVO:61388963 Keywords : ruthenium * asymmetric transfer hydrogenation * dihydroisoquinolines * sulfonamide Subject RIV: CC - Organic Chemistry Impact factor: 1.264, year: 2016

  16. Label triangulation

    International Nuclear Information System (INIS)

    May, R.P.

    1983-01-01

    Label Triangulation (LT) with neutrons allows the investigation of the quaternary structure of biological multicomponent complexes under native conditions. Provided that the complex can be fully separated into and reconstituted from its single - protonated and deuterated - components, small angle neutron scattering (SANS) can give selective information on shapes and pair distances of these components. Following basic geometrical rules, the spatial arrangement of the components can be reconstructed from these data. LT has so far been successfully applied to the small and large ribosomal subunits and the transcriptase of E. coli. (author)

  17. Ionic treatment for removal of sulfonamide and tetracycline classes of antibiotic

    International Nuclear Information System (INIS)

    Choi, Keun-Joo; Son, Hee-Jong; Kim, Seung-Hyun

    2007-01-01

    Self-decomposition and removal of antibiotics by ionic treatment was evaluated in this study. Seven sulfonamide classes (SA) and seven tetracycline classes (TA) of antibiotic were selected for this purpose. According to this study, self-decomposition of SAs and TAs was slow, and a considerable amount of antibiotics still remained after 15 days. Ionic treatment was effective for removal of SAs and TAs, but organic interference was observed. When dissolved organic (DOC) was present in raw water, the removal performance of antibiotics generally deteriorated due to competition with organics. SAs and TAs, which were present in ionic form at neutral pH, were removed through ion exchange. Their removal efficiencies were closely related to their chemical structure. Antibiotics with stronger electronegativity were easier to remove by ionic treatment. Equilibrium equations for removal of SAs and TAs by ionic treatment were also presented

  18. Recent advances in sample preparation techniques and methods of sulfonamides detection - A review.

    Science.gov (United States)

    Dmitrienko, Stanislava G; Kochuk, Elena V; Apyari, Vladimir V; Tolmacheva, Veronika V; Zolotov, Yury A

    2014-11-19

    Sulfonamides (SAs) have been the most widely used antimicrobial drugs for more than 70 years, and their residues in foodstuffs and environmental samples pose serious health hazards. For this reason, sensitive and specific methods for the quantification of these compounds in numerous matrices have been developed. This review intends to provide an updated overview of the recent trends over the past five years in sample preparation techniques and methods for detecting SAs. Examples of the sample preparation techniques, including liquid-liquid and solid-phase extraction, dispersive liquid-liquid microextraction and QuEChERS, are given. Different methods of detecting the SAs present in food and feed and in environmental, pharmaceutical and biological samples are discussed. Copyright © 2014 Elsevier B.V. All rights reserved.

  19. Synthesis and in silico studies of novel sulfonamides having oxadiazole ring: As β-glucuronidase inhibitors.

    Science.gov (United States)

    Taha, Muhammad; Baharudin, Mohd Syukri; Ismail, Nor Hadiani; Selvaraj, Manikandan; Salar, Uzma; Alkadi, Khaled A A; Khan, Khalid Mohammed

    2017-04-01

    Novel sulfonamides having oxadiazole ring were synthesized by multistep reaction and evaluated to check in vitro β-glucuronidase inhibitory activity. Luckily, except compound 13, all compounds were found to demonstrate good inhibitory activity in the range of IC 50 =2.40±0.01-58.06±1.60μM when compared to the standard d-saccharic acid 1,4-lactone (IC 50 =48.4±1.25μM). Structure activity relationship was also presented. However, in order to ensure the SAR as well as the molecular interactions of compounds with the active site of enzyme, molecular docking studies on most active compounds 19, 16, 4 and 6 was carried out. All derivatives were fully characterized by 1 H NMR, 13 C NMR and EI-MS spectroscopic techniques. CHN analysis was also presented. Copyright © 2017 Elsevier Inc. All rights reserved.

  20. Insights into functional-group-tolerant polymerization catalysis with phosphine-sulfonamide palladium (II) complexes

    KAUST Repository

    Jian, Zhongbao

    2014-12-08

    Two series of cationic palladium(II) methyl complexes {[(2-MeOC6H4)2PC6H4SO2NHC6H3(2,6-R1,R2)]PdMe}2[A]2 (X1+-A: R1=R2=H: H1+-A; R1=R2=CH(CH3)2: DIPP1+-A; R1=H, R2=CF3: CF31+-A; A=BF4 or SbF6) and neutral palladium(II) methyl complexes {[(2-MeOC6H4)2PC6H4SO2NC6H3(2,6-R1,R2)]PdMe(L)} (X1-acetone: L=acetone; X1-dmso: L=dimethyl sulfoxide; X1-pyr: L=pyridine) chelated by a phosphine-sulfonamide were synthesized and fully characterized. Stoichiometric insertion of methyl acrylate (MA) into all complexes revealed that a 2,1 regiochemistry dominates in the first insertion of MA. Subsequently, for the cationic complexes X1+-A, β-H elimination from the 2,1-insertion product X2+-AMA-2,1 is overwhelmingly favored over a second MA insertion to yield two major products X4+-AMA-1,2 and X5+-AMA. By contrast, for the weakly coordinated neutral complexes X1-acetone and X1-dmso, a second MA insertion of the 2,1-insertion product X2MA-2,1 is faster than β-H elimination and gives X3MA as major products. For the strongly coordinated neutral complexes X1-pyr, no second MA insertion and no β-H elimination (except for DIPP2-pyrMA-2,1) were observed for the 2,1-insertion product X2-pyrMA-2,1. The cationic complexes X1+-A exhibited high catalytic activities for ethylene dimerization, affording butenes (C4) with a high selectivity of up to 97.7% (1-butene: 99.3%). Differences in activities and selectivities suggest that the phosphine-sulfonamide ligands remain coordinated to the metal center in a bidentate fashion in the catalytically active species. By comparison, the neutral complexes X1-acetone, X1-dmso, and X1-pyr showed very low activity towards ethylene to give traces of oligomers. DFT analyses taking into account the two possible coordination modes (O or N) of the sulfonamide ligand for the cationic system CF31+ suggested that the experimentally observed high activity in ethylene dimerization is the result of a facile first ethylene insertion into the O-coordinated PdMe isomer and

  1. Synthesis and Antimicrobial Activity of Bis-4,6-sulfonamidated 5,7-Dinitrobenzofuroxans

    Directory of Open Access Journals (Sweden)

    Irina V. Galkina

    2014-01-01

    Full Text Available A new series of bis-4,6-sulfonamidated 5,7-dinitrbenzofuroxans  7–11 had been synthesized and tested for antimicrobial activity. The structures of new sulfanilamide derivatives were characterized by elemental analysis, IR spectroscopy, and mass spectrometry (MALDITOF. The synthesized compounds were tested for their in vitro antimicrobial activity using the disk diffusion method against Gram-positive bacteria Staphylococcus aureus; the Gram-negative bacteria Escherichia coli, Pseudomonas aeruginosa, and Proteus mirabilis; the fungal strain Aspergillus niger; and the yeast-like pathogenic fungus Candida albicans. Our results indicate that the compounds 7–11 exhibit potent antimicrobial activity. The stability of the compounds was evaluated by TG and DSC methods.

  2. Electrochemical degradation of sulfonamides at BDD electrode: Kinetics, reaction pathway and eco-toxicity evaluation

    Energy Technology Data Exchange (ETDEWEB)

    Fabiańska, Aleksandra; Białk-Bielińska, Anna; Stepnowski, Piotr [Faculty of Chemistry, University of Gdansk, ul. Wita Stwosza 63, 80-952 Gdansk (Poland); Stolte, Stefan [Faculty of Chemistry, University of Gdansk, ul. Wita Stwosza 63, 80-952 Gdansk (Poland); UFT-Centre of Environmental Research and Sustainable Technology, University of Bremen, Leobener Straße UFT, D-28359 Bremen (Germany); Siedlecka, Ewa Maria, E-mail: ewa.siedlecka@ug.edu.pl [Faculty of Chemistry, University of Gdansk, ul. Wita Stwosza 63, 80-952 Gdansk (Poland)

    2014-09-15

    Highlights: • SNs were electrochemically oxidized at BDD in one compartment reactor. • The efficiency of SN degradation was the highest in effluents from municipal WWTP. • The electro-degradation SNs based on oxidation but reduction was also possible. • Electrochemical oxidation of SNs led in some cases to mixtures toxic to L. minor. - Abstract: The investigation dealt with electrochemical oxidation of five sulfonamides (SNs): sulfadiazine (SDZ), sulfathiazole (STZ), sulfamerazine (SMR), sulfamethazine (SMN) and sulfadimethoxine (SDM) in aqueous solution at boron-doped diamond (BDD) electrode. All studied sulfonamides were degraded according to a pseudo first order kinetics. The structure of SNs had no significant effect on the values of pseudo first order rate constants. Increased degradation efficiency was observed in higher temperature and in acidic pH. Due to the presence of chlorine and nitrate SNs were more effectively oxidized from municipal wastewater treatment plant (WWTP) effluents than from pure supporting electrolyte Na{sub 2}SO{sub 4}. The intermediates identified by LC–MS and GC–MS analysis suggested that the hydroxyl radicals attack mainly the S-N bond, but also the aromatic ring systems (aniline, pyrimidine or triazole) of SNs. Finally, the toxicity of the SNs solutions and effluents after electrochemical treatment was assessed through the measurement of growth inhibition of green algae (Scenedesmus vacualatus) and duckweed (Lemna minor). Toxicity of SMR, STZ, SMN solutions before and after electrochemical oxidation and SDM solution after the process in L. minor test was observed. No significant toxicity of studied SNs was observed in algae test.

  3. Electrochemical degradation of sulfonamides at BDD electrode: Kinetics, reaction pathway and eco-toxicity evaluation

    International Nuclear Information System (INIS)

    Fabiańska, Aleksandra; Białk-Bielińska, Anna; Stepnowski, Piotr; Stolte, Stefan; Siedlecka, Ewa Maria

    2014-01-01

    Highlights: • SNs were electrochemically oxidized at BDD in one compartment reactor. • The efficiency of SN degradation was the highest in effluents from municipal WWTP. • The electro-degradation SNs based on oxidation but reduction was also possible. • Electrochemical oxidation of SNs led in some cases to mixtures toxic to L. minor. - Abstract: The investigation dealt with electrochemical oxidation of five sulfonamides (SNs): sulfadiazine (SDZ), sulfathiazole (STZ), sulfamerazine (SMR), sulfamethazine (SMN) and sulfadimethoxine (SDM) in aqueous solution at boron-doped diamond (BDD) electrode. All studied sulfonamides were degraded according to a pseudo first order kinetics. The structure of SNs had no significant effect on the values of pseudo first order rate constants. Increased degradation efficiency was observed in higher temperature and in acidic pH. Due to the presence of chlorine and nitrate SNs were more effectively oxidized from municipal wastewater treatment plant (WWTP) effluents than from pure supporting electrolyte Na 2 SO 4 . The intermediates identified by LC–MS and GC–MS analysis suggested that the hydroxyl radicals attack mainly the S-N bond, but also the aromatic ring systems (aniline, pyrimidine or triazole) of SNs. Finally, the toxicity of the SNs solutions and effluents after electrochemical treatment was assessed through the measurement of growth inhibition of green algae (Scenedesmus vacualatus) and duckweed (Lemna minor). Toxicity of SMR, STZ, SMN solutions before and after electrochemical oxidation and SDM solution after the process in L. minor test was observed. No significant toxicity of studied SNs was observed in algae test

  4. Development of a liquid chromatography–tandem mass spectrometry method for the determination of sulfonamides, trimethoprim and dapsone in honey and validation according to Commission Decision 2002/657/EC for banned compounds [corrected].

    Science.gov (United States)

    Economou, Anastasios; Petraki, Olympia; Tsipi, Despina; Botitsi, Eleni

    2012-08-15

    This work reports a sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for identification and quantification of seven sulfonamides, trimethoprim and dapsone in honey. The method is based on a solid-phase extraction (SPE) step of the target analytes with Oasis HLB cartridges after acidic hydrolysis of the honey sample to liberate the sugar-bound sulfonamides. Analysis was performed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) in the positive electro-spray ionization (ESI) mode with two different isotopically labeled internal standards with the view to improve the quantitative performance of the method. The method validation has been performed according to the Commission Decision 2002/657/EC; the average recoveries, measured at three concentration levels (1.5, 2.5 and 5.0 μg kg(-1)), have been estimated in the range 70 to 106% while the respective % relative standard deviations of the within-laboratory reproducibility ranged from 6 to 18%. Mean values of the expanded uncertainties calculated were in the range 22-41% at the 99% confidence level. Decision limit (CCα) and detection capability (CCβ) values were in the ranges 0.4-0.9 and 0.7-1.4 μg kg(-1), respectively. Matrix effects have been investigated demonstrating a moderate signal suppression/enhancement for most of the target compounds. The method described has been successfully applied to the analysis of honey samples; sulfamethoxazole, sulfathiazole and trimethoprim were detected in some cases. Copyright © 2012 Elsevier B.V. All rights reserved.

  5. Simultaneous neuron- and astrocyte-specific fluorescent marking

    Energy Technology Data Exchange (ETDEWEB)

    Schulze, Wiebke [Laboratory of Molecular Neuropharmacology, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871 (Japan); Hayata-Takano, Atsuko [Molecular Research Center for Children' s Mental Development, United Graduate School of Child Development, Osaka University, Kanazawa University, Hamamatsu University School of Medicine, Chiba University and University of Fukui, 2-2 Yamadaoka, Suita, Osaka 565-0871 (Japan); Kamo, Toshihiko [Laboratory of Molecular Neuropharmacology, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871 (Japan); Nakazawa, Takanobu, E-mail: takanobunakazawa-tky@umin.ac.jp [iPS Cell-based Research Project on Brain Neuropharmacology and Toxicology, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871 (Japan); Nagayasu, Kazuki [iPS Cell-based Research Project on Brain Neuropharmacology and Toxicology, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871 (Japan); Kasai, Atsushi; Seiriki, Kaoru [Laboratory of Molecular Neuropharmacology, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871 (Japan); Interdisciplinary Program for Biomedical Sciences, Institute for Academic Initiatives, Osaka University, 1-1 Yamadaoka, Suita, Osaka 565-0871 (Japan); Shintani, Norihito [Laboratory of Molecular Neuropharmacology, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871 (Japan); Ago, Yukio [Laboratory of Medicinal Pharmacology, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871 (Japan); Farfan, Camille [Laboratory of Molecular Neuropharmacology, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871 (Japan); and others

    2015-03-27

    Systematic and simultaneous analysis of multiple cell types in the brain is becoming important, but such tools have not yet been adequately developed. Here, we aimed to generate a method for the specific fluorescent labeling of neurons and astrocytes, two major cell types in the brain, and we have developed lentiviral vectors to express the red fluorescent protein tdTomato in neurons and the enhanced green fluorescent protein (EGFP) in astrocytes. Importantly, both fluorescent proteins are fused to histone 2B protein (H2B) to confer nuclear localization to distinguish between single cells. We also constructed several expression constructs, including a tandem alignment of the neuron- and astrocyte-expression cassettes for simultaneous labeling. Introducing these vectors and constructs in vitro and in vivo resulted in cell type-specific and nuclear-localized fluorescence signals enabling easy detection and distinguishability of neurons and astrocytes. This tool is expected to be utilized for the simultaneous analysis of changes in neurons and astrocytes in healthy and diseased brains. - Highlights: • We develop a method for the specific fluorescent labeling of neurons and astrocytes. • Neuron-specific labeling is achieved using Scg10 and synapsin promoters. • Astrocyte-specific labeling is generated using the minimal GFAP promoter. • Nuclear localization of fluorescent proteins is achieved with histone 2B protein.

  6. Simultaneous neuron- and astrocyte-specific fluorescent marking

    International Nuclear Information System (INIS)

    Schulze, Wiebke; Hayata-Takano, Atsuko; Kamo, Toshihiko; Nakazawa, Takanobu; Nagayasu, Kazuki; Kasai, Atsushi; Seiriki, Kaoru; Shintani, Norihito; Ago, Yukio; Farfan, Camille

    2015-01-01

    Systematic and simultaneous analysis of multiple cell types in the brain is becoming important, but such tools have not yet been adequately developed. Here, we aimed to generate a method for the specific fluorescent labeling of neurons and astrocytes, two major cell types in the brain, and we have developed lentiviral vectors to express the red fluorescent protein tdTomato in neurons and the enhanced green fluorescent protein (EGFP) in astrocytes. Importantly, both fluorescent proteins are fused to histone 2B protein (H2B) to confer nuclear localization to distinguish between single cells. We also constructed several expression constructs, including a tandem alignment of the neuron- and astrocyte-expression cassettes for simultaneous labeling. Introducing these vectors and constructs in vitro and in vivo resulted in cell type-specific and nuclear-localized fluorescence signals enabling easy detection and distinguishability of neurons and astrocytes. This tool is expected to be utilized for the simultaneous analysis of changes in neurons and astrocytes in healthy and diseased brains. - Highlights: • We develop a method for the specific fluorescent labeling of neurons and astrocytes. • Neuron-specific labeling is achieved using Scg10 and synapsin promoters. • Astrocyte-specific labeling is generated using the minimal GFAP promoter. • Nuclear localization of fluorescent proteins is achieved with histone 2B protein

  7. Design and synthesis of novel sulfonamide-containing bradykinin hB2 receptor antagonists. 1. Synthesis and SAR of alpha,alpha-dimethylglycine sulfonamides.

    Science.gov (United States)

    Fattori, Daniela; Rossi, Cristina; Fincham, Christopher I; Berettoni, Marco; Calvani, Federico; Catrambone, Fernando; Felicetti, Patrizia; Gensini, Martina; Terracciano, Rosa; Altamura, Maria; Bressan, Alessandro; Giuliani, Sandro; Maggi, Carlo A; Meini, Stefania; Valenti, Claudio; Quartara, Laura

    2006-06-15

    We recently published the extensive in vivo pharmacological characterization of MEN 16132 (J. Pharmacol. Exp. Ther. 2005, 616-623; Eur. J. Pharmacol. 2005, 528, 7), a member of the sulfonamide-containing human B(2) receptor (hB(2)R) antagonists. Here we report, in detail, how this family of compounds was designed, synthesized, and optimized to provide a group of products with subnanomolar affinity for the hB(2)R and high in vivo potency after topical administration to the respiratory tract. The series was designed on the basis of indications from the X-ray structures of the key structural motifs A and B present in known antagonists and is characterized by the presence of an alpha,alpha-dialkyl amino acid. The first lead (17) of the series was submitted to extensive chemical work to elucidate the structural requirements to increase hB(2) receptor affinity and antagonist potency in bioassays expressing the human B(2) receptor (hB(2)R). The following structural features were selected: a 2,4-dimethylquinoline moiety and a piperazine linker acylated with a basic amino acid. The representative lead compound 68 inhibited the specific binding of [(3)H]BK to hB(2)R with a pKi of 9.4 and antagonized the BK-induced inositolphosphate (IP) accumulation in recombinant cell systems expressing the hB(2)R with a pA(2) of 9.1. Moreover, compound 68 when administered (300 nmol/kg) intratracheally in the anesthetized guinea pig, was able to significantly inhibit BK-induced bronchoconstriction for up to 120 min after its administration, while having a lower and shorter lasting effect on hypotension.

  8. Synthesis and characterization of photoswitchable fluorescent silica nanoparticles

    NARCIS (Netherlands)

    Fölling, J.; Polyakova, S.; Belov, V.; van Blaaderen, A.; Bossi, M.L.; Hell, S.W.

    2008-01-01

    We have designed and synthesized a new functional (amino reactive) highly efficient fluorescent molecular switch (FMS) with a photochromic diarylethene and a rhodamine fluorescent dye. The reactive group in this FMS -N-hydroxysuccinimide ester- allows selective labeling of amino containing molecules

  9. Instrumentation and Fluorescent Chemistries Used in qPCR

    DEFF Research Database (Denmark)

    Josefsen, Mathilde Hartmann; Löfström, Charlotta; Hansen, Trine

    2012-01-01

    will be discussed from a user perspective leading to an instrument selection guide. Differences between fluorescent DNA binding dyes and target-specific fluorescently labeled primers or probes for detection of amplicon accumulation will be discussed, along with the properties and applications of the most frequently...

  10. Genotoxic, Cytotoxic, Antigenotoxic, and Anticytotoxic Effects of Sulfonamide Chalcone Using the Ames Test and the Mouse Bone Marrow Micronucleus Test.

    Directory of Open Access Journals (Sweden)

    Carolina Ribeiro E Silva

    Full Text Available Chalcones present several biological activities and sulfonamide chalcone derivatives have shown important biological applications, including antitumor activity. In this study, genotoxic, cytotoxic, antigenotoxic, and anticytotoxic activities of the sulfonamide chalcone N-{4-[3-(4-nitrophenylprop-2-enoyl]phenyl} benzenesulfonamide (CPN were assessed using the Salmonella typhimurium reverse mutation test (Ames test and the mouse bone marrow micronucleus test. The results showed that CPN caused a small increase in the number of histidine revertant colonies in S. typhimurium strains TA98 and TA100, but not statistically significant (p > 0.05. The antimutagenicity test showed that CPN significantly decreased the number of His+ revertants in strain TA98 at all doses tested (p 0.05. Additionally, CPN co-administered with MMC significantly increased PCE/NCE ratio at all doses tested, demonstrating its anticytotoxic effect. In summary, CPN presented genotoxic, cytotoxic, antigenotoxic, and anticytotoxic properties.

  11. Inhibition of the α-carbonic anhydrase from Vibrio cholerae with amides and sulfonamides incorporating imidazole moieties.

    Science.gov (United States)

    De Vita, Daniela; Angeli, Andrea; Pandolfi, Fabiana; Bortolami, Martina; Costi, Roberta; Di Santo, Roberto; Suffredini, Elisabetta; Ceruso, Mariangela; Del Prete, Sonia; Capasso, Clemente; Scipione, Luigi; Supuran, Claudiu T

    2017-12-01

    We discovered novel and selective sulfonamides/amides acting as inhibitors of the α-carbonic anhydrase (CA, EC 4.2.1.1) from the pathogenic bacterium Vibrio cholerae (VchCA). This Gram-negative bacterium is the causative agent of cholera and colonises the upper small intestine where sodium bicarbonate is present at a high concentration. The secondary sulfonamides and amides investigated here were potent, low nanomolar VchCA inhibitors whereas their inhibition of the human cytosolic isoforms CA I and II was in the micromolar range or higher. The molecules represent an interesting lead for antibacterial agents with a possibly new mechanism of action, although their CA inhibition mechanism is unknown for the moment.

  12. Alternate gram staining technique using a fluorescent lectin.

    Science.gov (United States)

    Sizemore, R K; Caldwell, J J; Kendrick, A S

    1990-01-01

    Fluorescence-labeled wheat germ agglutinin binds specifically to N-acetylglucosamine in the outer peptidoglycan layer of gram-positive bacteria. The peptidoglycan layer of gram-negative bacteria is covered by a membrane and is not labeled by the lectin. By exploiting this phenomenon, an alternative Gram staining technique has been developed. Images PMID:1697149

  13. Preparation and Characterization of Fluorescent SiO2 Microspheres

    Science.gov (United States)

    Xu, Cui; Zhang, Hao; Guan, Ruifang

    2018-01-01

    Fluorescent compound without typical fluorophores was synthesized with citric acid (CA) and aminopropyltriethoxysilane (APTS) firstly, and then it was grafted to the surface of the prepared SiO2 microspheres by chemical reaction. The fluorescent SiO2 microspheres with good fluorescent properties were obtained by optimizing the reaction conditions. And the morphology and structure of the fluorescent SiO2 microspheres have been characterized by scanning electron microscopy (SEM) and fourier transform infrared (FTIR) spectroscopy. The results showed that the preparation of fluorescent SiO2 microspheres have good monodispersity and narrow particle size distribution. Moreover, the fluorescent SiO2 microspheres can be applied to detect Fe3+ in aqueous solution, prepare fluorescent SiO2 rubber, and have potential to be applied in the fluorescent labeling and fingerprint appearing technique fields.

  14. Mechanistic link between uptake of sulfonamides and bacteriostatic effect: model development and application to experimental data from two soil microorganisms.

    Science.gov (United States)

    Focks, Andreas; Klasmeier, Jörg; Matthies, Michael

    2010-07-01

    Sulfonamides (SA) are antibiotic compounds that are widely used as human and veterinary pharmaceuticals. They are not rapidly biodegradable and have been detected in various environmental compartments. Effects of sulfonamides on microbial endpoints in soil have been reported from laboratory incubation studies. Sulfonamides inhibit the growth of sensitive microorganisms by competitive binding to the dihydropteroate-synthase (DHPS) enzyme of folic acid production. A mathematical model was developed that relates the extracellular SA concentration to the inhibition of the relative bacterial growth rate. Two factors--the anionic accumulation factor (AAF) and the cellular affinity factor (CAF)--determine the effective concentration of an SA. The AAF describes the SA uptake into bacterial cells and varies with both the extra- and intracellular pH values and with the acidic pKa value of an SA. The CAF subsumes relevant cellular and enzyme properties, and is directly proportional to the DHPS affinity constant for an SA. Based on the model, a mechanistic dose-response relationship is developed and evaluated against previously published data, where differences in the responses of Pseudomonas aeruginosa and Panthoea agglomerans toward changing medium pH values were found, most likely as a result of their diverse pH regulation. The derived dose-response relationship explains the pH and pKa dependency of mean effective concentration values (EC50) of eight SA and two soil bacteria based on AAF and CAF values. The mathematical model can be used to extrapolate sulfonamide effects to other pH values and to calculate the CAF as a pH-independent measure for the SA effects on microbial growth. Copyright (c) 2010 SETAC.

  15. Reduction in lipophilicity improved the solubility, plasma–protein binding, and permeability of tertiary sulfonamide RORc inverse agonists

    Energy Technology Data Exchange (ETDEWEB)

    Fauber, Benjamin P.; René, Olivier; de Leon Boenig, Gladys; Burton, Brenda; Deng, Yuzhong; Eidenschenk, Céline; Everett, Christine; Gobbi, Alberto; Hymowitz, Sarah G.; Johnson, Adam R.; La, Hank; Liimatta, Marya; Lockey, Peter; Norman, Maxine; Ouyang, Wenjun; Wang, Weiru; Wong, Harvey (Genentech); (Argenta)

    2014-08-01

    Using structure-based drug design principles, we identified opportunities to reduce the lipophilicity of our tertiary sulfonamide RORc inverse agonists. The new analogs possessed improved RORc cellular potencies with >77-fold selectivity for RORc over other nuclear receptors in our cell assay suite. The reduction in lipophilicity also led to an increased plasma–protein unbound fraction and improvements in cellular permeability and aqueous solubility.

  16. Sulfonamide-Resistant Bacteria and Their Resistance Genes in Soils Fertilized with Manures from Jiangsu Province, Southeastern China

    OpenAIRE

    Wang, Na; Yang, Xiaohong; Jiao, Shaojun; Zhang, Jun; Ye, Boping; Gao, Shixiang

    2014-01-01

    Antibiotic-resistant bacteria and genes are recognized as new environmental pollutants that warrant special concern. There were few reports on veterinary antibiotic-resistant bacteria and genes in China. This work systematically analyzed the prevalence and distribution of sulfonamide resistance genes in soils from the environments around poultry and livestock farms in Jiangsu Province, Southeastern China. The results showed that the animal manure application made the spread and abundance of a...

  17. Prevalence of Sulfonamide Resistance Genes in Bacterial Isolates from Manured Agricultural Soils and Pig Slurry in the United Kingdom▿

    OpenAIRE

    Byrne-Bailey, K. G.; Gaze, W. H.; Kay, P.; Boxall, A. B. A.; Hawkey, P. M.; Wellington, E. M. H.

    2008-01-01

    The prevalences of three sulfonamide resistance genes, sul1, sul2, and sul3 and sulfachloropyridazine (SCP) resistance were determined in bacteria isolated from manured agricultural clay soils and slurry samples in the United Kingdom over a 2-year period. Slurry from tylosin-fed pigs amended with SCP and oxytetracycline was used for manuring. Isolates positive for sul genes were further screened for the presence of class 1 and 2 integrons. Phenotypic resistance to SCP was significantly higher...

  18. Rh(III)-catalyzed oxidative olefination of N-(1-naphthyl)sulfonamides using activated and unactivated alkenes.

    Science.gov (United States)

    Li, Xuting; Gong, Xue; Zhao, Miao; Song, Guoyong; Deng, Jian; Li, Xingwei

    2011-11-04

    Rhodium(III)-catalyzed oxidative olefination of N-(1-naphthyl)sulfonamides has been achieved at the peri position. Three categories of olefins have been successfully applied. Activated olefins reacted to afford five-membered azacycles as a result of oxidative olefination-hydroamination. Unactivated olefins reacted to give the olefination product. 2-fold oxidative C-C and C-N coupling was achieved for allylbenzenes. © 2011 American Chemical Society

  19. Instant live-cell super-resolution imaging of cellular structures by nanoinjection of fluorescent probes.

    Science.gov (United States)

    Hennig, Simon; van de Linde, Sebastian; Lummer, Martina; Simonis, Matthias; Huser, Thomas; Sauer, Markus

    2015-02-11

    Labeling internal structures within living cells with standard fluorescent probes is a challenging problem. Here, we introduce a novel intracellular staining method that enables us to carefully control the labeling process and provides instant access to the inner structures of living cells. Using a hollow glass capillary with a diameter of <100 nm, we deliver functionalized fluorescent probes directly into the cells by (di)electrophoretic forces. The label density can be adjusted and traced directly during the staining process by fluorescence microscopy. We demonstrate the potential of this technique by delivering and imaging a range of commercially available cell-permeable and nonpermeable fluorescent probes to cells.

  20. Determination of sulfonamides in butter samples by ionic liquid magnetic bar liquid-phase microextraction high-performance liquid chromatography.

    Science.gov (United States)

    Wu, Lijie; Song, Ying; Hu, Mingzhu; Xu, Xu; Zhang, Hanqi; Yu, Aimin; Ma, Qiang; Wang, Ziming

    2015-01-01

    A novel, simple, and environmentally friendly pretreatment method, ionic liquid magnetic bar liquid-phase microextraction, was developed for the determination of sulfonamides in butter samples by high-performance liquid chromatography. The ionic liquid magnetic bar was prepared by inserting a stainless steel wire into the hollow of a hollow fiber and immobilizing ionic liquid in the micropores of the hollow fiber. In the extraction process, the ionic liquid magnetic bars were used to stir the mixture of sample and extraction solvent and enrich the sulfonamides in the mixture. After extraction, the analyte-adsorbed ionic liquid magnetic bars were readily isolated with a magnet from the extraction system. It is notable that the present method was environmentally friendly since water and only several microliters of ionic liquid were used in the whole extraction process. Several parameters affecting the extraction efficiency were investigated and optimized, including the type of ionic liquid, sample-to-extraction solvent ratio, the number of ionic liquid magnetic bars, extraction temperature, extraction time, salt concentration, stirring speed, pH of the extraction solvent, and desorption conditions. The recoveries were in the range of 73.25-103.85 % and the relative standard deviations were lower than 6.84 %. The experiment results indicated that the present method was effective for the extraction of sulfonamides in high-fat content samples.

  1. Conformer-Specific IR Spectroscopy of Laser-Desorbed Sulfonamide Drugs: Tautomeric and Conformational Preferences of Sulfanilamide and its Derivatives

    Science.gov (United States)

    Uhlemann, Thomas; Seidel, Sebastian; Müller, Christian W.

    2017-06-01

    Molecules containing the sulfonamide group R^{1}-SO_2-NHR^{2} have a longstanding history as antimicrobial agents. Even though nowadays they are not commonly used in treating humans anymore, they continue to be studied as effective inhibitors of metalloenzyme carbonic anhydrases. These enzymes are important targets for a variety of diseases, such as, for instance, breast cancer, glaucoma, and obesity. Here we present the results of our laser desorption single-conformation UV and IR study of sulfanilamide (NH_2Ph-SO_2-NHR, R=H), a variety of singly substituted derivatives, and their monohydrated complexes. Depending on the substituent, the sulfonamide group can either adopt an amino or an imino tautomeric form. The form prevalent in the crystal is not necessarily also the tautomeric form we identified in the molecular beam after laser desorbing the sample. Furthermore, we explored the effect of complexation with a single water molecule on the tautomeric and conformational preferences of the sulfonamides. Our conformer-specific IR spectra in the NH and OH stretch region (3200-3750 \\wn) suggest that the intra- and intermolecular interactions governing the structures of the monomers and water complexes are surprisingly diverse. We have undertaken both Quantum Theory of Atoms in Molecules (QTAIM) and Interacting Quantum Atoms (IQA) analyses of calculated electron densities to quantitatively characterize the nature and strengths of the intra- and intermolecular interactions prevalent in the monomer and water complex structures.

  2. Prevalence of sulfonamide resistance genes in bacterial isolates from manured agricultural soils and pig slurry in the United Kingdom.

    Science.gov (United States)

    Byrne-Bailey, K G; Gaze, W H; Kay, P; Boxall, A B A; Hawkey, P M; Wellington, E M H

    2009-02-01

    The prevalences of three sulfonamide resistance genes, sul1, sul2, and sul3 and sulfachloropyridazine (SCP) resistance were determined in bacteria isolated from manured agricultural clay soils and slurry samples in the United Kingdom over a 2-year period. Slurry from tylosin-fed pigs amended with SCP and oxytetracycline was used for manuring. Isolates positive for sul genes were further screened for the presence of class 1 and 2 integrons. Phenotypic resistance to SCP was significantly higher in isolates from pig slurry and postapplication soil than in those from preapplication soil. Of 531 isolates, 23% carried sul1, 18% sul2, and 9% sul3 only. Two percent of isolates contained all three sul genes. Class 1 and class 2 integrons were identified in 5% and 11.7%, respectively, of sul-positive isolates. In previous reports, sul1 was linked to class 1 integrons, but in this study only 8% of sul1-positive isolates carried the intI1 gene. Sulfonamide-resistant pathogens, including Shigella flexneri, Aerococcus spp., and Acinetobacter baumannii, were identified in slurry-amended soil and soil leachate, suggesting a potential environmental reservoir. Sulfonamide resistance in Psychrobacter, Enterococcus, and Bacillus spp. is reported for the first time, and this study also provides the first description of the genotypes sul1, sul2, and sul3 outside the Enterobacteriaceae and in the soil environment.

  3. Prevalence of Sulfonamide Resistance Genes in Bacterial Isolates from Manured Agricultural Soils and Pig Slurry in the United Kingdom▿

    Science.gov (United States)

    Byrne-Bailey, K. G.; Gaze, W. H.; Kay, P.; Boxall, A. B. A.; Hawkey, P. M.; Wellington, E. M. H.

    2009-01-01

    The prevalences of three sulfonamide resistance genes, sul1, sul2, and sul3 and sulfachloropyridazine (SCP) resistance were determined in bacteria isolated from manured agricultural clay soils and slurry samples in the United Kingdom over a 2-year period. Slurry from tylosin-fed pigs amended with SCP and oxytetracycline was used for manuring. Isolates positive for sul genes were further screened for the presence of class 1 and 2 integrons. Phenotypic resistance to SCP was significantly higher in isolates from pig slurry and postapplication soil than in those from preapplication soil. Of 531 isolates, 23% carried sul1, 18% sul2, and 9% sul3 only. Two percent of isolates contained all three sul genes. Class 1 and class 2 integrons were identified in 5% and 11.7%, respectively, of sul-positive isolates. In previous reports, sul1 was linked to class 1 integrons, but in this study only 8% of sul1-positive isolates carried the intI1 gene. Sulfonamide-resistant pathogens, including Shigella flexneri, Aerococcus spp., and Acinetobacter baumannii, were identified in slurry-amended soil and soil leachate, suggesting a potential environmental reservoir. Sulfonamide resistance in Psychrobacter, Enterococcus, and Bacillus spp. is reported for the first time, and this study also provides the first description of the genotypes sul1, sul2, and sul3 outside the Enterobacteriaceae and in the soil environment. PMID:19064898

  4. NOVEL FLUORESCENT PROBES FOR THE DOPAMINE TRANSPORTER

    DEFF Research Database (Denmark)

    Cha, J; Vægter, Christian Bjerggaard; Adkins, Erica

    -reactive rhodamine red derivatives. The resulting N-substituted (JHC 1-64) and 2-substituted (JHC 1-53) ligands showed high affinity binding to DAT expressed in HEK 293 cells (Ki= 6.4 and 29 nM, respectively). Their ability to selectively label the DAT was demonstrated by confocal laser scanning microscopy of HEK......To enable visualization of the dopamine transporter (DAT) through fluorescence technologies we have synthesized a novel series of fluorescently tagged analogs of cocaine. Previous structure-activity relationship (SAR) studies have demonstrated that the dopamine transporter (DAT) can tolerate...... in untransfected control cells. The possibility of using these ligands for direct labeling of the DAT in living cells represents a new and important approach for understanding cellular targeting and trafficking of the DAT. Moreover, these fluorescent ligands might also provide the molecular tools...

  5. Insights into functional-group-tolerant polymerization catalysis with phosphine-sulfonamide palladium(II) complexes.

    Science.gov (United States)

    Jian, Zhongbao; Falivene, Laura; Wucher, Philipp; Roesle, Philipp; Caporaso, Lucia; Cavallo, Luigi; Göttker-Schnetmann, Inigo; Mecking, Stefan

    2015-01-26

    Two series of cationic palladium(II) methyl complexes {[(2-MeOC6 H4 )2 PC6 H4 SO2 NHC6 H3 (2,6-R(1) ,R(2) )]PdMe}2 [A]2 ((X) 1(+) -A: R(1) =R(2) =H: (H) 1(+) -A; R(1) =R(2) =CH(CH3 )2 : (DIPP) 1(+) -A; R(1) =H, R(2) =CF3 : (CF3) 1(+) -A; A=BF4 or SbF6 ) and neutral palladium(II) methyl complexes {[(2-MeOC6 H4 )2 PC6 H4 SO2 NC6 H3 (2,6-R(1) ,R(2) )]PdMe(L)} ((X) 1-acetone: L=acetone; (X) 1-dmso: L=dimethyl sulfoxide; (X) 1-pyr: L=pyridine) chelated by a phosphine-sulfonamide were synthesized and fully characterized. Stoichiometric insertion of methyl acrylate (MA) into all complexes revealed that a 2,1 regiochemistry dominates in the first insertion of MA. Subsequently, for the cationic complexes (X) 1(+) -A, β-H elimination from the 2,1-insertion product (X) 2(+) -AMA-2,1 is overwhelmingly favored over a second MA insertion to yield two major products (X) 4(+) -AMA-1,2 and (X) 5(+) -AMA . By contrast, for the weakly coordinated neutral complexes (X) 1-acetone and (X) 1-dmso, a second MA insertion of the 2,1-insertion product (X) 2MA-2,1 is faster than β-H elimination and gives (X) 3MA as major products. For the strongly coordinated neutral complexes (X) 1-pyr, no second MA insertion and no β-H elimination (except for (DIPP) 2-pyrMA-2,1 ) were observed for the 2,1-insertion product (X) 2-pyrMA-2,1 . The cationic complexes (X) 1(+) -A exhibited high catalytic activities for ethylene dimerization, affording butenes (C4 ) with a high selectivity of up to 97.7 % (1-butene: 99.3 %). Differences in activities and selectivities suggest that the phosphine-sulfonamide ligands remain coordinated to the metal center in a bidentate fashion in the catalytically active species. By comparison, the neutral complexes (X) 1-acetone, (X) 1-dmso, and (X) 1-pyr showed very low activity towards ethylene to give traces of oligomers. DFT analyses taking into account the two possible coordination modes (O or N) of the sulfonamide ligand for the cationic system (CF3) 1(+) suggested

  6. Understanding Food Labels

    Science.gov (United States)

    ... Healthy eating for girls Understanding food labels Understanding food labels There is lots of info on food ... need to avoid because of food allergies. Other food label terms top In addition to the Nutrition ...

  7. Radioactive labelling with 125 I of infectious pancreatic necrosis virus

    International Nuclear Information System (INIS)

    Soler Ch, M.; Farias O, G.; Kuznar H, J.

    1993-01-01

    In order to understand the interaction between a cellular receptor and a ligand the photochemical crosslinking method has been widely used. This method has been utilized as an approach to determine the presence or absence of virus receptors in susceptible cells. Successful detection of crosslinks is achieved if one of the components, in the crosslinked product, has been radioactively labeled. The incorporation of a radioactive isotope, in the virus-receptor complex, enables the identification of the receptor. To undertake this study in the future, in this communication the radioactive labeling of virus particles is presented. The infectious necrosis pancreatic virus (IPN virus) was the chosen moiety to be in vitro labeled with 125 I using a direct method. Three oxidizing agents were used in the iodination procedure for comparison: an enzyme, lactoperoxidase and two chemical reagents, N-Chloro-benceno-sulfonamide (Iodo-Beads) and 1,3,4,6-Tetra chloro-3a,6a-diphenyl glycouril (Iodo-Gen). The results are analysed to select the method which guarantee the incorporation of 125 I in the viral capsid protein, while preserving its full infectivity. (author)

  8. Design, Synthesis, and Evaluation of Dihydrobenzo[cd]indole-6-sulfonamide as TNF-alpha Inhibitors

    Science.gov (United States)

    Deng, Xiaobing; Zhang, Xiaoling; Tang, Bo; Liu, Hongbo; Shen, Qi; Liu, Ying; Lai, Luhua

    2018-04-01

    Tumor necrosis factor-α (TNF-α) plays a pivotal role in inflammatory response. Dysregulation of TNF can lead to a variety of disastrous pathological effects, including auto-inflammatory diseases. Antibodies that directly targeting TNF-α have been proven effective in suppressing symptoms of these disorders. Compared to protein drugs, small molecule drugs are normally orally available and less expensive. Till now, peptide and small molecule TNF-α inhibitors are still in the early stage of development, and much more efforts should be made. In a previously study, we reported a TNF-α inhibitor, EJMC-1 with modest activity. Here, we optimized this compound by shape screen and rational design. In the first round, we screened commercial compound library for EJMC-1 analogs based on shape similarity. Out of the 68 compounds tested, 20 compounds showed better binding affinity than EJMC-1 in the SPR competitive binding assay. These 20 compounds were tested in cell assay and the most potent compound was 2-oxo-N-phenyl-1,2-dihydrobenzo[cd]indole-6-sulfonamide (S10) with an IC50 of 14 M, which was 2.2-fold stronger than EJMC-1. Based on the docking analysis of S10 and EJMC-1 binding with TNF-α, in the second round, we designed S10 analogues, purchased 7 of them and synthesized 7 new compounds. The best compound, 4e showed an IC50 value of 3 M in cell assay, which was 14-fold stronger than EJMC-1. 4e was among the most potent TNF-α organic compound inhibitors reported so far. Our study demonstrated that 2-oxo-N-phenyl-1,2-dihydrobenzo[cd]indole-6-sulfonamide analogues could be developed as potent TNF-α inhibitors. 4e can be further optimized for its activity and properties. Our study provides insights into designing small molecule inhibitors directly targeting TNF-α and for protein-protein interaction inhibitor design.

  9. Prevalence and characterization of plasmids carrying sulfonamide resistance genes among Escherichia coli from pigs, pig carcasses and human.

    Science.gov (United States)

    Wu, Shuyu; Dalsgaard, Anders; Hammerum, Anette M; Porsbo, Lone J; Jensen, Lars B

    2010-07-30

    Sulfonamide resistance is very common in Escherichia coli. The aim of this study was to characterize plasmids carrying sulfonamide resistance genes (sul1, sul2 and sul3) in E. coli isolated from pigs and humans with a specific objective to assess the genetic diversity of plasmids involved in the mobility of sul genes. A total of 501 E. coli isolates from pig feces, pig carcasses and human stools were tested for their susceptibility to selected antimicrobial. Multiplex PCR was conducted to detect the presence of three sul genes among the sulfonamide-resistant E. coli isolates. Fifty-seven sulfonamide-resistant E. coli were selected based on presence of sul resistance genes and subjected to conjugation and/or transformation experiments. S1 nuclease digestion followed by pulsed-field gel electrophoresis was used to visualize and determine the size of plasmids. Plasmids carrying sul genes were characterized by PCR-based replicon typing to allow a comparison of the types of sul genes, the reservoir and plasmid present. A total of 109/501 isolates exhibited sulfonamide resistance. The relative prevalences of sul genes from the three reservoirs (pigs, pig carcasses and humans) were 65%, 45% and 12% for sul2, sul1, and sul3, respectively. Transfer of resistance through conjugation was observed in 42/57 isolates. Resistances to streptomycin, ampicillin and trimethoprim were co-transferred in most strains. Class 1 integrons were present in 80% of sul1-carrying plasmids and 100% of sul3-carrying plasmids, but only in 5% of sul2-carrying plasmids. The sul plasmids ranged from 33 to 160-kb in size and belonged to nine different incompatibility (Inc) groups: FII, FIB, I1, FIA, B/O, FIC, N, HI1 and X1. IncFII was the dominant type in sul2-carrying plasmids (52%), while IncI1 was the most common type in sul1 and sul3-carrying plasmids (33% and 45%, respectively). Multireplicons were found associated with all three sul genes. Sul genes were distributed widely in E. coli isolated

  10. Prevalence and characterization of plasmids carrying sulfonamide resistance genes among Escherichia coli from pigs, pig carcasses and human

    Directory of Open Access Journals (Sweden)

    Hammerum Anette M

    2010-07-01

    Full Text Available Abstract Background Sulfonamide resistance is very common in Escherichia coli. The aim of this study was to characterize plasmids carrying sulfonamide resistance genes (sul1, sul2 and sul3 in E. coli isolated from pigs and humans with a specific objective to assess the genetic diversity of plasmids involved in the mobility of sul genes. Methods A total of 501 E. coli isolates from pig feces, pig carcasses and human stools were tested for their susceptibility to selected antimicrobial. Multiplex PCR was conducted to detect the presence of three sul genes among the sulfonamide-resistant E. coli isolates. Fifty-seven sulfonamide-resistant E. coli were selected based on presence of sul resistance genes and subjected to conjugation and/or transformation experiments. S1 nuclease digestion followed by pulsed-field gel electrophoresis was used to visualize and determine the size of plasmids. Plasmids carrying sul genes were characterized by PCR-based replicon typing to allow a comparison of the types of sul genes, the reservoir and plasmid present. Results A total of 109/501 isolates exhibited sulfonamide resistance. The relative prevalences of sul genes from the three reservoirs (pigs, pig carcasses and humans were 65%, 45% and 12% for sul2, sul1, and sul3, respectively. Transfer of resistance through conjugation was observed in 42/57 isolates. Resistances to streptomycin, ampicillin and trimethoprim were co-transferred in most strains. Class 1 integrons were present in 80% of sul1-carrying plasmids and 100% of sul3-carrying plasmids, but only in 5% of sul2-carrying plasmids. The sul plasmids ranged from 33 to 160-kb in size and belonged to nine different incompatibility (Inc groups: FII, FIB, I1, FIA, B/O, FIC, N, HI1 and X1. IncFII was the dominant type in sul2-carrying plasmids (52%, while IncI1 was the most common type in sul1 and sul3-carrying plasmids (33% and 45%, respectively. Multireplicons were found associated with all three sul genes

  11. Wide-field fluorescent microscopy and fluorescent imaging flow cytometry on a cell-phone.

    Science.gov (United States)

    Zhu, Hongying; Ozcan, Aydogan

    2013-04-11

    Fluorescent microscopy and flow cytometry are widely used tools in biomedical research and clinical diagnosis. However these devices are in general relatively bulky and costly, making them less effective in the resource limited settings. To potentially address these limitations, we have recently demonstrated the integration of wide-field fluorescent microscopy and imaging flow cytometry tools on cell-phones using compact, light-weight, and cost-effective opto-fluidic attachments. In our flow cytometry design, fluorescently labeled cells are flushed through a microfluidic channel that is positioned above the existing cell-phone camera unit. Battery powered light-emitting diodes (LEDs) are butt-coupled to the side of this microfluidic chip, which effectively acts as a multi-mode slab waveguide, where the excitation light is guided to uniformly excite the fluorescent targets. The cell-phone camera records a time lapse movie of the fluorescent cells flowing through the microfluidic channel, where the digital frames of this movie are processed to count the number of the labeled cells within the target solution of interest. Using a similar opto-fluidic design, we can also image these fluorescently labeled cells in static mode by e.g. sandwiching the fluorescent particles between two glass slides and capturing their fluorescent images using the cell-phone camera, which can achieve a spatial resolution of e.g. - 10 μm over a very large field-of-view of - 81 mm(2). This cell-phone based fluorescent imaging flow cytometry and microscopy platform might be useful especially in resource limited settings, for e.g. counting of CD4+ T cells toward monitoring of HIV+ patients or for detection of water-borne parasites in drinking water.

  12. Refractive index modulation in the polyurethane films containing diazo sulfonamide chromophores

    Energy Technology Data Exchange (ETDEWEB)

    Ortyl, E. [Insitute of Organic and Polymer Technology, WrocIaw University of Technology, 50-384 WrocIaw (Poland); Kucharski, S. [Insitute of Organic and Polymer Technology, WrocIaw University of Technology, 50-384 WrocIaw (Poland)]. E-mail: stanislaw.kucharski@pwr.wroc.pl; Gotszalk, T. [Faculty of Microsystem Electronics and Photonics, WrocIaw University of Technology, 50-384 WrocIaw (Poland)

    2005-05-23

    The series of photochromic polyurethanes was obtained by modification of precursor polymers prepared from 4,4'-diphenylmethane diisocyanate (MDI), hexamethylene 1,6-diisocyanate (HDI) or toluene 2,4-diisocyanate (TDI) and N,N'-di-(2-hydroxyethyl) aniline. The precursor polymers were functionalized by an azo-coupling reaction to form the polymers with different degrees of functionalization and various heterocyclic sulfonamide groups. Ellipsometric measurements showed a decrease of the refractive index during illumination of thin polymer films with white light. The change of real part of the refractive index was in the range of 0.0033-0.0296 depending on the polymer kind and chromophore content. It was found that photocurrent was generated in the polymer films deposited onto indium tin oxide (ITO) glass plates. For the polyurethanes containing sulfathiazole groups in side chains the current density was up to 180 nA/cm{sup 2}. The formation of diffraction grating in the polymer films was easily achieved using linearly polarized laser light (532 nm) in a standard two beam coupling (TBC) system. The diffraction efficiency of the first diffraction beam was dependent on the chromophore content reaching ca. 12% for the derivatives of sulfamethoxazole.

  13. Synthesis and description of intermolecular interactions in new sulfonamide derivatives of tranexamic acid

    Science.gov (United States)

    Ashfaq, Muhammad; Arshad, Muhammad Nadeem; Danish, Muhammad; Asiri, Abdullah M.; Khatoon, Sadia; Mustafa, Ghulam; Zolotarev, Pavel N.; Butt, Rabia Ayub; Şahin, Onur

    2016-01-01

    Tranexamic acid (4-aminomethyl-cyclohexanecarboxylic acid) was reacted with sulfonyl chlorides to produce structurally related four sulfonamide derivatives using simple and environmental friendly method to check out their three-dimensional behavior and van der Walls interactions. The molecules were crystallized in different possibilities, as it is/after alkylation at its O and N atoms/along with a co-molecule. All molecules were crystallized in monoclinic crystal system with space group P21/n, P21/c and P21/a. X-ray studies reveal that the molecules stabilized themselves by different kinds of hydrogen bonding interactions. The molecules are getting connected through O-H⋯O hydrogen bonds to form inversion dimers which are further connected through N-H⋯O interactions. The molecules in which N and O atoms were alkylated showed non-classical interaction and generated centro-symmetric R22(24) ring motif. The co-crystallized host and guest molecules are connected to each other via O-H⋯O interactions to generate different ring motifs. By means of the ToposPro software an analysis of the topologies of underlying nets that correspond to molecular packings and hydrogen-bonded networks in structures under consideration was carried out.

  14. The effect of miscellaneous oral dosage forms on the environmental pollution of sulfonamides in pig holdings.

    Science.gov (United States)

    Stahl, Jessica; Zessel, Katrin; Schulz, Jochen; Finke, Jan Henrik; Müller-Goymann, Christel Charlotte; Kietzmann, Manfred

    2016-04-01

    Due to antibiotic treatment of humans and animals, the prevalence of bacterial resistances increases worldwide. Especially in livestock farming, large quantities of faeces contaminated with antibiotics pose a risk of the carryover of the active ingredient to the environment. Accordingly, the aim of the present study was the evaluation of the benefit of different oral dosage forms (powder, pellets, granula) in pigs concerning the environmental pollution of sulfadiazine. Two subtherapeutic dosages were evaluated in powder mixtures to gain information about their potential to pollute the pig barn. Furthermore, a new group of pigs was kept in the stable after powder feeding of another pig group to determine the possible absorption of environmentally distributed antibiotics. Pigs were orally treated with three dosage forms. Simultaneously, sedimentation and airborne dust were collected and plasma and urine levels were determined. All formulations result in comparable plasma and urine levels, but massive differences in environmental pollution (powder > pellets, granula). Pigs housing in a contaminated barn exhibit traces of sulfadiazine in plasma and urine. Using pharmaceutical formulations like pellets or granula, the environmental pollution of sulfonamides can significantly be diminished due to massive dust reduction during feeding.

  15. Synthesis of piperazine sulfonamide analogs as diabetic-II inhibitors and their molecular docking study.

    Science.gov (United States)

    Taha, Muhammad; Irshad, Maryam; Imran, Syahrul; Chigurupati, Sridevi; Selvaraj, Manikandan; Rahim, Fazal; Ismail, Nor Hadiani; Nawaz, Faisal; Khan, Khalid Mohammed

    2017-12-01

    Piperazine Sulfonamide analogs (1-19) have been synthesized, characterized by different spectroscopic techniques and evaluated for α-amylase Inhibition. Analogs 1-19 exhibited a varying degree of α-amylase inhibitory activity with IC 50 values ranging in between 1.571 ± 0.05 to 3.98 ± 0.397 μM when compared with the standard acarbose (IC 50  = 1.353 ± 0.232 μM). Compound 1, 2, 3 and 7 showed significant inhibitory effects with IC 50 value 2.348 ± 0.444, 2.064 ± 0.04, 1.571 ± 0.05 and 2.118 ± 0.204 μM, respectively better than the rest of the series. Structure activity relationships were established. Molecular docking studies were performed to understand the binding interaction of the compounds. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  16. Fate of sulfonamides, macrolides, and trimethoprim in different wastewater treatment technologies

    International Nuclear Information System (INIS)

    Goebel, Anke; McArdell, Christa S.; Joss, Adriano; Siegrist, Hansruedi; Giger, Walter

    2007-01-01

    The elimination of sulfonamides, macrolides and trimethoprim from raw wastewater was investigated in several municipal wastewater treatment plants. Primary treatment provided no significant elimination for the investigated substances. Similar eliminations were observed in the secondary treatment of two conventional activated sludge (CAS) systems and a fixed-bed reactor (FBR). Sulfamethoxazole, including the fraction present as N 4 -acetyl-sulfamethoxazole, was eliminated by approximately 60% in comparison to about 80% in a membrane bioreactor (MBR) independently of the solid retention time (SRT), indicating a positive correlation of the observed elimination to the organic substrate concentration. The elimination for macrolides and trimethoprim varied significantly between the different sampling campaigns in the two CAS systems and in the FBR. In the MBR, these analytes were eliminated up to 50% at SRT of 16 ± 2 and 33 ± 3 d. Trimethoprim, clarithromycin and dehydro-erythromycin showed a higher elimination of up to 90% at a SRT of 60-80 d indicating a correlation with reduced substrate loading (SL). Together with the high SRT, the SL may lead to an increased biodiversity of the active biomass, resulting in a broader range of degradation pathways available. Two investigated sand filters showed different elimination behavior. One led to a significant elimination of most macrolides (17-23%) and trimethoprim (74 ± 14%), while no elimination was observed in the other sand filter investigated

  17. A survey of perfluoroalkyl sulfonamides in indoor and outdoor air using passive air samplers

    Energy Technology Data Exchange (ETDEWEB)

    Shoeib, M.; Harner, T. [Meteorological Service of Canada, Environment Canada (Canada); Wilford, B.; Jones, K. [Lancaster Univ. (United Kingdom). Environmental Science; Zhu, J. [Chemistry Research Division, Health Canada, Tunney' s Pasture, Ottawa (Canada)

    2004-09-15

    Perfluorooctane sulfonate (PFOS) has recently emerged as a priority environmental pollutant due to its widespread detection in biological samples from remote regions including the Arctic and the Mid-North Pacific Ocean. Because PFOS is fairly involatile, it is hypothesized that its occurrence in remote regions is the result of atmospheric transport of more volatile precursor compounds such as the perfluoroalkyl sulfonamides (PFASs). PFASs are used in variety of consumer products for water and oil resistance including surface treatments for fabric, upholstery, carpet, paper and leather. In a recent pilot study employing high volume air samples, indoor air concentrations of PFASs were approximately 100 times greater than outdoor levels. This is of significance because people typically spend about 90% of their time indoors 5 and this exposure may serve as an important uptake pathway. Indoor air also serves as a source of PFASs to the outside where PFASs are ultimately transported and distributed throughout the environment. The current study is intended to be a more comprehensive survey of indoor and outdoor air allowing more confident conclusions to be made. Passive air samplers comprised of polyurethane foam (PUF) disks were used. These are quiet, non-intrusive samplers that operate without the aid of a pump or electricity. Air movement delivers chemical to the sampler which has a high retention capacity for persistent organic pollutants (POPs). PUF disks samplers have been previously used successfully to monitor different classes of hydrophobic persistent organic pollutants POPs.

  18. Residual veterinary antibiotics in pig excreta after oral administration of sulfonamides.

    Science.gov (United States)

    Qiu, Jinrong; Zhao, Tao; Liu, Qingyun; He, Jinhua; He, Dechun; Wu, Genyi; Li, Yongtao; Jiang, Chengai; Xu, Zhencheng

    2016-04-01

    Sulfonamides (SAs) are applied widely as feed additives in the farming of livestock and poultry. It can lead to the excretion of large amounts of SAs in manure and result in persistent environmental pollution. We evaluated the fate of four SAs, sulfamerazine (SM1), sulfachloropyridazine (SCP), sulfadimoxine (SDM') and sulfaquinoxaline (SQ), from oral administration to excretion in urine and feces in pigs. The four SAs were added to homemade feed to make them reach the required concentration gradient, which were 0, 50 and 100 mg/kg (low, normal and high concentrations, respectively). In different treatments, excretions of the four SAs were 35.68-86.88 %. With regard to total excretion, the order was SQ > SCP > SM1 > SDM' for all treatments. The concentration of SAs in the feed had significant effects on the amount of the four SAs excreted every day. The concentration of SAs in feces and in the urine for different treatments was 15.03-26.55 and 14.54-69.22 %, respectively. In each treatment, excretions of SCP, SDM' and SQ in feces were lower than that in urine. The four SAs remained longer in urine than in feces. Excretions in urine and feces were lower if SAs were administered orally rather than by injection.

  19. Traceability of sulfonamide antibiotic treatment by immunochemical analysis of farm animal hair samples.

    Science.gov (United States)

    Adrian, Javier; Gratacós-Cubarsí, Marta; Sánchez-Baeza, Francisco; Garcia Regueiro, Jose-Antonio; Castellari, Massimo; Marco, M-Pilar

    2009-10-01

    The use of hair to trace use of unauthorized substances, therapeutic agents, or their misuse is becoming very attractive since residues can be detected for a long time after treatment. For this purpose, an indirect enzyme-linked immunosorbent assay (ELISA) has been evaluated for its capability to trace sulfonamide antibiotic treatment by analyzing cattle and pig hair samples. Pigmented and nonpigmented hair samples from control and sulfamethazine (SMZ)-treated pigs and calves were collected, extracted under different alkaline conditions, and analyzed by ELISA after just diluting the extracts with the assay buffer. Data analysis following the European recommendations for screening methods demonstrates that the ELISA can detect SMZ in hair samples with a limit of detection (90% of the zero dose (IC(90))) between 30 and 75 ng g(-1). The same samples have been analyzed by HPLC after a dual solid-phase extraction. The ELISA results matched very well those obtained by the chromatographic method, demonstrating that the immunochemical method can be used as a screening tool to trace animal treatments. Between the benefits of this method are the possibility to directly analyze hair extracts with sufficient detectability and its high-throughput capability. Preliminary validation data are reported using an experimental approach inspired on the Commission Decision 2002/657/EC criteria for screening methods.

  20. Noncovalent Labeling of Biomolecules with Red and Near- Infrared Dyes

    Directory of Open Access Journals (Sweden)

    Lucjan Strekowski

    2004-02-01

    Full Text Available Biopolymers such as proteins and nucleic acids can be labeled with a fluorescent marker to allow for their detection. Covalent labeling is achieved by the reaction of an appropriately functionalized dye marker with a reactive group on a biomolecule. The recent trend, however, is the use of noncovalent labeling that results from strong hydrophobic and/or ionic interactions between the marker and biomolecule of interest. The main advantage of noncovalent labeling is that it affects the functional activity of the biomolecule to a lesser extent. The applications of luminescent cyanine and squarylium dyes are reviewed.

  1. Issues in Data Labelling

    NARCIS (Netherlands)

    Cowie, Roddy; Cox, Cate; Martin, Jeam-Claude; Batliner, Anton; Heylen, Dirk K.J.; Karpouzis, Kostas; Cowie, Roddy; Pelachaud, Catherine; Petta, Paolo

    2011-01-01

    Labelling emotion databases is not a purely technical matter. It is bound up with theoretical issues. Different issues affect labelling of emotional content, labelling of the signs that convey emotion, and labelling of the relevant context. Linked to these are representational issues, involving time

  2. Antioxidant flavone analog functionalized fluorescent silica nanoparticles: Synthesis and exploration of their possible use as biomolecule sensor.

    Science.gov (United States)

    Das, Sreeparna; Batuta, Shaikh; Alam, Md Niharul; Fouzder, Chandrani; Kundu, Rakesh; Mandal, Debabrata; Begum, Naznin Ara

    2017-09-01

    For the first time, a synthetic fluorescent antioxidant flavone analog was successfully anchored onto the surface of the APTES-modified mesoporous silica nanoparticles (NPs) through sulfonamide linkage. The surface chemistry and morphology of the flavone modified fluorescent silica (FMFS) NPs were studied in detail. The flavone moiety when attached onto the FMFS NP surface, imparted its characteristic fluorescence and antioxidant activities to these NPs. Moreover, the NPs are highly biocompatible as evidenced from their cytotoxicity assay on normal lung cell (L132). The fluorescence activity of these biocompatible NPs was further utilized to study their interaction with a biomolecule, BSA (Bovine Serum Albumin). It was interesting to note that the fluorescence behavior of FMFS NPs completely changed on their binding with BSA. On the other hand, the intrinsic fluorescence activity of BSA was also significantly modified due to its interaction with FMFS NPs. Thus, the sensing and detection of biomolecules like BSA in presence of FMFS NPs can be accomplished by monitoring changes in the fluorescence behavior of either FMFS NPs or BSA. Furthermore, these FMFS NPs retained their intrinsic fluorescence behavior in the cellular medium which opens up their possible use as biocompatible cell imaging agents in future. Copyright © 2017 Elsevier B.V. All rights reserved.

  3. Mixed Map Labeling

    Directory of Open Access Journals (Sweden)

    Maarten Löffler

    2016-12-01

    Full Text Available Point feature map labeling is a geometric visualization problem, in which a set of input points must be labeled with a set of disjoint rectangles (the bounding boxes of the label texts. It is predominantly motivated by label placement in maps but it also has other visualization applications. Typically, labeling models either use internal labels, which must touch their feature point, or external (boundary labels, which are placed outside the input image and which are connected to their feature points by crossing-free leader lines. In this paper we study polynomial-time algorithms for maximizing the number of internal labels in a mixed labeling model that combines internal and external labels. The model requires that all leaders are parallel to a given orientation θ ∈ [0, 2π, the value of which influences the geometric properties and hence the running times of our algorithms.

  4. Sulfonamide antibiotic removal and nitrogen recovery from synthetic urine by the combination of rotating advanced oxidation contactor and methylene urea synthesis process.

    Science.gov (United States)

    Fukahori, S; Fujiwara, T; Ito, R; Funamizu, N

    2015-01-01

    The combination of nitrogen recovery and pharmaceutical removal processes for livestock urine treatment were investigated to suppress the discharge of pollutants and recover nitrogen as resources. We combined methylene urea synthesis from urea and adsorption and photocatalytic decomposition of sulfonamide antibiotic using rotating advanced oxidation contactor (RAOC) contained for obtaining both safe fertilizer and reclaimed water. The methylene urea synthesis could recover urea in synthetic urine, however, almost all sulfonamide antibiotic was also incorporated, which is unfavorable from a safety aspect if the methylene urea is to be used as fertilizer. Conversely, RAOC could remove sulfonamide antibiotic without consuming urea. It was also confirmed that the methylene urea could be synthesized from synthetic urine treated by RAOC. Thus, we concluded that RAOC should be inserted prior to the nitrogen recovery process for effective treatment of urine and safe use of methylene urea as fertilizer.

  5. Continuous degradation of a mixture of sulfonamides by Trametes versicolor and identification of metabolites from sulfapyridine and sulfathiazole

    International Nuclear Information System (INIS)

    Rodríguez-Rodríguez, Carlos E.; Jesús García-Galán, Ma.; Blánquez, Paqui; Díaz-Cruz, M. Silvia; Barceló, Damià; Caminal, Glòria; Vicent, Teresa

    2012-01-01

    Highlights: ► Degradation of sulfapyridine and sulfathiazole by Trametes versicolor was evaluated. ► The role of laccase and cytochrome P450 was determined. ► Degradation metabolites were identified for sulfapyridine (8) and sulfathiazole (5). ► A mixture of three sulfonamides was degraded in a continuous fluidized bed reactor. - Abstract: In this study, we assessed the degradation of the sulfonamides sulfapyridine (SPY) and sulfathiazole (STZ) by the white-rot fungus Trametes versicolor. Complete degradation was accomplished in fungal cultures at initial pollutant concentrations of approximately 10 mg L −1 , although a longer period of time was needed to completely remove STZ in comparison to SPY. When cytochrome P450 inhibitors were added to the fungal cultures, STZ degradation was partially suppressed, while no additional effect was observed for SPY. Experiments with purified laccase and laccase mediators caused the removal of greater than 75% of each antibiotic. Ultra-performance liquid chromatography-quadupole time of flight mass spectrometry (UPLC-QqTOF-MS) analyses allowed the identification of a total of eight degradation intermediates of SPY in both the in vivo and the laccase experiments, being its desulfonated moiety the commonly detected product. For STZ, a total of five products were identified. A fluidized bed reactor with T. versicolor pellets degraded a mixture of sulfonamides (SPY, STZ and sulfamethazine, SMZ) by greater than 94% each at a hydraulic residence time of 72 h. Because wastewater contains many diverse pollutants, these results highlight the potential of T. versicolor as a bioremediation agent not only for the removal of antibiotics but also for the elimination of a wide range of contaminants.

  6. Continuous degradation of a mixture of sulfonamides by Trametes versicolor and identification of metabolites from sulfapyridine and sulfathiazole

    Energy Technology Data Exchange (ETDEWEB)

    Rodriguez-Rodriguez, Carlos E., E-mail: CarlosEsteban.Rodriguez@uab.cat [Unitat asociada de Biocatalisi Aplicada IQAC-CSIC, Escola d' Enginyeria, Universitat Autonoma de Barcelona, 08193 Bellaterra, Barcelona (Spain); Centro de Investigacion en Contaminacion Ambiental, Universidad de Costa Rica, 2060 San Jose (Costa Rica); Jesus Garcia-Galan, Ma. [Departament de Quimica Ambiental, IDAEA-CSIC, C/Jordi Girona 18-26, 08034, Barcelona (Spain); Blanquez, Paqui [Departament d' Enginyeria Quimica, Escola d' Enginyeria, Universitat Autonoma de Barcelona, 08193 Bellaterra, Barcelona (Spain); Diaz-Cruz, M. Silvia [Departament de Quimica Ambiental, IDAEA-CSIC, C/Jordi Girona 18-26, 08034, Barcelona (Spain); Barcelo, Damia [Departament de Quimica Ambiental, IDAEA-CSIC, C/Jordi Girona 18-26, 08034, Barcelona (Spain); Catalan Institute for Water Research (ICRA), Parc Cientific i Tecnologic de la Universitat de Girona, C/Emili Grahit, 101 Edifici H2O, E-17003 Girona (Spain); Caminal, Gloria [Unitat asociada de Biocatalisi Aplicada IQAC-CSIC, Escola d' Enginyeria, Universitat Autonoma de Barcelona, 08193 Bellaterra, Barcelona (Spain); Vicent, Teresa [Departament d' Enginyeria Quimica, Escola d' Enginyeria, Universitat Autonoma de Barcelona, 08193 Bellaterra, Barcelona (Spain)

    2012-04-30

    Highlights: Black-Right-Pointing-Pointer Degradation of sulfapyridine and sulfathiazole by Trametes versicolor was evaluated. Black-Right-Pointing-Pointer The role of laccase and cytochrome P450 was determined. Black-Right-Pointing-Pointer Degradation metabolites were identified for sulfapyridine (8) and sulfathiazole (5). Black-Right-Pointing-Pointer A mixture of three sulfonamides was degraded in a continuous fluidized bed reactor. - Abstract: In this study, we assessed the degradation of the sulfonamides sulfapyridine (SPY) and sulfathiazole (STZ) by the white-rot fungus Trametes versicolor. Complete degradation was accomplished in fungal cultures at initial pollutant concentrations of approximately 10 mg L{sup -1}, although a longer period of time was needed to completely remove STZ in comparison to SPY. When cytochrome P450 inhibitors were added to the fungal cultures, STZ degradation was partially suppressed, while no additional effect was observed for SPY. Experiments with purified laccase and laccase mediators caused the removal of greater than 75% of each antibiotic. Ultra-performance liquid chromatography-quadupole time of flight mass spectrometry (UPLC-QqTOF-MS) analyses allowed the identification of a total of eight degradation intermediates of SPY in both the in vivo and the laccase experiments, being its desulfonated moiety the commonly detected product. For STZ, a total of five products were identified. A fluidized bed reactor with T. versicolor pellets degraded a mixture of sulfonamides (SPY, STZ and sulfamethazine, SMZ) by greater than 94% each at a hydraulic residence time of 72 h. Because wastewater contains many diverse pollutants, these results highlight the potential of T. versicolor as a bioremediation agent not only for the removal of antibiotics but also for the elimination of a wide range of contaminants.

  7. Sulfonamide-resistant bacteria and their resistance genes in soils fertilized with manures from Jiangsu Province, Southeastern China.

    Directory of Open Access Journals (Sweden)

    Na Wang

    Full Text Available Antibiotic-resistant bacteria and genes are recognized as new environmental pollutants that warrant special concern. There were few reports on veterinary antibiotic-resistant bacteria and genes in China. This work systematically analyzed the prevalence and distribution of sulfonamide resistance genes in soils from the environments around poultry and livestock farms in Jiangsu Province, Southeastern China. The results showed that the animal manure application made the spread and abundance of antibiotic resistance genes (ARGs increasingly in the soil. The frequency of sulfonamide resistance genes was sul1 > sul2 > sul3 in pig-manured soil DNA and sul2 > sul1 > sul3 in chicken-manured soil DNA. Further analysis suggested that the frequency distribution of the sul genes in the genomic DNA and plasmids of the SR isolates from manured soil was sul2 > sul1 > sul3 overall (p<0.05. The combination of sul1 and sul2 was the most frequent, and the co-existence of sul1 and sul3 was not found either in the genomic DNA or plasmids. The sample type, animal type and sampling time can influence the prevalence and distribution pattern of sulfonamide resistance genes. The present study also indicated that Bacillus, Pseudomonas and Shigella were the most prevalent sul-positive genera in the soil, suggesting a potential human health risk. The above results could be important in the evaluation of antibiotic-resistant bacteria and genes from manure as sources of agricultural soil pollution; the results also demonstrate the necessity and urgency of the regulation and supervision of veterinary antibiotics in China.

  8. Synthesis of some new heterocyclic compounds bearing a sulfonamide moiety and studying their combined anticancer effect with γ-radiation

    Inte