Structure-guided approach to site-specific fluorophore labeling of the lac repressor LacI.

Directory of Open Access Journals (Sweden)

Kalle Kipper

Full Text Available The lactose operon repressor protein LacI has long served as a paradigm of the bacterial transcription factors. However, the mechanisms whereby LacI rapidly locates its cognate binding site on the bacterial chromosome are still elusive. Single-molecule fluorescence imaging approaches are well suited for the study of these mechanisms but rely on a functionally compatible fluorescence labeling of LacI. Particularly attractive for protein fluorescence labeling are synthetic fluorophores due to their small size and favorable photophysical characteristics. Synthetic fluorophores are often conjugated to natively occurring cysteine residues using maleimide chemistry. For a site-specific and functionally compatible labeling with maleimide fluorophores, the target protein often needs to be redesigned to remove unwanted native cysteines and to introduce cysteines at locations better suited for fluorophore attachment. Biochemical screens can then be employed to probe for the functional activity of the redesigned protein both before and after dye labeling. Here, we report a mutagenesis-based redesign of LacI to enable a functionally compatible labeling with maleimide fluorophores. To provide an easily accessible labeling site in LacI, we introduced a single cysteine residue at position 28 in the DNA-binding headpiece of LacI and replaced two native cysteines with alanines where derivatization with bulky substituents is known to compromise the protein's activity. We find that the redesigned LacI retains a robust activity in vitro and in vivo, provided that the third native cysteine at position 281 is retained in LacI. In a total internal reflection microscopy assay, we observed individual Cy3-labeled LacI molecules bound to immobilized DNA harboring the cognate O1 operator sequence, indicating that the dye-labeled LacI is functionally active. We have thus been able to generate a functional fluorescently labeled LacI that can be used to unravel mechanistic

Comparative conventional- and quantum dot-labelling strategies for LPS binding site detection in Arabidopsis thaliana mesophyll protoplasts

Directory of Open Access Journals (Sweden)

Londiwe Siphephise Mgcina

2015-05-01

Full Text Available Lipopolysaccharide (LPS from Gram-negative bacteria is recognized as a microbe-associated molecular pattern (MAMP and not only induces an innate immune response in plants, but also stimulates the development of characteristic defense responses. However, identification and characterization of a cell surface LPS-receptor/binding site, as described in mammals, remains elusive in plants. As an amphiphilic, macromolecular lipoglycan, intact LPS potentially contains three MAMP-active regions, represented by the O-polysaccharide chain, the core and the lipid A. Binding site studies with intact labelled LPS were conducted in Arabidopsis thaliana protoplasts and quantified using flow cytometry fluorescence changes. Qdots, which allow non-covalent, hydrophobic labelling were used as a novel strategy in this study and compared to covalent, hydrophilic labelling with Alexa 488. Affinity for LPS-binding sites was clearly demonstrated by concentration-, temperature- and time-dependent increases in protoplast fluorescence following treatment with the labelled LPS. Moreover, this induced fluorescence increase was convincingly reduced following pre-treatment with excess unlabeled LPS, thereby indicating reversibility of LPS binding. Inhibition of the binding process is also reported using endo- and exocytosis inhibitors. Here, we present evidence for the anticipated presence of LPS-specific binding sites in Arabidopsis protoplasts, and furthermore propose Qdots as a more sensitive LPS-labelling strategy in comparison to the conventional Alexa 488 hydrazide label for binding studies.

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

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.

Site-specific confocal fluorescence imaging of biological microstructures in a turbid medium

International Nuclear Information System (INIS)

Saloma, Caesar; Palmes-Saloma, Cynthia; Kondoh, Hisato

1998-01-01

Normally transparent biological structures in a turbid medium are imaged using a laser confocal microscope and multiwavelength site-specific fluorescence labelling. The spatial filtering capability of the detector pinhole in the confocal microscope limits the number of scattered fluorescent photons that reach the photodetector. Simultaneous application of different fluorescent markers on the same sample site minimizes photobleaching by reducing the excitation time for each marker. A high-contrast grey-level image is also produced by summing confocal images of the same site taken at different fluorescence wavelengths. Monte Carlo simulations are performed to obtain the quantitative behaviour of confocal fluorescence imaging in turbid media. Confocal images of the following samples were also obtained: (i) 15 μm diameter fluorescent spheres placed 1.16 mm deep beneath an aqueous suspension of 0.0823 μm diameter polystyrene latex spheres, and (ii) hindbrain of a whole-mount mouse embryo (age 10 days) that was stained to fluoresce at 515 nm and 580 nm peak wavelengths. Expression of RNA transcripts of a gene within the embryo hindbrain was detected by a fluorescence-based whole-mount in situ hybridization procedure that we recently tested. (author)

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.

Construction of a photoactivatable profluorescent enzyme via propinquity labeling.

Science.gov (United States)

Lee, Hsien-Ming; Xu, Weichen; Lawrence, David S

2011-03-02

A strategy for the construction of a profluorescent caged enzyme is described. An active site-directed peptide-based affinity label was designed, synthesized, and employed to covalently label a nonactive site residue in the cAMP-dependent protein kinase. The modified kinase displays minimal catalytic activity and low fluorescence. Photolysis results in partial cleavage of the enzyme-bound affinity label, restoration of enzymatic activity (60-80%) and a strong fluorescent response (10-20 fold). The caged kinase displays analogous behavior in living cells, inducing a light-dependent loss of stress fibers that is characteristic of cAMP action. This strategy furnishes molecularly engineered enzymes that can be remotely controlled in time, space, and total activity.

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.

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.

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.

SimLabel: a graphical user interface to simulate continuous wave EPR spectra from site-directed spin labeling experiments.

Science.gov (United States)

Etienne, E; Le Breton, N; Martinho, M; Mileo, E; Belle, V

2017-08-01

Site-directed spin labeling (SDSL) combined with continuous wave electron paramagnetic resonance (cw EPR) spectroscopy is a powerful technique to reveal, at the residue level, structural transitions in proteins. SDSL-EPR is based on the selective grafting of a paramagnetic label on the protein under study, followed by cw EPR analysis. To extract valuable quantitative information from SDSL-EPR spectra and thus give reliable interpretation on biological system dynamics, numerical simulations of the spectra are required. Such spectral simulations can be carried out by coding in MATLAB using functions from the EasySpin toolbox. For non-expert users of MATLAB, this could be a complex task or even impede the use of such simulation tool. We developed a graphical user interface called SimLabel dedicated to run cw EPR spectra simulations particularly coming from SDSL-EPR experiments. Simlabel provides an intuitive way to visualize, simulate, and fit such cw EPR spectra. An example of SDSL-EPR spectra simulation concerning the study of an intrinsically disordered region undergoing a local induced folding is described and discussed. We believe that this new tool will help the users to rapidly obtain reliable simulated spectra and hence facilitate the interpretation of their results. Copyright © 2017 John Wiley & Sons, Ltd. Copyright © 2017 John Wiley & Sons, Ltd.

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.

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.

Ion Channel Conformation and Oligomerization Assessment by Site-Directed Spin Labeling and Pulsed-EPR.

Science.gov (United States)

Pliotas, Christos

2017-01-01

Mechanosensitive (MS) ion channels are multimeric integral membrane proteins that respond to increased lipid bilayer tension by opening their nonselective pores to release solutes and relieve increased cytoplasmic pressure. These systems undergo major conformational changes during gating and the elucidation of their mechanism requires a deep understanding of the interplay between lipids and proteins. Lipids are responsible for transmitting lateral tension to MS channels and therefore play a key role in obtaining a molecular-detail model for mechanosensation. Site-directed spin labeling combined with electron paramagnetic resonance (EPR) spectroscopy is a powerful spectroscopic tool in the study of proteins. The main bottleneck for its use relates to challenges associated with successful isolation of the protein of interest, introduction of paramagnetic labels on desired sites, and access to specialized instrumentation and expertise. The design of sophisticated experiments, which combine a variety of existing EPR methodologies to address a diversity of specific questions, require knowledge of the limitations and strengths, characteristic of each particular EPR method. This chapter is using the MS ion channels as paradigms and focuses on the application of different EPR techniques to ion channels, in order to investigate oligomerization, conformation, and the effect of lipids on their regulation. The methodology we followed, from the initial strategic selection of mutants and sample preparation, including protein purification, spin labeling, reconstitution into lipid mimics to the complete set-up of the pulsed-EPR experiments, is described in detail. © 2017 Elsevier Inc. All rights reserved.

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

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.

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.

Selective 15N labeling and direct observation by NMR of the active-site glutamine of Fe-containing superoxide dismutase

International Nuclear Information System (INIS)

Vance, Carrie K.; Kang, Young M.; Miller, Anne-Frances

1997-01-01

The glutamine in position 69 is one of only three conserved active-site amino acid differences between Fe- and Mn-containing superoxide dismutases (SODs). We have refined the conditions for extremely selective labeling of the side chains of glutamine with 15N, and thus obtained dramatically simplified spectra, despite the large size of Fe-SOD. The improved resolution afforded by such highly specific labeling permits the use of direct 15N detection to observe and assign Gln 69, even though its distance to the paramagnetic Fe2+ is only 5A. Selective glutamine side-chain labeling is inexpensive and has general utility for large (and paramagnet-containing) proteins

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.

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

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%).

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.

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.

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.

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

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.

Site-Directed Spin-Labeling of Nucleic Acids by Click Chemistry. Detection of Abasic Sites in Duplex DNA by EPR Spectroscopy

DEFF Research Database (Denmark)

Sigurdsson, Snorri; Vogel, Stefan; Shelke, Sandip

2010-01-01

and the nitroxide spin label. The spin label was used to detect, for the first time, abasic sites in duplex DNA by X-band CW-EPR spectroscopy and give information about other structural deformations as well as local conformational changes in DNA. For example, reduced mobility of the spin label in a mismatched pair...... label out of the duplex and toward the solution. Thus, reposition of the spin label, when acting as a mercury(II)-controlled mechanical lever, can be readily detected by EPR spectroscopy. The ease of incorporation and properties of the new spin label make it attractive for EPR studies of nucleic acids...

Direct imaging of glycans in Arabidopsis roots via click labeling of metabolically incorporated azido-monosaccharides.

Science.gov (United States)

Hoogenboom, Jorin; Berghuis, Nathalja; Cramer, Dario; Geurts, Rene; Zuilhof, Han; Wennekes, Tom

2016-10-10

Carbohydrates, also called glycans, play a crucial but not fully understood role in plant health and development. The non-template driven formation of glycans makes it impossible to image them in vivo with genetically encoded fluorescent tags and related molecular biology approaches. A solution to this problem is the use of tailor-made glycan analogs that are metabolically incorporated by the plant into its glycans. These metabolically incorporated probes can be visualized, but techniques documented so far use toxic copper-catalyzed labeling. To further expand our knowledge of plant glycobiology by direct imaging of its glycans via this method, there is need for novel click-compatible glycan analogs for plants that can be bioorthogonally labelled via copper-free techniques. Arabidopsis seedlings were incubated with azido-containing monosaccharide analogs of N-acetylglucosamine, N-acetylgalactosamine, L-fucose, and L-arabinofuranose. These azido-monosaccharides were metabolically incorporated in plant cell wall glycans of Arabidopsis seedlings. Control experiments indicated active metabolic incorporation of the azido-monosaccharide analogs into glycans rather than through non-specific absorption of the glycan analogs onto the plant cell wall. Successful copper-free labeling reactions were performed, namely an inverse-electron demand Diels-Alder cycloaddition reaction using an incorporated N-acetylglucosamine analog, and a strain-promoted azide-alkyne click reaction. All evaluated azido-monosaccharide analogs were observed to be non-toxic at the used concentrations under normal growth conditions. Our results for the metabolic incorporation and fluorescent labeling of these azido-monosaccharide analogs expand the possibilities for studying plant glycans by direct imaging. Overall we successfully evaluated five azido-monosaccharide analogs for their ability to be metabolically incorporated in Arabidopsis roots and their imaging after fluorescent labeling. This expands

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.

Elucidating the design principles of photosynthetic electron-transfer proteins by site-directed spin labeling EPR spectroscopy.

Science.gov (United States)

Ishara Silva, K; Jagannathan, Bharat; Golbeck, John H; Lakshmi, K V

2016-05-01

Site-directed spin labeling electron paramagnetic resonance (SDSL EPR) spectroscopy is a powerful tool to determine solvent accessibility, side-chain dynamics, and inter-spin distances at specific sites in biological macromolecules. This information provides important insights into the structure and dynamics of both natural and designed proteins and protein complexes. Here, we discuss the application of SDSL EPR spectroscopy in probing the charge-transfer cofactors in photosynthetic reaction centers (RC) such as photosystem I (PSI) and the bacterial reaction center (bRC). Photosynthetic RCs are large multi-subunit proteins (molecular weight≥300 kDa) that perform light-driven charge transfer reactions in photosynthesis. These reactions are carried out by cofactors that are paramagnetic in one of their oxidation states. This renders the RCs unsuitable for conventional nuclear magnetic resonance spectroscopy investigations. However, the presence of native paramagnetic centers and the ability to covalently attach site-directed spin labels in RCs makes them ideally suited for the application of SDSL EPR spectroscopy. The paramagnetic centers serve as probes of conformational changes, dynamics of subunit assembly, and the relative motion of cofactors and peptide subunits. In this review, we describe novel applications of SDSL EPR spectroscopy for elucidating the effects of local structure and dynamics on the electron-transfer cofactors of photosynthetic RCs. Because SDSL EPR Spectroscopy is uniquely suited to provide dynamic information on protein motion, it is a particularly useful method in the engineering and analysis of designed electron transfer proteins and protein networks. This article is part of a Special Issue entitled Biodesign for Bioenergetics--the design and engineering of electronic transfer cofactors, proteins and protein networks, edited by Ronald L. Koder and J.L. Ross Anderson. Copyright © 2016. Published by Elsevier B.V.

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)

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.

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

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.

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.

Site-Specific Biomolecule Labeling with Gold Clusters

OpenAIRE

Ackerson, Christopher J.; Powell, Richard D.; Hainfeld, James F.

2010-01-01

Site-specific labeling of biomolecules in vitro with gold clusters can enhance the information content of electron cryomicroscopy experiments. This chapter provides a practical overview of well-established techniques for forming biomolecule/gold cluster conjugates. Three bioconjugation chemistries are covered: Linker-mediated bioconjugation, direct gold–biomolecule bonding, and coordination-mediated bonding of nickel(II) nitrilotriacetic acid (NTA)-derivatized gold clusters to polyhistidine (...

Canonical Labelling of Site Graphs

Directory of Open Access Journals (Sweden)

Nicolas Oury

2013-06-01

Full Text Available We investigate algorithms for canonical labelling of site graphs, i.e. graphs in which edges bind vertices on sites with locally unique names. We first show that the problem of canonical labelling of site graphs reduces to the problem of canonical labelling of graphs with edge colourings. We then present two canonical labelling algorithms based on edge enumeration, and a third based on an extension of Hopcroft's partition refinement algorithm. All run in quadratic worst case time individually. However, one of the edge enumeration algorithms runs in sub-quadratic time for graphs with "many" automorphisms, and the partition refinement algorithm runs in sub-quadratic time for graphs with "few" bisimulation equivalences. This suite of algorithms was chosen based on the expectation that graphs fall in one of those two categories. If that is the case, a combined algorithm runs in sub-quadratic worst case time. Whether this expectation is reasonable remains an interesting open problem.

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.

Site-Specific Biomolecule Labeling with Gold Clusters

Science.gov (United States)

Ackerson, Christopher J.; Powell, Richard D.; Hainfeld, James F.

2013-01-01

Site-specific labeling of biomolecules in vitro with gold clusters can enhance the information content of electron cryomicroscopy experiments. This chapter provides a practical overview of well-established techniques for forming biomolecule/gold cluster conjugates. Three bioconjugation chemistries are covered: Linker-mediated bioconjugation, direct gold–biomolecule bonding, and coordination-mediated bonding of nickel(II) nitrilotriacetic acid (NTA)-derivatized gold clusters to polyhistidine (His)-tagged proteins. PMID:20887859

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.

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

Multiple Site-Directed and Saturation Mutagenesis by the Patch Cloning Method.

Science.gov (United States)

Taniguchi, Naohiro; Murakami, Hiroshi

2017-01-01

Constructing protein-coding genes with desired mutations is a basic step for protein engineering. Herein, we describe a multiple site-directed and saturation mutagenesis method, termed MUPAC. This method has been used to introduce multiple site-directed mutations in the green fluorescent protein gene and in the moloney murine leukemia virus reverse transcriptase gene. Moreover, this method was also successfully used to introduce randomized codons at five desired positions in the green fluorescent protein gene, and for simple DNA assembly for cloning.

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.

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.

Site-Specific Antibody Labeling by Covalent Photoconjugation of Z Domains Functionalized for Alkyne-Azide Cycloaddition Reactions.

Science.gov (United States)

Perols, Anna; Arcos Famme, Melina; Eriksson Karlström, Amelie

2015-11-01

Antibodies are extensively used in research, diagnostics, and therapy, and for many applications the antibodies need to be labeled. Labeling is typically performed by using amine-reactive probes that target surface-exposed lysine residues, resulting in heterogeneously labeled antibodies. An alternative labeling strategy is based on the immunoglobulin G (IgG)-binding protein domain Z, which binds to the Fc region of IgG. Introducing the photoactivable amino acid benzoylphenylalanine (BPA) into the Z domain makes it possible for a covalent bond to be be formed between the Z domain and the antibody on UV irradiation, to produce a site-specifically labeled product. Z32 BPA was synthesized by solid-phase peptide synthesis and further functionalized to give alkyne-Z32 BPA and azide-Z32 BPA for Cu(I) -catalyzed cycloaddition, as well as DBCO-Z32 BPA for Cu-free strain-promoted cycloaddition. The Z32 BPA variants were conjugated to the human IgG1 antibody trastuzumab and site-specifically labeled with biotin or fluorescein. The fluorescently labeled trastuzumab showed specific staining of the membranes of HER2-expressing cells in immunofluorescence microscopy. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

Fluorescence quenching of graphene oxide combined with the site-specific cleavage of restriction endonuclease for deoxyribonucleic acid demethylase activity assay

Energy Technology Data Exchange (ETDEWEB)

Ji, Lijuan; Qian, Yingdan; Wu, Ping; Zhang, Hui; Cai, Chenxin, E-mail: cxcai@njnu.edu.cn

2015-04-15

Highlights: • An approach for sensitive and selective DNA demethylase activity assay is reported. • This assay is based on the fluorescence quenching of GO and site-specific cleavage of endonuclease. • It can determine as low as 0.05 ng mL{sup −1} of MBD2 with a linear range of 0.2–300 ng mL{sup −1}. • It has an ability to recognize MBD2 from other possibly coexisting proteins and cancer cell extracts. • It can avoid false signals, requiring no bisulfite conversion, PCR amplification, radioisotope-labeling. - Abstract: We report on the development of a sensitive and selective deoxyribonucleic acid (DNA) demethylase (using MBD2 as an example) activity assay by coupling the fluorescence quenching of graphene oxide (GO) with the site-specific cleavage of HpaII endonuclease to improve the selectivity. This approach was developed by designing a single-stranded probe (P1) that carries a binding region to facilitate the interaction with GO, which induces fluorescence quenching of the labeled fluorophore (FAM, 6-carboxyfluorescein), and a sensing region, which contains a hemi-methylated site of 5′-CmCGG-3′, to specifically recognize the target (T1, a 32-mer DNA from the promoter region of p53 gene) and hybridize with it to form a P1/T1 duplex. After demethylation with MBD2, the duplex can be specifically cleaved using HpaII, which releases the labeled FAM from the GO surface and results in the recovery of fluorescence. However, this cleavage is blocked by the hemi-methylation of this site. Thus, the magnitude of the recovered fluorescence signal is related to the MBD2 activity, which establishes the basis of the DNA demethylase activity assay. This assay can determine as low as ∼(0.05 ± 0.01) ng mL{sup −1} (at a signal/noise of 3) of MBD2 with a linear range of 0.2–300 ng mL{sup −1} and recognize MBD2 from other possibly coexisting proteins and cancer cell extracts. The advantage of this assay is its ability to avoid false signals and no

Active site labeling of the guanine-7-methyltransferase

International Nuclear Information System (INIS)

Streaker, E.; Sitz, T.O.

1992-01-01

Studies on the guanine-7-methyltransferase have defined three domains in the active site: the S-adenosylmethionine (SAM) region, the cap region (GpppG), and the RNA binding domain (--NpNpNpNpNp---). The authors attempted to label the SAM binding domain by a photoaffinity label using 8-azido-SAM and another method using 3 H-SAM and long exposures to uv-light. Neither method was successful. The next approach was to attempt to label the cap-RNA binding domain (GpppGpNpNpNpNpN) by synthesizing RNA containing 8-azido-Ap using an in vitro transcription system and T7 RNA polymerase. The 8-azido-ATP inhibited the T7 RNA polymerase preventing the synthesis of RNA. As they were unable to synthesize the photoaffinity label, they next tried to synthesize an end labeled RNA and directly label by long exposures to uv-light. When the enzyme was incubated with 32 P-labeled RNA for 15 min at 37 degrees and then exposed to a germicidal lamp for various times at O degrees, optimal labeling occurred after 45 min. Various enzyme preparations were labeled by this method and two polypeptides were found to specifically bind the non-methylated mRNA analog. This labeling method should allow characterization of the subunit structure and generate information about the nature of the RNA binding domain

Determination of the shell growth direction during the formation of silica microcapsules by confocal fluorescence microscopy

NARCIS (Netherlands)

van Wijk, J.; Salari, J.W.O.; Meuldijk, J.; Klumperman, L.

2015-01-01

A novel procedure was developed to determine the direction of silica growth during the formation of a silica shell around aqueous microdroplets in water-in-oil Pickering emulsions. Two fluorescently labeled silica precursors were added consecutively and the resulting microcapsules were visualized

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

Institute of Scientific and Technical Information of China (English)

无

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.

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.

Dansyl labeling to modulate the relative affinity of bile acids for the binding sites of human serum albumin.

Science.gov (United States)

Rohacova, Jana; Sastre, German; Marin, M Luisa; Miranda, Miguel A

2011-09-08

Binding of natural bile acids to human serum albumin (HSA) is an important step in enterohepatic circulation and provides a measure of liver function. In this article, we report on the use of four dansyl (Dns) derivatives of cholic acid (ChA) to demonstrate a regiodifferentiation in their relative affinity for the two binding sites of HSA. Using both steady-state and time-resolved fluorescence, formation of Dns-ChA@HSA complexes was confirmed; the corresponding binding constants were determined, and their distribution between bulk solution and HSA microenvironment was estimated. By means of energy transfer from Trp to the Dns moiety, donor-acceptor distances were estimated (21-25 Å) and found to be compatible with both site 1 and site 2 occupancies. Nevertheless, titration using warfarin and ibuprofen as specific displacement probes clearly indicated that 3α- and 3β-Dns-ChA bind to HSA at site 2, whereas their C-7 regioisomers bind to HSA at site 1. Furthermore, the C-3-labeled compounds are displaced by lithocholic acid, whereas they are insensitive to ChA, confirming the assumption that the former binds to HSA at site 2. Thus, Dns labeling provides a useful tool to modulate the relative affinity of ChA to the major binding sites of HSA and, in combination with other fluorescent ChA analogs, to mimic the binding behavior of natural bile acids.

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.

Relocation of the disulfonic stilbene sites of AE1 (band 3) on the basis of fluorescence energy transfer measurements.

Science.gov (United States)

Knauf, Philip A; Law, Foon-Yee; Leung, Tze-Wah Vivian; Atherton, Stephen J

2004-09-28

Previous fluorescence resonance energy transfer (FRET) measurements, using BIDS (4-benzamido-4'-isothiocyanostilbene-2,2'-disulfonate) as a label for the disulfonic stilbene site and FM (fluorescein-5-maleimide) as a label for the cytoplasmic SH groups on band 3 (AE1), combined with data showing that the cytoplasmic SH groups lie about 40 A from the cytoplasmic surface of the lipid bilayer, would place the BIDS sites very near the membrane's inner surface, a location that seems to be inconsistent with current models of AE1 structure and mechanism. We reinvestigated the BIDS-FM distance, using laser single photon counting techniques as well as steady-state fluorescence of AE1, in its native membrane environment. Both techniques agree that there is very little energy transfer from BIDS to FM. The mean energy transfer (E), based on three-exponential fits to the fluorescence decay data, is 2.5 +/- 0.7% (SEM, N = 12). Steady-state fluorescence measurements also indicate BIDS to FM. These data indicate that the BIDS sites are probably over 63 A from the cytoplasmic SH groups, placing them near the middle or the external half of the lipid bilayer. This relocation of the BIDS sites fits with other evidence that the disulfonic stilbene sites are located farther toward the external membrane surface than Glu-681, a residue near the inner membrane surface whose modification affects the pH dependence and anion selectivity of band 3. The involvement of two relatively distant parts of the AE1 protein in transport function suggests that the transport mechanism requires coordinated large-scale conformational changes in the band 3 protein.

Site-selective 13C labeling of proteins using erythrose

International Nuclear Information System (INIS)

Weininger, Ulrich

2017-01-01

NMR-spectroscopy enables unique experimental studies on protein dynamics at atomic resolution. In order to obtain a full atom view on protein dynamics, and to study specific local processes like ring-flips, proton-transfer, or tautomerization, one has to perform studies on amino-acid side chains. A key requirement for these studies is site-selective labeling with 13 C and/or 1 H, which is achieved in the most general way by using site-selectively 13 C-enriched glucose (1- and 2- 13 C) as the carbon source in bacterial expression systems. Using this strategy, multiple sites in side chains, including aromatics, become site-selectively labeled and suitable for relaxation studies. Here we systematically investigate the use of site-selectively 13 C-enriched erythrose (1-, 2-, 3- and 4- 13 C) as a suitable precursor for 13 C labeled aromatic side chains. We quantify 13 C incorporation in nearly all sites in all 20 amino acids and compare the results to glucose based labeling. In general the erythrose approach results in more selective labeling. While there is only a minor gain for phenylalanine and tyrosine side-chains, the 13 C incorporation level for tryptophan is at least doubled. Additionally, the Phe ζ and Trp η2 positions become labeled. In the aliphatic side chains, labeling using erythrose yields isolated 13 C labels for certain positions, like Ile β and His β, making these sites suitable for dynamics studies. Using erythrose instead of glucose as a source for site-selective 13 C labeling enables unique or superior labeling for certain positions and is thereby expanding the toolbox for customized isotope labeling of amino-acid side-chains.

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.

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)

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.

Lesion-induced DNA weak structural changes detected by pulsed EPR spectroscopy combined with site-directed spin labelling.

Science.gov (United States)

Sicoli, Giuseppe; Mathis, Gérald; Aci-Sèche, Samia; Saint-Pierre, Christine; Boulard, Yves; Gasparutto, Didier; Gambarelli, Serge

2009-06-01

Double electron-electron resonance (DEER) was applied to determine nanometre spin-spin distances on DNA duplexes that contain selected structural alterations. The present approach to evaluate the structural features of DNA damages is thus related to the interspin distance changes, as well as to the flexibility of the overall structure deduced from the distance distribution. A set of site-directed nitroxide-labelled double-stranded DNA fragments containing defined lesions, namely an 8-oxoguanine, an abasic site or abasic site analogues, a nick, a gap and a bulge structure were prepared and then analysed by the DEER spectroscopic technique. New insights into the application of 4-pulse DEER sequence are also provided, in particular with respect to the spin probes' positions and the rigidity of selected systems. The lesion-induced conformational changes observed, which were supported by molecular dynamics studies, confirm the results obtained by other, more conventional, spectroscopic techniques. Thus, the experimental approaches described herein provide an efficient method for probing lesion-induced structural changes of nucleic acids.

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

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.

Environmentally Sensitive Fluorescent Sensors Based on Synthetic Peptides

Directory of Open Access Journals (Sweden)

Laurence Choulier

2010-03-01

Full Text Available Biosensors allow the direct detection of molecular analytes, by associating a biological receptor with a transducer able to convert the analyte-receptor recognition event into a measurable signal. We review recent work aimed at developing synthetic fluorescent molecular sensors for a variety of analytes, based on peptidic receptors labeled with environmentally sensitive fluorophores. Fluorescent indicators based on synthetic peptides are highly interesting alternatives to protein-based sensors, since they can be synthesized chemically, are stable, and can be easily modified in a site-specific manner for fluorophore coupling and for immobilization on solid supports.

Efficient one-step direct labelling of recombinant antibodies with technetium-99m

International Nuclear Information System (INIS)

Liberatore, M.; Neri, D.; Neri, G.; Pini, A.; Lurilli, A.P.; Ponzo, F.; Spampinato, G.; Padula, F.; Pala, A.; Colella, A.C.

1995-01-01

High-affinity bacterially expressed antibody fragments can nowadays be cloned from established hybridomas or, more conveniently, isolated directly from antibody libraries displayed on filamentous phage. Such antibodies can be tagged with C-terminal peptide tags containing one cysteine residue, which represents a convenient functionalisation site for a number of applications, including technetium-99m labelling. Here we describe a simple one-step method for 99m Tc labelling of cysteine-tagged recombinant antibodies with more than 50% radionuclide incorporation. The labelled antibodies displayed full retention of immuoreactivity and good stability. (orig.)

Efficient one-step direct labelling of recombinant antibodies with technetium-99m

Energy Technology Data Exchange (ETDEWEB)

Liberatore, M. [Dipartimento di Medicina Sperimentale, Sezione di Medicina Nucleare, Policlinico Umberto I, Universita di Roma `La Sapienza` (Italy); Neri, D. [Cambridge Centre for Protein Engineering - MRC Centre (United Kingdom); Neri, G. [Dipartimento di Biologia Molecolare, Universita di Siena (Italy); Pini, A. [Dipartimento di Biologia Molecolare, Universita di Siena (Italy); Lurilli, A.P. [Dipartimento di Medicina Sperimentale, Sezione di Medicina Nucleare, Policlinico Umberto I, Universita di Roma `La Sapienza` (Italy); Ponzo, F. [Dipartimento di Medicina Sperimentale, Sezione di Medicina Nucleare, Policlinico Umberto I, Universita di Roma `La Sapienza` (Italy); Spampinato, G. [Laboratorio di Biochimica degli Ormoni Sessuali, Il Instituto di Clinica Ostetrica e Ginecologica, Universita di Roma `La Sapienza` (Italy); Padula, F. [Laboratorio di Biochimica degli Ormoni Sessuali, Il Instituto di Clinica Ostetrica e Ginecologica, Universita di Roma `La Sapienza` (Italy); Pala, A. [Laboratorio di Biochimica degli Ormoni Sessuali, Il Instituto di Clinica Ostetrica e Ginecologica, Universita di Roma `La Sapienza` (Italy); Colella, A.C. [Dipartimento di Medicina Sperimentale, Sezione di Medicina Nucleare, Policlinico Umberto I, Universita di Roma `La Sapienza` (Italy)

1995-11-01

High-affinity bacterially expressed antibody fragments can nowadays be cloned from established hybridomas or, more conveniently, isolated directly from antibody libraries displayed on filamentous phage. Such antibodies can be tagged with C-terminal peptide tags containing one cysteine residue, which represents a convenient functionalisation site for a number of applications, including technetium-99m labelling. Here we describe a simple one-step method for {sup 99m}Tc labelling of cysteine-tagged recombinant antibodies with more than 50% radionuclide incorporation. The labelled antibodies displayed full retention of immuoreactivity and good stability. (orig.)

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...

Modeling membrane protein structure through site-directed ESR spectroscopy

NARCIS (Netherlands)

Kavalenka, A.A.

2009-01-01

Site-directed spin labeling (SDSL) electron spin resonance (ESR) spectroscopy is a
relatively new biophysical tool for obtaining structural information about proteins. This
thesis presents a novel approach, based on powerful spectral analysis techniques (multicomponent
spectral

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.

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.

Site-selective {sup 13}C labeling of proteins using erythrose

Energy Technology Data Exchange (ETDEWEB)

Weininger, Ulrich, E-mail: ulrich.weininger@physik.uni-halle.de [Lund University, Department of Biophysical Chemistry, Center for Molecular Protein Science (Sweden)

2017-03-15

NMR-spectroscopy enables unique experimental studies on protein dynamics at atomic resolution. In order to obtain a full atom view on protein dynamics, and to study specific local processes like ring-flips, proton-transfer, or tautomerization, one has to perform studies on amino-acid side chains. A key requirement for these studies is site-selective labeling with {sup 13}C and/or {sup 1}H, which is achieved in the most general way by using site-selectively {sup 13}C-enriched glucose (1- and 2-{sup 13}C) as the carbon source in bacterial expression systems. Using this strategy, multiple sites in side chains, including aromatics, become site-selectively labeled and suitable for relaxation studies. Here we systematically investigate the use of site-selectively {sup 13}C-enriched erythrose (1-, 2-, 3- and 4-{sup 13}C) as a suitable precursor for {sup 13}C labeled aromatic side chains. We quantify {sup 13}C incorporation in nearly all sites in all 20 amino acids and compare the results to glucose based labeling. In general the erythrose approach results in more selective labeling. While there is only a minor gain for phenylalanine and tyrosine side-chains, the {sup 13}C incorporation level for tryptophan is at least doubled. Additionally, the Phe ζ and Trp η2 positions become labeled. In the aliphatic side chains, labeling using erythrose yields isolated {sup 13}C labels for certain positions, like Ile β and His β, making these sites suitable for dynamics studies. Using erythrose instead of glucose as a source for site-selective {sup 13}C labeling enables unique or superior labeling for certain positions and is thereby expanding the toolbox for customized isotope labeling of amino-acid side-chains.

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.

Direct 99mTc labeling of monoclonal antibodies: radiolabeling and in vitro stability

International Nuclear Information System (INIS)

Garron, J.Y.; Moinereau, M.; Pasqualini, R.; Saccavini, J.C.

1991-01-01

Direct labeling involves 99m Tc binding to different donor groups on the protein, giving multiple binding sites of various affinities resulting in an in vivo instability. The stability has been considerably improved by activating the antibody using a controlled reduction reaction (using 2-aminoethanethiol). This reaction generates sulfhydryl groups, which are known to strongly bind 99m Tc. The direct 99m Tc antibody labeling method was explored using whole antibodies and fragments. Analytical methods were developed for routine evaluation of radiolabeling yield and in vitro stability. Stable direct antibody labeling with 99m Tc requires the generation of sulfhydryl groups, which show high affinity binding sites for 99m Tc. Such groups are obtained with 2-aminoethanethiol (AET), which induces the reduction of the intrachain or interchain disulfide bond, with no structural deterioration or any loss of immunobiological activity of the antibody. The development of fast, reliable analytical methods has made possible the qualitative and quantitative assessment of technetium species generated by the radiolabeling process. Labeling stability is determined by competition of the 99m Tc-antibody bond with three ligands, Chelex 100 (a metal chelate-type resin), free DTPA solution and 1% HSA solution. Very good 99m Tc-antibody stability is obtained with activated IgG (IgGa) and Fab' fragment, which makes these substances possible candidates for immunoscintigraphy use. (author)

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.

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)

Ultraviolet-fluorescent tattoo location of cutaneous biopsy site.

Science.gov (United States)

Chuang, Gary S; Gilchrest, Barbara A

2012-03-01

Cutaneous biopsies often heal with little or no scarring. Prior studies have shown an alarming percentage of patients who incorrectly identify biopsy sites at the time of surgery. To investigate the safety and utility of an ultraviolet (UV)-fluorescent tattoo for biopsy site identification. A preclinical proof of concept was established with skin culture. An UV-fluorescent tattoo was applied to discarded neonatal foreskin in culture medium. The stability of the tattooed skin was examined clinically and histologically. One patient with a recurrent basal cell carcinoma in a difficult-to-identify location underwent tattoo application at the time of biopsy to demarcate the site. The patient was monitored for tattoo reaction and referred for surgical excision. The cultured tissue exhibited stable UV fluorescence with daily washing. Tissue histology demonstrated tattoo particles lining the skin edge under fluorescent microscopy. The patient was reluctant to undergo another surgical procedure and instead returned to our clinic at 3 months and 17 months after the biopsy for management of other tumors. The patient had no symptoms of allergic reaction to the tattoo dye. The fluorescent tattoo remains invisible under visible light and visible only under Wood's light. The present study documents the utility of an UV-fluorescent tattoo to locate a biopsy site. © 2011 by the American Society for Dermatologic Surgery, Inc. Published by Wiley Periodicals, Inc.

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.)

Site-Specific Three-Color Labeling of α-Synuclein via Conjugation to Uniquely Reactive Cysteines during Assembly by Native Chemical Ligation.

Science.gov (United States)

Lee, Taehyung C; Moran, Crystal R; Cistrone, Philip A; Dawson, Philip E; Deniz, Ashok A

2018-04-12

Single-molecule fluorescence is widely used to study conformational complexity in proteins, and has proven especially valuable with intrinsically disordered proteins (IDPs). Protein studies using dual-color single-molecule Förster resonance energy transfer (smFRET) are now quite common, but many could benefit from simultaneous measurement of multiple distances through multi-color labeling. Such studies, however, have suffered from limitations in site-specific incorporation of more than two dyes per polypeptide. Here we present a fully site-specific three-color labeling scheme for α-synuclein, an IDP with important putative functions and links to Parkinson disease. The convergent synthesis combines native chemical ligation with regiospecific cysteine protection of expressed protein fragments to permit highly controlled labeling via standard cysteine-maleimide chemistry, enabling more global smFRET studies. Furthermore, this modular approach is generally compatible with recombinant proteins and expandable to accommodate even more complex experiments, such as by labeling with additional colors. Copyright © 2018 Elsevier Ltd. All rights reserved.

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

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

Selective disulfide reduction for labeling and enhancement of Fab antibody fragments.

Science.gov (United States)

Kirley, Terence L; Greis, Kenneth D; Norman, Andrew B

2016-11-25

Many methods have been developed for chemical labeling and enhancement of the properties of antibodies and their common fragments, including the Fab and F(ab') 2 fragments. Somewhat selective reduction of some antibody disulfide bonds has been previously achieved, yielding antibodies and antibody fragments that can be labeled at defined sites, enhancing their utility and properties. Selective reduction of the two hinge disulfide bonds present in F(ab') 2 fragments using mild reduction has been useful. However, such reduction is often not quantitative and results in the reduction of multiple disulfide bonds, and therefore subsequent multiple labeling or conjugation sites are neither homogenous nor stoichiometric. Here, a simple and efficient selective reduction of the single disulfide bond linking the partial heavy chain and the intact light chain which compose the Fab fragment is accomplished utilizing tris(2-carboxyethyl)phosphine (TCEP) immobilized on agarose beads. The resultant reduced cysteine residues were labeled with several cysteine-selective fluorescent reagents, as well as by cysteine-directed PEGylation. These two cysteine residues can also be re-ligated by means of a bifunctional cysteine cross-linking agent, dibromobimane, thereby both restoring a covalent linkage between the heavy and light chains at this site, far removed from the antigen binding site, and also introducing a fluorescent probe. There are many other research and clinical uses for these selectively partially reduced Fab fragments, including biotinylation, toxin and drug conjugation, and incorporation of radioisotopes, and this technique enables simple generation of very useful Fab fragment derivatives with many potential applications. Copyright © 2016 Elsevier Inc. All rights reserved.

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)

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.

Site directed spin labeling studies of Escherichia coli dihydroorotate dehydrogenase N-terminal extension

Energy Technology Data Exchange (ETDEWEB)

Couto, Sheila G. [Instituto de Fisica de Sao Carlos, Universidade de Sao Paulo, Av. Trabalhador Sao-carlense 400, C.P. 369, 13560-970, Sao Carlos, SP (Brazil); Grupo de Biofisica e Fisica Aplicada a Medicina, Instituto de Fisica, Universidade Federal de Goias, Campus Samambaia, C.P. 131, 74001-970, Goiania, GO (Brazil); Cristina Nonato, M. [Laboratorio de Cristalografia de Proteinas, Faculdade de Ciencias Farmaceuticas de Ribeirao Preto, Universidade de Sao Paulo, Av. do Cafe S/N, 14040-903, Ribeirao Preto, SP (Brazil); Costa-Filho, Antonio J., E-mail: ajcosta@ffclrp.usp.br [Instituto de Fisica de Sao Carlos, Universidade de Sao Paulo, Av. Trabalhador Sao-carlense 400, C.P. 369, 13560-970, Sao Carlos, SP (Brazil); Departamento de Fisica, Faculdade de Filosofia, Ciencias e Letras de Ribeirao Preto, Av. Bandeirantes 3900, 14040-901, Ribeirao Preto, SP (Brazil)

2011-10-28

Highlights: Black-Right-Pointing-Pointer EcDHODH is a membrane-associated enzyme and a promising target for drug design. Black-Right-Pointing-Pointer Enzyme's N-terminal extension is responsible for membrane association. Black-Right-Pointing-Pointer N-terminal works as a molecular lid regulating access to the protein interior. -- Abstract: Dihydroorotate dehydrogenases (DHODHs) are enzymes that catalyze the fourth step of the de novo synthesis of pyrimidine nucleotides. In this reaction, DHODH converts dihydroorotate to orotate, using a flavine mononucleotide as a cofactor. Since the synthesis of nucleotides has different pathways in mammals as compared to parasites, DHODH has gained much attention as a promising target for drug design. Escherichia coli DHODH (EcDHODH) is a family 2 DHODH that interacts with cell membranes in order to promote catalysis. The membrane association is supposedly made via an extension found in the enzyme's N-terminal. In the present work, we used site directed spin labeling (SDSL) to specifically place a magnetic probe at positions 2, 5, 19, and 21 within the N-terminal and thus monitor, by using Electron Spin Resonance (ESR), dynamics and structural changes in this region in the presence of a membrane model system. Overall, our ESR spectra show that the N-terminal indeed binds to membranes and that it experiences a somewhat high flexibility that could be related to the role of this region as a molecular lid controlling the entrance of the enzyme's active site and thus allowing the enzyme to give access to quinones that are dispersed in the membrane and that are necessary for the catalysis.

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.)

Simultaneous fluorescence light-up and selective multicolor nucleobase recognition based on sequence-dependent strong binding of berberine to DNA abasic site.

Science.gov (United States)

Wu, Fei; Shao, Yong; Ma, Kun; Cui, Qinghua; Liu, Guiying; Xu, Shujuan

2012-04-28

Label-free DNA nucleobase recognition by fluorescent small molecules has received much attention due to its simplicity in mutation identification and drug screening. However, sequence-dependent fluorescence light-up nucleobase recognition and multicolor emission with individual emission energy for individual nucleobases have been seldom realized. Herein, an abasic site (AP site) in a DNA duplex was employed as a binding field for berberine, one of isoquinoline alkaloids. Unlike weak binding of berberine to the fully matched DNAs without the AP site, strong binding of berberine to the AP site occurs and the berberine's fluorescence light-up behaviors are highly dependent on the target nucleobases opposite the AP site in which the targets thymine and cytosine produce dual emission bands, while the targets guanine and adenine only give a single emission band. Furthermore, more intense emissions are observed for the target pyrimidines than purines. The flanking bases of the AP site also produce some modifications of the berberine's emission behavior. The binding selectivity of berberine at the AP site is also confirmed by measurements of fluorescence resonance energy transfer, excited-state lifetime, DNA melting and fluorescence quenching by ferrocyanide and sodium chloride. It is expected that the target pyrimidines cause berberine to be stacked well within DNA base pairs near the AP site, which results in a strong resonance coupling of the electronic transitions to the particular vibration mode to produce the dual emissions. The fluorescent signal-on and emission energy-modulated sensing for nucleobases based on this fluorophore is substantially advantageous over the previously used fluorophores. We expect that this approach will be developed as a practical device for differentiating pyrimidines from purines by positioning an AP site toward a target that is available for readout by this alkaloid probe. This journal is © The Royal Society of Chemistry 2012

Flexibility of Enzymes Suspended in Organic Solvents Probed by Time-Resolved Fluorescence Anisotropy. Evidence That Enzyme Activity and Enantioselectivity Are Directly Related to Enzyme Flexibility

NARCIS (Netherlands)

Broos, Jaap; Visser, Antonie J.W.G.; Engbersen, Johan F.J.; Verboom, Willem; Hoek, Arie van; Reinhoudt, David N.

1995-01-01

A time-resolved fluorescence anisotropy study on the molecular flexibility of active-site labeled anthraniloyl-α-chymotrypsin, dansylsubtilisin Carlsberg, and native subtilisin Carlsberg, suspended in organic solvents, is described. The internal rotational mobility of the fluorophore in the

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.

Sensing site-specific structural characteristics and chirality using vibrational circular dichroism of isotope labeled peptides.

Science.gov (United States)

Keiderling, Timothy A

2017-12-01

Isotope labeling has a long history in chemistry as a tool for probing structure, offering enhanced sensitivity, or enabling site selection with a wide range of spectroscopic tools. Chirality sensitive methods such as electronic circular dichroism are global structural tools and have intrinsically low resolution. Consequently, they are generally insensitive to modifications to enhance site selectivity. The use of isotope labeling to modify vibrational spectra with unique resolvable frequency shifts can provide useful site-specific sensitivity, and these methods have been recently more widely expanded in biopolymer studies. While the spectral shifts resulting from changes in isotopic mass can provide resolution of modes from specific parts of the molecule and can allow detection of local change in structure with perturbation, these shifts alone do not directly indicate structure or chirality. With vibrational circular dichroism (VCD), the shifted bands and their resultant sign patterns can be used to indicate local conformations in labeled biopolymers, particularly if multiple labels are used and if their coupling is theoretically modeled. This mini-review discusses selected examples of the use of labeling specific amides in peptides to develop local structural insight with VCD spectra. © 2017 Wiley Periodicals, Inc.

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.

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.

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.

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

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...

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

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.

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.

Software-Supported USER Cloning Strategies for Site-Directed Mutagenesis and DNA Assembly

DEFF Research Database (Denmark)

Genee, Hans Jasper; Bonde, Mads Tvillinggaard; Bagger, Frederik Otzen

2015-01-01

USER cloning is a fast and versatile method for engineering of plasmid DNA. We have developed a user friendly Web server tool that automates the design of optimal PCR primers for several distinct USER cloning-based applications. Our Web server, named AMUSER (Automated DNA Modifications with USER...... cloning), facilitates DNA assembly and introduction of virtually any type of site-directed mutagenesis by designing optimal PCR primers for the desired genetic changes. To demonstrate the utility, we designed primers for a simultaneous two-position site-directed mutagenesis of green fluorescent protein...... (GFP) to yellow fluorescent protein (YFP), which in a single step reaction resulted in a 94% cloning efficiency. AMUSER also supports degenerate nucleotide primers, single insert combinatorial assembly, and flexible parameters for PCR amplification. AMUSER is freely available online at ....

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.

Robust, directed assembly of fluorescent nanodiamonds.

Science.gov (United States)

Kianinia, Mehran; Shimoni, Olga; Bendavid, Avi; Schell, Andreas W; Randolph, Steven J; Toth, Milos; Aharonovich, Igor; Lobo, Charlene J

2016-10-27

Arrays of fluorescent nanoparticles are highly sought after for applications in sensing, nanophotonics and quantum communications. Here we present a simple and robust method of assembling fluorescent nanodiamonds into macroscopic arrays. Remarkably, the yield of this directed assembly process is greater than 90% and the assembled patterns withstand ultra-sonication for more than three hours. The assembly process is based on covalent bonding of carboxyl to amine functional carbon seeds and is applicable to any material, and to non-planar surfaces. Our results pave the way to directed assembly of sensors and nanophotonics devices.

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.

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

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.

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...

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.

Dissecting the Catalytic Mechanism of Betaine-Homocysteine S-Methyltransferase Using Intrinsic Tryptophan Fluorescence and Site-Directed Mutagenesis

Energy Technology Data Exchange (ETDEWEB)

Castro, C.; Gratson, A.A.; Evans, J.C.; Jiracek, J.; Collinsova, M.; Ludwig, M.L.; Garrow, T.A. (ASCR); (UIUC); (Michigan)

2010-03-05

Betaine-homocysteine S-methyltransferase (BHMT) is a zinc-dependent enzyme that catalyzes the transfer of a methyl group from glycine betaine (Bet) to homocysteine (Hcy) to form dimethylglycine (DMG) and methionine (Met). Previous studies in other laboratories have indicated that catalysis proceeds through the formation of a ternary complex, with a transition state mimicked by the inhibitor S-({delta}-carboxybutyl)-l-homocysteine (CBHcy). Using changes in intrinsic tryptophan fluorescence to determine the affinity of human BHMT for substrates, products, or CBHcy, we now demonstrate that the enzyme-substrate complex reaches its transition state through an ordered bi-bi mechanism in which Hcy is the first substrate to bind and Met is the last product released. Hcy, Met, and CBHcy bind to the enzyme to form binary complexes with K{sub d} values of 7.9, 6.9, and 0.28 {micro}M, respectively. Binary complexes with Bet and DMG cannot be detected with fluorescence as a probe, but Bet and DMG bind tightly to BHMT-Hcy to form ternary complexes with K{sub d} values of 1.1 and 0.73 {micro}M, respectively. Mutation of each of the seven tryptophan residues in human BHMT provides evidence that the enzyme undergoes two distinct conformational changes that are reflected in the fluorescence of the enzyme. The first is induced when Hcy binds, and the second, when Bet binds. As predicted by the crystal structure of BHMT, the amino acids Trp44 and Tyr160 are involved in binding Bet, and Glu159 in binding Hcy. Replacing these residues by site-directed mutagenesis significantly reduces the catalytic efficiency (V{sub max}/K{sub m}) of the enzyme. Replacing Tyr77 with Phe abolishes enzyme activity.

Peritoneal macrophages mediated delivery of chitosan/siRNA nanoparticle to the lesion site in a murine radiation-induced fibrosis model

DEFF Research Database (Denmark)

Nawroth, Isabel; Alsner, Jan; Deleuran, B.W.

2013-01-01

of chitosan/siRNA nanoparticles directed towards silencing TNF alpha in local macrophage populations, but the mechanism for the therapeutic effect at the lesion site remains unclear. Methods. Using the same murine RIF model we utilized an optical imaging technique and fluorescence microscopy to investigate...... the uptake of chitosan/fluorescently labeled siRNA nanoparticles by peritoneal macrophages and their subsequent migration to the inflamed tissue in the RIF model. Results. We observed strong accumulation of the fluorescent signal in the lesion site of the irradiated leg up to 24 hours using the optical...... imaging system. We further confirm by immunohistochemical staining that Cy3 labeled siRNA resides in macrophages of the irradiated leg. Conclusion. We provide a proof-of-concept for host macrophage trafficking towards the inflamed region in a murine RIF model, which thereby suggests that the chitosan...

DIRECT AND INDIRECT FLUORESCENT-ANTIBODY TECHNIQUES FOR THE PSITTACOSIS-LYMPHOGRANULOMA VENEREUM-TRACHOMA GROUP OF AGENTS1

Science.gov (United States)

Ross, Martin R.; Borman, Earle K.

1963-01-01

Ross, Martin R. (Connecticut State Department of Health, Hartford) and Earle K. Borman. Direct and indirect fluorescent-antibody techniques for the psittacosis-lymphogranuloma venereum-trachoma group of agents. J. Bacteriol. 85:851–858. 1963.—Direct and indirect fluorescent-antibody (FA) techniques were developed for the detection of group antigen in infected tissue cultures and the titration of group antibody in human antiserum. The growth of the agent of meningopneumonitis (MP) in mouse embryo lung cell monolayers was followed by infectivity and complement-fixing (CF) antigen titrations, and cytological examination of FA stained cultures. Although infectivity and CF antigen reached a peak at 2 days and remained constant for an additional 3 days, only cells tested 2 to 3 days after infection were suitable for FA staining with labeled anti-MP serum because of excessive artifacts in the older cultures. Fluorescein isothiocyanate-labeled rooster and guinea pig anti-MP serums and human antipsittacosis serums were titrated in direct FA and hemagglutination-inhibition (HI) tests. The rooster conjugate showed brighter staining and higher antibody titers than the guinea pig or human conjugates and was more effective in detecting minimal amounts of virus antigen. FA staining reactions with 1 and 2 units of labeled rooster serum were inhibited by unlabeled rooster serum but clear-cut inhibition with human antipsittacosis serum could not be demonstrated. The indirect FA technique was successfully used for the titration of group antibody in human serum. A comparison of the indirect FA, HI, and CF tests showed the indirect FA technique to be intermediate in sensitivity between the HI and CF tests. None of the three tests showed significant cross reactions with human serums reactive for influenza A and B; parainfluenza 1, 2, and 3; respiratory syncytial virus; Q fever; or the primary atypical pneumonia agent. PMID:14044954

Global Structure of a Three-Way Junction in a Phi29 Packaging RNA Dimer Determined Using Site-Directed Spin Labeling

Energy Technology Data Exchange (ETDEWEB)

Zhang, Xiaojun; Tung, Chang-Shung; Sowa, Glenna; Hatmal, Ma' mon M.; Haworth, Ian S.; Qin, Peter Z.

2012-02-08

The condensation of bacteriophage phi29 genomic DNA into its preformed procapsid requires the DNA packaging motor, which is the strongest known biological motor. The packaging motor is an intricate ring-shaped protein/RNA complex, and its function requires an RNA component called packaging RNA (pRNA). Current structural information on pRNA is limited, which hinders studies of motor function. Here, we used site-directed spin labeling to map the conformation of a pRNA three-way junction that bridges binding sites for the motor ATPase and the procapsid. The studies were carried out on a pRNA dimer, which is the simplest ring-shaped pRNA complex and serves as a functional intermediate during motor assembly. Using a nucleotide-independent labeling scheme, stable nitroxide radicals were attached to eight specific pRNA sites without perturbing RNA folding and dimer formation, and a total of 17 internitroxide distances spanning the three-way junction were measured using Double Electron-Electron Resonance spectroscopy. The measured distances, together with steric chemical constraints, were used to select 3662 viable three-way junction models from a pool of 65 billion. The results reveal a similar conformation among the viable models, with two of the helices (HT and HL) adopting an acute bend. This is in contrast to a recently reported pRNA tetramer crystal structure, in which HT and HL stack onto each other linearly. The studies establish a new method for mapping global structures of complex RNA molecules, and provide information on pRNA conformation that aids investigations of phi29 packaging motor and developments of pRNA-based nanomedicine and nanomaterial.

Conformational change in full-length mouse prion: A site-directed spin-labeling study

International Nuclear Information System (INIS)

Inanami, Osamu; Hashida, Shukichi; Iizuka, Daisuke; Horiuchi, Motohiro; Hiraoka, Wakako; Shimoyama, Yuhei; Nakamura, Hideo; Inagaki, Fuyuhiko; Kuwabara, Mikinori

2005-01-01

The structure of the mouse prion (moPrP) was studied using site-directed spin-labeling electron spin resonance (SDSL-ESR). Since a previous NMR study by Hornemanna et al., [Hornemanna, Korthb, Oeschb, Rieka, Widera, Wuethricha, Glockshubera, Recombinant full-length murine prion protein, mPrP (23-231): purification and spectroscopic characterization, FEBS Lett. 413 (1997) 277-281] has indicated that N96, D143, and T189 in moPrP are localized in a Cu 2+ binding region, Helix1 and Helix2, respectively, three recombinant moPrP mutations (N96C, D143C, and T189C) were expressed in an Escherichia coli system, and then refolded by dialysis under low pH and purified by reverse-phase HPLC. By using the preparation, we succeeded in preserving a target cystein residue without alteration of the α-helix structure of moPrP and were able to apply SDSL-ESR with a methane thiosulfonate spin label to the full-length prion protein. The rotational correlation times (τ) of 1.1, 3.3, and 4.8 ns were evaluated from the X-band ESR spectra at pH 7.4 and 20 deg C for N96R1, D143R1, and T189R1, respectively. τ reflects the fact that the Cu 2+ binding region is more flexible than Helix1 or Helix2. ESR spectra recorded at various temperatures revealed two phases together with a transition point at around 20 deg C in D143R1 and T189R1, but not in N96R1. With the variation of pH from 4.0 to 7.8, ESR spectra of T189R1 at 20 deg C showed a gradual increase of τ from 2.9 to 4.8 ns. On the other hand, the pH-dependent conformational changes in N96R1 and D143R1 were negligible. These results indicated that T189 located in Helix2 possessed a structure sensitive to physiological pH changes; simultaneously, N96 in the Cu 2+ binding region and D143 in Helix1 were conserved

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.

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

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

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.

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.

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.

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.

Epoxyethylglycyl peptides as inhibitors of oligosaccharyltransferase: double-labelling of the active site.

Science.gov (United States)

Bause, E; Wesemann, M; Bartoschek, A; Breuer, W

1997-02-15

Pig liver oligosaccharyltransferase (OST) is inactivated irreversibly by a hexapeptide in which threonine has been substituted by epoxyethylglycine in the Asn-Xaa-Thr glycosylation triplet. Incubation of the enzyme in the presence of Dol-PP-linked [14C]oligosaccharides and the N-3,5-dinitrobenzoylated epoxy derivative leads to the double-labelling of two subunits (48 and 66 kDa) of the oligomeric OST complex, both of which are involved in the catalytic activity. Labelling of both subunits was blocked competitively by the acceptor peptide N-benzoyl-Asu-Gly-Thr-NHCH3 and by the OST inhibitor N-benzoyl-alpha,gamma-diaminobutyric acid-Gly-Thr-NHCH3, but not by an analogue derived from the epoxy-inhibitor by replacing asparagine with glutamine. Our data clearly show that double-labelling is an active-site-directed modification, involving inhibitor glycosylation at asparagine and covalent attachment of the glycosylated inhibitor, via the epoxy group, to the enzyme. Double-labelling of OST can occur as the result of either a consecutive or a syn-catalytic reaction sequence. The latter mechanism, during the course of which OST catalyses its own 'suicide' inactivation, is more likely, as suggested by indirect experimental evidence. The syn-catalytic mechanism corresponds with our current view of the functional role of the acceptor site Thr/Ser acting as a hydrogen-bond acceptor, not a donor, during transglycosylation.

Differential Isotope Labeling of Glycopeptides for Accurate Determination of Differences in Site-Specific Glycosylation.

Science.gov (United States)

Pabst, Martin; Benešová, Iva; Fagerer, Stephan R; Jacobsen, Mathias; Eyer, Klaus; Schmidt, Gregor; Steinhoff, Robert; Krismer, Jasmin; Wahl, Fabian; Preisler, Jan; Zenobi, Renato

2016-01-04

We introduce a stable isotope labeling approach for glycopeptides that allows a specific glycosylation site in a protein to be quantitatively evaluated using mass spectrometry. Succinic anhydride is used to specifically label primary amino groups of the peptide portion of the glycopeptides. The heavy form (D4(13)C4) provides an 8 Da mass increment over the light natural form (H4(12)C4), allowing simultaneous analysis and direct comparison of two glycopeptide profiles in a single MS scan. We have optimized a protocol for an in-solution trypsin digestion, a one-pot labeling procedure, and a post-labeling solid-phase extraction to obtain purified and labeled glycopeptides. We provide the first demonstration of this approach by comparing IgG1 Fc glycopeptides from polyclonal IgG samples with respect to their galactosylation and sialylation patterns using MALDI MS and LC-ESI-MS.

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.

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)

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.

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.

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).

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)

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

Stem cell monitoring with a direct or indirect labeling method

Energy Technology Data Exchange (ETDEWEB)

Kim, Min Hwan; Lee, Yong Jin [Molecular Imaging Research Center, Korea Institute of Radiological and Medical Sciences (KIRAMS), Seoul (Korea, Republic of)

2016-12-15

The molecular imaging techniques allow monitoring of the transplanted cells in the same individuals over time, from early localization to the survival, migration, and differentiation. Generally, there are two methods of stem cell labeling: direct and indirect labeling methods. The direct labeling method introduces a labeling agent into the cell, which is stably incorporated or attached to the cells prior to transplantation. Direct labeling of cells with radionuclides is a simple method with relatively fewer adverse events related to genetic responses. However, it can only allow short-term distribution of transplanted cells because of the decreasing imaging signal with radiodecay, according to the physical half-lives, or the signal becomes more diffuse with cell division and dispersion. The indirect labeling method is based on the expression of a reporter gene transduced into the cell before transplantation, which is then visualized upon the injection of an appropriate probe or substrate. In this review, various imaging strategies to monitor the survival and behavior change of transplanted stem cells are covered. Taking these new approaches together, the direct and indirect labeling methods may provide new insights on the roles of in vivo stem cell monitoring, from bench to bedside.

Comparison of infrared-excited up-converting phosphors and europium nanoparticles as labels in a two-site immunoassay

International Nuclear Information System (INIS)

Ukonaho, Telle; Rantanen, Terhi; Jaemsen, Laura; Kuningas, Katri; Paekkilae, Henna; Loevgren, Timo; Soukka, Tero

2007-01-01

Research in the field of immunoassays and labels used in the detection has been recently focused on particulate reporters, which possess very high specific activity that excludes the label as a sensitivity limiting factor. However, the large size and shape of the particulate labels may produce additional problems to immunoassay performance. The aim of this work was to study with two identical non-competitive two-site immunoassays whether up-converting phosphor (UCP) particles are comparable in performance with europium(III) chelate-dyed nanoparticles as particulate labels. In addition we strived to verify the common assumption of the photostability of up-converting phosphor particles supporting their potential applicability in imaging. Detection limits in two-site immunoassay for free prostate-specific antigen (free-PSA) were 0.53 ng L -1 and 1.3 ng L -1 using two different up-converting phosphors and 0.16 ng L -1 using europium(III) nanoparticle. Large size distribution and non-specific binding of up-converting phosphor particles caused assay variation in low analyte concentrations and limited the analytical detection limit. The non-specific binding was the major factor limiting the analytical sensitivity of the immunoassay. The results suggests the need for nanoscaled and uniformely sized UCP-particles to increace the sensitivity and applicability of up-converting phosphor particles. Anti-Stokes photoluminescence of up-converting phosphor particles did not photobleach when measured repeatedly, on the contrary, the time-resolved fluorescence of europium nanoparticles photobleached relatively rapidly

Chemoenzymatic site-specific labeling of influenza glycoproteins as a tool to observe virus budding in real time.

Directory of Open Access Journals (Sweden)

Maximilian Wei-Lin Popp

Full Text Available The influenza virus uses the hemagglutinin (HA and neuraminidase (NA glycoproteins to interact with and infect host cells. While biochemical and microscopic methods allow examination of the early steps in flu infection, the genesis of progeny virions has been more difficult to follow, mainly because of difficulties inherent in fluorescent labeling of flu proteins in a manner compatible with live cell imaging. We here apply sortagging as a chemoenzymatic approach to label genetically modified but infectious flu and track the flu glycoproteins during the course of infection. This method cleanly distinguishes influenza glycoproteins from host glycoproteins and so can be used to assess the behavior of HA or NA biochemically and to observe the flu glycoproteins directly by live cell imaging.

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.

Selective disulfide reduction for labeling and enhancement of Fab antibody fragments

International Nuclear Information System (INIS)

Kirley, Terence L.; Greis, Kenneth D.; Norman, Andrew B.

2016-01-01

Many methods have been developed for chemical labeling and enhancement of the properties of antibodies and their common fragments, including the Fab and F(ab’) 2 fragments. Somewhat selective reduction of some antibody disulfide bonds has been previously achieved, yielding antibodies and antibody fragments that can be labeled at defined sites, enhancing their utility and properties. Selective reduction of the two hinge disulfide bonds present in F(ab’) 2 fragments using mild reduction has been useful. However, such reduction is often not quantitative and results in the reduction of multiple disulfide bonds, and therefore subsequent multiple labeling or conjugation sites are neither homogenous nor stoichiometric. Here, a simple and efficient selective reduction of the single disulfide bond linking the partial heavy chain and the intact light chain which compose the Fab fragment is accomplished utilizing tris(2-carboxyethyl)phosphine (TCEP) immobilized on agarose beads. The resultant reduced cysteine residues were labeled with several cysteine-selective fluorescent reagents, as well as by cysteine-directed PEGylation. These two cysteine residues can also be re-ligated by means of a bifunctional cysteine cross-linking agent, dibromobimane, thereby both restoring a covalent linkage between the heavy and light chains at this site, far removed from the antigen binding site, and also introducing a fluorescent probe. There are many other research and clinical uses for these selectively partially reduced Fab fragments, including biotinylation, toxin and drug conjugation, and incorporation of radioisotopes, and this technique enables simple generation of very useful Fab fragment derivatives with many potential applications. - Highlights: • TCEP agarose is effective for selective reduction of a single Fab disulfide bond. • This disulfide is solvent accessible and distant from the antigen binding site. • A variety of buffers of varying pHs can be used, simplifying

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.

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.

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.

Design and Elementary Evaluation of a Highly-Automated Fluorescence-Based Instrument System for On-Site Detection of Food-Borne Pathogens

Directory of Open Access Journals (Sweden)

Zhan Lu

2017-02-01

Full Text Available A simple, highly-automated instrument system used for on-site detection of foodborne pathogens based on fluorescence was designed, fabricated, and preliminarily tested in this paper. A corresponding method has been proved effective in our previous studies. This system utilizes a light-emitting diode (LED to excite fluorescent labels and a spectrometer to record the fluorescence signal from samples. A rotation stage for positioning and switching samples was innovatively designed for high-throughput detection, ten at most in one single run. We also developed software based on LabVIEW for data receiving, processing, and the control of the whole system. In the test of using a pure quantum dot (QD solution as a standard sample, detection results from this home-made system were highly-relevant with that from a well-commercialized product and even slightly better reproducibility was found. And in the test of three typical kinds of food-borne pathogens, fluorescence signals recorded by this system are highly proportional to the variation of the sample concentration, with a satisfied limit of detection (LOD (nearly 102–103 CFU·mL−1 in food samples. Additionally, this instrument system is low-cost and easy-to-use, showing a promising potential for on-site rapid detection of food-borne pathogens.

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

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

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.

Tuning a Protein-Labeling Reaction to Achieve Highly Site Selective Lysine Conjugation.

Science.gov (United States)

Pham, Grace H; Ou, Weijia; Bursulaya, Badry; DiDonato, Michael; Herath, Ananda; Jin, Yunho; Hao, Xueshi; Loren, Jon; Spraggon, Glen; Brock, Ansgar; Uno, Tetsuo; Geierstanger, Bernhard H; Cellitti, Susan E

2018-04-16

Activated esters are widely used to label proteins at lysine side chains and N termini. These reagents are useful for labeling virtually any protein, but robust reactivity toward primary amines generally precludes site-selective modification. In a unique case, fluorophenyl esters are shown to preferentially label human kappa antibodies at a single lysine (Lys188) within the light-chain constant domain. Neighboring residues His189 and Asp151 contribute to the accelerated rate of labeling at Lys188 relative to the ≈40 other lysine sites. Enriched Lys188 labeling can be enhanced from 50-70 % to >95 % by any of these approaches: lowering reaction temperature, applying flow chemistry, or mutagenesis of specific residues in the surrounding protein environment. Our results demonstrated that activated esters with fluoro-substituted aromatic leaving groups, including a fluoronaphthyl ester, can be generally useful reagents for site-selective lysine labeling of antibodies and other immunoglobulin-type proteins. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Small fluorescence-activating and absorption-shifting tag for tunable protein imaging in vivo.

Science.gov (United States)

Plamont, Marie-Aude; Billon-Denis, Emmanuelle; Maurin, Sylvie; Gauron, Carole; Pimenta, Frederico M; Specht, Christian G; Shi, Jian; Quérard, Jérôme; Pan, Buyan; Rossignol, Julien; Moncoq, Karine; Morellet, Nelly; Volovitch, Michel; Lescop, Ewen; Chen, Yong; Triller, Antoine; Vriz, Sophie; Le Saux, Thomas; Jullien, Ludovic; Gautier, Arnaud

2016-01-19

This paper presents Yellow Fluorescence-Activating and absorption-Shifting Tag (Y-FAST), a small monomeric protein tag, half as large as the green fluorescent protein, enabling fluorescent labeling of proteins in a reversible and specific manner through the reversible binding and activation of a cell-permeant and nontoxic fluorogenic ligand (a so-called fluorogen). A unique fluorogen activation mechanism based on two spectroscopic changes, increase of fluorescence quantum yield and absorption red shift, provides high labeling selectivity. Y-FAST was engineered from the 14-kDa photoactive yellow protein by directed evolution using yeast display and fluorescence-activated cell sorting. Y-FAST is as bright as common fluorescent proteins, exhibits good photostability, and allows the efficient labeling of proteins in various organelles and hosts. Upon fluorogen binding, fluorescence appears instantaneously, allowing monitoring of rapid processes in near real time. Y-FAST distinguishes itself from other tagging systems because the fluorogen binding is highly dynamic and fully reversible, which enables rapid labeling and unlabeling of proteins by addition and withdrawal of the fluorogen, opening new exciting prospects for the development of multiplexing imaging protocols based on sequential labeling.

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.

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.

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.

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.

Characterization of fluorescence quenching in bifluorophoric protease substrates.

Science.gov (United States)

Packard, B Z; Toptygin, D D; Komoriya, A; Brand, L

1997-09-01

NorFES is a relatively rigid, bent undecapeptide which contains an amino acid sequence that is recognized by the serine protease elastase (AspAlaIleProNle downward arrow SerIleProLysGlyTyr ( downward arrow indicates the primary cleavage site)). Covalent attachment of a fluorophore on each side of NorFES's elastase cleavage site enables one to use a change of fluorescence intensity as a measure of enzymatic activity. In this study two bichromophoric NorFES derivatives, D-NorFES-A and D-NorFES-D, were prepared in which D (donor) was tetramethylrhodamine and A (acceptor) was rhodamine-X, two chromophores with characteristics suitable for energy transfer. Absorption and fluorescence spectra were obtained with both the intact and cleaved homodoubly, heterodoubly and singly labeled derivatives. It was found that both the homo and hetero doubly-labeled derivatives form ground-state complexes which exhibit exciton bands. The hetero labeled derivative exhibits little or no resonance energy transfer. Spectral measurements were also done in urea, which partially disrupts ground-state dimers.

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.

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

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)

Labeling proteins inside living cells using external fluorophores for microscopy.

Science.gov (United States)

Teng, Kai Wen; Ishitsuka, Yuji; Ren, Pin; Youn, Yeoan; Deng, Xiang; Ge, Pinghua; Lee, Sang Hak; Belmont, Andrew S; Selvin, Paul R

2016-12-09

Site-specific fluorescent labeling of proteins inside live mammalian cells has been achieved by employing Streptolysin O, a bacterial enzyme which forms temporary pores in the membrane and allows delivery of virtually any fluorescent probes, ranging from labeled IgG's to small ligands, with high efficiency (>85% of cells). The whole process, including recovery, takes 30 min, and the cell is ready to be imaged immediately. A variety of cell viability tests were performed after treatment with SLO to ensure that the cells have intact membranes, are able to divide, respond normally to signaling molecules, and maintains healthy organelle morphology. When combined with Oxyrase, a cell-friendly photostabilizer, a ~20x improvement in fluorescence photostability is achieved. By adding in glutathione, fluorophores are made to blink, enabling super-resolution fluorescence with 20-30 nm resolution over a long time (~30 min) under continuous illumination. Example applications in conventional and super-resolution imaging of native and transfected cells include p65 signal transduction activation, single molecule tracking of kinesin, and specific labeling of a series of nuclear and cytoplasmic protein complexes.

Direct photoaffinity labeling by nucleotides of the apparent catalytic site on the heavy chains of smooth muscle and Acanthamoeba myosins

International Nuclear Information System (INIS)

Maruta, H.; Korn, E.D.

1981-01-01

The heavy chains of Acanthamoeba myosins, IA, IB and II, turkey gizzard myosin, and rabbit skeletal muscle myosin subfragment-1 were specifically labeled by radioactive ATP, ADP, and UTP, each of which is a substrate or product of myosin ATPase activity, when irradiated with uv light at 0 0 C. With UTP, as much as 0.45 mol/mol of Acanthamoeba myosin IA heavy chain and 1 mol/mol of turkey gizzard myosin heavy chain was incorporated. Evidence that the ligands were associated with the catalytic site included the observations that reaction occurred only with nucleotides that are substrates or products of the ATPase activity; that the reaction was blocked by pyrophosphate which is an inhibitor of the ATPase activity; that ATP was bound as ADP; and that label was probably restricted to a single peptide following limited subtilisin proteolysis of labeled Acanthamoeba myosin IA heavy chain and extensive cleavage with CNBr and trypsin of labeled turkey gizzard myosin heavy chain

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

Development and utilization of a fluorescence-based receptor-binding assay for the site 5 voltage-sensitive sodium channel ligands brevetoxin and ciguatoxin.

Science.gov (United States)

McCall, Jennifer R; Jacocks, Henry M; Niven, Susan C; Poli, Mark A; Baden, Daniel G; Bourdelais, Andrea J

2014-01-01

Brevetoxins are a family of ladder-frame polyether toxins produced during blooms of the marine dinoflagellate Karenia brevis. Consumption of fish exposed to K. brevis blooms can lead to the development of neurotoxic shellfish poisoning. The toxic effects of brevetoxins are due to activation of voltage-sensitive sodium channels (VSSCs) in cell membranes. Binding of toxins has historically been measured using a radioligand competition assay that is fraught with difficulty. In this study, we developed a novel fluorescence-based binding assay for the brevetoxin receptor. Several fluorophores were conjugated to polyether brevetoxin-2 and used as the labeled ligand. Brevetoxin analogs were able to compete for binding with the fluorescent ligands. This assay was qualified against the standard radioligand receptor assay for the brevetoxin receptor. Furthermore, the fluorescence-based assay was used to determine relative concentrations of toxins in raw extracts of K. brevis culture, and to determine ciguatoxin affinity to site 5 of VSSCs. The fluorescence-based assay was quicker, safer, and far less expensive. As such, this assay can be used to replace the current radioligand assay and will be a vital tool for future experiments examining the binding affinity of various ligands for site 5 on sodium channels.

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.

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.

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.

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.

Direct fluorescence detection of VirE2 secretion by Agrobacterium tumefaciens

Science.gov (United States)

Yaakov, Noga; Barak, Yoav; Pereman, Idan; Christie, Peter J.; Elbaum, Michael

2017-01-01

VirE2 is a ssDNA binding protein essential for virulence in Agrobacterium tumefaciens. A tetracysteine mutant (VirE2-TC) was prepared for in vitro and in vivo fluorescence imaging based on the ReAsH reagent. VirE2-TC was found to be biochemically active as it binds both ssDNA and the acidic secretion chaperone VirE1. It was also biologically functional in complementing virE2 null strains transforming Arabidopsis thaliana roots and Nicotiana tabacum leaves. In vitro experiments demonstrated a two-color fluorescent complex using VirE2-TC/ReAsH and Alexa Fluor 488 labeled ssDNA. In vivo, fluorescent VirE2-TC/ReAsH was detected in bacteria and in plant cells at time frames relevant to transformation. PMID:28403156

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...

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...

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.

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.

Photoaffinity labeling of the pactamycin binding site on eubacterial ribosomes

International Nuclear Information System (INIS)

Tejedor, F.; Amils, R.; Ballesta, J.P.

1985-01-01

Pactamycin, an inhibitor of the initial steps of protein synthesis, has an acetophenone group in its chemical structure that makes the drug a potentially photoreactive molecule. In addition, the presence of a phenolic residue makes it easily susceptible to radioactive labeling. Through iodination, one radioactive derivative of pactamycin has been obtained with biological activities similar to the unmodified drug when tested on in vivo and cell-free systems. With the use of [ 125 I]iodopactamycin, ribosomes of Escherichia coli have been photolabeled under conditions that preserve the activity of the particles and guarantee the specificity of the binding sites. Under these conditions, RNA is preferentially labeled when free, small ribosomal subunits are photolabeled, but proteins are the main target in the whole ribosome. This indicates that an important conformational change takes place in the binding site on association of the two subunits. The major labeled proteins are S2, S4, S18, S21, and L13. These proteins in the pactamycin binding site are probably related to the initiation step of protein synthesis

Nanobodies: site-specific labeling for super-resolution imaging, rapid epitope-mapping and native protein complex isolation

Science.gov (United States)

Pleiner, Tino; Bates, Mark; Trakhanov, Sergei; Lee, Chung-Tien; Schliep, Jan Erik; Chug, Hema; Böhning, Marc; Stark, Holger; Urlaub, Henning; Görlich, Dirk

2015-01-01

Nanobodies are single-domain antibodies of camelid origin. We generated nanobodies against the vertebrate nuclear pore complex (NPC) and used them in STORM imaging to locate individual NPC proteins with nanobody sequence and labeled the resulting proteins with fluorophore-maleimides. As nanobodies are normally stabilized by disulfide-bonded cysteines, this appears counterintuitive. Yet, our analysis showed that this caused no folding problems. Compared to traditional NHS ester-labeling of lysines, the cysteine-maleimide strategy resulted in far less background in fluorescence imaging, it better preserved epitope recognition and it is site-specific. We also devised a rapid epitope-mapping strategy, which relies on crosslinking mass spectrometry and the introduced ectopic cysteines. Finally, we used different anti-nucleoporin nanobodies to purify the major NPC building blocks – each in a single step, with native elution and, as demonstrated, in excellent quality for structural analysis by electron microscopy. The presented strategies are applicable to any nanobody and nanobody-target. DOI: http://dx.doi.org/10.7554/eLife.11349.001 PMID:26633879

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.

Fluorescent antibody application in bioremediation procedures at the Savannah River Site

International Nuclear Information System (INIS)

Brigmon, R.L.; Richardson, B.S.; Franck, M.M.

1997-01-01

Direct Fluorescent Antibodies (DFA) and Most Probable Number (MPN) techniques are currently being employed at the Savannah River Site to monitor methanotrophic bacteria for the bioremediation of trichloroethylene (TCE) in field studies. Direct Fluorescent Antibodies were developed against various methanotrophic bacteria isolated from SRS as well as methanotrophic bacteria acquired from the American Type Culture Collection (ATCC). DFA's are anticipated to be more efficient for monitoring methanotroph activity than MPN's because of shorter processing time, lower cost, and the direct nature of the assay. The DFA method is a direct technique, in that samples are processed immediately and can be enumerated within an hour. The MPN method is indirect, since samples must be cultured for 6-8 weeks before measuring methane consumption and carbon dioxide production. Indirect methods are not highly selective and have limited application. The greatest advantage of a faster assay, is that bioremediation procedures utilizing methanotrophic bacteria could be amended. These amendments would be based on environmental monitoring with results in real time (1 hour). The elimination of the MPN technique and the use of DFA's will save significantly on both materials and labor. The data obtained from the DFA's and MPN's were statistically compared to each other and to total bacterial counts (AODC). The statistical analysis used was Analysis of Variants (ANOVA). Using this analysis, groundwater samples were found to be not significantly different; whereas soil were significantly different. These methods were employed on soil samples from the Southern Sector and ground water samples from the TCE-contaminated Sanitary Landfill at SRS. Acridine Orange Direct Counts were compared to show relative differences between total bacterial and methanotroph population

Photoacoustic-fluorescence in vitro flow cytometry for quantification of absorption, scattering and fluorescence properties of the cells

Science.gov (United States)

Nedosekin, D. A.; Sarimollaoglu, M.; Foster, S.; Galanzha, E. I.; Zharov, V. P.

2013-03-01

Fluorescence flow cytometry is a well-established analytical tool that provides quantification of multiple biological parameters of cells at molecular levels, including their functional states, morphology, composition, proliferation, and protein expression. However, only the fluorescence and scattering parameters of the cells or labels are available for detection. Cell pigmentation, presence of non-fluorescent dyes or nanoparticles cannot be reliably quantified. Herewith, we present a novel photoacoustic (PA) flow cytometry design for simple integration of absorbance measurements into schematics of conventional in vitro flow cytometers. The integrated system allow simultaneous measurements of light absorbance, scattering and of multicolor fluorescence from single cells in the flow at rates up to 2 m/s. We compared various combinations of excitation laser sources for multicolor detection, including simultaneous excitation of PA and fluorescence using a single 500 kHz pulsed nanosecond laser. Multichannel detection scheme allows simultaneous detection of up to 8 labels, including 4 fluorescent tags and 4 PA colors. In vitro PA-fluorescence flow cytometer was used for studies of nanoparticles uptake and for the analysis of cell line pigmentation, including genetically encoded melanin expression in breast cancer cell line. We demonstrate that this system can be used for direct nanotoxicity studies with simultaneous quantification of nanoparticles content and assessment of cell viability using a conventional fluorescent apoptosis assays.

Charting Monosynaptic Connectivity Maps by Two-Color Light-Sheet Fluorescence Microscopy

Directory of Open Access Journals (Sweden)

Christian J. Niedworok

2012-11-01

Full Text Available Cellular resolution three-dimensional (3D visualization of defined, fluorescently labeled long-range neuronal networks in the uncut adult mouse brain has been elusive. Here, a virus-based strategy is described that allowed fluorescent labeling of centrifugally projecting neuronal populations in the ventral forebrain and their directly, monosynaptically connected bulbar interneurons upon a single stereotaxic injection into select neuronal populations. Implementation of improved tissue clearing combined with light-sheet fluorescence microscopy permitted imaging of the resulting connectivity maps in a single whole-brain scan. Subsequent 3D reconstructions revealed the exact distribution of the diverse neuronal ensembles monosynaptically connected with distinct bulbar interneuron populations. Moreover, rehydratation of brains after light-sheet fluorescence imaging enabled the immunohistochemical identification of synaptically connected neurons. Thus, this study describes a method for identifying monosynaptic connectivity maps from distinct, virally labeled neuronal populations that helps in better understanding of information flow in neural systems.

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

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.

Gastric ulcer localization by direct in vivo labeling of sucralfate: work in progress

Energy Technology Data Exchange (ETDEWEB)

Pera, A.; Seevers, R.H.; Meyer, K.; Hall, C.; Bekerman, C.; Anderson, T.M.; Katzen, H.; Laakso, L.; Pinsky, S.M.

1985-09-01

The authors developed and evaluated a new procedure for imaging gastric ulcer disease with technetium 99m - labeled sucralfate. The new method employs direct in vivo labeling of sucralfate instead of in vitro labeling using human serum albumin, as previously reported in the literature. In 26 studies using humans with sucralfate labeled directly in vivo, 15 gave true-negative results. Of 14 studies using humans with in vitro labeled sucralfate, three gave true-negative results, three gave true-positive results, and the results of eight were either false-negative or could not be interpreted because of high levels of activity remaining in the stomach. They suggest that the direct in vivo labeling method significantly improves the sucralfate gastric ulcer imaging technique.

Gastric ulcer localization by direct in vivo labeling of sucralfate: work in progress

International Nuclear Information System (INIS)

Pera, A.; Seevers, R.H.; Meyer, K.; Hall, C.; Bekerman, C.; Anderson, T.M.; Katzen, H.; Laakso, L.; Pinsky, S.M.

1985-01-01

The authors developed and evaluated a new procedure for imaging gastric ulcer disease with technetium 99m - labeled sucralfate. The new method employs direct in vivo labeling of sucralfate instead of in vitro labeling using human serum albumin, as previously reported in the literature. In 26 studies using humans with sucralfate labeled directly in vivo, 15 gave true-negative results. Of 14 studies using humans with in vitro labeled sucralfate, three gave true-negative results, three gave true-positive results, and the results of eight were either false-negative or could not be interpreted because of high levels of activity remaining in the stomach. They suggest that the direct in vivo labeling method significantly improves the sucralfate gastric ulcer imaging technique

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.

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.

Directed supramolecular surface assembly of SNAP-tag fusion proteins

NARCIS (Netherlands)

Uhlenheuer, D.A.; Wasserberg, D.; Haase, C.; Nguyen, H.; Schenkel, J.H.; Huskens, J.; Ravoo, B.J.; Jonkheijm, P.; Brunsveld, L.

2012-01-01

Supramolecular assembly of proteins on surfaces and vesicles was investigated by site-selective incorporation of a supramolecular guest element on proteins. Fluorescent proteins were site-selectively labeled with bisadamantane by SNAP-tag technology. The assembly of the bisadamantane functionalized

Directed Supramolecular Surface Assembly of SNAP-tag Fusion Proteins

NARCIS (Netherlands)

Uhlenheuer, D.A.; Wasserberg, D.; Haase, C.; Nguyen, Hoang D.; Schenkel, J.H.; Huskens, Jurriaan; Ravoo, B.J.; Jonkheijm, Pascal; Brunsveld, Luc

2012-01-01

Supramolecular assembly of proteins on surfaces and vesicles was investigated by site-selective incorporation of a supramolecular guest element on proteins. Fluorescent proteins were site-selectively labeled with bisadamantane by SNAP-tag technology. The assembly of the bisadamantane functionalized

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.

Site-specific labeling of proteins with NMR-active unnatural amino acids

International Nuclear Information System (INIS)

Jones, David H.; Cellitti, Susan E.; Hao Xueshi; Zhang Qiong; Jahnz, Michael; Summerer, Daniel; Schultz, Peter G.; Uno, Tetsuo; Geierstanger, Bernhard H.

2010-01-01

A large number of amino acids other than the canonical amino acids can now be easily incorporated in vivo into proteins at genetically encoded positions. The technology requires an orthogonal tRNA/aminoacyl-tRNA synthetase pair specific for the unnatural amino acid that is added to the media while a TAG amber or frame shift codon specifies the incorporation site in the protein to be studied. These unnatural amino acids can be isotopically labeled and provide unique opportunities for site-specific labeling of proteins for NMR studies. In this perspective, we discuss these opportunities including new photocaged unnatural amino acids, outline usage of metal chelating and spin-labeled unnatural amino acids and expand the approach to in-cell NMR experiments.

Circular dichroism and site-directed spin labeling reveal structural and dynamical features of high-pressure states of myoglobin

Science.gov (United States)

Lerch, Michael T.; Horwitz, Joseph; McCoy, John; Hubbell, Wayne L.

2013-01-01

Excited states of proteins may play important roles in function, yet are difficult to study spectroscopically because of their sparse population. High hydrostatic pressure increases the equilibrium population of excited states, enabling their characterization [Akasaka K (2003) Biochemistry 42:10875–85]. High-pressure site-directed spin-labeling EPR (SDSL-EPR) was developed recently to map the site-specific structure and dynamics of excited states populated by pressure. To monitor global secondary structure content by circular dichroism (CD) at high pressure, a modified optical cell using a custom MgF2 window with a reduced aperture is introduced. Here, a combination of SDSL-EPR and CD is used to map reversible structural transitions in holomyoglobin and apomyoglobin (apoMb) as a function of applied pressure up to 2 kbar. CD shows that the high-pressure excited state of apoMb at pH 6 has helical content identical to that of native apoMb, but reversible changes reflecting the appearance of a conformational ensemble are observed by SDSL-EPR, suggesting a helical topology that fluctuates slowly on the EPR time scale. Although the high-pressure state of apoMb at pH 6 has been referred to as a molten globule, the data presented here reveal significant differences from the well-characterized pH 4.1 molten globule of apoMb. Pressure-populated states of both holomyoglobin and apoMb at pH 4.1 have significantly less helical structure, and for the latter, that may correspond to a transient folding intermediate. PMID:24248390

Site-specific conjugation and labelling of prostate antibody 7E11C5.3 (CYT-351) with technetium-99m

International Nuclear Information System (INIS)

Stalteri, M.A.; Mather, S.J.; Belinka, B.A.; Coughlin, D.J.; Chengazi, V.U.; Britton, K.E.

1997-01-01

Attachment of chelating agents to the sugar residues of antibodies for subsequent radiolabelling is an attractive approach since it may have less effect on the immunoreactivity than attachment through lysine residues, which are distributed throughout the antibody and may be present near the antigen binding site. We have attached a new hydrazide-linked chelator CYT-395 (Cytogen Corp., Princeton, N.J.) to the sugar residues of the anti-prostate monoclonal antibody 7E11C5.3 and optimised the conditions for labelling the conjugate with technetium-99m in order to compare the conjugate to 7E11C5.3 antibody labelled directly with technetium using a mercaptoethanol reduction technique. Labelling yields of 70%-90% were obtained at specific activities up to 2000 MBq/mg antibody. The stability of the technetium-labelled conjugate in plasma or to a challenge with 0.1 or 1.0 mM cysteine was similar to that of direct-labelled antibody. In nine patients with prostate cancer, the plasma clearance of the labelled conjugate followed a two-compartment model, with an average β-phase half-life of 31.4±3.9 h. The average urinary clearance at 24 h was 15.3±5.0% of the injected dose. In this group of patients there was no significant difference between the blood and urine clearance of the labelled conjugate, and the clearances of the direct-labelled antibody. (orig.). With 5 figs

DNA abasic site-selective enhancement of sanguinarine fluorescence with a large emission shift.

Directory of Open Access Journals (Sweden)

Fei Wu

Full Text Available Small molecules that can specifically bind to a DNA abasic site (AP site have received much attention due to their importance in DNA lesion identification, drug discovery, and sensor design. Herein, the AP site binding behavior of sanguinarine (SG, a natural alkaloid, was investigated. In aqueous solution, SG has a short-wavelength alkanolamine emission band and a long-wavelength iminium emission band. At pH 8.3, SG experiences a fluorescence quenching for both bands upon binding to fully matched DNAs without the AP site, while the presence of the AP site induces a strong SG binding and the observed fluorescence enhancement for the iminium band are highly dependent on the nucleobases flanking the AP site, while the alkanolamine band is always quenched. The bases opposite the AP site also exert some modifications on the SG's emission behavior. It was found that the observed quenching for DNAs with Gs and Cs flanking the AP site is most likely caused by electron transfer between the AP site-bound excited-state SG and the nearby Gs. However, the flanking As and Ts that are not easily oxidized favor the enhanced emission. This AP site-selective enhancement of SG fluorescence accompanies a band conversion in the dominate emission from the alkanolamine to iminium band thus with a large emission shift of about 170 nm. Absorption spectra, steady-state and transient-state fluorescence, DNA melting, and electrolyte experiments confirm that the AP site binding of SG occurs and the stacking interaction with the nearby base pairs is likely to prevent the converted SG iminium form from contacting with water that is thus emissive when the AP site neighbors are bases other than guanines. We expect that this fluorophore would be developed as a promising AP site binder having a large emission shift.

Monitoring Ras Interactions with the Nucleotide Exchange Factor Son of Sevenless (Sos) Using Site-specific NMR Reporter Signals and Intrinsic Fluorescence*

Science.gov (United States)

Vo, Uybach; Vajpai, Navratna; Flavell, Liz; Bobby, Romel; Breeze, Alexander L.; Embrey, Kevin J.; Golovanov, Alexander P.

2016-01-01

The activity of Ras is controlled by the interconversion between GTP- and GDP-bound forms partly regulated by the binding of the guanine nucleotide exchange factor Son of Sevenless (Sos). The details of Sos binding, leading to nucleotide exchange and subsequent dissociation of the complex, are not completely understood. Here, we used uniformly 15N-labeled Ras as well as [13C]methyl-Met,Ile-labeled Sos for observing site-specific details of Ras-Sos interactions in solution. Binding of various forms of Ras (loaded with GDP and mimics of GTP or nucleotide-free) at the allosteric and catalytic sites of Sos was comprehensively characterized by monitoring signal perturbations in the NMR spectra. The overall affinity of binding between these protein variants as well as their selected functional mutants was also investigated using intrinsic fluorescence. The data support a positive feedback activation of Sos by Ras·GTP with Ras·GTP binding as a substrate for the catalytic site of activated Sos more weakly than Ras·GDP, suggesting that Sos should actively promote unidirectional GDP → GTP exchange on Ras in preference of passive homonucleotide exchange. Ras·GDP weakly binds to the catalytic but not to the allosteric site of Sos. This confirms that Ras·GDP cannot properly activate Sos at the allosteric site. The novel site-specific assay described may be useful for design of drugs aimed at perturbing Ras-Sos interactions. PMID:26565026

Seeing the electroporative uptake of cell-membrane impermeable fluorescent molecules and nanoparticles

Science.gov (United States)

Kim, Kisoo; Kim, Jeong Ah; Lee, Soon-Geul; Lee, Won Gu

2012-07-01

This paper presents direct visualization of uptake directionality for cell-membrane impermeant fluorescent molecules and fluorescence-doped nanoparticles at a single-cell level during electroporation. To observe directly the uptake direction, we used microchannel-type electroporation that can generate a relatively symmetric and uniform electric field. For all the image frames during electroporation, fluorescence intensities that occurred at cell membranes in both uptake directions toward the electrodes have been sequentially recorded and quantitatively analyzed pixel by pixel. In our experiments, we found that fluorescent molecules, even not labeled to target biomolecules, had their own uptake direction with different intensities. It is also observed that the uptake intensity toward the cell membrane had a maximal value at a certain electric voltage, not at the highest value of voltages applied. The results also imply that the uptake direction of fluorescence-doped nanoparticles can be determined by a net surface charge of uptake materials and sizes in the electroporative environments. In summary, we performed a quantitative screening and direct visualization of uptake directionality for a set of fluorescent molecules and fluorescence-doped nanoparticles using electric-pulsation. Taking a closer look at the uptake direction of exogenous materials will help researchers to understand an unknown uptake phenomenon in which way foreign materials are inclined to move, and furthermore to design functional nanoparticles for electroporative gene delivery.This paper presents direct visualization of uptake directionality for cell-membrane impermeant fluorescent molecules and fluorescence-doped nanoparticles at a single-cell level during electroporation. To observe directly the uptake direction, we used microchannel-type electroporation that can generate a relatively symmetric and uniform electric field. For all the image frames during electroporation, fluorescence intensities

Nanodiamonds as multi-purpose labels for microscopy.

Science.gov (United States)

Hemelaar, S R; de Boer, P; Chipaux, M; Zuidema, W; Hamoh, T; Martinez, F Perona; Nagl, A; Hoogenboom, J P; Giepmans, B N G; Schirhagl, R

2017-04-07

Nanodiamonds containing fluorescent nitrogen-vacancy centers are increasingly attracting interest for use as a probe in biological microscopy. This interest stems from (i) strong resistance to photobleaching allowing prolonged fluorescence observation times; (ii) the possibility to excite fluorescence using a focused electron beam (cathodoluminescence; CL) for high-resolution localization; and (iii) the potential use for nanoscale sensing. For all these schemes, the development of versatile molecular labeling using relatively small diamonds is essential. Here, we show the direct targeting of a biological molecule with nanodiamonds as small as 70 nm using a streptavidin conjugation and standard antibody labelling approach. We also show internalization of 40 nm sized nanodiamonds. The fluorescence from the nanodiamonds survives osmium-fixation and plastic embedding making them suited for correlative light and electron microscopy. We show that CL can be observed from epon-embedded nanodiamonds, while surface-exposed nanoparticles also stand out in secondary electron (SE) signal due to the exceptionally high diamond SE yield. Finally, we demonstrate the magnetic read-out using fluorescence from diamonds prior to embedding. Thus, our results firmly establish nanodiamonds containing nitrogen-vacancy centers as unique, versatile probes for combining and correlating different types of microscopy, from fluorescence imaging and magnetometry to ultrastructural investigation using electron microscopy.

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.

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.

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.

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.

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.

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.

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.

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

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

Monitoring Ras Interactions with the Nucleotide Exchange Factor Son of Sevenless (Sos) Using Site-specific NMR Reporter Signals and Intrinsic Fluorescence.

Science.gov (United States)

Vo, Uybach; Vajpai, Navratna; Flavell, Liz; Bobby, Romel; Breeze, Alexander L; Embrey, Kevin J; Golovanov, Alexander P

2016-01-22

The activity of Ras is controlled by the interconversion between GTP- and GDP-bound forms partly regulated by the binding of the guanine nucleotide exchange factor Son of Sevenless (Sos). The details of Sos binding, leading to nucleotide exchange and subsequent dissociation of the complex, are not completely understood. Here, we used uniformly (15)N-labeled Ras as well as [(13)C]methyl-Met,Ile-labeled Sos for observing site-specific details of Ras-Sos interactions in solution. Binding of various forms of Ras (loaded with GDP and mimics of GTP or nucleotide-free) at the allosteric and catalytic sites of Sos was comprehensively characterized by monitoring signal perturbations in the NMR spectra. The overall affinity of binding between these protein variants as well as their selected functional mutants was also investigated using intrinsic fluorescence. The data support a positive feedback activation of Sos by Ras·GTP with Ras·GTP binding as a substrate for the catalytic site of activated Sos more weakly than Ras·GDP, suggesting that Sos should actively promote unidirectional GDP → GTP exchange on Ras in preference of passive homonucleotide exchange. Ras·GDP weakly binds to the catalytic but not to the allosteric site of Sos. This confirms that Ras·GDP cannot properly activate Sos at the allosteric site. The novel site-specific assay described may be useful for design of drugs aimed at perturbing Ras-Sos interactions. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

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.

Quantitative localization microscopy: effects of photophysics and labeling stoichiometry.

Directory of Open Access Journals (Sweden)

Robert P J Nieuwenhuizen

Full Text Available Quantification in localization microscopy with reversibly switchable fluorophores is severely hampered by the unknown number of switching cycles a fluorophore undergoes and the unknown stoichiometry of fluorophores on a marker such as an antibody. We overcome this problem by measuring the average number of localizations per fluorophore, or generally per fluorescently labeled site from the build-up of spatial image correlation during acquisition. To this end we employ a model for the interplay between the statistics of activation, bleaching, and labeling stoichiometry. We validated our method using single fluorophore labeled DNA oligomers and multiple-labeled neutravidin tetramers where we find a counting error of less than 17% without any calibration of transition rates. Furthermore, we demonstrated our quantification method on nanobody- and antibody-labeled biological specimens.

Labeling and stability of radiolabeled antibody fragments by a direct 99mTc-labeling method

International Nuclear Information System (INIS)

Pak, K.Y.; Nedelman, M.A.; Tam, S.H.; Wilson, E.; Daddona, P.E.

1992-01-01

The in vitro labeling and stability of 99m Tc-labeled antibody Fab' fragments prepared by a direct labeling technique were evaluated. Eight antibody fragments derived from murine IgG1 (N = 5), IgG2a (N = 2) and IgG3 (N = 1) isotypes were labeled with a preformed 99m Tc-D-glucarate complex. No loss of radioactivity incorporation was observed for all the 99m Tc-labeled antibody fragments after 24 h incubation at 37 o C. 99m Tc-labeled antibody fragments (IgG1, N = 2; IgG2a, n = 2; IgG3, N = 1) were stable upon challenge with DTPA, EDTA or acidic pH. Using the affinity chromatography technique, two of the 99m Tc-labeled antibody fragments displayed no loss of immunoreactivity after prolonged incubation in phosphate buffer up to 24 h at 37 o C. Bonding between 99m Tc and antibody fragments was elucidated by challenging with a diamide ditholate (N 2 S 2 ) compound. The Fab' with IgG2a isotype displayed tighter binding to 99m Tc in comparison to Fab' from IgG1 and IgG3 isotype in N 2 S 2 challenge and incubation with human plasma. The in vivo biodistribution of five 99m Tc-labeled fragments were evaluated in normal mice. (Author)

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.

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.

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

Affinity labeling and characterization of the active site histidine of glucosephosphate isomerase

International Nuclear Information System (INIS)

Gibson, D.R.; Gracy, R.W.; Hartman, F.C.

1980-01-01

N-bromoacetylethanolamine phosphate was found to act as a specific affinity label for the active center of glucosephosphate isomerase. The inactivation process followed pseudo-first order kinetics, was irreversible, and exhibited rate saturation kinetics with minimal half-lives of inactivation of 4.5 and 6.3 min for the enzyme isolated from human placenta and rabbit muscle, respectively. The pH dependence of the inactivation process closely paralleled the pH dependence of the overall catalytic process with pK/sub a/ values at pH 6.4 and 9.0. The stoichiometry of labeling of either enzyme, as determined with N-bromo[ 14 C 2 ]acetylethanolamine phosphate, was 1 eq of the affinity label/subunit of enzyme. After acid hydrolysis and amino acid analysis of the radioactive affinity-labeled human enzyme, only radioactive 3-carboxymethyl histidine was found. In the case of the rabbit enzyme, the only radioactive derivative obtained was 1-carboxymethyl histidine. Active site tryptic peptides were isolated by solvent extraction, thin layer peptide fingerprinting, and ion exchange chromatography before and after removal of the phosphate from the active site peptide. Amino acid analysis of the labeled peptides from the two species were very similar. Using high sensitivity methods for sequence analysis, the primary structure of the active site was established as Val-Leu-His-Ala-Glu-Asn-Val-Asp (Gly,Thr,Ser) Glu-Ile (Thr-Gly-His-Lys-Glx)-Tyr-Phe. Apparent sequence homology between the catalytic center of glucosephosphate isomerase and triosephosphate isomerase suggest that the two enzymes may have evolved from a common ancestral gene

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

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.

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.

A Conjugated Aptamer-Gold Nanoparticle Fluorescent Probe for Highly Sensitive Detection of rHuEPO-α

Directory of Open Access Journals (Sweden)

Zhaoyang Zhang

2011-11-01

Full Text Available We present here a novel conjugated aptamer-gold nanoparticle (Apt-AuNPs fluorescent probe and its application for specific detection of recombinant human erythropoietin-α (rHuEPO-α. In this nanobiosensor, 12 nm AuNPs function as both a nano-scaffold and a nano-quencher (fluorescent energy acceptor, on the surface of which the complementary sequences are linked (as cODN-AuNPs and pre-hybridized with carboxymethylfluorescein (FAM-labeled anti-rHuEPO-α aptamers. Upon target protein binding, the aptamers can be released from the AuNP surface and the fluorescence signal is restored. Key variables such as the length of linker, the hybridization site and length have been designed and optimized. Full performance evaluation including sensitivity, linear range and interference substances are also described. This nanobiosensor provides a promising approach for a simple and direct quantification of rHuEPO-α concentrations as low as 0.92 nM within a few hours.

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...

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.

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.

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).

Rhenium-186 direct labelling HIgG

International Nuclear Information System (INIS)

Lungu, V.; Mihailescu, G.; Dumitrescu, G.

2001-01-01

The aim of this study is to develop and improve existing radiolabelling techniques of peptides and monoclonal antibodies with 186 Re for achievement of potential agents for cancer targeted radiotherapy. There were selected methods and techniques for the direct labelling of intact HIgG by studding chemical and radiochemical processes of -S-S- bridges prereduction, reduction of 186 ReO 4 - and coupling reaction of rhenium with HIgG. The -S-S- bridges prereduction of HIgG to sulfhydryls was effected using different reducing agents: ascorbic acid, 2,3 dimercaptopropanol, cysteine, active hydrogen. The prereduction reactions are controlled by masic ratios of HIgG/reduction agent, pH, temperature and time of incubation. A pH=4.5 and a 24 hours incubation time are in the advantage of the prereduction yield. The labelling with 186 Re of prereduced HIgG with ascorbic acid or active hydrogen and 37 deg. C incubation in 22 hours releases 92% radiochemical purity. (author)

Covalent labeling of the beta-adrenergic ligand-binding site with para-(bromoacetamidyl)benzylcarazolol. A highly potent beta-adrenergic affinity label

International Nuclear Information System (INIS)

Dickinson, K.E.; Heald, S.L.; Jeffs, P.W.; Lefkowitz, R.J.; Caron, M.G.

1985-01-01

Para-(Bromoacetamidyl)benzylcarazolol (pBABC) was synthesized and found to be an extremely potent affinity label for beta-adrenergic receptors. Its interaction with mammalian (rabbit and hamster lung) and nonmammalian (turkey and frog erythrocyte) beta-adrenergic receptors was similar, displaying EC 50 values of 400-900 pM for inhibiting 125 I-cyanopindolol binding to these receptors. pBABC reduced the number of beta-adrenergic receptors in frog erythrocyte membranes, without any change in the affinity of the remaining sites for [ 125 I]iodocyanopindolol. pBABC has been radioiodinated. As assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, this affinity probe specifically labeled the beta-adrenergic peptide of a purified preparation of hamster lung, with high efficiency (approximately 40%) and with a pharmacological specificity characteristic of an interaction at the beta 2-adrenergic receptor ligand-binding site. Comparison of the proteolyzed products derived from purified receptor labeled with [ 125 I]pBABC and with the photoaffinity agent [ 125 I]p-azidobenzylcarazolol suggested that covalent labeling of the beta-adrenergic receptor by these probes occurs at similar domains of the beta-adrenergic receptor

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.

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.

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.

Human platelet vasopressin receptor identification by direct ultraviolet photoaffinity labeling

International Nuclear Information System (INIS)

Thibonnier, M.

1987-01-01

Tritiated vasopressin ([ 3 H]AVP) was directly crosslinked to its human platelet receptor by using an ultraviolet irradiation procedure. After preincubation with [ 3 H]AVP, the hydrodynamic parameters of the hormone-receptor complexes solubilized with 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate were derived from Sephacryl S-300 superfine gel filtration and from sucrose density gradient ultracentrifugation experiments. The following values were obtained: Stoke's radius = 5.48 +/- 0.1 nm, apparent sedimentation coefficient = 5.55 +/- 0.1 S, and calculated molecular weight = 132,000. On sodium dodecyl sulfate-8% polyacrylamide slab gel electrophoresis under reducing conditions, [ 3 H]AVP preferentially and specifically labeled a 125,000-dalton protein. The labeling of this protein was suppressed by addition of excess cold vasopressin, whereas angiotensin II did not inhibit incorporation of tritiated vasopressin in this protein. These results suggest that direct UV-photoaffinity labelling with [ 3 H]AVP is a suitable tool for the purification of the human platelet vasopressin receptor

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

Functionalization of embedded thiol-ene waveguides for evanescent wave induced fluorescence detection in a microfluidic device

DEFF Research Database (Denmark)

Feidenhans'l, Nikolaj Agentoft; Jensen, Thomas Glasdam; Lafleur, Josiane P.

2013-01-01

We demonstrate the use of functional surface groups inherently present on off-stoichiometric thiol−ene polymers, for site-specific immobilization of biomolecules and detection by evanescent wave-induced fluorescence. An optofluidic chip featuring an embedded thiol−ene waveguide was selectively...... functionalized with biotin using photografting. The biotin was used for immobilization of fluorescently labelled streptavidin, and experiments revealed a linear correlation between streptavidin concentration and fluorescent intensity. To further demonstrate the attractiveness of using thiol−ene for optofluidic...

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.

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

FRET-based localization of fluorescent protein insertions within the ryanodine receptor type 1.

Directory of Open Access Journals (Sweden)

Shweta A Raina

Full Text Available Fluorescent protein (FP insertions have often been used to localize primary structure elements in mid-resolution 3D cryo electron microscopic (EM maps of large protein complexes. However, little is known as to the precise spatial relationship between the location of the fused FP and its insertion site within a larger protein. To gain insights into these structural considerations, Förster resonance energy transfer (FRET measurements were used to localize green fluorescent protein (GFP insertions within the ryanodine receptor type 1 (RyR1, a large intracellular Ca(2+ release channel that plays a key role in skeletal muscle excitation contraction coupling. A series of full-length His-tagged GFP-RyR1 fusion constructs were created, expressed in human embryonic kidney (HEK-293T cells and then complexed with Cy3NTA, a His-tag specific FRET acceptor. FRET efficiency values measured from each GFP donor to Cy3NTA bound to each His tag acceptor site were converted into intermolecular distances and the positions of each inserted GFP were then triangulated relative to a previously published X-ray crystal structure of a 559 amino acid RyR1 fragment. We observed that the chromophoric centers of fluorescent proteins inserted into RyR1 can be located as far as 45 Å from their insertion sites and that the fused proteins can also be located in internal cavities within RyR1. These findings should prove useful in interpreting structural results obtained in cryo EM maps using fusions of small fluorescent proteins. More accurate point-to-point distance information may be obtained using complementary orthogonal labeling systems that rely on fluorescent probes that bind directly to amino acid side chains.

Murine leukemia virus (MLV replication monitored with fluorescent proteins

Directory of Open Access Journals (Sweden)

Bittner Alexandra

2004-12-01

Full Text Available Abstract Background Cancer gene therapy will benefit from vectors that are able to replicate in tumor tissue and cause a bystander effect. Replication-competent murine leukemia virus (MLV has been described to have potential as cancer therapeutics, however, MLV infection does not cause a cytopathic effect in the infected cell and viral replication can only be studied by immunostaining or measurement of reverse transcriptase activity. Results We inserted the coding sequences for green fluorescent protein (GFP into the proline-rich region (PRR of the ecotropic envelope protein (Env and were able to fluorescently label MLV. This allowed us to directly monitor viral replication and attachment to target cells by flow cytometry. We used this method to study viral replication of recombinant MLVs and split viral genomes, which were generated by replacement of the MLV env gene with the red fluorescent protein (RFP and separately cloning GFP-Env into a retroviral vector. Co-transfection of both plasmids into target cells resulted in the generation of semi-replicative vectors, and the two color labeling allowed to determine the distribution of the individual genomes in the target cells and was indicative for the occurrence of recombination events. Conclusions Fluorescently labeled MLVs are excellent tools for the study of factors that influence viral replication and can be used to optimize MLV-based replication-competent viruses or vectors for gene therapy.

Cross-Linked Fluorescent Supramolecular Nanoparticles as Finite Tattoo Pigments with Controllable Intradermal Retention Times.

Science.gov (United States)

Choi, Jin-Sil; Zhu, Yazhen; Li, Hongsheng; Peyda, Parham; Nguyen, Thuy Tien; Shen, Mo Yuan; Yang, Yang Michael; Zhu, Jingyi; Liu, Mei; Lee, Mandy M; Sun, Shih-Sheng; Yang, Yang; Yu, Hsiao-Hua; Chen, Kai; Chuang, Gary S; Tseng, Hsian-Rong

2017-01-24

Tattooing has been utilized by the medical community for precisely demarcating anatomic landmarks. This practice is especially important for identifying biopsy sites of nonmelanoma skin cancer (NMSC) due to the long interval (i.e., up to 3 months) between the initial diagnostic biopsy and surgical treatment. Commercially available tattoo pigments possess several issues, which include causing poor cosmesis, being mistaken for a melanocytic lesion, requiring additional removal procedures when no longer desired, and potentially inducing inflammatory responses. The ideal tattoo pigment for labeling of skin biopsy sites for NMSC requires (i) invisibility under ambient light, (ii) fluorescence under a selective light source, (iii) a finite intradermal retention time (ca. 3 months), and (iv) biocompatibility. Herein, we introduce cross-linked fluorescent supramolecular nanoparticles (c-FSNPs) as a "finite tattoo" pigment, with optimized photophysical properties and intradermal retention time to achieve successful in vivo finite tattooing. Fluorescent supramolecular nanoparticles encapsulate a fluorescent conjugated polymer, poly[5-methoxy-2-(3-sulfopropoxy)-1,4-phenylenevinylene] (MPS-PPV), into a core via a supramolecular synthetic approach. FSNPs which possess fluorescent properties superior to those of the free MPS-PPV are obtained through a combinatorial screening process. Covalent cross-linking of FSNPs results in micrometer-sized c-FSNPs, which exhibit a size-dependent intradermal retention. The 1456 nm sized c-FSNPs display an ideal intradermal retention time (ca. 3 months) for NMSC lesion labeling, as observed in an in vivo tattoo study. In addition, the c-FSNPs induce undetectable inflammatory responses after tattooing. We believe that the c-FSNPs can serve as a "finite tattoo" pigment to label potential malignant NMSC lesions.

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.

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

Photoaffinity labeling with cholesterol analogues precisely maps a cholesterol-binding site in voltage-dependent anion channel-1.

Science.gov (United States)

Budelier, Melissa M; Cheng, Wayland W L; Bergdoll, Lucie; Chen, Zi-Wei; Janetka, James W; Abramson, Jeff; Krishnan, Kathiresan; Mydock-McGrane, Laurel; Covey, Douglas F; Whitelegge, Julian P; Evers, Alex S

2017-06-02

Voltage-dependent anion channel-1 (VDAC1) is a highly regulated β-barrel membrane protein that mediates transport of ions and metabolites between the mitochondria and cytosol of the cell. VDAC1 co-purifies with cholesterol and is functionally regulated by cholesterol, among other endogenous lipids. Molecular modeling studies based on NMR observations have suggested five cholesterol-binding sites in VDAC1, but direct experimental evidence for these sites is lacking. Here, to determine the sites of cholesterol binding, we photolabeled purified mouse VDAC1 (mVDAC1) with photoactivatable cholesterol analogues and analyzed the photolabeled sites with both top-down mass spectrometry (MS), and bottom-up MS paired with a clickable, stable isotope-labeled tag, FLI -tag. Using cholesterol analogues with a diazirine in either the 7 position of the steroid ring (LKM38) or the aliphatic tail (KK174), we mapped a binding pocket in mVDAC1 localized to Thr 83 and Glu 73 , respectively. When Glu 73 was mutated to a glutamine, KK174 no longer photolabeled this residue, but instead labeled the nearby Tyr 62 within this same binding pocket. The combination of analytical strategies employed in this work permits detailed molecular mapping of a cholesterol-binding site in a protein, including an orientation of the sterol within the site. Our work raises the interesting possibility that cholesterol-mediated regulation of VDAC1 may be facilitated through a specific binding site at the functionally important Glu 73 residue. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

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.

Click strategies for single-molecule protein fluorescence.

Science.gov (United States)

Milles, Sigrid; Tyagi, Swati; Banterle, Niccolò; Koehler, Christine; VanDelinder, Virginia; Plass, Tilman; Neal, Adrian P; Lemke, Edward A

2012-03-21

Single-molecule methods have matured into central tools for studies in biology. Foerster resonance energy transfer (FRET) techniques, in particular, have been widely applied to study biomolecular structure and dynamics. The major bottleneck for a facile and general application of these studies arises from the need to label biological samples site-specifically with suitable fluorescent dyes. In this work, we present an optimized strategy combining click chemistry and the genetic encoding of unnatural amino acids (UAAs) to overcome this limitation for proteins. We performed a systematic study with a variety of clickable UAAs and explored their potential for high-resolution single-molecule FRET (smFRET). We determined all parameters that are essential for successful single-molecule studies, such as accessibility of the probes, expression yield of proteins, and quantitative labeling. Our multiparameter fluorescence analysis allowed us to gain new insights into the effects and photophysical properties of fluorescent dyes linked to various UAAs for smFRET measurements. This led us to determine that, from the extended tool set that we now present, genetically encoding propargyllysine has major advantages for state-of-the-art measurements compared to other UAAs. Using this optimized system, we present a biocompatible one-step dual-labeling strategy of the regulatory protein RanBP3 with full labeling position freedom. Our technique allowed us then to determine that the region encompassing two FxFG repeat sequences adopts a disordered but collapsed state. RanBP3 serves here as a prototypical protein that, due to its multiple cysteines, size, and partially disordered structure, is not readily accessible to any of the typical structure determination techniques such as smFRET, NMR, and X-ray crystallography.

Application of Fluorescence In Situ Hybridization (FISH) Technique for the Detection of Genetic Aberration in Medical Science

Science.gov (United States)

Ratan, Zubair Ahmed; Zaman, Sojib Bin; Haidere, Mohammad Faisal; Runa, Nusrat Jahan; Akter, Nasrin

2017-01-01

Fluorescence in situ hybridization (FISH) is a macromolecule recognition technique, which is considered as a new advent in the field of cytology. Initially, it was developed as a physical mapping tool to delineate genes within chromosomes. The accuracy and versatility of FISH were subsequently capitalized upon in biological and medical research. This visually appealing technique provides an intermediate degree of resolution between DNA analysis and chromosomal investigations. FISH consists of a hybridizing DNA probe, which can be labeled directly or indirectly. In the case of direct labeling, fluorescent nucleotides are used, while indirect labeling is incorporated with reporter molecules that are subsequently detected by fluorescent antibodies or other affinity molecules. FISH is applied to detect genetic abnormalities that include different characteristic gene fusions or the presence of an abnormal number of chromosomes in a cell or loss of a chromosomal region or a whole chromosome. It is also applied in different research applications, such as gene mapping or the identification of novel oncogenes. This article reviews the concept of FISH, its application, and its advantages in medical science.  PMID:28690958

Site directed recombination

Science.gov (United States)

Jurka, Jerzy W.

1997-01-01

Enhanced homologous recombination is obtained by employing a consensus sequence which has been found to be associated with integration of repeat sequences, such as Alu and ID. The consensus sequence or sequence having a single transition mutation determines one site of a double break which allows for high efficiency of integration at the site. By introducing single or double stranded DNA having the consensus sequence flanking region joined to a sequence of interest, one can reproducibly direct integration of the sequence of interest at one or a limited number of sites. In this way, specific sites can be identified and homologous recombination achieved at the site by employing a second flanking sequence associated with a sequence proximal to the 3'-nick.

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

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.

Fluorescent Biosensors Based on Single-Molecule Counting.

Science.gov (United States)

Ma, Fei; Li, Ying; Tang, Bo; Zhang, Chun-Yang

2016-09-20

fluorescence signals by specific in vitro/in vivo fluorescent labeling, and consequently, the fluorescent molecules indicate the presence of target molecules. The resultant fluorescence signals may be simply counted by either microfluidic device-integrated confocal microscopy or total internal reflection fluorescence-based single-molecule imaging. We have developed a series of single-molecule counting-based biosensors which can be classified as separation-free and separation-assisted assays. As a proof-of-concept, we demonstrate the applications of single-molecule counting-based biosensors for sensitive detection of various target biomolecules such as DNAs, miRNAs, proteins, enzymes, and intact cells, which may function as the disease-related biomarkers. Moreover, we give a summary of future directions to expand the usability of single-molecule counting-based biosensors including (1) the development of more user-friendly and automated instruments, (2) the discovery of new fluorescent labels and labeling strategies, and (3) the introduction of new concepts for the design of novel biosensors. Due to their high sensitivity, good selectivity, rapidity, and simplicity, we believe that the single-molecule counting-based fluorescent biosensors will indubitably find wide applications in biological research, clinical diagnostics, and drug discovery.

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.

Direct labelling of monomeric antibody fragments Fab' with 99mTc

International Nuclear Information System (INIS)

Li Jun; Wang Shizhen; Yang Ziyi

1994-01-01

Direct labelling method and conditions of monomeric antibody Fab' with 99m Tc were investigated. Polyclonal antibody IgG was digested with ficin to produce dimeric fragments F(ab') 2 , which was subsequently reduced to monomeric fragments Fab' with 2-mercaptoethylamine. Finally, Fab' was incubated with sodium gluconate (Sn(II)) kit solution and 99m TcO 4 - eluted at room temperature to form 99m Tc-Fab'. The labelling efficiency was 85%-95%. The stability of labelled products was satisfactory and the elimination rate was faster than 99m Tc-IgG

Identification of a trypsin-like site associated with acetylcholinesterase by affinity labelling with (/sup 3/H)diisopropyl fluorophosphate

Energy Technology Data Exchange (ETDEWEB)

Small, D.H.; Chubb, I.W.

1988-07-01

In addition to its ability to hydrolyze acetylcholine, purified eel acetylcholinesterase possesses a trypsin-like endopeptidase activity. The tryptic activity is associated with a serine residue at a site that is distinct from the esteratic site. To label both the esteratic and tryptic sites, the enzyme was incubated with the serine hydrolase inhibitor (/sup 3/H)diisopropyl fluorophosphate. This compound labelled the protein in a biphasic manner, with both slow and rapid labelling kinetics. The time course of the rapid phase was similar to the time course of inactivation of the esteratic activity. The time course of the slow phase was similar to the time course of inactivation of the tryptic activity. Labelling of the nonesteratic site was inhibited by the trypsin inhibitor N alpha-p-tosyl-L-lysine chloromethyl ketone. The total number of sites labelled by (/sup 3/H)diisopropyl fluorophosphate on eel acetylcholinesterase was 2.6 mol/280,000 g protein, whereas the number of tryptic sites was less (0.52 mol/280,000 g). The results suggest that a subpopulation of acetylcholinesterase molecules may possess tryptic activity. Extensive chromatography of the purified enzyme by ion-exchange and gel filtration failed to separate the labelled tryptic component from acetylcholinesterase. On sodium dodecyl sulfate-polyacrylamide gels, the labelled tryptic component comigrated with a polypeptide of 50,000 molecular weight, which is a major proteolytic digestion product derived from the intact acetylcholinesterase monomer. Because of its localization in many noncholinergic peptide-containing cells, acetylcholinesterase could act as a neuropeptide processing enzyme in these cells.

Diagnosis of canine rabies by the direct fluorescent antibody ...

African Journals Online (AJOL)

Diagnosis of canine rabies by the direct fluorescent antibody technique in Plateau State, Nigeria. DO Ehizibolo, EA Ogunsan, MJ Muhammad, CI Nwosuh, S Olaleye, OOC Chuckwu, MY Sugun, NM Sati, NE Waziri, OK Egwu, J Kamani, CA Meseko, SE Idachaba, GI Dogo ...

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

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.

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

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.

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

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.

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.

Membrane docking geometry of GRP1 PH domain bound to a target lipid bilayer: an EPR site-directed spin-labeling and relaxation study.

Directory of Open Access Journals (Sweden)

Huai-Chun Chen

Full Text Available The second messenger lipid PIP(3 (phosphatidylinositol-3,4,5-trisphosphate is generated by the lipid kinase PI3K (phosphoinositide-3-kinase in the inner leaflet of the plasma membrane, where it regulates a broad array of cell processes by recruiting multiple signaling proteins containing PIP(3-specific pleckstrin homology (PH domains to the membrane surface. Despite the broad importance of PIP(3-specific PH domains, the membrane docking geometry of a PH domain bound to its target PIP(3 lipid on a bilayer surface has not yet been experimentally determined. The present study employs EPR site-directed spin labeling and relaxation methods to elucidate the membrane docking geometry of GRP1 PH domain bound to bilayer-embedded PIP(3. The model target bilayer contains the neutral background lipid PC and both essential targeting lipids: (i PIP(3 target lipid that provides specificity and affinity, and (ii PS facilitator lipid that enhances the PIP(3 on-rate via an electrostatic search mechanism. The EPR approach measures membrane depth parameters for 18 function-retaining spin labels coupled to the PH domain, and for calibration spin labels coupled to phospholipids. The resulting depth parameters, together with the known high resolution structure of the co-complex between GRP1 PH domain and the PIP(3 headgroup, provide sufficient constraints to define an optimized, self-consistent membrane docking geometry. In this optimized geometry the PH domain engulfs the PIP(3 headgroup with minimal bilayer penetration, yielding the shallowest membrane position yet described for a lipid binding domain. This binding interaction displaces the PIP(3 headgroup from its lowest energy position and orientation in the bilayer, but the headgroup remains within its energetically accessible depth and angular ranges. Finally, the optimized docking geometry explains previous biophysical findings including mutations observed to disrupt membrane binding, and the rapid lateral

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.

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.

Electricity savings in Latvia through implementation of labelling directives. Final report

Energy Technology Data Exchange (ETDEWEB)

NONE

2002-01-01

Legislation on labelling of household appliances was enforced in Latvia July 2001 to implement the European labelling directives. The project 'Electricity Savings in Latvia through Implementation of labelling Directives' was started January 2001 in order to support the implementation of the directives to ensure that they got an immediate effect. The project has included the following activities: Information of the general public and of traders and importers. In relation to the general public the activities consisted of production of a widely spread information brochure, TV-spots and a TV competition and participation in a school project. Seminars and meetings with traders and importers were held to inform about the legislation before it was enforced and to create a network for discussion of practical implementation problems and planning of the information activities. Test and control activities. The purpose of the test and control activities was to strengthen the test and control capacities and to transfer experience from Denmark. This was achieved though workshops for as well inspectors from the Consumer Rights Protection Centre and for members of the voluntary Consumer Protection Clubs. Results: It is assumed that the project has been very successful in the sense that the information about labelling of appliances has been efficiently spread via the information brochure, which was printed in 100.000 copies and distributed in shops, via Latvenergo and the consumer clubs. Furthermore, a cartoon about 'Energy-eaters' produced as part of the project by a very talented Latvian film study (Environmental Film Studio) has been used as TV-spot and has been broadcasted 40 times, including in connection to a morning TV-competition on labelling of appliances. The participatory approach used in the project has resulted in a very dedicated participation from the different stake holders side. Thus there is basis for sustainability in the sense, that a network

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.

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...

Improved localization of cellular membrane receptors using combined fluorescence microscopy and simultaneous topography and recognition imaging

International Nuclear Information System (INIS)

Duman, M; Pfleger, M; Chtcheglova, L A; Neundlinger, I; Bozna, B L; Ebner, A; Schuetz, G J; Hinterdorfer, P; Zhu, R; Mayer, B; Rankl, C; Moertelmaier, M; Kada, G; Kienberger, F; Salio, M; Shepherd, D; Polzella, P; Cerundolo, V; Dieudonne, M

2010-01-01

The combination of fluorescence microscopy and atomic force microscopy has a great potential in single-molecule-detection applications, overcoming many of the limitations coming from each individual technique. Here we present a new platform of combined fluorescence and simultaneous topography and recognition imaging (TREC) for improved localization of cellular receptors. Green fluorescent protein (GFP) labeled human sodium-glucose cotransporter (hSGLT1) expressed Chinese Hamster Ovary (CHO) cells and endothelial cells (MyEnd) from mouse myocardium stained with phalloidin-rhodamine were used as cell systems to study AFM topography and fluorescence microscopy on the same surface area. Topographical AFM images revealed membrane features such as lamellipodia, cytoskeleton fibers, F-actin filaments and small globular structures with heights ranging from 20 to 30 nm. Combined fluorescence and TREC imaging was applied to detect density, distribution and localization of YFP-labeled CD1d molecules on α-galactosylceramide (αGalCer)-loaded THP1 cells. While the expression level, distribution and localization of CD1d molecules on THP1 cells were detected with fluorescence microscopy, the nanoscale distribution of binding sites was investigated with molecular recognition imaging by using a chemically modified AFM tip. Using TREC on the inverted light microscope, the recognition sites of cell receptors were detected in recognition images with domain sizes ranging from ∼ 25 to ∼ 160 nm, with the smaller domains corresponding to a single CD1d molecule.

Improved localization of cellular membrane receptors using combined fluorescence microscopy and simultaneous topography and recognition imaging

Energy Technology Data Exchange (ETDEWEB)

Duman, M; Pfleger, M; Chtcheglova, L A; Neundlinger, I; Bozna, B L; Ebner, A; Schuetz, G J; Hinterdorfer, P [Institute for Biophysics, University of Linz, Altenbergerstrasse 69, A-4040 Linz (Austria); Zhu, R; Mayer, B [Christian Doppler Laboratory for Nanoscopic Methods in Biophysics, Institute for Biophysics, University of Linz, Altenbergerstrasse 69, A-4040 Linz (Austria); Rankl, C; Moertelmaier, M; Kada, G; Kienberger, F [Agilent Technologies Austria GmbH, Aubrunnerweg 11, A-4040 Linz (Austria); Salio, M; Shepherd, D; Polzella, P; Cerundolo, V [Cancer Research UK Tumor Immunology Group, Weatherall Institute of Molecular Medicine, Nuffield Department of Medicine, University of Oxford, Oxford OX3 9DS (United Kingdom); Dieudonne, M, E-mail: ferry_kienberger@agilent.com [Agilent Technologies Belgium, Wingepark 51, Rotselaar, AN B-3110 (Belgium)

2010-03-19

The combination of fluorescence microscopy and atomic force microscopy has a great potential in single-molecule-detection applications, overcoming many of the limitations coming from each individual technique. Here we present a new platform of combined fluorescence and simultaneous topography and recognition imaging (TREC) for improved localization of cellular receptors. Green fluorescent protein (GFP) labeled human sodium-glucose cotransporter (hSGLT1) expressed Chinese Hamster Ovary (CHO) cells and endothelial cells (MyEnd) from mouse myocardium stained with phalloidin-rhodamine were used as cell systems to study AFM topography and fluorescence microscopy on the same surface area. Topographical AFM images revealed membrane features such as lamellipodia, cytoskeleton fibers, F-actin filaments and small globular structures with heights ranging from 20 to 30 nm. Combined fluorescence and TREC imaging was applied to detect density, distribution and localization of YFP-labeled CD1d molecules on {alpha}-galactosylceramide ({alpha}GalCer)-loaded THP1 cells. While the expression level, distribution and localization of CD1d molecules on THP1 cells were detected with fluorescence microscopy, the nanoscale distribution of binding sites was investigated with molecular recognition imaging by using a chemically modified AFM tip. Using TREC on the inverted light microscope, the recognition sites of cell receptors were detected in recognition images with domain sizes ranging from {approx} 25 to {approx} 160 nm, with the smaller domains corresponding to a single CD1d molecule.

DIRECT VISUALIZATION OF THE DOPAMINE TRANSPORTER IN CULTURED NEWBORN RAT MIDBRAIN NEURONS USING THE FLUORESCENT COCAINE ANALOGUE JHC 1-64

DEFF Research Database (Denmark)

Rasmussen, Søren; Vægter, Christian Bjerggaard; Cha, J

In this study we have established methods for visualization and tracking of the dopamine transporter (DAT) in cultured dopaminergic neurons in real time using a fluorescent cocaine analogue JHC 1-64 and confocal fluorescence microscopy. The initial binding experiments in HEK 293 cells stably...... 1-64 was prevented by excess concentrations of dopamine, cocaine, mazindol, or RTI-55, whereas the norepinephrine and/or serotonin transporter specific inhibitors desmethylimipramine and citalopram did not affect fluorescent labeling of the neurons. This strongly supports that JHC 1-64 specifically...

The Near-Term Viability and Benefits of eLabels for Patients, Clinical Sites, and Sponsors.

Science.gov (United States)

Smith-Gick, Jodi; Barnes, Nicola; Barone, Rocco; Bedford, Jeff; James, Jason R; Reisner, Stacy Frankovitz; Stephenson, Michael

2018-01-01

Current clinical trial labels are designed primarily to meet regulatory requirements. These labels have low patient and site utility, few are opened, and they have limited space and small fonts. As our world transitions from paper to electronic, an opportunity exists to provide patients with information about their investigational clinical trial product in a way that is more easily accessible, meets Health Authority requirements, and provides valuable additional information for the patient and caregiver. A TransCelerate initiative was launched to understand the current regulatory and technology landscape for the potential use an electronic label (eLabel) for investigational medicinal products (IMPs). Concepts and an example proof of concept were developed intended to show the "art of the possible" for a foundational eLabel and a "universal printed label." In addition, possible patient-centric enhancements were captured in the eLabel proof of concept. These concepts were shared with Health Authorities as well as patient and site advisory groups to gather feedback and subsequently enhance the concepts. Feedback indicated that the concept of an eLabel provides value and concepts should continue to be pursued. While the Health Authorities engaged with did not express issues with the use of an eLabel per se, the reduction in the content on the paper label is not possible in some geographic locations due to existing regulations. There is nothing that prevents transmitting the label electronically in conjunction with current conventional labeling. While there are still some regulatory barriers that need to be addressed for reducing what is on the paper label, advancement toward a more patient-centric approach benefits stakeholders and will enable a fully connected patient-centric experience. The industry must start now to build the foundation.

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.

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...

Completion of proteomic data sets by Kd measurement using cell-free synthesis of site-specifically labeled proteins.

Directory of Open Access Journals (Sweden)

Paul Majkut

Full Text Available The characterization of phosphotyrosine mediated protein-protein interactions is vital for the interpretation of downstream pathways of transmembrane signaling processes. Currently however, there is a gap between the initial identification and characterization of cellular binding events by proteomic methods and the in vitro generation of quantitative binding information in the form of equilibrium rate constants (Kd values. In this work we present a systematic, accelerated and simplified approach to fill this gap: using cell-free protein synthesis with site-specific labeling for pull-down and microscale thermophoresis (MST we were able to validate interactions and to establish a binding hierarchy based on Kd values as a completion of existing proteomic data sets. As a model system we analyzed SH2-mediated interactions of the human T-cell phosphoprotein ADAP. Putative SH2 domain-containing binding partners were synthesized from a cDNA library using Expression-PCR with site-specific biotinylation in order to analyze their interaction with fluorescently labeled and in vitro phosphorylated ADAP by pull-down. On the basis of the pull-down results, selected SH2's were subjected to MST to determine Kd values. In particular, we could identify an unexpectedly strong binding of ADAP to the previously found binding partner Rasa1 of about 100 nM, while no evidence of interaction was found for the also predicted SH2D1A. Moreover, Kd values between ADAP and its known binding partners SLP-76 and Fyn were determined. Next to expanding data on ADAP suggesting promising candidates for further analysis in vivo, this work marks the first Kd values for phosphotyrosine/SH2 interactions on a phosphoprotein level.

Hydrophilic interaction liquid chromatography of anthranilic acid-labelled oligosaccharides with a 4-aminobenzoic acid ethyl ester-labelled dextran hydrolysate internal standard.

Science.gov (United States)

Neville, David C A; Alonzi, Dominic S; Butters, Terry D

2012-04-13

Hydrophilic interaction liquid chromatography (HILIC) of fluorescently labelled oligosaccharides is used in many laboratories to analyse complex oligosaccharide mixtures. Separations are routinely performed using a TSK gel-Amide 80 HPLC column, and retention times of different oligosaccharide species are converted to glucose unit (GU) values that are determined with reference to an external standard. However, if retention times were to be compared with an internal standard, consistent and more accurate GU values would be obtained. We present a method to perform internal standard-calibrated HILIC of fluorescently labelled oligosaccharides. The method relies on co-injection of 4-aminobenzoic acid ethyl ester (4-ABEE)-labelled internal standard and detection by UV absorption, with 2-AA (2-aminobenzoic acid)-labelled oligosaccharides. 4-ABEE is a UV chromophore and a fluorophore, but there is no overlap of the fluorescent spectrum of 4-ABEE with the commonly used fluorescent reagents. The dual nature of 4-ABEE allows for accurate calculation of the delay between UV and fluorescent signals when determining the GU values of individual oligosaccharides. The GU values obtained are inherently more accurate as slight differences in gradients that can influence retention are negated by use of an internal standard. Therefore, this paper provides the first method for determination of HPLC-derived GU values of fluorescently labelled oligosaccharides using an internal calibrant. Copyright © 2012 Elsevier B.V. All rights reserved.

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.

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.

A low-cost method for visible fluorescence imaging.

Science.gov (United States)

Tarver, Crissy L; Pusey, Marc

2017-12-01

A wide variety of crystallization solutions are screened to establish conditions that promote the growth of a diffraction-quality crystal. Screening these conditions requires the assessment of many crystallization plates for the presence of crystals. Automated systems for screening and imaging are very expensive. A simple approach to imaging trace fluorescently labeled protein crystals in crystallization plates has been devised, and can be implemented at a cost as low as $50. The proteins β-lactoglobulin B, trypsin and purified concanavalin A (ConA) were trace fluorescently labeled using three different fluorescent probes: Cascade Yellow (CY), Carboxyrhodamine 6G (CR) and Pacific Blue (PB). A crystallization screening plate was set up using β-lactoglobulin B labeled with CR, trypsin labeled with CY, ConA labeled with each probe, and a mixture consisting of 50% PB-labeled ConA and 50% CR-labeled ConA. The wells of these plates were imaged using a commercially available macro-imaging lens attachment for smart devices that have a camera. Several types of macro lens attachments were tested with smartphones and tablets. Images with the highest quality were obtained with an iPhone 6S and an AUKEY Ora 10× macro lens. Depending upon the fluorescent probe employed and its Stokes shift, a light-emitting diode or a laser diode was used for excitation. An emission filter was used for the imaging of protein crystals labeled with CR and crystals with two-color fluorescence. This approach can also be used with microscopy systems commonly used to observe crystallization plates.

40 CFR 170.232 - Knowledge of labeling and site-specific information.

Science.gov (United States)

2010-07-01

... 40 Protection of Environment 23 2010-07-01 2010-07-01 false Knowledge of labeling and site-specific information. 170.232 Section 170.232 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) PESTICIDE PROGRAMS WORKER PROTECTION STANDARD Standard for Pesticide Handlers § 170.232 Knowledge...

"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.

Direct analysis of biological samples by total reflection X-ray fluorescence

International Nuclear Information System (INIS)

Lue M, Marco P.; Hernandez-Caraballo, Edwin A.

2004-01-01

The technique of total reflection X-ray fluorescence (TXRF) is well suited for the direct analysis of biological samples due to the low matrix interferences and simultaneous multi-element nature. Nevertheless, biological organic samples are frequently analysed after digestion procedures. The direct determination of analytes requires shorter analysis time, low reactive consumption and simplifies the whole analysis process. On the other hand, the biological/clinical samples are often available in minimal amounts and routine studies require the analysis of large number of samples. To overcome the difficulties associated with the analysis of organic samples, particularly of solid ones, different procedures of sample preparation and calibration to approach the direct analysis have been evaluated: (1) slurry sampling, (2) Compton peak standardization, (3) in situ microwave digestion, (4) in situ chemical modification and (5) direct analysis with internal standardization. Examples of analytical methods developed by our research group are discussed. Some of them have not been previously published, illustrating alternative strategies for coping with various problems that may be encountered in the direct analysis by total reflection X-ray fluorescence spectrometry

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.

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.

Effects of organizational scheme and labeling on task performance in product-centered and user-centered retail Web sites.

Science.gov (United States)

Resnick, Marc L; Sanchez, Julian

2004-01-01

As companies increase the quantity of information they provide through their Web sites, it is critical that content is structured with an appropriate architecture. However, resource constraints often limit the ability of companies to apply all Web design principles completely. This study quantifies the effects of two major information architecture principles in a controlled study that isolates the incremental effects of organizational scheme and labeling on user performance and satisfaction. Sixty participants with a wide range of Internet and on-line shopping experience were recruited to complete a series of shopping tasks on a prototype retail shopping Web site. User-centered labels provided a significant benefit in performance and satisfaction over labels obtained through company-centered methods. User-centered organization did not result in improved performance except when the label quality was poor. Significant interactions suggest specific guidelines for allocating resources in Web site design. Applications of this research include the design of Web sites for any commercial application, particularly E-commerce.

Fluorescence In Situ Hybridization with Peptide Nucleic Acid Probes for Rapid Identification of Candida albicans Directly from Blood Culture Bottles

Science.gov (United States)

Rigby, Susan; Procop, Gary W.; Haase, Gerhard; Wilson, Deborah; Hall, Geraldine; Kurtzman, Cletus; Oliveira, Kenneth; Von Oy, Sabina; Hyldig-Nielsen, Jens J.; Coull, James; Stender, Henrik

2002-01-01

A new fluorescence in situ hybridization (FISH) method that uses peptide nucleic acid (PNA) probes for identification of Candida albicans directly from positive-blood-culture bottles in which yeast was observed by Gram staining (herein referred to as yeast-positive blood culture bottles) is described. The test (the C. albicans PNA FISH method) is based on a fluorescein-labeled PNA probe that targets C. albicans 26S rRNA. The PNA probe is added to smears made directly from the contents of the blood culture bottle and hybridized for 90 min at 55°C. Unhybridized PNA probe is removed by washing of the mixture (30 min), and the smears are examined by fluorescence microscopy. The specificity of the method was confirmed with 23 reference strains representing phylogenetically related yeast species and 148 clinical isolates covering the clinically most significant yeast species, including C. albicans (n = 72), C. dubliniensis (n = 58), C. glabrata (n = 5), C. krusei (n = 2), C. parapsilosis (n = 4), and C. tropicalis (n = 3). The performance of the C. albicans PNA FISH method as a diagnostic test was evaluated with 33 routine and 25 simulated yeast-positive blood culture bottles and showed 100% sensitivity and 100% specificity. It is concluded that this 2.5-h method for the definitive identification of C. albicans directly from yeast-positive blood culture bottles provides important information for optimal antifungal therapy and patient management. PMID:12037084

Direct and indirect radioiodination of protein: comparative study of chemotactic peptide labeling

International Nuclear Information System (INIS)

Lavinas, Tatiana

2004-01-01

The development of simple methods for protein radioiodination have stimulated the use of radioiodinated peptides in vivo. There are two basic methods for labeling proteins with radioiodine: direct labeling, reaction of an electrophilic radioiodine with functional activated groups on protein, like the phenol ring in the tyrosine residue, and the conjugation of a previously radioiodinated molecule to the protein, referred as indirect method. The great problem related to the direct radioiodination of proteins is the in vivo dehalogenation. This problem can be minimized if a non-phenolic prosthetic group is used in the indirect radioiodination of the peptide. The ATE prosthetic group, N-succinimidyl 3-(tri-n-butylstannyl) benzoate, when radioiodinated by electrophilic iododestannilation produces N-succinimidyl 3-[ 123 l/ 131 l] iodine benzoate (SIB) that is subsequently conjugated to the protein by the acylation of the lysine group. There are many radiopharmaceuticals employed in scintigraphic images of infection and inflammation used with some limitations. These limitations stimulated the improvement of a new class of radiopharmaceuticals, the receptor-specific related labeled peptides, as the mediators of the inflammatory response, that presents high affinity by receptors expressed in the inflammation process, and fast clearance from blood and non-target tissues. One of these molecules is the synthetic chemotactic peptide fNleLFNIeYK that presents potent chemotaxis for leukocytes, with high affinity by the receptors presented in polymorphonuclear leukocytes and mononuclear phagocytes. The objective of this work included the synthesis of ATE prosthetic group and comparative radioiodination of the chemotactic peptide fNleLFNIeYK by direct and indirect methods, with radiochemical purity determination and evaluation of in vivo and in vitro stability of the compounds. This work presented an original contribution in the comparative biological distribution studies of the

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

Site-specific multipoint fluorescence measurement system with end-capped optical fibers.

Science.gov (United States)

Song, Woosub; Moon, Sucbei; Lee, Byoung-Cheol; Park, Chul-Seung; Kim, Dug Young; Kwon, Hyuk Sang

2011-07-10

We present the development and implementation of a spatially and spectrally resolved multipoint fluorescence correlation spectroscopy (FCS) system utilizing multiple end-capped optical fibers and an inexpensive laser source. Specially prepared end-capped optical fibers placed in an image plane were used to both collect fluorescence signals from the sample and to deliver signals to the detectors. The placement of independently selected optical fibers on the image plane was done by monitoring the end-capped fiber tips at the focus using a CCD, and fluorescence from specific positions of a sample were collected by an end-capped fiber, which could accurately represent light intensities or spectral data without incurring any disturbance. A fast multipoint spectroscopy system with a time resolution of ∼1.5 ms was then implemented using a prism and an electron multiplying charge coupled device with a pixel binning for the region of interest. The accuracy of our proposed system was subsequently confirmed by experimental results, based on an FCS analysis of microspheres in distilled water. We expect that the proposed multipoint site-specific fluorescence measurement system can be used as an inexpensive fluorescence measurement tool to study many intracellular and molecular dynamics in cell biology. © 2011 Optical Society of America

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...

A novel fluorescent retrograde neural tracer: cholera toxin B conjugated carbon dots

Science.gov (United States)

Zhou, Nan; Hao, Zeyu; Zhao, Xiaohuan; Maharjan, Suraj; Zhu, Shoujun; Song, Yubin; Yang, Bai; Lu, Laijin

2015-09-01

The retrograde neuroanatomical tracing method is a key technique to study the complex interconnections of the nervous system. Traditional tracers have several drawbacks, including time-consuming immunohistochemical or immunofluorescent staining procedures, rapid fluorescence quenching and low fluorescence intensity. Carbon dots (CDs) have been widely used as a fluorescent bio-probe due to their ultrasmall size, excellent optical properties, chemical stability, biocompatibility and low toxicity. Herein, we develop a novel fluorescent neural tracer: cholera toxin B-carbon dot conjugates (CTB-CDs). It can be taken up and retrogradely transported by neurons in the peripheral nervous system of rats. Our results show that CTB-CDs possess high photoluminescence intensity, good optical stability, a long shelf-life and non-toxicity. Tracing with CTB-CDs is a direct and more economical way of performing retrograde labelling experiments. Therefore, CTB-CDs are reliable fluorescent retrograde tracers.The retrograde neuroanatomical tracing method is a key technique to study the complex interconnections of the nervous system. Traditional tracers have several drawbacks, including time-consuming immunohistochemical or immunofluorescent staining procedures, rapid fluorescence quenching and low fluorescence intensity. Carbon dots (CDs) have been widely used as a fluorescent bio-probe due to their ultrasmall size, excellent optical properties, chemical stability, biocompatibility and low toxicity. Herein, we develop a novel fluorescent neural tracer: cholera toxin B-carbon dot conjugates (CTB-CDs). It can be taken up and retrogradely transported by neurons in the peripheral nervous system of rats. Our results show that CTB-CDs possess high photoluminescence intensity, good optical stability, a long shelf-life and non-toxicity. Tracing with CTB-CDs is a direct and more economical way of performing retrograde labelling experiments. Therefore, CTB-CDs are reliable fluorescent retrograde

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

Preparation of a pure 99mTc-F(ab')2 radioimmunoconjugate by direct labeling methods

International Nuclear Information System (INIS)

Griffiths, G.L.; Jones, A.L.; Hansen, H.J.; Goldenberg, D.M.

1994-01-01

Intact IgG and Fab' can be labeled directly with 99m Tc to give quantitative incorporation of radioactivity into the protein. With F(ab') 2 the reductive conditions yield a mixture of 99m Tc-F(ab') 2 and 99m Tc-Fab'. We now report a direct labeling method to produce only 99m Tc-F(ab') 2 in quantitative yield and contaminated with 99m Tc-Fab'. The properties, stability and biodistribution of the 99m Tc-F(ab') 2 have been compared to 99m Tc-Fab'. This new technology will allow us to compare technetium direct-labeled IgG, F(ab') 2 and Fab' derivatives of the same antibody for radioimmunodetection. (author)

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.

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.

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

Synthesis and characterization of 18F-labeled active site inhibited factor VII (ASIS)

DEFF Research Database (Denmark)

Erlandsson, Maria; Nielsen, Carsten Haagen; Jeppesen, Troels Elmer

2015-01-01

Activated factor VII blocked in the active site with Phe-Phe-Arg-chloromethyl ketone (active site inhibited factor VII (ASIS)) is a 50-kDa protein that binds with high affinity to its receptor, tissue factor (TF). TF is a transmembrane glycoprotein that plays an important role in, for example......, thrombosis, metastasis, tumor growth, and tumor angiogenesis. The aim of this study was to develop an 18F-labeled ASIS derivative to assess TF expression in tumors. Active site inhibited factor VII was labeled using N-succinimidyl-4-[18F]fluorobenzoate, and the [18F]ASIS was purified on a PD-10 desalting...... column. The radiochemical yield was 25 ± 6%, the radiochemical purity was >97%, and the pseudospecific radioactivity was 35 ± 9 GBq/µmol. The binding efficacy was evaluated in pull-down experiments, which monitored the binding of unlabeled ASIS and [18F]ASIS to TF and to a specific anti-factor VII...

Bacterial production of site specific {sup 13}C labeled phenylalanine and methodology for high level incorporation into bacterially expressed recombinant proteins

Energy Technology Data Exchange (ETDEWEB)

Ramaraju, Bhargavi; McFeeters, Hana; Vogler, Bernhard; McFeeters, Robert L., E-mail: robert.mcfeeters@uah.edu [University of Alabama in Huntsville, Department of Chemistry (United States)

2017-01-15

Nuclear magnetic resonance spectroscopy studies of ever larger systems have benefited from many different forms of isotope labeling, in particular, site specific isotopic labeling. Site specific {sup 13}C labeling of methyl groups has become an established means of probing systems not amenable to traditional methodology. However useful, methyl reporter sites can be limited in number and/or location. Therefore, new complementary site specific isotope labeling strategies are valuable. Aromatic amino acids make excellent probes since they are often found at important interaction interfaces and play significant structural roles. Aromatic side chains have many of the same advantages as methyl containing amino acids including distinct {sup 13}C chemical shifts and multiple magnetically equivalent {sup 1}H positions. Herein we report economical bacterial production and one-step purification of phenylalanine with {sup 13}C incorporation at the Cα, Cγ and Cε positions, resulting in two isolated {sup 1}H-{sup 13}C spin systems. We also present methodology to maximize incorporation of phenylalanine into recombinantly overexpressed proteins in bacteria and demonstrate compatibility with ILV-methyl labeling. Inexpensive, site specific isotope labeled phenylalanine adds another dimension to biomolecular NMR, opening new avenues of study.

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.

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...

Engineered, highly reactive substrates of microbial transglutaminase enable protein labeling within various secondary structure elements.

Science.gov (United States)

Rachel, Natalie M; Quaglia, Daniela; Lévesque, Éric; Charette, André B; Pelletier, Joelle N

2017-11-01

Microbial transglutaminase (MTG) is a practical tool to enzymatically form isopeptide bonds between peptide or protein substrates. This natural approach to crosslinking the side-chains of reactive glutamine and lysine residues is solidly rooted in food and textile processing. More recently, MTG's tolerance for various primary amines in lieu of lysine have revealed its potential for site-specific protein labeling with aminated compounds, including fluorophores. Importantly, MTG can label glutamines at accessible positions in the body of a target protein, setting it apart from most labeling enzymes that react exclusively at protein termini. To expand its applicability as a labeling tool, we engineered the B1 domain of Protein G (GB1) to probe the selectivity and enhance the reactivity of MTG toward its glutamine substrate. We built a GB1 library where each variant contained a single glutamine at positions covering all secondary structure elements. The most reactive and selective variants displayed a >100-fold increase in incorporation of a recently developed aminated benzo[a]imidazo[2,1,5-cd]indolizine-type fluorophore, relative to native GB1. None of the variants were destabilized. Our results demonstrate that MTG can react readily with glutamines in α-helical, β-sheet, and unstructured loop elements and does not favor one type of secondary structure. Introducing point mutations within MTG's active site further increased reactivity toward the most reactive substrate variant, I6Q-GB1, enhancing MTG's capacity to fluorescently label an engineered, highly reactive glutamine substrate. This work demonstrates that MTG-reactive glutamines can be readily introduced into a protein domain for fluorescent labeling. © 2017 The Protein Society.

Exploring the local conformational space of a membrane protein by site-directed spin labeling

NARCIS (Netherlands)

Stopar, D.; Strancar, J.; Spruijt, R.B.; Hemminga, M.A.

2005-01-01

Molecular modeling based on a hybrid evolutionary optimization and an information condensation algorithm, called GHOST, of spin label ESR spectra was applied to study the structure and dynamics of membrane proteins. The new method is capable of providing detailed molecular information about the

Labeling proteins on live mammalian cells using click chemistry.

Science.gov (United States)

Nikić, Ivana; Kang, Jun Hee; Girona, Gemma Estrada; Aramburu, Iker Valle; Lemke, Edward A

2015-05-01

We describe a protocol for the rapid labeling of cell-surface proteins in living mammalian cells using click chemistry. The labeling method is based on strain-promoted alkyne-azide cycloaddition (SPAAC) and strain-promoted inverse-electron-demand Diels-Alder cycloaddition (SPIEDAC) reactions, in which noncanonical amino acids (ncAAs) bearing ring-strained alkynes or alkenes react, respectively, with dyes containing azide or tetrazine groups. To introduce ncAAs site specifically into a protein of interest (POI), we use genetic code expansion technology. The protocol can be described as comprising two steps. In the first step, an Amber stop codon is introduced--by site-directed mutagenesis--at the desired site on the gene encoding the POI. This plasmid is then transfected into mammalian cells, along with another plasmid that encodes an aminoacyl-tRNA synthetase/tRNA (RS/tRNA) pair that is orthogonal to the host's translational machinery. In the presence of the ncAA, the orthogonal RS/tRNA pair specifically suppresses the Amber codon by incorporating the ncAA into the polypeptide chain of the POI. In the second step, the expressed POI is labeled with a suitably reactive dye derivative that is directly supplied to the growth medium. We provide a detailed protocol for using commercially available ncAAs and dyes for labeling the insulin receptor, and we discuss the optimal surface-labeling conditions and the limitations of labeling living mammalian cells. The protocol involves an initial cloning step that can take 4-7 d, followed by the described transfections and labeling reaction steps, which can take 3-4 d.

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.

High resolution x-ray fluorescence spectroscopy - a new technique for site- and spin-selectivity

International Nuclear Information System (INIS)

Wang, Xin

1996-12-01

X-ray spectroscopy has long been used to elucidate electronic and structural information of molecules. One of the weaknesses of x-ray absorption is its sensitivity to all of the atoms of a particular element in a sample. Through out this thesis, a new technique for enhancing the site- and spin-selectivity of the x-ray absorption has been developed. By high resolution fluorescence detection, the chemical sensitivity of K emission spectra can be used to identify oxidation and spin states; it can also be used to facilitate site-selective X-ray Absorption Near Edge Structure (XANES) and site-selective Extended X-ray Absorption Fine Structure (EXAFS). The spin polarization in K fluorescence could be used to generate spin selective XANES or spin-polarized EXAFS, which provides a new measure of the spin density, or the nature of magnetic neighboring atoms. Finally, dramatic line-sharpening effects by the combination of absorption and emission processes allow observation of structure that is normally unobservable. All these unique characters can enormously simplify a complex x-ray spectrum. Applications of this novel technique have generated information from various transition-metal model compounds to metalloproteins. The absorption and emission spectra by high resolution fluorescence detection are interdependent. The ligand field multiplet model has been used for the analysis of Kα and Kβ emission spectra. First demonstration on different chemical states of Fe compounds has shown the applicability of site selectivity and spin polarization. Different interatomic distances of the same element in different chemical forms have been detected using site-selective EXAFS

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.

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.

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.

Nanodiamond arrays on glass for quantification and fluorescence characterisation.

Science.gov (United States)

Heffernan, Ashleigh H; Greentree, Andrew D; Gibson, Brant C

2017-08-23

Quantifying the variation in emission properties of fluorescent nanodiamonds is important for developing their wide-ranging applicability. Directed self-assembly techniques show promise for positioning nanodiamonds precisely enabling such quantification. Here we show an approach for depositing nanodiamonds in pre-determined arrays which are used to gather statistical information about fluorescent lifetimes. The arrays were created via a layer of photoresist patterned with grids of apertures using electron beam lithography and then drop-cast with nanodiamonds. Electron microscopy revealed a 90% average deposition yield across 3,376 populated array sites, with an average of 20 nanodiamonds per site. Confocal microscopy, optimised for nitrogen vacancy fluorescence collection, revealed a broad distribution of fluorescent lifetimes in agreement with literature. This method for statistically quantifying fluorescent nanoparticles provides a step towards fabrication of hybrid photonic devices for applications from quantum cryptography to sensing.

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.

Fluorescence-intensity multiplexing: simultaneous seven-marker, two-color immunophenotyping using flow cytometry.

Science.gov (United States)

Bradford, Jolene A; Buller, Gayle; Suter, Michael; Ignatius, Michael; Beechem, Joseph M

2004-10-01

Conventional immuno-based multiparameter flow cytometric analysis has been limited by the requirement of a dedicated detection channel for each antibody-fluorophore set. To address the need to resolve multiple biological targets simultaneously, flow cytometers with as many as 10-15 detection channels have been developed. In this study, a new Zenon immunolabeling technology is developed that allows for multiple antigen detection per detection channel using a single fluorophore, through a unique method of fluorescence-intensity multiplexing. By varying the Zenon labeling reagent-to-antibody molar ratio, the fluorescence intensity of the antibody-labeled cellular targets can be used as a unique identifier. Although demonstrated in the present study with lymphocyte immunophenotyping, this approach is broadly applicable for any immuno-based multiplexed flow cytomety assay. Lymphocyte immunophenotyping of 38 clinical blood specimens using CD3, CD4, CD8, CD16, CD56, CD19, and CD20 antibodies was performed using conventional flow cytometric analysis and fluorescence-intensity multiplexing analysis. Conventional analysis measures a single antibody-fluorophore per photomultiplier tube (PMT). Fluorescence-intensity multiplex analysis simultaneously measures seven markers with two PMTs, using Zenon labeling reagent-antibody complexes in a single tube: CD19, CD4, CD8, and CD16 antibodies labeled with Zenon Alexa Fluor 488 Mouse IgG(1) labeling reagent and CD56, CD3, and CD20 antibodies labeled with Zenon R-Phycoerythrin (R-PE) Mouse IgG(1) or IgG(2b) labeling reagents. The lymphocyte immunophenotyping results from fluorescence-intensity multiplexing using Zenon labeling reagents in a single tube were comparable to results from conventional flow cytometric analysis. Simultaneous evaluation of multiple antigens using a single fluorophore can be performed using antibodies labeled with varying ratios of a Zenon labeling reagent. Labeling two sets of antibodies with different Zenon

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

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

A comparison of labelled antibody methods for the detection of virus antigens in cell monolayers

International Nuclear Information System (INIS)

Oram, J.D.; Crooks, A.J.

1979-01-01

A number of labelled antibody methods have been applied to the detection of Semliki Forest virus antigens after replication of the virus in monolayers of host cells in multi-well polystyrene plates. The importance of several reaction variables has been investigated and the sensitivity of the methods compared for different periods of virus replication. Direct assays with radio-labelled antibody (RLA) and indirect assays peroxidase-antiperoxidase complexes (PAP) were equally sensitive. Direct and indirect assays using enzyme-linked antibodies (ELA) were slightly less sensitive than the direct RLA and PAP methods but were more sensitive than the indirect RLA or fluorescent antibody (FLA) methods. Direct assays using ELA were more rapid and easier to perform than the other assay methods. (Auth.)

FRET study of membrane proteins: determination of the tilt and orientation of the N-terminal domain of M13 major coat protein

NARCIS (Netherlands)

Nazarov, P.V.; Koehorst, R.B.M.; Vos, W.L.; Apanasovich, V.V.; Hemminga, M.A.

2007-01-01

A formalism for membrane protein structure determination was developed. This method is based on steady-state FRET data and information about the position of the fluorescence maxima on site-directed fluorescent labeled proteins in combination with global data analysis utilizing simulation-based

Active site-directed alkylation of Na+-K+-ATPase by digitalis sulphonate derivatives of different lipophilicity.

Science.gov (United States)

Fricke, U.; Klaus, W.; Rogatti, M.

1981-01-01

1 Sulphonate derivatives of k-strophanthidin and digitoxigenin were tested as active site-directed labels of Na+-K+-adenosine triphosphatase (Na+-ATPase) from guinea-pig heart. 2 Lipophilicity ranged between P = 93 for strophanthidin-3-tosyloxy-acetate (STA) and P = 3028 for digitoxigenin-3-tosyloxy-acetate (DTA). 3 Although the alkylating moiety of STA and DTA was identical, the reversibility of Na+-K+-ATPase inhibition varied appreciably (82% and 35% respectively). 4 It is concluded that lipophilicity contributes considerably to the irreversible binding of alkylating cardiotonic steroids to myocardial Na+-K+-ATPase. PMID:6261865

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.

Genetically-directed, cell type-specific sparse labeling for the analysis of neuronal morphology.

Directory of Open Access Journals (Sweden)

Thomas Rotolo

Full Text Available In mammals, genetically-directed cell labeling technologies have not yet been applied to the morphologic analysis of neurons with very large and complex arbors, an application that requires extremely sparse labeling and that is only rendered practical by limiting the labeled population to one or a few predetermined neuronal subtypes.In the present study we have addressed this application by using CreER technology to non-invasively label very small numbers of neurons so that their morphologies can be fully visualized. Four lines of IRES-CreER knock-in mice were constructed to permit labeling selectively in cholinergic or catecholaminergic neurons [choline acetyltransferase (ChAT-IRES-CreER or tyrosine hydroxylase (TH-IRES-CreER], predominantly in projection neurons [neurofilament light chain (NFL-IRES-CreER], or broadly in neurons and some glia [vesicle-associated membrane protein2 (VAMP2-IRES-CreER]. When crossed to the Z/AP reporter and exposed to 4-hydroxytamoxifen in the early postnatal period, the number of neurons expressing the human placental alkaline phosphatase reporter can be reproducibly lowered to fewer than 50 per brain. Sparse Cre-mediated recombination in ChAT-IRES-CreER;Z/AP mice shows the full axonal and dendritic arbors of individual forebrain cholinergic neurons, the first time that the complete morphologies of these very large neurons have been revealed in any species.Sparse genetically-directed, cell type-specific neuronal labeling with IRES-creER lines should prove useful for studying a wide variety of questions in neuronal development and disease.

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.

Rapid Identification of Staphylococcus aureus Directly from Blood Cultures by Fluorescence In Situ Hybridization with Peptide Nucleic Acid Probes

Science.gov (United States)

Oliveira, Kenneth; Procop, Gary W.; Wilson, Deborah; Coull, James; Stender, Henrik

2002-01-01

A new fluorescence in situ hybridization (FISH) method with peptide nucleic acid (PNA) probes for identification of Staphylococcus aureus directly from positive blood culture bottles that contain gram-positive cocci in clusters (GPCC) is described. The test (the S. aureus PNA FISH assay) is based on a fluorescein-labeled PNA probe that targets a species-specific sequence of the 16S rRNA of S. aureus. Evaluations with 17 reference strains and 48 clinical isolates, including methicillin-resistant and methicillin-susceptible S. aureus species, coagulase-negative Staphylococcus species, and other clinically relevant and phylogenetically related bacteria and yeast species, showed that the assay had 100% sensitivity and 96% specificity. Clinical trials with 87 blood cultures positive for GPCC correctly identified 36 of 37 (97%) of the S. aureus-positive cultures identified by standard microbiological methods. The positive and negative predictive values were 100 and 98%, respectively. It is concluded that this rapid method (2.5 h) for identification of S. aureus directly from blood culture bottles that contain GPCC offers important information for optimal antibiotic therapy. PMID:11773123

Multimodal quantitative phase and fluorescence imaging of cell apoptosis

Science.gov (United States)

Fu, Xinye; Zuo, Chao; Yan, Hao

2017-06-01

Fluorescence microscopy, utilizing fluorescence labeling, has the capability to observe intercellular changes which transmitted and reflected light microscopy techniques cannot resolve. However, the parts without fluorescence labeling are not imaged. Hence, the processes simultaneously happen in these parts cannot be revealed. Meanwhile, fluorescence imaging is 2D imaging where information in the depth is missing. Therefore the information in labeling parts is also not complete. On the other hand, quantitative phase imaging is capable to image cells in 3D in real time through phase calculation. However, its resolution is limited by the optical diffraction and cannot observe intercellular changes below 200 nanometers. In this work, fluorescence imaging and quantitative phase imaging are combined to build a multimodal imaging system. Such system has the capability to simultaneously observe the detailed intercellular phenomenon and 3D cell morphology. In this study the proposed multimodal imaging system is used to observe the cell behavior in the cell apoptosis. The aim is to highlight the limitations of fluorescence microscopy and to point out the advantages of multimodal quantitative phase and fluorescence imaging. The proposed multimodal quantitative phase imaging could be further applied in cell related biomedical research, such as tumor.

Hybrid Imaging Labels: Providing the Link Between Mass Spectrometry-Based Molecular Pathology and Theranostics

Science.gov (United States)

Buckle, Tessa; van der Wal, Steffen; van Malderen, Stijn J.M.; Müller, Larissa; Kuil, Joeri; van Unen, Vincent; Peters, Ruud J.B.; van Bemmel, Margaretha E.M.; McDonnell, Liam A.; Velders, Aldrik H.; Koning, Frits; Vanhaeke, Frank; van Leeuwen, Fijs W. B.

2017-01-01

Background: Development of theranostic concepts that include inductively coupled plasma mass spectrometry (ICP-MS) and laser ablation ICP-MS (LA-ICP-MS) imaging can be hindered by the lack of a direct comparison to more standardly used methods for in vitro and in vivo evaluation; e.g. fluorescence or nuclear medicine. In this study a bimodal (or rather, hybrid) tracer that contains both a fluorescent dye and a chelate was used to evaluate the existence of a direct link between mass spectrometry (MS) and in vitro and in vivo molecular imaging findings using fluorescence and radioisotopes. At the same time, the hybrid label was used to determine whether the use of a single isotope label would allow for MS-based diagnostics. Methods: A hybrid label that contained both a DTPA chelate (that was coordinated with either 165Ho or 111In) and a Cy5 fluorescent dye was coupled to the chemokine receptor 4 (CXCR4) targeting peptide Ac-TZ14011 (hybrid-Cy5-Ac-TZ4011). This receptor targeting tracer was used to 1) validate the efficacy of (165Ho-based) mass-cytometry in determining the receptor affinity via comparison with fluorescence-based flow cytometry (Cy5), 2) evaluate the microscopic binding pattern of the tracer in tumor cells using both fluorescence confocal imaging (Cy5) and LA-ICP-MS-imaging (165Ho), 3) compare in vivo biodistribution patterns obtained with ICP-MS (165Ho) and radiodetection (111In) after intravenous administration of hybrid-Cy5-Ac-TZ4011 in tumor-bearing mice. Finally, LA-ICP-MS-imaging (165Ho) was linked to fluorescence-based analysis of excised tissue samples (Cy5). Results: Analysis with both mass-cytometry and flow cytometry revealed a similar receptor affinity, respectively 352 ± 141 nM and 245 ± 65 nM (p = 0.08), but with a much lower detection sensitivity for the first modality. In vitro LA-ICP-MS imaging (165Ho) enabled clear discrimination between CXCR4 positive and negative cells, but fluorescence microscopy was required to determine the

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.

Opalescence and fluorescence properties of indirect and direct resin materials.

Science.gov (United States)

Song, Sang-Hoon; Yu, Bin; Ahn, Jin-Soo; Lee, Yong-Keun

2008-08-01

To measure the opalescence and fluorescence properties of indirect and direct resin materials before and after polymerization, and to determine the influence of the material and shade group combination on these properties. BelleGlass NG (BG, indirect resin) and Estelite Sigma (ES, direct resin), each composed in 3 shade groups (EN, OD and TL for BG; BS, AS and OP for ES) out of a total of 16 shades were investigated. Resin material was packed into a mold (the BEC condition) and polymerized with a light-polymerization unit (CWL). Secondary polymerization (CIC) was performed for BG. Color was measured in the BEC, CWL, and CIC conditions, and the opalescence parameter (OP) and fluorescence parameter (FL) were calculated. For the OP, the mean for BG material was 24.3 before polymerization, which changed to 19.9 after polymerization (CIC). In the case of ES, the mean OP before polymerization was 25.6, which changed to 12.4 after polymerization (CWL). For the FL, the mean FL for BG was 2.5 before polymerization, which changed to 0.7 after polymerization. In the case of ES, the mean FL before polymerization was 1.2, which did not change after polymerization. Material and shade group combination influenced the OP and FL values (popalescence and fluorescence properties of resin materials varied depending on the material, shade group, and polymerization. Clinically, these properties should be considered when neighboring teeth are restored with different types of material.

Selective labelling of stromal cell-derived factor 1α with carboxyfluorescein to study receptor internalisation.

Science.gov (United States)

Bellmann-Sickert, Kathrin; Baumann, Lars; Beck-Sickinger, Annette G

2010-10-01

SDF1α plays an important role in the regeneration of injured tissue after ischemia or stroke by inducing the migration of progenitor cells. In order to study the function of this therapeutically relevant chemokine site-specific protein labelling is of great interest. However, modification of SDF1α is complicated because of its complex tertiary structure. Here, we describe the first site-specific fluorescent modification of SDF1α by EPL. We recombinantly expressed SDF1α (1-49) by intein-mediated protein expression. The C-terminal peptide SDF1α (50-68) was synthesised by SPPS and selectively labelled with carboxyfluorescein at Lys(56). In a cell migration assay, M-[K(56)(CF)]SDF1α showed a clear potency to induce chemotaxis of human T-cell leukaemia cells. Microscopic analysis on HEK293 cells transfected with the CXCR4 revealed specific binding of the fluorescent ligand. Furthermore, receptor-induced internalisation of the ligand could be visualised. These results show that site-specific modification of SDF1α yields in a biologically functional molecule that allows the characterisation of CXCR4 production of cells on a molecular level. © 2010 European Peptide Society and John Wiley & Sons, Ltd.

Direct labeling of doxorubicin with technetium-99m. Its optimization, characterization and quality control

International Nuclear Information System (INIS)

Rizvi, F.A.; Bokhari, T.H.; Roohi, S.; Mushtaq, A.

2012-01-01

Doxorubicin (DOX) is an anthracycline antineoplastic and one of the most potent and widely used drugs in clinical oncology. It is used in the treatment of a wide variety of cancers. The aim of this study was the direct labeling of DOX with 99m Tc; its optimization, characterization and quality control of the radiolabeled DOX. Labeling efficiency was determined by paper chromatography. More than 92% labeling was obtained at pH 6-7, 10-12 μg stannous chloride and 200 μg of DOX. The stability of 99m Tc-DOX was studied up to 5 h. All the experiments were performed at room temperature (25 ± 2 deg C). The characterization of the labeling compound was performed by HPLC and electrophoresis. Electrophoresis indicates that labeled DOX has no charge and HPLC shows single specie of labeled compound. (author)

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.

Multi-color imaging of fluorescent nanodiamonds in living HeLa cells using direct electron-beam excitation.

Science.gov (United States)

Nawa, Yasunori; Inami, Wataru; Lin, Sheng; Kawata, Yoshimasa; Terakawa, Susumu; Fang, Chia-Yi; Chang, Huan-Cheng

2014-03-17

Multi-color, high spatial resolution imaging of fluorescent nanodiamonds (FNDs) in living HeLa cells has been performed with a direct electron-beam excitation-assisted fluorescence (D-EXA) microscope. In this technique, fluorescent materials are directly excited with a focused electron beam and the resulting cathodoluminescence (CL) is detected with nanoscale resolution. Green- and red-light-emitting FNDs were employed for two-color imaging, which were observed simultaneously in the cells with high spatial resolution. This technique could be applied generally for multi-color immunostaining to reveal various cell functions. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Histochemical evidence for the differential surface labeling, uptake, and intracellular transport of a colloidal gold-labeled insulin complex by normal human blood cells.

Science.gov (United States)

Ackerman, G A; Wolken, K W

1981-10-01

A colloidal gold-labeled insulin-bovine serum albumin (GIA) reagent has been developed for the ultrastructural visualization of insulin binding sites on the cell surface and for tracing the pathway of intracellular insulin translocation. When applied to normal human blood cells, it was demonstrated by both visual inspection and quantitative analysis that the extent of surface labeling, as well as the rate and degree of internalization of the insulin complex, was directly related to cell type. Further, the pathway of insulin (GIA) transport via round vesicles and by tubulo-vesicles and saccules and its subsequent fate in the hemic cells was also related to cell variety. Monocytes followed by neutrophils bound the greatest amount of labeled insulin. The majority of lymphocytes bound and internalized little GIA, however, between 5-10% of the lymphocytes were found to bind considerable quantities of GIA. Erythrocytes rarely bound the labeled insulin complex, while platelets were noted to sequester large quantities of the GIA within their extracellular canalicular system. GIA uptake by the various types of leukocytic cells appeared to occur primarily by micropinocytosis and by the direct opening of cytoplasmic tubulo-vesicles and saccules onto the cell surface in regions directly underlying surface-bound GIA. Control procedures, viz., competitive inhibition of GIA labeling using an excess of unlabeled insulin in the incubation medium, preincubation of the GIA reagent with an antibody directed toward porcine insulin, and the incorporation of 125I-insulin into the GIA reagent, indicated the specificity and selectivity of the GIA histochemical procedure for the localization of insulin binding sites.

Histochemical evidence for the differential surface labeling, uptake, and intracellular transport of a colloidal gold-labeled insulin complex by normal human blood cells

International Nuclear Information System (INIS)

Ackerman, G.A.; Wolken, K.W.

1981-01-01

A colloidal gold-labeled insulin-bovine serum albumin (GIA) reagent has been developed for the ultrastructural visualization of insulin binding sites on the cell surface and for tracing the pathway of intracellular insulin translocation. When applied to normal human blood cells, it was demonstrated by both visual inspection and quantitative analysis that the extent of surface labeling, as well as the rate and degree of internalization of the insulin complex, was directly related to cell type. Further, the pathway of insulin (GIA) transport via round vesicles and by tubulo-vesicles and saccules and its subsequent fate in the hemic cells was also related to cell variety. Monocytes followed by neutrophils bound the greatest amount of labeled insulin. The majority of lymphocytes bound and internalized little GIA, however, between 5-10% of the lymphocytes were found to bind considerable quantities of GIA. Erythrocytes rarely bound the labeled insulin complex, while platelets were noted to sequester large quantities of the GIA within their extracellular canalicular system. GIA uptake by the various types of leukocytic cells appeared to occur primarily by micropinocytosis and by the direct opening of cytoplasmic tubulo-vesicles and saccules onto the cell surface in regions directly underlying surface-bound GIA. Control procedures, viz., competitive inhibition of GIA labeling using an excess of unlabeled insulin in the incubation medium, preincubation of the GIA reagent with an antibody directed toward porcine insulin, and the incorporation of 125I-insulin into the GIA reagent, indicated the specificity and selectivity of the GIA histochemical procedure for the localization of insulin binding sites

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.

On-site preparation of technetium-99m labeled human serum albumin for clinical application

International Nuclear Information System (INIS)

Wang Yuhfeng; Chuang Meihua; Cham Thauming; Chung Meiing; Chiu Jainnshiun

2007-01-01

Technetium-99m labeled human serum albumin (Tc-99m HSA) is an important radiopharmaceutical for clinical applications, such as cardiac function tests or protein-losing gastroenteropathy assessment. However, because of transfusion-induced infectious diseases, the safety of serum products is a serious concern. In this context, serum products acquired from patients themselves are the most ideal tracer. However, the development of rapid separation and easy clinical labeling methods is not yet well established. Under such situation, products from the same ethnic group or country are now recommended by the World Health Organization as an alternative preparation. This article describes the on-site preparation of Tc-99m HSA from locally supplied serum products. Different formulations were prepared and the labeling efficiency and stability were examined. Radio-labeling efficiencies were more than 90% in all preparation protocols, except for one that omitted the stannous solution. The most cost-effective protocol contained HSA 0.1 mg, treated with stannous fluoride 0.2 mg, and mixed with Tc-99m pertechnetate 30 mCi. A biodistribution study was performed in rats using a gamma camera immediately after intravenous administration of radiolabeled HSA. Tissue/organ uptake was obtained by measuring the radioactivity in organs after sacrificing the rats at timed intervals. The biologic half-life was about 32 min, determined from sequential venous blood collections. These data indicate that our preparation of Tc-99m HSA is useful and potentially applicable clinically. In addition, this on-site preparation provides the possibility of labeling a patient's own serum for subsequent clinical application. (author)

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).

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

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.

Direct spectrometry: a new alternative for measuring the fluorescence of composite resins and dental tissues.

Science.gov (United States)

da Silva, Tm; de Oliveira, Hpm; Severino, D; Balducci, I; Huhtala, Mfrl; Gonçalves, Sep

2014-01-01

The aim of this study was to evaluate the fluorescence intensity of different composite resins and compare those values with the fluorescence intensity of dental tissues. Different composite resins were used to make 10 discs (2 mm in depth and 4 mm in diameter) of each brand, divided into groups: 1) Z (Filtek Z350, 3M ESPE), 2) ES (Esthet-X, Dentsply), 3) A (Amelogen Plus, Ultradent), 4) DVS (Durafill-VS, Heraeus Kulzer) with 2 mm composite resin for enamel (A2), 5) OES ([Esthet-X] opaque-OA [1 mm] + enamel-A2 [1 mm]); 6) ODVSI ([Charisma-Opal/Durafill-VSI], opaque-OM (1 mm) + translucent [1mm]), and 7) DVSI ([Durafill- VSI] translucent [2 mm]). Dental tissue specimens were obtained from human anterior teeth cut in a mesiodistal direction to obtain enamel, dentin, and enamel/dentin samples (2 mm). The fluorescence intensity of specimens was directly measured using an optic fiber associated with a spectrometer (Ocean Optics USB 4000) and recorded in graphic form (Origin 8.0 program). Data were submitted to statistical analysis using Dunnet, Tukey, and Kruskall-Wallis tests. Light absorption of the composite resins was obtained in a spectral range from 250 to 450 nm, and that of dental tissues was between 250 and 300 nm. All composite resins were excited at 398 nm and exhibited maximum emissions of around 485 nm. Fluorescence intensity values for all of the resins showed statistically significant differences (measured in arbitrary units [AUs]), with the exception of groups Z and DVS. Group DVSI had the highest fluorescence intensity values (13539 AU), followed by ODVS (10440 AU), DVS (10146 AU), ES (3946 AU), OES (3841 AU), A (3540 AU), and Z (1146 AU). The fluorescence intensity values for the composite resins differed statistically from those of dental tissues (E=1380 AU; D=6262 AU; E/D=3251 AU). The opacity interfered with fluorescence intensity, and group Z demonstrated fluorescence intensity values closest to that of tooth enamel. It is concluded that the

Optical imaging of non-fluorescent nanodiamonds in live cells using transient absorption microscopy.

Science.gov (United States)

Chen, Tao; Lu, Feng; Streets, Aaron M; Fei, Peng; Quan, Junmin; Huang, Yanyi

2013-06-07

We directly observe non-fluorescent nanodiamonds in living cells using transient absorption microscopy. This label-free technology provides a novel modality to study the dynamic behavior of nanodiamonds inside the cells with intrinsic three-dimensional imaging capability. We apply this method to capture the cellular uptake of nanodiamonds under various conditions, confirming the endocytosis mechanism.

A highly selective and sensitive fluorescent chemosensor and its application for rapid on-site detection of Al3 +

Science.gov (United States)

Yue, Xiao-li; Wang, Zhao-qing; Li, Chao-rui; Yang, Zheng-yin

2018-03-01

In this paper, a simple naphthalene-based derivative (HL) has been designed and synthesized as a Al3 +-selective fluorescent chemosensor based on the PET mechanism. HL exhibited high selectivity and sensitivity towards Al3 + over other commonly coexisting metal ions in ethanol with a detection limit of 2.72 nM. The 1:1 binding stoichiometry of the complex (HL-Al3 +) was determined from the Job's plot based on fluorescence titrations and the ESI-MS spectrum data. Moreover, the binding site of HL with Al3 + was assured by the 1H NMR titration experiment. The binding constant (Ka) of the complex (HL-Al3 +) was calculated to be 5.06 × 104 M- 1 according to the Benesi-Hildebrand equation. In addition, the recognizing process of HL towards Al3 + was chemically reversible by adding Na2EDTA. Importantly, HL could directly and rapidly detect aluminum ion through the filter paper without resorting to additional instrumental analysis.

Local mobility in lipid domains of supported bilayers characterized by atomic force microscopy and fluorescence correlation spectroscopy.

Energy Technology Data Exchange (ETDEWEB)

Frankel, Daniel J.; Buranda, T. (University of New Mexico, Albuquerque, NM); Burns, Alan Richard

2005-01-01

Fluorescence correlation spectroscopy (FCS) is used to examine mobility of labeled probes at specific sites in supported bilayers consisting of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) lipid domains in 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). Those sites are mapped beforehand with simultaneous atomic force microscopy and submicron confocal fluorescence imaging, allowing characterization of probe partitioning between gel DPPC and disordered liquid DOPC domains with corresponding topography of domain structure. We thus examine the relative partitioning and mobility in gel and disordered liquid phases for headgroup- and tailgroup-labeled GM1 ganglioside probes and for headgroup- and tailgroup-labeled phospholipid probes. For the GM1 probes, large differences in mobility between fluid and gel domains are observed; whereas unexpected mobility is observed in submicron gel domains for the phospholipid probes. We attribute the latter to domain heterogeneities that could be induced by the probe. Furthermore, fits to the FCS data for the phospholipid probes in the DOPC fluid phase require two components (fast and slow). Although proximity to the glass substrate may be a factor, local distortion of the probe by the fluorophore could also be important. Overall, we observe nonideal aspects of phospholipid probe mobility and partitioning that may not be restricted to supported bilayers.

Optimization of in vitro cell labeling methods for human umbilical cord-derived mesenchymal stem cells.

Science.gov (United States)

Tao, R; Sun, T-J; Han, Y-Q; Xu, G; Liu, J; Han, Y-F

2014-01-01

Human umbilical cord-derived mesenchymal stem cells (hUCMSCs) are a novel source of seed cells for cell therapy and tissue engineering. However, in vitro labeling methods for hUCMSCs need to be optimized for better detection of transplanted cells. To identify the most stable and efficient method for labeling hUCMSCs in vitro. hUCMSCs were isolated using a modified enzymatic digestion procedure and cultured. hUCMSCs of passage three (P3) were then labeled with BrdU, PKH26, or lentivirus-GFP and passaged further. Cells from the first labeled passage (LP1), the fourth labeled passage (LP4) and later passages were observed using a fluorescence microscope. The differentiation potential of LP4 cells was assessed by induction with adipogenic and osteogenic medium. Flow cytometry was used to measure the percentage of labeled cells and the percentage of apoptotic or dead cells. The labeling efficiencies of the three hUCMSC-labeling methods were compared in vitro. BrdU, PKH26, and lentivirus-GFP all labeled LP1 cells with high intensity and clarity. However, the BrdU labeling of the LP4 cells was vague and not localized to the cell nuclei; LP9 cells were not detected under a fluorescence microscope. There was also a significant decrease in the fluorescence intensity of PKH26-labeled LP4 cells, and LP11 cells were not detected under a fluorescence microscope. However, the fluorescence of LP4 cells labeled with lentivirus-GFP remained strong, and cells labeled with lentivirus-GFP were detected up to LP14 under a fluorescence microscope. Statistical analyses indicated that percentages of LP1 cells labeled with PKH26 and lentivirus-GFP were significantly higher than that of cells labeled with BrdU (p 0.05) was observed between the death rates of labeled and unlabeled cells. Lentivirus-GFP is a valid method for long-term in vitro labeling, and it may be used as a long-term hUCMSC tracker following transplantation in vivo.

99mTc labelled peptides for imaging of peripheral receptors

International Nuclear Information System (INIS)

Mustanser, J.; Anjum, A.

2001-01-01

Several peptides are being used as radiopharmaceuticals for receptor imaging scintigraphy. The peptide receptors are found in the tumours of various sites in the human body. Somatostatin is one of those, which is expressed by a variety of tumours say in brain cortex, medullary carcinoma of thyroid, adrenal glands, pancreas and gut. Therefore neuropeptides based on somatostatin analogues are labelled with different radionuclide, 123 I and 111 In. Efforts are underway to label RC-160 (an analogue of somatostatin) with 99m Tc because of its favourable radiation dosimetry, short half-life, low price, high count rate and better diagnostic efficacy. In this project various methods of labelling RC-160 with different radionuclides 125 I and 99m Tc have been studied in detail. Radioiodination of RC-160 was tried with 125 I using the iodogen method as directed and then with Chloramine T method. Labelling of RC-160 peptide with 99m Tc was done using two different aspects. Direct labelling with 99m Tc and indirect labelling with 99m Tc using double chelating agents. Radiochemical quality control was carried out applying instant thin layer chromatography using ITLC-SG strips in 85% of methanol. Later the HPLC analysis was used for its evaluation. To label RC-160 with 99m Tc the approach of direct labelling was attempted first. 46% labelling could be achieved with 95% of radiochemical purity. The biodistribution of 99m Tc-RC-160 complex in rats has also been studied to determine uptake in various sites of somatostatin receptors. Eventually, attempt was made to synthesize biomolecule by conjugating Boc protected RC-160 with benzoyl MAG-3. As a result 80% of Boc-RC-160 went under conjugation with benzoyl MAG-3. (author)

Efficient processing of fluorescence images using directional multiscale representations.

Science.gov (United States)

Labate, D; Laezza, F; Negi, P; Ozcan, B; Papadakis, M

2014-01-01

Recent advances in high-resolution fluorescence microscopy have enabled the systematic study of morphological changes in large populations of cells induced by chemical and genetic perturbations, facilitating the discovery of signaling pathways underlying diseases and the development of new pharmacological treatments. In these studies, though, due to the complexity of the data, quantification and analysis of morphological features are for the vast majority handled manually, slowing significantly data processing and limiting often the information gained to a descriptive level. Thus, there is an urgent need for developing highly efficient automated analysis and processing tools for fluorescent images. In this paper, we present the application of a method based on the shearlet representation for confocal image analysis of neurons. The shearlet representation is a newly emerged method designed to combine multiscale data analysis with superior directional sensitivity, making this approach particularly effective for the representation of objects defined over a wide range of scales and with highly anisotropic features. Here, we apply the shearlet representation to problems of soma detection of neurons in culture and extraction of geometrical features of neuronal processes in brain tissue, and propose it as a new framework for large-scale fluorescent image analysis of biomedical data.

(125I)LSD labels 5-TCsub(IC) recognition sites in pig choriod plexus membranes. Comparison with (3H)mesulergine and (3H)5-HT binding

International Nuclear Information System (INIS)

Hoyer, D.; Srivatsa, S.; Pazos, A.; Engel, G.; Palacios, J.M.

1986-01-01

The mammalian choroid plexus is enriched in a newly described serotonin recognition site, the binding characteristics of ( 125 I)LSD, ( 3 H)mesulergine and ( 3 H)serotonin to pig choroid plexus membranes were compared. These ligands labelled with high affinity a similar number of sites. The binding profiles of the sites labelled with these radioligands are indistinguishable as illustrated by highly significant correlation parameters. These sites are very similar to those labelled by Nsub(I)-methyl-2-( 125 I)LSD in pig and rat choroid plexus membranes. The data demonstrate that these ligands label 5-HTsub(IC) recognition sites in the pig and rat choroid plexus membranes. (author)

Photoaffinity labeling of the lumenal K+ site of the gastric (H+ + K+)-ATPase

International Nuclear Information System (INIS)

Keeling, D.J.; Fallowfield, C.; Lawrie, K.M.; Saunders, D.; Richardson, S.; Ife, R.J.

1989-01-01

A photoaffinity label for the lumenal K+ site of the gastric (H+ + K+)-ATPase has been identified. Seven azido derivatives based upon the reversible K+ site inhibitor SCH 28080 were studied, one of which, m-ATIP (8-(3-azidophenylmethoxy)-1,2,3-trimethylimidazo[1,2-a] pyridinium iodide), was subsequently synthesized in radiolabeled form. In the absence of UV irradiation, m-ATIP inhibited K+ -stimulated ATPase activity in lyophilized gastric vesicles competitively with respect to K+, with a Ki value of 2.4 microM at pH 7.0. Irradiation of lyophilized gastric vesicles at pH 7.0 with [ 14 C]m-ATIP in the presence of 0.2 mM ATP resulted in a time-dependent inactivation of ATPase activity that was associated with an incorporation of radioactivity into a 100-kDa polypeptide representing the catalytic subunit of the (H+ + K+)-ATPase. Both inactivation and incorporation were blocked in the presence of 10 mM KCl but not with 10 mM NaCl, consistent with interaction at the K+ site. The level of incorporation required to produce complete inhibition of ATPase activity was 1.9 +/- 0.2 times the number of catalytic phosphorylation sites in the same preparation. Tryptic digestion of gastric vesicle membranes, labeled with [ 14 C]m-ATIP, failed to release the radioactivity from the membranes suggesting that the site of interaction was close to or within the membrane-spanning sections of this ion pump

In vivo target bio-imaging of Alzheimer's disease by fluorescent zinc oxide nanoclusters.

Science.gov (United States)

Lai, Lanmei; Zhao, Chunqiu; Su, Meina; Li, Xiaoqi; Liu, Xiaoli; Jiang, Hui; Amatore, Christian; Wang, Xuemei

2016-07-21

Alzheimer's disease (AD) is an irreversible neurodegenerative disease which is difficult to cure. When Alzheimer's disease occurs, the level of zinc ions in the brain changes, and the relevant amount of zinc ions continue decreasing in the cerebrospinal fluid and plasma of Alzheimer's patients with disease exacerbation. In view of these considerations, we have explored a new strategy for the in vivo rapid fluorescence imaging of Alzheimer's disease through target bio-labeling of zinc oxide nanoclusters which were biosynthesized in vivo in the Alzheimer's brain via intravenous injection of zinc gluconate solution. By using three-month-old and six-month-old Alzheimer's model mice as models, our observations demonstrate that biocompatible zinc ions could pass through the blood-brain barrier of the Alzheimer's disease mice and generate fluorescent zinc oxide nanoclusters (ZnO NCs) through biosynthesis, and then the bio-synthesized ZnO NCs could readily accumulate in situ on the hippocampus specific region for the in vivo fluorescent labeling of the affected sites. This study provides a new way for the rapid diagnosis of Alzheimer's disease and may have promising prospects in the effective diagnosis of Alzheimer's disease.

Pharmacokinetics on a microscale: visualizing Cy5-labeled oligonucleotide release from poly(n-butylcyanoacrylate nanocapsules in cells

Directory of Open Access Journals (Sweden)

Tomcin S

2014-11-01

Full Text Available Stephanie Tomcin,1 Grit Baier,1 Katharina Landfester,1 Volker Mailänder1,21Max Planck Institute for Polymer Research, 2University Medical Center of the Johannes Gutenberg University, III Medical Clinic, Mainz, GermanyAbstract: For successful design of a nanoparticulate drug delivery system, the fate of the carrier and cargo need to be followed. In this work, we fluorescently labeled poly(n-butylcyanoacrylate (PBCA nanocapsules as a shell and separately an oligonucleotide (20 mer as a payload. The nanocapsules were formed by interfacial anionic polymerization on aqueous droplets generated by an inverse miniemulsion process. After uptake, the PBCA capsules were shown to be round-shaped, endosomal structures and the payload was successfully released. Cy5-labeled oligonucleotides accumulated at the mitochondrial membrane due to a combination of the high mitochondrial membrane potential and the specific molecular structure of Cy5. The specificity of this accumulation at the mitochondria was shown as the uncoupler dinitrophenol rapidly diminished the accumulation of the Cy5-labeled oligonucleotide. Importantly, a fluorescence resonance energy transfer investigation showed that the dye-labeled cargo (Cy3/Cy5-labeled oligonucleotides reached its target site without degradation during escape from an endosomal compartment to the cytoplasm. The time course of accumulation of fluorescent signals at the mitochondria was determined by evaluating the colocalization of Cy5-labeled oligonucleotides and mitochondrial markers for up to 48 hours. As oligonucleotides are an ideal model system for small interfering RNA PBCA nanocapsules demonstrate to be a versatile delivery platform for small interfering RNA to treat a variety of diseases.Keywords: drug delivery, mitochondria, miniemulsion, colocalization

Synthesis and optical properties of pyrrolidinyl peptide nucleic acid carrying a clicked Nile red label

Directory of Open Access Journals (Sweden)

Nattawut Yotapan

2014-09-01

Full Text Available DNA or its analogues with an environment-sensitive fluorescent label are potentially useful as a probe for studying the structure and dynamics of nucleic acids. In this work, pyrrolidinyl peptide nucleic acid (acpcPNA was labeled at its backbone with Nile red, a solvatochromic benzophenoxazine dye, by means of click chemistry. The optical properties of the Nile red-labeled acpcPNA were investigated by UV–vis and fluorescence spectroscopy in the absence and in the presence of DNA. In contrast to the usual quenching observed in Nile red-labeled DNA, the hybridization with DNA resulted in blue shifting and an enhanced fluorescence regardless of the neighboring bases. More pronounced blue shifts and fluorescence enhancements were observed when the DNA target carried a base insertion in close proximity to the Nile red label. The results indicate that the Nile red label is located in a more hydrophobic environment in acpcPNA–DNA duplexes than in the single-stranded acpcPNA. The different fluorescence properties of the acpcPNA hybrids of complementary DNA and DNA carrying a base insertion are suggestive of different interactions between the Nile red label and the duplexes.

Rapid on-site detection of Acidovorax avenae subsp. citrulli by gold-labeled DNA strip sensor.

Science.gov (United States)

Zhao, Wenjun; Lu, Jie; Ma, Wenwei; Xu, Chuanlai; Kuang, Hua; Zhu, Shuifang

2011-06-15

Acidovorax avenae subsp. citrulli (AAC) is one of the most harmful diseases in cucurbit production. A rapid and sensitive DNA strip sensor was constructed based on gold nanoparticle-labeled oligonucleotide probes for the detection of AAC. Both the qualitative and semi-quantitative detections of target DNA were successfully achieved using the developed DNA strip sensor. The qualitative limit of detection (LOD) of the strip sensor was determined as 4 nM. The LOD for the semi-quantitative detection was calculated to be 0.48 nM in the range of 0-10 nM. The genomic DNA was detected directly using the DNA strip sensor without any further treatment. This DNA strip sensor is a potentially useful tool for rapid on-site DNA screening. Copyright © 2011 Elsevier B.V. All rights reserved.

Direct and indirect fluorescent detection of tetracyclines using dually emitting carbon dots

International Nuclear Information System (INIS)

Qu, Fei; Sun, Zhe; Liu, Dongya; Zhao, Xianen; You, Jinmao

2016-01-01

The authors describe dual-emission carbon nanodots containing blue emitters (BE; peak emission at 385 nm under 315 nm excitation) and yellow emitters (YE; peak emission at 530 nm under 365 nm excitation), and how they can be applied to direct and indirect determination of tetracyclines (TCs). The direct detection scheme is based on the finding that tetracycline (TET), oxytetracycline, chlortetracycline and doxycycline quench the two emissions of the carbon dots. While direct determination is rapid and convenient, it cannot differentiate between TCs. The indirect detection scheme, in contrast, is based on the finding that Al (III) ions enhance the fluorescence of the YE in the carbon dots, and that they cause a blue shift in emission. It is, however, known that TET forms a strong complex with Al (III), and this can inhibit the interaction between Al (III) and the YE, so that the fluorescence of YE is not enhanced and blue-shifted by Al (III) in the presence of TET. This finding is exploited in a fluorescence turn-on/off assay for TET that can distinguish TET from other TCs. The linear range of indirect determination for TET extends from 1 nM to 30 μM, and the limit of detection is 0.52 nM. The indirect method was successfully applied to the determination of TET in spiked milk, fish and pork, and recoveries ranged from 91.7 to 102 %. (author)

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

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.

Neutrophil labeling with [99mTc]-technetium stannous colloid is complement receptor 3-mediated and increases the neutrophil priming response to lipopolysaccharide

International Nuclear Information System (INIS)

Gallagher, Hayley; Ramsay, Stuart C.; Barnes, Jodie; Maggs, Jacqueline; Cassidy, Nathan; Ketheesan, Natkunam

2006-01-01

Introduction: [ 99m Tc]-technetium stannous colloid (TcSnC)-labeled white cells are used to image inflammation. Neutrophil labeling with TcSnC is probably phagocytic, but the phagocytic receptor involved is not known. We hypothesised that complement receptor 3 (CR3) plays a key role. Phagocytic labeling could theoretically result in neutrophil activation or priming, affecting the behaviour of labeled cells. Fluorescence-activated cell sorter (FACS) analysis side scatter measurements can assess neutrophil activation and priming. Methods: We tested whether TcSnC neutrophil labeling is CR3-mediated by assessing if neutrophil uptake of TcSnC was inhibited by a monoclonal antibody (mAb) directed at the CD11b component of CR3. We tested if TcSnC-labeled neutrophils show altered activation or priming status, comparing FACS side scatter in labeled and unlabeled neutrophils and examining the effect of lipopolysaccharide (LPS), a known priming agent. Results: Anti-CD11b mAb reduced neutrophil uptake of TcSnC in a dose-dependent fashion. Labeled neutrophils did not show significantly increased side scatter compared to controls. LPS significantly increased side scatter in control cells and labeled neutrophils. However, the increase was significantly greater in labeled neutrophils than unlabeled cells. Conclusions: Neutrophil labeling with TcSnC is related to the function of CR3, a receptor which plays a central role in phagocytosis. TcSnC labeling did not significantly activate or prime neutrophils. However, labeled neutrophils showed a greater priming response to LPS. This could result in labeled neutrophils demonstrating increased adhesion on activated endothelium at sites of infection

Identification of corticotropin-releasing factor (CRF) target cells and effects of dexamethasone on binding in anterior pituitary using a fluorescent analog of CRF

DEFF Research Database (Denmark)

Schwartz, J; Billestrup, Nils; Perrin, M

1986-01-01

A fluorescein-conjugated bioactive analog of corticotropin-releasing factor (CRF) was synthesized and used to label cells that have high affinity CRF-binding sites. Of cultured bovine anterior pituitary cells, 6.1 +/- 0.6% were visible by fluorescence microscopy after incubation with the analog......-binding sites and suggest that binding of CRF to anterior pituitary cells is altered by glucocorticoids....

Drug-laden 3D biodegradable label using QR code for anti-counterfeiting of drugs.

Science.gov (United States)

Fei, Jie; Liu, Ran

2016-06-01

Wiping out counterfeit drugs is a great task for public health care around the world. The boost of these drugs makes treatment to become potentially harmful or even lethal. In this paper, biodegradable drug-laden QR code label for anti-counterfeiting of drugs is proposed that can provide the non-fluorescence recognition and high capacity. It is fabricated by the laser cutting to achieve the roughness over different surface which causes the difference in the gray levels on the translucent material the QR code pattern, and the micro mold process to obtain the drug-laden biodegradable label. We screened biomaterials presenting the relevant conditions and further requirements of the package. The drug-laden microlabel is on the surface of the troches or the bottom of the capsule and can be read by a simple smartphone QR code reader application. Labeling the pill directly and decoding the information successfully means more convenient and simple operation with non-fluorescence and high capacity in contrast to the traditional methods. Copyright © 2016 Elsevier B.V. All rights reserved.

Fluorescence Imaging of Fast Retrograde Axonal Transport in Living Animals

Directory of Open Access Journals (Sweden)

Dawid Schellingerhout

2009-11-01

Full Text Available Our purpose was to enable an in vivo imaging technology that can assess the anatomy and function of peripheral nerve tissue (neurography. To do this, we designed and tested a fluorescently labeled molecular probe based on the nontoxic C fragment of tetanus toxin (TTc. TTc was purified, labeled, and subjected to immunoassays and cell uptake assays. The compound was then injected into C57BL/6 mice (N = 60 for in vivo imaging and histologic studies. Image analysis and immunohistochemistry were performed. We found that TTc could be labeled with fluorescent moieties without loss of immunoreactivity or biologic potency in cell uptake assays. In vivo fluorescent imaging experiments demonstrated uptake and retrograde transport of the compound along the course of the sciatic nerve and in the spinal cord. Ex vivo imaging and immunohistochemical studies confirmed the presence of TTc in the sciatic nerve and spinal cord, whereas control animals injected with human serum albumin did not exhibit these features. We have demonstrated neurography with a fluorescently labeled molecular imaging contrast agent based on the TTc.

Label-free and live cell imaging by interferometric scattering microscopy.

Science.gov (United States)

Park, Jin-Sung; Lee, Il-Buem; Moon, Hyeon-Min; Joo, Jong-Hyeon; Kim, Kyoung-Hoon; Hong, Seok-Cheol; Cho, Minhaeng

2018-03-14

Despite recent remarkable advances in microscopic techniques, it still remains very challenging to directly observe the complex structure of cytoplasmic organelles in live cells without a fluorescent label. Here we report label-free and live-cell imaging of mammalian cell, Escherischia coli , and yeast, using interferometric scattering microscopy, which reveals the underlying structures of a variety of cytoplasmic organelles as well as the underside structure of the cells. The contact areas of the cells attached onto a glass substrate, e.g. , focal adhesions and filopodia, are clearly discernible. We also found a variety of fringe-like features in the cytoplasmic area, which may reflect the folded structures of cytoplasmic organelles. We thus anticipate that the label-free interferometric scattering microscopy can be used as a powerful tool to shed interferometric light on in vivo structures and dynamics of various intracellular phenomena.

Fluorescence confocal endomicroscopy in biological imaging

Science.gov (United States)

Delaney, Peter; Thomas, Steven; Allen, John; McLaren, Wendy; Murr, Elise; Harris, Martin

2007-02-01

In vivo fluorescence microscopic imaging of biological systems in human disease states and animal models is possible with high optical resolution and mega pixel point-scanning performance using optimised off-the-shelf turn-key devices. There are however various trade-offs between tissue access and instrument performance when miniaturising in vivo microscopy systems. A miniature confocal scanning technology that was developed for clinical human endoscopy has been configured into a portable device for direct hand-held interrogation of living tissue in whole animal models (Optiscan FIVE-1 system). Scanning probes of 6.3mm diameter with a distal tip diameter of 5.0mm were constructed either in a 150mm length for accessible tissue, or a 300mm probe for laparoscopic interrogation of internal tissues in larger animal models. Both devices collect fluorescence confocal images (excitation 488 nm; emission >505 or >550 nm) comprised of 1024 x 1204 sampling points/image frame, with lateral resolution 0.7um; axial resolution 7um; FOV 475 x 475um. The operator can dynamically control imaging depth from the tissue surface to approx 250um in 4um steps via an internally integrated zaxis actuator. Further miniaturisation is achieved using an imaging contact probe based on scanning the proximal end of a high-density optical fibre bundle (~30,000 fibres) of small animal organs, albeit at lower resolution (30,000 sampling points/image). In rodent models, imaging was performed using various fluorescent staining protocols including fluorescently labelled receptor ligands, labelled antibodies, FITC-dextrans, vital dyes and labelled cells administered topically or intravenously. Abdominal organs of large animals were accessed laparoscopically and contrasted using i.v. fluorescein-sodium. Articular cartilage of sheep and pigs was fluorescently stained with calcein-AM or fluorescein. Surface and sub-surface cellular and sub-cellular details could be readily visualised in vivo at high

Specificity of N-terminal methionyl peptidase: analysis by site-directed mutagenesis

International Nuclear Information System (INIS)

Kasper, T.J.; Boissel, J.P.; Bunn, H.F.

1987-01-01

The start site of eukaryotic translation is normally an AUG codon. The corresponding N-terminal methionine is most often removed when the nascent chain reaches about 30 residues. Data from a survey of 1764 eukaryotic protein sequences suggest that the residue adjacent to the initiator Met determines Met cleavage. In order to investigate the mechanism of this reaction, the authors have prepared oligonucleotide-directed mutants of human β-globin from gapped heteroduplexes of a T3/T7 plasmid containing a globin cDNA clone. To date, the authors have produced mutants encoding for 15 of 19 possible amino acid replacements at position 1 in the β-globin chain. These mutants have been confirmed by dideoxy sequencing, transcribed in vitro, and translated in a rabbit reticulocyte lysate in the presence of 35 S-methionine. Labeled translation products were then isolated by cation exchange HPLC, and tryptic peptides were analyzed by RP-HPLC. Thus far, this structural analysis has shown that for β-1 Val, Ala, and Ser, the initiator Met is cleaved, whereas for β-1 Lys, Met, Glu, Trp, Asn, Tyr, and Glu, initiator Met is retained. For β-1 Leu initiator Met is cleaved with a frequency of about 50%. These results are consistent with the data obtained from the previous survey. The expression of site-directed mutants in a cell-free system can also be used to investigate other N-terminal processing events, such as acetylation and myristylation

Fluorogen-activating proteins: beyond classical fluorescent proteins

Directory of Open Access Journals (Sweden)

Shengnan Xu

2018-05-01

Full Text Available Fluorescence imaging is a powerful technique for the real-time noninvasive monitoring of protein dynamics. Recently, fluorogen activating proteins (FAPs/fluorogen probes for protein imaging were developed. Unlike the traditional fluorescent proteins (FPs, FAPs do not fluoresce unless bound to their specific small-molecule fluorogens. When using FAPs/fluorogen probes, a washing step is not required for the removal of free probes from the cells, thus allowing rapid and specific detection of proteins in living cells with high signal-to-noise ratio. Furthermore, with different fluorogens, living cell multi-color proteins labeling system was developed. In this review, we describe about the discovery of FAPs, the design strategy of FAP fluorogens, the application of the FAP technology and the advances of FAP technology in protein labeling systems. KEY WORDS: Fluorogen activating proteins, Fluorogens, Genetically encoded sensors, Fluorescence imaging, Molecular imaging

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)

Synthesis of coumarin or ferrocene labeled nucleosides via Staudinger ligation

Directory of Open Access Journals (Sweden)

Kois Pavol

2006-11-01

Full Text Available Abstract Background Reaction of azides with triaryl phosphines under mild conditions gives iminophosphoranes which can react with almost any kind of electrophilic reagent, e.g. aldehydes/ketones to form imines or esters to form amides. This so-called Staudinger ligation has been employed in a wide range of applications as a general tool for bioconjugation including specific labeling of nucleic acids. Results A new approach for the preparation of labeled nucleosides via intermolecular Staudinger ligation is described. Reaction of azidonucleosides with triphenylphosphine lead to iminophosphorane intermediates, which react subsequently with derivatives of coumarin or ferrocene to form coumarin or ferrocene labeled nucleosides. Fluorescent properties of coumarin labeled nucleosides are determined. Conclusion New coumarin and ferrocene labeled nucleosides were prepared via intermolecular Staudinger ligation. This reaction joins the fluorescent coumarin and biospecific nucleoside to the new molecule with promising fluorescent and electrochemical properties. The isolated yields of products depend on the structure of azidonucleoside and carboxylic acids. A detailed study of the kinetics of the Staudinger ligation with nucleoside substrates is in progress.

Isozyme-specific fluorescent inhibitor of glutathione s-transferase omega 1.

Science.gov (United States)

Son, Junghyun; Lee, Jae-Jung; Lee, Jun-Seok; Schüller, Andreas; Chang, Young-Tae

2010-05-21

Recently, the glutathione S-transferase omega 1 (GSTO1) is suspected to be involved in certain cancers and neurodegenerative diseases. However, profound investigation on the pathological roles of GSTO1 has been hampered by the lack of specific methods to determine or modulate its activity in biological systems containing other isoforms with similar catalytic function. Here, we report a fluorescent compound that is able to inhibit and monitor the activity of GSTO1. We screened 43 fluorescent chemicals and found a compound (6) that binds specifically to the active site of GSTO1. We observed that compound 6 inhibits GSTO1 by covalent modification but spares other isoforms in HEK293 cells and demonstrated that compound 6 could report the activity of GSTO1 in NIH/3T3 or HEK293 cells by measuring the fluorescence intensity of the labeled amount of GSTO1 in SDS-PAGE. Compound 6 is a useful tool to study GSTO1, applicable as a specific inhibitor and an activity reporter.

Optofluidic fluorescent imaging cytometry on a cell phone.

Science.gov (United States)

Zhu, Hongying; Mavandadi, Sam; Coskun, Ahmet F; Yaglidere, Oguzhan; Ozcan, Aydogan

2011-09-01

Fluorescent microscopy and flow cytometry are widely used tools in biomedical sciences. Cost-effective translation of these technologies to remote and resource-limited environments could create new opportunities especially for telemedicine applications. Toward this direction, here we demonstrate the integration of imaging cytometry and fluorescent microscopy on a cell phone using a compact, lightweight, and cost-effective optofluidic attachment. In this cell-phone-based optofluidic imaging cytometry platform, fluorescently labeled particles or cells of interest are continuously delivered to our imaging volume through a disposable microfluidic channel that is positioned above the existing camera unit of the cell phone. The same microfluidic device also acts as a multilayered optofluidic waveguide and efficiently guides our excitation light, which is butt-coupled from the side facets of our microfluidic channel using inexpensive light-emitting diodes. Since the excitation of the sample volume occurs through guided waves that propagate perpendicular to the detection path, our cell-phone camera can record fluorescent movies of the specimens as they are flowing through the microchannel. The digital frames of these fluorescent movies are then rapidly processed to quantify the count and the density of the labeled particles/cells within the target solution of interest. We tested the performance of our cell-phone-based imaging cytometer by measuring the density of white blood cells in human blood samples, which provided a decent match to a commercially available hematology analyzer. We further characterized the imaging quality of the same platform to demonstrate a spatial resolution of ~2 μm. This cell-phone-enabled optofluidic imaging flow cytometer could especially be useful for rapid and sensitive imaging of bodily fluids for conducting various cell counts (e.g., toward monitoring of HIV+ patients) or rare cell analysis as well as for screening of water quality in

Neutrophil labeling with [{sup 99m}Tc]-technetium stannous colloid is complement receptor 3-mediated and increases the neutrophil priming response to lipopolysaccharide

Energy Technology Data Exchange (ETDEWEB)

Gallagher, Hayley [School of Veterinary and Biomedical Sciences, James Cook University, Townsville, Queensland 4811 (Australia); Ramsay, Stuart C. [School of Medicine, James Cook University, Townsville, Queensland (Australia) and Townsville Nuclear Medicine, Mater Hospital, Townsville, Queensland 4812 (Australia)]. E-mail: stuart.ramsey@jcu.edu.au; Barnes, Jodie [School of Veterinary and Biomedical Sciences, James Cook University, Townsville, Queensland 4811 (Australia); Maggs, Jacqueline [Department of Nuclear Medicine, Townsville Hospital, Townsville, Queensland 4814 (Australia); Cassidy, Nathan [Townsville Nuclear Medicine, Mater Hospital, Townsville, Queensland 4812 (Australia); Ketheesan, Natkunam [School of Veterinary and Biomedical Sciences, James Cook University, Townsville, Queensland 4811 (Australia); School of Medicine, James Cook University, Townsville, Queensland (Australia)

2006-04-15

Introduction: [{sup 99m}Tc]-technetium stannous colloid (TcSnC)-labeled white cells are used to image inflammation. Neutrophil labeling with TcSnC is probably phagocytic, but the phagocytic receptor involved is not known. We hypothesised that complement receptor 3 (CR3) plays a key role. Phagocytic labeling could theoretically result in neutrophil activation or priming, affecting the behaviour of labeled cells. Fluorescence-activated cell sorter (FACS) analysis side scatter measurements can assess neutrophil activation and priming. Methods: We tested whether TcSnC neutrophil labeling is CR3-mediated by assessing if neutrophil uptake of TcSnC was inhibited by a monoclonal antibody (mAb) directed at the CD11b component of CR3. We tested if TcSnC-labeled neutrophils show altered activation or priming status, comparing FACS side scatter in labeled and unlabeled neutrophils and examining the effect of lipopolysaccharide (LPS), a known priming agent. Results: Anti-CD11b mAb reduced neutrophil uptake of TcSnC in a dose-dependent fashion. Labeled neutrophils did not show significantly increased side scatter compared to controls. LPS significantly increased side scatter in control cells and labeled neutrophils. However, the increase was significantly greater in labeled neutrophils than unlabeled cells. Conclusions: Neutrophil labeling with TcSnC is related to the function of CR3, a receptor which plays a central role in phagocytosis. TcSnC labeling did not significantly activate or prime neutrophils. However, labeled neutrophils showed a greater priming response to LPS. This could result in labeled neutrophils demonstrating increased adhesion on activated endothelium at sites of infection.

Stable non-covalent labeling of layered silicate nanoparticles for biological imaging.

Science.gov (United States)

Mortimer, Gysell M; Jack, Kevin S; Musumeci, Anthony W; Martin, Darren J; Minchin, Rodney F

2016-04-01

Layered silicate nanoparticles (LSN) are widely used in industrial applications and consumer products. They also have potential benefits in biomedical applications such as implantable devices and for drug delivery. To study how nanomaterials interact with cells and tissues, techniques to track and quantify their movement through different biological compartments are essential. While radiolabels can be very sensitive, particularly for in vivo studies, fluorescent labeling has been preferred in recent years because of the array of methods available to image and quantify fluorescent nanoparticles. However, labeling can be problematic, especially if it alters the physical properties of the nanomaterial. Herein is described a novel non-covalent labeling technique for LSN using readily available fluorescent dimeric cyanine dyes without the need to use excess amounts of dye to achieve labeling, or the need for removal of unbound dye. The approach utilizes the cationic binding properties of layered silicate clays and the multiple quaternary nitrogens associated with the dyes. Preparation of YOYO-1 labeled LSN with optimal dispersion in aqueous media is presented. The utilization of the labeled particles is then demonstrated in cell binding and uptake studies using flow cytometry and confocal microscopy. The labeled LSN are highly fluorescent, stable and exhibit identical physical properties with respect to the unlabeled nanoparticles. The general approach described here is applicable to other cyanine dyes and may be utilized more widely for labeling nanoparticles that comprise a crystalline plate structure with a high binding capacity. Crown Copyright © 2016. Published by Elsevier B.V. All rights reserved.

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.

Direct identification of amyloids by label-free quantitative LC-MS

DEFF Research Database (Denmark)

Dueholm, Morten Simonsen; Danielsen, Heidi Nolsøe; Hansen, Susan Hove

adhesive and therefore bind to pipette tips and other consumables. Pure cultures, large sample volumes and high productivity of amyloids are therefore required for successful purification. We here present a quantitative proteomics technique that allow direct identification of functional amyloid candidates......Direct identification of amyloids by label-free quantitative LC-MS H. N. Danielsen, S. H. Hansen, F.-A. Herbst, P. H. Nielsen, M. S. Dueholm Amyloids are highly ordered fibrillar protein polymers used by organisms from all domains of life due to their exceptional properties. We have previously...... in complex samples based on their structural stability in the presence of increasing concentrations of formic acid....

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.

Activation of 5-[125I]iodonaphthyl-1-azide via excitation of fluorescent (N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)) lipid analogs in living cells. A potential tool for identification of compartment-specific proteins and proteins involved in intracellular transport and metabolism of lipids

International Nuclear Information System (INIS)

Rosenwald, A.G.; Pagano, R.E.; Raviv, Y.

1991-01-01

We describe a new technique for analysis of proteins located near fluorescent lipid analogs in intact living cells using the membrane-permeant, photoactivatable probe, 5-[ 125 I]iodonaphthyl-1-azide ([ 125 I]INA). [ 125 I] INA can be activated directly with UV light or indirectly through excitation of adjacent fluorophores (photosensitizers) with visible light to modify nearby proteins covalently with 125 I. In this report we demonstrate that fluorescent phospholipids and sphingolipids containing N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-6-aminocaproic acid serve as appropriate photosensitizers for [ 125 I]INA. Using Chinese hamster ovary fibroblasts, we optimized the labeling conditions with respect to lipid concentration and time of irradiation and then examined the profiles of cellular proteins that were labeled when fluorescent analogs of ceramide, sphingomyelin, and phosphatidic acid were used as photosensitizers in living cells. The use of different fluorescent lipids, which label different subcellular compartments of cells as determined by fluorescence microscopy, derivatized different sets of cellular proteins with 125 I. The labeled proteins were subsets of the total set of proteins available for derivatization as determined by direct activation of [ 125 I]INA. Most proteins labeled by this procedure were pelleted by centrifugation of cell lysates at high speed (260,000 x g), but several soluble proteins were also labeled under these conditions. The implications of using this technique for identification of compartment-specific proteins and proteins involved in lipid metabolism and transport are discussed

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

Inulin-125I-tyramine, an improved residualizing label for studies on sites of catabolism of circulating proteins

International Nuclear Information System (INIS)

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

1988-01-01

Residualizing labels for protein, such as dilactitol-125I-tyramine (125I-DLT) and cellobiitol-125I-tyramine, have been used to identify the tissue and cellular sites of catabolism of long-lived plasma proteins, such as albumin, immunoglobulins, and lipoproteins. The radioactive degradation products formed from labeled proteins are relatively large, hydrophilic, resistant to lysosomal hydrolases, and accumulate in lysosomes in the cells involved in degradation of the carrier protein. However, the gradual loss of the catabolites from cells (t1/2 approximately 2 days) has limited the usefulness of residualizing labels in studies on longer lived proteins. We describe here a higher molecular weight (Mr approximately 5000), more efficient residualizing glycoconjugate label, inulin-125I-tyramine (125I-InTn). Attachment of 125I-InTn had no effect on the plasma half-life or tissue sites of catabolism of asialofetuin, fetuin, or rat serum albumin in the rat. The half-life for hepatic retention of degradation products from 125I-InTn-labeled asialofetuin was 5 days, compared to 2.3 days for 125I-DLT-labeled asialofetuin. The whole body half-lives for radioactivity from 125I-InTn-, 125I-DLT-, and 125I-labeled rat serum albumin were 7.5, 4.3, and 2.2 days, respectively. The tissue distribution of degradation products from 125I-InTn-labeled proteins agreed with results of previous studies using 125I-DLT, except that a greater fraction of total degradation products was recovered in tissues. Kinetic analyses indicated that the average half-life for retention of 125I-InTn degradation products in tissues is approximately 5 days and suggested that in vivo there are both slow and rapid routes for release of degradation products from cells

Comparison of stabilities in translucency, fluorescence and opalescence of direct and indirect composite resins.

Science.gov (United States)

Yu, Bin; Lee, Young-Keun

2013-01-01

To evaluate translucency, fluorescence and opalescence stabilities of direct and indirect composite resins after aging. One direct (16 shades) and two indirect composite resins (16 and 26 shades) were investigated. Resins were filled in a mold (1 mm thick) and light cured; post-curings were performed for indirect resins. Color was measured before and after 5,000 cycles of thermocycling on a reflection spectrophotometer in reflectance and transmittance modes to calculate parameters for translucency (TP), fluorescence (FL) and opalescence (OP). Differences in the changes of TP, FL and OP after aging by the type of resin were determined by t test, and those were also determined by one-way ANOVA with the factor of the brand or the shade group (P resins; and were -2.0 to 1.8, -0.9 to 0.4 and -2.9 to 3.7, respectively, for indirect resins. Changes in TP were not significantly different by the type of resin, but those in FL and OP were different (P = 0.05). Changes in optical parameters were influenced by the brand or the shade group of the resins (P resins varied depending on type, brand or shade group. Aging significantly affected fluorescence and opalescence, but not translucency, of indirect resins compared to those of direct resins.

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.

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...

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.

Fluorescence guided lymph node biopsy in large animals using direct image projection device

Science.gov (United States)

Ringhausen, Elizabeth; Wang, Tylon; Pitts, Jonathan; Akers, Walter J.

2016-03-01

The use of fluorescence imaging for aiding oncologic surgery is a fast growing field in biomedical imaging, revolutionizing open and minimally invasive surgery practices. We have designed, constructed, and tested a system for fluorescence image acquisition and direct display on the surgical field for fluorescence guided surgery. The system uses a near-infrared sensitive CMOS camera for image acquisition, a near-infra LED light source for excitation, and DLP digital projector for projection of fluorescence image data onto the operating field in real time. Instrument control was implemented in Matlab for image capture, processing of acquired data and alignment of image parameters with the projected pattern. Accuracy of alignment was evaluated statistically to demonstrate sensitivity to small objects and alignment throughout the imaging field. After verification of accurate alignment, feasibility for clinical application was demonstrated in large animal models of sentinel lymph node biopsy. Indocyanine green was injected subcutaneously in Yorkshire pigs at various locations to model sentinel lymph node biopsy in gynecologic cancers, head and neck cancer, and melanoma. Fluorescence was detected by the camera system during operations and projected onto the imaging field, accurately identifying tissues containing the fluorescent tracer at up to 15 frames per second. Fluorescence information was projected as binary green regions after thresholding and denoising raw intensity data. Promising results with this initial clinical scale prototype provided encouraging results for the feasibility of optical projection of acquired luminescence during open oncologic surgeries.

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.

Fluorescence reporters for Hfq oligomerization and RNA annealing

Science.gov (United States)

Panja, Subrata; Woodson, Sarah A.

2015-01-01

Fluorescence spectroscopy is a sensitive technique for detecting protein-protein, protein-RNA and RNA-RNA interactions, requiring only nanomolar concentrations of labeled components. Fluorescence anisotropy provides information about the assembly of multi-subunit proteins, while molecular beacons provide a sensitive and quantitative reporter for base pairing between complementary RNAs. Here we present a detailed protocol for labeling Hfq protein with cyanine 3-maleimide and dansyl chloride to study the protein oligomerization and RNA binding by semi-native polyacrylamide gel electrophoresis (PAGE) and fluorescence anisotropy. We also present a detailed protocol for measuring the rate of annealing between a molecular beacon and a target RNA in the presence of Hfq using a stopped-flow spectrometer. PMID:25579597

Gold nanoparticle enhanced fluorescence anisotropy for the assay of single nucleotide polymorphisms (SNPs) based on toehold-mediated strand-displacement reaction.

Science.gov (United States)

Wang, Xinyi; Zou, Mingjian; Huang, Hongduan; Ren, Yuqian; Li, Limei; Yang, Xiaoda; Li, Na

2013-03-15

We developed a highly differentiating, homogeneous gold nanoparticle (AuNP) enhanced fluorescence anisotropic method for single nucleotide polymorphism (SNP) detection at nanomolar level using toehold-mediated strand-displacement reaction. The template strand, containing a toehold domain with an allele-specific site, was immobilized on the surface of AuNPs, and the solution fluorescence anisotropy was markedly enhanced when the fluorescein-labeled blocking DNA was attached to the AuNP via hybridization. Strand-displacement by the target ssDNA strand resulted in detachment of fluorescein-labeled DNA from AuNPs, and thus decreased fluorescence anisotropy. The drastic kinetic difference in strand-displacement from toehold design was used to distinguish between the perfectly matched and the single-base mismatched strands. Free energy changes were calculated to elucidate the dependence of the differentiation ability on the mutation site in the toehold region. A solid negative signal change can be obtained for single-base mismatched strand in the dynamic range of the calibration curve, and a more than 10-fold signal difference can still be observed in a mixed solution containing 100 times the single-base mismatched strand, indicating the good specificity of the method. This proposed method can be performed with a standard spectrofluorimeter in a homogeneous and cost-effective manner, and has the potential to be extended to the application of fluorescence anisotropy method of SNP detection. Copyright © 2012 Elsevier B.V. All rights reserved.

Predictable efficacy for splenic artery embolization with sites of sequestration using 99mTc labeled platelets in immunogenic idiopathic thrombocytopenic purpura

International Nuclear Information System (INIS)

Tian Aiping

1992-01-01

28 patients with chronic Immunogenic (Idiopathic) Thrombocytopenic purpura (ITP) were determined of the site of sequestration using 99m Tc labeled platelets. Among them, partial splenic artery embolization were done in 20 patients. 7/8 spleen type cases and 1/6 hepato-spleen hepatic type cases have their platelets counts raised above 100 x 10 9 /L. The separation of platelets and its 99m Tc labelling method were also discussed. Above results suggested that the determination of sequestration site of the 99m Tc labelled platelets have some help for the splenic artery embolization therapy in ITP

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

Direct observation of labelled aerosols deposition into the respiratory tract of the rat

International Nuclear Information System (INIS)

Duport, P.

1977-01-01

With a new process the deposition of labelled aerosols into the respiratory tract of the rat can be directly observed. A qualitative convergence between the theoretical retention and real retention for a large scale of aerosol dimensions, is found out [fr

Fluorescence lidar measurements at the archaeological site House of Augustus at Palatino, Rome

Science.gov (United States)

Raimondi, Valentina; Alisi, Chiara; Barup, Kerstin; Bracciale, Maria Paola; Broggi, Alessandra; Conti, Cinzia; Hällström, Jenny; Lognoli, David; Palombi, Lorenzo; Santarelli, Maria Laura; Sprocati, Anna Rosa

2013-10-01

Early diagnostics and documentation fulfill an essential role for an effective planning of conservation and restoration of cultural heritage assets. In particular, remote sensing techniques that do not require the use of scaffolds or lifts, such as fluoresence lidar, can provide useful information to obtain an overall assessment of the status of the investigated surfaces and can be exploited to address analytical studies in selected areas. Here we present the results of a joint Italian-Swedish project focused on documenting and recording the status of some sections of the part closed to the public by using fluorescence hyperspectral imaging lidar. The lidar used a tripled-frequency Nd:YAG laser emitting at 355 nm as excitation source and an intensified, gated 512x512-pixel CCD as detector. The lidar had imaging capabilities thanks to a computer-controlled scanning mirror. The fluorescence characteristics of fresco wall paintings were compared to those of fresco fragments found at the same archaeological site and separately examined in the lab using FT-IR and Raman techniques for the identification of pigments. The fluorescence lidar was also used to remotely detect the growth of phototrophic biodeteriogens on the walls. The fluorescence lidar data were compared with results from biological sampling, cultivation and laboratory analysis by molecular techniques.

(/sup 3/H)MK-801 labels a site on the N-methyl-D-aspartate receptor channel complex in rat brain membranes

Energy Technology Data Exchange (ETDEWEB)

Wong, E H; Knight, A R; Woodruff, G N

1988-01-01

The potent noncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist (/sup 3/H)MK-801 bound with nanomolar affinity to rat brain membranes in a reversible, saturable, and stereospecific manner. The affinity of (/sup 3/H)MK-801 was considerably higher in 5 mM Tris-HCl (pH 7.4) than in previous studies using Krebs-Henseleit buffer. (/sup 3/H)MK-801 labels a homogeneous population of sites in rat cerebral cortical membranes with KD of 6.3 nM and Bmax of 2.37 pmol/mg of protein. This binding was unevenly distributed among brain regions, with hippocampus greater than cortex greater than olfactory bulb = striatum greater than medulla-pons, and the cerebellum failing to show significant binding. Detailed pharmacological characterization indicated (/sup 3/H)MK-801 binding to a site which was competitively and potently inhibited by known noncompetitive NMDA receptor antagonists, such as phencyclidine, thienylcyclohexylpiperidine (TCP), ketamine, N-allylnormetazocine (SKF 10,047), cyclazocine, and etoxadrol, a specificity similar to sites labelled by (/sup 3/H)TCP. These sites were distinct from the high-affinity sites labelled by the sigma receptor ligand (+)-(/sup 3/H)SKF 10,047. (/sup 3/H)MK-801 binding was allosterically modulated by the endogenous NMDA receptor antagonist Mg2+ and by other active divalent cations. These data suggest that (/sup 3/H)MK-801 labels a high-affinity site on the NMDA receptor channel complex, distinct from the NMDA recognition site, which is responsible for the blocking action of MK-801 and other noncompetitive NMDA receptor antagonists.

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

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.

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

2007-01-01

The gram-positive lactic acid bacterium Tetragenococcus halophilus catalyzes the decarboxylation of l-aspartate (Asp) with release of l-alanine (Ala) and CO2. 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-β-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. PMID:17660287

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

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....

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.

ML-MG: Multi-label Learning with Missing Labels Using a Mixed Graph

KAUST Repository

Wu, Baoyuan

2015-12-07

This work focuses on the problem of multi-label learning with missing labels (MLML), which aims to label each test instance with multiple class labels given training instances that have an incomplete/partial set of these labels (i.e. some of their labels are missing). To handle missing labels, we propose a unified model of label dependencies by constructing a mixed graph, which jointly incorporates (i) instance-level similarity and class co-occurrence as undirected edges and (ii) semantic label hierarchy as directed edges. Unlike most MLML methods, We formulate this learning problem transductively as a convex quadratic matrix optimization problem that encourages training label consistency and encodes both types of label dependencies (i.e. undirected and directed edges) using quadratic terms and hard linear constraints. The alternating direction method of multipliers (ADMM) can be used to exactly and efficiently solve this problem. To evaluate our proposed method, we consider two popular applications (image and video annotation), where the label hierarchy can be derived from Wordnet. Experimental results show that our method achieves a significant improvement over state-of-the-art methods in performance and robustness to missing labels.

Electroadsorption-assisted direct determination of trace arsenic without interference using transmission X-ray fluorescence spectroscopy.

Science.gov (United States)

Jiang, Tian-Jia; Guo, Zheng; Liu, Jin-Huai; Huang, Xing-Jiu

2015-08-18

An analytical technique based on electroadsorption and transmission X-ray fluorescence (XRF) for the quantitative determination of arsenic in aqueous solution with ppb-level limits of detection (LOD) is proposed. The approach uses electroadsorption to enhance the sensitivity and LOD of the arsenic XRF response. Amine-functionalized carbonaceous microspheres (NH2-CMSs) are found to be the ideal materials for both the quantitative adsorption of arsenic and XRF analysis due to the basic amine sites on the surface and their noninterference in the XRF spectrum. In electroadsorptive X-ray fluorescence (EA-XRF), arsenic is preconcentrated by a conventional three-electrode system with a positive electricity field around the adsorbents. Then, the quantification of arsenic on the adsorbents is achieved using XRF. The electroadsorption preconcentration can realize the fast transfer of arsenic from the solution to the adsorbents and improve the LOD of conventional XRF compared with directly determining arsenic solution by XRF alone. The sensitivity of 0.09 cnt ppb(-1) is obtained without the interferences from coexisted metal ions in the determination of arsenic, and the LOD is found to be 7 ppb, which is lower than the arsenic guideline value of 10 ppb given by the World Health Organization (WHO). These results demonstrated that XRF coupled with electroadsorption was able to determine trace arsenic in real water sample.

Estimation of presampling MTF in CR systems by using direct fluorescence and its problems

International Nuclear Information System (INIS)

Ono, Kitahei; Inatsu, Hiroshi; Harao, Mototsugu; Itonaga, Haruo; Miyamoto, Hideyuki

2001-01-01

We proposed a method for practical estimation of the presampling modulation transfer function (MTF) of a computed radiography (CR) system by using the MTFs of an imaging plate and the sampling aperture. The MTFs of three imaging plates (GP-25, ST-VN, and RP-1S) with different photostimulable phosphors were measured by using direct fluorescence (the light emitted instantaneously by x-ray exposure), and the presampling MTFs were estimated from these imaging plate MTFs and the sampling aperture MTF. Our results indicated that for imaging plate RP-1S the measured presampling MTF was significantly superior to the estimated presampling MTF at any spatial frequency. This was because the estimated presampling MTF was degraded by the diffusion of direct fluorescence in the protective layer of the imaging plate's surface. Therefore, when the presampling MTF of the imaging plate with a thick protective layer is estimated, correction for the thickness of the protective layer should be carried out. However, the estimated presampling MTF of imaging plates with a thin protective layer were almost the same as the measured presampling MTF, except in the high spatial frequency range. Therefore, we consider this estimation method to be useful and practical, because the spatial resolution property of a CR system can be obtained simply from the imaging plate MTF measured with direct fluorescence. (author)

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

Location of the higher affinity copper site on human hemoglobin by the use of the spin label technique

International Nuclear Information System (INIS)

Tabak, M.; Louro, S.R.W.

1983-11-01

Addition of copper (II) ions to Cys β-93 maleimide spin-labelled human hemoglobin A produces a dramatic decrease in the amplitude of the spin-label ESR spectra. This effect was analyzed in the framework of Leigh's theory which permits interspin distances to be deduced from the effect of dipolar coupling on the ESR spectra and led to an estimate of 9A as the distance between the label and the higher affinity copper site. Taking into account the previous results which suggest that four nitrogen atoms coordinate with copper, and that the N terminal val β-1 and His β-2 residues are involved, the location of the higher affinity copper site is proposed to be at the β 1 β 2 interface of the hemoglobin molecule, involving the N terminal of one β subunit and the C terminal of the other. (Author) [pt

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.

HaloTag protein-mediated specific labeling of living cells with quantum dots

International Nuclear Information System (INIS)

So, Min-kyung; Yao Hequan; Rao Jianghong

2008-01-01

Quantum dots emerge as an attractive alternative to small molecule fluorophores as fluorescent tags for in vivo cell labeling and imaging. This communication presents a method for specific labeling of live cells using quantum dots. The labeling is mediated by HaloTag protein expressed at the cell surface which forms a stable covalent adduct with its ligand (HaloTag ligand). The labeling can be performed in one single step with quantum dot conjugates that are functionalized with HaloTag ligand, or in two steps with biotinylated HaloTag ligand first and followed by streptavidin coated quantum dots. Live cell fluorescence imaging indicates that the labeling is specific and takes place at the cell surface. This HaloTag protein-mediated cell labeling method should facilitate the application of quantum dots for live cell imaging

Near-infrared fluorescence glucose sensing based on glucose/galactose-binding protein coupled to 651-Blue Oxazine

Energy Technology Data Exchange (ETDEWEB)

Khan, Faaizah; Pickup, John C., E-mail: john.pickup@kcl.ac.uk

2013-08-30

Highlights: •We showed that the NIR fluorophore, 651-Blue Oxazine, is solvatochromic (polarity sensitive). •Blue Oxazine was covalently attached to mutants of glucose/galactose-binding protein (GBP). •Fluorescence intensity of GBP-Blue Oxazine increased with addition of glucose. •Fluorescence from bead-immobilised GBP-Blue Oxazine was detectable through skin in vitro. •This shows proof-of-concept for non-invasive glucose sensing using GBP-Blue Oxazine. -- Abstract: Near-infrared (NIR) fluorescent dyes that are environmentally sensitive or solvatochromic are useful tools for protein labelling in in vivo biosensor applications such as glucose monitoring in diabetes since their spectral properties are mostly independent of tissue autofluorescence and light scattering, and they offer potential for non-invasive analyte sensing. We showed that the fluorophore 651-Blue Oxazine is polarity-sensitive, with a marked reduction in NIR fluorescence on increasing solvent polarity. Mutants of glucose/galactose-binding protein (GBP) used as the glucose receptor were site-specifically and covalently labelled with Blue Oxazine using click chemistry. Mutants H152C/A213R and H152C/A213R/L238S showed fluorescence increases of 15% and 21% on addition of saturating glucose concentrations and binding constants of 6 and 25 mM respectively. Fluorescence responses to glucose were preserved when GBP-Blue Oxazine was immobilised to agarose beads, and the beads were excited by NIR light through a mouse skin preparation studied in vitro. We conclude GBP-Blue Oxazine shows proof-of-concept as a non-invasive continuous glucose sensing system.

Theory for site-site pair distribution functions of molecular fluids. II. Approximations for the Percus--Yevick site-site direct correlation functions

International Nuclear Information System (INIS)

Johnson, E.

1977-01-01

A theory for site-site pair distribution functions of molecular fluids is derived from the Ornstein-Zernike equation. Atom-atom pair distribution functions of this theory which were obtained by using different approximations for the Percus-Yevick site-site direct correlation functions are compared

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

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.

Scanning fluorescent microscopy is an alternative for quantitative fluorescent cell analysis.

Science.gov (United States)

Varga, Viktor Sebestyén; Bocsi, József; Sipos, Ferenc; Csendes, Gábor; Tulassay, Zsolt; Molnár, Béla

2004-07-01

Fluorescent measurements on cells are performed today with FCM and laser scanning cytometry. The scientific community dealing with quantitative cell analysis would benefit from the development of a new digital multichannel and virtual microscopy based scanning fluorescent microscopy technology and from its evaluation on routine standardized fluorescent beads and clinical specimens. We applied a commercial motorized fluorescent microscope system. The scanning was done at 20 x (0.5 NA) magnification, on three channels (Rhodamine, FITC, Hoechst). The SFM (scanning fluorescent microscopy) software included the following features: scanning area, exposure time, and channel definition, autofocused scanning, densitometric and morphometric cellular feature determination, gating on scatterplots and frequency histograms, and preparation of galleries of the gated cells. For the calibration and standardization Immuno-Brite beads were used. With application of shading compensation, the CV of fluorescence of the beads decreased from 24.3% to 3.9%. Standard JPEG image compression until 1:150 resulted in no significant change. The change of focus influenced the CV significantly only after +/-5 microm error. SFM is a valuable method for the evaluation of fluorescently labeled cells. Copyright 2004 Wiley-Liss, Inc.

Site-Selective Ribosylation of Fluorescent Nucleobase Analogs Using Purine-Nucleoside Phosphorylase as a Catalyst: Effects of Point Mutations

Directory of Open Access Journals (Sweden)

Alicja Stachelska-Wierzchowska

2015-12-01

Full Text Available Enzymatic ribosylation of fluorescent 8-azapurine derivatives, like 8-azaguanine and 2,6-diamino-8-azapurine, with purine-nucleoside phosphorylase (PNP as a catalyst, leads to N9, N8, and N7-ribosides. The final proportion of the products may be modulated by point mutations in the enzyme active site. As an example, ribosylation of the latter substrate by wild-type calf PNP gives N7- and N8-ribosides, while the N243D mutant directs the ribosyl substitution at N9- and N7-positions. The same mutant allows synthesis of the fluorescent N7-β-d-ribosyl-8-azaguanine. The mutated form of the E. coli PNP, D204N, can be utilized to obtain non-typical ribosides of 8-azaadenine and 2,6-diamino-8-azapurine as well. The N7- and N8-ribosides of the 8-azapurines can be analytically useful, as illustrated by N7-β-d-ribosyl-2,6-diamino-8-azapurine, which is a good fluorogenic substrate for mammalian forms of PNP, including human blood PNP, while the N8-riboside is selective to the E. coli enzyme.