Peptide-Conjugated Quantum Dots Act as the Target Marker for Human Pancreatic Carcinoma Cells

Directory of Open Access Journals (Sweden)

Shuang-ling Li

2016-03-01

Full Text Available Background/Aims: In the present study, we describe a novel and straightforward approach to produce a cyclic- arginine-glycine-aspartic (RGD-peptide-conjugated quantum dot (QD probe as an ideal target tumor biomarker. Due to its specific structure, the probe can be used for targeted imaging of pancreatic carcinoma cells. Methods: Pancreatic carcinoma cells were routinely cultured and marked with QD-RGD probe. The QD-RGD probe on the fluorescence-labeled cancer cell was observed by fluorescence microscopy and laser confocal microscopy. Cancer cell viability was detected by MTT assay after culturing with QD-RGD probe. Results: Fluorescence microscopy and laser confocal microscopy displayed that 10nmol/L QD-RGD probe was able to effectively mark pancreatic carcinoma cells. In comparison with organic dyes and fluorescent proteins, the quantum dot-RGD probe had unique optical and electronic properties. Conclusion: QD-RGD probe has a low cytotoxicity with an excellent optical property and biocompatibility. These findings support further evaluation of QD-RGD probes for the early detection of pancreatic cancer.

Synthesis of two tritium-labeled derivatives of a vasopressin antagonist peptide

International Nuclear Information System (INIS)

Landvatter, S.W.; Heys, J.R.

1986-01-01

SK and F 101926, a potent vasopressin antagonist, has been tritium labeled in the tyrosine residue via exchange followed by solid phase coupling to a hexapeptide. The peptide thus obtained was subsequently coupled with a PMP residue, cleaved from the resin with HF, oxidized by ferricyanide and purified by HPLC giving the desired cyclic peptide. Alternatively, a labeled PMP residue can be prepared via reduction starting from phenol. Conversion of the labeled cyclohexanone to PMP followed by solid phase coupling to a heptapeptide can then afford PMP labeled peptide. 3 refs

Optimizing labeling conditions for cysteine-based peptides with 99mTc

International Nuclear Information System (INIS)

Sabahnoo, Hamideh; Hosseinimehr, Seyed Jalal

2016-01-01

Radiolabelled peptides have attracted a great deal of attention due to their wide applicability in the development of target-specific radiopharmaceuticals. They can easily be used in diagnostic imaging as carriers for the delivery of radionuclides to tumors as well as for therapy. Previous investigations revealed that technetium(V) could form stable complexes with peptide-based ligands of N 3 S type such as Cys-Gly-Gly-Gly. Herein, a targeting HER-2 receptor peptide was labeled with technetium- 99m ( 99m Tc) with two different types of tetrapeptide-based ligands, Cys-Gly-Gly-Gly and Cys-Ser-Ser-Ser. The effect of experimental parameters in the labeling procedure such as type of buffer solutions, pH of media, and type of exchange ligands were optimized toward obtaining maximum labeling yield. The optimum labeling conditions were different for two peptides. Shelf life of both labeled peptides was determined by analytical reversed-phase high-performance liquid chromatography (RP-HPLC) and thin layer chromatography (TLC) that showed radiochemical yield up to 95% even after 4 h. (author)

In-capillary self-assembly and proteolytic cleavage of polyhistidine peptide capped quantum dots

Energy Technology Data Exchange (ETDEWEB)

Wang, Jianhao; Li, Jingyan; Li, Jinchen; Liu, Feifei [School of Pharmaceutical Engineering and Life Science, Changzhou University, Changzhou, Jiangsu, 213164 (China); Zhou, Xiang; Yao, Yi [Changzhou Qianhong Bio-pharma Co. Ltd, Changzhou 213164, Jiangsu (China); Wang, Cheli [School of Pharmaceutical Engineering and Life Science, Changzhou University, Changzhou, Jiangsu, 213164 (China); Qiu, Lin, E-mail: linqiupjj@gmail.com [School of Pharmaceutical Engineering and Life Science, Changzhou University, Changzhou, Jiangsu, 213164 (China); Jiang, Pengju, E-mail: pengju.jiang@gmail.com [School of Pharmaceutical Engineering and Life Science, Changzhou University, Changzhou, Jiangsu, 213164 (China); State Key Laboratory of Pharmaceutical Biotechnology, Nanjing, Jiangsu (China)

2015-10-01

A new method using fluorescence coupled capillary electrophoresis (CE-FL) for monitoring self-assembly and proteolytic cleavage of hexahistidine peptide capped quantum dots (QDs) inside a capillary has been developed in this report. QDs and the ATTO 590-labeled hexahistidine peptide (H6-ATTO) were injected into a capillary, sequentially. Their self-assembly inside the capillary was driven by a metal-affinity force which yielded a new fluorescence signal due to Förster resonance energy transfer (FRET). The highly efficient separation of fluorescent complexes and the FRET process were analyzed using CE-FL. The self-assembly of QDs and biomolecules was found to effectively take place inside the capillary. The kinetics of the assembly was monitored by CE-FL, and the approach was extended to the study of proteolytic cleavage of surface conjugated peptides. Being the first in-depth analysis of in-capillary nanoparticle–biomolecule assembly, the novel approach reported here provides inspiration to the development of QD-based FRET probes for biomedical applications. - Highlights: • We examined the self-assembly QDs with H6-ATTO inside a capillary. • We prove CE-FL to be a powerful method to resolve QDs-H6-ATTO complex. • We achieve chromatographic separation of QDs-H6-ATTO complex. • We discovered a novel strategy for the online detection of thrombin. • This technique integrated “injection, mixing, reaction, separation and detection”.

Photoluminescence of patterned CdSe quantum dot for anti-counterfeiting label on paper

International Nuclear Information System (INIS)

Isnaeni,; Yulianto, Nursidik; Suliyanti, Maria Margaretha

2016-01-01

We successfully developed a method utilizing colloidal CdSe nanocrystalline quantum dot for anti-counterfeiting label on a piece of glossy paper. We deposited numbers and lines patterns of toluene soluble CdSe quantum dot using rubber stamper on a glossy paper. The width of line pattern was about 1-2 mm with 1-2 mm separation between lines. It required less than one minute for deposited CdSe quantum dot on glossy paper to dry and become invisible by naked eyes. However, patterned quantum dot become visible using long-pass filter glasses upon excitation of UV lamp or blue laser. We characterized photoluminescence of line patterns of quantum dot, and we found that emission boundaries of line patterns were clearly observed. The error of line size and shape were mainly due to defect of the original stamper. The emission peak wavelength of CdSe quantum dot was 629 nm. The emission spectrum of deposited quantum dot has full width at half maximum (FWHM) of 30-40 nm. The spectra similarity between deposited quantum dot and the original quantum dot in solution proved that our stamping method can be simply applied on glossy paper without changing basic optical property of the quantum dot. Further development of this technique is potential for anti-counterfeiting label on very important documents or objects.

Photoluminescence of patterned CdSe quantum dot for anti-counterfeiting label on paper

Energy Technology Data Exchange (ETDEWEB)

Isnaeni,, E-mail: isnaeni@lipi.go.id; Yulianto, Nursidik; Suliyanti, Maria Margaretha [Research Center for Physics, Indonesian Institute of Sciences, Building 442, Kawasan Puspiptek, South Tangerang,Banten 15314 Indonesia (Indonesia)

2016-03-11

We successfully developed a method utilizing colloidal CdSe nanocrystalline quantum dot for anti-counterfeiting label on a piece of glossy paper. We deposited numbers and lines patterns of toluene soluble CdSe quantum dot using rubber stamper on a glossy paper. The width of line pattern was about 1-2 mm with 1-2 mm separation between lines. It required less than one minute for deposited CdSe quantum dot on glossy paper to dry and become invisible by naked eyes. However, patterned quantum dot become visible using long-pass filter glasses upon excitation of UV lamp or blue laser. We characterized photoluminescence of line patterns of quantum dot, and we found that emission boundaries of line patterns were clearly observed. The error of line size and shape were mainly due to defect of the original stamper. The emission peak wavelength of CdSe quantum dot was 629 nm. The emission spectrum of deposited quantum dot has full width at half maximum (FWHM) of 30-40 nm. The spectra similarity between deposited quantum dot and the original quantum dot in solution proved that our stamping method can be simply applied on glossy paper without changing basic optical property of the quantum dot. Further development of this technique is potential for anti-counterfeiting label on very important documents or objects.

Synthesis of radioiodinated labeled peptides

International Nuclear Information System (INIS)

Matloobi, M.; Rafii, H.; Beigi, D.; Khalaj, A.; Kamali-Dehghan, M.

2003-01-01

Optimization of radioiodination of peptides is covered by both a direct method in which a constituent tyrosine residue is labeled and indirect method by using an iodinated derivative (SIB) of N succinimidyl 3-(tri-n-butylstannyl) benzoate (ATE) as the intermediate. Radioiodination of IgG and FMLF were performed by direct method using Chloramine-T as an oxidant but since Formyl-Methyl-Leucyl-Phenylalanine, FMLF, does not lend itself for direct radioiodination we performed labeling of FMLF by indirect method via radioiodined SIB at different pH. (author)

Optimizing labeling conditions for cysteine-based peptides with {sup 99m}Tc

Energy Technology Data Exchange (ETDEWEB)

Sabahnoo, Hamideh; Hosseinimehr, Seyed Jalal, E-mail: sjhosseinim@yahoo.com [Department of Radiopharmacy, Faculty of Pharmacy, Pharmaceutical Sciences Research Center, Mazandaran University of Medical Sciences, Sari (Iran, Islamic Republic of)

2016-07-01

Radiolabelled peptides have attracted a great deal of attention due to their wide applicability in the development of target-specific radiopharmaceuticals. They can easily be used in diagnostic imaging as carriers for the delivery of radionuclides to tumors as well as for therapy. Previous investigations revealed that technetium(V) could form stable complexes with peptide-based ligands of N{sub 3}S type such as Cys-Gly-Gly-Gly. Herein, a targeting HER-2 receptor peptide was labeled with technetium-{sup 99m} ({sup 99m}Tc) with two different types of tetrapeptide-based ligands, Cys-Gly-Gly-Gly and Cys-Ser-Ser-Ser. The effect of experimental parameters in the labeling procedure such as type of buffer solutions, pH of media, and type of exchange ligands were optimized toward obtaining maximum labeling yield. The optimum labeling conditions were different for two peptides. Shelf life of both labeled peptides was determined by analytical reversed-phase high-performance liquid chromatography (RP-HPLC) and thin layer chromatography (TLC) that showed radiochemical yield up to 95% even after 4 h. (author)

DOTA-TATE peptides labelling with Lutetium 177: Preliminary study

International Nuclear Information System (INIS)

Aliaga, Eleazar; Robles, Anita; Ramos, Bertha; Martinez, Flor

2014-01-01

he peptide DOTA-TATE was labeled with lutetium 177 according to the methodology provided under the regional project RLA/6/074, sponsored by the IAEA. The labeling was done in 0.26 M gentisic acid solution in 0.8 M sodium acetate buffer, pH 5, at 100 °C for 30 minutes in a dry heating block. The radiochemical purity was assessed by thin layer chromatography, using ITLC SG strips and a mixture of 0.15 M ammonium acetate - methanol (1:1) as solvent. The radiolabeled peptide 177 Lu-DOTA-TATE reached a radiochemical purity of 98 % with a specific activity of 2,8 mCi/µg of peptide. (authors).

Technetium-99m labelled antimicrobial peptides discriminate between bacterial infections and sterile inflammations

International Nuclear Information System (INIS)

Welling, M.M.; Pauwels, E.K.J.; Paulusma-Annema, A.; Nibbering, P.H.; Balter, H.S.

2000-01-01

The aim of this study was to select technetium-99m labelled peptides that can discriminate between bacterial infections and sterile inflammations. For this purpose, we first assessed the binding of various 99m Tc-labelled natural or synthetic peptides, which are based on the sequence of the human antimicrobial peptide ubiquicidin (UBI) or human lactoferrin (hLF), to bacteria and to leucocytes in vitro. In order to select peptides that preferentially bind to bacteria over host cells, radiolabelled peptides were injected into mice intraperitoneally infected with Klebsiella pneumoniae (K. pneumoniae) and the amount of radioactivity associated with the bacteria and with the leucocytes was quantitated. The next phase focussed on discrimination between bacterial infections and sterile inflammatory processes using 99m Tc-labelled peptides in mice intramuscularly infected with various bacteria (e.g. multi-drug-resistant Staphylococcus aureus) and in animals that had been injected with lipopolysaccharides (LPS) of bacterial origin to create a sterile inflammatory process. Also, we studied the distribution of 99m Tc-labelled UBI 29-41 and UBI 18-35 in rabbits having an experimental thigh muscle infection with K. pneumoniae and in rabbits injected with LPS. Based on the results of our in vitro and in vivo binding assays, two peptides, i.e. UBI 29-41 and UBI 18-35, were selected as possible candidates for infection imaging. The radiolabelled peptides can detect infections with both gram-positive and gram-negative bacteria in mice as early as 5-30 min after injection, with a target-to-non-target (T/NT) ratio between 2 and 3; maximum T/NT ratios were seen within 1 h after injection. In rabbits, high T/NT ratios (>5) for 99m Tc-labelled UBI 29-41 were observed from 1 h after injection. No accumulation of the selected 99m Tc-labelled UBI-derived peptides was observed in thighs of mice and rabbits previously injected with LPS. Scintigraphic investigation into the biodistribution of

Technetium-99m labelled antimicrobial peptides discriminate between bacterial infections and sterile inflammations

Energy Technology Data Exchange (ETDEWEB)

Welling, M.M.; Pauwels, E.K.J. [Dept. of Radiology, Leiden University Medical Center (LUMC) (Netherlands); Paulusma-Annema, A.; Nibbering, P.H. [Dept. of Infectious Diseases, Leiden University Medical Center (Netherlands); Balter, H.S. [Centro Investigaciones Nucleares, Univ. of the Republic Uruguay, Montevideo (Uruguay)

2000-03-01

The aim of this study was to select technetium-99m labelled peptides that can discriminate between bacterial infections and sterile inflammations. For this purpose, we first assessed the binding of various {sup 99m}Tc-labelled natural or synthetic peptides, which are based on the sequence of the human antimicrobial peptide ubiquicidin (UBI) or human lactoferrin (hLF), to bacteria and to leucocytes in vitro. In order to select peptides that preferentially bind to bacteria over host cells, radiolabelled peptides were injected into mice intraperitoneally infected with Klebsiella pneumoniae (K. pneumoniae) and the amount of radioactivity associated with the bacteria and with the leucocytes was quantitated. The next phase focussed on discrimination between bacterial infections and sterile inflammatory processes using {sup 99m}Tc-labelled peptides in mice intramuscularly infected with various bacteria (e.g. multi-drug-resistant Staphylococcus aureus) and in animals that had been injected with lipopolysaccharides (LPS) of bacterial origin to create a sterile inflammatory process. Also, we studied the distribution of {sup 99m}Tc-labelled UBI 29-41 and UBI 18-35 in rabbits having an experimental thigh muscle infection with K. pneumoniae and in rabbits injected with LPS. Based on the results of our in vitro and in vivo binding assays, two peptides, i.e. UBI 29-41 and UBI 18-35, were selected as possible candidates for infection imaging. The radiolabelled peptides can detect infections with both gram-positive and gram-negative bacteria in mice as early as 5-30 min after injection, with a target-to-non-target (T/NT) ratio between 2 and 3; maximum T/NT ratios were seen within 1 h after injection. In rabbits, high T/NT ratios (>5) for {sup 99m}Tc-labelled UBI 29-41 were observed from 1 h after injection. No accumulation of the selected {sup 99m}Tc-labelled UBI-derived peptides was observed in thighs of mice and rabbits previously injected with LPS. Scintigraphic investigation

Pengaruh Customer Relationship Management (CRM) Terhadap Customer Satisfaction Dan Customer Loyalty Pada Pelanggan Sushi Tei Surabaya

OpenAIRE

Chandra Dewi, Ayu Abriyanti

2015-01-01

Manajemen hubungan pelanggan (CRM) merupakan bagian dari strategi pemasaran untuk memuaskan, dan menjaga loyalitas pelanggan. CRM dapat menjadi stimulus terhadap kepuasan dan loyalitas konsumen, bahwa CRM yang tepat berdampak pada kepuasan dan selanjutnya akan mempengaruhi loyalitas pelanggan. Populasi penelitian adalah seluruh pelanggan Sushi Tei Surabaya, dan sampel sebanyak 86 pelanggan Sushi Tei yang dipilih dengan teknik purposive sampling, dan teknik analisis yang digunakan adalah Gener...

Feasibility and availability of 68Ga-labelled peptides

International Nuclear Information System (INIS)

Decristoforo, Clemens; Pickett, Roger D.; Verbruggen, Alfons

2012-01-01

68 Ga has attracted tremendous interest as a radionuclide for PET based on its suitable half-life of 68 min, high positron emission yield and ready availability from 68 Ge/ 68 Ga generators, making it independent of cyclotron production. 68 Ga-labelled DOTA-conjugated somatostatin analogues, including DOTA-TOC, DOTA-TATE and DOTA-NOC, have driven the development of technologies to provide such radiopharmaceuticals for clinical applications mainly in the diagnosis of somatostatin receptor-expressing tumours. We summarize the issues determining the feasibility and availability of 68 Ga-labelled peptides, including generator technology, 68 Ga generator eluate postprocessing methods, radiolabelling, automation and peptide developments, and also quality assurance and regulatory aspects. 68 Ge/ 68 Ga generators based on SnO 2 , TiO 2 or organic matrices are today routinely supplied to nuclear medicine departments, and a variety of automated systems for postprocessing and radiolabelling have been developed. New developments include improved chelators for 68 Ga that could open new ways to utilize this technology. Challenges and limitations in the on-site preparation and use of 68 Ga-labelled peptides outside the marketing authorization track are also discussed. (orig.)

125I-labeling and purification of peptide hormones and bovine serum albumin

International Nuclear Information System (INIS)

Nemeth, J; Jakab, B.; Szilvassy, Z.; Oroszi, G.; Roeth, E.; Magyarlaki, M.; Farkas, B.

2002-01-01

The iodination and separation of various diagnostically and/or experimentally important peptides including (Tyr 1 )-somatostatin-14, rat Tyr-α-calcitonin gene-related peptide (23-37), motilin and vasoactive intestinal peptide, furthermore bovine serum albumin are described. All species were iodinated by the iodogen method. The 125 I-labeled peptide products were separated by reversed-phase HPLC, the specific activities of mono-iodinated forms are near identical with the theoretical value. The labeled bovine serum albumin was separated by Sephadex G-100 gel filtration. (author)

Tritium labelling of PACAP-38 using a synthetic diiodinated precursor peptide

DEFF Research Database (Denmark)

Pedersen, Martin Holst Friborg; Baun, Michael

2012-01-01

In the interest of developing efficient methods for tritium labelling peptides, we here demonstrate the successful labelling of PACAP-38 (pituitary adenylate cyclase-activating polypeptide), a 38-mer peptide, using a synthetic diiodinated PACAP-38 precursor. In this example, we employ standard hy...... hydrogenation chemistry with the use of a heterogeneous palladium catalyst and carrier-free tritium gas on a tritium manifold system....

Characterization of VCAM-1-binding peptide-functionalized quantum dots for molecular imaging of inflamed endothelium.

Directory of Open Access Journals (Sweden)

Yun Chen

Full Text Available Inflammation-induced activation of endothelium constitutes one of the earliest changes during atherogenesis. New imaging techniques that allow detecting activated endothelial cells can improve the identification of persons at high cardiovascular risk in early stages. Quantum dots (QDs have attractive optical properties such as bright fluorescence and high photostability, and have been increasingly studied and developed for bio-imaging and bio-targeting applications. We report here the development of vascular cell adhesion molecule-1 binding peptide (VCAM-1 binding peptide functionalized QDs (VQDs from amino QDs. It was found that the QD fluorescence signal in tumor necrosis factor [Formula: see text] (TNF-[Formula: see text] treated endothelial cells in vitro was significantly higher when these cells were labeled with VQDs than amino QDs. The VQD labeling of TNF-[Formula: see text]-treated endothelial cells was VCAM-1 specific since pre-incubation with recombinant VCAM-1 blocked cells' uptake of VQDs. Our ex vivo and in vivo experiments showed that in the inflamed endothelium, QD fluorescence signal from VQDs was also much stronger than that of amino QDs. Moreover, we observed that the QD fluorescence peak was significantly blue-shifted after VQDs interacted with aortic endothelial cells in vivo and in vitro. A similar blue-shift was observed after VQDs were incubated with recombinant VCAM-1 in tube. We anticipate that the specific interaction between VQDs and VCAM-1 and the blue-shift of the QD fluorescence peak can be very useful for VCAM-1 detection in vivo.

Tritium labeling of amino acids and peptides with liquid and solid tritium

International Nuclear Information System (INIS)

Peng, C.T.; Hua, R.L.; Souers, P.C.; Coronado, P.R.

1988-01-01

Amino acids and peptides were labeled with liquid and solid tritium at 21 K and 9 K. At these low temperatures radiation degradation is minimal, and tritium incorporation increases with tritium concentration and exposure time. Ring saturation in L-phenyl-alanine does not occur. Peptide linkage in oligopeptides is stable toward tritium. Deiodination in 3-iodotyrosine and 3,5-diiodotyrosine occurs readily and proceeds in steps by losing one iodine atom at a time. Nickel and noble metal supported catalysts when used as supports for dispersion of the substrate promote tritium labeling at 21 K. Our study shows that both liquid and solid tritium are potentially useful agents for labeling peptides and proteins. 11 refs., 1 fig., 3 tabs

Tritium labeling of amino acids and peptides with liquid and solid tritium

International Nuclear Information System (INIS)

Souers, P.C.; Coronado, P.R.; Peng, C.T.; Hua, R.L.

1988-01-01

Amino acids and peptides were labeled with liquid and solid tritium at 21/degree/K and 9/degree/K. At these low temperatures radiation degradation is minimal, and tritium incorporation increases with tritium concentration and exposure time. Ring saturation in L-phenylalanine does not occur. Peptide linkage in oligopeptides is stable toward tritium. Deiodination in 3-iodotyrosine and 3,5-diiodotyrosine occurs readily and proceeds in steps by losing one iodine atom at a time. Nickel and noble metal supported catalysts when used as supports for dispersion of the substrate promote tritium labeling at 21 K. Our study shows that both liquid and solid tritiums are potentially useful agents for labeling peptides and proteins

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)

Feasibility and availability of ⁶⁸Ga-labelled peptides.

Science.gov (United States)

Decristoforo, Clemens; Pickett, Roger D; Verbruggen, Alfons

2012-02-01

(68)Ga has attracted tremendous interest as a radionuclide for PET based on its suitable half-life of 68 min, high positron emission yield and ready availability from (68)Ge/(68)Ga generators, making it independent of cyclotron production. (68)Ga-labelled DOTA-conjugated somatostatin analogues, including DOTA-TOC, DOTA-TATE and DOTA-NOC, have driven the development of technologies to provide such radiopharmaceuticals for clinical applications mainly in the diagnosis of somatostatin receptor-expressing tumours. We summarize the issues determining the feasibility and availability of (68)Ga-labelled peptides, including generator technology, (68)Ga generator eluate postprocessing methods, radiolabelling, automation and peptide developments, and also quality assurance and regulatory aspects. (68)Ge/(68)Ga generators based on SnO(2), TiO(2) or organic matrices are today routinely supplied to nuclear medicine departments, and a variety of automated systems for postprocessing and radiolabelling have been developed. New developments include improved chelators for (68)Ga that could open new ways to utilize this technology. Challenges and limitations in the on-site preparation and use of (68)Ga-labelled peptides outside the marketing authorization track are also discussed.

PRELIMINARY CONSIDERATIONS ON SUSHI AS POTENTIALLY HAZARDOUS FOOD

OpenAIRE

G. Liuzzo; P. Bonilauri; R. Leonelli; A. Serraino; S. Bentley

2011-01-01

The Authors studied physicochemical properties (pH and Aw) of samples of Nigiri sushi and their ingredients along their shelf life, integrating those results with a predictive microbiological model, in order to determine or to rule out the growth of Listeria monocytogenes above the thresholds set by Reg.(EU) 2073/2005. Results point towards substantial containment of the target biological hazard, even though the prevention of thermal abuse is a keypoint in increasing safety.

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.

111In and 90Y labelled peptide radiopharmaceuticals for diagnosis and therapy

International Nuclear Information System (INIS)

Obenaus, E.; Crudo, J.L.; Castiglia, S.G. de

2004-01-01

Full text: The application of 111In-labelled octreotide to target somatostatin receptors on tumour cells has gained acceptance as a diagnostic procedure for demonstrating neuroendocrine and other SSTR-positive tumors. A further advance in the field of somatostatin analogues is the development of macrocyclic chelators that also bind beta particle emitters like 90Y and 177Lu. [90Y-DOTA, Tyr3] octreotide and [177Lu-DOTA, Tyr3] octreotate are currently being tried as the therapeutic agents. The aim of this work was to label two somatostatin analogues (Tyr3 Octreotide and Lanreotide) with 111In and 90Y using DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) as a chelating agent. DOTA in the form of the tri-t-butyl ester was coupled to the Lys5 (BOC) protected Tyr3octreotide and Lys5 (BOC) protected lanreotide in N,N-dimethylformamide, in a three-step reaction involving conjugation, using HATU and diisopropylethylamine as coupling reagents, de-protection with trifluoroacetic and HPLC purification of the conjugates. The DOTALAN and DOTATOC were labelled with 111In and 90Y and assayed by HPLC. In both cases solutions of gentisic acid in sodium acetate 0.4M pH=5.0 and conjugated peptides were added to the 111In or 90Y chloride. The final solution was heated at 90 deg. C for 25 minutes. We tried different amounts of the two peptide complexes per mCi of 111In/90Y and quality control of the labelled products was performed with the following HPLC system: reverse phase column, flow rate 1ml-min, UV/radiometric detection and a gradient solvent: solvent A: acetonitrile, solvent B: water TFA 0.1%, gradient: 0-3min 100% B, linear increase of eluent A to 50% from 3-13 min, 13-18 min 50% A, 18-20 min linear increase of eluent A to 70%. Serum stability was determined after 4 and 24 hr. incubation of the labeled peptide in human serum at 37 deg. C. After precipitation of proteins with acetonitrile the incubation mixture was analyzed by HPLC. Normal Wistar rats were

Feasibility and availability of {sup 68}Ga-labelled peptides

Energy Technology Data Exchange (ETDEWEB)

Decristoforo, Clemens [Innsbruck Medical University, Department of Nuclear Medicine, Innsbruck (Austria); European Directorate of Quality of Medicines, Group 14, Radioactive Compounds, The European Pharmacopeia, Strasbourg (France); Pickett, Roger D. [GE Healthcare, Little Chalfont (United Kingdom); European Directorate of Quality of Medicines, Group 14, Radioactive Compounds, The European Pharmacopeia, Strasbourg (France); Verbruggen, Alfons [University of Leuven, Laboratory of Radiopharmacy, Department of Pharmaceutical Sciences, Leuven (Belgium); European Directorate of Quality of Medicines, Group 14, Radioactive Compounds, The European Pharmacopeia, Strasbourg (France)

2012-02-15

{sup 68}Ga has attracted tremendous interest as a radionuclide for PET based on its suitable half-life of 68 min, high positron emission yield and ready availability from {sup 68}Ge/{sup 68}Ga generators, making it independent of cyclotron production. {sup 68}Ga-labelled DOTA-conjugated somatostatin analogues, including DOTA-TOC, DOTA-TATE and DOTA-NOC, have driven the development of technologies to provide such radiopharmaceuticals for clinical applications mainly in the diagnosis of somatostatin receptor-expressing tumours. We summarize the issues determining the feasibility and availability of {sup 68}Ga-labelled peptides, including generator technology, {sup 68}Ga generator eluate postprocessing methods, radiolabelling, automation and peptide developments, and also quality assurance and regulatory aspects. {sup 68}Ge/{sup 68}Ga generators based on SnO{sub 2}, TiO{sub 2} or organic matrices are today routinely supplied to nuclear medicine departments, and a variety of automated systems for postprocessing and radiolabelling have been developed. New developments include improved chelators for {sup 68}Ga that could open new ways to utilize this technology. Challenges and limitations in the on-site preparation and use of {sup 68}Ga-labelled peptides outside the marketing authorization track are also discussed. (orig.)

Tetrazine-Containing Amino Acid for Peptide Modification and Live Cell Labeling.

Directory of Open Access Journals (Sweden)

Zhongqiu Ni

Full Text Available A novel amino acid derivative 3-(4-(1, 2, 4, 5-tetrazine-3-yl phenyl-2-aminopropanoic acid was synthesized in this study. The compound possessed better water-solubility and was synthesized more easily compared with the well-known and commercially available 3-(p-benzylamino-1, 2, 4, 5-tetrazine. Tetrazine-containing amino acid showed excellent stability in biological media and might be used for cancer cell labeling. Moreover, the compound remained relatively stable in 50% TFA/DCM with little decomposition after prolonged exposure at room temperature. The compound could be utilized as phenylalanine or tyrosine analogue in peptide modification, and the tetrazine-containing peptide demonstrated more significant biological activity than that of the parent peptide. The combination of tetrazine group and amino acid offered broad development prospects of the bioorthogonal labeling and peptide synthesis.

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

Synthesis of carbon nanohorns/chitosan/quantum dots nanocomposite and its applications in cells labeling and in vivo imaging

Energy Technology Data Exchange (ETDEWEB)

Li, Jing; He, Zhe [Chemistry Department, Northeastern University, Shenyang 110819 (China); Guo, Changrun [College of Life Sciences, Jilin University, Changchun 130023 (China); Wang, Liping, E-mail: wanglp@jlu.edu.cn [College of Life Sciences, Jilin University, Changchun 130023 (China); Xu, Shukun, E-mail: xushukun46@126.com [Chemistry Department, Northeastern University, Shenyang 110819 (China)

2014-01-15

Due to the unique optical and chemical features of quantum dots and the special structural advantages of carbon nanohorns, it is highly desirable to synthesize nanohorns/quantum dots nanocompsite which can be applied in cell labeling and in vivo imaging. Here, we report a new method which uses chitosan as connector to synthesize nanohorns/chitosan/quantum dots fluorescent nanocomposite. Further more, the synthesized nanocomposite demonstrated strong red fluorescence and had been successfully used in Hela cells labeling and in vivo imaging of Caenorhabditis elegans (C. elegans). -- Highlights: Carbon nanohorn/chitosan/QDs nanocomposite was prepared by covalent linkage The nanocomposite was successfully used in the labeling of HeLa cells The nanocomposite was used for in vivo imaging with C. elegans as animal mode.

Preparation of ⁶⁸Ga-labelled DOTA-peptides using a manual labelling approach for small-animal PET imaging.

Science.gov (United States)

Romero, Eduardo; Martínez, Alfonso; Oteo, Marta; García, Angel; Morcillo, Miguel Angel

2016-01-01

(68)Ga-DOTA-peptides are a promising PET radiotracers used in the detection of different tumours types due to their ability for binding specifically receptors overexpressed in these. Furthermore, (68)Ga can be produced by a (68)Ge/(68)Ga generator on site which is a very good alternative to cyclotron-based PET isotopes. Here, we describe a manual labelling approach for the synthesis of (68)Ga-labelled DOTA-peptides based on concentration and purification of the commercial (68)Ga/(68)Ga generator eluate using an anion exchange-cartridge. (68)Ga-DOTA-TATE was used to image a pheochromocytoma xenograft mouse model by a microPET/CT scanner. The method described provides satisfactory results, allowing the subsequent (68)Ga use to label DOTA-peptides. The simplicity of the method along with its implementation reduced cost, makes it useful in preclinical PET studies. Copyright © 2015 Elsevier Ltd. All rights reserved.

A 99Tcm labeled HYNIC peptide 'tracer' libraries on continuous cellulose membrane supports

International Nuclear Information System (INIS)

Zeng Jun; Liu Ciyi; Xie Wenhui; Hu Silong; Jin Xiumu

2007-01-01

Objective: The interference of bifunctional ligands with activities of small peptides has long been recognized. To solve the problem, the hydrazine-nicotinamide (HYNIC) conjugated peptide 'tracer' libraries were synthesized on a continuous cellulose membrane support and the 99 Tc m labeled heat shock protein 70 (HSP70) binding peptides were identified by screening libraries with HSP70. Methods: Octapeptide libraries were prepared by manual spot synthesis. HYNIC peptides were C terminally attached to cellulose via a (β-Ala) 2 spacer. For screening, the cellulose membranes were incubated with human HSP70 (or biotin labeled HSP70) after nonspecific blocking. Alkaline phosphatase labeled streptavidin and Ab against HSP70 were used for the detection of HSP70 binding. Human lung cancer cell lines (A549 and H460) were cultured in RPMI1640 medium supplemented with 10% fetal calf serum and antibiotics. For in vivo test, 2 x l0 5 cells were subcutaneously transplanted into the chest of female nude mice. Results: Quality control of HYNIC peptide libraries was good as carried out by 99 Tc m labeling. Because peptide NLLRLTG had high affinity for HSP70 family members, 99 Tc m -HYNIC-NLLRLTG was used as the control. Fifteen HYNIC peptides were found with HSP70 binding property. Among them, eight peptides had higher uptake (percentage activity of injection dose pergram of tissue, %ID/g) values than 99 Tc m -HYNIC-NLLRLTG in tumor. 99 Tc m -HYNIC-QGVLTGTR had the best distribution in tumors. Six hours after injection, the %ID/g values of 99 Tc m HYNIC-QGVLTGTR and 99 Tc m -HYNIC-NLLRLTG in tumor were (1.15±0.32)% ID/g and (0.75±0.24)% ID/g respectively. In vivo replace studies and heat shock stress of tumors demonstrated that 99 Tc m -HYNIC-QGVLTGTR was the HSP70 binding peptide compound, but not 99 Tc m -HYNIC-NLLRLTG. Conclusions: The identification of 99 Tc m labeled HSP70 binding peptides from HYNIC conjugated octapeptide libraries facilitated the hypothesis of the 'tracer

Solid-phase peptide quantitation assay using labeled monoclonal antibody and glutaraldehyde fixation

International Nuclear Information System (INIS)

Kasprzyk, P.G.; Cuttitta, F.; Avis, I.; Nakanishi, Y.; Treston, A.; Wong, H.; Walsh, J.H.; Mulshine, J.L.

1988-01-01

A solid-phase radioimmunoassay utilizing iodinated peptide-specific monoclonal antibody as a detection system instead of labeled peptide has been developed. Regional specific monoclonal antibodies to either gastrin-releasing peptide or gastrin were used as models to validate the general application of our modified assay. Conditions for radioactive labeling of the monoclonal antibody were determined to minimize oxidant damage, which compromises the sensitivity of other reported peptide quantitation assays. Pretreatment of 96-well polyvinyl chloride test plates with a 5% glutaraldehyde solution resulted in consistent retention of sufficient target peptide on the solid-phase matrix to allow precise quantitation. This quantitative method is completed within 1 h of peptide solid phasing. Pretreatment of assay plates with glutaraldehyde increased binding of target peptide and maximized antibody binding by optimizing antigen presentation. The hypothesis that glutaraldehyde affects both peptide binding to the plate and orientation of the peptide was confirmed by analysis of several peptide analogs. These studies indicate that peptide binding was mediated through a free amino group leaving the carboxy-terminal portion of the target peptide accessible for antibody binding. It was observed that the length of the peptide also affects the amount of monoclonal antibody that will bind. Under the optimal conditions, results from quantitation of gastrin-releasing peptide in relevant samples agree well with those from previously reported techniques. Thus, we report here a modified microplate assay which may be generally applied for the rapid and sensitive quantitation of peptide hormones

Technetium-99m somatostatin analogues: effect of labelling methods and peptide sequence

International Nuclear Information System (INIS)

Decristoforo, C.; Mather, S.J.

1999-01-01

In this paper the preclinical evaluation of the somatostatin analogue RC160 labelled with technetium-99m using bifunctional chelators (BFCs) based on the hydrazinonicotinamide (HYNIC) and N 3 S system is described and a comparison made with [Tyr 3 ]-octreotide (TOC). Conjugates of both peptides with HYNIC, and of RC160 with benzoyl-MAG 3 and an N 3 S-adipate derivative were prepared and radiolabelling performed at high specific activities using tricine, tricine/nicotinic acid and ethylenediamine-N,N'-diacetic adic (EDDA) as co-ligands for HYNIC conjugates. All conjugates and 99m Tc-labelled peptides showed preserved binding affinity for the somatostatin receptor (IC50, Kd 99m Tc-RC160 derivatives compared with 99m Tc-EDDA/HYNIC-[Tyr 3 ]-octreotide (0.2%-3.5%ID/g vs 9.7%ID/g) and correlated well with the reduced internalisation rate for RC160 derivatives. Our results show that the selection of the labelling approach as well as the right choice of the peptide structure are crucial for labelling peptides with 99m Tc to achieve complexes with favourable biodistribution. Despite the relatively low tumour uptake compared with 99m Tc-EDDA/HYNIC-[Tyr 3 ]-octreotide, 99m Tc-RC160 could play a role in imaging tumours that do not bind octreotide derivatives. (orig.)

Efficient and Selective Chemical Labeling of Electrochemically Generated Peptides Based on Spirolactone Chemistry

NARCIS (Netherlands)

Zhang, Tao; Niu, Xiaoyu; Yuan, Tao; Tessari, Marco; de Vries, Marcel P.; Permentier, Hjalmar P.; Bischoff, Rainer

2016-01-01

Specific digestion of proteins is an essential step for mass spectrometry-based proteomics, and the chemical labeling of the resulting peptides is often used for peptide enrichment or the introduction of desirable tags. Cleavage of the peptide bond following electrochemical oxidation of Tyr or Trp

123I labelled vasoactive intestinal peptide: Optimization of the radioiodination method, in vivo and in vitro assays

International Nuclear Information System (INIS)

Pozzi, O.R.; Sajaroff, E.O.; Edreira, M.; Gomez, S.I.; Manzini, A.

2002-01-01

In the framework of the CRP, our country has worked on the optimization of synthesis, quality control, in vitro and in vivo evaluation of 123 I radiopharmaceuticals based on peptides. We have worked on selective labelling procedures using prosthetic groups with the goal to create a strong carbon-halogen bond, which will be resistant to in vivo dehalogenation and other catabolic processes. The method utilizes the labelling agent, reactive with ε-amino lysine groups, N-succinimidyl 3-iodobenzoate. This conjugation agent was radiolabelled by using an organometallic intermediate to facilitate the reaction. The organometallic N-succinimidyl 3-(tri-nbutylstannyl) benzoate (ATE) was made in a three-step synthesis pathway. The yields for the reactions of this synthetic pathway were: 56.4% for the first reaction, 67% for the second, and 58% for the ATE (469 mg, 0.92 mmol). Because of only 0.1 μmol of ATE is needed for the labelling of peptides, from one batch of organic synthesis we obtained ATE to make more than 9000 labelling. The N-succinimidyl 3-(tri-n-butylstannyl) benzoate (ATE) was radiolabelled in 55-85% radiochemical yield to obtain the N-succinimidyl 3-iodobenzoate ( [ 131 I]SIB ). Parameters like reactive concentration and isolation method of the labelling agent were studied. The labelling agent [ 131 I]SIB was subsequently conjugated to a human IgG and a peptide. A chemotactic peptide was used as a model peptide. A potent chemotactic peptide N-formyl-norleucyl-leucyl-phenylalanyl-norleucyltyrosyl- lysine (fNleLFNleYK) was derivatized by reaction with the labelling agent in 59-75% of radiochemical yield. This derivatized peptide bound specifically to human polymorphonuclear leukocytes in vitro and exhibited biological activity in a superoxide production assay. Binding affinity IC 50 : 36 nM, in the displacing of [ 3 H]fMLF binding, and IC 50 : 68 nM, in the displacing of the fNleLFNleYK-[ 131 I]SIB conjugate, for the derivatized peptide were obtained. Because

44Sc for labeling of DOTA- and NODAGA-functionalized peptides: preclinical in vitro and in vivo investigations.

Science.gov (United States)

Domnanich, Katharina A; Müller, Cristina; Farkas, Renata; Schmid, Raffaella M; Ponsard, Bernard; Schibli, Roger; Türler, Andreas; van der Meulen, Nicholas P

2017-01-01

Recently, 44 Sc (T 1/2  = 3.97 h, Eβ + av  = 632 keV, I = 94.3 %) has emerged as an attractive radiometal candidate for PET imaging using DOTA-functionalized biomolecules. The aim of this study was to investigate the potential of using NODAGA for the coordination of 44 Sc. Two pairs of DOTA/NODAGA-derivatized peptides were investigated in vitro and in vivo and the results obtained with 44 Sc compared with its 68 Ga-labeled counterparts.DOTA-RGD and NODAGA-RGD, as well as DOTA-NOC and NODAGA-NOC, were labeled with 44 Sc and 68 Ga, respectively. The radiopeptides were investigated with regard to their stability in buffer solution and under metal challenge conditions using Fe 3+ and Cu 2+ . Time-dependent biodistribution studies and PET/CT imaging were performed in U87MG and AR42J tumor-bearing mice. Both RGD- and NOC-based peptides with a DOTA chelator were readily labeled with 44 Sc and 68 Ga, respectively, and remained stable over at least 4 half-lives of the corresponding radionuclide. In contrast, the labeling of NODAGA-functionalized peptides with 44 Sc was more challenging and the resulting radiopeptides were clearly less stable than the DOTA-derivatized matches. 44 Sc-NODAGA peptides were clearly more susceptible to metal challenge than 44 Sc-DOTA peptides under the same conditions. Instability of 68 Ga-labeled peptides was only observed if they were coordinated with a DOTA in the presence of excess Cu 2+ . Biodistribution data of the 44 Sc-labeled peptides were largely comparable with the data obtained with the 68 Ga-labeled counterparts. It was only in the liver tissue that the uptake of 68 Ga-labeled DOTA compounds was markedly higher than for the 44 Sc-labeled version and this was also visible on PET/CT images. The 44 Sc-labeled NODAGA-peptides showed a similar tissue distribution to those of the DOTA peptides without any obvious signs of in vivo instability. Although DOTA revealed to be the preferred chelator for stable coordination of 44

Radiopharmacological evaluation of 18F-labeled phosphatidylserine-binding peptides for molecular imaging of apoptosis

International Nuclear Information System (INIS)

Wuest, Melinda; Perreault, Amanda; Kapty, Janice; Richter, Susan; Foerster, Christian; Bergman, Cody; Way, Jenilee; Mercer, John; Wuest, Frank

2015-01-01

Introduction: Radiolabeled phosphatidylserine (PS)-binding peptides represent an innovative strategy for molecular imaging of apoptosis with positron emission tomography (PET). The goal of this study was the radiopharmacological evaluation of radiolabeled peptides for their binding to PS on apoptotic cancer cells, involving metabolic stability, cellular uptake, biodistribution, and dynamic PET imaging experiments. Methods: Binding of peptides LIKKPF, PGDLSR, FBz-LIKKPF, FBz-PGDLSR, FBAM-CLIKKPF and FBAM-CPGDLSR to PS was analyzed in a newly developed radiometric binding assay using 64 Cu-labeled wild-type annexin-V as radiotracer. Radiolabeling of most potent peptides with fluorine-18 was carried out with thiol-selective prosthetic group [ 18 F]FBAM to give [ 18 F]FBAM-CLIKKPF and [ 18 F]FBAM-CPGDLSR. [ 18 F]FBAM-labeled peptides were studied in camptothecin-induced apoptotic human T lymphocyte Jurkat cells, and in a murine EL4 tumor model of apoptosis using dynamic PET imaging and biodistribution. Results: Peptides LIKKPF and PGDLSR inhibited binding of 64 Cu-labeled annexin-V to immobilized PS in the millimolar range (IC 50 10–15 mM) compared to annexin-V (45 nM). Introduction of FBAM prosthetic group slightly increased inhibitory potencies (FBAM-CLIKKPF: IC 50 = 1 mM; FBAM-CPGDLSR: IC 50 = 6 mM). Radiolabeling succeeded in good radiochemical yields of 50–54% using a chemoselective alkylation reaction of peptides CLIKKPF and CPGDLSR with [ 18 F]FBAM. In vivo metabolic stability studies in mice revealed 40–60% of intact peptides at 5 min p.i. decreasing to 25% for [ 18 F]FBAM-CLIKKPF and less than 5% for [ 18 F]FBAM-CPGDLSR at 15 min p.i.. Cell binding of [ 18 F]FBAM-CLIKKPF in drug-treated Jurkat cells was significantly higher compared to untreated cells, but this was not observed for [ 18 F]FBAM-CPGDLSR. Dynamic PET imaging experiments showed that baseline uptake of [ 18 F]FBAM-CLIKKPF in EL4 tumors was higher (SUV 5min 0.46, SUV 60min 0.13) compared to

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

Science.gov (United States)

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

2015-12-01

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

Recommended administered activities for {sup 68}Ga-labelled peptides in paediatric nuclear medicine

Energy Technology Data Exchange (ETDEWEB)

Machado, J.S.; Beykan, S.; Lassmann, M. [University Hospital Wuerzburg, Department of Nuclear Medicine, Wuerzburg (Germany); Herrmann, K. [University Hospital Wuerzburg, Department of Nuclear Medicine, Wuerzburg (Germany); David Geffen School of Medicine at UCLA, Department of Molecular and Medical Pharmacology, Los Angeles, CA (United States)

2016-10-15

The aim of this study was to establish a method for determining administered activities for {sup 68}Ga-labelled peptides. Dose calculations were based on the weight-independent effective dose model proposed by the EANM paediatric dosage card for use in paediatric nuclear medicine. Previously published time-integrated activity coefficients for {sup 68}Ga-DOTATATE, {sup 68}Ga-DOTATOC and {sup 68}Ga-pentixafor were used to calculate age-independent effective doses. Consequently, the corresponding weight-dependent effective dose coefficients were rescaled according to the formalism of the EANM dosage card to determine the radiopharmaceutical class of {sup 68}Ga-labelled peptides (''multiples'') and to calculate the baseline activities based on an upper limit for administered activity (185 MBq) in an adult. All calculated normalization factors suggest that the {sup 68}Ga-labelled peptides are class ''B'' radiopharmaceuticals. The baseline activity for all compounds is 12.8 MBq. In analogy to {sup 18}F-fluoride, we recommend a minimum activity of 14 MBq. For paediatric nuclear medicine applications involving {sup 68}Ga-labelled peptides, we suggest determining administered activities based on the formalism proposed in this work. The corresponding effective doses from these procedures will remain age-independent. (orig.)

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.

Optimization of synthesis and quality control procedures for the preparation of 18F-labelled peptides

International Nuclear Information System (INIS)

Amartey, J.K.

2002-01-01

Radiohalogenation via prosthetic groups has provided a useful route for labelling proteins, peptides and drug molecules. This method is the only option available as far as molecules that are not amenable to the classical radiohalogenation reactions are concerned. This pertains to proteins and peptides lacking tyrosyl groups in their structure. More importantly, radiofluorination by electrophilic method has not been developed for labelling these macromolecules. The need to optimize methods and techniques to enable efficient labelling and fully exploit the potential biochemical application of these molecules prompted this investigation. Reaction conditions were optimized to prepare ethyl 4-[ 18 F]-benzoate from an ammonium precursor, ethyl 4-trimethylammoniumbenzoate.triflate in excellent yield. The fluorinated ester was hydrolyzed quantitatively to the acid. The acid was then converted to the activated N-succinimidylfluorobenzoate (SFB) using O- (Nsuccinimidyl)- tetramethyluroniumtetrafluoroborate also typically in greater than 90% radiochemical yield. The activated ester was purified either by HPLC or SEPPAK cartridge and was conjugated to a potent chemotactic peptide (Formyl-Nle-Leu-Phe-Nle-Tyr-Lys) as a model in acetonitrile. The conjugate was purified chromatographically or by SEPPAK cartridges. To ascertain the retention of biological activity of the peptide after these chemical manipulations, the superoxide production assay was employed. The purified [ 19 F]-peptide conjugate specifically bound and activated human polymorphonuclear leukocytes to generate superoxide in a dose dependent manner. Biodistribution in normal mice showed that the conjugated peptide did not suffer any significant dehalogenation in vivo. This was indicated by the low uptake of activity in bone. The methodology developed with the chemotactic peptide was used to label RC-160 (cyclic-D-Phe-Cys-Tyr-D-Trp-Lys (Boc)- Val-Cys-Trp-NH2) a SST analog. The conjugate peptide inhibited the growth of

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

Science.gov (United States)

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

2015-06-01

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

Functional characterization and axonal transport of quantum dot labeled BDNF

OpenAIRE

Xie, Wenjun; Zhang, Kai; Cui, Bianxiao

2012-01-01

Brain derived neurotrophic factor (BDNF) plays a key role in the growth, development and maintenance of the central and peripheral nervous systems. Exogenous BDNF activates its membrane receptors at the axon terminal, and subsequently sends regulation signals to the cell body. To understand how BDNF signal propagates in neurons, it is important to follow the trafficking of BDNF after it is internalized at the axon terminal. Here we labeled BDNF with bright, photostable quantum dot (QD-BDNF) a...

Yttrium-labelled peptides for therapy of NET

Energy Technology Data Exchange (ETDEWEB)

Bodei, Lisa; Grana, Chiara M.; Chinol, Marco; Baio, Silvia M.; Paganelli, Giovanni [European Institute of Oncology, Division of Nuclear Medicine, Milan (Italy); Cremonesi, Marta [European Institute of Oncology, Division of Medical Physics, Milan (Italy); Severi, Stefano [Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori, Radiometabolic Unit, Meldola (FC) (Italy)

2012-02-15

Peptide receptor radionuclide therapy (PRRT) consists in the systemic administration of a synthetic peptide, labelled with a suitable beta-emitting radionuclide, able to irradiate tumours and their metastases via the internalization through a specific receptor, overexpressed on the cell membrane. After 15 years of experience, we can state that PRRT with {sup 90}Y-labelled peptides is generally well tolerated. Acute side effects are usually mild, some of which are related to the co-administration of amino acids, such as nausea. Others are related to the radiopeptide, such as fatigue or the exacerbation of an endocrine syndrome, which rarely occurs in functioning tumours. Chronic and permanent effects on target organs, particularly the kidneys and the bone marrow, are generally mild if the necessary precautions are taken. Currently, the potential risk to kidney and red marrow limits the amount of radioactivity that may be administered. However, when tumour masses are irradiated with adequate doses, volume reduction may be observed. {sup 90}Y-octreotide has been the most widely used radiopeptide in the first 8-10 years of experience. Unfortunately, all of the published results derive from different and inhomogeneous phase I/II studies. Hence, a direct comparison is virtually impossible to date. Nevertheless, even with these limitations, objective responses are registered in 10-34% of patients. The optimal timing of {sup 90}Y-DOTATOC in the management of somatostatin receptor (SSTR)-positive tumours and the way in which it should be integrated with other treatments have yet to be defined, and prospective phase II/III trials comparing the efficacy and toxicity of different schemes of {sup 90}Y-DOTATOC administration are still warranted. (orig.)

Yttrium-labelled peptides for therapy of NET

International Nuclear Information System (INIS)

Bodei, Lisa; Grana, Chiara M.; Chinol, Marco; Baio, Silvia M.; Paganelli, Giovanni; Cremonesi, Marta; Severi, Stefano

2012-01-01

Peptide receptor radionuclide therapy (PRRT) consists in the systemic administration of a synthetic peptide, labelled with a suitable beta-emitting radionuclide, able to irradiate tumours and their metastases via the internalization through a specific receptor, overexpressed on the cell membrane. After 15 years of experience, we can state that PRRT with 90 Y-labelled peptides is generally well tolerated. Acute side effects are usually mild, some of which are related to the co-administration of amino acids, such as nausea. Others are related to the radiopeptide, such as fatigue or the exacerbation of an endocrine syndrome, which rarely occurs in functioning tumours. Chronic and permanent effects on target organs, particularly the kidneys and the bone marrow, are generally mild if the necessary precautions are taken. Currently, the potential risk to kidney and red marrow limits the amount of radioactivity that may be administered. However, when tumour masses are irradiated with adequate doses, volume reduction may be observed. 90 Y-octreotide has been the most widely used radiopeptide in the first 8-10 years of experience. Unfortunately, all of the published results derive from different and inhomogeneous phase I/II studies. Hence, a direct comparison is virtually impossible to date. Nevertheless, even with these limitations, objective responses are registered in 10-34% of patients. The optimal timing of 90 Y-DOTATOC in the management of somatostatin receptor (SSTR)-positive tumours and the way in which it should be integrated with other treatments have yet to be defined, and prospective phase II/III trials comparing the efficacy and toxicity of different schemes of 90 Y-DOTATOC administration are still warranted. (orig.)

Quantification of pharmaceutical peptides using selenium as an elemental detection label

DEFF Research Database (Denmark)

Møller, Laura Hyrup; Gabel-Jensen, Charlotte; Franzyk, Henrik

2014-01-01

analysis of cell samples by LC-ICP-MS showed mainly uptake of the intact peptides, while the amount of intact peptides in cell lysates was semi-quantitatively determined. The selenium-containing penetratin analogues were to some extent degraded in pure cell medium, while an extensive degradation......The aim of the present work was to demonstrate how selenium labelling of a synthetic cell-penetrating peptide may be employed in evaluation of stability and quantitative estimation of cellular uptake by inductively coupled plasma mass spectrometry (ICP-MS). Two analogues of the cell...

Rhenium 188 labelling of peptide conjugates

International Nuclear Information System (INIS)

Melendez-Alafort, Laura

2001-01-01

Many human tumours express high levels, of somatostatin receptors. In order to make possible a radiotherapeutic treatment of this kind for tumour a series of somatostatin analogues that can tightly chelate beta emitting isotopes have been developed in recent years. The work carried out for this thesis has been aimed towards development of a new therapeutic radiopharmaceutical for treatment of somatostatin receptor positive tumours. The first chapters describe work with technetium-99m to establish the labelling and analytical conditions for a somatostatin analogue, [Tyr 3 ]-octreotide (TOC), as a precursor to undertaking labelling studies with the beta emitter rhenium-188. 6-Hydrazinopyridine-3-carboxylic acid (HYNIC) was conjugated to TOC and labelled with 99m using different coligands. Then the stability, receptor binding and biodistribution of each complex were assessed. 99m Tc-HYNIC-TOC using EDDA as coligand showed the best characteristics, and was superior for tumour imaging in humans than the commercially available 111 In-DTPA-octreotide. The conditions for labelling the HYNIC-TOC conjugate with 188 Re were then optimised using tricine as a co-ligand. A labelling yield of ∼80% was achieved. After purification however, the stability of the complex was low. The use of other coligand systems which had proved useful for 99m Tc labelling was explored, but yields were very poor. Other chelators such as diethylenetriamine pentaacetic acid (DTPA), dimercaptosuccinic acid (DMSA) and mercaptoacetyltriglycine (MAG 3 ) were studied as potential co-ligand agents to label the HYNIC-TOC conjugate with 188 Re but, again low yields of the labelled peptide complexes were achieved. A novel 188 Re-HYNIC complex was prepared in high yields using N-N-disubstituted dithiocarbamates as coligands. However to date, the specific activities achieved with this system are relatively low. The use of the [ 99m Tc(CO) 3 (H 2 O) 3 ] complex to label the HYNIC-TOC conjugate was investigated

How chelators can modified in vitro and in vivo response of indirect labelled BPTI peptide

International Nuclear Information System (INIS)

Crudo, J.L.; Obenaus, E.R.; Zapata, A.M.; Castiglia, S.G. de

2002-01-01

Aim: the aim of this work was to compare labeling methods for BPTI and in vitro and in vivo properties when it was labeled with 99mTc via two different chelators HYNIC and DTPA. BPTI (provided by Dr. Hnatowich) was used as a model for future labeling of HNE2, an analog peptide used in infection imaging. Materials and Methods: conjugation reaction was carried out at molar ratio of 5:1 between the chelators (NHS-HYNIC and cDTPA) and the peptide BPTI. After Biogel P4 purification, the first four fractions were labeled with 99mTc. The one with the best labelling efficiency was selected for testing each conjugated product. DTPA-BPTI was labeled with 99mTc at pH 5.2. HYNIC-BPTI was labeled with 99mTc using tricine as coligand. 99mTc-DTPA-BPTI was purified by C18 Sep-Pack cartridge due to its impurities. Both 99mTc-labelled BPTI were analysed by reverse phase high performance liquid chromatography (RP-HPLC). Stability of the labeled peptides was asses ed by incubating at 37 0 C with PBS 0.05M pH 7.2, for 24h. 99mTc-peptides were tested for instability toward cysteine, binding to serum protein and trypsin. Bio distributions in normal NIH mice were carried out for both labeled peptides at 2 h post injection (p.i.). Results: Radiochemical purity of 99mTc-((Tricine)HYNIC-BPTI) and 99mTc-DTPA-BPTI determined by RP-HPLC was higher than 95 % and 55 % respectively. The range of specific activity of these products was between 0.07-0.6-MBq/μg. The radiochemical purity of the C18 Sp-Pack purified 99mTc-DTPA-BPTI was 77 %. The dissociation value for 99mTc-((Tricine)HYNIC-BPTI)) was less than 10 % in PBS at 24 h. The results of a cysteine challenge assay of labelled BPTI showed anomalous behaviour for HYNIC conjugate. The activity bound to serum protein of 99mTc-((Tricine)HYNIC-BPTI) was 20 % higher than the value of 99mTc-DTPA-BPTI. G75 radiometric elution profile of 99mTc-((Tricine)HYNIC-BPTI)) : trypsin (Molar ratio 1:10) showed positive binding. Biodistribution in NIH normal mice

Synthesis of stable isotopically labeled peptides with filter-assisted enzymatic labeling for the diagnosis of hepatitis B virus infection utilizing mass spectrometry-based proteomics strategy

International Nuclear Information System (INIS)

Tsai, Hsing-Fen; Hsiao, He-Hsuan

2017-01-01

A facile method for the preparation of stable isotopically labeled peptides was developed by means of filter-assisted tryptic "1"6O/"1"8O water labeling, which could be directly applied to the determination of hepatitis B virus infection from human serum with tandem mass spectrometry. Tryptic peptides of hepatitis B surface antigen or hepatitis B e antigen from different subtypes of hepatitis B virus were synthesized with traditional solid-phase peptide synthesis as potential biomarkers. Trypsin catalyzed oxygen-18 exchange at their amidated c-terminus of arginine or lysine residue. The protease catalyzed oxygen-18 to oxygen-16 back exchange reaction was eliminated due to the complete removal of trypsin by the centrifugal filter containing a thin membrane associated with molecular weight cut-off of 10 KDa. The synthetic isotopic peptides were spiked into trichloroacetic acid/acetone precipitated human serum as internal standards and were selectively detected with multiplexed parallel reaction monitoring on a hybrid quadrupole-orbitrap mass spectrometer. The limit of detection for all synthetic peptides were in the range of 0.09 fmol–1.13 fmol. The results indicated that the peptide YLWEWASVR derived from hepatitis B surface antigen was quantified approximately 200 fmol per μl serum and may serve as a diagnostic biomarker for the detection of hepatitis B virus infected disease. - Highlights: • Facile synthesis of an inexpensive and highly reproducible stable isotopically labeled peptides. • Complete incorporation of two "1"8O atoms into synthesized peptides with filter-assisted enzymatic labeling. • Targeted analysis with parallel reaction monitoring assay for the disease diagnosis.

Synthesis of stable isotopically labeled peptides with filter-assisted enzymatic labeling for the diagnosis of hepatitis B virus infection utilizing mass spectrometry-based proteomics strategy

Energy Technology Data Exchange (ETDEWEB)

Tsai, Hsing-Fen; Hsiao, He-Hsuan, E-mail: hhhsiao@dragon.nchu.edu.tw

2017-03-01

A facile method for the preparation of stable isotopically labeled peptides was developed by means of filter-assisted tryptic {sup 16}O/{sup 18}O water labeling, which could be directly applied to the determination of hepatitis B virus infection from human serum with tandem mass spectrometry. Tryptic peptides of hepatitis B surface antigen or hepatitis B e antigen from different subtypes of hepatitis B virus were synthesized with traditional solid-phase peptide synthesis as potential biomarkers. Trypsin catalyzed oxygen-18 exchange at their amidated c-terminus of arginine or lysine residue. The protease catalyzed oxygen-18 to oxygen-16 back exchange reaction was eliminated due to the complete removal of trypsin by the centrifugal filter containing a thin membrane associated with molecular weight cut-off of 10 KDa. The synthetic isotopic peptides were spiked into trichloroacetic acid/acetone precipitated human serum as internal standards and were selectively detected with multiplexed parallel reaction monitoring on a hybrid quadrupole-orbitrap mass spectrometer. The limit of detection for all synthetic peptides were in the range of 0.09 fmol–1.13 fmol. The results indicated that the peptide YLWEWASVR derived from hepatitis B surface antigen was quantified approximately 200 fmol per μl serum and may serve as a diagnostic biomarker for the detection of hepatitis B virus infected disease. - Highlights: • Facile synthesis of an inexpensive and highly reproducible stable isotopically labeled peptides. • Complete incorporation of two {sup 18}O atoms into synthesized peptides with filter-assisted enzymatic labeling. • Targeted analysis with parallel reaction monitoring assay for the disease diagnosis.

HYNIC a bifunctional prosthetic group for the labelling of peptides with 99mTc and 18FDG

International Nuclear Information System (INIS)

Sepideh Khoshbakht; Omid Sabzevari; Mohsen Amini; Faramarz Mehrnejad; Kimia Tabib; Soraya Shahhosseini; Shahid Beheshti University of Medical Sciences, Tehran

2016-01-01

With regard to high reactivity and chemoselectivity of HYNIC towards carbonyl of acyclic form of 18 FDG and its stable complexes with 99m Tc, in this study, LIKKPF as the model peptide was conjugated with HYNIC and labelled with 99m Tc (RCP[90 %) and 18 FDG for the first time. The RCP of [70 % was achieved for labelling with 18 FDG, in the presence of glucose (50-250 lg/mL). Our results showed the high potential of HYNIC conjugated peptides for labelling with 99m Tc and 18 FDG as 18 F-fluorinated prosthetic group, to be clinically accepted for the radiolabelling of peptides. (author)

Confirmation of hydrazone formation in HYNIC-peptide conjugate preparation, and its hydrolysis during labeling with 99mTc

International Nuclear Information System (INIS)

Gandomkar, M.; Najafi, R.; Shafiei, M.; Ebrahimi, S.E.S.

2007-01-01

Because of its monodenticity, 6-hydrazinopyridine-3-carboxylic acid (HYNIC) is of interest as a bifunctional chelator for labeling peptide with 99m Tc. Here, we confirm the formation of hydrazone in HYNIC-conjugated peptide. The preparative HPLC was used to purify the HYNIC conjugated somatostatin-based peptide and the result showed two peaks, even after two consecutive purifications. Analysis of these peaks by mass spectrometry indicated the presence of hydrazone, produced during preparation conjugate. Further, we have shown that presence of hydrazone really does not matter because under 99m Tc-labeling conditions, hydrazone is hydrolyzed back to HYNIC that then chelates 99m Tc. A HYNIC-peptide conjugate freeze-dried kit was also prepared in a mildly acidic or neutral condition with a final pH of 6-7. The kit was then labeled by 99m Tc and incubated in 100 dec. C for 10 min, and a labeling yield of >95% was obtained

Mobility of TOAC spin-labelled peptides binding to the Src SH3 domain studied by paramagnetic NMR

Energy Technology Data Exchange (ETDEWEB)

Lindfors, Hanna E. [Leiden University, Leiden Institute of Chemistry, Gorlaeus Laboratories (Netherlands); Koning, Peter E. de; Wouter Drijfhout, Jan [Leiden University Medical Centre, Department of Immunohematology and Blood Transfusion (Netherlands); Venezia, Brigida; Ubbink, Marcellus [Leiden University, Leiden Institute of Chemistry, Gorlaeus Laboratories (Netherlands)], E-mail: m.ubbink@chem.leidenuniv.nl

2008-07-15

Paramagnetic relaxation enhancement provides a tool for studying the dynamics as well as the structure of macromolecular complexes. The application of side-chain coupled spin-labels is limited by the mobility of the free radical. The cyclic, rigid amino acid spin-label TOAC (2,2,6,6-Tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid), which can be incorporated straightforwardly by peptide synthesis, provides an attractive alternative. In this study, TOAC was incorporated into a peptide derived from focal adhesion kinase (FAK), and the interaction of the peptide with the Src homology 3 (SH3) domain of Src kinase was studied, using paramagnetic NMR. Placing TOAC within the binding motif of the peptide has a considerable effect on the peptide-protein binding, lowering the affinity substantially. When the TOAC is positioned just outside the binding motif, the binding constant remains nearly unaffected. Although the SH3 domain binds weakly and transiently to proline-rich peptides from FAK, the interaction is not very dynamic and the relative position of the spin-label to the protein is well-defined. It is concluded that TOAC can be used to generate reliable paramagnetic NMR restraints.

Mobility of TOAC spin-labelled peptides binding to the Src SH3 domain studied by paramagnetic NMR

International Nuclear Information System (INIS)

Lindfors, Hanna E.; Koning, Peter E. de; Wouter Drijfhout, Jan; Venezia, Brigida; Ubbink, Marcellus

2008-01-01

Paramagnetic relaxation enhancement provides a tool for studying the dynamics as well as the structure of macromolecular complexes. The application of side-chain coupled spin-labels is limited by the mobility of the free radical. The cyclic, rigid amino acid spin-label TOAC (2,2,6,6-Tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid), which can be incorporated straightforwardly by peptide synthesis, provides an attractive alternative. In this study, TOAC was incorporated into a peptide derived from focal adhesion kinase (FAK), and the interaction of the peptide with the Src homology 3 (SH3) domain of Src kinase was studied, using paramagnetic NMR. Placing TOAC within the binding motif of the peptide has a considerable effect on the peptide-protein binding, lowering the affinity substantially. When the TOAC is positioned just outside the binding motif, the binding constant remains nearly unaffected. Although the SH3 domain binds weakly and transiently to proline-rich peptides from FAK, the interaction is not very dynamic and the relative position of the spin-label to the protein is well-defined. It is concluded that TOAC can be used to generate reliable paramagnetic NMR restraints

Optimizing labelling conditions of 213Bi-DOTATATE for preclinical applications of peptide receptor targeted alpha therapy.

Science.gov (United States)

Chan, Ho Sze; de Blois, Erik; Konijnenberg, Mark W; Morgenstern, Alfred; Bruchertseifer, Frank; Norenberg, Jeffrey P; Verzijlbergen, Fred J; de Jong, Marion; Breeman, Wouter A P

2017-01-01

213 Bismuth ( 213 Bi, T 1/2 = 45.6 min) is one of the most frequently used α-emitters in cancer research. High specific activity radioligands are required for peptide receptor radionuclide therapy. The use of generators containing less than 222 MBq 225 Ac (actinium), due to limited availability and the high cost to produce large-scale 225 Ac/ 213 Bi generators, might complicate in vitro and in vivo applications though.Here we present optimized labelling conditions of a DOTA-peptide with an 225 Ac/ 213 Bi generator (< 222 MBq) for preclinical applications using DOTA-Tyr 3 -octreotate (DOTATATE), a somatostatin analogue. The following labelling conditions of DOTATATE with 213 Bi were investigated; peptide mass was varied from 1.7 to 7.0 nmol, concentration of TRIS buffer from 0.15 mol.L -1 to 0.34 mol.L -1 , and ascorbic acid from 0 to 71 mmol.L -1 in 800 μL. All reactions were performed at 95 °C for 5 min. After incubation, DTPA (50 nmol) was added to stop the labelling reaction. Besides optimizing the labelling conditions, incorporation yield was determined by ITLC-SG and radiochemical purity (RCP) was monitored by RP-HPLC up to 120 min after labelling. Dosimetry studies in the reaction vial were performed using Monte Carlo and in vitro clonogenic assay was performed with a rat pancreatic tumour cell line, CA20948. At least 3.5 nmol DOTATATE was required to obtain incorporation ≥ 99 % with 100 MBq 213 Bi (at optimized pH conditions, pH 8.3 with 0.15 mol.L -1 TRIS) in a reaction volume of 800 μL. The cumulative absorbed dose in the reaction vial was 230 Gy/100 MBq in 30 min. A minimal final concentration of 0.9 mmol.L -1 ascorbic acid was required for ~100 MBq (t = 0) to minimize radiation damage of DOTATATE. The osmolarity was decreased to 0.45 Osmol/L.Under optimized labelling conditions, 213 Bi-DOTATATE remained stable up to 2 h after labelling, RCP was ≥ 85 %. In vitro showed a negative correlation between ascorbic acid

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

Synthesis of water-soluble scaffolds for peptide cyclization, labeling, and ligation

NARCIS (Netherlands)

Smeenk, L.E.J.; Dailly, N.; Hiemstra, H.; van Maarseveen, J.H.; Timmerman, P.

2012-01-01

The synthesis and applications of water-soluble scaffolds that conformationally constrain side chain unprotected linear peptides containing two cysteines are described. These scaffolds contain a functionality with orthogonal reactivity to be used for labeling and ligation. This is illustrated by the

Labeling of Complex III peptides in beef heart mitochondria and submitochondrial particles by diazonium bensene [35S]sulfonate

International Nuclear Information System (INIS)

Mendel-Hartvig, I.; Nelson, B.D.

1978-01-01

The labeling of Complex III peptides in DABS-treated mitochondria and submitochondrial particles from beef heart is reported. The results are consistent with the labeling of the two core proteins on the matrix surface of the inner membrane, and of a 29 000 molecular weight peptide on the cytoplasmic surface. (Auth.)

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)

Confirmation of hydrazone formation in HYNIC-peptide conjugate preparation, and its hydrolysis during labeling with {sup 99m}Tc

Energy Technology Data Exchange (ETDEWEB)

Gandomkar, M. [Radioisotope Division, Nuclear Research Center, Atomic Energy Organization of Iran (AEOI), Tehran (Iran, Islamic Republic of)]. E-mail: msgandomkar@yahoo.com; Najafi, R. [Radioisotope Division, Nuclear Research Center, Atomic Energy Organization of Iran (AEOI), Tehran (Iran, Islamic Republic of); Shafiei, M. [Radioisotope Division, Nuclear Research Center, Atomic Energy Organization of Iran (AEOI), Tehran (Iran, Islamic Republic of); Ebrahimi, S.E.S. [Department of Medicinal Chemistry, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran (Iran, Islamic Republic of)

2007-07-15

Because of its monodenticity, 6-hydrazinopyridine-3-carboxylic acid (HYNIC) is of interest as a bifunctional chelator for labeling peptide with {sup 99m}Tc. Here, we confirm the formation of hydrazone in HYNIC-conjugated peptide. The preparative HPLC was used to purify the HYNIC conjugated somatostatin-based peptide and the result showed two peaks, even after two consecutive purifications. Analysis of these peaks by mass spectrometry indicated the presence of hydrazone, produced during preparation conjugate. Further, we have shown that presence of hydrazone really does not matter because under {sup 99m}Tc-labeling conditions, hydrazone is hydrolyzed back to HYNIC that then chelates {sup 99m}Tc. A HYNIC-peptide conjugate freeze-dried kit was also prepared in a mildly acidic or neutral condition with a final pH of 6-7. The kit was then labeled by {sup 99m}Tc and incubated in 100 dec. C for 10 min, and a labeling yield of >95% was obtained.

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.

Synthesis and characterization of C@CdS dots in aqueous solution and their application in labeling human gastric carcinoma cells

Energy Technology Data Exchange (ETDEWEB)

Dong, Wei, E-mail: dongwei5873@126.com [Shenyang Medical College, Department of Chemistry (China); Zhou, Siqi [Fengtian Hospital Affiliated to Shenyang Medical College, ICU (China); Dong, Yan [Shenyang Pharmaceutical University, Experiment Center of Traditional Chinese Medicine Department (China); Wang, Jingwen; Liu, Shuang; Zhu, Pengxia [Shenyang Medical College, Department of Chemistry (China)

2015-03-15

Colloidal carbon spheres coated with cadmium sulfide nanoparticle quantum dots (C@CdS dots) with the particle size smaller than 50 nm were synthesized by an aqueous approach. The effects of different reaction times, temperatures, and pH values were carefully investigated to optimize the synthesis conditions. The as-prepared C@CdS dots were linked with mouse anti-human carcinoembryonic antigen antibody and goat anti-mouse immunoglobulin (IgG) to directly and indirectly label fixed human gastric carcinoma cells, respectively. The cytotoxicity of the C@CdS dots was also tested using the human gastric carcinoma cells. No apparent cytotoxicity was observed, which suggested the potential application of the as-prepared C@CdS dots in bioimaging.

Influence of quantum dot labels on single molecule movement in the plasma membrane

DEFF Research Database (Denmark)

Clausen, Mathias P.; Lagerholm, B. Christoffer

2011-01-01

Single particle tracking results are very dependent on the probe that is used. In this study we have investigated the influence that functionalized quantum dots (QDs) have on the recorded movement in single molecule tracking experiments of plasma membrane species in live cells. Potential issues...... for simultaneous investigations of different plasma membrane species in order to discriminate the effect of the label from differences in movement of the target molecules....

[The Qualitative Analysis of the Amide Derivative of HLDF-6 Peptide and Its Metabolites with the Use of Tritium- and Deuterium-Labeled Derivatives].

Science.gov (United States)

Zolotarev, A; Dadayan, A K; Kost, N V; Voevodina, M E; Sokolov, O Y; Kozik, V S; Shram, S I; Azev, V N; Bocharov, E V; Bogachouk, A P; Lipkin, V M; Myasoedov, N F

2015-01-01

The goal of the study was to elaborate the pharmacokinetics methods of the amide derivative of peptide HLDF-6 (TGENHR-NH2) and its range of nootropic and neuroprotective activity is wide. The hexapeptide 41TGENHR46 is a fragment of the HDLF differentiation factor. It forms the basis for the development of preventive and therapeutic preparations for treating cerebrovascular and neurodegenerative conditions. Pharmacokinetic and molecular mechanisms of the action of the HLDF-6 peptide were studied using tritium- and deuterium-labeled derivatives of this peptide, produced with the use of the high-temperature solid-state catalytic isotope exchange reaction (HSCIE). This reaction was employed to produce the tritium-labeled peptide [3H]TGENHR-NH2 with a molar radioactivity of 230 Ci/mmol and the deuterium-labeled peptide [2H]TGENHR-NH2 with an average deuterium incorporation equal to 10.5 atoms. It was shown by the NMR spectroscopy that the isotope label distribution over the labeled peptide's molecule was uniform, which allowed qualitative analysis ofboth the peptide itself and its fragments in the organism's tissues to be conducted. The newly developed pharmacokinetics method makes it possible to avoid almost completely losses of the peptides under study due to biodegradation during the analysis of tissues. These labeled peptides were used in mice, rats and rabbits to study the pharmacokinetics of the peptide and to calculate the values of its principal pharmacokinetic parameters. Characteristics of its pharmacokinetic profile in the blood were obtained, the hypothesis of pharmacokinetics linearity tested, its metabolism analyzed and its bioavailability value, 34%, calculated. It has been shown that the studied TGENHR-NH2 peptide shows high resistance to hydrolysis in the blood plasma, with dipeptidyl aminopeptidases making the largest contribution to its hydrolysis.

Comparative biodistribution of 12 {sup 111}In-labelled gastrin/CCK2 receptor-targeting peptides

Energy Technology Data Exchange (ETDEWEB)

Laverman, Peter; Joosten, Lieke; Eek, Annemarie; Roosenburg, Susan; Oyen, Wim J.G.; Boerman, Otto C. [Radboud University Nijmegen Medical Centre, Department of Nuclear Medicine, Nijmegen (Netherlands); Peitl, Petra Kolenc [University Medical Centre Ljubljana, Department of Nuclear Medicine, Ljubljana (Slovenia); Maina, Theodosia [National Center for Scientific Research Demokritos, Molecular Radiopharmacy, Institute of Radioisotopes-Radiodiagnostic Products, Athens (Greece); Maecke, Helmut [University Hospital Freiburg, Department of Nuclear Medicine, Freiburg (Germany); Aloj, Luigi [Fondazione ' ' G. Pascale' ' , Department of Nuclear Medicine, Istituto Nazionale Tumouri, Naples (Italy); Guggenberg, Elisabeth von [Innsbruck Medical University, Department of Nuclear Medicine, Innsbruck (Austria); Sosabowski, Jane K. [Queen Mary, University of London, Centre for Molecular Oncology and Imaging, Institute of Cancer, Barts and The London School of Medicine and Dentistry, London (United Kingdom); Jong, Marion de [Erasmus MC, Department of Nuclear Medicine, Rotterdam (Netherlands); Reubi, Jean-Claude [University of Berne, Institute of Pathology, Berne (Switzerland)

2011-08-15

Cholecystokinin 2 (CCK-2) receptor overexpression has been demonstrated in various tumours such as medullary thyroid carcinomas and small-cell lung cancers. Due to this high expression, CCK-2 receptors might be suitable targets for radionuclide imaging and/or radionuclide therapy. Several CCK-2 receptor-binding radiopeptides have been developed and some have been tested in patients. Here we aimed to compare the in vivo tumour targeting properties of 12 {sup 111}In-labelled 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)-conjugated gastrin/CCK2 receptor-binding peptides. Two CCK8-based peptides and ten gastrin-based peptide analogues were tested. All peptides were conjugated with DOTA and labelled with {sup 111}In. Biodistribution studies were performed in mice with subcutaneous CCK2/gastrin receptor-expressing tumours and with receptor-negative tumours contralaterally. Biodistribution was studied by counting dissected tissues at 1 and 4 h after injection. Both the CCK analogues displayed relatively low tumour uptake (approximately 2.5%ID/g) as compared to minigastrin analogues. Two linear minigastrin peptides (MG0 and sargastrin) displayed moderate tumour uptake at both 1 and 4 h after injection, but also very high kidney uptake (both higher than 48%ID/g). The linear MG11, lacking the penta-Glu sequence, showed lower tumour uptake and also low kidney uptake. Varying the N-terminal Glu residues in the minigastrin analogues led to improved tumour targeting properties, with PP-F11 displaying the optimal biodistribution. Besides the monomeric linear peptides, a cyclized peptide and a divalent peptide were tested. Based on these studies, optimal peptides for peptide receptor radionuclide targeting of CCK2/gastrin receptor-expressing tumours were the linear minigastrin analogue with six D-Glu residues (PP-F11), the divalent analogue MGD5 and the cyclic peptide cyclo-MG1. These peptides combined high tumour uptake with low kidney retention, and may

Transformation of cell-derived microparticles into quantum-dot-labeled nanovectors for antitumor siRNA delivery.

Science.gov (United States)

Chen, Gang; Zhu, Jun-Yi; Zhang, Zhi-Ling; Zhang, Wei; Ren, Jian-Gang; Wu, Min; Hong, Zheng-Yuan; Lv, Cheng; Pang, Dai-Wen; Zhao, Yi-Fang

2015-01-12

Cell-derived microparticles (MPs) have been recently recognized as critical intercellular information conveyors. However, further understanding of their biological behavior and potential application has been hampered by the limitations of current labeling techniques. Herein, a universal donor-cell-assisted membrane biotinylation strategy was proposed for labeling MPs by skillfully utilizing the natural membrane phospholipid exchange of their donor cells. This innovative strategy conveniently led to specific, efficient, reproducible, and biocompatible quantum dot (QD) labeling of MPs, thereby reliably conferring valuable traceability on MPs. By further loading with small interference RNA, QD-labeled MPs that had inherent cell-targeting and biomolecule-conveying ability were successfully employed for combined bioimaging and tumor-targeted therapy. This study provides the first reliable and biofriendly strategy for transforming biogenic MPs into functionalized nanovectors. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Dynamic PET and Optical Imaging and Compartment Modeling using a Dual-labeled Cyclic RGD Peptide Probe

OpenAIRE

Zhu, Lei; Guo, Ning; Li, Quanzheng; Ma, Ying; Jacboson, Orit; Lee, Seulki; Choi, Hak Soo; Mansfield, James R.; Niu, Gang; Chen, Xiaoyuan

2012-01-01

Purpose: The aim of this study is to determine if dynamic optical imaging could provide comparable kinetic parameters to that of dynamic PET imaging by a near-infrared dye/64Cu dual-labeled cyclic RGD peptide. Methods: The integrin αvβ3 binding RGD peptide was conjugated with a macrocyclic chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) for copper labeling and PET imaging and a near-infrared dye ZW-1 for optical imaging. The in vitro biological activity of RGD-C(DOTA)...

CdSe/ZnS Quantum Dot-Labeled Lateral Flow Strips for Rapid and Quantitative Detection of Gastric Cancer Carbohydrate Antigen 72-4

Science.gov (United States)

Yan, Xinyu; Wang, Kan; Lu, Wenting; Qin, Weijian; Cui, Daxiang; He, Jinghua

2016-03-01

Carbohydrate antigen 72-4 (CA72-4) is an important biomarker associated closely with diagnosis and prognosis of early gastric cancer. How to realize quick, sensitive, specific, and quantitative detection of CA72-4 in clinical specimens has become a great requirement. Herein, we reported a CdSe/ZnS quantum dot-labeled lateral flow test strip combined with a charge-coupled device (CCD)-based reader was developed for rapid, sensitive, and quantitative detection of CA72-4. Two mouse monoclonal antibodies (mAbs) against CA72-4 were employed. One of them was coated as a test line, while another mAb was labeled with quantum dots and coated onto conjugate pad. The goat anti-mouse IgG was immobilized as a control line. After sample was added, a sandwich structure was formed with CA72-4 and these two mAbs. The fluorescent signal from quantum dots (QD)-labeled mAb in sandwich structure was related to the amount of detected CA72-4. A CCD-based reader was used to realize quantitative detection of CA72-4. Results showed that developed QD-labeled lateral flow strips to detect CA72-4 biomarker with the sensitivity of 2 IU/mL and 10 min detection time. One hundred sera samples from clinical patients with gastric cancer and healthy people were used to confirm specificity of this strip method; results showed that established strip method own 100 % reproducibility and 100 % specificity compared with Roche electrochemiluminescence assay results. In conclusion, CdSe/ZnS quantum dot-labeled lateral flow strips for detection of CA72-4 could realize rapid, sensitive, and specific detection of clinical samples and could own great potential in clinical translation in near future.

Preparation of carbon quantum dots with a high quantum yield and the application in labeling bovine serum albumin

Energy Technology Data Exchange (ETDEWEB)

Liu, Pengpeng; Zhang, Changchang; Liu, Xiang, E-mail: liuxiang@ahut.edu.cn; Cui, Ping, E-mail: cokecp@sohu.com

2016-04-15

Graphical abstract: - Highlights: • Cheap carbon quantum dots (CQDs) with a high quantum yield were prepared. • The preparation process and surface functionalization on CQDs are rather facile. • Such functionalized CQDs can be attached to BSA covalently. • This predicts that some biomolecules can be labeled by the fluorescent CQDs. - Abstract: An economic and green approach of manufacturing carbon quantum dots (CQDs) with a high quantum yield (denoted with HQY-CQDs) and the application in labeling bovine serum albumin (BSA) were described in detail in this work. Firstly, the cheap resources of citric acid and glycine were pyrolysed in drying oven for preparing the CQDs. Then the product was immersed in tetrahydrofuran for 8 h. HQY-CQDs were obtained by removing tetrahydrofuran from the supernate and were evaluated that they possessed a much higher quantum yield compared with that without dealing with tetrahydrofuran and a wonderful photo-bleaching resistance. Such HQY-CQDs could be functionalized by N-hydroxysuccinimide and successively combined with BSA covalently. Thus fluorescent labeling on BSA was realized. The HQY-CQDs were demonstrated with transmission electron microscopy and the chemical modification with N-hydroxysuccinimide was proved by infrared and X-ray photoelectron spectra. Labeling BSA with the HQY-CQDs was confirmed by gel electrophoresis and fluorescence imaging.

Antisense Oligonucleotides Internally Labeled with Peptides Show Improved Target Recognition and Stability to Enzymatic Degradation

DEFF Research Database (Denmark)

Taskova, Maria; Madsen, Charlotte S.; Jensen, Knud J.

2017-01-01

Specific target binding and stability in diverse biological media is of crucial importance for applications of synthetic oligonucleotides as diagnostic and therapeutic tools. So far, these issues have been addressed by chemical modification of oligonucleotides and by conjugation with a peptide, m...... and makes internally labeled POCs an exciting object of study, i.e., showing high target specificity and simultaneous stability in biological media.......Specific target binding and stability in diverse biological media is of crucial importance for applications of synthetic oligonucleotides as diagnostic and therapeutic tools. So far, these issues have been addressed by chemical modification of oligonucleotides and by conjugation with a peptide......, most often at the terminal position of the oligonucleotide. Herein, we for the first time systematically investigate the influence of internally attached short peptides on the properties of antisense oligonucleotides. We report the synthesis and internal double labeling of 21-mer oligonucleotides...

High Sensitivity Detection of CdSe/ZnS Quantum Dot-Labeled DNA Based on N-type Porous Silicon Microcavities.

Science.gov (United States)

Lv, Changwu; Jia, Zhenhong; Lv, Jie; Zhang, Hongyan; Li, Yanyu

2017-01-01

N-type macroporous silicon microcavity structures were prepared using electrochemical etching in an HF solution in the absence of light and oxidants. The CdSe/ZnS water-soluble quantum dot-labeled DNA target molecules were detected by monitoring the microcavity reflectance spectrum, which was characterized by the reflectance spectrum defect state position shift resulting from changes to the structures' refractive index. Quantum dots with a high refractive index and DNA coupling can improve the detection sensitivity by amplifying the optical response signals of the target DNA. The experimental results show that DNA combined with a quantum dot can improve the sensitivity of DNA detection by more than five times.

Limitations to the use of radioactively labelled substrates for studying peptide transport in microorganisms

International Nuclear Information System (INIS)

Payne, J.W.; Nisbet, T.M.

1980-01-01

The authors wished to investigate the stoicheiometry of energy coupling to peptide transport in whole cells of several organisms, a study that requires accurate measurements of the rate and amount of peptide translocation. They show that using radioactively-labelled substrates can lead to severe miscalculation of these parameters and produce misleading data on the kinetics of uptake. These conclusions are based on comparative studies using the fluorescamine and dansyl procedures. (Auth.)

Intracellular bimodal nanoparticles based on quantum dots for high-field MRI at 21.1 T.

Science.gov (United States)

Rosenberg, Jens T; Kogot, Joshua M; Lovingood, Derek D; Strouse, Geoffrey F; Grant, Samuel C

2010-09-01

Multimodal, biocompatible contrast agents for high magnetic field applications represent a new class of nanomaterials with significant potential for tracking of fluorescence and MR in vitro and vivo. Optimized for high-field MR applications-including biomedical imaging at 21.1 T, the highest magnetic field available for MRI-these nanoparticles capitalize on the improved performance of chelated Dy(3+) with increasing magnetic field coupled to a noncytotoxic Indium Phosphide/Zinc Sulfide (InP/ZnS) quantum dot that provides fluorescence detection, MR responsiveness, and payload delivery. By surface modifying the quantum dot with a cell-penetrating peptide sequence coupled to an MR contrast agent, the bimodal nanomaterial functions as a self-transfecting high-field MR/optical contrast agent for nonspecific intracellular labeling. Fluorescent images confirm sequestration in perinuclear vesicles of labeled cells, with no apparent cytotoxicity. These techniques can be extended to impart cell selectivity or act as a delivery vehicle for genetic or pharmaceutical interventions. 2010 Wiley-Liss, Inc.

A molecular beacon microarray based on a quantum dot label for detecting single nucleotide polymorphisms.

Science.gov (United States)

Guo, Qingsheng; Bai, Zhixiong; Liu, Yuqian; Sun, Qingjiang

2016-03-15

In this work, we report the application of streptavidin-coated quantum dot (strAV-QD) in molecular beacon (MB) microarray assays by using the strAV-QD to label the immobilized MB, avoiding target labeling and meanwhile obviating the use of amplification. The MBs are stem-loop structured oligodeoxynucleotides, modified with a thiol and a biotin at two terminals of the stem. With the strAV-QD labeling an "opened" MB rather than a "closed" MB via streptavidin-biotin reaction, a sensitive and specific detection of label-free target DNA sequence is demonstrated by the MB microarray, with a signal-to-background ratio of 8. The immobilized MBs can be perfectly regenerated, allowing the reuse of the microarray. The MB microarray also is able to detect single nucleotide polymorphisms, exhibiting genotype-dependent fluorescence signals. It is demonstrated that the MB microarray can perform as a 4-to-2 encoder, compressing the genotype information into two outputs. Copyright © 2015 Elsevier B.V. All rights reserved.

In vivo study of immunogenicity and kinetic characteristics of a quantum dot-labelled baculovirus.

Science.gov (United States)

Wang, Meng; Zheng, Zhenhua; Meng, Jin; Wang, Han; He, Man; Zhang, Fuxian; Liu, Yan; Hu, Bin; He, Zike; Hu, Qinxue; Wang, Hanzhong

2015-09-01

Nanomaterials conjugated with biomacromolecules, including viruses, have great potential for in vivo applications. Therefore, it is important to evaluate the safety of nanoparticle-conjugated macromolecule biomaterials (Nano-mbio). Although a number of studies have assessed the risks of nanoparticles and macromolecule biomaterials in living bodies, only a few of them investigated Nano-mbios. Here we evaluated the in vivo safety profile of a quantum dot-conjugated baculovirus (Bq), a promising new Nano-mbio, in mice. Each animal was injected twice intraperitoneally with 50 μg virus protein labelled with around 3*10(-5)nmol conjugated qds. Control animals were injected with PBS, quantum dots, baculovirus, or a mixture of quantum dots and baculovirus. Blood, tissues and body weight were analysed at a series of time points following both the first and the second injections. It turned out that the appearance and behaviour of the mice injected with Bq were similar to those injected with baculovirus alone. However, combination of baculovirus and quantum dot (conjugated or simply mixed) significantly induced stronger adaptive immune responses, and lead to a faster accumulation and longer existence of Cd in the kidneys. Thus, despite the fact that both quantum dot and baculovirus have been claimed to be safe in vivo, applications of Bq in vivo should be cautious. To our knowledge, this is the first study examining the interaction between a nanoparticle-conjugated virus and a living body from a safety perspective, providing a basis for in vivo application of other Nano-mbios. Copyright © 2015 Elsevier Ltd. All rights reserved.

End-labeling of peptide nucleic acid with osmium complex. Voltammetry at carbon and mercury electrodes

Czech Academy of Sciences Publication Activity Database

Paleček, Emil; Trefulka, Mojmír; Fojta, Miroslav

2009-01-01

Roč. 11, č. 2 (2009), s. 359-362 ISSN 1388-2481 R&D Projects: GA AV ČR(CZ) KAN400310651; GA MŠk(CZ) LC06035 Institutional research plan: CEZ:AV0Z50040507; CEZ:AV0Z50040702 Keywords : peptide nucleic acid end-labeling * osmium tetroxide complexes * electroactive labels Subject RIV: BO - Biophysics Impact factor: 4.243, year: 2009

Secretory overexpression and isotopic labeling of the chimeric relaxin family peptide R3/I5 in Pichia pastoris.

Science.gov (United States)

Guo, Yu-Qi; Wu, Qing-Ping; Shao, Xiao-Xia; Shen, Ting; Liu, Ya-Li; Xu, Zeng-Guang; Guo, Zhan-Yun

2015-06-01

Relaxin family peptides are a group of peptide hormones with divergent biological functions. Mature relaxin family peptides are typically composed of two polypeptide chains with three disulfide linkages, rendering their preparation a challenging task. In the present study, we established an efficient approach for preparation of the chimeric relaxin family peptide R3/I5 through secretory overexpression in Pichia pastoris and in vitro enzymatic maturation. A designed single-chain R3/I5 precursor containing the B-chain of human relaxin-3 and the A-chain of human INSL5 was overexpressed in PichiaPink strain 1 by high-density fermentation in a two-liter fermenter, and approximately 200 mg of purified precursor was obtained from one liter of the fermentation supernatant. We also developed an economical approach for preparation of the uniformly (15)N-labeled R3/I5 precursor by culturing in shaking flasks, and approximately 15 mg of purified (15)N-labeled precursor was obtained from one liter of the culture supernatant. After purification by cation ion-exchange chromatography and reverse-phase high performance liquid chromatography, the R3/I5 precursor was converted to the mature two-chain form by sequential treatment with endoproteinase Lys-C and carboxypeptidase B. The mature R3/I5 peptide had an α-helix-dominated conformation and retained full receptor-binding and receptor activation activities. Thus, Pichia overexpression was an efficient approach for sample preparation and isotopic labeling of the chimeric R3/I5 peptide. This approach could also be extended to the preparation of other relaxin family peptides in future studies.

An improved method of 18F peptide labeling: hydrazone formation with HYNIC-conjugated c(RGDyK)

International Nuclear Information System (INIS)

Lee, Yun-Sang; Jeong, Jae Min; Kim, Hyung Woo; Chang, Young Soo; Kim, Young Joo; Hong, Mee Kyung; Rai, Ganesha B.; Chi, Dae Yoon; Kang, Won Jun; Kang, Joo Hyun; Lee, Dong Soo; Chung, June-Key; Lee, Myung Chul; Suh, Young-Ger

2006-01-01

Radiolabeled α v β 3 -integrin antagonists are increasingly investigated as a means of imaging angiogenesis. Several methods of labeling α v β 3 -integrin binding peptide with 18 F have been reported recently. In the present study, we devised a straightforward means for labeling Arg-Gly-Asp (RGD) peptide with 18 F via hydrazone formation between c(RGDyK)-hydrazinonicotinic acid (HYNIC) (3) and 4-[ 18 F]-fluorobenzaldehyde ([ 18 F]4). The resulting reaction mixture was purified by HPLC to give 4'-[ 18 F]-fluorobenzylidenehydrazone-6-nicotinamide-c(RGDyK) ([ 18 F]5). The conjugation efficiency of 3 and 4 to form [ 18 F]5 was 95.2%, and the radiochemical purity of [ 18 F]5 after purification was >99%. The specific activity of [ 18 F]5 estimated by radio-HPLC was 20.5 GBq/μmol (end of synthesis). Competitive binding assay of c(RGDyK) (1) and 5 was performed using [ 125 I]iodo-c(RGDyK) as a radioligand, and K i values were found to be 2.8 and 21.7 nM, respectively. For the biodistribution study, the angiogenic mouse model was established by inducing unilateral ischemia on the left hindlimbs of ICR mice after femoral artery ablation. Seven days after inducing ischemia, [ 18 F]5 was administered to the mice through the tail vein. Ischemic muscle uptake of [ 18 F]5 was significantly higher than that of normal muscle (P 18 F]5. Here, we successfully labeled RGD peptide with 18 F via hydrazone formation between 3 and 4, resulting to [ 18 F]5. [ 18 F]5 was found to have high affinity for α v β 3 -integrin and to accumulate specifically in ischemic hindlimb muscle of mice. We suggest that 18 F labeling via formation of hydrazone between HYNIC peptide and [ 18 F]4 is a useful method for labeling c(RGDyK), which can be applied for imaging angiogenesis

99mTc labelled peptide for imaging of peripheral receptors

International Nuclear Information System (INIS)

Mishra, A.K.; Mishra, P.; Chuttani, K.; Sharma, R.K.; Lazar Mathew, T.

2001-01-01

Conjugates of somatostatin analogues, RC-160 with different bifunctional chelators to label with 99m Tc, were synthesized. Conjugates with hydrazinonicotinamide (HYNIC) and compounds (benzoyl MAG-3 and CITC-DTPA) were prepared on a small scale with high purity and evaluated as different types of chelators on RC-160. Stability studies performed under physiological conditions showed high stability. Peptide conjugates could be labelled at high specific activities (307inCi/umol) with 99m Tc and different transchelator were used for the HYNIC conjugates. The resulting radiolabelled with ( 99m Tc and 1251) complexes were highly stable and showed binding affinity to somatostatin receptors in the nanomolar range. The radioconjugates were administered to rabbits and mice in order to study their in vivo stability, biokinetics and biodistribution. (author)

Dual-Quantum-Dots-Labeled Lateral Flow Strip Rapidly Quantifies Procalcitonin and C-reactive Protein

Science.gov (United States)

Qi, XiaoPing; Huang, YunYe; Lin, ZhongShi; Xu, Liang; Yu, Hao

2016-03-01

In the article, a dual-quantum-dots-labeled (dual-QDs-labeled) lateral flow strip (LFS) method was developed for the simultaneous and rapid quantitative detection of procalcitonin (PCT) and C-reactive protein (CRP) in the blood. Two QD-antibody conjugates with different fluorescence emission spectra were produced and sprayed on the LFS to capture PCT and CRP in the blood. Furthermore, a double antibody sandwich method for PCT and, meanwhile, a competitive inhibition method for CRP were employed in the LFS. For PCT and CRP in serum assayed by the dual-QDs-labeled LFS, their detection sensitivities reached 0.1 and 1 ng/mL, respectively, and their linear quantitative detection ranges were from 0.3 to 200 ng/mL and from 50 to 250 μg/mL, respectively. There was little evidence that the PCT and CRP assays would be interfered with each other. The correlations for testing CRP and PCT in clinical samples were 99.75 and 97.02 %, respectively, between the dual-QDs-labeled LFS we developed and commercial methods. The rapid quantification of PCT and CRP on dual-QDs-labeled LFS is of great clinical value to distinguish inflammation, bacterial infection, or viral infection and to provide guidance for the use of antibiotics or other medicines.

Analysis of the differentially expressed low molecular weight peptides in human serum via an N-terminal isotope labeling technique combining nano-liquid chromatography/matrix-assisted laser desorption/ionization mass spectrometry.

Science.gov (United States)

Leng, Jiapeng; Zhu, Dong; Wu, Duojiao; Zhu, Tongyu; Zhao, Ningwei; Guo, Yinlong

2012-11-15

Peptidomics analysis of human serum is challenging due to the low abundance of serum peptides and interference from the complex matrix. This study analyzed the differentially expressed (DE) low molecular weight peptides in human serum integrating a DMPITC-based N-terminal isotope labeling technique with nano-liquid chromatography and matrix-assisted laser desorption/ionization mass spectrometry (nano-LC/MALDI-MS). The workflow introduced a [d(6)]-4,6-dimethoxypyrimidine-2-isothiocyanate (DMPITC)-labeled mixture of aliquots from test samples as the internal standard. The spiked [d(0)]-DMPITC-labeled samples were separated by nano-LC then spotted on the MALDI target. Both quantitative and qualitative studies for serum peptides were achieved based on the isotope-labeled peaks. The DMPITC labeling technique combined with nano-LC/MALDI-MS not only minimized the errors in peptide quantitation, but also allowed convenient recognition of the labeled peptides due to the 6 Da mass difference. The data showed that the entire research procedure as well as the subsequent data analysis method were effective, reproducible, and sensitive for the analysis of DE serum peptides. This study successfully established a research model for DE serum peptides using DMPITC-based N-terminal isotope labeling and nano-LC/MALDI-MS. Application of the DMPITC-based N-terminal labeling technique is expected to provide a promising tool for the investigation of peptides in vivo, especially for the analysis of DE peptides under different biological conditions. Copyright © 2012 John Wiley & Sons, Ltd.

Labeling bombesin-like peptide with 99mTc via hydrazinonicotinamide. Description of optimized radiolabeling conditions

International Nuclear Information System (INIS)

Yurt Lambrecht, F.; Durkan, K.; Bayrak, E.

2010-01-01

Bombesin (BNN)-like peptides have very high binding affinity for the gastrin-releasing peptide (GRP) receptor. The goal of the current study was to optimize the labeling conditions of a new 99m Tc-radiolabeled BNN-like peptide based on the bifunctional chelating ligand HYNIC using different co-ligands (EDDA and tricine). The radiolabeling conditions (pH, amount of co-ligand, amount of stannous chloride, temperature and reaction time) for newly-formed 99m Tc-tricine-HYNIC-Q-Litorin and 99m Tc-EDDA-HYNIC-Q-Litorin were optimized and evaluated by RHPLC and RTLC. Radiochemical yields for 99m Tc-tricine-HYNIC-Q-Litorin and 99m Tc-EDDA-HYNIC-Q-Litorin were 98.0 ± 1.7 and 97.5 ± 2.5%, respectively. When EDDA was used as co-ligand, the labeling of 99m Tc-EDDA-HYNIC-Q-Litorin was optimal in the following reaction mixture: HYNIC-peptide: EDDA: 10 μg/5 mg, pH 3, SnCl 2 concentration: 12 μg/0.1 mL, reaction temperature: 100 deg C, reaction time: 15 min. Besides, the optimum conditions were HYNIC-peptide:tricine: 10 μg/50 mg, pH 5, SnCl 2 concentration: 12 μg/0.1 mL, reaction temperature: 100 deg C, reaction time: 15 min for preparing 99m Tc-tricine-HYNIC-Q-Litorin. The manufactured 99m Tc-HYNIC-Q-Litorin conjugates may offer new possibilities for imaging cancer cells expressing bombesin receptors. (author)

One-step, green, and economic synthesis of water-soluble photoluminescent carbon dots by hydrothermal treatment of wheat straw, and their bio-applications in labeling, imaging, and sensing

Energy Technology Data Exchange (ETDEWEB)

Yuan, Ming; Zhong, Ruibo; Gao, Haiyang; Li, Wanrong; Yun, Xiaoling; Liu, Jingran; Zhao, Xinmin; Zhao, Guofen; Zhang, Feng, E-mail: fengzhang1978@hotmail.com

2015-11-15

Graphical abstract: Water-soluble photoluminescent carbon dots can be synthesized simply by a green, economic and one-pot hydrothermal treatment of wheat straw with ∼20% yield, in addition to the compact size and robust photostability they are experimentally demonstrated for multiplexed applications such as sensing ions and labeling and imaging for inorganic nanostructures, cells and even nematodes. The converting biomass wastes to promising biocompatible nanomaterials could be a “one-stone-two-birds” strategy to other carbon-containing biomass waste for a highly effectively carbon recycling use and sustainable energy and environment future. - Highlights: • Photoluminescent carbon dots can be synthesized by wheat straw with about 20% yield. • Carbon dots can be used for both nonliving and living labeling, imaging, and sensing. • Carbon dots can be used as a fluorescent ink. - Abstract: The use of biomass as renewable and sustainable energy source has attracted the attention of politics and research and development (R&D) facilities around the world. Agricultural straw acts as a typical biowaste, which still needs highly effective recycling to save the biomass urgently at present. Photoluminescent carbon dots (C-dots) are novel biocompatible nanomaterials that have been proved to be produced from many carbon-abundant materials and hold great promise for the modern nanobiomedicine. In order to realize a “one-stone-two-birds” strategy, we report a green, economic, one-pot method in this article for synthesizing photoluminescent C-dots by hydrothermal treatment of wheat straw. Using X-ray photoelectron spectroscopy (XPS) and X-ray diffraction (XRD), we show that the as-prepared C-dots are amorphous in structure and are mainly composed of carbon. Their tiny size (<2 nm), combined with the characteristic excitation-dependent relatively bright emission, and robust photostability made the C-dots a potential biocompatible nanomaterial for bio-applications. We

Catalytic center of lecithin:cholesterol acyltransferase: isolation and sequence of diisopropyl fluorophosphate-labeled peptides

Energy Technology Data Exchange (ETDEWEB)

Park, Y.B.; Yueksel, U.G.; Gracy, R.W.; Lacko, A.G.

1987-02-27

Lecithin:cholesterol acyltransferase (LCAT) was purified from hog plasma and subsequently reacted with (/sup 3/H)-Diisopropyl fluorophosphate (DFP). The labeled enzyme was digested with pepsin and the peptides separated by high performance liquid chromatography (HPLC). Two radioactive peptides were isolated, subjected to automated amino acid sequencing and yielded the following data: A) Ile-Ser-Leu-Gly-Ala-Pro-Trp-Gly-Gly-Ser, and B) Tyr-Ile-Phe-Asp-x-Gly-Phe-Pro-Tyr-x-Asp-Pro-Val. Both of these sequences represent very highly conserved regions of the enzyme when compared to the sequence of human LCAT. Peptide (A) is considered to represent the catalytic center of LCAT based on comparisons with data reported in the literature.

Quantum dot labeling and tracking of cultured limbal epithelial cell transplants in-vitro

Science.gov (United States)

Genicio, Nuria; Paramo, Juan Gallo; Shortt, Alex J.

2015-01-01

PURPOSE Cultured human limbal epithelial cells (HLEC) have shown promise in the treatment of limbal stem cell deficiency but little is known about their survival, behaviour and long-term fate post transplantation. The aim of this research was to evaluate, in-vitro, quantum dot (QDot) technology as a tool for tracking transplanted HLEC. METHODS In-vitro cultured HLEC were labeled with Qdot nanocrystals. Toxicity was assessed using live-dead assays. The effect on HLEC function was assessed using colony forming efficiency assays and expression of CK3, P63alpha and ABCG2. Sheets of cultured HLEC labeled with Qdot nanocrystals were transplanted onto decellularised human corneo-scleral rims in an organ culture model and observed to investigate the behaviour of transplanted cells. RESULTS Qdot labeling had no detrimental effect on HLEC viability or function in-vitro. Proliferation resulted in a gradual reduction in Qdot signal but sufficient signal was present to allow tracking of cells through multiple generations. Cells labeled with Qdots could be reliably detected and observed using confocal microscopy for at least 2 weeks post transplantation in our organ culture model. In addition it was possible to label and observe epithelial cells in intact human corneas using the Rostock corneal module adapted for use with the Heidelberg HRA. CONCLUSIONS This work demonstrates that Qdots combined with existing clinical equipment could be used to track HLEC for up to 2 weeks post transplantation, however, our model does not permit the assessment of cell labeling beyond 2 weeks. Further characterisation in in-vivo models are required. PMID:26024089

Detection of human spermatozoal peptides after conjugation to 125I-labelled human serum albumin

International Nuclear Information System (INIS)

Metler, L.; Skrabei, H.; Czuppon, A.B.

1981-01-01

Human spermatozoal peptides, liberated during autolysis of the cells, were fractionated by gel-filtration chromatography and thin-layer chromatography. After conjugation to 125 I-labelled human serum albumin, all fractions were assayed with rabbit antihuman spermatozoa antiserum. In earlier publications, human sperm-immobilizing and sperm-agglutinating sera were used for the detection of solubilized spermatozoal antigen. The low sensitivity of these tests necessitated a more sensitive test. The purpose of this work is to describe a solid-phase radioimmunoassay for the detection of antigenic peptides

99mTc labelled peptides for imaging peripheral receptors

International Nuclear Information System (INIS)

Gil, M.C.; Chandia, V.M.; Errazu, X.

2001-01-01

Radiolabelling of somatostatin analogues as RC-160 and TOC with 99m Tc, using direct and bifunctional chelating methods as well as quality control and evaluation methods, has been accomplished following the techniques and recommendation of the first and second RCMs. Synthesis of bifunctional chelating agents, such as Bz-MAG-3, is routinely produced in our laboratory. Synthesis of HYNIC and HYNIC-MAG-3 is in progress. Radioiodination of RC-160 using chloramine-T and iodogen methods were also studied in order to get experience with the different techniques used to evaluate the labelled peptides. (author)

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.

Hydrogen atom scrambling in selectively labeled anionic peptides upon collisional activation by MALDI tandem time-of-flight mass spectrometry

DEFF Research Database (Denmark)

Bache, Nicolai; Rand, Kasper Dyrberg; Roepstorff, Peter

2008-01-01

have now measured the level of hydrogen scrambling in a deprotonated, selectively labeled peptide using MALDI tandem time-of-flight mass spectrometry. Our results conclusively show that hydrogen scrambling is prevalent in the deprotonated peptide upon collisional activation. The amide hydrogens ((1)H....../(2)H) have migrated extensively in the anionic peptide, thereby erasing the original regioselective deuteration pattern obtained in solution....

111In-labeled lactam bridge-cyclized alpha-melanocyte stimulating hormone peptide analogues for melanoma imaging.

Science.gov (United States)

Miao, Yubin; Gallazzi, Fabio; Guo, Haixun; Quinn, Thomas P

2008-02-01

The purpose of this study was to examine the influence of the lactam bridge cyclization on melanoma targeting and biodistribution properties of the radiolabeled conjugates. Two novel lactam bridge-cyclized alpha-MSH peptide analogues, DOTA-CycMSH (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid-c[Lys-Nle-Glu-His-DPhe-Arg-Trp-Gly-Arg-Pro-Val-Asp]) and DOTA-GlyGlu-CycMSH (DOTA-Gly-Glu-c[Lys-Nle-Glu-His-DPhe-Arg-Trp-Gly-Arg-Pro-Val-Asp]), were synthesized and radiolabeled with (111)In. The internalization and efflux of (111)In-labeled CycMSH peptides were examined in B16/F1 melanoma cells. The melanoma targeting properties, pharmacokinetics, and SPECT/CT imaging of (111)In-labeled CycMSH peptides were determined in B16/F1 melanoma-bearing C57 mice. Both (111)In-DOTA-CycMSH and (111)In-DOTA-GlyGlu-CycMSH exhibited fast internalization and extended retention in B16/F1 cells. The tumor uptake values of (111)In-DOTA-CycMSH and (111)In-DOTA-GlyGlu-CycMSH were 9.53+/-1.41% injected dose/gram (% ID/g) and 10.40+/-1.40% ID/g at 2 h postinjection, respectively. Flank melanoma tumors were clearly visualized with (111)In-DOTA-CycMSH and (111)In-DOTA-GlyGlu-CycMSH by SPECT/CT images at 2 h postinjection. Whole-body clearance of the peptides was fast, with greater than 90% of the radioactivities cleared through urinary system by 2 h postinjection. There was low radioactivity (<0.8% ID/g) accumulated in blood and normal organs except kidneys at all time points investigated. Introduction of a negatively charged linker (-Gly-Glu-) into the peptide sequence decreased the renal uptake by 44% without affecting the tumor uptake at 4 h postinjection. High receptor-mediated melanoma uptakes coupled with fast whole-body clearance in B16/F1 melanoma-bearing C57 mice demonstrated the feasibility of using (111)In-labeled lactam bridge-cyclized alpha-MSH peptide analogues as a novel class of imaging probes for receptor-targeting melanoma imaging.

Technetium-99m labelled fluconazole and antimicrobial peptides for imaging of Candida albicans and Aspergillus fumigatus infections

Energy Technology Data Exchange (ETDEWEB)

Lupetti, Antonella [Department of Infectious Diseases, Leiden University Medical Center (LUMC), Leiden (Netherlands); Dipartimento di Patologia Sperimentale, Biotecnologie Mediche, Univ. di Pisa (Italy); Welling, Mick M. [Department of Radiology, Division of Nuclear Medicine, LUMC, Leiden (Netherlands); Mazzi, Ulderico [Dipartimento di Scienze Farmaceutiche, Universita degli Studi di Padova (Italy); Nibbering, Peter H. [Department of Infectious Diseases, Leiden University Medical Center (LUMC), Leiden (Netherlands); Pauwels, Ernest K.J. [Department of Radiology, Division of Nuclear Medicine, LUMC, Leiden (Netherlands); Department of Radiology, Leiden University Medical Center (LUMC) (Netherlands)

2002-05-01

The aim of this study was to investigate whether technetium-99m labelled fluconazole can distinguish fungal from bacterial infections. Fluconazole was labelled with {sup 99m}Tc and radiochemical analysis showed less than 5% impurities. The labelling solution was injected into animals with experimental infections. For comparison, we used two peptides for infection detection, i.e. UBI 29-41 and hLF 1-11, and human IgG, all labelled with {sup 99m}Tc. Mice were infected with Candida albicans or injected with heat-killed C. albicans or lipopolysaccharides to induce sterile inflammation. Also, mice were infected with Staphylococcus aureus or Klebsiella pneumoniae. Next, accumulation of {sup 99m}Tc-fluconazole and {sup 99m}Tc-labelled peptides/IgG at affected sites was determined scintigraphically. {sup 99m}Tc-fluconazole detected C. albicans infections (T/NT ratio=3.6{+-}0.47) without visualising bacterial infections (T/NT ratio=1.3{+-}0.04) or sterile inflammatory processes (heat-killed C. albicans: T/NT ratio=1.3{+-}0.2; lipopolysaccharide: T/NT ratio=1.4{+-}0.1). C. albicans infections were already seen within the first hour after injection of {sup 99m}Tc-fluconazole (T/NT ratio=3.1{+-}0.2). A good correlation (R{sup 2}=0.864; P<0.05) between T/NT ratios for this tracer and the number of viable C. albicans was found. Although {sup 99m}Tc-UBI 29-41 and {sup 99m}Tc-hLF 1-11 were able to distinguish C. albicans infections from sterile inflammatory processes in mice, these {sup 99m}Tc-labelled peptides did not distinguish these fungal infections from bacterial infections. It is concluded that {sup 99m}Tc-fluconazole distinguishes infections with C. albicans from bacterial infections and sterile inflammations. (orig.)

Using DNA barcoding to track seafood mislabeling in Los Angeles restaurants.

Science.gov (United States)

Willette, Demian A; Simmonds, Sara E; Cheng, Samantha H; Esteves, Sofia; Kane, Tonya L; Nuetzel, Hayley; Pilaud, Nicholas; Rachmawati, Rita; Barber, Paul H

2017-10-01

Seafood mislabeling is common in both domestic and international markets. Studies on seafood fraud often report high rates of mislabeling (e.g., >70%), but these studies have been limited to a single sampling year, which means it is difficult to assess the impact of stricter governmental truth-in-labeling regulations. We used DNA barcoding to assess seafood labeling in 26 sushi restaurants in Los Angeles over 4 years. Seafood from 3 high-end grocery stores were also sampled (n = 16) in 2014. We ordered 9 common sushi fish from menus, preserved tissue samples in 95% ethanol, extracted the genomic DNA, amplified and sequenced a portion of the mtDNA COI gene, and identified the resulting sequence to known fish sequences from the National Center for Biotechnology Information nucleotide database. We compared DNA results with the U.S. Food and Drug Administration (FDA) list of acceptable market names and retail names. We considered sushi-sample labels that were inconsistent with FDA names mislabeled. Sushi restaurants had a consistently high percentage of mislabeling (47%; 151 of 323) from 2012 to 2015, yet mislabeling was not homogenous across species. Halibut, red snapper, yellowfin tuna, and yellowtail had consistently high (15%). All sampled sushi restaurants had at least one case of mislabeling. Mislabeling of sushi-grade fish from high-end grocery stores was also identified in red snapper, yellowfin tuna, and yellowtail, but at a slightly lower frequency (42%) than sushi restaurants. Despite increased regulatory measures and media attention, we found seafood mislabeling continues to be prevalent. © 2017 Society for Conservation Biology.

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.

Synthesis and labelling of organo-metallic prosthetic groups used for indirect radioiodination of peptides and proteins

International Nuclear Information System (INIS)

Pozzi, Oscar R.; Castiglia, Silvia G.

1999-01-01

In the framework of an IAEA co-ordinated research programme the prosthetic compound ATE [N-succidinimil 3-(tri-n-butylstannyl) benzoate] has been synthesized and it has been labelled with 131 I and 125 I. Its structure has been confirmed by NMR and mass spectrometry. The labelled ATE has been conjugated with human immunoglobulin G with a yield of 41%-57%. Indirect radioiodination of peptides is currently prepared. (author)

In vitro cell studies of technetium-99m labeled RGD-HYNIC peptide, a comparison of tricine and EDDA as co-ligands

International Nuclear Information System (INIS)

Su Zifen; He, Jiang; Rusckowski, Mary; Hnatowich, Donald J.

2003-01-01

The level of α V β 3 integrins on endothelial cells is elevated in angiogenesis. The high binding specificity to α V β 3 integrins of peptides containing Arg-Gly-Asp (RGD) residues suggests that the radiolabeled RGD peptides may be useful as tumor specific imaging agents. In this research, cyclised peptides containing Arg-Gly-Asp (RGD) and Arg-Gly-Glu (RGE, as control) residues were conjugated with HYNIC and labeled with 99m Tc. Objective: The goal was to evaluate the influence of co-ligand, either tricine or ethylenediamine-N,N'-diacetic acid (EDDA) on protein and integrin binding and on cellular uptake in culture. Methods: The n-octanol/water partition coefficient, binding to bovine serum albumin (BSA) and human umbilical vein endothelial (HUVE) cells, and cell lysate distributions of the radiolabeled peptides were evaluated. Results: The co-ligands had a significant effect on the labeling efficiency of the HYNIC conjugates and on certain properties of the 99m Tc complexes. The labeling efficiency with tricine was 10 fold higher and BSA binding was over 8 fold greater compared to EDDA. Both RGD labels showed higher (6 to 28 fold) binding to HUVE cells than that of the RGE labels, indicating binding specificity. After cell-lysis, only a small percentage of the total RGD label that accumulated in the cells was found bound to cellular proteins (9% of RGD/tricine and 5% of RGD/EDDA), implying that over 90% of the radiolabeled peptides were internalized for both radiolabeled RGDs. The number of the RGD molecules bound to proteins was estimated to be approximately three per cell, suggesting that only a small number of α V β 3 integrin proteins are expressed on the cells. Conclusions: Apart from the differences in radiolabeling, the only important effect of substituting EDDA for tricine as co-ligand on the HYNIC-peptides was the lower degree of serum protein binding. In spite of the lower serum protein binding potential, in vivo tumor accumulation of the RGD

In vitro cell studies of technetium-99m labeled RGD-HYNIC peptide, a comparison of tricine and EDDA as co-ligands.

Science.gov (United States)

Su, Zi-Fen; He, Jiang; Rusckowski, Mary; Hnatowich, Donald J

2003-02-01

The level of alpha(V)beta(3) integrins on endothelial cells is elevated in angiogenesis. The high binding specificity to alpha(V)beta(3) integrins of peptides containing Arg-Gly-Asp (RGD) residues suggests that the radiolabeled RGD peptides may be useful as tumor specific imaging agents. In this research, cyclised peptides containing Arg-Gly-Asp (RGD) and Arg-Gly-Glu (RGE, as control) residues were conjugated with HYNIC and labeled with (99m)Tc. The goal was to evaluate the influence of co-ligand, either tricine or ethylenediamine-N,N'-diacetic acid (EDDA) on protein and integrin binding and on cellular uptake in culture. The n-octanol/water partition coefficient, binding to bovine serum albumin (BSA) and human umbilical vein endothelial (HUVE) cells, and cell lysate distributions of the radiolabeled peptides were evaluated. The co-ligands had a significant effect on the labeling efficiency of the HYNIC conjugates and on certain properties of the (99m)Tc complexes. The labeling efficiency with tricine was 10 fold higher and BSA binding was over 8 fold greater compared to EDDA. Both RGD labels showed higher (6 to 28 fold) binding to HUVE cells than that of the RGE labels, indicating binding specificity. After cell-lysis, only a small percentage of the total RGD label that accumulated in the cells was found bound to cellular proteins (9% of RGD/tricine and 5% of RGD/EDDA), implying that over 90% of the radiolabeled peptides were internalized for both radiolabeled RGDs. The number of the RGD molecules bound to proteins was estimated to be approximately three per cell, suggesting that only a small number of alpha(V)beta(3) integrin proteins are expressed on the cells. Apart from the differences in radiolabeling, the only important effect of substituting EDDA for tricine as co-ligand on the HYNIC-peptides was the lower degree of serum protein binding. In spite of the lower serum protein binding potential, in vivo tumor accumulation of the RGD/EDDA may not be improved

Noninvasive imaging of malignant tumors using laminin peptide fragments YIGSR labeled with Technetium-99m

International Nuclear Information System (INIS)

Qin, G.M.; Zhang, Y.X.; Hu, J.; An, R.; Gao, Z.R.; Cao, G.X.; Hnatowich, D.J.

2002-01-01

The radiopharmaceuticals that localize specifically at certain sites (such as peptides directed against receptors expressed on tumor cells or antibodies with high binding affinities for bacterial determinants) may be expected to display greater specificity of localization. Peptides, which diffuse rapidly into target lesions and clear rapidly elsewhere, may be expected to enjoy a pharmacokinetic advantage over those, such as antibodies, which accumulate and clear more slowly. The laminin peptide fragments YIGSR is known to bind to a 67-kDa laminin receptor. This receptor is understood to be expressed at higher than normal levels in malignant tumor cells, particularly those of breast and colon carcinomas. Methods 1 peptide conjugation and labeling A 2.5 mg/mL solution of YIGSR in 0.1 M N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) buffer, pH8.0, and a fresh 10mg/mL solution of NHS-S-acetyl-MAG 3 in dimethylformamide dried over molecular sieve were prepared. 2 biodistribution and imaging studies A colony of KM mice (15-20g) were inoculated with 1x10 6 Ehrlich (breast) carcinoma tumor cells in the right thigh, and the tumors were allowed to grow for 6-7 days to a size of 1.0-1.5 cm in diameter. Biodistribution studies were performed in 40 KM mice after 50 μCi per mouse of 99m Tc-labeled YIGSR were injected intravenously. A total of 10 mice were injected intravenously in the tail vein with 1-2 mCi of 99m Tc-labeled YIGSR, immobilized with ketamine hydrochloride and imaged periodically from 0.5 hr to 24 hr with a gamma camera. The identical imaging procedure was also performed in mice with sterile infection/inflammation lesions to evaluate the specificity. Results Essentially complete conjugation was achieved by reverse-phase Sep-Pak C18 chromatography analysis. The highest accumulation of label was in the kidney first, with the liver and small bowel next. The injected activity localized in the lesion as early as 15 min and reached a saturation value at 3

Quantitation of peptides and proteins by matrix-assisted laser desorption/ionization mass spectrometry using (18)O-labeled internal standards

DEFF Research Database (Denmark)

Mirgorodskaya, O A; Kozmin, Y P; Titov, M I

2000-01-01

A method for quantitating proteins and peptides in the low picomole and sub-picomole range has been developed using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) with internal (18)O-labeled standards. A simple procedure is proposed to produce such internal standards for...... inhibitor, were quantified by MALDI-time-of-flight (TOF) mass spectrometry.......A method for quantitating proteins and peptides in the low picomole and sub-picomole range has been developed using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) with internal (18)O-labeled standards. A simple procedure is proposed to produce such internal standards...

Comparative assessment of a 99mTc labeled H1299.2-HYNIC peptide bearing two different co-ligands for tumor-targeted imaging.

Science.gov (United States)

Torabizadeh, Seyedeh Atekeh; Abedi, Seyed Mohammad; Noaparast, Zohreh; Hosseinimehr, Seyed Jalal

2017-05-01

Peptides are a class of targeting agents that bind to cancer-specific cell surfaces. Since they specifically target cancer cells, they could be used as molecular imaging tools. In this study, the 15-mer peptide Ac-H1299.2 (YAAWPASGAWTGTAP) was conjugated with HYNIC via lysine amino acid on C-terminus and labeled with 99m Tc using tricine and EDDA/tricine as the co-ligands. These radiotracers were evaluated for potential utilization in diagnostic imaging of ovarian cancer cells (SKOV-3). The cell-specificity of these radiolabeled peptides was determined based on their binding on an ovarian cancer cell line (SKOV-3), and displaying a low affinity for lung adenocarcinoma cell line (A549) and breast cancer cell line (MCF7). Biodistribution studies were conducted in normal mice as well as in nude mice bearing SKOV-3 ovarian cancer xenografts. HYNIC-peptide was labeled with 99m Tc with more than 99% efficiency and showed high stability in buffer and serum. We observed nanomolar binding affinities for both radiolabeled peptides. The tumor uptakes were 3.27%±0.46% and 1.55%±0.20% for tricine and 2.34±1.1% and 1.09%±0.18% for EDDA/tricine at 1 and 4h after injection, respectively. A higher tumor to background ratio and lower radioactivity in the blood were observed for EDDA/tricine co-ligands, leading to clear tumor visualization in imaging with injection of this peptide. This new 99m Tc-labeled peptide selectively targeted ovarian cancer and introduction of a (EDDA/tricine) as a co-ligand improved the pharmacokinetics of 99m Tc-labeled H1299.2 for tumor imaging in animals. Copyright © 2017 Elsevier Ltd. All rights reserved.

Gamma scintigraphy imaging of murine invasive pulmonary aspergillosis with a {sup 111}In-labeled cyclic peptide

Energy Technology Data Exchange (ETDEWEB)

Yang Zhi [Department of Experimental Diagnostic Imaging, Infection Control and Employee Health, University of Texas M.D. Anderson Cancer Center, Houston, TX 77030 (United States); Kontoyiannis, Dimitrios P. [Department of Infectious Diseases, Infection Control and Employee Health, University of Texas M.D. Anderson Cancer Center, Houston, TX 77030 (United States); Wen Xiaoxia; Xiong Chiyi; Zhang Rui [Department of Experimental Diagnostic Imaging, Infection Control and Employee Health, University of Texas M.D. Anderson Cancer Center, Houston, TX 77030 (United States); Albert, Nathaniel D. [Department of Infectious Diseases, Infection Control and Employee Health, University of Texas M.D. Anderson Cancer Center, Houston, TX 77030 (United States); Li Chun [Department of Experimental Diagnostic Imaging, Infection Control and Employee Health, University of Texas M.D. Anderson Cancer Center, Houston, TX 77030 (United States)], E-mail: cli@mdanderson.org

2009-04-15

Introduction: Invasive pulmonary aspergillosis (IPA) is a leading cause of infection-associated death in immunosuppressed patients. Early detection and early administration of antifungal therapy are critical factors in improving outcome for patients with IPA. Here, we evaluated the imaging properties of a {sup 111}In-labeled cyclic peptide targeted to Aspergillus fumigatus in an immunosuppressed murine model of IPA. Methods: A cyclic peptide c(CGGRLGPFC)-NH{sub 2} was labeled with {sup 111}In by means of diethylenetriaminepentaacetic acid (DTPA). Two days after intranasal inoculation of 17.5x10{sup 6} conidia of A. fumigatus, mice were injected {sup 111}In-DTPA-c(CGGRLGPFC)-NH{sub 2} intravenously. Biodistribution data were obtained at 2 h, and {gamma}-images were acquired at 10 min and 2 h after radiotracer injection. Healthy mice were used as controls. In addition, a group of infected mice were co-injected with the radiotracer and unlabeled c(CGGRLGPFC)-NH{sub 2} to evaluate the inhibition of radiotracer's binding to infected lungs. Autoradiographs of lungs from infected and healthy mice were compared with corresponding photographs of transaxial sections of the lung tissues stained for A. fumigatus hyphae. Results: The labeling efficiency was >98%, with specific radioactivity of up to 74 MBq/nmol peptide. Significantly higher uptake of {sup 111}In-DTPA-c(CGGRLGPFC)-NH{sub 2} was observed in the lungs of mice infected with A. fumigatus than in those of healthy mice (0.37{+-}0.06 %ID/g vs. 0.14{+-}0.02 %ID/g, P=.00044). Simultaneous injection with unlabeled peptide reduced radioactivity in the infected lungs by 41% (P=.0037). Increased radioactivity in the lungs of infected mice was visible in {gamma} images at both 10 min and 2 h after radiotracer injection. Moreover, autoradiography confirmed radiotracer uptake in infected lungs, but not in the lungs of healthy mice or infected mice co-injected with unlabeled peptide. Conclusions: {gamma}-Imaging with {sup

99mTc labelled peptides for imaging of peripheral receptors. Final report of a co-ordinated research project. 1995-1999

International Nuclear Information System (INIS)

2001-04-01

two IAEA-TECDOCs. Even though a few 99m Tc monoclonal antibodies are now used for diagnostic imaging, it is generally acknowledged that receptor specific peptides will provide better carriers for 99m Tc for in vivo receptor imaging. The possible peptides are, however, too numerous and information from other sources, such as the pharmaceutical industry, has to be relied upon to narrow down the choice for labelling and evaluation. One such peptide, octreotide, an analogue of the neuropeptide somatostatin, developed by the pharmaceutical industry for therapy of neuroendocrine tumours, was used as a carrier for 123 I and 111 In for imaging such tumours. 111 In-octreotide soon became a regular, commercially available and probably the first peptide based radiopharmaceutical successfully used for imaging somatostatin positive tumours. There are several obvious advantages of using 99m Tc in place of 111 In and soon development of a 99m Tc labelled octreotide analogue attracted the attention of several radiopharmaceutical research groups. Keeping in mind the possibility of a wider reach for a 99m Tc octreotide agent and based on the recommendations of an Advisory Group meeting, the IAEA initiated a CRP in 1995 on 99m Tc Labelled Peptides for Imaging Peripheral Receptors. The techniques and methodologies involved in 99m Tc labelling of peptides with high specific activity and their evaluation are also more complex and their mastering could be expected to pave the way for further research and development of other 99m Tc labelled peptides for various applications in Member States. Fourteen laboratories from Europe, Latin America, the United States of America and Asia took part in the CRP which was concluded at. the end of 1999. Efforts in the CRP were focused on evaluating different methods of 99m Tc labelling of a few selected octreotide derivatives and evaluating their receptor specificity by biochemical techniques. Based on the experience of a number of the participants, it

Identification of quantum dots labeled metallothionein by fast scanning laser-induced breakdown spectroscopy

International Nuclear Information System (INIS)

Konecna, Marie; Novotny, Karel; Krizkova, Sona; Blazkova, Iva; Kopel, Pavel; Kaiser, Jozef; Hodek, Petr; Kizek, Rene

2014-01-01

The technique described in this paper allows detection of quantum dots (QDs) specifically deposited on the polystyrene surface by laser-induced breakdown spectroscopy (LIBS). Using LIBS, the distribution of QDs or their conjugates with biomolecules deposited on the surface can be observed, regardless of the fact if they exhibit fluorescence or not. QDs deposited on the specific surface of polystyrene microplate in the form of spots are detected by determination of the metal included in the QDs structure. Cd-containing QDs (CdS, CdTe) stabilized with mercaptopropionic (MPA) or mercaptosuccinic (MSA) acid, respectively, alone or in the form of conjugates with metallothionein (MT) biomolecule are determined by using the 508.58 nm Cd emission line. The observed absolute detection limit for Cd in CdTe QDs conjugates with MT in one spot was 3 ng Cd. Due to the high sensitivity of this technique, the immunoanalysis in combination with LIBS was also investigated. Cd spatial distribution in sandwich immunoassay was detected. - Highlights: • We describe determination of biomolecules labeled with quantum dots by LIBS. • LIBS and immunoassay are applied for the determination of metallothionein. • Metallothionein amount detected by LIBS is 10-times lower compared to ELISA

Identification of quantum dots labeled metallothionein by fast scanning laser-induced breakdown spectroscopy

Energy Technology Data Exchange (ETDEWEB)

Konecna, Marie [Central European Institute of Technology, Brno University of Technology, Technicka 3058/10, CZ-616 00 Brno (Czech Republic); Department of Chemistry and Biochemistry, Faculty of Agronomy, Mendel University in Brno, Zemedelska 1, CZ-613 00 Brno (Czech Republic); Novotny, Karel [Central European Institute of Technology, Masaryk University, Kamenice 753/5, CZ-625 00 Brno (Czech Republic); Krizkova, Sona [Central European Institute of Technology, Brno University of Technology, Technicka 3058/10, CZ-616 00 Brno (Czech Republic); Department of Chemistry and Biochemistry, Faculty of Agronomy, Mendel University in Brno, Zemedelska 1, CZ-613 00 Brno (Czech Republic); Blazkova, Iva [Department of Chemistry and Biochemistry, Faculty of Agronomy, Mendel University in Brno, Zemedelska 1, CZ-613 00 Brno (Czech Republic); Kopel, Pavel [Central European Institute of Technology, Brno University of Technology, Technicka 3058/10, CZ-616 00 Brno (Czech Republic); Department of Chemistry and Biochemistry, Faculty of Agronomy, Mendel University in Brno, Zemedelska 1, CZ-613 00 Brno (Czech Republic); Kaiser, Jozef [Central European Institute of Technology, Brno University of Technology, Technicka 3058/10, CZ-616 00 Brno (Czech Republic); Institute of Physical Engineering, Brno University of Technology, Technicka 2, CZ-616 69 Brno (Czech Republic); Hodek, Petr [Department of Biochemistry, Faculty of Science, Charles University in Prague, Hlavova 2030/8, CZ-128 00 Prague,Czech Republic (Czech Republic); Kizek, Rene [Central European Institute of Technology, Brno University of Technology, Technicka 3058/10, CZ-616 00 Brno (Czech Republic); Department of Chemistry and Biochemistry, Faculty of Agronomy, Mendel University in Brno, Zemedelska 1, CZ-613 00 Brno (Czech Republic); and others

2014-11-01

The technique described in this paper allows detection of quantum dots (QDs) specifically deposited on the polystyrene surface by laser-induced breakdown spectroscopy (LIBS). Using LIBS, the distribution of QDs or their conjugates with biomolecules deposited on the surface can be observed, regardless of the fact if they exhibit fluorescence or not. QDs deposited on the specific surface of polystyrene microplate in the form of spots are detected by determination of the metal included in the QDs structure. Cd-containing QDs (CdS, CdTe) stabilized with mercaptopropionic (MPA) or mercaptosuccinic (MSA) acid, respectively, alone or in the form of conjugates with metallothionein (MT) biomolecule are determined by using the 508.58 nm Cd emission line. The observed absolute detection limit for Cd in CdTe QDs conjugates with MT in one spot was 3 ng Cd. Due to the high sensitivity of this technique, the immunoanalysis in combination with LIBS was also investigated. Cd spatial distribution in sandwich immunoassay was detected. - Highlights: • We describe determination of biomolecules labeled with quantum dots by LIBS. • LIBS and immunoassay are applied for the determination of metallothionein. • Metallothionein amount detected by LIBS is 10-times lower compared to ELISA.

Synthesis, Characterization, and Initial Biological Evaluation of [99m Tc]Tc-Tricarbonyl-labeled DPA-α-MSH Peptide Derivatives for Potential Melanoma Imaging.

Science.gov (United States)

Gao, Feng; Sihver, Wiebke; Bergmann, Ralf; Belter, Birgit; Bolzati, Cristina; Salvarese, Nicola; Steinbach, Jörg; Pietzsch, Jens; Pietzsch, Hans-Jürgen

2018-06-06

α-Melanocyte stimulating hormone (α-MSH) derivatives target the melanocortin-1 receptor (MC1R) specifically and selectively. In this study, the α-MSH-derived peptide NAP-NS1 (Nle-Asp-His-d-Phe-Arg-Trp-Gly-NH 2 ) with and without linkers was conjugated with 5-(bis(pyridin-2-ylmethyl)amino)pentanoic acid (DPA-COOH) and labeled with [ 99m Tc]Tc-tricarbonyl by two methods. With the one-pot method the labeling was faster than with the two-pot method, while obtaining similarly high yields. Negligible trans-chelation and high stability in physiological solutions was determined for the [ 99m Tc]Tc-tricarbonyl-peptide conjugates. Coupling an ethylene glycol (EG)-based linker increased the hydrophilicity. The peptide derivatives displayed high binding affinity in murine B16F10 melanoma cells as well as in human MeWo and TXM13 melanoma cell homogenates. Preliminary in vivo studies with one of the [ 99m Tc]Tc-tricarbonyl-peptide conjugates showed good stability in blood and both renal and hepatobiliary excretion. Biodistribution was performed on healthy rats to gain initial insight into the potential relevance of the 99m Tc-labeled peptides for in vivo imaging. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Photoaffinity labeling of human serum vitamin D binding protein and chemical cleavage of the labeled protein: Identification of an 11.5-kDa peptide containing the putative 25-hydroxyvitamin D3 binding site

International Nuclear Information System (INIS)

Ray, R.; Holick, M.F.; Bouillon, R.; Baelen, H.V.

1991-01-01

In this paper, the authors describe photoaffinity labeling and related studies of human serum vitamin D binding protein (hDBP) with 25-hydroxyvitamin D 3 3β-3'-[N-(4-azido-2-nitrophenyl)amino]propyl ether (25-ANE) and its radiolabeled counterpart, i.e., 25-hydroxyvitamin D 3 3β-3'-[N-(4-azido-2-nitro-[3,5- 3 H]phenyl)amino]propyl ether ( 3 H-25-ANE). They have carried out studies to demonstrate that (1) 25-ANE competes with 25-OH-D 3 for the binding site of the latter in hDBP and (2) 3 H-25-ANE is capable of covalently labeling the hDBP molecule when exposed ot UV light. Treatment of a sample of purified hDBP, labeled with 3 H-25-ANE, with BNPS-skatole produced two Coomassie Blue stained peptide fragments, and the majority of the radioactivity was assoicated with the smaller of the two peptide fragments (16.5 kDa). On the other hand, cleavage of the labeled protein with cyanogen bromide produced a peptide (11.5 kDa) containing most of the covalently attached radioactivity. Considering the primary amino acid structure of hDBP, this peptide fragment (11.5 kDa) represents the N-terminus through residue 108 of the intact protein. Thus, the results tentatively identify this segment of the protein containing the binding pocket for 25-OH-D 3

Efficient preparation of {sup 99m}Tc(III) '4+1' mixed-ligand complexes for peptide labeling with high specific activity

Energy Technology Data Exchange (ETDEWEB)

Kunstler, Jens-Uwe [Biotectid GmbH, Deutscher Platz 5c, 04103 Leipzig (Germany); Seidel, Gesine [Institute of Radiopharmacy, Forschungszentrum Dresden-Rossendorf, P.O. Box 510 119, 01314 Dresden (Germany); Pietzsch, Hans-Jurgen, E-mail: h.j.pietzsch@fzd.d [Institute of Radiopharmacy, Forschungszentrum Dresden-Rossendorf, P.O. Box 510 119, 01314 Dresden (Germany)

2010-09-15

An improved labeling procedure for peptides attached to organometallic {sup 99m}Tc(III) '4+1' mixed-ligand complexes in which the radiometal is coordinated by a tripodal tetradentate chelator 2,2',2''-nitrilotriethanethiol (NS{sub 3}) and a monodentate isocyanide ligand is presented. The labeling procedure was evaluated by the synthesis of [{sup 99m}Tc(NS{sub 3})(L2-RGD)]. The containing radiopharmaceutically interesting RGD-peptide cyclo[Arg-Gly-Asp-D-Tyr-Lys] was modified with 4-isocyanobutanoic acid (L2) as linker conjugated to N{sup 6}-Lys to get the monodentate ligand L2-RGD. The structural identity of the {sup 99m}Tc-conjugate was confirmed by comparison to a Re reference compound. The Tc- and Re-conjugates had matching retention times under identical HPLC conditions. The {sup 99m}Tc-labeling was performed in a novel one-step procedure using the eluate of a {sup 99}Mo/{sup 99m}Tc generator, NS{sub 3}, the isocyanide modified peptide, SnCl{sub 2}, Na{sub 2}EDTA, mannitol and ascorbic acid in the reaction mixture. Using optimized reagents it is possible to label 50 nmol peptide with {sup 99m}Tc within 60 min at room temperature with a radiochemical yield higher than 95% and a specific activity of {approx}20 GBq/{mu}mol.

Determination of bovine lactoferrin in dairy products by ultra-high performance liquid chromatography–tandem mass spectrometry based on tryptic signature peptides employing an isotope-labeled winged peptide as internal standard

Energy Technology Data Exchange (ETDEWEB)

Zhang, Jingshun [Zhejiang Provincial Center for Disease Control and Prevention, Hangzhou 310051 (China); Lai, Shiyun [Beingmate Research Institute, Beingmate Baby and Child Food Co., Ltd., Hangzhou 310007 (China); Cai, Zengxuan [Zhejiang Provincial Center for Disease Control and Prevention, Hangzhou 310051 (China); Chen, Qi [Beingmate Research Institute, Beingmate Baby and Child Food Co., Ltd., Hangzhou 310007 (China); Huang, Baifen [Zhejiang Provincial Center for Disease Control and Prevention, Hangzhou 310051 (China); Ren, Yiping, E-mail: renyiping@263.net [Zhejiang Provincial Center for Disease Control and Prevention, Hangzhou 310051 (China)

2014-06-01

Highlights: • A UHPLC–MS/MS method for quantification of bovine lactoferrin was developed. • Tryptic fragment LRPVAAEIYGTK was chosen as signature peptide of bovine lactoferrin. • A winged peptide containing isotopically-labeled signature peptide was designed as internal standard. • The method for determining lactoferrin does not discriminate between the different forms of lactoferrin. • Meet the growing demand to quantify bovine lactoferrin in different dairy products. Abstract: A new and sensitive determination method was developed for bovine lactoferrin in dairy products including infant formulas based on the signature peptide by ultra high-performance liquid chromatography and triple-quadrupole tandem mass spectrometry under the multiple reaction monitoring mode. The simple pretreatment procedures included the addition of a winged peptide containing the isotope-labeled signature peptide as internal standard, followed by an enzymatic digestion with trypsin. The signature peptide was chosen and identified from the tryptic hydrolyzates of bovine lactoferrin by ultra high-performance liquid chromatography and quadrupole-time-of-flight tandem mass spectrometry based on sequence database search. Analytes were separated on an ACQUITY UPLC BEH 300 C18 column and monitored by MS/MS in seven minutes. Quantitative result bias due to matrix effect and tryptic efficiency was corrected through the use of synthetic isotope-labeled standards. The limit of detection and limit of quantification were 0.3 mg/100 g and 1.0 mg/100 g, respectively. Bovine lactoferrin within the concentration range of 10–1000 nmol L⁻¹ showed a strong linear relationship with a linear correlation coefficient (r) of >0.998. The intra- and inter-day precision of the method were RSD < 6.5% and RSD < 7.1%, respectively. Excellent repeatability (RSD < 6.4%) substantially supported the application of this method for the determination of bovine lactoferrin in dairy samples. The present method

Determination of bovine lactoferrin in dairy products by ultra-high performance liquid chromatography–tandem mass spectrometry based on tryptic signature peptides employing an isotope-labeled winged peptide as internal standard

International Nuclear Information System (INIS)

Zhang, Jingshun; Lai, Shiyun; Cai, Zengxuan; Chen, Qi; Huang, Baifen; Ren, Yiping

2014-01-01

Highlights: • A UHPLC–MS/MS method for quantification of bovine lactoferrin was developed. • Tryptic fragment LRPVAAEIYGTK was chosen as signature peptide of bovine lactoferrin. • A winged peptide containing isotopically-labeled signature peptide was designed as internal standard. • The method for determining lactoferrin does not discriminate between the different forms of lactoferrin. • Meet the growing demand to quantify bovine lactoferrin in different dairy products. - Abstract: A new and sensitive determination method was developed for bovine lactoferrin in dairy products including infant formulas based on the signature peptide by ultra high-performance liquid chromatography and triple-quadrupole tandem mass spectrometry under the multiple reaction monitoring mode. The simple pretreatment procedures included the addition of a winged peptide containing the isotope-labeled signature peptide as internal standard, followed by an enzymatic digestion with trypsin. The signature peptide was chosen and identified from the tryptic hydrolyzates of bovine lactoferrin by ultra high-performance liquid chromatography and quadrupole-time-of-flight tandem mass spectrometry based on sequence database search. Analytes were separated on an ACQUITY UPLC BEH 300 C18 column and monitored by MS/MS in seven minutes. Quantitative result bias due to matrix effect and tryptic efficiency was corrected through the use of synthetic isotope-labeled standards. The limit of detection and limit of quantification were 0.3 mg/100 g and 1.0 mg/100 g, respectively. Bovine lactoferrin within the concentration range of 10–1000 nmol L −1 showed a strong linear relationship with a linear correlation coefficient (r) of >0.998. The intra- and inter-day precision of the method were RSD < 6.5% and RSD < 7.1%, respectively. Excellent repeatability (RSD < 6.4%) substantially supported the application of this method for the determination of bovine lactoferrin in dairy samples. The present

Quantum dot coating of baculoviral vectors enables visualization of transduced cells and tissues

International Nuclear Information System (INIS)

Zhao, Ying; Lo, Seong Loong; Zheng, Yuangang; Lam, Dang Hoang; Wu, Chunxiao; Han, Ming Yong; Wang, Shu

2013-01-01

Highlights: •The use of quantum dot (QD)-labeled viral vectors for in vivo imaging is not well investigated. •A new method to label enveloped baculovirus with glutathione-capped CdTe QDs is developed. •The labeling enables the identification of transduced, cultured cells based on fluorescence. •The labeling also allows evaluation of viral transduction in a real-time manner in living mice. •The method has the potential to assess viral vector-based gene therapy protocols in future. -- Abstract: Imaging of transduced cells and tissues is valuable in developing gene transfer vectors and evaluating gene therapy efficacy. We report here a simple method to use bright and photostable quantum dots to label baculovirus, an emerging gene therapy vector. The labeling was achieved through the non-covalent interaction of glutathione-capped CdTe quantum dots with the virus envelope, without the use of chemical conjugation. The quantum dot labeling was nondestructive to viral transduction function and enabled the identification of baculoviral vector-transduced, living cells based on red fluorescence. When the labeled baculoviral vectors were injected intravenously or intraventricularly for in vivo delivery of a transgene into mice, quantum dot fluorescence signals allow us monitor whether or not the injected tissues were transduced. More importantly, using a dual-color whole-body imaging technology, we demonstrated that in vivo viral transduction could be evaluated in a real-time manner in living mice. Thus, our method of labeling a read-to-use gene delivery vector with quantum dots could be useful towards the improvement of vector design and will have the potential to assess baculovirus-based gene therapy protocols in future

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.

18F- and 11C-labelling of quantum dots with n.c.a. [18F]fluoroethyltosylate and [11C]methyliodide. A feasibility study

International Nuclear Information System (INIS)

Patt, M.; Schildan, A.; Habermann, B.; Mishchenko, O.; Patt, J.T.; Sabri, O.

2010-01-01

Quantum dots functionalized on the outer surface with either amino- or carboxyl functions were labelled with [ 18 F]fluoroethyltosylate and [ 11 C]methyliodide in order to use the positron emitter-labelled fluorescence agents for multimodality imaging techniques, i.e. fluorescence imaging and positron emission tomography. 18 F-Labelling of both compounds was realized with yields up to 5% as determined by size exclusion chromatography, which is twice as much as reported in literature before [1]. 11 C-Labelling of amino- and carboxyl-QDs proceeded with good yields (up to 45 and 35%, respectively) under optimized reaction conditions. In general for both QD-types and both labelling agents the labelling yield increased with the amount of QDs used in the reaction as well as with reaction time and reaction temperature. (author)

Synthesis of positron labeled photoactive compounds: 18F labeled aryl azides for positron labeling of biochemical molecules

International Nuclear Information System (INIS)

Hashizume, Kazunari; Hashimoto, Naota; Miyake, Yoshihiro

1995-01-01

The authors have prepared various [ 18 F] fluorine labeled aryl azides as a novel photoactive compounds suitable for positron labeling of biochemical molecules. The introduction of fluorine substituents to aryl azides can be expected to have dramatic effects on their nature and reactivity toward photolysis. Positron labeled reagents for labeling proteins or peptides have recently attracted considerable attention due to their wide applicability in biochemistry and positron emission tomography (PET). Various labeled azide compounds are often used in biochemistry for radiolabeling biological molecules by photolysis, but there have been no reports on the preparation or use of fluorine-18 labeled azides. The authors now report a novel synthesis of 18 F-labeled aryl azides which will have wide application in the biochemistry and nuclear medicine as a means for 18 F-fluorine labeling for proteins, peptides, and nucleic acids. 2 tabs

Labeling of the peptide DOTA-tyr3-octreotate with radioiodine and biodistribution and AR42J neuroendocrine tumor affinity study in mice

International Nuclear Information System (INIS)

Nagamati, Lucio Takeshi

2006-01-01

Neuroendocrine tumors are rare and affect mainly the gastrointestinal tract but other systems are also affected like the skin, lungs and the nervous system. They are rich in type 2 somatostatin (SM) receptors (SSTR2) and may secrete hormones in excess. Synthetic SM derivative peptides are of great utility because presented bigger half life when compared to SM and can be used to clinical improvement of these patients due to its tumoral inhibitory action. The labeling of these peptides with radioisotopes allowed the acquisition of images with favourable cost-efficiency relationship and use in therapy. The peptide, DOTATyr3- octreotate (DOTATATE), has much more affinity for the SSTR2 receptor than the peptide commercially used nowadays, is easily radioiodinated and has a favourable biodistribution for diagnosis and treatment due to the presence of the chelator DOTA. We have studied the influence of various factors on the radiochemical purity of the labeled compound as labeling stability, absorbed dose estimation and biodistribution in normal and AR42J cell tumor-bearing Swiss and Nude mice. We observed easy and stable peptide radioiodination at peptide/radioiodine ( 131 I) ratio of 2.73 that produced a radiochemical species with retention time of 22.7 minutes at high performance liquid chromatography and presented a favourable biodistribution and dosimetry for imaging and therapy of patients with neuroendocrine tumors, just the opposite result observed the radioiodinated compounds without a chelator as described in the literature. Other molar peptide/radioiodine ratios did not showed good results, with various radiochemical species and unfavourable biodistribution. A possible dosimetric study in patients with neuroendocrine tumors may be carried out in the near future. (author)

Site-specific Orientation of an α-helical Peptide Ovispirin-1 from Isotope Labeled SFG Spectroscopy

Science.gov (United States)

Ding, Bei; Laaser, Jennifer E.; Liu, Yuwei; Wang, Pengrui; Zanni, Martin T.; Chen, Zhan

2013-01-01

Sum-frequency generation (SFG) vibrational spectroscopy is often used to probe the backbone structures and orientations of polypeptides at surfaces. Using the ovispirin-1 polypeptide at the solid/liquid interface of polystyrene, we demonstrate for the first time that SFG can probe the polarization response of a single isotope labeled residue. To interpret the spectral intensities, we simulated the spectra using an excitonic Hamiltonian approach. We show that the polarization dependence of either the label or the unlabeled amide I band alone does not provide sufficient structural constraints to obtain both the tilt and the twist of the ovispirin helix at a solid/liquid interface, but that both can be determined from the polarization dependence of the complete spectrum. For ovispirin, the detailed analysis of the polarized SFG experimental data shows that the helix axis is tilted at roughly 138 degrees from the surface normal, and the transition dipole of the isotope labeled C=O group is tilted at 23 degrees from the surface normal, with the hydrophobic region facing the polystyrene surface. We further demonstrated that the Hamiltonian approach is able to address the coupling effect and the structural disorder. For comparison, we also collected the FTIR spectrum of ovispirin under similar conditions, which reveals the enhanced sensitivity of SFG for structural studies of single monolayer peptide surfaces. Our study provides insight into how structural and environmental effects appear in SFG spectra of the amide I band and establishes that SFG of isotope labeled peptides will be a powerful technique for elucidating secondary structures with residue-by-residue resolution. PMID:24228619

Site-specific orientation of an α-helical peptide ovispirin-1 from isotope-labeled SFG spectroscopy.

Science.gov (United States)

Ding, Bei; Laaser, Jennifer E; Liu, Yuwei; Wang, Pengrui; Zanni, Martin T; Chen, Zhan

2013-11-27

Sum-frequency generation (SFG) vibrational spectroscopy is often used to probe the backbone structures and orientations of polypeptides at surfaces. Using the ovispirin-1 polypeptide at the solid/liquid interface of polystyrene, we demonstrate for the first time that SFG can probe the polarization response of a single-isotope-labeled residue. To interpret the spectral intensities, we simulated the spectra using an excitonic Hamiltonian approach. We show that the polarization dependence of either the label or the unlabeled amide I band alone does not provide sufficient structural constraints to obtain both the tilt and the twist of the ovispirin helix at a solid/liquid interface, but that both can be determined from the polarization dependence of the complete spectrum. For ovispirin, the detailed analysis of the polarized SFG experimental data shows that the helix axis is tilted at roughly 138° from the surface normal, and the transition dipole of the isotope-labeled C═O group is tilted at 23° from the surface normal, with the hydrophobic region facing the polystyrene surface. We further demonstrate that the Hamiltonian approach is able to address the coupling effect and the structural disorder. For comparison, we also collected the FTIR spectrum of ovispirin under similar conditions, which reveals the enhanced sensitivity of SFG for structural studies of single monolayer peptide surfaces. Our study provides insight into how structural and environmental effects appear in SFG spectra of the amide I band and establishes that SFG of isotope-labeled peptides will be a powerful technique for elucidating secondary structures with residue-by-residue resolution.

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.

In vitro and in vivo documentation of quantum dots labeled Trypanosoma cruzi--Rhodnius prolixus interaction using confocal microscopy.

Science.gov (United States)

Feder, Denise; Gomes, Suzete A O; de Thomaz, André A; Almeida, Diogo B; Faustino, Wagner M; Fontes, Adriana; Stahl, Cecília V; Santos-Mallet, Jacenir R; Cesar, Carlos L

2009-12-01

Semiconductor quantum dots (QDs) are highly fluorescent nanocrystals markers that allow long photobleaching and do not destroy the parasites. In this paper, we used fluorescent core shell quantum dots to perform studies of live parasite-vector interaction processes without any observable effect on the vitality of parasites. These nanocrystals were synthesized in aqueous medium and physiological pH, which is very important for monitoring live cells activities, and conjugated with molecules such as lectins to label specific carbohydrates involved on the parasite-vector interaction. These QDs were successfully used for the study of in vitro and in vivo interaction of Trypanosoma cruzi and the triatomine Rhodnius prolixus. These QDs allowed us to acquire real time confocal images sequences of live T. cruzi-R. prolixus interactions for an extended period, causing no damage to the cells. By zooming to the region of interest, we have been able to acquire confocal images at the three to four frames per second rate. Our results show that QDs are physiological fluorescent markers capable to label living parasites and insect vector cells. QDs can be functionalized with lectins to specifically mark surface carbohydrates on perimicrovillar membrane of R. prolixus to follow, visualize, and understand interaction between vectors and its parasites in real-time.

In vivo labelling of acetyl-aspartyl peptides in mouse brain from intracranially and intracranially and intraperitoneally administered acetyl-L-[U-14C]aspartate

International Nuclear Information System (INIS)

Sinichkin, A.; Sterri, S.; Edminson, P.D.; Reichelt, K.L.; Kvamme, E.

1977-01-01

Following intracranial and intraperitoneal injection of acetyl-L-[U- 14 C]aspartate into mice about 5% and 0.7% of the radioactivity, respectively, was recovered from the brain after 30 min. On chromatographic separation of the cationic and anionic compounds on a Dowex 50 column, the former fraction contained about 60% of the radioactivity, predominantly as labelled asparate and glutamate. The anionic compounds, containing 20% of the labelled compounds, were fractionated in several chromatographic systems and resolved into a great variety of labelled peptidic compounds of which five acetyl-[U 14 ]aspartyl peptides, containing two to four amino acids, were purified. One of these, acetyl-aspartyl glutamine, has not previously been found in brain. (author)

Label-free detection of biomolecular interaction — DNA — Antimicrobial peptide binding

DEFF Research Database (Denmark)

Fojan, Peter; Jensen, Kasper Risgaard; Gurevich, Leonid

2011-01-01

the molecule. In particular, surface plasmon resonance (SPR) sensors have been already demonstrated suitable for food-safety control, label-free screening for various disease markers in bodily fluids, as well as for real-time continuous monitoring of drug levels in intensive care environment. We envisage...... of plasmon based biosensors to the study of the interaction of Antimicrobial peptide IL4 and DNA. Our results indicate high affinity binding between IL4 and DNA thereby preventing DNA replication and eventually killing the affected cell. We speculate that this is common for a large class of Antimicrobial...

Imaging tumor endothelial marker 8 using an 18F-labeled peptide

International Nuclear Information System (INIS)

Quan, Qimeng; Yang, Min; Gao, Haokao; Zhu, Lei; Lin, Xin; Guo, Ning; Chen, Xiaoyuan; Zhang, Guixiang; Eden, Henry S.; Niu, Gang

2011-01-01

Tumor endothelial marker 8 (TEM8) has been reported to be upregulated in both tumor cells and tumor-associated endothelial cells in several cancer types. TEM8 antagonists and TEM8-targeted delivery of toxins have been developed as effective cancer therapeutics. The ability to image TEM8 expression would be of use in evaluating TEM8-targeted cancer therapy. A 13-meric peptide, KYNDRLPLYISNP (QQM), identified from the small loop in domain IV of protective antigen of anthrax toxin was evaluated for TEM8 binding and labeled with 18 F for small-animal PET imaging in both UM-SCC1 head-and-neck cancer and MDA-MB-435 melanoma models. A modified ELISA showed that QQM peptide bound specifically to the extracellular vWA domain of TEM8 with an IC 50 value of 304 nM. Coupling 4-nitrophenyl 2- 18 F-fluoropropionate with QQM gave almost quantitative yield and a high specific activity (79.2 ± 7.4 TBq/mmol, n = 5) of 18 F-FP-QQM at the end of synthesis. 18 F-FP-QQM showed predominantly renal clearance and had significantly higher accumulation in TEM8 high-expressing UM-SCC1 tumors (2.96 ± 0.84 %ID/g at 1 h after injection) than TEM8 low-expressing MDA-MB-435 tumors (1.38 ± 0.56 %ID/g at 1 h after injection). QQM peptide bound specifically to the extracellular domain of TEM8. 18 F-FP-QQM peptide tracer would be a promising lead compound for measuring TEM8 expression. Further efforts to improve the affinity and specificity of the tracer and to increase its metabolic stability are warranted. (orig.)

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.

PET imaging of {alpha}{sub v}{beta}{sub 3} integrin expression in tumours with {sup 68}Ga-labelled mono-, di- and tetrameric RGD peptides

Energy Technology Data Exchange (ETDEWEB)

Dijkgraaf, Ingrid; Franssen, Gerben M.; Oyen, Wim J.G.; Boerman, Otto C. [Radboud University Nijmegen Medical Centre, Department of Nuclear Medicine, P.O. Box 9101, Nijmegen (Netherlands); Yim, Cheng-Bin [Radboud University Nijmegen Medical Centre, Department of Nuclear Medicine, P.O. Box 9101, Nijmegen (Netherlands); Utrecht University, Department of Medicinal Chemistry and Chemical Biology, Utrecht Institute for Pharmaceutical Sciences, Utrecht (Netherlands); Schuit, Robert C. [VU University Medical Centre, Department of Nuclear Medicine and PET Research, P.O. Box 7057, Amsterdam (Netherlands); Luurtsema, Gert [University Medical Center Groningen, Department of Nuclear Medicine and Molecular Imaging, Hanzeplein 1, P.O. Box 30.001, Groningen (Netherlands); Liu, Shuang [Purdue University, School of Health Sciences, West Lafayette, IN (United States)

2011-01-15

Due to the restricted expression of {alpha}{sub v}{beta}{sub 3} in tumours, {alpha}{sub v}{beta}{sub 3} is considered a suitable receptor for tumour targeting. In this study the {alpha}{sub v}{beta}{sub 3}-binding characteristics of {sup 68}Ga-labelled monomeric, dimeric and tetrameric RGD peptides were determined and compared with their {sup 111}In-labelled counterparts. A monomeric (E-c(RGDfK)), a dimeric (E-[c(RGDfK)]{sub 2}) and a tetrameric (E{l_brace}E[c(RGDfK)]{sub 2}{r_brace}{sub 2}) RGD peptide were synthesised, conjugated with DOTA and radiolabelled with {sup 68}Ga. In vitro {alpha}{sub v}{beta}{sub 3}-binding characteristics were determined in a competitive binding assay. In vivo {alpha}{sub v}{beta}{sub 3}-targeting characteristics of the compounds were assessed in mice with subcutaneously growing SK-RC-52 xenografts. In addition, microPET images were acquired using a microPET/CT scanner. The IC{sub 50} values for the Ga(III)-labelled DOTA-E-c(RGDfK), DOTA-E-[c(RGDfK)]{sub 2} and DOTA-E{l_brace}E[c(RGDfK)]{sub 2}{r_brace}{sub 2} were 23.9 {+-} 1.22, 8.99 {+-} 1.20 and 1.74 {+-} 1.18 nM, respectively, and were similar to those of the In(III)-labelled mono-, di- and tetrameric RGD peptides (26.6 {+-} 1.15, 3.34 {+-} 1.16 and 1.80 {+-} 1.37 nM, respectively). At 2 h post-injection, tumour uptake of the {sup 68}Ga-labelled mono-, di- and tetrameric RGD peptides (3.30 {+-} 0.30, 5.24 {+-} 0.27 and 7.11 {+-} 0.67%ID/g, respectively) was comparable to that of their {sup 111}In-labelled counterparts (2.70 {+-} 0.29, 5.61 {+-} 0.85 and 7.32 {+-} 2.45%ID/g, respectively). PET scans were in line with the biodistribution data. On all PET scans, the tumour could be clearly visualised. The integrin affinity and the tumour uptake followed the order of DOTA-tetramer > DOTA-dimer > DOTA-monomer. The {sup 68}Ga-labelled tetrameric RGD peptide has excellent characteristics for imaging of {alpha}{sub v} {beta}{sub 3} expression with PET. (orig.)

Specific labeling of the thyroxine binding site in thyroxine-binding globulin: determination of the amino acid composition of a labeled peptide fragment isolated from a proteolytic digest of the derivatized protein.

Science.gov (United States)

Tabachnick, M; Perret, V

1987-08-01

[125I] Thyroxine has been covalently bound to the thyroxine binding site in thyroxine-binding globulin by reaction with the bifunctional reagent, 1,5-difluoro-2,4-dinitrobenzene. An average of 0.47 mol of [125I] thyroxine was incorporated per mol protein; nonspecific binding amounted to 8%. A labeled peptide fragment was isolated from a proteolytic digest of the derivatized protein by HPLC and its amino acid composition was determined. Comparison with the amino acid sequence of thyroxine-binding globulin indicated partial correspondence of the labeled peptide with two possible regions in the protein. These regions also coincide with part of the barrel structure present in the closely homologous protein, alpha 1-antitrypsin.

Are radiogallium-labelled DOTA-conjugated somatostatin analogues superior to those labelled with other radiometals?

Energy Technology Data Exchange (ETDEWEB)

Antunes, P.; Ginj, M.; Zhang, H.; Maecke, H. [University Hospital Basel, Division of Radiological Chemistry, Basel (Switzerland); Waser, B.; Reubi, J.C. [University of Bern, Institute of Pathology, Bern (Switzerland); Baum, R.P. [Zentralklinik Bad Berka, Department of Nuclear Medicine/PETCT-Center, Bad Berka (Germany)

2007-07-15

Gallium-68 is a metallic positron emitter with a half-life of 68 min that is ideal for the in vivo use of small molecules, such as [{sup 68}Ga-DOTA,Tyr{sup 3}]octreotide, in the diagnostic imaging of somatostatin receptor-positive tumours. In preclinical studies it has shown a striking superiority over its {sup 111}In-labelled congener. The purpose of this study was to evaluate whether third-generation somatostatin-based, radiogallium-labelled peptides show the same superiority. Peptides were synthesised on solid phase. The receptor affinity was determined by in vitro receptor autoradiography. The internalisation rate was studied in AR4-2J and hsst-HEK-transfected cell lines. The pharmacokinetics was studied in a rat xenograft tumour model, AR4-2J. All peptides showed high affinities on hsst2, with the highest affinity for the Ga{sup III}-complexed peptides. On hsst3 the situation was reversed, with a trend towards lower affinity of the Ga{sup III} peptides. A significantly increased internalisation rate was found in sst2-expressing cells for all {sup 67}Ga-labelled peptides. Internalisation into HEK-sst3 was usually faster for the {sup 111}In-labelled peptides. No internalisation was found into sst5. Biodistribution studies employing [{sup 67}Ga-DOTA,1-Nal{sup 3}]octreotide in comparison to [{sup 111}In-DOTA,1-Nal{sup 3}]octreotide and [{sup 67}Ga-DOTA,Tyr{sup 3}]octreotide showed a significantly higher and receptor-mediated uptake of the two{sup 67}Ga-labelled peptides in the tumour and somatostatin receptor-positive tissues. A patient study illustrated the potential advantage of a broad receptor subtype profile radiopeptide over a high-affinity sst2-selective radiopeptide. This study demonstrates that {sup 67/68}Ga-DOTA-octapeptides show distinctly better preclinical, pharmacological performances than the {sup 111}In-labelled peptides, especially on sst2-expressing cells and the corresponding animal models. They may be excellent candidates for further

Are radiogallium-labelled DOTA-conjugated somatostatin analogues superior to those labelled with other radiometals?

International Nuclear Information System (INIS)

Antunes, P.; Ginj, M.; Zhang, H.; Maecke, H.; Waser, B.; Reubi, J.C.; Baum, R.P.

2007-01-01

Gallium-68 is a metallic positron emitter with a half-life of 68 min that is ideal for the in vivo use of small molecules, such as [ 68 Ga-DOTA,Tyr 3 ]octreotide, in the diagnostic imaging of somatostatin receptor-positive tumours. In preclinical studies it has shown a striking superiority over its 111 In-labelled congener. The purpose of this study was to evaluate whether third-generation somatostatin-based, radiogallium-labelled peptides show the same superiority. Peptides were synthesised on solid phase. The receptor affinity was determined by in vitro receptor autoradiography. The internalisation rate was studied in AR4-2J and hsst-HEK-transfected cell lines. The pharmacokinetics was studied in a rat xenograft tumour model, AR4-2J. All peptides showed high affinities on hsst2, with the highest affinity for the Ga III -complexed peptides. On hsst3 the situation was reversed, with a trend towards lower affinity of the Ga III peptides. A significantly increased internalisation rate was found in sst2-expressing cells for all 67 Ga-labelled peptides. Internalisation into HEK-sst3 was usually faster for the 111 In-labelled peptides. No internalisation was found into sst5. Biodistribution studies employing [ 67 Ga-DOTA,1-Nal 3 ]octreotide in comparison to [ 111 In-DOTA,1-Nal 3 ]octreotide and [ 67 Ga-DOTA,Tyr 3 ]octreotide showed a significantly higher and receptor-mediated uptake of the two 67 Ga-labelled peptides in the tumour and somatostatin receptor-positive tissues. A patient study illustrated the potential advantage of a broad receptor subtype profile radiopeptide over a high-affinity sst2-selective radiopeptide. This study demonstrates that 67/68 Ga-DOTA-octapeptides show distinctly better preclinical, pharmacological performances than the 111 In-labelled peptides, especially on sst2-expressing cells and the corresponding animal models. They may be excellent candidates for further development for clinical studies. (orig.)

Radiopharmaceutical development of radiolabelled peptides

Energy Technology Data Exchange (ETDEWEB)

Fani, Melpomeni; Maecke, Helmut R. [University Hospital Freiburg, Department of Nuclear Medicine, Freiburg (Germany)

2012-02-15

Receptor targeting with radiolabelled peptides has become very important in nuclear medicine and oncology in the past few years. The overexpression of many peptide receptors in numerous cancers, compared to their relatively low density in physiological organs, represents the molecular basis for in vivo imaging and targeted radionuclide therapy with radiolabelled peptide-based probes. The prototypes are analogs of somatostatin which are routinely used in the clinic. More recent developments include somatostatin analogs with a broader receptor subtype profile or with antagonistic properties. Many other peptide families such as bombesin, cholecystokinin/gastrin, glucagon-like peptide-1 (GLP-1)/exendin, arginine-glycine-aspartic acid (RGD) etc. have been explored during the last few years and quite a number of potential radiolabelled probes have been derived from them. On the other hand, a variety of strategies and optimized protocols for efficient labelling of peptides with clinically relevant radionuclides such as {sup 99m}Tc, M{sup 3+} radiometals ({sup 111}In, {sup 86/90}Y, {sup 177}Lu, {sup 67/68}Ga), {sup 64/67}Cu, {sup 18}F or radioisotopes of iodine have been developed. The labelling approaches include direct labelling, the use of bifunctional chelators or prosthetic groups. The choice of the labelling approach is driven by the nature and the chemical properties of the radionuclide. Additionally, chemical strategies, including modification of the amino acid sequence and introduction of linkers/spacers with different characteristics, have been explored for the improvement of the overall performance of the radiopeptides, e.g. metabolic stability and pharmacokinetics. Herein, we discuss the development of peptides as radiopharmaceuticals starting from the choice of the labelling method and the conditions to the design and optimization of the peptide probe, as well as some recent developments, focusing on a selected list of peptide families, including somatostatin

Peptide radiopharmaceuticals in nuclear medicine

International Nuclear Information System (INIS)

Blok, D.; Vermeij, P.; Feitsma, R.I.J.; Pauwels, E.J.K.

1999-01-01

This article reviews the labelling of peptides that are recognised to be of interest for nuclear medicine or are the subject of ongoing nuclear medicine research. Applications and approaches to the labelling of peptide radiopharmaceuticals are discussed, and drawbacks in their development considered. (orig.)

{sup 18}F-labeled RGD peptide: initial evaluation for imaging brain tumor angiogenesis

Energy Technology Data Exchange (ETDEWEB)

Chen Xiaoyuan; Park, Ryan; Shahinian, Anthony H.; Tohme, Michel; Khankaldyyan, Vazgen; Bozorgzadeh, Mohammed H.; Bading, James R.; Moats, Rex; Laug, Walter E.; Conti, Peter S. E-mail: pconti@usc.edu

2004-02-01

Brain tumors are highly angiogenesis dependent. The cell adhesion receptor integrin {alpha}{sub v}{beta}{sub 3} is overexpressed in glioma and activated endothelial cells and plays an important role in brain tumor growth, spread and angiogenesis. Suitably labeled {alpha}{sub v}{beta}{sub 3}-integrin antagonists may therefore be useful for imaging brain tumor associated angiogenesis. Cyclic RGD peptide c(RGDyK) was labeled with {sup 18}F via N-succinimidyl-4-[{sup 18}F]fluorobenzoate through the side-chain {epsilon}-amino group of the lysine residue. The radiotracer was evaluated in vivo for its tumor targeting efficacy and pharmacokinetics in subcutaneously implanted U87MG and orthotopically implanted U251T glioblastoma nude mouse models by means of microPET, quantitative autoradiography and direct tissue sampling. The N-4-[{sup 18}F]fluorobenzoyl-RGD ([{sup 18}F]FB-RGD) was produced in less than 2 h with 20-25% decay-corrected yields and specific activity of 230 GBq/{mu}mol at end of synthesis. The tracer showed very rapid blood clearance and both hepatobiliary and renal excretion. Tumor-to-muscle uptake ratio at 30 min was approximately 5 in the subcutaneous U87MG tumor model. MicroPET imaging with the orthotopic U251T brain tumor model revealed very high tumor-to-brain ratio, with virtually no uptake in the normal brain. Successful blocking of tumor uptake of [{sup 18}F]FB-RGD in the presence of excess amount of c(RGDyK) revealed receptor specific activity accumulation. Hence, N-4-[{sup 18}F]fluorobenzoyl labeled cyclic RGD peptide [{sup 18}F]FB-RGD is a potential tracer for imaging {alpha}{sub v}{beta}{sub 3}-integrin positive tumors in brain and other anatomic locations.

Quantum dot nanoparticle conjugation, characterization, and applications in neuroscience

Science.gov (United States)

Pathak, Smita

Quantum dot are semiconducting nanoparticles that have been used for decades in a variety of applications such as solar cells, LEDs and medical imaging. Their use in the last area, however, has been extremely limited despite their potential as revolutionary new biological labeling tools. Quantum dots are much brighter and more stable than conventional fluorophores, making them optimal for high resolution imaging and long term studies. Prior work in this area involves synthesizing and chemically conjugating quantum dots to molecules of interest in-house. However this method is both time consuming and prone to human error. Additionally, non-specific binding and nanoparticle aggregation currently prevent researchers from utilizing this system to its fullest capacity. Another critical issue that has not been addressed is determining the number of ligands bound to nanoparticles, which is crucial for proper interpretation of results. In this work, methods to label fixed cells using two types of chemically modified quantum dots are studied. Reproducible non-specific artifact labeling is consistently demonstrated if antibody-quantum dot conditions are less than optimal. In order to explain this, antibodies bound to quantum dots were characterized and quantified. While other groups have qualitatively characterized antibody functionalized quantum dots using TEM, AFM, UV spectroscopy and gel electrophoresis, and in some cases have reported calculated estimates of the putative number of total antibodies bound to quantum dots, no quantitative experimental results had been reported prior to this work. The chemical functionalization and characterization of quantum dot nanocrystals achieved in this work elucidates binding mechanisms of ligands to nanoparticles and allows researchers to not only translate our tools to studies in their own areas of interest but also derive quantitative results from these studies. This research brings ease of use and increased reliability to

Core–shell quantum dots: Properties and applications

Energy Technology Data Exchange (ETDEWEB)

Vasudevan, D., E-mail: vasudevand@rediffmail.com [Electrodics and electrocatalysis division, CSIR-CECRI, Karaikudi 630006 (India); Gaddam, Rohit Ranganathan [Amity Institute of Nanotechnology, Amity University, Noida 201301 (India); Trinchi, Adrian; Cole, Ivan [CSIRO Materials Science and Engineering, Clayton South MDC, 3169 (Australia)

2015-07-05

Fluorescent quantum dots (QDs) are semiconducting nanocrystals (NCs) that find numerous applications in areas, such as bio labelling, sensors, lasers, light emitting diodes and medicine. Core–shell quantum dots were developed to improve the photoluminescence efficiency of single quantum dots. Capping their surface with organic ligands as well as their extraction into aqueous media enables their use in sensing applications. The current review highlights the importance and applications of core shell quantum dots as well as their surface modifications and applications in the field of medicine and as sensors for chemical and biochemical analysis.

Core–shell quantum dots: Properties and applications

International Nuclear Information System (INIS)

Vasudevan, D.; Gaddam, Rohit Ranganathan; Trinchi, Adrian; Cole, Ivan

2015-01-01

Fluorescent quantum dots (QDs) are semiconducting nanocrystals (NCs) that find numerous applications in areas, such as bio labelling, sensors, lasers, light emitting diodes and medicine. Core–shell quantum dots were developed to improve the photoluminescence efficiency of single quantum dots. Capping their surface with organic ligands as well as their extraction into aqueous media enables their use in sensing applications. The current review highlights the importance and applications of core shell quantum dots as well as their surface modifications and applications in the field of medicine and as sensors for chemical and biochemical analysis

PET imaging of alphavbeta integrin expression in tumours with Ga-labelled mono-, di- and tetrameric RGD peptides

NARCIS (Netherlands)

Dijkgraaf, I.; Yim, C.B.; Franssen, G.M.; Schuit, R.C.; Luurtsema, G.; Liu, S.; Oyen, W.J.G.; Boerman, O.C.

2011-01-01

PURPOSE: Due to the restricted expression of alpha(v)beta(3) in tumours, alpha(v)beta(3) is considered a suitable receptor for tumour targeting. In this study the alpha(v)beta(3)-binding characteristics of (68)Ga-labelled monomeric, dimeric and tetrameric RGD peptides were determined and compared

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.

Acetone-Linked Peptides: A Convergent Approach for Peptide Macrocyclization and Labeling.

Science.gov (United States)

Assem, Naila; Ferreira, David J; Wolan, Dennis W; Dawson, Philip E

2015-07-20

Macrocyclization is a broadly applied approach for overcoming the intrinsically disordered nature of linear peptides. Herein, it is shown that dichloroacetone (DCA) enhances helical secondary structures when introduced between peptide nucleophiles, such as thiols, to yield an acetone-linked bridge (ACE). Aside from stabilizing helical structures, the ketone moiety embedded in the linker can be modified with diverse molecular tags by oxime ligation. Insights into the structure of the tether were obtained through co-crystallization of a constrained S-peptide in complex with RNAse S. The scope of the acetone-linked peptides was further explored through the generation of N-terminus to side chain macrocycles and a new approach for generating fused macrocycles (bicycles). Together, these studies suggest that acetone linking is generally applicable to peptide macrocycles with a specific utility in the synthesis of stabilized helices that incorporate functional tags. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Uptake of silica covered Quantum Dots into living cells: Long term vitality and morphology study on hyaluronic acid biomaterials

International Nuclear Information System (INIS)

D'Amico, Michele; Fiorica, Calogero; Palumbo, Fabio Salvatore; Militello, Valeria; Leone, Maurizio; Dubertret, Benoit; Pitarresi, Giovanna; Giammona, Gaetano

2016-01-01

Quantum Dots (QDs) are promising very bright and stable fluorescent probes for optical studies in the biological field but water solubility and possible metal bio-contamination need to be addressed. In this work, a simple silica-QD hybrid system is prepared and the uptake in bovine chondrocytes living cells without any functionalization of the external protective silica shield is demonstrated. Moreover, long term treated cells vitality (up to 14 days) and the transfer of silica-QDs to the next cell generations are here reported. Confocal fluorescence microscopy was also used to determine the morphology of the so labelled cells and the relative silica-QDs distribution. Finally, we employ silica-QD stained chondrocytes to characterize, as proof of concept, hydrogels obtained from an amphiphilic derivative of hyaluronic acid (HA-EDA-C _1_8) functionalized with different amounts of the RGD peptide. - Highlights: • Non functionalized silica-quantum dots fluorescent nanoparticles uptake is observed. • Morphology studies of such cells could be done by confocal fluorescence microscopy. • Labelled chondrocytes are viable until at least 14 days. • RGD functionalized Hyaluronic Acid hydrogels are studied as cell scaffolds. • Chondrocyte are promptly attached on RGD-functionalized hydrogels.

Uptake of silica covered Quantum Dots into living cells: Long term vitality and morphology study on hyaluronic acid biomaterials

Energy Technology Data Exchange (ETDEWEB)

D' Amico, Michele [Dip. Biomedico di Medicina Interna e Specialistica, Universitá degli Studi di Palermo, Piazza delle Cliniche, 2, 90127 Palermo (Italy); Dip. di Fisica e Chimica, Universitá degli Studi di Palermo, Viale delle Scienze, Ed. 18, 90128 Palermo (Italy); Fiorica, Calogero, E-mail: calogero.fiorica@unipa.it [Dip. di Scienze e Tecnologie Molecolari e Biomolecolari, Sezione di Chimica e Tecnologie Farmaceutiche, Universitá degli Studi di Palermo, Via Archirafi, 28, 90136 Palermo (Italy); Palumbo, Fabio Salvatore [Dip. di Scienze e Tecnologie Molecolari e Biomolecolari, Sezione di Chimica e Tecnologie Farmaceutiche, Universitá degli Studi di Palermo, Via Archirafi, 28, 90136 Palermo (Italy); Militello, Valeria; Leone, Maurizio [Dip. di Fisica e Chimica, Universitá degli Studi di Palermo, Viale delle Scienze, Ed. 18, 90128 Palermo (Italy); Dubertret, Benoit [Laboratoire de Physique et d’Etude des Matèriaux, ESPCI-ParisTech, PSL Research University, Sorbonne Universitè UPMC Univ. Paris 06, CNRS, 10 rue Vauquelin, 75005 Paris (France); Pitarresi, Giovanna; Giammona, Gaetano [Dip. di Scienze e Tecnologie Molecolari e Biomolecolari, Sezione di Chimica e Tecnologie Farmaceutiche, Universitá degli Studi di Palermo, Via Archirafi, 28, 90136 Palermo (Italy)

2016-10-01

Quantum Dots (QDs) are promising very bright and stable fluorescent probes for optical studies in the biological field but water solubility and possible metal bio-contamination need to be addressed. In this work, a simple silica-QD hybrid system is prepared and the uptake in bovine chondrocytes living cells without any functionalization of the external protective silica shield is demonstrated. Moreover, long term treated cells vitality (up to 14 days) and the transfer of silica-QDs to the next cell generations are here reported. Confocal fluorescence microscopy was also used to determine the morphology of the so labelled cells and the relative silica-QDs distribution. Finally, we employ silica-QD stained chondrocytes to characterize, as proof of concept, hydrogels obtained from an amphiphilic derivative of hyaluronic acid (HA-EDA-C {sub 18}) functionalized with different amounts of the RGD peptide. - Highlights: • Non functionalized silica-quantum dots fluorescent nanoparticles uptake is observed. • Morphology studies of such cells could be done by confocal fluorescence microscopy. • Labelled chondrocytes are viable until at least 14 days. • RGD functionalized Hyaluronic Acid hydrogels are studied as cell scaffolds. • Chondrocyte are promptly attached on RGD-functionalized hydrogels.

Quantum dots as optical labels for ultrasensitive detection of polyphenols.

Science.gov (United States)

Akshath, Uchangi Satyaprasad; Shubha, Likitha R; Bhatt, Praveena; Thakur, Munna Singh

2014-07-15

Considering the fact that polyphenols have versatile activity in-vivo, its detection and quantification is very much important for a healthy diet. Laccase enzyme can convert polyphenols to yield mono/polyquinones which can quench Quantum dots fluorescence. This phenomenon of charge transfer from quinones to QDs was exploited as optical labels to detect polyphenols. CdTe QD may undergo dipolar interaction with quinones as a result of broad spectral absorption due to multiple excitonic states resulting from quantum confinement effects. Thus, "turn-off" fluorescence method was applied for ultrasensitive detection of polyphenols by using laccase. We observed proportionate quenching of QDs fluorescence with respect to polyphenol concentration in the range of 100 µg to 1 ng/mL. Also, quenching of the photoluminescence was highly efficient and stable and could detect individual and total polyphenols with high sensitivity (LOD-1 ng/mL). Moreover, proposed method was highly efficient than any other reported methods in terms of sensitivity, specificity and selectivity. Therefore, a novel optical sensor was developed for the detection of polyphenols at a sensitive level based on the charge transfer mechanism. Copyright © 2014 Elsevier B.V. All rights reserved.

Direct and indirect radioiodination of protein: comparative study of chemotactic peptide labeling; Radioiodacao de proteina por via direta e indireta: estudo comparativo da marcacao de peptideo quimiotatico

Energy Technology Data Exchange (ETDEWEB)

Lavinas, Tatiana

2004-07-01

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-[{sup 123}l/{sup 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

Synthesis of radioactively labelled CdSe/CdS/ZnS quantum dots for in vivo experiments

Directory of Open Access Journals (Sweden)

Gordon M. Stachowski

2014-12-01

Full Text Available During the last decades of nanoparticles research, many nanomaterials have been developed for applications in the field of bio-labelling. For the visualization of transport processes in the body, organs and cells, luminescent quantum dots (QDs make for highly useful diagnostic tools. However, intercellular routes, bio-distribution, metabolism during degradation or quantification of the excretion of nanoparticles, and the study of the biological response to the QDs themselves are areas which to date have not been fully investigated. In order to aid in addressing those issues, CdSe/CdS/ZnS QDs were radioactively labelled, which allows quantification of the QD concentration in the whole body or in ex vivo samples by γ-counting. However, the synthesis of radioactively labelled QDs is not trivial since the coating process must be completely adapted, and material availability, security and avoidance of radioactive waste must be considered. In this contribution, the coating of CdSe/CdS QDs with a radioactive 65ZnS shell using a modified, operator-safe, SILAR procedure is presented. Under UV illumination, no difference in the photoluminescence of the radioactive and non-radioactive CdSe/CdS/ZnS colloidal solutions was observed. Furthermore, a down-scaled synthesis for the production of very small batches of 5 nmol QDs without loss in the fluorescence quality was developed. Subsequently, the radio-labelled QDs were phase transferred by encapsulation into an amphiphilic polymer. γ-counting of the radioactivity provided confirmation of the successful labelling and phase transfer of the QDs.

Next-generation detection of antigen-responsive T cells using DNA barcode-labeled peptide-major histocompatibility complex I multimers

DEFF Research Database (Denmark)

Bentzen, Amalie Kai; Marquard, Andrea Marion; Lyngaa, Rikke Birgitte

2016-01-01

sample using >1000 different peptide-MHC multimers labeled with individual DNA barcodes.After isolation of MHC multimer binding T cells their recognition are revealed by amplification andsequencing of the MHC multimer-associated DNA barcodes. The relative frequency of the sequencedDNA barcodes...... originating from a given peptide-MHC motif relates to the size of the antigenresponsiveT cell population. We have demonstrated the use of large panels of >1000 DNA barcodedMHC multimers for detection of rareT cell populations of virus and cancer-restricted origin in various tissues and compared...

Epidemiology and whole genome sequencing of an ongoing point-source Salmonella Agona outbreak associated with sushi consumption in western Sydney, Australia 2015.

Science.gov (United States)

Thompson, C K; Wang, Q; Bag, S K; Franklin, N; Shadbolt, C T; Howard, P; Fearnley, E J; Quinn, H E; Sintchenko, V; Hope, K G

2017-07-01

During May 2015, an increase in Salmonella Agona cases was reported from western Sydney, Australia. We examine the public health actions used to investigate and control this increase. A descriptive case-series investigation was conducted. Six outbreak cases were identified; all had consumed cooked tuna sushi rolls purchased within a western Sydney shopping complex. Onset of illness for outbreak cases occurred between 7 April and 24 May 2015. Salmonella was isolated from food samples collected from the implicated premise and a prohibition order issued. No further cases were identified following this action. Whole genome sequence (WGS) analysis was performed on isolates recovered during this investigation, with additional S. Agona isolates from sporadic-clinical cases and routine food sampling in New South Wales, January to July 2015. Clinical isolates of outbreak cases were indistinguishable from food isolates collected from the implicated sushi outlet. Five additional clinical isolates not originally considered to be linked to the outbreak were genomically similar to outbreak isolates, indicating the point-source contamination may have started before routine surveillance identified an increase. This investigation demonstrated the value of genomics-guided public health action, where near real-time WGS enhanced the resolution of the epidemiological investigation.

Molecular imaging of neuroendocrine tumors using 68Ga-labeled peptides (Somatostatin receptor PET/CT)

International Nuclear Information System (INIS)

Baum, R.P.; Prasad, V.; Hoersch, D.

2009-01-01

Receptor PET/CT using 68 Ga-labeled somatostatin analogues (DOTA-NOC, DOTA-TOC or DOTA-TATE) enables the highly sensitive molecular imaging of neuroendocrine tumors (NETs) based on the expression of somatostatin receptors and even the detection of receptor subtypes. Our experience after more than 3000 studies shows that receptor PET/CT has a significantly higher tumor detection rate than conventional scintigraphy (even in SPECT/CT technique), and that tumor lesions can be very accurately localized. By calculating standardized uptake values (SUV) - which are reproducible and investigator-independent - patients can be selected for peptide receptor radiotherapy and also the course after therapy can be controlled. Receptor-PET/CT is the most sensitive imaging modality for the detection of unknown primary tumors (CUP syndrome), which is especially true for the detection of neuroendocrine tumors of the pancreas and small bowel; whole-body staging (''one stop shop'') as well as restaging and selection of patients for peptide receptor radiotherapy can be performed using a patient-friendly procedure (examination finished within one hour) exposing the patient to less radiation than whole-body CT scanning. The 68 Ge/ 68 Ga generator has proved very reliable over the years - even in a hospital environment. The effective costs for 68 Ga labeled somatostatin analogues might be less than for scintigraphic agents, provided a certain number of studies per year are performed. The development of new tumor-specific peptides as well as of other DOTA- or NOTA-coupled radiopharmaceuticals opens a new avenue into the future: finally, the 68 Ga generator could play a similar important role for PET/CT as did the 99m Tc-Generator for conventional gamma camera imaging over the last decades. (orig.)

Sub-cellular localisation of a 15N-labelled peptide vector using NanoSIMS imaging

Science.gov (United States)

Römer, Winfried; Wu, Ting-Di; Duchambon, Patricia; Amessou, Mohamed; Carrez, Danièle; Johannes, Ludger; Guerquin-Kern, Jean-Luc

2006-07-01

Dynamic SIMS imaging is proposed to map sub-cellular distributions of isotopically labelled, exogenous compounds. NanoSIMS imaging allows the characterisation of the intracellular transport pathways of exogenous molecules, including peptide vectors employed in innovative therapies, using stable isotopes as molecular markers to detect the compound of interest. Shiga toxin B-subunit (STxB) was chosen as a representative peptide vector. The recombinant protein ( 15N-STxB) was synthesised in Escherichia coli using 15NH 4Cl as sole nitrogen source resulting in 15N enrichment in the molecule. Using the NanoSIMS 50 ion microprobe (Cameca), different ion species ( 12C 14N -, 12C 15N -, 31P -) originating from the same sputtered micro volume were simultaneously detected. High mass resolving power enabled the discrimination of 12C 15N - from its polyatomic isobars of mass 27. We imaged the membrane binding and internalisation of 15N-STxB in HeLa cells at spatial resolutions of less than 100 nm. Thus, the use of rare stable isotopes like 15N with dynamic SIMS imaging permits sub-cellular detection of isotopically labelled, exogenous molecules and imaging of their transport pathways at high mass and spatial resolution. Application of stable isotopes as markers can replace the large and chemically complex tags used for fluorescence microscopy, without altering the chemical and physical properties of the molecule.

Use of a nitrotryptophan-containing peptide for photoaffinity labeling the pancreatic cholecystokinin receptor

International Nuclear Information System (INIS)

Klueppelberg, U.G.; Gaisano, H.Y.; Powers, S.P.; Miller, L.J.

1989-01-01

The authors report the preparation and characterization of a new type of intrinsic photoaffinity labeling probe, on the basis of the incorporation of a photolabile nitrotryptophan into a biologically relevant domain of a peptide. The model system used was the pancreatic cholecystokinin (CCK) receptor, previously affinity labeled with a variety of probes. Those studies have suggested that an M r = 85,000-95,000 protein is more likely to be labeled as the site of covalent attachment approaches the receptor-binding domain of this hormone. Indeed, CCK has a Trp in the center of its receptor-binding region, and replacement of that residue with 6-nitrotryptophan resulted in a photolabile probe which affinity labeled the same M r = 85,000-95,000 pancreatic membrane protein. This probe, 125 I-D-Tyr-Gly-[(Nle 28,31 ,6-NO 2 -Trp 30 )CCK-26-33], was synthesized by solid-phase and solution techniques and characterized by mass spectrometry. Following oxidative iodination, it was purified on HPLC to 2000 Ci/mmol. Binding to pancreatic membranes was rapid, temperature dependent, reversible, saturable, and specific and was with high affinity. While its binding affinity was only 3-fold lower than that of native CCK-8, this probe was 70-fold less potent than native hormone in stimulating amylase secretion and equally efficacious to native hormone

Dynamic PET and Optical Imaging and Compartment Modeling using a Dual-labeled Cyclic RGD Peptide Probe

Directory of Open Access Journals (Sweden)

Lei Zhu, Ning Guo, Quanzheng Li, Ying Ma, Orit Jacboson, Seulki Lee, Hak Soo Choi, James R. Mansfield, Gang Niu, Xiaoyuan Chen

2012-01-01

Full Text Available Purpose: The aim of this study is to determine if dynamic optical imaging could provide comparable kinetic parameters to that of dynamic PET imaging by a near-infrared dye/64Cu dual-labeled cyclic RGD peptide.Methods: The integrin αvβ3 binding RGD peptide was conjugated with a macrocyclic chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA for copper labeling and PET imaging and a near-infrared dye ZW-1 for optical imaging. The in vitro biological activity of RGD-C(DOTA-ZW-1 was characterized by cell staining and receptor binding assay. Sixty-min dynamic PET and optical imaging were acquired on a MDA-MB-435 tumor model. Singular value decomposition (SVD method was applied to compute the dynamic optical signal from the two-dimensional optical projection images. Compartment models were used to quantitatively analyze and compare the dynamic optical and PET data.Results: The dual-labeled probe 64Cu-RGD-C(DOTA-ZW-1 showed integrin specific binding in vitro and in vivo. The binding potential (Bp derived from dynamic optical imaging (1.762 ± 0.020 is comparable to that from dynamic PET (1.752 ± 0.026.Conclusion: The signal un-mixing process using SVD improved the accuracy of kinetic modeling of 2D dynamic optical data. Our results demonstrate that 2D dynamic optical imaging with SVD analysis could achieve comparable quantitative results as dynamic PET imaging in preclinical xenograft models.

Dynamic PET and Optical Imaging and Compartment Modeling using a Dual-labeled Cyclic RGD Peptide Probe.

Science.gov (United States)

Zhu, Lei; Guo, Ning; Li, Quanzheng; Ma, Ying; Jacboson, Orit; Lee, Seulki; Choi, Hak Soo; Mansfield, James R; Niu, Gang; Chen, Xiaoyuan

2012-01-01

The aim of this study is to determine if dynamic optical imaging could provide comparable kinetic parameters to that of dynamic PET imaging by a near-infrared dye/(64)Cu dual-labeled cyclic RGD peptide. The integrin α(v)β(3) binding RGD peptide was conjugated with a macrocyclic chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) for copper labeling and PET imaging and a near-infrared dye ZW-1 for optical imaging. The in vitro biological activity of RGD-C(DOTA)-ZW-1 was characterized by cell staining and receptor binding assay. Sixty-min dynamic PET and optical imaging were acquired on a MDA-MB-435 tumor model. Singular value decomposition (SVD) method was applied to compute the dynamic optical signal from the two-dimensional optical projection images. Compartment models were used to quantitatively analyze and compare the dynamic optical and PET data. The dual-labeled probe (64)Cu-RGD-C(DOTA)-ZW-1 showed integrin specific binding in vitro and in vivo. The binding potential (Bp) derived from dynamic optical imaging (1.762 ± 0.020) is comparable to that from dynamic PET (1.752 ± 0.026). The signal un-mixing process using SVD improved the accuracy of kinetic modeling of 2D dynamic optical data. Our results demonstrate that 2D dynamic optical imaging with SVD analysis could achieve comparable quantitative results as dynamic PET imaging in preclinical xenograft models.

One pot synthesis of highly luminescent polyethylene glycol anchored carbon dots functionalized with a nuclear localization signal peptide for cell nucleus imaging.

Science.gov (United States)

Yang, Lei; Jiang, Weihua; Qiu, Lipeng; Jiang, Xuewei; Zuo, Daiying; Wang, Dongkai; Yang, Li

2015-04-14

Strong blue fluorescent polyethylene glycol (PEG) anchored carbon nitride dots (CDs@PEG) with a high quantum yield (QY) of 75.8% have been synthesized by a one step hydrothermal treatment. CDs with a diameter of ca. 6 nm are well dispersed in water and present a graphite-like structure. Photoluminescence (PL) studies reveal that CDs display excitation-dependent behavior and are stable under various test conditions. Based on the as-prepared CDs, we designed novel cell nucleus targeting imaging carbon dots functionalized with a nuclear localization signal (NLS) peptide. The favourable biocompatibilities of CDs and NLS modified CDs (NLS-CDs) are confirmed by in vitro cytotoxicity assays. Importantly, intracellular localization experiments in MCF7 and A549 cells demonstrate that NLS-CDs could be internalized in the nucleus and show blue light, which indicates that CDs may serve as cell nucleus imaging probes.

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.

An assessment tumor targeting ability of 177Lu labeled cyclic CCK analogue peptide by binding with cholecystokinin receptor

Directory of Open Access Journals (Sweden)

Eun-Ha Cho

2016-07-01

Full Text Available The cholecystokinin (CCK receptor is known as a receptor that is overexpressed in many human tumors. The present study was designed to investigate the targeting ability of cyclic CCK analogue in AR42J pancreatic cells. The CCK analogues, DOTA-K(glucose-Gly-Trp-Nle-Asp-Phe (DOTA-glucose-CCK and DOTA-Nle-cyclo(Glu-Trp-Nle-Asp-Phe-Lys-NH2 (DOTA-[Nle]-cCCK, were synthesized and radiolabeled with 177Lu, and competitive binding was evaluated. The binding appearance of synthesized peptide with AR42J cells was evaluated by confocal microscopy. And bio-distribution was performed in AR42J xenografted mice. Synthesized peptides were prepared by a solid phase synthesis method, and their purity was over 98%. DOTA is the chelating agent for 177Lu-labeling, in which the peptides were radiolabeled with 177Lu by a high radiolabeling yield. A competitive displacement of 125I-CCK8 on the AR42J cells revealed that the 50% inhibitory concentration value (IC50 was 12.3 nM of DOTA-glucose-CCK and 1.7 nM of DOTA-[Nle]-cCCK. Radio-labeled peptides were accumulated in AR42J tumor in vivo, and %ID/g of the tumor was 0.4 and 0.9 at 2 h p.i. It was concluded that 177Lu-DOTA-[Nle]-cCCK has higher binding affinity than 177Lu-DOTA-glucose-CCK and can be a potential candidate as a targeting modality for a CCK receptor over-expressing tumors.

Protease-activated quantum dot probes

International Nuclear Information System (INIS)

Chang, Emmanuel; Miller, Jordan S.; Sun, Jiantang; Yu, William W.; Colvin, Vicki L.; Drezek, Rebekah; West, Jennifer L.

2005-01-01

We have developed a novel nanoparticulate luminescent probe with inherent signal amplification upon interaction with a targeted proteolytic enzyme. This construct may be useful for imaging in cancer detection and diagnosis. In this system, quantum dots (QDs) are bound to gold nanoparticles (AuNPs) via a proteolytically degradable peptide sequence to non-radiatively suppress luminescence. A 71% reduction in luminescence was achieved with conjugation of AuNPs to QDs. Release of AuNPs by peptide cleavage restores radiative QD photoluminescence. Initial studies observed a 52% rise in luminescence over 47 h of exposure to 0.2 mg/mL collagenase. These probes can be customized for targeted degradation simply by changing the sequence of the peptide linker

Ultrasmall visible-to-near-infrared emitting silver-sulfide quantum dots for cancer detection and imaging

Science.gov (United States)

Tang, Rui; Xu, Baogang; Shen, Duanwen; Sudlow, Gail; Achilefu, Samuel

2018-02-01

The large size of many near infrared (NIR) fluorescent nanoparticles prevents rapid extravasation from blood vessels and subsequent diffusion to tumors. This confines in vivo uptake to the peritumoral space and results in high liver retention. We developed a viscosity modulated approach to synthesize ultrasmall silver sulfide quantum dots (QDs) with distinct tunable light emission from visible to near-infrared in spectrum and a QD core diameter between less than 5 nm. Further functionalization of these Ag2S QDs with different type of molecules such as targeting peptides, retains monodisperse, relatively small water soluble QDs without loss of the functionality of the peptide's high binding affinity to cancerous tumor. Fluorescence and electron microscopy showed that selective integrin-mediated internalization was observed only in cancer cells treated with the peptide-labeled QDs, demonstrating that the unlabeled hydrophilic nanoparticles exhibit characteristics of negatively charged fluorescent dye molecules, which typically do not internalize in cells. The biodistribution profiles of intravenously administered QDs in different mouse models of cancer reveal an exceptionally high tumor-to-liver uptake ratio, suggesting that the small sized QDs evaded conventional opsonization and subsequent high uptake in the liver and spleen. The seamless tunability of the QDs over a wide spectral range with only a small increase in size, as well as the ease of labeling the bright and non-cytotoxic QDs with biomolecules, provides a platform for multiplexing information, tracking the trafficking of single molecules in cells, and selectively targeting disease biomarkers in living organisms without premature QD opsonization in circulating blood.

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).

Production of the antimicrobial peptide UBI 29-41 labelled with 99mTc by an indirect method

International Nuclear Information System (INIS)

Nevares, Noemi; Crudo, Jose L.; Zapata, Miguel; Castiglia, Silvia G. de

2003-01-01

The infection processes are a major problem in human health causing a high number of human deaths all around the world. Diagnostic imaging in nuclear medicine is an attractive option in the detection of infection processes due to its sensitivity. The antimicrobial peptides are very important in the development of new radiopharmaceuticals, since their antimicrobial activity towards a great variety of microorganisms have been proven. The aim of this work was to obtain the antimicrobial peptide UBI 29-41 labelled with technetium 99 m, by an indirect method via NHS-Hynic and tricine as a coligand, and evaluate its stability and its ability to discriminate between infection and inflammation sites. The radiochemical purity of the labeling procedure was 95.5±1,2 %. The cysteine challenge showed a great stability of the 99mTc UBI-Hynic, and the stability in human serum showed that the 81% of the radioactivity remained bounded to UBI-Hynic at 48 hs of incubation. The bio distribution's studies showed main elimination via kidney of 99mTc UBI-Hynic and the target/non target ratio was 1,81 for infected mice and 1,16 for inflamed mice. (author)

Melanoma targeting with [99mTc(N)(PNP3)]-labeled α-melanocyte stimulating hormone peptide analogs: Effects of cyclization on the radiopharmaceutical properties

International Nuclear Information System (INIS)

Carta, Davide; Salvarese, Nicola; Morellato, Nicolò; Gao, Feng; Sihver, Wiebke; Pietzsch, Hans Jurgen; Biondi, Barbara; Ruzza, Paolo; Refosco, Fiorenzo; Carpanese, Debora; Rosato, Antonio; Bolzati, Cristina

2016-01-01

The purpose of this study was to evaluate the effect of cyclization on the biological profile of a [ 99m Tc(N)(PNP3)]-labeled α-melanocyte stimulating hormone peptide analog. A lactam bridge-cyclized H-Cys-Ahx-βAla 3 -c[Lys 4 -Glu-His-D-Phe-Arg-Trp-Glu 10 ]-Arg 11 -Pro-Val-NH 2 (NAP―NS2) and the corresponding linear H-Cys-Ahx-βAla-Nle-Asp-His-D-Phe-Arg-Trp-Gly-NH 2 (NAP―NS1) peptide were synthetized, characterized by ESI-MS spectroscopy and their melanocortin-1 receptor (MC1R) binding affinity was determined in B16/F10 melanoma cells. The consistent [ 99m Tc(N)(PNP3)]-labeled compounds were readily obtained in high specific activity and their stability and biological properties were assessed. As an example, the chemical identity of [ 99m Tc(N)(NAP–NS1)(PNP3)] + was confirmed by carrier added experiments supported by radio/UV HPLC analysis combined with ESI(+)-MS. Compared with the linear peptide, cyclization negatively affected the biological properties of NAP–NS2 peptide by reducing its binding affinity for MC1R and by decreasing the overall excretion rate of the corresponding [ 99m Tc(N)(PNP3)]-labeled peptide from the body as well as its in vivo stability. [ 99m Tc(N)(NAP–NS1)(PNP3)] + was evaluated for its potential as melanoma imaging probe in murine melanoma model. Data from in vitro and in vivo studies on B16/F10 melanoma model of [ 99m Tc(N)(NAP–NS1)(PNP3)] + clearly evidenced that the radiolabeled linear peptide keeps its biological properties up on the conjugation to the [ 99m Tc(N)(PNP3)]-building block. The progressive increase of the tumor-to-nontarget ratios over the time indicates a quite stable interaction between the radio-complex and the MC1R.

Development of New Tritium Labelling Methods for Peptides & Investigation of Guest-Host Mediated Electrocyclization and Sigma-Tropic Rearrangement Reactions

DEFF Research Database (Denmark)

Pedersen, Martin Holst Friborg

The main parts of the work presented here is Part I; which have involved the installation of a Tritium Chemistry Facility for the synthesis of radiolabelled compounds with tritium, and Part II; the development of new tritium labelling methods for peptides. The intention of Part I is to supply bac...

Assigning Significance in Label-Free Quantitative Proteomics to Include Single-Peptide-Hit Proteins with Low Replicates

OpenAIRE

Li, Qingbo

2010-01-01

When sample replicates are limited in a label-free proteomics experiment, selecting differentially regulated proteins with an assignment of statistical significance remains difficult for proteins with a single-peptide hit or a small fold-change. This paper aims to address this issue. An important component of the approach employed here is to utilize the rule of Minimum number of Permuted Significant Pairings (MPSP) to reduce false positives. The MPSP rule generates permuted sample pairings fr...

Imaging GABAc Receptors with Ligand-Conjugated Quantum Dots

Directory of Open Access Journals (Sweden)

Ian D. Tomlinson

2007-01-01

Full Text Available We report a methodology for labeling the GABAc receptor on the surface membrane of intact cells. This work builds upon our earlier work with serotonin-conjugated quantum dots and our studies with PEGylated quantum dots to reduce nonspecific binding. In the current approach, a PEGylated derivative of muscimol was synthesized and attached via an amide linkage to quantum dots coated in an amphiphilic polymer derivative of a modified polyacrylamide. These conjugates were used to image GABAC receptors heterologously expressed in Xenopus laevis oocytes.

Quantitative phosphoproteomics using acetone-based peptide labeling: Method evaluation and application to a cardiac ischemia/reperfusion model

Science.gov (United States)

Wijeratne, Aruna B.; Manning, Janet R.; Schultz, Jo El J.; Greis, Kenneth D.

2013-01-01

Mass spectrometry (MS) techniques to globally profile protein phosphorylation in cellular systems that are relevant to physiological or pathological changes have been of significant interest in biological research. In this report, an MS-based strategy utilizing an inexpensive acetone-based peptide labeling technique known as reductive alkylation by acetone (RABA) for quantitative phosphoproteomics was explored to evaluate its capacity. Since the chemistry for RABA-labeling for phosphorylation profiling had not been previously reported, it was first validated using a standard phosphoprotein and identical phosphoproteomes from cardiac tissue extracts. A workflow was then utilized to compare cardiac tissue phosphoproteomes from mouse hearts not expressing FGF2 vs. hearts expressing low molecular weight fibroblast growth factor-2 (LMW FGF2) to relate low molecular weight fibroblast growth factor-2 (LMW FGF2) mediated cardioprotective phenomena induced by ischemia/reperfusion (I/R) injury of hearts, with downstream phosphorylation changes in LMW FGF2 signaling cascades. Statistically significant phosphorylation changes were identified at 14 different sites on 10 distinct proteins including some with mechanisms already established for LMW FGF2-mediated cardioprotective signaling (e.g. connexin-43), some with new details linking LMW FGF2 to the cardioprotective mechanisms (e.g. cardiac myosin binding protein C or cMyBPC), and also several new downstream effectors not previously recognized for cardio-protective signaling by LMW FGF2. Additionally, one of the phosphopeptides, cMyBPC/pSer-282, identified was further verified with site-specific quantification using an SRM (selected reaction monitoring)-based approach that also relies on isotope labeling of a synthetic phosphopeptide with deuterated acetone as an internal standard. Overall, this study confirms that the inexpensive acetone-based peptide labeling can be used in both exploratory and targeted quantification

Detection of KatG Gen Mutation on Mycobacterium Tuberculosis by Means of PCR-Dot Blot Hybridization with 32P Labeled Oligonucleotide Probe Methods

International Nuclear Information System (INIS)

Maria Lina R; Budiman Bela; Andi Yasmon

2009-01-01

Handling and controlling of tuberculosis, a disease caused by Mycobacterium tuberculosis (MTB), is now complicated since there are many MTBs that are resistant against anti-tuberculosis drugs such as isoniazid. The drug resistance could occurred due to the inadequate and un-regular drug utilization that cause gene mutation of the drug target such as katG gene for isoniazid. The molecular biology techniques such as the PCR- dot blot hybridization with radioisotope ( 32 P) labeled oligonucleotide probe, has been reported as a technique that is more sensitive and rapid for detection of gene mutations related with drug resistances. Hence, the aim of this study was to apply the PCR- dot blot hybridization technique using 32 P labeled oligonucleotide probe for detection of single mutation at codon 315 of katG gene of MTBs that rise the isoniazid resistance. In this study, we used 89 sputum specimens and a standard MTB (MTB H 37 RV) as a control. DNA extractions were performed by the BOOM method and the phenol chloroform for sputum samples and standard MTB, respectively. Primers used for PCR technique were Pt8 and Pt9 and RTB59 and RTB36 for detecting tuberculosis causing Mycobacterium and the existence of katG gene, respectively. Both of the primers are specific for IS6110 region and katG gene, respectively. PCR products were detected by an agarose gel electrophoresis technique. Dot blot hybridization with 32 P-oligonucleotide probe 315mu was performed to detect mutation at codon 315 of tested samples. Results of the PCR using primer Pt8 and Pt9 showed that all sputum specimens had positive results. Mutation detection by PCR- dot blot hybridization with 32 P-oligonucleotide probe 315mu, revealed that 11 of 89 tested samples had a mutation at their codon 315 of katG gene. Based upon these results, it is concluded that PCR-dot blot hybridization with 32 P-oligonucleotide probe is a technique that is rapid and highly specific and sensitive for detection of mutation at codon

Optimization of labelling PSMA-HBED-CC peptide with 68Ga

International Nuclear Information System (INIS)

Alcarde, Lais F.; Dias, Luis A.P.; Massicano, Adriana V.F.; Mengatti, Jair; Araujo, Elaine B. de

2015-01-01

Early detection of metastases or recurrent prostate cancer (PC) lesions is of clinical relevance in terms of clinical staging, prognosis and therapy management. When PC is not treated, it is potentially lethal. Clinical methods for diagnosis of PC include the dosage of prostatic specific antigen (PSA) and the rectal touch. Unfortunately, these initial procedures are not specific for PC detection. The level of PSA, in about 20 to 30% of the cases is high, due to benign pathologies, that result in false positive and unneeded biopsy. The prostatic specific membrane antigen (PSMA) is a type II transmembrane glycoprotein and differs from the PSA that is a free protein in blood. High levels of PSMA are observed in almost all prostatic pathologies and low levels were observed in brain, kidneys, salivary glands and small intestine. This fact stimulated the development of PSMA inhibitor molecules that could be used as a vector for imaging tumor agents and that could perfuse in the tumor microvasculature. Recent studies suggest that the chelator HBED-CC contributes intrinsically for the labelling of the PSMA inhibitor peptide based in urea - Glu-urea-Lys (Ahx) – to the pharmacophore group. This work describes the study of labelling conditions of PSMA-HBED-CC with 68 Ga and determined the ideal conditions to obtaining the high radiochemical purity (≥ 95%) and stability, without final purification, and stimulates the in vitro and in vivo evaluation to determine the potential of the radiopharmaceutical for clinical application. (author)

An integrated study on antimicrobial activity and ecotoxicity of quantum dots and quantum dots coated with the antimicrobial peptide indolicidin

Directory of Open Access Journals (Sweden)

Galdiero E

2016-08-01

Full Text Available Emilia Galdiero,1 Antonietta Siciliano,1 Valeria Maselli,1 Renato Gesuele,1 Marco Guida,1 Domenico Fulgione,1 Stefania Galdiero,2 Lucia Lombardi,3 Annarita Falanga2 1Department of Biology, University of Naples “Federico II”, Naples, Italy; 2Department of Pharmacy and Cirpeb, University of Naples “Federico II”, Naples, Italy; 3Department of Experimental Medicine, Second University of Naples, Naples, Italy Abstract: This study attempts to evaluate the antimicrobial activity and the ecotoxicity of quantum dots (QDs alone and coated with indolicidin. To meet this objective, we tested the level of antimicrobial activity on Gram-positive and Gram-negative bacteria, and we designed an ecotoxicological battery of test systems and indicators able to detect different effects using a variety of end points. The antibacterial activity was analyzed against Staphylococcus aureus (ATCC 6538, Pseudomonas aeruginosa (ATCC 1025, Escherichia coli (ATCC 11229, and Klebsiella pneumoniae (ATCC 10031, and the results showed an improved germicidal action of QDs-Ind. Toxicity studies on Daphnia magna indicated a decrease in toxicity for QDs-Ind compared to QDs alone, lack of bioluminescence inhibition on Vibrio fisheri, and no mutations in Salmonella typhimurium TA 100. The comet assay and oxidative stress experiments performed on D. magna showed a genotoxic and an oxidative damage with a dose–response trend. Indolicidin retained its activity when bound to QDs. We observed an enhanced activity for QDs-Ind. The presence of indolicidin on the surface of QDs was able to decrease its QDs toxicity. Keywords: peptide, quantum dots, ecotoxicity, antimicrobial activity, oxidative stress, genotoxicity

Labelling and quality control of 99mTc labelled somatostatin analogues

International Nuclear Information System (INIS)

Poramatikul, N.; Sangsuriyan, J.; Kongpeth, P.; Ngamprayad, T.; Laloknam, S.; Permtermsin, C.; Madsomboon, N.

2001-01-01

To standardize interlaboratory reproducibility, iodination of RC-160 with 125 I and direct labelling of RC-160 with 99m Tc, quality control and binding assay were performed. Two conjugated peptides, HYNIC-RC-160 and MAG-3-RC-160, were synthesized. The conjugated peptides were radiolabelled with 99m Tc via co-ligands; 99m Tc-MAG-3-RC-160 via glucoheptonate, 99m Tc-HYNIC-RC-160 via EDDA and tricine. Conditions for labelling were optimized. Analytical and purification methods for the labelled products were developed. Radiochemical purity test of 99m Tc labelled peptides was performed by HPLC with gradient elution of 0.1%TFA/water and acetonitrile, or by ITLC-SG in saline and in 50% acetonitrile. The contaminants in 99m Tc radiolabelled product were separated by elution from SEPPAK C-18 cartridge by 0.1% acetic acid and the pure product was eluted out of SEPPAK column by 50% acetonitrile with about 68% recovery. Stability of the purified 99m Tc-MAG3-RC-160 stored at -20 deg. C was more than 72 h. 99m Tc-MAG-3-RC-160 showed a high equilibrium dissociation constant with K D of 26 pmole/mg protein and B max of 7.9 mM. (author)

Tunable ultrasmall visible-to-extended near-infrared emitting silver sulfide quantum dots for integrin-targeted cancer imaging.

Science.gov (United States)

Tang, Rui; Xue, Jianpeng; Xu, Baogang; Shen, Duanwen; Sudlow, Gail P; Achilefu, Samuel

2015-01-27

The large size of many near-infrared (NIR) fluorescent nanoparticles prevents rapid extravasation from blood vessels and subsequent diffusion to tumors. This confines in vivo uptake to the peritumoral space and results in high liver retention. In this study, we developed a viscosity modulated approach to synthesize ultrasmall silver sulfide quantum dots (QDs) with distinct tunable light emission from 500 to 1200 nm and a QD core diameter between 1.5 and 9 nm. Conjugation of a tumor-avid cyclic pentapeptide (Arg-Gly-Asp-DPhe-Lys) resulted in monodisperse, water-soluble QDs (hydrodynamic diameter < 10 nm) without loss of the peptide's high binding affinity to tumor-associated integrins (KI = 1.8 nM/peptide). Fluorescence and electron microscopy showed that selective integrin-mediated internalization was observed only in cancer cells treated with the peptide-labeled QDs, demonstrating that the unlabeled hydrophilic nanoparticles exhibit characteristics of negatively charged fluorescent dye molecules, which typically do not internalize in cells. The biodistribution profiles of intravenously administered QDs in different mouse models of cancer reveal an exceptionally high tumor-to-liver uptake ratio, suggesting that the small sized QDs evaded conventional opsonization and subsequent high uptake in the liver and spleen. The seamless tunability of the QDs over a wide spectral range with only a small increase in size, as well as the ease of labeling the bright and noncytotoxic QDs with biomolecules, provides a platform for multiplexing information, tracking the trafficking of single molecules in cells, and selectively targeting disease biomarkers in living organisms without premature QD opsonization in circulating blood.

Templated self-assembly of quantum dots from aqueous solution using protein scaffolds

Energy Technology Data Exchange (ETDEWEB)

Blum, Amy Szuchmacher [Center for Bio/Molecular Science and Engineering, Naval Research Laboratory, 4555 Overlook Avenue SW, Washington, DC 20375 (United States); Soto, Carissa M [Center for Bio/Molecular Science and Engineering, Naval Research Laboratory, 4555 Overlook Avenue SW, Washington, DC 20375 (United States); Wilson, Charmaine D [Geo-Centers, Incorporated, Newton, MA 02459 (United States); Whitley, Jessica L [Geo-Centers, Incorporated, Newton, MA 02459 (United States); Moore, Martin H [Center for Bio/Molecular Science and Engineering, Naval Research Laboratory, 4555 Overlook Avenue SW, Washington, DC 20375 (United States); Sapsford, Kim E [George Mason University, 10910 University Boulevard, Manassas, VA 20110 (United States); Lin, Tianwei [Department of Molecular Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037 (United States); Chatterji, Anju [Department of Molecular Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037 (United States); Johnson, John E [Department of Molecular Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037 (United States); Ratna, Banahalli R [Center for Bio/Molecular Science and Engineering, Naval Research Laboratory, 4555 Overlook Avenue SW, Washington, DC 20375 (United States)

2006-10-28

Short, histidine-containing peptides can be conjugated to lysine-containing protein scaffolds to controllably attach quantum dots (QDs) to the scaffold, allowing for generic attachment of quantum dots to any protein without the use of specially engineered domains. This technique was used to bind quantum dots from aqueous solution to both chicken IgG and cowpea mosaic virus (CPMV), a 30 nm viral particle. These quantum dot-protein assemblies were studied in detail. The IgG-QD complexes were shown to retain binding specificity to their antigen after modification. The CPMV-QD complexes have a local concentration of quantum dots greater than 3000 nmol ml{sup -1}, and show a 15% increase in fluorescence quantum yield over free quantum dots in solution.

Evaluation of protein acylation agents for the radioiodination of peptides: Application to labelling octreotide

International Nuclear Information System (INIS)

Zalutsky, M.; Vaidyanathan, G.

2002-01-01

The purpose of this study was to investigate the utility of two acylation agents originally developed for protein labelling - N-succinimidyl 3-[ 131 I]iodobenzoate and N-succinimidyl 5-[ 131 I]iodopyridine-3- carboxylate - for the radioiodination of peptides. Because of the widespread interest in imaging and treating malignancies that overexpress somatostatin receptors, octreotide was selected as the model peptide. Using these reagents, octreotide was coupled to 3-iodobenzoyl and 3-iodonicotinoyl templates, yielding [N-(3-iodobenzoyl)- D-Phe 1 ]octreotide (IBO) and [N-(3-iodonicotinoyl)-D-Phe 1 ]octreotide (INO), respectively. The IC 50 values for the binding of IBO and INO to somatostatin receptor expressing CA20948 rat pancreatic tumour membranes were 0.90 nM and 0.13 nM, respectively, compared with 0.35 nM for octreotide itself. Yields for the preparation of [ 131 I]IBO and [ 131 I]INO from N-succinimidyl 3-[ 131 I]iodobenzoate and N-succinimidyl 5-[ 131 I]iodopyridine-3- carboxylate, were 35-50%. In vitro assays with AR42J rat pancreatic tumour cells demonstrated considerably higher receptor-specific retention of cell-internalized radioiodine activity for [ 131 I]INO compared with [ 125 I]IBO. A tissue distribution study with both conjugates revealed low levels of activity in the thyroid, consistent with a low degree of deiodination of these radioiodinated peptide conjugates. (author)

Preliminary study on the inhibition of nuclear internalization of Tat peptides by conjugation with a receptor-specific peptide and fluorescent dyes

Science.gov (United States)

Shen, Duanwen; Liang, Kexiang; Ye, Yunpeng; Tetteh, Elizabeth; Achilefu, Samuel

2006-02-01

Numerous studies have shown that basic Tat peptide (48-57) internalized non-specifically in cells and localized in the nucleus. However, localization of imaging agents in cellular nucleus is not desirable because of the potential mutagenesis. When conjugated to the peptides that undergo receptor-mediated endocytosis, Tat peptide could target specific cells or pathologic tissue. We tested this hypothesis by incorporating a somatostatin receptor-avid peptide (octreotate, Oct) and two different fluorescent dyes, Cypate 2 (Cy2) and fluorescein 5'-carboxlic acid (5-FAM), into the Tat-peptide sequence. In addition to the Cy2 or 5-FAM-labeled Oct conjugated to Tat peptide (Tat) to produce Tat-Oct-Cypate2 or Tat-Oct-5-FAM, we also labeled the Tat the Tat peptide with these dyes (Tat-Cy2 and Tat-5-FAM) to serve as positive control. A somatostatin receptor-positive pancreatic tumor cell line, AR42J, was used to assess cell internalization. The results show that Tat-5-FAM and Tat-Cypate2 localized in both nucleus and cytoplasm of the cells. In contrast to Tat-Oct-Cypate2, which localized in both the cytoplasm and nucleus, Tat-Oct-5-FAM internalized in the cytoplasm but not in the nucleus of AR42J cells. The internalizations were inhibited by adding non-labeled corresponding peptides, suggesting that the endocytoses of each group of labeled and the corresponding unlabeled compounds occurred through a common pathway. Thus, fluorescent probes and endocytosis complex between octreotate and somatostatin receptors in cytoplasm could control nuclear internalization of Tat peptides.

Metabolism and pharmacokinetic of cyclo-peptides and peptides. Use of radioelement and stable isotopes

International Nuclear Information System (INIS)

Aninat, C.

2003-10-01

More and more peptides and proteins are used in therapeutic. Three mainly techniques are used for pharmacokinetic and metabolism studies: immunoassay, radioactively labeled molecules and mass spectrometry. In the first part of this work, we have used uniformly labelled peptides (C-peptide and insulin) with stables ( 13 C, 15 N, and 13 C/ 15 N) or radioactive ( 14 C) isotopes to investigated these kind of studies. These works are based on isotope dilution mass spectrometry assay. In a second time we have investigated the metabolism of a particular cyclo-peptides families composed of two amino acids: the diketo-piperazine. These compounds are found in mammals and in microorganisms. There are not recognized by proteolytic enzymes. We have estimated if the main enzymes implicated in the metabolism of xenobiotics, the P450 cytochrome mono-oxygenases, were able to recognized them

The Real maccoyii: Identifying Tuna Sushi with DNA Barcodes – Contrasting Characteristic Attributes and Genetic Distances

Science.gov (United States)

Lowenstein, Jacob H.; Amato, George; Kolokotronis, Sergios-Orestis

2009-01-01

Background The use of DNA barcodes for the identification of described species is one of the least controversial and most promising applications of barcoding. There is no consensus, however, as to what constitutes an appropriate identification standard and most barcoding efforts simply attempt to pair a query sequence with reference sequences and deem identification successful if it falls within the bounds of some pre-established cutoffs using genetic distance. Since the Renaissance, however, most biological classification schemes have relied on the use of diagnostic characters to identify and place species. Methodology/Principal Findings Here we developed a cytochrome c oxidase subunit I character-based key for the identification of all tuna species of the genus Thunnus, and compared its performance with distance-based measures for identification of 68 samples of tuna sushi purchased from 31 restaurants in Manhattan (New York City) and Denver, Colorado. Both the character-based key and GenBank BLAST successfully identified 100% of the tuna samples, while the Barcode of Life Database (BOLD) as well as genetic distance thresholds, and neighbor-joining phylogenetic tree building performed poorly in terms of species identification. A piece of tuna sushi has the potential to be an endangered species, a fraud, or a health hazard. All three of these cases were uncovered in this study. Nineteen restaurant establishments were unable to clarify or misrepresented what species they sold. Five out of nine samples sold as a variant of “white tuna” were not albacore (T. alalunga), but escolar (Lepidocybium flavorunneum), a gempylid species banned for sale in Italy and Japan due to health concerns. Nineteen samples were northern bluefin tuna (T. thynnus) or the critically endangered southern bluefin tuna (T. maccoyii), though nine restaurants that sold these species did not state these species on their menus. Conclusions/Significance The Convention on International Trade

Specific dot-immunobinding assay for detection and enumeration of Thiobacillus ferrooxidans

International Nuclear Information System (INIS)

Arredondo, R.; Jerez, C.A.

1989-01-01

A specific and very sensitive dot-immunobinding assay for the detection and enumeration of the bioleaching microorganism Thiobacillus ferrooxidans was developed. Nitrocellulose spotted with samples was incubated with polyclonal antisera against whole T. ferrooxidans cells and then in 125 I-labeled protein A or 125 I-labeled goat antirabbit immunoglobulin G; incubation was followed by autoradiography. Since a minimum of 10 3 cells per dot could be detected, the method offers the possibility of simultaneous processing of numerous samples in a short time to monitor the levels of T. ferrooxidans in bioleaching operations

Therapeutic Efficacy with Treatment-related Toxicities of 177Lu-labeled Bombesin Derivative for the Peptide Receptor Radiotherapy

International Nuclear Information System (INIS)

Lim, Jae Cheong; Cho, Eun Ha; Lee, So Young

2015-01-01

The gastrin-releasing peptide receptor (GRPR) has been shown to be overexpressed in many human tumours, including breast cancer, prostate cancer, small cell lung cancer, ovarian cancers, endometrial cancers, and gastrointestinal stromal tumors. In particular, GRPR expression is high in 83 % of invasive primary prostatic carcinomas. These results suggest that 177 Lu-labeled bombesin derivative has promising characteristics as a novel nuclear medicine, especially for the treatment of GRPR over-expressing prostate tumors

Human C-peptide. Pt. 1

International Nuclear Information System (INIS)

Beischer, W.; Keller, L.; Maas, M.; Schiefer, E.; Pfeiffer, E.F.

1976-01-01

Synthetic human C-peptide bearing a tyrosine group at its amino end is labelled with 125 iodine using chloramin T or hydrogen peroxide and lactoperoxidase. The results of the two methods are compared. Antiserum to synthetic human C-peptide (without tyrosine), which was partially coupled to rabbit albumin, is raised in guinea pigs and goats. Goats show to be superior to guinea pips concerning antibody production. The so-called 'hook effect' phenomenon is observed when setting up the standard curves for the radioimmunoassay. Monotonically decreasing standard curves are obtained on dilution of antiserum with a high antibody titer which was produced by repeated immunization in goats. Free C-peptide and C-peptide bound to antiserum are separated using the anion exchange resin amberlite. Using this separation technique we excluded unspecific binding of labelled C-peptide to protein fractions in serum of diabetics. The sensitivity of our radioimmunoassay is approx. 0.3 ng C-peptide/ml serum. Intra- and interassay variability are below 10%. Human proinsulin is the only substance found to crossreact with the antiserum. (orig.) [de

Quantum Dots Encapsulated with Canine Parvovirus-Like Particles Improving the Cellular Targeted Labeling.

Directory of Open Access Journals (Sweden)

Dan Yan

Full Text Available Quantum dots (QDs have a promising prospect in live-cell imaging and sensing because of unique fluorescence features. QDs aroused significant interest in the bio-imaging field through integrating the fluorescence properties of QDs and the delivery function of biomaterial. The natural tropism of Canine Parvovirus (CPV to the transferrin receptor can target specific cells to increase the targeting ability of QDs in cell imaging. CPV virus-like particles (VLPs from the expression of the CPV-VP2 capsid protein in a prokaryotic expression system were examined to encapsulate the QDs and deliver to cells with an expressed transferrin receptor. CPV-VLPs were used to encapsulate QDs that were modified using 3-mercaptopropionic acid. Gel electrophoresis, fluorescence spectrum, particle size, and transmission electron microscopy verified the conformation of a complex, in which QDs were encapsulated in CPV-VLPs (CPV-VLPs-QDs. When incubated with different cell lines, CPV-VLPs-QDs significantly reduced the cytotoxicity of QDs and selectively labeled the cells with high-level transferrin receptors. Cell-targeted labeling was achieved by utilizing the specific binding between the CPV capsid protein VP2 of VLPs and cellular receptors. CPV-VLPs-QDs, which can mimic the native CPV infection, can recognize and attach to the transferrin receptors on cellular membrane. Therefore, CPV-VLPs can be used as carriers to facilitate the targeted delivery of encapsulated nanomaterials into cells via receptor-mediated pathways. This study confirmed that CPV-VLPs can significantly promote the biocompatibility of nanomaterials and could expand the application of CPV-VLPs in biological medicine.

Technetium-99m-labeled Arg-Gly-Asp-conjugated alpha-melanocyte stimulating hormone hybrid peptides for human melanoma imaging

Energy Technology Data Exchange (ETDEWEB)

Yang Jianquan; Guo Haixun [College of Pharmacy, University of New Mexico, Albuquerque, NM 87131 (United States); Miao Yubin, E-mail: ymiao@salud.unm.ed [College of Pharmacy, University of New Mexico, Albuquerque, NM 87131 (United States); Cancer Research and Treatment Center, University of New Mexico, Albuquerque, NM 87131 (United States); Department of Dermatology, University of New Mexico, Albuquerque, NM 87131 (United States)

2010-11-15

Introduction: The purpose of this study was to examine whether {sup 99m}Tc-labeled Arg-Gly-Asp (RGD)-conjugated alpha-melanocyte stimulating hormone ({alpha}-MSH) hybrid peptide targeting both melanocortin-1 (MC1) and {alpha}{sub v{beta}3} integrin receptors was superior in melanoma targeting to {sup 99m}Tc-labeled {alpha}-MSH or RGD peptide targeting only the MC1 or {alpha}{sub v{beta}3} integrin receptor. Methods: RGD-Lys-(Arg{sup 11})CCMSH, RAD-Lys-(Arg{sup 11})CCMSH and RGD-Lys-(Arg{sup 11})CCMSHscramble were designed to target both MC1 and {alpha}{sub v{beta}3} integrin receptors, MC1 receptor only and {alpha}{sub v{beta}3} integrin receptor only, respectively. The MC1 or {alpha}{sub v{beta}3} integrin receptor binding affinities of three peptides were determined in M21 human melanoma cells. The melanoma targeting properties of {sup 99m}Tc-labeled RGD-Lys-(Arg{sup 11})CCMSH, RAD-Lys-(Arg{sup 11})CCMSH and RGD-Lys-(Arg{sup 11})CCMSHscramble were determined in M21 human melanoma-xenografted nude mice. Meanwhile, the melanoma uptake of {sup 99m}Tc-RGD-Lys-(Arg{sup 11})CCMSH was blocked with various non-radiolabeled peptides in M21 melanoma xenografts. Results: RGD-Lys-(Arg{sup 11})CCMSH displayed 2.0 and 403 nM binding affinities to both MC1 and {alpha}{sub v{beta}3} integrin receptors, whereas RAD-Lys-(Arg{sup 11})CCMSH or RGD-Lys-(Arg{sup 11})CCMSHscramble lost their {alpha}{sub v{beta}3} integrin receptor binding affinity by greater than 248-fold or MC1 receptor binding affinity by more than 100-fold, respectively. The melanoma uptake of {sup 99m}Tc-RGD-Lys-(Arg{sup 11})CCMSH was 2.49 and 2.24 times (P < .05) the melanoma uptakes of {sup 99m}Tc-RAD-Lys-(Arg{sup 11})CCMSH and {sup 99m}Tc-RGD-Lys-(Arg{sup 11})CCMSHscramble at 2 h post-injection, respectively. Either RGD or (Arg{sup 11})CCMSH peptide co-injection could block 42% and 57% of the tumor uptake of {sup 99m}Tc-RGD-Lys-(Arg{sup 11})CCMSH, whereas the coinjection of RGD+(Arg{sup 11})CCMSH peptide mixture

Luminescent passive-oxidized silicon quantum dots as biological staining labels and their cytotoxicity effects at high concentration

International Nuclear Information System (INIS)

Fujioka, Kouki; Manabe, Noriyoshi; Hanada, Sanshiro; Hoshino, Akiyoshi; Yamamoto, Kenji; Hiruoka, Masaki; Sato, Keisuke; Hirakuri, Kenji; Miyasaka, Ryosuke; Tilley, Richard D; Manome, Yoshinobu

2008-01-01

Semiconductor quantum dots (QDs) hold some advantages over conventional organic fluorescent dyes. Due to these advantages, they are becoming increasingly popular in the field of bioimaging. However, recent work suggests that cadmium based QDs affect cellular activity. As a substitute for cadmium based QDs, we have developed photoluminescent stable silicon quantum dots (Si-QDs) with a passive-oxidation technique. Si-QDs (size: 6.5 ± 1.5 nm) emit green light, and they have been used as biological labels for living cell imaging. In order to determine the minimum concentration for cytotoxicity, we investigated the response of HeLa cells. We have shown that the toxicity of Si-QDs was not observed at 112 μg ml -1 and that Si-QDs were less toxic than CdSe-QDs at high concentration in mitochondrial assays and with lactate dehydrogenase (LDH) assays. Especially under UV exposure, Si-QDs were more than ten times safer than CdSe-QDs. We suggest that one mechanism for the cytotoxicity is that Si-QDs can generate oxygen radicals and these radicals are associated with membrane damages. This work has demonstrated the suitability of Si-QDs for bioimaging in lower concentration, and their cytotoxicity and one toxicity mechanism at high concentration

Rosette Assay: Highly Customizable Dot-Blot for SH2 Domain Screening.

Science.gov (United States)

Ng, Khong Y; Machida, Kazuya

2017-01-01

With a growing number of high-throughput studies, structural analyses, and availability of protein-protein interaction databases, it is now possible to apply web-based prediction tools to SH2 domain-interactions. However, in silico prediction is not always reliable and requires experimental validation. Rosette assay is a dot blot-based reverse-phase assay developed for the assessment of binding between SH2 domains and their ligands. It is conveniently customizable, allowing for low- to high-throughput analysis of interactions between various numbers of SH2 domains and their ligands, e.g., short peptides, purified proteins, and cell lysates. The binding assay is performed in a 96-well plate (MBA or MWA apparatus) in which a sample spotted membrane is incubated with up to 96 labeled SH2 domains. Bound domains are detected and quantified using a chemiluminescence or near-infrared fluorescence (IR) imaging system. In this chapter, we describe a practical protocol for rosette assay to assess interactions between synthesized tyrosine phosphorylated peptides and a library of GST-tagged SH2 domains. Since the methodology is not confined to assessment of SH2-pTyr interactions, rosette assay can be broadly utilized for ligand and drug screening using different protein interaction domains or antibodies.

Automated selected reaction monitoring software for accurate label-free protein quantification.

Science.gov (United States)

Teleman, Johan; Karlsson, Christofer; Waldemarson, Sofia; Hansson, Karin; James, Peter; Malmström, Johan; Levander, Fredrik

2012-07-06

Selected reaction monitoring (SRM) is a mass spectrometry method with documented ability to quantify proteins accurately and reproducibly using labeled reference peptides. However, the use of labeled reference peptides becomes impractical if large numbers of peptides are targeted and when high flexibility is desired when selecting peptides. We have developed a label-free quantitative SRM workflow that relies on a new automated algorithm, Anubis, for accurate peak detection. Anubis efficiently removes interfering signals from contaminating peptides to estimate the true signal of the targeted peptides. We evaluated the algorithm on a published multisite data set and achieved results in line with manual data analysis. In complex peptide mixtures from whole proteome digests of Streptococcus pyogenes we achieved a technical variability across the entire proteome abundance range of 6.5-19.2%, which was considerably below the total variation across biological samples. Our results show that the label-free SRM workflow with automated data analysis is feasible for large-scale biological studies, opening up new possibilities for quantitative proteomics and systems biology.

Noninvasive imaging of tumor integrin expression using 18F-labeled RGD dimer peptide with PEG4 linkers

International Nuclear Information System (INIS)

Liu, Zhaofei; Liu, Shuanglong; Wang, Fan; Liu, Shuang; Chen, Xiaoyuan

2009-01-01

Various radiolabeled Arg-Gly-Asp (RGD) peptides have been previously investigated for tumor integrin α v β 3 imaging. To further develop RGD radiotracers with enhanced tumor-targeting efficacy and improved in vivo pharmacokinetics, we designed a new RGD homodimeric peptide with two PEG 4 spacers (PEG 4 = 15-amino-4,7,10,13-tetraoxapentadecanoic acid) between the two monomeric RGD motifs and one PEG 4 linker on the glutamate α-amino group ( 18 F-labeled PEG 4 -E[PEG 4 -c(RGDfK)] 2 , P-PRGD2), as a promising agent for noninvasive imaging of integrin expression in mouse models. P-PRGD2 was labeled with 18 F via 4-nitrophenyl 2- 18 F-fluoropropionate ( 18 F-FP) prosthetic group. In vitro and in vivo characteristics of the new dimeric RGD peptide tracer 18 F-FP-P-PRGD2 were investigated and compared with those of 18 F-FP-P-RGD2 ( 18 F-labeled RGD dimer without two PEG 4 spacers between the two RGD motifs). The ability of 18 F-FP-P-PRGD2 to image tumor vascular integrin expression was evaluated in a 4T1 murine breast tumor model. With the insertion of two PEG 4 spacers between the two RGD motifs, 18 F-FP-P-PRGD2 showed enhanced integrin α v β 3 -binding affinity, increased tumor uptake and tumor-to-nontumor background ratios compared with 18 F-FP-P-RGD2 in U87MG tumors. MicroPET imaging with 18 F-FP-P-PRGD2 revealed high tumor contrast and low background in tumor-bearing nude mice. Biodistribution studies confirmed the in vivo integrin α v β 3 -binding specificity of 18 F-FP-P-RGD2. 18 F-FP-P-PRGD2 can specifically image integrin α v β 3 on the activated endothelial cells of tumor neovasculature. 18 F-FP-P-PRGD2 can provide important information on integrin expression on the tumor vasculature. The high integrin binding affinity and specificity, excellent pharmacokinetic properties and metabolic stability make the new RGD dimeric tracer 18 F-FP-P-PRGD2 a promising agent for PET imaging of tumor angiogenesis and for monitoring the efficacy of antiangiogenic

Optimization of synthesis and quality control procedures for the preparation of 18F and 123I labelled peptides for nuclear medicine

International Nuclear Information System (INIS)

2002-09-01

The general scope of this CRP focused on the optimization of syntheses, quality control, in vitro and in vivo evaluation of 18 F and 123 I radiopharmaceuticals based on peptides with known or anticipated clinical potential. Selective labelling procedures using prosthetic groups were applied to both fluorine and iodine. Studies included investigation on the fate of the label, stability in vivo, biodistribution and pharmacokinetic studies in rodents and in cell culture. With respect to 123 I, the work aimed at developing a simplified labelling kit using solid state systems. The first Research Co-ordination Meeting (RCM) that was held in August 1997 took up and decided on the criteria for selecting the peptides and agreed upon a set of recommended laboratory protocols for the CRP participants to follow and further optimize. Eight scientists from reputed laboratories from Argentina, Brazil, China, Germany, Greece, the Islamic Republic of Iran, Saudi Arabia and the United States of America participated in the CRP. Three RCMs were held where the participants presented their scientific results: August 1997 in Sao Paulo, Brazil, April 1999 in Athens, Greece, and November 2000 in Shanghai, People's Republic of China. Reports describing the research work of all participants are included herein. Each of the report has been indexed separately

[3H]Azidodantrolene photoaffinity labeling, synthetic domain peptides and monoclonal antibody reactivity identify the dantrolene binding sequence on RyR1

Energy Technology Data Exchange (ETDEWEB)

Paul-Pletzer, Kalanethee; Yamamoto, Takeshi; Bhat, Manju B.; Ma, Jianjie; Ikemoto, Noriaki; Jimenez, Leslie S.; Morimoto, Hiromi; Williams, Philip G.; Parness, Jerome

2002-06-14

Dantrolene is a drug that suppresses intracellular Ca2+ release from sarcoplasmic reticulum in normal skeletal muscle and is used as a therapeutic agent in individuals susceptible to malignant hyperthermia. Though its precise mechanism of action has not been elucidated, we have identified the N-terminal region (amino acids 1-1400) of the skeletal muscle isoform of the ryanodine receptor (RyR1), the primary Ca2+ release channel in sarcoplasmic reticulum, as a molecular target for dantrolene using the photoaffinity analog [3H]azidodantrolene(1). Here, we demonstrate that heterologously expressed RyR1 retains its capacity to be specifically labeled with [3H]azidodantrolene,indicating that muscle specific factors are not required for this ligand-receptor interaction. Synthetic domain peptides of RyR1, previously shown to affect RyR1 function in vitro and in vivo, were exploited as potential drug binding site mimics and used in photoaffinity labeling experiments. Only DP1 and DP1-2, peptide s containing the amino acid sequence corresponding to RyR1 residues 590-609, were specifically labeled by [3H]azidodantrolene. A monoclonal anti-RyR1 antibody which recognizes RyR1 and its 1400 amino acid N-terminal fragment, recognizes DP1 and DP1-2 in both Western blots and immunoprecipitation assays, and specifically inhibits [3H]azidodantrolene photolabeling of RyR1 and its N-terminal fragment in sarcoplasmic reticulum. Our results indicate that synthetic domain peptides can mimic a native, ligand binding conformation in vitro, and that the dantrolene binding site and the epitope for the monoclonal antibody on RyR1 are equivalent and composed of amino-acids 590-609.

Comparative study of colloidal gold and quantum dots as labels for multiplex screening tests for multi-mycotoxin detection

Energy Technology Data Exchange (ETDEWEB)

Foubert, Astrid, E-mail: astrid.foubert@hotmail.com; Beloglazova, Natalia V.; De Saeger, Sarah

2017-02-22

Quantum dots (QDs) and colloidal gold nanoparticles (CG) were evaluated as labels for multiplex lateral flow immunoassay (LFIA) for determination of mycotoxins deoxynivalenol (DON), zearalenone (ZEN) and T2/HT2-toxin (T2/HT2) in cereal matrices. Both developed assays were based on the same immunoreagents (except for the labels), therefore their analytical characteristics could be objectively compared. For both LFIAs antigens (DON-ovalbumin (OVA), ZEN-OVA and T2-OVA) and rabbit anti-mouse immunoglobulin were immobilized on a nitrocellulose membrane as three test lines and one control line, respectively. Depending on the LFIA, monoclonal antibodies (mAb) against DON, ZEN and T2 were conjugated with CdSeS/ZnS QDs or CG. T2 and HT2 were detected by one test line (T2-OVA) with an anti-T2 mAb which showed 110% cross-reactivity with HT2. Both tests were developed in accordance with the legal limits and were developed in such a way that they had the same cut-off limits of 1000 μg kg{sup −1}, 80 μg kg{sup −1} and 80 μg kg{sup −1} for DON, ZEN and T2/HT2, respectively in order to allow a correct comparison. Applicability of these assays was demonstrated by analysis of naturally contaminated wheat samples. The results demonstrate that both the LFIAs can be used as rapid, cost-effective and convenient qualitative tool for on-site screening for simultaneous detection of DON, ZEN and HT2/T2 in wheat without special instrumentation. However, the QD-based LFIA consumed less immunoreagents and was more sensitive and economically beneficial. In addition, the results were easier to interpret, resulting in a lower false negative rate (<5%) which was in good agreement with Commission Decision 2002/657/EC regarding the performance of analytical methods intended for screening purposes. - Highlights: • Development of colloidal gold- and quantum dot-based multiplex lateral flow immunoassay. • Lateral flow immunoassays allow simultaneous detection of four mycotoxins. �

Summarization on the synthesis and radionuclide-labeling of peptide nucleic acid for an oligonucleotide analogue

International Nuclear Information System (INIS)

Song, Hongtao; Zhang, Huaming; Gao, Hui

2009-04-01

Peptide nucleic acid (PNA), which is one kind of antisense nucleic acid compounds and an oligonucleotide analogue that binds strongly to DNA and RNA in a sequence specific manner, has its unique advantages in the field of molecular diagnostics and treatment of diseases. Now, people gradually attach more importance to PNA. To optimize the application of PNA in genetic re- search and therapy, a great number of backbone modifications on the newly- type structures of PNA were synthesized to improve its physicochemical proper- ties, such as hybridization speciality, solubility in biofluid, or cell permeability. The modified PNA labeled with radionuclides, which can obtain the aim at specific target and minimal non-target trauma, has important role in research and application of tumorous genitherapy. Here a review on the basic synthesis idea and several primary synthetic methods of PNA analogs was given, and also correlative studies and expectation on the compounds belonging to PNA series labeled with radionuclides were included. (authors)

PET imaging of alpha(v)beta(3) integrin expression in tumours with Ga-68-labelled mono-, di- and tetrameric RGD peptides

NARCIS (Netherlands)

Dijkgraaf, Ingrid; Yim, Cheng-Bin; Franssen, Gerben M.; Schuit, Robert C.; Luurtsema, Gert; Liu, Shuang; Oyen, Wim J. G.; Boerman, Otto C.

Due to the restricted expression of alpha(v)beta(3) in tumours, alpha(v)beta(3) is considered a suitable receptor for tumour targeting. In this study the alpha(v)beta(3)-binding characteristics of Ga-68-labelled monomeric, dimeric and tetrameric RGD peptides were determined and compared with their

Aptamer-Modified Semiconductor Quantum Dots for Biosensing Applications.

Science.gov (United States)

Wen, Lin; Qiu, Liping; Wu, Yongxiang; Hu, Xiaoxiao; Zhang, Xiaobing

2017-07-28

Semiconductor quantum dots have attracted extensive interest in the biosensing area because of their properties, such as narrow and symmetric emission with tunable colors, high quantum yield, high stability and controllable morphology. The introduction of various reactive functional groups on the surface of semiconductor quantum dots allows one to conjugate a spectrum of ligands, antibodies, peptides, or nucleic acids for broader and smarter applications. Among these ligands, aptamers exhibit many advantages including small size, high chemical stability, simple synthesis with high batch-to-batch consistency and convenient modification. More importantly, it is easy to introduce nucleic acid amplification strategies and/or nanomaterials to improve the sensitivity of aptamer-based sensing systems. Therefore, the combination of semiconductor quantum dots and aptamers brings more opportunities in bioanalysis. Here we summarize recent advances on aptamer-functionalized semiconductor quantum dots in biosensing applications. Firstly, we discuss the properties and structure of semiconductor quantum dots and aptamers. Then, the applications of biosensors based on aptamer-modified semiconductor quantum dots by different signal transducing mechanisms, including optical, electrochemical and electrogenerated chemiluminescence approaches, is discussed. Finally, our perspectives on the challenges and opportunities in this promising field are provided.

Aptamer-Modified Semiconductor Quantum Dots for Biosensing Applications

Directory of Open Access Journals (Sweden)

Lin Wen

2017-07-01

Full Text Available Semiconductor quantum dots have attracted extensive interest in the biosensing area because of their properties, such as narrow and symmetric emission with tunable colors, high quantum yield, high stability and controllable morphology. The introduction of various reactive functional groups on the surface of semiconductor quantum dots allows one to conjugate a spectrum of ligands, antibodies, peptides, or nucleic acids for broader and smarter applications. Among these ligands, aptamers exhibit many advantages including small size, high chemical stability, simple synthesis with high batch-to-batch consistency and convenient modification. More importantly, it is easy to introduce nucleic acid amplification strategies and/or nanomaterials to improve the sensitivity of aptamer-based sensing systems. Therefore, the combination of semiconductor quantum dots and aptamers brings more opportunities in bioanalysis. Here we summarize recent advances on aptamer-functionalized semiconductor quantum dots in biosensing applications. Firstly, we discuss the properties and structure of semiconductor quantum dots and aptamers. Then, the applications of biosensors based on aptamer-modified semiconductor quantum dots by different signal transducing mechanisms, including optical, electrochemical and electrogenerated chemiluminescence approaches, is discussed. Finally, our perspectives on the challenges and opportunities in this promising field are provided.

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.

Nitriles form mixed-coligand complexes with 99mTc-HYNIC-Peptide

International Nuclear Information System (INIS)

Liu Guozheng; Wescott, Charles; Sato, Aaron; Wang Yi; Liu Ning; Zhang Yumin; Rusckowski, Mary; Hnatowich, Donald J.

2002-01-01

Using a 12-amino acid peptide conjugated with HYNIC as a model, we investigated nitriles as possible coligands for labeling with 99m Tc. After the preparation of the 99m Tc labeled HYNIC-peptide using tricine as coligand, the addition of acetonitile was found by reverse phase HPLC to block further coligand exchange with ethylenediamine diacetic acid (EDDA) at room temperature. The addition of this nitrile changed the pharmacokinetics of the 99m Tc labeled peptide in normal mice towards faster clearance and significant differences in accumulation in most tissues sampled. By replacing acetonitrile with cyanoacetate, a nitrile not present in the HPLC eluant, it was possible to show the existence of a new, more hydrophilic, species by reverse phase HPLC. We conclude that nitriles can act as coligands for HYNIC-conjugated peptides labeled with 99m Tc and tricine. Furthermore, the presence of acetonitrile during Sep-Pak or HPLC purification may inadvertently generate a mixed tricine/acetonitile coligand 99m Tc-HYNIC-peptide complex

Efficient Subcellular Targeting to the Cell Nucleus of Quantum Dots Densely Decorated with a Nuclear Localization Sequence Peptide.

Science.gov (United States)

Maity, Amit Ranjan; Stepensky, David

2016-01-27

Organelle-targeted drug delivery can enhance the efficiency of the intracellularly acting drugs and reduce their toxicity. We generated core-shell type CdSe-ZnS quantum dots (QDs) densely decorated with NLS peptidic targeting residues using a 3-stage decoration approach and investigated their endocytosis and nuclear targeting efficiencies. The diameter of the generated QDs increased following the individual decoration stages (16.3, 18.9, and 21.9 nm), the ζ-potential became less negative (-33.2, -17.5, and -11.9 mV), and characteristic changes appeared in the FTIR spectra following decoration with the linker and NLS peptides. Quantitative analysis of the last decoration stage revealed that 37.9% and 33.2% of the alkyne-modified NLS groups that were added to the reaction mix became covalently attached or adsorbed to the QDs surface, respectively. These numbers correspond to 63.6 and 55.7 peptides conjugated or adsorbed to a single QD (the surface density of 42 and 37 conjugated and adsorbed peptides per 1000 nm(2) of the QDs surface), which is higher than in the majority of previous studies that reported decoration efficiencies of formulations intended for nuclear-targeted drug delivery. QDs decorated with NLS peptides undergo more efficient endocytosis, as compared to other investigated QDs formulations, and accumulated to a higher extent in the cell nucleus or in close vicinity to it (11.9%, 14.6%, and 56.1% of the QDs endocytosed by an average cell for the QD-COOH, QD-azide, and QD-NLS formulations, respectively). We conclude that dense decoration of QDs with NLS residues increased their endocytosis and led to their nuclear targeting (preferential accumulation in the cells nuclei or in close vicinity to them). The experimental system and research tools that were used in this study allow quantitative investigation of the mechanisms that govern the QDs nuclear targeting and their dependence on the formulation properties. These findings will contribute to the

Physical-chemical quality of fish used for making sushi and sashimi tuna and salmon marketed in Rio de Janeiro, BrazilQualidade físico-química do pescado utilizado na elaboração de sushis e sashimis de atum e salmão comercializados no município do Rio de Janeiro, Brasil

Directory of Open Access Journals (Sweden)

Bruna Leal Rodrigues

2012-10-01

Full Text Available This study aimed to determine the physical-chemical quality of fish used in a random sampling of 54 samples of sushi and sashimi tuna and salmon obtained in restaurants with the support of Sanitary Surveillance of the city of Rio de Janeiro from July to August 2011. The temperature were measured at the time of sampling. We carried out physical and chemical tests to determine pH, ammonia, H2S, total volatile bases (TVB, trimethylamine (TMA and histamine. All samples were exposed to inadequate temperature. Only one of the samples was found above the recommended pH value by law. Five were positive for ammonia and two positive results for H2S. The average results of TVB ranged between 9.43 and 12.37 mg N-BVT/100g and the amount of TMA ranged between 0.57 and 0.96 mg N-TMA/100g, being within the recommended standard. The histamine was found in five samples, ranging between 1 and 10 mg/100g. Considering the samples studied, seven (12,96% showed some abnormalities on the evaluated parameters. In conclusion, the consumption of sushi and sashimi can cause risk to consumer health. Therefore, we suggest that a continuous attention from the authorities should be held on this type of food due to their high perishability and risk to public health. O presente estudo objetivou determinar a qualidade físico-química do pescado utilizado em uma amostragem aleatória de 54 sushis e sashimis de atum e salmão obtidos em restaurantes com apoio da Vigilância Sanitária da cidade do Rio de Janeiro no período de julho a agosto de 2011. A temperatura foi mensurada no momento da coleta. Realizou-se provas físico-químicas de determinação de pH, amônia, H2S, bases voláteis totais (BVT, trimetilamina (TMA e histamina. Todas as amostras apresentavam-se expostas em temperatura inadequada. Apenas uma das amostras apresentou pH acima do limite preconizado pela legislação. Cinco apresentaram resultados positivos para amônia e duas resultados positivos para H2S. Os

Exploring the Potential of (99m)Tc(CO)3-Labeled Triazolyl Peptides for Tumor Diagnosis.

Science.gov (United States)

Gaonkar, Raghuvir H; Ganguly, Soumya; Baishya, Rinku; Dewanjee, Saikat; Sinha, Samarendu; Gupta, Amit; Ganguly, Shantanu; Debnath, Mita C

2016-04-01

In recent years the authors have reported on (99m)Tc(CO)3-labeled peptides that serve as carriers for biomolecules or radiopharmaceuticals to the tumors. In continuation of that work they report the synthesis of a pentapeptide (Met-Phe-Phe-Gly-His; pep-1), a hexapeptide (Met-Phe-Phe-Asp-Gly-His; pep-2), and a tetrapeptide (Asp-Gly-Arg-His; pep-3) and the attachment of 3-amino-1,2,4-triazole to the β carboxylic function of the aspartic acid unit of pep-2 and pep-3. The pharmacophores were radiolabeled in high yields with [(99m)Tc(CO)3(H2O)3](+) metal aqua ion, characterized for their stability in serum and saline, as well as in His solution, and found to be substantially stable. B16F10 cell line binding studies showed favorable uptake and internalization. In vivo behavior of the radiolabeled triazolyl peptides was assessed in mice bearing induced tumor. The (99m)Tc(CO)3-triazolyl pep-3 demonstrated rapid urinary clearance and comparatively better tumor uptake. Imaging studies showed visualization of the tumor using (99m)Tc(CO)3-triazolyl pep-3, but due to high abdominal background, low delineation occurred. Based on the results further experiments will be carried out for targeting tumor with triazolyl peptides.

A Novel Quantum Dots-Based Point of Care Test for Syphilis

Science.gov (United States)

Yang, Hao; Li, Ding; He, Rong; Guo, Qin; Wang, Kan; Zhang, Xueqing; Huang, Peng; Cui, Daxiang

2010-05-01

One-step lateral flow test is recommended as the first line screening of syphilis for primary healthcare settings in developing countries. However, it generally shows low sensitivity. We describe here the development of a novel fluorescent POC (Point Of Care) test method to be used for screening for syphilis. The method was designed to combine the rapidness of lateral flow test and sensitiveness of fluorescent method. 50 syphilis-positive specimens and 50 healthy specimens conformed by Treponema pallidum particle agglutination (TPPA) were tested with Quantum Dot-labeled and colloidal gold-labeled lateral flow test strips, respectively. The results showed that both sensitivity and specificity of the quantum dots-based method reached up to 100% (95% confidence interval [CI], 91-100%), while those of the colloidal gold-based method were 82% (95% CI, 68-91%) and 100% (95% CI, 91-100%), respectively. In addition, the naked-eye detection limit of quantum dot-based method could achieve 2 ng/ml of anti-TP47 polyclonal antibodies purified by affinity chromatography with TP47 antigen, which was tenfold higher than that of colloidal gold-based method. In conclusion, the quantum dots were found to be suitable for labels of lateral flow test strip. Its ease of use, sensitiveness and low cost make it well-suited for population-based on-the-site syphilis screening.

Micro-PET Imaging of αvβ3-Integrin Expression with 18F-Labeled Dimeric RGD Peptide

Directory of Open Access Journals (Sweden)

Xiaoyuan Chen

2004-04-01

Full Text Available The αv integrins, which act as cell adhesion molecules, are closely involved with tumor invasion and angiogenesis. In particular, αvβ3 integrin, which is specifically expressed on proliferating endothelial cells and tumor cells, is a logical target for development of a radiotracer method to assess angiogenesis and anti-angiogenic therapy. In this study, a dimeric cyclic RGD peptide E[c(RGDyK]2 was labeled with 18F (t1/2 = 109.7 min by using a prosthetic 4-[18F]fluorobenzoyl moiety to the amino group of the glutamate. The resulting [18F]FB-E[c(RGDyK]2, with high specific activity (200–250 GBq/μmol at the end of synthesis, was administered to subcutaneous U87MG glioblastoma xenograft models for micro-PET and autoradiographic imaging as well as direct tissue sampling to assess tumor targeting efficacy and in vivo kinetics of this PET tracer. The dimeric RGD peptide demonstrated significantly higher tumor uptake and prolonged tumor retention in comparison with a monomeric RGD peptide analog [18F]FB-c(RGDyK. The dimeric RGD peptide had predominant renal excretion, whereas the monomeric analog was excreted primarily through the biliary route. Micro-PET imaging 1 hr after injection of the dimeric RGD peptide exhibited tumor to contralateral background ratio of 9.5 ± 0.8. The synergistic effect of polyvalency and improved pharmacokinetics may be responsible for the superior imaging characteristics of [18F]FB-E[c(RGDyK]2.

Influence of prosthetic radioiodination on the chemical and biological behavior of chemotactic peptides labeled at high specific activity

International Nuclear Information System (INIS)

Pozzi, Oscar R.; Sajaroff, Elisa O.; Edreira, Martin M.

2006-01-01

The influence of radioiodination made through prosthetic group N-succinimidyl-3-[ 131 I]iodo-benzoate ([ 131 I]SIB) on the behavior of small peptides was investigated using as model the chemotactic hexapeptide Nα-for-Nle-Leu-Phe-Nle-Tyr-Lys. No carrier added labeled peptide was isolated by reverse-phase HPLC (RP-HPLC) with coupling efficiencies up to 59-75%. Biodistribution in normal and infected C57 mice showed mainly a hepatobiliary clearance, a very low thyroid uptake and the highest uptake at the infection site was within 1h of injection. Superoxide production and competitive binding assays studies in human polymorphonuclear leukocytes showed a preserved biological activity and high-affinity specific binding. However, the results indicated that the changes observed in the receptor-binding properties with an IC 50 almost twice than the unlabeled peptide and the increasing in the hepatobiliary excretion could be the consequence of the increased lipophicity observed due to the presence of the prosthetic group together with a strong influence of the radioisotope per se

Influence of prosthetic radioiodination on the chemical and biological behavior of chemotactic peptides labeled at high specific activity

Energy Technology Data Exchange (ETDEWEB)

Pozzi, Oscar R. [National Atomic Energy Commission, Ezeiza Atomic Centre, Buenos Aires (Argentina)]. E-mail: oscar.pozzi@duke.edu; Sajaroff, Elisa O. [National Atomic Energy Commission, Ezeiza Atomic Centre, Buenos Aires (Argentina); Edreira, Martin M. [National Atomic Energy Commission, Ezeiza Atomic Centre, Buenos Aires (Argentina)

2006-06-15

The influence of radioiodination made through prosthetic group N-succinimidyl-3-[{sup 131}I]iodo-benzoate ([{sup 131}I]SIB) on the behavior of small peptides was investigated using as model the chemotactic hexapeptide N{alpha}-for-Nle-Leu-Phe-Nle-Tyr-Lys. No carrier added labeled peptide was isolated by reverse-phase HPLC (RP-HPLC) with coupling efficiencies up to 59-75%. Biodistribution in normal and infected C57 mice showed mainly a hepatobiliary clearance, a very low thyroid uptake and the highest uptake at the infection site was within 1h of injection. Superoxide production and competitive binding assays studies in human polymorphonuclear leukocytes showed a preserved biological activity and high-affinity specific binding. However, the results indicated that the changes observed in the receptor-binding properties with an IC{sub 50} almost twice than the unlabeled peptide and the increasing in the hepatobiliary excretion could be the consequence of the increased lipophicity observed due to the presence of the prosthetic group together with a strong influence of the radioisotope per se.

Investigating the Effect of Ligand Amount and Injected Therapeutic Activity: A Simulation Study for 177Lu-Labeled PSMA-Targeting Peptides

Science.gov (United States)

Schuchardt, Christiane; Kulkarni, Harshad R.; Shahinfar, Mostafa; Singh, Aviral; Glatting, Gerhard; Baum, Richard P.; Beer, Ambros J.

2016-01-01

In molecular radiotherapy with 177Lu-labeled prostate specific membrane antigen (PSMA) peptides, kidney and/or salivary glands doses limit the activity which can be administered. The aim of this work was to investigate the effect of the ligand amount and injected activity on the tumor-to-normal tissue biologically effective dose (BED) ratio for 177Lu-labeled PSMA peptides. For this retrospective study, a recently developed physiologically based pharmacokinetic model was adapted for PSMA targeting peptides. General physiological parameters were taken from the literature. Individual parameters were fitted to planar gamma camera measurements (177Lu-PSMA I&T) of five patients with metastasizing prostate cancer. Based on the estimated parameters, the pharmacokinetics of tumor, salivary glands, kidneys, total body and red marrow was simulated and time-integrated activity coefficients were calculated for different peptide amounts. Based on these simulations, the absorbed doses and BEDs for normal tissue and tumor were calculated for all activities leading to a maximal tolerable kidney BED of 10 Gy2.5/cycle, a maximal salivary gland absorbed dose of 7.5 Gy/cycle and a maximal red marrow BED of 0.25 Gy15/cycle. The fits yielded coefficients of determination > 0.85, acceptable relative standard errors and low parameter correlations. All estimated parameters were in a physiologically reasonable range. The amounts (for 25−29 nmol) and pertaining activities leading to a maximal tumor dose, considering the defined maximal tolerable doses to organs of risk, were calculated to be 272±253 nmol (452±420 μg) and 7.3±5.1 GBq. Using the actually injected amount (235±155 μg) and the same maximal tolerable doses, the potential improvement for the tumor BED was 1–3 fold. The results suggest that currently given amounts for therapy are in the appropriate order of magnitude for many lesions. However, for lesions with high binding site density or lower perfusion, optimizing the

Particularities in the biodistribution and pharmacokinetic of labeled peptide with 99mTc in regional administration of patient with cervix cancer

International Nuclear Information System (INIS)

Palau San Pedro, A.; Lopez Diaz, A.; Martin Escuela, J. M.; Galvez Perez, E.

2013-01-01

This study had as objective characterize the biodistribution pharmacokinetic and dosimetry of labeled peptide with 9 9mTc in two dose levels, prepared in 2ml, starting from its intratumoral injection in patient with cervix cancer. The protocol selection to use, the correction and calculate methods were analysis object keeping in mind that antecedents of studies of this type didn't exist and that the administration intratumoral can originate new problems not foreseen in conventional intravenous studies. This study carried out mensurations of sensibility that should be corrected in a particular way. A careful protocol of acquisition was designed able to detect the behavior of the radio-peptide in the time, with a serial gathering of samples of blood and urine until the 24 hours, as well as images of the whole body up to 48h. For the quantification of the images they were necessary also the classic corrections of background and of overlapping of structures. The labeled peptide with 9 9mTc administered for intralesional way, like it was of waiting it presented a very high reception tumoral, being this maxim in the first images, however the product was absorbed quickly in blood, reaching its maximum levels in most of the patients as much in serum as in total blood, in the first 5-15 minutes of having administered. (Author)

Human C-peptide. Pt. 1. Radioimmunoassay

Energy Technology Data Exchange (ETDEWEB)

Beischer, W; Keller, L; Maas, M; Schiefer, E; Pfeiffer, E F [Ulm Univ. (Germany, F.R.). Abt. Innere Medizin, Endokrinologie und Stoffwechsel

1976-08-01

Synthetic human C-peptide bearing a tyrosine group at its amino end is labelled with /sup 125/iodine using chloramin T or hydrogen peroxide and lactoperoxidase. The results of the two methods are compared. Antiserum to synthetic human C-peptide (without tyrosine), which was partially coupled to rabbit albumin, is raised in guinea pigs and goats. Goats show to be superior to guinea pips concerning antibody production. The so-called 'hook effect' phenomenon is observed when setting up the standard curves for the radioimmunoassay. Monotonically decreasing standard curves are obtained on dilution of antiserum with a high antibody titer which was produced by repeated immunization in goats. Free C-peptide and C-peptide bound to antiserum are separated using the anion exchange resin amberlite. Using this separation technique we excluded unspecific binding of labelled C-peptide to protein fractions in serum of diabetics. The sensitivity of our radioimmunoassay is approx. 0.3 ng C-peptide/ml serum. Intra- and interassay variability are below 10%. Human proinsulin is the only substance found to crossreact with the antiserum.

Fourier transform spectra of quantum dots

Science.gov (United States)

Damian, V.; Ardelean, I.; Armăşelu, Anca; Apostol, D.

2010-05-01

Semiconductor quantum dots are nanometer-sized crystals with unique photochemical and photophysical properties that are not available from either isolated molecules or bulk solids. These nanocrystals absorb light over a very broad spectral range as compared to molecular fluorophores which have very narrow excitation spectra. High-quality QDs are proper to be use in different biological and medical applications (as fluorescent labels, the cancer treatment and the drug delivery). In this article, we discuss Fourier transform visible spectroscopy of commercial quantum dots. We reveal that QDs produced by Evident Technologies when are enlightened by laser or luminescent diode light provides a spectral shift of their fluorescence spectra correlated to exciting emission wavelengths, as shown by the ARCspectroNIR Fourier Transform Spectrometer. In the final part of this paper we show an important biological application of CdSe/ZnS core-shell ODs as microbial labeling both for pure cultures of cyanobacteria (Synechocystis PCC 6803) and for mixed cultures of phototrophic and heterotrophic microorganisms.

Novel Tc-99m labeled ELR-containing 6-mer peptides for tumor imaging in epidermoid carcinoma xenografts model. A pilot study

International Nuclear Information System (INIS)

Kim, Dae-Weung; Kim, Woo-Hyoung; Kim, Myoung-Hyoun; Kim, Chang-Guhn

2013-01-01

ELR-containing peptides targeting CXCR2 could be the excellent candidate for targeting ligand of molecular tumor imaging. In this study, we had developed two ELR-containing 6-mer peptides and evaluated the diagnostic performance of Tc-99m labeled 6-mer peptides as a molecular imaging agent in murine models bearing KB epidermoid carcinoma. Peptides were synthesized using Fmoc solid phase peptide synthesis. Radiolabeling efficiency with Tc-99m was evaluated using instant thin-layer chromatography. In KB epidermoid cancer-bearing mice, gamma images had acquired and tumor-to-muscle uptake ratio was calculated. Competition and biodistribution studies had performed. Two 6-mer peptides, ELR-ECG and ECG-ELR were successfully synthesized. After radiolabeling procedures with Tc-99m, the complex Tc-99m ELR-ECG and Tc-99m ECG-ELR were prepared in high yield. In the gamma camera imaging of murine model, Tc-99m ELR-ECG was substantially accumulated in the subcutaneously engrafted tumor and tumor uptake had been suppressed by the free ELR co-injection. However, Tc-99m ECG-ELR was minimally accumulated in the tumor. Two ELR-containing 6-mer peptides, ELR-ECG and ECG-ELR, were developed as a molecular imaging agent to target CXCR2 of epidermoid carcinoma. Tc-99m ELR-ECG had showed significant uptake in tumor and it was good candidate for a tumor imaging. (author)

Kit for instant 99mTc labeling of the antimicrobial peptide ubiquicidin 29-41

International Nuclear Information System (INIS)

Ferro-Flores, G.; Arteaga de Murphy, C.; Pedraza-Lopez, M.; Palomares-Rodriguez, P.; Melendez-Alafort, L.

2005-01-01

The ubiquicidin 29-41 fragment (UBI) is a cationic antimicrobial peptide. An instant kit formulation was developed for the preparation of 99m Tc-UBI 29-41 in high radiochemical yield and its use as an infection imaging agent in humans was evaluated. The components were selected to produce a direct 99m Tc labeling, presumably to the amine groups of Lys and Arg7. 99m Tc-UBI 29-41 obtained from the lyophilized kit showed radiochemical purity of >97% with an average target/non-target ratio of 2.3±0.6 in positive infection sites at 2 hours. Kits were stable at 4 deg C for over 6 months. (author)

Development of labelled biomolecules for targeted radiotherapy

International Nuclear Information System (INIS)

Arteaga de Murphy, C.

2000-01-01

The scope of the co-ordinated research project (Dec 15 1997) included the following activities: 1) develop coupling techniques using bifunctional chelating agents for monoclonal antibodies and peptides, 2) optimised radiolabelling procedures and reaction parameters using Sm-153 and Re-188, 3) investigate direct methods of labelling monoclonal antibodies and peptides with Re-188. 4) initiate animal distribution studies. The modifications specified for the period 1999/02/15 to 2000/02/14 are as follows: a) continue with the optimisation of Re-188-peptide labelling, b) continue with the work to prepare a kit, c) in-vivo and in-vitro studies, d) lanreotide labelling. The group formed by researchers from several Mexican Institutions have worked together and in different aspects of the CRP in order to fulfil the proposed aims (our published work listed)

Development of labelled biomolecules for targeted radiotherapy

Energy Technology Data Exchange (ETDEWEB)

Arteaga de Murphy, C [Instituto Nacional de la Nutricion Salvador Zubiran, Departmento de Medicina Nuclear, Mexico D.F. (Mexico). E-mail: cmurphy at data.net.mx

2000-07-01

The scope of the co-ordinated research project (Dec 15 1997) included the following activities: 1) develop coupling techniques using bifunctional chelating agents for monoclonal antibodies and peptides, 2) optimised radiolabelling procedures and reaction parameters using Sm-153 and Re-188, 3) investigate direct methods of labelling monoclonal antibodies and peptides with Re-188. 4) initiate animal distribution studies. The modifications specified for the period 1999/02/15 to 2000/02/14 are as follows: a) continue with the optimisation of Re-188-peptide labelling, b) continue with the work to prepare a kit, c) in-vivo and in-vitro studies, d) lanreotide labelling. The group formed by researchers from several Mexican Institutions have worked together and in different aspects of the CRP in order to fulfil the proposed aims (our published work listed)

PNA Peptide chimerae

DEFF Research Database (Denmark)

Koch, T.; Næsby, M.; Wittung, P.

1995-01-01

Radioactive labelling of PNA has been performed try linking a peptide segment to the PNA which is substrate for protein kinase A. The enzymatic phosphorylation proceeds in almost quantitative yields....

Effects of the Amino Acid Linkers on the Melanoma-Targeting and Pharmacokinetic Properties of Indium-111-labeled Lactam Bridge-Cyclized α-MSH Peptides

Science.gov (United States)

Guo, Haixun; Yang, Jianquan; Gallazzi, Fabio; Miao, Yubin

2011-01-01

The purpose of this study was to examine the profound effects of the amino acid linkers on the melanoma targeting and pharmacokinetic properties of novel 111In-labeled lactam bridge-cyclized DOTA-[X]-CycMSHhex {1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid-[X]-c[Asp-His-dPhe-Arg-Trp-Lys]-CONH2, X=GlyGlyNle, GlyGluNle or NleGlyGlu} peptides. Methods Three novel DOTA-GGNle-CycMSHhex, DOTA-GENle-CycMSHhex and DOTA-NleGE-CycMSHhex peptides were designed and synthesized. The melanocortin-1 (MC1) receptor binding affinities of the peptides were determined in B16/F1 melanoma cells. The melanoma targeting and pharmacokinetic properties of 111In-DOTA-GGNle-CycMSHhex and 111In-DOTA-GENle-CycMSHhex were determined in B16/F1 melanoma-bearing C57 mice. Results DOTA-GGNle-CycMSHhex and DOTA-GENle-CycMSHhex displayed 2.1 and 11.5 nM MC1 receptor binding affinities, whereas DOTA-NleGE-CycMSHhex showed 873.4 nM MC1 receptor binding affinity. The introduction of the -GlyGly- linker maintained high melanoma uptake while decreased the renal and liver uptakes of 111In-DOTA-GlyGlyNle-CycMSHhex. The tumor uptake values of 111In-DOTA-GGNle-CycMSHhex were 19.05 ± 5.04 and 18.6 ± 3.56 % injected dose/gram (%ID/g) at 2 and 4 h post-injection. 111In-DOTA-GGNle-CycMSHhex exhibited 28, 32 and 42% less renal uptake values than 111In-DOTA-Nle-CycMSHhex we reported previously, and 61, 65 and 68% less liver uptake values than 111In-DOTA-Nle-CycMSHhex at 2, 4 and 24 h post-injection, respectively. Conclusion The amino acid linkers exhibited the profound effects on the melanoma targeting and pharmacokinetic properties of the 111In-labeled lactam bridge-cyclized α-MSH peptides. Introduction of the -GlyGly- linker maintained high melanoma uptake while reducing the renal and liver uptakes of 111In-DOTA-GlyGlyNle-CycMSHhex, highlighting its potential as an effective imaging probe for melanoma detection, as well as a therapeutic peptide for melanoma treatment when labeled with a therapeutic

Prediction of Impending Type 1 Diabetes through Automated Dual-Label Measurement of Proinsulin:C-Peptide Ratio.

Directory of Open Access Journals (Sweden)

Annelien Van Dalem

Full Text Available The hyperglycemic clamp test, the gold standard of beta cell function, predicts impending type 1 diabetes in islet autoantibody-positive individuals, but the latter may benefit from less invasive function tests such as the proinsulin:C-peptide ratio (PI:C. The present study aims to optimize precision of PI:C measurements by automating a dual-label trefoil-type time-resolved fluorescence immunoassay (TT-TRFIA, and to compare its diagnostic performance for predicting type 1 diabetes with that of clamp-derived C-peptide release.Between-day imprecision (n = 20 and split-sample analysis (n = 95 were used to compare TT-TRFIA (AutoDelfia, Perkin-Elmer with separate methods for proinsulin (in-house TRFIA and C-peptide (Elecsys, Roche. High-risk multiple autoantibody-positive first-degree relatives (n = 49; age 5-39 were tested for fasting PI:C, HOMA2-IR and hyperglycemic clamp and followed for 20-57 months (interquartile range.TT-TRFIA values for proinsulin, C-peptide and PI:C correlated significantly (r2 = 0.96-0.99; P<0.001 with results obtained with separate methods. TT-TRFIA achieved better between-day %CV for PI:C at three different levels (4.5-7.1 vs 6.7-9.5 for separate methods. In high-risk relatives fasting PI:C was significantly and inversely correlated (rs = -0.596; P<0.001 with first-phase C-peptide release during clamp (also with second phase release, only available for age 12-39 years; n = 31, but only after normalization for HOMA2-IR. In ROC- and Cox regression analysis, HOMA2-IR-corrected PI:C predicted 2-year progression to diabetes equally well as clamp-derived C-peptide release.The reproducibility of PI:C benefits from the automated simultaneous determination of both hormones. HOMA2-IR-corrected PI:C may serve as a minimally invasive alternative to the more tedious hyperglycemic clamp test.

18F-Fluoroglucosylation of peptides, exemplified on cyclo(RGDfK)

International Nuclear Information System (INIS)

Hultsch, Christina; Schottelius, Margret; Auernheimer, Joerg; Alke, Andrea; Wester, Hans-Juergen

2009-01-01

Oxime formation between an aminooxy-functionalized peptide and an 18 F-labelled aldehyde has recently been introduced as a powerful method for the rapid one-step chemoselective synthesis of radiofluorinated peptides. Here, the potential of using routinely produced and thus readily available [ 18 F]fluorodeoxyglucose ([ 18 F](FDG)) as the aldehydic prosthetic group was investigated using an aminooxyacetyl-conjugated cyclic RGD peptide (cyclo(RGDfK)(Aoa-Boc)) as a model peptide. The use of [ 18 F]FDG from routine production ([ 18 F]FDGTUM) containing an excess of d-glucose did not allow the radiosynthesis of [ 18 F]FDG-RGD in activities >37 MBq in reasonable yield, rendering the direct use of clinical grade [ 18 F]FDG for the routine clinical synthesis of 18 F-labelled peptides impossible. Using no-carrier-added (n.c.a.) [ 18 F]FDG obtained via HPLC separation of [ 18 F]FDGTUM from excess glucose, however, afforded [ 18 F]FDG-RGD in yields of 56-93% (decay corrected) and activities up to 37 MBq. Suitable reaction conditions were 20 min at 120 C and pH 2.5, and a peptide concentration of 5 mM. In a preliminary in vivo biodistribution study in M21 melanoma-bearing nude mice, [ 18 F]FDG-RGD showed increased tumour accumulation compared to the ''gold standard'' [ 18 F]galacto-RGD (2.18 vs 1.49 %iD/g, respectively, at 120 min after injection), but also slightly increased uptake in non-target organs, leading to comparable tumour/organ ratios for both compounds.??These data demonstrate that chemoselective 18 F-labelling of aminooxy-functionalized peptides using n.c.a. [ 18 F]FDG represents a radiofluorination/glycosylation strategy that allows preparation of 18 F-labelled peptides in high yield with suitable pharmacokinetics. As soon as the necessary n.c.a. preparation of [ 18 F]FDG prior to reaction with the Aoa-peptide can be implemented in a fully automated [ 18 F]FDG-synthesis, [ 18 F]fluoroglucosylation of peptides may represent a promising alternative to currently

(18)F-Fluoroglucosylation of peptides, exemplified on cyclo(RGDfK).

Science.gov (United States)

Hultsch, Christina; Schottelius, Margret; Auernheimer, Jörg; Alke, Andrea; Wester, Hans-Jürgen

2009-09-01

Oxime formation between an aminooxy-functionalized peptide and an (18)F-labelled aldehyde has recently been introduced as a powerful method for the rapid one-step chemoselective synthesis of radiofluorinated peptides. Here, the potential of using routinely produced and thus readily available [(18)F]fluorodeoxyglucose ([(18)F]FDG) as the aldehydic prosthetic group was investigated using an aminooxyacetyl-conjugated cyclic RGD peptide (cyclo(RGDfK(Aoa-(Boc)) as a model peptide. The use of [(18)F]FDG from routine production ([(18)F]FDGTUM) containing an excess of D: -glucose did not allow the radiosynthesis of [(18)F]FDG-RGD in activities >37 MBq in reasonable yield, rendering the direct use of clinical grade [(18)F]FDG for the routine clinical synthesis of (18)F-labelled peptides impossible. Using no-carrier-added (n.c.a.) [(18)F]FDG obtained via HPLC separation of [(18)F]FDGTUM from excess glucose, however, afforded [(18)F]FDG-RGD in yields of 56-93% (decay corrected) and activities up to 37 MBq. Suitable reaction conditions were 20 min at 120 degrees C and pH 2.5, and a peptide concentration of 5 mM. In a preliminary in vivo biodistribution study in M21 melanoma-bearing nude mice, [(18)F]FDG-RGD showed increased tumour accumulation compared to the "gold standard" [(18)F]galacto-RGD (2.18 vs 1.49 %iD/g, respectively, at 120 min after injection), but also slightly increased uptake in non-target organs, leading to comparable tumour/organ ratios for both compounds. These data demonstrate that chemoselective (18)F-labelling of aminooxy-functionalized peptides using n.c.a. [(18)F]FDG represents a radiofluorination/glycosylation strategy that allows preparation of (18)F-labelled peptides in high yield with suitable pharmacokinetics. As soon as the necessary n.c.a. preparation of [(18)F]FDG prior to reaction with the Aoa-peptide can be implemented in a fully automated [(18)F]FDG-synthesis, [(18)F]fluoroglucosylation of peptides may represent a promising alternative to

Iodine-131 labelled octreotide: not an option for somatostatin receptor therapy

International Nuclear Information System (INIS)

Bakker, W.H.; Breeman, W.A.P.; Pluijm, M.E. van der; Jong, M. de; Visser, T.J.; Krenning, E.P.

1996-01-01

This study deals with the radioiodination of very small amounts of peptide on a therapeutic scale, the required purification procedures after radioiodination, and the influence of high beta fluxes from 131 I on a peptide during radioiodination and purification. Based on the regularly used therapeutic doses of 131 I in cancer treatment and out previous experience with [ 111 In-DTPA-D-Phe 1 ]-octreotide, it was assumed that a minimal effective therapeutic dose of 3.7 GBq 131 I has to be coupled to a maximum of ∼100 μg peptide, representing only a slight excess of peptide over 131 I. This contrasts with non-peptide radiopharmaceuticals in which high compound to radionuclide ratios are usually used. Labelling at low peptide to radionuclide ratios (low labelling yields) results in the formation of di-iodinated compounds, whereas at high peptide to radionuclide ratios mono-iodinated products of low specific activity are formed. Thus, after radioiodination the desired mono-iodinated peptide has to be separated form unreacted iodide, and from di-iodinated and unreacted peptide, as both compounds compete for the receptors. Possible radiolysis of the peptide during labelling and separation steps were investigated by irradiating 30 μg unlabelled peptide with 370 MBq 131 I in a small volume. The peptide composition of the incubation mixtures was investigated by high-performance liquid chromatography after irradiation for 30 min to 24 h. The results showed that the peptide was degraded with a half-life of less than 1 h. During the preparation of a real therapeutic dose (at much higher β-flux) the peptide will be degraded even faster during the various steps required. In conclusion, intact mono-iodinated 131 I-labelled somatostatin analogues for peptide receptor therapy will be difficult to obtain. (orig./VHE)

Green biosynthesis of biocompatible CdSe quantum dots in living Escherichia coli cells

International Nuclear Information System (INIS)

Yan, Zhengyu; Qian, Jing; Su, Yilong; Ai, Xiaoxia; Wu, Shengmei; Gu, Yueqing

2014-01-01

A green and efficient biosynthesis method to prepare fluorescence-tunable biocompatible cadmium selenide quantum dots using Escherichia coli cells as biological matrix was proposed. Decisive factors in biosynthesis of cadmium selenide quantum dots in a designed route in Escherichia coli cells were elaborately investigated, including the influence of the biological matrix growth stage, the working concentration of inorganic reactants, and the co-incubation duration of inorganic metals to biomatrix. Ultraviolet-visible, photoluminescence, and inverted fluorescence microscope analysis confirmed the unique optical properties of the biosynthesized cadmium selenide quantum dots. The size distribution of the nanocrystals extracted from cells and the location of nanocrystals foci in vivo were also detected seriously by transmission electron microscopy. A surface protein capping layer outside the nanocrystals was confirmed by Fourier transform infrared spectroscopy measurements, which were supposed to contribute to reducing cytotoxicity and maintain a high viability of cells when incubating with quantum dots at concentrations as high as 2 μM. Cell morphology observation indicated an effective labeling of living cells by the biosynthesized quantum dots after a 48 h co-incubation. The present work demonstrated an economical and environmentally friendly approach to fabricating highly fluorescent quantum dots which were expected to be an excellent fluorescent dye for broad bio-imaging and labeling. (papers)

Optimization of synthesis and quality control procedures for the preparations of 18F and 123I labelled peptides

International Nuclear Information System (INIS)

Archimandritis, S.C.; Potamianos, S.; Varvarigou, A.D.

2002-01-01

Radiolabelled biomolecules like proteins and peptides, are playing now days an important role in experimental and clinical Nuclear Medicine. Radioiodination techniques remain important, with improvements accounting for high purity, specific activity and better in vivo stability. Radioiodination using prosthetic groups is the method of choice in cases where the molecules are lacking of thyrosyl groups in their structure and are also sensitive to circulating dehalogenase enzymes. This investigation was based on the need to optimize labelling and quality control techniques for these molecules. The N-succinimidyliodobenzoate (SIB) was used in this study as the prosthetic group for the radioiodination. Optimization of SIB synthesis and modification of the protocol resulted in an improved mean yield of SIB. The combination of TLC and column chromatography using silica gel proved suitable in identifying SIB. Furthermore, the ability of SIB to couple to protein was also used to confirm the presence of SIB. In this case, SEC and ITLC-SG proved suitable to confirm protein binding of SIB. Column chromatography using silica gel containing Sep-Pak was appropriate for SIB purification. Concerning SIB conjugation to peptides, high radioiodination yields were only possible for peptides with amino-containing-side-chain amino acids. Furthermore, lysine containing peptides retained stability, at 4 deg. C, for at least 24 h and reverse phase HPLC proved the most suitable technique for assessing conjugation of SIB to peptide. The biological evaluation of the radiolabelled product was made in normal mice. SIB and SIB-peptide conjugates were tested comparatively and a number of tentative but interesting inferences were drawn. SIB and its peptide conjugates exhibited good in vivo stability as evidenced by low thyroid accumulation and were cleared via the kidneys. A time dependant decrease in the% dose per gram of tissue indicates possible adrenal metabolism of SIB and SIB-peptide

A visible light photoelectrochemical sensor for tumor marker detection using tin dioxide quantum dot-graphene as labels.

Science.gov (United States)

Wang, Yanhu; Li, Meng; Zhu, Yuanna; Ge, Shenguang; Yu, Jinghua; Yan, Mei; Song, Xianrang

2013-12-07

In this paper, a simple and sensitive sandwich-type photoelectrochemical (PEC) immunosensor for measurement of biomarkers on a gold nanoparticle-modified indium tin oxide (ITO) electrode through electrodeposition for point-of-care testing was developed by using a tin dioxide quantum dot-graphene nanocomposite (G-SnO2) as an excellent label with amplification techniques. The capture antibody (Ab1) was firstly immobilized on the gold nanoparticle-modified ITO electrode due to the covalent conjugation, then the antigen and the AuNP/PDDA-G-SnO2 nanocomposite nanoparticle labeled signal antibody (Ab2) were conjugated successively to form a sandwich-type immunocomplex through a specific interaction. Under irradiation with a common ultraviolet lamp (∼365 nm, price $50), the SnO2 NPs were excited and underwent charge-separation to yield electrons (e(-)) and holes (h(+)). As the holes were scavenged by ascorbic acid (AA), the electrons were transferred to the ITO electrode through RGO to generate a photocurrent. The photocurrents were proportional to the CEA concentrations, and the linear range of the developed immunosensor was from 0.005 to 10 ng mL(-1) with a detection limit of 0.036 pg mL(-1). The proposed sensor shows high sensitivity, stability, reproducibility, and can become a promising platform for other biomolecular detection.

Polyacrylamide gel electrophoretic preparation of labelled and non-labelled peptides for radioimmunoassay

International Nuclear Information System (INIS)

Besch, W.; Woltanski, K.P.; Keilacker, H.; Kohnert, K.D.

1986-01-01

Radioiodinated polypeptide hormones, such as insulin, glucagon, human growth hormone, and human C-peptide are employed for radioimmunoassays for investigation of hormonal alterations in states of disturbed carbohydrate metabolism. Iodination was performed using chloramine T. Iodination products of these polypeptide hormones and, for preparation of standard material, native human C-peptide from cadaver pancreases were fractionated by polyacrylamide gel electrophoresis at pH 8.9. Disc electrophoresis in 24 cm long gel rods resulted in stable tracers with high specific activity as well as homogeneous standard material being highly suitable for radioimmunoassays. (author)

γ-Preprotachykinin-(72-92)-peptide amide: An endogenous preprotachykinin I gene-derived peptide that preferentially binds to neurokinin-2 receptors

International Nuclear Information System (INIS)

Dam, T.V.; Takeda, Y.; Krause, J.E.; Escher, E.; Quirion, R.

1990-01-01

The presence of N-terminally extended forms of neurokinin A has recently been reported in the mammalian brain. Among them, gamma-preprotachykinin-(72-92)-peptide amide [gamma-PPT-(72-92)-NH2], a peptide derived by posttranslational processing of gamma-preprotachykinin, is most prominent. We report here that this peptide most likely acts on neurokinin-2 receptor sites since neurokinin A (a putative neurokinin-2 agonist) and gamma-PPT-(72-92)-NH2 are potent competitors of 125I-labeled gamma-PPT-(72-92)-NH2 binding whereas selective neurokinin-1 and -3 agonists are not. Moreover, the distribution of 125I-labeled gamma-PPT-(72-92)-NH2 and 125I-labeled neurokinin A binding sites are very similar in rat brain. On the other hand, 125I-labeled Bolton-Hunter-substance P (a neurokinin-1 ligand) and 125I-labeled Bolton-Hunter-eledoisin (a neurokinin-3 ligand) binding sites are differentially located in this tissue. Thus, it appears that gamma-PPT-(72-92)-NH2 binds to neurokinin-2 receptors and should be considered as a putative endogenous ligand for this receptor class

Microfluidic bead-based multienzyme-nanoparticle amplification for detection of circulating tumor cells in the blood using quantum dots labels

Energy Technology Data Exchange (ETDEWEB)

Zhang, He, E-mail: mzhang_he@126.com; Fu, Xin; Hu, Jiayi; Zhu, Zhenjun

2013-05-24

Graphical abstract: A microfluidic beads-based nucleic acid sensor for sensitive detection of circulating tumor cells (CTCs) in the blood using multienzyme-nanoparticle amplification and quantum dots labels was developed. The chip-based CTCs analysis could detect reverse transcription-polymerase chain reaction (RT-PCR) products of tumor cell as low as 1 tumor cell (e.g. CEA expressing cell) in 1 mL blood sample. This microfluidic beads-based nucleic acid sensor is a promising platform for disease-related nucleic acid molecules at the lowest level at their earliest incidence. -- Highlights: •Combination of microfluidic bead-based platform and enzyme–probe–AuNPs is proposed. •The developed nucleic acid sensor could respond to 5 fM of tumor associated DNA. •Microfluidic platform and multienzyme-labeled AuNPs greatly enhanced sensitivity. •The developed nucleic acid sensor could respond to RT-PCR products of tumor cell as low as 1 tumor cell in 1 mL blood sample. •We report a sensitive nucleic acid sensor for detection of circulating tumor cells. -- Abstract: This study reports the development of a microfluidic bead-based nucleic acid sensor for sensitive detection of circulating tumor cells in blood samples using multienzyme-nanoparticle amplification and quantum dot labels. In this method, the microbeads functionalized with the capture probes and modified electron rich proteins were arrayed within a microfluidic channel as sensing elements, and the gold nanoparticles (AuNPs) functionalized with the horseradish peroxidases (HRP) and DNA probes were used as labels. Hence, two signal amplification approaches are integrated for enhancing the detection sensitivity of circulating tumor cells. First, the large surface area of Au nanoparticle carrier allows several binding events of HRP on each nanosphere. Second, enhanced mass transport capability inherent from microfluidics leads to higher capture efficiency of targets because continuous flow within micro

Compact and highly stable quantum dots through optimized aqueous phase transfer

Science.gov (United States)

Tamang, Sudarsan; Beaune, Grégory; Poillot, Cathy; De Waard, Michel; Texier-Nogues, Isabelle; Reiss, Peter

2011-03-01

A large number of different approaches for the aqueous phase transfer of quantum dots have been proposed. Surface ligand exchange with small hydrophilic thiols, such as L-cysteine, yields the lowest particle hydrodynamic diameter. However, cysteine is prone to dimer formation, which limits colloidal stability. We demonstrate that precise pH control during aqueous phase transfer dramatically increases the colloidal stability of InP/ZnS quantum dots. Various bifunctional thiols have been applied. The formation of disulfides, strongly diminishing the fluorescence QY has been prevented through addition of appropriate reducing agents. Bright InP/ZnS quantum dots with a hydrodynamic diameter <10 nm and long-term stability have been obtained. Finally we present in vitro studies of the quantum dots functionalized with the cell-penetrating peptide maurocalcine.

Large-scale detection of antigen-specific T cells using peptide-MHC-I multimers labeled with DNA barcodes

DEFF Research Database (Denmark)

Bentzen, Amalie Kai; Marquard, Andrea Marion; Lyngaa, Rikke Birgitte

2016-01-01

-major histocompatibility complex (MHC) multimers labeled with individual DNA barcodes to screen >1,000 peptide specificities in a single sample, and detect low-frequency CD8 T cells specific for virus- or cancer-restricted antigens. When analyzing T-cell recognition of shared melanoma antigens before and after adoptive...... cell therapy in melanoma patients, we observe a greater number of melanoma-specific T-cell populations compared with cytometry-based approaches. Furthermore, we detect neoepitope-specific T cells in tumor-infiltrating lymphocytes and peripheral blood from patients with non-small cell lung cancer...

Fluorescence Quenching of CdSe/ZnS Quantum Dots by Using Black Hole Quencher Molecules Intermediated With Peptide for Biosensing Application.

Science.gov (United States)

Pillai, Sreenadh Sasidharan; Yukawa, Hiroshi; Onoshima, Daisuke; Biju, Vasudevanpillai; Baba, Yoshinobu

2015-12-17

Quantum dots (QDs) have recently been investigated as fluorescent probes for detecting a very small number of biomolecules and live cells; however, the establishment of molecular imaging technology with on-off control of QD fluorescence remains to be established. Here we have achieved the fluorescence off state of QDs with the conjugation of black hole quencher (BHQ) molecules intermediated with peptide by using streptavidin-QDs585 and biotin-pep-BHQ-1. The fluorescence of streptavidin-QDs585 was decreased by the addition of biotin-pep-BHQ-1 in a dose-dependent manner. It has been suggested that the decrease in QDs585 fluorescence occurred through a Förster resonance energy transfer (FRET) mechanism from the analysis of fluorescence intensity and lifetime of streptavidin-QDs585 and QDs585-pep-BHQ-1. QDs585 fluorescence could be quenched by more than 60% efficiency in this system. The sequence of intermediate peptide (pep) was GPLGVRGK, which can be cleaved by matrix metalloproteinases (MMPs) produced by cancer cells. QDs585-pep-BHQ-1 is thus expected to detect the MMP production by the recovery of QDs585 fluorescence as a new bioanalytical agent for molecular imaging.

Quantum dot conjugates in a sub-micrometer fluidic channel

Science.gov (United States)

Stavis, Samuel M.; Edel, Joshua B.; Samiee, Kevan T.; Craighead, Harold G.

2010-04-13

A nanofluidic channel fabricated in fused silica with an approximately 500 nm square cross section was used to isolate, detect and identify individual quantum dot conjugates. The channel enables the rapid detection of every fluorescent entity in solution. A laser of selected wavelength was used to excite multiple species of quantum dots and organic molecules, and the emission spectra were resolved without significant signal rejection. Quantum dots were then conjugated with organic molecules and detected to demonstrate efficient multicolor detection. PCH was used to analyze coincident detection and to characterize the degree of binding. The use of a small fluidic channel to detect quantum dots as fluorescent labels was shown to be an efficient technique for multiplexed single molecule studies. Detection of single molecule binding events has a variety of applications including high throughput immunoassays.

Quantum dot conjugates in a sub-micrometer fluidic channel

Science.gov (United States)

Stavis, Samuel M [Ithaca, NY; Edel, Joshua B [Brookline, MA; Samiee, Kevan T [Ithaca, NY; Craighead, Harold G [Ithaca, NY

2008-07-29

A nanofluidic channel fabricated in fused silica with an approximately 500 nm square cross section was used to isolate, detect and identify individual quantum dot conjugates. The channel enables the rapid detection of every fluorescent entity in solution. A laser of selected wavelength was used to excite multiple species of quantum dots and organic molecules, and the emission spectra were resolved without significant signal rejection. Quantum dots were then conjugated with organic molecules and detected to demonstrate efficient multicolor detection. PCH was used to analyze coincident detection and to characterize the degree of binding. The use of a small fluidic channel to detect quantum dots as fluorescent labels was shown to be an efficient technique for multiplexed single molecule studies. Detection of single molecule binding events has a variety of applications including high throughput immunoassays.

A rapid and convenient method for specific 11C-labelling of synthetic polypeptides containing methionine

International Nuclear Information System (INIS)

Laengstroem, B.; Sjoeberg, S.; Ragnarsson, U.

1981-01-01

11 C-labelling of methionine residues in a synthetic peptide via the preparation of the corresponding protected, pure homocysteine peptide has been investigated. Complete deprotection of the peptide and specific methylation of the homocysteine residue can be performed in one step in liquid ammonia. As a first application of this method the synthesis of the tripeptide, Z-Gly-L-Hcy(Bzl)-Gly-O-Bzl, and its conversion to Gly-Met-Gly and the corresponding labelled Gly-([ 11 C]-methyl)-Met-Gly, is reported. Starting with the protected peptide the labelling was performed in 20 +- 5 min (starting with 11 CO 2 ), yielding the labelled peptide in 92 +- 5 % radiochemical yield. Analyses and preparative LC can be performed within 6 min. (author)

Enabling biomedical research with designer quantum dots

NARCIS (Netherlands)

Tomczak, N.; Janczewski, D.; Dorokhin, D.V.; Han, M-Y; Vancso, Gyula J.; Navarro, Melba; Planell, Josep A.

2012-01-01

Quantum Dots (QDs) are a new class of semiconductor nanoparticulate luminophores, which are actively researched for novel applications in biology and nanomedicine. In this review, the recent progress in the design and applications of QD labels for in vitro and in vivo imaging of cells is presented.

Development of labelled biomolecules for targeted radiotherapy. Mexico

International Nuclear Information System (INIS)

Arteaga de Murphy, Consuelo

2000-01-01

The scope of the co-ordinated research project (Dec 15 1997) included the following activities: 1) develop coupling techniques using bifunctional chelating agents for monoclonal antibodies and peptides; 2) optimise radiolabelling procedures and reaction parameters using Sm-153 and Re-188; 3) investigate direct methods of labelling monoclonal antibodies and peptides with Re-188; 4) initiate animal distribution studies. The modifications specified for the period 1999/02/15 to 2000/02/14 are as follows: a) continue with the optimisation of Re-188-peptide labelling; b) continue with the work to prepare a kit; c) in-vivo and in-vitro studies; d) Lanreotide labelling. The group formed by researchers from several Mexican institutions have worked together and in different aspects of the CRP in order to fulfil the proposed aims

Biocompatibility of quantum dots (CdSe/ZnS ) in human amniotic membrane-derived mesenchymal stem cells in vitro.

Science.gov (United States)

Wang, Gongping; Zeng, Guangwei; Wang, Caie; Wang, Huasheng; Yang, Bo; Guan, Fangxia; Li, Dongpeng; Feng, Xiaoshan

2015-06-01

Amniotic membrane-derived mesenchymal stem cells (hAM-dMSCs) are a potential source of mesenchymal stem cells which could be used to repair skin damage. The use of mesenchymal stem cells to repair skin damage requires safe, effective and biocompatible agents to evaluate the effectiveness of the result. Quantum dots (QDs) composed of CdSe/ZnS are semiconductor nanocrystals with broad excitation and narrow emission spectra, which have been considered as a new chemical and fluorescent substance for non-invasively labeling different cells in vitro and in vivo. This study investigated the cytotoxic effects of QDs on hAM-dMSCs at different times following labeling. Using 0.75, 1.5 and 3.0 μL between quantum dots, labeled human amniotic mesenchymal stem cells were collected on days 1, 2 and 4 and observed morphological changes, performed an MTT cell growth assay and flow cytometry for mesenchymal stem cells molecular markers. Quantum dot concentration 0.75 μg/mL labeled under a fluorescence microscope, cell morphology was observed, The MTT assay showed cells in the proliferative phase. Flow cytometry expression CD29, CD31, CD34, CD44, CD90, CD105 and CD106. Within a certain range of concentrations between quantum dots labeled human amniotic mesenchymal stem cells has good biocompatibility.

Analysis of peptide uptake and location of root hair-promoting peptide accumulation in plant roots.

Science.gov (United States)

Matsumiya, Yoshiki; Taniguchi, Rikiya; Kubo, Motoki

2012-03-01

Peptide uptake by plant roots from degraded soybean-meal products was analyzed in Brassica rapa and Solanum lycopersicum. B. rapa absorbed about 40% of the initial water volume, whereas peptide concentration was decreased by 75% after 24 h. Analysis by reversed-phase HPLC showed that number of peptides was absorbed by the roots during soaking in degraded soybean-meal products for 24 h. Carboxyfluorescein-labeled root hair-promoting peptide was synthesized, and its localization, movement, and accumulation in roots were investigated. The peptide appeared to be absorbed by root hairs and then moved to trichoblasts. Furthermore, the peptide was moved from trichoblasts to atrichoblasts after 24 h. The peptide was accumulated in epidermal cells, suggesting that the peptide may have a function in both trichoblasts and atrichoblasts. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.

Efficacy of NGR peptide-modified PEGylated quantum dots for crossing the blood-brain barrier and targeted fluorescence imaging of glioma and tumor vasculature.

Science.gov (United States)

Huang, Ning; Cheng, Si; Zhang, Xiang; Tian, Qi; Pi, Jiangli; Tang, Jun; Huang, Qing; Wang, Feng; Chen, Jin; Xie, Zongyi; Xu, Zhongye; Chen, Weifu; Zheng, Huzhi; Cheng, Yuan

2017-01-01

Delivery of imaging agents to brain glioma is challenging because the blood-brain barrier (BBB) functions as a physiological checkpoint guarding the central nervous system from circulating large molecules. Moreover, the ability of existing probes to target glioma has been insufficient and needs to be improved. In present study, PEG-based long circulation, CdSe/ZnS quantum dots (QDs)-based nanoscale and fluorescence, asparagines-glycine-arginine peptides (NGR)-based specific CD13 recognition were integrated to design and synthesize a novel nanoprobe by conjugating biotinylated NGR peptides to avidin-PEG-coated QDs. Our data showed that the NGR-PEG-QDs were nanoscale with less than 100 nm and were stable in various pH (4.0~8.0). These nanomaterials with non-toxic concentrations could cross the BBB and target CD13-overexpressing glioma and tumor vasculature in vitro and in vivo, contributing to fluorescence imaging of this brain malignancy. These achievements allowed groundbreaking technological advances in targeted fluorescence imaging for the diagnosis and surgical removal of glioma, facilitating potential transformation toward clinical nanomedicine. Copyright © 2016 Elsevier Inc. All rights reserved.

Identification of human embryonic progenitor cell targeting peptides using phage display.

Directory of Open Access Journals (Sweden)

Paola A Bignone

Full Text Available Human pluripotent stem (hPS cells are capable of differentiation into derivatives of all three primary embryonic germ layers and can self-renew indefinitely. They therefore offer a potentially scalable source of replacement cells to treat a variety of degenerative diseases. The ability to reprogram adult cells to induced pluripotent stem (iPS cells has now enabled the possibility of patient-specific hPS cells as a source of cells for disease modeling, drug discovery, and potentially, cell replacement therapies. While reprogramming technology has dramatically increased the availability of normal and diseased hPS cell lines for basic research, a major bottleneck is the critical unmet need for more efficient methods of deriving well-defined cell populations from hPS cells. Phage display is a powerful method for selecting affinity ligands that could be used for identifying and potentially purifying a variety of cell types derived from hPS cells. However, identification of specific progenitor cell-binding peptides using phage display may be hindered by the large cellular heterogeneity present in differentiating hPS cell populations. We therefore tested the hypothesis that peptides selected for their ability to bind a clonal cell line derived from hPS cells would bind early progenitor cell types emerging from differentiating hPS cells. The human embryonic stem (hES cell-derived embryonic progenitor cell line, W10, was used and cell-targeting peptides were identified. Competition studies demonstrated specificity of peptide binding to the target cell surface. Efficient peptide targeted cell labeling was accomplished using multivalent peptide-quantum dot complexes as detected by fluorescence microscopy and flow cytometry. The cell-binding peptides were selective for differentiated hPS cells, had little or no binding on pluripotent cells, but preferential binding to certain embryonic progenitor cell lines and early endodermal hPS cell derivatives. Taken

Neuropeptides labelled with [sup 35]S

Energy Technology Data Exchange (ETDEWEB)

Kaspersen, F.M.; Nispen, J.W. van; Sperling, E.M.G.; Vader, J.F. (Organon Int BV, Oss (Netherlands))

1993-01-01

Methionine and cysteine containing peptides can be labelled with [sup 35]S by coupling of [sup 35]S-cysteine or [sup 35]S-methionine with a large excess of suitability protected peptide precursors. This is illustrated for [pGlu[sup 4], Cyt[sup 6

A label-free electrochemical immunosensor for the detection of cardiac marker using graphene quantum dots (GQDs).

Science.gov (United States)

Tuteja, Satish K; Chen, Rui; Kukkar, Manil; Song, Chung Kil; Mutreja, Ruchi; Singh, Suman; Paul, Ashok K; Lee, Haiwon; Kim, Ki-Hyun; Deep, Akash; Suri, C Raman

2016-12-15

A label-free immunosensor based on electrochemical impedance spectroscopy has been developed for the sensitive detection of a cardiac biomarker myoglobin (cMyo). Hydrothermally synthesized graphene quantum dots (GQDs) have been used as an immobilized template on screen printed electrodes for the construction of an impedimetric sensor platform. The GQDs-modified electrode was conjugated with highly specific anti-myoglobin antibodies to develop the desired immunosensor. The values of charge transfer resistance (Rct) were monitored as a function of varying antigen concentration. The Rct value of the immunosensor showed a linear increase (from 0.20 to 0.31kΩ) in the range of 0.01-100ng/mL cMyo. The specific detection of cMyo was also made in the presence of other competing proteins. The limit of detection for the proposed immunosensor was estimated as 0.01ng/mL which is comparable to the standard ELISA techniques. Copyright © 2016 Elsevier B.V. All rights reserved.

Molecular imaging of a cell-penetrating peptide labeled fluorescein-5-isothiocyanate and MR contrast agents: gadopentetate dimeglumine

International Nuclear Information System (INIS)

Liu Min; Guo Youmin; Duan Xiaoyi; Guo Xiaojuan; Yang Junle; Xu Min

2006-01-01

Objective: To study the value of a new intracellular contrast agent--cell penetrating peptide labeled Fluorescein-5-isothiocyanate (FITC) and MRI contrast agent, Gadopentetate dimeglumine in molecular imaging. Methods: A new cell penetration peptides (CPPs)sequence LAGRRRRRRRRRK were synthesized in solid phase on the base of arginine (9) and were labelled with FITC (CPP 13 -FITC) and Gd - DTPA (CPP 13 -DTPA-Gd). Hepatic carcinoma cell line-HEPG 2 and mouse bone marrow stem cell was respectively stained by CPP 13 -FITC for different time intervals for observing the uptake and intracellular distribution. HEPG 2 in three l00 mm 2 culture plates was respectively incubated with CPP 13 -DTPA-Gd, Gd- DTPA and Dulbecco minimum essential medium for 30 min and imaged by 1.5 T MRI for studying the intracellular uptake and T 1 WI signal characteristics. Results: The peptide was synthesized by the manual solid-phase method successfully. The calculated molecular weight was 1792.78 and the chemical purity was over 95%. By inverted fluorescence microscope, HEPG 2 and mouse stem cell could transport CPP-FITC in cytoplasm and nuclear in 10 min. By MR imaging, CPP-DTPA-Gd could be uptake by HEPG 2 in 30 min and had a short T 1 short T 2 signal, furthermore. T 1 WI signal intensity ratio between in-tube (Ii) and out-tube (Io) in three groups of three scan slices were shown below: Iil/Io of group 1 (Group 1 was the cell incubated by CPP 13 -DTPA-Gd ) respectively was 2.84, 2.60, 2. 48; Iil/Io of group 2 (Group 2 was the cell incubated by DTPA-Gd) respectively is 1.15, 1.11, 1.12; Iil/Io of group 3 (Group 3 was the controled cell ) respectively was 1.15, 1.11, 1.11. By ANVOA analysis, the signal intensity among group 1, group 2 and group 3 had significant difference(F (1,2) = 201.88 P (1,3) =206.37 P (2,3) =0.529 P=0.507). Conclusion: The new constructed cell penetration peptide on the base of the polyargnine can translocate cell by carting FITC and MRI contrast agent-DTPA-Gd and the

Species Distribution and Prevalence of Putative Virulence Factors in Mesophilic Aeromonas spp. Isolated from Fresh Retail Sushi

Directory of Open Access Journals (Sweden)

Sunniva Hoel

2017-05-01

Full Text Available Aeromonas spp. are ubiquitous bacteria that have received increasing attention as human pathogens because of their widespread occurrence in food, especially seafood and vegetables. The aim of this work was to assess the species identity and phylogenetic relationship of 118 Aeromonas strains isolated from fresh retail sushi from three producers, and to characterize the isolates with respect to genetic and phenotypic virulence factors. We also evaluate the potential hazard associated with their presence in ready-to-eat seafood not subjected to heat treatment. Mesophilic Aeromonas salmonicida was most prevalent (74%, followed by A. bestiarum (9%, A. dhakensis (5%, A. caviae (5%, A. media (4%, A. hydrophila (2%, and A. piscicola (1%. All isolates were considered potentially pathogenic due to the high prevalence of genes encoding hemolysin (hlyA (99%, aerolysin (aerA (98%, cytotoxic enterotoxin (act (86%, heat-labile cytotonic enterotoxin (alt (99%, and heat-stable cytotonic enterotoxin (ast (31%. The shiga-like toxins 1 and 2 (stx-1 and stx-2 were not detected. Moreover, there was heterogeneity in toxin gene distribution among the isolates, and the combination of act/alt/hlyA/aerA was most commonly detected (63%. β-hemolysis was species-dependent and observed in 91% of the isolates. All A. media and A. caviae strains were non-hemolytic. For isolates belonging to this group, lack of hemolysis was possibly related to the absence of the act gene. Swimming motility, linked to adhesion and host invasion, occurred in 65% of the isolates. Partial sequencing of the gyrB gene demonstrated its suitability as a genetic marker for Aeromonas species identification and for assessment of the phylogenetic relationship between the isolates. The gyrB sequence divergence within a given species ranged from 1.3 to 2.9%. A. bestiarum, A. salmonicida, and A. piscicola were the most closely related species; their sequences differed by 2.7–3.4%. The average gyrB sequence

Effects of the amino acid linkers on the melanoma-targeting and pharmacokinetic properties of 111In-labeled lactam bridge-cyclized alpha-MSH peptides.

Science.gov (United States)

Guo, Haixun; Yang, Jianquan; Gallazzi, Fabio; Miao, Yubin

2011-04-01

The purpose of this study was to examine the profound effects of the amino acid linkers on the melanoma-targeting and pharmacokinetic properties of (111)In-labeled lactam bridge-cyclized DOTA-[X]-CycMSH(hex) {1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid-[X]-c[Asp-His-dPhe-Arg-Trp-Lys]-CONH(2); X = GGNle, GENle, or NleGE; GG = -Gly-Gly- and GE = -Gly-Glu-} peptides. Three novel peptides (DOTA-GGNle-CycMSH(hex), DOTA-GENle-CycMSH(hex), and DOTA-NleGE-CycMSH(hex)) were designed and synthesized. The melanocortin-1 (MC1) receptor-binding affinities of the peptides were determined in B16/F1 melanoma cells. The melanoma-targeting and pharmacokinetic properties of (111)In-DOTA-GGNle-CycMSH(hex) and (111)In-DOTA-GENle-CycMSH(hex) were determined in B16/F1 melanoma-bearing C57 mice. DOTA-GGNle-CycMSH(hex) and DOTA-GENle-CycMSH(hex) displayed 2.1 and 11.5 nM MC1 receptor-binding affinities, whereas DOTA-NleGE-CycMSH(hex) showed 873.4 nM MC1 receptor-binding affinity. The introduction of the -GG- linker maintained high melanoma uptake while decreasing kidney and liver uptake of (111)In-DOTA-GGNle-CycMSH(hex). The tumor uptake of (111)In-DOTA-GGNle-CycMSH(hex) was 19.05 ± 5.04 and 18.6 ± 3.56 percentage injected dose per gram at 2 and 4 h after injection, respectively. (111)In-DOTA-GGNle-CycMSH(hex) exhibited 28%, 32%, and 42% less kidney uptake than (111)In-DOTA-Nle-CycMSH(hex) we reported previously, and 61%, 65%, and 68% less liver uptake than (111)In-DOTA-Nle-CycMSH(hex) at 2, 4, and 24 h after injection, respectively. The amino acid linkers exhibited profound effects on the melanoma-targeting and pharmacokinetic properties of the (111)In-labeled lactam bridge-cyclized α-melanocyte-stimulating hormone peptides. Introduction of the -GG- linker maintained high melanoma uptake while reducing kidney and liver uptake of (111)In-DOTA-GGNle-CycMSH(hex), highlighting its potential as an effective imaging probe for melanoma detection, as well as a therapeutic peptide

In vivo cation exchange in quantum dots for tumor-specific imaging.

Science.gov (United States)

Liu, Xiangyou; Braun, Gary B; Qin, Mingde; Ruoslahti, Erkki; Sugahara, Kazuki N

2017-08-24

In vivo tumor imaging with nanoprobes suffers from poor tumor specificity. Here, we introduce a nanosystem, which allows selective background quenching to gain exceptionally tumor-specific signals. The system uses near-infrared quantum dots and a membrane-impermeable etchant, which serves as a cation donor. The etchant rapidly quenches the quantum dots through cation exchange (ionic etching), and facilitates renal clearance of metal ions released from the quantum dots. The quantum dots are intravenously delivered into orthotopic breast and pancreas tumors in mice by using the tumor-penetrating iRGD peptide. Subsequent etching quenches excess quantum dots, leaving a highly tumor-specific signal provided by the intact quantum dots remaining in the extravascular tumor cells and fibroblasts. No toxicity is noted. The system also facilitates the detection of peritoneal tumors with high specificity upon intraperitoneal tumor targeting and selective etching of excess untargeted quantum dots. In vivo cation exchange may be a promising strategy to enhance specificity of tumor imaging.The imaging of tumors in vivo using nanoprobes has been challenging due to the lack of sufficient tumor specificity. Here, the authors develop a tumor-specific quantum dot system that permits in vivo cation exchange to achieve selective background quenching and high tumor-specific imaging.

Application on Gold Nanoparticles-Dotted 4-Nitrophenylazo Graphene in a Label-Free Impedimetric Deoxynivalenol Immunosensor

Directory of Open Access Journals (Sweden)

Christopher Edozie Sunday

2015-02-01

Full Text Available In this paper, we report a new concept to construct a label-free electrochemical inhibition-based immunosensor for the detection of the mycotoxin deoxynivalenol (DON in cereal samples. The electrochemical impedance spectroscopy of tris(bipyridine ruthenium (II chloride was used as a marker enhanced with gold nanoparticles-dotted 4-nitrophenylazo functionalized graphene (AuNp/G/PhNO2 nanocatalyst mediated in Nafion on a glassy carbon electrode. Under the optimized conditions, the formation of immunocomplexes inhibited electron flow and increased the charge transfer resistance of the sensing interface linearly. The change in impedance was proportional to DON concentrations in the range of 6–30 ng/mL with a sensitivity and detection limit of 32.14 ΩL/ng and 0.3 µg/mL, respectively, which compares favorably with the ELISA result. The proposed sensor had a stability of 80.3%, good precision and selectivity in DON standard solution containing different interfering agents, indicating promising application prospect for this strategy in designing impedimetric, electrochemiluminescent, voltammetric or amperometric sensors.

Peptides for Specific Intracellular Delivery and Targeting of Nanoparticles: Implications for Developing Nanoparticle-Mediated Drug Delivery

Science.gov (United States)

2010-01-01

less in size and span an array of compositions including met- als, semiconductor quantum dots (QDs), oxides, polymers , vesicles (e.g., micelles...93,98] Polymers Poly(lactic-co-glycolic acid), poly(amido amine) and dendrimers Biocompatibility, biodegradability and controlled release High drug...Secondary interactions Covalent atachment to surface ligand Thiolated peptide Biotinylated peptide Aminated peptide Sulfo-NHS Tetravalent streptavidin

Label-free SPR detection of gluten peptides in urine for non-invasive celiac disease follow-up.

Science.gov (United States)

Soler, Maria; Estevez, M-Carmen; Moreno, Maria de Lourdes; Cebolla, Angel; Lechuga, Laura M

2016-05-15

Motivated by the necessity of new and efficient methods for dietary gluten control of celiac patients, we have developed a simple and highly sensitive SPR biosensor for the detection of gluten peptides in urine. The sensing methodology enables rapid and label-free quantification of the gluten immunogenic peptides (GIP) by using G12 mAb. The overall performance of the biosensor has been in-depth optimized and evaluated in terms of sensitivity, selectivity and reproducibility, reaching a limit of detection of 0.33 ng mL(-1). Besides, the robustness and stability of the methodology permit the continuous use of the biosensor for more than 100 cycles with excellent repeatability. Special efforts have been focused on preventing and minimizing possible interferences coming from urine matrix enabling a direct analysis in this fluid without requiring extraction or purification procedures. Our SPR biosensor has proven to detect and identify gluten consumption by evaluating urine samples from healthy and celiac individuals with different dietary gluten conditions. This novel biosensor methodology represents a novel approach to quantify the digested gluten peptides in human urine with outstanding sensitivity in a rapid and non-invasive manner. Our technique should be considered as a promising opportunity to develop Point-of-Care (POC) devices for an efficient, simple and accurate gluten free diet (GFD) monitoring as well as therapy follow-up of celiac disease patients. Copyright © 2015 Elsevier B.V. All rights reserved.

Radiolabelled peptides for oncological diagnosis

Energy Technology Data Exchange (ETDEWEB)

Laverman, Peter; Boerman, Otto C.; Oyen, Wim J.G. [Radboud University Nijmegen Medical Centre, Department of Nuclear Medicine, Nijmegen (Netherlands); Sosabowski, Jane K. [Queen Mary University of London, Centre for Molecular Oncology, Barts Cancer Institute, London (United Kingdom)

2012-02-15

Radiolabelled receptor-binding peptides targeting receptors (over)expressed on tumour cells are widely under investigation for tumour diagnosis and therapy. The concept of using radiolabelled receptor-binding peptides to target receptor-expressing tissues in vivo has stimulated a large body of research in nuclear medicine. The {sup 111}In-labelled somatostatin analogue octreotide (OctreoScan trademark) is the most successful radiopeptide for tumour imaging, and was the first to be approved for diagnostic use. Based on the success of these studies, other receptor-targeting peptides such as cholecystokinin/gastrin analogues, glucagon-like peptide-1, bombesin (BN), chemokine receptor CXCR4 targeting peptides, and RGD peptides are currently under development or undergoing clinical trials. In this review, we discuss some of these peptides and their analogues, with regard to their potential for radionuclide imaging of tumours. (orig.)

Synthesis, characterization and inhibitory activities of (4-N3[3,5-3H]Phe10)PKI(6-22)amide and its precursors: photoaffinity labeling peptides for the active site of cyclic AMP-dependent protein kinase.

Science.gov (United States)

Katz, B M; Lundquist, L J; Walsh, D A; Glass, D B

1989-06-01

PKI(6-22)amide is a 17 residue peptide corresponding to the active portion of the heat-stable inhibitor of cAMP-dependent protein kinase. The peptide is a potent (Ki = 1.6 nM), competitive inhibitor of the enzyme. The photoreactive peptide analog (4-azidophenylalanine10)PKI(6-22)amide was synthesized in both its non-radiolabeled and tritiated forms by chemical modification of precursor peptides that were prepared by stepwise solid-phase synthesis. (4-Amino[3,5-3H]phenylalanine10)PKI(6-22)amide, the precursor for the radiolabeled arylazide peptide, was obtained by catalytic reduction of the corresponding peptide containing the 3,5-diiodo-4-aminophenylalanine residue at position 10. The purified PKI peptides were analyzed by HPLC, amino acid analysis, and u.v. spectra. In the dark, (4-azidophenylalanine10)PKI(6-22)amide inhibited the catalytic subunit of cAMP-dependent protein kinase with a Ki value of 2.8 nM. The photoreactivity of the arylazide peptide was demonstrated by time-dependent u.v. spectral changes on exposure to light. Photolysis of the catalytic subunit (4-azido[3,5-3H]phenylalanine10)PKI(6-22)amide complex resulted in specific covalent labeling of the enzyme. The data indicate that this peptide is a useful photoaffinity labeling reagent for the active site of the protein kinase.

Effect of shells on photoluminescence of aqueous CdTe quantum dots

International Nuclear Information System (INIS)

Yuan, Zhimin; Yang, Ping

2013-01-01

Graphical abstract: Size-tunable CdTe coated with several shells using an aqueous solution synthesis. CdTe/CdS/ZnS quantum dots exhibited high PL efficiency up to 80% which implies the promising applications for biomedical labeling. - Highlights: • CdTe quantum dots were fabricated using an aqueous synthesis. • CdS, ZnS, and CdS/ZnS shells were subsequently deposited on CdTe cores. • Outer ZnS shells provide an efficient confinement of electron and hole inside the QDs. • Inside CdS shells can reduce the strain on the QDs. • Aqueous CdTe/CdS/ZnS QDs exhibited high stability and photoluminescence efficiency of 80%. - Abstract: CdTe cores with various sizes were fabricated in aqueous solutions. Inorganic shells including CdS, ZnS, and CdS/ZnS were subsequently deposited on the cores through a similar aqueous procedure to investigate the effect of shells on the photoluminescence properties of the cores. In the case of CdTe/CdS/ZnS quantum dots, the outer ZnS shell provides an efficient confinement of electron and hole wavefunctions inside the quantum dots, while the middle CdS shell sandwiched between the CdTe core and ZnS shell can be introduced to obviously reduce the strain on the quantum dots because the lattice parameters of CdS is situated at the intermediate-level between those of CdTe and ZnS. In comparison with CdTe/ZnS core–shell quantum dots, the as-prepared water-soluble CdTe/CdS/ZnS quantum dots in our case can exhibit high photochemical stability and photoluminescence efficiency up to 80% in an aqueous solution, which implies the promising applications in the field of biomedical labeling

Protein labelling with stable isotopes: strategies

International Nuclear Information System (INIS)

Lirsac, P.N.; Gilles, N.; Jamin, N.; Toma, F.; Gabrielsen, O.; Boulain, J.C.; Menez, A.

1994-01-01

A protein labelling technique with stable isotopes has been developed at the CEA: a labelled complete medium has been developed, performing as well as the Luria medium, but differing from it because it contains not only free aminated acids and peptides, but also sugars (96% of D-glucopyrannose) and labelled nucleosides. These precursors are produced from a labelled photosynthetic micro-organisms biomass, obtained with micro-algae having incorporated carbon 13, nitrogen 15 and deuterium during their culture. Labelling costs are reduced. 1 fig., 1 tab., 3 refs

{sup 18}F-Fluoroglucosylation of peptides, exemplified on cyclo(RGDfK)

Energy Technology Data Exchange (ETDEWEB)

Hultsch, Christina; Schottelius, Margret; Auernheimer, Joerg; Alke, Andrea; Wester, Hans-Juergen [Technische Universitaet Muenchen, Nuklearmedizinische Klinik und Poliklinik, Klinikum rechts der Isar, Muenchen (Germany)

2009-09-15

Oxime formation between an aminooxy-functionalized peptide and an {sup 18}F-labelled aldehyde has recently been introduced as a powerful method for the rapid one-step chemoselective synthesis of radiofluorinated peptides. Here, the potential of using routinely produced and thus readily available [{sup 18}F]fluorodeoxyglucose ([{sup 18}F](FDG)) as the aldehydic prosthetic group was investigated using an aminooxyacetyl-conjugated cyclic RGD peptide (cyclo(RGDfK)(Aoa-Boc)) as a model peptide. The use of [{sup 18}F]FDG from routine production ([{sup 18}F]FDGTUM) containing an excess of d-glucose did not allow the radiosynthesis of [{sup 18}F]FDG-RGD in activities >37 MBq in reasonable yield, rendering the direct use of clinical grade [{sup 18}F]FDG for the routine clinical synthesis of {sup 18}F-labelled peptides impossible. Using no-carrier-added (n.c.a.) [{sup 18}F]FDG obtained via HPLC separation of [{sup 18}F]FDGTUM from excess glucose, however, afforded [{sup 18}F]FDG-RGD in yields of 56-93% (decay corrected) and activities up to 37 MBq. Suitable reaction conditions were 20 min at 120 C and pH 2.5, and a peptide concentration of 5 mM. In a preliminary in vivo biodistribution study in M21 melanoma-bearing nude mice, [{sup 18}F]FDG-RGD showed increased tumour accumulation compared to the ''gold standard'' [{sup 18}F]galacto-RGD (2.18 vs 1.49 %iD/g, respectively, at 120 min after injection), but also slightly increased uptake in non-target organs, leading to comparable tumour/organ ratios for both compounds.??These data demonstrate that chemoselective {sup 18}F-labelling of aminooxy-functionalized peptides using n.c.a. [{sup 18}F]FDG represents a radiofluorination/glycosylation strategy that allows preparation of {sup 18}F-labelled peptides in high yield with suitable pharmacokinetics. As soon as the necessary n.c.a. preparation of [{sup 18}F]FDG prior to reaction with the Aoa-peptide can be implemented in a fully automated [{sup 18}F]FDG-synthesis, [{sup 18}F

Vasoactive intestinal peptide (VIP) labelling with iodine-131 by direct method

International Nuclear Information System (INIS)

Colturato, M.T.; Silva, C.P.G. da; Araujo, E.B.

2002-01-01

The Vasoactive Intestinal Peptide (VIP) is a 28-amino acid polypeptide with a great numbers of receptors in tumoral cells, including adenocarcinomas and pancreatic and colon carcinomas. The VIP molecule contains two tyrosine residues, in positions 10 and 22, that are theoretically equally susceptible to iodination, The VIP was labeled with 131-iodine by direct method using Iodogen as oxidant agent: 15.03 mmol VIP + 0.10 nmol KI + [ 131 I]NaI + 13.9 mmol Iodogen; the final volume was adjust to 100 μL using 0.2 M phosphate buffer, pH 7.5 and the reaction proceed with stirring for 30 minutes at room temperature. The radiochemical purity was determined by electrophoresis (Whatman 1MM paper; 0.05 M barbital buffer; pH 8.6; 150 V; 40 minutes) that indicates low percent of free 131-iodine. The high performance liquid chromatography (HPLC) system using RPC 18 , 10 μm, 4 x 250mm column, was able to separate the different radiochemical species, only when an isocratic mixture of acetonitrile: 0.1% trifluoroacetic acid (27:73) was used, with 0.5 mL/min. flux. (author)

Radiometallating antibodies and biologically active peptides

International Nuclear Information System (INIS)

Mercer-Smith, J.A.; Roberts, J.C.; Lewis, D.; Newmyer, S.L.; Schulte, L.D.; Burns, T.P.; Mixon, P.L.; Jeffery, A.L.; Schreyer, S.A.; Cole, D.A.; Figard, S.D.; Lennon, V.A.; Hayashi, M.; Lavallee, D.K.

1990-01-01

We have developed methods to radiolabel large molecules, using porphyrins as bifunctional chelating agents for radiometals. The porphyrins are substituted with an N-benzyl group to activate them for radiometallation under mild reaction conditions. Porphyrins that have on functional group for covalent attachment to other molecules cannot cause crosslinking. We have examined the labeling chemistry for antibodies, and we have also developed methods to label smaller biologically active molecules, such as autoantigenic peptides. The autoantigenic peptides, fragments of the acetylcholine receptor, are under investigation for myasthenia gravis research. The methods of covalent attachment of these bifunctional chelating agents to large molecules and the radiometallation chemistry will be discussed

Radioiodination of vasoactive intestinal peptide (VIP)

International Nuclear Information System (INIS)

Wang, Y.; Wang, L.; Yin, D.

2002-01-01

In recent years, increasing biochemical and radiochemical research has been performed to develop radiolabelled peptides as specific ligands for tumour associated receptors. VIP, a 28-amino acid peptide containing two tyrosines and three lysines, has demonstrated that various tumour cells express significantly higher amounts of VIP-receptors and could be applied to the clinic diagnosis. For these purposes, radiohalogenation of VIP by direct and indirect method was studied. Direct labelling works well for radioiodine but is limited to dehalogenation of labelling products in vivo. Conjugate labelling methods including Boltonhunter and wood reagents were developed but introduction of such a molecule to peptides may lead to the decrease of biological activity in vivo. In order to resolve these problems, N-Succinimidyl-3-(tri-nbutylstannyl) benzoate (ATE) was elected for the radioiodination of VIP and already employed to radioiodination of IgG successfully. The in vitro stability and biological activity would be compared in these two methods. Vasoactive intestinal peptide (VIP) and human immunoglobulin (IgG) were radioiodinated by direct and indirect methods. Iodogen was employed in direct method and N-Succinimidyl-3-(tri-n-butylstannyl) benzoate (ATE) was applied as a prosthetic group in the conjugation labelling. The subject of our study was optimizing the radiohalogenation of IgG and VIP followed by separation and analysis of reaction products. The advantages and disadvantages were illustrated by comparing the in vitro stability and biological activity in these two methods. Na 123 I was prepared by nuclear reaction of 124 Te(p, 2n) 123 I using cyclone-30. More than 95% of radiochemical purity, more than 95% of radionuclide purity and about 100 mCi/mL of radioactivity concentration were obtained. ATE was supplied by Dr. Pozzi and radioiodinated with iodogen and 96% of labelling efficiency was obtained. The stability of radioactive S 125 IB kept well in dark at 4

Comparison of biological properties of 111In-labeled dimeric cyclic RGD peptides

International Nuclear Information System (INIS)

Zheng, Yumin; Ji, Shundong; Tomaselli, Elena; Yang, Yong; Liu, Shuang

2015-01-01

Introduction: In this study two 111 In-labeled dimeric cyclic RGD peptides, 111 In(DOTA-Galacto-RGD 2 ) and 111 In(DOTA-3P-RGD 2 ), were evaluated as radiotracers for breast tumor imaging. The objective was to evaluate the impact of SAA, PEG 2 and 1,2,3-triazole linkers as compare to PEG 4 on the tumor uptake and excretion kinetics of 111 In radiotracers. Methods: DOTA-Galacto-RGD 2 was prepared by conjugation of Galacto-RGD 2 with DOTA-OSu in the presence of diisopropylethylamine. Its integrin α v β 3 binding affinity was determined using a whole-cell displacement assay against 125 I-echistatin bound to U87MG glioma cells, and was compared with those of c(RGDfK), DOTA-3P-RGD 2 and DOTA-3P-RGK 2 (a nonsense peptide conjugate with “scrambled” RGK sequences). 111 In(DOTA-Galacto-RGD 2 ) and 111 In(DOTA-3P-RGD 2 ) were prepared and evaluated for their tumor-targeting capability and biodistribution properties in athymic nude mice bearing MDA-MB-435 breast tumor xenografts. Planar imaging studies were performed to demonstrate the utility of 111 In(DOTA-Galacto-RGD 2 ) and 111 In(DOTA-3P-RGD 2 ) for breast tumor imaging. Results: IC 50 values of DOTA-Galacto-RGD 2 , DOTA-3P-RGD 2 , and DOTA-3P-RGK 2 were calculated to be 27 ± 2, 29 ± 4, 596 ± 48 nM, respectively. The tumor uptake values of 111 In(DOTA-Galacto-RGD 2 ) (6.79 ± 0.98, 6.56 ± 0.56, 4.17 ± 0.61 and 1.09 ± 0.13 %ID/g at 1, 4, 24 and 72 hours p.i., respectively) were almost identical to those of 111 In(DOTA-3P-RGD 2 ) (6.17 ± 1.65, 5.94 ± 0.84, 3.40 ± 0.50 and 0.99 ± 0.20 %ID/g, respectively). 111 In(DOTA-Galacto-RGD 2 ) had a faster clearance from blood and muscle than 111 In(DOTA-3P-RGD 2 ), leading to higher tumor/blood and tumor/muscle ratios. 111 In(DOTA-3P-RGD 2 ) had lower liver uptake and better tumor/liver ratios than 111 In(DOTA-Galacto-RGD 2 ). The tumor uptake of 111 In(DOTA-Galacto-RGD 2 ) and 111 In(DOTA-3P-RGD 2 ) was both integrin α v β 3 and RGD-specific. Imaging data suggest

Covalent dye attachment influences the dynamics and conformational properties of flexible peptides.

Directory of Open Access Journals (Sweden)

Manuel P Luitz

Full Text Available Fluorescence spectroscopy techniques like Förster resonance energy transfer (FRET and fluorescence correlation spectroscopy (FCS have become important tools for the in vitro and in vivo investigation of conformational dynamics in biomolecules. These methods rely on the distance-dependent quenching of the fluorescence signal of a donor fluorophore either by a fluorescent acceptor fluorophore (FRET or a non-fluorescent quencher, as used in FCS with photoinduced electron transfer (PET. The attachment of fluorophores to the molecule of interest can potentially alter the molecular properties and may affect the relevant conformational states and dynamics especially of flexible biomolecules like intrinsically disordered proteins (IDP. Using the intrinsically disordered S-peptide as a model system, we investigate the impact of terminal fluorescence labeling on the molecular properties. We perform extensive molecular dynamics simulations on the labeled and unlabeled peptide and compare the results with in vitro PET-FCS measurements. Experimental and simulated timescales of end-to-end fluctuations were found in excellent agreement. Comparison between simulations with and without labels reveal that the π-stacking interaction between the fluorophore labels traps the conformation of S-peptide in a single dominant state, while the unlabeled peptide undergoes continuous conformational rearrangements. Furthermore, we find that the open to closed transition rate of S-peptide is decreased by at least one order of magnitude by the fluorophore attachment. Our approach combining experimental and in silico methods provides a benchmark for the simulations and reveals the significant effect that fluorescence labeling can have on the conformational dynamics of small biomolecules, at least for inherently flexible short peptides. The presented protocol is not only useful for comparing PET-FCS experiments with simulation results but provides a strategy to minimize the

Labeling of vasoactive intestinal peptide (VIP) and VIP 10-28 fragment with radioiodine by direct method. Comparative study of the kinetics biodistribution and affinity for neuroendocrine tumor cells

International Nuclear Information System (INIS)

Colturato, Maria Tereza

2005-01-01

In the progress of the Nuclear Medicine, many protein based radiopharmaceuticals have been developed in the last years using antibodies and, more recently, biologically active natural peptides or similar synthetic peptides. In the search for agents with specificity for the target tissue in tumors detection, it was verified that small sequences of amino acids may interact with selective sites, with homogenous distribution, fast accumulation in tissues and fast blood clearance when compared to the antibodies. Among the peptides used in the diagnosis of tumors, Vasoactive Intestinal Peptide (VIP) has been studied. VIP labeled with iodine-123 is applied in the images of intestinal adenocarcinoma and endocrine tumors. The molecule of VIP contains two tyrosine residues, in the positions 10 and 22 that are, theoretically, equally susceptible to radioiodination for direct method. The objective of this work was to produce VIP labeled with radioiodine (iodine-123), in order to introduce to the brazilian medical class this radiopharmaceutical of interest for the diagnosis and recurrence of tumors that express specific receptors. In an unpublished way, the work studied the labeling and the kinetic distribution of the VIP fragment (VIP 10-28) and verified its potential as radiopharmaceutical applied in the identification of tumors that express VIP receptors. After the choice of the appropriated technique for labeling VIP and VIP 10-28 with radioiodine, using Ceremonial T as oxidant agent and sodium metabisulfite as reducing agent, the quality control procedures were accomplished (electrophoresis and high performance liquid chromatography, HPLC) for radiochemical purity determination as well as the separation of the radiochemical species obtained. Labeling and quality control procedures applied were efficient and accurate. [ 131 I]VIP and [ 131 l]VIP 10-28 were obtained with high radiochemical purity (> 95%). The purification studies to remove free radioiodine in the labeling

99mTc labelled peptides for imaging of peripheral receptors

International Nuclear Information System (INIS)

Mikolajczak, R.; Markiewicz, A.; Deptula, C.Z.; Zulczyk, W.; Birnbaum, G.; Zakrezewska, E.; Wozniak, I.

2001-01-01

The first trials of 99m Tc labelling by direct method using dithionite as a reducing agent (prepared in the freeze-dried form) gave the yields of around 30%. RC-160 labelling with 125 I by chloramine-T method resulted in 40-80% labelling yield. Our efforts were focused on BFC approach. HYNIC-TOC and HYNIC-RC-160 conjugates obtained in our laboratory were successfully labelled with 99m Tc with the yields over 90%. HPLC and TLC methods were applied for quality control (QC) of the labelled preparation. Methods of in vitro (stability and protein binding) testing of the labelled preparations were adopted to our laboratory conditions. First attempts on dry kit formulation based on HYNIC-TOC conjugates with tricine, tricine/nicotinic acid and EDDA were described. Various amounts of tin (II) (as SnCl 2 ) were added to the kits. Incubation conditions (time, temperature) were investigated. The kits were tested for labelling yield and radiochemical purity. It was shown that the results are at the same level or better than obtained in liquid phase but the procedure of labelling is significantly easier. Kit produced with tricine as co-ligand was labelled with 97% labelling yield after 30 min of incubation at room temperature, which is considered acceptable for diagnostic radiopharmaceutical preparation. Tricine/nicotinic acid kit requires heating to get labelling of around 95%. Similarly EDDA kit gives around 70% labelling after 30 min incubation at 80 deg. C. Further experiments on optimal kit composition and stability are required. Results of DOTA-RC-160 labelling with 90 Y show that this isotope, manufactured by Radioisotope Centre POLATOM, can be successfully used for medical applications. (author)

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

Single-Residue Sensitivity in Neutron Reflectivity and Resonant X-ray Reflectivity from Langmuir Monolayers of Synthetic Peptides

Science.gov (United States)

Strzalka, Joseph; Satija, Sushil; Dimasi, Elaine; Kuzmenko, Ivan; Gog, Thomas; Blasie, J. Kent

2004-03-01

Labeling groups with ^2H to distinguish them in the scattering length density (SLD) profile constitutes the chief advantage of neutron reflectivity (NR) in studying Langmuir monolayers (LM) of lipids and proteins. Solid phase synthesis (SPPS) permits the labeling of a single residue in a peptide. Recent work demonstrates the sensitivity of NR to single ^2H-labeled residues in LM of vectorially oriented α -helical bundle peptides. NR requires comparison of isomorphic samples of all-^1H and ^2H-labeled peptides. Alternately, resonant x-ray reflectivity (RXR) uses only one sample. RXR exploits energy-dependent changes in the scattering factor from heavy atoms to distinguish them within the SLD profile. Peptides may be labeled by SPPS (e.g. Br-Phe), or may have inherent labels (e.g. Fe in heme proteins). As test cases, we studied LM of Br-labeled lipids and peptides with RXR. Both approaches require a model-independent means of obtaining SLD profiles from the reflectivity data. We have applied box-refinement to obtain the gradient SLD profile. This is fit uniquely with a sum of Gaussians and integrated analytically [Blasie et al., PRB 67 224201 (2003)] to provide the SLD profile. Label positions can then be determined to sub-Ångstrom accuracy. This work supported by the NIH (GM55876).

The human complement inhibitor Sushi Domain-Containing Protein 4 (SUSD4) expression in tumor cells and infiltrating T cells is associated with better prognosis of breast cancer patients

OpenAIRE

Englund, Emelie; Reitsma, Bart; King, Ben C.; Escudero-Esparza, Astrid; Owen, Sioned; Orimo, Akira; Okroj, Marcin; Anagnostaki, Lola; Jiang, Wen G.; Jirström, Karin; Blom, Anna M.

2015-01-01

Background: The human Sushi Domain-Containing Protein 4 (SUSD4) was recently shown to function as a novel inhibitor of the complement system, but its role in tumor progression is unknown. \\ud \\ud Methods: Using immunohistochemistry and quantitative PCR, we investigated SUSD4 expression in breast cancer tissue samples from two cohorts. The effect of SUSD4 expression on cell migration and invasion was studied in vitro using two human breast cancer cell lines overexpressing SUSD4. \\ud \\ud Result...

Comparison of a Label-Free Quantitative Proteomic Method Based on Peptide Ion Current Area to the Isotope Coded Affinity Tag Method

Directory of Open Access Journals (Sweden)

Young Ah Goo

2008-01-01

Full Text Available Recently, several research groups have published methods for the determination of proteomic expression profiling by mass spectrometry without the use of exogenously added stable isotopes or stable isotope dilution theory. These so-called label-free, methods have the advantage of allowing data on each sample to be acquired independently from all other samples to which they can later be compared in silico for the purpose of measuring changes in protein expression between various biological states. We developed label free software based on direct measurement of peptide ion current area (PICA and compared it to two other methods, a simpler label free method known as spectral counting and the isotope coded affinity tag (ICAT method. Data analysis by these methods of a standard mixture containing proteins of known, but varying, concentrations showed that they performed similarly with a mean squared error of 0.09. Additionally, complex bacterial protein mixtures spiked with known concentrations of standard proteins were analyzed using the PICA label-free method. These results indicated that the PICA method detected all levels of standard spiked proteins at the 90% confidence level in this complex biological sample. This finding confirms that label-free methods, based on direct measurement of the area under a single ion current trace, performed as well as the standard ICAT method. Given the fact that the label-free methods provide ease in experimental design well beyond pair-wise comparison, label-free methods such as our PICA method are well suited for proteomic expression profiling of large numbers of samples as is needed in clinical analysis.

Development of a formulation lyophilized for the obtention of a antimicrobial peptide Ubiquicidine labelled with 99m Tc

International Nuclear Information System (INIS)

Palomares R, P.; Hernandez B, C.A.; Contreras N, G.; Garcia P, M.L.; Pantoja H, I.E.; Ferro F, G.

2004-01-01

The 99m Tc-UBI 29-41 are a labelled fragment of the antimicrobial human peptide Ubiquicidine proposed as a new radiopharmaceutical able to differentiate an infectious process of an inflammatory one through the gamma graphic image. It has been demonstrated that the 99m Tc-UBI 29-41 unite to bacteria in vitro and that accumulates in infection sites in human with minimum captivation in inflammation sites. In this work the development of a pharmaceutical lyophilized formulation is presented for the instantaneous marked one of the UBI 29-41 with 99m Tc. The selection of the components of the formulation settled down by means of the employment of an experimental design of 3 factors with mixed levels, evaluating the effect of the diluent type, concentration of tinny chloride and the reaction volume. The obtained formulations showed to be stable until for 6 months, being obtained complexes of the radiolabelled peptide with radiochemical purity > 95 % in sterile form and apirogen. The developed pharmaceutical form, will facilitate the routinary use of this new radiopharmaceutical in the diverse hospital departments of nuclear medicine. (Author)

Label-free peptide aptamer based impedimetric biosensor for highly sensitive detection of TNT with a ternary assembly layer.

Science.gov (United States)

Li, Yanyan; Zhao, Manru; Wang, Haiyan

2017-11-01

We report a label-free peptide aptamer based biosensor for highly sensitive detection of TNT which was designed with a ternary assembly layer consisting of anti-TNT peptide aptamer (peptamer), dithiothreitol (DTT), and 6-mercaptohexanol (MCH), forming Au/peptamer-DTT/MCH. A linear relationship between the change in electron transfer resistance and the logarithm of the TNT concentration from 0.44 to 18.92 pM, with a detection limit of 0.15 pM, was obtained. In comparison, the detection limit of the aptasensor with a common binary assembly layer (Au/peptamer/MCH) was 0.15 nM. The remarkable improvement in the detection limit could be ascribed to the crucial role of the ternary assembly layer, providing an OH-richer hydrophilic environment and a highly compact surface layer with minimal surface defects, reducing the non-covalent binding (physisorption) of the peptamer and non-specific adsorption of TNT onto the electrode surface, leading to high sensitivity, and which can serve as a general sensing platform for the fabrication of other biosensors.

A dual-color flow cytometry protocol for the simultaneous detection of Vibrio parahaemolyticus and Salmonella typhimurium using aptamer conjugated quantum dots as labels

Energy Technology Data Exchange (ETDEWEB)

Duan, Nuo; Wu, Shijia [State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi 214122 (China); Yu, Ye [Zhangjiagang Entry-Exit Inspection and Quarantine Bureau, Zhangjiangang 215600 (China); Ma, Xiaoyuan; Xia, Yu; Chen, Xiujuan; Huang, Yukun [State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi 214122 (China); Wang, Zhouping, E-mail: wangzp@jiangnan.edu.cn [State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi 214122 (China)

2013-12-04

Graphical abstract: -- Highlights: •Two bacteria were simultaneously detected using QD-apt as labels by flow cytometry. •QD-apt were used for recognition and fluorescence detection of two bacteria. •The method was applied successfully for bacteria detection in real samples. -- Abstract: A sensitive, specific method for the collection and detection of pathogenic bacteria was demonstrated using quantum dots (QDs) as a fluorescence marker coupled with aptamers as the molecular recognition element by flow cytometry. The aptamer sequences were selected using a bacterium-based SELEX strategy in our laboratory for Vibrio parahaemolyticus and Salmonella typhimurium that, when applied in this method, allows for the specific recognition of the bacteria from complex mixtures including shrimp samples. Aptamer-modified QDs (QD-apt) were employed to selectively capture and simultaneously detect the target bacteria with high sensitivity using the fluorescence of the labeled QDs. The signal intensity is amplified due to the high photostability of QDs nanoparticles, resulting in improved sensitivity over methods using individual dye-labeled probes. This proposed method is promising for the sensitive detection of other pathogenic bacteria in food stuff if suitable aptamers are chosen. The method may also provide another potential platform for the application of aptamer-conjugated QDs in flow cytometry.

A dual-color flow cytometry protocol for the simultaneous detection of Vibrio parahaemolyticus and Salmonella typhimurium using aptamer conjugated quantum dots as labels

International Nuclear Information System (INIS)

Duan, Nuo; Wu, Shijia; Yu, Ye; Ma, Xiaoyuan; Xia, Yu; Chen, Xiujuan; Huang, Yukun; Wang, Zhouping

2013-01-01

Graphical abstract: -- Highlights: •Two bacteria were simultaneously detected using QD-apt as labels by flow cytometry. •QD-apt were used for recognition and fluorescence detection of two bacteria. •The method was applied successfully for bacteria detection in real samples. -- Abstract: A sensitive, specific method for the collection and detection of pathogenic bacteria was demonstrated using quantum dots (QDs) as a fluorescence marker coupled with aptamers as the molecular recognition element by flow cytometry. The aptamer sequences were selected using a bacterium-based SELEX strategy in our laboratory for Vibrio parahaemolyticus and Salmonella typhimurium that, when applied in this method, allows for the specific recognition of the bacteria from complex mixtures including shrimp samples. Aptamer-modified QDs (QD-apt) were employed to selectively capture and simultaneously detect the target bacteria with high sensitivity using the fluorescence of the labeled QDs. The signal intensity is amplified due to the high photostability of QDs nanoparticles, resulting in improved sensitivity over methods using individual dye-labeled probes. This proposed method is promising for the sensitive detection of other pathogenic bacteria in food stuff if suitable aptamers are chosen. The method may also provide another potential platform for the application of aptamer-conjugated QDs in flow cytometry

Peptides and proteins

Energy Technology Data Exchange (ETDEWEB)

Bachovchin, W.W.; Unkefer, C.J.

1994-12-01

Advances in magnetic resonance and vibrational spectroscopy make it possible to derive detailed structural information about biomolecular structures in solution. These techniques are critically dependent on the availability of labeled compounds. For example, NMR techniques used today to derive peptide and protein structures require uniformity {sup 13}C-and {sup 15}N-labeled samples that are derived biosynthetically from (U-6-{sup 13}C) glucose. These experiments are possible now because, during the 1970s, the National Stable Isotope Resource developed algal methods for producing (U-6-{sup 13}C) glucose. If NMR techniques are to be used to study larger proteins, we will need sophisticated labelling patterns in amino acids that employ a combination of {sup 2}H, {sup 13}C, and {sup 15}N labeling. The availability of these specifically labeled amino acids requires a renewed investment in new methods for chemical synthesis of labeled amino acids. The development of new magnetic resonance or vibrational techniques to elucidate biomolecular structure will be seriously impeded if we do not see rapid progress in labeling technology. Investment in labeling chemistry is as important as investment in the development of advanced spectroscopic tools.

Experimental studies of colon carcinoma imaging with 99Tcm labeled neurotension peptide

International Nuclear Information System (INIS)

Zhang Kaijun; Zhang Yongxue; An Rui; Gao Zairong

2004-01-01

Objective: To prepare neurotension (NT) peptide labeled with 99 Tc m for the early diagnosis of colon carcinoma and to evaluate the advantages of the tracer. Methods: Biodistribution studies were performed at 3 and 12 h, respectively after injection of 99 Tc m -NT, and tissue distribution analysis after receptor-blocking was performed at 3 h in nude mice bearing colon carcinoma. Imaging with 99 Tc m -NT was performed at different time points in nude mice bearing colon carcinoma, and imaging after receptor-blocking was also performed at 3 h. The affinity of 99 Tc m -NT binding to the cell of colon carcinoma was studied in vitro. Results: The affinity constant of 99 Tc m -NT binding to the cells of colon carcinoma was obtained (Kd=0.91 nmol/L). The labeling yield of 99 Tc m -NT was more than 94% and the complex was stable in vitro and in vivo. Biodistribution analysis in nude mice bearing colon carcinoma showed that 99 Tc m -NT was excreted chiefly from the kidney, the ratios of tumor to muscle at 3 and 12 h were 3.25 ± 1.02 and 4.15 ± 1.46, respectively. In mice pretreated with unlabeled NT, the uptake of 99 Tc m -NT decreased in the tumor, the ratio of tumor to muscle at 3 h (1.21 ± 0.62) was significantly different from that of the mice without unlabeled NT treatment. Tumor lesion was detected with 99 Tc m -NT earlier (the lesion image showed up at 0.5 h postinjection), the ratio of tumor to contralateral limb at 3 h postinjection was 2.68 ± 0.44 obtained by technique of region of interest (ROI) . The ratio at 3 h was 1.14 ± 0.36 and that was significantly different from the ratio at 3 h postinjection in mice pretreated with unlabeled NT. Conclusion: The results of all studies in vitro and in vivo indicate that this labeling procedure of 99 Tc m -NT is simple and its specific binding to the cells of colon carcinoma is high, and it is a promising method for diagnosis of colon carcinoma

Synthesis, characterization and applications of carboxylated and polyethylene-glycolated bifunctionalized InP/ZnS quantum dots in cellular internalization mediated by cell-penetrating peptides.

Science.gov (United States)

Liu, Betty R; Winiarz, Jeffrey G; Moon, Jong-Sik; Lo, Shih-Yen; Huang, Yue-Wern; Aronstam, Robert S; Lee, Han-Jung

2013-11-01

Semiconductor nanoparticles, also known as quantum dots (QDs), are widely used in biomedical imaging studies and pharmaceutical research. Cell-penetrating peptides (CPPs) are a group of small peptides that are able to traverse cell membrane and deliver a variety of cargoes into living cells. CPPs deliver QDs into cells with minimal nonspecific absorption and toxic effect. In this study, water-soluble, monodisperse, carboxyl-functionalized indium phosphide (InP)/zinc sulfide (ZnS) QDs coated with polyethylene glycol lipids (designated QInP) were synthesized for the first time. The physicochemical properties (optical absorption, fluorescence and charging state) and cellular internalization of QInP and CPP/QInP complexes were characterized. CPPs noncovalently interact with QInP in vitro to form stable CPP/QInP complexes, which can then efficiently deliver QInP into human A549 cells. The introduction of 500nM of CPP/QInP complexes and QInP at concentrations of less than 1μM did not reduce cell viability. These results indicate that carboxylated and polyethylene-glycolylated (PEGylated) bifunctionalized QInP are biocompatible nanoparticles with potential for use in biomedical imaging studies and drug delivery applications. Copyright © 2013 Elsevier B.V. All rights reserved.

Optical properties and the use of CdSe quantum dot for biolabeling applications

International Nuclear Information System (INIS)

Tran Hong Nhung; Nguyen Thi Van; Vu Xuan Hoa; Pham Minh Tan; Tong Kim Thuan; Tran Thi Thu Thuy; Jean Claude Brochon; Patrick Tauc

2008-01-01

The quantum dots CdSe type Qtracker 565 and 605 of Quantum Dot Company have been investigated by size, chemical structure and optical properties. The Qtracker 605 QDs were introduced into Lipomyces Starkeyi yeast cells. It was found that for the young cells (36 h of culture), the labeling QDs are mainly located in vacuoles, and the emission remains narrow with the maximum is clearly around 605 nm. For age cells (96 h of culture), the labeling QDs are concentrated in the cell cytoplasm, the emission is broaden with the maximum shifted to 580 nm. The live cell image was still observed after two months of introduction. The Qtracker 605 QDs were also successfully introduced into mouse blood cancerous cells. (author)

Rapid purification of radioiodinated peptides with Sep-Pak reversed phase cartridges and HPLC

International Nuclear Information System (INIS)

Miller, J.J.; Schultz, G.S.; Levy, R.S.

1984-01-01

A simple, rapid method is described for the purification of radioiodinated peptides for use in radioimmuno- and in radioreceptor assays. Iodinated reaction mixtures are applied directly onto Sep-Pak disposable, reversed phase cartridges equilibrated with phosphate buffer. Unreacted 125-iodide and other non-peptide reaction components are eluted with buffer. The peptide fraction is then eluted with 70% buffer:30% acetonitrile. The peptide fraction is further purified by reversed phase high pressure liquid chromatography to separate the native peptide and the mono- and diiodo-derivatives. In this study the method is used to prepare 125-iodide-labeled monoiodo-leucine enkephalin and monoiodo-angiotensin II, which are free of the parent peptides and diiodo-derivatives and are of maximum obtainable specific radioactivity. The usefulness of these labeled peptides in radioimmuno- and radioreceptor assays is demonstrated by their binding to specific antibodies and receptors, respectively. (author)

Interaction between a "processed" ovalbumin peptide and Ia molecules

DEFF Research Database (Denmark)

Buus, S; Colon, S; Smith, C

1986-01-01

The binding of 125I-labeled immunogenic peptides to purified Ia molecules in detergent solution was examined by equilibrium dialysis. We used the chicken ovalbumin peptide ovalbumin-(323-339)-Tyr, which is immunogenic in the BALB/c mouse and restricted to I-Ad. 125I-labeled ovalbumin-(323-339)-Tyr......-Ak but not to I-Ek, I-Ad, or I-Ed. Thus, a specific interaction between Ia and antigen that correlates with the major histocompatibility complex restriction was demonstrated, strongly arguing in favor of a determinant selection hypothesis for such restriction....

EBprot: Statistical analysis of labeling-based quantitative proteomics data.

Science.gov (United States)

Koh, Hiromi W L; Swa, Hannah L F; Fermin, Damian; Ler, Siok Ghee; Gunaratne, Jayantha; Choi, Hyungwon

2015-08-01

Labeling-based proteomics is a powerful method for detection of differentially expressed proteins (DEPs). The current data analysis platform typically relies on protein-level ratios, which is obtained by summarizing peptide-level ratios for each protein. In shotgun proteomics, however, some proteins are quantified with more peptides than others, and this reproducibility information is not incorporated into the differential expression (DE) analysis. Here, we propose a novel probabilistic framework EBprot that directly models the peptide-protein hierarchy and rewards the proteins with reproducible evidence of DE over multiple peptides. To evaluate its performance with known DE states, we conducted a simulation study to show that the peptide-level analysis of EBprot provides better receiver-operating characteristic and more accurate estimation of the false discovery rates than the methods based on protein-level ratios. We also demonstrate superior classification performance of peptide-level EBprot analysis in a spike-in dataset. To illustrate the wide applicability of EBprot in different experimental designs, we applied EBprot to a dataset for lung cancer subtype analysis with biological replicates and another dataset for time course phosphoproteome analysis of EGF-stimulated HeLa cells with multiplexed labeling. Through these examples, we show that the peptide-level analysis of EBprot is a robust alternative to the existing statistical methods for the DE analysis of labeling-based quantitative datasets. The software suite is freely available on the Sourceforge website http://ebprot.sourceforge.net/. All MS data have been deposited in the ProteomeXchange with identifier PXD001426 (http://proteomecentral.proteomexchange.org/dataset/PXD001426/). © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Quantum dots as mineral- and matrix-specific strain gages for bone biomechanical studies

Science.gov (United States)

Zhu, Peizhi; Xu, Jiadi; Morris, Michael; Ramamoorthy, Ayyalusamy; Sahar, Nadder; Kohn, David

2009-02-01

We report the use of quantum dots (Qdots) as strain gages in the study of bone biomechanics using solid state nuclear magnetic resonance (NMR) spectroscopy. We have developed solid state NMR sample cells for investigation of deformations of bone tissue components at loads up to several Mega Pascal. The size constraints of the NMR instrumentation limit the bone specimen diameter and length to be no greater than 2-3 mm and 30 mm respectively. Further, magic angle spinning (MAS) solid state NMR experiments require the use of non-metallic apparatus that can be rotated at kilohertz rates. These experimental constraints preclude the use of standard biomechanical measurement systems. In this paper we explore the use of quantum dot center of gravity measurement as a strain gage technology consistent with the constraints of solid state NMR. We use Qdots that bind calcium (625 nm emission) and collagen (705 nm emission) for measurement of strain in these components. Compressive loads are applied to a specimen in a cell through a fine pitch screw turned with a mini-torque wrench. Displacement is measured as changes in the positions of arrays of quantum dots on the surface of a specimen. Arrays are created by spotting the specimen with dilute suspensions of Qdots. Mineral labeling is achieved with 705 nm carboxylated dots and matrix labeling with 565 nm quantum dots conjugated to collagen I antibodies. After each load increment the new positions of the quantum dots are measured by fluorescence microscopy. Changes in Qdot center of gravity as a function of applied load can be measured with submicron accuracy.

Inhibition of 125I-labeled ristocetin binding to Micrococcus luteus cells by the peptides related to bacterial cell wall mucopeptide precursors: quantitative structure-activity relationships

International Nuclear Information System (INIS)

Kim, K.H.; Martin, Y.; Otis, E.; Mao, J.

1989-01-01

Quantitative structure-activity relationships (QSAR) of N-Ac amino acids, N-Ac dipeptides, and N-Ac tripeptides in inhibition of 125 I-labeled ristocetin binding to Micrococcus luteus cell wall have been developed to probe the details of the binding between ristocetin and N-acetylated peptides. The correlation equations indicate that (1) the binding is stronger for peptides in which the side chain of the C-terminal amino acid has a large molar refractivity (MR) value, (2) the binding is weaker for peptides with polar than for those with nonpolar C-terminal side chains, (3) the N-terminal amino acid in N-Ac dipeptides contributes 12 times that of the C-terminal amino acid to binding affinity, and (4) the interactions between ristocetin and the N-terminal amino acid of N-acetyl tripeptides appear to be much weaker than those with the first two amino acids

National Bureau of Standards health physics radioactive material shipment survey, packaging, and labelling program under ICAO/IATA and DOT regulations

International Nuclear Information System (INIS)

Sharp, D.R.; Slaback, L.A.

1984-01-01

NBS routinely ships many radionuclides in small to moderate activities, with many shipments containing mixtures of radionuclides in a variety of combinations. The ICAO/IATA shipping regulations (and the new DoT regulations on their model) specify individual shipping parameters for every radionuclide. As a result, quality control in the shipment of these radioactive packages has become difficult to maintain. The authors have developed a computer program that will guide a Health Physics technician through package surveys and give exact packaging and labelling instructions. The program is a 27 kilobyte user-friendly BASIC program that runs on an Epson-HX20 notebook computer with microcassette drive and 16 kilobyte memory expansion unit. This small computer is more manageable than the regulation books for which it will be substituted and will be used in routine radioactive shipments

DNA nanosensor based on biocompatible graphene quantum dots and carbon nanotubes.

Science.gov (United States)

Qian, Zhao Sheng; Shan, Xiao Yue; Chai, Lu Jing; Ma, Juan Juan; Chen, Jian Rong; Feng, Hui

2014-10-15

An ultrasensitive nanosensor based on fluorescence resonance energy transfer (FRET) between biocompatible graphene quantum dots and carbon nanotubes for DNA detection was reported. We take advantage of good biocompatibility and strong fluorescence of graphene quantum dots, base pairing specificity of DNA and unique fluorescence resonance energy transfer between graphene quantum dots and carbon nanotubes to achieve the analysis of low concentrations of DNA. Graphene quantum dots with high quantum yield up to 0.20 were prepared and served as the fluorophore of DNA probe. FRET process between graphene quantum dots-labeled probe and oxidized carbon nanotubes is easily achieved due to their efficient self-assembly through specific π-π interaction. This nanosensor can distinguish complementary and mismatched nucleic acid sequences with high sensitivity and good reproducibility. The detection method based on this nanosensor possesses a broad linear span of up to 133.0 nM and ultralow detection limit of 0.4 nM. The constructed nanosensor is expected to be highly biocompatible because of all its components with excellent biocompatibility. Copyright © 2014 Elsevier B.V. All rights reserved.

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.

Imaging of urokinase-type plasminogen activator receptor expression using a 64Cu-labeled linear peptide antagonist by microPET

DEFF Research Database (Denmark)

Li, Zi-Bo; Niu, Gang; Wang, Hui

2008-01-01

for positron emission tomography (PET) imaging. A linear, high-affinity uPAR-binding peptide antagonist AE105 was conjugated with 1,4,7,10-tetraazadodecane-N,N',N'',N'''-tetraacetic acid (DOTA) and labeled with (64)Cu for microPET imaging of mice bearing U87MG human glioblastoma (uPAR positive) and MDA-MB-435...... human breast cancer (uPAR negative). RESULTS: Surface plasmon resonance measurements show that AE105 with DOTA conjugated at the alpha-amino group (DOTA-AE105) has high affinity toward uPAR. microPET imaging reveals a rapid and high accumulation of (64)Cu-DOTA-AE105 in uPAR-positive U87MG tumors (10.......8 +/- 1.5%ID/g at 4.5 hours, n = 3) but not in uPAR-negative MDA-MB-435 tumors (1.2 +/- 0.6%ID/g at 4.5 hours, n = 3). Specificity of this peptide-based imaging of uPAR was validated by further control experiments. First, a nonbinding variant of AE105 carrying a single amino acid replacement (Trp...

Introduction of an 8-aminooctanoic acid linker enhances uptake of 99mTc-labeled lactam bridge-cyclized α-MSH peptide in melanoma.

Science.gov (United States)

Guo, Haixun; Miao, Yubin

2014-12-01

The purpose of this study was to examine the effects of amino acid, hydrocarbon, and polyethylene glycol (PEG) linkers on the melanoma targeting and imaging properties of (99m)Tc-labeled lactam bridge-cyclized HYNIC-linker-Nle-CycMSHhex (hydrazinonicotinamide-linker-Nle-c[Asp-His-DPhe-Arg-Trp-Lys]-CONH2) peptides. Four novel peptides (HYNIC-GGGNle-CycMSHhex, HYNIC-GSGNle-CycMSHhex, HYNIC-PEG2Nle-CycMSHhex, and HYNIC-AocNle-CycMSHhex) were designed and synthesized. The melanocortin-1 receptor binding affinities of the peptides were determined in B16/F1 melanoma cells. The biodistribution of (99m)Tc(ethylenediaminediacetic acid [EDDA])-HYNIC-GGGNle-CycMSHhex, (99m)Tc(EDDA)-HYNIC-GSGNle-CycMSHhex, (99m)Tc(EDDA)-HYNIC-PEG2Nle-CycMSHhex, and (99m)Tc(EDDA)-HYNIC-AocNle-CycMSHhex were determined in B16/F1 melanoma-bearing C57 mice at 2 h after injection to select a lead peptide for further evaluation. The melanoma targeting and imaging properties of (99m)Tc(EDDA)-HYNIC-AocNle-CycMSHhex were further examined because of its high melanoma uptake. The inhibitory concentrations of 50% (IC50) for HYNIC-GGGNle-CycMSHhex, HYNIC-GSGNle-CycMSHhex, HYNIC-PEG2Nle-CycMSHhex, and HYNIC-AocNle-CycMSHhex were 0.7 ± 0.1, 0.8 ± 0.09, 0.4 ± 0.08, and 0.3 ± 0.06 nM, respectively, in B16/F1 melanoma cells. Among these four (99m)Tc-labeled peptides, (99m)Tc(EDDA)-HYNIC-AocNle-CycMSHhex displayed the highest melanoma uptake (22.3 ± 1.72 percentage injected dose/g) at 2 h after injection. (99m)Tc(EDDA)-HYNIC-AocNle-CycMSHhex exhibited high tumor-to-normal-organ uptake ratios except for the kidneys. The tumor-to-kidney uptake ratios of (99m)Tc(EDDA)-HYNIC-AocNle-CycMSHhex were 3.29, 3.63, and 6.78 at 2, 4, and 24 h, respectively, after injection. The melanoma lesions were clearly visualized by SPECT/CT using (99m)Tc(EDDA)-HYNIC-AocNle-CycMSHhex as an imaging probe at 2 h after injection. High melanoma uptake and fast urinary clearance of (99m)Tc(EDDA)-HYNIC-AocNle-CycMSHhex highlighted its

Introduction of an 8-Aminooctanoic Acid Linker Enhances the melanoma uptake of Tc-99m-labeled Lactam Bridge-Cyclized Alpha-MSH Peptide

Science.gov (United States)

Guo, Haixun; Miao, Yubin

2015-01-01

The purpose of this study was to examine the effects of amino acid, hydrocarbon and polyethylene glycol (PEG) linkers on melanoma targeting and imaging properties of 99mTc-labeled lactam bridge-cyclized HYNIC-linker-Nle-CycMSHhex {hydrazinonicotinamide-linker-Nle-c[Asp-His-DPhe-Arg-Trp-Lys]-CONH2} peptides. Methods four novel peptides {HYNIC-GGGNle-CycMSHhex, HYNIC-GSGNle-CycMSHhex, HYNIC-PEG2Nle-CycMSHhex and HYNIC-AocNle-CycMSHhex} were designed and synthesized. The melanocortin-1 (MC1) receptor binding affinities of the peptides were determined in B16/F1 melanoma cells. The biodistribution of 99mTc(EDDA)-HYNIC-GGGNle-CycMSHhex, 99mTc(EDDA)-HYNIC-GSGNle-CycMSHhex, 99mTc(EDDA)-HYNIC-PEG2Nle-CycMSHhex and 99mTc(EDDA)-HYNIC-AocNle-CycMSHhex were determined in B16/F1 melanoma-bearing C57 mice at 2 h post-injection to select a lead peptide for further evaluation. The melanoma targeting and imaging properties of 99mTc(EDDA)-HYNIC-AocNle-CycMSHhex were further examined because of its high melanoma uptake. Results The IC50 values of HYNIC-GGGNle-CycMSHhex, HYNIC-GSGNle-CycMSHhex, HYNIC-PEG2Nle-CycMSHhex, and HYNIC-AocNle-CycMSHhex were 0.7 ± 0.1, 0.8 ± 0.09, 0.4 ± 0.08, and 0.3 ± 0.06 nM in B16/F1 melanoma cells, respectively. Among these four 99mTc-labeled peptides, 99mTc(EDDA)-HYNIC-AocNle-CycMSHhex displayed the highest melanoma uptake (22.3 ± 1.72% ID/g) at 2 h post-injection. 99mTc(EDDA)-HYNIC-AocNle-CycMSHhex exhibited high tumor to normal organ uptake ratios except for the kidneys. The tumor/kidney uptake ratios of 99mTc(EDDA)-HYNIC-AocNle-CycMSHhex were 3.29, 3.63 and 6.78 at 2, 4 and 24 h post-injection. The melanoma lesions were clearly visualized by single photon emission computed tomography (SPECT)/CT using 99mTc(EDDA)-HYNIC-AocNle-CycMSHhex as an imaging probe at 2 h post-injection. Conclusion High melanoma uptake and fast urinary clearance of 99mTc(EDDA)-HYNIC-AocNle-CycMSHhex highlighted its potential for metastatic melanoma detection in the future

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.

Somatostatin analogues labelled with 99mTc

International Nuclear Information System (INIS)

Obenaus, E.; Crudo, J.; Edreira, M.; Viaggi, M.; De Castiglia, S.G.

2001-01-01

The aim of the present work was to study the biological and radiochemical behaviour of two somatostatin analogues, the RC-160 and Tyr3Octreotide(TOC) peptides when labelling with 99m Tc by two methods: direct and indirect using S-benzoyl- mercaptoacetyl triglycine (MAG-3) and hydrazinonicotinamide (HYNIC) as chelating agents. RC-160 was labelled with 125I (30% labelling yield) in order to examine its receptor specificity and to study the biodistribution in normal animals. A total binding of 30% and a non specific binding lower than 10% was obtained. On the other hand, the RC-160 was labelled with 99m Tc by a direct method (70% labelling yield), using sodium ascorbate and dithionite in order to reduce the peptide and 99m Tc, respectively. The synthesis of RC-160 with S-benzoyl MAG-3 and TOC with HYNIC, for labelling with 99m Tc are also described. The conjugates were prepared on a small scale and labelled with the radionuclide using tricine as co-ligands for HYNIC conjugates. Chromatographic studies were performed using HPLC system and radiochemical purities higher than 75% and 95% were obtained respectively. Biodistributions studies in normal Wistar rats were performed and results were correlated with chromatographic and protein binding properties. Lower lipophilicity of the labelled conjugates resulted in a higher renal excretion. HYNIC-TOC complex showed promising results when labelling with 99m Tc using tricine as co-ligand although higher stability should be found for ternary co-ligands compared to tricine. (author)

Chimeric opioid peptides: Tools for identifying opioid receptor types

International Nuclear Information System (INIS)

Xie, G.; Miyajima, A.; Yokota, T.; Arai, K.; Goldstein, A.

1990-01-01

The authors synthesized several chimeric [125J-labelled] peptides in which the N-terminal nine residues of dynorphin-32, a peptide selective for the κ opioid receptor, were replaced by opioid peptides selective for other opioid receptor types. Each chimeric peptide retained the high affinity and type selectivity characteristic of its N-terminal sequence. The common C-terminal two-thirds of the chimeric peptides served as an epitope recognized by the same monoclonal antibody. When bound to receptors on a cell surface or membrane preparation, these peptides could still bind specifically to the monoclonal antibody. These chimeric peptides should be useful for isolating μ, δ, and κ opioid receptors and for identifying opioid receptors on transfected cells in expression cloning procedures. The general approach using chimeric peptides should be applicable to other peptide receptors

The effect of macrocyclic chelators on the targeting properties of the 68Ga-labeled gastrin releasing peptide receptor antagonist PEG2-RM26

International Nuclear Information System (INIS)

Varasteh, Zohreh; Mitran, Bogdan; Rosenström, Ulrika; Velikyan, Irina; Rosestedt, Maria; Lindeberg, Gunnar; Sörensen, Jens; Larhed, Mats; Tolmachev, Vladimir; Orlova, Anna

2015-01-01

Introduction: Overexpression of gastrin-releasing peptide receptors (GRPR) has been reported in several cancers. Bombesin (BN) analogs are short peptides with a high affinity for GRPR. Different BN analogs were evaluated for radionuclide imaging and therapy of GRPR-expressing tumors. We have previously investigated an antagonistic analog of BN (D-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH 2 , RM26) conjugated to NOTA via a PEG 2 spacer (NOTA-PEG 2 -RM26) labeled with 68 Ga, 111 In and Al 18 F. 68 Ga-labeled NOTA-PEG 2 -RM26 showed high tumor-to-organ ratios. Methods: The influence of different macrocyclic chelators (NOTA, NODAGA, DOTA and DOTAGA) on the targeting properties of 68 Ga-labeled PEG 2 -RM26 was studied in vitro and in vivo. Results: All conjugates were labeled with generator-produced 68 Ga with high yields and demonstrated high stability and specific binding to GRPR. The IC 50 values of nat Ga-X-PEG 2 -RM26 (X = NOTA, DOTA, NODAGA, DOTAGA) were 2.3 ± 0.2, 3.0 ± 0.3, 2.9 ± 0.3 and 10.0 ± 0.6 nM, respectively. The internalization of the conjugates by PC-3 cells was low. However, the DOTA-conjugated analog demonstrated a higher internalization rate compared to other analogs. GRPR-specific uptake was found in receptor-positive normal tissues and PC-3 xenografts for all conjugates. The biodistribution of the conjugates was influenced by the choice of the chelator moiety. Although all radiotracers cleared rapidly from the blood, [ 68 Ga]Ga-NOTA-PEG 2 -RM26 showed significantly lower uptake in lung, muscle and bone compared to the other analogs. The uptake in tumors (5.40 ± 1.04 %ID/g at 2 h p.i.) and the tumor-to-organ ratios (25 ± 3, 157 ± 23 and 39 ± 4 for blood, muscle and bone, respectively) were significantly higher for the NOTA-conjugate than the other analogs. Conclusions: Chelators had a clear influence on the biodistribution and targeting properties of 68 Ga-labeled antagonistic BN analogs. Positively charged [ 68 Ga]Ga-NOTA-PEG 2 -RM26 provided

Quantum Dot-Based Luminescent Oxygen Channeling Assay for Potential Application in Homogeneous Bioassays.

Science.gov (United States)

Zhuang, Si-Hui; Guo, Xin-Xin; Wu, Ying-Song; Chen, Zhen-Hua; Chen, Yao; Ren, Zhi-Qi; Liu, Tian-Cai

2016-01-01

The unique photoproperties of quantum dots are promising for potential application in bioassays. In the present study, quantum dots were applied to a luminescent oxygen channeling assay. The reaction system developed in this study was based on interaction of biotin with streptavidin. Carboxyl-modified polystyrene microspheres doped with quantum dots were biotinylated and used as acceptors. Photosensitizer-doped carboxyl-modified polystyrene microspheres were conjugated with streptavidin and used as donors. The results indicated that the singlet oxygen that was released from the donor beads diffused into the acceptor beads. The acceptor beads were then exited via thioxene, and were subsequently fluoresced. To avoid generating false positives, a high concentration (0.01 mg/mL) of quantum dots is required for application in homogeneous immunoassays. Compared to a conventional luminescent oxygen channeling assay, this quantum dots-based technique requires less time, and would be easier to automate and miniaturize because it requires no washing to remove excess labels.

Peptide ligands for targeting the extracellular domain of EGFR: Comparison between linear and cyclic peptides.

Science.gov (United States)

Williams, Tyrslai M; Sable, Rushikesh; Singh, Sitanshu; Vicente, Maria Graca H; Jois, Seetharama D

2018-02-01

Colorectal cancer (CRC) is the third most common solid internal malignancy among cancers. Early detection of cancer is key to increasing the survival rate of colorectal cancer patients. Overexpression of the EGFR protein is associated with CRC. We have designed a series of peptides that are highly specific for the extracellular domain of EGFR, based on our earlier studies on linear peptides. The previously reported linear peptide LARLLT, known to bind to EGFR, was modified with the goals of increasing its stability and its specificity toward EGFR. Peptide modifications, including D-amino acid substitution, cyclization, and chain reversal, were investigated. In addition, to facilitate labeling of the peptide with a fluorescent dye, an additional lysine residue was introduced onto the linear (KLARLLT) and cyclic peptides cyclo(KLARLLT) (Cyclo.L1). The lysine residue was also converted into an azide group in both a linear and reversed cyclic peptide sequences cyclo(K(N3)larllt) (Cyclo.L1.1) to allow for subsequent "click" conjugation. The cyclic peptides showed enhanced binding to EGFR by SPR. NMR and molecular modeling studies suggest that the peptides acquire a β-turn structure in solution. In vitro stability studies in human serum show that the cyclic peptide is more stable than the linear peptide. © 2017 John Wiley & Sons A/S.

Biological evaluation of 177Lu-labeled DOTA-Ala(SO3H)-Aminooctanoyl-Gln-Trp-Ala-Val-N methyl Gly-His-Statine-Leu-NH2 for gastrin-releasing peptide receptor-positive prostate tumor targeting

International Nuclear Information System (INIS)

Lim, Jae Cheong; Cho, Eun Ha; Kim, Jin Joo; Choi, Sang Mu; Lee, So young; Nam, Sung Soo; Park, Ul Jae; Park, Soo Hyun

2015-01-01

Bombesin binds with selectivity and high affinity to a Gastrin-releasing peptide receptor (GRPR), which is highly overexpressed in prostate cancer cells. The present study describes the in vitro and in vivo biological characteristics of DOTA-Ala(SO 3 H)-Aminooctanoyl-Gln-Trp-Ala-Val-N methyl Gly-His-Statine-Leu-NH 2 (DOTA-sBBNA), an antagonist analogue of bombesin peptide for the targeting of GRPR. DOTA-sBBNA was synthesized and labeled with 177 Lu as previously published. A saturation assay on PC-3 human prostate cancer cells revealed that the Kd value of the radiolabeled peptide was 1.88 nM with a maximum binding capacity (Bmax) of 289.3 fmol/10 6 cells. The radio-peptide slowly internalized, and 24.4 ± 0.5% of the total binding was internalized in 4 hr. Biodistribution studies were conducted in healthy and PC-3 xenografted balb/c mice, which showed high uptake and retention of tumor-associated radioactivity in PC-3 xenografted mice. The tumor-to-blood ratio was 126.02 ± 9.36 at 1.5 hr p.i., and was increased to 216.33 ± 61.58 at 24 hr p.i., which means that the radiolabeled peptide was highly accumulated in a tumor and rapidly cleared from the blood pool. The GRPR is also over-expressed in Korean prostate cancer patients. These results suggest that this 177 Lu-labeled peptide has promising characteristics for application in nuclear medicine, namely for the diagnosis and treatment of GRPR over-expressing prostate tumors

Peptide receptor radionuclide therapy with 90Y/177Lu-labelled peptides for inoperable head and neck paragangliomas (glomus tumours)

International Nuclear Information System (INIS)

Puranik, Ameya D.; Kulkarni, Harshad R.; Singh, Aviral; Baum, Richard P.

2015-01-01

Head and neck paragangliomas (HNPGLs) are rare tumours arising from autonomic nervous system ganglia. Although surgery offers the best chance of complete cure, there is associated morbidity due to the crucial location of these tumours. Radiotherapy arrests tumour growth and provides symptomatic improvement, but has long-term consequences. These tumours express somatostatin receptors (SSTR) and hence peptide receptor radionuclide therapy (PRRT) is now a treatment option. We assessed the molecular, morphological and clinical responses of inoperable HNPGLs to PRRT. Nine patients with inoperable HNPGL assessed between June 2006 and June 2014 were included. Four patients had a solitary lesion, four had multifocal involvement and one had distant metastases (bone and lungs). The patients were treated with PRRT using 90 Y/ 177 Lu-labelled peptides after positive confirmation of SSTR expression on 68 Ga-DOTATOC PET/CT. All patients received two to four courses of PRRT. Subsequent serial imaging with 68 Ga-DOTATOC PET/CT was carried out every 6 months to assess response to treatment. Clinical (symptomatic) response was also assessed. Based on molecular response (EORTC) criteria, four of the nine patients showed a partial molecular response to treatment seen as significant decreases in SUV max , accompanied by a reduction in tumour size. Five patients showed stable disease on both molecular and morphological criteria. Six out of nine patients were symptomatic at presentation with manifestations of cranial nerve involvement, bone destruction at the primary site and metastatic bone pain. Molecular responses were correlated with symptomatic improvement in four out of these six patients; while two patients showed small reductions in tumour size and SUV max . The three asymptomatic patients showed no new lesions or symptomatic worsening. PRRT was effective in all patients, with no disease worsening seen, either in the form of neurological symptoms or distant spread. Though these

Correlative Light- and Electron Microscopy Using Quantum Dot Nanoparticles.

Science.gov (United States)

Killingsworth, Murray C; Bobryshev, Yuri V

2016-08-07

A method is described whereby quantum dot (QD) nanoparticles can be used for correlative immunocytochemical studies of human pathology tissue using widefield fluorescence light microscopy and transmission electron microscopy (TEM). To demonstrate the protocol we have immunolabeled ultrathin epoxy sections of human somatostatinoma tumor using a primary antibody to somatostatin, followed by a biotinylated secondary antibody and visualization with streptavidin conjugated 585 nm cadmium-selenium (CdSe) quantum dots (QDs). The sections are mounted on a TEM specimen grid then placed on a glass slide for observation by widefield fluorescence light microscopy. Light microscopy reveals 585 nm QD labeling as bright orange fluorescence forming a granular pattern within the tumor cell cytoplasm. At low to mid-range magnification by light microscopy the labeling pattern can be easily recognized and the level of non-specific or background labeling assessed. This is a critical step for subsequent interpretation of the immunolabeling pattern by TEM and evaluation of the morphological context. The same section is then blotted dry and viewed by TEM. QD probes are seen to be attached to amorphous material contained in individual secretory granules. Images are acquired from the same region of interest (ROI) seen by light microscopy for correlative analysis. Corresponding images from each modality may then be blended to overlay fluorescence data on TEM ultrastructure of the corresponding region.

Synthesis of Tc-99m labeled 1,2,3-triazole-4-yl c-met binding peptide as a potential c-met receptor kinase positive tumor imaging agent.

Science.gov (United States)

Kim, Eun-Mi; Joung, Min-Hee; Lee, Chang-Moon; Jeong, Hwan-Jeong; Lim, Seok Tae; Sohn, Myung-Hee; Kim, Dong Wook

2010-07-15

The mesenchymal-epithelial transition factor (c-Met), which is related to tumor cell growth, angiogenesis and metastases, is known to be overexpressed in several tumor types. In this study, we synthesized technetium-99m labeled 1,2,3-triazole-4-yl c-Met binding peptide (cMBP) derivatives, prepared by solid phase peptide synthesis and the 'click-to-chelate' protocol for the introduction of tricarbonyl technetium-99m, as a potential c-Met receptor kinase positive tumor imaging agent, and evaluated their in vitro c-Met binding affinity, cellular uptake, and stability. The (99m)Tc labeled cMBP derivatives ([(99m)Tc(CO)(3)]12, [(99m)Tc(CO)(3)]13, and [(99m)Tc(CO)(3)]14) were prepared in 85-90% radiochemical yields. The cold surrogate cMBP derivatives, [Re(CO)(3)]12, [Re(CO)(3)]13, and [Re(CO)(3)]14, were shown to have high binding affinities (0.13 microM, 0.06 microM, and 0.16 microM, respectively) to a purified cMet/Fc chimeric recombinant protein. In addition, the in vitro cellular uptake and inhibition studies demonstrated the high specific binding of these (99m)Tc labeled cMBP derivatives ([(99m)Tc(CO)(3)]12-14) to c-Met receptor positive U87MG cells. 2010 Elsevier Ltd. All rights reserved.

The interplay of T1- and T2-relaxation on T1-weighted MRI of hMSCs induced by Gd-DOTA-peptides.

Science.gov (United States)

Cao, Limin; Li, Binbin; Yi, Peiwei; Zhang, Hailu; Dai, Jianwu; Tan, Bo; Deng, Zongwu

2014-04-01

Three Gd-DOTA-peptide complexes with different peptide sequence are synthesized and used as T1 contrast agent to label human mesenchymal stem cells (hMSCs) for magnetic resonance imaging study. The peptides include a universal cell penetrating peptide TAT, a linear MSC-specific peptide EM7, and a cyclic MSC-specific peptide CC9. A significant difference in labeling efficacy is observed between the Gd-DOTA-peptides as well as a control Dotarem. All Gd-DOTA-peptides as well as Dotarem induce significant increase in T1 relaxation rate which is in favor of T1-weighted MR imaging. Gd-DOTA-CC9 yields the maximum labeling efficacy but poor T1 contrast enhancement. Gd-DOTA-EM7 yields the minimum labeling efficacy but better T1 contrast enhancement. Gd-DOTA-TAT yields a similar labeling efficacy as Gd-DOTA-CC9 and similar T1 contrast enhancement as Gd-DOTA-EM7. The underlying mechanism that governs T1 contrast enhancement effect is discussed. Our results suggest that T1 contrast enhancement induced by Gd-DOTA-peptides depends not only on the introduced cellular Gd content, but more importantly on the effect that Gd-DOTA-peptides exert on the T1-relaxation and T2-relaxation processes/rates. Both T1 and particularly T2 relaxation rate have to be taken into account to interpret T1 contrast enhancement. In addition, the interpretation has to be based on cellular instead of aqueous longitudinal and transverse relaxivities of Gd-DOTA-peptides. Copyright © 2014 Elsevier Ltd. All rights reserved.

Visualization of plasma membrane compartmentalization by high-speed quantum dot tracking

DEFF Research Database (Denmark)

Clausen, M. P.; Lagerholm, B. C.

2013-01-01

In this study, we have imaged plasma membrane molecules labeled with quantum dots in live cells using a conventional wide-field microscope with high spatial precision at sampling frequencies of 1.75 kHz. Many of the resulting single molecule trajectories are sufficiently long (up to several...

Dynamic PET and SPECT imaging with radioiodinated, amyloid-reactive peptide p5 in mice: a positive role for peptide dehalogenation.

Science.gov (United States)

Martin, Emily B; Kennel, Stephen J; Richey, Tina; Wooliver, Craig; Osborne, Dustin; Williams, Angela; Stuckey, Alan; Wall, Jonathan S

2014-10-01

Dynamic molecular imaging provides bio-kinetic data that is used to characterize novel radiolabeled tracers for the detection of disease. Amyloidosis is a rare protein misfolding disease that can affect many organs. It is characterized by extracellular deposits composed principally of fibrillar proteins and hypersulfated proteoglycans. We have previously described a peptide, p5, which binds preferentially to amyloid deposits in a murine model of reactive (AA) amyloidosis. We have determined the whole body distribution of amyloid by molecular imaging techniques using radioiodinated p5. The loss of radioiodide from imaging probes due to enzymatic reaction has plagued the use of radioiodinated peptides and antibodies. Therefore, we studied iodine-124-labeled p5 by using dynamic PET imaging of both amyloid-laden and healthy mice to assess the rates of amyloid binding, the relevance of dehalogenation and the fate of the radiolabeled peptide. Rates of blood pool clearance, tissue accumulation and dehalogenation of the peptide were estimated from the images. Comparisons of these properties between the amyloid-laden and healthy mice provided kinetic profiles whose differences may prove to be indicative of the disease state. Additionally, we performed longitudinal SPECT/CT imaging with iodine-125-labeled p5 up to 72h post injection to determine the stability of the radioiodinated peptide when bound to the extracellular amyloid. Our data show that amyloid-associated peptide, in contrast to the unbound peptide, is resistant to dehalogenation resulting in enhanced amyloid-specific imaging. These data further support the utility of this peptide for detecting amyloidosis and monitoring potential therapeutic strategies in patients. Copyright © 2014 Elsevier Inc. All rights reserved.

Label-free fluorimetric detection of CEA using carbon dots derived from tomato juice.

Science.gov (United States)

Miao, Hong; Wang, Lan; Zhuo, Yan; Zhou, Zinan; Yang, Xiaoming

2016-12-15

A facile-green strategy to synthesize carbon dots (CDs) with a quantum yield (QY) of nearly 13.9% has been built up, while tomato juice served as the carbon source. Interestingly, not only the precursor of CDs and the whole synthesis procedure were environmental-friendly, but this type of CDs also exhibited multiple advantages including high fluorescent QY, excellent photostability, non-toxicity and satisfactory stability. Significantly, a label-free sensitive assay for detecting carcinoembryonic antigen (CEA) in a continuous and recyclable way has been proposed on the basis of adsorption and desorption of aptamers by the surface of CDs through a competitive mechanism. To be specific, the richness of carboxyl groups of the CDs enabled strong adsorption of ssDNA to the surface of CDs through π-π stacking interactions, resulting in the effective fluorescence quenching by forming CDs-aptamer complexes. The stronger binding affinity between CEA and CEA-aptamer than the π-π stacking interactions has been taken advantage to achieve immediate recovery of the fluorescence of CDs once CEA was introduced. Thereby, quantitative evaluation of CEA concentration in a broad range from 1ngmL(-1) to 0.5ngmL(-1) with the detection limit of 0.3ngmL(-1) was realized in this way. This strategy can be applied in a recyclable way, broadening the sensing application of CDs with biocompatibility. Besides, the CDs were used for cell imaging, potentiating them towards diverse purposes. Copyright © 2016 Elsevier B.V. All rights reserved.

Cell-penetrating antimicrobial peptides - prospectives for targeting intracellular infections

DEFF Research Database (Denmark)

Bahnsen, Jesper S; Franzyk, Henrik; Sayers, Edward J

2015-01-01

PURPOSE: To investigate the suitability of three antimicrobial peptides (AMPs) as cell-penetrating antimicrobial peptides. METHODS: Cellular uptake of three AMPs (PK-12-KKP, SA-3 and TPk) and a cell-penetrating peptide (penetratin), all 5(6)-carboxytetramethylrhodamine-labeled, were tested in He......La WT cells and analyzed by flow cytometry and confocal microscopy. Furthermore, the effects of the peptides on eukaryotic cell viability as well as their antimicrobial effect were tested. In addition, the disrupting ability of the peptides in the presence of bilayer membranes of different composition...... the cellular viability to an unacceptable degree. TPk showed acceptable uptake efficiency, high antimicrobial activity and relatively low toxicity, and it is the best potential lead peptide for further development....

A novel facile method of labeling octreotide with (18)F-fluorine.

Science.gov (United States)

Laverman, Peter; McBride, William J; Sharkey, Robert M; Eek, Annemarie; Joosten, Lieke; Oyen, Wim J G; Goldenberg, David M; Boerman, Otto C

2010-03-01

Several methods have been developed to label peptides with (18)F. However, in general these are laborious and require a multistep synthesis. We present a facile method based on the chelation of (18)F-aluminum fluoride (Al(18)F) by 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA). The method is characterized by the labeling of NOTA-octreotide (NOTA-d-Phe-cyclo[Cys-Phe-d-Trp-Lys-Thr-Cys]-Throl (MH(+) 1305) [IMP466]) with (18)F. Octreotide was conjugated with the NOTA chelate and labeled with (18)F in a 2-step, 1-pot method. The labeling procedure was optimized with regard to the labeling buffer, peptide, and aluminum concentration. Radiochemical yield, specific activity, in vitro stability, and receptor affinity were determined. Biodistribution of (18)F-IMP466 was studied in AR42J tumor-bearing mice and compared with that of (68)Ga-labeled IMP466. In addition, small-animal PET/CT images were acquired. IMP466 was labeled with Al(18)F in a single step with 50% yield. The labeled product was purified by high-performance liquid chromatography to remove unbound Al(18)F and unlabeled peptide. The radiolabeling, including purification, was performed in 45 min. The specific activity was 45,000 GBq/mmol, and the peptide was stable in serum for 4 h at 37 degrees C. Labeling was performed at pH 4.1 in sodium citrate, sodium acetate, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, and 2-(N-morpholino)ethanesulfonic acid buffer and was optimal in sodium acetate buffer. The apparent 50% inhibitory concentration of the (19)F-labeled IMP466 determined on AR42J cells was 3.6 nM. Biodistribution studies at 2 h after injection showed a high tumor uptake of (18)F-IMP466 (28.3 +/- 5.2 percentage injected dose per gram [%ID/g]; tumor-to-blood ratio, 300 +/- 90), which could be blocked by an excess of unlabeled peptide (8.6 +/- 0.7 %ID/g), indicating that the accumulation in the tumor was receptor-mediated. Biodistribution of (68)Ga-IMP466 was similar to that of (18)F-IMP466. (18)F

Autoradiographic localization of a gluten peptide during organ culture of human duodenal mucosa

International Nuclear Information System (INIS)

Fluge, G.; Aksnes, L.

1983-01-01

An 125I-labeled subfraction of Frazer's fraction III (molecular weight, 8,000) was added to the culture medium during organ culture of duodenal biopsies from two patients with celiac disease in exacerbation. The isotope-labeled gluten peptide was localized by autoradiography after 6, 12, and 24 h of culture. At 6 h, labeling was located mainly in the basal layers of the biopsies. The tissue was well preserved. After 12 h in culture, the labeling had spread to the lamina propria and the crypts. A few grains were located over enterocytes and desquamated cells. Moderate histological signs of toxicity were observed. After 24 h, there was marked toxic deterioration, comparable to that seen after culture with alpha-gliadin. Labeling had spread throughout the entire section. There seemed to be no specificity of the binding, for the entire section was affected. Culture with the identical gluten fraction, in the radionegative state, produced histological deterioration comparable to that seen after exposure to the isotope-labeled peptide. Gluten peptides are presented to the target cells in a unique way during organ culture, different from in vivo conditions. This may influence the results when the organ culture method is used to investigate the pathogenesis of celiac disease

Autoradiographic localization of a gluten peptide during organ culture of human duodenal mucosa

Energy Technology Data Exchange (ETDEWEB)

Fluge, G.; Aksnes, L.

1983-01-01

An 125I-labeled subfraction of Frazer's fraction III (molecular weight, 8,000) was added to the culture medium during organ culture of duodenal biopsies from two patients with celiac disease in exacerbation. The isotope-labeled gluten peptide was localized by autoradiography after 6, 12, and 24 h of culture. At 6 h, labeling was located mainly in the basal layers of the biopsies. The tissue was well preserved. After 12 h in culture, the labeling had spread to the lamina propria and the crypts. A few grains were located over enterocytes and desquamated cells. Moderate histological signs of toxicity were observed. After 24 h, there was marked toxic deterioration, comparable to that seen after culture with alpha-gliadin. Labeling had spread throughout the entire section. There seemed to be no specificity of the binding, for the entire section was affected. Culture with the identical gluten fraction, in the radionegative state, produced histological deterioration comparable to that seen after exposure to the isotope-labeled peptide. Gluten peptides are presented to the target cells in a unique way during organ culture, different from in vivo conditions. This may influence the results when the organ culture method is used to investigate the pathogenesis of celiac disease.

Development of radioactively labelled cancer seeking biomolecules for targeted radiotherapy

International Nuclear Information System (INIS)

Varvarigou, A.D.; Archimandritis, S.C.

2000-01-01

Within the framework of the above project we are studying the labelling of biomolecules, peptides and antibodies, with radionuclides emitting β - and γ radiation. More specifically, for the time being, we have investigated the labelling of peptides with Re-188 and of antibodies with Sm-153 and Re-188. The radiolabelled derivatives are further evaluated in vivo for possible application in Oncology. For these radiobiological studies we are trying to apply ectopic and orthotopic tumour animal models and to develop, in collaboration with other national and foreign institutes, proper imaging devices for small animal imaging

Quantum Dot-based Immunohistochemistry for Pathological Applications

Directory of Open Access Journals (Sweden)

Li Zhou

2016-01-01

Full Text Available Quantum dots (QDs are novel light emitting semiconductor nanocrystals with diameter ranging from 2 to 20 nm. In comparison with traditional organic dyes and fluorescent proteins, QDs possess unique optical properties including extremely high fluorescence efficiency and minimal photobleaching which make them emerge as a new class of fluorescent labels for molecular imaging and biomedical analysis. Herein, recent advances in fundamental mechanisms and pathological applications of QD were reviewed.

In vitro comparison of renal handling and uptake of two somatostatin receptor-specific peptides labeled with indium-111

International Nuclear Information System (INIS)

Trejtnar, F.; Novy, Z.; Petrik, M.; Laznickova, A.; Melicharova, L.; Vankova, M.; Laznicek, M.

2008-01-01

Radiolabeled receptor-specific somatostatin analogs labeled with gamma- or beta-emitting radionuclides are useful for scintigraphic imaging and/or therapy of selected neuroendocrine tumors. However, significant renal uptake may result in radiotoxicological injury of the kidney and can limit clinical application of the agents. The aim of the study was to analyze renal handling, rate, and mechanism of renal accumulation of two somatostatin receptor-targeted peptides, [DOTA 0 , Tyr 3 , Thr 8 ]-octreotide (DOTA-TATE) and [DOTA 0 , 1-Nal 3 ]-octreotide (DOTA-NOC), labeled with indium-111 using in vitro methods. The perfused rat kidney and freshly isolated rat renal cells were used as experimental models. The perfusion was performed in a recirculation regimen at constant pressure with solution containing bovine albumin, erythrocytes, and a mixture of essential substrates. The renal cells were isolated from rat kidneys using two-phase collagenase perfusion. Accumulation studies were used to evaluate the renal uptake of the peptides and to compare their accumulation with that of passively or actively transported model drugs. The influence of selected inhibitors of receptor-mediated endocytosis and the inhibition of energy-dependent transport processes on the uptake were also investigated using isolated renal cells. The renal clearance of 111 In-DOTA-NOC in the perfused rat kidney was significantly lower than that of 111 In-DOTA-TATE. Reverse situation was found in the case of renal retention. Pretreatment of the perfused kidney with maleate markedly decreased the renal retention. 111 In-DOTA-NOC was accumulated in the isolated renal cells at a higher rate than 111 In-DOTA-TATE (ratio 3:1). The uptake of the radiopeptides in renal cells was higher than the uptake of not only the passively transported sucrose but also actively transported and accumulated methylglucose. The rank order of potency to inhibit the uptake by active endocytosis was approximately aprotinin

Biosynthesis of amidated joining peptide from pro-adrenocorticotropin-endorphin

Energy Technology Data Exchange (ETDEWEB)

Cullen, E.I.; Mains, R.E. (Johns Hopkins Univ. School of Medicine, Baltimore, MD (USA))

1987-09-01

Joining peptide is the major alpha-amidated product of pro-ACTH/endorphin (PAE) in AtT-20 corticotropic tumor cells. To study intracellular joining peptide synthesis, affinity purified antibodies directed against gamma-MSH, joining peptide, and ACTH were used to immunoprecipitate extracts from biosynthetically labeled AtT-20 cells. Immunoprecipitates were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by tryptic peptide mapping on HPLC. In steady labeling experiments, radioactivity in amidated joining peptide (JP) increased roughly linearly with time, in the manner of a final product, whereas radioactivity associated with PAE (1-94)NH2 reached a constant value after 2-4 h, indicating that PAE(1-94)NH2 is an intermediate in the biosynthesis of JP. Radioactivity appeared in ACTH(1-39) well before JP, consistent with a cleavage order in which ACTH is cleaved from PAE(1-95) before JP sequences are cleaved from PAE(1-74). This conclusion was supported by tryptic peptide analyses of immunoprecipitates, which indicated that less than 5% of JP-related material is cleaved from PAE(1-74) before being cleaved from ACTH-related sequences. After a pulse label, radioactivity in PAE(1-94)NH2 reached a peak value after 1 h of chase and declined with a half-life of less than 1 h. Amidated JP increased to a constant level after 2 h of chase. Enough radiolabeled PAE(1-94)NH2 was detected to account for about half of the radioactivity found in amidated JP, indicating that about half of JP-related material is first cleaved from PAE(1-95) before being amidated. This result was corroborated using HPLC purification to determine both amidated and glycine-extended forms of JP.

Biosynthesis of amidated joining peptide from pro-adrenocorticotropin-endorphin

International Nuclear Information System (INIS)

Cullen, E.I.; Mains, R.E.

1987-01-01

Joining peptide is the major alpha-amidated product of pro-ACTH/endorphin (PAE) in AtT-20 corticotropic tumor cells. To study intracellular joining peptide synthesis, affinity purified antibodies directed against gamma-MSH, joining peptide, and ACTH were used to immunoprecipitate extracts from biosynthetically labeled AtT-20 cells. Immunoprecipitates were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by tryptic peptide mapping on HPLC. In steady labeling experiments, radioactivity in amidated joining peptide (JP) increased roughly linearly with time, in the manner of a final product, whereas radioactivity associated with PAE (1-94)NH2 reached a constant value after 2-4 h, indicating that PAE(1-94)NH2 is an intermediate in the biosynthesis of JP. Radioactivity appeared in ACTH(1-39) well before JP, consistent with a cleavage order in which ACTH is cleaved from PAE(1-95) before JP sequences are cleaved from PAE(1-74). This conclusion was supported by tryptic peptide analyses of immunoprecipitates, which indicated that less than 5% of JP-related material is cleaved from PAE(1-74) before being cleaved from ACTH-related sequences. After a pulse label, radioactivity in PAE(1-94)NH2 reached a peak value after 1 h of chase and declined with a half-life of less than 1 h. Amidated JP increased to a constant level after 2 h of chase. Enough radiolabeled PAE(1-94)NH2 was detected to account for about half of the radioactivity found in amidated JP, indicating that about half of JP-related material is first cleaved from PAE(1-95) before being amidated. This result was corroborated using HPLC purification to determine both amidated and glycine-extended forms of JP

Identifying and quantifying proteolytic events and the natural N terminome by terminal amine isotopic labeling of substrates.

Science.gov (United States)

Kleifeld, Oded; Doucet, Alain; Prudova, Anna; auf dem Keller, Ulrich; Gioia, Magda; Kizhakkedathu, Jayachandran N; Overall, Christopher M

2011-09-22

Analysis of the sequence and nature of protein N termini has many applications. Defining the termini of proteins for proteome annotation in the Human Proteome Project is of increasing importance. Terminomics analysis of protease cleavage sites in degradomics for substrate discovery is a key new application. Here we describe the step-by-step procedures for performing terminal amine isotopic labeling of substrates (TAILS), a 2- to 3-d (depending on method of labeling) high-throughput method to identify and distinguish protease-generated neo-N termini from mature protein N termini with all natural modifications with high confidence. TAILS uses negative selection to enrich for all N-terminal peptides and uses primary amine labeling-based quantification as the discriminating factor. Labeling is versatile and suited to many applications, including biochemical and cell culture analyses in vitro; in vivo analyses using tissue samples from animal and human sources can also be readily performed. At the protein level, N-terminal and lysine amines are blocked by dimethylation (formaldehyde/sodium cyanoborohydride) and isotopically labeled by incorporating heavy and light dimethylation reagents or stable isotope labeling with amino acids in cell culture labels. Alternatively, easy multiplex sample analysis can be achieved using amine blocking and labeling with isobaric tags for relative and absolute quantification, also known as iTRAQ. After tryptic digestion, N-terminal peptide separation is achieved using a high-molecular-weight dendritic polyglycerol aldehyde polymer that binds internal tryptic and C-terminal peptides that now have N-terminal alpha amines. The unbound naturally blocked (acetylation, cyclization, methylation and so on) or labeled mature N-terminal and neo-N-terminal peptides are recovered by ultrafiltration and analyzed by tandem mass spectrometry (MS/MS). Hierarchical substrate winnowing discriminates substrates from the background proteolysis products and

Radiolabeled peptides: experimental and clinical applications

International Nuclear Information System (INIS)

Thakur, M.L.; Pallela, V.R.

1998-01-01

Radiolabeled receptor specific biomolecules hold unlimited potential in nuclear medicine. During the past few years much attention has been drawn to the development radiolabeled peptides for a variety of diagnostic applications, as well as for therapy of malignant tumors. Although only one peptide, In-111-DTPA-(D)-Phe 1 -octreotide, is available commercially for oncologic imaging, many more have been examined in humans with hematological disorders, and the early results appear to be promising. Impetus generated by these results have prompted investigators to label peptides with such radionuclides as Tc-99m, I-123, F-18, Cu-64, and Y-90. This review is intended to highlight the qualities of peptides, summarize the clinical results, and address some important issues associated with radiolabeling of highly potent peptides. While doing so, various methods of radiolabeling have been described, and their strengths and weaknesses have also been discussed. (author)

Peptides for radiotherapy of neuroendocrine cancers

International Nuclear Information System (INIS)

Melendez A, L.

2002-01-01

During the last decade there has been a resurgence of interest in therapeutic nuclear medicine, due to the limitation of conventional or external beam radiotherapy in the treatment of secondary or metastatic cancer sites outside of the primary treatment area. Some of the human tumours that produce metastases express high levels of somatostatin receptors. In order to make possible the diagnostic and radiotherapeutic treatment of these kind of tumours, various somatostatin analogue peptides have been developed in recent years. Peptides have become an important class of radiopharmaceuticals,due to its unique ability to detect specific sites as receptors or enzymes. This paper describes the work with 99m Tc to establish the labelling and analytical conditions for a somatostatin analogue as a precursor, to undertake a therapeutic radiopharmaceutical labelled with 188 Re for treatment of somatostatin receptor positive tumours. (Author)

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.

Dual-purpose linker for alpha helix stabilization and imaging agent conjugation to glucagon-like peptide-1 receptor ligands.

Science.gov (United States)

Zhang, Liang; Navaratna, Tejas; Liao, Jianshan; Thurber, Greg M

2015-02-18

Peptides display many characteristics of efficient imaging agents such as rapid targeting, fast background clearance, and low non-specific cellular uptake. However, poor stability, low affinity, and loss of binding after labeling often preclude their use in vivo. Using glucagon-like peptide-1 receptor (GLP-1R) ligands exendin and GLP-1 as a model system, we designed a novel α-helix-stabilizing linker to simultaneously address these limitations. The stabilized and labeled peptides showed an increase in helicity, improved protease resistance, negligible loss or an improvement in binding affinity, and excellent in vivo targeting. The ease of incorporating azidohomoalanine in peptides and efficient reaction with the dialkyne linker enable this technique to potentially be used as a general method for labeling α helices. This strategy should be useful for imaging beta cells in diabetes research and in developing and testing other peptide targeting agents.

Structural Characterization of Peptide Antibodies

DEFF Research Database (Denmark)

Chailyan, Anna; Marcatili, Paolo

2015-01-01

The role of proteins as very effective immunogens for the generation of antibodies is indisputable. Nevertheless, cases in which protein usage for antibody production is not feasible or convenient compelled the creation of a powerful alternative consisting of synthetic peptides. Synthetic peptides...... can be modified to obtain desired properties or conformation, tagged for purification, isotopically labeled for protein quantitation or conjugated to immunogens for antibody production. The antibodies that bind to these peptides represent an invaluable tool for biological research and discovery....... To better understand the underlying mechanisms of antibody-antigen interaction here we present a pipeline developed by us to structurally classify immunoglobulin antigen binding sites and to infer key sequence residues and other variables that have a prominent role in each structural class....

Highly sensitive polymerase chain reaction-free quantum dot-based quantification of forensic genomic DNA

International Nuclear Information System (INIS)

Tak, Yu Kyung; Kim, Won Young; Kim, Min Jung; Han, Eunyoung; Han, Myun Soo; Kim, Jong Jin; Kim, Wook; Lee, Jong Eun; Song, Joon Myong

2012-01-01

Highlights: ► Genomic DNA quantification were performed using a quantum dot-labeled Alu sequence. ► This probe provided PCR-free determination of human genomic DNA. ► Qdot-labeled Alu probe-hybridized genomic DNAs had a 2.5-femtogram detection limit. ► Qdot-labeled Alu sequence was used to assess DNA samples for human identification. - Abstract: Forensic DNA samples can degrade easily due to exposure to light and moisture at the crime scene. In addition, the amount of DNA acquired at a criminal site is inherently limited. This limited amount of human DNA has to be quantified accurately after the process of DNA extraction. The accurately quantified extracted genomic DNA is then used as a DNA template in polymerase chain reaction (PCR) amplification for short tandem repeat (STR) human identification. Accordingly, highly sensitive and human-specific quantification of forensic DNA samples is an essential issue in forensic study. In this work, a quantum dot (Qdot)-labeled Alu sequence was developed as a probe to simultaneously satisfy both the high sensitivity and human genome selectivity for quantification of forensic DNA samples. This probe provided PCR-free determination of human genomic DNA and had a 2.5-femtogram detection limit due to the strong emission and photostability of the Qdot. The Qdot-labeled Alu sequence has been used successfully to assess 18 different forensic DNA samples for STR human identification.

Non-invasive glucagon-like peptide-1 receptor imaging in pancreas with (18)F-Al labeled Cys(39)-exendin-4.

Science.gov (United States)

Mi, Baoming; Xu, Yuping; Pan, Donghui; Wang, Lizhen; Yang, Runlin; Yu, Chunjing; Wan, Weixing; Wu, Yiwei; Yang, Min

2016-02-26

Glucagon-like peptide-1 receptor (GLP-1R) is abundantly expressed on beta cells and may be an ideal target for the pancreas imaging. Monitoring the GLP-1R of pancreas could be benefit for understanding the pathophysiology of diabetes. In the present study, (18)F-Al labeled exendin-4 analog, (18)F-Al-NOTA-MAL-Cys(39)-exendin-4, was evaluated for PET imaging GLP-1R in the pancreas. The targeting of (18)F-Al labeled exendin-4 analog was examined in healthy and streptozotocin induced diabetic rats. Rats were injected with (18)F-Al-NOTA-MAL-Cys(39)-exendin-4 and microPET imaging was performed at 1 h postinjection, followed by ex vivo biodistribution. GLP-1R expression in pancreas was determined through post mortern examinations. The pancreas of healthy rats was readily visualized after administration of (18)F-Al-NOTA-MAL-Cys(39)-exendin-4, whereas the pancreas of diabetic rats, as well as those from rats co-injected with excess of unlabeled peptides, was barely visible by microPET. At 60 min postinjection, the pancreatic uptakes were 1.02 ± 0.15%ID/g and 0.23 ± 0.05%ID/g in healthy and diabetic rats respectively. Under block, the pancreatic uptakes of non-diabetic rats reduced to 0.21 ± 0.07%ID/g at the same time point. Biodistribution data and IHC staining confirmed the findings of the microPET imaging. The favorable preclinical data indicated that (18)F-Al-NOTA-MAL-Cys(39)-exendin-4may be suitable for non-invasive monitoring functional pancreatic beta cells. Copyright © 2016 Elsevier Inc. All rights reserved.

Quantum Dots

Science.gov (United States)

Tartakovskii, Alexander

2012-07-01

Part I. Nanostructure Design and Structural Properties of Epitaxially Grown Quantum Dots and Nanowires: 1. Growth of III/V semiconductor quantum dots C. Schneider, S. Hofling and A. Forchel; 2. Single semiconductor quantum dots in nanowires: growth, optics, and devices M. E. Reimer, N. Akopian, M. Barkelid, G. Bulgarini, R. Heeres, M. Hocevar, B. J. Witek, E. Bakkers and V. Zwiller; 3. Atomic scale analysis of self-assembled quantum dots by cross-sectional scanning tunneling microscopy and atom probe tomography J. G. Keizer and P. M. Koenraad; Part II. Manipulation of Individual Quantum States in Quantum Dots Using Optical Techniques: 4. Studies of the hole spin in self-assembled quantum dots using optical techniques B. D. Gerardot and R. J. Warburton; 5. Resonance fluorescence from a single quantum dot A. N. Vamivakas, C. Matthiesen, Y. Zhao, C.-Y. Lu and M. Atature; 6. Coherent control of quantum dot excitons using ultra-fast optical techniques A. J. Ramsay and A. M. Fox; 7. Optical probing of holes in quantum dot molecules: structure, symmetry, and spin M. F. Doty and J. I. Climente; Part III. Optical Properties of Quantum Dots in Photonic Cavities and Plasmon-Coupled Dots: 8. Deterministic light-matter coupling using single quantum dots P. Senellart; 9. Quantum dots in photonic crystal cavities A. Faraon, D. Englund, I. Fushman, A. Majumdar and J. Vukovic; 10. Photon statistics in quantum dot micropillar emission M. Asmann and M. Bayer; 11. Nanoplasmonics with colloidal quantum dots V. Temnov and U. Woggon; Part IV. Quantum Dot Nano-Laboratory: Magnetic Ions and Nuclear Spins in a Dot: 12. Dynamics and optical control of an individual Mn spin in a quantum dot L. Besombes, C. Le Gall, H. Boukari and H. Mariette; 13. Optical spectroscopy of InAs/GaAs quantum dots doped with a single Mn atom O. Krebs and A. Lemaitre; 14. Nuclear spin effects in quantum dot optics B. Urbaszek, B. Eble, T. Amand and X. Marie; Part V. Electron Transport in Quantum Dots Fabricated by

Study of pharmacokinetics and biodistribution of radiolabelled receptor specific peptides in laboratory animals

International Nuclear Information System (INIS)

Laznickova, A.; Laznicek, M.; Trejtnar, F.; Maecke, H.R.; Mather, S.J.

2001-01-01

Somatostatin analogues labelled with different radionuclides could be employed for visualization or treatment of somatostatin receptor-positive tumours. An octapeptide 111 In [DTPA] octreotide is a synthetic radiolabelled somatostatin analogue which is currently in clinical use for detecting small neuroendocrine tumours and metastases not detectable by conventional means. However, several other somatostatin analogues have been under development and testing. The aim of this study was to radiolabel selected somatostatin receptor-binding octapeptides by different radionuclides and to report the results of their biodistribution in rats. The study was focused on the direct labelling of vapreotide (RC-160) with 99m Tc, on the conjugates of octreotide with DFO (desferrioxamine) for labelling with 67 Ga, and on the conjugates of octreotide and TOC with DOTA (tetraazacyclo-dodecane tetraacetic acid) for labelling with 88 Y. In the present study, 88 Y isotope instead of 90 Y was used as a label as 88 Y exhibits a longer half life of decay and emits gamma radiation which can be much more easily detected in biological samples than beta emission. The labelling of octreotide analogues with metal radionuclides using derived bifunctional chelates was simple, straightforward and consistently resulted in high radiochemical purity of the product. On the other hand, the application of the direct labelling method for labelling of RC-160 with 99m Tc was difficult because all procedures had to be made under nitrogen atmosphere and an attainment of high yield proved to be highly dependent on the accurate observation of reaction conditions. The labelling efficiency makes an immediate use of the radiolabelled RC-160 for biological studies impossible and it is necessary to involve the purification step into the labelling procedure. All radiolabelled receptor specific peptides under study exhibited rapid radioactivity clearance from the blood and most organs and tissues. On the other hand

The preparation and characterization of peptide's lung cancer imaging agent

International Nuclear Information System (INIS)

Liu Jianfeng; Chu Liping; Wang Yan; Wang Yueying; Liu Jinjian; Wu Hongying

2010-01-01

Objective: To screen in vivo lung cancer specific binding seven peptides by T7 phage display peptide library, so as to prepare peptide's lung cancer early diagnostic agent. Methods: Use phage display in vivo technology, the 7-peptide phage that binding the lung cancer specifically was obtained, then the DNA sequence was measured and the seven peptide was synthesized. After labeled by 125 I, the seven peptide was injected into mice via vein and the distribution was observed. Results: One peptide was obtained by four rounds screening, and the peptide can bind lung cancer tissue specifically. Two hours after injection get the best imaging of lung cancer, metabolism of peptide in mice is fast, the distribution in vivo is decrease six hours and almost disappear 20 hours after injection. Conclusion: The peptide can image and diagnose lung cancer better. (authors)

Design of a dual-function peptide probe as a binder of angiotensin II and an inducer of silver nanoparticle aggregation for use in label-free colorimetric assays.

Science.gov (United States)

Okochi, Mina; Kuboyama, Masashi; Tanaka, Masayoshi; Honda, Hiroyuki

2015-09-01

Label-free colorimetric assays using metallic nanoparticles have received much recent attention, for their application in simple and sensitive methods for detection of biomolecules. Short peptide probes that can bind to analyte biomolecules are attractive ligands in molecular nanotechnology; however, identification of biological recognition motifs is usually based on trial-and-error experiments. Herein, a peptide probe was screened for colorimetric detection of angiotensin II (Ang II) using a mechanism for non-crosslinking aggregation of silver nanoparticles (AgNPs). The dual-function peptides, which bind to the analyte and induce AgNP aggregation, were identified using a two-step strategy: (1) screening of an Ang II-binding peptide from an Ang II receptor sequence library, using SPOT technology, which enable peptides synthesis on cellulose membranes via an Fmoc method and (2) selection of peptide probes that effectively induce aggregation of AgNPs using a photolinker modified peptide array. Using the identified peptide probe, KGKNKRRR, aggregation of AgNPs was detected by observation of a pink color in the absence of Ang II, whereas AgNPs remained dispersed in the presence of Ang II (yellow). The color changes were not observed in the presence of other hormone molecules. Ang II could be detected within 15 min, with a detection limit of 10 µM, by measuring the ratio of absorbance at 400 nm and 568 nm; the signal could also be observed with the naked eye. These data suggest that the peptide identified here could be used as a probe for simple and rapid colorimetric detection of Ang II. This strategy for the identification of functional peptides shows promise for the development of colorimetric detection of various diagnostically important biomolecules. Copyright © 2015 Elsevier B.V. All rights reserved.

One-pot synthesis of stable water soluble Mn:ZnSe/ZnS core/shell quantum dots

Energy Technology Data Exchange (ETDEWEB)

Zhang Hao; Gao Xue; Liu Siyu; Su Xingguang, E-mail: suxg@jlu.edu.cn [College of Chemistry, Jilin University, Department of Analytical Chemistry (China)

2013-06-15

In this paper, Mn:ZnSe/ZnS core/shell-doped quantum dots (d-dots) with 3-mercaptopropionic acid as the stabilizer are successfully synthesized through a simple one-pot synthesis procedure in aqueous solution. The average diameter of Mn:ZnSe/ZnS core/shell d-dots is about 2.9 nm, which is lager than that of Mn:ZnSe cores (about 1.9 nm). The optical features and structure of the obtained Mn:ZnSe/ZnS core/shell quantum dots have been characterized by UV-Vis and fluorescence spectroscopy, X-ray diffraction, transmission electron microscopy, and X-ray photoelectron spectroscopy. The photostability against UV irradiation and chemical stability against H{sub 2}O{sub 2} etching have been studied, and the results showed that the prepared Mn:ZnSe/ZnS core/shell d-dots are more stable than CdTe quantum dots prepared in aqueous solution. Finally, the resulting core/shell quantum dots are used as fluorescent label in human osteoblast-like HepG2 cell imaging.

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

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

Energy Technology Data Exchange (ETDEWEB)

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

2015-09-03

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

Synthesis of two S-(methyl-3H)-labelled enkephalins and S-(methyl-14C) substance P

International Nuclear Information System (INIS)

Naegren, K.; Laangstroem, B.; Franzen, H.M.; Ragnarsson, U.

1988-01-01

The synthesis of 3 H-labelled Met-enkephalin and Tyr-D-Ala-Gly-Phe-Met-NH 2 (DALA) and 14 C-labelled Substance P (SP) from previously described, fully protected intermediates is reported. The labelled peptides were prepared by methylation with ( 3 H)- or ( 14 C)methyl iodide of the sulphide anions formed on deprotection of the corresponding S-benzyl-homocysteine precursors with sodium in liquid ammonia. After purification by LC, the labelled peptides were obtained in radiochemical yields in the range of 9 to 24% with a radiochemical purity higher than 97%. The specific radioactivities of the 3 H- and 14 C- labelled products, corresponding to the labelled methyl iodides used, were 80 mCi/μmol and 60 μCi/μmol, respectively. (author)

Development of radioactively labelled cancer seeking biomolecules for targeted radiotherapy. Greece

International Nuclear Information System (INIS)

Varvarigou, Alexandra D.; Archimandritis, Spyridon C.

2000-01-01

Within the framework of the above project we are studying the labelling of biomolecules, peptides and antibodies, with radionuclides emitting β - and γ radiation. More specifically, for the time being, we have investigated the labelling of peptides with Re-188 and of antibodies with Sm-153 and Re-188. The radiolabelled derivatives are further evaluated in vivo for possible application in Oncology. For these radiobiological studies we are trying to apply ectopic and orthotopic tumour animal models and to develop, in collaboration with other national and foreign institutes, proper imaging devices for small animal imaging

A Dual-Purpose Linker for Alpha Helix Stabilization and Imaging Agent Conjugation to Glucagon-Like Peptide-1 Receptor Ligands

Science.gov (United States)

Zhang, Liang; Navaratna, Tejas; Liao, Jianshan; Thurber, Greg M.

2016-01-01

Peptides display many characteristics of efficient imaging agents such as rapid targeting, fast background clearance, and low non-specific cellular uptake. However, poor stability, low affinity, and loss of binding after labeling often preclude their use in vivo. Using the glucagon-like peptide-1 receptor (GLP-1R) ligands exendin and GLP-1 as a model system, we designed a novel alpha helix stabilizing linker to simultaneously address these limitations. The stabilized and labeled peptides showed an increase in helicity, improved protease resistance, negligible loss or an improvement in binding affinity, and excellent in vivo targeting. The ease of incorporating azidohomoalanine in peptides and efficient reaction with the dialkyne linker enables this technique to potentially be used as a general method for labeling alpha helices. This strategy should be useful for imaging beta cells in diabetes research and in developing and testing other peptide targeting agents. PMID:25594741

Reproduction-associated immunoreactive peptides in the nervous systems of prosobranch gastropods.

Science.gov (United States)

Ram, J L; Gallardo, C S; Ram, M L; Croll, R P

1998-12-01

Antibodies against reproductive peptides of Aplysia and Lymnaea were used to localize homologous immunoreactive peptides in the nervous systems of three prosobranch species: Busycon canaliculatum, Concholepas concholepas, and Tegula atra. Positive control experiments in L. stagnalis demonstrated the broad species range of the anti-egg-laying hormone (anti-ELH) antibody used in this study, and showed binding of anti-alpha-caudodorsal-cell peptide (anti-alpha-CDCP) to the same cells in cerebral and buccal ganglia. Dot immunoassays with synthetic ELH confirmed the reactivity and sensitivity (concholepas and T atra, ELH-like immunoreactivity was found in cerebral ganglia, and in T. atra in fibers in the cerebral ganglia and cerebral-pedal connectives. Thus, cerebral ganglia are the major locus of the ELH-like immunoreactivity in prosobranchs.

Synthesis of tritium labeled Ac-[Nle4, D-Phe7]-α-MSH/sub 4-11/-NH2: a superpotent melanotropin with prolonged biological activity

International Nuclear Information System (INIS)

Wilkes, B.D.; Hruby, V.J.; Yamamura, H.I.; Akiyama, K.; Castrucci, A.M. de; Hadley, M.E.; Andrews, J.R.; Wan, Y.P.

1984-01-01

Ac-[Nle 4 , D-Phe 7 ]-α-MSH/sub 4-11/-NH 2 an octapeptide, is a melanotropin analogue (Ac-Nle-Glu-His-D-Phe-Arg-Trp-Gly-Lys-NH 2 ), which is a superpotent agonist of frog and lizard skin melanocytes and mouse S 91 (Cloudman) melanoma cells. This melanotropin possesses ultraprolonged activity on melanocytes, both in vitro and in vivo, and the peptide is resistant to inactivation by serum enzymes. The tritium-labeled congener was prepared by direct incorporation of [ 3 H]-labeled norleucine into the peptide. The melanotropic activity of the labeled peptide is identical to the unlabeled analogue. This labeled peptide should be useful for studies on the localization and characterization of melanotropin receptors

The EIPeptiDi tool: enhancing peptide discovery in ICAT-based LC MS/MS experiments

Directory of Open Access Journals (Sweden)

Tradigo Giuseppe

2007-07-01

Full Text Available Abstract Background Isotope-coded affinity tags (ICAT is a method for quantitative proteomics based on differential isotopic labeling, sample digestion and mass spectrometry (MS. The method allows the identification and relative quantification of proteins present in two samples and consists of the following phases. First, cysteine residues are either labeled using the ICAT Light or ICAT Heavy reagent (having identical chemical properties but different masses. Then, after whole sample digestion, the labeled peptides are captured selectively using the biotin tag contained in both ICAT reagents. Finally, the simplified peptide mixture is analyzed by nanoscale liquid chromatography-tandem mass spectrometry (LC-MS/MS. Nevertheless, the ICAT LC-MS/MS method still suffers from insufficient sample-to-sample reproducibility on peptide identification. In particular, the number and the type of peptides identified in different experiments can vary considerably and, thus, the statistical (comparative analysis of sample sets is very challenging. Low information overlap at the peptide and, consequently, at the protein level, is very detrimental in situations where the number of samples to be analyzed is high. Results We designed a method for improving the data processing and peptide identification in sample sets subjected to ICAT labeling and LC-MS/MS analysis, based on cross validating MS/MS results. Such a method has been implemented in a tool, called EIPeptiDi, which boosts the ICAT data analysis software improving peptide identification throughout the input data set. Heavy/Light (H/L pairs quantified but not identified by the MS/MS routine, are assigned to peptide sequences identified in other samples, by using similarity criteria based on chromatographic retention time and Heavy/Light mass attributes. EIPeptiDi significantly improves the number of identified peptides per sample, proving that the proposed method has a considerable impact on the protein

Intramolecular migration of amide hydrogens in protonated peptides upon collisional activation

DEFF Research Database (Denmark)

Jørgensen, Thomas J. D.; Gårdsvoll, H.; Ploug, M.

2005-01-01

Presently different opinions exist as to the degree of scrambling of amide hydrogens in gaseous protonated peptides and proteins upon collisional activation in tandem mass spectrometry experiments. This unsettled controversy is not trivial, since only a very low degree of scrambling is tolerable...... if collision-induced dissociation (CID) should provide reliable site-specific information from (1)H/(2)H exchange experiments. We have explored a series of unique, regioselectively deuterium-labeled peptides as model systems to probe for intramolecular amide hydrogen migration under low-energy collisional...... are protected against exchange with the solvent, while the amide hydrogens of the nonbinding sequences exchange rapidly with the solvent. We have utilized such long-lived complexes to generate peptides labeled with deuterium in either the binding or nonbinding region, and the expected regioselectivity...

Labelling and quality control of somatostatin analogues with 99mTc

International Nuclear Information System (INIS)

Verdera, S.; Balter, H.; Rodriguez, G.; Robles, A.; Oliver, P.; Laiz, J.; Souto, B.

2001-01-01

Techniques and methodologies for labelling peptides with 99m Tc and methods for their purification, chemical, radiochemical and biological controls were evaluated. With the purpose of gaining experience, labelling with 125 I was also studied. RC-160 was labelled with 125 I using iodogen as well as chloramine-T method. Higher yields were obtained with chloramine-T method (60%), rendering 125 I-peptide with 98% of radiochemical purity, with specific activity of 240 μCi/μg - 274 μCi/μg. The product was stable for five weeks (at -20 deg. C). For somatostatin receptors studies rat brain cortex membrane was prepared. Maximum binding capacity was 24.7% and Kaff for the binding of RC-160 to receptor was estimated as 2.0x10 10 M -1 . Other peptides as β-(2-Naphthyl)- D Ala-Cys-Tyr- D Trp-Lys-Val-Cys-Thr amide (N-9642, Σ) and mouse epidermal growth factor (mEGF) were also labelled by means of limiting chloramine-T method. In case of mEGF the availability of membrane receptors allowed us to experiment in mice as well as in vitro. The reaction yields were up to 60% and 70% respectively. Biodistribution of 125 I-mEGF in a mouse with adenoma demonstrated preferential uptake in tumour (21,7% injected dose). The radioimmunoassay system gave 39% maximum binding (MB) and 50% displacement (ED 50 ) for 10 ng/mL unlabelled mEGF. Direct method and BFC's for labelling peptides with 99m Tc were investigated and purification and quality controls studies were performed by TLC, HPLC (UV and gamma detection). RC-160 was labelled by a direct method using sodium dithionite as reducing agent with radiochemical purity >95%. The product was stable up to six hours (at RT). Considerable adsorption problems were observed. Biological behavior was in accordance with the compounds' lipophilicity. The synthesis of TOC conjugates with HYNIC as BFC was done with 45%±5% (n=3) yield. Labelling of HYNIC-TOC with tricine as co-ligand was conducted with up to 90% yield. Studies of RC-160 labelling using

Synthesis of 35S-labeled caerulein (FI 6934)

International Nuclear Information System (INIS)

Uemura, Ieaki; Murakami, Hironori

1975-01-01

FI6934 (Caerulein) is a biologically active decapeptide extracted from the skin of Australian amphibians (Hyla caerulea). For metabolic study of FI6934, we have attempted to label the sulfo group of tyrosine of FI6934 with 35 S. The starting decapeptide was sulfonated with an excess of pyridine-N-sulfonate 35 S, and the 35 S-labelled peptide resulted was deacetylated by hydrolysis in alkaline, purified by paper chromatography to obtained radio chemically pure 35 S-labelled FI6934. The 35 S-labelled FI6934 was identified as a standard FI6934 in physiological activity to contract guinea pig gallbladders. (author)

RAPID AND AUTOMATED PROCESSING OF MALDI-FTICR/MS DATA FOR N-METABOLIC LABELING IN A SHOTGUN PROTEOMICS ANALYSIS.

Science.gov (United States)

Jing, Li; Amster, I Jonathan

2009-10-15

Offline high performance liquid chromatography combined with matrix assisted laser desorption and Fourier transform ion cyclotron resonance mass spectrometry (HPLC-MALDI-FTICR/MS) provides the means to rapidly analyze complex mixtures of peptides, such as those produced by proteolytic digestion of a proteome. This method is particularly useful for making quantitative measurements of changes in protein expression by using (15)N-metabolic labeling. Proteolytic digestion of combined labeled and unlabeled proteomes produces complex mixtures that with many mass overlaps when analyzed by HPLC-MALDI-FTICR/MS. A significant challenge to data analysis is the matching of pairs of peaks which represent an unlabeled peptide and its labeled counterpart. We have developed an algorithm and incorporated it into a compute program which significantly accelerates the interpretation of (15)N metabolic labeling data by automating the process of identifying unlabeled/labeled peak pairs. The algorithm takes advantage of the high resolution and mass accuracy of FTICR mass spectrometry. The algorithm is shown to be able to successfully identify the (15)N/(14)N peptide pairs and calculate peptide relative abundance ratios in highly complex mixtures from the proteolytic digest of a whole organism protein extract.

Quantum dots trace lymphatic drainage from the mouse eye

Energy Technology Data Exchange (ETDEWEB)

Tam, Alex L C; Gupta, Neeru; Zhang Zhexue; Yuecel, Yeni H, E-mail: yucely@smh.ca [Department of Ophthalmology and Vision Sciences, University of Toronto, M5T 2S8 (Canada)

2011-10-21

Glaucoma is a leading cause of blindness in the world, often associated with elevated eye pressure. Currently, all glaucoma treatments aim to lower eye pressure by improving fluid exit from the eye. We recently reported the presence of lymphatics in the human eye. The lymphatic circulation is known to drain fluid from organ tissues and, as such, lymphatics may also play a role in draining fluid from the eye. We investigated whether lymphatic drainage from the eye is present in mice by visualizing the trajectory of quantum dots once injected into the eye. Whole-body hyperspectral fluorescence imaging was performed in 17 live mice. In vivo imaging was conducted prior to injection, and 5, 20, 40 and 70 min, and 2, 6 and 24 h after injection. A quantum dot signal was observed in the left neck region at 6 h after tracer injection into the eye. Examination of immunofluorescence-labelled sections using confocal microscopy showed the presence of a quantum dot signal in the left submandibular lymph node. This is the first direct evidence of lymphatic drainage from the mouse eye. The use of quantum dots to image this lymphatic pathway in vivo is a novel tool to stimulate new treatments to reduce eye pressure and prevent blindness from glaucoma.

Quantum dots trace lymphatic drainage from the mouse eye

International Nuclear Information System (INIS)

Tam, Alex L C; Gupta, Neeru; Zhang Zhexue; Yuecel, Yeni H

2011-01-01

Glaucoma is a leading cause of blindness in the world, often associated with elevated eye pressure. Currently, all glaucoma treatments aim to lower eye pressure by improving fluid exit from the eye. We recently reported the presence of lymphatics in the human eye. The lymphatic circulation is known to drain fluid from organ tissues and, as such, lymphatics may also play a role in draining fluid from the eye. We investigated whether lymphatic drainage from the eye is present in mice by visualizing the trajectory of quantum dots once injected into the eye. Whole-body hyperspectral fluorescence imaging was performed in 17 live mice. In vivo imaging was conducted prior to injection, and 5, 20, 40 and 70 min, and 2, 6 and 24 h after injection. A quantum dot signal was observed in the left neck region at 6 h after tracer injection into the eye. Examination of immunofluorescence-labelled sections using confocal microscopy showed the presence of a quantum dot signal in the left submandibular lymph node. This is the first direct evidence of lymphatic drainage from the mouse eye. The use of quantum dots to image this lymphatic pathway in vivo is a novel tool to stimulate new treatments to reduce eye pressure and prevent blindness from glaucoma.

A label-free silicon quantum dots-based photoluminescence sensor for ultrasensitive detection of pesticides.

Science.gov (United States)

Yi, Yinhui; Zhu, Gangbing; Liu, Chang; Huang, Yan; Zhang, Youyu; Li, Haitao; Zhao, Jiangna; Yao, Shouzhuo

2013-12-03

Sensitive, rapid, and simple detection methods for the screening of extensively used organophosphorus pesticides and highly toxic nerve agents are in urgent demand. A novel label-free silicon quantum dots (SiQDs)-based sensor was designed for ultrasensitive detection of pesticides. This sensing strategy involves the reaction of acetylcholine chloride (ACh) with acetylcholinesterase (AChE) to form choline that is in turn catalytically oxidized by choline oxidase (ChOx) to produce betaine and H2O2 which can quench the photoluminescence (PL) of SiQDs. Upon the addition of pesticides, the activity of AChE is inhibited, leading to the decrease of the generated H2O2, and hence the PL of SiQDs increases. By measuring the increase in SiQDs PL, the inhibition efficiency of pesticide to AChE activity was evaluated. It was found that the inhibition efficiency was linearly dependent on the logarithm of the pesticides concentration. Consequently, pesticides, such as carbaryl, parathion, diazinon, and phorate, were determined with the SiQDs PL sensing method. The lowest detectable concentrations for carbaryl, parathion, diazinon, and phorate reached 7.25 × 10(-9), 3.25 × 10(-8), 6.76 × 10(-8), and 1.9 × 10(-7) g/L, respectively, which were much lower than those previously reported. The detecting results of pesticide residues in food samples via this method agree well with those from high-performance liquid chromatography. The simple strategy reported here should be suitable for on-site pesticides detection, especially in combination with other portable platforms.

Quadra-quantum Dots and Related Patterns of Quantum Dot Molecules:

Directory of Open Access Journals (Sweden)

Somsak Panyakeow

2010-10-01

Full Text Available Abstract Laterally close-packed quantum dots (QDs called quantum dot molecules (QDMs are grown by modified molecular beam epitaxy (MBE. Quantum dots could be aligned and cross hatched. Quantum rings (QRs created from quantum dot transformation during thin or partial capping are used as templates for the formations of bi-quantum dot molecules (Bi-QDMs and quantum dot rings (QDRs. Preferable quantum dot nanostructure for quantum computation based on quantum dot cellular automata (QCA is laterally close-packed quantum dot molecules having four quantum dots at the corners of square configuration. These four quantum dot sets are called quadra-quantum dots (QQDs. Aligned quadra-quantum dots with two electron confinements work like a wire for digital information transmission by Coulomb repulsion force, which is fast and consumes little power. Combination of quadra-quantum dots in line and their cross-over works as logic gates and memory bits. Molecular Beam Epitaxial growth technique called ‘‘Droplet Epitaxy” has been developed for several quantum nanostructures such as quantum rings and quantum dot rings. Quantum rings are prepared by using 20 ML In-Ga (15:85 droplets deposited on a GaAs substrate at 390°C with a droplet growth rate of 1ML/s. Arsenic flux (7–8×10-6Torr is then exposed for InGaAs crystallization at 200°C for 5 min. During droplet epitaxy at a high droplet thickness and high temperature, out-diffusion from the centre of droplets occurs under anisotropic strain. This leads to quantum ring structures having non-uniform ring stripes and deep square-shaped nanoholes. Using these peculiar quantum rings as templates, four quantum dots situated at the corners of a square shape are regrown. Two of these four quantum dots are aligned either or , which are preferable crystallographic directions of quantum dot alignment in general.

A review on syntheses, properties, characterization and bioanalytical applications of fluorescent carbon dots

International Nuclear Information System (INIS)

Zuo, Pengli; Lu, Xiuhua; Sun, Zhigang; Guo, Yuhan; He, Hua

2016-01-01

Carbon dots (C-dots) are a kind of fluorescent nanoparticles that are strongly fluorescent, non-blinking, and can be easily synthesized at low cost. Their emission color can be tuned by varying the excitation wavelength. Their properties make them strong competitors to semiconductor quantum dots. Synthetic approaches for C-dots can be classified into two categories, viz. top-down and bottom-up methods. Surface passivated and functionalized C-dots can be utilized to sense pH values, metal ions and organic molecules. Owing to their low cytotoxicity, biocompatibility and impressive photostability, long-term observations become possible. C-dots also show promise as labels and for bioimaging. This review (with 142 refs.) is divided into several sections. The first covers commonly used methods for preparation of C-dots including laser ablation, arc discharge, electrochemical methods, pyrolytic processes, template based methods, microwave assisted methods, chemical oxidation methods, reverse micelle based methods, etc. The first section also covers methods for surface functionalization and passivation. We continue by discussing the spectroscopic properties and other physical and chemical properties of C-dots (fluorescence, up-conversion fluorescence, methods for enhancing photoluminescence, effects of pH value, cytotoxicity, etc.). Another section covers the characterization including TEM and XRD. Applications in biology are summarized and subdivided into in vitro imaging, in vivo imaging, chemical probe, quantitation of biomacromolecules, but also in drug delivery, photoacoustic imaging and anticancer therapy. We finally discuss current challenges and perspectives in this promising field. (author)

Label-Free Potentiometry for Detecting DNA Hybridization Using Peptide Nucleic Acid and DNA Probes

Science.gov (United States)

Goda, Tatsuro; Singi, Ankit Balram; Maeda, Yasuhiro; Matsumoto, Akira; Torimura, Masaki; Aoki, Hiroshi; Miyahara, Yuji

2013-01-01

Peptide nucleic acid (PNA) has outstanding affinity over DNA for complementary nucleic acid sequences by forming a PNA-DNA heterodimer upon hybridization via Watson-Crick base-pairing. To verify whether PNA probes on an electrode surface enhance sensitivity for potentiometric DNA detection or not, we conducted a comparative study on the hybridization of PNA and DNA probes on the surface of a 10-channel gold electrodes microarray. Changes in the charge density as a result of hybridization at the solution/electrode interface on the self-assembled monolayer (SAM)-formed microelectrodes were directly transformed into potentiometric signals using a high input impedance electrometer. The charge readout allows label-free, reagent-less, and multi-parallel detection of target oligonucleotides without any optical assistance. The differences in the probe lengths between 15- to 22-mer dramatically influenced on the sensitivity of the PNA and DNA sensors. Molecular type of the capturing probe did not affect the degree of potential shift. Theoretical model for charged rod-like duplex using the Gouy-Chapman equation indicates the dominant effect of electrostatic attractive forces between anionic DNA and underlying electrode at the electrolyte/electrode interface in the potentiometry. PMID:23435052

Label-Free Potentiometry for Detecting DNA Hybridization Using Peptide Nucleic Acid and DNA Probes

Directory of Open Access Journals (Sweden)

Yuji Miyahara

2013-02-01

Full Text Available Peptide nucleic acid (PNA has outstanding affinity over DNA for complementary nucleic acid sequences by forming a PNA-DNA heterodimer upon hybridization via Watson-Crick base-pairing. To verify whether PNA probes on an electrode surface enhance sensitivity for potentiometric DNA detection or not, we conducted a comparative study on the hybridization of PNA and DNA probes on the surface of a 10-channel gold electrodes microarray. Changes in the charge density as a result of hybridization at the solution/electrode interface on the self-assembled monolayer (SAM-formed microelectrodes were directly transformed into potentiometric signals using a high input impedance electrometer. The charge readout allows label-free, reagent-less, and multi-parallel detection of target oligonucleotides without any optical assistance. The differences in the probe lengths between 15- to 22-mer dramatically influenced on the sensitivity of the PNA and DNA sensors. Molecular type of the capturing probe did not affect the degree of potential shift. Theoretical model for charged rod-like duplex using the Gouy-Chapman equation indicates the dominant effect of electrostatic attractive forces between anionic DNA and underlying electrode at the electrolyte/electrode interface in the potentiometry.

Novel aspects of application of cadmium telluride quantum dots nanostructures in radiation oncology

Science.gov (United States)

Fazaeli, Yousef; Zare, Hakimeh; Karimi, Shokufeh; Rahighi, Reza; Feizi, Shahzad

2017-08-01

In the last two decades, quantum dots nanomaterials have garnered a great deal of scientific interest because of their unique properties. Quantum dots (QDs) are inorganic fluorescent nanocrystals in the size range between 1 and 20 nm. Due to their structural properties, they possess distinctive properties and behave in different way from crystals in macro scale, in many branches of human life. Cadmium telluride quantum dots (CdTe QDs) were labeled with 68Ga radio nuclide for fast in vivo targeting and coincidence imaging of tumors. Using instant paper chromatography, the physicochemical properties of the Cadmium telluride quantum dots labeled with 68Ga NPs (68Ga@ CdTe QDs) were found high enough stable in organic phases, e.g., a human serum, to be reliably used in bioapplications. In vivo biodistribution of the 68Ga@ CdTe QDs nanoconposite was investigated in rats bearing fibro sarcoma tumor after various post-injection periods of time. The 68Ga NPs exhibited a rapid as well as high tumor uptake in a very short period of time (less than 10 min), resulting in an efficient tumor targeting/imaging agent. Meantime, the low lipophilicity of the 68Ga NPs caused to their fast excretion throughout the body by kidneys (as also confirmed by the urinary tract). Because of the short half-life of 68Ga radionuclide, the 68Ga@ CdTe QDs with an excellent tumor targeting/imaging and fast washing out from the body can be suggested as one of the most effective and promising nanomaterials in nanotechnology-based cancer diagnosis and therapy.

Peptide receptor radionuclide therapy with {sup 90}Y/{sup 177}Lu-labelled peptides for inoperable head and neck paragangliomas (glomus tumours)

Energy Technology Data Exchange (ETDEWEB)

Puranik, Ameya D.; Kulkarni, Harshad R.; Singh, Aviral; Baum, Richard P. [Zentralklinik Bad Berka, THERANOSTICS Centre for Molecular Radiotherapy and Molecular Imaging, ENETS Center of Excellence, Bad Berka (Germany)

2015-07-15

Head and neck paragangliomas (HNPGLs) are rare tumours arising from autonomic nervous system ganglia. Although surgery offers the best chance of complete cure, there is associated morbidity due to the crucial location of these tumours. Radiotherapy arrests tumour growth and provides symptomatic improvement, but has long-term consequences. These tumours express somatostatin receptors (SSTR) and hence peptide receptor radionuclide therapy (PRRT) is now a treatment option. We assessed the molecular, morphological and clinical responses of inoperable HNPGLs to PRRT. Nine patients with inoperable HNPGL assessed between June 2006 and June 2014 were included. Four patients had a solitary lesion, four had multifocal involvement and one had distant metastases (bone and lungs). The patients were treated with PRRT using {sup 90}Y/{sup 177}Lu-labelled peptides after positive confirmation of SSTR expression on {sup 68}Ga-DOTATOC PET/CT. All patients received two to four courses of PRRT. Subsequent serial imaging with {sup 68}Ga-DOTATOC PET/CT was carried out every 6 months to assess response to treatment. Clinical (symptomatic) response was also assessed. Based on molecular response (EORTC) criteria, four of the nine patients showed a partial molecular response to treatment seen as significant decreases in SUV{sub max}, accompanied by a reduction in tumour size. Five patients showed stable disease on both molecular and morphological criteria. Six out of nine patients were symptomatic at presentation with manifestations of cranial nerve involvement, bone destruction at the primary site and metastatic bone pain. Molecular responses were correlated with symptomatic improvement in four out of these six patients; while two patients showed small reductions in tumour size and SUV{sub max}. The three asymptomatic patients showed no new lesions or symptomatic worsening. PRRT was effective in all patients, with no disease worsening seen, either in the form of neurological symptoms or

Improved Immunoassay Sensitivity in Serum as a Result of Polymer-Entrapped Quantum Dots: 'Papaya Particles'

NARCIS (Netherlands)

Ranzoni, A.; den Hamer, A.; Karoli, T.; Buechler, J.; Cooper, M.A.

2015-01-01

Fluorescent labels are widely employed in biomarker quantification and diagnostics, however they possess narrow Stokes shifts and can photobleach, limiting multiplexed detection applications and compromising sensitivity. In contrast, quantum dots do not photobleach and have much wider Stokes shifts,

Fluorescent CdSe/ZnS nanocrystal-peptide conjugates for long-term, nontoxic imaging and nuclear targeting in living cells

International Nuclear Information System (INIS)

Chen, Fanqing; Gerion, Daniele

2004-01-01

One of the biggest challenges in cell biology is the imaging of living cells. For this purpose, the most commonly used visualization tool is fluorescent markers. However, conventional labels, such as organic fluorescent dyes or green fluorescent proteins (GFP), lack the photostability to allow the tracking of cellular events that happen over minutes to days. In addition, they are either toxic to cells (dyes), or difficult to construct and manipulate (GFP). We report here the use of a new class of fluorescent labels, silanized CdSe/ZnS nanocrystal-peptide conjugates, for imaging the nuclei of living cells. CdSe/ZnS nanocrystals, or so called quantum dots (qdots), are extremely photostable, and have been used extensively in cellular imaging of fixed cells. However, most of the studies about living cells so far have been concerned only with particle entry into the cytoplasm or the localization of receptors on the cell membrane. Specific targeting of qdots to the nucleus of living cells ha s not been reported in previous studies, due to the lack of a targeting mechanism and proper particle size. Here we demonstrate for the first time the construction of a CdSe/ZnS nanocrystal-peptide conjugate that carries the SV40 large T antigen nuclear localization signal (NLS), and the transfection of the complex into living cells. By a novel adaptation of commonly used cell transfection techniques for qdots, we were able to introduce and retain the NLS-qdots conjugate in living cells for up to a week without detectable negative cellular effects. Moreover, we can visualize the movement of the CdSe/ZnS nanocrystal-peptide conjugates from cytoplasm to the nucleus, and the accumulation of the complex in the cell nucleus, over a long observation time period. This report opens the door for using qdots to visualize long-term biological events that happen in the cell nucleus, and provides a new nontoxic, long-term imaging platform for cell nuclear processes

A novel route to radioiodinated [{sup 123}I]-N-succinimidyl-3-iodobenzoate, a reagent for radioiodination of bioactive peptides

Energy Technology Data Exchange (ETDEWEB)

Al-Jammaz, I.; Al-Otaibi, B.; Amartey, J.K. E-mail: amarty@kfshrc.edu.sa

2002-11-01

Radiolabeled peptides continue to emerge as potential radiopharmaceuticals for targeting several diseases such as cancer, infection and inflammation and even tissue and organ rejection. The classical method for labeling these molecules has been the electrophilic route. Evidence suggests that most molecules labeled via this route perturb their biological activity. Moreover, this method is not applicable to peptides lacking a tyrosine moiety in their structure. Hence, there is the need to develop alternate methods such as the prosthetic approach. We have optimized a solid-state radioiodination by exchange to produce [{sup 123}I]-metaiodobenzylguanidine ([{sup 123}I]-mIBG). The mIBG served as a precursor to obtain an activated N-succinimidyl ester for efficient coupling to amine functions in peptides, preferably the lysine group(s). The method was used to label a model chemotactic peptide and evaluated in vivo.

Quadra-Quantum Dots and Related Patterns of Quantum Dot Molecules: Basic Nanostructures for Quantum Dot Cellular Automata Application

Directory of Open Access Journals (Sweden)

Somsak Panyakeow

2010-10-01

Full Text Available Laterally close-packed quantum dots (QDs called quantum dot molecules (QDMs are grown by modified molecular beam epitaxy (MBE. Quantum dots could be aligned and cross hatched. Quantum rings (QRs created from quantum dot transformation during thin or partial capping are used as templates for the formations of bi-quantum dot molecules (Bi-QDMs and quantum dot rings (QDRs. Preferable quantum dot nanostructure for quantum computation based on quantum dot cellular automata (QCA is laterally close-packed quantum dot molecules having four quantum dots at the corners of square configuration. These four quantum dot sets are called quadra-quantum dots (QQDs. Aligned quadra-quantum dots with two electron confinements work like a wire for digital information transmission by Coulomb repulsion force, which is fast and consumes little power. Combination of quadra-quantum dots in line and their cross-over works as logic gates and memory bits. Molecular Beam Epitaxial growth technique called 'Droplet Epitaxy' has been developed for several quantum nanostructures such as quantum rings and quantum dot rings. Quantum rings are prepared by using 20 ML In-Ga (15:85 droplets deposited on a GaAs substrate at 390'C with a droplet growth rate of 1ML/s. Arsenic flux (7'8'10-6Torr is then exposed for InGaAs crystallization at 200'C for 5 min. During droplet epitaxy at a high droplet thickness and high temperature, out-diffusion from the centre of droplets occurs under anisotropic strain. This leads to quantum ring structures having non-uniform ring stripes and deep square-shaped nanoholes. Using these peculiar quantum rings as templates, four quantum dots situated at the corners of a square shape are regrown. Two of these four quantum dots are aligned either or, which are preferable crystallographic directions of quantum dot alignment in general.

Peptides in headlock--a novel high-affinity and versatile peptide-binding nanobody for proteomics and microscopy.

Science.gov (United States)

Braun, Michael B; Traenkle, Bjoern; Koch, Philipp A; Emele, Felix; Weiss, Frederik; Poetz, Oliver; Stehle, Thilo; Rothbauer, Ulrich

2016-01-21

Nanobodies are highly valuable tools for numerous bioanalytical and biotechnical applications. Here, we report the characterization of a nanobody that binds a short peptide epitope with extraordinary affinity. Structural analysis reveals an unusual binding mode where the extended peptide becomes part of a β-sheet structure in the nanobody. This interaction relies on sequence-independent backbone interactions augmented by a small number of specificity-determining side chain contacts. Once bound, the peptide is fastened by two nanobody side chains that clamp it in a headlock fashion. Exploiting this unusual binding mode, we generated a novel nanobody-derived capture and detection system. Matrix-coupled nanobody enables the fast and efficient isolation of epitope-tagged proteins from prokaryotic and eukaryotic expression systems. Additionally, the fluorescently labeled nanobody visualizes subcellular structures in different cellular compartments. The high-affinity-binding and modifiable peptide tag of this system renders it a versatile and robust tool to combine biochemical analysis with microscopic studies.

Photoaffinity labeling of myosin subfragment-one-with 3'(2')-O-(4-benzoyl)benzoyl adenosine 5'-triphosphate

International Nuclear Information System (INIS)

Mahmood, R.

1985-01-01

The photoaffinity analogue 3'(2')-O-(4-benzoyl)benzoyl adenosine 5'-triphosphate (Bz 2 ATP) contains the photoreactive benzophenone group esterified at the 2' or 3' hydroxyl groups of ribose. MgBz 2 ADP has a single binding site on skeletal myosin chymotryptic subfragment-one (SF 1 ) with a binding constant of 3.2 x 10 5 M -1 . Bz 2 ATP is also a substrate for the ATPase activity of SF 1 in the presence of different cations. The irradiation of SF 1 with [ 3 H]Bz 2 ATP photoinactivates the ATPase activity with concomitant incorporation of the analogue into the enzyme. Polyacrylamide gel electrophoresis of photolabeled SF 1 after milk trypsin digestion shows that all three tryptic peptides, 25 K, 50K, and 20 K, and both light chains are labeled. The presence of ATP during irradiation reduces labeling of the 50 K peptide only indicating that the other peptides are non-specifically labeled. To reduce the non-specific labeling [ 3 H]Bz 2 ATP is trapped on SF 1 by cross-linking the two reactive thiols, SH 1 and SH 2 , by N,N'-p-phenylene dimaleimide or Co(II)/Co(III) phenanthroline complexes. The Co(II)/Co(III) phenanthroline modified [ 14 C]Bz 2 ATP-SF 1 , after proteolytic digestion, yields five labeled peptides which were purified by gel filtration and high performance liquid chromatography

The preparation and identification of peptide imaging agent of lung cancer

International Nuclear Information System (INIS)

Chu Liping; Wang Yan; Wang Yueying; Liu Jinjian; Wu Hongying; Liu Jianfeng

2010-01-01

Objective: To screen in vivo lung cancer specific binding 7-peptide from T7 phage display random peptide library and prepare peptide imaging agent in early in early diagnosis of lung cancer. Methods: Used phage display in vivo technology to get the 7-peptide phage that can bind the lung cancer specifically, then sequenced and synthesized 7-peptide. After being labeled by 125 I, this 7-peptide was injected into mice via vein and the distribution in the mice tumor mold was observed. Results: One 7-peptide was obtained after four rounds of screening, and the peptide could bind lung cancer tissue specifically. Metabolism of this peptide in mice was fast and imaging of lung cancer was best two hours later after injection. The distribution in vivo decreased and almost disappeared after six hours. Conclusion: This 7-peptide could be used to image and diagnose of lung cancer effectively. (authors)

Simple Identification of Human Taenia Species by Multiplex Loop-Mediated Isothermal Amplification in Combination with Dot Enzyme-Linked Immunosorbent Assay

Science.gov (United States)

Nkouawa, Agathe; Sako, Yasuhito; Okamoto, Munehiro; Ito, Akira

2016-01-01

For differential detection of Taenia solium, Taenia saginata, and Taenia asiatica, loop-mediated isothermal amplification (LAMP) assay targeting the cytochrome c oxidase subunit 1 gene has been recently developed and shown to be sensitive, specific, and effective. However, to achieve differential identification, one specimen requires three reaction mixtures containing a primer set of each Taenia species separately, which is complex and time consuming and increases the risk of cross-contamination. In this study, we developed a simple differential identification of human Taenia species using multiplex LAMP (mLAMP) in combination with dot enzyme-linked immunosorbent assay (dot-ELISA). Forward inner primers of T. solium, T. saginata, and T. asiatica labeled with fluorescein isothiocyanate (FITC), digoxigenin (DIG), and tetramethylrhodamine (TAMRA), respectively, and biotin-labeled backward inner primers were used in mLAMP. The mLAMP assay succeeded in specific amplification of each respective target gene in a single tube. Furthermore, the mLAMP product from each species was easily distinguished by dot-ELISA with an antibody specific for FITC, DIG, or TAMRA. The mLAMP assay in combination with dot-ELISA will make identification of human Taenia species simpler, easier, and more practical. PMID:27044566

Photodissociative Cross-Linking of Non-covalent Peptide-Peptide Ion Complexes in the Gas Phase

Science.gov (United States)

Nguyen, Huong T. H.; Andrikopoulos, Prokopis C.; Rulíšek, Lubomír; Shaffer, Christopher J.; Tureček, František

2018-05-01

We report a gas-phase UV photodissociation study investigating non-covalent interactions between neutral hydrophobic pentapeptides and peptide ions incorporating a diazirine-tagged photoleucine residue. Phenylalanine (Phe) and proline (Pro) were chosen as the conformation-affecting residues that were incorporated into a small library of neutral pentapeptides. Gas-phase ion-molecule complexes of these peptides with photo-labeled pentapeptides were subjected to photodissociation. Selective photocleavage of the diazirine ring at 355 nm formed short-lived carbene intermediates that underwent cross-linking by insertion into H-X bonds of the target peptide. The cross-link positions were established from collision-induced dissociation tandem mass spectra (CID-MS3) providing sequence information on the covalent adducts. Effects of the amino acid residue (Pro or Phe) and its position in the target peptide sequence were evaluated. For proline-containing peptides, interactions resulting in covalent cross-links in these complexes became more prominent as proline was moved towards the C-terminus of the target peptide sequence. The photocross-linking yields of phenylalanine-containing peptides depended on the position of both phenylalanine and photoleucine. Density functional theory calculations were used to assign structures of low-energy conformers of the (GLPMG + GLL*LK + H)+ complex. Born-Oppenheimer molecular dynamics trajectory calculations were used to capture the thermal motion in the complexes within 100 ps and determine close contacts between the incipient carbene and the H-X bonds in the target peptide. This provided atomic-level resolution of potential cross-links that aided spectra interpretation and was in agreement with experimental data. [Figure not available: see fulltext.

Effects of amino acids on melanoma targeting and clearance properties of Tc-99m-labeled Arg-X-Asp-conjugated α-melanocyte stimulating hormone peptides.

Science.gov (United States)

Flook, Adam M; Yang, Jianquan; Miao, Yubin

2013-11-14

The purpose of this study was to examine the effects of amino acids on melanoma targeting and clearance properties of new (99m)Tc-labeled Arg-X-Asp-conjugated α-melanocyte stimulating hormone (α-MSH) peptides. RSD-Lys-(Arg(11))CCMSH {c[Arg-Ser-Asp-DTyr-Asp]-Lys-Cys-Cys-Glu-His-dPhe-Arg-Trp-Cys-Arg-Pro-Val-NH2}, RNleD-Lys-(Arg(11))CCMSH, RPheD-Lys-(Arg(11))CCMSH, and RdPheD-Lys-(Arg(11))CCMSH peptides were synthesized and evaluated for their melanocortin-1 (MC1) receptor binding affinities in B16/F1 melanoma cells. The biodistribution of (99m)Tc-RSD-Lys-(Arg(11))CCMSH, (99m)Tc-RFD-Lys-(Arg(11))CCMSH, and (99m)Tc-RfD-Lys-(Arg(11))CCMSH were determined in B16/F1 melanoma-bearing C57 mice. The substitution of Gly with Ser, Phe, and dPhe increased the MC1 receptor binding affinities of the peptides, whereas the substitution of Gly with Nle decreased the MC1 receptor binding affinity of the peptide. (99m)Tc-RSD-Lys-(Arg(11))CCMSH exhibited the highest melanoma uptake (18.01 ± 4.22% ID/g) and the lowest kidney and liver uptake among these (99m)Tc-peptides. The B16/F1 melanoma lesions could be clearly visualized by SPECT/CT using (99m)Tc-RSD-Lys-(Arg(11))CCMSH as an imaging probe. It is desirable to reduce the renal uptake of (99m)Tc-RSD-Lys-(Arg(11))CCMSH to facilitate its potential therapeutic application.

Symmetric analysis, categorization, and optical spectrum of ideal pyramid quantum dots

Science.gov (United States)

Li, Wei; Belling, Samuel W.

2017-11-01

Self-assembled quantum dots possess an intrinsic geometric symmetry. Applying group representation theory, we systematically analyze the symmetric properties of the bound states for ideal pyramid quantum dots, which neglect band mixing and strain effects. We label each bound state by its symmetry group’s corresponding irreducible representation and define a concept called the quantum dots’ symmetry category. A class of quantum dots with the same irreducible representation sequence of bound states are characterized as belonging to a specific symmetry category. This category concept generally describes the symmetric order of Hilbert space or wavefunction space. We clearly identify the connection between the symmetry category and the geometry of quantum dots by the symmetry category graph or map. The symmetry category change or transition corresponds to an accidental degeneracy of the bound states. The symmetry category and category transition are observable from the photocurrent spectroscopy or optical spectrum. For simplicity’s sake, in this paper, we only focus on inter-subband transition spectra, but the methodology can be extended to the inter-band transition cases. We predict that from the spectral measurements, the quantum dots’ geometric information may be inversely extracted.

Design and evaluation of new Tc-99m-labeled lactam bridge-cyclized alpha-MSH peptides for melanoma imaging.

Science.gov (United States)

Guo, Haixun; Gallazzi, Fabio; Miao, Yubin

2013-04-01

The purpose of this study was to examine the melanoma targeting and imaging properties of new (99m)Tc-labeled lactam bridge-cyclized alpha-melanocyte stimulating hormone (α-MSH) peptides using bifunctional chelating agents. MAG3-GGNle-CycMSH(hex), AcCG3-GGNle-CycMSH(hex), and HYNIC-GGNle-CycMSH(hex) peptides were synthesized, and their melanocortin-1 (MC1) receptor binding affinities were determined in B16/F1 melanoma cells. The biodistribution of (99m)Tc-MAG3-GGNle-CycMSH(hex), (99m)Tc-AcCG3-GGNle-CycMSH(hex), (99m)Tc(CO)3-HYNIC-GGNle-CycMSH(hex), and (99m)Tc(EDDA)-HYNIC-GGNle-CycMSH(hex) were determined in B16/F1 melanoma-bearing C57 mice at 2 h postinjection to select a lead peptide for further evaluation. The melanoma targeting and imaging properties of (99m)Tc(EDDA)-HYNIC-GGNle-CycMSH(hex) were further examined because of its high melanoma uptake and fast urinary clearance. The IC50 values of MAG3-GGNle-CycMSH(hex), AcCG3-GGNle-CycMSH(hex), and HYNIC-GGNle-CycMSH(hex) were 1.0 ± 0.05, 1.2 ± 0.19, and 0.6 ± 0.04 nM in B16/F1 melanoma cells, respectively. Among these four (99m)Tc-peptides, (99m)Tc(EDDA)-HYNIC-GGNle-CycMSH(hex) exhibited the highest melanoma uptake (14.14 ± 4.90% ID/g) and fastest urinary clearance (91.26 ± 1.96% ID) at 2 h postinjection. (99m)Tc(EDDA)-HYNIC-GGNle-CycMSH(hex) showed high tumor to normal organ uptake ratios except for the kidneys. The tumor/kidney uptake ratios of (99m)Tc(EDDA)-HYNIC-GGNle-CycMSH(hex) were 2.50 and 3.55 at 4 and 24 h postinjection. The melanoma lesions were clearly visualized by SPECT/CT using (99m)Tc(EDDA)-HYNIC-GGNle-CycMSH(hex) as an imaging probe at 2 h postinjection. Overall, high melanoma uptake coupled with fast urinary clearance of (99m)Tc(EDDA)-HYNIC-GGNle-CycMSH(hex) highlighted its potential for metastatic melanoma detection in the future.

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

Optimization of iTRAQ labelling coupled to OFFGEL fractionation as a proteomic workflow to the analysis of microsomal proteins of Medicago truncatula roots

Directory of Open Access Journals (Sweden)

Abdallah Cosette

2012-06-01

Full Text Available Abstract Background Shotgun proteomics represents an attractive technical framework for the study of membrane proteins that are generally difficult to resolve using two-dimensional gel electrophoresis. The use of iTRAQ, a set of amine-specific isobaric tags, is currently the labelling method of choice allowing multiplexing of up to eight samples and the relative quantification of multiple peptides for each protein. Recently the hyphenation of different separation techniques with mass spectrometry was used in the analysis of iTRAQ labelled samples. OFFGEL electrophoresis has proved its effectiveness in isoelectric point-based peptide and protein separation in solution. Here we describe the first application of iTRAQ-OFFGEL-LC-MS/MS on microsomal proteins from plant material. The investigation of the iTRAQ labelling effect on peptide electrofocusing in OFFGEL fractionator was carried out on Medicago truncatula membrane protein digests. Results In-filter protein digestion, with easy recovery of a peptide fraction compatible with iTRAQ labelling, was successfully used in this study. The focusing quality in OFFGEL electrophoresis was maintained for iTRAQ labelled peptides with a higher than expected number of identified peptides in basic OFFGEL-fractions. We furthermore observed, by comparing the isoelectric point (pI fractionation of unlabelled versus labelled samples, a non-negligible pI shifts mainly to higher values. Conclusions The present work describes a feasible and novel protocol for in-solution protein digestion in which the filter unit permits protein retention and buffer removal. The data demonstrates an impact of iTRAQ labelling on peptide electrofocusing behaviour in OFFGEL fractionation compared to their native counterpart by the induction of a substantial, generally basic pI shift. Explanations for the occasionally observed acidic shifts are likewise presented.

Imaging and Manipulating Energy Transfer Among Quantum Dots at Individual Dot Resolution.

Science.gov (United States)

Nguyen, Duc; Nguyen, Huy A; Lyding, Joseph W; Gruebele, Martin

2017-06-27

Many processes of interest in quantum dots involve charge or energy transfer from one dot to another. Energy transfer in films of quantum dots as well as between linked quantum dots has been demonstrated by luminescence shift, and the ultrafast time-dependence of energy transfer processes has been resolved. Bandgap variation among dots (energy disorder) and dot separation are known to play an important role in how energy diffuses. Thus, it would be very useful if energy transfer could be visualized directly on a dot-by-dot basis among small clusters or within films of quantum dots. To that effect, we report single molecule optical absorption detected by scanning tunneling microscopy (SMA-STM) to image energy pooling from donor into acceptor dots on a dot-by-dot basis. We show that we can manipulate groups of quantum dots by pruning away the dominant acceptor dot, and switching the energy transfer path to a different acceptor dot. Our experimental data agrees well with a simple Monte Carlo lattice model of energy transfer, similar to models in the literature, in which excitation energy is transferred preferentially from dots with a larger bandgap to dots with a smaller bandgap.

Alpha radioisotopes Ac-225 and Bi-213: a production and labelling of antibodies and peptides for clinical use

Energy Technology Data Exchange (ETDEWEB)

Bruchertseifer, Frank, E-mail: frank.bruchertseifer@ec.europa.eu [European Commission, Joint Research Centre, Karlsruhe (Germany)

2017-07-01

Full text: In various preclinical and clinical works the potential of the alpha emitters {sup 225}Ac and {sup 213}Bi as therapeutic radionuclides for application in targeted alpha therapy of cancer and infectious diseases was demonstrated. Both alpha emitters are available with high specific activity from established radionuclide generators. Their favorable chemical and physical properties have led to the conduction of a large number of preclinical studies and several clinical trials, demonstrating the feasibility, safety and therapeutic efficacy of targeted alpha therapy with {sup 225}Ac and {sup 213}Bi. This presentation will give an overview about the methods for the production of {sup 225}Ac and {sup 213}Bi, the {sup 225}Ac/{sup 213}Bi radionuclide generator systems, labelling of peptides and antibodies with {sup 225}Ac and {sup 213}Bi and relevant in vivo and in vitro works. (author)

Detection and correction of blinking bias in image correlation transport measurements of quantum dot tagged macromolecules

DEFF Research Database (Denmark)

Durisic, Nela; Bachir, Alexia I; Kolin, David L

2007-01-01

Semiconductor nanocrystals or quantum dots (QDs) are becoming widely used as fluorescent labels for biological applications. Here we demonstrate that fluorescence fluctuation analysis of their diffusional mobility using temporal image correlation spectroscopy is highly susceptible to systematic e...

Biological Evaluation of 99mTc-HYNIC-EDDA/tricine-(Ser)-D4 Peptide for Tumor Targeting.

Science.gov (United States)

Kazemi, Ziba; Zahmatkesh, Mona Haddad; Abedi, Seyed Mohammad; Hosseinimehr, Seyed Jalal

2017-08-24

D4 small peptide (Leu-Ala-Arg-Leu-Leu-Thr) was selected as an appropriate agent for specific targeting of epidermal growth factor receptor (EGFR). The aim of study was to investigate the 99mTc-labeled D4 peptide for non-small cell lung tumor targeting. HYNIC-(Ser)3-D4 peptide was labeled with 99mTc using mixture of tricine and ethylenediamine diacetic acid (EDDA) as co-ligands. The in vitro cellular uptake of radiolabeled peptide was evaluated by blocking test on human non-small cell lung cancer (A-549) cell line and its biodistribution was evaluated in A-549 xenografted nude mice. This conjugated peptide was labeled with 99mTc in high radiochemical purity and it was highly stable in buffer and serum. The un-blocked to blocked cellular radioactivity ratio was 4- fold that showed a specific binding of this radiolabeled peptide on A-549 cell. Animal biodistribution in A-549 xenografted nude mice showed rapid clearance from blood and other non-target organs. Tumor uptake values as %ID/g (percentage of injection dose per gram of tissue) were 2.47% and 1.30% at 1 and 4 h after injection. This study showed the 99mTc-EDDA/tricine-HYNIC-(Ser)3-D4 peptide had tumor targeting on the non-small cell lung tumor. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Development of novel radiogallium-labeled bone imaging agents using oligo-aspartic acid peptides as carriers.

Directory of Open Access Journals (Sweden)

Kazuma Ogawa

Full Text Available (68Ga (T 1/2 = 68 min, a generator-produced nuclide has great potential as a radionuclide for clinical positron emission tomography (PET. Because poly-glutamic and poly-aspartic acids have high affinity for hydroxyapatite, to develop new bone targeting (68Ga-labeled bone imaging agents for PET, we used 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA as a chelating site and conjugated aspartic acid peptides of varying lengths. Subsequently, we compared Ga complexes, Ga-DOTA-(Aspn (n = 2, 5, 8, 11, or 14 with easy-to-handle (67Ga, with the previously described (67Ga-DOTA complex conjugated bisphosphonate, (67Ga-DOTA-Bn-SCN-HBP. After synthesizing DOTA-(Aspn by a Fmoc-based solid-phase method, complexes were formed with (67Ga, resulting in (67Ga-DOTA-(Aspn with a radiochemical purity of over 95% after HPLC purification. In hydroxyapatite binding assays, the binding rate of (67Ga-DOTA-(Aspn increased with the increase in the length of the conjugated aspartate peptide. Moreover, in biodistribution experiments, (67Ga-DOTA-(Asp8, (67Ga-DOTA-(Asp11, and (67Ga-DOTA-(Asp14 showed high accumulation in bone (10.5 ± 1.5, 15.1 ± 2.6, and 12.8 ± 1.7% ID/g, respectively but were barely observed in other tissues at 60 min after injection. Although bone accumulation of (67Ga-DOTA-(Aspn was lower than that of (67Ga-DOTA-Bn-SCN-HBP, blood clearance of (67Ga-DOTA-(Aspn was more rapid. Accordingly, the bone/blood ratios of (67Ga-DOTA-(Asp11 and (67Ga-DOTA-(Asp14 were comparable with those of (67Ga-DOTA-Bn-SCN-HBP. In conclusion, these data provide useful insights into the drug design of (68Ga-PET tracers for the diagnosis of bone disorders, such as bone metastases.

Radio peptide imaging and therapy

International Nuclear Information System (INIS)

Buscombe, Jonh

1997-01-01

Full text. The concept of the magic bullet retains its attraction to us. If only we could take a drug or radioisotope and inject this intravenously and then will attach to the target cancer. This may allow imaging if labelled with a radio pharmaceutical or possibly even effective therapy. Initially work was started using antibodies of mouse origin. These have shown some utility in targeting tumors but there are problems in that these are essentially non-human proteins, often derived from mice. This leads to the formation of antibodies against that antibody so that repeat administrations lead to reduced efficacy and possibly may carry a risk anaphylaxis for the patient. Two different methods have evolved to deal with this situation. Either make antibodies more human or use smaller fragments, so that they are less likely to cause allergic reactions. The second method is to try and use a synthetic peptide. This will contain a series of amino acids which recognize a certain cell receptor. For example the somatostatin analogue Octreotide is an 8 amino acid peptide which has the same biological actions as natural somatostatin but an increased plasma half life. To this is added a linker a good example being DTPA and then radioisotope for example In-111. There we can have the complex In-111-DTPA-Octreotide which can be used to image somatostatin receptors in vivo. The main advantage over antibodies is that the cost production is less and many different variation of peptides for a particular receptor can be manufactured and assessed to find which is the optimal agent tumour imaging at a fraction of the cost of antibody production. There are two main approaches. Firstly to take a natural peptide hormone such as insulin or VIP and label by a simple method such as iodination with I-123. A group in Vienna have done it and shown good uptake of I-123 Insulin in primary hepatomas and of I-123 VIP in pancreatic cancers. Many natural peptide hormones however have a short plasma half

Labeling of DOTATATE with 131-iodine for therapy application

International Nuclear Information System (INIS)

Araujo, E.B.; Nagamati, L.T.; Caldeira Filho, J.S.; Colturato, M.T.; Silva, C.P.G. da

2004-01-01

Full text: Peptide receptor radiotherapy (PRRT) and peptide receptor imaging (PRI) of malignant neoplasms have become a primary focus of interest in nuclear medicine. [111In]-DTPA-D-Phe1-octreotide is routinely used as diagnostic tool and promising therapeutic results have been reported with [90Y] DOTA-Tyr3-octreotide in patients with somatostatin (sst) receptor-positive advanced tumours. The radio-iodinated analogue, [123I] Tyr3-octreotide was the first sst-directed radiotracer to be clinically evaluated. The diagnostic and therapeutic usefulness of radio-iodinated sst ligands has been limited by their unfavourable biokinetics, in vivo deiodination and resulting dosimetry. The radio-iodination of sst derivatives is often time-consuming multi step procedure and needs final product purification. However, comparative studies with the radioiodinated sst analogues Tyr3-octreotide and Tyr3-Thr8-octreotide (octreotate) showed that the substitution of Thr(ol)8 by Thr8 reduces the lipophilicity and also dramatically improves the biodistribution in nude mice bearing AR42J rat pancreatic tumour xenografts. Favourable pharmacokinetic of DOTA-Tyr3-octreotate labeled with 90Y and 177Lu was observed, including rapid renal clearance and high focal uptake in sst receptor positive tumors. This work studied the labelling of DOTA-Tyr3-octreotate (Pichem) with 131-iodine (Nordion/CNEN - 2.9 x 1016 Bq/mol), quality control and purification procedures to evaluate the production viability of 131I-labeled sst analogue in radiotherapeutic amounts. 131I radiolabeling of DOTA-Tyr3-octreotate was performed using the Chloramine T method. A solution of 1.5-10 mg of peptide in 40 ml of PBS (0.1M phosphate buffered saline pH 7.5) was transferred to an Eppendorf. After the addition of 5 ml of Chloramine T solution (5 mg/PBS) and 5-10 ml of radioiodine solution (37-740 MBq, molar peptide to radionuclide ratios varying from 0.8 to 45), the cap was carefully vortexed and the labelling reaction was

Non-invasive glucagon-like peptide-1 receptor imaging in pancreas with {sup 18}F-Al labeled Cys{sup 39}-exendin-4

Energy Technology Data Exchange (ETDEWEB)

Mi, Baoming [Department of Nuclear Medicine, The First Affiliated Hospital of Soochow University, Suzhou, Jiangsu, 215006 (China); Department of Nuclear Medicine, Affiliated Hospital of Jiangnan University (Wuxi 4th People' s Hospital), Wuxi, Jiangsu, 214062 (China); Xu, Yuping [Key Laboratory of Nuclear Medicine, Ministry of Health, Jiangsu Key Laboratory of Molecular Nuclear Medicine, Jiangsu Institute of Nuclear Medicine, Wuxi, Jiangsu, 214063 (China); Nanjing Medical University, Nanjing, Jiangsu, 210029 (China); Pan, Donghui; Wang, Lizhen; Yang, Runlin [Key Laboratory of Nuclear Medicine, Ministry of Health, Jiangsu Key Laboratory of Molecular Nuclear Medicine, Jiangsu Institute of Nuclear Medicine, Wuxi, Jiangsu, 214063 (China); Yu, Chunjing; Wan, Weixing [Department of Nuclear Medicine, Affiliated Hospital of Jiangnan University (Wuxi 4th People' s Hospital), Wuxi, Jiangsu, 214062 (China); Wu, Yiwei, E-mail: wuyiwei3988@gmail.com [Department of Nuclear Medicine, The First Affiliated Hospital of Soochow University, Suzhou, Jiangsu, 215006 (China); Yang, Min, E-mail: ymzfk@yahoo.com.hk [Key Laboratory of Nuclear Medicine, Ministry of Health, Jiangsu Key Laboratory of Molecular Nuclear Medicine, Jiangsu Institute of Nuclear Medicine, Wuxi, Jiangsu, 214063 (China); Nanjing Medical University, Nanjing, Jiangsu, 210029 (China)

2016-02-26

Purpose: Glucagon-like peptide-1 receptor (GLP-1R) is abundantly expressed on beta cells and may be an ideal target for the pancreas imaging. Monitoring the GLP-1R of pancreas could be benefit for understanding the pathophysiology of diabetes. In the present study, {sup 18}F-Al labeled exendin-4 analog, {sup 18}F-Al-NOTA-MAL-Cys{sup 39}-exendin-4, was evaluated for PET imaging GLP-1R in the pancreas. Methods: The targeting of {sup 18}F-Al labeled exendin-4 analog was examined in healthy and streptozotocin induced diabetic rats. Rats were injected with {sup 18}F-Al-NOTA-MAL-Cys{sup 39}-exendin-4 and microPET imaging was performed at 1 h postinjection, followed by ex vivo biodistribution. GLP-1R expression in pancreas was determined through post mortern examinations. Results: The pancreas of healthy rats was readily visualized after administration of {sup 18}F-Al-NOTA-MAL-Cys{sup 39}-exendin-4, whereas the pancreas of diabetic rats, as well as those from rats co-injected with excess of unlabeled peptides, was barely visible by microPET. At 60 min postinjection, the pancreatic uptakes were 1.02 ± 0.15%ID/g and 0.23 ± 0.05%ID/g in healthy and diabetic rats respectively. Under block, the pancreatic uptakes of non-diabetic rats reduced to 0.21 ± 0.07%ID/g at the same time point. Biodistribution data and IHC staining confirmed the findings of the microPET imaging. Conclusion: The favorable preclinical data indicated that {sup 18}F-Al-NOTA-MAL-Cys{sup 39}-exendin-4may be suitable for non-invasive monitoring functional pancreatic beta cells.

Biomolecule labelling by 186 Re

International Nuclear Information System (INIS)

Lungu, Valeria Viorica; Mihailescu, Gabriela; Dumitrescu, Gabriela

1998-01-01

The aim of this study is to develop and improve the existing radiolabelling techniques of peptides and monoclonal antibodies with 186 Re and 188 Re as potential agents for cancer targeted radiotherapy. We selected the following methods and techniques for direct labelling of peptides and monoclonal antibody: 1. Prereduction of -S-S- bridges of biomolecule to sulfhydryls using reducing agents: ascorbic acid, cysteine, active hydrogen, 2,3 dimercaptopropanol. The prereduction reactions are controlled by massic ratios of reduction agents/biomolecule, pH, temperature and time of incubation; 2. Reduction of 186 Re O 4 - stannous chloride in acid and alkaline pH; 3. Coupling reaction of 186 Re (red) with the biomolecule controlled by the time and temperature of incubation, the influence of pH regarding the binding of 186 Re to the biomolecules. The quality control was effected by chromatography techniques (paper and elution gel chromatography) on labeled biomolecule before and after purification. The elution gel chromatography was spectrophotometricaly monitored at 280 nm. In the same time the radioactivity of samples was measured using a gamma counter. All the results confirm in vitro stability of labeled biomolecule. The biological evaluation studies regarding accumulation and biological affinity will be controlled by scintigraphy method. Biodistribution studies will be effected to Walker tumor bearing animals at 4 and 24 hours after injections. (authors)

Peptides in headlock – a novel high-affinity and versatile peptide-binding nanobody for proteomics and microscopy

Science.gov (United States)

Braun, Michael B.; Traenkle, Bjoern; Koch, Philipp A.; Emele, Felix; Weiss, Frederik; Poetz, Oliver; Stehle, Thilo; Rothbauer, Ulrich

2016-01-01

Nanobodies are highly valuable tools for numerous bioanalytical and biotechnical applications. Here, we report the characterization of a nanobody that binds a short peptide epitope with extraordinary affinity. Structural analysis reveals an unusual binding mode where the extended peptide becomes part of a β-sheet structure in the nanobody. This interaction relies on sequence-independent backbone interactions augmented by a small number of specificity-determining side chain contacts. Once bound, the peptide is fastened by two nanobody side chains that clamp it in a headlock fashion. Exploiting this unusual binding mode, we generated a novel nanobody-derived capture and detection system. Matrix-coupled nanobody enables the fast and efficient isolation of epitope-tagged proteins from prokaryotic and eukaryotic expression systems. Additionally, the fluorescently labeled nanobody visualizes subcellular structures in different cellular compartments. The high-affinity-binding and modifiable peptide tag of this system renders it a versatile and robust tool to combine biochemical analysis with microscopic studies. PMID:26791954

Specific Affinity Enrichment of Electrochemically Cleaved Peptides Based on Cu(II)-Mediated Spirolactone Tagging

NARCIS (Netherlands)

Zhang, Tao; de Vries, Marcel P.; Permentier, Hjalmar P.; Bischoff, Rainer

2017-01-01

Specific digestion of proteins is an essential step for mass spectrometry-based proteomics, and the chemical labeling of the resulting peptides is often used for peptide enrichment or the introduction of desirable tags. Electrochemical oxidation yielding specific cleavage C-terminal to tyrosine

Reducing renal uptake of 9Y- and 177Lu-labeled alpha-melanocyte stimulating hormone peptide analogues

International Nuclear Information System (INIS)

Miao Yubin; Fisher, Darrell R.; Quinn, Thomas P.

2006-01-01

Objective: The purpose of this study was to improve the tumor-to-kidney uptake ratios of 9 Y- and 177 Lu-[1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid-Re-Cys 3,4,1 , D-Phe 7 , Arg 11 ]α-melanocyte stimulating hormone 3-13 {DOTA-Re(Arg 11 )CCMSH} through coupling a negatively charged glutamic acid (Glu) to the peptide sequence. Methods: A new peptide of DOTA-Re(Glu 2 , Arg 11 )CCMSH was designed, synthesized and labeled with 9 Y and 177 Lu. Pharmacokinetics of 9 Y- and 177 Lu-DOTA-Re(Glu 2 , Arg 11 )CCMSH was determined in B16/F1 murine melanoma-bearing C57 mice. Results: 9 Y- and 177 Lu-DOTA-Re(Glu 2 , Arg 11 )CCMSH exhibited significantly (P 9 Y- and 177 Lu-DOTA-Re(Arg 11 )CCMSH at 30 min and at 2, 4 and 24 h after dose administration. The renal uptake values of 9 Y- and 177 Lu-DOTA-Re(Glu 2 , Arg 11 )CCMSH were 28.16% and 28.81% of those of 9 Y- and 177 Lu-DOTA-Re(Arg 11 )CCMSH, respectively, at 4 h postinjection. 9 Y- and 177 Lu-DOTA-Re(Glu 2 , Arg 11 )CCMSH displayed higher tumor-to-kidney uptake ratios than 9 Y- and 177 Lu-DOTA-Re(Arg 11 )CCMSH at 30 min and at 2, 4 and 24 h after dose administration. The tumor-to-kidney uptake ratio of 9 Y- and 177 Lu-DOTA-Re(Glu 2 , Arg 11 )CCMSH was 2.28 and 1.69 times of 9 Y- and 177 Lu-DOTA-Re(Arg 11 )CCMSH, respectively, at 4 h postinjection. The 9 Y- and 177 Lu-DOTA-Re(Glu 2 , Arg 11 )CCMSH activity accumulation was low in normal organs except for kidney. Conclusions: Coupling a negatively charged amino acid (Glu) to the CCMSH peptide sequence dramatically reduced the renal uptake values and increased the tumor-to-kidney uptake ratios of 9 Y- and 177 Lu-DOTA-Re(Glu 2 , Arg 11 )CCMSH, facilitating their potential applications as radiopharmaceuticals for targeted radionuclide therapy of melanoma

Novel aspects of application of cadmium telluride quantum dots nanostructures in radiation oncology

Energy Technology Data Exchange (ETDEWEB)

Fazaeli, Yousef; Feizi, Shahzad [Nuclear Science and Technology Research Institute (NSTRI), Radiation Application Research School, Karaj (Iran, Islamic Republic of); Zare, Hakimeh; Karimi, Shokufeh [Yazd University, Department of Physics, Yazd (Iran, Islamic Republic of); Rahighi, Reza [Sharif University of Technology, Department of Physics, Tehran (Iran, Islamic Republic of)

2017-08-15

In the last two decades, quantum dots nanomaterials have garnered a great deal of scientific interest because of their unique properties. Quantum dots (QDs) are inorganic fluorescent nanocrystals in the size range between 1 and 20 nm. Due to their structural properties, they possess distinctive properties and behave in different way from crystals in macro scale, in many branches of human life. Cadmium telluride quantum dots (CdTe QDs) were labeled with {sup 68}Ga radio nuclide for fast in vivo targeting and coincidence imaging of tumors. Using instant paper chromatography, the physicochemical properties of the Cadmium telluride quantum dots labeled with {sup 68}Ga NPs ({sup 68}Ga rate at CdTe QDs) were found high enough stable in organic phases, e.g., a human serum, to be reliably used in bioapplications. In vivo biodistribution of the {sup 68}Ga rate at CdTe QDs nanoconposite was investigated in rats bearing fibro sarcoma tumor after various post-injection periods of time. The {sup 68}Ga NPs exhibited a rapid as well as high tumor uptake in a very short period of time (less than 10 min), resulting in an efficient tumor targeting/imaging agent. Meantime, the low lipophilicity of the {sup 68}Ga NPs caused to their fast excretion throughout the body by kidneys (as also confirmed by the urinary tract). Because of the short half-life of {sup 68}Ga radionuclide, the {sup 68}Ga rate at CdTe QDs with an excellent tumor targeting/imaging and fast washing out from the body can be suggested as one of the most effective and promising nanomaterials in nanotechnology-based cancer diagnosis and therapy. (orig.)

Development of direct competitive biomimetic immunosorbent assay based on quantum dot label for determination of trichlorfon residues in vegetables.

Science.gov (United States)

Liu, Qiurui; Jiang, Mingdi; Ju, Zeliang; Qiao, Xuguang; Xu, Zhixiang

2018-06-01

A direct competitive biomimetic immunosorbent assay method based on molecularly imprinted polymer was developed for the determination of trichlorfon. A CdSe/ZnS quantum dot label was used as the marker. The hydrophilic imprinted film was synthesized directly on the surface of a 96-well plate, and characterized by Fourier-transform infrared spectroscopy and thermo-gravimetric analyses. The method exhibited high stability, selectivity, and sensitivity. Under optimal conditions, the limits of detection and sensitivity of the biomimetic immunosorbent assay method were 9.0 μg L -1 and 5.0 mg L -1 (0.1 mg kg -1 and 62.5 mg kg -1 for vegetable sample), respectively. Low cross-reactivity values of 19.2% and 15.6% were obtained for the structural analogues. Spinach and rape samples spiked with trichlorfon were extracted and determined by this method with recoveries ranging from 83.6% to 91.1%. The method was applied for the detection of trichlorfon residues in leek and cucumber samples, and results correlated well with those obtained using GC. Copyright © 2018 Elsevier Ltd. All rights reserved.

Novel isotopic N, N-Dimethyl Leucine (iDiLeu) Reagents Enable Absolute Quantification of Peptides and Proteins Using a Standard Curve Approach

Science.gov (United States)

Greer, Tyler; Lietz, Christopher B.; Xiang, Feng; Li, Lingjun

2015-01-01

Absolute quantification of protein targets using liquid chromatography-mass spectrometry (LC-MS) is a key component of candidate biomarker validation. One popular method combines multiple reaction monitoring (MRM) using a triple quadrupole instrument with stable isotope-labeled standards (SIS) for absolute quantification (AQUA). LC-MRM AQUA assays are sensitive and specific, but they are also expensive because of the cost of synthesizing stable isotope peptide standards. While the chemical modification approach using mass differential tags for relative and absolute quantification (mTRAQ) represents a more economical approach when quantifying large numbers of peptides, these reagents are costly and still suffer from lower throughput because only two concentration values per peptide can be obtained in a single LC-MS run. Here, we have developed and applied a set of five novel mass difference reagents, isotopic N, N-dimethyl leucine (iDiLeu). These labels contain an amine reactive group, triazine ester, are cost effective because of their synthetic simplicity, and have increased throughput compared with previous LC-MS quantification methods by allowing construction of a four-point standard curve in one run. iDiLeu-labeled peptides show remarkably similar retention time shifts, slightly lower energy thresholds for higher-energy collisional dissociation (HCD) fragmentation, and high quantification accuracy for trypsin-digested protein samples (median errors <15%). By spiking in an iDiLeu-labeled neuropeptide, allatostatin, into mouse urine matrix, two quantification methods are validated. The first uses one labeled peptide as an internal standard to normalize labeled peptide peak areas across runs (<19% error), whereas the second enables standard curve creation and analyte quantification in one run (<8% error).

Radiometallating antibodies and autoantigenic peptides

International Nuclear Information System (INIS)

Mercer-Smith, J.A.; Lewis, D.; Cole, D.A.; Newmyer, S.L.; Schulte, L.D.; Mixon, P.L.; Schreyer, S.A.; Burns, T.P.; Roberts, J.C.; Figard, S.D.; McCormick, D.J.; Lennon, V.A.; Hayashi, M.; Lavallee, D.K.

1991-01-01

We have developed methods to radiolabel large molecules, using porphyrins as bifunctional chelating agents for radiometals. The porphyrins are substituted with an N- benzyl group to activate them for radiometallation under mild reaction conditions. Porphyrins that have one functional group for covalent attachment to other molecules cannot cause crosslinking. We have examined the labeling chemistry for antibodies and have developed methods to label smaller biologically active molecules, such as autoantigenic peptides (fragments of the acetylcholine receptor), which are pertinent to myasthenia gravis research. The methods of covalent attachment of these bifunctional chelating agents to large molecules, the radiometallation chemistry, and biological characterization of the radiolabeled compounds will be discussed

Ribonuclease S dynamics measured using a nitrile label with 2D IR vibrational echo spectroscopy.

Science.gov (United States)

Bagchi, Sayan; Boxer, Steven G; Fayer, Michael D

2012-04-05

A nitrile-labeled amino acid, p-cyanophenylalanine, is introduced near the active site of the semisynthetic enzyme ribonuclease S to serve as a probe of protein dynamics and fluctuations. Ribonuclease S is the limited proteolysis product of subtilisin acting on ribonuclease A, and consists of a small fragment including amino acids 1-20, the S-peptide, and a larger fragment including residues 21-124, the S-protein. A series of two-dimensional vibrational echo experiments performed on the nitrile-labeled S-peptide and the RNase S are described. The time-dependent changes in the two-dimensional infrared vibrational echo line shapes are analyzed using the center line slope method to obtain the frequency-frequency correlation function (FFCF). The observations show that the nitrile probe in the S-peptide has dynamics that are similar to, but faster than, those of the single amino acid p-cyanophenylalanine in water. In contrast, the dynamics of the nitrile label when the peptide is bound to form ribonuclease S are dominated by homogeneous dephasing (motionally narrowed) contributions with only a small contribution from very fast inhomogeneous structural dynamics. The results provide insights into the nature of the structural dynamics of the ribonuclease S complex. The equilibrium dynamics of the nitrile labeled S-peptide and the ribonuclease S complex are also investigated by molecular dynamics simulations. The experimentally determined FFCFs are compared to the FFCFs obtained from the molecular dynamics simulations, thereby testing the capacity of simulations to determine the amplitudes and time scales of protein structural fluctuations on fast time scales under thermal equilibrium conditions.

Fluorescence Stability of Mercaptopropionic Acid Capped Cadmium Telluride Quantum Dots in Various Biochemical Buffers.

Science.gov (United States)

Borse, Vivek; Kashikar, Adisha; Srivastava, Rohit

2018-04-01

Quantum dots are the semiconductor nanocrystals having unique optical and electronic properties. Quantum dots are category of fluorescent labels utilized for biological tagging, biosensing, bioassays, bioimaging and in vivo imaging as they exhibit very small size, signal brightness, photostability, tuning of light emission range, longer photoluminescence decay time as compared to organic dyes. In this work, we have synthesized and characterized mercaptopropionic acid capped cadmium telluride quantum dots (MPA-CdTe QDs) using hydrothermal method. The study further reports fluorescence intensity stability of quantum dots suspended in different buffers of varying concentration (1-100 mM), stored at various photophysical conditions. Fluorescence intensity values were reduced with increase in buffer concentration. When the samples were stored at room temperature in ambient light condition the quantum dots suspended in different buffers lost the fluorescence intensity after day 15 (except TRIS II). Fluorescence intensity values were found stable for more than 30 days when the samples were stored in dark condition. Samples stored in refrigerator displayed modest fluorescence intensity even after 300 days of storage. Thus, storage of MPA-CdTe QDs in refrigerator may be the suitable choice to maintain its fluorescence stability for longer time for further application.

Europium-labeled epidermal growth factor and neurotensin: novel probes for receptor-binding studies.

Science.gov (United States)

Mazor, Ohad; Hillairet de Boisferon, Marc; Lombet, Alain; Gruaz-Guyon, Anne; Gayer, Batya; Skrzydelsky, Delphine; Kohen, Fortune; Forgez, Patricia; Scherz, Avigdor; Rostene, William; Salomon, Yoram

2002-02-01

We investigated the possibility of labeling two biologically active peptides, epidermal growth factor (EGF) and neurotensin (NT), with europium (Eu)-diethylenetriaminepentaacetic acid. More specifically, we tested them as probes in studying receptor binding using time-resolved fluorescence of Eu3+. The relatively simple synthesis yields ligands with acceptable binding characteristics similar to isotopically labeled derivatives. The binding affinity (Kd) of labeled Eu-EGF to human A431 epidermal carcinoid cells was 3.6 +/- 1.2 nM, similar to the reported Kd values of EGF, whereas the Kd of Eu-NT to human HT29 colon cancer cells (7.4 +/- 0.5 nM) or to Chinese hamster ovary (CHO) cells transfected with the high-affinity NT receptor (CHO-NT1) were about 10-fold higher than the Kd values of NT. The bioactivity of the Eu-labeled EGF as determined by stimulation of cultured murine D1 hematopoietic cell proliferation was nearly the same as that obtained with native EGF. The maximal stimulation of Ca2+ influx with NT and Eu-NT in CHO-NT1 cells was similar, but the respective K0.5 values were 20 pM and 1 nM, corresponding to differences in the binding affinities previously described. The results of these studies indicate that Eu labeling of peptide hormones and growth factor molecules ranging from 10(3) to 10(5) Da can be conveniently accomplished. Importantly, the Eu-labeled products are stable for approximately 2 years and are completely safe for laboratory use compared to the biohazardous radioligands. Thus, Eu-labeled peptides present an attractive alternative for commonly used radiolabeled ligands in biological studies in general and in receptor assays in particular.

Investigation of solid-phase hydrogenation of amino acids and peptides

International Nuclear Information System (INIS)

Zolotarev, Yu.A.; Myasoedov, N.F.; Zajtsev, D.A.; Lubnin, M.Yu.; Tatur, V.Yu.; Kozik, V.S.; Dorokhova, E.M.; Rozenberg, S.N.

1990-01-01

The possibility of synthesizing amino acids and peptides multiply labelled with tritium or deuterium by the method of solid-phase isotopic exchange with gaseous hydrogen isotopes was verified. Establishment of the isotopic hydrogen equilibrium between the gaseous phase and the solid phase formed by the amino acid molecules was found experimentally. The activation energy of the isotopic exchange is 13 kcal/mol. A mathematical model was set up for the isotopic exchange with a probable substitution of hydrogen atoms. Uniformly labelled amino acids were obtained in a high optical purity and with 80 to 90% hydrogen substitution by deuterium and tritium. Tritiated peptides were prepared in high yields at molar activities of 1.5 to 3.7 TBq/mmol. (author). 4 tabs

Dual integrin and gastrin-releasing peptide receptor targeted tumor imaging using 18F-labeled PEGylated RGD-bombesin heterodimer 18F-FB-PEG3-Glu-RGD-BBN.

Science.gov (United States)

Liu, Zhaofei; Yan, Yongjun; Chin, Frederic T; Wang, Fan; Chen, Xiaoyuan

2009-01-22

Radiolabeled RGD and bombesin peptides have been extensively investigated for tumor integrin alpha(v)beta(3) and GRPR imaging, respectively. Due to the fact that many tumors are both integrin and GRPR positive, we designed and synthesized a heterodimeric peptide Glu-RGD-BBN, which is expected to be advantageous over the monomeric peptides for dual-receptor targeting. A PEG(3) spacer was attached to the glutamate alpha-amino group of Glu-RGD-BBN to enhance the (18)F labeling yield and to improve the in vivo kinetics. PEG(3)-Glu-RGD-BBN possesses the comparable GRPR and integrin alpha(v)beta(3) receptor-binding affinities as the corresponding monomers, respectively. The dual-receptor targeting properties of (18)F-FB-PEG(3)-Glu-RGD-BBN were observed in PC-3 tumor model. (18)F-FB-PEG(3)-Glu-RGD-BBN with high tumor contrast and favorable pharmacokinetics is a promising PET tracer for dual integrin and GRPR positive tumor imaging. This heterodimer strategy may also be an applicable method to develop other molecules with improved in vitro and in vivo characterizations for tumor diagnosis and therapy.

GLP-1 and exendin-4 for imaging endocrine pancreas. A review. Labelled glucagon-like peptide-1 analogues: past, present and future

International Nuclear Information System (INIS)

Hubalewska-Dydejczyk, A.; Sowa-Staszczak, A.; Tomaszuk, M.; Stefańska, A.

2015-01-01

Glucagon-like peptide 1 (GLP-1) receptors expression has been found on many types of cancer cells. In case of benign insulinoma the density of those receptors is even higher than the density of somatostatin receptors. This article presents the results of clinical trials proving the utility of GLP-1 receptors imaging. Scintigraphy or positron emission tomography with the use of GLP-1 analogues labelled with appropriate radioisotopes ( 111I n, 99m Tc, 68 Ga, 18 F or 64 Cu) seem to be superior compared with other available techniques in diagnosis of hardly detectable benign insulinoma. While surgery is the only effective therapy for insulinoma patients, therefore proper preoperative localization of the tumor allows sparing operation. Glucagon-like peptide 1 receptors might become also a target for imaging of other tumors such as gastrinoma, pheochromocytoma and medullary thyroid cancer (MTC), which also were shown to over express this type of receptors. However, studies with larger groups of patients are required to prove the clinical usefulness of this indication. Moreover GLP-1 receptor imaging seems to be a potential tool to evaluate pancreatic beta cell mass (BCM). It may be useful in the early diagnosis of beta cell loss in preclinical phases of diabetes. The panceratic beta cells imaging may influence the prophylaxis of diabetes and management of diabetic patients. Presented results of clinical trials prove that glucagon-like peptide 1 receptor imaging might become helpful diagnostic strategy particularly in case of patients with benign insulinoma tumors, but also patients with gastrinoma, pheochromocytoma, medullary thyroid cancer and diabetes.

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

INTERNALIZATION OF ANTIMICROBIAL PEPTIDE ACIPENSIN 1 INTO HUMAN TUMOR CELLS

Directory of Open Access Journals (Sweden)

E. S. Umnyakova

2016-01-01

Full Text Available Search for new compounds providing delivery of drugs into infected or neoplastic cells, is an important direction of biomedical research. Cell-penetrating peptides are among those compounds, due to their ability to translocate through membranes of eukaryotic cells, serving as potential carriers of various therapeutic agents to the target cells. The aim of present work was to investigate the ability of acipensin 1, an antimicrobial peptide of innate immune system, for in vitro penetration into human tumor cells. Acipensin 1 is a cationic peptide that we have previously isolated from leukocytes of the Russian sturgeon, Acipenser gueldenstaedtii. Capability of acipensin 1 to enter the human erytroleukemia K-562 cells has been investigated for the first time. A biotechnological procedure for producing a recombinant acipensin 1 peptide has been developed. The obtained peptide was conjugated with a fluorescent probe BODIPY FL. By means of confocal microscopy, we have shown that the tagged acipensin 1 rapidly enters into K-562 cells and can be detected in the intracellular space within 5 min after its addition to the cell culture. Using flow cytometry technique, penetration kinetics of the labeled peptide into K-562 cells (at nontoxic micromolar concentrations has been studied. We have observed a rapid internalization of the peptide to the target cells, thus confirming the results of microscopic analysis, i.e, the labeled acipensin was detectable in K-562 cells as soon as wihin 2-3 seconds after its addition to the incubation medium. The maximum of fluorescence was reached within a period of approx. 45 seconds, with further “plateau” at the terms of >100 seconds following cell stimulation with the test compound. These data support the concept, that the antimicrobial peptides of innate immunity system possess the features of cell-penetrating peptides, and allow us to consider the studied sturgeon peptide a promising template for development of new

Synthesis of /sup 35/S-labeled caerulein (FI 6934)

Energy Technology Data Exchange (ETDEWEB)

Uemura, I; Murakami, H [Nomura Research Institute, Kanagawa (Japan). Life Sciences Division

1975-05-01

FI6934 (Caerulein) is a biologically active decapeptide extracted from the skin of Australian amphibians (Hyla caerulea). For metabolic study of FI6934, we have attempted to label the sulfo group of tyrosine of FI6934 with /sup 35/S. The starting decapeptide was sulfonated with an excess of pyridine-N-sulfonate/sup 35/S, and the /sup 35/S-labelled peptide resulted was deacetylated by hydrolysis in alkaline, purified by paper chromatography to obtained radio chemically pure /sup 35/S-labelled FI6934. The /sup 35/S-labelled FI6934 was identified as a standard FI6934 in physiological activity to contract guinea pig gallbladders.

99m Tc-HYNIC-(Ser)3 -J18 peptide: A radiotracer for non-small-cell lung cancer targeting.

Science.gov (United States)

Shaghaghi, Zahra; Abedi, Seyed Mohammad; Hosseinimehr, Seyed Jalal

2018-02-14

Radiolabeled peptide could be a useful tool for the diagnosis of non-small-cell lung cancer (NSCLC). In this study, HYNIC-(Ser) 3 -J18 peptide was labeled with 99m Tc using EDDA/tricine as coligands. The in vitro and in vivo studies of this radiolabeled peptide were performed for cellular-specific binding and tumor targeting in A-549 cells and tumor-bearing mice, respectively. The high radiochemical purity was obtained and this radiolabeled peptide exhibited high stability in buffer and serum. The radiolabeled peptide showed high affinity for the A-549 cells with a dissociation constant value (K D ) of 4.4 ± 0.8 nm. The tumor-muscles ratios were 2.7 and 4.4 at 1 and 2 hr after injection of 99m Tc-(EDDA/tricine)-HYNIC-(Ser) 3 -J18 in tumor-bearing mice. The tumor uptake was decreased after preinjection with non-labeled peptide for this radiolabeled peptide in blocking experiment. The results of this study showed the 99m Tc-(EDDA/tricine)-(Ser) 3 -HYNIC-J18 peptide might be a promising radiolabeled peptide for NSCLC targeting. © 2018 John Wiley & Sons A/S.

Cytosolic antibody delivery by lipid-sensitive endosomolytic peptide

Science.gov (United States)

Akishiba, Misao; Takeuchi, Toshihide; Kawaguchi, Yoshimasa; Sakamoto, Kentarou; Yu, Hao-Hsin; Nakase, Ikuhiko; Takatani-Nakase, Tomoka; Madani, Fatemeh; Gräslund, Astrid; Futaki, Shiroh

2017-08-01

One of the major obstacles in intracellular targeting using antibodies is their limited release from endosomes into the cytosol. Here we report an approach to deliver proteins, which include antibodies, into cells by using endosomolytic peptides derived from the cationic and membrane-lytic spider venom peptide M-lycotoxin. The delivery peptides were developed by introducing one or two glutamic acid residues into the hydrophobic face. One peptide with the substitution of leucine by glutamic acid (L17E) was shown to enable a marked cytosolic liberation of antibodies (immunoglobulins G (IgGs)) from endosomes. The predominant membrane-perturbation mechanism of this peptide is the preferential disruption of negatively charged membranes (endosomal membranes) over neutral membranes (plasma membranes), and the endosomolytic peptide promotes the uptake by inducing macropinocytosis. The fidelity of this approach was confirmed through the intracellular delivery of a ribosome-inactivation protein (saporin), Cre recombinase and IgG delivery, which resulted in a specific labelling of the cytosolic proteins and subsequent suppression of the glucocorticoid receptor-mediated transcription. We also demonstrate the L17E-mediated cytosolic delivery of exosome-encapsulated proteins.

Simple Identification of Human Taenia Species by Multiplex Loop-Mediated Isothermal Amplification in Combination with Dot Enzyme-Linked Immunosorbent Assay.

Science.gov (United States)

Nkouawa, Agathe; Sako, Yasuhito; Okamoto, Munehiro; Ito, Akira

2016-06-01

For differential detection of Taenia solium, Taenia saginata, and Taenia asiatica, loop-mediated isothermal amplification (LAMP) assay targeting the cytochrome c oxidase subunit 1 gene has been recently developed and shown to be sensitive, specific, and effective. However, to achieve differential identification, one specimen requires three reaction mixtures containing a primer set of each Taenia species separately, which is complex and time consuming and increases the risk of cross-contamination. In this study, we developed a simple differential identification of human Taenia species using multiplex LAMP (mLAMP) in combination with dot enzyme-linked immunosorbent assay (dot-ELISA). Forward inner primers of T. solium, T. saginata, and T. asiatica labeled with fluorescein isothiocyanate (FITC), digoxigenin (DIG), and tetramethylrhodamine (TAMRA), respectively, and biotin-labeled backward inner primers were used in mLAMP. The mLAMP assay succeeded in specific amplification of each respective target gene in a single tube. Furthermore, the mLAMP product from each species was easily distinguished by dot-ELISA with an antibody specific for FITC, DIG, or TAMRA. The mLAMP assay in combination with dot-ELISA will make identification of human Taenia species simpler, easier, and more practical. © The American Society of Tropical Medicine and Hygiene.

Electronic transport through a quantum dot chain with strong dot-lead coupling

International Nuclear Information System (INIS)

Liu, Yu; Zheng, Yisong; Gong, Weijiang; Gao, Wenzhu; Lue, Tianquan

2007-01-01

By means of the non-equilibrium Green function technique, the electronic transport through an N-quantum-dot chain is theoretically studied. By calculating the linear conductance spectrum and the local density of states in quantum dots, we find the resonant peaks in the spectra coincides with the eigen-energies of the N-quantum-dot chain when the dot-lead coupling is relatively weak. With the increase of the dot-lead coupling, such a correspondence becomes inaccurate. When the dot-lead coupling exceeds twice the interdot coupling, such a mapping collapses completely. The linear conductance turn to reflect the eigen-energies of the (N-2)- or (N-1)-quantum dot chain instead. The two peripheral quantum dots do not manifest themselves in the linear conductance spectrum. More interestingly, with the further increase of the dot-lead coupling, the system behaves just like an (N-2)- or (N-1)-quantum dot chain in weak dot-lead coupling limit, since the resonant peaks becomes narrower with the increase of dot-lead coupling

Improved segmental isotope labeling of proteins and application to a larger protein

International Nuclear Information System (INIS)

Otomo, Takanori; Teruya, Kenta; Uegaki, Koichi; Yamazaki, Toshio; Kyogoku, Yoshimasa

1999-01-01

A new isotope labeling technique for peptide segments in a protein sample was recently established using the protein splicing element intein [Yamazaki et al. (1998) J. Am. Chem. Soc., 120, 5591-5592]. This method makes it possible to observe signals of a selected amino (N-) or carboxyl (C-) terminal region along a peptide chain. However, there is a problem with the yield of the segmentally labeled protein. In this paper, we report an increase in the yield of the protein that enables the production of sufficient amounts of segmentally 13 C/ 15 N-labeled protein samples. This was achieved by improvement of the expression level of the N-terminal fragment in cells and the efficiency of refolding into the active splicing conformation. The N-terminal fragment was expressed as a fused protein with the cellulose binding domain at its N-terminus, which was expressed as an insoluble peptide in cells and the expression level was increased. Incubation with 2.5 M urea and 50% glycerol increased the efficiency of the refolding greatly, thereby raising the final yields of the ligated proteins. The feasibility of application of the method to a high-molecular-weight protein was demonstrated by the results for a maltose binding protein consisting of 370 amino acids. All four examined joints in the maltose binding protein were successfully ligated to produce segmentally labeled protein samples

Printer model for dot-on-dot halftone screens

Science.gov (United States)

Balasubramanian, Raja

1995-04-01

A printer model is described for dot-on-dot halftone screens. For a given input CMYK signal, the model predicts the resulting spectral reflectance of the printed patch. The model is derived in two steps. First, the C, M, Y, K dot growth functions are determined which relate the input digital value to the actual dot area coverages of the colorants. Next, the reflectance of a patch is predicted as a weighted combination of the reflectances of the four solid C, M, Y, K patches and their various overlays. This approach is analogous to the Neugebauer model, with the random mixing equations being replaced by dot-on-dot mixing equations. A Yule-Neilsen correction factor is incorporated to account for light scattering within the paper. The dot area functions and Yule-Neilsen parameter are chosen to optimize the fit to a set of training data. The model is also extended to a cellular framework, requiring additional measurements. The model is tested with a four color xerographic printer employing a line-on-line halftone screen. CIE L*a*b* errors are obtained between measurements and model predictions. The Yule-Neilsen factor significantly decreases the model error. Accuracy is also increased with the use of a cellular framework.

Water accessibility in a membrane-inserting peptide comparing Overhauser DNP and pulse EPR methods

Energy Technology Data Exchange (ETDEWEB)

Segawa, Takuya F., E-mail: takuya.segawa@alumni.ethz.ch; Doppelbauer, Maximilian; Garbuio, Luca; Doll, Andrin; Polyhach, Yevhen O.; Jeschke, Gunnar, E-mail: gjeschke@ethz.ch [Laboratory of Physical Chemistry, ETH Zurich, Vladimir-Prelog-Weg 2, CH-8093 Zurich (Switzerland)

2016-05-21

Water accessibility is a key parameter for the understanding of the structure of biomolecules, especially membrane proteins. Several experimental techniques based on the combination of electron paramagnetic resonance (EPR) spectroscopy with site-directed spin labeling are currently available. Among those, we compare relaxation time measurements and electron spin echo envelope modulation (ESEEM) experiments using pulse EPR with Overhauser dynamic nuclear polarization (DNP) at X-band frequency and a magnetic field of 0.33 T. Overhauser DNP transfers the electron spin polarization to nuclear spins via cross-relaxation. The change in the intensity of the {sup 1}H NMR spectrum of H{sub 2}O at a Larmor frequency of 14 MHz under a continuous-wave microwave irradiation of the nitroxide spin label contains information on the water accessibility of the labeled site. As a model system for a membrane protein, we use the hydrophobic α-helical peptide WALP23 in unilamellar liposomes of DOPC. Water accessibility measurements with all techniques are conducted for eight peptides with different spin label positions and low radical concentrations (10–20 μM). Consistently in all experiments, the water accessibility appears to be very low, even for labels positioned near the end of the helix. The best profile is obtained by Overhauser DNP, which is the only technique that succeeds in discriminating neighboring positions in WALP23. Since the concentration of the spin-labeled peptides varied, we normalized the DNP parameter ϵ, being the relative change of the NMR intensity, by the electron spin concentration, which was determined from a continuous-wave EPR spectrum.

The quantum mechanical description of the dot-dot interaction in ionic colloids

International Nuclear Information System (INIS)

Morais, P.C.; Qu, Fanyao

2007-01-01

In this study the dot-dot interaction in ionic colloids is systematically investigated by self-consistently solving the coupled Schroedinger and Poisson equations in the frame of finite difference method (FDM). In a first approximation the interacting two-dot system (dimer) is described using the picture of two coupled quantum wells. It was found that the dot-dot interaction changes the colloid characteristic by changing the hopping coefficient (t) and consequently the nanodot surface charge density (σ). The hopping coefficient and the surface charge density were investigated as a function of the dot size and dot-dot distance

Core–shell Fe3O4–Au magnetic nanoparticles based nonenzymatic ultrasensitive electrochemiluminescence immunosensor using quantum dots functionalized graphene sheet as labels

International Nuclear Information System (INIS)

Liu, Weiyan; Zhang, Yan; Ge, Shenguang; Song, Xianrang; Huang, Jiadong; Yan, Mei; Yu, Jinghua

2013-01-01

Graphical abstract: Core–shell Fe 3 O 4 –Au magnetic nanoparticles and P-GS@QDs were prepared to immobilize Ab 1 and Ab 2 respectively and combined to fabricate a novel sandwich-type ECL immunosensor for detecting CA125 at low concentration. Highlights: ► ECL immunosensor for CA125 based on a microfluidic strategy with a homemade ECL cell was proposed. ► Core–shell Fe 3 O 4 –Au magnetic nanoparticles were employed as the carriers of the primary antibodies. ► CdTe quantum dots functionalized graphene sheet were used for signal amplification. -- Abstract: In this paper, a novel, low-cost electrochemiluminescence (ECL) immunosensor using core–shell Fe 3 O 4 –Au magnetic nanoparticles (AuMNPs) as the carriers of the primary antibody of carbohydrate antigen 125 (CA125) was designed. Graphene sheet (GS) with property of good conductivity and large surface area was a captivating candidate to amplify ECL signal. We successively synthesized functionalized GS by loading large amounts of quantum dots (QDs) onto the poly (diallyldimethyl-ammonium chloride) (PDDA) coated graphene sheet (P-GS@QDs) via self-assembly electrostatic reactions, which were used to label secondary antibodies. The ECL immunosensors coupled with a microfluidic strategy exhibited a wide detection range (0.005–50 U mL −1 ) and a low detection limit (1.2 mU mL −1 ) with the help of an external magnetic field to gather immunosensors. The method was evaluated with clinical serum sample, receiving good correlation with results from commercially available analytical procedure

Peptide Based Radiopharmaceuticals: Specific Construct Approach

Energy Technology Data Exchange (ETDEWEB)

Som, P; Rhodes, B A; Sharma, S S

1997-10-21

The objective of this project was to develop receptor based peptides for diagnostic imaging and therapy. A series of peptides related to cell adhesion molecules (CAM) and immune regulation were designed for radiolabeling with ^99mTc and evaluated in animal models as potential diagnostic imaging agents for various disease conditions such as thrombus (clot), acute kidney failure, and inflection/inflammation imaging. The peptides for this project were designed by the industrial partner, Palatin Technologies, (formerly Rhomed, Inc.) using various peptide design approaches including a newly developed rational computer assisted drug design (CADD) approach termed MIDAS (Metal ion Induced Distinctive Array of Structures). In this approach, the biological function domain and the ^99mTc complexing domain are fused together so that structurally these domains are indistinguishable. This approach allows construction of conformationally rigid metallo-peptide molecules (similar to cyclic peptides) that are metabolically stable in-vivo. All the newly designed peptides were screened in various in vitro receptor binding and functional assays to identify a lead compound. The lead compounds were formulated in a one-step ^99mTc labeling kit form which were studied by BNL for detailed in-vivo imaging using various animals models of human disease. Two main peptides usingMIDAS approach evolved and were investigated: RGD peptide for acute renal failure and an immunomodulatory peptide derived from tuftsin (RMT-1) for infection/inflammation imaging. Various RGD based metallopeptides were designed, synthesized and assayed for their efficacy in inhibiting ADP-induced human platelet aggregation. Most of these peptides displayed biological activity in the 1-100 µM range. Based on previous work by others, RGD-I and RGD-II were evaluated in animal models of acute renal failure. These earlier studies showed that after acute ischemic injury the renal cortex displays

Human acid β-glucosidase: isolation and amino acid sequence of a peptide containing the catalytic site

International Nuclear Information System (INIS)

Dinur, T.; Osiecki, K.M.; Legler, G.; Gatt, S.; Desnick, R.J.; Grabowski, G.A.

1986-01-01

Human acid β-glucosidase (D-glucosyl-N-acylsphingosine glucohydrolase, EC 3.2.1.45) cleaves the glucosidic bonds of glucosylceramide and synthetic β-glucosides. The deficient activity of this hydrolase is the enzymatic defect in the subtypes and variants of Gaucher disease, the most prevalent lysosomal storage disease. To isolate and characterize the catalytic site of the normal enzyme, brominated 3 H-labeled conduritol B epoxide ( 3 H-Br-CBE), which inhibits the enzyme by binding covalently to this site, was used as an affinity label. Under optimal conditions 1 mol of 3 H-Br-CBE bound to 1 mol of pure enzyme protein, indicating the presence of a single catalytic site per enzyme subunit. After V 8 protease digestion of the 3 H-Br-CBE-labeled homogeneous enzyme, three radiolabeled peptides, designated peptide A, B, or C, were resolved by reverse-phase HPLC. The partial amino acid sequence (37 residues) of peptide A (M/sub r/, 5000) was determined. The sequence of this peptide, which contained the catalytic site, had exact homology to the sequence near the carboxyl terminus of the protein, as predicted from the nucleotide sequence of the full-length cDNA encoding acid β-glucosidase

Sensitive Bioanalysis Based on in-Situ Droplet Anodic Stripping Voltammetric Detection of CdS Quantum Dots Label after Enhanced Cathodic Preconcentration

Directory of Open Access Journals (Sweden)

Xiaoli Qin

2016-08-01

Full Text Available We report a protocol of CdS-labeled sandwich-type amperometric bioanalysis with high sensitivity, on the basis of simultaneous chemical-dissolution/cathodic-enrichment of the CdS quantum dot biolabel and anodic stripping voltammetry (ASV detection of Cd directly on the bioelectrode. We added a microliter droplet of 0.1 M aqueous HNO3 to dissolve CdS on the bioelectrode and simultaneously achieved the potentiostatic cathodic preconcentration of Cd by starting the potentiostatic operation before HNO3 addition, which can largely increase the ASV signal. Our protocol was used for immunoanalysis and aptamer-based bioanalysis of several proteins, giving limits of detection of 4.5 fg·mL−1 for human immunoglobulin G, 3.0 fg·mL−1 for human carcinoembryonic antigen (CEA, 4.9 fg·mL−1 for human α-fetoprotein (AFP, and 0.9 fM for thrombin, which are better than many reported results. The simultaneous and sensitive analysis of CEA and AFP at two screen-printed carbon electrodes was also conducted by our protocol.

De novo sequencing of two novel peptides homologous to calcitonin-like peptides, from skin secretion of the Chinese Frog, Odorrana schmackeri

Directory of Open Access Journals (Sweden)

Geisa P.C. Evaristo

2015-09-01

Full Text Available An MS/MS based analytical strategy was followed to solve the complete sequence of two new peptides from frog (Odorrana schmackeri skin secretion. This involved reduction and alkylation with two different alkylating agents followed by high resolution tandem mass spectrometry. De novo sequencing was achieved by complementary CID and ETD fragmentations of full-length peptides and of selected tryptic fragments. Heavy and light isotope dimethyl labeling assisted with annotation of sequence ion series. The identified primary structures are GCD[I/L]STCATHN[I/L]VNE[I/L]NKFDKSKPSSGGVGPESP-NH2 and SCNLSTCATHNLVNELNKFDKSKPSSGGVGPESF-NH2, i.e. two carboxyamidated 34 residue peptides with an aminoterminal intramolecular ring structure formed by a disulfide bridge between Cys2 and Cys7. Edman degradation analysis of the second peptide positively confirmed the exact sequence, resolving I/L discriminations. Both peptide sequences are novel and share homology with calcitonin, calcitonin gene related peptide (CGRP and adrenomedullin from other vertebrates. Detailed sequence analysis as well as the 34 residue length of both O. schmackeri peptides, suggest they do not fully qualify as either calcitonins (32 residues or CGRPs (37 amino acids and may justify their classification in a novel peptide family within the calcitonin gene related peptide superfamily. Smooth muscle contractility assays with synthetic replicas of the S–S linked peptides on rat tail artery, uterus, bladder and ileum did not reveal myotropic activity.

Somatostatin analogues labelled with 99mTc

International Nuclear Information System (INIS)

Obenaus, Esteban R.; Crudo, Jose L.; Edreira, Martin M.; Castiglia, Silvia G.

1999-01-01

Biological and radiochemical studies have been carried out on two labelled somatostatin analogues, the peptide RC-150 and the Tyr 3 -Octreotide. Both analogues have been labelled with 99m Tc using the direct and the indirect method and MAG-3 and HYNIC as chelating agents. By the direct method RC-150 was labelled using sodium ascorbate and dithionite as reducing agents. The radiochemical purity was 70%. By the indirect method, in the case of RC-160 with MAG-3 a radiochemical purity higher than 70% was attained while a purity of 100% was reached in the case of Tyr 3 -Octreotide with HYNIC. The biological distribution of HYNIC-Tyr 3 -Octreotide has been studied in rats. (author)

Quantum dots

International Nuclear Information System (INIS)

Kouwenhoven, L.; Marcus, C.

1998-01-01

Quantum dots are man-made ''droplets'' of charge that can contain anything from a single electron to a collection of several thousand. Their typical dimensions range from nanometres to a few microns, and their size, shape and interactions can be precisely controlled through the use of advanced nanofabrication technology. The physics of quantum dots shows many parallels with the behaviour of naturally occurring quantum systems in atomic and nuclear physics. Indeed, quantum dots exemplify an important trend in condensed-matter physics in which researchers study man-made objects rather than real atoms or nuclei. As in an atom, the energy levels in a quantum dot become quantized due to the confinement of electrons. With quantum dots, however, an experimentalist can scan through the entire periodic table by simply changing a voltage. In this article the authors describe how quantum dots make it possible to explore new physics in regimes that cannot otherwise be accessed in the laboratory. (UK)

Nanogel-quantum dot hybrid nanoparticles for live cell imaging

International Nuclear Information System (INIS)

Hasegawa, Urara; Nomura, Shin-ichiro M.; Kaul, Sunil C.; Hirano, Takashi; Akiyoshi, Kazunari

2005-01-01

We report here a novel carrier of quantum dots (QDs) for intracellular labeling. Monodisperse hybrid nanoparticles (38 nm in diameter) of QDs were prepared by simple mixing with nanogels of cholesterol-bearing pullulan (CHP) modified with amino groups (CHPNH 2 ). The CHPNH 2 -QD nanoparticles were effectively internalized into the various human cells examined. The efficiency of cellular uptake was much higher than that of a conventional carrier, cationic liposome. These hybrid nanoparticles could be a promising fluorescent probe for bioimaging

A Probabilistic Framework for Peptide and Protein Quantification from Data-Dependent and Data-Independent LC-MS Proteomics Experiments

Science.gov (United States)

Richardson, Keith; Denny, Richard; Hughes, Chris; Skilling, John; Sikora, Jacek; Dadlez, Michał; Manteca, Angel; Jung, Hye Ryung; Jensen, Ole Nørregaard; Redeker, Virginie; Melki, Ronald; Langridge, James I.; Vissers, Johannes P.C.

2013-01-01

A probability-based quantification framework is presented for the calculation of relative peptide and protein abundance in label-free and label-dependent LC-MS proteomics data. The results are accompanied by credible intervals and regulation probabilities. The algorithm takes into account data uncertainties via Poisson statistics modified by a noise contribution that is determined automatically during an initial normalization stage. Protein quantification relies on assignments of component peptides to the acquired data. These assignments are generally of variable reliability and may not be present across all of the experiments comprising an analysis. It is also possible for a peptide to be identified to more than one protein in a given mixture. For these reasons the algorithm accepts a prior probability of peptide assignment for each intensity measurement. The model is constructed in such a way that outliers of any type can be automatically reweighted. Two discrete normalization methods can be employed. The first method is based on a user-defined subset of peptides, while the second method relies on the presence of a dominant background of endogenous peptides for which the concentration is assumed to be unaffected. Normalization is performed using the same computational and statistical procedures employed by the main quantification algorithm. The performance of the algorithm will be illustrated on example data sets, and its utility demonstrated for typical proteomics applications. The quantification algorithm supports relative protein quantification based on precursor and product ion intensities acquired by means of data-dependent methods, originating from all common isotopically-labeled approaches, as well as label-free ion intensity-based data-independent methods. PMID:22871168

Optimised labeling, preclinical and initial clinical aspects of CCK-2 receptor-targeting with 3 radiolabeled peptides

Energy Technology Data Exchange (ETDEWEB)

Breeman, Wouter A.P. [Department of Nuclear Medicine, Erasmus MC Rotterdam' s 3015 CE Rotterdam (Netherlands)], E-mail: w.a.p.breeman@erasmusmc.nl; Froeberg, A.C.; Blois, E. de; Gameren, A. van; Melis, M.; Jong, M. de [Department of Nuclear Medicine, Erasmus MC Rotterdam' s 3015 CE Rotterdam (Netherlands); Maina, T.; Nock, B.A. [Molecular Radiopharmacy Section, I/R-RP, NCSR ' Demokritos' , Athens (Greece); Erion, J.L. [BioSynthema Inc., St. Louis, MO (United States); Maecke, H.R. [Radiological Chemistry, University Hospital Basel (Switzerland); Krenning, E.P. [Department of Nuclear Medicine, Erasmus MC Rotterdam' s 3015 CE Rotterdam (Netherlands); Department of Internal Medicine, Erasmus MC, Rotterdam (Netherlands)

2008-11-15

Medullary thyroid carcinoma (MTC) expresses CCK-2 receptors. {sup 111}In-labeled DOTA-DGlu-Ala-Tyr-Gly-Trp-Met-Asp-Phe-NH{sub 2} (DOTA-MG11), DOTA-DAsp-Tyr-Nle-Gly-Trp-Nle-Asp-Phe-NH{sub 2} (DOTA-CCK), and {sup 99m}Tc-labeled N{sub 4}-Gly-DGlu-(Glu){sub 5}-Ala-Tyr-Gly-Trp-Met-Asp-Phe-NH{sub 2} ({sup 99m}Tc-Demogastrin 2) are analogs developed for CCK-2 receptor-targeted scintigraphy. All 3 radiolabeled analogs were selected on the basis of their high CCK-2 receptor affinity and their good in vitro serum stability, with in vitro serum t{sub 1/2} values of several hours. Radiolabeling of DOTA-peptides with {sup 111}In requires a heating procedure, typically in the range of 80 deg. - 100 deg. C up to 30 min. Following this procedure with DOTA-MG11 resulted in a >98 % incorporation of {sup 111}In, however, with a radiochemical purity (RCP) of <50 %. The decrease in RCP was found to be due to oxidation of the methionine residue in the molecule. Moreover, this oxidized compound lost its CCK-2 receptor affinity. Therefore, conditions during radiolabeling were optimised: labeling of DOTA-MG11 and DOTA-CCK with {sup 111}In involved 5 min heating at 80 deg. C and led to an incorporation of {sup 111}In of >98 %. In addition, all analogs were radiolabeled in the presence of quenchers to prevent radiolysis and oxidation resulting in a RCP of >90 %. All 3 radiolabeled analogs were i.v. administered to 6 MTC patients: radioactivity cleared rapidly by the kidneys, with no significant differences in the excretion pattern of the 3 radiotracers. All 3 radiolabeled analogs exhibited a low in vivo stability in patients, as revealed during analysis of blood samples, with the respective t{sub 1/2} found in the order of minutes. In patient blood, the rank of radiopeptide in vivo stability was: {sup 99m}Tc-Demogastrin 2 (t{sub 1/2} 10-15 min)>{sup 111}In-DOTA-CCK (t{sub 1/2}{approx}5-10 min)>{sup 111}In-DOTA-MG11 (t{sub 1/2}<5 min)

Peptide deformylase as an antibacterial drug target: assays for detection of its inhibition in Escherichia coli cell homogenates and intact cells.

Science.gov (United States)

Apfel, C M; Evers, S; Hubschwerlen, C; Pirson, W; Page, M G; Keck, W

2001-04-01

An assay was developed to determine the activity of peptide deformylase (PDF) inhibitors under conditions as close as possible to the physiological situation. The assay principle is the detection of N-terminal [35S]methionine labeling of a protein that contains no internal methionine. If PDF is active, the deformylation of the methionine renders the peptide a substrate for methionine aminopeptidase, resulting in the removal of the N-terminal methionine label. In the presence of a PDF inhibitor, the deformylation is blocked so that the N-formylated peptide is not processed and the label is detected. Using this assay, it is possible to determine the PDF activity under near-physiological conditions in a cell-free transcription-translation system as well as in intact bacterial cells.

Measuring protein synthesis using metabolic ²H labeling, high-resolution mass spectrometry, and an algorithm.

Science.gov (United States)

Kasumov, Takhar; Ilchenko, Serguey; Li, Ling; Rachdaoui, Nadia; Sadygov, Rovshan G; Willard, Belinda; McCullough, Arthur J; Previs, Stephen

2011-05-01

We recently developed a method for estimating protein dynamics in vivo with heavy water ((2)H(2)O) using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) [16], and we confirmed that (2)H labeling of many hepatic free amino acids rapidly equilibrated with body water. Although this is a reliable method, it required modest sample purification and necessitated the determination of tissue-specific amino acid labeling. Another approach for quantifying protein kinetics is to measure the (2)H enrichments of body water (precursor) and protein-bound amino acid or proteolytic peptide (product) and to estimate how many copies of deuterium are incorporated into a product. In the current study, we used nanospray linear trap Fourier transform ion cyclotron resonance mass spectrometry (LTQ FT-ICR MS) to simultaneously measure the isotopic enrichment of peptides and protein-bound amino acids. A mathematical algorithm was developed to aid the data processing. The most notable improvement centers on the fact that the precursor/product labeling ratio can be obtained by measuring the labeling of water and a protein (or peptide) of interest, thereby minimizing the need to measure the amino acid labeling. As a proof of principle, we demonstrate that this approach can detect the effect of nutritional status on albumin synthesis in rats given (2)H(2)O. Copyright © 2011 Elsevier Inc. All rights reserved.

Investigation of peptide based surface functionalization for copper ions detection using an ultrasensitive mechanical microresonator

DEFF Research Database (Denmark)

Cagliani, Alberto; Fischer, Lee MacKenzie; Rasmussen, Jakob Lyager

2011-01-01

In the framework of developing a portable label-free sensor for multi arrayed detection of heavy metals in drinking water, we present a mechanical resonator-based copper ions sensor, which uses a recently synthesized peptide Cysteine–Glycine–Glycine–Histidine (CGGH) and the l-Cysteine (Cys) peptide...

Decision peptide-driven: a free software tool for accurate protein quantification using gel electrophoresis and matrix assisted laser desorption ionization time of flight mass spectrometry.

Science.gov (United States)

Santos, Hugo M; Reboiro-Jato, Miguel; Glez-Peña, Daniel; Nunes-Miranda, J D; Fdez-Riverola, Florentino; Carvallo, R; Capelo, J L

2010-09-15

The decision peptide-driven tool implements a software application for assisting the user in a protocol for accurate protein quantification based on the following steps: (1) protein separation through gel electrophoresis; (2) in-gel protein digestion; (3) direct and inverse (18)O-labeling and (4) matrix assisted laser desorption ionization time of flight mass spectrometry, MALDI analysis. The DPD software compares the MALDI results of the direct and inverse (18)O-labeling experiments and quickly identifies those peptides with paralleled loses in different sets of a typical proteomic workflow. Those peptides are used for subsequent accurate protein quantification. The interpretation of the MALDI data from direct and inverse labeling experiments is time-consuming requiring a significant amount of time to do all comparisons manually. The DPD software shortens and simplifies the searching of the peptides that must be used for quantification from a week to just some minutes. To do so, it takes as input several MALDI spectra and aids the researcher in an automatic mode (i) to compare data from direct and inverse (18)O-labeling experiments, calculating the corresponding ratios to determine those peptides with paralleled losses throughout different sets of experiments; and (ii) allow to use those peptides as internal standards for subsequent accurate protein quantification using (18)O-labeling. In this work the DPD software is presented and explained with the quantification of protein carbonic anhydrase. Copyright (c) 2010 Elsevier B.V. All rights reserved.

Record Charge Carrier Diffusion Length in Colloidal Quantum Dot Solids via Mutual Dot-To-Dot Surface Passivation.

Science.gov (United States)

Carey, Graham H; Levina, Larissa; Comin, Riccardo; Voznyy, Oleksandr; Sargent, Edward H

2015-06-03

Through a combination of chemical and mutual dot-to-dot surface passivation, high-quality colloidal quantum dot solids are fabricated. The joint passivation techniques lead to a record diffusion length for colloidal quantum dots of 230 ± 20 nm. The technique is applied to create thick photovoltaic devices that exhibit high current density without losing fill factor. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Multimodality Imaging with Silica-Based Targeted Nanoparticle Platforms

International Nuclear Information System (INIS)

Lewis, Jason S.

2012-01-01

Objectives: To synthesize and characterize a C-Dot silica-based nanoparticle containing 'clickable' groups for the subsequent attachment of targeting moieties (e.g., peptides) and multiple contrast agents (e.g., radionuclides with high specific activity) (1,2). These new constructs will be tested in suitable tumor models in vitro and in vivo to ensure maintenance of target-specificity and high specific activity. Methods: Cy5 dye molecules are cross-linked to a silica precursor which is reacted to form a dye-rich core particle. This core is then encapsulated in a layer of pure silica to create the core-shell C-Dot (Figure 1) (2). A 'click' chemistry approach has been used to functionalize the silica shell with radionuclides conferring high contrast and specific activity (e.g. 64Cu and 89Zr) and peptides for tumor targeting (e.g. cRGD and octreotate) (3). Based on the selective Diels-Alder reaction between tetrazine and norbornene, the reaction is bioorthogonal, highyielding, rapid, and water-compatible. This radiolabeling approach has already been employed successfully with both short peptides (e.g. octreotate) and antibodies (e.g. trastuzumab) as model systems for the ultimate labeling of the nanoparticles (1). Results: PEGylated C-Dots with a Cy5 core and labeled with tetrazine have been synthesized (d = 55 nm, zeta potential = -3 mV) reliably and reproducibly and have been shown to be stable under physiological conditions for up to 1 month. Characterization of the nanoparticles revealed that the immobilized Cy5 dye within the C-Dots exhibited fluorescence intensities over twice that of the fluorophore alone. The nanoparticles were successfully radiolabeled with Cu-64. Efforts toward the conjugation of targeting peptides (e.g. cRGD) are underway. In vitro stability, specificity, and uptake studies as well as in vivo imaging and biodistribution investigations will be presented. Conclusions: C-Dot silica-based nanoparticles offer a robust, versatile, and multi

Peptide functionalized gold nanoparticles: the influence of pH on binding efficiency

Science.gov (United States)

Harrison, Emma; Hamilton, Jeremy W. J.; Macias-Montero, Manuel; Dixon, Dorian

2017-07-01

We report herein on the synthesis of mixed monolayer gold nanoparticles (AuNPs) capped with both polyethylene glycol (PEG) and one of three peptides. Either a receptor-mediated endocytosis peptide, an endosomal escape pathway (H5WYG) peptide or the Nrp-1 targeting RGD peptide (CRGDK) labeled with FITC. All three peptides have a thiol containing cysteine residue which can be used to bind the peptides to the AuNPs. In order to investigate the influence of pH on peptide attachment, PEGylated AuNPs were centrifuged, the supernatant removed, and the nanoparticles were then re-suspended in a range of pH buffer solutions above, below and at the respective isoelectric points of the peptides before co-functionalization. Peptide attachment was investigated using dynamic light scattering, Ultra-violet visible spectroscopy (UV/Vis), FTIR and photo luminescence spectroscopy. UV/Vis analysis coupled with protein assay results and photoluminescence of the FITC tagged RGD peptide concluded that a pH of ∼8 optimized the cysteine binding and stability, irrespective of the peptide used.

Nonlinear Dot Plots.

Science.gov (United States)

Rodrigues, Nils; Weiskopf, Daniel

2018-01-01

Conventional dot plots use a constant dot size and are typically applied to show the frequency distribution of small data sets. Unfortunately, they are not designed for a high dynamic range of frequencies. We address this problem by introducing nonlinear dot plots. Adopting the idea of nonlinear scaling from logarithmic bar charts, our plots allow for dots of varying size so that columns with a large number of samples are reduced in height. For the construction of these diagrams, we introduce an efficient two-way sweep algorithm that leads to a dense and symmetrical layout. We compensate aliasing artifacts at high dot densities by a specifically designed low-pass filtering method. Examples of nonlinear dot plots are compared to conventional dot plots as well as linear and logarithmic histograms. Finally, we include feedback from an expert review.

Molecular imaging of neuroendocrine tumors using {sup 68}Ga-labeled peptides (Somatostatin receptor PET/CT); Molekulare Bildgebung neuroendokriner Tumoren mit {sup 68}Ga-markierten Peptiden (Somatostatinrezeptor-PET/CT)

Energy Technology Data Exchange (ETDEWEB)

Baum, R.P.; Prasad, V. [Zentralklinik Bad Berka GmbH (Germany). Klinik fuer Nuklearmedizin/PET-Zentrum; Hoersch, D. [Zentralklinik Bad Berka GmbH (Germany). Klinik fuer Innere Medizin, Gastroenterologie, Onkologie, Endokrionologie

2009-06-15

Receptor PET/CT using {sup 68}Ga-labeled somatostatin analogues (DOTA-NOC, DOTA-TOC or DOTA-TATE) enables the highly sensitive molecular imaging of neuroendocrine tumors (NETs) based on the expression of somatostatin receptors and even the detection of receptor subtypes. Our experience after more than 3000 studies shows that receptor PET/CT has a significantly higher tumor detection rate than conventional scintigraphy (even in SPECT/CT technique), and that tumor lesions can be very accurately localized. By calculating standardized uptake values (SUV) - which are reproducible and investigator-independent - patients can be selected for peptide receptor radiotherapy and also the course after therapy can be controlled. Receptor-PET/CT is the most sensitive imaging modality for the detection of unknown primary tumors (CUP syndrome), which is especially true for the detection of neuroendocrine tumors of the pancreas and small bowel; whole-body staging (''one stop shop'') as well as restaging and selection of patients for peptide receptor radiotherapy can be performed using a patient-friendly procedure (examination finished within one hour) exposing the patient to less radiation than whole-body CT scanning. The {sup 68}Ge/{sup 68}Ga generator has proved very reliable over the years - even in a hospital environment. The effective costs for {sup 68}Ga labeled somatostatin analogues might be less than for scintigraphic agents, provided a certain number of studies per year are performed. The development of new tumor-specific peptides as well as of other DOTA- or NOTA-coupled radiopharmaceuticals opens a new avenue into the future: finally, the {sup 68}Ga generator could play a similar important role for PET/CT as did the {sup 99m}Tc-Generator for conventional gamma camera imaging over the last decades. (orig.)

Technetium-99m as alternative to produce somatostatin-labeled derivatives: comparative biodistribution evaluation with 111In-DTPA-octreotide

International Nuclear Information System (INIS)

Melo, Ivani B.; Buchpiguel, Carlos Alberto; Ueda, Laura T.; Araujo, Elaine B. de; Muramoto, Emiko; Barboza, Marycel F. de; Mengatti, Jair; Silva, Constancia P.G. da

2008-01-01

Synthetic somatostatin (SST) analogues have been used in the preparation of receptor-specific radiopharmaceuticals for diagnostic and therapy of neuroendocrine (NE) tumors. 111 In-DTPA-Octreotide (OctreoScan®) has found useful for imaging a range of tumors, including NE cancer, carcinoide and lymphoma. Unfortunately, 111 In is a high-cost cyclotron produced radioisotope with gamma emission not so suitable for scintigraphic images and for dosimetry like 99m Tc. This work studied the labeling conditions with 99m Tc and biological distribution in Swiss mice of two SST analogs (HYNIC-Tyr 3 -Octreotide and HYNICTyr 3 - Octreotate) and compared the biodistribution pattern with 111 In-DTPA-Octreotide. 99 mTc-HYNIC-Tyr 3 - Octreotate ( 99m Tc-HYNIC-TATE) and 99m Tc-HYNIC-Tyr 3 -Octreotide ( 99m Tc-HYNIC-OCT) were produced by labeling conditions using tricine and EDDA as coligands. 111 In-DTPA-Octreotide ( 111 In-DTPA-OCT) was produced by labeling DTPA-Octreotide with 111 InCl 3 (Nordion). Radiochemical purity of labeled preparations was determined by ITLC-SG. Biological distribution studies were performed after injection of radiopharmaceuticals on Swiss mice. Labeling procedures resulted on high radiochemical yield for all three preparations and the labeled products presented high in vitro stability. Biological distribution studies evidenced similar general biodistribution of 99m Tc-labeled peptides when compared with indium-labeled peptide with fast blood clearance and elimination by urinary tract. Kidneys uptake of 99 mTc-HYNIC-TATE are similar to 111 In-DTPA-Octreotide, and both are significantly higher than 99 mTc-HYNIC-OCT. All labeled peptides presented similar uptake on liver, but the retention in time at intestines, particularly at large intestine, was more expressive for 111 In-labeled peptide. The %ID of 99m Tc-HYNIC-OCT and 99m Tc-HYNIC-TATE in organs with high density of SST receptors like pancreas and adrenals were significant and similar to obtained for 111