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ABSTRACT: Sulfur mustard (SM) is a hazardous chemical warfare agent that has been used in several military conflicts. SM is also considered as a major threat to civilians because of its existing stockpiles and easy production. Analysis of exposure biomarkers in biological samples collected from suspected victims is a useful tool for early diagnosis of SM poisoning. In this study, a sensitive and rapid quantitative method with ultra high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was developed for simultaneous determination of seven SM plasma biomarkers, including its oxidative, hydrolysis and β-lyase metabolites. A simple one-step protein precipitation with acetonitrile-methanol (4:1) was used for sample preparation. A full validation was conducted with respect to specificity, linearity, recovery, matrix effect, precision, accuracy and stability. The lower limits of quantification for the seven metabolites ranged from 0.01μgL(-1) to 5μgL(-1). The intraday relative standard deviation was less than 7.0%, and the interday deviation was less than 9.1%. The recoveries varied in the range from 82.8% to 118%. This method has been successfully applied to a toxicokinetic study for obtaining the plasma profiles of seven metabolites in SM-exposed rats, following a single subcutaneous dose of 3.3mgkg(-1). All the targeted compounds were detected in rat plasma. bis-β-Chloroethyl sulfoxide (SMO), thiodiglycol (TDG), thiodiglycol sulfoxide (TDGO), 1,1'-sulfonylbis-[2-S-(N-acetylcysteinyl)ethane (SBSNAE), 1,1'-sulfonylbis-[2-(methylsulfinyl)ethane] (SBMSE) and 1-methylsulfinyl-2-[2-(methylthio)ethylsulfonyl]ethane (MSMTESE) were found to be the major metabolites in rat plasma. The time windows for the detection of these metabolites were varied in the range of 5min to 48h after exposure. The method provides a useful tool for short-term diagnosis of SM poisoning.
Journal of chromatography. B, Analytical technologies in the biomedical and life sciences 01/2013; 917-918C:100-107. · 2.78 Impact Factor
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ABSTRACT: In this paper, denatured bovine serum albumin (dBSA) has been developed as a useful scaffold for appending multiple oligonucleotides (ODNs), and the resulted novel dBSA-ODNs covalent hybrid can be self-assembled to the surface of CdTe quantum dots (QDs) via complexation with unoccupied coordination metal sites on QDs' surface. The formed QDs/dBSA-ODNs bioconjugate could keep a long-term stability in aqueous solution while maintaining its fluorescence quantum yield and spectral properties. Furthermore, the QDs/dBSA-ODNs also can be readily hybridized with an anti-rHuEPO-α aptamers linked AuNPs (Apt-AuNPs) to form a fluorescent resonance energy transfer-based nano-biosensing system for highly sensitive and selective detection of rHuEPO-α protein lowered to 53.6pM in a rapid, simple manner.
Biosensors & bioelectronics 01/2013; 43C:446-452. · 5.43 Impact Factor
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ABSTRACT: Aptamers are a series of high-affinity and high-specificity oligoneucleotides (single-stranded DNA or RNA) to the target,
usually selected by the combinatorial chemistry SELEX technique (systematic evolution of ligands by exponential enrichment).
Aptamers have proved to be one kind of novel functional molecules in life science and chemistry. After being labeled by signaling
groups, the aptamer probe can conveniently transfer the characteristics of aptamer-target recognition to a form of high-sensitive
signal, and the high-affinity, high-specificity measurements of metal ion, organic molecules, nucleic acid, proteins, or cells
become possible. This article summarizes the recent advances of aptamer probes in different sensing fields, with special emphasis
on aptamer probes as fluorescent sensors.
Science in China Series B Chemistry 04/2012; 51(3):193-204. · 1.20 Impact Factor
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ABSTRACT: A sensitive method for the determination of the organophosphorus nerve agents sarin, soman and VX adducts with tyrosine residue of albumin in rat plasma has been developed and validated using liquid chromatography-isotope dilution tandem mass spectrometry (LC-IDMS/MS). O-(O-Alkyl methylphosphonyl) tyrosine adducts and their deuterated products that were used as the internal standards were synthesised to establish the quantitative isotope-dilution method. Protein purification and solid-phase extraction (SPE) were applied to improve the recovery efficiency, reduce interference and achieve high sensitivity. The method provided a detection limit of 0.01 ng/mL for sarin and soman adducts and 0.05 ng/mL for the VX adduct. The value of the intra-day relative standard deviation over the calibration range was less than 6.16% (n=6), and that of the inter-day was less than 12.7% (n=6). The recovery varied from 86% to 111%. This sensitive method was successfully applied to the analysis of adducts in rat plasma after nerve agent exposure, and the results demonstrated the dose-effect relationships.
Journal of chromatography. A 03/2012; 1229:164-71. · 4.19 Impact Factor
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ABSTRACT: The N-terminal valine adduct (HETE-Val) in globin is believed to behave as a long-lived biomarker after exposure to sulfur mustard (HD). Development of a highly sensitive method for monitoring HETE-Val, particularly at low HD exposure levels or for retrospective detection, would be a significant achievement. In this study, by improving the sample preparation method, a sensitive NCI-GC/MS method was established for the analysis of HETE-Val in globin after HD exposure. To optimize and investigate the sample preparation method, all the relevant HETE-Val chemicals were synthesized, purified, and characterized. By carrying out optimized solid phase extraction (SPE) cleanup followed by modified Edman degradation results in a low detection level and clean baseline. The minimum detectable exposure level of human blood (in vitro) to HD is 20 nmol/L (S/N>3). The interday and intraday precisions of the proposed method were found to be acceptable with less than a 15% relative standard deviation (RSD). A nearly linear dose-effect relationship was observed between HETE-Val and a HD exposure concentration range of 0.1-120 μmol/L. The percentage of HD that reacted with N-terminal valine in globin obtained from human blood (in vitro) was quantified using the proposed method.
Talanta 08/2011; 85(2):1154-9. · 3.79 Impact Factor
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ABSTRACT: Here, a simple label-free colorimetric sensing method for organophosphate (OP) nerve agents and pesticide based on catalytic reaction of acetylcholine esterase (AChE) and the aggregation of lipoic acid (LA) capped AuNPs has been established, which is highly sensitive with a limit of detection (LOD) lowered to pM level. In this method, only the AChE hydrolysis product of acetylthiocholine (ATCh), i.e., cationic thiocholine (TCh) can induce the aggregation of LA capped AuNPs along with a distinct color change from red to steel-blue. When OPs as enzyme inhibitors exist, the generation of TCh can be suppressed and the color change of LA capped AuNPs is gradually diminished according to different concentrations of OPs. The feasibility of this method has been demonstrated by sensitive measurement of OP nerve agents and pesticide in a spiked fruit sample with reliable results. This distinct and rapid colorimetric response enables us to readily probe OPs without more technical demand.
Biosensors & bioelectronics 07/2011; 28(1):152-7. · 5.43 Impact Factor
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ABSTRACT: The analysis of biological samples can provide qualitative and quantitative evidence of exposure to sulfur mustard (SM). N7-(2-hydroxyethylthioethyl) guanine (N7-HETEG) is the most abundant DNA adduct and one of the biomarkers of SM exposure. For the detection and validation of SM exposure, a sensitive high performance liquid chromatography-tandem mass spectrometry (HPLC-ESI-MS/MS) method for the determination of N7-HETEG in a biomedical sample was developed, using N7-benzylguanine as the internal standard. The method was validated and showed the limit of detection of 300 pg/mL (S/N > or =10) and limit of quantitation of 850 pg/mL (S/N > or =20). The linear range was 300 pg/mL-1.28 microg/mL, with both the values of intraday and interday relative standard deviations of less than 10% (n =7) over the calibration range. The recoveries varied from 100% to 132%. The method was applied for the determination of N7-HETEG in the lung of SD rats caused by dermal exposure to SM in vivo. Under the exposure to SM of 5.5, 11, 22 and 45 mg/kg, respectively, (0.56 +/- 0.16), (0.67 +/- 0.12), (1.36 +/- 0.68) and (5.14 +/- 0.92) ng of N7-HETEG per gram of SD rat lung tissue were found after four days of exposure. The amount of N7-HETEG increased with the exposed doses, and it can act as a biomarker of sulfur mustard exposure.
Se pu = Chinese journal of chromatography / Zhongguo hua xue hui 04/2011; 29(4):325-9.
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ABSTRACT: We present here a novel conjugated aptamer-gold nanoparticle (Apt-AuNPs) fluorescent probe and its application for specific detection of recombinant human erythropoietin-α (rHuEPO-α). In this nanobiosensor, 12 nm AuNPs function as both a nano-scaffold and a nano-quencher (fluorescent energy acceptor), on the surface of which the complementary sequences are linked (as cODN-AuNPs) and pre-hybridized with carboxymethylfluorescein (FAM)-labeled anti-rHuEPO-α aptamers. Upon target protein binding, the aptamers can be released from the AuNP surface and the fluorescence signal is restored. Key variables such as the length of linker, the hybridization site and length have been designed and optimized. Full performance evaluation including sensitivity, linear range and interference substances are also described. This nanobiosensor provides a promising approach for a simple and direct quantification of rHuEPO-α concentrations as low as 0.92 nM within a few hours.
Sensors 01/2011; 11(11):10490-501. · 1.74 Impact Factor
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ABSTRACT: A lectin-mediated affinity chromatographic SELEX technique was developed to generate functional ssDNA aptamers for recombinant human erythropoietin-α (rHuEPO-α), an important pharmaceutical glycoprotein for the first time. Secondary structure analysis of the aptamer clones from sequential 6th, 7th, and 8th rounds showed that a certain fragment 'CGAGAT' in the 3' primer region could be used to trace increasing evolution stringency from its hybridization at different locations, in which a specific hybridization with its complement in the 5' primer region (aptamer 807) was evolved as the prevalent one with the simplest and most common motif. Characteristics of the aptamers with lower Gibbs' free energies and K(d) values (nM) were investigated. For aptamer 813, the minimer 813-42nt formed by the random and primer regions was indispensable for the specific binding with rHuEPO-α. While for aptamer 807, only the random region, that is, 807-39nt, was the functional motif. Further experiments of methylation, site-directed mutation and length variation showed that the loop of aptamer 807-39nt was the key region for binding with rHuEPO-α, and the stem should be considered as a stabilizing part. Lower cross-reactivity of aptamer 807-39nt was observed with human normal urothelium tissues than the anti-EPO monoclonal antibody AE7A5. Aptamer 807-39nt also exhibited a specific recognition for human bladder carcinoma cells and human urothelium tumors, which might provide a novel way to probe such tumors with overexpressed EPOs.
Bioorganic & medicinal chemistry 10/2010; 18(22):8016-25. · 2.82 Impact Factor
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ABSTRACT: Recombinant human erythropoietin (rHuEPO) is one kind of important hematopoietic growth factor, which is widely used in anaemia treatment as well as sometimes abused by endurance athletes. Based on a set of anti-rHuEPO-α aptamers successfully in vitro isolated in our laboratory, we herein describe a novel magnetic beads-based aptameric real-time PCR assay for the accurate quantification of rHuEPO-α, which combined the specific recognition with amplification capability of aptamers. Two detection strategies, termed 'recognition-after-hybridization' and 'recognition-before-hybridization' respectively, were constructed and compared. Strategy B, i.e.'recognition-before-hybridization', was finally adopted as the preferred one to measure rHuEPO-α. A limit of detection (LOD) of 1 pmol/L rHuEPO-α and a wide dynamic range from 6 pmol/L to 100 nmol/L were obtained for physiological buffer. Furthermore, a LOD of 6 pmol/L was achieved for more complicated matrix-half diluted artificial urine. These results indicate that the anti-rHuEPO-α aptamer fits the high sensitive detection of rHuEPO-α very well. The use of the aptamer with magnetic beads-based real-time PCR allows a direct and novel assay for rHuEPO-α.
The Analyst 09/2010; 135(11):2924-9. · 4.23 Impact Factor
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ABSTRACT: Recombinant human erythropoietin-alpha (rHuEPO-alpha) has been widely used in clinic for anemia treatment. The detection and quantification of rHuEPO-alpha is essential for monitoring this widespread recombinant glycoprotein pharmaceutical. In this paper, we developed a new affinity probe capillary electrophoresis/laser-induced fluorescence (APCE/LIF) method for the detection of rHuEPO-alpha by using a specific single-stranded DNA aptamer probe for the first time. In this method, the complex of aptamer-rHuEPO-alpha and the free aptamer can be well separated and identified by their migration and fluorescence intensity after systematic optimization. The existence of sodium cation in the sample buffer and running buffer played a critical role for stabilizing complex and enhancing the separation efficiency, additionally, suitable high voltage and sample buffer additives were also important for improving the peak height of the complex. Under the optimized conditions, the method was successfully applied for the quantification of rHuEPO-alpha in physiological buffer, artificial urine and human serum. The linear range for rHuEPO-alpha was from 0.2 to 100 nM and the limit of detection was 0.2 nM (i.e. 7.4 ng/mL). Further binding experiments using fluorescein isothiocyanate-labeled rHuEPO-alpha (F-rHuEPO-alpha) and N-deglycosylated F-rHuEPO-alpha demonstrated that the oligosaccharides moiety was of importance in the specific interaction between rHuEPO-alpha and its aptamer.
Journal of chromatography. A 08/2010; 1217(35):5635-41. · 4.19 Impact Factor
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ABSTRACT: A new rapid, sensitive method for protein determination using capillary electrophoresis and specific colorimetric reaction of bicinchoninic acid (BCA) was established, assisted by microwave incubation. With 60 mmol/L boric acid buffer (pH 9.5) and inclusion additive of beta-cyclodextrin, the complex of BCA-Cu+ and free BCA molecules were efficiently separated. The peak intensity of BCA-Cu+ was higher than those of native proteins about two orders of magnitude at a low wavelength of 200 nm. The linear ranges of this method were from 2 to 200 mg/L for transferrin, and 2 to 100 mg/L for ricin. The limits of detection for transferrin and ricin were 0.33 and 0.37 mg/L, respectively. This method was also successfully applied in the determination of some ricin samples in the First International Proficiency Test on the quantification of ricin.
Se pu = Chinese journal of chromatography / Zhongguo hua xue hui 07/2010; 28(7):682-7.
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ABSTRACT: The aim of this work is to describe the first example of aptameric molecular beacon (MB)-based probe for the detection of recombinant human erythropoietin (rHuEPO-alpha) in physiological buffer, using a novel 35 nt ssDNA aptamer (807-35 nt) originally isolated by Systematic Evolution of Ligands by Exponential enrichment (SELEX) technique in our laboratory. Both "Signal-on" and "Signal-off" MB modes were developed, respectively, in which the conformational alteration of aptamer before and after binding to rHuEPO-alpha can be demonstrated in terms of the correspondingly fluorescent changes. Comparing with "Signal-off" mode, "Signal-on" mode provided higher sensitivity, while with the addition of target rHuEPO-alpha, quenching between fluorescent 807-35 nt aptamer (F-Apt) and a short quencher-labeled complementary sequence (QDNA) was disturbed by the specific binding between rHuEPO-alpha and F-Apt. QDNA was thus loosened and released from F-Apt, leading to a consequently full fluorescent restoration. Systematic optimization of parameters in "Signal-on" mode were carried out, the choice of QDNA length, the hybridization site of a small supplementary DNA (SDNA) stabilizer, and the existence of Mg(2+) cation played essential roles for the performance characterization. A convenient and sensitive determination of rHuEPO-alpha with a LOD of 0.4 nM was achieved.
Talanta 12/2009; 80(2):985-90. · 3.79 Impact Factor
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ABSTRACT: A method for the identification of Mycobacterium species using reversed-phase high performance liquid chromatography (RP-HPLC) was developed. The fingerprints library was constructed on the basis of RP-HPLC chromatograms of the mycolic acids derivatives from 49 Mycobacterium species cultures. Two inoculation loops of Mycobacterium, for fast growth with one week incubation or for slow growth with three weeks incubation, were saponified for 1 h and stocked at 4 degrees C. The mycolic acids from each culture of Mycobacterium species were acidified, extracted, derivatized, and analyzed by the RP-HPLC method. On the basis of the HPLC patterns of relative retention time and relative peak height, the identifications of Mycobacterium species were performed. This established method has a good precision of retention times with the relative standard deviations (RSDs) ranging from 0.13% to 1.07%. The mycolic acids fingerprints library of HPLC patterns was set up, including 49 species that were recorded in "Bergey's Manual of Systematic Bacteriology". Three patterns were observed from the chromatographic behaviors of mycolic acids derivatives, including single peak-cluster, double peak-clusters, triple and multiple peak-clusters. Forty-one species were successfully identified according to the different relative retention times of the peaks and the relative peak heights. The established method can identify Mycobacterium species with rapidity and high reliability.
Se pu = Chinese journal of chromatography / Zhongguo hua xue hui 10/2008; 26(5):534-9.
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ABSTRACT: In the World Anti-doping Agency 2008 Prohibited List, the prohibited substances of S2 item are hormones and related substances, which are belonging to the endogenous biomacromolecules. How to identify the substances derived from endogenous secretion or exogenous administration is the main problem in doping control analysis and attracts more attention. The present report summarizes the main analytical strategies, including indirect blood tests and direct detection approaches developed to identify the presence of erythropoietin (EPO) and human growth hormone (hGH), which have wide pharmaceutical applications and thus been fully examined. The recent physico-chemical or immunoanalytical methodologies on the discrimination of recombinant and endogenous proteins are emphasized.
Se pu = Chinese journal of chromatography / Zhongguo hua xue hui 08/2008; 26(4):437-43.
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ABSTRACT: ACE technique provides an effective tool for the separation and identification of disease-related biomarkers in clinical analysis. In recent years, a couple of synthetic DNA or RNA oligonucleotides, known as aptamers, rival the specificity and affinity for targets to antibodies and are employed as one kind of powerful affinity probe in ACE. In this work, based on high affinity between antithrombin aptamer and thrombin (their dissociation constant is 0.5 nM), a carboxyfluorescein-labeled 29-nucleotide (nt) aptamer (F29-mer) was used and an aptamer-based affinity probe CE (aptamer-based APCE) method was successfully established for high-sensitive detection and quantitative analysis of thrombin. Experimental conditions including incubation temperature and time, buffer composition, and concentration of cations were investigated and optimized. Under the optimized condition, the linear range was from 0 to 400 nM and the LOD was 2 nM (74 ng/mL, S/N = 3), i.e., 40 amol, both in running buffer and in 5% v/v human serum. This LOD is the lowest one than those achieved by the previous APCE methods but based on a 15-mer aptamer. This approach offers a promising method for the rapid, selective, and sensitive detection of thrombin in practical utility. Further binding experiments using one carboxyfluorescein-labeled aptamer and the other nonlabeled aptamer or vice versa were carried out to deduce the formation of ternary complex when these two aptamers coexisted in the free solution with thrombin.
Electrophoresis 07/2008; 29(12):2570-7. · 3.30 Impact Factor
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ABSTRACT: Aptamers which specifically recognize targets are selected from random oligonucleotide library using systematic evolution
of ligands by exponential enrichment (SELEX). In this paper, capillary electrophoresis (CE) as a separation approach has been
introduced to SELEX procedure. The high efficiency of CE gives rise to greatly shorten the selection procedure. The results
from enzyme-linked assay and dot blot experiment show that an enrichment pool has been obtained after four rounds selection,
which can specifically recognize ricin.
Frontiers of Chemistry in China 09/2007; 2(4):431-435.
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Jiankun Qie,
Jinbo Ma,
Liangyou Wang,
Xiaoyu Xu,
Jianquan Zheng,
Sijian Dong, Jianwei Xie,
Huixian Sun,
Wenxia Zhou,
Chunhui Qi,
Xiunan Zhao,
Yongxiang Zhang,
Keliang Liu
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ABSTRACT: Site-specific mono-PEGylations were performed in different conformational regions of Thymosin alpha 1 (T alpha 1) by introducing one cysteine residue into the chosen site and coupling with thiol-specific mPEG-MAL reagent. Results demonstrated that PEGylated sites and regions influenced the conformations and pharmacokinetic profiles of the peptide greatly with following order: alpha-helix, beta-turn, random coil and terminals, but little on the immunoactivity.
Drug metabolism letters. 09/2007; 1(3):232-40.
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Jiankun Qie,,
Jinbo Ma,,
Liangyou Wang,,
Xiaoyu Xu,,
Jianquan Zheng,,
Sijian Dong,, Jianwei Xie,,
Huixian Sun,,
Wenxia Zhou,,
Chunhui Qi,,
Xiunan Zhao,,
Yongxiang Zhang,,
Keliang Liu,
[show abstract]
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ABSTRACT: Site-specific mono-PEGylations were performed in different conformational regions of Thymosin alpha 1 (Tα1) by introducing one cysteine residue into the chosen site and coupling with thiol-specific mPEG-MAL reagent. Results demonstrated that PEGylated sites and regions influenced the conformations and pharmacokinetic profiles of the peptide greatly with following order: α-helix, β-turn, random coil and terminals, but little on the immunoactivity.
Drug Metabolism Letters 07/2007; 1(3):232-340.
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ABSTRACT: Abrin toxin as the target protein, belongs to class II ribosome-inactivating proteins family, has high toxicity to eukaryotic cells. Here, we firstly report the DNA aptamers, isolated by in vitro selection, recognize abrin toxin with high affinity and specificity, and have the advantage of no cross-reaction with structure-similar protein ricin toxin over antibodies. Then, a highly selective and sensitive aptamer-based abrin assay was established using a molecular light switching reagent [Ru(phen)(2)(dppz)](2+) with a limit of detection of 1 nM and a wide linear range from 1 to 400 nM with the correlation coefficient of 0.993. This assay can be successfully directly performed not only in physiological buffer but also in more complicated biological matrix, such as diluted serum.
Biosensors and Bioelectronics 06/2007; 22(11):2456-63. · 5.60 Impact Factor
