共查询到20条相似文献,搜索用时 31 毫秒
1.
Ligand-tagged liposomes, obtained by covalent conjugation of ligands to the liposomal surface, have been widely used as detection reagents in bioanalytical assays. A non-covalent conjugation method where IgG was attached to protein G-tagged liposomes has been recently utilized. To enlarge the application of non-covalent methods to a greater variety of ligands, including peptides, proteins, and nucleic acids, we developed and optimized a new method for the preparation of NeutrAvidin-tagged liposomes with subsequent attachment of biotinylated ligands. Two assays were used to investigate the feasibility of NeutrAvidin-tagged liposomes. The first assay was a competitive immunoassay for detecting rabbit antibodies, while the second assay was a sandwich hybridization assay for detecting a synthetic target: a DNA fragment of Erwinia amylovora. To produce the immunoliposomes for the detection of rabbit IgG, NeutrAvidin was covalently tagged to the liposomal surface at four different starting molar percentages (0.1, 0.2, 0.4, and 0.8). The biotinylated goat anti-rabbit IgG at three different molar ratios of biotin to IgG (5, 10, and 20) were then attached to the NeutrAvidin-tagged liposomes by using two different molar ratios of goat anti-rabbit IgG to NeutrAvidin (1 and 5). After the comparison of all 24 combinations, the best result was obtained with the 0.1 starting molar percentage of NeutrAvidin, 20 as the molar ratio of biotin to goat IgG, and 1 as molar ratio of IgG to NeutrAvidin. Under these optimized conditions, the limit of detection (LOD) for rabbit IgG was 38 pmol/mL. Moreover, the best combination for the sandwich hybridization assay was with the 0.1 starting molar percentage of NeutrAvidin-tagged liposomes and when the molar ratio of biotinylated reporter probe to NeutrAvidin was equal to 1. The LOD for the synthetic target DNA fragment of E. amylovora was ca. 30 pmol/mL. Both assays could be completed in about 30 min without the requirement of sophisticated equipment or techniques. Therefore, these two assays have successfully demonstrated the feasibility of NeutrAvidin-tagged liposomal nanovesicles as a universal reagent for the attachment of different types of biotinylated ligands in a fast and easy coupling process. In addition, these ligand-tagged liposomes have the potential for wide use in different types of bioanalytical assays. 相似文献
2.
Jana Beinhauer Liangqiao Bian Hui Fan Marek Šebela Maciej Kukula Jose A. Barrera Kevin A. Schug 《Analytica chimica acta》2015
Estrone (E1), estradiols (α/β-E2), and estriol (E3) are four major metabolically active estrogens exerting strong biological activities at very low circulating concentrations. This paper reports a sensitive and efficient method with automated, on-line clean-up and detection to determine trace estrogens in a small volume of serum samples using liquid chromatography–electrospray ionization–tandem mass spectrometry directly, without off-line liquid–liquid or solid-phase extraction pretreatments. Serum aliquots (charcoal stripped fetal bovine serum, 100 μL) were spiked with four estrogen standards and their corresponding isotope-labeled internal standards, then bulk derivatized with 2-fluoro-1-methyl-pyridium p-toluenesulfonate (2-FMP) to establish the calibration curves and perform method validation. Calibration was established in the concentration ranges of 5–1000 pg mL−1, and demonstrated good linearity of R2 from 0.9944 to 0.9997 for the four derivatized estrogens. The lower detection limits obtained were 3–7 pg mL−1. Good accuracy and precision in the range of 86–112% and 2.3–11.9%, respectively, were observed for the quality control (QC) samples at low, medium, and high concentration levels. The stability tests showed that the derivatized serum samples were stable 8 h after derivatization at room temperature and at least to 48 h if stored at −20 °C. The method was applied to measure trace estrogens in real human and bovine serum samples, and three of four estrogen compounds studied were observed and quantified. 相似文献
3.
A gas chromatography–mass spectrometric (GC–MS) method has been established for the determination of hydrazine in drinking water and surface water. This method is based on the derivatization of hydrazine with ortho-phthalaldehyde (OPA) in water. The following optimum reaction conditions were established: reagent dosage, 40 mg mL−1 of OPA; pH 2; reaction for 20 min at 70 °C. The organic derivative was extracted with methylene chloride and then measured by GC–MS. Under the established condition, the detection and the quantification limits were 0.002 μg L−1 and 0.007 μg L−1 by using 5.0-mL of surface water or drinking water, respectively. The calibration curve showed good linearity with r2 = 0.9991 (for working range of 0.05–100 μg L−1) and the accuracy was in a range of 95–106%, and the precision of the assay was less than 13% in water. Hydrazine was detected in a concentration range of 0.05–0.14 μg L−1 in 2 samples of 10 raw drinking water samples and in a concentration range of 0.09–0.55 μg L−1 in 4 samples of 10 treated drinking water samples. 相似文献
4.
Luiz L. Mafra Jr. Claude Léger Stephen S. Bates Michael A. Quilliam 《Journal of chromatography. A》2009,1216(32):6003-6011
Quantitation of trace levels of domoic acid (DA) in seawater samples usually requires labour-intensive protocols involving chemical derivatization with 9-fluorenylmethylchloroformate and liquid chromatography with fluorescence detection (FMOC–LC–FLD). Procedures based on LC–MS have been published, but time-consuming and costly solid-phase extraction pre-concentration steps are required to achieve suitable detection limits. This paper describes an alternative, simple and inexpensive LC method with ultraviolet detection (LC–UVD) for the routine analysis of trace levels of DA in seawater without the use of sample pre-concentration or derivatization steps. Qualitative confirmation of DA identity in dubious samples can be achieved by mass spectrometry (LC–MS) using the same chromatographic conditions. Addition of an ion-pairing/acidifying agent (0.15% trifluoroacetic acid) to sample extracts and the use of a gradient elution permitted the direct analysis of large sample volumes (100 μl), resulting in both high selectivity and sensitivity (limit of detection = 42 pg ml−1 by LC–UVD and 15 pg ml−1 by LC–MS). Same-day precision varied between 0.4 and 5%, depending on the detection method and DA concentration. Mean recoveries of spiked DA in seawater by LC–UVD were 98.8% at 0.1–10 ng ml−1 and 99.8% at 50–1000 ng ml−1. LC–UVD exhibited strong correlation with FMOC–LC–FLD during inter-laboratory analysis of Pseudo-nitzschia multiseries cultures containing 60–2000 ng DA ml−1 (r2 > 0.99), but more variable results were obtained by LC–MS (r2 = 0.85). This new technique was used to confirm the presence of trace DA levels in low-toxicity Pseudo-nitzschia spp. isolates (0.2–1.6 ng ml−1) and in whole-water field samples (0.3–5.8 ng ml−1), even in the absence of detectable Pseudo-nitzschia spp. cells in the water column. 相似文献
5.
Zhanguang Chen Yulong LeiZhisen Liang Fengji LiLidong Liu Changlong LiFang Chen 《Analytica chimica acta》2012
As a kind of glycoprotein, carcinoembryonic antigen (CEA) is the important tumor marker for clinical diagnosis of the presence or recurrence of cancer. In this work, a novel label-free resonance light scattering (RLS) spectral CEA assay was developed based on the combination of highly selective immunoreaction and ultrasensitive RLS technique. In Tris–HCl buffer solution (pH 7.5), the specific immunoreaction between CEA antigen and mouse anti-CEA formed immune complexes which had a maximum RLS spectral peak at 389.0 nm, with the existence of physiological saline and polyethylene glycol 20,000 (PEG 20,000). Under the optimal conditions, the magnitude of enhanced RLS intensity (ΔIRLS) was proportional to the concentration of CEA in the range from 0.1 to 60 ng mL−1, with a detection limit (LOD, 3σ) of 0.03 ng mL−1. The characteristics of RLS, the CEA immunocomplex, the immune response, the ratio of CEA antigen and mouse anti-CEA, and the optimum conditions of the immunoreaction have been investigated. The CEA concentrations of 20 serum specimens detected by the developed assay showed consistent results in comparison with those obtained by commercially available enzyme-linked immunosorbent assay (ELISA) kit. And this method has many satisfying merits including label-free, sensitivity and high selectivity. 相似文献
6.
In previous studies we have developed a simple electrokinetically-controlled lab-on-a-chip for heterogeneous immunoassay. In that method, all the sequential operations in an immunoassay, such as reagent loading and washing, were performed automatically by electrokinetically controlling the flow in an H-shaped microchannel. Here, we demonstrated further development of a high-throughput immunoassay microfluidic chip, and the application of the new immunoassay microfluidic chip in assaying human serum. The microfluidic immunoassay analyzed ten samples in parallel in 22 min. Bacterial antibodies in samples were captured by antigens pre-patterned on the bottom wall of a microchannel and then bound with TRITC-labeled detection antibodies to generate fluorescent signals. With optimized surface concentration of antigen, the assay detected Escherichia coli O157:H7 antibody and Helicobacter pylori antibody from buffer solutions in concentration ranges of 0.02-10 μg mL−1 and 0.1-50 μg mL−1, respectively. Human sera that were E. coli-positive or H. pylori-positive were accurately distinguished from respective negative controls. Moreover, the two antibodies, anti-E. coli and anti- H. pylori antibodies, could be simultaneously detected from human serum. This electrokinetically-controlled immunoassay shows an excellent potential for efficiently detecting multiple pathogenic infections in clinical environments. 相似文献
7.
Hyun-Jin Jung Won-Yong Lee Bong Chul Chung Man Ho Choi 《Journal of chromatography. A》2009,1216(9):1463-1468
An efficient analytical method for simultaneous determination of 12 SFEs in serum is described. The method involves solid-phase extraction to isolate of SFEs from interfering species, especially cholesteryl esters, conversion to trimethylsilyl (TMS) ether derivatives for the direct analysis by gas chromatography–mass spectrometry (GC–MS) using a high temperature MXT-1 (Silcosteel-treated stainless steel) capillary column. All SFEs as their TMS derivatives were well separated with excellent peak shapes within 12 min. Overall recoveries ranged from 88% to 119%, with a detection limits for SFEs ranged from 2 to 30 μg L−1. The linearity as correlation coefficient was higher than 0.99 except for pregnenolone-3-arachidate (r2 = 0.98) in the concentration range of 5–3000 μg L−1. Ten serum samples obtained from volunteers were also analyzed and quantitatively determined of DHEA-3-palmitate and pregnenolone-3-stearate in 1.8–1195.8 μg L−1 concentration. The devised high temperature GC–MS method could be useful for identification of SFEs in biological specimens including serum. 相似文献
8.
We have developed a new fluorescent probe of thioglycolic acid (TGA)-capped CdTe quantum dots (QDs) complexed with a model drug, meso-tetrakis(N-methylpyridinium-4-yl)porphyrin (TMPyP) for detecting deoxyribonucleic acids (DNAs). This probe operates with an “Off–On” mode: TMPyP quenches the photoluminescence (PL) of QDs via a photo induced electron-transfer (PIET) process; the presence of DNA can break the QD/TMPyP complexation, interrupting the PIET process, and switch on the PL of QDs. Sensitive detection of DNA with the detection limit of 0.16 nM and a linear detection range of 0.25–6.0 nM are achieved. Importantly, this probe can be used to distinguish the binding modes of DNA–TMPyP interactions, exhibiting the DNA sequence-dependent PL recovery behaviors. The obtained binding constant for poly(dA)·poly(dT) is ∼3.30 × 107 L mol−1, which is approximately one order of magnitude larger than those for native DNAs and poly(dG)·poly(dC). Furthermore, the thymine bases preferential of the TMPyP–DNA interaction is proved by this probe. 相似文献
9.
We report a magnetic-particle (MMP)-based chemiluminescence enzyme immunoassay (CLEIA) for free prostate-specific antigen (f-PSA) in human serum. In this method, the f-PSA is sandwiched between the anti-PSA antibody coated MMPs and alkaline phosphatase (ALP)-labeled anti-f-PSA antibody. The signal produced by the emitted photons from the chemiluminescent substrate (4-methoxy-4-(3-phosphatephenyl)-spiro-(1,2-dioxetane-3,2′-adamantane)) is directly proportional to the amount of f-PSA in a sample. The present MMP-based assay can detect f-PSA in the range of 0.1–30 ng mL−1 with the detection limit of 0.1 ng mL−1. The linear detection range could match the concentration range within the “diagnostic gray zone” of serum f-PSA levels (4–10 ng mL−1). The detection limit was sufficient for measuring clinically relevant f-PSA levels (>4 ng mL−1). Furthermore, the method was highly selective; it was unaffected by cross-reaction with human glandular kallikrein-2, a kallikrein-like serine protease that is 80% similar to f-PSA. The proposed method was finally applied to determine f-PSA in 40 samples of human sera. Results obtained using the method showed high correlation with those obtained using a commercially available microplate CLEIA kit (correlation coefficient, 0.9821). This strategy shows great potential application in the fabrication of diagnostic kits for determining f-PSA in serum. 相似文献
10.
An electrochemiluminescence (ECL) biosensor for simultaneous detection of adenosine and thrombin in one sample based on bifunctional aptamer and N-(aminobutyl)-N-(ethylisoluminol) functionalized gold nanoparticles (ABEI-AuNPs) was developed. A streptavidin coated gold nanoparticles modified electrode was utilized to immobilize biotinylated bifunctional aptamer (ATA), which consisted of adenosine and thrombin aptamer. The ATA performed as recognition element of capture probe. For adenosine detection, ABEI-AuNPs labeled hybridization probe with a partial complementary sequence of ATA reacted with ATA, leading to a strong ECL response of N-(aminobutyl)-N-(ethylisoluminol) enriched on ABEI-AuNPs. After recognition of adenosine, the hybridization probe was displaced by adenosine and ECL signal declined. The decrease of ECL signal was in proportion to the concentration of adenosine over the range of 5.0 × 10−12–5.0 × 10−9 M with a detection limit of 2.2 × 10−12 M. For thrombin detection, thrombin was assembled on ATA modified electrode via aptamer–target recognition, another aptamer of thrombin tagged with ABEI-AuNPs was bounded to another reactive site of thrombin, producing ECL signals. The ECL intensity was linearly with the concentration of thrombin from 5 × 10−14 M to 5 × 10−10 M with a detection limit of 1.2 × 10−14 M. In the ECL biosensor, adenosine and thrombin can be detected when they coexisted in one sample and a multi-analytes assay was established. The sensitivity of the present biosensor is superior to most available aptasensors for adenosine and thrombin. The biosensor also showed good selectivity towards the targets. Being challenged in real plasma sample, the biosensor was confirmed to be a good prospect for multi-analytes assay of small molecules and proteins in biological samples. 相似文献
11.
Molecular beacons (MBs) are oligonucleotide probes that fluoresce upon hybridization. The development of a real-time polymerase chain reaction (PCR) assay to detect the presence of Escherichia coli using these fluorogenic reporter molecules is reported. MBs were designed to recognize a 19-bp region of the uid A gene, coding for an enzyme β-glucuronidase. The specificity of the MB-based PCR assay was evaluated for various E. coli strains as well as bacteria species that are present in nature. The capability of the assay to detect E. coli in drinking water and produce was demonstrated. Positive detection of E. coli was demonstrated when >101 CFU mL−1 (colony forming unit) was present in the water samples and fresh produce after 18 h of enrichment. These assays could be carried out entirely in sealed PCR tubes, enabling rapid and semiautomated detection of E. coli in food and environmental samples. 相似文献
12.
This study aims to use solid phase microextraction (SPME), a simple tool to investigate diffusion rate (time) constant of selected pharmaceuticals in gel and fish muscle by comparing desorption rate of diffusion of the drugs in both agarose gel prepared with phosphate-buffered saline (PBS; pH 7.4) and fish muscle. The gel concentration (agarose gel model) that could be used to simulate tissue matrix (fish muscle) for free diffusion of drugs under in vitro and in vivo conditions was determined to model mass transfer phenomena between fibre polymer coating and environmental matrix such that partition coefficients and desorption time constant (diffusion coefficient) can be determined. SPME procedure involves preloading the extraction phase (fibre) with the standards from spiked PBS for 1 h via direct extraction. Subsequently, the preloaded fibre is introduced to the sample such fish or agarose gel for specified time ranging from 0.5 to 60 h. Then, fibre is removed at specified time and desorbed in 100 μL of desorption solution (acetonitrile: water 1:1) for 90 min under agitation speed of 1000 rpm. The samples extract were immediately injected to the instrument and analysed using liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS). The limit of detection of the method in gel and fish muscle was 0.01–0.07 ng mL−1 and 0.07–0.34 ng g−1, respectively, while the limit quantification was 0.10–0.20 ng mL−1 in gel samples and 0.40–0.97 ng g−1 in fish sample. The reproducibility of the method was good (5–15% RSD). The results suggest that kinetics of desorption of the compounds in fish tissue and different viscosity of gel can be determined using desorption time constant. In this study, desorption time constant which is directly related to desorption rate (diffusion kinetics) of selected drugs from the fibre to the gel matrix is faster as the viscosity of the gel matrix reduces from 2% (w/v) to 0.8% (w/v). As the concentration of gel reduces, viscosity of the gel will be reduced therefore allowing faster diffusion which invariably affect desorption time constant. Also, desorption time constant of model drugs in the fish muscle and 0.8–0.9% (w/v) gel model are similar based on free diffusion of studied compounds. In addition, in vitro and in vivo desorption time constant comparison shows that desorption time constant in an in vivo system (live fish muscle) is generally higher than an in vitro system (dead fish muscle) except for sertraline and nordiazepam. This study demonstrates SPME as a simple investigative tool to understand kinetics of desorption in an in vivo system with a goal to measure desorption rate of pharmaceuticals in fish. 相似文献
13.
Satish K. Pandey Aaydha C. Vinayaka Dharam B. Rishi Praveen Rishi C. Raman Suri 《Analytica chimica acta》2014
Typhoid fever is a life threatening bacterial infection that remains a major global health concern. This continued high burden associated with significant morbidity and mortality rate demands specific and rapid detection technique. This work reports a new sandwich type fluorescence immunoassay format using polymyxin B, a cationic receptor molecule, as a binder agent while anti-Vi antibody served as the capturing agent for specifically detecting Salmonella enterica serovar Typhi. Anti-Vi IgG antibody raised against Vi–BSA conjugate revealed affinity of 7.779 nM−1 signifying immunodominancy of O-acetyls groups in Vi polysaccharide. The detection limit of the developed assay was around 101 cells mL−1 of Vi expressing Salmonella enterica serovar Typhi with a correlation coefficient (R2) equal to 0.97. Positive response obtained for all the tested serovar Typhi clinical isolates as well as the pathogen spiked blood samples recommended specificity and accuracy of Vi antigen as a biomarker during typhoid fever. The intra- and inter-assay precision with Vi spiked samples were satisfactory revealing coefficient of variance (CV%) with a mean of 4.05% and 5.97% respectively. This may be the novel attempt and constructive report on the fluorescence based detection of Vi antigen of serovar Typhi in the epidemic as well as pandemic outbreaks. 相似文献
14.
Saluma Samanman Apon Numnuam Warakorn Limbut Proespichaya Kanatharana Panote Thavarungkul 《Analytica chimica acta》2015
For the first time, a simple and highly sensitive label-free electrochemical carcinoembryonic antigen (CEA) immunosensor based on a cryogel electrode has been developed and tested. The as-prepared nanocomposite combined the advantages of the graphene, AuNPs and chitosan (AuNPs–GP–CS) together with the ease of preparing a cryogel coupled to a silver deposition, to act as a redox mediator, on a Au electrode. Under the optimal conditions, the decrease of the cyclic voltammetry (CV) silver peak current was proportional to the CEA concentration over a range of from 1.0 × 10−6 to 1.0 ng mL−1 with a detection limit of 2.0 × 10−7 ng mL−1. This AuNPs–GP–CS cryogel electrode gave a 1.7 times higher sensitivity and 25 times lower detection limit than the non-cryogel electrode. Moreover, the proposed electrochemical immunosensor exhibited good selectivity, reproducibility and stability. When applied to analyse clinical serum samples, the data determined by the developed immunosensor were in agreement with those obtained by the current hospital analysis system (enzyme linked fluorescent assay) (P > 0.05), to indicate that the immunosensor would be potentially useful for clinical diagnostics. 相似文献
15.
Dan Zhu Yurong Yan Pinhua Lei Bo Shen Wei Cheng Huangxian Ju Shijia Ding 《Analytica chimica acta》2014
A novel electrochemical sensing strategy was developed for ultrasensitive and rapid detection of Salmonella by combining the rolling circle amplification with DNA–AuNPs probe. The target DNA could be specifically captured by probe 1 on the sensing interface. Then the circularization mixture was added to form a typical sandwich structure. In the presence of dNTPs and phi29 DNA polymerase, the RCA was initiated to produce micrometer-long single-strand DNA. Finally, the detection probe (DNA–AuNPs) could recognize RCA product to produce enzymatic electrochemical signal. Under optimal conditions, the calibration curve of synthetic target DNA had good linearity from 10 aM to 10 pM with a detection limit of 6.76 aM (S/N = 3). The developed method had been successfully applied to detect Salmonella as low as 6 CFU mL−1 in real milk sample. This proposed strategy showed great potential for clinical diagnosis, food safety and environmental monitoring. 相似文献
16.
Guodong Xiao Guowen Li Liang Chen Zijia Zhang Jun-Jie Yin Tao Wu Zhihong Cheng Xiaohui Wei Zhengtao Wang 《Journal of chromatography. A》2010,1217(34):5470-5476
A combinative method using high-speed counter-current chromatography (HSCCC) and thin layer chromatography (TLC) as an antioxidant autographic assay was developed to separate antioxidant components from the fruits of Psoralea corylifolia. Under the guidance of TLC bioautography, eight compounds including five flavonoids and three coumarins were successfully separated from the fruits of P. corylifolia by HSCCC with an optimized two-phase solvent system, n-hexane–ethyl acetate–methanol–water (1:1.1:1.3:1, v/v/v/v). The separation produced 5.91 mg psoralen, 6.26 mg isopsoralen, 3.19 mg psoralidin, 0.92 mg corylifol A, and 2.43 mg bavachinin with corresponding purities of 99.5, 99.8, 99.4, 96.4, and 99.0%, as well as three sub-fractions, in a single run from 250 mg ethyl acetate fraction of P. corylifolia extract. Following an additional clean-up step by preparative TLC, 0.4 mg 8-prenyldaidzein (purity 91.7%), 4.18 mg neobavaisoflavone (purity 97.4%) and 4.36 mg isobavachalcone (purity 96.8%) were separated from the three individual sub-fractions. The structures of the isolated compounds were identified by 1H NMR and 13C NMR. The results of antioxidant activity estimation by electron spin resonance (ESR) method showed that psoralidin was the most active antioxidant with an IC50 value of 44.7 μM. This is the first report on simultaneous separation of eight compounds from P. corylifolia by HSCCC. 相似文献
17.
A highly sensitive amperometric nanobiosensor has been developed by integration of glucose oxidase (GOx) with a gold nanowires array (AuNWA) by cross-linking with a mixture of glutaraldehyde (GLA) and bovine serum albumin (BSA). An initial investigation of the morphology of the synthesized AuNWA by field emission scanning electron microscopy (FESEM) and field emission transmission electron microscopy (FETEM) revealed that the nanowires array was highly ordered with rough surface, and the electrochemical features of the AuNWA with/without modification were also investigated. The integrated AuNWA–BSA–GLA–GOx nanobiosensor with Nafion membrane gave a very high sensitivity of 298.2 μA cm−2 mM−1 for amperometric detection of glucose, while also achieving a low detection limit of 0.1 μM, and a wide linear range of 5–6000 μM. Furthermore, the nanobiosensor exhibited excellent anti-interference ability towards uric acid (UA) and ascorbic acid (AA) with the aid of Nafion membrane, and the results obtained for the analysis of human blood serum indicated that the device is capable of glucose detection in real samples. 相似文献
18.
Mara Mirasoli Angela Buragina Luisa Stella Dolci Massimo Guardigli Patrizia Simoni Angel Montoya Elisabetta Maiolini Stefano Girotti Aldo Roda 《Analytica chimica acta》2012
Simple, rapid and highly sensitive assays, possibly allowing on-site analysis, are required in the security and forensic fields or to obtain early signs of environmental pollution. Several bioanalytical methods and biosensors based on portable devices have been developed for this purpose. Among them, Lateral Flow ImmunoAssays (LFIAs) offer the advantages of rapidity and ease of use and, thanks to the high specificity of antigen–antibody binding, allow greatly simplifying and reducing sample pre-analytical treatments. However, LFIAs usually employ colloidal gold or latex beads as labels and they rely on the formation of colored bands visible by the naked eye. With this assay format, only qualitative or semi-quantitative information can be obtained and low sensitivity is achieved. Recently, the use of enzyme-catalyzed chemiluminescence detection in LFIA has been proposed to overcome these problems. In this work, we describe the development of a quantitative CL-LFIA assay for the detection of 2,4,6-trinitrotoluene (TNT) in real samples. Thanks to the use of a portable imaging device for CL signal measurement based on a thermoelectrically cooled CCD camera, the analysis could be performed directly on-field. A limit of detection of 0.2 μg mL−1 TNT was obtained, which is five times lower than that obtained with a previously described colloidal gold-based LFIA developed employing the same immunoreagents. The dynamic range of the assay extended up to 5 μg mL−1 TNT and recoveries ranging from 97% to 111% were obtained in the analysis of real samples (post blast residues obtained from controlled explosion). 相似文献
19.
Five different haptens of the N-methylcarbamate insecticide metolcarb were designed and synthesized. All of the haptens were conjugated with ovalbumin (OVA) for the coating antigen, and one hapten containing all of the structure of metolcarb was conjugated with bovine serum albumin (BSA) for the immunogen. Two polyclonal antisera were raised against the BSA conjugate, and ten antibody/coating conjugate combinations were selected for studies of assay sensitivity and specificity for metolcarb. A class-specific combination was found, with the I50 of the assay ranged from 0.64 to 20.98 μg mL−1 for seven tested N-methylcarbamate insecticides except for pirimicarb. Considering titer, I50 and cross-reactivity of all combinations of antibody/coating conjugate, a competitive indirect enzyme-linked immunosorbent assay (ELISA) in a homologous system, whose limit of detection (LoD) reached 1.4 ng mL−1, was presented. The results of competitive ELISAs indicated that coating hapten structure can significantly affect not only assay sensitivity but also its specificity. 相似文献
20.
A highly sensitive microchip electrophoresis (MCE) method with chemiluminescence (CL) detection was developed for the determination of biogenic amines including agmatine (Agm), epinephrine (E), dopamine (DA), tyramine, and histamine in human urine samples. To achieve a high assay sensitivity, the targeted analytes were pre-column labeled by a CL tagging reagent, N-(4-aminobutyl)-N-ethylisoluminol (ABEI). ABEI-tagged biogenic amines after MCE separation reacted with hydrogen peroxide in the presence of horseradish peroxidase (HRP), producing CL emission. Since no CL reagent was added to the running buffer, the background of the CL detection was extremely low, resulting in a significant improvement in detection sensitivity. Detection limits (S/N = 3) were in the range from 5.9 × 10−8 to 7.7 × 10−8 M for the biogenic amines tested, which were at least 10 times lower than those of the MCE–CL methods previously reported. Separation of a urine sample on a 7 cm glass/poly(dimethylsiloxane) (PDMS) microchip channel was completed within 3 min. Analysis of human urine samples found that the levels of Agm, E and DA were in the ranges of 2.61 × 10−7 to 4.30 × 10−7 M, 0.81 × 10−7 to 1.12 × 10−7 M, and 8.76 × 10−7 to 11.21 × 10−7 M (n = 4), respectively. 相似文献