Gold nanoparticle-based immunochromatographic assay for the detection of 7-aminoclonazepam in urine

An analytical system of immunochromatographic assay based on gold nanoparticles was developed for the detection of 7-aminoclonazepam (7-ACLZ) in human urine. The qualitative assay was based on the competitive immunoassay using anti-7-ACLZ polyclonal antibody (PcAb) and a detector reagent that contains colloidal gold particles coated with anti-7-ACLZ PcAb. Nitrocellulose membrane was separately immobilised with goat anti-rabbit IgG (control line) and 7-ACLZ-OVA conjugate (test line). The sensitivity of the strip was tested for detecting 7-ACLZ spiked in urine and each specimen was independently measured by liquid chromatography tandem mass spectrometry. Good correlation was showed by the recovery results. The limit of detection for the strip test in urine was 100 ng mL^?1. The assay can be applied to the rapid detection of 7-ACLZ with the short testing time.     

Colloidal gold nanoparticle probe-based immunochromatographic assay for the rapid detection of chromium ions in water and serum samples

An immunochromatographic assay (ICA) using gold nanoparticles coated with monoclonal antibody (McAb) for the detection of chromium ions (Cr) in water and serum samples was developed, optimized and validated. Gold nanoparticles coated with affinity-purified monoclonal antibodies against isothiocyanobenzyl-EDTA (iEDTA)-chelated Cr³⁺ were used as the detecting reagent in this completive immunoassay-based one-step test strip. The ICA was investigated to measure chromium speciation (Cr³⁺ and Cr⁶⁺ ions) in water samples. Chromium standard samples of 0–80 ng mL⁻¹ in water were determined by the test strips. The results showed that the visual lowest detection limit (LDL) of the test strip was 50.0 ng mL⁻¹. A portable colorimetric lateral flow reader was used for the quantification of Cr. The results indicated that the linear range of the ICA with colorimetric detection was 5–80 ng mL⁻¹. The ICA was also validated for the detection of chromium ions in serum samples. The test trips showed high stability in that they could be stored at 37 °C for at least 12 weeks without significant loss of activity. The test strip also showed good selectivity for Cr detection with negligible interference from other heavy metals. Because of its low cost and short testing time (within 5 min), the test strip is especially suitable for on-site large-scale screening of Cr-polluted water samples, biomonitoring of Cr exposure, and many other field applications.     

氟苯尼考胶体金免疫层析定量检测方法的建立

建立了定量检测氟苯尼考的胶体金免疫层析方法.对胶体金标记抗体时溶液pH和抗体浓度、金标抗体用量、检测线上抗原浓度以及检测时间进行了优化.采用胶体金试纸条读取仪测定试纸条检测线和质控线的信号强度,以标准品的浓度为横坐标,阳性样本和阴性样本的检测线/质控线的信号比值(Bx/B0)为纵坐标建立标准曲线.结果表明,胶体金免疫层析试纸定量检测氟苯尼考的线性范围为0.1~1.5 ng/mL,检出限为0.08 ng/mL,检测时间为15 min.本方法具有简便、快速和可定量等特点,适于大批量样品的现场筛查.     

Visual and on-site detection of mercury(II) ions on lateral flow strips using DNA-functionalized gold nanoparticles

A test strip for detection of Hg(2+) in aqueous solution based on the DNA-functionalized gold nanoparticles (DNA-AuNPs) was developed and evaluated. When Hg(2+) ions were introduced, the biotinylated DNA(2) hybridized with thiolated DNA(1) functionalized on the AuNPs (DNA(1)-AuNPs) to form mismatch complexes through thymine-Hg(2+)-thymine (T-Hg(2+)-T) coordination. The formed mismatch complexes and excess DNA(1)-AuNPs could be captured on the test line formed by streptavidin and the control line formed by DNA(3)-BSA, respectively. Two red lines appeared due to the accumulation of AuNPs, enabling visual detection of Hg(2+) with a detection limit of about 6 nM. The assay results can be obtained within 5 min. The results show that the test strip has excellent sensitivity and selectivity for detection of Hg(2+); thus it holds a great potential for rapid, on-site and real time detection of Hg(2+).     

孔雀石绿的磁性免疫层析快速检测方法研究

开发了一种适用于现场快速检测孔雀石绿(MG)的免疫层析试纸条,在超顺磁性纳米微球上偶联MG单克隆抗体作为检测探针,分别将孔雀石绿完全抗原(MG-B SA)和羊抗鼠IgG喷涂于NC膜的T线和C线.结果 发现,T线最佳喷涂量为0.25 mg/mL,抗体最佳偶联量为20 μg,构建的试纸条可在25 min内实现养殖用水及鱼肉...     

Urchin-like gold nanoparticle-based immunochromatographic strip test for rapid detection of fumonisin B<Subscript>1</Subscript> in grains

An immunochromatographic strip (ICS) using urchin-like gold nanoparticles (UGNs) for sensitive detection of fumonisin B₁ (FB₁) was developed to meet the requirement for rapidly monitoring FB₁ in grain samples. The sensitivity of the ICS was 5.0 ng/mL, which represents a fourfold increase in sensitivity over conventional strip preparation using colloidal gold as the antibody-labeled probe. Analysis of FB₁ in grain samples showed that data obtained from the strip tests were in a good agreement with those obtained from HPLC and enzyme-linked immunosorbent assays (ELISAs). This qualitative test did not require any specialized equipment, and the detection time was less than 5 min, which is suitable for on-site testing of FB₁ in grain samples. Overall, to our knowledge, this is the first report of using a UGN as the antibody-labeled probe for sensitive detection of FB₁ in grains using an ICS.

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Graphical Abstract Preparation of ICS using conventional colloidal gold and urchin-like gold nanoparticle, respectively

     

Colloidal gold-based immunochromatographic assay for detection of diethylstilbestrol residues

One-step membrane-based competitive colloidal gold-based immunoassays in immunochromatographic formats for the rapid detection of diethylstilbestrol (DES) were developed. Nitro-cellulose membrane strip was separately coated with goat anti-rabbit IgG (control line) and DES hapten-ovalubumin conjugate (test line). Anti-DES polyclonal antibody labeled with colloidal gold particles was first incubated with DES. A positive reaction as a result of the remaining antibody-gold conjugate combining with antigen coated on the membrane was obvious by visual detection, with detection limits for immunochromatographic of 0.5 microg/kg for detecting DES standard solution, and the limit of detection was 5 microg/kg for detecting the DES spiked in swine pork and liver. The assay time for test was less than 5 min, suitable for rapid testing on-site.     

Immunochromatographic lateral flow strip for on-site detection of bisphenol A

We have developed a lateral flow assay (LFA) for the detection of bisphenol A (BPA) in water samples. Antibody against BPA was labeled with gold nanoparticles, and these conjugates were used as the recognition probes for the construction of an LFA strip. The diameter of the gold nanoparticles, the amount of antibody, the pH of the buffer, and the categories of the conjugation pad were optimized. The resulting method has a (visual) detection limit of 5 ppb, and of 0.92 ppb if used in combination with professional software. This LFA displays excellent specificity and was applied to spiked water samples with satisfactory results.

Development of colloidal gold-based flow-through and lateral-flow immunoassays for the rapid detection of the insecticide carbaryl

One-step membrane-based competitive colloidal gold-based immunoassays in flow-through and lateral-flow formats for the rapid detection of carbaryl were developed. Nitro-cellulose membrane strip was separately coated with goat anti-rabbit IgG (control line) and carbaryl hapten-OVA conjugate (test line). Anti-carbaryl antibody labeled with colloidal gold particles was firstly incubated with carbaryl. A positive reaction as a result of the remaining antibody-gold conjugate combining with antigen coated on the membrane was obvious by visual detection, with detection limits for flow-through and lateral flow of 50 and l00 μg/L, respectively. The assay time for both tests was less than 5 min, suitable for rapid testing on-site.     

Competitive immunochromatographic assay for the detection of the organophosphorus pesticide chlorpyrifos

An immunochromatographic assay (ICA) based on competitive antigen-coated format using colloidal gold as the label was developed for the detection of the organophosphorus insecticide chlorpyrifos. The ICA test strip consisted of a membrane with a detection zone, a sample pad and an absorbent pad. The membrane was separately coated with chlorpyrifos Hapten-OVA conjugate (test line) and anti-mouse IgG (control line). Based on the fact that the competition is between the migrating analyte in the sample and the analyte hapten immobilized on the test strip for the binding sites of the antibody-colloidal gold (Ab-CG) conjugate migrating on the test strip, this study suggests that the relative migration speed between the two migrating substances is a critically important factor for the sensitive detection by competitive ICA. This criterion was utilized for the confirmation of appropriateness of a nitrocellulose (NC) membrane for chlorpyrifos ICA. The detection limit of the ICA for chlorpyrifos standard and chlorpyrifos spiked into agricultural samples were 10 and 50 ng mL(-1), respectively. The assay time for the ICA test was less than 10 min, suitable for rapid on-site testing of chlorpyrifos.     

Lateral flow immunodipstick for visual detection of aflatoxin B1 in food using immuno-nanoparticles composed of a silver core and a gold shell

An immunodipstick assay with a lateral flow strip was developed for fast screening of food for aflatoxin B₁ (AFB₁) using the respective monoclonal antibody immobilized on nanoparticles with a silver core and a gold shell (AgAu) as detection reagent. The membrane-based immunodipstick consisted of a test line containing AFB₁ conjugated to bovine serum albumin, and a control line with goat anti-mouse IgG. One to two colored lines are formed on the membrane by using the red AgAu nanoparticles coated with anti-AFB₁ as signaling reagents. Under optimal conditions, the dipstick exhibits a lower visual detection limit of 0.1 ng?mL^?1 of AFB₁. Compared to the use of pure gold nanoparticles, the AgAu nanoparticles strongly enhance the sensitivity of the assay, and the reproducibility and stability are comparable. The assay was evaluated with naturally contaminated samples including rice, wheat, sunflower, cotton, chillies, and almonds, and a good correlation was found with data obtained with a commercially available enzyme-linked immunosorbent assay. The simple and non-instrumental dipstick method may further be extended to the screening of other mycotoxins in food.     

Development of a liposome-based immunochromatographic strip assay for the detection of Salmonella

Salmonella species are ubiquitous human pathogens which pose a dangerous threat to the elderly and children worldwide. In this study, to develop a more efficient assay procedure for the rapid detection of Salmonella Typhimurium, an immunochromatographic strip assay was developed using immunoliposome (anti-Salmonella IgG-tagged) encapsulated with sulforhodamine B (SRB). The detection sensitivity of the developed immunochromatographic assay was compared with a commercial immunochromatographic test strip using colloidal gold nanoparticles. The liposomes were prepared through a reverse-phase evaporation method by using a lipid and phospholipid mixture and SRB, a fluorescence dye, which was encapsulated in the phospholipid bilayer. Furthermore, the outer surface of the SRB-encapsulated liposome was conjugated with antibody (affinity-purified polyclonal goat anti-Salmonella IgG) to form an immunoliposome (size, 223 nm), used as the analytical reagent in the developed immunoassay. For this strip assay, a plastic-backed nitrocellulose strip was immobilized with two antibody zones. The lower zone of the strip referred to Salmonella antigen capture zone (test line), while the other zone served as a positive control (control line). The lower zone was coated with affinity-purified polyclonal goat anti-Salmonella IgG, while the upper zone was coated with rabbit anti-goat IgG. During capillary migration of the wicking solution (diluted liposome and Salmonella culture, each 50 μl), Salmonella was captured with surface-bound immunoliposomes at the antigen capture zone, while the unbound liposomes migrated upward and bound to another zone. The color density of the antigen capture zone was directly proportional to the amount of S. Typhimurium in the test sample. As a result, the detection limit of the immunochromatographic strip assay developed in this study against S. Typhimurium was found to be 10² CFU/ml, which was significantly higher than the detection limit (10⁷ CFU/ml) of the commercial immunochromatographic test strip assay.     

A novel enhancement assay for immunochromatographic test strips using gold nanoparticles

The immunochromatographic assay is a well-known and convenient diagnostic system. In this report, the development of a novel enhancement assay for the test strips is described. Additionally, this highly sensitive immunochromatographic assay was applied to detect human chorionic gonadotropin hormone (HCG) as the model case. The primary antibody-conjugated gold nanoparticles were used as the enhancer of the standard method. The primary antibodies were immobilized within a defined detection zone (test line) on the diagnostic nitrocellulose membrane. The secondary antibodies were conjugated with colloidal gold nanoparticles. In combination with an effective sample pretreatment, the gold-conjugated antibodies and the primary antibodies formed a sandwich complex with the target protein. Within the test line, the sandwich complex was immobilized, and furthermore, concentrated by the enhancer resulting in a localized surface plasmon resonance (LSPR) phenomenon and a distinct red color on the test line. The intensity of color of the red test line (signal intensity), which correlated directly with the concentration of the target protein in the standard or spiked samples, was assessed visually and by computer image analysis using a three-determination analysis. Under optimum conditions, the limit of detection (LOD) for HCG assay was 1 pg/mL. When using human serum, 10 pg/mL of HCG could be detected. We have also spiked total prostate-specific antigen (TPSA) in female serum. The LOD for TPSA was determined as 0.2 ng/mL. With this method, the quantitative determination of the target protein could be completed in less than 15 min. Our novel immunochromatographic strips using the enhancing method based on LSPR of gold nanoparticles are useful as a rapid and simple screening method for the detection of important analytes for medical applications, environmental monitoring, food control, and biosecurity.      

A competitive immunoassay for ultrasensitive detection of Hg in water,human serum and urine samples using immunochromatographic test based on surface-enhanced Raman scattering

An immunochromatographic test (ICT) strip was developed for ultrasensitive competitive immunoassay of Hg²⁺. This strategy was achieved by combining the easy-operation and rapidity of ICT with the high sensitivity of surface-enhanced Raman scattering (SERS). Monoclonal antibody (mAb) against Hg²⁺ and Raman active substance 4-mercaptobenzoic acid (MBA) dual labelled gold nanoparticles (GNPs) were prepared as an immunoprobe. The Raman scattering intensity of MBA on the test line of the ICT strip was measured for quantitative determination of Hg²⁺. The ICT was able to directly detect Hg²⁺ without complexing due to the specific recognition of the mAb with Hg²⁺. The IC₅₀ and limit of detection (LOD) of the assay for Hg²⁺ detection were 0.12 ng mL⁻¹ and 0.45 pg mL⁻¹, respectively. There was no cross-reactivity (CR) of the assay with other nineteen ions and the ICT strips could be kept for 5 weeks without loss of activity. The recoveries of the assay for water, human serum and urine samples spiked with Hg²⁺ were in range of 88.3–107.3% with the relative standard deviations (RSD) of 1.5–9.5% (n = 3). The proposed ICT was used for the detection of Hg²⁺ in urine samples collected from Occupational Disease Hospital and the results were confirmed by cold-vapor atomic fluorescence spectroscopy (CV-AFS). The assay exhibited high sensitivity, selectivity, stability, precision and accuracy, demonstrating a promising method for the detection of trace amount of Hg²⁺ in environmental water samples and biological serum and urine samples.     

基于新型量子点荧光微球的氯霉素免疫层析试纸条的制备和应用

以羧基化CdTe/ZnSe量子点荧光微球为标记物,通过1-乙基-(3-二甲基氨基丙基)碳二亚胺/N-羟基琥珀酰亚胺(EDC/NHS)活化法将氯霉素(CAP)单克隆抗体与量子点荧光微球偶联制备荧光探针.氯霉素全抗原(CAP-HS-BSA)及羊抗鼠二抗分别喷涂硝酸纤维素膜,形成检测线(T线)和质控线(C线),组装成新型氯霉素量子点荧光微球免疫层析试纸条,建立了快速、定量检测牛奶中CAP的方法.本研究开发的量子点荧光微球试纸条可在15 min内完成牛奶样品中CAP的定量检测,线性范围为0.1~100.0μg/L,检出限为0.1μg/L.牛奶样品CAP的加标回收率为93.3%~97.9%,相对标准偏差在4.9%~6.9%之间.     

菊酯类农药广谱型免疫层析试纸条的研究及应用

建立了菊酯类农药广谱型免疫层析检测方法,可同时检测水果、蔬菜中12种甲氰菊酯、溴氰菊酯、氯氰菊酯、三氟氯氰菊酯农药残留。以粒径20 nm的胶体金标记羊抗小鼠IgG抗体于金标垫上,分别固定包被原(检测线,T线)、兔抗山羊 IgG 抗体(质控线, C 线)于硝酸纤维素膜( NC 膜)上,与吸水垫及聚氯乙烯( PVC)底板组合成层析试纸条。10 g样品经乙腈提取,PBS稀释4倍,取100μL与菊酯类农药的单克隆抗体混合后,直接用于试纸条检测。结果表明,试纸条对甲氰菊酯、溴氰菊酯、氯氰菊酯、三氟氯氰菊酯农药的裸眼观察检测灵敏度为0.5,5.0,5.0和5.0μg/mL,检测时长为8~10 min,采用QuEChERS方法,方法检测灵敏度可提高16倍。对蔬菜和水果的方法验证表明,除辣椒基质干扰呈假阳性外,其它样本的检测结果均与GC方法结果一致。试纸条采用金标羊抗小鼠IgG二抗的方法,使检测结果的重现性更好、更稳定,且抗基质干扰能力强,为菊酯类农药的累积毒性检测提供了新方法。     

基于网状混合自组装膜的压电免疫传感器及其应用

结合纳米金及混合自组装技术, 制备了一种新型网状混合膜, 提出了一种新的生物分子固定化方法, 研制了一种用于检测人血清抗精子抗体的压电免疫传感器. 首先, 将纳米金溶胶、巯基丙酸和1,6-二巯基己烷按一定的比例混合制得网状混合自组装膜, 然后将此膜组装到压电石英晶振的金电极表面, 经EDC/NHS活化后, 再将抗原固定到电极上, 实现对抗精子抗体的检测. 结果表明, 该方法能明显提高抗体抗原结合效率, 从而提高传感器的灵敏度, 并降低传感界面的非特异性吸附. 将此传感器应用于人血清抗精子抗体的检测, 线性范围为10~800 mU/mL, 检出限为7 mU/mL. 此传感器为抗精子抗体的临床检测提供了新平台.     

Colloidal Nanogold-Based Immunochromatographic Strip Test for the Detection of Digoxin Toxicity

Digoxin is widely used as a cardiac glycoside drug in the treatment of various heart conditions. Because it is a toxic drug, it should be regularly monitored in the serum of patients under treatment. In this study, colloidal nanogold is synthesized and the preparation of nanogold-labeled monoclonal antibody probe to digoxin is described under optimal conditions. In addition, an immunochromatographic (IC) method for digoxin analysis employing nanogold-labeled probe is developed. With this technique, it requires only 5 min to complete the quantitative detection of digoxin. The detection time is decreased 20–30 times in comparison to radioimmunoassay (RIA). The sensitivity to digoxin was about 2 ng/ml by naked eye, which is within the therapeutic and toxic ranges of digoxin. The results of serum samples obtained by IC strip were in agreement with those obtained by RIA. The IC strip was sufficiently sensitive and accurate to be used for the rapid detection of digoxin in serum samples.     

五氯酚免疫层析检测试纸条的研究

利用胶体金免疫层析技术建立了一种快速检测五氯酚(PCP)残留的方法。采用柠檬酸三钠还原法制备大小一致、分布均匀、粒径为20 nm的胶体金颗粒,以此标记五氯酚抗体,制备金标抗体。将五氯酚包被抗原和羊抗鼠二抗分别结合于硝酸纤维膜上,依次将型号Millipore135硝酸纤维膜、型号VL78金标垫、型号SB06样品垫及吸水纸组装于PVC底板上,组装成胶体金免疫层析检测试纸条。通过试纸条上颜色的深浅,检测样品中PCP的残留量。试纸条检出限为10 ng/mL,检测时间为5 min。该方法检测所需试剂已预先包被在试纸条上,操作简单、重复性好、成本低廉,可用于五氯酚的现场快速检测。     

基于免疫胶体金试纸条检测盐酸克伦特罗的便携式光电型传感器

利用免疫竞争抗制原理制备检测盐酸克伦特罗的免疫纳米金试纸条,盐酸克伦特罗与CLB-BSA复合物竞争性结合胶体金上抗体的有限位点,从而使试纸条上的检测带显色减弱或消失.将显色后的试纸条插入自行研制的光电型传感器的测试孔内,根据反射光的强弱计算出克伦特罗的浓度.结果表明:用光电传感方法检测盐酸克伦特罗的线性范围为1～10 μg/L,检出限为0.05 μg/L,回收率为85.5％～96.0％.此光电型传感器检测克伦特罗具有简便、快速、灵敏、特异性强的优点.