一种基于纳米金介导生物沉积铂催化氢还原的电化学免疫分析新方法

本文发展了一种基于纳米金介导生物沉积铂并以铂催化氢还原伏安法进行检测的高灵敏电化学免疫分析新方法。该方法采用夹心免疫分析模式,实现了人免疫球蛋白（HIgG）的测定。首先在聚苯乙烯微孔板中固定羊抗HIgG捕获抗体,HIgG捕获后,碱性磷酸酶标记的HIgG抗体修饰的纳米金探针通过与HIgG的形成的夹心复合物而结合在微孔板上。结合的碱性磷酸酶催化抗坏血酸磷酸酯底物水解产生抗坏血酸,后者在纳米金上介导下还原铂离子沉积于纳米金表面。沉积的金属铂用王水溶解并电富集于玻碳电极上。通过测定铂催化氢还原产生的阴极电流,可实现HIgG的高灵敏分析。催化氢还原电流与HIgG浓度对数在0.1~100ng/ml之间呈线性相关性,检测限达22pg/ml。由于铂催化氢还原的高灵敏度及纳米金介导的生物沉积放大反应,该法具有较高的分析灵敏度,且免疫分析微孔板模式使得该法可同时用于大量样品的分析。     

金纳米簇信号放大的电化学免疫传感器

通过抗原抗体的特异性识别作用以及金纳米簇(AuNCs)探针和金标银染的双重信号放大作用,构建了一种新的电化学免疫传感器,对人的免疫球蛋白(IgG)进行了检测。受贻贝分泌的黏附蛋白启示,首先将聚多巴胺薄膜修饰在铟锡氧化物电极(ITO)上,并对一抗抗体进行固定,通过观察电化学阻抗的变化来监控免疫传感器的构建过程。将待检测的IgG抗原组装在该电极上并与AuNCs标记的二抗反应,最后经银染反应,用溶出伏安法对IgG的含量进行定量检测,其灵敏度达到0.5 ng/L。该方法可应用于实际血清样品中IgG含量的测定。     

基于铜增强纳米金标记物结合磁分离的溶出伏安免疫分析法用于蛋白质的高灵敏测定

应用铜原位化学放大纳米金颗粒的信号增强特性, 并结合磁分离技术, 提出了一种高灵敏的溶出伏安免疫分析方法. 实验中以人IgG为模式蛋白质, 将抗体修饰的SiO₂@Fe₃O₄核壳型磁性纳米颗粒和纳米金标抗体悬浊液混合, 用以均相免疫识别人IgG, 借助外加磁场分离纯化, 在免疫复合物悬浊液中加入铜增强试剂进行沉积放大反应, 再将铜用稀硝酸溶解并进行溶出伏安分析检测. 结果表明, 与基于固相反应的金属免疫分析法相比, 所提出的基于均相反应和磁分离原理的方法具有操作简单、分析时间短等优点. 该方法显示出明显增强的人IgG检测性能, 其线性检测范围为01~1000 ng/mL, 检出限为73 pg/mL. 此外, 将其用于实际样品的回收率测定, 结果令人满意.     

基于纳米金／壳聚糖掺杂碳纳米管修饰金电极非标记免疫传感器的研究

应用吸附法将羊抗人IgG抗体直接固定于纳米金(GNPs)/壳聚糖(Chit)掺杂碳纳米管(CNTs)修饰的金电极表面,制备了用于人IgG抗原检测的非标记电化学免疫传感器.利用循环伏安法和交流阻抗研究了修饰电极表面的电化学特性,用差分脉冲伏安法研究了测试底液的pH值对免疫传感器性能的影响.实验表明,在含不同浓度人IgG的...     

微孔板蛋白芯片可视化检测方法的研究

将微孔板生物芯片制备技术与银增强显色方法相结合,建立了一种高通量蛋白免疫分析新方法。研究采用夹心法反应原理,将人IgG捕获抗体以微阵列形式点样于96孔板底部制备成蛋白微阵列,并依次与人IgG、生物素标记二抗及链亲和素胶体金反应,最后用银增强法显色。采用平板式扫描仪对微孔板显色结果进行快速扫描成像,图像采用luxscan 3.0软件处理。结果表明:微孔板蛋白芯片的最低检测量可达0.6 ng/mL,线性范围10 ng/mL~100μg/mL,相关系数为0.98,重复性较好(CV10%)。     

免疫纳米金催化氯金酸与羟胺反应-共振散射光谱检测痕量青霉素G

小粒径的金和免疫金纳米粒子对氯金酸-盐酸羟胺这一反应具有较强的催化作用,其产物在580 nm处有一共振散射峰。以半抗原青霉素G为模型,用粒径为9 nm的金纳米微粒标记羊抗兔青霉素噻唑蛋白抗体制备了青霉素G的免疫纳米金共振散射光谱探针。在pH5.4的柠檬酸-磷酸氢二钠缓冲溶液中,青霉素G与金标兔抗青霉素发生特异性结合生成胶体金免疫复合物,离心分离。取适量金标羊抗兔青霉素的上层清液做催化剂,在pH3.36盐酸-柠檬酸钠缓冲溶液－40μg/mL氯金酸－21.6μg/mL的盐酸羟胺条件下,进行催化反应后金纳米粒径增大,在580nm共振散射强度处增强。随着青霉素G浓度c增大,上层清液中免疫金纳米微粒数量降低,I580nm值降低。其降低值△I580nm与c在0.15-225 ng/mL范围内成线性关系,其回归方程为△I580nm=0.28c+5.16,检出限为0. 05 ng/mL。该法用于牛奶中青霉素G的检测,结果较好。     

基于丝网印刷电极的甲胎蛋白电化学免疫传感器

报道了一种基于金纳米粒子(AuNPs)双重信号放大的高灵敏电化学免疫传感器,并应用于肝癌标志物甲胎蛋白(AFP)的检测。通过在丝网印刷电极(SPE)表面电沉积AuNPs提高电极的重现性,利用AuNPs的吸附作用固定AFP抗体,用于捕获样品中的待测AFP抗原,并进一步与固定了辣根过氧化物酶(HRP)标记检测抗体的纳米金免疫探针发生特异性结合,所形成的夹心免疫复合物可以催化底物得到响应电流。用扫描电镜(SEM)和微分脉冲伏安法(DPV)等技术研究电极组装过程以及电极的化学性质,讨论了影响免疫传感器性能的因素。在最优实验条件下,传感器的峰电流信号与AFP浓度在2.5~30ng/mL范围内呈良好的线性关系,检出限为0.16ng/mL。该传感器具有灵敏度高、成本低、仪器体积小的优点,具有较好的应用前景。     

一种新的测定痕量IgG的纳米金标免疫共振散射光谱探针

在pH8.5条件下，用粒径为9nm的金纳米微粒标记羊抗人IgG获得IgG金标免疫探针。在pH5.8磷酸氢二钠-柠檬酸缓冲溶液中及聚乙二醇存在下，金标记羊抗人IgG与人IgG产生特异性结合,羊抗人IgG包裹的金纳米微粒释放出来，聚集形成较大的微粒，导致体系在580 nm处的共振散射峰增强。IgG浓度在1.3~1.5×10³ ng·mL^-1范围内与580 nm处的共振散射光强度增加值呈线性，方法的检出限为0.78ng.mL^-1。该法用于定量分析人血清IgG,，简便快速，结果令人满意。     

基于铕螯合物纳米粒子标记的时间分辨荧光免疫法检测嗜水气单胞菌

以铕螯合物荧光纳米粒子标记嗜水气单胞菌菌株B15兔抗IgG,得到纳米粒子-IgG复合物,建立了新的嗜水气单胞菌双抗夹心时间分辨荧光免疫检测(TRFIA)法。采用正交试验优化了兔抗IgG的包被时间和包被浓度,以及纳米粒子-IgG的免疫反应时间和稀释度。偶合了铕螯合物的纳米粒子发光强度大,光稳定性高,因此灵敏度被改善。方法的检测限为1.0×10~3 CFU/mL,在1.0×10~3～5.0×10~8 CFU/mL范围内线性关系良好(相关系数0.990),板内和板间变异系数分别为2.70%和9.62%。将该方法用于实际样品的检测,阳性检出率为100%。     

基于二氧化硅-硫堇纳米复合物构建高灵敏癌胚抗原电流型免疫传感器

利用电沉积纳米金(AuNPs)修饰玻碳电极(GCE)表面并通过AuNPs固定癌胚抗原(CEA)的捕获抗体(Ab1),以牛血清白蛋白(BSA)封闭非特异性吸附位点;以γ-(2,3环氧丙氧)丙基三甲氧基硅烷(GPMS)作交联剂,将单分散的SiO_2纳米粒子与电子媒介体硫堇(Thi)结合成SiO_2-Thi纳米复合物,偶联辣根过氧化物酶(HRP)标记的CEA二抗(HRP-Ab2)作为电化学免疫检测信号,构建了具有信号放大效应的电流型免疫传感器并用于CEA的高灵敏检测。在CEA存在下,进行电化学酶联夹心免疫反应。在含有H2O2的磷酸盐缓冲溶液(PBS)中,标记在SiO_2-Thi纳米复合物上的HRP能催化H_2O_2氧化电子媒介体Thi,产生增强的还原峰电流,从而提高检测CEA的峰电流响应信号,进而实现对CEA的高灵敏电化学酶联夹心免疫分析。在最优实验条件下,该免疫传感器的差分脉冲伏安(DPV)还原峰电流与CEA质量浓度的对数在0.01~20ng/mL范围内呈良好的线性关系,检出限为3pg/mL(S/N=3)。该传感器对血清样品进行加标回收实验,回收率为97.3%~105.7%,可初步用于临床对CEA的检测。     

Electrochemical Metalloimmunoassay Based on Enlargement of Gold Nanoparticle Label Treated with Ag Enhancement System

A novel sensitive electrochemical immunoassay with colloidal gold as the antibody labeling tag and subse-quent signal amplification by silver enhancement is described. Colloidal gold was treated by a light-sensitive silver enhancement system which made silver deposit on the surface of colloidal gold(form Au/Ag core-shell structure), followed by the release of the metallic silver atoms anchored on the antibody by oxidative dissolu-tion of them in an acidic solution and the indirect determination of the dissolved Ag ions by anodic stripping voltamrnetry(ASV) at a carbon fiber microelectrode. The electrochemical signal is directly proportional to the amount of analyte(goat IgG) in the standard or a sample, The method was evaluated by means of a non-competitive heterogeneous immunoassay of immunoglobulin G(IgG) with a concentration as low as 0. 2 ng/mL. The high performance of the method is related to the sensitive ASV determination of silver( I ) at a car-bon fiber microelectrode and to the release of a large number of Ag^ ions from each silver shell anchored on the analyte (goat IgG).     

A new strategy for electrochemical immunoassay based on enzymatic silver deposition on agarose beads

A novel, sensitive electrochemical immunoassay in a homogeneously dispersed medium is described herein based on the unique features of agarose beads and the special amplified properties of biometallization. The immunochemical recognition event between human immunoglobulin G (IgG) and goat anti-human IgG antibody is chosen as the model system to demonstrate the proposed immunoassay approach. Avidin-agarose beads rapidly react with the biotinylated goat anti-human IgG antibody to form agarose beads-goat anti-human IgG conjugate (agarose bead-Ab). Agarose bead-Ab, alkaline phosphatase conjugated goat anti-human IgG antibody (ALP-Ab) and the human IgG analyte are mixed to form sandwich-type immunocomplex followed by the addition of the enzymatic silver deposition solution to deposit silver onto the surface of proteins and agarose beads. The silver deposited are dissolved and quantified by anodic stripping voltammetry. The influence of relevant experimental variables was examined and optimized. The logarithm of the anodic stripping peak current depended linearly on the logarithm of the concentration of human IgG in the range from 1 to 1000 ng/ml. A detection limit as low as 0.5 ng/ml human IgG was attained by 3σ-rule. The R.S.D. of the approach is 9.65% for eight times determination of 10 ng/ml human IgG under same conditions. Optical microscope and TEM graphs were also utilized to characterize agarose beads and silver nanoparticles formed.     

Gold nanoparticle-based immunoassay by using non-stripping chemiluminescence detection

A novel microplate-compatible chemiluminescence (CL) immunoassay has been developed for the determination of human immunoglobulin G (IgG) based on the luminol-AgNO(3)-gold nanoparticles CL system. Polystyrene microtiter plates were used for both immunoreactions and CL measurements. The primary antibody, goat-anti-human IgG, was first immobilized on polystyrene microwells. Then the antigen (human IgG) and the gold-labeled second antibody were connected to the microwells successively to form a sandwich-type immunocomplex. The gold label could trigger the reaction between luminol and AgNO(3), accompanied by light emission. Under the optimized conditions, the CL intensity of the system was linear with the logarithm of the concentration of human IgG in the range from 25 to 5000 ng mL(-1), with a detection limit of 12.8 ng mL(-1) ( approximately 80 pM) at a signal to noise ratio of three (S/N = 3). Compared with other reported CL immunoassay method based on gold labels, the proposed CL protocol avoids a strict stripping procedure or difficult to control synthesis processes, making the method more simple, time-saving and easily automated. The present CL method is promising for the determination of clinically important bioactive analytes.     

Antibody-biotemplated HgS nanoparticles: extremely sensitive labels for atomic fluorescence spectrometric immunoassay

In this work, antibody goat anti-human IgG as a scaffold was employed for the synthesis and biofunctionalization of HgS nanoparticles (NPs) via a facile one-pot process. After a complete sandwich-type immunoreaction among primary antibody, human IgG and secondary antibody labeled with HgS NPs, a large number of mercury ions released from captured HgS NPs dissolution were quantitatively detected by chemical vapor generation atomic fluorescence spectrometry (CVG-AFS). Taking advantage of the signal amplification property of HgS NPs and the high sensitivity of CVG-AFS, the assay detected human IgG with a limit of detection (S/N = 3) of 0.6 ng mL(-1) (4.0 fmol mL(-1) or 0.4 fmol) and the response was linear over a dynamic range from 1.0 to 5.0 × 10(4) ng mL(-1) with a correlation coefficient of 0.996. A relative standard deviation (RSD) of 1.0 × 10(2) ng mL(-1) human IgG was 1.5% for within-batch (intra-assay) and 4.5% for between-batch (inter-assay). Other proteins, such as goat anti-rabbit IgG, goat anti-human IgG, rabbit anti-human IgG, carcinoembryonic (CEA), α-fetoprotein (AFP), human serum albumin (HSA) and bovine serum albumin (BSA) did not significantly interfere with the assay for human IgG. The analytical result of HgS NPs with AFS-based immunoassay technology for the quantification of human IgG in human serum from patients is in good agreement with the result obtained by conventional immunoturbidimetric method. The consequence shows that the novel immunosensor possessed satisfactory precision, extremely high sensitivity, high selectivity and could be applied for the quantification analysis of real samples.     

Copper-enhanced gold nanoparticle tags for electrochemical stripping detection of human IgG

We demonstrate herein a novel electrochemical protocol for quantification of human IgG based on the precipitation of copper on gold nanoparticle tags and a subsequent electrochemical stripping detection of the dissolved copper. The immunoassay was conducted by following the typical procedure for sandwich-type immunoreaction. Goat anti-human IgG was immobilized on the wells of microtiter plates. The human IgG analyte was first captured by the primary antibody and then sandwiched by secondary antibody labeled with gold nanoparticles. The copper enhancer solution was then added to deposite copper on the gold nanoparticle tags. After dissolved with HNO₃, the released copper ions were then quantified by ASV. The detection limit is 0.5 ng/mL by 3σ-rule. In order to investigate the feasibility of the newly developed technique to be applied for clinical analysis, several standard human IgG serum specimens were also examined by the method. To our knowledge, the copper enhancing procedure is the first time to be developed for immunoassay. The new strategy of using copper-enhanced gold nanoparticle tags for electrochemical stripping detection holds great promise for immunoassay and DNA detection.     

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.     

QDs-labeled microspheres for the adsorption of rabbit immunoglobulin G and fluoroimmunoassay

This paper presents a study on the adsorption of rabbit immunoglobulin G onto CdTe quantum dots (QDs)/polystyrene microspheres. The adsorption appears to be sensitive to pH conditions and ionic strength. Maximum adsorption for protein was obtained near the isoelectric point. Adsorption isotherm analysis demonstrated that the electrostatic interaction plays an important role in the adsorption of protein. The thickness of adsorbed layer calculated from the maximal adsorption amounts (q(m)) is 6.5 nm, which indicates that the rabbit IgG molecules exist between the side-on and end-on mode in the monolayer. The bio-functional rabbit IgG/fluorescent microspheres were further used for the detection of antibody in fluoroimmunoassays. This approach allowed detection of goat anti-rabbit IgG in the range of 1-100 ng/mL.     

A new and selective and sensitive nanogold-labeled immunoresoance scattering spectral assay for trace prealbumin

A gold-labeled immunoresonance scattering spectral probe for trace prealbumin (PA) was prepared by using gold nanoparticles in size of 9.0 nm to label goat anti-human prealbumin polyclonal antibody. The immune reaction between the gold-labeled antibody and prealbumin took place in pH 7.6 Na₂HPO₄-NaH₂PO₄ buffer solution. In the presence of polyethylene glycol PEG-10000, the labeled gold nanoparticles were released and aggregated which brought the resonance scattering intensity (I_RS) at 580 nm to enhance greatly. The ΔI_RS is proportional to the prealbumin concentration in the range from 16.67 to 666.67 ng/mL, with a detection limit of 4.1 ng/mL. This simple, sensitive and selective assay was applied to determination of prealbumin in human plasma, with satisfactory results.     

MCM-41 mesoporous material modified carbon paste electrode for the determination of cardiac troponin I by anodic stripping voltammetry

An anodic stripping voltammetric method for the determination of cardiac troponin I (cTnI) at a MCM-41 mesoporous material modified carbon paste electrode (MCM-MCPE) was investigated. The test was based on the dual monoclonal antibody “sandwich” principle using colloidal gold as a labeled substrate. Four main steps were carried out to obtain the analytical signal, i.e. electrode preparation, immunoreaction, silver enhancement, and anodic stripping voltammetric detection. The anodic stripping peak current increased linearly with the concentration of cTnI over the range of 0.8-5.0 ng/ml. A detection limit of 0.5 ng/ml was obtained. The established method was applied to detect cTnI in acute myocardial infarction (AMI) samples using routine enzyme-linked immunoadsorbent assay (ELISA) for comparison analysis, and good results were obtained.