基于胶体金标记的阳极溶出伏安免疫分析用于免疫球蛋白G的测定

提出了一种基于胶体金标记的阳极溶出伏安免疫分析方法。免疫反应在聚苯乙烯微孔板中以夹心分析模式进行,通过物理吸附将兔抗人免疫球蛋白G(IgG)抗体固定于微孔板上,与相应抗原IgG发生免疫反应后,再通过夹心模式捕获相应的纳米金标记的羊抗人IgG抗体,然后再与金标羊抗人IgG抗体和金标兔抗羊二抗形成的免疫复合物反应,在微孔板上进一步引入大量的纳米金,将金溶解后,在碳糊电极上用阳极溶出伏安法(ASV)对金离子进行检测,溶出峰电流的大小间接与待分析物IgG的浓度成正比。对免疫分析的一些实验条件进行了优化。阳极溶出峰电流与IgG的对数浓度在1.1~1 143 ng/mL范围内呈良好的线性关系,检出限为1 ng/mL。将该方法应用于人血清中IgG浓度的测定,取得了满意结果。     

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).     

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.     

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.     

Ultrasensitive electrochemical immunosensor of carcinoembryonic antigen based on gold-label silver-stain signal amplification

A novel gold-label silver-stain electrochemical immunosensor based on polythionine-gold nanoparticles (PTh-Au NPs) modified glassy carbon electrode (GCE) as a platform and secondary antibody labeled Au NPs (Ab₂-Au NPs) as immumoprobe for carcinoembryonic antigen (CEA) detection. The sandwich-type biosensor adopted anodic stripping voltammetry to detect silver stripping signal when the Ab₂-Au NPs of the formed immunocomplexes were stained with silver.     

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.     

Direct detection of myoglobin in whole blood using a disposable amperometric immunosensor

An amperometric immunosensor for the rapid detection of myoglobin in whole blood was developed. Due to its rapid kinetics, myoglobin is a useful biochemical marker for the early assessment of acute myocardial infarction (AMI). A one-step indirect sandwich assay was employed using a polyclonal goat anti-human cardiac myoglobin antibody with monoclonal mouse anti-myoglobin and goat anti-mouse IgG conjugated to alkaline phosphatase (AP), as the detecting antibodies. The final sensor required 30 min for incubation. The standard curve was linear between 85 and 925 ng/ml. The intra- and inter-assay coefficients of variation were below 8%. No cross-reactivity of the antibodies was found with other cardiac proteins. The overall performance of the sensor, rapid analysis time, wide working range, good precision and specificity demonstrate its potential usefulness for early assessment of AMI.     

Protein array for assist diagnosis of acute myocardial infarction

A nanogold probe immunoassay for cardiac troponin I (cTnI) combining the concepts of the one-step dual monoclonal antibody “sandwich” principle, the low density protein array, and silver enhancement on the gold particle is described. Two main substrates, namely the capture antibody (IgG1) coated supporting nitrocellulose membrane and the colloidal gold-labeled detection antibody (cAu–IgG2), were prepared before the detection. The detection procedure involved two steps, i.e. immunoreaction and silver amplification. The assay needs only small amounts of serum samples of patients. The detection results could be easily imaged with a simple flatbed scanner or even the naked eye. The whole detection procedure of the assay could be fulfilled within 40 min (much faster than the routine enzyme-linked immunosorbent assay (ELISA) that takes usually at least 3 h for a turnaround test). The detection limit of cTnI was found to be 1 ng/ml. The detecting results of cTnI in serum samples were similar to those detected by ELISA.     

PDMS microfludic device for optical detection of protein immunoassay using gold nanoparticles

A simple but highly specific immunoassay system for goat anti-human IgG has been developed using gold nanoparticles and microfluidic techniques. The assay is based on the deposition of gold nanoparticles that are coated with protein antigens in the presence of their corresponding antibodies to microfluidic channel surface. The effects of time accumulation, the flow velocity, and the concentration of antibodies to the red light absorption percentage (RAP) of deposition were investigated with an ordinary optical microscope. By controlling the reaction time and flow velocity, a dynamic range of 3 orders of magnitude and a detection sensitivity of 10 ng ml(-1) of goat anti-human IgG were achieved. Because of its simplicity and flexibility, this new technique should be useful for fast, highthroughput screening of antibodies in clinical diagnostic applications.     

An Enzyme-Linked Immunosorbent Assay (Elisa) for Measurement of Human Igg Subclass Levels in Serum

Abstract

We established an enzyme-linked immunosorbent assay (ELISA) to quantify human IgG subclasses in serum. IgG subclasses captured with subclass-specific mouse MoAbs could be detected with enzyme-labeled goat anti-human IgG. We screened the MoAbs which were suitable for the method from the commercially available MoAbs raised against human IgG subclasses. The specificity of the present ELISA for each subclass was clarified by the experiments as follows: (a) identification of individual IgG subclasses purified from total IgG by protein A column chromatography and (b) identification of a certain specific subclass which increased relatively in monoclonal (M)-proteinemia-patient sera compared with normal. We could detect each subclass with the sensitivity of 4.0 ng/ml for IgG₁, 13 ng/ml for IgG₂, 0.5 ng/ml for IgG₃ and 0.4 ng/ml for IgG₄ using pooled normal serum calibrated against a reference serum 67/86 from the World Health Organization (WHO) as a standard.     

Anodic‐Stripping Voltammetric Immunoassay for Ultrasensitive Detection of Low‐Abundance Proteins Using Quantum Dot Aggregated Hollow Microspheres

A new anodic‐stripping voltammetric immunoassay protocol for detection of IgG1, as a model protein, was designed by using CdS quantum dot (QD) layer‐by‐layer assembled hollow microspheres (QDHMS) as molecular tags. Initially, monoclonal anti‐human IgG1 specific antibodies were anchored on amorphous magnetic beads preferably selective to capture F_ab of IgG1 analyte from the sample. For detection, monoclonal anti‐human IgG1 (F_c‐specific) antibodies were covalently coupled to the synthesized QDHMS. In a sandwich‐type immunoassay format, subsequent anodic‐stripping voltammetric detection of cadmium released under acidic conditions from the coupled QDs was conducted at an in situ prepared mercury film electrode. The immunoassay combines highly efficient magnetic separation with signal amplification by the multilayered QD labels. The dynamic concentration range spanned from 1.0 fg mL^?1 to 1.0 μg mL^?1 of IgG1 with a detection limit of 0.1 fg mL^?1. The electrochemical immunoassay showed good reproducibility, selectivity, and stability. The analysis of clinical serum specimens revealed good accordance with the results obtained by an enzyme‐linked immunosorbent assay method. The new immunoassay is promising for enzyme‐free, and cost‐effective analysis of low‐abundance biomarkers.     

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。     

Immunoassay based on peroxidase silver conjugate for the determination of human immunoglobulins against tick-borne borreliosis (lyme disease) by voltammetry and spectrophotometry

In this work two strategies for the synthesis of peroxidase silver conjugates for the qualitative and quantitative determination of immunoglobulins (IgG) to ixodid tick-borne borreliosis (ITBB) (Lyme disease) in human serum were proposed. The first approach for Ab-HRP@AgNP conjugate synthesis involved silver nanoparticles (Ag NPs) capped with a commercial peroxidase conjugate (Ab-HRP) by passive adsorption. The second strategy was based on the initial coupling of Ag NPs with human anti-species antibodies (Ab) by passive adsorption followed by the introduction of horseradish peroxidase (HRP) enzyme into the reaction mixture as a blocking reagent for Ab@AgNP@HRP conjugate synthesis. The formation of peroxidase silver conjugates was proved by UV/Vis spectroscopy and Transmission Electron Microscopy (TEM). The catalytic activity of Ab-HRP@AgNP and Ab@AgNP@HRP conjugates was evaluated by Michaelis-Menten kinetics. A commercially available 96-well microtiter plate with recombinant antigens to ITBB was used as a platform for immobilization of analyzed IgG. The HRP in Ab-HRP@AgNP conjugate was found to retain a sufficient level of activity for interaction with the H₂O₂ substrate to form an intensely colored reaction product. Therefore Ab-HRP@AgNP conjugate can be used in enzyme-linked immunosorbent assay (ELISA) with spectrophotometric detection of 3,3’,5,5’-Tetramethylbenzidine (TMB Ox) for quantitative determination of IgG to ITBB in human serum in the concentration range 12.5–800 ng ml⁻¹ with LOD 2 ng ml⁻¹. Ab@AgNP@HRP conjugate is recommended for the electrochemical determination of IgG to ITBB in human serum at LOD 3 ng ml⁻¹ with registration of silver oxidation by linear sweep anodic stripping voltammetry (LSASV). Ag NPs in Ab-HRP@AgNP and Ab@AgNP@HRP conjugates do not change electrochemical activity during storage and can be used as an electrochemical label in LSASV method in case of HRP inactivation. The immunoassay based on peroxidase silver conjugates expands the analytical potential for the determination of IgG to ITBB especially during the period of increasing incidence.     

壳聚糖-纳米金复合膜修饰电化学发光免疫传感器检测人免疫球蛋白G

利用壳聚糖与纳米金良好的生物相容性及蛋白固定能力,制备了兼具导电性和透光性的人免疫球蛋白G(IgG)修饰膜,用于修饰玻碳电极,研制了新型电化学发光免疫传感器,并通过扫描电镜(SEM)及交流阻抗技术(EIS)考查了传感器表面性质.基于竞争免疫分析模式,以Ru(bpy)32+标记的羊抗人IgG为发光示踪物,采用新型共反应剂二丁基乙醇胺(DBAE)对光信号进行放大,建立了人IgG的检测方法,线性范围20ngmL-1～1.0μgmL-1,检测限6.5ngmL-1.将该电化学发光传感器应用于人血中IgG的检测,结果令人满意.     

Electrochemical Dissolution of Silver Nanoparticles and Its Application in Metalloimmunoassay

This work demonstrates the use of silver nanoparticles as a simple electrochemical biolabel to induce 10⁶ signal enhancement. We propose a mechanism of measuring the silver nanoparticles on a specific screen‐printed planar carbon electrode, without the requirement for the harsh oxidant or toxic reagents described in prior‐art for gold sol methods. The possible measurement process was validated with orthogonal techniques such as UV/Vis spectroscopy, dynamic light scattering and by anodic stripping voltammetry (ASV). The findings were utilized to develop a novel electrochemical sandwich immunoassay where the analyte concentration is directly proportional to ASV oxidation peak of silver. This technique in the future is envisaged to form the foundation of a generic Point of Care platform. The assay was applied to cardiac marker: myoglobin with detection limit of 3 ng/mL.     

A novel immunoassay based on the dissociation of immunocomplex and fluorescence quenching by gold nanoparticles

This study reports a novel, simple and sensitive immunoassay using fluorescence quenching caused by gold nanoparticles coated with antibody. The method is based on a non-competitive heterogeneous immunoassay of human IgG conducted by the typical procedure of sandwich immunocomplex formation. Goat anti-human IgG was first adsorbed on polystyrene microwells, and human IgG analyte was captured by the primary antibody and then sandwiched by antibody labeled with gold nanoparticles. The sandwich-type immunocomplex was subsequently dissociated by the mixed solution of sodium hydroxide and trisodium citrate, the solution obtained, which contains gold nanoparticles coated with antibody, was used to quench fluorescence. The fluorescence intensity of fluorescein at 517 nm was inversely proportional to the logarithm of the concentration of human IgG in the dynamic range of 10-5000 ng mL⁻¹ with a detection limit of 4.7 ng mL⁻¹. The electrochemical experiments and the UV-vis measurements were applied to demonstrate whether the immunoglod was dissociated completely and whether the gold nanoparticles aggregated after being dissociated, respectively. The proposed system can be extended to detect target molecules such as other kinds of antigen and DNA strands, and has broad potential applications in disease diagnosis.     

磁性无机-生物复合粒子敏感膜新型电流型免疫传感器的研究

合成了磁性Fe3O4纳米粒子,利用3-氨基丙基三乙氧基硅烷(APS)进行硅烷化,形成表面带有氨基的磁性Fe3O4纳米复合粒子,再用戊二醛将羊抗人免疫球蛋白G抗体(anti-IgG)固定在该磁性粒子表面,通过磁力将其修饰于固体石蜡碳糊电极表面制作成免疫传感器。与标记HRP的二抗体anti-IgG结合,以对苯二酚作为电子媒介体,实现对人免疫球蛋白G(IgG)的定量检测。IgG测定线性范围为2.5~400μg/L,检出限为0.75μg/L。该免疫传感器制作简单,成本低,表面更新方便,可用于临床血清检测。     

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.     

Microdrop analysis of a bead-based immunoassay

The progress to electrochemical detection of a microbead-based immunoassay in small volumes has led to a reduced assay time and lower detection limits. Three electrochemical techniques are described for an immunoassay with detection in a microdrop. The techniques are amperometric detection with a rotating disk electrode (RDE), a microelectrode, and an interdigitated array (IDA) electrode. An enzyme-labeled sandwich immunoassay with mouse IgG as the model analyte is used to demonstrate the three techniques. The microbead assay is carried out in a test tube using a magnet to control bead collection. Once the immunocomplex is formed on the microbead, the beads are transferred to a microdrop where the enzyme, either alkaline phosphatase or β-galactosidase, generates 4-aminophenol (PAP). PAP is oxidized at the electrode with an applied potential of +290 mV vs. Ag/AgCl. For all three techniques, the upper limit of the dynamic range was 1000 ng/ml mouse IgG, and the detection limits were: 50 ng/ml for the RDE, 40 ng/ml for the microelectrode, and 26 ng/ml for the IDA electrode.