基于酶催化沉积放大的电化学阻抗法用于人免疫球蛋白M的检测

提出了一种基于酶催化沉积放大的电化学免疫分析方法。先通过免疫夹心反应,将酶标抗体(羊抗人IgM-HRP)固定到电极表面。通过HRP催化双氧水氧化3,3′-二氨基联苯胺在电极表面形成不溶性沉积物,从而放大电化学检测信号。实验通过蛋白A实现抗体的定向固定。考察了包被抗体的浓度和包被溶液的pH对抗体固定的影响,以及酶标抗体的用量和沉积时间对免疫分析的影响。传感器信号响应与人免疫球蛋白M(IgM)浓度在2.1~670μg/L范围内具有良好的线性关系;检出限达0.08μg/L。     

基于金纳米链标记抗体及HRP信号增强的电流型免疫传感器

酶联免疫分析将抗原-抗体反应的特异性与酶的高效催化放大作用相结合,因而极大地提高了免疫分析的灵敏度~([1]).本研究将均匀分散的碳纳米管修饰到预处理的电极表面,通过电化学还原氯金酸制成碳纳米管-纳米金复合基质用以固载抗体.同时制备了金纳米链(Au NCs)并标记二抗,结合辣根过氧化物酶(HRP),采用双抗体夹心分析模式,以心血管疾病标志物血清人N末端脑钠尿肽前体(NT-proBNP)为免疫蛋白模型,构建了高灵敏电流型酶免疫传感器.     

基于双层酶信号放大及纳米功能界面的微囊藻毒素电化学免疫传感器

设计了一种基于纳米复合膜双层酶信号放大的竞争型电化学免疫传感器,用于检测痕量微囊藻毒素-LR(Microcystin-(leucine-arginine),MCLR)。在多壁碳纳米管修饰玻碳电极上电沉积金纳米粒子形成纳米复合膜,作为MCLR抗体(anti-MCLR)和辣根过氧化物酶(HRP)的固定化载体;引入HRP作为封闭剂或者通过抗体捕获HRP标记的MCLR(HRP-MCLR),起封闭活性位点及协同催化的作用。利用MCLR与HRPMCLR对纳米复合膜所固载抗体活性位点的竞争结合模式,采用微分脉冲伏安法(DPV)测定电极界面上HRP酶催化过氧化氢(H_2O_2)氧化对苯二酚(HQ)产生的还原电流,实现MCLR的定量检测。此传感器具有良好的特异性、稳定性与灵敏度,线性检测范围为0.1~100.0 μg/L,检出限为0.038 μg/L(S/N=3),对实际水样的加标回收率为72.9%~117.3%。     

基于酶催化银沉积质量放大的血吸虫压电免疫传感器的研究

发展了一种基于酶催化金属银沉积信号放大的新型高灵敏气相压电免疫传感检测技术.先将血吸虫抗原(SjAg)共价固定在石英晶体表面,制备得到血吸虫压电免疫传感器.检测时,在晶振上滴加不同浓度的待测血吸虫抗体,再将碱性磷酸酶标记的二抗通过夹心方式结合到传感器表面.然后利用碱性磷酸酶催化磷酸化的抗坏血酸酯水解从而还原硝酸银,使金属银沉积在晶振表面上,放大传感器的质量响应信号.实验结果表明该传感检测方法可显著提高气相压电免疫传感器的检测灵敏度,传感器对血吸虫抗体的响应线性范围在1～225 ng/mL,检测下限为1 ng/mL.     

红细胞标记抗体的化学发光免疫分析

用红细胞代替辣根过氧化物酶作为双抗体夹心免疫分析中第二抗体的标记物, 建立了一种红细胞标记抗体的免疫化学发光测定乙型肝炎病毒表面抗原的新方法. 在免疫反应完成后, 结合了抗原-抗体免疫复合物的致敏红细胞在低渗溶液中溶血, 释放出血红蛋白. 基于血红蛋白对鲁米诺-H2O2体系化学发光具有催化作用的原理, 采用化学发光法测定血红蛋白含量. 测得的血红蛋白发光强度与待测抗原浓度呈线性关系. 采用这种方法可检测出0.5 ng/mL的乙型肝炎病毒表面抗原. 将该方法与酶联免疫吸附分析(ELISA)结合起来对乙型肝炎患者血清乙肝病毒表面抗原(HBsAg)进行检测, 两者符合率均为97%, 表明本法具有良好的灵敏度和特异性, 可用于临床标本测试.     

麻疹病毒抗体的酶免疫电化学分析法研究

建立了测定麻疹病毒抗体的酶免疫电化学分析法。其原理是在异相酶联免疫之后用极谱法检测酶促产物。本法采用小型三电极系统“ＤＭＥ－Ｐｔ－Ａｇ／ＡｇＣｌ”直接与商品酶联板配套使用，简化了操作步骤。对测定麻疹抗体的合适条件以及灵敏度，特异性等作了考察。结果表明，本法特异性强，灵敏度高，检测范围宽，不受样品颜色、浊度、杂散光及酶联板自身光吸收不均一的影响；应用于测定三批共１１８份血清样品，阳性率分别为１５％（６／４０），６２．５％（２５／４０）和３１．５８％（１２／３８），用４６份样品对本法和ＥＬＩＳＡ作相关性检验，其相关性良好（γ＝０．７７．ｔ＿ｒ＝１０．８４，Ｐ＜０．００１，相关显著）。     

基于酪胺信号放大的新型免疫传感器

将酪胺应用于酶联免疫分析,建立了一种新的高灵敏伏安型免疫传感器。利用纳米金的静电吸咐和己二硫醇、巯基乙胺的自组装,将羊抗人IgG抗体固定到金电极表面上,以辣根过氧化物酶标记羊抗人IgG抗体为酶标抗体,以生物素化酪胺为酶底物,利用催化酪胺沉积反应,在传感界面沉积大量生物素,使原始信号得到几何级数的放大。结果表明,通过生物素化酪胺催化放大后,制得的免疫传感器对H2O2的催化能力增大近20倍,检测hIgG在1.5μg/L～22 mg/L范围内有良好的线性关系,检出限为0.1μg/L。用于实际试样的回收率的测定,结果良好。     

3,3',5,5'-四甲基联苯胺-H2O2-辣根过氧化物酶伏安酶联免疫分析法测定人血清免疫球蛋白E

近年来,电化学免疫分析法由于成功地将免疫反应的高选择性和电化学测定的高灵敏度相结合而越来越受到人们的重视[1]．酶联电化学免疫分析法的测定灵敏度与放射免疫法相近而又不必使用放射性同位素[2],充分显示了该法在临床检验中的优越性和发展前景．本文利用HRP催化TMB－H2O2的反应,以金电极为工作电极,用示差脉冲伏安法检测酶催化产物,建立了TMB－H2O2－HRP酶联免疫示差脉冲伏安分析体系,并成功地用于人血清IgE的测定。实验表明,本法较ELISA显色光度测定法的灵敏度高4倍,且具有更宽的线性范围,样品溶液基体对测定不产…     

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。     

以金纳米颗粒-二氧化钛纳米线阵列为支架的电化学免疫传感的构建及其应用

首先以掺杂氟的二氧化锡导电玻璃为基片,采用水热法制备出二氧化钛纳米线阵列,然后使用电化学沉积法将金纳米颗粒修饰于二氧化钛纳米线阵列表面以增强其导电性和生物相容性.将纳米金-二氧化钛纳米线阵列用作电化学免疫传感器支架,利用纳米金与癌胚抗原抗体之间的静电吸附作用,将包被抗体负载于电极表面制得检测癌胚抗原的电流型免疫传感器.同样,水热法还被用于制备圆柱形二氧化钛纳米棒,以辛二酸双(3-磺基-N-羟基琥珀酰亚胺酯)钠盐为双氨基交联剂,将辣根过氧化物酶和信号抗体一起固定于二氧化钛纳米棒表面作为示踪标记物.扫描电子显微镜、透射电子显微镜和X射线衍射分析仪均被用于分析上述材料的结构、形貌和组成.通过夹心型免疫反应,示踪标记物上信号抗体被定量负载于免疫传感器表面.通过一个以过氧化氢为媒介体的辣根过氧化物酶催化反应,差示脉冲伏安法被用于癌胚抗原的定量测定.结果表明,该癌胚抗原免疫传感器线性范围为0.01~120μg/L,检测限为6 ng/L.     

3D Dendritic Nanostructure of Silver‐Array: Preparation,Growth Mechanism and Application in Nitrate Sensor

A 3D dendritic nanostructure of silver‐array (DSA) was prepared via electrodeposition on an interdigitated array (IDA) microelectrode. For preparation of this nanostructure, the optimal parameters such as deposition potential, deposition time, the electrode bandwidth and gap size were systematically investigated. It was found that the edge effect of the IDA microelectrode is a key factor for controlling the 3D dendritic growth of silver. Furthermore, the formation of the 3D dendritic morphology was discussed from the aspect of electrochemical nucleation theory and nonequilibrium growth dynamics to give a deep understanding of its growth mechanism. For its potential practical application, we showed that the as‐prepared 3D DSA nanomaterial exhibited high electrocatalytic reduction ability to nitrate in neutral solution and excellent performance for nitrate determination. An amperometric nitrate microsensor based on the 3D DSA was obtained.     

Amplified Electrochemical Immunoassay Using HRP Labeled Protein as an Inhibitor to Silver Deposition in the Presence of H2O2

A novel electrochemical immunosensor with amplification effect based on the enzyme inhibition of silver deposition was proposed. In this method, the capture antibody was first immobilized onto a gold electrode via a self-assembled layer. After a sandwich immunoreaction, HRP labeled antibody was bound to the gold electrode. The HRP on the electrode inhibited silver deposition when the electrode was incubated in hydroquinone-H2O2 solution and silver ion solution. The linear sweep voltammetry was chosen to detect the deposited silver and the result showed that the peak current was linearly proportional to the content of IgG in the range of 50 to 2500 ng/mL with a detection limit of 35 ng/mL.     

Comparison of Planar and 3‐D Interdigitated Electrodes as Electrochemical Impedance Biosensors

It has been reported that the introduction of a dielectric barrier between adjacent digits of an interdigitated electrode array can improve the sensitivity of the array as an electrochemical impedance biosensor. Here we present an in‐depth analysis of the impedance in planar interdigitated electrodes and 3‐D interdigitated electrodes (with dielectric barriers). The analysis indicates that the planar geometry not only provides lower impedance but also a higher change impedance as a result of molecular immobilization on the electrode array surface.     

基于三维纳米银修饰电极的硝酸根微型传感芯片研究

研制一种基于金叉指微电极阵列(IDA)的电流型硝酸根离子(NO-3)微传感电极芯片.基于微机电系统(Micro-Electro-Mechanical Systems,MEMS)工艺制备金IDA微电极,通过电化学沉积技术在IDA微电极表面修饰三维枝状结构纳米银敏感膜,利用敏感膜对硝酸根离子良好的电催化还原性能,采用脉冲方波伏安(SWV)电化学测量方法,实现对硝酸根离子在25～1000μmol/L浓度范围内的快速检测,灵敏度达9.5 nA/(μmol/L),线性度为99.98%,检测下限为10μmol/L.考察水体中常见的NO-2,F-,3PO 4-,SO 42-,2CO3-,NH+4,Na+和K+等离子对该传感芯片的干扰性能,传感芯片表现出较好的抗干扰性能.制备的三维枝状结构纳米银修饰IDA微电极可实现水环境(pH 5.0～9.0)中NO-3的电化学检测,对应用于自然水环境中硝酸根离子的现场检测具有积极意义.     

Glucose sensing based on interdigitated array microelectrode.

A micro glucose sensor consisting of an interdigitated array gold microelectrode was developed. The interdigitated array structure, which has 10 microns band width and 10 microns band gap, was fabricated in a small region (2.5 x 5 mm2) on a quartz substrate. Glucose oxidase was chemically fixed onto the electrode surface through self-assembled monolayer of 11-mercaptoundecanoic acid; ferroceneacetic acid was used as electron mediator. Electrochemical properties of the glucose oxidase-immobilized microelectrode were investigated by cyclic voltammogram measurements. Results confirmed that the reductive ferroceneacetic acid generated at counter electrode diffuses through a narrow band gap (10 microns) and can reach the working electrode surface.     

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.     

Indirect voltammetric detection of fluoride ions in toothpaste on a comb-shaped interdigitated microelectrode array

A novel technique based on dynamic electrochemistry for the detection of fluoride ions was developed. It is based on its strong complexation with ferric ion. Formed fluoroferric complex is cathodically inactive at the potential of the reduction of free ferric aquo ion. The voltammetric and amperometric response of platinum comb-shaped interdigitated microelectrode array is decreased after fluoride addition. This decrease serves for the quantification of fluoride ions added to the solution. The detection limit of 4.5 × 10⁻⁵ mol dm⁻³ was achieved when one of the segments of interdigitated microelectrode array (IDA) was used as an indicating electrode. The detection limit is about one order of magnitude lower than in the case of conventional platinum macroelectrode. In comparison with ISE electrodes this method is faster and also avoiding large error resulting from the antilogarithmization of ISE Nerstian response. The method was applied to the analysis of toothpaste.     

Studies of electrochemical quantized capacitance charging of surface ensembles of silver nanoparticles

CTAB-stabilized silver nanoparticles were synthesized by NaBH₄ reduction. The as-prepared nanoparticles can be self-assembled on 3-mercaptopropionic acid (MPA) modified gold electrode, which was supported strongly by XPS measurements. Exceptional long-term stability of the as-prepared colloidal silver aqueous solution and the desorption of silver nanoparticle ensemble on MPA after alcohol rinsing proved that these CTAB molecules adsorbed on silver core formed interdigitated bilayer structure. DPV and differential capacitance measurements were performed to characterize the as-prepared silver nanoparticle ensemble, and the interesting quantized capacitance charging behaviors were observed.     

电泳沉积制备氧化石墨烯-银电极及其氧还原催化性能

通过在1-甲基-2-吡咯烷酮（NMP）中超声剥离氧化石墨制备出稳定的氧化石墨烯（GO）分散液,添加AgNO₃使氧化石墨烯吸附Ag+而带正电荷。采用电泳沉积法使GO沉积到阴极的玻璃碳电极上,Ag+被电化学还原为单质银,均匀的分散在GO片层当中。通过AFM、SEM、Raman、XRD及元素面扫分析对制备电极的形貌、结构进行表征。在碱性环境中进行氧还原测试,结果表明GO+Ag电极的氧还原起始电位较玻璃碳电极最大正移228mV,还原电流密度最大为7.564mA·cm^-2,是玻璃碳电极的3.4倍。通过不同转速下的线性扫描曲线绘制Koutechy-Levich图,计算氧还原反应的电子转移数为3.3。     

Electrochemical Detection of Schistosoma Japonicum Antibody Using Biocatalytic Deposition

Abstract

An ultrasensitive electrochemical immunoassay based on biocatalytic deposition has been proposed for the detection of Schistosoma japonicum antibody (SjAb) in infected rabbit serum. Schistosoma japonicum antigen (SjAg) was immobilized on the gold electrode surface via glutaraldehyde crosslink and then incubated with infected rabbit serum containing SjAb; finally, the goat anti-rabbit IgG labeled with alkaline phosphatase was sandwiched to form the immunocomplex on the gold electrode surface. The alkaline phosphatase converted nonelectroactive substrate into the reducing agent and the latter, in turn, reduced metal ions to form electroactive metallic product on the electrode surface. Linear sweep voltammetry (LSV) was used to quantify the amount of the deposited silver and give the analytical signal for SjAb. Assay conditions such as the antigen concentration and enzymatic silver deposition time were optimized. The electrochemical immunosensor was able to realize a reliable determination of SjAb in the dilution range from 1:5000 to 1:100 with a detection limit of 1:6457 of dilution ratio. The feasibility of the proposed immunosensor for possible clinical applications was also investigated by analyzing real serum samples.