基于放大鲁米诺电致化学发光构建的夹心型超灵敏免疫传感器

本文以金铂纳米合金(Au-PtNPs)修饰的玻碳电极作为一抗(Ab1)甲胎蛋白抗体(anti-AFP)的固载界面,葡萄糖氧化酶(GOD)负载纳米金修饰的还原态石墨烯(AuNPs@rGr-GOD)来标记二抗,构建超灵敏的基于放大鲁米诺(Luminol)电致化学发光(ECL)的免疫传感器。在含适量葡萄糖的Luminol检测底液中,构建的免疫传感器可有效放大ECL信号。实验结果表明,制备的免疫传感器对甲胎蛋白(AFP)的检测在0.001～200ng/mL范围内呈现良好的线性响应,其检测限低至0.3pg/mL。该传感器可用于实际血清样本的检测。     

基于酶-抗体共固定二氧化锆纳米信号探针的瘦肉精安培免疫传感器研究

制备了基于酶-抗体共固定二氧化锆(ZrO2)纳米探针进行信号放大的盐酸特伦克罗瘦肉精(CLB)检测安培免疫传感器。首先,利用ZrO2纳米粒子负载葡萄糖氧化酶(Glucose oxidase,GOD)和CLB抗体(Clen-buterol antibody,anti-CLB),制得纳米探针(ZNPs/GOD-anti-CLB signal probe,简称ZNPG)。同时将聚二烯丙基二甲基氯化铵[Poly(diallyldimethylammonium chloride),PDDA]、氯化血红素(Hemin)和纳米金(AuNPs)层层组装到多壁碳纳米管(MWCNTs)修饰的丝网印刷电极(SPCE)表面,制得SPCE│MWCNTs/(PDDA/Hemin/AuNPs)n电流型传感器,可对H2O2产生还原催化电流。检测CLB时,将样品和ZNPG一起加入预先包埋好CLB的96孔板底部。样品CLB与板底CLB共同竞争结合孔溶液中的纳米探针ZNPG。反应结束后加入葡萄糖,板底ZNPG-CLB免疫复合物上的GOD催化葡萄糖氧化产生H2O2,可通过传感器检测。电流下降值(ΔI)与样品CLB成反比,可用于CLB的定量分析。由于ZNPG上具有高的GOD酶标记密度,故可以显著放大检测信号,提高检测灵敏度。在最佳条件下(pH 7.0,温育时间30 min,温育温度37℃),此传感器对CLB的检测范围为0.003～100μg/L,检测下限为1 ng/L,比酶联免疫分析法(ELISA)法灵敏度提高2个数量级,在猪饲料样品中进行加标回收实验,平均回收率达93.6%,相对标准偏差(RSD)小于2.5%,精密度好。同时该传感器具有可重复使用、灵敏快速,适合于食物样品中痕量CLB现场检测。     

串联的纳米传感器用于癌细胞中miRNA的超灵敏检测

MicroRNA(miRNA)的检测在癌症诊断和治疗中具有重要意义。本研究设计了DNA序列,组装了3组金纳米粒子-量子点复合物,基于此叠加构建了以熵驱动催化循环为模型的单重、双重和多重循环放大的3种纳米传感器。叠加的传感器中,上层复合物催化循环解组装输出的产物作为下层复合物的催化链,引发其解组装,释放更多荧光信号,实现了多重放大。传感器中每叠加一个核酸催化循环体系,检测限降低一个数量级,最终构建的传感器的检测限达到fmol/L级。本研究通过串联熵驱动的核酸催化循环体系,构建了动态可调的、可定量检测细胞中不同表达水平miRNA的新型纳米传感器。     

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。     

基于场放大进样及Au纳米粒子双重富集用于大肠杆菌检测的毛细管电泳电化学免疫分析法

利用Au纳米粒子作为辣根过氧化物酶(HRP)标记抗体的载体,结合电堆积预富集技术,发展了一种基于场放大进样及Au纳米粒子双重富集的毛细管电泳电化学免疫分析技术用于大肠杆菌的检测.大肠杆菌与酶标抗体免疫反应后直接进行场放大进样预富集,免疫样品快速迁移并堆积在毛细管入口端,同时带负电荷的金纳米粒子向阳极端迁移,在样品与缓冲溶液的界面处吸附样品离子.金纳米粒子作为多酶载体使检测信号进一步放大.以标记在抗体上的HRP催化H2O2氧化邻苯二胺产生的电流信号来检测大肠杆菌.同常规电动进样毛细管电泳相比,该双重富集技术可使灵敏度提高1400倍.该方法对大肠杆菌检测的线性范围为2.0～2000.0 cfu mL-1,检出限为1.0 cfu mL-1,实现了对扇贝样品中大肠杆菌的快速、灵敏检测.     

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.     

Au纳米标记物增强电化学免疫分析大肠杆菌的研究

通过在Au纳米颗粒表面修饰辣根过氧化酶(HRP)标记的大肠杆菌抗体制备了一种新型的Au纳米标记物, 并将该纳米标记物应用于增强电化学免疫分析大肠杆菌. 经过酶联免疫反应后, Au纳米标记物、免疫磁性颗粒(IMB)和大肠杆菌形成了IMB/抗体-大肠杆菌-Au纳米标记物的三明治式免疫复合物. 以3,3,5,5-四甲基联苯二胺(TMB)溶液作为底物, 采用电化学与流动注射检测(FIA)相结合的技术测定HRP的活性. 检测到的电流大小与免疫复合物上HRP的量成正比, 从而与大肠杆菌的浓度成正比. Au纳米颗粒增加了HRP的负载量, 增强了电化学信号, 大大提高了大肠杆菌的检测灵敏度. 实验结果表明, 大肠杆菌浓度在 1.0×102～5.0×104 cfu•mL－1范围内与电流大小成线性相关, 最低检测限达50 cfu•mL－1, 若对大肠杆菌样品溶液进行预浓缩, 将得到更宽的检测范围和更低的检测限. 本方法总的分析时间比其他方法短, 在1 h内就能完成对大肠杆菌样品的快速检测.     

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。     

基于纳米金/硫堇层层自组装的新型电流型酶-癌胚抗原免疫传感器

将Nafion 膜固定在金电极(Au)表面, 通过静电吸附和共价键合作用将硫堇(Thi)和纳米金颗粒(nano-Au)层层自组装到Nafion膜修饰的金电极表面. 再通过形成的纳米金单层吸附癌胚抗体(anti-CEA), 最后用辣根过氧化物酶(HRP)代替牛血清白蛋白(BSA)封闭电极上的非特异性吸附位点, 并同时起到放大响应电流信号的作用, 从而制得高灵敏、高稳定电流型酶-癌胚抗原(CEA)免疫传感器. 通过循环伏安和交流阻抗考察了电极表面的电化学特性, 并对该免疫传感器的性能进行了详细的研究. 该传感器对CEA检测的线性范围为2.5~80.0 ng/mL, 检测限为0.90 ng/mL.     

基于电活性铁氰化镍和金属模拟酶协同催化的蛋白质电化学检测

该文设计了一种基于金属纳米模拟酶协同催化产生不溶性沉淀的“signal-off”型电化学免疫传感器用于超灵敏检测甲胎蛋白(AFP）。通过夹心免疫法将具有辣根过氧化物活性的二抗耦合物空心纳米金-铂钯纳米颗粒(HAuNPs-PtPdNPs-Ab2）固载在电活性物质铁氰化镍纳米颗粒修饰的电极上。在H2O2存在下,以AFP捕获的二抗耦合物中的HAuNPs和PtPdNPs作为辣根过氧化物模拟酶催化4-氯-1-萘酚(4-CN）,并在电极界面生成不溶且不导电的沉淀物苯并-4-氯己二烯酮(4-CD）,有效阻碍了电子传递,电化学信号显著降低,可用于AFP的定量检测。实验表明,该传感器对0.1 pg/mL~200 ng/mL AFP表现出良好的检测线性,检出限(S/N=3）为33 fg/mL。该传感策略具有协同催化作用,可提供一种新的多重信号放大方法用于改善传感器的灵敏度。     

Ultrasensitive electrochemical immunoassay based on graphene oxide-Ag composites for rapid determination of clenbuterol

We report the development of an ultrasensitive amperometric biosensor based on Ag nanoparticles-decorated graphene oxide nanosheets (GO) (Ag-GO) for the rapid detection of clenbuterol (CLB). The morphology and structure of the Ag-GO labeled CLB (Ag-GO-CLB) were characterized by transmission electron microscope (TEM), atomic force microscope (AFM), and ultraviolet-visible spectroscope (UV-vis). The immunosensor was prepared by covalently immobilizing capture antibodies on a multi-walled carbon nanotubes-modified glassy carbon electrode. Through competitive immunoreactions, the Ag-GO-CLB nanocomposites were captured on the immunosensor and the silver was measured by positive differential pulse voltammetry (DPV) in KCl solution for the detection of antigen. The experimental results show a linear response over the range from 0.01 to 10.0 ng mL(-1) with a lower detection limit of 6.8 pg mL(-1) (signal-to-noise ratio of 3). The Ag-GO based immunosensor offers a simple and convenient route for metal-immunoassay labels, which can avoid the complicated and time-consuming dissolving of metal component for ultrasensitive determination. Moreover, the electrochemical immunoassay shows acceptable specificity and stability and is suitable for the determination of CLB in real samples.     

基于纳米金固定半抗原的电化学免疫传感器灵敏检测克伦特罗

研制了一种基于纳米金固定半抗原的间接竞争电化学免疫传感器,可灵敏检测克伦特罗.在金电极表面组装1,6-己二硫醇单分子膜,通过Au-S共价作用连接纳米金颗粒,通过吸附作用固定克伦特罗牛血清白蛋白偶联物.样品中的待测组分与固定化的克伦特罗偶联物竞争结合单克隆抗体,碱性磷酸酯酶标记的二抗选择性地与电极表面捕获的一抗反应,进而催化底物1-萘酚磷酸酯水解生成1-萘酚,在电极表面氧化产生电信号.在优化的实验条件下,克伦特罗浓度在0.1～1000 μg/L范围内与电流强度线性相关,线性方程为I(A)-8.79× 10-7-2.66× 10-7logC (μg/L),相关系数0.9960,检出限达20 ng/L.同时测定了猪肉及猪肝样品中克伦特罗含量,相对标准偏差平均值为7.0％,加标回收率在89.1％～105.6％之间,与传统的间接竞争酶联免疫吸附法对照,结果无显著性差异.     

Sensitive electrochemical immunoassay of carcinoembryonic antigen with signal dual-amplification using glucose oxidase and an artificial catalase

A new dual-amplification strategy of electrochemical signal based on the catalytic recycling of the product was developed for the antigen-antibody interaction by glucose oxidase (GOD)- conjugated gold-silver hollow microspheres (AuAgHSs) coupled with an artificial catalase, Prussian blue nanoparticles (PB), on a graphene-based immunosensing platform. The first signal amplification introduced in this study was based on the labeled GOD on the AuAgHSs toward the catalytic oxidation of glucose. The generated H(2)O(2) was catalytically reduced by the immobilized PB on the graphene nanosheets with the second amplification. With a sandwich-type immunoassay format, carcinoembryonic antigen (CEA) was monitored as a model analyte by using the synthesized AuAgHSs as labels in pH 6.0 phosphate buffer containing 10mM glucose. Under optimal conditions, the electrochemical immunosensor exhibited a wide dynamic range of 0.005-50 ng mL(-1) with a low detection limit (LOD) of 1.0 pg mL(-1) CEA (at 3σ). Both the intra- and inter-assay coefficients of variation (CVs) were lower than 10%. The specificity and stability of the immunosensor were acceptable. In addition, the assay was evaluated for clinical serum specimens, and received a good correlation with those obtained by the referenced electrochemiluminescent (ECL).     

A novel lable-free electrochemical immunosensor for carcinoembryonic antigen based on gold nanoparticles-thionine-reduced graphene oxide nanocomposite film modified glassy carbon electrode

In this paper, gold nanoparticle-thionine-reduced graphene oxide (GNP-THi-GR) nanocomposites were prepared to design a label-free immunosensor for the sensitive detection of carcinoembryonic antigen (CEA). The nanocomposites with good biocompatibility, excellent redox electrochemical activity and large surface area were coated onto the glassy carbon electrode (GCE) surface and then CEA antibody (anti-CEA) was immobilized on the electrode to construct the immunosensor. The morphologies and electrochemistry of the formed nanocomposites were investigated by using scanning electron microscopy (SEM), ultraviolet-visible (UV-vis) spectrometry, electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). CV and differential pulse voltammetry (DPV) studies demonstrated that the formation of antibody-antigen complexes decreased the peak current of THi in the GNP-THi-GR nanocomposites. The decreased currents were proportional to the CEA concentration in the range of 10-500 pg/mL with a detection limit of 4 pg/mL. The proposed method was simple, fast and inexpensive for the determination of CEA at very low levels.     

Label-free Microcantilever Immunosensor Based on a Competitive Immunoassay for the Determination of Clenbuterol

A label-free microcantilever immunosensor based on a competitive immunoassay is reported for the determination of clenbuterol. The immunosensor was fabricated by modifying clenbuterol–ovalbumin on the gold surface of the microcantilever with crossing linkage by L-cysteine and glutaraldehyde. Atomic force microscopy was utilized to characterize the construction of immunosensor and to measure the deflection of the microcantilever. The deflection response of the microcantilever was in negatively proportional to the concentration of clenbuterol from 1.0?×?10^?2 to 20?µg/L with a limit of detection of 1.0?×?10^?2?µg/L. The fabricated immunosensor was used to determine clenbuterol in pork samples with satisfactory results. In addition, the results were in accordance with those obtained by high-performance liquid chromatography. The reported immunosensor displayed high sensitivity and specificity together with excellent repeatability and reliability.     

A Label-Free Electrochemical Immunosensor for Clostridium Difficile Toxin B Based on One-Step Immobilization of Thionine in a Silica Matrix

A label-free amperometric immunosensor was fabricated to test clostridium difficile toxin B. Multi-walled carbon nanotubes were modified on the surface of a glassy carbon electrode by electrodeposition. A sol-gel method was developed to encapsulate thionine in an electrochemically induced three-dimensional porous silica matrix by a one-step process. Gold nanoparticle layers were constructed by covalent bonds and electrostatic adsorption with thionine. The clostridium difficile toxin B antibody was immobilized on the gold nanoparticles to construct the immunosensor. Cyclic voltammetry and differential pulse voltammetry demonstrated that the formation of antibody-antigen complexes decreased the peak current of thionine. The morphologies of the nanocomposites were investigated by scanning electron microscopy and ultraviolet-visible spectrometry. The electrode was shown to be sensitive and specific to detect clostridium difficile toxin B from 1.0 to 80.0 ng/mL with a limit of detection of 0.3 ng/mL.     

Ultrasensitive electrochemiluminescence immunosensor using PtAg@carbon nanocrystals composites as labels and carbon nanotubes-chitosan/gold nanoparticles as enhancer

An ultrasensitive electrochemiluminescence (ECL) immunosensor was developed using PtAg@carbon nanocrystals (CNCs) as excellent labels based on carbon nanotubes-chitosan/AuNPs (CNT-CHIT/AuNPs) composite modified screen-printed carbon electrodes (SPCEs) for prostate protein antigen (PSA) detection. The CNCs were obtained simply by electro-oxidation of graphite with abundant carboxyl groups at their surfaces. The PtAg bimetallic nanocomposites with hierarchically hollow structures were fabricated through simple replacement reaction using dealloyed nanoporous silver (NPS) as both a template and reducing agent. Structure characterization was obtained by means of transmission electron microscope (TEM) and scanning electron microscope (SEM) images. The PtAg@CNCs composites exhibit a 6 times higher ECL intensity than the pure CNCs labeled anti-PSA. The as-prepared CNT-CHIT/AuNPs composite can attach more antibody than pure CNTs. Due to the dual-amplification techniques, the concentrations of PSA were obtained in the range from 1 pg mL(-1) to 50 ng mL(-1) with a detection limit of 0.6 pg mL(-1). Finally, the as-proposed ECL immunosensor has the advantages of high sensitivity, specificity and stability and could become a promising technique for tumor marker detection.     

基于花状铂纳米颗粒构建的电致化学发光免疫传感器用于检测载脂蛋白A1

采用一锅合成法制备了新型的具有大比表面积的花状铂纳米颗粒（PtNFs）,并构建了一个高灵敏电致化学发光（ECL）免疫传感器用于检测载脂蛋白A1（Apo-A1）. 该PtNFs用于吸附二抗（anti-Apo-A1）,并用葡糖糖氧化酶（GOD）封闭其表面的非特异性位点,最终制备了PtNFs@anti-Apo-A1@GOD信号探针. 当Apo-A1存在时,通过夹心免疫反应将制备的信号探针捕获于电极表面,并将所制得的电极置于含有葡萄糖的过硫酸根底液中检测. GOD催化葡萄糖产生H₂O₂,H₂O₂在PtNFs的催化下分解并在电极表面原位产生O₂,所产生的O₂能够催化过硫酸根-氧气体系的电致化学发光反应,放大发光信号,提高检测灵敏度. 该传感器在0.1ng•mL^-1 ~ 100 ng•mL^-1范围内对Apo-A1有良好的线性响应,检测下限达到0.03ng•mL^-1,有望应用于临床分析诊断.     

基于双层纳米金修饰的高灵敏电位型乙肝表面抗原免疫传感器研究

基于电沉积和层层自组装技术，提出了一种新的生物分子固定化方法，研制成一种高灵敏电位型乙肝表面抗原免疫传感器。利用L-半胱胺酸（LCys）的双官能团结合双层纳米金，从而通过比表面积大，生物相容性好的纳米金胶吸附大量抗体，同时用聚乙烯醇缩丁醛（PVB）薄膜的笼效应把乙肝表面抗体（HBsAb）和纳米金固定在玻碳电极上，从而制得了高灵敏度、高稳定性的电位型免疫传感器。采用循环伏安法（CV）对电极的层层自组装过程进行了考察，并对该免疫传感器的性能进行了详细的研究。该免疫传感器线性范围是8．5～256．0ng／mL，线性相关系数为0．9978，灵敏度为89．0，检出限为3．1ng／mL。已用于病人的血清样品分析。     

Label‐Free Detection of Bisphenol A Using a Potentiometric Immunosensor

A potentiometric immunosensor for the label‐free detection of bisphenol A (BPA) was developed by covalently immobilizing a polyclonal antibody (PAb) onto the surface of a carboxylated poly(vinyl chloride) (PVC‐COOH) membrane. The immunosensor was characterized using scanning electron microscopy (SEM), X‐ray photoelectron spectroscopy (XPS), and electrochemical impedance spectroscopy. The immobilization of PAb and its interaction with antigen (Ag) and BPA were also confirmed by quartz crystal microbalance (QCM) studies. Experimental parameters affecting the immuno‐interaction between PAb and its Ag or BPA were examined in terms of pH, antibody concentration, and temperature. The immunosensor showed a specific recognition of BPA with less interference than 4.8% from other common phenolic compounds. A calibration plot was obtained between 1.0 and 30.0 ng/mL and the detection limit was determined to be 0.6 ng/mL. The proposed immunosensor was applied for a real water sample spiked with BPA and the recoveries were in the range between 102.5 and 105.7%.