基于树枝状分子及功能化金纳米粒子的电化学免疫传感器检测污泥中大肠杆菌

利用树枝状分子-金纳米粒子复合物修饰电极和金纳米粒子标记物构建电化学免疫传感器,用于污泥中大肠杆菌的检测.首先在玻碳电极表面电聚合对氨基苯甲酸,通过共价作用结合第Ⅳ代氨基末端的树枝状分子(G4-PAMAM),并在其内部载入金纳米粒子,制备修饰电极(GCE/p-ABA/PAMAM (AuNPs)),用于固定大肠杆菌.采用硫堇作为电活性物质包被金纳米粒子,用于标记二抗制备金纳米粒子标记物(Ab2-Au-Th).通过抗原-抗体之间的特异性识别作用,将一抗、金纳米粒子标记物依次修饰在电极表面,用差分脉冲伏安法测定硫堇产生的电流信号,实现对大肠杆菌的检测.在优化的实验条件下,响应电流与大肠杆菌浓度的对数在1.0×102～1.0×106 cfu/mL范围内呈线性关系,检出限为70 cfu/mL(S/N=3).利用本方法检测污水处理厂的不同污泥样品中的大肠杆菌,回收率为89.4％～ 105.8％.     

金纳米粒子和钼氧化物复合膜修饰电极的制备及应用

利用循环伏安法将金纳米粒子和钼氧化物共同电沉积在玻碳电极表面,制备了金纳米粒子和钼氧化物复合膜修饰电极,利用SEM和XPS研究了MoOx/AuNPs复合膜的表面形态,并研究其修饰电极对葡萄糖的电催化氧化过程. 首次提出了阳极扫描极化反向催化伏安法,即在反向扫描过程中纯的催化氧化电流通过扣减背景电流的方法被提取出来. 显著提高电流测量灵敏度改善了信噪比. 制备的MoOx/AuNPs复合膜修饰电极在0.01-4.0 mmol/L对葡萄糖具有线性响应,电流灵敏度为2.35 mA·L/（mmol·cm2）,检测限为9.01 μmol/L（信噪比为3）.     

基于聚对苯二甲酸乙二醇酯薄膜的石墨烯/金纳米粒子/葡萄糖氧化酶柔性电极的制备及检测葡萄糖

采用化学气相沉积法生长多晶石墨烯(Graphene, G),转移至聚对苯二甲酸乙二醇酯(PET)薄膜表面,通过控制金溶胶蒸发速率,在多晶石墨烯表面组装均匀分布的亚单层金纳米粒子(AuNPs);然后修饰巯基乙酸,通过共价交联反应将葡萄糖氧化酶固定于AuNPs表面,构建基于PET膜的石墨烯/金纳米粒子/葡萄糖氧化酶(G/AuNPs/GOD)柔性电极.此电极在工作电位0.6 V(vs.SCE电极)、pH 7.0磷酸盐缓冲溶液、室温25℃条件下,差分脉冲伏安法响应电流与被测葡萄糖浓度在0.05~10.55 mmol/L范围内呈线性关系,线性方程为I(108A)=0.2629 C(mmol/L)+1.4149,线性相关系数 r=0.9955,检出限1 μmol/L (3σ). G/AuNPs/GOD柔性电极的制备可为特定环境和可穿戴设备的葡萄糖检测提供了新的途径和方法,拓展了葡萄糖检测的应用范围.     

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。     

基于磁性Fe_3O_4/Au-Pt纳米复合物构建甲胎蛋白免疫传感器的研究

制备了磁性Fe3O4/Au-Pt纳米复合物,将该纳米复合物修饰在玻碳电极表面,用牛血清蛋白封闭电极表面非特异性吸附位点。通过循环伏安法对电极的制备过程以及响应性能进行表征。实验结果表明,该修饰电极对甲胎蛋白(AFP)有良好的电流响应,在甲胎蛋白浓度为0.05~1.0ng/mL和1.0~100.0ng/mL两个范围内,分别呈良好的线性,检出限为0.01ng/mL。该免疫传感器制备方法简单,灵敏度较高,有望应用于临床医学中检测人体血清中甲胎蛋白含量。     

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.     

纳米金颗粒增强信号的电化学生物传感器用于谷胱甘肽和半胱氨酸的检测

构建了一种高灵敏检测谷胱甘肽(GSH)和半胱氨酸(Cys)的新型电化学生物传感器.先将富含T碱基的DNA1和DNA2探针分别修饰在金电极和纳米金颗粒(AuNPs)上,再加入Hg2+,通过形成T-Hg2+-T结构使AuNPs结合到金电极表面.当加入GSH(或Cys)后,GSH(或Cys)可以竞争结合T-Hg2+-T结构中的Hg2+,使AuNPs离开电极表面.由于AuNPs上修饰的DNA探针能够静电吸附大量电活性物质六氨合钌(RuHex),因此该过程可引起计时电量信号的显著变化,据此实现了GSH(或Cys)的高灵敏检测.该传感器的检出限达10 pmol/L,比荧光法或比色法降低了2～3个数量级.实验结果表明,该传感器具有较好的选择性.     

基于硫化镍的C-反应蛋白免疫传感器的研制

本文合成了负载金纳米颗粒(Au NPs)的NiS纳米材料,通过壳聚糖(CHIT)将其固定在玻碳电极表面作为电化学生物传感器的固定基质。将C-反应蛋白(CRP)抗体固定到修饰过的玻碳电极表面,利用二茂铁甲酸标记CRP抗体,构建夹心型CRP生物传感器。采用差分脉冲伏安法(DPV)检测标记物二茂铁甲酸在0.3V左右的特征峰信号,该电流与培育的CRP抗原量成正比,从而实现对CRP的定量检测。传感器检测CRP的线性范围为0.01~500ng/mL,线性相关系数为0.9939,检测限为3.3pg/mL。     

纳米金/Nafion/石墨烯修饰玻碳电极检测对苯二酚的研究

采用一步电化学共还原的方法将纳米金(AuNPs)、Nafion、电化学还原石墨烯(ERGO)修饰到玻碳电极(GCE)表面,制成修饰电极AuNPs/Nafion/ERGO/GCE。以扫描电镜对其进行表征,用循环伏安法和微分脉冲伏安法研究对苯二酚在该修饰电极上的电催化行为。优化了实验参数,对苯二酚在2.0～100μmol/L及100～800μmol/L浓度范围内与其氧化峰电流呈良好的线性关系,检出限为0.3μmol/L。用该修饰电极成功地进行了实际水样中对苯二酚含量的测定。     

碳量子点/纳米金@碳纳米管修饰电极同时检测鸟嘌呤和腺嘌呤

制备了一种碳量子点(CQDs)/金纳米颗粒(AuNPs)@羟基化多壁碳纳米管(MWCNT-OHs)复合膜修饰电极用于鸟嘌呤(GA)和腺嘌呤(AE)的同时检测.与裸电极和其它修饰电极相比,复合膜修饰电极能显著提高GA和AE的氧化峰电流及峰电位差,能够对GA和AE同时高灵敏检测.研究了GA和AE在复合膜修饰电极上的电化学行为,结果表明,在0.2 mol/L PBS(pH 7.0)中,GA和AE的氧化峰电流与浓度分别在1～200 μmol/L和2～80μmol/L范围内呈良好的线性关系,检测限分别为0.9和1.8 μmol/L.将该修饰电极用于人体血清样品中GA和AE的同时电化学检测,加标回收率在90.4％～107.4％之间,证明了该修饰电极在生物样品分析领域的应用潜力.     

Dual Amplified Electrochemical Immunosensor for Hepatitis B Virus Surface Antigen Detection Using Hemin/G‐Quadruplex Immobilized onto Fe3O4‐AuNPs or (Hemin‐Amino‐rGO‐Au) Nanohybrid

A sensitive electrochemical immunosensor for Hepatitis B virus surface antigen (HBsAg) detection was fabricated based on hemin/G‐quadruplex interlaced onto Fe₃O₄‐AuNPs or hemin ‐amino‐reduced graphene oxide nanocomposite (H‐amino‐rGO‐Au). G‐quadruplex DNAzyme, which is composed of hemin and guanine‐rich nucleic acid, is an effective signal amplified tool for its outstanding peroxidase activity and Fe₃O₄‐AuNPs or (H‐amino‐rGO‐Au) nanocomposites with quasi‐enzyme activity provide appropriate support for the immobilization of hemin/G‐quadruplex. The target protein was sandwiched between the primary antibody immobilized on the GO and secondary antibody immobilized on the Fe₃O₄‐AuNPs or (H‐amino‐rGO‐Au) nanocomposites and glutaraldehyde was used as linking agent for the immobilization of primary antibody on the surface of GO. Both Fe₃O₄‐AuNPs and H‐amino‐rGO‐Au nanocomposite and also hemin/G‐quadruplex can cooperate the electrocatalytic reduction of H₂O₂ in the presence of methylene blue as mediator. The proposed immunosensor has a wide linear dynamic range of 0.1 pg/ml to 300 pg/ml with a detection limit of 60 fg/ml when Fe₃O₄‐AuNPs was used for immobilization of hemin/G‐quadruplex, while the dynamic range and DL were 0. 1–1000 pg/mL and 10 fg/mL, respectively in the presence of H‐amino‐rGO‐ Au nanocomposite as platform for immobilizing of hemin/G‐quadruplex. The proposed immunosensor was also used for analysis of HBsAg in spiked human serum samples with satisfactory results.     

Label-free sandwich type of immunosensor for hepatitis C virus core antigen based on the use of gold nanoparticles on a nanostructured metal oxide surface

We report on the construction of a label-free electrochemical immunosensor for detecting the core antigen of the hepatitis C virus (HCV core antigen). A glassy carbon electrode (GCE) was modified with a nanocomposite made from gold nanoparticles, zirconia nanoparticles and chitosan, and prepared by in situ reduction. The zirconia nanoparticles were first dispersed in chitosan solution, and then AuNPs were prepared in situ on the ZrO₂-chitosan composite. In parallel, a nanocomposite was synthesized from AuNPs, silica nanoparticles and chitosan, and conjugated to a secondary antibody. The properties of the resulting nanocomposites were investigated by UV-visible photometry and transmission electron microscopy, and the stepwise assembly process was characterized by means of cyclic voltammetry and electrochemical impedance spectroscopy. An sandwich type of immunosensor was developed which displays high sensitivity to the HCV core antigen in the concentration range between 2 and 512?ng?mL^?1, with a detection limit of 0.17?ng?mL^?1 (at S/N?=?3). This immunosensor provides an alternative approach towards the diagnosis of HCV.

A label-free electrochemical immunosensor based on multi-functionalized graphene oxide for ultrasensitive detection of microcystin-LR

In this paper, a label-free electrochemical immunosensor for ultrasensitive detection of microcystin-leucine-arginine (MC-LR) based on multi-functionalized graphene oxide was constructed. The graphene oxide has a large surface area for the immobilization of the antibody. Meanwhile, the introduction of the AuNPs and 1-butyl-3-methylimidazolium hexafluorophosphate could enhance the response of the current by improving the electrical conductivity. Thus the electrochemical immunosensor could be prepared through a one-step process and differential pulse voltammetry was employed to detect sensitively MC-LR. Under optimal conditions, the current response of the immunosensor decreased proportionally to the logarithmic concentrations of MC-LR in the range of 0.1–1000 ng/mL with a detection limit of 0.1 ng/mL (S/N = 3). This one-step label-free electrochemical immunosensor showed good performance in specificity, stability, reproducibility, and application.     

Sensitive electrochemical immunosensor array for the simultaneous detection of multiple tumor markers

A novel electrochemical immunosensor array for the simultaneous detection of multiple tumor markers was developed by incorporating electrochemically addressing immobilization and one signal antibody strategy. As a proof-of-principle, an eight-electrode array including six carbon screen-printed working electrodes was used as a base array for the analysis of two important tumor markers, carcinoembryonic antigen (CEA) and α-fetoprotein (AFP) and a horseradish peroxidase-labeled antibody was employed as a signal antibody. The immunosensor in the array was fabricated in sequence by covalently coupling the capture antibody onto the surface of the desired working electrode, which was firstly electrochemically addressably grafted with an aminophenyl group by reduction of in situ generated aminophenyl diazonium cation generated from p-phenylenediamine, using glutaraldehyde as cross-linker. This allowed the selective immobilization of the capture antibody at the desired position on a single array via an electrochemical operation. The immunoassay in sandwich mode was performed by specifically binding the targets, second antibodies and one signal antibody to the immunosensor array. The result showed that the steady current density was directly proportional to the concentration of target CEA/AFP in the range from 0.10 to 50 ng mL(-1) with a detection limit of 0.03 ng mL(-1) for CEA and 0.05 ng mL(-1) for AFP (S/N = 3), respectively. This work demonstrates that the employment of an electrochemically addressing method for the fabrication of an immunosensor array and one signal antibody is a promising approach for the determination of multiple tumor markers in clinical samples.     

Disposable electrochemical immunosensor by using carbon sphere/gold nanoparticle composites as labels for signal amplification

This work designed a simple, sensitive, and low-cost immunosensor for the detection of protein marker by using a carbon sphere/gold nanoparticle (CNS/AuNP) composite as an electrochemical label. The nanoscale carbon spheres, prepared with a hydrothermal method by using glucose as raw material, were used to load AuNPs for labeling antibody by electrostatic interaction, which provided a feasible pathway for electron transfer due to the remarkable conductivity. The disposable immunosensor was constructed by coating a polyethylene glycol (PEG) film on a screen-printed carbon-working electrode and then immobilizing capture antibody on the film. With a sandwich-type immunoassay format, the analyte and then the CNS/AuNP-labeled antibody were successively bound to the immunosensor. The bound AuNPs were finally electro-oxidized in 0.1 M HCl to produce AuCl(4)(-) for differential pulse voltammetric (DPV) detection. The high-loading capability of AuNPs on CNS for the sandwich-type immunorecognition led to obvious signal amplification. By using human immunoglobulin?G (IgG) as model target, the DPV signal of AuNPs after electro-oxidized at optimal potential of +1.40?V for 40?s showed a wide linear dependence on the logarithm of target concentration ranging from 10?pg mL(-1) to 10?ng mL(-1). The detection limit was around 9?pg mL(-1). The immunosensor showed excellent analytical performance with cost effectivity, good fabrication reproducibility, and acceptable precision and accuracy, providing significant potential application in clinical analysis.     

Biomolecule‐Doped Organic/Inorganic Hybrid Nanocomposite Film for Label‐Free Electrochemical Immunoassay of α‐1‐Fetoprotein

A new electrochemical immunosensor for the detection of α‐1‐fetoprotien (AFP) was developed based on AFP antibody (anti‐AFP)‐functionalized organic/inorganic hybrid nanocomposite membrane. To fabricate such a hybrid composite membrane, 3,4,9,10‐perylenetetracarboxylic acid‐bound thionine molecules (PTCTH) were initially doped into titania colloids (TiO₂), and then gold nanoparticles and anti‐AFP were immobilized onto the composite film in turn. Comparison with the electrode fabricated only with thionine not 3,4,9,10‐perylenetetracarboxylic acid, the immunosensor with PTCTH exhibited high sensitivity and fast electron transfer. The presence of gold nanoparticles provided a good microenvironment for the immobilization of biomolecules, enhanced the surface coverage of protein, and improved the sensitivity of the immunosensor. The modified process was characterized by scanning electron microscope (SEM), cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The surface topography of the membrane was investigated by scanning electron microscopy (SEM). Under optimal conditions, the proposed immunosensor exhibited a wide linear range from 2.5 to 200.0 ng/mL towards AFP with a detection limit of 0.5 ng/mL (S/N=3). The stability, reproducibility and precision of the immunosensor were acceptable. Comparison with the conventional enzyme‐linked immunosorbent assay (ELISA), the present method did not require more labeled procedures and washing steps. Significantly, the detection methodology provides a promising approach for other proteins or biosecurities.     

Regenerable Leptin Immunosensor Based on Protein G Immobilized Au‐Pyrrole Propylic Acid‐Polypyrrole Nanocomposite

A regenerable, labelless electrochemical immunosensor is investigated. In this work, pyrrole (Py) and pyrrole propylic acid (Pa) were co‐electropolymerized in the presence of gold nanoparticles to form a porous, conductive, stable and hydrophilic nanocomposite, followed by the covalent attachment of protein G to capture an antibody as the probe for the immunoassay. The regeneration of the sensor was achieved by rinsing the electrodes with 0.1 M glycine buffer (pH 2.7). The binding and dissociation of the antibody with protein G and optimization of the efficient immobilization were studied by impedance and optical measurements, respectively. The charge transfer resistance obtained from the impedance measurements is used to study the interaction between antibody‐protein G and antibody‐antigen. The immunosensor performance and its regenerability were evaluated by using anti‐leptin IgG as the probe protein to detect leptin in 0.01 M PBS, and its specificity was tested in 1% human serum. The leptin impedimetric immunosensor exhibits a detection dynamic range of 10–100 000 ng/mL with 10 ng/mL detection limit in 0.01 M PBS+1% serum solutions. This work proves the feasibility to make a sensitive, regenerative electrochemical immunosensor, which could be very useful for environmental control and food analysis.     

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.     

Ultrasensitive electrochemiluminescence immunoassay for tumor marker detection using functionalized Ru-silica@nanoporous gold composite as labels

In this work, we reported a simple and sensitive sandwich-type electrochemiluminescence (ECL) immunosensor for carcinoembryonic antigen (CEA) on a gold nanoparticles (AuNPs) modified glassy carbon electrode (GCE). The Ru-silica (Ru(bpy)(3)(2+)-doped silica) capped nanoporous gold (NPG) (Ru-silica@NPG) composite was used as an excellent label with amplification techniques. The NPG was prepared with a simple dealloying strategy, by which silver was dissolved from silver/gold alloys in nitric acid. The primary antibody was immobilized on the AuNPs modified electrode through l-cysteine and glutaraldehyde, and then the antigen and the functionalized Ru-silica@NPG composite labeled secondary antibody were conjugated successively to form a sandwich-type immunocomplex through the specific interaction. The concentrations of CEA were obtained in the range from 1 pg mL(-1) to 10 ng mL(-1) with a detection limit of 0.8 pg mL(-1). The as-proposed ECL immunosensor has the advantages of high sensitivity, specificity and stability and could become a promising technique for tumor marker detection.