基于脂质体信号增强癌胚抗原电化学免疫传感器的研究

研究了在玻碳电极利用巯基乙胺固定纳米金、然后纳米金固载癌胚抗体(Ab1),采用脂质体包裹电子媒介体硫堇,脂质体周围联接标记辣根过氧化物酶(HRP)的癌胚抗体(Ab2)对其传感器进行信号放大,通过循环伏安法考察了该免疫传感器的电化学特性,在优化的实验条件下,该免疫传感器的峰电流随着检测溶液中癌胚抗原(CEA)浓度的增大而增大,并在0.05～200 ng/mL CEA范围内呈现线性关系,回归方程为:Δi=0.20+0.24ρ(ng/mL);检测限为:18pg/mL(R=0.9947)。该免疫传感器可用于临床上对CEA的检测。     

基于Cu-TPA的电化学生物传感器对黄曲霉毒素B1的检测

利用对苯二甲酸铜(Cu-TPA)能产生强的电化学信号设计了一种灵敏的电化学生物传感器, 并将其用于测定黄曲霉毒素B1(AFB1). 信号探针中的Cu-TPA含有可产生电化学信号的Cu(Ⅱ), 当加入一定量的AFB1后, AFB1与探针中特定的适配体结合, 使信号探针脱落, 电化学信号降低. 根据电化学信号值的变化实现了对AFB1的检测. 在最佳条件下, 该传感器的检出限为4.2×10 ^-6 ng/mL(S/N=3), 线性范围为10 ^-5~10 ng/mL. 将该传感器用于啤酒中AFB1的检测, 回收率为95%~106%.     

电化学免疫传感器测定牛奶中的青霉素

利用共价键合法,将新亚甲蓝(NMB)与辣根过氧化酶(HRP)标记的青霉素多克隆抗体(Ab*)修饰于玻碳电极表面,制成电流型免疫传感器;考察了该传感器的电化学行为和对H2O2的电化学响应.结果表明,NMB作为介体能有效地传递电子,传感器对H2O2具有很好的催化作用.用此传感器对牛奶中的青霉素进行检测,其线性范围为0.25～3.00ng/mL (R=0.984),检出限为0.298ng/mL.     

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。     

癌胚抗原快速无分离电化学免疫检测

通过同时固定硫堇和HRP标记癌胚抗原(CEA)抗体于电化学预处理的玻碳电极表面,制备了新型无需分离的CEA快速电化学检测免疫传感器.CEA测定的两段线性范围为0.5～3.0和3.0～167 ng/mL,检测下限为0.1 ng/mL.该传感器具有良好的准确性、精密度、制备重复性和稳定性.该方法缩短了分析时间,降低了测定成本,适用于临床CEA的快速测定.     

基于纳米银与有机多孔材料/纳米金修饰的甲胎蛋白免疫传感器研究

在金电极表面电沉积银为氧化还原探针,利用有机多孔材料(PTC-NH2)、纳米金(nano-Au)固载甲胎蛋白抗体(anti-AFP),制备出用于检测甲胎蛋白(AFP)的安培型免疫传感器。通过交流阻抗技术、循环伏安法研究了电极的电化学特性,考察了孵育时间、测试液pH值等实验条件对传感器性能的影响,并利用扫描电子显微镜(SEM)对电极的修饰过程进行了表征。该传感器对AFP有良好的电流响应,线性范围分别为1.0～20.0ng/mL和20.0～60.0 ng/mL,检测限为0.6 ng/mL。     

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。     

基于砷和汞离子标记SiO2@Au纳米探针的超敏多元免疫分析方法研究

构建了一种基于As3+和Hg2+标记SiO2@Au复合纳米探针(NPs),以及氢化物发生-原子荧光(HGAFS)同时检测癌胚抗原(CEA)和糖蛋白19-9(CA 19-9)的超灵敏多元免疫分析方法。采用氨基化SiO2(核)@Au NPs(壳)分别吸附As3+和Hg2+,并标记CEA和CA 19-9的第二抗体(Ab2),由此获得了富As(或Hg)型信号探针(As(Hg)-SiO2@Au-Ab2)。基于夹心免疫分析方法,将两种信号探针和对应的抗原以及96孔板上一抗,在板底形成两种免疫复合物(Ab 1/Ag/As(Hg)-SiO2@Au-Ab2),利用HG-AFS同时检测As3+和Hg2+,其与CEA和CA 19-9的含量对数值成正比,可用于定量分析。此类探针不仅具有良好的分散性、还具有出色的信号放大效果。优化了探针合成和最佳反应条件,并对探针制备过程进行了表征。本方法与CEA和CA19-9分别在0.001~100μg/L和0.01~80 U/mL范围内呈线性关系,检出限分别为0.5 ng/L和0.005 U/mL(3σ),低于标准ELISA方法3个数量级。用于血清样品中CEA和CA 19-9同时检测,并与标准ELISA方法对照,结果一致。此免疫分析方法灵敏、简便、一次可实现多个肿瘤标志物检测,适合于基层卫生部门进行恶性肿瘤早期筛查。     

构建免标记电流型免疫传感器用于检测血清中癌胚抗原(CEA)

构建了灵敏的铁氰化钾-壳聚糖-戊二醛信号体系,并以此为信号指示剂,建立了稳定、准确的免标记电化学免疫传感器用于血清中癌胚抗原(CEA)的检测。信号体系和Nafion分别修饰于玻碳电极表面,并固定CEA抗体,分别用原子力显微镜(AFM)和循环伏安法(CV)对电极修饰过程的形貌和电化学行为进行表征。结果表明,循环伏安的电流响应值与固定在电极表面的CEA浓度直接相关,且CEA浓度的对数值在0.005~40.0 ng/mL范围内与电流的降低值呈良好的线性关系,检出限为1.23 pg/mL。方法具有良好的特异性,能准确检测血清样本中CEA的浓度。     

基于聚2,6-二氨基吡啶膜及纳米金修饰的癌胚抗原免疫传感器研究

在玻碳电极表面电聚合2,6-二氨基吡啶(pPA), 利用硫堇(Thi)、纳米金(nano-Au)固载癌胚抗体, 制得稳定性好、灵敏度较高、线性范围宽的电流型免疫传感器. 通过循环伏安法考察了该免疫传感器的电化学特性, 在优化的实验条件下, 该免疫传感器的峰电流随着检测溶液中癌胚抗原(CEA)浓度的增大而减小, 并在0.5～20和20～160 ng/mL CEA范围内呈现出良好的线性关系, 检测下限为0.2 ng/mL. 该免疫传感器具有制作简单、重现性好、线性范围宽等优点, 可用于临床上对CEA的检测.     

Electrochemical Biosensor Based on Nanocomposites Film of Thiol Graphene‐Thiol Chitosan/Nano Gold for the Detection of Carcinoembryonic Antigen

In this paper, a thiol graphene‐thiol chitosan‐gold nanoparticles (thGP‐thCTS‐AuNPs) nanocomposites film with porous structure was fabricated by electrochemically depositing on glassy carbon electrode (GCE), which exhibited good biocompatibility and improved conductivity, to construct immunosensor free label for detection of carcinoembryonic antigen (CEA). The electrochemical behavior of this immunosensor was investigated by cyclic voltammetry. Under the optimum conditions, the immunosensor revealed a good amperometric response to CEA in two linear ranges (0.3–8.0 ng mL^?1 and 8.0–100 ng mL^?1) with a detection limit of 0.03 ng mL^?1. The results indicated that the immunosensor has the advantages of good selectivity, high sensitivity, and good stability for the determination of CEA.     

Sandwich-type amperometric immunosensor for human immunoglobulin G using antibody-adsorbed Au/SiO2 nanoparticles

A novel sandwich-type electrochemical immunosensor for human immunoglobulin G (hIgG) was developed using Au/SiO₂ nanoparticles (NPs) with adsorbed horseradish peroxidase-anti-hIgG as the secondary antibody layer. The signal readout is based on the amperometric response to the catalytic reduction of hydrogen peroxide at an AuNPs-polythionine modified glassy carbon electrode. Under optimized conditions, the linear range is from 0.1 to 200 ng·mL^?1, with a detection limit of 0.035 ng·mL^?1 (at an S/N of 3). The immunosensor exhibited a performance that is better than that based on Au/SiO₂NPs-excluded secondary antibody.     

Signal‐Enhanced Amperometric Immunosensor Based on Ferrocene‐Branched Poly(allylamine)/Multiwalled Carbon Nanotubes Redox‐Active Composite

A signal‐enhanced immunosensor has been developed by self‐assembling Au NPs onto a ferrocene‐branched poly(allylamine)/multiwalled carbon nanotubes (PAA‐Fc/MWNTs) modified electrode for the sensitive determination of hepatitis B surface antigen (HBsAg) as a model protein. The formation of PAA‐Fc/MWNTs composite not only effectively avoided the leakage of Fc and retained its electrochemical activity, but also enhanced the conductivity and charge‐transport properties of the composite. Further adsorption of Au NPs into the PAA matrix provided both the interactive sites for the immobilization of hepatitis B surface antibody (HBsAb) and a favorable microenvironment to maintain its activity. Tests performed with this immunosensor showed a specific response to HBsAg in the range of 0.1–350.0 ng mL^?1 with a detection limit of 0.03 ng mL^?1.     

A photoelectrochemical immunosensor based on Au-doped TiO2 nanotube arrays for the detection of α-synuclein

α‐Synuclein (α‐SYN) is a very important neuronal protein that is associated with Parkinson’s disease. In this paper, we utilized Au‐doped TiO₂ nanotube arrays to design a photoelectrochemical immunosensor for the detection of α‐SYN. The highly ordered TiO₂ nanotubes were fabricated by using an electrochemical anodization technique on pure Ti foil. After that, a photoelectrochemical deposition method was exploited to modify the resulting nanotubes with Au nanoparticles, which have been demonstrated to facilitate the improvement of photocurrent responses. Moreover, the Au‐doped TiO₂ nanotubes formed effective antibody immobilization arrays and immobilized primary antibodies (Ab₁) with high stability and bioactivity to bind target α‐SYN. The enhanced sensitivity was obtained by using {Ab₂‐Au‐GOx} bioconjugates, which featured secondary antibody (Ab₂) and glucose oxidase (GOx) labels linked to Au nanoparticles for signal amplification. The GOx enzyme immobilized on the prepared immunosensor could catalyze glucose in the detection solution to produce H₂O₂, which acted as a sacrificial electron donor to scavenge the photogenerated holes in the valence band of TiO₂ nanotubes upon irradiation of the other side of the Ti foil and led to a prompt photocurrent. The photocurrents were proportional to the α‐SYN concentrations, and the linear range of the developed immunosensor was from 50 pg mL^?1 to 100 ng mL^?1 with a detection limit of 34 pg mL^?1. The proposed method showed high sensitivity, stability, reproducibility, and could become a promising technique for protein detection.     

FeS2−AuNPs Nanocomposite as Mimicking Enzyme for Constructing Signal‐off Sandwich‐type Electrochemical Immunosensor Based on Electroactive Nickel Hexacyanoferrate as Matrix

In this work, a novel sandwich‐type electrochemical immunosensor with electroactive nickel hexacyanoferrate nanoparticles (NiHCFNPs) as matrix was constructed for α‐fetoprotein (AFP) detection in a signal‐off manner by using FeS₂?AuNPs nanocomposite catalyzed insoluble precipitation to significantly inhibit the electrochemical signal. Initially, the NiHCFNPs with excellent electrochemical property was modified on the electrodeposited nano‐Au electrode to obtain a strong initial electrochemical signal. Subsequently, another nano‐Au layer was formed for immobilization of capture antibody (Ab₁). In the presence of target AFP, the prepared FeS₂?AuNPs‐Ab₂ bioconjugate could be specifically recognized and immobilized on electrode through the sandwich‐type immunoreaction. The FeS₂ with large specific surface areas were used as scaffolds to load abundant mimicking enzyme AuNPs. With the help of hydrogen peroxide (H₂O₂), FeS₂?AuNPs with peroxidase‐like activity accelerated the 4‐chloro‐1‐naphthol (4‐CN) oxidation with generation of insoluble precipitation on electrode, which would greatly hinder the electron transfer and thus caused the decrease of electrochemical signal for quantitative determination of AFP. This approach achieved a wide dynamic linear range from 0.0001 to 100 ng mL^?1 with an ultralow limit detection of 0.028 pg mL^?1. Especially, the proposed AFP immunosensor can be applied to detect human serum samples with satisfactory results, indicating a potential application in clinical monitoring of tumor biomarkers.     

A Sensitive Electrochemical Immunosensor for α‐Fetoprotein Detection with Colloidal Gold‐Based Dentritical Enzyme Complex Amplification

A sensitive and specific electrochemical immunosensor was developed with α‐fetoprotein (AFP) as the model analyte by using gold nanoparticle label for enzymatic catalytic amplification. A self‐assembled monolayer membrane of mercaptopropionic acid (MPA) was firstly formed on the electrode surface through gold‐sulfur interaction. Monoclonal mouse anti‐human AFP was covalently immobilized to serve as the capture antibody. In the presence of the target human AFP, gold nanoparticles coated with polyclonal rabbit anti‐human AFP were bound to the electrode via the formation of a sandwiched complex. With the introduction of goat anti‐rabbit IgG conjugated with alkaline phosphatase, the dentritical enzyme complex was formed through selective interaction of the secondary antibodies with the colloidal gold‐based primary antibody at the electrode, thus affording the possibility of signal amplification for AFP detection. Current response arising from the oxidation of enzymatic product was significantly amplified by the dentritical enzyme complex. The current signal was proportional to the concentration of AFP from 1.0 ng mL^?1 to 500 ng mL^?1 with a detection limit of 0.8 ng mL^?1. This system could be extended to detect other target molecules with the corresponding antibody pairs.     

High Sensitive Detection of Prostate Specific Antigen by Using Ferrocene Cored Asymmetric PAMAM Dendrimer Interface Screen Printed Electrodes

In this study, electrochemical immunosensors were developed for the detection of prostate specific antigen (PSA) using ferrocene (Fc) and polyamidoamine dendrimer (PAMAM) constructs. The biosensor fabrication was designed by modifying the screen‐printed gold electrode (Au) with ferrocene cored dendrimers (FcPAMAM) synthesized in three different generations. The self‐assembled monolayer principle was followed, to obtain sensitive, selective and disposable electrodes. Therefore, the Au electrodes were modified with cysteamine (Cys) to obtain a functional surface for FcPAMAM dendrimers to bind. Dendrimer generations were attached to this surface using a cross‐linker (glutaraldehyde) so that a suitable surface was obtained for binding of biological components. The Monoclonal PSA antibody (anti‐PSA) was immobilized on the Au electrode surface which coated with dendrimer, and (Au/Cys/FcPAMAM/anti‐PSA) biosensing electrode was obtained. The PSA detection performances of electrochemical impedance spectroscopy (EIS) and Amperometry based immunosensors exhibited very low detection limits; 0.001 ng mL^?1 and 0.1 pg mL^?1, respectively. In addition, EIS and Amperometry based biosensors using Au/Cys/FcPAMAM/anti‐PSA sensing electrode were represented excellent linear ranges of 0.01 ng mL^?1 to 100 ng mL^?1 and 0.001 ng mL^?1 to 100 ng mL^?1. In order to determine the applicability recovery and selectivity tests were performed using three different proteins in human serum.     

A Highly Sensitive Electrochemical Impedance Spectroscopy Immunosensor for Determination of 1‐Pyrenebutyric Acid Based on the Bifunctionality of Nafion/Gold Nanoparticles Composite Electrode

A simple, highly sensitive and label‐free electrochemical impedance spectroscopy (EIS) immunosensor was developed using Nafion and gold nanoparticles (nano‐Au/Nafion) composites for the determination of 1‐pyrenebutyric acid (PBA). Under the optimal conditions, the amount of immobilized antibody was significantly improved on the nano‐Au/Nafion electrode due to the synergistic effect and biocompatibility of Nafion film and gold nanoparticles composites. The results showed that the sensitivity and stability of nano‐Au/Nafion composite electrode for PBA detection were much better than those of nano‐Au modified glassy carbon electrode (nano‐Au/GCE). The plot of increased electron transfer resistances (R_ets) against the logarithm of PBA concentration is linear over the range from 0.1 to 150 ng·mL^?1 with the detection limit of 0.03 ng·mL^?1. The selectivity and accuracy of the proposed EIS immunosensor were evaluated with satisfactory results.     

Electrochemical Immunoanalysis for Carcinoembryonic Antigen Based on Multilayer Architectures of Gold Nanoparticles and Polycation Biomimetic Interface on Glassy Carbon Electrode

This paper describes a layer‐by‐layer (LBL) self‐assembly process of chitosan (CTS) and gold nanoparticles (Au) on the pretreated negatively charged glassy carbon (GC) electrode to fabricate electrochemistry immunosensor with a nontoxic biomimetic interface, which provided an environment similar to a native system and allowed more freedom in orientation for immobilization of carcinoembryonic antibody (anti‐CEA) to monitor carcinoembryonic antigen (CEA). UV‐vis spectroscope, atomic force microscopy (AFM), and cyclic voltammetric (CV) measurements were used to follow the multilayer film formation. The performance of the biominetic interface and factors influencing the assay system were investigated in detail. The differential pulse voltammetry (DPV) current response is used for the CEA concentration assay. The dynamic range was from 0.50 to 80.00 ng mL^?1 with a detection limit of 0.27 ng mL^?1 at 3σ. In addition, the experiment results indicate that immobilization described in this proposed method exhibits a good sensitivity, selectivity, and stability.     

Ultrasensitive amperometric immunosensor for the determination of carcinoembryonic antigen based on a porous chitosan and gold nanoparticles functionalized interface

An immunosensor has been fabricated for direct amperometric determination of carcinoembryonic antigen. It is based on a biocompatible composite film composed of porous chitosan (pChit) and gold nanoparticles (GNPs). Firstly, a pChit film was formed on a glassy carbon electrode by means of electrodeposition. Then, thionine as a redox probe was immobilized on the pChit film modified electrode using glutaraldehyde as a cross-linker. Finally, GNPs were adsorbed on the electrode surface to assemble carcinoembryonic antibody (anti-CEA). The surface morphology of the pChit films was studied by means of a scanning electron microscope. The immunosensor was further characterized by cyclic voltammetry and electrochemical impedance spectroscopy. The electrochemical behaviors and factors influencing the performance of the resulting immunosensors were studied in detail. Results showed that the pChit films can enhance the surface coverage of antibodies and improve the sensitivity of the immunosensor. Under optimal conditions, the immunosensor was highly sensitive to CEA with a detection limit of 0.08 ng·mL^?1 at three times the background noise and linear ranges of 0.2～10.0 ng·mL^?1 and 10.0～160 ng·mL^?1. Moreover, the immunosensor exhibited high selectivity, good reproducibility and stability.