Photoelectrochemical immunosensor for label-free detection and quantification of anti-cholera toxin antibody

We demonstrate herein a newly developed photoelectrochemical immunosensor for the determination of anti-cholera toxin antibody by using a photosensitive biotinylated polypyrrole film. The latter was generated by electro-oxidation of a biotinylated tris(bipyridyl) ruthenium(II) complex bearing pyrrole groups. The photoexcitation of this modified electrode potentiostated at 0.5 V vs SCE, in the presence of an oxidative quencher, pentaaminechloro cobalt(III) chloride (15 mM), led to a cathodic photocurrent. As a result of the affinity interactions, a layer of biotinylated cholera toxin was firmly bound to the functionalized polypyrrole film via avidin bridges. The resulting modified electrodes were tested as immunosensors for the detection of the corresponding antibody from 0 to 200 microg mL(-)(1). The antibody concentration was measured through the decrease in photocurrent intensity resulting from its specific binding onto the polymeric coating, the detection limit being 0.5 microg mL(-)(1).     

Electropolymerized biotinylated poly (pyrrole–viologen) film as platform for the development of reagentless impedimetric immunosensors

We describe herein the synthesis and electrochemical polymerization of a viologen monomer functionalized by a biotin group and the use of its redox properties for the impedimetric sensing of protein binding. The electrochemical oxidation of the pyrrole group at 0.85 V in CH₃CN led to a biotinylated polypyrrole film allowing the successive anchoring of an avidin layer and a biotinylated cholera toxin layer. EIS performed at ?0.45 V/SCE without redox probe in solution showed the specific immobilization of the anti-cholera toxin antibody.     

Application of polypyrrole/GOx film to glucose biosensor based on electrochemical-surface plasmon resonance technique

A surface plasmon resonance (SPR) based biosensor for glucose is presented in which a thin gold film modified with polypyrrole and glucose oxidase (PPy-GOx) acts as the sensor chip. It is based on SPR response to the change of refractive index of PPy-GOx film by the enzymatic catalytic reaction. The co-electropolymerization of pyrrole and GOx was carried out under cyclic voltammetric conditions, and simultaneously monitored by in-situ SPR. It has been revealed that the enzymatic reaction between GOx and PPy in the presence of glucose can lead to distinct changes in the SPR signal. From the experiments, a linear relationship was obtained in the range 1–100 μmol L^?1 between glucose concentration and the rate of redox transformation of PPy. The detection limit was 0.5 μmol L^?1 (S/N?=?3) and recoveries were 95.2–102.7%.     

电化学合成吡咯与己内酰胺导电共聚物

采用恒电位方法实现了吡咯与己内酰胺在导电玻璃电极上的直接电化学共聚,聚合反应在含有0.1mol/L吡咯和1.5 mol/L己内酰胺的硝基甲烷电解质溶液中进行,外加电位控制在1.2 V以上.聚合产物中聚吡咯与聚己内酰胺链段的组成可通过调节合成电位加以控制.共聚物的形貌、结构与性质采用扫描电子显微镜、热重分析、红外光谱等手...     

纳米级微带金电极上葡萄糖氧化酶的固定.性质及应用

实现了葡萄糖氧化酶以及葡萄糖氧化酶和电子传递媒体Fe(CN)^3^-~6同时在纳米级微带电极上的固定,用红外光谱和循环伏安对GOD/PPy微电极进行了表征, 研究了微带金电极上聚吡咯恒电位形成过程的动力学及葡萄糖氧化酶对其动力学过程的影响,探讨了微酶电极GOD/Fe(CN)^3^-~6/PPy对葡萄糖氧化的催化作用, 考察了PPy膜厚度和溶液中氧的存在对GOD/Fe(CN)^3^-~6/PPy微电极测定葡萄糖的影响.     

Continuous Immunoassay for Sulfamethazine by Surface Plasmon Resonance-Based Biosensor

Regeneration of the sensor chip surface is difficult in many surface plasmon resonance (SPR) biosensor assays. Improper regeneration will reduce life span of the sensor chip and decrease the quality of the data. Considering the advantages of reducing the regeneration frequency, a theoretically feasible continuous SPR biosensor immunoassay for sulfamethazine (SMT) was developed. In the continuous inhibitive immunoassay, the sensor chip surface is regenerated only once after a definite number of tests instead of every test. The SMT-bovine serum albumin (BSA) conjugate was covalently immobilized to a carboxymethyldextran modified gold film. The immobilization conditions of the antigen were studied and the working dilution of the antibody was optimized. The antibody was mixed with SMT of different concentrations prepared with PBS buffer to construct the calibration curve. The limit of detection was 0.5 ng mL^?1. The continuous SPR biosensor assay was proved to be simpler and more practical than a normal one.     

Gold Nanoparticle‐Based Redox Signal Enhancement for Sensitive Detection of Human Chorionic Gonadotropin Hormone

A sensitive immunosensor for the detection of pregnancy marker, human chorionic gonadotropin hormone (hCG), was developed using the direct electrical detection of Au nanoparticles. We utilized disposable screen‐printed carbon strips (SPCSs) for the development of our immunosensor, which provided cost‐effective tests with the required antigen sample volume as small as 2 μL. After the recognition reaction between the surface‐immobilized primary antibody and hCG, the captured antigen was sandwiched with a secondary antibody that was labeled with Au nanoparticles. Au nanoparticles were exposed to a preoxidation process at 1.2 V for 40 s, which was subsequently followed with a reduction scan on the same surface using differential pulse voltammetry (DPV). We could observe Au nanoparticle‐labeled antigen‐antibody complexes immobilized on the surface of SPCS using scanning electron microscopy (SEM). Additionally, the number of Au nanoparticles on the immunosensor was determined using SEM images, and showed a linear relationship with the current intensity obtained from the DPV measurements with a detection limit of 36 pg/mL hCG (612 fM, 3.6×10^?4 IU/mL). Our immunosensor system, a combination of the screen‐printing technology with Au nanoparticles provides a promising biosensor for various applications in life sciences.     

Fabrication of DNA-protein conjugate layer on gold-substrate and its application to immunosensor

The fabrication of antibody thin film using both protein G and oligonucleotide was carried out by self-assembly (SA) technique for immunosensor. A mixture of 11-mercaptoundecanoic acid (MUA) and oligonucleotide with thiol (SH) end group was self-assembled of gold (Au) surface for two-dimensional (2D) configuration. Protein G was chemically adsorbed on the 11-MUA surface, and then the antibody was immobilized on the protein G region. On the immobilized single-stranded DNA, the complementary DNA–antibody conjugate was hybridized for the oriented immobilization of antibody. The formation of self-assembled 11-MUA/oligonucleotide layer, protein G immobilization, antibody layer, and antigen binding was investigated using surface plasmon resonance (SPR). The topographies of the fabricated surfaces were observed by atomic force microscopy (AFM). When compared with the amount of antigen binding on the antibody thin film fabricated by protein G only, the proposed biosurface fabricated with both protein G and oligonucleotide showed better binding capacity, which implicates the improvement of the detection limit.     

Capacitance Polypyrrole‐based Impedimetric Immunosensor for Interleukin‐10 Cytokine Detection

In the present study, we developed a novel label‐free capacitance impedimetric immunosensor based on the immobilization of the human monoclonal antibody anti‐interleukin‐10 (anti‐IL‐10 mAb) onto polypyrrole (PPy)‐modified silicon nitride (Si₃N₄) substrates. The immunosensor was used for the detection of the recombinant interleukin‐10 antigen (rh IL‐10) that may be secreted in patients at the early stage of inflammation. The immunosensor was created by chemical deposition of PPy conducting layer on pyrrole?silane (SPy)‐treated Si/SiO₂/Si₃N₄ substrates (Si/SiO₂/Si₃N₄?SPy), followed by anti‐IL‐10 mAb immobilization through carboxyl‐functionalized diazonium (CMA) protocol and carbodiimide chemistry. The surface characterization and the biofunctionalization steps were characterized by SEM, FTIR and cyclic voltammetry (CV) while the detection process was carried out by using electrochemical impedance spectroscopy (EIS) analyses. The created immunosensor showed two linear fittings (R²=0.999) for the detection of rh IL‐10 within the concentration range from 1–50 pg/mL. It exhibited high sensitivity (0.1128 (pg/mL)^?1) with a very low limit of detection (LOD)=0.347 pg/mL, more particularly, at the low concentration range (1–10 pg/mL). Thus, this developed polypyrrole‐based immunosensor represents a promising strategy for creation of miniaturized label‐free, fast and highly sensitive biosensors for diagnosis of inflammation biomarkers at very low concentrations with reduced cost.     

Preparation and application of triangular silver nanoplates/chitosan composite in surface plasmon resonance biosensing

This paper reports the utilization of triangular silver nanoplates (TSNPs) to enhance the sensitivity of surface plasmon resonance (SPR) biosensor. TSNPs modified with 3-mercaptopropinic acid (MPA) were simply mixed with chitosan and glutaraldehyde to form TSNPs/chitosan composite. The composite was deposited on Au film as immobilization substrate for SPR biosensor. The novel structures of TSNPs are preserved against etching by MPA and chitosan polymer. Moreover, chitosan cross-linked by glutaraldehyde enables antibody to be immobilized on fabricated substrate directly via Schiff alkali reaction. In the optimized conditions, the resulting biosensor based on TSNPs/chitosan composite shows a satisfactory response to bovine IgG in the concentration range of 0.075–40.00 μg mL⁻¹. While the biosensor based on chitosan without TSNPs shows a response in the concentration range of 0.6–40 μg mL⁻¹ and the biosensor based on Au film shows a response in the concentration range of 2.5–40 μg mL⁻¹. The experiment results show that the sensitivity of SPR biosensor based on TSNPs/chitosan composite was significantly enhanced and the immobilization procedure of antibody was simplified.     

Binding kinetics of human cellular prion detection by DNA aptamers immobilized on a conducting polypyrrole

We developed a biosensor based on the surface plasmon resonance (SPR) method for the study of the binding kinetics and detection of human cellular prions (PrP^C) using DNA aptamers as bioreceptors. The biosensor was formed by immobilization of various biotinylated DNA aptamers on a surface of conducting polypyrrole modified by streptavidin. We demonstrated that PrP^C interaction with DNA aptamers could be followed by measuring the variation of the resonance angle. This was studied using DNA aptamers of various configurations, including conventional single-stranded aptamers that contained a rigid double-stranded supporting part and aptamer dimers containing two binding sites. The kinetic constants determined by the SPR method suggest strong interaction of PrP^C with various DNA aptamers depending on their configuration. SPR aptasensors have a high selectivity to PrP^C and were regenerable by a brief wash in 0.1 M NaOH. The best limit of detection (4 nM) has been achieved with this biosensor based on DNA aptamers with one binding site but containing a double-stranded supporting part.

Electrochemical Evaluation of the Interaction between Avidin and Biotin at Biotinylated Polypyrrole Electrode Using a Redox Marker

The interaction between avidin and biotin was evaluated electrochemically by monitoring the change in the electrode response of redox markers. Biotin was immobilized on the electrode surface by means of the electrochemical polymerization of biotinylated pyrrole and pyrrole. When avidin was introduced onto the biotinylated polypyrrole electrode surface, the large change in the electrode response of the redox marker was detected. The fact that the change in the electrode response of a marker ion could be attributed to the electrostatic interaction between avidin on the electrode surface and the redox marker ion present in a solution was verified by replacing avidin with NutrAvidin. At a pH lower than the isoelectric point of avidin, the electrode response of ferrocyanide as an anionic marker ion increased linearly within the range of 5.0×10^?9 ?3.0×10^?8 M avidin. The relative standard deviation at 1.5×10^?8 M avidin was about 5.4% (n=5). The detection of biotin was also performed using a competitive reaction between biotin in solution and biotin that had been immobilized on the electrode surface in the form of the biotinylated polypyrrole.     

Amperometric Immunosensor for Prostate Specific Antigen Based on Gold Nanoparticles/Ionic Liquid/Chitosan Hybrid Film

The hybrid film of gold nanoparticles/ionic liquid-chitosan (GNP/IL-Ch) was first prepared by a simple one-step synthesis and used as an efficient immobilization matrix to fabricate an immunosensor. The GNP/IL-Ch film not only prevents the leakage of the IL units, but also produces a well-defined voltammetric signal due to the synergistic effects of the IL units and GNPs. By immobilizing an alkaline phosphatase (ALP)-labeled antibody in GNP/IL-Ch film, a sensitive amperometric immunosensor has been developed for prostate specific antigen (PSA). Under the optimized conditions, the immunosensor exhibits a linear range from 1.0 to 80 ng mL^?1 of PSA.     

核壳型Fe_3O_4/Au粒子的制备及其在表面等离子体子共振传感器中的应用

合成了核壳型Fe3O4/Au复合粒子,并对其形貌、光学性质进行了表征.通过外加磁场将Fe3O4/Au复合粒子与兔抗人IgG的偶联体固定于表面等离子体子共振(SPR)传感器的金基底膜上,形成了Fe3O4/Au/抗IgG敏感膜.与传统的通过巯基丙酸连接蛋白的方式相比,磁场作用固定的Fe3O4/Au/抗IgG敏感膜制备简单,易洗脱,具有良好的再生性,且在一定程度上提高了传感器的灵敏度.并对人IgG进行了测定,结果表明,传感器对于浓度范围在1.25～20.00μg·mL-1的人IgG有良好的信号响应.     

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.     

One step electrochemically deposited nanocomposite film of chitosan-carbon nanotubes-gold nanoparticles for carcinoembryonic antigen immunosensor application

A simple and controllable one-step electrodeposition method for the preparation of a chitosan-carbon nanotubes-gold nanoparticles (CS-CNTs-GNPs) nanocomposite film was used to fabricate an immunosensor for detection of carcinoembryonic antigen (CEA). The porous three-dimensional CS-CNTs-GNPs nanocomposite film, which offered a large specific surface area for immobilization of antibodies, exhibited improved conductivity, high stability and good biocompatibility. The morphology of the formed nanocomposite film was investigated by scanning electron microscopy (SEM), and the electrochemical behaviors of the immunosensor were characterized by electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). Under the optimal conditions, the proposed immunosensor could detect CEA in two linear ranges from 0.1 to 2.0 ng mL⁻¹ and from 2.0 to 200.0 ng mL⁻¹, with a detection limit of 0.04 ng mL⁻¹. The immunosensor based on CS-CNTs-GNPs nanocomposite film as the antibody immobilization matrix could exhibit good sensitivity, stability, and reproducibility for the determination of CEA.     

Highly conducting gold nanoparticles-graphene nanohybrid films for ultrasensitive detection of carcinoembryonic antigen

A new label-free amperometric immunosensor was developed for detection of carcinoembryonic antigen (CEA) based on chitosan-ferrocene (CS-Fc) and nano-TiO₂ (CS-Fc + TiO₂) complex film and gold nanoparticles-graphene (Au-Gra) nanohybrid. CS-Fc + TiO₂ composite membrane was first modified on a bare glass carbon electrode. Then Au-Gra nanohybrid was formed on the CS-Fc + TiO₂ membrane by self-assembly strategy. Next, further immobilization of anti-CEA was constructed according to the strong interaction between Au-Gra and the amido groups of anti-CEA. Since Au-Gra nanohybrid films provided a congenial microenvironment for the immobilization of biomolecules, the surface coverage of antibody protein could be enhanced and the sensitivity of the immunosensor has been improved. The good electronic conductive characteristic might be attributed to the synergistic effect of graphene nanosheets and Au NPs. The modified process was characterized by scanning electron microscope (SEM) and cyclic voltammetry (CV). Under optimized conditions, the resulting biosensor displayed good amperometric response to CEA with linear range from 0.01 to 80 ng/mL and a detection limit of 3.4 pg/mL (signal/noise = 3). The results demonstrated that the immunosensor has advantages of high conduction, sensitivity, and long life time. This assay approach showed a great potential in clinical applications and detection of low level proteins.     

Enhanced surface plasmon resonance immunoassay for human complement factor 4

Using an enhanced surface plasmon resonance (SPR) immunosensor, we have determined the concentration of human complement factor 4 (C4). Antibody protein was concentrated into a carboxymethyldextran-modified gold surface by electrostatic attraction force and a simultaneous covalent immobilization of antibody based on amine coupling reaction took place. The sandwich method was applied to enhance the response signal and the specificity of antigen binding assay. The antibody immobilized surface had good response to C4 in the range of 0.02-20 μg/ml by this enhanced immunoassay. The regeneration effect by pH 2 glycine-HCl buffer was also investigated. The same antibody immobilized surface could be used more than 80 cycles of C4 binding and regeneration. In addition, the ability to determinate C4 directly from serum sample without any purification was investigated. The sensitivity, specificity and reproducibility of the enhanced immunoassay are satisfactory. The results clearly demonstrate the advantages of the enhanced SPR technique for C4 immunoassay.     

基于铁氰化钾电子媒介体及醛基吡啶盐的电化学免疫传感器

采用聚二烯丙基二甲基氯化铵(PDDA)将铁氰化钾电子媒介体固定在电极表面,构建免标记的电化学免疫传感器. 醛基吡啶盐不仅作为基底物质直接固定抗体,还可以很好地增强电极表面的导电性能. 将构建的传感器用于肿瘤标志物甲胎蛋白的检测. 其线性范围为0.01-20 ng·mL^-1,检测下限为0.004 ng·mL^-1（3 S/N）. 此传感器的构建简单方便、无标记、特异性好,为甲胎蛋白及其他肿瘤标志物提供了新的检测方法.     

Electrochemical copolymerization of pyrrole and 2,2-bithiophene and semiconducting characterization of the resulting copolymer films by electrochemical impedance spectroscopy and photoelectrochemistry

The electrochemical copolymerization of pyrrole and bithiophene was studied at a polymerization potential of 1.1 V for various monomer ratios. The cyclic voltammograms showed that the electrochemical properties of the resulting copolymer films changed gradually from those of polypyrrole to polybithiophene with an increase in concentration of bithiophene in the initial electrolyte. The evidence for copolymer formation is based on the analytical results of electrospray ionization mass spectroscopy, thermoanalysis, and Raman spectroscopy. The results showed that cooligomers and homooligomers were found in the electrolyte after copolymerization. The difference between the morphology of a copolymer of pyrrole and bithiophene and a polymer mixture of polypyrrole and polythiophene was demonstrated by scanning electron microscopy. Electrochemical impedance and photocurrent measurements were carried out in order to achieve information on the semiconducting properties of the homopolymers and copolymers obtained. A model of a very thin layer of polypyrrole formed immediately on the electrode surface covered by a thicker copolymer film was developed to explain the results.Dedicated to Zbigniew Galus on the occasion of his 70^th birthday.