Ultrasensitive potentiometric immunosensor based on SA and OCA techniques for immobilization of HBsAb with colloidal Au and polyvinyl butyral as matrixes

A novel potentiometric immunosensor for detection of hepatitis B surface antigen (HBsAg) has been developed by means of self-assembly (SA) and opposite-charged adsorption (OCA) techniques to immobilize hepatitis B surface antibody (HBsAb) on a platinum electrode. A cleaned platinum electrode was first pretreated in the presence of 10% HNO3 and 2.5% K2CrO4 solution and held at -1.5 V (vs SCE) for 1 min to make it negatively charged and then immersed in a mixing solution containing hepatitis B surface antibody, colloidal gold (Au), and polyvinyl butyral (PVB). Finally, HBsAb was successfully immobilized onto the surface of the negatively charged platinum electrode modified nanosized gold and PVB sol-gel matrixes. The modified procedure was characterized by electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). The immobilized hepatitis B surface antibody exhibited direct electrochemical behavior toward hepatitis B surface antigen (HBsAg). The performance and factors influencing the performance of the resulting immunosensor were studied in detail. More than 95.7% of the results of the human serum samples obtained by this method were in agreement with those obtained by enzyme-linked immunosorbent assays (ELISAs). The resulting immunosensor exhibited fast potentiometric response (<3 min) to HBsAg. The detection limit of the immunosensor was 2.3 ng.mL(-1), and the linear range was from 8 to 1280 ng.mL(-1). Moreover, the studied immunosensor exhibited high sensitivity, good reproducibility, and long-term stability (>6 months).     

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。     

Highly sensitive potentiometric immunosensor for hepatitis B surface antigen diagnosis

Immunosenors are of great interest because oftheir potential utility as specific, simple, label-free anddirect detection techniques and reductions in size, costand time of analysis compared with conventional im-munoassay techniques. The immunoassays with …     

Direct and Rapid Detection of Diphtherotoxin via Potentiometric Immunosensor Based on Nanoparticles Mixture and Polyvinyl Butyral as Matrixes

In this paper a novel potentiometric immunosensor for direct and rapid detection of diphtherotoxin (D‐Ag) has been developed by means of self‐assembly of monoclonal diphtheria antibody (D‐Ab) onto a platinum electrode based on nanoparticles mixture (containing gold nanoparticles and silica nanoparticles) and polyvinyl butyral (PVB) as matrixes. At first, D‐Ab was absorbed onto the surface of nanoparticles mixture, and then they were entrapped into polyvinyl butyral sol‐gel network on a platinum electrode. The detection is based on the change in the potentiometric response before and after the antigen‐antibody reaction in a phosphate buffer solution (pH 7.0). The immobilized D‐Ab exhibited direct potentiometric response toward D‐Ag. In comparison to the conventional applied methods, this strategy could allow antibodies immobilized with higher loading amount and better retained immunoactivity, as demonstrated by potentiometric response, cyclic voltammetry and electrochemical impedance spectroscopy of the immunosensor. The immunosensor with nanoparticles mixture exhibited much higher sensitivity, better reproducibility, and long‐term stability than that with gold nanoparticles or silica nanoparticles alone. The linear range was from 5.0×10^?3 to 1.2 μg?mL^?1 with a detection limit of 1.1×10^?3 μg?mL^?1. Up to 16 successive assay cycles with retentive sensitivity were achieved for the probes regenerated with in 0.2 mol?L^?1 glycine‐hydrochloric acid (Gly‐HCl) buffer solution and 0.25 mol?L^?1 NaCl. Moreover, the immunosensor with nanoparticles mixture was applied to evaluate a number of practical specimens with potentiometric results in acceptable agreement with those given by the ELISA method, implying a promising alternative approach for detecting diphtherotoxin in the clinical diagnosis.     

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.     

Disposable immunoassay for hepatitis B surface antigen based on a graphene paste electrode functionalized with gold nanoparticles and a Nafion-cysteine conjugate

We report on the modification of a graphene paste electrode with gold nanoparticles (AuNPs) and a Nafion-L-cysteine composite film, and how this electrode can serve as a platform for the construction of a novel electrochemical immunosensor for the detection of hepatitis B surface antigen (HBsAg). To obtain the immunosensor, an antibody against HBsAg was immobilized on the surface of the electrode, and this process was followed by cyclic voltammetry and electrochemical impedance spectroscopy. The peak currents of a hexacyanoferrate redox system decreased on formation of the antibody-antigen complex on the surface of the electrode. Then increased electrochemical response is thought to result from a combination of beneficial effects including the biocompatibility and large surface area of the AuNPs, the high conductivity of the graphene paste electrode, the synergistic effects of composite film, and the increased quantity of HBsAb adsorbed on the electrode surface. The differential pulse voltammetric responses of the hexacyanoferrate redox pair are proportional to the concentration of HBsAg in the range from 0.5–800?ng?mL^?1, and the detection limit is 0.1?ng?mL^?1 (at an S/N of 3). The immunosensor is sensitive and stable.

A Label‐Free Amperometric Immunosensor Based on Redox‐Active Ferrocene‐Branched Chitosan/Multiwalled Carbon Nanotubes Conductive Composite and Gold Nanoparticles

A novel immunosensor has been developed by self‐assembling Au NPs onto a ferrocene‐branched chitosan/multiwalled carbon nanotubes (CS‐Fc/MWCNTs) modified electrode for the sensitive determination of hepatitis B surface antigen (HBsAg). The formation of CS‐Fc effectively avoids the leakage of Fc and retains its electrochemical activity. Incorporation of MWCNTs and Au NPs into CS‐Fc further increases the electrochemical active Fc in the CS films and provides interactive sites for the immobilization of HBsAb. The morphologies and electrochemistry of the formed biofilm were investigated by using scanning electron microscopy and electrochemical techniques. The immunosensor exhibits a specific response to HBsAg in the range of 1.0–420 ng mL^?1. Excellent analytical performance, fabrication reproducibility and operational stability of the proposed immunosensor indicated its promising application in clinical diagnostics.     

Fabrication, characterization, and application of potentiometric immunosensor based on biocompatible and controllable three-dimensional porous chitosan membranes

A novel three-dimensional porous chitosan membrane material was prepared as a matrix to encapsulate hepatitis B surface antibody (HBsAb) for fabrication of immunosensors. The porous chitosan matrix was prepared by electrodepositing a designer nanocomposite solution of chitosan-encapsulated silica nanoparticle hybrid film on an ITO electrode, and then removing the silica nanoparticles with HF solution. Using HBsAb as a model, the potentiometric immunosensor was constructed by linking HBsAb molecules to the three-dimensional porous chitosan film using glutaraldehyde as a cross-linker. Scanning electron microscopy was used to investigate the surface morphology of the three-dimensional porous chitosan films. Cyclic voltammograms and electrochemical impedance spectroscopy were used to probe the interfacial properties of the immunosensor. Results showed that the fabricated immunosensor with three-dimensional porous structure possessed high surface area, good mechanical stability, and good hydrophilicity, which provided a biocompatible microenvironment for maintaining the bioactivity of the immobilized protein and increased the protein loading. Therefore, the present immunosensor exhibits a wide linear range from 6.85 to 708 ng mL(-1) with a low detection limit of 3.89 ng mL(-1) for the detection of hepatitis B surface antigen (HBsAg). This work implied that the biocompatible and controllable three-dimensional porous chitosan membrane possessed potential applications for biosensing.     

Gold nanoparticle-labeled detection antibodies for use in an enhanced electrochemical immunoassay of hepatitis B surface antigen in human serum

The application of gold nanoparticle-based electrochemical immunoassays have been extensively studied for the detection of hepatitis B surface antigen (HBsAg), but most often they exhibit low sensitivity. We describe the fabrication of a new electrochemical immunoassay for signal amplification of the antigen-antibody reaction combined with the nanogold-based bio-barcode technique. Hepatitis B surface antibody (HBsAb) was initially immobilized on a nanogold/thionine/DNA-modified gold electrode, and then a sandwich-type immunoassay format was employed for the detection of HBsAg using nanogold-codified horseradish peroxidase-HBsAb conjugates as secondary antibodies. Under optimal conditions, the current response of the sandwich-type immunocomplex relative to the H₂O₂ system was proportional to HBsAg concentration in the range from 0.5 to 650 ng·mL^?1 with a detection limit of 0.1 ng·mL^?1 (S/N?=?3). The precision, reproducibility and stability of the immunosensor were acceptable. Subsequently, the immunosensors were used to assay HBsAg in human serum specimens. Analytical results were in agreement with those obtained by the standard chemiluminescence enzyme-linked immunosorbent assay.     

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.     

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.     

Sandwich Immunoassay for Hepatitis C Virus Non-Structural 5A Protein Using a Glassy Carbon Electrode Modified with an Au-MoO3/Chitosan Nanocomposite

A novel electrochemical immunosensor was prepared for the detection of the hepatitis C virus non-structural 5A protein. A glassy carbon electrode was modified with an Au-MoO₃/Chitosan nanocomposite that warranted good conductivity and biocompatibility. Mesoporous silica with a large specific surface served as a nanocarrier for horseradish peroxidase and the polyclonal antibody as the reporter probe. The immunosensor was characterized by scanning electron microscopy, electrochemical impedance spectroscopy, and cyclic voltammetry. Following the sandwich-type immunoreaction, horseradish peroxidase was efficiently captured on the surface of the electrode to catalyze the decomposition of hydrogen peroxide. The analytical signal was obtained as an amperometric i-t curve (chronoamperometry). The assay reported here had a wide detection range (1 ng mL^?1 ?50 µg mL^?1) and detection limit as low as 1 ng mL^?1 of hepatitis C virus non-structural 5A protein. The electrochemical biosensor experiments showed excellent reproducibility, high selectivity, and outstanding stability for the determination of hepatitis C virus non-structural 5A protein, and it was successfully applied to the detection of the analyte in real serum samples.     

Fabrication of Immunosensor Based on Polyaniline,Fullerene‐C60 and Palladium Nanoparticles Nanocomposite: An Electrochemical Detection Tool for Prostate Cancer

Present work demonstrates the fabrication of new and facile sandwich‐type electrochemical immunosensor based on palladium nanoparticles (PdNPs), polyaniline (PANI) and fullerene‐C₆₀ nanocomposite film modified glassy carbon electrode (PdNP@PANI‐C₆₀/GCE) for ultrasensitive detection of Prostate‐specific antigen (PSA) biomarker. PdNP@PANI‐C₆₀ was electrochemically synthesized on GCE and used as an electroactive substrate. PdNP@PANI‐C₆₀ was characterized by scanning electron microscopy (SEM), energy‐dispersive X‐ray spectroscopy (EDS), cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). Primary antibody anti‐PSA (Ab1) was covalently immobilized on PdNP@PANI‐C₆₀/GCE using NHS/EDC linkers. In the presence of PSA antigen, horseradish peroxidase secondary antibody (HRP‐Ab2) was brought into the surface of the electrode, developing stable amplified signals of H₂O₂ reduction. Under the optimal conditions, a linear curve for determination of PSA at the proposed immunosensor was 1.6×10^?4 ng.mL^?1 to 38 ng.mL^?1 with a limit of detection (LOD) of 1.95×10^?5 ng.mL^?1. The proposed immunosensor was successfully validated in serum and urine samples towards PSA detection with satisfactory and acceptable results.     

溶胶-凝胶-HBsAb膜免疫传感器的研制与应用

采用溶胶-凝胶(Sol-gel)技术,成功地将乙肝表面抗体(HBsAb)包埋于Sol-gel中,再滴涂于铂盘电极表面,制成溶胶-凝胶-HBsAb膜非标记免疫传感器.根据抗原与抗体特异性结合形成的免疫复合物使敏感膜有效扩散截面积减小的特性,提出了利用铁氰化钾作为氧化还原探针间接检测乙肝表面抗原(HBsAg)的新方法.用循环伏安法(CV)对电极逐层修饰过程进行了表征,并探讨了对HBsAg定量检测的可行性及其响应机理.采用差示脉冲伏安法(DPV)检测人体血清中的HBsAg.线性范围5～320μg/L,线性相关系数r=0.997.该传感器响应迅速,灵敏度高,稳定性好.于4℃干态保存14d,其响应信号基本不变.将其用于108例临床血清检验,与酶联免疫吸附法(ELISA)的符合率为87.5％.     

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 novel amperometric immunosensor based on three-dimensional sol-gel network and nanoparticle self-assemble technique

A novel hepatitis B surface antigen (HBsAg) immunosensor has been developed by self-assembling gold nanoparticles to a thiol-containing sol-gel network. A cleaned gold electrode was first immersed in a hydrolyzed mercaptopropyltrimethoxysilane (MPS) sol-gel solution to assemble three-dimensional silica gel, and then gold nanoparticles were chemisorbed onto the thiol groups of the sol-gel network. Finally, hepatitis B surface antibody (HBsAb) was adsorbed onto the surface of the gold nanoparticles. Thus, an interfacial design of bare gold electrode (BGE)/MPS/Au/HBsAb was prepared to detect HBsAg in human serum based on the specific reaction of HBsAb and HBsAg. The electrochemistry of ferricyanide redox reaction was used as a marker to probe the interface and as a redox probe to determinate HBsAg. The main conditions of the assembly of MPS sol-gel, gold nanoparticles, the immobilization of HBsAb, and incubation time were investigated in detail. Compared with the glutaraldehyde binding approach, the antibodies immobilized by this method present larger amount and higher immunoactivity. The linearity of HBsAg in the range of 2-360 ng/mL with the correlation coefficient of 0.998 was obtained. This immunosensor system was evaluated on several clinical sample, the analytical results obtained by this method were in agreement with those detected by the enzyme-linked immunosorbent assay (ELISA) method, indicating a promising alternative tool for clinical diagnosis. Moreover, the studied immunosensor exhibited good reproducibility, long-term stability, high sensitivity and specificity.     

Heparin‐gold Nanoparticles and Liquid Crystal Applied in Label‐free Electrochemical Immunosensor for Prostate‐specific Antigen

A label‐free electrochemical immunosensor based on the liquid crystal (E)‐1‐decyl‐4‐[(4‐decyloxyphenyl)diazenyl]pyridinium bromide (Br−Py), together with heparin‐stabilized gold nanoparticles (AuNP‐Hep) and Nafion is proposed for the determination of prostate‐specific antigen (PSA). The Br−Py liquid crystal presented redox properties and good film‐forming abilities on the electrode surface, and thus it is a suitable alternative as a redox probe for a label‐free electrochemical immunosensor, which could simplify the analysis methodology. The stepwise construction of the immunosensor and the incubation process (immunocomplex formation) were characterized by voltammetry and electrochemical impedance spectroscopy. The proposed immunosensor could directly detect PSA concentrations in the incubation samples, based on the suppression of the Br−Py redox peak (‘base peak’) current. After optimization, the immunosensor exhibited a linear response to PSA concentrations in the range of 0.1 to 50 ng mL⁻¹, with a calculated detection limit of 0.08 ng mL⁻¹. The reproducibility (coefficient of variance less than 3.0 %), selectivity and accuracy of the methodology were adequate. The immunosensor was satisfactorily applied in the quantification of PSA in human blood plasma samples.     

Biometallization‐Based Electrochemical Magnetoimmunosensing Strategy for Avian Influenza A (H7N9) Virus Particle Detection

A highly sensitive electrochemical immunosensor for avian influenza A (H7N9) virus (H7N9 AIV) detection was proposed by using electrochemical magnetoimmunoassay coupled with biometallization and anodic stripping voltammetry. This strategy could accumulate the enzyme‐generated product on the surface of the magneto electrode by means of silver deposition, which amplified the detection signal about 80 times. The use of magnetic beads (MBs) and the magneto electrode could also amplify the detection signal. Furthermore, a bi‐electrode signal transduction system was introduced into this immunosensor, which is also beneficial to the immunoassay. A concentration as low as 0.011 ng mL^?1 of H7N9 AIV could be detected in about 1.5 h with good specificity. This study not only provides a simple and sensitive approach for virus detection but also offers an effective signal enhancement strategy for the development of highly sensitive MB‐based electrochemical immunoassays.     

An Immunosensor for Ultrasensitive Detection of 1‐Pyrenebutyric Acid with Enhanced Electrochemical Performance Based on a Graphene‐Ionic Liquid Doped Chitosan Film Modified Glassy Carbon Electrode

This paper describes a highly sensitive and label‐free electrochemical immunosensor for the detection of 1‐pyrenebutyric acid (PBA) which is based on a graphene (GS), chitosan (CS), and ionic liquid (IL) composite modified glassy carbon electrode (GS‐CS‐IL/GCE). The modification process was monitored by transmission electron microscopy (TEM) and cyclic voltammetry (CV). Due to the synergistic effects of GS, CS, and IL, the biosensor exhibits excellent selectivity to PBA. The current response of the proposed immunosensor decreases linearly at two concentration ranges from 0.01 to 5 and from 5 to 150 ng mL^?1 with a detection limit of 0.01 ng mL^?1.     

Amplified Electrochemical Immunosensor for Calmodulin Detection Based on Gold‐Silver‐Graphene Hybrid Nanomaterials and Enhanced Gold Nanorods Labels

Calmodulin (CaM) is an important intracellular calcium‐binding protein. It plays a critical role in a variety of biological and biochemical processes. In this paper, a new electrochemical immunosensing protocol for sensitive detection of CaM was developed by using gold‐silver‐graphene (AuAgGP) hybrid nanomaterials as protein immobilization matrices and gold nanorods (GNRs) as enhanced electrochemical labels. Electrode was first modified with thionine‐chitosan film to provide an immobilization support for gold‐silver‐graphene hybrid nanomaterials. The hybrid materials formed an effective matrix for binding of CaM with high density and improved the electrochemical responses as well. Gold nanorods were prepared for the fabrication of enhanced labels (HRP‐Ab₂‐GNRs), which provided a large capacity for HRP‐Ab₂ immobilization and a facile pathway for electron transfer. With two‐step immunoassay format, the HRP‐Ab₂‐GNRs labels were introduced onto the electrode surface, and produced electrochemical responses by catalytic reaction of HRP toward enzyme substrate of hydrogen peroxide (H₂O₂) in the presence of thionine. The proposed immunosensor showed an excellent analytical performance for the detection of CaM ranging from 50 pg mL^?1 to 200 ng mL^?1 with a detection limit of 18 pg mL^?1. The immunosensor has also been successfully applied to the CaM analysis in two cancer cells (HepG2 and MCF‐7) with high sensitivity, which has shown great potency for improving clinic diagnosis and treatment for cancer study.