Amperometric Immunosensor Based on Gold Nanoparticles/Fe3O4-FCNTs-CS Composite Film Functionalized Interface for Carbofuran Detection

In this paper, a novel amperometric immunosensor for the determination of carbofuran based on gold nanoparticles (GNPs), magnetic Fe₃O₄ nanoparticles-functionalized multiwalled carbon nanotubes-chitosan (Fe₃O₄-FCNTs-CS), and bovine serum albumin (BSA) composite film was proposed. First, GNPs were immobilized onto the glassy carbon electrode (GCE) surface, and then the magnetic Fe₃O₄ nanoparticles mixed with chitosan-functionalized multiwall carbon nanotubes (CS-FCNTs) homogeneous composite (CS-FCNTs-Fe₃O₄) was immobilized onto the GNPs layer by electrostatic interactions between amino groups of CS and GNPs. Because chitosan (CS) contains many amino groups, it can absorb more antibodies. FCNTs have high surface area, high electrical conductivity, and it can enhance the electron transfer rate; Magnetite (Fe₃O₄) nanoparticles can provide a favorable microenvironment for biomolecules immobilization due to their good biocompatibility, strong superparamagnetic property, and low toxicity; and GNPs possess high surface-to-volume reaction, stability, and high conductivity. Gold Nanoparticles/Fe₃O₄-FCNTs-CS composite film was constructed onto the GCE surface, which had significant synergistic effects toward immunoreaction signal amplification. The stepwise assembly process was characterized by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS), respectively. Under the optimal conditions, the current response was proportional to the concentration of carbofuran ranging from 1.0 ng/mL to 100.0 ng/mL and from 100.0 ng/mL to 200 µg/mL with the detection limit 0.032 ng/mL. The proposed immunosensor exhibited good accuracy, high sensitivity, and stability, and it can be used for detection of carbofuran pesticide.     

基于自组装电化学免疫传感器对污泥中PCB77的检测

构建了一种基于L-半胱氨酸、 壳聚糖、 戊二醛和纳米金层层自组装技术的新型无标记、 高灵敏电流型免疫传感器, 并用于检测3,3',4,4'-四氯联苯(PCB77). 利用示差脉冲法研究了修饰电极表面的电化学特性以及测试溶液的pH值、 孵育时间和温度对免疫传感器性能的影响. 实验结果表明, 该免疫传感器在含不同浓度PCB77的磷酸盐缓冲溶液(PBS, pH=7.4)中于35 ℃下孵育30 min后, 在含有5 mmol/L K₃Fe(CN)₆/K₄Fe(CN)₆(摩尔比1:1)和0.1 mol/L KCl的PBS溶液(pH=6.0)中测定, 响应电流与PCB77浓度在0.1～160 ng/mL范围内呈良好的线性关系, R=0.9964, 检出限为0.01 ng/mL. 该传感器制备简单、 灵敏度高、 稳定性好, 可以重复使用. 将其用于检测实际污泥样品中的PCB77, 回收率为95%～112%.     

Disposable Amperometric A-fetoprotein Immunosensor Based on the Biocompatible Silk Protein Membranes-Modified Indium Tin Oxide Electrodes

A simple and disposable electrochemical immunosensor for detection of 68 kDa alpha-fetoprotein (AFP) was fabricated based on films of silk fibroin protein membrane (SFPM)/Prussian blue (PB)/deposition of gold nanoparticles (DpAu). First, DpAu and PB were electrochemically deposited successively on the surface of indium tin oxide (ITO) electrode. Then, SFPM with excellent biocompatibility was modified on the surface of PB/DpAu/ITO. The SFPM could form a stable matrix on the electrode surface for the deposition of immunoactive agents. More importantly, the SFPM could prevent the possible leakage of electron mediator and enhance the stability of immunosensor. Scanning electron microscopy (SEM), electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) were used to characterize the assembly process of the modified electrode. The linear range of the proposed immunosensor extended from 1.0 to 200.0 ng/mL for detection of AFP with a detection limit of 0.6 ng/mL. Moreover, the CV test demonstrated the immunosensor exhibited acceptable reproducibility and stability. This composite membrane could be applied for the detection of different biomarkers, diagnosis, and monitoring of carcinoma.

[Supplementary materials are available for this article. Go to the publisher's online edition of Analytical Letters for the following free supplemental resources: additional figures.]     

Fabrication of an electrochemical immunosensor for α-fetoprotein based on a poly-L-lysine-single-walled carbon nanotubes/Prussian blue composite film interface

Single-walled carbon nanotubes functionalized with poly-L-lysine (PLL-SWCNTs) were successfully prepared and were used as a biocompatible platform to immobilize α-fetoprotein antibody (anti-AFP) which was labeled with horseradish peroxidase (HRP). Then, anti-AFP-HRP/PLL-SWCNT nanocomposites were coated onto a Prussian blue (PB) film-modified glassy carbon electrode surface. Glutaraldehyde was used to further stabilize the biosensing interface through a cross-linking step. All unspecific sites were blocked by bovine serum albumin to fabricate a novel electrochemical immunosensor for α-fetoprotein determination. The immunosensor was characterized by voltammetry and electrochemical impedance spectroscopy. Based on the catalytic current response of H₂O₂, the experimental conditions for α-fetoprotein determination were optimized. Under optimal conditions, the current response was linearly related to α-fetoprotein concentration in the range of 0.05～10.0 and 10.0～50.0 ng/mL with a detection limit of 0.011 ng/mL. The immunosensor was successfully used for the determination of α-fetoprotein in human blood plasma. The results were satisfied with that obtained with ELISA, demonstrating a good accuracy of the immunosensor.     

QCM‐Based Immunosensor for the Determination of Ochratoxin A

Abstract

A piezoelectric immunosensor based on a competitive format was developed for determination of ochratoxin A (OTA) concentration. Surface modifications via two self‐assembled monolayers (SAMs) were investigated respectively and a better result was obtained with the SAM of 16‐mercaptohexadecanoic acid (16‐MHDA). The quartz crystal microbalance (QCM)‐based immunosensor was fabricated by immobilizing anti‐OTA antibodies onto the surface of the 16‐MHDA‐modified electrode, and allowing competition between free OTA and that conjugated with BSA to occur. The assay exhibited a working range of 50–1000 ng/mL and a detection limit of 16.1 ng/mL. Studies of interference and matrix effects were performed to evaluate the feasibility of the developed immunosensor for the direct analysis of OTA in real samples. Recoveries were conducted at 50, 200, and 1000 ng/g and were determined to be in the range of 142%–76%. The OTA assay is specific. No cross‐reactivates were observed with citrinin.     

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。     

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.     

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。     

A Dendrimer Supported Electrochemical Immunosensor for the Detection of Alpha‐feto protein – a Cancer Biomarker

The electrochemical detection of alpha‐feto protein based on novel gold nanoparticles‐ poly(propylene imine) dendrimer platform is reported. The platform was prepared by co‐electrodeposition of gold nanoparticles and generation 3 poly (propylene imine) dendrimer on a glassy carbon electrode. Each modifying step was characterised by cyclic voltammetry and electrochemical impedance spectroscopy. The electrochemical measurements showed that the platform was stable, conducting and exhibited reversible electrochemistry. Results obtained from the electrochemical impedance spectroscopy interrogation in [Fe(CN)₆^3−/4−] redox probe showed a marked reduction in charge transfer resistance (R_ct) after each modification step. The immunosensor was prepared by immobilisation of a probe anti‐alpha feto protein (AFP) on the platform for 3 hrs at 35 °C followed by blocking the surface with bovine serum albumin to minimise non‐specific binding. The prepared immunosensor was used to detect AFP over a wide concentration range from 0.005 to 500 ng/mL and detection limits of 0.0022 and 0.00185 ng/mL were obtained for SWV and EIS measurements respectively. The immunosensor gave good stability over a period of fourteen days when stored at 4 °C.     

Sensitive detection of thyroid stimulating hormone by inkjet printed microchip with a double signal amplification strategy

In this paper, we reported a sensitive electrochemical immunosensor coupling protein A/G@magnetic beads and an ALP-based enzymatic-electrochemical reaction on the inkjet printing microchips for the determination of thyroid stimulating hormone.     

A novel immunosensor for carcinoembryonic antigen based on poly(diallyldimethylammonium chloride) protected prussian blue nanoparticles and double-layer nanometer-sized gold particles

A highly sensitive amperometric immunosensor has been developed for the detection of carcinoembryonic antigen (CEA). It is based on (a) Prussian Blue nanoparticles coated with poly(diallyldimethylammonium chloride) (P-PB) and (b) double-layer gold nanocrystals. The sensor was obtained by first electrodepositing porous gold nanocrystals on the glassy carbon electrode (GCE), and then by modifying the electrode with the coated P-PB. Subsequently, colloidal gold nanoparticles (nano-Au) were adsorbed onto the GCE by electrostatic interactions between the negatively charged nano-Au and the positively charged P-PB to immobilize CEA antibodies. Finally, bovine serum albumin was employed to block possible remaining active sites and to prevent the non-specific adsorption on the nano-Au. This immunosensor was characterized by cyclic voltammetry and scanning electron microscopy. The working range was adjusted to two concentration ranges, viz. from 0.5 to 10 ng.mL^?1, and from 10 to 120 ng.mL^?1 of CEA, with a detection limit of 0.2 ng.mL^?1 at three times the background noise.     

A novel α-fetoprotein-MIP immunosensor based on AuNPs/PTh modified glass carbon electrode

A new alpha-fetoprotein-MIP (AFP-MIP) immunosensor based on glass carbon electrode (GCE) modified with polythionine (PTh) and gold nanoparticles (AuNPs) was successfully prepared for the sensitive detection of AFP. The AFP-MIP immunosensor presented a facile preparation, low sample consumption, and good stability, and could become a new promising method for the detection of AFP.     

Chitosan/AuNPs Modified Graphene Electrochemical Sensor for Label‐Free Human Chorionic Gonadotropin Detection

A new immunosensor is presented for human chorionic gonadotropin (hCG), made by electrodepositing chitosan/gold‐nanoparticles over graphene screen‐printed electrode (SPE). The antibody was covalently bound to CS via its Fc‐terminal. The assembly was controlled by electrochemical Impedance Spectroscopy (EIS) and followed by Fourier Transformed Infrared (FTIR). The hCG‐immunosensor displayed linear response against the logarithm‐hCG concentration for 0.1–25 ng/mL with limit of detection of 0.016 ng/mL. High selectivity was observed in blank urine and successful detection of hCG was also achieved in spiked samples of real urine from pregnant woman. The immunosensor showed good detection capability, simplicity of fabrication, low‐cost, high sensitivity and selectivity.     

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.     

A novel impedance immunosensor based on O-phenylenediamine modified gold electrode to analyze abscisic acid

<正>An impedance immunosensor based on O-phenylenediamine modified gold electrode for the determination of phytohormone abscisic acid(ABA) was proposed.The operating pH,absorption time,absorption temperature and concentration of anti-ABA antibody were investigated to optimize the analytical performance.The calibration curve for the determination of ABA was obtained from this impedance immunosensor under optimal conditions.The results showed that the detection limit at about 1 ng/mL in the range of 10-5000 ng/mL.     

A novel electrochemical immunosensor based on colabeled silica nanoparticles for determination of total prostate specific antigen in human serum

A novel electrochemical immunosensor using functionalized silica nanoparticles (Si NPs) as protein tracer has been developed for the detection of prostate specific antigen (PSA) in human serum. The immunosensor was carried out based on a heterogeneous sandwich procedure. The PSA capture antibody was immobilized on the gold electrode via glutaraldehyde crosslink. After reaction with the antigen in human serum, Si NPs colabeled with detection antibody and alkaline phosphatase (ALP) was sandwiched to form the immunocomplex on the gold electrode. ALP carried by Si NPs convert nonelectroactive substrate into the reducing agent and the latter, in turn, reduce metal ions to form electroactive metallic product on the electrode. Linear sweep voltammetry (LSV) was used to quantify the amount of the deposited silver and give the analytical signal for PSA. The parameters including the concentration of the ALP used to functionalize the Si NPs and the enzyme catalytic reaction time have been studied in detail and optimized. Under the optimum conditions of immunoreaction and electrochemical detection, the electrochemical immunosensor was able to realize a reliable determination of PSA in the range of 1–35 ng/mL with a detection limit of 0.76 ng/mL. For six human serum samples, the results performed with the electrochemical immunosensor were in good agreement with those obtained by chemiluminescent microparticle immunoassay (CMIA), indicating that the electrochemical immunosensor could satisfy the need of practical sample detection.     

A sensitive amperometric immunosensor for alpha-fetoprotein based on carbon nanotube/DNA/Thi/nano-Au modified glassy carbon electrode

A novel amperometric immunosensor for the determination of alpha-fetoprotein (AFP) was constructed using films of multi-wall carbon nanotubes/DNA/thionine/gold nanoparticles (nano-Au). Firstly, multi-wall carbon nanotubes (MWCNT) dispersed in poly(diallydimethlammonium chloride) (PDDA) were immobilized on the nano-Au film which was electrochemically deposited on the surface of glassy carbon electrode. Then a negatively charged DNA film was absorbed on the positively charged PDDA. Subsequently, thionine was attached to the electrode via the electrostatic interaction between thionine and the DNA. Finally, the nano-Au was retained on the thionine film for immobilization of AFP antibody (anti-AFP). The modification process was characterized by cyclic voltammetry (CV) and scanning electron microscope (SEM). The factors possibly influenced the performance of the proposed immunosensors were studied in detail. Under optimal conditions, the proposed immunosensor exhibited good electrochemical behavior to AFP in a two concentration ranges: 0.01–10.0 and 10.0–200.0 ng/mL with a relatively low detection limit of 0.04 ng/mL at three times the background noise. Moreover, the selectivity, repeatability and stability of the proposed immunosensor were acceptable.     

Electrochemical Detection of β-Lactoglobulin Allergen Using Titanium Dioxide/Carbon Nanochips/Gold Nanocomposite-based Biosensor

A label-free electrochemical immunosensor was developed for the ultra-sensitive detection of β-lactoglobulin (β-LG). The novel nanocomplex of carbon nanochips, colloidal gold nanoparticles and titanium dioxide nanoparticles TiO₂/CNC/AuC were constructed on conducting polymer, chitosan, and were characterised using scanning electron microscopy (SEM) and energy-dispersive X-ray spectroscopy (EDX). This nanocomplex interface was studied using cyclic voltammetry (CV) and showed great improvement at the gold electrode surface with enhanced electrochemical performance, sensitivity and selectivity for β-lactoglobulin. Under optimal parameters, the square wave voltammetry (SWV) response curve was determined from 0.01 pg/mL to 500 pg/mL using [Fe(CN)₆]^3−/4−] redox probe. The calibration plot illustrates a linear relationship between log β-LG concentration and SWV current, with the limit of detection determined to be 0.01 pg/mL. This immunosensor displayed high sensitivity, selectivity, reproducibility and stability, and can be utilised for the detection of β-LG in real food samples.     

Detection of Multiple Herbicide Residues Using A Planar Array Evanescent Field Immunosensor

ABSTRACT

A planar array evanescent immunosensor, equipped with a charge-coupled device (CCD) as a detector was used to simultaneously detect pesticides, paraoxon, carbofuran and tetrachloroethylene. Wells approximately 2.5 mm in diameter were formed on glass slides using a photoactivated optical adhesive. Coating haptens were covalently attached to the bottoms of the circular wells to form a sensing surface. The detection of the hapten was completed through competitive immuno-reaction between the coasting haptens, and haptens in solution, with the corresponding antibody in solution. The identification and amount of antibody bound at each location on the slide were determined by quantitative image analysis. Concentrations as low as 0.5ng/mL of paraoxon, 0.Ing/mL of carbofuran, and 0.2ng/mL of tetrachloroethlene could be measured.     

A Reagentless Amperometric Immunosensor for Alpha‐Fetoprotein Based on Gold Nanoparticles/TiO2 Colloids/Prussian Blue Modified Platinum Electrode

A novel reagentless amperometric immunosensor for the determination of alpha‐fetoprotein (AFP) was prepared by immobilizing TiO₂ colloids on Prussian blue (PB) modified platinum electrode, which yielded a positively charged interface with strong adsorption to deposit gold nanoparticles for immobilization of alpha‐fetoprotein antibody (anti‐AFP). The factors influencing the performance of the proposed immunosensors were studied in detail. Under the optimized conditions, cyclic voltammograms determination of AFP showed a specific response in two concentration ranges from 3.0 to 30.0 ng/mL and from 30.0 to 300.0 ng/mL with a detection limit of 1.0 ng/mL at a signal‐to‐noise ratio of 3. The proposed immunosensor exhibited high selectivity, good reproducibility, long‐term stability (>2 months) and good repeatability.