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.     

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.     

生物素-亲和素系统在压电免疫传感器中质量放大作用研究

生物素-亲和素系统(BiotinAvidinSystem,BAS)是70年代后期迅速发展起来的一种新型生物放大系统,具有高灵敏度、高特异性以及简便、经济、快速等优点,在现代生物免疫学领域中已得到广泛的应用^[1].     

Use of 3-(4-hydroxyphenyl)propionic acid as electron donating compound in a potentiometric aflatoxin M1-immunosensor

We developed a potentiometric aflatoxin M₁-immunosensor which utilizes 3-(4-hydroxyphenyl)propionic acid (p-HPPA) as electron donating compound for horseradish peroxidase (HRP; EC 1.11.1.7). The assay system consists of a polypyrrole-surface-working electrode coated with a polyclonal anti-M₁ antibody (pAb-AFM₁), a Ag/AgCl reference electrode and a HRP-aflatoxin B₁ conjugate (HRP-AFB₁ conjugate).To optimize the potentiometric measuring system p-HPPA as well as related compounds serving as electron donating compounds were compared. Also the influence of different buffer systems, varying pH and substrate concentrations on signal intensity was investigated. Our results suggest that reaction conditions that favor the formation of Pummerer's type ketones lead to an increase in signal intensity rather than formation of fluorescent dye. Comparison with commercial ready-to-use HRP electron donating compounds such as 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), o-phenylenediamine (OPD) or 3,3′,5,5′-tetramethylbenzidine (TMB) showed that only 34%, 77% and 49% of the signal intensity of p-HPPA were reached, respectively.The optimized assay had a detection limit of 40 pg mL⁻¹ and allowed detection of 500 pg mL⁻¹ (FDA action limit) aflatoxin M₁ (AFM₁) in pasteurized milk and UHT-milk containing 0.3-3.8% fat within 10 min without any sample treatment. The working range was between 250 and 2000 pg mL⁻¹ AFM₁.     

基于复合纳米微粒修饰电极的氯霉素快速检测用磁场可控一次性安培免疫传感器研究

在丝网印刷碳电极(SPCE)表面涂敷一层碳纳米管(CNT)/二茂铁(Fc)/Nafion膜,进而通过外磁场作用将包被了氯霉素-牛血清蛋白(CAP-BSA)的纳米金磁微粒[Fe3O4(核)/Au(壳),简称GMP]吸附到其表面,构建了可用于CAP检测的磁场可控一次性安培免疫传感器.采用扫描电镜(SEM)、X射线荧光光谱(XRFS)表征了免疫传感器的制备过程,采用循环伏安法(CV)和示差脉冲伏安法(DPV)研究了免疫传感器的电化学性质,结合竞争性免疫分析法测定了CAP浓度.免疫传感器在含有100ng·mL-1anti-CAP的25μLpH=6.0磷酸盐缓冲溶液(PBS)中加入不同浓度的CAP,并在25℃下温育6min,DPV还原峰电流上升百分比(CI%)与CAP浓度在0.2～80.0ng·mL―1范围内呈线性关系,检测限为0.11ng·mL―1.用于牛奶样品中CAP检测并和标准HPLC方法对照,结果一致,回收率在88%～104%之间.该免疫传感器灵敏快速、外磁场可控、样品用量小、可抛弃,有望用于食品中痕量CAP快速检测.     

多层自组装硫脲和纳米金电流型萘免疫传感器的研究

建立了基于硫脲和纳米金层层自组装技术的无标记、高灵敏电流型免疫传感器,用于萘的检测。利用循环伏安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。实验表明,此免疫传感器在含不同浓度萘抗原的PBS溶液(pH7.4)中37℃下孵育30 min后,在pH7.4的测试底液中测定,响应电流与萘浓度在0.5～100μg/L范围内有良好的线性关系,r=0.9986,检出限为0.08μg/L。此传感器制备简单,灵敏度高,稳定性好,可以重复使用。应用于实际水样中萘的测定,回收率为94.3%～107.0%。     

A Photoelectrochemical Immunosensor for Prostate Specific Antigen Detection Based on Graphdiyne Oxide Conjugated with Horseradish Peroxidase

Graphdiyne (GDY) was a novel flat material with sp and sp² hybridized carbon atoms. It exhibited good biocompatibility. The application of GDY in PEC immunosensor was very limited. Thus, a novel photoelectrochemical sensor for the sensitive detection of prostate specific antigen (PSA) was proposed by using GDY oxide (GDYO) conjugated with horseradish peroxidase (HRP) and secondary antibody for photocurrent signal inhibition. GDYO was prepared by oxidation of honeycomb-like nanotubes composed of numerous GDY nanosheets. It showed high loading capacity for HRP and the catalytic activity of HRP could be remained. With reduced graphene oxide-CdS (rGO-CdS) as photoelectrochemical sensing platform, a sandwich-type photoelectrochemical (PEC) immunosensor was thus fabricated. The immunosensor presented a wide linear concentration range of 10 fg mL⁻¹–20.0 ng mL⁻¹ with a detection limit (LOD) of 3.5 fg mL⁻¹. Moreover, the PEC immunosensor displayed ideal reproducibility, stability, and selectivity, which was a promising platform for the detection of other important tumor targets.     

Two immunoassay formats for fully automated CRP detection in human serum

Immunoassays are a proven approach towards fast, sensitive, cost-effective and easy-to-use analytical systems which are able to measure a variety of interesting analytes, especially in medical diagnostics. Herein, we report two assay formats, binding inhibition and sandwich assay format, for detection of C-reactive protein (CRP) in human serum. Both assays were characterised and compared with respect to their suitability and adaption into a complete sensor system. An automated, optical biosensor system, based on evanescent field technology, was used to carry out a full threefold calibration in each case. Owing to the resulting working ranges, 0.044–2.9 mg L⁻¹ and 0.13–22.9 mg L⁻¹, respectively, the assays qualify for use in detecting high-sensitivity CRP (C-reactive protein).     

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。     

非标记型河虾免疫传感器的制备及应用

四乙氧基硅在HCl催化下水解形成硅溶胶。将硅溶胶与河虾抗体混合均匀后,涂于玻碳电极表面制备得非标记型河虾抗体免疫传感器。采用Fe(CN)63-/4-的磷酸盐缓冲溶液(pH 6.5)为测试底液,研究此传感器在免疫反应中的循环伏安和交流阻抗特性。结果显示免疫反应后传感器的循环伏安图上未出现新的氧化-还原峰,表明该免疫反应属于非氧化还原过程。采用交流阻抗监测电极表面,发现传感器阻抗值随虾过敏原浓度的增加而增大。对交流阻抗图进行分析,结果表明电极表面的免疫反应是一个受电子转移控制的过程。当虾过敏原浓度在0~10 ng/mL之间,电极表面电子转移阻抗的增加值与虾过敏原浓度呈线性关系,检出限为0.1 ng/mL(S/N=3)。该方法已成功应用于河虾制品中过敏原的测定。