Electrochemical Immunosensors for Food Analysis: A Review of Recent Developments

Electrochemical immunosensors have the potential to transform analytical procedures within the food industry by providing highly specific, rapid, and inexpensive determination of pathogens. In this paper, recent advances in this area are outlined. In particular, attention is paid to new methods that have been developed for the modification of working electrode surfaces. Many advances have been related to the use of novel nanomaterials such as carbon nanotubes, graphene, and metallic nanoparticles, often used in conjunction with each other or polymers. The use of these materials has generally provided superior sensor sensitivity. The application of immunosensors to the detection of a range of pathogens in real samples is then investigated to establish whether they provide solutions in practical applications.     

Multiplexed Ultrasensitive Determination of Adrenocorticotropin and Cortisol Hormones at a Dual Electrochemical Immunosensor

A novel dual electrochemical immunosensor for the multiplexed determination of adrenocorticotropin (ACTH) and cortisol is reported. Aminophenylboronic acid‐modified dual screen‐printed carbon electrodes were prepared on which the corresponding ACTH and cortisol antibodies were immobilized. Competitive immunoassays involved biotinylated ACTH and alkaline phosphatase labelled streptavidin, or alkaline phosphatase labelled cortisol. Differential pulse voltammetry upon 1‐naphtyl phosphate addition was employed to monitor the affinity reactions. The ranges of linearity were 5.0×10^?5?0.1 and 0.1?500 ng/mL for ACTH and cortisol. The usefulness of the dual immunosensor was demonstrated by analyzing certified human serum samples with good recoveries.     

Recent Advances of Carbon Nanotubes‐based Electrochemical Immunosensors for the Detection of Protein Cancer Biomarkers

An efficient electrochemical immunosensor can offer the potential for the detection of protein cancer biomarkers due to its high sensitivity, low cost and possible integration in compact analytical devices. In the last several years, researchers have developed various electrochemical immunoassay methods for the detection of protein cancer biomarkers. Significant progresses have been made in the study of electrochemical immunosensor that based on CNTs, especially in the fields of clinical screening and diagnosis of cancer field. This is because CNTs possess unique structural, mechanical and electronic properties that can decrease over‐potential and improve the sensitivity of electrochemical immunosensor. This paper reviews recent advances in the different modified strategies of constructing electrochemical immunosensor based on CNTs for detecting protein cancer biomarkers. CNTs or CNTs hybrid nanomaterials modified electrodes have been firstly introduced as the sensing platforms for the detection of protein cancer biomarkers. On the other hand, CNTs or functional CNTs used as labels in sandwich‐type electrochemical immunosensors have been systematically summarized. These novel strategies and the general principles could increase the sensitivity of the immunosensor, thereby overcoming the limitations of its application in the biosensing field.     

丙肝电化学免疫传感器在血清检测中的应用

丙型肝炎病毒(hepatitis C virus,HCV)是丙肝的致病因子,该病毒自70年代发现以来,就一直困扰着人类。丙肝已成为近年来最为严重的传染病之一。目前临床诊断丙肝的主要方法有血清学非特异性检测[1]和特异性检测(酶联免疫分析方法、重组免疫印迹、聚合酶链反应[2]等)。血清学非特     

基于纳米金固定半抗原的电化学免疫传感器灵敏检测克伦特罗

研制了一种基于纳米金固定半抗原的间接竞争电化学免疫传感器,可灵敏检测克伦特罗.在金电极表面组装1,6-己二硫醇单分子膜,通过Au-S共价作用连接纳米金颗粒,通过吸附作用固定克伦特罗牛血清白蛋白偶联物.样品中的待测组分与固定化的克伦特罗偶联物竞争结合单克隆抗体,碱性磷酸酯酶标记的二抗选择性地与电极表面捕获的一抗反应,进而催化底物1-萘酚磷酸酯水解生成1-萘酚,在电极表面氧化产生电信号.在优化的实验条件下,克伦特罗浓度在0.1～1000 μg/L范围内与电流强度线性相关,线性方程为I(A)-8.79× 10-7-2.66× 10-7logC (μg/L),相关系数0.9960,检出限达20 ng/L.同时测定了猪肉及猪肝样品中克伦特罗含量,相对标准偏差平均值为7.0％,加标回收率在89.1％～105.6％之间,与传统的间接竞争酶联免疫吸附法对照,结果无显著性差异.     

基于丝网印刷电极的甲胎蛋白电化学免疫传感器

报道了一种基于金纳米粒子(AuNPs)双重信号放大的高灵敏电化学免疫传感器,并应用于肝癌标志物甲胎蛋白(AFP)的检测。通过在丝网印刷电极(SPE)表面电沉积AuNPs提高电极的重现性,利用AuNPs的吸附作用固定AFP抗体,用于捕获样品中的待测AFP抗原,并进一步与固定了辣根过氧化物酶(HRP)标记检测抗体的纳米金免疫探针发生特异性结合,所形成的夹心免疫复合物可以催化底物得到响应电流。用扫描电镜(SEM)和微分脉冲伏安法(DPV)等技术研究电极组装过程以及电极的化学性质,讨论了影响免疫传感器性能的因素。在最优实验条件下,传感器的峰电流信号与AFP浓度在2.5~30ng/mL范围内呈良好的线性关系,检出限为0.16ng/mL。该传感器具有灵敏度高、成本低、仪器体积小的优点,具有较好的应用前景。     

人血清中雌三醇的磁性微粒子放射免疫分析法研究

在传统的放射免疫分析的基础上,以磁性微粒子为二抗分离剂和固相一抗为结合反应剂,建立了两种快速、超灵敏、简便的血清及其它样品中雌三醇含量的分析方法。雌三醇测定的曲线范围0.3~30μg/L;磁性微粒子二抗分离法批内和批间相对标准偏差分别为4.3%和8.7%;回收率为104%~114%;健全性为0.9989。磁性微粒子固相一抗法批内和批间相对标准偏差分别为4.4%和7.6%;回收率为96%~115%;健全性为0.9986。两种方法相比,磁性微粒子固相一抗法具有更为简便、快捷的优点。两种磁性微粒子的放射免疫分析不仅缩短了反应时间,其准确性和精密度也均满足临床及环境研究中的检验要求,且灵敏度较现有方法提高了一个数量级。     

Electrochemical immunosensor for rapid and sensitive determination of estradiol

This work describes the preparation of an electrochemical immunosensor for estradiol based on the surface modification of a screen printed carbon electrode with grafted p-aminobenzoic acid followed by covalent binding of streptavidin (Strept) and immobilization of biotinylated anti-estradiol (anti-estradiol-Biotin). The hormone determination was performed by applying a competitive immunoassay with peroxidase-labelled estradiol (HRP–estradiol) and measurement of the amperometric response at −200 mV using hydroquinone (HQ) as redox mediator. The calibration curve for estradiol exhibited a linear range between 1 and 250 pg mL⁻¹ (r = 0.990) and a detection limit of 0.77 pg mL⁻¹ was achieved. Cross-reactivity studies with other hormones related with estradiol at physiological concentration levels revealed the practical specificity of the developed method for estradiol. A good reproducibility, with RSD = 5.9% (n = 8) was also observed. The operating stability of a single bioelectrode modified with anti-estradiol-Biotin-Strept was nine days when it was stored at 8 °C under humid conditions between measurements. The developed immunosensor was applied to the analysis of certified serum and spiked urine samples with good results.     

Polyaniline Nanowires-Based Electrochemical Immunosensor for Label Free Detection of Japanese Encephalitis Virus

Polyaniline (PANI) conducting polymers have attracted increasing interest as a transducer material for biosensors applications. In this study, we demonstrate the use of PANI nanowires (NWs) as immobilization platforms in the configuration of an electrochemical immunosensor for label free detection of Japanese encephalitis virus. The PANI NWs were synthesized on the surface of an interdigitated platinum (Pt) microelectrode via electrochemical growth. The morphology and characteristics of the PANI NWs on the Pt microelectrode were verified by scanning electron microscopy and Fourier transform infrared spectroscopy. The anti- Japanese encephalitis virus polyclonal IgG antibody was then covalently immobilized on the PANI NWs-coated Pt microelectrode by using 1-ethyl-3-(3-dimethyaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimde (NHS). The detection of Japanese encephalitis virus antigens was analyzed by electrochemical impedance spectroscopy (EIS). The developed PANI NWs-based electrochemical immunosensor could detect the Japanese encephalitis virus with a detection limit below 10 ng/ml. The results from EIS analysis also indicate that when the PANI NWs were exposed to nonspecific molecules, a negligible response was found, and it did not impact to the specificity of the sensor in the virus detection. This work shows the potential use of PANI NWs in electrochemical immunosensors for label free detection of other pathogens and small biomolecules.     

Gold Nanoparticle‐labeled Electrochemical Immunoassay Using Open Circuit Potential for Human Chorionic Gonadotropin Detection

This study presents a new approach for an electrochemical immunoassay using gold nanoparticle (AuNP)‐labeled antibodies and pre‐oxidation and reduction processes, followed by open circuit potential (OCP) measurement. Detection of the pregnancy marker, human chorionic gonadotropin hormone (hCG), was used as a model. After preparation of a sandwich‐type immunosystem, the pre‐oxidation and reduction processes were applied, followed by OCP detection. The applied potential and time period were studied for the optimization of pre‐oxidation and reduction processes. We observed that the pre‐oxidation potential of 1.2 V for 60 s and reduction potential of −0.2 V for 30 s provided the highest OCP signal. The detection limit was 79 pg/mL using the optimal conditions. This system could be applied to a simplified and miniaturized diagnostic system for integration in compact analytical devices.     

基于AuNPs/PDDA-GO纳米复合物的电化学免疫传感器的构建及对SirT1蛋白的检测

基于AuNPs/PDDA-GO纳米复合物制备了一种新型电化学免疫传感器, 并将其用于SirT1的检测. 首先, 在电极表面修饰复合材料AuNPs/PDDA-GO, 然后将目标蛋白SirT1固定到修饰了AuNPs/PDDA-GO的电极表面, 再通过特异性免疫反应结合一抗(Ab₁)和辣根过氧化酶标记的二抗分子(HRP-Ab₂), 最后用示差脉冲伏安法检测电流信号, 实现了对SirT1蛋白水平的测定. 在优化的实验条件下, SirT1蛋白的浓度在0.1~100 ng/mL范围内与响应电流呈良好线性关系, 检出限为0.029 ng/mL.     

睾酮电化学检测的研究进展

睾酮作为人体内一种重要的类固醇激素,在调节和促进人体机能等方面发挥着重要作用。睾酮的检测因在临床诊断和体育运动等方面具有重要作用而备受关注。该文对目前睾酮的主要检测方法进行了概述,包括常规仪器分析、免疫学检测方法和电化学检测方法,并对电化学方法特别是电化学免疫传感器对睾酮的检测进行了综述和展望。     

A disposable immunosensor for detection of 17beta-estradiol in non-extracted bovine serum

This paper reports the assembly of a disposable immunosensor based on the direct competitive enzyme-linked immunosorbent assay (ELISA), for simple and fast measurement of 17β-estradiol (17β-E₂) in bovine serum, using screen-printed electrodes (SPEs) and a Palm-Sens portable electrochemical detector. The immunosensor strip was assembled immobilising, by passive adsorption, anti-rabbit IgG onto the surface of the working SPE electrode. After the interaction between anti-rabbit IgG and rabbit anti-17β-E₂ polyclonal antibodies (PAb), the competition was performed using 17β-estradiol-alkaline phosphatase conjugate (17β-E₂-AP) synthesised in our laboratory. The enzymatic substrate used for signal generation was 1-naphthylphosphate and its conversion to an electroactive product (1-naphthol) was measured using differential pulse voltammetry (DPV). To develop a prototype for field measurements, the entire competitive protocol has been optimised directly in a blank non-extracted bovine serum.According to the new EU criteria established by the Commission Decision 2002/657/EC for qualitative and quantitative screening methods, the detection capability (CCβ), was determined. The CCβ value resulted below the action limit (40 pg mL⁻¹) fixed for 17β-E_2.Spiked and real samples were analysed using the electrochemical immunostrips obtaining precision values (relative standard deviation, R.S.D.%) ranging from 8.6 to 17.0% and a recovery (R%) from 88.2 to 120.0%.Results obtained on real samples were confirmed by liquid chromatography coupled on-line with tandem mass spectrometry (LC-MS/MS) using an atmospheric pressure chemical ionisation (APCI) source and a heated nebulizer (HN) interface; this is the method currently used to confirm illegal hormone administration for regulatory purposes. The disposable immunosensor appears suitable as a screening tool for field analysis of bovine serum estradiol.     

一种新的压电免疫传感器中生物分子固定化方法的研究

生物分子固定化或传感界面设计技术是研制压电免疫传感器的关键之一。本文 结合自组装单分子膜（SAMs）和聚电解质静电吸附组装技术，提出了一种新的压电 免疫传感器中生物分子固定化方法，研制成一种检测补体C_3的压电免疫传感器。 先在石英晶振的金电极表面组装一层胱胺SAMs，再在膜上组装带相反电荷的聚苯磺 酸钠（PSS）单层膜，通过静电吸附作用固定抗体（抗原），实现对相应抗原（抗 体）的检测。利用扫描电镜技术，从形态上考察了晶振组装胱氨SAMs与PSS及固定 补体C_3抗体后的表面形貌。研究了抗体的固定化条件，探讨了传感器采用这种固 定化方法的响应与再生性能，并与戊二醛键合固定法进行比较。结果表明，这种固 定化方法不仅对蛋白质类生物分子的固定化具有普适性，而且对所固定的生物分子 的活性影响小，传感器的响应的频移值大，灵敏度高，选择性和再生性能均较好。     

Amperometric Magnetoimmunosensors for Direct Determination of D‐Dimer in Human Serum

Two different D‐dimer disposable amperometric immunosensing designs based on indirect competitive or sandwich formats and the use of carboxylic acid‐modified magnetic beads (COOH‐MBs) and screen‐printed carbon electrodes (SPCEs) have been developed and compared. In both approaches, the resulting modified MBs were magnetically captured on the surface of a SPCE which was used as the transducer for the electrochemical detection at ?0.20 V upon addition of H₂O₂, and hydroquinone (HQ). Both configurations exhibited linear ranges of clinical usefulness and detection limits quite below the clinical threshold (0.5 µg mL^?1 D‐dimer). The sandwich configuration has been successfully tested with serum samples.     

Electrochemical immunosensor based on nanoporpus gold loading thionine for carcinoembryonic antigen

Nanoporous gold (NPG) has recently received considerable attention in analytical electrochemistry because of its good conductivity and large specific surface area. A facile layer-by-layer assembly technique fabricated NPG was used to construct an electrochemical immunosensor for carcinoembryonic antigen (CEA). NPG was fabricated on glassy carbon (GC) electrode by alternatively assembling gold nanoparticles (AuNPs) and silver nanoparticles (AgNPs) using 1,4-benzenedimethanethiol as a cross-linker, and then AgNPs were dissolved with HNO₃. The thionine was absorbed into the NPG and then gold nanostructure was electrodeposited on the surface through the electrochemical reduction of gold chloride tetrahydrate (HAuCl₄). The anti-CEA was directly adsorbed on gold nanostructure fixed on the GC electrode. The linear range of the immunosensor was from 10 pg mL⁻¹ to 100 ng mL⁻¹ with a detection limit of 3 pg mL⁻¹ (S/N = 3). The proposed immunosensor has high sensitivity, wide linear range, low detection limit, and good selectivity. The present method could be widely applied to construct other immunosensors.     

基于新型聚钙羧酸修饰电极的甲胎蛋白电化学免疫传感器研究

通过电聚合制得新型聚钙羧酸修饰电极并用于构建检测甲胎蛋白（AFP）的高灵敏电化学免疫传感器. 采用扫描电镜（SEM）、电化学交流阻抗（EIS）观察、表征修饰电极和AFP单克隆抗体（Ab1）固定前后的差异. 固定Ab1的电极与一定浓度的AFP、辣根过氧化物酶联AFP单克隆抗体（HRP-Ab2）反应,形成夹心型免疫复合物. 辣根过氧化物酶（HRP）催化3,3',5,5'-四甲基联苯胺（TMB）底物产生电流信号,实现AFP浓度的测定. 本检测方法灵敏度高,重现性好.     

以溴甲酚紫为电化学探针测定血清白蛋白

常见的蛋白质定量分析方法、电化学行为及其分析应用已有报道．本文基于酸性条件下蛋白质可与溴甲酚紫(BP)结合生成一种超分子复合物,使溴甲酚紫还原峰电流降低,建立了一种测定蛋白质的新方法,并用于测定人血清样品中的白蛋白含量．     

Voltammetric Label‐free Immunosensors for the Diagnosis of Cystic Echinococcosis

Cystic echinococcosis (CE) or hydatid disease is a parasitic infection caused by Echinococcus granulosus. Early serodiagnosis and continuous monitoring of the disease is very important for medical treatment. Here, we report the detecting of both echinococcus antigen and antibody for the diagnosis of hydatid disease using square wave voltammetry (SWV)‐based immunosensors. The gold electrodes were functionalized using cysteamine/phenylene diisothiocyanate linkers and used for the immunosensors fabrication. The hydatid antigen and antibody immunosensors were constructed by the immobilization of either purified rabbit polyclonal antibody or recombinant antigen B (AgB), respectively on the functionalized gold electrodes surfaces. The detection in both cases was achieved by following the change in the SWV reduction peak current of the ferro/ferricyanide redox couple upon antibody or antigen binding. These immunosensors enabled the detection of echinococcus antigen and antibody within a concentration range of 1 pg.mL^?1 to 1 μg.mL^?1 with detection limits of 0.4 pg.mL^?1 and 0.3 pg.mL^?1, respectively. A preliminary application of the developed immunosensor was performed in spiked serum sample showing good recovery percentages ranging from 102 to 110 % for both hydatid antibody and antigen detection. This easy‐to‐use, sensitive, and low cost quantitative method holds great promise for the early diagnosis of hydatid disease and thus, better managements and treatment outcomes.