基于丝网印刷电极的阻抗型免疫传感器用于检测烯效唑

采用循环伏安法在丝网印刷碳电极表面电聚合聚苯胺/壳聚糖复合膜,利用静电吸附作用固定烯效唑抗体,制备了检测烯效唑的阻抗型免疫传感器.采用循环伏安法和电化学阻抗谱对电极修饰过程进行了表征,并考察了缓冲溶液pH值、温育温度及时间对免疫反应的影响.在优化的条件下研究了免疫传感器的性能.此免疫传感器具有灵敏度高、特异性强、重现性和稳定性好的特点,对烯效唑的线性检测范围为0.01 ～100 mg/L,相关系数为0.999,检出限为8μg/L(3σ).白菜样品中添加0.05,0.10和1.0 mg/kg烯效唑,平均回收率为98.5％ ～ 104.6％,相对标准偏差为3.3％ ～4.3％,检测结果与气相色谱法的检测结果相符.     

Fabrication of a Sensitive Impedance Biosensor of DNA Hybridization Based on Gold Nanoparticles Modified Gold Electrode

In this work, we report on the preparation of a simple, sensitive DNA impedance sensor. Firstly gold nanoparticles were electrodeposited on the surface of a gold electrode, and then probe DNA was immobilized on the surface of gold nanoparticles through a 5′‐thiol‐linker. Electrochemical impedance spectroscopy (EIS) was used to investigate probe DNA immobilization and hybridization. Compared to the bare gold electrode, the gold nanoparticles modified electrode could improve the density of probe DNA attachment and the sensitivity of DNA sensor greatly. The difference of electron transfer resistance (ΔR_et) was linear with the logarithm of complementary oligonucleotides sequence concentrations in the range of 2.0×10^?12 to 9.0×10^?8 M, and the detection limit was 6.7×10^?13 M. In addition, the DNA sensor showed a fairly good reproducibility and stability during repeated regeneration and hybridization cycles.     

乳品中金黄色葡萄球菌肠毒素B免疫传感检测方法的研究

本文利用自制的金黄色葡萄球菌肠毒素B(SEB)抗体构建了一种L-半胱氨酸和纳米金的双层自组装免疫传感器,采用循环伏安法及交流阻抗法对传感器进行表征与测定,并对各项相关条件进行优化,最终建立了SEB检测的标准曲线,线性范围分别在2～10 ng/mL和10～100 ng/mL,相关系数分别为0.9939和0.9926,检出限(S/N=3)为0.667 ng/mL,乳品检测回收率在84.3%～93.2%之间。该传感器特异性良好,稳定性好,可再生使用,可应用于乳品中SEB的快速检测。     

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.     

An Electrochemical Immunosensor for C‐Reactive Protein Based on Multi‐Walled Carbon Nanotube‐Modified Electrodes

C‐reactive protein (CRP) is a nonspecific biomarker of inflammation and infection that can be used as a predictive risk marker of cardiovascular disease in asymptomatic individuals or as a prognostic marker of recurrent ischemia and death among patients with coronary heart disease or stroke. We developed a sensitive, disposable, and easy to operate sensor based on heterogeneous sandwich immunoassay for high sensitivity CRP (hs‐CRP) measurement. We used screen‐printed electrodes modified with multi‐walled carbon nanotubes and protein A to ensure the oriented immobilization of anti‐CRP antibodies. CRP was quantitatively measured down to a concentration of 0.5 ng mL^?1, enabling high dilution of the samples before measurement and consequently reducing interferences present in the serum. The sensor developed is suitable for point of care detection of hs‐CRP at clinically relevant concentrations for the diagnosis of both conventional and low‐grade inflammations.     

The Electrochemical Behavior of Gold Nanoparticle‐Tantalum(V) Phthalocyanine Composites: Applications Towards the Electroanalysis of Bisphenol A

The formation of gold nanoparticle (AuNP) composites with tantalum phthalocyanines (TaPc) complexes { 1a and 1b (Figure 1 )} is reported. The TaPc‐AuNPs conjugates were characterised by atomic force microscopy (AFM) and transmission electron microscopy. The AFM analyses show that conjugates of TaPc with AuNPs are more aggregated when compared to AuNPs alone. The conjugates and TaPc complexes were immobilized on a gold electrode by drop and dry method and these were characterized by electrochemical impedance spectroscopy. The charge transfer behaviour of AuNPs was enhanced in the presence of TaPc complexes. All the modified electrodes showed electrocatalytic oxidation of bisphenol A. The limits of detection for complexes 1a and 1 b were 4.78×10^?10 and 2.76×10^?10 mol L^?1, respectively.     

One‐Step Electrochemical Immunoassay for Carcinoembryonic Antigen in Human via Back‐Filling Immobilization of Gold Nanoparticles on DNA‐Modified Gold Electrodes

A new amperometric immunobiosensor for carcinoembryonic antigen (CEA) determination in human serum was developed via encapsulation of horseradish peroxidase‐labeled carcinoembryonic antibody (HRP‐anti‐CEA) in a gold nanoparticles/DNA composite architecture. The presences of gold nanoparticles provided a congenial microenvironment for the immobilized biomolecules and decreased the electron transfer impedance, leading to a direct electrochemical behavior of the immobilized HRP. The formation of the antibody–antigen complex by a simple one‐step immunoreaction between the immobilized HRP‐anti‐CEA and CEA in sample solution introduced a barrier of direct electrical communication between the immobilized HRP and the gold electrode surface. Under optimal conditions, the current change obtained from the labeled HRP relative to H₂O₂ system was proportional to the CEA concentration in two linear ranges from 0.5 to 15 ng/mL and 15 to 300 ng/mL with a detection limit of 0.1 ng/mL (at 3δ). The precision and reproducibility are acceptable with the intraassay CV of 6.3% and 4.7% at 8 and 60 ng/mL CEA, respectively. The storage stability of the proposed immunosensor is acceptable in a pH 7.0 PBS at 4 °C for 9 days. Moreover, the proposed immunosensors were used to analyze CEA in human serum specimens. Analytical results of clinical samples show the developed immunoassay has a promising alternative approach for detecting CEA in the clinical diagnosis.     

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.     

Lightweight ITO Electrodes Decorated with Gold Nanostructures for Electrochemical Applications

Gold nanostructures were fabricated on a transparent indium tin oxide (ITO) coated PET substrate by an electrodeposition technique from a potassium gold (III) chloride solution for two different types of applications. It was found that the optical transparency of lightweight ITO electrodes could be maintained by depositing isolated gold nanostructures while opening up the use of these electrodes for inner sphere electron reactions, such as hydroquinone oxidation, which are not possible at ITO electrodes. For practical applications the adhesion of gold to the ITO electrode was improved by modifying the ITO surface with 3‐mercaptopropyl‐trimethoxysilane (MPS). Compared to Au/ITO, the Au/MPS/ITO electrode showed vastly improved electrochemical activity toward various electron transfer reactions when subjected to mechanical stress. The biosensing properties of the Au/MPS/ITO electrode was also investigated by studying the detection of immobilized DNA on the Au/MPS/ITO electrode via electrochemical impedance spectroscopy (EIS).     

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.     

离子液体保护聚苯胺纳米金复合膜传感器的制备及其在检测乳制品中金黄色葡萄球菌毒素B的应用

采用原位合成法制得聚苯胺修饰纳米金的复合膜,并采用紫外、红外及透射电镜对其进行表征,以离子液体1,3-二丁基-咪唑六氟磷酸盐为保护复合膜的溶剂,成功制得离子液体保护的聚苯胺纳米金复合膜免疫传感器,并应用于乳品中金黄色葡萄球菌肠毒素B(SEB)的检测。在Fe(CN)63-/4-溶液中,采用循环伏安法(CV)及交流阻抗法(EIS)对传感器进行表征与测定,建立了SEB检测标准曲线,Y=933.46x+1399.8,线性范围为0.1～8.0μg/L,相关系数R2=0.9932,检出限明显降低(0.033μg/L,S/N=3)。结果表明,本传感器特异性良好,乳品检测回收率在88%～119%之间,稳定性好,可应用于乳品中的SEB快速检测。     

Electrochemical Detection of Avian Influenza Virus Genotype Using Amino‐ssDNA Probe Modified Gold Electrodes

A DNA biosensor for the detection of specific oligonucleotide sequences of Avian Influenza Virus type H5N1 has been proposed. The NH₂‐ssDNA probe was deposited onto a gold electrode surface to form an amide bond between the carboxyl group of thioacid and the amino group from ssDNA probe. The signals generated as a result of hybridization were registered in square wave voltammetry and electrochemical impedance spectroscopy in the presence of [Fe(CN)₆]^3?/4? as a redox marker. The genosensor is capable to determine 20‐mer and 180‐bp (PCR products) oligonucleotides complementary sequences with detection limit in the fM range. The genosensor displays good selectivity and sensitivity. The 20‐mer as well as 180‐bp oligonucleotides without a complementary sequence generate very low signal.     

Electrochemical Immunosensor for Ultrasensitive Detection of Human Chorionic Gonadotropin Based on Pd@SBA‐15

A simple and sensitive electrochemical immunosensor was conducted for the determination of human chorionic gonadotropin (hCG) with Pd@SBA‐15. Thionine (TH) was selected as an electron transfer mediator, and modified onto the electrode together with functionalized graphene nanomaterial (HSO₃^?GS) through electrostatic adsorption. Then Pd@SBA‐15 was immobilized onto the as‐prepared film for biomolecules anchoring. Pd@SBA‐15 composites not only retain the good biocompatibility of the SBA‐15, but also exhibit an excellent catalytic activity of Pd and low Pd leaching. hCG antibody was immobilized onto the composite film for the detection of hCG. hCG can be determined in the range of 0.01–16.00 ng/mL and the detection limit is 8.60 pg/mL. The method has been applied to the analysis of hCG in human serum samples with satisfactory results.     

Electrochemical Impedance Characterization of Nafion‐Coated Carbon Film Resistor Electrodes for Electroanalysis

Carbon film disk electrodes with Nafion coatings have been characterized by electrochemical impedance spectroscopy (EIS) with a view to a better understanding of their advantages and limitations in electroanalysis, particularly in anodic stripping voltammetry of metal ions. After initial examination by cyclic voltammetry, spectra were recorded over the full potential range in acetate buffer solution at the bare electrodes, electrodes electrochemically pretreated in acid solution, and Nafion‐coated pretreated electrodes in the presence and absence of dissolved oxygen. EIS equivalent circuit analysis clearly demonstrated the changes between these electrode assemblies. In order to simulate anodic stripping voltammetry conditions, spectra were also obtained in the presence of cadmium and lead ions in solution at Nafion‐coated electrodes, both after metal ion deposition and following re‐oxidation. Permanent changes to the structure of the Nafion film occurred, which has implications for use of these electrode assemblies in anodic stripping voltammetry at relatively high trace metal ion concentrations.     

基于四氧化三铁磁性纳米粒子的微囊藻毒素免疫传感器研究

采用化学共沉淀法制备四氧化三铁(Fe3O4)磁性纳米粒子(MNPs),依次用3-氨基丙基三乙氧基硅烷(APTS)、丁二酸酐(SAH)对Fe3O4 MNPs表面进行修饰,得到羧基功能化的核壳型磁性纳米粒子(Fe3O4@APTS·SAH MNPs),分别采用透射电镜(TEM)、磁滞回线、X射线光电子能谱(XPS)和傅里叶红外光谱(FTIR)对其进行了表征.将此纳米粒子修饰在自制的磁性玻碳电极(MGCE)表面,用1-(3-二氨基丙基)-3-乙基碳二亚胺盐酸盐(EDC)和N-羟基琥珀酰亚胺(NHS)活化纳米粒子表面的羧基,通过与氨基的共价交联,将抗微囊藻毒素-(亮氨酸-精氨酸)(MCLR)抗体(anti-MCLR)固定于该修饰电极上,用牛血清白蛋白(BSA)封闭非特异性吸附位点,构建了一种检测MCLR的电流型免疫传感器.采用直接竞争免疫反应模式,在标记物辣根过氧化物酶(HRP)的MCLR (MCLR-HRP)存在下,利用差分脉冲伏安法(DPV)测定溶液中的微囊藻毒素.在优化的实验条件下,免疫传感器对MCLR的线性测定范围为0.05 ～ 100 μg/L,检出限为0.01 μg/L(S/N=3).构建的免疫传感器呈现出良好的重现性、稳定性和特异性.将本传感器用于实际水样的测定,加标回收率为94.3％ ～ 99.5％.     

检测禽流感H5亚型病毒的阻抗型免疫研究

研制了一种可用于H5亚型禽流感病毒快速检测的阻抗型免疫传感器。通过蛋白A将H5N1表面抗原血凝素(HA)的单克隆抗体固定于金叉指阵列微电极表面,并与待测溶液中的目标抗原H5N1进行免疫反应。在[Fe(CN)6]3"/4"溶液中进行电化学阻抗谱扫描,表征电极的表面修饰及抗原捕获过程。当H5N1病毒浓度在21～26 HA unit/50μL范围时,其浓度的对数值与叉指阵列微电极的电子传递阻抗的变化值呈线性关系,相关系数为0.9885;检出限为20 HA unit/50μL,检测时间为1 h。此传感器特异性好,灵敏度高,可以重复使用,在病原微生物快速检测领域具有良好的应用前景。     

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.     

Electrochemical Immunoassay for Carbohydrate Antigen‐125 Based on Polythionine and Gold Hollow Microspheres Modified Glassy Carbon Electrodes

A new electrochemical immunosensor for the detection of carbohydrate antigen‐125 (CA125), a carcinoma antigen, was developed by immobilization CA125 antibody (anti‐CA125) on gold hollow microspheres and porous polythionine (PTH) modified glassy carbon electrodes (GCE). The gold hollow microspheres provided a biocompatible microenvironment for proteins, and greatly amplified the coverage of anti‐CA125 molecules on the electrode surface. The performance and factors influencing the immunosensor were investigated in detail. The detection is based on the current change before and after the antigen‐antibody interaction. Under optimal conditions, the amperometric changes were proportional to CA125 concentration ranging from 4.5 to 36.5 U/mL with a detection limit of 1.3 U/mL (at 3σ). The CA125 immunosensor exhibited good precision, high sensitivity, acceptable stability, accuracy and reproducibility. The as‐prepared immunosensors were used to analyze CA125 in human serum specimens. Analytical results suggest that the developed immunoassay has a promising alternative approach for detecting CA125 in the clinical diagnosis.     

Sandwich-type ELISA Impedimetric Immunosensor for Early Detection of Prostate-specific Antigen (PSA) in Human Serum

Early detection of cancer is vital for the successful treatment of the disease. Hence, a rapid and sensitive diagnosis is essential before the cancer is spread out to the other body organs. A gold millielectrode (GME) functionalized with a mixed (16-MHA+EG₃SH) self-assembled monolayer (SAM) was used to fabricate a point-of-care immunosensor for the detection of the prostate cancer biomarker: prostate-specific antigen (PSA) in human serum samples. An organic matrix (Amine-PEG₃-biotin/Avidin) was used to reduce non-specific protein adsorption on the electrode surface. A 16-MHA/EG₃SH/Amine-PEG₃-biotin/Avidin GME was exposed to solutions with different sandwich-type immunocomplex (^BtnAb-Ag_PSA-^HRPAb) concentrations and its response was measured using the electrochemical impedance spectroscopy (EIS) technique. This approach was compared with regard to a reference ELISA immunoassay. The obtained results showed that the immunosensor proposed in this work qualifies for its pre-clinical application in the quantification of PSA in serum samples of a representative Mexican population.     

Cathodized Gold Nanoparticle‐Modified Graphite Pencil Electrode for Non‐Enzymatic Sensitive Voltammetric Detection of Glucose

A highly sensitive enzymeless electrochemical glucose sensor has been developed based on the simply prepared cathodized gold nanoparticle‐modified graphite pencil electrode (AuNP‐GPE). Cyclic voltammetry (CV) experiments show that AuNP‐GPE is able to oxidize glucose partially at low potential (around −0.27) whereas the bare GPE cannot oxidize glucose in the entire tested potential windows. Besides, fructose and sucrose cannot be oxidized at potential lower than +0.1 V at AuNP‐GPE. As a result, the glucose oxidation peak at around −0.27 V is suitable enough for selective detection of glucose in the presence of fructose and sucrose. Cathodization of AuNP‐GPE under optimum condition (‐1.0 V for 30 s) in the same glucose solution before voltammetric measurement enhanced glucose oxidation peak current around −0.27 V to achieve an efficient electrochemical sensor for glucose with a detection limit of 12 μM and dynamic range between 0.05 to 5.0 mM with a good linearity (R²= 0.999). Almost no interference effect was observed for sensing of glucose in the presence of ascorbic acid, alanine, phenylalanine, fructose, sucrose, and NaCl.