基于纳米金修饰的两种无汞型重金属微传感器的对比研究

采用微加工技术制备了集成有工作电极和对电极的两种重金属微传感电极芯片,工作电极表面采用电沉积法修饰纳米金(Gold nanoparticles,GNPs),由半胱氨酸(L-cysteine,Cys)和天冬氨酸(L-aspartic acid,Asp)修饰制备Asp/Cys/GNPs/微传感电极芯片,并利用原位镀锡膜(Sn film)的方法,制成Sn/GNPs/微传感电极芯片。采用方波伏安法和方波溶出伏安法考察了两种微传感电极芯片对重金属离子Cu2+,Pb2+和Zn2+的响应特性。Asp/Cys/GNPs/微传感电极芯片可有效识别Cu2+和Pb2+,线性范围为5～2000μg/L,检出限为1μg/L;Sn/GNPs/微传感电极芯片可有效识别Cu2+,Pb2+和Zn2+,线性检测范围分别为5～500μg/L,5～500μg/L和10～500μg/L,检出限分别为2,3和5μg/L。相比而言,Asp/Cys/GNPs/微传感电极芯片具有较宽的检测范围,而Sn/AuNPs/微传感电极芯片具有较高的灵敏度,两种传感器绿色环保、制备简单、更新简便、易于集成,在水质在线监测方面具有应用前景。     

用于氨氮检测的薄层氨气微传感芯片及其系统研究

设计并制备了一种具有微池薄液层结构的氨气微传感芯片,并构建了以此微传感芯片为敏感单元的氨氮检测系统,探索了使用安培型氨气微传感器检测氨氮的方法。此微传感芯片采用MEMS工艺制备,通过电化学方法在微电极表面修饰了对氨具有良好电催化氧化性能的纳米铂,提高了传感器的灵敏度。在芯片的SU-8微池中滴入微量碳酸丙烯酯(PC),形成可使氨气迅速扩散到电极表面的薄层电解液,使传感器具有较快的响应速度。使用自行设计的氨氮检测系统对氨氮进行检测,考察了氨氮检测的浓度响应特性、时间响应特性、重复性及选择性。氨氮检测系统的线性范围为0.1～5.0 mg/L;检测下限为0.1 mg/L;响应时间小于1 min;重复性偏差为4.0%。     

基于电流脉冲沉积法的硝酸根微传感器研究

基于微加工技术(Microfabrication technology)制备微传感电极并进行电化学表面修饰,研制出一种用于水体中NO#3浓度检测的电化学微传感器。微传感器以两电极传感芯片为信号转换部件,使用电流脉冲沉积法在铂质工作电极表面制备微观形貌呈枝簇状的铜质敏感材料,利用铜质材料对酸性溶液中NO#3的电催化还原特性,测量还原电流的大小,实现对NO#3浓度的检测。采用扫描电子显微镜(SEM)和X射线衍射分析(XRD)技术对敏感膜进行表征和监测,探索高活性铜质敏感膜的制备方法;使用微传感器对硝酸盐标准样品进行检测,在低浓度范围(12.5~200μmol/L),响应灵敏度为0.1422μA/(μmol/L);高浓度范围(200~3000μmol/L),响应灵敏度为0.0984μA/(μmol/L),均表现出较高的检测灵敏度;使用微传感器对北京等地的实际湖库水样进行检测,结果与专业水质检测机构采用紫外分光光度法的测试结果偏差在#3.9%~15.4%之间,两者具有一定的相关性,表明微传感器能够用于实际水样中NO#3浓度的测量。     

检测猪肉中地西泮的分子印迹仿生传感器的研制

在一次性丝网印刷电极上原位制备地西泮的分子印迹膜,将丝网印刷电极通过电极插口与便携式电导仪相连接,组装成检测地西泮残留的电导型传感器,建立了检测地西泮的标准曲线并测试了实际肉类样品中的地西泮含量.通过扫描电镜分析了该膜的表征,与非印迹膜相比,印迹膜表面形成了大量印迹微孔.本传感器装置对地西泮具有很高的灵敏度和特异性,检出限为0.008 mg/L,线性范围为0.039～1.25 mg/L,基于肉品的检测回收率为91.3%～95.0%,可实现现场快速检测.     

基于丝网印刷工艺的安培型酶免疫传感器的研究

研究了一种一次性安培型免疫传感器,检测辣根过氧化物酶(HRP)。用丝网印刷工艺制备三电极结构的传感器,工作电极是碳电极,银电极是伪参比电极。采用电荷传递系数、评估厚膜电极的一致性。通过戊二醛交联法,把抗HRP酶抗体固定在电极表面。加入底物H2O2和电子介体邻苯二胺,分别用安培法和比色法进行检测和比较分析。安培法测定HRP的测量范围10μg/L~10 mg/L,检出限10μg/L;比色法测定范围为2~40 mg/L,检出限2 mg/L。同时分析了湿度对传感器寿命的影响。     

纳米氧化锡修饰的微催化燃烧式氢气传感器的研制

研制了一种基于多孔纳米氧化锡(SnO₂)催化剂的微催化燃烧式气体传感芯片(Pellistor). 基于微机电系统(Micro- Electro-Mechanical Systems, MEMS)工艺制备硅基封闭膜式微催化燃烧式传感器, 通过气相沉积技术在Pt微加热电极和高温绝缘层表面制备三维纳米氧化锡催化膜, 利用催化膜对氢气良好的催化特性, 采用惠斯通电桥电路进行测量, 实现对空气环境中氢气在0～4%浓度范围内的快速检测, 响应时间和恢复时间分别达到0.65 s和2.32 s, 灵敏度达75.4 mV/1% H₂, 线性度为99.4%. 考察200 天内该传感芯片对氢气的检测能力, 传感芯片表现出良好的稳定性, 精确度保持在95%以上. 在绝缘层高温性能稳定的条件下, 将三维纳米氧化锡应用于微催化燃烧式传感器的氢气检测, 对催化燃烧式传感器性能的改进具有重要的意义.     

用于细胞检测的微电极传感器设计及传感分析

本文针对肿瘤细胞的活性检测、神经细胞的神经递质检测与巨噬细胞等的氧化损伤检测等细胞检测中的核心问题,简要介绍电化学生化传感器和传感方法在细胞检测领域的应用和发展,重点对不同微电极结构的电化学传感器的设计制作、细胞检测方法及应用进展进行了综述。电化学生化传感器从单一检测电极向集成多功能和阵列式电极发展,从单个电极传感检测模式向芯片集成微电极式传感系统发展,而在其生物相容性、检测限和检测效率等方面尚需进一步提升和拓展。基于微机电系统(MEMS)技术制作的微电极研制,电极表面的多种化学和生物修饰的敏感膜研究,从硅基到聚合物柔性基底电极的材料拓展,小体积、植入式、可穿戴式的电化学生化传感器研制等是目前发展的方向,其在临床检验、精准医疗、运动健康监测、老年健康服务等诸多领域中显示出巨大的应用前景。     

基于三维纳米银修饰电极的硝酸根微型传感芯片研究

研制一种基于金叉指微电极阵列(IDA)的电流型硝酸根离子(NO-3)微传感电极芯片.基于微机电系统(Micro-Electro-Mechanical Systems,MEMS)工艺制备金IDA微电极,通过电化学沉积技术在IDA微电极表面修饰三维枝状结构纳米银敏感膜,利用敏感膜对硝酸根离子良好的电催化还原性能,采用脉冲方波伏安(SWV)电化学测量方法,实现对硝酸根离子在25～1000μmol/L浓度范围内的快速检测,灵敏度达9.5 nA/(μmol/L),线性度为99.98%,检测下限为10μmol/L.考察水体中常见的NO-2,F-,3PO 4-,SO 42-,2CO3-,NH+4,Na+和K+等离子对该传感芯片的干扰性能,传感芯片表现出较好的抗干扰性能.制备的三维枝状结构纳米银修饰IDA微电极可实现水环境(pH 5.0～9.0)中NO-3的电化学检测,对应用于自然水环境中硝酸根离子的现场检测具有积极意义.     

基于分子印迹膜的检测牛奶中琥珀酸氯霉素残留传感方法的研究

采用光聚合法在一次性丝网印刷电极上制备琥珀酸氯霉素分子印迹膜,然后将丝网印刷电极通过电极插口与电化学分析装置相连接,组装成检测琥珀酸氯霉素残留的电化学传感仪.使用与传感装置相连接的记录仪记录响应的结果.采用本传感仪建立了检测氯霉素的标准曲线并测试了实际牛奶样品中氯霉素含量.电镜学观察表明,与非印迹膜相比,在印迹膜表面形成大量直径约为100 nm的印迹微孔.本传感仪装置检测琥珀酸氯霉素具有很高的灵敏度和特异性,检出限为2×10-9 mol/L,检测线性范围为1×10-8～1.2×10-5 mol/L,基于牛奶样品的检测回收率介于93.5%～95.5%之间.     

基于分子印迹膜修饰丝网印刷电极的地西泮电化学传感器

以地西泮为模板分子,采用循环伏安法在一次性丝网印刷电极表面原位电聚合形成聚邻苯二胺膜,洗脱除去模板分子后得到地西泮分子印迹膜修饰丝网印刷电极。利用差示脉冲法对印迹膜和非印迹膜进行评价,表征了电极表面膜的电化学性质。以KI为印迹电极和底液间的探针,建立了一种间接检测地西泮的传感方法。该传感器的敏感元件为修饰有分子印迹膜的丝网印刷电极,其制备和更换非常方便。用于电化学检测时,样品的富集时间为3min,地西泮的浓度在2.0×10-7～1.0×10-5mol/L范围内与峰电流呈良好的线性关系,检出限为2.5×10-8mol/L,基于猪肉样品的加标回收率为92%～95%。将该传感器初步用于实际样品分析,结果满意。     

FET immunosensor for hemoglobin A1c using a gold nanofilm grown by a seed-mediated technique and covered with mixed self-assembled monolayers

A micro FET-based immunosensor was developed for the determination of hemoglobin-A1c (HbA1c). The HbA1c/hemoglobin ratio is an important index in diabetes control. The sensor was fabricated by Complementary Metal-Oxide-Semiconductor Transistor (CMOS) and Micro Electronic Mechanical System (MEMS) techniques. The antibodies were immobilized via mixed self-assembled monolayers (SAMs) on a gold nanofilm. The nanofilm was deposited on a gold electrode by seed-mediated growth and gave a uniform and well distributed coverage. Nonspecific sites and interferences by noise were eliminated by covering the AuNPs with mixed SAMs. Compared to the immunosensor fabricated via the mixed SAMs method without gold nanofilm, the immunosensor displays a more than 2-fold sensitivity. The immunosensor is capable of detecting HbA1c and hemoglobin in hemolyzed and diluted whole blood, and results showed good agreement with the established clinical method.

Surface Plasmon Resonance Immunosensor for Anionic Surfactants Based on an Indirect Competitive Immunoreaction

Abstract

A highly sensitive surface plasmon resonance immunosensor for the determination of linear alkylbenzene sulfonate (LAS) was fabricated. The method is based on an indirect competitive reaction of an anti‐LAS antibody in a sample solution with LAS immobilized on a sensor chip and with LAS in the sample solution. A sensor chip immobilized with LAS was prepared by utilizing an electrostatic interaction between an LAS conjugate, LAS–horseradish peroxidase (LAS‐HRP), and a self‐assembled monolayer of 11‐amino‐1‐undecanethiol hydrochloride, which was preliminary prepared on a gold thin film of the sensor chip. The quantitative determination of LAS in the concentration range 10–1000 ppb was achieved by using the proposed immunosensor.     

PoPD Modified ITO Based Capacitive Immunosensor for Sulphathiazole

A capacitive immunosensor for determination of sulphathaizole (STZ) has been developed on polymer coated indium tin oxide glass chip (ITO). The immunosensor chip was fabricated by polymerizing, ortho‐ phenylenediamine (o PD) on ITO followed by surface modification with anti‐sulphathiazole antibody. The developed immunosensor chip was characterized by using Atomic force microscopy (AFM), Cyclicvoltammetry (CV) and Electrochemical impedance spectroscopy (EIS). The capacitive measurement of the developed immunosensor was performed by using EIS in spiked drinking water and milk. The developed sensor showed liner detection range 0.1‐100 μgL⁻¹for STZ with a limit of detection 0.01 μgL⁻¹ in water with recovery between 95–106 %. The biosensor showed excellent selectivity and storage stability upto 4 weeks when preserved at 4 °C.     

Surface plasmon resonance immunosensor for histamine based on an indirect competitive immunoreaction

The use of a surface plasmon resonance immunosensor for the analysis of histamine (β-imidazole ethylamine) is described. The method is based on an indirect competitive reaction of an anti-histamine antibody in a sample solution with histamine immobilized on a sensor chip and with histamine in the sample solution. A sensor chip immobilized with histamine was prepared using a self-assembly monolayer of 11-mercaptoundecanoic acid (11-MUA) as an anchor membrane, followed by an amino-coupling reaction with histamine after activation of the 11-MUA layer on the sensor chip by treatment with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide and N-hydroxysuccinimide. The sensor chip can be reused, after regeneration with a 10 mM HCl solution, which dissociates the anti-histamine antibody complex from histamine on the sensor chip. The affinity constants for the immunocomplex of the anti-histamine antibody with histamine in the solution and for that of the anti-histamine antibody with histamine immobilized on the sensor chip were calculated to be 1.5 × 10⁷ and 7.2 × 10⁵ M⁻¹, respectively, by assuming a Langmuir-type adsorption of the anti-histamine antibody to histamine immobilized on the sensor chip. The detection limit of the method was determined to be 3 ppb.     

Surface plasmon resonance immunosensor for IgE analysis using two types of anti-IgE antibodies with different active recognition sites.

A simple and novel method for the determination of an IgE antibody based on a surface plasmon resonance immunosensor for the diagnosis of an allergy is described. The method involves the use of an anti-IgE(D) antibody and an anti-IgE(H) antibody, which reacts with the Ce2 domain and the Ce3 domain of the IgE antibody. The anti-IgE(D) antibody was immobilized on the gold surface of a sensor chip by physical adsorption. An IgE antibody sample was incubated by adding it to an anti-IgE(H) antibody solution to form an anti-IgE(H) immunocomplex through a reaction of the Ce3 domain of the IgE antibody. The incubated solution was introduced onto the sensor chip and the immunocomplex of the IgE-anti-IgE(H) then reacted with the anti-IgE(D) antibody immobilized on the sensor chip through the Ce2 domain of the IgE antibody part of the IgE-anti-IgE(H) immunocomplex. The detection limit of the present method for the determination of the IgE antibody was about 10 ppb. The affinity constants for the anti-IgE(H) antibody immunocomplex with the IgE antibody in solution and that of the anti-IgE(H) antibody immunocomplex with the IgE antibody immobilized on the sensor chip by a biotin-streptavidin interaction were estimated to be 4.1 x 10(7) M(-1) and 5.8 x 10(6) M(-1), respectively. The affinity constant for the immunocomplex of the anti-IgE(H) antibody with the IgE antibody with the anti-IgE(D) immobilized on the sensor chip was estimated to be 4.9 x 10(7) M(-1), 20-times larger than the affinity constant for the IgE antibody immunocomplex with the anti-IgE(D) antibody immobilized on the sensor chip, based on a direct immunoassay method of the IgE antibody under the same experimental conditions.     

Flow immunoassay of trinitrophenol based on a surface plasmon resonance sensor using a one-pot immunoreaction with a high molecular weight conjugate

A surface plasmon resonance (SPR) immunosensor based on a competitive immunoreaction for the determination of trinitrophenol (TNP) is described. A goat anti-mouse IgG (1st antibody), which recognizes an Fc moiety of an antibody, was immobilized on a gold film of an SPR sensor chip by physical adsorption. A TNP solution containing a fixed concentration of a mouse anti-TNP monoclonal antibody (2nd antibody) and a TNP-keyhole limpet hemocyanin (KLH) conjugate was incubated in one-pot and introduced into the sensor chip. The TNP-KLH conjugate competes with TNP for binding with the 2nd antibody. The resulting complex of the 2nd antibody with the TNP-KLH conjugate was bound to the 1st antibody, which is immobilized on the sensor chip. The SPR sensor signal based on resonance angle shift is dependent on the concentration of TNP in the incubation solution in the range from 25 ppt to 25 ppb, and the coefficient of variation of the SPR signals for the 25 ppb TNP solution was determined to be 13% (n = 4). The experimental results for the adsorption constant of the 1st antibody on the sensor chip and the binding constant of the 1st antibody complex with the 2nd antibody are discussed, together with theoretical considerations.     

β-Lactamase label-based potentiometric biosensor for α-2 interferon detection

An effective immunosensor for α-2 interferon detection based on pH-sensitive field effect transistor (pH-FET) has been developed. A specific sensing element was fabricated by immobilizing α-2 interferon on the gate of a pH-FET. The interaction of anti-interferon antibodies labelled with β-lactamase with interferon–pH-FET (in the presence of specific enzyme substrate) leads to a local pH-change at the surface of transducer and produces an electrochemical signal which is proportional to the conjugate concentration. The main performance characteristics of the sensor obtained (sensitivity, dynamic range, operational and storage stability) were estimated. The effect of pH, buffer concentration and ionic strength on the immunosensor response as well as conditions of immunosensor regeneration were studied. For the determination of the interferon concentration in a sample solution the competitive electrochemical immunoassay has been employed. A linear response of the sensor on α-2 interferon concentration is obtained in the range 10–100 μg/ml. This gives the possibility to detect α-2 interferon in a non-diluted cultivated broth. The data of the competitive electrochemical immunoassay are available within 30 min and are in good accordance with the enzyme-linked immunosorbent assays.     

Enhanced sensitivity of self-assembled-monolayer-based SPR immunosensor for detection of benzaldehyde using a single-step multi-sandwich immunoassay

This paper describes the fabrication and sensing characteristics of a self-assembled monolayer (SAM)-based surface plasmon resonance (SPR) immunosensor for detection of benzaldehyde (BZ). The functional sensing surface was fabricated by the immobilization of a benzaldehyde–ovalbumin conjugate (BZ–OVA) on Au-thiolate SAMs containing carboxyl end groups. Covalent binding of BZ–OVA on SAM was found to be dependent on the composition of the base SAM, and it is improved very much with the use of a mixed monolayer strategy. Based on SPR angle measurements, the functional sensor surface is established as a compact monolayer of BZ–OVA bound on the mixed SAM. The BZ–OVA-bound sensor surface undergoes immunoaffinity binding with anti-benzaldehyde antibody (BZ-Ab) selectively. An indirect inhibition immunoassay principle has been applied, in which analyte benzaldehyde solution was incubated with an optimal concentration of BZ-Ab for 5 min and injected over the sensor chip. Analyte benzaldehyde undergoes immunoreaction with BZ-Ab and makes it inactive for binding to BZ–OVA on the sensor chip. As a result, the SPR angle response decreases with an increase in the concentration of benzaldehyde. The fabricated immunosensor demonstrates a low detection limit (LDL) of 50 ppt (pg mL⁻¹) with a response time of 5 min. Antibodies bound to the sensor chip during an immunoassay could be detached by a brief exposure to acidic pepsin. With this surface regeneration, reusability of the same sensor chip for as many as 30 determination cycles has been established. Sensitivity has been enhanced further with the application of an additional single-step multi-sandwich immunoassay step, in which the BZ-Ab bound to the sensor chip was treated with a mixture of biotin-labeled secondary antibody, streptavidin and biotin–bovine serum albumin (Bio–BSA) conjugate. With this approach, the SPR sensor signal increased by ca. 12 times and the low detection limit improved to 5 ppt with a total response time of no more than ca. 10 min. Figure A single-step multi-sandwich immunoassay step increases SPR sensor signal by ca. 12 times affording a low detection limit for benzaldehyde of 5 ppt     

Fluorescence-based assay with enzyme amplification on a micro-flow immunosensor chip for monitoring coplanar polychlorinated biphenyls

Detection of pollutants is of significant importance for environmental protection. However, conventional monitoring methods are often time-consuming, and require expensive equipments. Biosensors based on enzyme linked immunosorbent assay (ELISA) provide an alternative method to conventional ones. In this research, the reduction in the size of ELISA utilizing micro-chemical reaction is described in a micro-flow immunosensor chip. The immunosensor chips were fabricated by micro-electromechanical system (MEMS) technology. The quantitative determination of coplanar polychlorinated biphenyls (Co-PCBs) was performed by using a micro-flow immunosensor chip. Polystyrene beads were used as the solid substrate for the immobilization of Co-PCB antibody. The antibody-immobilized beads were introduced into the flow channel. As a competitive ELISA, sample solution mixed with horseradish peroxidase (HRP) conjugated antigen, and non-HRP conjugated antigen was allowed to react in the flow channel. After the antigen-antibody reaction, addition of phosphate buffer solution containing hydrogen peroxide and the fluorogenic substrate produced a fluorescent dye, which was monitored with the resulting change in the fluorescence intensity. By using our micro-flow immunosensor chip, it was possible to determine the sensing range of Co-PCB derivatives up to 0.1 ppt in 30 s. This immunosensor chip had a wide linear range for Co-PCB detection from 0.1 pg/ml to 1.0 μg/ml. The regression analysis provided the correlation coefficients of r = 0.982−0.964 with good reproducibility and precision. In a series of five measurements with immunosensor chips prepared with a new batch of antibody-immobilized polystyrene beads, a relative standard deviation of 21.3% was obtained. Our immunosensor chip design reported here has the potential to be implemented to several different detection methodologies for numerous analytes.     

Membrane-immobilized haptoglobin as affinity matrix for a hemoglobin-A1c immunosensor

An amperometric immunosensor for hemoglobin-A1c (HbA1c) determination has been developed utilizing membrane-immobilized haptoglobin as affinity matrix fixed in front of a Pt-working electrode. The HbA1c assay was carried out in a two-step procedure including the selective hemoglobin enrichment on the sensor surface and the specific HbA1c detection by a glucose oxidase (GOx) labeled anti-HbA1c antibody. Hydrogen peroxide generated by the enzyme label was oxidized at +600 mV versus Ag/AgCl. A standard curve for HbA1c was obtained with a linear range between 0 and 25% HbA1c of total hemoglobin which correspond to 7.8–39 nM. ELISA studies confirmed the advantage of a sandwich-type format with haptoglobin as capture molecule for selective hemoglobin binding over the direct adsorption method. Results by the sandwich immunoassay showed a linear correlation within the clinically relevant range 5–20% (CV < 3). For sensor application the immobilization procedure of haptoglobin onto CDI-activated cellulose membranes was optimized.