电聚高分子膜固定化酶生物传感器及其进展

本文将电化学聚合高分子膜固定膜酶制备的生物传感器分为以下三种主要类型，并分别 就其发展概况和发展方向进行了评述。即：以溶解氧为电子受体的的生物传感器（第一代电流型生物传感器）；以非氧介全为电子受体的生物传感器（第二代电流型生物传感器）和电子在酶和聚合高分膜之间直接进行转移的传感器（第三代电流型生物传感器）。     

辣根过氧化物酶在表面活性剂膜中的直接电化学

利用3种表面活性剂分别将辣根过氧化氢酶固定在裂解石墨棱面（edge-plane pyrolytic graphite,EPG)电极表面,研究了辣根过氧化物酶（HRP）中Fe(Ⅲ）／Fe（Ⅱ）电对与电极之间的直接电子传递过程以及酶催化双氧化还原过程。实验结果表明：（1）表面活性剂是一种固定酶的理想材料;（2）这种体系可能构造第三代生物传感器,对解释生物体代谢过程具有理论意义,对制备第三代生物传感器具有应用价值。     

酶电极电子转移的途径及研究进展

酶的活性中心与电极表面的电子转移直接影响酶电极的性能和特征。自1962年第一个酶电极报道以来,科学家们不断探索新的方式实现酶与电极间电子转移并取得了较大的进展,使生物传感器由第一代依靠氧与葡萄糖氧化酶中的活性中心反应测量氧的消耗为原理,发展到第三代实现酶的活性中心与电极表面之间的直接电子转移,即所谓的"无试剂电化学生物传感器"。然而,探究酶电极内在电子转移机理以及设计能够满足不同应用要求并适合大规模量产、价格合适的酶电极仍然是研究的热点。本文综述了主要的电子转移方式以及相应的优缺点,以及笔者团队开发的使用氧化还原聚合物实现电子转移的方法,并对其应用前景进行展望。     

酶电极电子转移途径的研究进展

酶的活性中心与电极表面的电子转移直接影响酶电极的性能和特征。自1962年第一个酶电极报道以来,科学家们不断探索新的方式实现酶与电极间电子转移并取得了较大的进展,使生物传感器由第一代依靠氧与葡萄糖氧化酶中的活性中心反应测量氧的消耗为原理,发展到第三代实现酶的活性中心与电极表面之间的直接电子转移,即所谓的"无试剂电化学生物传感器"。然而,探究酶电极内在电子转移机理以及设计能够满足不同应用要求并适合大规模量产、价格合适的酶电极仍然是研究的热点。本文综述了主要的电子转移方式以及相应的优缺点,以及笔者团队开发的使用氧化还原聚合物实现电子转移的方法,并对其应用前景进行展望。     

纳米颗粒增强的葡萄糖生物传感器

研制的纳米增强葡萄糖传感器是用纳米憎水Ａｕ颗粒。亲水Ａｕ颗粒、憎水ＳｉＯ_２颗粒以及Ａｕ和－ＳｉＯ_2颗粒混合与聚乙烯醇缩丁醛（ＰＶＢ）构成复合固酶膜基质,用溶胶－凝胶法固定葡萄糖氧化酶（ＧＯＤ）,组成葡萄糖生物传感器．实验表明,纳米颗粒可以大幅度提高固定化酶的催化活性,响应电流从相应浓度的几十纳安增强到几千纳安,电极响应迅速, １ｍｉｎ达到稳态,探讨了纳米颗粒效应在固定化酶中所起的作用,开辟了制备直接电子传递第三代生物传感器的新途径和纳米颗粒应用的新领域。     

蛋白质直接电化学研究及其应用

蛋白质直接电化学研究在生物电化学中具有重要地位，对于蛋白质结构．功能研究、蛋白质电子传递过程的热力学和动力学研究都有着重要意义，而且是研制第三代电化学生物传感器的基础。本文对在裸电极、分子自组装修饰电极和模拟生物膜修饰电极上进行蛋白质直接电化学的研究及相关应用进行简要综述。     

固定在硬脂酸Langmuir-Blodgett膜中的辣根过氧化酶修饰电极的直接电化学行为

近年来,氧化还原蛋白质的直接电子转移反应引起了越来越多研究者的兴趣~([1]),研究氧化还原蛋白质的直接电子转移反应,不仅对于探索生命体内的生理作用机理等理论研究具有重要意义,而且为制备基于氧化还原蛋白质直接电化学行为的第三代生物传感器奠定了技术基础.本文研究了硬脂酸(SA)Langmuir-Blodgett(LB)膜固定的辣根过氧化物酶(HRP)在金电极(Au)上的直接电化学行为.     

蛋白质直接电化学研究及其应用

蛋白质直接电化学研究在生物电化学中具有重要地位,对于蛋白质结构-功能研究、蛋白质电子传递过程的热力学和动力学研究都有着重要意义,而且是研制第三代电化学生物传感器的基础.本文对在裸电极、分子自组装修饰电极和模拟生物膜修饰电极上进行蛋白质直接电化学的研究及相关应用进行简要综述.     

蛋白质直接电化学研究及其应用

蛋白质直接电化学研究在生物电化学中具有重要地位,对于蛋白质结构-功能研究、蛋白质电子传递过程的热力学和动力学研究都有着重要意义,而且是研制第三代电化学生物传感器的基础。本文对在裸电极、分子自组装修饰电极和模拟生物膜修饰电极上进行蛋白质直接电化学的研究及相关应用进行简要综述。     

生物电化学简介

简单介绍了生物电化学研究领域的概况。包括：生物膜与生物界面模拟研究（ＳＡＭ膜模拟生物膜的电化学、液／液界面模拟生物膜的电化学），用于生命科学的电化学技术（电脉冲基因直接导入、电场加速作物生长、癌症的电化学疗法、电化学控制药物释放、在体研究的电化学方法、生物分子的电化学行为）和电化学生物传感器（酶电极传感器、微生物电极传感器、电化学免疫传感器、组织电极与细胞器电极传感器、电化学ＤＮＡ传感器）。     

Amperometric Biosensors Based on Direct Electron Transfer Enzymes

The accurate determination of analyte concentrations with selective, fast, and robust methods is the key for process control, product analysis, environmental compliance, and medical applications. Enzyme-based biosensors meet these requirements to a high degree and can be operated with simple, cost efficient, and easy to use devices. This review focuses on enzymes capable of direct electron transfer (DET) to electrodes and also the electrode materials which can enable or enhance the DET type bioelectrocatalysis. It presents amperometric biosensors for the quantification of important medical, technical, and environmental analytes and it carves out the requirements for enzymes and electrode materials in DET-based third generation biosensors. This review critically surveys enzymes and biosensors for which DET has been reported. Single- or multi-cofactor enzymes featuring copper centers, hemes, FAD, FMN, or PQQ as prosthetic groups as well as fusion enzymes are presented. Nanomaterials, nanostructured electrodes, chemical surface modifications, and protein immobilization strategies are reviewed for their ability to support direct electrochemistry of enzymes. The combination of both biosensor elements—enzymes and electrodes—is evaluated by comparison of substrate specificity, current density, sensitivity, and the range of detection.     

Carbon-decorated ZnO nanowire array: A novel platform for direct electrochemistry of enzymes and biosensing applications

We report here the direct electron transfer of GOD and a novel glucose biosensor based on carbon-decorated ZnO(C–ZnO) nanowire array electrode. The C–ZnO nanowire array provides a novel platform for fast direct electrochemistry of GOD, and its based biosensor shows very high sensitivity and low detection limit. Based on the direct electrochemistry of horseradish peroxidase (HRP), the H₂O₂ biosensing application is further demonstrated using this new C–ZnO array architecture. The high conductivity of carbon and good electron transfer capability of ZnO nanowires, along with their low cost and biocompatibility make the C–ZnO nanowire array a promising platform for direct electrochemistry of enzymes and mediator-free enzymatic biosensors.     

Mb/AuNPs/MWCNTs/GC电极对H2O2电催化性能的研究

采用还原法制备了AuNPs/MWCNTs复合材料,并构建了氧化还原蛋白质的固定化和生物传感界面AuNPs/MWCNTs/GC电极.以肌红蛋白(Myoglobin,Mb)为例,研究了固定化蛋白质在AuNPs/MWCNTs/GC电极上的直接电化学.结果表明,AuNPs/MWCNTs复合材料不仅能有效地促进Mb与电极表面的直接电子转移,而且能很好地保持固定化Mb的生物催化活性.Mb/AuNPs/MWCNTs/GC电极对H2O2具有良好的电催化还原性能,其线性响应范围为1～138μmol·L-1,检测限为0.32μmol·L-1(S/N=3),并具有较低的米氏常数(0.143 mmol·L-1).该电极操作简单,响应迅速,稳定性和重现性好,有望用于蛋白质的固定化及第三代生物传感器的制备.     

Strategies for "wiring" redox-active proteins to electrodes and applications in biosensors, biofuel cells, and nanotechnology

In this tutorial review the basic approaches to establish electrochemical communication between redox-active proteins and electrodes are elucidated and examples for applications in electrochemical biosensors, biofuel cells and nanotechnology are presented. The early stage of protein electrochemistry is described giving a short overview over electron transfer (ET) between electrodes and proteins, followed by a brief introduction into experimental procedures for studying proteins at electrodes and possible applications arising thereof. The article starts with discussing the electrochemistry of cytochrome c, the first redox-active protein, for which direct reversible ET was obtained, under diffusion controlled conditions and after adsorption to electrodes. Next, examples for the electrochemical study of redox enzymes adsorbed on electrodes and modes of immobilization are discussed. Shortly the experimental approach for investigating redox-active proteins adsorbed on electrodes is outlined. Possible applications of redox enzymes in electrochemical biosensors and biofuel cells working by direct ET (DET) and mediated ET (MET) are presented. Furthermore, the reconstitution of redox active proteins at electrodes using molecular wire-like units in order to "wire" the proteins to the electrode surface and possible applications in nanotechnology are discussed.     

Recent trends in enzyme engineering aiming to improve bioelectrocatalysis proceeding with direct electron transfer

Among the known types of electrochemical biosensors, the third generation based on the ability of some enzymes to direct electron transfer (DET) is the most promising one. The enzyme property to DET is depending on its capability to electron transfer from enzymatically reduced built-in native cofactor (flavin mononucleotide, flavin adenine dinucleotide, pyrroloquinoline quinone, or heme) to a conductive surface directly for single cofactor enzymes or through a native structural electron acceptor (heme or copper-containing prosthetic groups) for multicofactor enzymes. Thus, there are two possibilities to use such type enzymes: to find a natural source of the enzyme with these properties; or to construct the recombinant chimeric analogs using the gene-engineering techniques. The modern molecular genetics opens the possibility to be independent of million-year natural evolution and engineer the specific enzymes for scientific and technological needs. This brief review is focused mostly on the recent publications on application of DET-capable engineered enzymes for the third-generation electrochemical biosensors.     

Recent developments, characteristics, and potential applications of electrochemical biosensors.

The objective of this study is to analyze the technical importance, performance, techniques, advantages, and disadvantages of the biosensors in general and of the electrochemical biosensors in particular. A product of reaction diffuses to the transducer in the first generation biosensors (based on Clark biosensors). The mediated biosensors or second generation biosensors use specific mediators between the reaction and the transducer to improve sensitivity. The second generation biosensors involve two steps: first, there is a redox reaction between enzyme and substrate that is reoxidized by the mediator, and eventually the mediator is oxidized by the electrode. No normal product or mediator diffusion is directly involved in the third generation biosensors, direct biosensors. Based on the type of transducer, current biosensors are divided into optical, mass, thermal, and electrochemical sensors. They are used in medical diagnostics, food quality controls, environmental monitoring, and other applications. These biosensors are also grouped under two broad categories of sensors: direct and indirect detection systems. Moreover, these systems could be further grouped into continuous or batch operation. Therefore, amperometric biosensors and their current applications are focused on more in detail since they are the most commonly used biosensors in monitoring and diagnosing tests in clinical analysis. Problems related to the commercialization of medical, environmental, and industrial biosensors as well as their performance characteristics, their competitiveness in comparison to the conventional analytical tools, and their costs determine the future development of these biosensors.     

Catalytic electrooxidation of NADH for dehydrogenase amperometric biosensors

The developments in the techniques of NADH catalytic oxidation relevant for incorporation in amperometric biosensors with dehydrogenase enzymes are reviewed with special emphasis in the years following 1990. The review stresses the direct electro-catalytic methods of NAD⁺ recycling as opposed to enzymatic regeneration of the coenzyme. These developments are viewed and evaluated from a mechanistic perspective of recycling of NADH to enzymatically active NAD⁺, and from the point of view of development of technologically useful reagentless dehydrogenase biosensors. An effort is made to propose a method for the standardization of evaluation of new mediating and direct coenzyme recycling schemes. A perspective is given for the requirements that have to be met for successful biosensor development incorporating dehydrogenase enzymes that open the analytical possibilities to a number of new analytes. The intrinsic limitations of the system are finally discussed and a view of the future of the field is presented.     

Electrochemistry and electroanalytical applications of carbon nanotubes: a review.

This review addresses recent developments in electrochemistry and electroanalytical chemistry of carbon nanotubes (CNTs). CNTs have been proved to possess unique electronic, chemical and structural features that make them very attractive for electrochemical studies and electrochemical applications. For example, the structural and electronic properties of the CNTs endow them with distinct electrocatalytic activities and capabilities for facilitating direct electrochemistry of proteins and enzymes from other kinds of carbon materials. These striking electrochemical properties of the CNTs pave the way to CNT-based bioelectrochemistry and to bioelectronic nanodevices, such as electrochemical sensors and biosensors. The electrochemistry and bioelectrochemistry of the CNTs are summarized and discussed, along with some common methods for CNT electrode preparation and some recent advances in the rational functionalization of the CNTs for electroanalytical applications.     

Direct electron transfer between heme-containing enzymes and electrodes as basis for third generation biosensors

Direct electron transfer (DET) between redox enzymes and electrodes found the basis for third generation biosensors. Recent investigations in the authors’ laboratories on the bioelectrochemistry of heme-containing proteins and enzymes, primarily peroxidases, but also cellobiose dehydrogenase, will be reviewed.