胆红素氧化酶的生物电化学特性研究进展

从胆红素氧化酶的结构、来源、生物电催化的反应机制（铜离子活性中心的氧化还原电势、直接和介体参与的电子传递）、修饰电极的材料、固定化技术以及在生物燃料电池中的应用等方面介绍了胆红素氧化酶,提出了胆红素氧化酶的结构与来源、生物电催化,胆红素氧化酶燃料电池和胆红素氧化酶在电化学中的应用。     

“直接电子传递”源自碳纳米管与杂质的协同作用

多壁碳纳米管被众多研究者视为生物燃料电池或酶传感器直接电子传递的根源。但新的实验证据表明,碳纳米管中的金属杂质起着至关重要的作用。葡萄糖氧化酶FAD氧化还原峰在碳纳米管中金属杂质被回流去除之后消失,重新添加金属杂质之后其氧化还原峰又再次出现。所以酶电极的直接电子传递是金属杂质和碳纳米管协同作用的结果。     

酶的固定化技术

酶是一种高活性的生物催化剂,虽然其在酶生物燃料电池和传感器等方面的应用十分广泛,但酶在工业上的广泛应用一直受到很多限制,因为酶在水溶液中一般很不稳定.酶的固定化技术与合理设计、酶生物传感器的结构仍然是酶生物燃料电池和传感器领域今后研究的重要方向.如果通过酶的固定化技术的改进能使酶在使用过程中提高其稳定性,保持催化活性将使酶生物传感器得到更大的发展.     

纳米材料在生物燃料电池中的应用

酶生物燃料电池的寿命短以及能量密度低都与酶的稳定性、电子迁移速率和酶载量相关。采用纳米粒子、纳米纤维和介孔介质作为酶固定化的支持物,由于纳米材料巨大的表面可以增加酶载量和促进反应的发生,从而提高生物燃料电池的能量密度。将纳米材料应用于酶生物燃料电池的酶催化剂的固定,在完善电池性能上具有很大的发展潜力。     

酶定向固定化方法及应用的研究进展

传统固定化方法常会导致酶活性大幅度下降,回收率较低,而酶定向固定由于固定化后可完全暴露其活性部位,因而可以保持较高的酶活回收率。本文主要综述了定向固定化酶的两种方法,分别为共价定向固定和非共价定向固定。其中非共价定向固定化主要是抗体与抗原、亲和素/链霉亲和素和生物素以及组氨酸标签与Co2+/Ni2+之间的亲和作用;共价定向固定化主要是通过半胱氨酸残基上的巯基与载体作用。简述了其在生物传感器、分子识别、酶生物燃料电池及酶纯化方面的应用。最后指出今后的主要研究方向为探索新的定向固定化标签以降低对酶活性部位的影响,应用新的载体以提高固定化酶酶活回收率,优化固定化过程及简化固定化步骤等。     

介孔二氧化硅载体在酶蛋白固定化中的应用

介孔二氧化硅材料因制备简单、生物相容性良好、比表面积和孔径可控、表面修饰基团多样等特点,被广泛应用于酶蛋白的固定化.主要综述了介孔二氧化硅对水解酶、氧化还原酶等酶蛋白的固定化效果及其影响固定化效果的因素,展望了介孔二氧化硅在酶蛋白固定化中的应用前景及存在的挑战,为介孔二氧化硅更好地应用于固定化酶制剂的制备奠定基础.     

酶型生物燃料电池的研究进展

本文综述了近期构建酶型生物燃料电池的电极材料、酶的固定方法以及酶型生物燃料电池应用的研究进展,分析了酶型生物燃料电池构建和应用面临的问题与挑战,展望了酶型生物燃料电池今后发展的方向。     

浅谈酶电极表面的电子传递

蛋白质和酶可以固定在电极上，由于其催化效率高、受环境限制小等优点而具有更广泛的用途。本文以介绍酶修饰电极中酶与电极间电子传递过程，探讨酶在电极上的电化学行为及生物催化作用，为生命过程中蛋白质电子传递过程的研究提供理论依据。     

纤维素在固定化酶的研究进展

分析了酶及固定化酶研究进展及工业化应用瓶颈,并对无机载体、合成聚合物载体、天然聚合物载体等当前酶固定化载体材料和种类及其特点进行总结,重点介绍了纤维素作为一种无毒、可再生、可降解、生物相容性好的天然高分子材料用于固定酶载体的研究进展,阐述了纤维素在固定化酶技术工业应用亟需解决的问题及未来的发展趋势。     

氧化石墨烯和过渡金属碳/氮化合物固定化酶

二维纳米材料具有高机械强度和比表面积、大量表面官能团、良好的亲水性及生物相容性,是固定化酶的良好载体。本文选取经典的氧化石墨烯（GO）以及新型的过渡金属碳/氮化合物（MXenes）,分别介绍了它们的制备方法和结构、物理和化学性质,综述了它们在固定化酶领域的应用研究,并进行了比较。文中指出：GO由石墨烯经化学氧化再剥离制得,MXenes由其前体经刻蚀制得,不同的氧化或刻蚀方法制得的材料在组成、结构、性能等方面存在差异。GO表面的可反应官能团更多,包括羟基、羧基和环氧基,故在固定化酶领域应用广泛。MXenes固定化酶则主要利用表面的羟基反应或负电荷吸附,目前主要用于制备生物传感器。最后指出这两种材料还存在制备效率低、纳米片易聚集、循环利用性差等问题。今后的发展方向是要开发更为简单和安全的材料制备方法,探索更为有效的插层和剥离手段以及改善固定化酶的回收策略,进一步推进二维纳米材料在固定化酶领域的应用。     

Analytical solution of amperometric enzymatic reactions based on Homotopy perturbation method

Electrochemical polymerization is a simple and direct technique often employed for immobilizing redox enzymes at an electrode surface. Besides these, it allows precise control over the amount, spatial distribution and orientation of the enzymes. Analytical expressions pertaining to the immobilization of enzyme by electrochemical polymerization on the electrode surface were obtained by Homotopy perturbation method (HPM). This expression further distinguishes the product of the enzyme reaction at the electrode surface from those occurring alongside the polymer employed for immobilization. These analytical results are compared with the available limiting case results and they are found to be in good agreement.     

Gold nanoparticle-based electrochemical biosensors

The unique properties of gold nanoparticles to provide a suitable microenvironment for biomolecules immobilization retaining their biological activity, and to facilitate electron transfer between the immobilized proteins and electrode surfaces, have led to an intensive use of this nanomaterial for the construction of electrochemical biosensors with enhanced analytical performance with respect to other biosensor designs. Recent advances in this field are reviewed in this article. The advantageous operational characteristics of the biosensing devices designed making use of gold nanoparticles are highlighted with respect to non-nanostructured biosensors and some illustrative examples are commented. Electrochemical enzyme biosensors including those using hybrid materials with carbon nanotubes and polymers, sol-gel matrices, and layer-by-layer architectures are considered. Moreover, electrochemical immunosensors in which gold nanoparticles play a crucial role in the electrode transduction enhancement of the affinity reaction as well as in the efficiency of immunoreagents immobilization in a stable mode are reviewed. Similarly, recent advances in the development of DNA biosensors using gold nanoparticles to improve DNA immobilization on electrode surfaces and as suitable labels to improve detection of hybridization events are considered. Finally, other biosensors designed with gold nanoparticles oriented to electrically contact redox enzymes to electrodes by a reconstitution process and to the study of direct electron transfer between redox proteins and electrode surfaces have also been treated.     

New electrochemical techniques applied to medicine and biology

Rapid and reversible protein-electrode interaction has been achieved through the appropriate design of electrode surfaces and choice of electrolyte conditions. This enabled the direct (unmediated) electrochemistry of redox proteins at a range of electrode materials to be examined. Coupling of the electrode reaction to enzymes, for which the redox proteins act as cofactors, extended the study to systems of biological significance. Electrochemical techniques are finding increasing application in clinical analysis and a new type of amperometric enzyme electrode has been devised which exploits ferrocene derivatives as mediators between enzyme and electrode. The synthesis of ferrocene-drug conjugates permitted the development of an amperometric immunoelectrode.     

生物材料修饰的碳糊电极及其应用

就酶、细胞器、动植物组织和微生物细胞等生物材料修饰的碳糊电极进行综述,着重归纳了酶与底物的作用原理,以及氧化还原介体对促进酶电活性中心与电极之间直接导电连接的原理。     

Electrochemical evaluation of electron transfer kinetics of high and low redox potential laccases on gold electrode surface

Laccases and other multicopper oxidases are reported to be able to carry out direct electron transfer reactions when immobilized onto electrode surface. This allows detailed research of their electron transfer mechanisms. We have recently characterized the kinetic properties of four laccases in homogenous solution and immobilized onto an electrode surface with respect to a set of different redox mediators. In this paper we report the direct electron transfer of four purified laccases from Trametes hirsuta (ThL), Trametes versicolor (TvL), Melanocarpus albomyces (r-MaL) and Rhus vernicifera (RvL), by trapping the proteins within an electrochemically inert polymer of tributylmethyl phosphonium chloride coating a gold electrode surface. In particular, we have characterized the steps involved in the laccases electron transfer mechanism as well as the factors limiting each step. During the voltammetric experiments, non-turnover Faradic signals with midpoint potential of about 790 and 400 mV were observed for high potential laccases, ThL and TvL, corresponding to redox transformations of the T1 site and the T2/T3 cluster of the enzyme, respectively, whereas low redox potential laccases r-MaL and RvL shown a redox couple with a midpoint potential around 400 mV. The electrocatalytic properties of these laccase modified electrodes for the reduction of oxygen have been evaluated demonstrating significative direct electron transfer kinetics. The biocatalytic activity of laccases was also monitored in the presence of a well known inhibitor, sodium azide. On the basis of the experimental results, a hypothesis about the electronic pathway for intramolecular electron transfer characterizing laccases has been proposed.     

Electrochemical sensors based on conducting polymer—polypyrrole

Conducting polymers can be exploited as an excellent tool for the preparation of nanocomposites with nano-scaled biomolecules. Polypyrrole (Ppy) is one of the most extensively used conducting polymers in design of bioanalytical sensors. In this review article significant attention is paid to immobilization of biologically active molecules within Ppy during electrochemical deposition of this polymer. Such unique properties of this polymer as prevention of some undesirable electrochemical interactions and facilitation of electron transfer from some redox enzymes are discussed. Recent advances in application of polypyrrole in immunosensors and DNA sensors are presented. Some new electrochemical target DNA and target protein detection methods based on changes of semiconducting properties of electrochemically generated Ppy doped by affinity agents are introduced. Recent progress and problems in development of molecularly imprinted polypyrrole are considered.     

面向生物能源的酶固定化的计算机模拟

利用酶固定化技术来对生物质发酵获取生物能源已显得日益重要。酶与表面间的相互作用强烈影响固定化酶的取向,进而影响催化效率。本文采用并行退火蒙特卡洛（PTMC）方法对三种生物能源用酶（脂肪酶、纤维素酶和氢化酶）在不同的带电表面和离子强度下的吸附取向进行了模拟研究。模拟结果发现三种酶的吸附主要由静电吸引力主导,并且很大程度上与蛋白表面带电氨基酸的分布和溶液离子的静电屏蔽有关。脂肪酶和氢化酶在带负电表面上吸附,其活性位和电子转移通道分别为朝向溶液和靠近表面,而纤维素酶则在带正电表面上取得较优的吸附取向。本文研究结果可为工业用酶以合理的取向在载体材料表面固定化提供一定的指导。     

介孔材料中酶固定化的影响因素

介孔材料因具有均一可调的孔径、高的比表面积和规整的孔道结构以及易于表面功能化等优点,可广泛用于生物大分子分离、酶固定化等方面。酶固定化易受介孔材料的影响,本文综述了介孔材料的孔径、表面特性、形貌及溶液的pH等因素对酶固定化的影响,并对改性材料中酶固定化的发展前景进行了预测。     

Direct electrochemistry and electrocatalysis of horseradish peroxidase based on halloysite nanotubes/chitosan nanocomposite film

The novel halloysite nanotubes/chitosan (HNTs/Chi) composite films were firstly explored to utilize for the immobilization of horseradish peroxidase (HRP) and their bioelectrochemical properties were studied, in which the biopolymer chitosan was used as a binder to increase film adherence on glassy carbon (GC) electrode. UV-vis and FTIR spectroscopy demonstrated that HRP in the composite film could retain its native secondary structure. A pair of well-defined redox peaks of HRP was obtained at the HRP/HNTs/Chi composite film-modified electrode, exhibiting its fast direct electron transfer (DET). Furthermore, the immobilized HRP displayed its good electrocatalytic activity for the reduction of hydrogen peroxide (H₂O₂). The results demonstrate that the HNTs/Chi composite film may improve the enzyme loading with the retention of bioactivity and greatly promote the direct electron transfer, which can be attributed to its unique tubular structure, high specific surface area, and good biocompatibility.