Hengge Elisabeth, Steyskal Eva-Maria, Dennig Alexander, Nachtnebel Manfred, Fitzek Harald, Würschum Roland, Nidetzky Bernd
Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, Petersgasse 12, Graz, 8010, Austria.
Institute of Materials Physics, Graz University of Technology, Petergasse 16, Graz, 8010, Austria.
Small Methods. 2025 Mar;9(3):e2400844. doi: 10.1002/smtd.202400844. Epub 2024 Sep 19.
Enzyme-modified electrodes are core components of electrochemical biosensors for diagnostic and environmental analytics and have promising applications in bioelectrocatalysis. Despite huge research efforts spanning decades, design of enzyme electrodes for superior performance remains challenging. Nanoporous gold (npAu) represents advanced electrode material due to high surface-to-volume ratio, tunable porosity, and intrinsic redox activity, yet its coupling with enzyme catalysis is complex. Here, the study reports a flexible-modular approach to modify npAu with functional enzymes by combined material and protein engineering and use a tailored assortment of surface and in-solution methodologies for characterization. Self-assembled monolayer (SAM) of mercaptoethanesulfonic acid primes the npAu surface for electrostatic adsorption of the target enzyme (flavocytochrome P450 BM3; CYT102A1) that is specially equipped with a cationic protein module for directed binding to anionic surfaces. Modulation of the SAM surface charge is achieved by electrochemistry. The electrode-adsorbed enzyme retains well the activity (33%) and selectivity (complete) from in-solution. Electrochemically triggered nanoscale stirring in the internal porous network of npAu-SAM enhances speed (2.5-fold) and yield (3.0-fold) of the enzyme immobilization. Biocatalytic reaction is fueled from the electrode via regeneration of its reduced coenzyme (NADPH). Collectively, the study presents a modular design of npAu-based enzyme electrode that can support flexible bioelectrochemistry applications.
酶修饰电极是用于诊断和环境分析的电化学生物传感器的核心组件，在生物电催化方面具有广阔的应用前景。尽管经过了数十年的大量研究工作，但设计高性能的酶电极仍然具有挑战性。纳米多孔金（npAu）由于其高比表面积、可调孔隙率和固有氧化还原活性而成为先进的电极材料，但其与酶催化的耦合较为复杂。在此，该研究报告了一种灵活的模块化方法，通过材料和蛋白质工程相结合，用功能酶修饰npAu，并使用一系列定制的表面和溶液内方法进行表征。巯基乙磺酸的自组装单分子层（SAM）对npAu表面进行预处理，以实现目标酶（黄素细胞色素P450 BM3；CYT102A1）的静电吸附，该酶特别配备了一个阳离子蛋白模块，用于定向结合阴离子表面。通过电化学实现SAM表面电荷的调制。电极吸附的酶在溶液中很好地保留了活性（33%）和选择性（完全保留）。npAu-SAM内部多孔网络中的电化学触发纳米级搅拌提高了酶固定化的速度（2.5倍）和产率（3.0倍）。生物催化反应通过其还原辅酶（NADPH）的再生从电极获得燃料。总体而言，该研究提出了一种基于npAu的酶电极的模块化设计，可支持灵活的生物电化学应用。
