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Design of amperometric biosensors and biofuel cells by the reconstitution of electrically contacted enzyme electrodes

机译：通过电接触酶电极的重构设计电流型生物传感器和生物燃料电池

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The electrical contacting of redox enzymes with electrodes is the most fundamental requirement for the development of amperometric biosensors and biofuel cell elements. For the effective electrical communication of redox enzymes with electrodes the use of electron relay units that transport the electrons between the enzyme redox center and the conducting surface is essential. Also, the structural alignment of the redox enzyme units in respect to the electrode in a configuration where the enzyme redox center is in close proximity to the conductive surface is needed. The present report summarizes the reconstitution paradigm developed by our laboratory in the last decade as a versatile method to electrically contact redox enzymes with electrodes, and as a generic approach to develop amperometric biosensors and biofuel cell elements. The process is based on the reconstitution of the apo-enzyme on a relay-cofactor monolayer on thin film-functionalized electrode. Different relay units were used to electrically communicate flavin adenine dinucleotide (FAD)-containing enzymes (flavoenzymes) or pyrroloquinoline quinone (PQQ)-containing enzymes with electrodes. This included molecular redox-active relays, molecular redox-active 'shuttles', redox-active polymers (e.g., polyaniline), An nanoparticles, and carbon nanotubes. The reconstitution of different apo-enzymes on these relay-cofactor-functionalized electrodes led to unprecedented efficient electrical contacting between the redox centers of the enzymes and the electrodes. Besides very sensitive amperometric biosensors that emerged from this method, the resulting amperometric biosensors revealed high selectivity and specificity. A related approach to establish electrical contact between redox enzymes dependent on diffusional cofactors and electrodes and to develop an integrated bioelectrocatalytically active enzyme electrode was developed. The method involved assembly of a relay-cofactor diad on the electrode, and the surface crosslinking of an affinity complex generated between the enzyme and the surface-confined cofactor units. This method was successfully applied to electrically contact nicotinamide adenine dinucleotide (phosphate) NAD(P)(+)-dependent enzymes and cytochrome c-dependent enzymes. For example, enzyme-modified electrodes for the bioelectrocatalyzed oxidation of alcohol, lactate and malate were fabricated by the electrical contacting of the respective NAD(P)(+)-dependent dehydrogenases. Similarly, the bioelectrocatalytic reduction of O-2 was accomplished by an integrated cytochrome c/cytochrome oxidase-functionalized electrode. The electrically contacted enzyme electrodes were also used to develop noncompartmentalized biofuel cell elements. Biofuel cell elements consisting of electrically contacted reconstituted enzyme electrodes were constructed. Glucose or alcohol were used in these systems as fuel substrates and O-2 as oxidizer.
机译：氧化还原酶与电极的电接触是发展电流型生物传感器和生物燃料电池元件的最基本要求。为了使氧化还原酶与电极有效地电连通，必须使用在酶氧化还原中心和导电表面之间传输电子的电子中继单元。此外，在酶氧化还原中心与导电表面非常接近的构造中，需要氧化还原酶单元相对于电极的结构对准。本报告总结了我们实验室在过去十年中开发的重构范例，它是一种使氧化还原酶与电极电接触的通用方法，并且是开发电流型生物传感器和生物燃料电池元件的通用方法。该方法基于脱脂酶在薄膜功能化电极上的中继辅因子单层上的重构。使用不同的继电器单元将含黄素腺嘌呤二核苷酸（FAD）的酶（flavoenzymes）或含吡咯并喹啉醌（PQQ）的酶与电极进行电通讯。这包括分子氧化还原活性继电器，分子氧化还原活性“穿梭”，氧化还原活性聚合物（例如聚苯胺），纳米颗粒和碳纳米管。这些中继辅因子功能化的电极上不同脱辅基酶的重构导致了酶的氧化还原中心与电极之间空前有效的电接触。除了从该方法中出现的非常灵敏的安培生物传感器之外，所得的安培生物传感器还显示出高选择性和特异性。开发了一种相关的方法，以建立依赖于扩散辅因子的氧化还原酶与电极之间的电接触，并开发集成的生物电催化活性酶电极。该方法包括在电极上组装辅助辅因子二单元组，以及在酶和表面限制的辅因子单元之间产生的亲和复合物的表面交联。此方法已成功地应用于电接触烟酰胺腺嘌呤二核苷酸（磷酸盐）NAD（P）（+）依赖酶和细胞色素c依赖酶。例如，通过相应的NAD（P）（+）依赖性脱氢酶的电接触，制备了用于乙醇，乳酸和苹果酸的生物电催化氧化的酶修饰电极。同样，O-2的生物电催化还原是通过集成的细胞色素c /细胞色素氧化酶功能化电极完成的。电接触的酶电极也被用于开发无隔室的生物燃料电池元件。构建了由电接触的重组酶电极组成的生物燃料电池元件。在这些系统中，葡萄糖或酒精被用作燃料基质，O-2被用作氧化剂。

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