State Key Laboratory of Bioelectronics, School of Chemistry and Chemical Engineering, Southeast University, Nanjing 211189, PR China.
State Key Laboratory of Bioelectronics, School of Chemistry and Chemical Engineering, Southeast University, Nanjing 211189, PR China.
Biosens Bioelectron. 2014 May 15;55:307-12. doi: 10.1016/j.bios.2013.12.042. Epub 2013 Dec 27.
The construction of nanodevices coupled with an integrated real-time detection system for evaluation of the function of biomolecules in biological processes, and enzymatic reaction kinetics occurring at the confined space or interface is a significant challenge. In this work, a nanochannel-enzyme system in which the enzymatic reaction could be investigated with an electrochemical method was constructed. The model system was established by covalently linking glucose oxidase (GOD) onto the inner wall of the nanochannels of the porous anodic alumina (PAA) membrane. An Au disc was attached at the end of the nanochannels of the PAA membrane as the working electrode for detection of H2O2 product of enzymatic reaction. The effects of ionic strength, amount of immobilized enzyme and pore diameter of the nanochannels on the enzymatic reaction kinetics were illustrated. The GOD confined in nanochannels showed high stability and reactivity. Upon addition of glucose to the nanochannel-enzyme system, the current response had a calibration range span from 0.005 to 2 mM of glucose concentration. The apparent Michaelis-Menten constant (K(m)(app)) of GOD confined in nanochannel was 0.4 mM. The presented work provided a platform for real-time monitoring of the enzyme reaction kinetics confined in nanospaces. Such a nanochannel-enzyme system could also help design future biosensors and enzyme reactors with high sensitivity and efficiency.
构建纳米器件并结合集成实时检测系统，用于评估生物过程中生物分子的功能和发生在受限空间或界面的酶反应动力学，这是一个重大挑战。在这项工作中，构建了一种纳米通道-酶系统，可以用电化学方法研究酶反应。该模型系统是通过将葡萄糖氧化酶（GOD）共价连接到多孔阳极氧化铝（PAA）膜的纳米通道内表面来建立的。在 PAA 膜的纳米通道末端连接一个 Au 盘作为工作电极，用于检测酶反应的 H2O2 产物。研究了离子强度、固定化酶量和纳米通道孔径对酶反应动力学的影响。限制在纳米通道内的 GOD 表现出高稳定性和反应性。当将葡萄糖添加到纳米通道-酶系统中时，电流响应具有从 0.005 到 2 mM 葡萄糖浓度的校准范围。限制在纳米通道内的 GOD 的表观米氏常数（K(m)(app)）为 0.4 mM。本工作为实时监测限制在纳米空间内的酶反应动力学提供了一个平台。这种纳米通道-酶系统还有助于设计具有高灵敏度和高效率的未来生物传感器和酶反应器。
