Enzymatic reactivity of glucose oxidase confined in nanochannels

•We establish a biocompatible system that enzyme is confined in PAA nanochannels.•The real-time electrochemical response to glucose is detected by our system.•Ionic strength, enzyme amount and pore diameter could affect the enzymatic reaction.•The steric and electrostatic effects on the reaction in nanochannel are investigated.

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 (Kmapp) 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.