Quantitative Comparison of Enzyme Immobilization Strategies for Glucose Biosensing in Real‐Time Using Fast‐Scan Cyclic Voltammetry Coupled with Carbon‐Fiber Microelectrodes

Electrochemical monitoring of non‐electroactive species requires a biosensor that is stable and selective, with sensitivity to physiological concentrations of targeted analytes. We have combined glucose oxidase‐modified carbon‐fiber microelectrodes with fast‐scan cyclic voltammetry for real‐time measurements of glucose fluctuations in brain tissue. Work presented herein quantitatively compares three approaches to enzyme immobilization on the microelectrode surface—physical adsorption, hydrogel entrapment, and entrapment in electrospun nanofibers. The data suggest that each of these methods can be used to create functional microbiosensors. Immobilization of glucose oxidase by physical adsorption generates a biosensor with poor sensitivity to glucose and unstable performance. Entrapment of glucose oxidase in poly(vinyl alcohol) nanofibers generates microbiosensors that are effective for glucose measurements over a large linear range, and that may be particularly useful when targeting glucose concentrations in excess of 3 mm, such as in blood. Hydrogel entrapment is the most effective in terms of sensitivity and stability. These microbiosensors can be used for simultaneous monitoring of glucose and dopamine in real time. The findings outlined herein should be applicable to other oxidase enzymes, and thus they are broadly important for the development of new tools for real‐time measurements of fluctuating molecules that are not inherently electroactive. Glucose oxidase‐modified carbon‐fiber microelectrodes can be combined with fast‐scan cyclic voltammetry for real‐time measurements of glucose fluctuations in brain tissue. Work presented herein quantitatively compares three approaches to enzyme immobilization on the microelectrode surface—physical adsorption, hydrogel entrapment, and entrapment in electrospun nanofibers. The data suggest that each of these methods can be used to create functional microbiosensors, but hydrogel entrapment is the most effective in terms of sensitivity and stability.