Probing the influence of crosslinkers on the properties, response, and degradation of enzymatic hydrogels for electrochemical glucose biosensing through fluorescence analysis.

Drop-cast crosslinked hydrogels are a common platform for enzymatic electrochemical biosensors. Despite the widespread use of these complex systems, there are still several questions about how their physicochemical properties affect their performance, stability, and reproducibility. In this work, first-generation faradaic biosensors composed of glucose oxidase and branched polyethyleneimine (BPEI) are prepared using either glutaraldehyde (GA) or ethylene glycol diglycidyl ether (EGDGE) as crosslinkers. While EGDGE gels present an increasing electrochemical response with increasing crosslinker concentration, the current of GA gels decreases at high crosslinker concentration probably due to the hampered diffusion on tightly networked gels. We compared different strategies to use fluorescence microscopy to gain insight into the gel structure either by labeling the gel components with fluorophores or taking advantage of the intrinsic fluorescence of the imines formed upon crosslinking with GA. By monitoring the fluorescence of the crosslinking bonds and the electrochemical response, we demonstrate that hydrolysis, a common hydrogel degradation mechanism, is not responsible for the loss of electrical current over time in gels prepared with glutaraldehyde. Most hydrogel-based electrochemical biosensor studies do not perform specific experiments to determine the cause of the degradation and instead just infer it from the dependence of the current on the preparation conditions (most commonly concentrations). We show that, by taking advantage of several analytical techniques, it is possible to gain more knowledge about the degradation mechanisms and design better enzymatic biosensors.