Multienzyme, Multistep Biosensor Produced through Kinetic Doping

The recently developed kinetic doping technique has shown promise in loading individual enzymes for use as a biosensor. In this study, the first example of kinetic doping to produce a biosensor loaded with more than one enzyme and using a multistep reaction pathway for detection is presented. Glucose oxidase (GOD) is shown to load both individually and together with horseradish peroxidase (HRP) with the tandem action of the two enzymes proving to be effective at detecting glucose in solution. Using a calculation based on known maximum loadings and experimentally determined activities, the final dual-enzyme thin films of known volume are shown to contain 1.8 ± 0.1 mmol/L of HRP and 0.22 ± 0.01 mmol/L of GOD, which represent 33 and 92% of loading efficiencies that each enzyme is known to be, respectively, capable of in a singularly loaded thin film. With the high loading afforded by the kinetic doping process under benign conditions, the thin films are able to load both enzymes all at once in an amount sufficient to function as an efficient biosensor. The most advantageous aspects of this process are its ease of production, involving only a few steps to produce highly loaded thin films that require no additional processing to function as intended, as well as the protein friendly environment that exists in the sol-gel film at the time of enzyme loading. This removes many typical restrictions on immobilizing protein and opens up a wider range of enzymes amenable to the process that enables the fabrication of more complex multistep biosensors utilizing a large array of proteins in the foreseeable future.