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.

Kinetically Doped Silica Sol-Gel Optical Biosensors: Expanding Potential Through Dip-Coating.

Kinetic doping has previously been shown to be an effective method of doping silica sol-gel thin films with an enzyme to construct biosensors. Until now, kinetic doping has only been applied to films produced through the spin-coating method. In this study, we present the use of dip-coating to produce thin films kinetically doped for biosensor development. In this way, kinetically doped biosensors may benefit from the increased range of substrate material shapes and sizes that may be easily coated through dip-coating but not spin-coating. The biosensors produced through dip-coating continue to show enhanced performance over more conventional enzyme loading methods with horseradish peroxidase and cytochrome C samples, showing an increase of 2400× and 1300× in enzyme concentration over that in their loading solutions, respectively. These correspond to enzyme concentrations of 5.37 and 10.57 mmol/L all while preserving a modest catalytic activity for the detection of hydrogen peroxide by horseradish peroxidase. This leads to a 77% and 88% increase in the total amount of horseradish peroxidase and cytochrome C, respectively, over that from coating the same glass coverslip via spin-coating methods.

Silica Sol-Gel Optical Biosensors: Ultrahigh Enzyme Loading Capacity on Thin Films via Kinetic Doping.

Easy to use and easy to produce biosensors would have a huge range of applications. To reach this goal many see the incorporation of a protein into a sol-gel network as one of the most viable options. The current most prevalent technique of predoping presents inherent limits on the concentration possible for the resulting thin film. In this study we demonstrate a new process utilizing the newly developed kinetic doping method to load silica sol-gel thin films with cytochrome C (CytC) and horseradish peroxidase (HRP). Both enzymes are shown to successfully load and have a concentration increase over their original loading solution by factors of 1300× and 2600×, respectively. Furthermore, each enzyme once loaded retained the ability to act as a catalyst for the detection of hydrogen peroxide. Ultimately the CytC- and HRP-loaded thin films were found to have enzyme concentrations of 11 ± 1 mM and 6.0 ± 0.4 mM, respectively, a considerable step up from any doping method reported in the past.