Molecular Crowding and a Minimal Footprint at a Gold Nanoparticle Support Stabilize Glucose Oxidase and Boost its Activity.

Abstract

Enzymes conjugated to nanomaterials are used in the design of various biotechnologies. In development of biosensors, sur-face modifications with the enzyme glucose oxidase (GOx) serve to aid the detection of blood glucose. In order to optimize sensor effectiveness, the enzyme tertiary structure needs to be preserved upon immobilization to retain the enzyme s catalytic activity. Due to the nature of GOx, it suffers from a tendency to denature when immobilized at a solid surface, methods to optimize enzyme stability are of great importance. Here, we introduce the study of the interaction of GOx to the highly curved surface of 20 nm gold nanoparticles (AuNP) while adsorbing a monolayer coating of enzyme as determined by floccu-lation assays and quantification of immobilized GOx at the nanoparticle surface. Enzyme crowding was determined by com-paring the numbero fo enzyme that bind to how many can physically fit.These measurements show how placing a monolayer of enzyme where the enzyme spreads thin at the AuNP surface still provides stable catalytic performance up to the 14 days compared to enzymes free in solution. Moreover, by increasing enzyme density via increasing the amount of GOx present in solution during the the GOx-AuNP conjugatation step creates a molecularly crowded environment at the highly curved nano-particle surface. This limits the size of the enzyme footprint for attachment, and shows that the activity per enzyme can be enhanced up to 300 %. This is of great importance for implementing stable and sensitive sensor technologies that are con-structed by enzyme-based nanoparticle scaffolds. Here, we show by using the conditions that maintain GOx structure and function when limiting the enzyme coating to an ultrathin layer, the design and construction of ultrafast responding diagnos-tic sensor technology for glucose can be achieved, which is crucial for monitoring rapid fluctuations of, for instance, glucose in the brain.