Freezing-directed construction of enzyme/nano interfaces: Reagentless conjugation, superior activity, and better stability

Immobilizing enzyme to nano interfaces has demonstrated to be a favorable strategy for prompting the industrialized application of enzyme. Despite tremendous endeavor has been devoted to using gold nanoparticles (AuNPs) as conjugation matrix due to its fascinating physico-chemical properties, main-taining enzymatic activity while circumventing cumbersome modification remains a formidable chal-lenge. Herein, the freezing-directed conjugation of enzyme/nano interfaces was constructed without extra reagent. As the proof of concept, glucose oxidase (GOx) was chosen as model enzyme. The one-pot con-jugation process can be facilely completed at -20 degrees C under aqueous solution. Moreover, with the loading of GOx on AuNP at freezing, the enzyme exhibited superior catalytic activity and stability upon ther-mal and pH perturbation. The mechanism of boosted activity was then discussed in detail. It was found that higher loading density under freezing condition and more enzyme tending to bind AuNPs via Au-S bond were the main factors for the superior activity. More importantly, this methodology was universal and can also be applied to other enzyme which contains natural cysteine, such as horseradish peroxi-dase (HRP) and papain. This facile conjugation strategy accompanied by remarkable bioactivity expand the possibilities for enzymatic biosensing, microdevice and even drug delivery. (c) 2023 Published by Elsevier B.V. on behalf of Chinese Chemical Society and Institute of Materia Medica, Chinese Academy of Medical Sciences.