A cascade nanoreactor based on metal azolate framework integrated natural enzyme for α-glucosidase activity assay and inhibitor screening

Abstract

Enzyme cascades have attracted widespread attention owing to the exceptional specificity and efficient signals transduction, however, constrained by the high cost and limited stability of bio-enzymes. In this study, a novel mimic multienzyme nanoreactor (GOx@MAF-7(Fe), i.e. GMF) was developed through a one-step encapsulation of glucose oxidase (GOx) into a metal azolate framework, MAF-7(Fe). Benefiting from the synergistic effect of GOx and the exceptional peroxidase-like (POD) activity of MAF-7(Fe), GMF enabled a robust cascade catalytic reaction for colorimetric sensing. The unique structural and functional properties of MAF-7(Fe) not only facilitated efficient enzyme immobilization but also enhanced the stability of GOx, outperforming free enzymes in terms of storage and thermal tolerance. The GMF-based platform demonstrated high sensitivity and selectivity in glucose response. More importantly, by integrating α-glucosidase (α-Glu) into a three-enzyme cascade system, a colorimetric assay was successfully developed for α-Glu activity with a detection limit of 0.25 mU/mL, surpassing most existing methods. This platform was further applied for α-Glu inhibitor screening, with acarbose as a model inhibitor, and achieved precise quantification of inhibition efficiency (IC₅₀ = 60.06 nM). This work not only establishes a versatile and efficient sensing platform for diabetes-related biomolecule detection but also pioneers a novel strategy for enzyme immobilization and multienzyme cascade construction, opening new avenues for multifunctional material design in biomedical research.