Cobalt Single-Atom Oxidase Mimics for Glucose Oxidase-Free Cascade Colorimetric Detection of α-Glucosidase Activity

The precise evaluation of α-glucosidase (α-Glu) activity is important for diabetes-related diagnosis and drug screening. However, conventional nanozyme-based α-Glu assays rely on multienzyme cascade systems involving glucose oxidase (GOx), which increase assay complexity and introduce instability associated with hydrogen peroxide (H2O2). Herein, a GOx-free cascade colorimetric platform based on an atomically dispersed cobalt nanozyme (Co-NC) was established for α-Glu activity detection and inhibitor evaluation. The Co-NC catalyst exhibited intrinsic oxidase-like activity and directly activated dissolved oxygen to trigger the oxidative coupling of hydroquinone (HQ) with 4-aminoantipyrine (4-AAP). By coupling the α-Glu-mediated hydrolysis of α-Arbutin with the Co-NC-mediated chromogenic reaction, the enzymatic event was converted into a measurable optical response at 507 nm without the involvement of GOx and the unstable intermediate H2O2. The biosensor achieved sensitive quantification of α-Glu with a limit of detection of 0.19 mU/mL and satisfactory performance in serum samples. In addition, the method was successfully applied to the evaluation of three commercial α-Glu inhibitors. The proposed Co-NC-based cascade strategy provides a practical and reliable tool for α-Glu monitoring and the proof-of-concept evaluation of representative α-Glu inhibitors.