Distinguished by the coupled catalysis-facilitated high turnover and admirable specificity, enzyme cascades have sparked tremendous attention in bioanalysis. However, three-enzyme cascade-based versatile platforms have rarely been explored without resorting to tedious immobilization procedures. Herein, we have demonstrated that formamide-converted transition metal–nitrogen–carbon (f-MNC, M = Fe, Cu, Mn, Co, Zn) with a high loading of atomically dispersed active sites possesses intrinsic peroxidase-mimetic activity following the activity order of f-FeNC > f-CuNC > f-MnNC > f-CoNC > f-ZnNC. Ulteriorly, benefitting from the greatest catalytic performance and explicit catalytic mechanism of f-FeNC, versatile enzyme cascade-based colorimetric bioassays for ultrasensitive detection of diabetes-related glucose and α-glucosidase (α-Glu) have been unprecedentedly devised using f-FeNC-triggered chromogenic reaction of 3,3′,5,5′-tetramethylbenzidine as an amplifier. Notably, several types of α-Glu substrates can be effectively utilized in this three-enzyme cascade-based α-Glu assay, and it can be further employed for screening α-Glu inhibitors that are used as antidiabetic and antiviral drugs. These versatile assays can also be extended to detect other H2O2-generating or -consuming biomolecules and other bioenzymes that are capable of catalyzing glucose generation procedures. These nanozyme-involved multienzyme cascades without intricate enzyme-engineering techniques may provide a concept to facilitate the deployment of nanozymes in celestial versatile bioassay fabrication, disease diagnosis, and biomedicine.

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