Multienzyme Cascades Based on Highly Efficient Metal-Nitrogen- Carbon Nanozymes for Construction of Versatile Bioassays

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 tran-sition 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 alpha-glucosidase (alpha-Glu) have been unprecedentedly devised using f-FeNC-triggered chromogenic reaction of 3,3 ' ,5,5 '-tetramethylbenzidine as an amplifier. Notably, several types of alpha-Glu substrates can be effectively utilized in this three-enzyme cascade-based alpha-Glu assay, and it can be further employed for screening alpha-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.

Automated summary

Enzyme cascades have attracted attention in bioanalysis, but versatile platforms based on three-enzyme cascades have been rarely explored. In this study, a formamide-converted transition metal-nitrogen-carbon material was used to develop a three-enzyme cascade platform without immobilization. This platform was utilized for ultrasensitive detection of diabetes-related substances and alpha-glucosidase, and for screening antidiabetic and antiviral drugs.