A cascade system constructed with dual-enzyme active carbon dot nanozymes for dual-color sensing of alkaline phosphatase in gels.

The controllable synthesis of multi-enzymic active carbon dots (CDs) and the construction of a multi-channel, highly sensitive enzyme-linked detection platform are of great significance in biochemical analysis. This paper reports the synthesis of a novel CDs nanozyme (Cu/Co-CDs) with yellow fluorescence and both laccase-like and peroxidase-like activities through a one-step hydrothermal route via a bimetallic doping strategy. The catalytic mechanism and the enzyme kinetic parameters of the Cu/Co-CDs were investigated through optimization of synthesis conditions and various characterization techniques. Two colorimetric sensing platforms were constructed for highly sensitive detection of alkaline phosphatase (ALP) activity via cascade catalysis. On one hand, thanks to the laccase-like activity of Cu/Co-CDs, a cascade catalysis reaction between ALP and the CDs convert disodium phenyl phosphate into a red quinone imine product, establishing a red color-enhanced detection mode with a detection limit of 0.06 U/L. On the other hand, the peroxidase-like activity of Cu/Co-CDs coupled with ALP can convert L-ascorbic acid-2-phosphate to ascorbic acid, inhibiting the oxidative coloration of TMB, and thus establishing a blue color-reduced detection mode, and a detection limit of 0.08 U/L. Furthermore, a portable hydrogel sensing system, with the assistance of the RGB readout function of a smartphone, was further constructed to enable an on-site visual detection of ALP. This research provides a general strategy for the controllable synthesis of multi-enzyme active CDs and also expands the application prospects of multi-enzyme active nanozymes in biosensing and point-of-care testing.