Cascade reaction biosensor based on Cu/N co-doped two-dimensional carbon-based nanozyme for the detection of lactose and β-galactosidase

Developing high-performance nanozyme for the construction of high-sensitivity biosensor platform is of great significance. Herein, we have designed and synthesized a Cu/N co-doped carbon-based nanozyme by anchoring Cu atoms on two-dimensional carbon nanosheet (Cu/NC NS). The Cu/NC NS possessed ultra-high Cu loading, superior peroxidase-like catalytic activity, great stability and reusability. Significantly, the Cu/NC NS exhibited high affinity towards H2O2 and 3,3',5,5'-tetramethylbenzidine (TMB) under acidic condition, which could induce obvious color changes of TMB in the presence of low H2O2 concentration. By integrating the Cu/NC NS with β-galactosidase (β-Gal) and galactose oxidase (Gal Ox), a multienzyme cascade colorimetric sensing system was established for the detection of lactose and β-Gal, which realizing the assay of lactose and β-Gal in the linear ranges of 0.1-1.4 mM and 0.025-0.2 U/mL, respectively. And the limit of detection for lactose and β-Gal were 0.03 mM and 0.01 U/mL, respectively. Furthermore, this work achieved the accurate detection of the content of lactose in milk and the activity of β-Gal in human serum, exploiting a novel application for nanozyme in biosensing.