AuNPs/CaHF NPs/N-GDY as bifunctional nanozyme breaking pH limitation for miRNA-21 sensitive detection at physiological pH

Nanozyme cascade have garnered substantial interest in recent years due to their distinctive properties. However, the conventional stepwise cascade reaction undergoes tedious two-step operation process owing to the incompatibility of reaction conditions. Moreover, most of reported nanozymes exhibit favorable catalytic performance only in acidic medium, which greatly restricts their usage especially in biochemical analysis. To address above challenges, we developed gold nanoparticles/calcium hexacyanoferrate (Ⅲ)/nitrogen-doped graphitic alkyne (AuNPs/CaHF NPs/N-GDY) nanozyme with superior cascade catalytic activity at neutral pH comparable to that of acidic. Specifically, AuNPs/CaHF NPs/N-GDY simultaneously possessed glucose oxidase-like (GOx) and peroxidase-like (HRP) activities, which could induce one-step cascade reaction in the presence of glucose, resulting in 5-fold enhancement in catalytic efficiency compared with conventional two-step cascade reaction. Besides, tripedal DNA walker was equipped with sufficient walking legs to walk on directional and highly controllable stepped track, reducing the possibility of derailment and boosting walking efficiency. As a proof of concept, a novel electrochemical biosensor was constructed for miRNA-21 sensitive detection at physiological pH, and successfully applied in human serum samples as well as practical intracellular analysis, offering great potential in biomedical research and clinical diagnosis.