Versatile electrochemical biosensor based on bi-enzyme cascade biocatalysis spatially regulated by DNA architecture

The regulation of biocatalytic cascades in microenvironments for high performance and extended applications is still challenging. Herein, we develop a rolling circle amplification (RCA)-based one-pot method to prepare the micron-sized DNA flowers (DFs), which achieve the co-encapsulation and spatial regulation of bi-enzyme molecules, glucose oxidase (GOx) and horseradish peroxidase (HRP). In this system, GOx and HRP are integrated into the DFs simultaneously during RCA with the bridging of magnesium between enzyme residues and phosphate backbones on DFs. The cascade of GOx/HRP is regulated with the formation of highly ordered and hydrogenbonded water environment in the cavity of DFs, resulting in an enhanced cascade catalytic efficiency compared with that in homogeneous solution. Moreover, the high density of DNA scaffold ensures the encapsulation of GOx/HRP with high efficiency. Accordingly, a glucose electrochemical biosensor with amplified signal response is fabricated using the as-prepared GOx/HRP DFs as biosensing interface, realizing sensitive detection of glucose. Further, through designing the complementary sequence of aptamer into the programmable circular template of RCA, the bi-enzyme co-encapsulated DFs are versatilely applied to sensitive and selective detection of cancerous exosomes and thrombin in signal-on and signal-off' modes, respectively, which are further applied to the analysis of complex biological samples successfully. Overall, the encapsulation of multienzyme with DFs proposes a promising strategy to regulate the microenvironment of biocatalytic cascades, which hold great potential in biotechnology, bioanalysis and disease diagnosis.

Automated summary

A one-pot method based on RCA is developed to prepare micron-sized DNA flowers (DFs) with co-encapsulation and spatial regulation of bi-enzyme molecules, GOx and HRP. The cascade of GOx/HRP is regulated in the highly ordered water environment of DFs, leading to enhanced catalytic efficiency. The versatile application of bi-enzyme co-encapsulated DFs in sensitive and selective detection of cancerous exosomes and thrombin shows great potential in bioanalysis and disease diagnosis.