Amplified electrochemical biosensing based on bienzymatic cascade catalysis confined in a functional DNA structure

Herein, an amplified and renewable electrochemical biosensor was developed via bienzymatic cascade catalysis of glucose oxidase (GOx) and horseradish peroxidase (HRP), which were confined in a functional Y-shaped DNA nanostructure oriented by a dual-thiol-ended hairpin probe (dSH-HP) with a paired stem as a rigid scaffold and unpaired loop as enclosed binding platform. For proof-of-concept assay of sequence-specific biomarker DNA related to Alzheimer's disease (aDNA), GOx and redox ferrocene-modified HRP (Fc@HRP) were chemically conjugated in two enzyme strands (GOx-ES1 and Fc@HRP-ES2), respectively. The repeated recycling of aDNA was powered by the displacement of GOx-ES1 by aDNA and exonuclease III (ExoIII)-assisted cleavage reaction for amplified output of numerous GOx-ES1 as dependent transducers, together with Fc@HRP-ES2 which was simultaneously hybridized with dSH-HP to assemble this DNA structure. Rationally, the bienzymatic cascade catalysis was motivated through GOx-catalyzed glucose oxidization to in situ generate hydrogen peroxide (H₂O₂) and overlapped HRP-catalyzed H₂O₂ decomposition to promote the electron transfer, producing significantly enhanced electrochemical signal of Fc with an ultrahigh sensitivity down to 0.22 fM of aDNA. Benefited from the unique design of dSH-HP-oriented bienzymatic cascades, this one-step strategy without non-specific blockers passivation was simple and renewable, and would pave a promising avenue for sensitive electrochemical assay of biomolecules.