PolyA-Bridged Capture Probe Architecture Enables High-Efficiency DNA Hybridization for Multiplex Biosensing Applications

Abstract

DNA target-probe hybridization is a critical recognition and combination process for establishing high-performance biosensors. However, in conventional self-assembly strategies, surface-anchored capture probes exhibit heterogeneous molecular conformations and limit the kinetics of DNA hybridization at the interface. As a result, the response speed and practicability of electrochemical biosensors are quite limited, especially in real samples. Interfacial regulation of the molecular conformation using artificial DNA nanostructures has been widely recognized as a promising strategy to improve the accessibility and activity of capture probes. This study introduces a significantly simplified molecular regulatory structure on the surface of the gold electrode consisting of a probe-polyA-probe (PAP) sequence and a capture probe (CP). The PAP sequence has a central polyA fragment anchoring to the gold electrode and two flanking probes for hybridization with the two ends of the capture probe, forming a bridged CP (BCP). Upon dual-terminal hybridization with PAP, the capture probe underwent structural linearization through opposing directional extension, thereby markedly enhancing the steric accessibility and subsequent hybridization efficiency. By establishing a BCP biosensor, we achieved rapid and sensitive detection of DNA hybridization from 1 fM to 1 nM. More importantly, the platform demonstrated valuable versatility in the construction of both a gap hybridization biosensor for microRNA and a DNAzyme biosensor for Pb²⁺. The BCP biosensor exhibited exceptional biorecognition capability, achieving rapid DNA hybridization kinetics in only 3 min and a remarkable hybridization efficiency of 95.56%. Based on its high sensitivity, operational simplicity, and broad applicability, our BCP biosensor has shown an avenue for the development of novel electrochemical biosensors for molecular diagnostics and environmental monitoring.