Single-enzyme-driven cascade amplification integrating target recycling and DNA walker for highly sensitive nucleic acid detection

Abstract

Highly sensitive nucleic acid detection is essential for biomedical diagnostics and bioanalytical applications, yet remains challenging due to the limited amplification efficiency and system complexity of existing sensing strategies. Herein, we report a simplified yet efficient cascade amplification strategy for nucleic acid detection by exploiting the multifunctionality of Exonuclease III (Exo III). In this design, a single Exo III enzyme simultaneously drives two mechanistically distinct amplification processes. In the first stage, target DNA triggers Exo III-assisted strand displacement, enabling efficient target recycling and generation of abundant trigger strands. In the second stage, the released strands activate an Exo III-powered DNA walker amplification process on gold nanoparticle surfaces, resulting in amplified fluorescence signal. Benefiting from the synergistic integration of solution-phase target recycling and interface-confined DNA walker-based amplification, the proposed system achieves high sensitivity with reduced enzymatic and operational complexity, and the limit of detection is 48.8 pM. This work provides a versatile and sequence-independent cascade amplification paradigm for advanced nucleic acid biosensing applications.