Construction of ultrafast-walking stochastic 3D dual-DNA walker nanoprobes for highly sensitive fluorescence detection of extracellular vesicles for cancer diagnosis

Abstract

Extracellular vesicles (EVs) serve as a promising biomarker for the early screening and monitoring of cancer through non-invasive testing. However, achieving rapid and sensitive detection of tumor-derived extracellular vesicles (TEVs) remains a significant challenge. In this study, we report an ultrafast-walking stochastic three-dimensional (3D) dual-DNA walker nanoprobes (D-DWN)-based fluorescence biosensor for the detection of EVs. The D-DWN facilitates autonomous movement on AuNP at high speeds, powered by Exo III, which results in the release of fluorophores that act as an analytical signal for the detection of target EVs. Compared to traditional DNA walkers, our proposed D-DWN demonstrates superior reaction kinetics, achieving saturation within 20 min. This advancement enables highly sensitive detection of EVs, with a linear response range from 8 × 10⁵ to 1.28 × 10⁷ particles/μL and a limit of detection of 1.18 particles/μL. Furthermore, clinical samples can be analyzed using the D-DWN to differentiate patients with cancer from healthy individuals. This work is anticipated to provide an effective tool for the accurate detection of EVs, offering potential for early cancer diagnosis and postoperative response prediction.