DNA Tetrahedron-Mediated Sensing Platform on a Single Gold Nanowire: Special Signal Amplification, Self-Referencing Capability, and Cross-Validated Exosome Detection for Liver Cancer Liquid Biopsy

Exosomes have emerged as promising biomarkers for noninvasive cancer diagnosis, while highly sensitive, reliable, and convenient assays remain challenging, particularly with conventional single-signal approaches. Herein, we report an exosome-tailored dual-mode self-referencing biosensing platform on a single gold nanowire (AuNW) substrate for the accurate and sensitive quantification of HepG2-derived exosomes, integrating surface-enhanced Raman scattering (SERS) and electrochemical readouts on a AuNW substrate with dual-functional Au nanotag amplification. The platform features three exosome-specific design strategies: CD63 aptamer‑functionalized DNA tetrahedrons (AD‑NTH) with well‑controlled probe orientation, spacing, and accessibility to enhance exosome capture efficiency and mitigate interfacial steric hindrance; dual recognition via CD63‑mediated capture and AFP‑mediated signal tagging for improved specificity toward HepG2‑derived exosomes; and a ratiometric SERS/electrochemical dual‑mode readout on a unified substrate to enhance signal reliability. This self-referencing mechanism effectively suppresses nonspecific interference and signal fluctuation, while the dual-functional Au nanotags provide amplified signal outputs. The platform achieves detection limits of 8.41 × 102 particles mL−1 (SERS) and 1.07 × 103 particles mL−1 (electrochemical). Clinical evaluation using serum samples from 10 liver cancer patients and 10 healthy volunteers shows statistically significant differentiation (P < 0.0001), supporting its promising application in noninvasive early screening and diagnosis of liver cancer.