3D DNA walker-powered biological field-effect-transistors (BioFETs) for ultrasensitive cancer exosome detection

Exosomes (30–150 nm) are phospholipid nanovesicles that carry molecular cargo reflecting their cellular origin, making them promising non-invasive biomarkers for cancer detection. Herein, we report the first 3D DNA walker-powered graphene field-effect transistor (GFET) biosensing platform for ultrasensitive detection of HER2-positive breast cancer exosomes. The assay integrates a two-stage, cascade amplification strategy. First, specific recognition of HER2-positive exosomes induces aptamer displacement, thereby activating DNAzyme‑powered 3D DNA walkers that catalytically cleave substrate strands in the presence of Zn 2+ , continuously releasing single-stranded DNA (ssDNA) reporters. Second, the released ssDNA is captured by hairpin probes at the GFET gate interface, increasing the local negative charge within the Debye screening length and producing a shift in the charge neutrality point voltage (V cnp ). By measuring the signal change, the platform enables quantitative detection within ~1.5 h and achieves a limit of detection (LOD) of 1.57 particles µL −1 . Furthermore, this DNA walker-powered GFET platform was validated using clinical plasma samples and successfully distinguished HER2-positive from HER2-negative breast cancer patients. • First GFET BioFET integrating 3D DNA walkers for ultrasensitive detection of HER2-positive breast cancer exosomes • DNAzyme-driven autonomous walking enabled catalytic cleavage and continuous ssDNA reporter generation • Two-stage amplification enriched ssDNA reporters at the GFET interface to reduce ionic screening and enhance transduction. • Achieved a detection limit of 1.57 particles µL⁻¹ with high specificity for HER2-positive exosomes • Validated with clinical plasma samples, enabling discrimination between HER2-positive and HER2-negative breast cancer patients