Label-free electrical detection of salivary extracellular vesicles using an anti-CD63-functionalized gold...

Salivary extracellular vesicles (sEVs) are accessible candidates for non-invasive biomarker analysis, but their measurement commonly requires multistep, label-dependent workflows. Here a label-free gold extended-gate field-effect transistor (EG-FET) functionalized with an antibody that binds specifically to the Cluster of Differentiation 63 (anti-CD63), was developed for electrical detection of isolated salivary sEVs. Gold gates were modified with an 11-mercaptoundecanoic acid self-assembled monolayer and [1-Ethyl-3-(3-Dimethylaminopropyl) carbodiimide/N-Hydroxysuccinimide] EDC/NHS-mediated antibody immobilization. Binding-induced changes were quantified as the ratio of areas under transfer curves (AUC) acquired before and after incubation. Inter-chip coefficient of variation decreased from 10.5% for bare gates to 1.5% after antibody immobilization. The AUC ratio decreased with sEV concentration across 0.01X-1X (R² = 0.980); using the blank mean minus three standard deviations, the estimated detection threshold was approximately 0.1X (about 9.8 × 10⁷ particles/mL for the representative stock). Anti-CD63 gates exposed to 0.1X sEVs produced a significant response, whereas Phosphate-Buffered Saline (PBS), Bovine Serum Albumin (BSA), and heterologous-antibody controls remained near baseline. Four independent sessions across a 16-day interval yielded coefficients of variation of 1.4% for PBS and 1.7% for sEV measurements. Response was retained when isolated sEVs were spiked into 0.2X clarified saliva. These proof-of-concept findings support reproducible label-free sEV detection while defining the need for direct whole-saliva interference testing, storage studies, and validation in clinically characterized cohorts.