Gap-Plasmon Metasurface Combined with Bio-Barcode of CD63 Nanoflares for SERS Detection of Cancerous Exosomes

Exosomes (Exos) have garnered significant attention as promising noninvasive biomarkers for early cancer diagnosis due to their rich molecular information derived from parent cells. However, the sensitive and accurate detection of cancerous Exos remains challenging because of their low abundance compared to Exos from healthy cells. Herein, a sensitive and "signal-off" platform for the analysis of cancerous Exos was developed using surface-enhanced Raman spectroscopy (SERS) as a readout method. A well-ordered plasmonic gold nanoparticles nanomembrane (denoted as AuNPs-NM) was designed as the SERS substrate with high reproducibility, which can produce a dense and uniform "hot spots" distribution of gap-plasmons. A bio-barcode of AuNPs decorated with the biotin-CD63 aptamer and its complementary strand (named as CD63 nanoflares) was then designed with the recognition and signal enhancement ability to boost the SERS signal by further forming "hot spots" between AuNPs-NM and CD63 nanoflares. Upon incubation with Exos, the "hot spots" between AuNPs-NM and CD63 nanoflares are destroyed, owing to the preferred binding affinity between the CD63 protein on the surface of Exos and the biotin-CD63 aptamer, which results in a significant decrease in the SERS signal. The proposed SERS platform demonstrated a wide linear relationship for Exos ranging from 1 × 10⁶ to 2 × 10⁸ particles/mL, with a limit of detection (LOD) as low as 4.7 × 10⁵ particles/mL. As a proof of concept, this SERS platform was successfully applied to distinguish cervical cancer patients from healthy individuals by analyzing Exos in clinical serum samples, enabling early and noninvasive cancer diagnosis.