Surface enhanced Raman scattering tag enabled ultrasensitive molecular identification of Hippocampus trimaculatus based on DNA barcoding

Rapid and precise DNA barcode-based identification of biological species holds significant potential for pharmaceutical authentication and biomedical diagnostics. Herein, we present a polymerase chain reaction (PCR)–surface-enhanced Raman scattering (SERS) platform that integrates SERS tags for ultrasensitive and fast authentication of Hippocampus trimaculatus, a high-value traditional Chinese medicine (TCM). The SERS tags are composed of gold nanostars, near-infrared cyanine7 Raman reporters and carboxylated polystyrene shells, which achieve single-particle detection sensitivity under 780 nm irradiation. The tags also show excellent colloidal and SERS stability under physiologically relevant conditions (e.g., phosphate buffer saline, serum, 1 mM NaCl, and pH 1–12), with signal variations less than 5 %. The carboxylated polystyrene shells enable efficient DNA functionalization. Leveraging these advancements, the PCR-SERS assay detects genomic DNA (gDNA) at concentrations as low as 10 copies/μL within 20 thermal cycles, with remarkable specificity for Hippocampus trimaculatus over four common adulterant species. Notably, the method reduces amplification requirements to 5 thermal cycles (detection limit of 10⁶ copies/μL) while completing the entire workflow in less than 30 min (conventional qPCR, 20–30 cycles, 1–2 h). Beyond TCM verification, this PCR-SERS platform holds broad applicability for rapid nucleic acid detection in fields ranging from environmental eDNA monitoring to point-of-care diagnostics.