CRISPR/Cas12a and CHA-based SERS platform for ultrasensitive nucleic acid detection

Highly sensitive nucleic acid detection is essential for analytical applications. Conventional methods often require complex pre-amplification procedures, limiting their practical utility in screening. Developing nucleic acid detection strategies with high sensitivity and selectivity, without target gene pre-amplification, remains a significant challenge.

This study integrates CRISPR/Cas12a recognition, catalytic hairpin assembly (CHA) amplification, and surface-enhanced Raman spectroscopy (SERS). CRISPR/Cas12a recognizes target nucleic acids and cleaves single-stranded DNA (ssDNA), thereby blocking the toehold-mediated strand displacement reaction (TSDR) and triggering CHA. Hairpin probe HP₁ with C-Ag⁺-C structures bridges CHA, releasing Ag⁺ through cyclic amplification. Ag⁺ induces charge transfer and aggregation of AgNPs@4-ABT, generating strong SERS signals. The platform achieved femtomolar sensitivity and high selectivity in detecting pCaMV35S, with 96.4% accuracy in maize seeds and 100% in maize leaves.

This strategy eliminates the need for pre-amplification of target genes by combining CRISPR's targeting feature, CHA, and ultrasensitive SERS detection. It demonstrates excellent performance in genetically modified organism screening, seed quality testing, and leaf sample analysis, providing a promising tool for food safety and agricultural regulation.