Single-Nucleotide Variation Analysis in Oral Squamous Cell Carcinoma-Related ctDNA by dCas9/sgRNA Recognition-Mediated Proximity Ligation-Triggered Terminal Hairpin Formation and Self-Priming Amplification

Circulating tumor DNA (ctDNA) represents a promising noninvasive biomarker for cancer diagnosis, including oral cancer. However, its clinical translation is currently limited by the lack of precise and reliable detection techniques. In this study, we developed a novel fluorescent biosensor for the detection of single-nucleotide variations in ctDNA, which integrates dual dCas9/sgRNA complexes for target recognition, proximity ligation-initiated terminal hairpin formation and self-priming amplification (PS-THSP), and Cas12a/crRNA-mediated signal output. A key innovation of this design is its multilayered specificity strategy, combining mutation-specific recognition by dual dCas9/sgRNA, proximity-dependent ligation, and Cas12a/crRNA-assisted verification of PS-THSP amplicons. This integrated approach offers a significant advance over existing CRISPR/Cas-based methods that rely primarily on signal amplification. Furthermore, the biosensor achieves high sensitivity through the synergistic coupling of PS-THSP amplification and Cas12a trans-cleavage activity, enabling a broad dynamic range spanning 6 orders of magnitude and a detection limit as low as 0.12 fM within 120 min. When applied to serum samples, the biosensor reliably detected ctDNA with high accuracy, demonstrating its strong potential for clinical cancer diagnostics.