CRISPR/Cas12a-based transition state molecular switch for low-background detection of HPV-16 on a microfluidic...

Human papillomavirus type 16 (HPV-16) is a high-risk oncogenic subtype of HPV, strongly associated with the pathogenesis of multiple cancers. Researchers have developed many detection methods for HPV-16, among which, the detection method based on microfluidic has the characteristics of high efficiency and high sensitivity. However, non-specific adsorption remains a critical challenge, often leading to elevated background signals. Here, we propose an On-Chip assay integrated by transition state molecular switch based on CRISPR-Cas12a (OCTMS-CRISPR) for stable, sensitive, and low-background fluorescence detection of HPV-16. This system leverages a highly integrated molecular switch and Cas12a to perform dual-screening, while the dissociation products of the molecular switch activate trans-cleavage activity. Our data suggest that OCTMS-CRISPR suppresses background signals on microfluidic chip while maintaining the specificity and sensitivity of trans-cleavage. For demonstration, we detected five HPV subtypes and base mismatches at varying positions and quantities. The LOD can reach 7.64 pM (average fluorescence intensity) and 9.91fM (pixel counting), showing great potential in the field of biosensing and DNA chips.