Quencher-free CRISPR-based molecular detection using an amphiphilic DNA fluorescence probe

Rapid, sensitive, and specific nucleic acid detection methods play crucial roles in clinical diagnostics and healthcare. Here, we report a novel amphiphilic DNA fluorescence probe for CRISPR-based nucleic acid detection. Unlike conventional fluorophore-quencher probe detection system, our amphiphilic DNA fluorescence probe features a hydrophobic Cy5 fluorophore head and a hydrophilic single-stranded DNA (ssDNA) tail. By combining the amphiphilic DNA fluorescence probe with a paper-based microfluidic device, we developed a quencher-free, CRISPR-based detection system for target nucleic acid quantification. In the presence of the target nucleic acid, the activated CRISPR-Cas12a enzyme cleaves the hydrophilic ssDNA tail of the amphiphilic DNA fluorescence probe, releasing the hydrophobic Cy5 head and altering the wettability of the CRISPR reaction solution. When the CRISPR reaction product is applied to the paper-based microfluidic device, the migration of the cleaved Cy5 head along the hydrophilic microfluidic channel is limited. The higher the target nucleic acid concentration, the shorter the fluorescence diffusion distance, enabling visual quantification of the nucleic acid target. We used human papillomavirus-16 (HPV-16) DNA as a model to evaluate the analytical performance of the system. Furthermore, we validated its clinical feasibility by testing clinical swab samples, achieving results comparable to the traditional PCR method. Our quencher-free CRISPR-based detection system shows potential for simple, affordable, and sensitive clinical diagnostics of HPV-associated cancer and other infectious diseases.