Highly sensitive and specific detection of human papillomavirus type 16 using CRISPR/Cas12a assay coupled with an enhanced single nanoparticle dark-field microscopy imaging technique

Human papillomavirus (HPV) is a prevalent sexually transmitted pathogen associated with cervical cancer. Detecting high-risk HPV (hr-HPV) infections is crucial for cervical cancer prevention, particularly in resource-limited settings. Here, we present a highly sensitive and specific sensor for HPV-16 detection based on CRISPR/Cas12a coupled with enhanced single nanoparticle dark-field microscopy (DFM) imaging techniques. Ag–Au satellites were assembled through the hybridization of AgNPs-based spherical nucleic acid (Ag-SNA) and AuNPs-based spherical nucleic acid (Au-SNA), and their disassembly upon target-mediated cleavage by the Cas12a protein was monitored using DFM for HPV-16 quantification. To enhance the cleavage efficiency and detection sensitivity, the composition of the ssDNA sequences on Ag-SNA and Au-SNA was optimized. Additionally, we explored using the SynSed technique (synergistic sedimentation of Brownian motion suppression and dehydration transfer) as an alternative particle transfer method in DFM imaging to traditional electrostatic deposition. This addresses the issue of inconsistent deposition efficiency of Ag–Au satellites and their disassembly due to their size and charge differences. The sensor achieved a remarkable limit of detection (LOD) of 10 fM, lowered by 9-fold compared to traditional electrostatic deposition methods. Clinical testing in DNA extractions from 10 human cervical swabs demonstrated significant response differences between the positive and negative samples. Our sensor offers a promising solution for sensitive and specific HPV-16 detection, with implications for cancer screening and management.