Guozhen Liu

CRISPR/Cas on Microfluidic Paper-Based Analytical Devices for Point-of-Care Screening of Cervical Cancer

Highly sensitive point-of-care early screening for high-risk human papillomavirus (HPV) infections is urgently needed, particularly in resource-limited settings. Nucleic acid amplification methods, especially CRISPR/Cas-based biosensors, have emerged as promising tools for sensitive HPV detection; however, current approaches typically rely on tedious tube-based formats coupled with lateral flow assays for signal readout in point-of-care testing (POCT). Here, we developed customized microfluidic paper-based analytical devices (μPADs) with valves that seamlessly integrated recombinase polymerase amplification (RPA) with CRISPR/Cas12a biosensing (RPA-CRISPR/Cas12a) on the filter paper substrate. This innovation achieved sensitive and cost-effective high-risk HPV detection in POCT. The RPA-CRISPR/Cas12a system with a linear reporter on μPADs, enabled fluorescence detection of the E7 gene, achieving a sensitivity of 1 pM at approximately 1 h. The sensitivity was further enhanced by introducing a circular reporter into the fluorescence-based RPA-CRISPR/Cas12a system on μPADs, enabling detection of the E7 gene with a detection limit of 1 fM and an assay time of 35 min. The system was validated using 50 cervical swab clinical samples, demonstrating 95% sensitivity and 100% specificity when compared to qPCR. This sample-to-answer detection platform holds significant promise for early screening of high-risk HPV infections in point-of-care scenarios.

T7-assisted special rolling circular amplification platform for point-of-care cervical cancer screening

Cervical cancer, primarily caused by high-risk human papillomavirus (HPV) infections, remains a leading cause of mortality among women in underdeveloped and developing countries. Conventional screening methods, necessitating professional equipment and trained personnel, are impractical in resource-limited settings, underscoring the need for a point-of-care (POC) detection platform. Isothermal nucleic acid amplification techniques (NAATs) are widely used in POC applications due to their cost-effectiveness and instrument-free nature. In this study, a novel one-pot dual amplification system, T7-MSSRCA was developed, by integrating the minimum secondary structure rolling circular amplification (MSS-RCA) with T7 Exonuclease. This system achieved an amplification time of 15 min and a total diagnostic time of approximately 1 h. Moreover, signal detection using both fluorescence and lateral flow assay (LFA) strips was achieved through reporter probe modifications. By employing a human chorionic gonadotropin (hCG)-modified reporter probe, the T7-MSSRCA system facilitated quantitative POC detection via pregnancy test strips. The system demonstrated impressive sensitivities for synthesized HPV16 targets, achieving 1 fM with fluorescent output and 10 fM with paper strips. When tested with 50 cervical swab samples, T7-MSSRCA exhibited a sensitivity of 0.95 and a specificity of 0.9333 compared to RT-PCR measurements.