Liu Shixian, Yu Tian, Song Libing, Kalantar-Zadeh Kourosh, Liu Guozhen
Integrated Devices and Intelligent Diagnosis (ID2) Laboratory, CUHK(SZ)-Boyalife Joint Laboratory of Regenerative Medicine Engineering, School of Medicine, The Chinese University of Hong Kong, Shenzhen 518172, China.
Department of Experimental Research State Key Laboratory of Oncology in South China, Collaborative Innovation Center for Cancer Medicine, Sun Yat-sen University Cancer Center, Guangzhou 510060, China.
ACS Sens. 2025 Jun 27;10(6):4569-4579. doi: 10.1021/acssensors.5c00863. Epub 2025 May 19.
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.
迫切需要对高危型人乳头瘤病毒（HPV）感染进行高灵敏度的即时早期筛查，尤其是在资源有限的环境中。核酸扩增方法，特别是基于CRISPR/Cas的生物传感器，已成为用于灵敏检测HPV的有前景的工具；然而，目前的方法通常依赖于繁琐的基于试管的形式，并结合侧向流动分析以进行即时检测（POCT）中的信号读出。在此，我们开发了定制的带有阀门的微流控纸基分析装置（μPAD），该装置在滤纸基质上无缝集成了重组酶聚合酶扩增（RPA）与CRISPR/Cas12a生物传感（RPA-CRISPR/Cas12a）。这一创新实现了POCT中灵敏且经济高效的高危型HPV检测。μPAD上带有线性报告分子的RPA-CRISPR/Cas12a系统能够对E7基因进行荧光检测，在约1小时内实现了1 pM的灵敏度。通过在μPAD上基于荧光的RPA-CRISPR/Cas12a系统中引入环状报告分子，灵敏度进一步提高，能够检测E7基因，检测限为1 fM，检测时间为35分钟。该系统使用50份宫颈拭子临床样本进行了验证，与qPCR相比，灵敏度为95%，特异性为100%。这个从样本到答案的检测平台在即时检测场景中对高危型HPV感染的早期筛查具有重大前景。
