Yang Rui, Guan Xin, Zhang Jiongyu, Moon Jeong, Guo Chong, Jia Zhengyang, Hou Chengyu, Ganbaatar Uyanga, Tricarico Steven, Ma Anson W K, Liu Changchun
Department of Biomedical Engineering, University of Connecticut Health Center, Farmington, CT, 06030, United States; Department of Biomedical Engineering, University of Connecticut, Storrs, CT, 06269, United States.
Department of Biomedical Engineering, University of Connecticut Health Center, Farmington, CT, 06030, United States.
Biosens Bioelectron. 2025 Mar 1;271:117054. doi: 10.1016/j.bios.2024.117054. Epub 2024 Dec 9.
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.
快速、灵敏且特异的核酸检测方法在临床诊断和医疗保健中发挥着关键作用。在此，我们报道了一种用于基于CRISPR的核酸检测的新型两亲性DNA荧光探针。与传统的荧光团-淬灭剂探针检测系统不同，我们的两亲性DNA荧光探针具有一个疏水的Cy5荧光团头部和亲水的单链DNA（ssDNA）尾部。通过将两亲性DNA荧光探针与纸基微流控装置相结合，我们开发了一种无淬灭剂的、基于CRISPR的用于目标核酸定量的检测系统。在目标核酸存在的情况下，激活的CRISPR-Cas12a酶切割两亲性DNA荧光探针的亲水ssDNA尾部，释放出疏水的Cy5头部并改变CRISPR反应溶液的润湿性。当将CRISPR反应产物应用于纸基微流控装置时，切割后的Cy5头部沿亲水微流控通道的迁移受到限制。目标核酸浓度越高，荧光扩散距离越短，从而能够对核酸目标进行可视化定量。我们使用人乳头瘤病毒16型（HPV-16）DNA作为模型来评估该系统的分析性能。此外，我们通过检测临床拭子样本验证了其临床可行性，获得了与传统PCR方法相当的结果。我们的无淬灭剂的基于CRISPR的检测系统在HPV相关癌症和其他传染病的简单、经济且灵敏的临床诊断方面显示出潜力。
