Shu Taowei, Yin Xueer, Xiong Qiangyuan, Hua Changyi, Bu Junjie, Yang Ke, Zhao Jun, Liu Yong, Zhu Ling, Zhu Cancan
School of Biomedical Engineering, Anhui Medical University, Hefei, 230032, PR China; Anhui Institute of Optics and Fine Mechanics, Hefei Institutes of Physical Science, Chinese Academy of Sciences, Hefei, 230031, PR China.
Anhui Institute of Optics and Fine Mechanics, Hefei Institutes of Physical Science, Chinese Academy of Sciences, Hefei, 230031, PR China; University of Science and Technology of China, Hefei, 230026, PR China.
Biosens Bioelectron. 2025 Apr 15;274:117178. doi: 10.1016/j.bios.2025.117178. Epub 2025 Jan 18.
Pathogen nucleic acid detection technology based on isothermal amplification and CRISPR/Cas12a system offers advantages in terms of high sensitivity, high specificity, and rapidity. However, this method has not been widely applied because of its shortcomings in utilizing conventional instruments, which cannot satisfy the requirements for Point of Care Testing (POCT), such as integration, convenience, and miniaturization. In this study, we developed an integrated lift-heater centrifugal microfluidic platform (Lift-CM) to automate the processes of isothermal amplification and CRISPR/Cas12a detection. A spatially encoded centrifugal microfluidic disc (SEC-disc) was employed to physically separate the amplification and detection processes while expanding the number of targets. The design of the dual-temperature and lift-heating centrifugal mechanism of the Lift-CM platform ensures that there is no manual intervention during amplification and detection processes. A smartphone-based app enables the setting of key parameters and monitoring of the experimental process, presenting results through a generated report that includes real-time fluorescence curves. We analyzed the plasmids of the Crimean-Congo hemorrhagic fever virus, Ebola virus, and five influenza viruses using different amplification methods (RPA/LAMP) to demonstrate the good compatibility of the Lift-CM platform with various amplification schemes. In clinical validation, the detection of H3N2-positive samples was completed within 30 min, and the results were highly consistent with qPCR results. This portable and compact platform offers a novel alternative solution for both clinical and at-home pathogen nucleic acid detection in the future.
基于等温扩增和CRISPR/Cas12a系统的病原体核酸检测技术在灵敏度高、特异性高和快速性方面具有优势。然而，由于该方法在使用传统仪器方面存在不足，无法满足即时检测（POCT）对集成化、便利性和小型化的要求，因此尚未得到广泛应用。在本研究中，我们开发了一种集成式升降加热离心微流控平台（Lift-CM），以实现等温扩增和CRISPR/Cas12a检测过程的自动化。采用空间编码离心微流控盘（SEC盘）在扩大目标数量的同时，对扩增和检测过程进行物理分离。Lift-CM平台的双温度和升降加热离心机制设计确保了扩增和检测过程无需人工干预。基于智能手机的应用程序能够设置关键参数并监控实验过程，通过生成的包含实时荧光曲线的报告呈现结果。我们使用不同的扩增方法（RPA/LAMP）分析了克里米亚-刚果出血热病毒、埃博拉病毒和五种流感病毒的质粒，以证明Lift-CM平台与各种扩增方案具有良好的兼容性。在临床验证中，30分钟内完成了H3N2阳性样本的检测，结果与qPCR结果高度一致。这个便携且紧凑的平台为未来临床和家庭病原体核酸检测提供了一种新颖的替代解决方案。
