Li Jun, Gao Zehang, Jia Chunping, Cai Gaozhe, Feng Shilun, Wu Man, Zhao Hui, Yu Juan, Bao Fang, Cong Hui, Bian Xiaojun, Zhao Jianlong
College of Food Science and Technology, International Research Center for Food and Health, Shanghai Ocean University, Shanghai 201306, China.
State Key Laboratory of Transducer Technology, Shanghai Institute of Microsystem and Information Technology, Chinese Academy of Sciences, Shanghai 200050, China.
Anal Chem. 2024 Aug 13. doi: 10.1021/acs.analchem.4c00990.
Respiratory pathogens pose significant challenges to public health, demanding efficient diagnostic methods. This study presents an integrated microfluidic chip for the simultaneous detection of multiple respiratory pathogens. The chip integrates magnetic bead-based nucleic acid extraction and purification, acoustic streaming-driven mixing, liquid equalization, and multiplex PCR amplification with in situ fluorescence detection. Nucleic acid extraction takes only 12 min, yielding results comparable to commercial kits. Efficient mixing of magnetic beads is achieved through a combination of designed micropillars and bubble-trapping array structures. The micropillars maintain the aqueous phase in the mixing chamber, while the bubble-trapping arrays enable stable formation of bubbles, serving as a micromixer under the acoustic field. To prevent cross-contamination, an oil-encapsulated water droplet system is incorporated throughout nucleic acid extraction and PCR amplification. This assay displays remarkable multiplex analysis capability on a single chip, enabling the simultaneous detection of 12 common respiratory pathogens with a low detection limit of 10 copies/μL. Moreover, this method demonstrates excellent practical applicability in clinical nasal samples. Compared to many microfluidic chip-based molecular biology methods, the assay exhibits comparable or superior multipathogen analysis capability, sensitivity, and speed, completing the sample-to-answer process in approximately 70 min. This integrated microfluidic device offers a promising multiplex molecular diagnosis platform for on-site simultaneous detection of multiple pathogens.
呼吸道病原体对公共卫生构成重大挑战，需要高效的诊断方法。本研究提出了一种用于同时检测多种呼吸道病原体的集成微流控芯片。该芯片集成了基于磁珠的核酸提取和纯化、声流驱动混合、液体均衡以及原位荧光检测的多重PCR扩增。核酸提取仅需12分钟，结果与商业试剂盒相当。通过设计的微柱和气泡捕获阵列结构的组合实现了磁珠的高效混合。微柱在混合腔中保持水相，而气泡捕获阵列使气泡稳定形成，在声场下充当微混合器。为防止交叉污染，在整个核酸提取和PCR扩增过程中采用了油包水液滴系统。该检测方法在单个芯片上具有显著的多重分析能力，能够同时检测12种常见呼吸道病原体，检测限低至10拷贝/μL。此外，该方法在临床鼻拭子样本中显示出优异的实际适用性。与许多基于微流控芯片的分子生物学方法相比，该检测方法具有相当或更优的多病原体分析能力、灵敏度和速度，在约70分钟内完成从样本到结果的过程。这种集成微流控装置为现场同时检测多种病原体提供了一个有前景的多重分子诊断平台。
