Department of Mechanical and Aerospace Engineering, West Virginia University, Morgantown, WV, USA.
C. Eugene Bennett Department of Chemistry, West Virginia University, Morgantown, WV, USA.
Biomed Microdevices. 2023 Aug 29;25(3):34. doi: 10.1007/s10544-023-00675-x.
Accurate, rapid, and multiplexed nucleic acid detection is critical for environmental and biomedical monitoring. In recent years, CRISPR-Cas12a has shown great potential in improving the performance of DNA biosensing. However, the nonspecific trans-cleavage activity of Cas12a complicates the multiplexing capability of Cas12a biosensing. We report a 3D-printed composable microfluidic plate (cPlate) device that utilizes miniaturized wells and microfluidic loading for a multiplexed CRISPR-Cas12a assay. The device easily combines loop-mediated isothermal amplification (LAMP) and CRISPR-Cas12a readout in a simple and high-throughput workflow with low reagent consumption. To ensure the maximum performance of the device, the concentration of Cas12a and detection probe was optimized, which yielded a four-fold sensitivity improvement. Our device demonstrates sensitive detection to the fg mL level for four waterborne pathogens including shigella, campylobacter, cholera, and legionella within 1 h, making it suitable for low-resource settings.
准确、快速和多重核酸检测对于环境和生物医学监测至关重要。近年来，CRISPR-Cas12a 在提高 DNA 生物传感性能方面显示出巨大的潜力。然而，Cas12a 的非特异性转切割活性使 Cas12a 生物传感的多重检测能力变得复杂。我们报告了一种 3D 打印可组合微流控板（cPlate）设备，该设备利用微型化孔和微流加载进行多重 CRISPR-Cas12a 分析。该设备可轻松结合环介导等温扩增（LAMP）和 CRISPR-Cas12a 读取，采用简单、高通量的工作流程，试剂消耗低。为了确保设备的最佳性能，我们优化了 Cas12a 和检测探针的浓度，从而将灵敏度提高了四倍。我们的设备在 1 小时内对包括志贺氏菌、弯曲杆菌、霍乱弧菌和军团菌在内的四种水传播病原体进行了 fg mL 级别的敏感检测，使其适用于资源有限的环境。
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