A Multiplexed CRISPR-Cas12a-based Microfluidic Device for Waterborne Pathogen Detection

Description:
Accurate, rapid, and multiplexed nucleic acid detection is critical for environmental and biomedical monitoring. Waterborne pathogens are one of the leading causes of diarrheal infections in areas that lack adequate sanitation. However, diarrheal disease is largely preventable through rapid detection of these pathogens in drinking water sources. Determining if drinking water is contaminated requires screening multiple species of bacteria such as shigella, campylobacter, cholera, and legionella. 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 CRIPSR-Cas12a readout in a simple and high-throughput workflow with low reagent consumption. To ensure maximum performance of the device, the concentration of Cas12a and detection probe were optimized, which yielded a four-fold sensitivity improvement. Our device demonstrates high sensitivity detection to the pg/mL level for four waterborne pathogens within 1 hour, making it suitable for low-resource settings.