Abstract
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− 1 level for four waterborne pathogens including shigella, campylobacter, cholera, and legionella within 1 h, making it suitable for low-resource settings.
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Acknowledgements
This work was partly supported by the National Institute of Health (R01GM135432) and National Science Foundation (ECCS-2144216). KC acknowledges NSF Graduate Research Fellowship Program (GRFP) grant (#13300542). We acknowledge the use of WVU BioNano Shared Research Facilities.
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K.C. and P.L. conceived and planned the experiments. K.C., J.W., B.F., and B.B. carried out the experiments. K.C., J.W., B.F., and B.B. contributed to the device preparation. K.C. and P.L. contributed to the interpretation of the results. K.C. took the lead in writing the manuscript. All authors reviewed and edited the manuscript.
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Curtin, K., Wang, J., Fike, B. et al. A 3D printed microfluidic device for scalable multiplexed CRISPR-cas12a biosensing. Biomed Microdevices 25, 34 (2023). https://doi.org/10.1007/s10544-023-00675-x
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DOI: https://doi.org/10.1007/s10544-023-00675-x