A 3D printed microfluidic device for scalable multiplexed CRISPR-cas12a biosensing
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. Graphical Abstract.
| Medienart: |
Artikel |
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| Erscheinungsjahr: |
2023 |
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| Erschienen: |
2023 |
| Enthalten in: |
Zur Gesamtaufnahme - volume:25 |
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| Enthalten in: |
Biomedical microdevices - 25(2023), 3 vom: 29. Aug. |
| Sprache: |
Englisch |
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| Beteiligte Personen: |
Curtin, Kathrine [VerfasserIn] |
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| Links: |
Volltext [lizenzpflichtig] |
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| Themen: |
Biosensor |
| Anmerkungen: |
© The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature 2023. Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law. |
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| doi: |
10.1007/s10544-023-00675-x |
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| funding: |
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| 520 | |a 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. Graphical Abstract | ||
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