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A 3D printed microfluidic device for scalable multiplexed CRISPR-cas12a biosensing.

Kathrine Curtin1, Jing Wang2, Bethany J Fike2

  • 1Department of Mechanical and Aerospace Engineering, West Virginia University, Morgantown, WV, USA.

Biomedical Microdevices
|August 29, 2023
PubMed
Summary

This study introduces a 3D-printed microfluidic device for multiplexed CRISPR-Cas12a DNA detection. The system enables rapid, sensitive identification of waterborne pathogens, improving biosensing capabilities for environmental monitoring.

Keywords:
BiosensorCRISPRCas12aMicrofluidicMolecular diagnosisWaterborne pathogen

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Area of Science:

  • Biotechnology
  • Molecular Biology
  • Environmental Science

Background:

  • Nucleic acid detection is vital for monitoring environmental and biomedical samples.
  • CRISPR-Cas12a technology offers advanced DNA biosensing but faces multiplexing challenges due to nonspecific activity.

Purpose of the Study:

  • To develop a novel microfluidic device for multiplexed CRISPR-Cas12a assays.
  • To overcome limitations in CRISPR-Cas12a multiplexing for enhanced DNA detection.

Main Methods:

  • A 3D-printed composable microfluidic plate (cPlate) device was engineered for miniaturized wells and microfluidic loading.
  • The device integrates loop-mediated isothermal amplification (LAMP) with CRISPR-Cas12a readout in a high-throughput workflow.
  • Optimization of Cas12a and probe concentrations was performed to maximize assay sensitivity.

Main Results:

  • The optimized device achieved a four-fold improvement in sensitivity.
  • Sensitive detection of four waterborne pathogens (Shigella, Campylobacter, Cholera, Legionella) at the fg mL⁻¹ level was demonstrated.
  • The assay provided results within one hour, showcasing high efficiency.

Conclusions:

  • The developed cPlate device facilitates simple, high-throughput, and low-reagent-consumption multiplexed CRISPR-Cas12a assays.
  • This technology is suitable for sensitive pathogen detection in low-resource settings, enhancing environmental and public health monitoring.
  • The device addresses challenges in CRISPR-Cas12a multiplexing, paving the way for improved biosensing applications.