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

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.