Developing an all-in-one microfluidic device for multiplexed and equipment-free CRISPR-Dx at the point of care

Abstract

CRISPR-based diagnostics (CRISPR-Dx) are a growing technology for sensitive and specific nucleic acid detection without the accessibility restrictions of qPCR. However, there is currently no platform that achieves highly multiplexed CRISPR-Dx without the need for specialized equipment, preventing efficient deployment in point-of-care or point-of-need settings. Here, we make significant progress toward addressing this gap in the field: we design, develop, and test several iterations of a microfluidic chip capable of 10-plex CRISPR-Dx with lateral flow (LF) integrated for equipment-free result readout. We confirm the efficacy of our device in facilitating stop-and-start fluid flow and on-chip mixing in colorimetric flow control tests. To make our device compatible with biological diagnostics, we also develop novel processes for lyophilized reagent storage and post-reaction fluid dilution. We make strides toward the validation of our device and workflow in a CRISPRDx setting, successfully detecting synthetic tuberculosis (TB) and monkeypox (Mpox) DNA targets to achieve a new level of combined multiplexing and equipment-free accessibility in CRISPR-Dx.