Developing a Microfluidic Device for Multiplexed CRISPR-Based Disease Detection in Point-Of-Need Settings

Abstract

Multiplexing is an important frontier for point-of-need CRISPR-based detection. Here we demonstrate the development and validation of a microfluidic device for multiplexed RPA (recombinase polymerase amplification) and Cas13 CRISPR-based detection. We analyzed the performance of three disposable-syringe-driven flow distribution geometries (tree, comb and valve), and designed a hybrid geometry for 100-plex detection. In addition, we developed a manual rotation-induced magnetic mixing process for efficient on-chip mixing of reaction chambers, implemented a process for on-chip storage of reagents using lyophilized pellets, and explored the integration of on-chip lateral flow for a naked-eye visual readout. We validated the performance of the 16-plex comb geometry, the 16-plex valve geometry and the 100-plex hybrid geometry with CRISPR-based assays designed to target mpox (monkeypox), using synthetic DNA targets. This work makes significant strides towards the development of a totally equipment-free multiplexed device for the detection of pathogens in point-of-need settings.