A CRISPR/Cas12a-based digital microfluidic platform for parallel detection of foodborne pathogens

Rapid and automated on-chip detection of multiple foodborne bacterial pathogens remains challenging because conventional molecular assays require multistep liquid handling and are difficult to integrate for parallel analysis. Here, we developed an EWOD-based DMF platform integrating RPA with CRISPR/Cas12a for parallel detection of S. aureus, E. coli O157:H7, L. monocytogenes, and C. sakazakii. The 16-site DMF chip was organized into four pathogen-specific detection modules, each containing a positive control, a negative control, and two replicate sample sites, enabling programmable droplet manipulation and sequential on-chip amplification and fluorescence detection without external pumps or complex valves.

Assay conditions were systematically optimized for fluidic compatibility and CRISPR/Cas12a performance. Following off-chip sample preparation and genomic DNA extraction, the automated on-chip RPA amplification and CRISPR/Cas12a detection workflow was completed within approximately 50 min. Under these conditions, the platform achieved detection limits of 10³, 10², 10, and 10² CFU/mL for S. aureus, E. coli O157:H7, L. monocytogenes, and C. sakazakii, respectively. No apparent cross-reactivity was observed among 35 food-associated bacterial strains. In artificially contaminated milk, milk powder, and chicken samples, comparable detection limits were obtained after off-chip sample processing, despite matrix-dependent variations in fluorescence intensity.

These results demonstrate that integrating EWOD-based digital microfluidics with RPA and CRISPR/Cas12a enables rapid, parallelized, and programmable molecular detection of multiple foodborne bacterial pathogens on a compact platform. By integrating droplet manipulation, amplification, and fluorescence detection on-chip, the proposed system reduces reliance on external fluidic components and provides a promising basis for further development toward automated and integrated food-safety testing.