Simultaneous Detection of Multiple Respiratory Pathogens Using an Integrated Microfluidic Chip.

Respiratory pathogens pose significant challenges to public health, demanding efficient diagnostic methods. This study presents an integrated microfluidic chip for the simultaneous detection of multiple respiratory pathogens. The chip integrates magnetic bead-based nucleic acid extraction and purification, acoustic streaming-driven mixing, liquid equalization, and multiplex PCR amplification with in situ fluorescence detection. Nucleic acid extraction takes only 12 min, yielding results comparable to commercial kits. Efficient mixing of magnetic beads is achieved through a combination of designed micropillars and bubble-trapping array structures. The micropillars maintain the aqueous phase in the mixing chamber, while the bubble-trapping arrays enable stable formation of bubbles, serving as a micromixer under the acoustic field. To prevent cross-contamination, an oil-encapsulated water droplet system is incorporated throughout nucleic acid extraction and PCR amplification. This assay displays remarkable multiplex analysis capability on a single chip, enabling the simultaneous detection of 12 common respiratory pathogens with a low detection limit of 10 copies/μL. Moreover, this method demonstrates excellent practical applicability in clinical nasal samples. Compared to many microfluidic chip-based molecular biology methods, the assay exhibits comparable or superior multipathogen analysis capability, sensitivity, and speed, completing the sample-to-answer process in approximately 70 min. This integrated microfluidic device offers a promising multiplex molecular diagnosis platform for on-site simultaneous detection of multiple pathogens.