A Droplet-Free and Oil-Free Microfluidics Based on Self-Pumping Hydrogel for Point-of-Care Multiplex Digital Nucleic...

Digital PCR (dPCR) enables rapid and accurate nucleic acid quantification and has been widely applied in clinical diagnostics, pathogen screening, environmental monitoring, and food safety. However, current dPCR platforms based on droplet microfluidic chip cannot been applied for in-field test and multiplexing, due to their complex fluidic manipulation. Here, we report a self-pumping hydrogel chip for multiplex digital nucleic acid detection that exploits the intrinsic water-absorbing behavior of hydrogel to drive autonomous fluid transport while simultaneously leveraging the cross-linking hydrogel network for digital amplification. This strategy eliminates external pumps and intricate microfabrication, allowing a fully passive operation with high sensitivity and single-copy quantitative accuracy. The ready-to-use hydrogel chip with pre-embedded reagents for isothermal loop-mediated isothermal amplification exhibits excellent storage stability and inhibitor tolerance. Furthermore, integration of four spatially separated detection zones allows simultaneous digital quantification of Vibrio parahemolyticus, Salmonella typhi, and Staphylococcus aureus in a single assay within 35 min on-site. This self-pumping hydrogel chip offers a simple, low-cost, and scalable platform that advances digital nucleic acid analysis toward portable and high-throughput point-of-care testing.