Cationic Microgel-Enhanced Paper Microfluidic Device for Rapid and Sensitive On-Site Detection of Waterborne Pathogens

A foldable paper-based microfluidic device is presented that seamlessly integrates the three essential stages of nucleic-acid amplification testing (NAAT), including cell lysis/DNA extraction, loop-mediated isothermal amplification (LAMP), and end-point readout by colorimetric detection. The core of the microdevice is a glass-fiber sample pad functionalized with a cationic microgel that selectively adsorbs and concentrates genomic DNA liberated from intact bacterial cells by one-step thermal lysis. LAMP reagents are predried in discrete reaction chambers, enabling on-chip amplification at 65 °C without additional reagent handling. After 45 min, amplification products are visualized colorimetrically with methylene blue (MB), allowing naked-eye discrimination of positive and negative results. The device accurately detects two major waterborne pathogens: <i>Salmonella</i> spp. and <i>Escherichia coli</i> O157:H7 in both laboratory buffer and spiked real-world samples (drinking water, pomegranate juice, and milk). Limits of detection were 10 CFU/mL (colony forming unit) for <i>E. coli</i> and 10² CFU/mL for <i>Salmonella</i> spp., achieved within 1 h 15 min total assay time. Shelf life studies show that analytical performance is retained at ∼50% of the initial signal for at least 8 days at -20 °C and 4 days at 4 °C, underscoring the platform's suitability for field deployment. Overall, this origami-style microdevice integrates high analytical sensitivity with true sample-to-answer simplicity, providing a promising platform for rapid point-of-care detection of waterborne pathogens in resource-limited settings.