Handheld RPA-based molecular POCT system for rapid, low-cost 8-plexed detection of respiratory pathogens at home.

During seasonal influenza or emerging respiratory outbreaks, rapid home-based multiplex molecular point-of-care testing (POCT) for respiratory pathogens is crucial for early diagnosis and intervention, particularly in vulnerable populations. However, existing POCT systems, primarily designed for clinical settings, are often too complex, costly, and reliant on trained operators, limiting their suitability for home use. To overcome these barriers, we introduce a microfluidic cartridge-based system leveraging recombinase polymerase amplification (RPA) for multiplexed detection of respiratory pathogens in home environments. The microfluidic cartridge is designed with three parallel channels-each integrating a lysis chamber, an RPA chamber preloaded with lyophilized reagents, and an air storage chamber. Each detection channel enables extraction-free, single-channel 3-plex RPA assays, and by combining three-channel parallel detection, the system achieves simultaneous identification of eight respiratory pathogens and one internal control in under 25 min. A novel pneumatic pressure pumping strategy ensures precise flow control through dynamic bladder compression, paired with microchannel hydraulic resistance matching to guarantee uniform volumetric distribution and synchronized flow across all channels. Furthermore, a dynamic mixing method promotes homogeneous mixing of RPA reagents with lysed samples a bidirectional flow between the lysis and RPA chambers, enhancing assay reliability. Our microfluidic design enables significant miniaturization, yielding a compact, lightweight system (<1 kg) suitable for handheld or desktop use. Its low power consumption (3 W) and remarkable cost-effectiveness ($1.4 per test) enhance the system's practicality and accessibility for home settings. Validation with 356 nasopharyngeal swabs further confirms its robust performance, achieving high sensitivity (>97%) and specificity (>99%), ensuring reliable at-home diagnosis of respiratory co-infections without requiring professional operation.