A pump-free gravity-driven microfluidic chip for rapid RPA-LFS-based detection of Magnaporthe oryzae AvrPi9 gene

We present a pump-free, gravity-assisted microfluidic lab-on-a-chip platform for the rapid detection of the rice blast pathogen Magnaporthe oryzae by targeting the AvrPi9 gene. The system integrates precise thermal control, programmable fluidic sequencing, and lateral-flow readout, enabling low-power diagnostics without complex instrumentation. Temperature regulation is achieved using a LinkIt 7697 development board, coupled with a proportional-integral-derivative (PID) controller, to drive a Peltier element. This maintains a stable reaction environment at 39 ± 0.5 °C for recombinase polymerase amplification (RPA). The microfluidic chip (65 mm × 34 mm × 5 mm) is fabricated via laser cutting and hot pressing, with the reaction chamber validated for spatial temperature uniformity through infrared thermal imaging. To enhance operational reliability in field settings, the platform utilizes a manual pin-actuated puncture mechanism to initiate fluidic transport. After a 5-minute isothermal amplification, the physical piercing of a sealing membrane opens strategic air vents, inducing a capillary-driven flow and pressure imbalance that facilitates the pump-free transport of 30 µL of RPA products into the lateral flow strip (LFS). This deterministic mechanical gating replaces complex actuators, ensuring a 100% success rate for vent opening. Visual results appear within 2 min, with a total assay time of approximately 15 min, achieving a detection limit of 10 pg/µL for AvrPi9. The system demonstrates high specificity, with no cross-reactivity to non-target pathogens, including Bipolaris oryzae, Sarocladium oryzae, and Ephelis sp., owing to strategic primer mismatches. This robust, pin-actuated, valve-free system highlights the potential for reliable, low-power, field-deployable nucleic acid diagnostics in point-of-care settings. While this study serves primarily as a hardware engineering proof-of-concept focusing on pump-free fluidic transportation, it establishes a foundational architecture for decentralized molecular diagnostics. Future translational development will focus on integrating raw matrix sample preparation to bypass the current limitation of requiring purified nucleic acid inputs.