A hand-powered positive pressure-driven sample injection digital RPA-CRISPR-Cas12a chip

Chamber-based digital nucleic acid assays allow absolute quantification with high analytical sensitivity. However, sample introduction and digital discretization mainly rely on negative pressure drive, and this injection method limits its application, especially in on-site detection. But their use outside laboratory settings is still limited by the way samples are partitioned, which often depends on vacuum systems, centrifugation or specialized actuation. Positive-pressure loading is simple and well suited to portable operation, yet it is difficult to apply directly to chamber-based digital assays because air backflow and sample trailing can disrupt stable compartmentalization. This study proposed a hand-powered positive pressure-driven sample injection digital RPA-CRISPR-Cas12a chip for on-site food-safety testing. In this study, the two problems were addressed by combining photocurable oil fixation with branch-channel optimization, enabling rapid and stable discretization in a 15 120 chamber-based microfluidic digital chip using only a hand-operated syringe. Sample loading and partitioning were completed within 2 min without vacuum or centrifugal loading equipment. After integration with a one-pot digital RPA-CRISPR-Cas12a assay targeting hlyA, the system achieved absolute quantification of Listeria monocytogenes with a detection limit of 10⁰ copies μL^-1 and good linearity across dilutions (R² = 0.99973). The tests conducted on the milk samples containing the standard substances further proved that this method could be used for on-site testing under resource-limited conditions. Overall, this study provides a feasible strategy for implementing positive-pressure digitization in a portable chamber-based digital microfluidic system, which expands the utility of chamber-based digital assays for portable and instrument-free on-site food-safety testing.