A high-speed sequential liquid compartmentalization method for digital loop-mediated isothermal amplification in a...

Accurate and rapid quantification of nucleic acid targets is crucial for molecular diagnostics, particularly in resource-limited settings where simple and robust technologies are required. This study presents a high-throughput digital loop-mediated isothermal amplification (dLAMP) platform for the absolute quantification of nucleic acids in a sample, using a microfluidic device comprising ten thousand nanoliter-scale reaction microchambers. The polydimethylsiloxane (PDMS)-based device achieved complete liquid compartmentalization within 60 s in a single operation using an electronic pipette, without requiring surface modification, pre-degassing, pre-priming, or external pumping systems, which are typically necessary in conventional methods. The aqueous sample/reagent mixture was reliably compartmentalized using fluorinated oil, with 97% of the microchambers successfully filled to at least 80% of their designed volume, exhibiting excellent volumetric uniformity (CV = 0.07). Fluorescent LAMP assays targeting Salmonella and cannabis exhibited strong correlations between estimated and true DNA concentrations (R² > 0.98), although quantification was consistently underestimated. Correction factors of 1000 and 10 000 were required for synthetic Salmonella and cannabis DNA, respectively, whereas only 10 were needed for cannabis seed-derived DNA, indicating these discrepancies were due to the intrinsic performance of the LAMP assays rather than device limitations. The dLAMP device also enabled the successful detection of cannabis seed DNA in the presence of 10 ng μL^-1 humic acid, which inhibits amplification in conventional turbidity-based LAMP, demonstrating its robustness for point-of-care testing (POCT) applications. The distinctive compartmentalization strategy of the pipette-operated dLAMP platform enables high scalability without compromising operational simplicity, achieving high throughput, wide dynamic range, and accurate quantification.