SANDWiCH: An Oil-Free High-Throughput Microwell-Array Platform for Digital Loop-Mediated Isothermal Amplification

Abstract

Digital nucleic acid amplification tests enable absolute molecular quantification by partitioning samples into independent reactors, but scalable digital loop-mediated isothermal amplification (LAMP) remains limited by oil-based compartmentalization, droplet stability requirements, and assembly-induced sample spreading in open microwell arrays. Here, we present serially-assembled nanodrop detection with a chip (SANDWiCH), an oil-free, high-throughput microwell-array digital LAMP platform designed for contamination-resistant multi-sample analysis. SANDWiCH integrates a 3 × 8 array layout, micropillar-assisted capillary loading, pressure-sensitive adhesive film sealing, and alignment-guided serial assembly to confine each sample to its assigned array before compartmentalization. This design enables spontaneous partitioning of reaction mixtures into 10,000 microwells, each with a volume of 7.85 pL, per array without pumps, actuators, or oil overlays, allowing 24 samples to be processed in parallel under identical amplification conditions. By varying the number of microwell-array units assigned per sample, the platform provides a tunable balance between assay throughput and quantification accuracy; the limit of quantification was reduced from 30.2 copies/μL in the highest-throughput mode to 1.3 copies/μL in the highest-accuracy mode. Using Plasmodium vivax as a target, quantitative analysis showed excellent agreement with theoretical predictions based on the Poisson distribution across six orders of magnitude (Pearson correlation coefficient, r = 0.991). Clinical validation with 60 blood-derived nucleic acid samples yielded 100% (95% CI, 88.4%−100%) sensitivity and 100% (95% CI, 88.4%−100%) specificity, while requiring only 0.29 μL of reagent per sample.