ThermiQuant™ MegaScan: High-throughput isothermal reactor with quantitative colorimetric readout for paper-based nucleic acid amplification tests

Abstract Isothermal nucleic acid amplification tests (NAATs), such as loop-mediated isothermal amplification (LAMP) implemented on microfluidic paper-based analytical devices (µPADs), enable inexpensive and rapid (≤60 min) colorimetric molecular diagnostics; however, no existing instrument supports high-throughput (>100 reactions) quantitative analysis of colorimetric isothermal assays on paper substrates under controlled laboratory conditions. To address this gap, we developed ThermiQuant™ MegaScan, a scanner- and water-bath–based platform that accommodates a 160-reaction µPAD cartridge, maintains uniform incubation at 65 ± 0.5 °C, and enables real-time imaging every 30 s. We also developed accompanying software, Amplimetrics™, for automated µPAD detection and kinetic colorimetric analysis. Using paper-based colorimetric LAMP targeting the SARS-CoV-2 orf7ab region, the assay achieved a limit of detection at 95% probability (LOD95) of 34 copies per reaction (5 copies/µL) and a limit of quantification (LOQ) of 250 copies per reaction (33 copies/µL) using purified synthetic DNA targets, and achieved 72% sensitivity and 100% specificity relative to digital PCR (dPCR) for diluted human nasopharyngeal (NP) swab virus samples. We further evaluated the effects of viral and universal transport media (VTM/UTM) on assay performance and found that linear calibration derived from synthetic targets do not reliably translate to clinical samples in these media. Together, these results establish ThermiQuant™ MegaScan as a high-throughput laboratory research platform for standardized evaluation, optimization, and benchmarking of paper-based colorimetric nucleic acid amplification assays. Competing Interest Statement A US patent related to aspects of this work has been filed (Application No. US20250222460A1). Inventors: Mohit S. Verma, Nafisa Rafiq, and Bibek Raut. Applicant/Assignee: Purdue Research Foundation. Status: Pending. The patent covers all aspect of the technology developed in this manuscript. MSV and JPS have interests in Krishi, Inc., which is a startup company developing molecular assays. Krishi, Inc. did not fund this work. M.S.V. has interests in Simply Experiment LLC, which did not fund this work either. The other authors do not have a competing interest. Footnotes Additional data was included to strengthen the manuscript.