A solid-state heater-imager for quantitative evaluation of colorimetric isothermal nucleic acid amplification on paper.

Maintaining precise isothermal conditions in portable nucleic acid amplification tests (NAATs) is critical for reproducible results but remains challenging with conventional single-sided thin-film heaters, which exhibit temperature gradients and strong dependence on ambient conditions. To close this gap, we engineered ThermiQuant™ VitroMini, a dual-sided heater design that achieves volumetric-level temperature uniformity using thin-film heaters while preserving optical transparency for real-time colorimetric loop-mediated isothermal amplification (LAMP) analysis on microfluidic paper-based analytical devices (μPADs). The device integrates two independently regulated indium tin oxide (ITO) heaters (8 Ω each) controlled by independent proportional-integral-derivative (PID) algorithms. Heaters were evaluated under controlled ambient environments of 4 °C (refrigerated), 23 °C (room temperature), and 50 °C (oven). Analytical tests were performed using a colorimetric LAMP assay targeting the SARS-CoV-2 orf7ab gene on μPADs preloaded with dried LAMP reagents, with time-lapse images (30 seconds interval) analyzed via Amplimetrics™ software. VitroMini maintained 65 ± 0.5 °C across 4 to 50 °C ambient conditions and achieved a limit of detection of 34 copies per reaction (4.5 copies per μL) and limit of quantification of 1000 copies per reaction (133 copies per μL), with quantification time (Tq) linearly correlated with log10 DNA concentration. Dual-sided heating eliminated temperature bias, condensation artifacts, and ambient-dependent variability while preserving optical transparency for real-time quantitative LAMP reaction. ThermiQuant™ VitroMini bridges the gap between benchtop volumetric heaters and portable diagnostic devices, offering a compact, low-power platform for quantitative colorimetric molecular analysis on paper with potential for decentralized and field-deployable applications.