ThermiQuantTM AquaStream: A Portable Instrument for Quantitative Colorimetric Isothermal Nucleic Acid Amplification Reactions in Paper and Tube Formats

Microfluidic paper-based analytical devices ({micro}PADs) are an attractive format for colorimetric nucleic acid amplification tests (NAATs) because they enable low-cost, portable diagnostics in resource-limited settings. However, researchers often optimize assays in liquid reactions in tubes before translating them to {micro}PADs. Since both formats require separate instruments for incubation and real-time sensing, direct comparison of reactions between the two formats is difficult. To address these cross-platform limitations, we developed ThermiQuant AquaStream, a portable benchtop device (15 x 20 x 16 cm, [~]5 kg; cost: USD 327) that supports seamless colorimetric loop-mediated isothermal amplification (LAMP) reactions in both {micro}PADs and tubes under a common workflow. The system enables real-time reaction tracking (every 30 seconds) through onboard image processing, precise isothermal control ({+/-}0.5 {degrees}C) using a repurposed consumer-grade sous-vide heater, and medium-throughput (24 tubes or 42 {micro}PADs). Testing with synthetic SARS-CoV-2 orf7ab DNA fragments demonstrated a limit of detection (LoD) of 50 copies per reaction in both formats (6.7 copies/{micro}L for {micro}PADs; 10 copies/{micro}L for tubes). Standard calibration curves based on quantification time (Tq) and log10 DNA concentration showed strong full range linearity in tubes (from 106 to 50 copies/reaction, R2 = 0.98) and piecewise linearity in {micro}PADs, with quantification reliability only above 1,000 copies/reaction (133 copies/{micro}L, R2 = 0.99), which we set as the limit of quantification, LoQ. By unifying assay optimization across tube and {micro}PAD formats, ThermiQuant AquaStream provides an affordable and versatile benchtop tool for molecular diagnostics for One Health applications in the clinic, on farms, and in the field.