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bioRxiv · 10.64898/2026.01.15.699357

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

Raut, B.·Palla, G.·Wang, J.·Campbell, S.·Kumar, V.·Ra, K.·Verma, M. S.
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Abstract

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 colorimetric 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 corresponding to a 95% probability of detection (LOD95) of 110 copies per reaction in tube (22 copies/{micro}L) and 39 copies per reaction in {micro}PADs (5 copies/{micro}L), estimated using probit regression. In both formats the limit of quantification (LOQ), defined as the lowest concentration yielding a coefficient of variation (CV) of Tq [≤] 10%, was 250 copies/reaction resulting in a strong linear (R2 = 0.98 & R2 = 0.96 respectively for tube and {micro}PADs) standard calibration curves. ThermiQuant AquaStream provides an affordable and versatile benchtop platform capable of supporting both tube- and {micro}PAD-based colorimetric LAMP assays, serving as a proof-of-concept research tool for assay development and molecular diagnostics in One Health settings such as clinics, farms, and field environments.

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Raut, B., Palla, G., Wang, J., Campbell, S., Kumar, V., Ra, K., Verma, M. S.. 2026-01-15. ThermiQuantTM AquaStream: A Portable Instrument for Quantitative Colorimetric Isothermal Nucleic Acid Amplification Reactions in Paper and Tube Formats. https://doi.org/10.64898/2026.01.15.699357

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