Loha K.Boonkoom T.Pitakjakpipop H.Alam I.Treetong A.Boonbanjong P.Chatnuntawech I.Teerapittayanon S.Keyser U.F.Schulte A.Japrung D.Mahidol University2025-10-032025-10-032025-09-26ACS Sensors Vol.10 No.9 (2025) , 7014-7024https://repository.li.mahidol.ac.th/handle/123456789/112407Rolling Circle Amplification (RCA) is a robust isothermal nucleic acid amplification technique widely used in molecular diagnostics. In this study, we combine RCA with solid-state nanopore sensing to monitor the amplification process at the single-molecule level using miR-21 as a model biomarker. This label-free platform enables detailed analysis of amplification kinetics and structural transitions over time. Changes in translocation dwell time and current blockage were evaluated across RCA incubation periods (30 min, 1 h, 2 h), revealing time-dependent increases consistent with the generation of longer and more complex DNA concatemers. These findings were validated by Urea-PAGE and atomic force microscopy (AFM), while Mfold-based secondary structure predictions further supported the evolution of more stable and folded configurations. Additionally, a custom-developed signal extraction application facilitated reproducible event classification and visualization. Overall, this integrated approach provides new insights into RCA behavior and highlights the potential of nanopore-based sensing for the development of sensitive, structure-resolved diagnostic tools.Chemical EngineeringPhysics and AstronomyArticleSCOPUS10.1021/acssensors.5c020392-s2.0-10501711217423793694