A stem-engineering strategy for thermally stable aptamer sensors: application to smartphone-based visual detection of berberine.

The prolonged or excessive use of berberine (Ber) can induce drug resistance and adverse reactions, necessitating the development of simple, sensitive, and rapid on-site detection methods. In contrast to conventional sequence truncation, we rationally extended the stem region of the aptamer to elevate its melting temperature, complemented by loop-region base mutagenesis. The obtained aptamer (BBRE7M9) exhibits substantially enhanced thermal stability, which is 6 °C and 10 °C higher than those of BBR38S-3A10C and BBR4S3, respectively. Its dissociation equilibrium constant remained stable at 1.42-1.86 μM across 22-40 °C. Circular dichroism, molecular docking, site-directed mutagenesis, and displacement assays collectively indicated that Ber binds within a hydrophobic minor groove constituted by C8, A9, T10, T17, and T18. This confined microenvironment restricts intramolecular motions and suppresses non-radiative transitions, triggering a fluorescence turn-on response. Upon integration with a smartphone-based portable dark-box platform, the sensor exhibited a linear response over 0-800 nM with a detection limit of 53.9 nM and satisfactory selectivity. This stem-elongation approach demonstrates a practical route to enhancing aptamer thermal stability, and the mechanistic elucidation of the key binding sites provides a structural basis for designing berberine-aptamer-based turn-on fluorescent probes. More broadly, this strategy may also serve as a reference for optimizing other aptamer sensors that suffer from similar temperature-induced signal attenuation.