A synergistic dual-mode biosensor for methotrexate detection leveraging FRET and Ag NP-based UV absorption.

A novel dual-mode sensing platform (DMSP) integrating fluorescence and ultraviolet (UV) detection was developed for the sensitive and accurate quantification of methotrexate (MTX). This approach combines a FRET-based fluorescent "turn-on" mechanism, employing FAM as the fluorophore and BHQ as the quencher, with a UV absorption mode utilizing the unique surface plasmon resonance properties of silver nanoparticles (Ag NPs). The fluorescence mode enables highly sensitive detection via target-induced structural switching, while the AgNP-based UV channel provides a complementary, concentration-dependent signal at 405 nm, allowing cross-validation and improved reliability. The platform demonstrates a good linear detection range (0.5-20 µM), with detection limits of 0.56 µM and 0.62 µM for the fluorescence and UV modes, respectively, in serum samples. It also exhibits excellent specificity against structurally similar molecules. This dual-mode strategy offers a robust and practical alternative for therapeutic drug monitoring of MTX in complex biological environments.