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

Abstract

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.