PlasmoBridge: Stable hotspot engineering and targeted Surface-enhanced Raman spectroscopy (SERS) monitoring of methotrexate

Surface-enhanced Raman spectroscopy (SERS) offers unique molecular fingerprinting capability and enhanced sensitivity. However, the application of SERS in therapeutic drug monitoring (TDM) is limited due to the occurrence of unstable plasmonic hotspots, poor reproducibility, and the lack of molecular specificity. In this study, we present “PlasmoBridge”, a dual-function aptamer–nanoparticle assembly that bridges adjacent silver nanoparticles with thiolated methotrexate (MTX) aptamers. This design generates uniform nanogaps (∼6.5 nm) that enable stable and reproducible hotspot formation while selectively enriching MTX molecules in the electromagnetic field. Consequently, PlasmoBridge achieves ultrasensitive MTX detection (10 −8  M) with relative standard deviations below 10 % and the requisite linearity across clinically relevant concentration ranges. Integrating liquid–liquid and solid-phase extraction delivers recoveries in serum that exceed 90 %. Convolutional neural network–assisted spectral analysis serves to further enhance quantitative accuracy (R 2  > 0.99). In a preclinical osteosarcoma model, PlasmoBridge-guided TDM preserved antitumor efficacy while reducing systemic toxicity via optimized dosing. When compared with enzyme-multiplied immunoassay technique (EMIT) and liquid chromatography-tandem mass spectrometry (LC–MS/MS) procedures, PlasmoBridge uniquely balances sensitivity, speed, and clinical practicality, enabling near-real-time and point-of-care monitoring. Moreover, this generalizable biosensing framework, combining nanoscale engineering, molecular recognition, and AI-driven quantification, offers a promising route to precision pharmacotherapy of a range of small-molecule drugs.