Metabolite microextraction on surface-enhanced Raman scattering nanofibres and D2O probing accelerate antibiotic susceptibility testing

Rapid antibiotic susceptibility tests (AST) are vital for the effective treatment of disease, necessitating the development of analytical tools to address unmet needs in healthcare. Leveraging the sensitivity of plasmonic nanosensors and isotopic labelling has the potential to accelerate AST. Here, we developed surface-enhanced Raman scattering (SERS)-based nanofibre sensors and heavy water [deuterium oxide (D₂O)] labelling (SERS-DIP) for detecting the minimum inhibitory concentration (MIC) and AST for trimethoprim (TMP) against <i>E. coli</i>. SERS-DIP rapidly detected the MIC of TMP for the susceptible strain in 2 h. TMP-resistant cells retained the metabolic activity regardless of TMP levels, confirming the resistance phenotype. Kinetic analysis of D uptake by resistant cells treated with TMP (2 × MIC) revealed increasing D levels proportional to peak redshifts over time, confirmed by machine learning-driven data exploration. Our results demonstrate the utility of nanofibre-enabled SERS-DIP for robust AST, uncovering new spectral biomarkers that may impact clinical medicine.