Methotrexate Detection in Serum at Clinically Relevant Levels with Electrochemically Assisted SERS on a Benchtop, Custom Built Raman Spectrometer

Therapeutic drug monitoring (TDM) is an essential clinical practice for optimizing drug dosing, thereby preventing adverse effects of drugs with a narrow therapeutic window, slow clearance, or high interperson pharmacokinetic variability. Monitoring methotrexate (MTX) during high-dose MTX (HD-MTX) therapy is necessary to avoid potentially fatal side effects caused by delayed elimination.

Despite the efficacy of HD-MTX treatment, its clinical application in resource-limited settings is constrained due to the relatively high cost and time of analysis with conventional analysis methods. In this work, we developed (i) an electrochemically assisted surface-enhanced Raman spectroscopy (SERS) method for detecting MTX in human serum at a clinically relevant concentration range and (ii) a benchtop, Raman detection system with an integrated potentiostat, software, and data analysis unit that enables mapping of small areas of SERS substrates and quantitative SERS-based analysis.

In the assay, by promoting electrostatic attraction between gold-coated nanopillar SERS substrates and MTX molecules in aqueous samples, a detection limit of 0.13 μM with a linear range of 0.43-2 μM was achieved in PBS. The implemented sample cleanup through gel filtration proved to be highly effective, resulting in a similar detection limit (0.55 μM) and linear range (1.81-5 μM) for both PBS and serum.

The developed and optimized assay could also be used on the in-house built, Raman device. We showed that MTX detection can be carried out in less than 30 min with the Raman device, paving the way toward the TDM of MTX at the point-of-need and in resource-limited environments.