Miniature multi-channel SPR instrument for methotrexate monitoring in clinical samples

A multi-channel fully integrated SPR biosensor was applied for the analysis of an anti-cancer drug, methotrexate (MTX) as a potential analytical tool used in clinical chemistry laboratories for therapeutic drug monitoring (TDM). MTX concentrations in a patient's serum undergoing chemotherapy treatments can be determined by surface plasmon resonance (SPR) sensing using folic acid-functionalized gold nanoparticles (FA-AuNP) in competition with MTX for the bioreceptor, human dihydrofolate reductase (hDHFR) immobilized on the SPR sensor chip. To validate this biosensor, 13 nm FA-AuNP were shown to interact with immobilized hDHFR in the absence of MTX and this interaction was inhibited in the presence of MTX. The sensor was calibrated for MTX in phosphate buffer at different dynamic range by varying nanoparticle sizes (5, 13, 23 nm) and by modifying the Kd of the bioreceptor using wild-type and mutant hDHFR. Furthermore, initial binding rate data analyzes demonstrated quantitative and fast sensor response under 60 s. This MTX assay was subsequently adapted to a fully integrated multi-channel SPR system built in-house and calibrated in human serum with a dynamic range of 28-500 nM. The SPR system was applied to analyzes of actual clinical samples and the results are in good agreement with fluorescence polarization immunoassay (FPIA) and LC-MS/MS. Finally, the prototype system was tested by potential clinical users in a hospital setting at the biochemistry laboratory of a Montreal hospital (Hôpital Maisonneuve-Rosemont).