Detection of methotrexate using surface plasmon resonance biosensors for chemotherapy monitoring

Methotrexate (MTX) cancer therapy requires therapeutic drug monitoring (TDM) for following its levels in a patient during the course of treatment in order to maximize efficacy while minimizing side effects. Despite its narrow therapeutic window, MTX remains until this date, one of the most employed chemotherapy agents. Existing TDM analytical techniques for MTX are costly, time-consuming and labor intensive which are not suitable to promptly generate results within the therapy timeframe. To provide rapid MTX quantification for TDM, a strategy is proposed based on a competitive assay featuring gold nanoparticles and surface plasmonic coupling. More specifically, the inhibition of MTX with its molecular receptor, human dihydrofolate reductase (hDHFR), an enzyme associated with proliferative diseases, is explored. Free MTX mixed with folic acid-functionalized gold nanoparticles (FA-AuNP) are in competition for hDHFR binding sites immobilized on a SPR active surface or free in solution. FA-AuNP binding to hDHFR provides signal amplification which is inversely proportional to the concentration of MTX. Surface plasmon resonance (SPR) is commonly used as a spectroscopic technique for the interrogation of biomolecular interactions. Current commercial SPR instruments are laboratory-based, bulky, expensive, lack sensitivity in complex matrix and have not shown adaptability in clinical settings. In addition, SPR analysis of small molecules such as drugs has not been extensively explored due to lack of sensitivity. The recent advances in materials science and surface chemistry exploiting gold nanoparticle integration for SPR response enhancement and peptide surface chemistry have shown potential in overcoming the poor sensitivity and surface-fouling limitations for crude biofluids analysis. These novel concepts of SPR technology are incorporated with a miniaturized fully integrated SPR prototype to conduct fast, reliable and sensitive analysis to monitor MTX levels of a patient undergoing chemotherapy. The objective of the thesis is to explore different strategies in improving drug analysis in a complex matrix using SPR biosensors and to put in perspective of the potential of SPR biosensors as a useful TDM tool in clinical laboratories or at a point-of-care situation.   To achieve these objectives, a colorimetric solution-based MTX competitive assay is first established with FA-AuNP. Then, the solution-based MTX competitive assay is translated onto a SPR platform. For both developed assays, sensitivity, selectivity, detection limit, dynamic range optimization as well as analysis of methotrexate in complex matrix are inspected. Furthermore, the SPR prototype is validated by its equivalent performance to existing SPR systems and by its utility in executing MTX analysis in actual serum samples from patients undergoing chemotherapy. The concentrations of MTX obtained by SPR biosensing are compared to standard techniques: fluorescence polarization immunoassay (FPIA) and liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) in order to confirm the feasibility of SPR biosensors as a useful clinical tool for performing rapid MTX concentration evaluation. Finally, the successful deployment of the prototype to a hospital laboratory demonstrates enormous prospective of SPR biosensors in clinical use.

Keyphrases: SPR, Gold nanoparticles, Surface plasmon resonance, Localized surface plasmon resonance, Methotrexate, Human dihydrofolate reductase, Therapeutic drug monitoring, Small molecule detection, Plasmonic coupling, Biosensors, Point-of-care device