A Differential Electrochemical Sensor for Resolving Metabolite Cross-Reactivity Toward Selective Chemotherapy Monitoring.

Despite its widespread use, high-dose methotrexate (MTX) regimen presents a risk of life-threatening nephrotoxicity. Consequently, rigorous monitoring of MTX levels at predefined time points is highly recommended to assess toxicity and guide dosing of rescue therapy such as glucarpidase. Upon administration, glucarpidase rapidly converts MTX into its inactive metabolite 2,4-diamino-N10-methylpteroic acid (DAMPA) which displays structural similarity to MTX, causing an overestimation of the actual MTX levels. Therefore, patients receiving glucarpidase necessitate analytical methods capable of specifically monitoring MTX concentrations to inform the need for further rescue treatment. However, existing point-of-care tools lack this selectivity, leaving clinicians reliant on time-consuming and lab-bound chromatographic assays for decision-making. Here, we introduce a differential electrochemical aptamer-based biosensor that enables simultaneous measurements of MTX and DAMPA concentrations even after glucarpidase intervention by exploiting, rather than avoiding, aptamer cross-reactivity. DAMPA levels are directly reported by a DAMPA-specific aptamer, whereas MTX concentrations are inferred via a mathematical deconvolution of the cross-reactive aptamer signal. We anticipate that our work will establish a translational framework for leveraging cross-reactive biosensors to measure drug metabolism in real time toward selective chemotherapy and beyond.