Exploiting Cross-Reactivity for Selective Chemotherapy Monitoring: A Differential Electrochemical Aptamer Approach

Despite its widespread use, high-dose methotrexate (MTX) regimen presents a risk of lifethreatening 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 decisionmaking. Here, we introduce a differential electrochemical aptamer-based biosensor that enables simultaneous real-time monitoring of MTX and DAMPA concentrations 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 advance point-of-care MTX monitoring during glucarpidase-assisted rescue therapy and support informed clinical decision-making in chemotherapy. More broadly, we envision this work will also establish a translational framework for leveraging cross-reactive biosensors to monitor drug metabolism in real time.