Structure-switching Aptamer Nanosensor for Therapeutic Drug Monitoring of Methotrexate in Diluted Serum

Abstract

Methotrexate (MTX) is an anticancer drug widely used in clinical practice. Due to its narrow therapeutic index and significant interindividual variability, there is an urgent need for therapeutic drug monitoring (TDM) to mitigate adverse effects. This study aims to establish a simple, rapid, and highly sensitive fluorescent assay for MTX. A rapid fluorescence detection strategy was developed using highly specific MTXstructure- switching nucleic acid adaptors; the linear range, limit of detection, and specificity of the method were systematically evaluated, and the method’s performance was validated in a complex biological matrix containing 80% fetal bovine serum (FBS) and 20% human serum. The linear detection range of this method for MTX in buffer is 0.5 μM to 6 μM, with a detection limit of 0.034 μM; effective detection remains achievable in 80% FBS, with a detection limit of 0.33 μM. Additionally, stable detection is possible in 20% human serum, and the method exhibits excellent specificity and is not affected by the presence of various interfering substances. The MTX fluorescence assay developed in this study offers advantages, including ease of use, high sensitivity, and good selectivity. It can effectively measure MTX in complex biological matrices and holds significant potential for clinical drug monitoring in MTX therapy. The validated sensor enables rapid MTX monitoring in biological matrices, supporting clinical individualized medication.