Single-atom anchored TiO₂ photoelectrochemical aptasensor for sensitive detection of methotrexate

Methotrexate (MTX) is a widely used chemotherapeutic agent with a narrow therapeutic window, necessitating precise and rapid therapeutic drug monitoring (TDM) to optimize efficacy and minimize toxicity. Here, we report a novel photoelectrochemical (PEC) aptasensor based on atomically dispersed gold anchored on TiO₂ nanotube arrays (Au SA/TiO₂) for label-free and selective detection of MTX in complex biological matrices. The Au SA sites serve as discrete anchoring points for thiolate MTX-specific DNA aptamers, enabling a “one-atom–one-aptamer” configuration that minimizes nonspecific adsorption and preserves aptamer conformational flexibility. Upon MTX binding, the aptamer undergoes a structure-switching event that enhances electronic coupling with the electrode, leading to a measurable photocurrent increase. The sensor exhibits a wide linear detection range from 0.1 to 100 μM, a low detection limit of 0.03 μM, and excellent selectivity against common antibiotics. Validation in 50% fetal bovine serum yielded recovery rates of 96.2–104.3%, and the sensor retained over 90% of its initial response after 15 days. This work highlights the potential of single-atom engineered interfaces for next-generation PEC biosensing in clinical TDM.