Folate-functionalized CoFe2O4 nanozyme inspired by folate antagonist effect for methotrexate detection with high anti-interference ability and recyclability

Methotrexate (MTX) is a widely used chemotherapeutic drug to treat cancer and autoimmune diseases, which has a narrow therapeutic window and can cause systemic toxicity. The monitoring of MTX is therefore of crucial importance in clinical practice and precision medication. However, current detection techniques suffer from limitations in cost and time effectiveness, making them unsuitable for out-of-lab and point-of-care testing. Inspired by the antimetabolite mechanism of MTX, a signal-on colorimetric sensing platform based on CoFe2O4 nanozyme functionalized with folic acid (FA@CoFe2O4) was elaborately developed to monitor MTX. Due to the ability of the immobilized folate to be reduced by dihydrofolate reductase (DHFR) to tetrahydrofolate, it poses competitive effect on the chromogenic substrate by its strong reducibility when FA@CoFe2O4 exerts its peroxidase-like activity, thereby inhibiting the discoloration of the substrate. Once MTX is introduced, the active sites of DHFR will preferentially bind to it, leading to suppression in the generation of tetrahydrofolate and further restoring the substrate's color rendering. Based on this principle, the developed sensing strategy exhibited a broad range from 0.1 μM to 40 μM towards MTX detection. In addition, the magnetic property endowed the FA@CoFe2O4 nanozyme excellent recyclability and allowed the MTX detection with high anti-interference ability from other drugs and coexisting antioxidants. The feasibility of the method was validated by monitoring MTX level in human serums. This work paves a new way towards therapeutic drug monitoring that serves personalized administration.