Discovery of a Highly Specific Anti-methotrexate (MTX) DNA Aptamer for Antibody-Independent MTX Detection.

High-dose methotrexate (MTX) therapy is used to treat a wide variety of cancers such as leukemia and lymphoma, while the resulting high blood concentration of MTX faces a risk of life-threatening side effects, so it is essential to monitor the concentration carefully. Currently, the MTX concentration is measured using antibody-based kits in a clinical setting; however, the heterogeneity and batch-to-batch variation of antibodies potentially compromise the detection limit. Here, we developed MTX detection systems with chemically synthesizable homogeneous oligonucleotides. Microbead-assisted capillary electrophoresis (MACE)-SELEX against MTX successfully identified MS7 with a similar level of specificity to anti-MTX antibodies within three rounds. The 3'-end of MS7 was coupled to a peroxidase-like hemin-DNAzyme to construct a bifunctional oligonucleotide for MTX sensing, where MTX in 50% human serum was detected with a limit of detection (LoD) of 118 nM. Furthermore, amplifying the DNAzyme region with rolling circle amplification significantly improved the sensitivity with an LoD of 290 pM. Presented oligonucleotide-based MTX detection systems will pave the way for antibody-independent MTX detection with reliability and less cost in the laboratory and the clinic.