Magnetic separation-assisted DNAzyme walker-based nanosensor for point-of-care therapeutic drug monitoring of methotrexate.

Methotrexate (MTX) is a widely used antimetabolite drug, mainly used in the treatment of a variety of cancer. Given the low therapeutic index and significant individual variability of MTX, it was critical to perform therapeutic drug monitoring (TDM) to minimize the side effects. Here, we designed a rapid and sensitive fluorescence/colorimetric assay for the detection of MTX in diluted human serum. After the aptamer binds specifically to MTX, the Walking strand cleaves the Hairpin strand and releases a large amount of fluorescent signal, and the color of the gold nanoparticles changes after the addition of sodium chloride. The change of color could be visualized by eyes to achieve point-of-care TDM of MTX. The quantitative detection of MTX concentration is carried out through the collection of fluorescent signals or absorptions. The fluorescence method can detect MTX in the range of 0.05-1000 μM with the detection limit of 0.0243 μM, meanwhile, the colorimetric method can detect MTX in the range of 0.01-100 μM with the detection limit of 0.0097 μM. The magnetic separation-assisted DNAzyme walker-based nanosensor exhibited good sensitivity, selectivity and stability for detecting MTX in serum and achieve point-of-care methods of TDM of MTX and the nanocarriers has demonstrated significant potential for clinical application.