Aptamer/proximity hybridization-based label-free and highly sensitive colorimetric detection of methotrexate via polymerization/nicking recycling amplifications

Methotrexate (MTX) is one of the commonly used therapeutic drugs for treating various tumors and autoimmune diseases. However, high dose usage of MTX may cause severe side effects and the monitoring of MTX is therefore critical. By coupling a new MTX aptamer-based proximity hybridization with polymerization/nicking reaction (PNR) recycling amplifications, we develop here a sensitive and label-free colorimetric approach for MTX detection in diluted human serums. The MTX molecules can bind and switch the conformation of aptamers in the DNA duplex probes to initiate subsequent proximity hybridization-induced PNR recycling processes for the yield of a great deal of G-quadruplexes with the assistance of two single-stranded assistant DNA sequences. Hemin subsequently combines with these G-quadruplexes to produce lots of G-quadruplex/hemin horseradish peroxidase (HRP) mimicking DNAzymes, which then catalyze intensified color transition of the substrate solution to exhibit highly magnified UV–Vis absorption for label-free and ultrasensitive detection of MTX at concentration as low as 5.66 nM in the range of 10 nM to 1 μM. High selectivity of the developed method also enables it to monitor low levels of MTX in diluted serum samples, which offers such a method enormous potentials for convenient and highly sensitive detection of other small molecule drugs for various clinical applications.