A Fluorescent Aptasensor for Sensitive and Selective Determination of Epigenetic Cancer Biomarker N1-Methyladenosine in Urine Samples

N1-methyladenosine (m1A) level in urine increases in the presence of cancer and is associated with the tumor size and stage. In the present study, we aimed to develop a method for rapid, sensitive and accurate determination of m1A in urine samples. The capture systematic evolution of ligands by exponential enrichment (SELEX) method was used to isolate aptamers that could selectively bind to m1A. We successfully isolated two sequences that have high selectivity toward m1A. The affinities against m1A were determined by isothermal titration calorimetry (ITC) and thioflavin T (ThT) assays. The N1MA1a aptamer has a Kd of 1.9 +/- 0.1 mu m determined by the ThT assay and 0.75 +/- 0.04 mu m determined by ITC. A strand-displacement biosensor was designed by labeling the aptamer with a carboxy fluorescein (FAM) and hybridizing it with a quencher-labeled complementary DNA strand. Using this biosensing system, m1A was detected with a detection limit of 1.9 mu m. The system shows high selectivity to m1A and high tolerance to adenosine, cytidine, guanosine, thymidine, uridine and N6-methyladenosine (m6A) as well as urine constituents at their real levels in urine. The sensor has been applied to five different human urine samples showing quantitative recovery values, which indicates practical potential of this aptamer-based biosensor.