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 K_d of 1.9±0.1 µm determined by the ThT assay and 0.75±0.04 µm 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 µm. 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.