Capture-SELEX of DNA aptamers for label-free detection of epinephrine and norepinephrine in urine

Epinephrine (EP) and norepinephrine (NE) are two essential catecholamine neurotransmitters. Accurate monitoring of their levels is crucial for clinical diagnosis. In this study, we employed the library-immobilized SELEX technique to select DNA aptamers for EP and NE. A representative aptamer, named EP-1, showed a strong affinity to EP and a slightly weaker binding to NE, with dissociation constant (K_d) values of 12.2 μM and 36.6 μM, respectively, as determined by the thioflavin T (ThT) assay. The K_d values obtained from isothermal titration calorimetry (ITC) were 9.4 μM EP and 58.1 μM NE, comparable to those measured from ThT fluorescence. The EP-1 aptamer showed no apparent binding to other neurotransmitters or biomolecules, indicating excellent selectivity. When NE was used as the target, although DNA sequences were enriched, the most enriched sequence showed no binding using ITC, ThT or DNA strand displacement assays. A comparison was made with a previously reported dopamine aptamer, and the results showed that the functional groups on the side chain of target molecules influence the binding of this aptamer. Furthermore, we developed a label-free fluorescent aptasensor for the simultaneous detection of EP and NE in urine samples, with limits of detection of 0.41 μM and 0.83 μM, respectively. These results indicate that it has a good application prospect in the diagnosis of neurodegenerative and other diseases.