Comparative Aptamer Discovery Providing Insights into Functional-Group Effects on the Recognition of Serotonin-Pathway Analytes

DNA aptamers are attractive molecular receptors for biosensing. Herein, we combined capture-SELEX, deep sequencing, binding characterization, and sequence-guided engineering to establish and compare DNA aptamers against three serotonin-pathway analytes: serotonin (5-hydroxytryptamine, 5-HT), its biosynthetic precursor 5-hydroxytryptophan (5-HTP), and its major metabolite 5-hydroxyindoleacetic acid (5-HIAA). Selection of 5-HT recovered the previously reported aptamer as the dominant sequence family, enabling family-guided structural analysis and truncation to a compact scaffold. Selections against 5-HTP and 5-HIAA yielded distinct aptamer families. Isothermal titration calorimetry measured dissociation constants of 8.1 μM for 5-HTP and 59.8 μM for 5-HIAA. Comparative analysis revealed distinct affinities among the three analytes, with functional-group-level affinity following the order: primary amine > amino acid backbone > carboxyl group. Finally, representative aptamers were tested using thioflavin T fluorescence for label-free fluorescent biosensors in serum- and synthetic urine-containing matrices, achieving limits of detection of 87 nM for 5-HT, 6 μM for 5-HTP, and 64 μM for 5-HIAA. These results expand the repertoire of aptamer receptors for serotonin-pathway analysis and establish an example of molecular recognition of closely related small molecules.