Label-Free Fluorometric Split Aptasensor for the Specific Detection of Uric Acid from Xanthine and Hypoxanthine

Xanthine and hypoxanthine are metabolic precursors of uric acid (UA), and their molecular structures and chemical properties are similar to those of UA. Consequently, accurately distinguishing between them has long posed a substantial challenge. In this study, library immobilization was employed to screen for aptamers for UA. An aptamer designated Apt2 exhibits a dissociation constant (K_d) of 14.11 μM for UA, demonstrating low affinity for xanthine and negligible affinity for hypoxanthine. DG10 and DG11 are critical sites for maintaining its affinity and selectivity. Microscale thermophoresis (MST) analysis yielded a K_d value of 461 nM for Apt2, while circular dichroism (CD) spectroscopy indicated that Apt2 might adopt a parallel G-quadruplex conformation. Based on its conserved sequence and secondary structure, Apt2 was systematically split and tailored to generate two split aptamers: Apt2-T4/Apt-T5 and Apt2-T4-T3/Apt2-T5. The K_d values of these split aptamers, as measured by thioflavin T (ThT) fluorescence, were 48.78 μM and 38.72 μM, respectively. To facilitate sensor development, the kinetic and thermodynamic characteristics of these aptamers were evaluated. The results indicated that Apt2-T4-T3/Apt2-T5 binds rapidly to UA, and the melting temperature (Tm) of Apt2-T4-T3/Apt2-T5 was determined to be 40.48 ± 0.18 °C. Finally, we developed a label-free fluorometric split aptasensor based on Apt2-T4-T3/Apt2-T5, which achieved a detection limit of 0.60 μM and exhibited a strong linear response within the UA concentration range of 3.15-200 μM. Subsequently, the aptasensor was applied to detect UA in real urine samples.