2-Aminopurine Fluorescence Spectroscopy for Probing a Glucose Binding Aptamer

Glucose is the most important analyte for biosensors. Recently a DNA aptamer was reported allowing binding-based detection. However, due to a relatively weak binding affinity, it is difficult to perform binding assays to understand the property of this aptamer. In this work, we replaced the only adenine base in the aptamer binding pocket with a 2-aminopurine (2AP) and used fluorescence spectroscopy to study glucose binding. In the selection buffer, glucose increased the 2AP fluorescence with a K_d of 15.0 mM glucose, which was comparable with the 10 mM K_d previously reported using the strand displacement assay. The binding required two Na⁺ ions or one Mg²⁺ that cannot be replaced by Li⁺ or K⁺ . The binding was weaker at higher temperature and its van't Hoff plot indicated enthalpy-driven binding. While other monosaccharides failed to achieve saturated binding even at high concentrations, two glucose-containing disaccharides, namely trehalose and sucrose, reached a similar fluorescence level as glucose although with over 10-fold higher K_d values. Detection limits in both the selection buffer (0.9 mM) and in artificial interstitial fluids (6.0 mM) were measured.