The binding of insulin to the insulin binding aptamer (IBA) was investigated with different spectroscopy techniques (UV/vis absorption, fluorescence, resonance light-scattering spectra (RLS), synchronous fluorescence, and three-dimensional fluorescence). The thermodynamic parameters (Kb, ΔH, ΔG, and ΔS) of the insulin-IBA complex were further investigated by temperature-dependent fluorescence measurement over the range of 25-37 °C. The results of synchronous fluorescence, resonance light scattering (RLS) and three-dimensional fluorescence spectra showed that IBA would alter the structure of insulin. Folding of The dual-labeled insulin binding aptamer, FL-IBA, carrying 6-carboxyfluorescein (FAM) and 6-carboxytetramethylrhodamine (TAMRA) labels at termini of the insulin binding aptamer (IBA), into G-quadruplex leading to the contact quenching occurs as a result of the formation of a nonfluorescent complex between donor and acceptor. Significant fluorescent signal change when 2 FL-IBA was bound to insulin is attributed to a conformational change in FL-IBA from a loose random coil to a compact G-quadruplex. Based on fluorescence quenching of IBA folding, a simple and sensitive insulin aptamer-based biosensor in the range of 2-70 nM was proposed.

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