Exploring Fluorescence Spectral Shifts in Aptamer-Intercalating Cyanine Dye Complexes upon Binding to Specific Small Molecules

DNA intercalating cyanine dyes, such as SYBR Green I (SG) and OliGreen (OG), are widely used in developing label-free, fluorescent aptamer-based biosensors. Despite their widespread use for direct analyte detection through changes in fluorescence intensity, the effects of dye concentrations and the specific nature of their interactions have been inadequately explored. Here, we reported how dye-to-base ratios (dbrs) influence the fluorescent response of DNA intercalating dyes in aptamer systems targeting adenosine triphosphate (ATP) and l-argininamide (LAA). We initially examined the fluorescence spectral shifts of an ATP aptamer (ABA) with SG across varying dbrs, observing an emission shift to longer wavelengths as the dbrs increased. Subsequently, systematic analysis of the ATP aptamer and SG complex (ABA/SG) at different target concentrations revealed a “signal-off” phenomenon at a very low dbr of 0.1, which transitioned to a blue shift in the fluorescence spectra at higher dbr values of 0.7 and 2.0. Further extending our research, we explored the use of OG as a ratiometric probe for detecting l-argininamide, noting similar spectral shifts to shorter wavelengths upon target binding. Absorption spectroscopy, circular dichroism (CD), and meticulously designed control studies were employed to elucidate the spectral shift phenomenon comprehensively. Our findings underscore the significant impact of dye selection and concentration on the performance of fluorescence aptasensors and demonstrate that clear spectral shifts, indicative of target binding, occur upon binding to targets, particularly at higher dye loading; however, excessive dye concentrations can perturb the aptamer structure, reducing its binding affinity. We believe that our findings will provide new insights into designing aptamer-based fluorescence assays for the sensitive and specific detection of small molecules.