This study reports a fluorescence microscope-imaging assay for determining the binding characteristics of single-stranded DNA aptamers selected against the antibacterial agent, triclosan. The imaging assay utilises fluorescently labelled aptamers and target-immobilised matrices. Upon binding of triclosan-specific aptamers to triclosan-conjugated matrices, the binding complex was visualised and the image was captured with the aid of a fluorescence microscope. Subsequently, the fluorescent intensities of aptamer-bound matrices were analysed using dedicated image-processing software and correlated to known concentrations of selected input aptamers. Thus, by plotting fluorescence intensities against different aptamer concentrations, binding isotherms were generated to determine aptamer Kd values. The imaging assay was applied to characterise the binding affinities and specificities of ten triclosan-specific aptamers H1-H10. One of the candidate aptamers, H6, showed a Kd value of 378 nM, which was comparable with previously published Kd values for aptamer-generated against triclosan analogous. In addition, the utility of the imaging assay for aptamer characterisation was compared with a commonly used affinity column-binding assay. It was concluded that the imaging assay was superior to alternative assays in terms of accuracy, simplicity, and reproducibility.
Keywords: Aptamers; Circular dichroism; Competitive-binding assays; Fluorescence microscopy; SELEX; Triclosan.