Selection and characterisation of triclosan-specific aptamers using a fluorescence microscope-imaging assay

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 K_d 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 K_d value of 378 nM, which was comparable with previously published K_d 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.