Selection and characterisation of triclosan-specific aptamers using a fluorescence microscope-imaging assay
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RESEARCH PAPER
Selection and characterisation of triclosan-specific aptamers using a fluorescence microscope-imaging assay Shiwei Li 1,2 & Mark Clarkson 2 & Kenneth McNatty 1 Received: 30 June 2020 / Revised: 29 July 2020 / Accepted: 4 August 2020 / Published online: 11 August 2020 # Springer-Verlag GmbH Germany, part of Springer Nature 2020
Abstract 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 targetimmobilised 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 . SELEX . Triclosan . Fluorescence microscopy . Circular dichroism . Competitive-binding assays
Introduction Triclosan (TCS) is a broad-spectrum antimicrobial and preservative agent used in many personal care, veterinary, industrial, and household products [1]. It has been estimated that the annual usage of TCS is about 300 and 450 tons in the USA and Europe [2], respectively. The intensive use of TCS increases the potential for general population to be exposed to TCS through ingestion or dermal contact. TCS has been identified in human breast milk [3], blood [4], and urine [5] samples. A cause for concern in this regard is that many studies
Electronic supplementary material The online version of this article (https://doi.org/10.1007/s00216-020-02863-7) contains supplementary material, which is available to authorized users. * Shiwei Li [email protected] 1
School of Biological Sciences, Faculty of Science, Victoria University of Wellington, Wellington 6140, New Zealand
2
Callaghan Innovation, 69 Gracefield Road, Lower Hutt 5010, New Zealand
show that TCS is capable of exerting adverse effects on hormonal homeostasis and metabolic mechanisms [6–8]. For example, Kumar et al. (2009) have reported that oral administrations of TCS at doses of 10 and 20 mg/kg/day inhibit testicular androgen productio
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