Selection and characterisation of single-stranded DNA aptamers for triclosan

Abstract

<p>Triclosan (TCS) is a chlorinated organic compound which, due to its antibacterial properties in vitro, has found widespread usages in many medical and consumer products such as textiles, plastics and personal care products. Humans are directly and chronically exposed to TCS via dermal and mucosal contact from the use of TCS-formulated products such as soap and toothpaste. TCS is classified as an environmental contaminant by the European Union Water Framework Directive, whose mandatory goal is to develop new and simple-to-use analytical methodologies capable of measuring low concentrations of TCS and that are suitable for high-throughput detection. Synthetically-derived single-stranded oligonucleotides, also known as aptamers, are superior candidates for the development of sensitive and high-throughput biosensing strategies. Biosensors utilising aptamers as molecular recognition elements have showed great promise in a variety of diagnostic and therapeutic applications, especially for the detection of small molecular weight organic compounds such as TCS. The aim of this thesis was to develop aptamers as new capture reagents for TCS, as the first step towards the development of an alternative, user-friendly, diagnostic technique for monitoring TCS in both environmental and biological samples. The objectives of the thesis were to: [i] produce by in vitro selection procedures, TCS binding single-stranded DNA (ssDNA) aptamers; [ii] characterise the selected aptamers and determine their equilibrium dissociation constant (Kd) values and; [iii] evaluate the applicability of the selected aptamers in a biosensing platform. To achieve these objectives, ssDNA aptamers capable of binding TCS were generated in vitro using a sequential approach known as systematic evolution of ligands by exponential enrichment (SELEX). An affinity column-based SELEX strategy together with a variety of SELEX modifications such as negative and counter selections, real-time amplification and fluorescence quantification were explored for finding TCS specific aptamers. A total of 20 TCS aptamers, ten from 8 rounds of a basic-SELEX procedure, and the other ten from 10 rounds of a revised-SELEX procedure were generated. In general, these aptamers showed acceptable levels of sensitivity and specificity to TCS, and the best binding aptamer demonstrated a Kd value of 378 nM. The Kd value is comparable to published Kd values for compounds that share similar chemical structures to TCS. In addition, a novel fluorescent-based imaging method was developed in this dissertation. The method developed provides an alternative approach for monitoring SELEX progression and has the potential to simplify the way to characterise the binding properties of an aptamer to its cognate target. The utility of this method was compared with commonly used methods such as dot blot and fluorescent binding assays. The performance of the new imaging method was superior to the existing methods in terms of accuracy, simplicity and reproducibility. Furthermore, the best binding TCS aptamer was evaluated for its utility in an aptamer-based biosensor. The developed aptasensor, utilising a TCS aptamer as the recognition element and gold nanoparticles (AuNPs) as the signal reporter, was capable of detecting TCS in spiked-water samples at concentrations ranging from 20–750 nM with a visual detection limit of 150 nM. In conclusion, methods were developed to select, refine, and characterise ssDNA aptamers capable of binding to TCS, and these aptamers have the potential to offer a sensitive, simple-to-use, and user-friendly analytical method for TCS detection.</p>