Novel Strategies for Gold Nanoparticle Based Aptamer Selection

While many successful biotechnological tools that enable the sensing of compounds within environmental and biological systems have been produced to date, the rapid detection of small molecules in solution remains challenging without the utilisation of highly specialised, expensive equipment.

Aptamers are antibody-like bioreceptors that are generated entirely by in vitro methodologies. Their use in biosensors has gained much attention due to the ability to generate them to bind small, sometimes toxic molecules that other bioreceptors are incapable of detecting. Much work has gone into generating aptamers for such targets, only to find that without drastic modification, many don’t function well within the biosensors they were designed for, despite their target molecule binding capabilities. In light of these findings, aptamer research has shifted towards producing aptamers  with qualities that make them more functional within their designated biosensing system.

The overall aim of this study was to design an aptamer generation protocol using a modified approach to systematic evolution of ligands by exponential enrichment (SELEX) that utilised gold nanoparticles (AuNP) to test its compatibility in a AuNP biosensing assay. The aptamer generated should possess the ability detect the small molecule it was generated for. My hypothesis was that utilizing AuNP in the partitioning stage of the aptamer generation process would result in aptamers that outperform those generated through traditional methods when implemented in a AuNP sensor.

Following lengthy optimisation experiments, a novel SELEX protocol involving AuNP was developed for the generation of aptamers that bound methamphetamine. Using this methodology, a random nucleotide library underwent enrichment in five selection rounds. From this, individual oligonucleotide sequences (i.e. aptamer candidates) were selected to undergo characterisation using a plethora of methods including a AuNP-based assay and isothermal titration calorimetry (ITC).

A AuNP-based characterisation assay was first optimised using a previously-published methamphetamine binding aptamer (MS-03) generated using a traditional matrix-based selection system as a tool to screen for successful aptamer candidates. This methodology identified one candidate (OM5-C6) which exhibited a superior response to methamphetamine in this AuNP assay than the MS-03 aptamer. The OM5-C6 demonstrated detection of methamphetamine at a concentration of 2 μM methamphetamine, supporting the hypothesis of the study. Interestingly, despite OM5-C6 having a higher response in the AuNP assay compared to MS-03, it exhibited aweaker affinity for methamphetamine as measured by ITC. This revealed interesting thermodynamic qualities of the aptamer and further supported our hypothesis that the selection method employed should consider the end use sensing system the aptamer is being designed for.

Finally, this novel AuNP SELEX method was used to generate aptamers for two additional but divergent target molecules, namely glyphosate and the ACE-2 receptor binding domain (RBD) of SARS-CoV-2 spike protein. Whilst reasonable enrichment of the random library was observed following selection rounds with glyphosate, no glyphosate binding aptamers were identified. The direct interactive properties of the peptide, RBD of SARS-CoV-2 spike protein, with AuNP meant that this AuNP SELEX method may be unsuitable for all protein target molecules.

This PhD study demonstrated the capability of a novel AuNP based SELEX and resulted in the development of an aptamer capable of binding methamphetamine which demonstrated lower detection limits in an AuNP sensor.