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Ooi, Jessica Sui Ying (2025) Design and development of optical biosensors coupling gold nanoparticles or silver nanoclusters with hybridisation chain reaction. PhD thesis, University of Nottingham.

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Abstract

Optical biosensors play a crucial role in detecting specific analytes, including nucleic acids, proteins, metabolites and metal ions. Label-free optical biosensors utilising noble metal nanoparticles have the potential to revolutionise detection of cancer due to their high selectivity, sensitivity, and rapid results.

Among the ribonucleic acids (RNA) that have emerged as putative cancer biomarkers, microRNAs (miRs) stand out. However, their low abundance in biological samples necessitates amplification techniques for accurate detection.

In this thesis, the overarching goal is to develop label-free optical biosensors utilising hybridisation chain reaction (HCR) coupled with gold nanoparticles (AuNPs) or silver nanoclusters (AgNCs) as a signal transducer for facile miR detection. HCR is an enzyme-free nucleic acid amplification technique that can be performed under isothermal conditions. A pair of deoxyribonucleic acid (DNA) probes in hairpin conformation were designed as “amplifiers” to simultaneously detect and amplify miR-665, culminating in the formation of nicked double stranded DNA. These DNA hairpins consist of a double-stranded region (stem) and a single-stranded loop and tail (termed ‘toehold) region.

AuNPs are known to aggregate in the presence of salt, resulting in a visible colour change from red to blue. DNA hairpin probes can adsorb to AuNPs, thereby stabilising them against salt-induced aggregation (SIA). In the first project, the effects of the length and sequence of DNA hairpin stem and toehold on their ability to stabilise AuNPs against SIA was investigated. The results showed that hairpins with longer stems generally provided better stabilisation of AuNPs. For hairpins with a 14-base pair (bp) stem and 8-nucleotide (nt) homopolymeric toehold, a stabilisation trend of adenine (A) > cytosine (C) > guanine (G) > thymine (T) was observed. No improvement was observed for 14- and 18-bp hairpins with toeholds having more than eight adenines (-8A). Hairpins with toeholds comprising at least 50% A (with C or G making up the rest) could also result in strong stabilisation. Finally, hairpins with G-rich toeholds (≥ 6G) could form G-quadruplexes and should be avoided due to their reduced AuNPs stabilisation ability. These results served as guidelines to design DNA hairpins which will give strong AuNPs stabilisation and ideally improve the signal-to-background ratio of colorimetric HCR-AuNPs biosensors.

Subsequently, a HCR-AuNPs biosensor to detect miR-665 was developed by applying the aforementioned guidelines in conjunction with HCR hairpin design guidelines. When stabilised by DNA hairpins, the AuNPs were able to withstand aggregation. In contrast, upon HCR triggered by miR-65, the DNA hairpins were utilised and double-stranded HCR products were formed which did not stabilise AuNPs. HCR feasibility was first tested computationally with NUPACK software, and then verified experimentally using gel electrophoresis. The HCR-AuNPs biosensor successfully detected miR-665 with a detection limit of 10.7 nM and a detection range of 5–100 nM, yielding colorimetric results visible to the naked eye within two hours, without the need for enzymes and complicated steps. In addition, this biosensor was highly selective for miR-665 compared to other miRs implicated in ovarian cancer.

The final project explores the feasibility of developing a biosensor that can be more easily customised to detect different miR targets, without the need to constantly redesign HCR hairpins. By utilising a capture probe and ‘universal’ HCR hairpin probes, one of which acts as template for fluorescent AgNCs formation, a proof-of-concept HCR-AgNCs biosensor for miR-665 detection was developed. It had a detection limit of 41 nM and wide detection range of 50–400 nM, yielding ratiometric fluorescence signals within 5.5 hours. This biosensor was highly selective for miR-665 compared to other miRs implicated in ovarian cancer and was capable of single base discrimination. The modular design of the capture probe and DNA hairpins, as well as tuneability of AgNCs fluorescence emission, positions this biosensor for more facile customisation in future miR detection applications.

Item Type:	Thesis (University of Nottingham only) (PhD)
Supervisors:	New, Siu Yee Pung, Yuh Fen
Keywords:	nucleic acid amplification; gold nanoparticles; silver nanoclusters; biosensors; DNA hairpin; hybridisation chain reaction; microRNA; ovarian cancer
Subjects:	R Medicine > RS Pharmacy and materia medica
Faculties/Schools:	University of Nottingham, Malaysia > Faculty of Science and Engineering — Science > School of Pharmacy
Item ID:	78811
Depositing User:	Ooi, Jessica
Date Deposited:	08 Feb 2025 04:40
Last Modified:	08 Feb 2025 04:40
URI:	https://eprints.nottingham.ac.uk/id/eprint/78811

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