Wong, Zheng Wei
(2023)
Design and development of DNA biosensors based on silver nanoclusters and hybridisation chain reaction.
PhD thesis, University of Nottingham.
Abstract
Biosensors that couple hybridisation chain reaction amplification and fluorogenic silver nanoclusters (HCR-AgNCs) have the potential to enable simple and cost-effective detection of target analytes. These biosensors feature non-enzymatic amplification at mild conditions with a strong label-free fluorescence response. Whilst the proof-of-concept of these sensors have been promising, the design and developmental stages of HCR-AgNCs biosensors remain a challenge due to the lack of studies within the field itself.
The primary aim of this thesis was to design and develop new HCR-AgNCs biosensors for specific detection of short nucleic acid sequences (i.e., DNA and microRNA (miR)). To achieve this, three novel HCR-AgNCs biosensors were developed by designing DNA probes with assistance of computational software, then validated via lab-based experiments such as feasibility tests, condition optimisation, as well as performance analysis in both buffer and complex samples.
A ratiometric fluorescence HCR-AgNCs biosensor was first developed for miR detection, where emission of AgNCs changed from red to yellow in the absence and presence of miR-155. The ratiometric analysis has reduced the possibility of false measurement and increased the sensitivity of the assay, with limit of detection (LOD) measured at 1.18 fM. In the next study, a HCR biosensor was integrated with an externalised AgNCs probes via an ‘end-to-end cluster transfer’ mechanism, for DNA detection. This strategy simplified the designing process of DNA hairpins, ensured HCR predictability, and formed specific AgNCs species. In addition, the HCR-AgNCs biosensor exhibited high sensitivity towards the DNA target, with qualitative LOD at 1 pM and quantitative LOD at 3.35 fM. In the final study, magnetic-assistance was integrated into a ratiometric fluorescence HCR-AgNCs biosensor for the detection of miR-144-3p. The addition of magnetic beads (MBs) into the HCR-AgNCs biosensor facilitated a quick extraction and direct analysis of miR-144-3p, with LOD measured at 4.88 fM and satisfactory detection in raw human serum sample.
The overall findings from these works conclude that by carefully designing the DNA probes used to construct HCR-AgNCs, one can develop a functional and stable HCRAgNCs biosensor without overcomplicating the detection process. Besides that, the concept of proximity-dependent AgNCs, introduced in each of the proposed biosensors, addressed some of the practical limitations posed by conventional HCR-AgNCs biosensors. Moreover, the implementation of different sensing mechanism and modifications (i.e., externalisation of AgNCs and MBs addition) in these studies highlight the versatility of HCR-AgNCs assays for different applications.
Ultimately, the design and development efforts of HCR-AgNCs biosensors in this work can inspire further insights and research into this class of biosensors and facilitate extensive biosensing applications in the field of clinical diagnostics and beyond.
| Item Type: |
Thesis (University of Nottingham only)
(PhD)
|
| Supervisors: |
New, Siu Yee
Lee, Mei Kee
Ng, Jeck Fei |
| Keywords: |
biosensors; nucleic acid sequences; DNA; fluorescent; silver nanoclusters |
| 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: |
73641 |
| Depositing User: |
Wong, Zheng
|
| Date Deposited: |
22 Jul 2023 04:40 |
| Last Modified: |
31 Jul 2024 04:30 |
| URI: |
https://eprints.nottingham.ac.uk/id/eprint/73641 |
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