Abstract
dc:description.abstractThis thesis endeavours to advance the field of biosensor development by introducing innovative DNA immobilisation strategies, with a primary focus on designing a new generation of biosensors utilising DNA as a biorecognition element. In Chapter 2, the exploration of a copper (II) (Cu (II)) specific DNA cleaving DNAzyme was undertaken towards the development of a sensitive Cu (II) sensor. Despite the promising utility of DNA cleaving DNAzymes, their slow kinetics limit their application in biosensors. Through studies in solution, it was discovered that the addition of polydopamine (PDA) and gold (Au) nanoparticles significantly enhances the rate of Cu (II) mediated cleavage, making this DNAzyme more suitable for biosensing applications. In Chapter 3, an electrochemical Cu (II) sensor is presented utilising the Cu (II) specific DNAzyme immobilised on the surface of a gold electrode. A self-polymerising PDA coating was employed to entrap both the DNAzyme and gold nanoparticles, resulting in a Cu (II) sensor with a low limit of detection (180 nM) and a linear range of 1 – 100 μM. This represents a significant step towards low-cost, remote environmental monitoring. Notably, the sensor can be fabricated in just 2 hours, delivers results in 15 minutes, and exhibits an excellent shelf life with a modest decay in signal over 2 months of storage under ambient conditions. Chapter 4 explores the use of an electrohydrodynamic (EHD) inkjet printer capable of depositing femtolitre volumes for the preparation of DNA microarrays. While such printers have been utilised for creating biomolecule patterns on various surfaces, few studies have investigated their impact on current DNA microarray functionalisation techniques that should render single stranded DNA covalently bound to a surface. Work presented here reveals that the femtolitre inkjet printing strategy is incompatible with the widely used silane (3-Glycidyloxypropyl)trimethoxysilane (GOPTS) surface functionalisation chemistry on both conductive (Silicon Dioxide) and non-conductive (Indium Tin Oxide) surfaces. Aside from being a lengthy protocol (over 24 hours), binding of EHD printed DNA is demonstrated to be largely mediated by non-specific adsorption due to unavoidable drying of the droplets of DNA ink. In addition, the requirement of surface blocking, wherein the remaining functional surface is rendered unreactive, is demonstrated to be required to prevent non-specific binding of the target DNA upon hybridisation. To overcome these challenges, Chapter 5 explores the creation of polymethyl methacrylate (PMMA) - azide microarrays, employing copper mediated click chemistry for the selective grafting of alkyne-modified ssDNA. This strategy results in covalent binding of DNA in 10 minutes, hybridisation with its complementary target in 2 hours, requires no blocking of the remaining surface, and permits substrate reuse, offering substantial advantages over conventional silane methods. In summary, this thesis is aimed at advancing biosensor manufacturing through the introduction of novel, easy-to-perform and useful DNA immobilisation strategies. The exploration of a Cu (II)-specific DNAzyme, enhanced by PDA and gold, has led to the development of a sensitive and rapid electrochemical Cu (II) sensor with remarkable attributes. Furthermore, the systematic investigation of an EHD inkjet printer for DNA microarray fabrication provides a comprehensive overview of the conventional techniques in microarray design, highlighting crucial factors in sensor design. The findings offer valuable insights and serve as a reference document, aiding researchers in conducting similar work and furthering the progress in this specialised domain. Finally, the development of a PMMA-azide ink and DNA attachment through click chemistry offers a valuable alternative protocol for the next generation of biosensing technologies utilising EHD printers.
Degree
thesis:*- Name dc:type.qualificationname
- Doctor of Philosophy (PhD)
- Level dc:type.qualificationlevel
- Doctoral
- Grantor dc:publisher.institution
- University of Cambridge
- Year dc:date.issued
- 2024
Author and committee
dc:creator, dc:contributor.*- Author dc:creator
-
- Etheridge, William
- Advisors dc:contributor.advisor
-
- Fruk, Ljiljana
- Brossard, Frederic
Subjects
dc:subject × 5Rights
dc:rightsIdentifiers
dc:identifier.*- DOI dc:identifier.doi
- https://doi.org/10.17863/CAM.111888
- OAI identifier oai:identifier
- oai:www.repository.cam.ac.uk:1810/373502