Thesis
Enhancement of electrochemical sensor performance through the optimisation of nucleic acid probe architecture
- Creator
- Rights statement
- Awarding institution
- University of Strathclyde
- Date of award
- 2024
- Thesis identifier
- T16902
- Person Identifier (Local)
- 201886296
- Qualification Level
- Qualification Name
- Department, School or Faculty
- Abstract
- A key aim in the field of diagnostics is to engineer instrumentation that fulfils three primary aims. This includes enhancing the sensitivity of a device, or improve the ability to determine minimal concentrations of analyte in a complex sample. Secondly, devices must be capable of producing a signal readout in response to the presence or absence of the target analyte in a short time window. Thirdly, manufactured devices must be feasibly deployed to a point of care setting at a low cost, often in challenging environments Electrochemical methods can serve as the workhorse in achieving such goals, with its power in discriminating variations to a series of properties that describe a bioelectric interface. Simply, these interfaces are composed of an immobilised biomolecule upon a metal transducer surface that is capable of the capture, or detection, of a desired molecular target. In the case of nucleic acid detection, immobilised receptor nucleic acids, or DNA probes, serve as the detection element of the system. These DNA probes are engineered to share complementarity to a desired nucleic acid target, and in the presence of such a target, will capture the analyte by hybridisation through Watson-Crick base pairing laws. These hybridisation events change the interfacial properties of the transducer, and by electrochemical techniques, devices can translate such derivations in to a signal read out for the user. Molecular self-assembly is a process whereby molecules spontaneously form organised structures, governed by the inherent interactions between the local constituents. It is this principle that drives the formation of immobilised DNA probes in a “DNA Self-Assembled Monolayer”. This technique allows for a simple method of bioelectric interface construction. Conventionally, these DNA probes are single-stranded linear elements. However, an increasing number of publications are exploring ever more complex probe geometries in biosensing applications. Despite this, there is a distinct lack of contributions to the literature detailing whether such advanced probe architectures may provide a meaningful solution to current problems facing low cost point of care devices. To this end, this thesis attempts to explore key metrics of biosensor performance with an ever-increasing bioelectric interface complexity. Here, increasing complexity is achieved by the introduction of higher order probe architectures, or by the introduction of DNA nanostructures free in solution, which may serve as signal amplifiers. The first section of this work provides an extensive literature review. This begins with exploring the need for rapid PoC diagnostic technologies, with a particular focus on tackling antimicrobial resistance. This is followed by a detailing of current nucleic acid detection methods, the advent of DNA nanotechnology, and its recent advances and emerging applications. Thereafter, the DNA origami method is described, and its power and application in biosensor design is discussed. Finally, an account of key theoretical concepts governing electrochemical methods is provided. Experimental chapters then follow, detailing the development and testing of a series of electrochemical biosensor designs, each with an increasing degree of probe complexity. The first of which explores a class of 1D and 2D probes. These linear and hairpin probes are thoroughly interrogated to explore potential improvements in both sensitivity, and specificity. Within this chapter, successful enhancement in sensor selectivity was observed with a hairpin probe architecture against a linear probe. Sensitivity to complementary target was deemed comparable between both probe apparatus; therefore, translation of the hairpin based bioelectric interface to a microelectrode platform was undertaken. This was successfully shown to boost sensitivity in accordance with literature reports, while maintaining the enhanced selectivity inherent to hairpin probe structures. The second experimental chapter focuses on the introduction of tetrahedral DNA nanostructures to electrochemical biosensor apparatus. Three distinct strategies where explored. Firstly, a designed tetrahedron serves as the immobilised probe. Secondly, the same tetrahedron was modified to harbour an electroactive redox tag producing a “signal off” biosensor design. Finally, a novel approach is detailed, using free tetrahedra in solution to serve as signal amplifiers by boosting impedance following their tethering to the surface by a complementary target oligonucleotide. A valuable proof of concept is established here in the ability of nanostructures to serve as inexpensive and powerful methods of signal amplification negating the need for complex and costly chemistries common to other strategies. The third experimental chapter builds upon the signal amplification strategy described above, with the introduction of a novel, and highly programmable DNA origami tile. In a first for the electrochemical biosensor field, this chapter reports on a series of tile nanostructure designs capable of effectively crosslinking to a linear probe DNA functionalised transducer, with the presence of a complementary target serving as the linking tether. With this approach, growth in the impedance of the interface contributes to a significant improvement in sensor limit of detection, and importantly remains highly effective in a DNA rich complex media, proving its potential in future PoC devices. The final section of experimental work here focuses on a novel sensing approach, with a divergence from nucleic acid detection, to the successful electrochemical interrogation of environmental conditions by a switchable DNA nanostructure. Here, a DNA origami “zipper” has been designed to be responsive to environmental stimuli, specifically pH. Such a sensing application is of need given the known alterations in local pH conditions associated with both bacterial growth, and a series of human pathologies. This zipper structure was successfully immobilised as part of mixed SAM, forming a bioelectric interface capable of discriminated local pH conditions across a broad and clinically relevant pH range.
- Advisor / supervisor
- Corrigan, Damion K.
- Resource Type
- DOI
- Date Created
- 2022
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