DNA-modified surfaces and frameworks for sensing and imaging applications

Dismorr, Jack (2024). DNA-modified surfaces and frameworks for sensing and imaging applications. University of Birmingham. Ph.D.

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Abstract

Nucleic acids act as biomarkers for a host of diseases and conditions. Various technologies,almost all employing some form of modified DNA, have been developed to sense extracellular nucleic acids. An important component of many nucleic acid biomarkers are single nucleotidevariants (SNVs). SNVs are positions in DNA in which a single nucleotide or base pair is altered,and include single nucleotide polymorphisms, point deletions, and epigenetic mutations. Some SNVs are directly or indirectly linked to certain diseases, and therefore their detection can offer valuable diagnostic and prognostic information. Currently, fluorescence-based methods are the dominant approaches used to sense or map SNVs. Whilst effective, their drawbacks include the need for careful experimental design to avoid false-positives, the inability to interrogate the nature of a mutation (i.e., to determine which other base is present), and their often complex and time-consuming nature. Electrochemical sensing of SNVs offers an alternative to fluorescence, with the potential for greater synchronisation with our increasingly digital, device-led world. The bulk of this thesis reports on the development of a surface-immobilised heterobimetallic DNA probe capable of electrochemically distinguishing between nucleobases at a single site (i.e., SNVs) in target DNA strands. Two redox-active complexes, a copper cyclidene macrocycle and a ferrocene unit, are incorporated into DNA using automated solid-phase synthesis. The copper cyclidene is incorporated internally and the ferrocene is appended to the 5′ end of the probe, with each producing a distinct electrochemical signal allowing for a ratiometric sensing approach. Key properties of the probe, including sensitivity, stability, and regeneration capability are determined. Clinically relevant SNV mutations associated with cancer and COVID-19 are detected using the bimetallic probe. Additionally, investigations into the SNV sensingmechanism of the copper cyclidene, efforts to improve the sensitivity of the probe, and the expansion of targets to include RNA biomarkers are described. Also included in this thesis is the functionalisation of metal-organic frameworks (MOFs) with fluorescently-modified DNA. MOFs are crystalline materials composed of metal ions or clusters connected through organic linkers, the different combinations of which allow for the tuning of a MOF’s properties. The functionalisation of two different MOFs with complementary fluorescently-modified DNA, and efforts to bind them together via DNA hybridisation to create a single material that combines the properties of each individual MOF, are described herein.

Type of Work:

Thesis (Doctorates > Ph.D.)

Award Type:

Doctorates > Ph.D.

Supervisor(s):