New biosensors for metabolic imaging

dc.check.embargoformatEmbargo not applicable (If you have not submitted an e-thesis or do not want to request an embargo)en
dc.check.infoNot applicableen
dc.check.reasonNot applicableen
dc.check.typeNo Embargo Required
dc.contributor.advisorDmitriev, Ruslanen
dc.contributor.authorO'Donnell, Neil
dc.contributor.funderScience Foundation Irelanden
dc.description.abstractTissue engineering is a multi-disciplinary field that involves three-dimensional cell and tissue models, live cell microscopy and related imaging modalities, along with fluorescence and phosphorescence-based biosensors. These technologies can work together in developing biologically relevant 3D tissue models for the modelling of complex physiological and diseased states. One of the main challenges facing such models is the lack of non-invasive strategies for quantitative real time monitoring of cellular and tissue physiology, metabolism and viability, that are compatible with live cell microscopy. This thesis presents the design and development of new biosensor, scaffold and nanoparticle materials, with the aim of facilitating quantitative metabolic imaging in cell and tissue culture, via fluorescence lifetime microscopy and phosphorescence lifetime microscopy. Thus, we have developed protein-based biosensor probes, sensitive to pH and calcium in intensity and fluorescence lifetime modalities for the labelling of cellulose scaffold materials, producing a hybrid scaffold material for tissue engineering applications. This was done by genetically engineering of recombinant proteins expressing the cellulose-binding domain (CBD) CenA protein, derived from the fungus C. fimi, fused to pH-sensitive enhanced cyan fluorescent protein (ECFP) and enhanced yellow fluorescent protein (EYFP), forming CBD-ECFP and CBD-EYFP biosensors, respectively. A third biosensor was also developed with CBD and the genetically encoded calcium indicator known as circularly permutated EGFP (cpEGFP)/M13/Calmodulin (CaM) fusion protein (GCaMP2) forming CBD-GCaMP2. For all three CBD constructs we observed responses in fluorescence intensity to changes in calcium for GCaMP2 and pH for both CBD-ECFP and CBD-EYFP, achieving efficient and stable labelling of various cellulose scaffolds including nanofibrillar, GrowDex, bacterial cellulose and decellularised plant materials. CBD-ECFP labelled GrowDex produced a biosensor scaffold material capable of supporting the growth of 3D cultured human colon cancer cells HCT116, with the ability to measure real-time changes in extracellular pH. The developed labelling strategy allows for the design of biosensor scaffold materials with potential multi-parametric fluorescence lifetime microscopy modalities, which can be used to achieve the controlled production of 3D tissue models with measurable pH and metabolic gradients. Intracellular metabolic imaging is currently dominated by synthetic nanoparticle constructs that suffer from suboptimal intracellular staining, along with high toxicity and immunogenicity. Here we developed several self-assembling protein nanoparticle constructs based on viral like particle, elastin like polypeptide-cowpea chlorotic mottle virus capsid protein (ELP-CP) and protein nanocage ferritin. Such constructs hold promise due to their biological nature making them more biocompatible and biodegradable, thereby reducing toxic and immunogenic effects. Such self-assembling protein nanoparticles are also amenable to multiple strategies of functionalisation such as metallochelate coupling, genetic engineering, chemical modification, and encapsulation. We evaluated metallochelate coupling to design intracellular O2-sensitive biosensors, where oligohistidine-tagged recombinant proteins are bound to nitrilotriacetate (NTA) or iminodiacetic acid (IDA) groups on dyes and small molecules. The NTA or IDA groups form a complex with transition metal ions such as: Zn2+, Ni2+, Co2+, or Cu2+. This complex then co-ordinates to histidine amino acids on the recombinant protein. We successfully produced ratiometric phosphorescent probes from enhanced green fluorescent protein (EGFP), enhanced monomeric blue fluorescent protein 2 (mTagBFP2) and Discosoma red fluorescent protein (DsRed express) coupled to tetracarboxylic platinum (II)-coproporphyrin I (PtCP) PtCP-NTA. Such complexes can be used for ratiometric-based measurements of O2, where fluorescent proteins (FPs) can be used as O2-insensitive references. Most notably we demonstrated the first example of a phosphorescent O2-senstive viral like particle (VLP) structure, ELPCP-H6-PtCP and in comparing to commercial O2-sensitive probe MitoXpress, we observed higher phosphorescence brightness, similar lifetime responses and increased sensitivity in response to O2. The potential to couple a range of FPs or self-assembling protein nanoparticles to O2 sensitive phosphorescent dyes demonstrates that metallochelate coupling is a highly attractive strategy in the design of new intracellular O2 sensors. Using genetic engineering and encapsulation strategies we successful produced both pH and O2- sensitive ferritin nanoparticles. Genetic engineering enabled the expression of multiple cell targeting and penetrating peptides, such as bactenecin 7 and α-enolase, along with fluorescent proteins EGFP or ECFP, without affecting spectral properties of the fluorescent proteins or ferritin self-assembly. Genetic engineered ECFP-FTN construct demonstrated pH sensitivity in fluorescence intensity and lifetime across a physiological range of pH, potentially allowing for applications in fluorescence lifetime microscopy-based measurements of intracellular pH. Through the strategy of pH dependent disassembly and reassembly encapsulation of phosphorescent O2 sensitive probe Pt-Glc, we successfully produced O2-sensitive horse ferritin-based (hoFTN) nanoparticles. The resulting hoFTNPt-Glc displayed a higher phosphorescence intensity signal than free Pt-Glc, possibly due to the concentrated number of Pt-Glc molecules in close proximity within the ferritin structure, and demonstrated responses to oxygenation, increasing phosphorescence intensity when deoxygenated. However, in characterisation of hoFTN-Pt-Glc with MEF cells we observed poor intracellular staining confined to endosomes, similar to free Pt-Glc. These results showed that encapsulation here does not improve intracellular staining or phosphorescence lifetime responses. Despite poor characterisation of ferritin constructs in HCT116 and MEF cell lines, the strategies evaluated here show promise and demonstrate an interchangeable approach to functionalising self-assembling protein nanoparticles and fluorescent proteins for applications in fluorescence lifetime microscopy and phosphorescence lifetime microscopy-based quantitative and ratiometric live cell imaging.en
dc.description.statusNot peer revieweden
dc.description.versionAccepted Version
dc.identifier.citationO'Donnell, N. 2018. New biosensors for metabolic imaging. PhD Thesis, University College Cork.en
dc.publisherUniversity College Corken
dc.relation.projectinfo:eu-repo/grantAgreement/SFI/SFI Starting Investigator Research Grant (SIRG)/13/SIRG/2144/IE/Development of Bionic Sensor Materials for Metabolic Imaging in Regenerative Medicine/en
dc.rights© 2018, Neil O'Donnell.en
dc.subjectLive cell imagingen
dc.subjectSelf assembling protein nanoparticlesen
dc.subjectMetallochelate couplingen
dc.subjectFluorescence lifetime microscopyen
dc.subjectCellulose based scaffoldsen
dc.subjectBionic sensor materialsen
dc.subjectMetabolic imagingen
dc.subjectRegenerative medicineen
dc.subjectThree-dimensional tissue modelsen
dc.titleNew biosensors for metabolic imagingen
dc.typeDoctoral thesisen
Original bundle
Now showing 1 - 1 of 1
Thumbnail Image
5.19 MB
Adobe Portable Document Format
Full Text E-thesis
License bundle
Now showing 1 - 1 of 1
Thumbnail Image
5.62 KB
Item-specific license agreed upon to submission