New phosphorescence based probes and techniques for the analysis of cellular oxygen and respiration

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dc.check.embargoformatNot applicableen
dc.check.infoNo embargo requireden
dc.check.opt-outNoen
dc.check.reasonNo embargo requireden
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dc.contributor.advisorPapkovsky, Dmitri B.en
dc.contributor.authorKondrashina, Alina
dc.contributor.funderEuropean Commissionen
dc.date.accessioned2015-11-20T13:17:36Z
dc.date.available2015-11-20T13:17:36Z
dc.date.issued2014
dc.date.submitted2014
dc.description.abstractReal time monitoring of oxygenation and respiration is on the cutting edge of bioanalysis, including studies of cell metabolism, bioenergetics, mitochondrial function and drug toxicity. This thesis presents the development and evaluation of new luminescent probes and techniques for intracellular O2 sensing and imaging. A new oxygen consumption rate (OCR) platform based on the commercial microfluidic perfusion channel μ-slides compatible with extra- and intracellular O2 sensitive probes, different cell lines and measurement conditions was developed. The design of semi-closed channels allowed cell treatments, multiplexing with other assays and two-fold higher sensitivity to compare with microtiter plate. We compared three common OCR platforms: hermetically sealed quartz cuvettes for absolute OCRs, partially sealed with mineral oil 96-WPs for relative OCRs, and open 96-WPs for local cell oxygenation. Both 96-WP platforms were calibrated against absolute OCR platform with MEF cell line, phosphorescent O2 probe MitoXpress-Intra and time-resolved fluorescence reader. Found correlations allow tracing of cell respiration over time in a high throughput format with the possibility of cell stimulation and of changing measurement conditions. A new multimodal intracellular O2 probe, based on the phosphorescent reporter dye PtTFPP, fluorescent FRET donor and two-photon antennae PFO and cationic nanoparticles RL-100 was described. This probe, called MM2, possesses high brightness, photo- and chemical stability, low toxicity, efficient cell staining and high-resolution intracellular O2 imaging with 2D and 3D cell cultures in intensity, ratiometric and lifetime-based modalities with luminescence readers and FLIM microscopes. Extended range of O2 sensitive probes was designed and studied in order to optimize their spectral characteristics and intracellular targeting, using different NPs materials, delivery vectors, ratiometric pairs and IR dyes. The presented improvements provide useful tool for high sensitive monitoring and imaging of intracellular O2 in different measurement formats with wide range of physiological applications.en
dc.description.sponsorshipEuropean Commission (Marie Curie Program)en
dc.description.statusNot peer revieweden
dc.description.versionAccepted Version
dc.format.mimetypeapplication/pdfen
dc.identifier.citationKondrashina, A. 2014. New phosphorescence based probes and techniques for the analysis of cellular oxygen and respiration. PhD Thesis, University College Cork.en
dc.identifier.endpage137
dc.identifier.urihttps://hdl.handle.net/10468/2080
dc.language.isoenen
dc.publisherUniversity College Corken
dc.rights© 2014, Alina Kondrashina.en
dc.rights.urihttp://creativecommons.org/licenses/by-nc-nd/3.0/en
dc.subjectCellular oxygenen
dc.subjectPhosphorescent nanoparticlesen
dc.subjectIntracellular probesen
dc.subjectCell respirationen
dc.thesis.opt-outfalse
dc.titleNew phosphorescence based probes and techniques for the analysis of cellular oxygen and respirationen
dc.typeDoctoral thesisen
dc.type.qualificationlevelDoctoralen
dc.type.qualificationnamePhD (Science)en
ucc.workflow.supervisord.papkovsky@ucc.ie
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