Development of new photoluminescent sensing materials, systems and applications for cell analysis

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dc.contributor.advisorPapkovsky, Dmitri B.
dc.contributor.authorLi, Liangen
dc.contributor.funderScience Foundation Ireland
dc.contributor.funderAgilent Technologies
dc.date.accessioned2025-02-04T13:41:57Z
dc.date.available2025-02-04T13:41:57Z
dc.date.issued2024
dc.date.submitted2024
dc.description.abstractCell analysis using optochemical sensors represents large and important niche in life and biomedical science. Sensor calibration and detection platforms are critical during sensor fabrication, characterization and application. However, existing calibration setups, platforms, and systems have limitations with respect to their robustness, analytical performance and capabilities. In this project, several new photoluminescent pH and oxygen (O2) sensing materials, platforms and systems were developed, evaluated comparatively and then applied for bio-analysis particularly for samples containing cells. The first experimental section describes a new barometric calibration setup which provides accurate, convenient and versatile O2 calibration. Using this setup, a detailed characterization of several sensor types based on PtBP dye (sensor stickers, membrane inserts, liquid probe NanO2-IR) was conducted in phosphorescence lifetime (PLT) mode by handheld sensor readers, and their key optional parameters (τ0, Ksv, T-coefficients, etc) and calibration equations were determined and cross-compared. The second experimental section evaluates comparatively the PLT measurements of several Pt-porphyrin based oxygen sensors on three different platforms: the time-revolved fluorescence reader Victor2, luminescence spectrometer Cary Eclipse, and confocal laser-scanning FLIM/PLIM-TCSPC microscope. The merits and limitations of each platform, including instrumental factors, analytical performance, lifetime calculation methods, potential sources of error were assessed. The third experimental section presents a new photoluminescent materials for cell analysis based on the substituted phosphorescent Pt(II)- or Pd(II)-porphyrin indicator dyes bearing dual O2 and pH sensing functionality (MePor-SB). The study includes screening of different host matrices for the sensor, synthesis and evaluation of new MePor-SB derivatives with altered protonation behaviour, development of ratiometric version of the pH sensor, photoluminescent signal enhancement options, deposition of sensor coatings on common cell analysis substrates and their demonstration in OCR/ECAR measurements with relevant cell models (PC 12 cells). The fourth experimental section, describes novel solid-state pH sensors for cell analysis based on hydrophobic protonable metal-free porphyrins, octaethylporphine (OEP), and octaethylporphine-ketone (OEPK), as fluorescent pH indicators. The internally referenced ratiometric intensity and nanosecond lifetime-based versions of these pH sensors were developed and also multiplexed with the O2 sensors based on phosphorescent PtOEP dye. The key parameters of the pH sensor were optimized, including the type and concentration of proton transfer agent, working pH range and pKa, dye concentrations and cross-talk with the O2 sensor (Pt-OEP). The optimised two-analyte sensors with stable internally referenced calibrations, convenient spectral characteristics and low cytotoxicity were coated on microplates and demonstrated with cultured cells and 3D spheroids made of HCT 116 cells, measuring their ECAR/OCR and responses to stimulation. Finally, the planar solid-state pH sensors on polyester films (Mylar) were used to measure extracellular pH in grafted tumours produced by CT26 cells and freshly excised from animals on a portable FLIM imager. The new pH sensors and dual pH/O2 sensors showed high stability (>1 year of storage), fast response and stable calibration, being well-suited for detailed metabolic studies of biological samples on widely available laboratory equipment.en
dc.description.statusNot peer revieweden
dc.description.versionAccepted Versionen
dc.format.mimetypeapplication/pdfen
dc.identifier.citationLi, L. 2024. Development of new photoluminescent sensing materials, systems and applications for cell analysis. PhD Thesis, University College Cork.
dc.identifier.endpage148
dc.identifier.urihttps://hdl.handle.net/10468/16956
dc.language.isoenen
dc.publisherUniversity College Corken
dc.relation.projectScience Foundation Ireland (Grant SFI/12/RC/2276_P2)
dc.relation.projectAgilent Technologies (Agilent Fund, Act-UR Project #4474)
dc.rights© 2024, Liang Li.
dc.rights.urihttps://creativecommons.org/licenses/by/4.0/
dc.subjectOptochemical oxygen and pH sensorsen
dc.subjectFluorescent and phosphorescent porphyrinsen
dc.subjectCell metabolism analysisen
dc.subjectFLIM/PLIM lifetime microscopy and sensing;en
dc.subjectBarometric oxygen calibrationen
dc.subjectECAR and OCR measurementsen
dc.subjectTumour pH imagingen
dc.titleDevelopment of new photoluminescent sensing materials, systems and applications for cell analysisen
dc.typeDoctoral thesisen
dc.type.qualificationlevelDoctoralen
dc.type.qualificationnamePhD - Doctor of Philosophyen
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