Nanoparticle-based intra-cellular diagnostics
Korzeniowska, Barbara (2012) Nanoparticle-based intra-cellular diagnostics. PhD thesis, Dublin City University.
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An in-depth understanding of biochemical processes occurring within the cell is a key factor for early diagnosis of disease and identification of appropriate treatment. Intracellular sensing using fluorescent nanoparticles (NPs) is a potentially useful tool for real-time, in vivo monitoring of important cellular analytes.
This work is focused on synthesis of organically modified-silicate (ORMOSIL) optical nanosensors for the quantitative analysis of oxygen concentration and pH sensing inside the cell. The structure of the sensor consists of a biofriendly silica matrix with encapsulated oxygen/pH-sensitive dyes. The optical probes used in this work are the oxygen-sensitive ([Ru(dpp)3]2+) complex and pH-sensitive fluorescein isothiocyanate (FITC) coencapsulated with the ATTO488 and Texas Red as the reference dyes, respectively. In order to obtain silica-based NPs, the Stöber method was used. The NPs were characterised using techniques such as Transmission Electron Microscopy (TEM), Dynamic Light Scattering (DLS), fluorescence and other spectroscopic techniques.
The second part of this work focuses on the introduction of the NPs into the cell and intracellular sensing. In this work the oxygen and pH nanosensors are introduced in a number of established human and mouse cell lines. Internalization of NPs within the cell is investigated using fluorescence confocal microscopy techniques. The detection of the optical signal is based on both ratiometric and fluorescence lifetime – based measurements carried out on the wide-field and confocal microscopes with fluorescence lifetime imaging platforms. After the NP calibration, the response of the cell to the different extracellular oxygen concentration is investigated.
Oxygen and pH sensing is the starting point for this intracellular diagnostics research. The silica-based NPs, thanks to the flexible processing conditions, allow for tailoring of pore size and hydrophilic-hydrophobic balance. The possibility to control these two parameters makes the NPs a very promising tool for a better understanding of many processes in living cells.
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