Développement et application de nano-architectures cœur-coquille fluorescentes pour la mesure du pH
|Abstract:||Ionic gradient changes are essential in almost every cellular and bacterial metabolic processes. Therefore, any malfunction that perturbs these ion concentrations can induce major illnesses in the affected physiological solution or biological tissue. Amongst all the chemical species influenced by cell metabolism, protons are key to many events; enzyme activity, protein structural changes, and activation of membrane transporters. In this doctoral thesis, pH-sensitive fluorescent nanoparticles were developed and grafted on planar substrates in order to probe the culture medium of different cells in vitro. These innovative analytical tools for biomedical research offer the possibility to image quantitative pH values in microscopy with both spatial (~1 μm) and temporal resolutions (<300 ms). As such, the sensors were optimized in a structural parametric study with regards to the nanoarchitecture design. These particles comprise a metallic core with plasmonic properties to enhance the fluorescence of molecules incorporated into a porous silica shell. The core size, the spacing layer thickness, the spectral overlap between the plasmon and the fluorescence excitation/emission bands, and the sensitive moieties were carefully chosen to take advantage of the plasmonic range of enhancement in concentric architectures. Since the covalent encapsulation of molecules is highly adaptable for many silanized fluorophores, core-multishell architectures were synthesized as a proof of concept which allows simultaneous measurements of pH and halides (Cl-, Br-, I-), and signal normalization with an internal reference onto the same nanoparticle. The ratiometric correction compensates for fluctuations in light intensity and experimental errors with sensor concentration, photobleaching and environmental effects as a function of time. Finally, this analytical strategy was applied in a spectroscopic study of fluorescein-doped nanoparticles as quantitative pH-sensitive markers implemented in biological cell cultures. Particularly, the core-shell nanoarchitectures were functionalized with a silane component and immobilized at the surface of a silica substrate by way of complementary click coupling. These improved microscope coverslips were evaluated as a fluorescent sensing surface for the culture of adhesive excitable cells – e.g. cardiac fibroblasts and neurons, but also for the growth of bacterial biofilms and the study of multiphase interfaces in microfluidic devices.|
|Document Type:||Thèse de doctorat|
|Open Access Date:||3 July 2018|
|Collection:||Thèses et mémoires|
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