Rocca, Francesco (2014) Engineering & characterization of a gfp-based biosensor for ph and chloride intracellular measurements. PhD thesis, University of Trento.
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ClopHensor, a new fluorescent ratiometric GFP-based biosensor, is a powerful tool for non-invasive pH and chloride quantification in cells. ClopHensor is a chimeric construct, with the pH- and chloride-sensing E2GFP linked to the reference red protein DsRed-monomer, whose fluorescence is used as reference signal. E2GFP dissociation constant of about 50 mM (at pH=7.3) makes it ideal for quantifying physiological chloride concentration. However, chloride affinity of E2GFP strongly depends on pH value in solution: precise chloride measurement requires also a pH measurement. By ratio-imaging technique, three different excitation wavelengths are necessary for a pH and chloride concentration estimation. With the goal to reduce the number of excitation wavelengths required for ratio-imaging technique, in this thesis I present a detailed study of H148G-V224L-E2GFP, selected among several E2GFP-variants for its improved photophysic and spectroscopic characteristics. H148G-V224L-E2GFP exhibits a chloride affinity and a pH sensitivity similar to ClopHensor. Its emission spectra interestingly display two distinct emission peaks at 480 nm and 520 nm after excitation at 415 nm. Importantly, fluorescence emission spectra collected at various pH values also display a clear isosbestic point at 495 nm. This property allows the innovative possibility of pH and chloride concentration determination using only two excitation wavelengths. Moreover, while being chloride independent, the 520-to-495 (nm) ratio displays a pKa value of about 7.3, centered in the physiological pH range. These characteristics make it ideal for quantifying intracellular pH changes and chloride fluxes in physiological conditions. Applications in living cells of this new biosensor demonstrated its usefulness for ratio-imaging analysis. H148G-V224L-E2GFP+DsRed was successfully expressed in neuron-like cells, as proof-of-concept that ratio-imaging analysis can be performed also in neuron-like cells. These results are very promising for H148G-V224L-E2GFP+DsRed future expression in brain neurons, where chloride plays a crucial role in neuronal activity. Purified H148G-V224L-E2GFP was successfully uploaded in polymeric vaterite nanospheres to characterize their endocytosis pathways in cells.
|Item Type:||Doctoral Thesis (PhD)|
|Subjects:||Area 02 - Scienze fisiche > FIS/07 FISICA APPLICATA (A BENI CULTURALI, AMBIENTALI, BIOLOGIA E MEDICINA)|
|Repository Staff approval on:||06 May 2014 16:47|
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