Detección selectiva de analitos de interés biológico a través de nuevas sondas fluorescentes. Diseño, síntesis y caracterización fotofísica de las mismas. Aplicación en medios biológicos

Resumen

The main research topic of this Thesis is the development of new probes for specifically analytes detection. For this aim, Fluorescence has been the technique of choice because it achieves high level of sensitivity, specificity, simplicity and wide concentration range. Furthermore, it is also a non-invasive method and it is characterized by its low toxicity. There are some biologically relevant analytes that are interesting to detect due to the fact that their deregulation is related to metabolic changes that promote damage in living organism. In this work it has been selected three analytes with clinical potential application, these are: phosphate, biothiols and acetate ions. Phoshate anions participate in signal transduction and energy storage of energy in cells and extracellular media. Apart from phosphate anions, biothiols are also widely present in living organisms and are relevant because changes in their normal levels cause cellular disorders and consequently oxidative stress. The assay of phosphate intracellular measurement as a marker of bone cell differentiation and bone deposition combined with biothiol levels as a marker of cell stress would be a very helpful tool to detect pathological processes that combine both pathologies. In this work, it is described the design of a new molecule with the ability to measure these two parameters that has not been described to date. For this goal, it has been synthesized a new xanthene-derived molecule that specifically detects phosphate and biothiols simultaneously. This dual probe reacts with biothiols by a thiolysis reaction at near neutral pH that makes changes in fluorescence intensity. The resulting released fluorescent moiety by the thiolysis, reacts simultaneously with phosphate anions in the excited state that elicits changes in fluorescent decay times. This new fluorescent probe has been tested in solution by using steady-state and time-resolved fluorescence and intracellularly by using Fluorescence Imaging Microscopy (FLIM) in HeLa (Human epithelioid cervix carcinoma cells). With the aim of studying in more depth cellular stress, this work reports also the design of a new molecule designed to optimize the intracellular biothiols detection for biomedical application and its use in bioimaging. Many cellular stresses are associated with oxidative stress and the response against these disorders cover a wide range of molecular changes that includes biothiols synthesis as a protective defence against its damaging effects. As a consequence, cellular stress study could let us understand better the molecular bases of cell damage. More specifically, it is described oxidative stress induced by light exposition in photoreceptor-derived (661W) cells. It is shown that light exposure time is related with fluorescence intensity response and as a result, with the presence of biothiols and oxidative stress. This field could be extended to develop tests for antioxidants drugs to prevent from oxidative diseases or treat them. Furthermore, we have focused our attention on acetate as a potential analyte that is related to several epigenetic alterations, fundamentally due to changes in chromatin acetylation. It has been designed and synthesized some fluorescent compounds, based on the family of the so-cold Tokyo green dyes, as acetate fluorescent probes thanks to an excited state proton transfer (ESPT) reaction mediated by acetate. In order to achieve this aim, it is necessary that the fluorophore has a similar pka value to acetate. Once the compounds were synthesized, some experiments that allowed us to select the best candidate for acetate detection. It has been also studied the reaction in the excited state and calculated the rate constants that rule the process. Moreover, the study has been carried out further and it has been checked the selected compound functionality in artificial serum in order to use this compound in a future as a probe for the diagnosis of some cancer in which acetate is involved. In order to conduct this research, steady-state and time-resolved measurements has been performed.