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
Metadatos
Afficher la notice complèteAuteur
Puente Muñoz, VirginiaEditorial
Universidad de Granada
Departamento
Universidad de Granada. Departamento de FisicoquímicaMateria
Físicoquímica Espectroscopía Química analítica Fluorimetría Marcadores bioquímicos Estrés oxidativo
Materia UDC
543 544 2210.03 2210.07
Date
2017Fecha lectura
2017-07-17Referencia bibliográfica
Puente Muñoz, V. 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. Granada: Universidad de Granada, 2017. [http://hdl.handle.net/10481/47430]
Patrocinador
Tesis Univ. Granada. Programa Oficial de Doctorado en FarmaciaRésumé
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 a 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 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 cells 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 timeresolved
fluorescence and intracellularly by using Fluorescence Imaging Microscopy (FLIM) in
HeLa (Human epithelioid cervix carcinoma cells). 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
protective defence against its damaging effects. As a consequence, cellular stress study could
let us understand better the molecular bases of cell damage. In this work it is also reported the
design of a new molecule designed to optimize the intracellular biothiols detection for
biomedical application and its use in bioimaging. More deeply, 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 socold
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,
they were performed some experiments that allowed 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.