Publication: Monitorización de biomarcadores de envejecimiento
en muestras clínicas con inmunosensores electroquímicos
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Publication Date
2021-01-21
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Universidad Complutense de Madrid
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Abstract
El envejecimiento es un deterioro funcional fisiológico dependiente del tiempo, que parece ser impulsado por la acumulación progresiva de defectos moleculares aleatorios en células y tejidos a lo largo de la vida. Durante el envejecimiento, se desarrolla un perfil inflamatorio crónico, estéril y de bajo grado, denominado “inflammageing”, que se confirma por el aumento de los niveles de citoquinas pro-inflamatorias y la disminución de los niveles de citoquinas anti-inflamatorias, siendo el resultado, a largo plazo, de una estimulación fisiológica crónica del sistema inmune innato, que puede ser perjudicial en la edad adulta, contribuyendo a la patogénesis de enfermedades relacionadas con la edad. El objetivo principal de esta Tesis Doctoral es el diseño, preparación y aplicación de diferentes plataformas electroquímicas inmunosensoras con características analíticas mejoradas para la determinación individual o múltiple de biomarcadores de envejecimiento en muestras clínicas. Con este propósito, se desarrollaron varias configuraciones utilizando la técnica del grafting o injertado electroquímico, ácido poli(pirrolpropiónico) y diferentes nanomateriales de carbono para la modificación de los electrodos, así como nuevas estrategias de inmovilización, y poli-HRP-Strept y nanotubos de carbono híbridos como estrategia electroactiva de amplificación de la señal. Todos los inmunosensores desarrollados se aplicaron a la determinación de diferentes biomarcadores de envejecimiento escasamente estudiados para los que no existían configuraciones similares o, cuando existían, las características analíticas de las metodologías previas se mejoraron significativamente. Todo este trabajo de investigación se llevó a cabo mediante la aplicación de la metodología habitual seguida para el diseño de inmunosensores, la elección de la estrategia de reconocimiento, la optimización de las variables experimentales, los estudios de caracterización, la construcción de la curva de calibrado y la obtención de las características analíticas y los estudios de repetibilidad, reproducibilidad y selectividad. En todos los casos, la utilidad analítica de las configuraciones desarrolladas se demostró mediante su aplicación a la determinación de estos biomarcadores en muestras clínicas, tales como un material de referencia certificado, suero o saliva. Finalmente, los resultados obtenidos empleando los inmunosensores desarrollados se compararon con los proporcionados por los respectivos inmunoensayos ELISA comerciales, no obteniéndose diferencias significativas entre los resultados proporcionados por ambos métodos, lo que demuestra la utilidad de las configuraciones desarrolladas...
Ageing is a time-dependent physiological functional decline, which appears to be driven by the progressive accumulation through the life of a variety of random molecular defects that build up within cells and tissues. During ageing, a chronic, sterile and low-grade inflammation, called “inflammageing”, is developed as the long-term result of a chronic physiological stimulation of the innate immune system, which can become damaging in the elderly, contributing to the pathogenesis of age-related diseases. Likewise, this stage results in an inflammatory profile, which is confirmed by the high levels of pro-inflammatory cytokines and the low concentration of anti-inflammatory ones. This change mainly may be attributed to the alterations in the immune system. The main objective of this Doctoral Thesis is the design, preparation and application of different electrochemical immunosensing platforms with improved analytical characteristics for individual or multiple determination of ageing biomarkers in clinical samples. For this purpose, several configurations were developed using electrochemical grafting, poly(pyrrolepropionic) acid and different carbon nanomaterials for electrode modification, as well as novel immobilization strategies, and poly-HRP-Strept and carbon nanotubes-modified hybrids as carrier nanotags for signal amplification. All the immunosensor designed were applied to the determination of different scarcely studied “inflammageing” biomarkers for which there were no similar configurations or, when existing, the analytical characteristics of the methodology were significantly improved. All this research work was carried out by application of the usual methodology of bioelectroanalysis, which involves the design of the immunosensor and the recognition strategy, optimization of the experimental variables, characterization studies, construction of the calibration plot and establishment of the analytical characteristics, followed by the studies of repeatability, reproducibility and selectivity of the method. In all cases, the analytical usefulness of the configurations designed was demonstrated by applying them to the determination of physiological concentrations of these species in clinical samples, such as certified biological reference material, serum and/or saliva. Finally, the results were compared with those obtained by the respective commercial ELISA immunoassays, with no significant differences between the results provided by both methods, which shows the usefulness of the configurations developed...
Ageing is a time-dependent physiological functional decline, which appears to be driven by the progressive accumulation through the life of a variety of random molecular defects that build up within cells and tissues. During ageing, a chronic, sterile and low-grade inflammation, called “inflammageing”, is developed as the long-term result of a chronic physiological stimulation of the innate immune system, which can become damaging in the elderly, contributing to the pathogenesis of age-related diseases. Likewise, this stage results in an inflammatory profile, which is confirmed by the high levels of pro-inflammatory cytokines and the low concentration of anti-inflammatory ones. This change mainly may be attributed to the alterations in the immune system. The main objective of this Doctoral Thesis is the design, preparation and application of different electrochemical immunosensing platforms with improved analytical characteristics for individual or multiple determination of ageing biomarkers in clinical samples. For this purpose, several configurations were developed using electrochemical grafting, poly(pyrrolepropionic) acid and different carbon nanomaterials for electrode modification, as well as novel immobilization strategies, and poly-HRP-Strept and carbon nanotubes-modified hybrids as carrier nanotags for signal amplification. All the immunosensor designed were applied to the determination of different scarcely studied “inflammageing” biomarkers for which there were no similar configurations or, when existing, the analytical characteristics of the methodology were significantly improved. All this research work was carried out by application of the usual methodology of bioelectroanalysis, which involves the design of the immunosensor and the recognition strategy, optimization of the experimental variables, characterization studies, construction of the calibration plot and establishment of the analytical characteristics, followed by the studies of repeatability, reproducibility and selectivity of the method. In all cases, the analytical usefulness of the configurations designed was demonstrated by applying them to the determination of physiological concentrations of these species in clinical samples, such as certified biological reference material, serum and/or saliva. Finally, the results were compared with those obtained by the respective commercial ELISA immunoassays, with no significant differences between the results provided by both methods, which shows the usefulness of the configurations developed...
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Tesis inédita de la Universidad Complutense de Madrid. Facultad de Ciencias Químicas, leída el 08-05-2020