Síntesis de hidrogeles biofuncionalizados con BSA-CD para biosensado óptico mediante holografía

Abstract

[ES] Los biosensores vestibles (wearables) están suscitando gran interés por su potencial para transformar los conceptos clásicos de diagnóstico médico y seguimiento continuo de la salud. En este sentido, los biosensores que trabajan en formato sin marcaje (label-free) resultan altamente atractivos. En este trabajo, se plantea la puesta a punto de un biosensor óptico sin marcaje basado en hidrogeles holográficos. Este tipo de sistemas utiliza un hidrogel que reconoce específicamente el analito a detectar y sobre el que se graba una red de difracción holográfica, de manera que el cambio en el patrón de difracción de la red grabada se pueda correlacionar con la concentración de analito. Esta investigación se centra en un hidrogel holográfico modelo que incorpore ciclodextrinas modificadas con BSA (albúmina de suero bovino) como elemento de biorreconocimiento de anticuerpos anti-BSA. En el proceso de fabricación del hidrogel las ciclodextrinas se ensartan en cadenas lineales que se entrecruzan y dan lugar a hidrogeles con más plasticidad y resistencia mecánica que los convencionales. Tras optimizar las composiciones y procedimientos de síntesis y fabricación para obtener materiales con las propiedades adecuadas, se estudiará su capacidad para realizar biosensado específico utilizando tanto técnicas de fluorescencia como la monitorización óptica sin marcaje.

[EN] Label-free optical biosensors have emerged as a technology that has captured the attention of the research community thanks to its potential applications and its ability to detect biomolecular interactions without the need for labels. Its application is especially relevant in medicine, food safety and environmental monitoring, ensuring the accurate detection of biomarkers, pathogens or contaminants, which makes label-free biosensing a high-impact technology for the welfare of society. Hydrogels are cross-linked hydrophilic systems capable of retaining a large amount of water in their hydrated state, maintaining their three-dimensional structure. This ability can be exploited by etching a diffraction grating within the hydrogel to transform it into an optical transducer since, once the specific recognition between the bioreceptor and the analyte of interest takes place, a change in the refractive index of the material or in the periodicity of the grating occurs and, consequently, causes a change in the diffraction pattern that can be measured and related to the analyte concentration. Therefore, the aim of this work is focused on the development of a label-free optical biosensor based on holographic hydrogels. For this purpose, the synthesis and characterization of hydrogels based on slip ring polymers, namely pseudopolyrotaxane, has been carried out. The model hydrogel integrates BSA-modified cyclodextrins as biorecognition elements of the analyte of interest, in this case, anti-BSA. During the fabrication of the hydrogel, the BSA-conjugated cyclodextrins were threaded into the linear chains that make up the pseudopolyrotaxane, thus obtaining more plastic hydrogels that are highly resistant to mechanical stress. In addition, the structural and mechanical characterization of the hydrogel, as well as its specific biorecognition capacity by means of fluorescence assays, has been proposed. In parallel, a comparison in terms of sensitivity and mechanical resistance between photochemically activated hydrogels and thermally activated hydrogels has been established. Moreover, the two techniques used so far to generate diffraction gratings have been explored. The replica molding technique has been the fabrication strategy mainly analyzed and optimized with the aim of obtaining surface relief gratings (SRGs) and, on the other hand, the fabrication of volume gratings (VHGs) in hydrogels has also been evaluated for their subsequent application in non-marking optical biosensing.