New immunochemical approaches for multiplexed diagnostics

Resumen

The central topic of this thesis has been the study, development and validation of diverse multiplexed diagnostics platforms and techniques that can be applied to a wide range of topics, from environmental monitoring to clinical diagnosis. A key feature of multiplexed systems consists in not only be able to detect multiple analyte but also to discriminate between each one of them. Multiplexation has been accomplished through different approaches and using distinct technology platforms Modulating antibody selectivity, using labels showing distinct optical or electrochemical properties, or particular technological platforms are the most important elements investigated to accomplish multiplexation being (i) spatial separation of detection sites, by means of different spots, wells, regions of a channel network or electrode arrays, or (ii) the use of various labels such as enzymes, redox molecules, beads or dyes, the most studied strategies. The thesis is divided in two blocks: one dedicated to developing multiplexed platforms which could be potential options as monitoring tools and; the second block is focused on novel strategies based on plasmonic nanoparticles and flexible polymers for sensing applications. The first chapter of the thesis is focused on the preparation and characterization of immunoreagents for the detection of two different pyrethroids: cypermethrin and deltamethrin; including the hapten synthesis and the antibody production. Both pyrethroids are commonly used as insecticides and monitoring its presence in water sample have been proposed during the last years. The immunoreagents produced have been implemented in the development of a single-analyte ELISA and in a multianalyte ELISA platform, demonstrating the feasibility of using the same reagents developed in various assay formats. Seawater has been chosen as the interest matrix since during the last decades, marine pollution has been recognized as a worldwide problem and the state of the oceans has become a great concern to governmental agencies. During the next two chapters two different multiplexed strategies have been explored: an ELISA multianalyte and a multiplexed microarray platform. Due to the huge variety of pollutants that are interesting to monitor, a preliminary selection of the pollutants that would be detected was done. The selection of targets analytes was based on different parameters: (1) Their impact in offshore aquaculture, (2) Their actual use in aquaculture facilities, (3) The possibility of using them as markers of quality water; and (4) Their interest for being persistent in environment and bioaccumulate Families of environmental contaminants with different chemical natures were selected, being representatives of herbicides, antibiotics, hormones, insecticides, industrial contaminants and algal toxins, the final chosen in this thesis. The second block has been carried on through progressing in two different lines. On one hand, a photosensible dual-system based on plasmonic nanoparticles as a possible and innovative diagnostic tool for pathogenic bacteria involved in respiratory disease and sepsis has been explored. The thesis has been currently focused in the functionalization of gold nanoparticles with fluorescent molecules and the characterization of its release by light-related mechanisms, taking profit of the inner ability of nanoplasmonics gold nanoparticles possess. On the other hand, the exploration and the preliminary validation of a new diagnostic platform based on nonrigid PDMS chips has been addressed. The study of this new material and the multiple possibilities the flexible material offer regarding microfluidics design, miniaturization and functionalization could be useful for the potential development of future multiplexed platforms. Those chips have been implemented as a new and promising diagnostic tool for the detection of a biomarker related to cardiovascular diseases initial stages, C-reactive protein.