Study of substrate modulation and bioreceptor anchoring for the development of high performance microarrays

Resumen

The present PhD thesis is focused on the study of new approaches able to improve the performance of microarrays. Aspects such as the nature of the surfaces and the probes, functionalization of the substrates, probe printing, immobilization and target detection were considered in the fabrication process. Within all these features, modulation of the surface behavior and probe anchoring were the most challenging aspects, as the interface is key for the immobilization of the receptors and the later detection, which will determine the performance of the final device. In this work, two microarray types have been developed, one for oligonucleotides and another one for antibodies. Then, a characterization of the reached achievements is done. All the routes have in common the use of light to catalyze the attachment of bioreceptors on the surface substrates, employing click-chemistry reactions. In the first chapter, the state of the art of microarray technology is overviewed, with special focus in the main aspects of microarray design. In the second chapter, the goals for this PhD thesis are settled. These general objectives are addressed in the following experimental chapters. In the third chapter, the effect of hydrophobicity and probe multi-point attachment on the microarray performance are studied. Thus, modulation of glass slide surfaces with alkenyl and alkynyl motifs for the anchoring of mono and multithiolated oligonucleotide probes by thiol-ene and thiol-yne photocoupling reactions, respectively, was accomplished. Surfaces modified with the most hydrophobic silane (alkynyl), or anchoring polythiolated probes, revealed better performances. These microarray systems were applied to the discrimination of SNPs and to detect bacterial genome PCR products. In the fourth chapter, a rational design for the preparation of microarrays of antibodies, is done. The immobilization approach displays the oriented anchoring of thiol-bearing antibody fragments to alkenylated glass slides by thiol-ene photocoupling reaction. Multiplexed detection of cardiac biomarkers is demonstrated. The designed microarray shows higher recognition capacity in comparison to whole antibody microarrays. In the fifth chapter, improvement of a novel methodology for the anchoring of thiolated oligonucleotides has been developed. Due to the interest on modifying highly hydrophobic surfaces, a new photoinduced reaction is set up. Thanks to the features of the named "fluor-thiol photocoupling reaction", immobilization of thiolated probes to surfaces containing C-F bonds in a fast, easy and biocompatible with aqueous media way, was achieved. Hydrophobicity of the surfaces was controlled to get successful hybridizations. Because of the high hydrophobicity of the surfaces, a huge confinement of the probes is accomplished, which allows the approximation of the analytes only where the probe is linked, keeping a high repulsion in the remaining surface. The perfluorinated glass slides improved the immobilization densities and detection capacity, regarding to the alkenylated and alkynylated surfaces, and allowed the discrimination of SNPs and detection of bacterial PCR products, as well. In the sixth chapter, other surfaces different than glass are explored. Thus, polyvinylidene fluoride membranes were employed as substrates for the development of oligonucleotide microarrays. Therefore, a fast, easy and mild functionalization process by UV irradiation and organosilane chemistry, was developed. Then, alkenyl functionalized and non-functionalized membranes were applied to microarray technology by covalent anchoring through thiol-ene and fluor-thiol photocoupling reactions, respectively. Promising results were obtained with both surfaces.