Nanophotonic Biosensors for Deciphering Cell Regulation Pathways
Doctorat en Biotecnologia Departament de Genètica i Microbiologia – Facultat de Biociències Nanophotonic Biosensors for Deciphering Cell Regulation Pathways Doctoral Thesis – 2016 César Sánchez Huertas Author Prof. Laura M. Lechuga Prof. Antoni Villaverde Director Tutor Abstract This Doctoral Thesis focuses on the development of innovative biosensor devices as alternative analytical techniques for the evaluation of different gene regulating pathways in order to obtain a more informative and accurate diagnosis and follow-up therapy of cancer. We propose the use of a novel nanophotonic biosensor for a rapid, highly sensitive and direct analysis of these regulating routes without the need of labeling or amplification steps. Different genetic and epigenetic disorders associated with cancer appearance and progression are studied taking advantage of circulating nucleic acids as target biomarkers. For the label-free detection and evaluation of these biomarkers, we have employed two different optical biosensors: (i) the well-known Surface Plasmon Resonance (SPR) biosensor, and (ii) a novel nanotechnology-based interferometric device, the Bimodal Waveguide (BiMW) biosensor. First, an in-depth study of different biofunctionalization strategies on both platforms is presented. Several surface chemistry procedures have been optimized for an efficient immobilization of nucleic acids as biorecognition elements that ensure a highly sensitive target detection with maximum selectivity and reproducibility, especially for the direct analysis of complex human samples such as urine or serum. The optimized strategies were applied for the evaluation of specific and clinically relevant gene regulation pathways, such as RNA alternative splicing events, micro-RNA regulation, or DNA methylation processes. All the developed methodologies have been assessed in terms of selectivity, sensitivity and reproducibility, and in some cases, they have been validated with real samples.
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