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Novel sensors technologies applied to force spectroscopy in molecular biology

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[spa] 	
La fuerza juega un papel esencial en todos los campos de la biología. Las fuerzas experimentadas y generadas por las biomoléculas son múltiples en la naturaleza y pueden ir desde los subpiconewtons hasta varios nanonewtons. La medida de estas fuerzas con alta precisión proporciona información acerca de la estructura, la dinámica, las interacciones intra e intermoleculares y las propiedades mecánicas de las biomoléculas. Se han desarrollado diferentes técnicas para hacer frente a esta tarea, en particular a nivel de moléculas individuales. El principal objetivo en este trabajo de tesis ha sido el desarrollo de tecnologías de sensores aplicadas a la espectroscopia de fuerzas y la demostración de sus posibilidades en estudios moleculares reales. La microscopía de sonda de barrido (SPM) es una tecnología de rápido crecimiento que ha sido la fuente para el desarrollo de una inmensa variedad de aplicaciones para investigar materiales y moléculas a la nanoescala. A partir del análisis de las principales limitaciones de los nanosensores basados en tuning fork (TF) por un lado y la espectroscopia de fuerzas convencional con puntas de AFM por otro lado, se han determinado las principales consideraciones para los desarrollos tecnológicos. Una vez que la tecnología se ha desarrollado, se han llevado a cabo diferentes experimentos biológicos con el objetivo de demostrar las posibilidades de las tecnologías desarrolladas en aplicaciones reales: (i) Diferentes técnicas de imagen de biomoléculas se han comparado con sensores TF. La muestra estudiada ha sido una monocapa auto-ensamblada (SAM) de anticuerpos microestructurada. (ii) Se han realizado medidas cuantitativas de interacción molecular entre el sistema biotina- estreptavidina midiendo las energías de adhesión a diferentes velocidades de tracción y los resultados se han comparado con los obtenidos con medidas de AFM. (iii) Medidas de desplegamiento de la proteína titina se han realizado con la nueva técnica desarrollada de espectroscopia de fuerzas a velocidades alcanzadas por simulación (~4 milímetros por segundo) pudiendo comparar los resultados experimentales con simulaciones.

# metadata.dc.description.abstract[].value
[eng] Force plays an essential role in all fields of biology. Measurement of these forces with high precision provides information about the structure, dynamics, intra and intermolecular interactions, and the mechanical properties of the biomolecules and, in general, about molecular basis of diverse biological phenomena. Different techniques have been developed to address this task, particularly at the single molecule level. The main objective accomplished in this work of thesis is the development of sensors technologies applied to force spectroscopy measurements and the demonstration of its possibilities in real molecular studies. Scanning probe microscopy (SPM) is a fast growing technology that has been the source for the development of an immense variety of applications to investigate materials and molecules at nanoscale. As another technology, SPM is constantly improving through different advances in instrumentation level and the emergence of new applications. From the analysis of the main limitations of quartz tuning fork (QTF) based nanosensors on one side and the conventional force spectroscopy with cantilever tips on the other side, the main considerations have been determined for the technological developments on force microscopy applications. One of the main limitations of tuning fork probes is that they are usually custom-made because no commercial probes suitable for a wide range of experiments are available. The custom-made devices show considerable variation in dynamic response, poor lateral resolution and the characterization of the sensors remains unclear for force quantification. A new controller is developed to ensure the same dynamic response of different sensors in order to maintain the conditions in which the measurements are conducted. Also, a new method to improve lateral resolution of the QTF probes when working in liquid is proposed in this thesis based on attaching a standard AFM tip to the end of the fiber probe which has been previously sharpened. A method to calculate the spring constant of the QTF based sensors from easily measurable parameters is presented in this thesis. The method is based on a finite element analysis (FEA) model which includes the electrical part and can be used to calculate the spring constant of a QTF accurately for quantitative measurements. Results obtained in real biological experiments are promising and show the possibilities of the shear force microscopy improvements developed in this work of thesis. In a first experiment, a self-assembled monolayer (SAM) of micropatterned antibodies was imaged with three different techniques and in a second experiment, a molecular interaction analysis was done between biotin- streptavidin complex and results are compared with those obtained with AFM tip. The main problem for comparing steered molecular dynamics (SMD) simulation results with experimental data is that SMD simulations were restricted to nanosecond timescales (due to the high computational demand of all-atomistic simulations). A high-speed force spectroscopy methodology has been developed to achieve rates comparable to SMD simulations. The validation of the technique is performed with titin unfolding measurements allowing the direct comparison of experimental and simulated forces.

# metadata.dc.source[].value
Tesis Doctorals - Departament - Electrònica

# metadata.dc.title[].value
Novel sensors technologies applied to force spectroscopy in molecular biology