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Novel surface energy sensor for detecting protein adsorption and subsequent conformation change Clark, Alison Jane

Abstract

This thesis describes the development of a novel experimental technique to measure the change in surface energy of a sensing membrane as molecules adsorb to its surface. The sensor is constructed from a thin elastomeric membrane mounted on an annular support that is immersed in an aqueous solution. The sensor is acoustically actuated to resonate in a selected mode of oscillation and the change of resonant frequency of the sensor as a function of time is monitored. These sensing membranes have a low inherent tension such that the surface energy on its two interfaces dominate the membrane tension and the adsorption of molecules decreases the surface energy and thus reduces the resonant frequency of the membrane. The adsorption behaviour of two types of molecules were investigated; surfactants and proteins. While the sensor responds quickly (~l-2 minutes) to the adsorption of the small surfactant molecules, it exhibits a long time response over many hours to the adsorption of protein molecules. This long time response is attributed to the slow conformation change of the protein molecules once they have adsorbed. An auxiliary method to measure the amount of protein molecules on the surface of the membrane was devised to run simultaneously with the observations of resonant frequency. This technique employed fluorescent excitation of tagged protein molecules in an optical evanescent field. This measurement confirmed that the population of protein molecules on the surface did indeed reach a steady-state value within 30 minutes, in turn confirming the sensitivity of the sensing membrane to molecular conformation change. A step-wise kinetic protein adsorption model was developed and compared to the experimental data generated in the simultaneous measurement described above. This model was able to successfully describe the puzzling kinetics of protein adsorption to, and desorption from, the sensing membrane. This required a key, non-obvious term in the rate equations - the molecules in the bulk solution are found to make an important contribution to both the desorption of bound molecules and also their slow conformation change. This observation provides an effective and self-consistent explanation for the previously conflicting notions of adsorption isotherms and irreversibility.

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