Control of nanometre-scale topography of solid surfaces has opened the possibility to tailor the interactions between materials and biomolecules maintaining the biological functions of these molecules, a crucial aspect for diverse biomaterial applications. However, the primary mechanisms that dictate protein adsorption to topographical nanostructures are often poorly understood. We have addressed this question by scrutinizing the catalytic activity of immobilized serine-protease trypsin as product of its adsorption properties on cluster-assembled nanostructured titania films. Both, adsorption and activity of surface-bound enzymes were evaluated in parallel using innovative microarray-based methodology developed in this PhD work. Trypsin adsorption analysis demonstrated an increment with roughness of Langmuir parameters – saturation uptake and equilibrium dissociation constant, that exceeded the contribution predicted by increase in sample specific area. This finding was interpreted by the clustering of protein molecules inside titania surface nanopores, a model proposed in our previous study of three non-enzymatic proteins. The growth of adsorbed trypsin activity with roughness was attributed to the increase in specific area of titania films, whereas the drop in specific activity resulted from steric hindrance of trypsin clustered inside titania nanopores. This study has shed light on the topographical determinants of trypsin adsorption on nanostructured titania surfaces and its impact on trypsin activity. A novel method was developed elucidating the obstacles and specifics of protease immobilization by physisorption and suggesting possible routes to solve them. This methodology is directly applicable in biomaterial screening with respect to the functionality of immobilized enzymes and can be extended beyond the trypsin-nanostructured titania model.

DESIGN OF A HIGH-THROUGHPUT METHODFOR THE ASSESSMENT OF ENZYME ACTIVITY UPON ADSORPTION ONCLUSTER-ASSEMBLED NANOSTRUCTURED TITANIUM OXIDE FILMS / L. Gailite ; supervisor: P. Milani ; external co-supervisor: Y. Shacham-Diamand. UNIVERSITA' DEGLI STUDI DI MILANO, 2013 Mar 04. 23. ciclo, Anno Accademico 2011. [10.13130/gailite-lasma_phd2013-03-04].

DESIGN OF A HIGH-THROUGHPUT METHODFOR THE ASSESSMENT OF ENZYME ACTIVITY UPON ADSORPTION ONCLUSTER-ASSEMBLED NANOSTRUCTURED TITANIUM OXIDE FILMS

L. Gailite
2013

Abstract

Control of nanometre-scale topography of solid surfaces has opened the possibility to tailor the interactions between materials and biomolecules maintaining the biological functions of these molecules, a crucial aspect for diverse biomaterial applications. However, the primary mechanisms that dictate protein adsorption to topographical nanostructures are often poorly understood. We have addressed this question by scrutinizing the catalytic activity of immobilized serine-protease trypsin as product of its adsorption properties on cluster-assembled nanostructured titania films. Both, adsorption and activity of surface-bound enzymes were evaluated in parallel using innovative microarray-based methodology developed in this PhD work. Trypsin adsorption analysis demonstrated an increment with roughness of Langmuir parameters – saturation uptake and equilibrium dissociation constant, that exceeded the contribution predicted by increase in sample specific area. This finding was interpreted by the clustering of protein molecules inside titania surface nanopores, a model proposed in our previous study of three non-enzymatic proteins. The growth of adsorbed trypsin activity with roughness was attributed to the increase in specific area of titania films, whereas the drop in specific activity resulted from steric hindrance of trypsin clustered inside titania nanopores. This study has shed light on the topographical determinants of trypsin adsorption on nanostructured titania surfaces and its impact on trypsin activity. A novel method was developed elucidating the obstacles and specifics of protease immobilization by physisorption and suggesting possible routes to solve them. This methodology is directly applicable in biomaterial screening with respect to the functionality of immobilized enzymes and can be extended beyond the trypsin-nanostructured titania model.
4-mar-2013
Settore FIS/07 - Fisica Applicata(Beni Culturali, Ambientali, Biol.e Medicin)
nanometre-scale topography ; protein adsorption ; enzyme immobilization ; protein microarrays
MILANI, PAOLO
MILANI, PAOLO
Doctoral Thesis
DESIGN OF A HIGH-THROUGHPUT METHODFOR THE ASSESSMENT OF ENZYME ACTIVITY UPON ADSORPTION ONCLUSTER-ASSEMBLED NANOSTRUCTURED TITANIUM OXIDE FILMS / L. Gailite ; supervisor: P. Milani ; external co-supervisor: Y. Shacham-Diamand. UNIVERSITA' DEGLI STUDI DI MILANO, 2013 Mar 04. 23. ciclo, Anno Accademico 2011. [10.13130/gailite-lasma_phd2013-03-04].
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/2434/219065
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