Paper BI+AS-ThA9
Exploiting Protein-Polyelectrolyte Interactions to Control and Tune Protein Immobilization at Interfaces. Applications in Biocatalysis and Separation Technology

Thursday, November 2, 2017, 5:00 pm, Room 12

INTRODUCTION: For many applications in biomedical science and biotechnology, it is challenging to control and tune the nature, amount, and activity of proteins at interfaces. Since proteins are polyampholytes, they do interact with polyelectrolytes (PE), in a way which strongly depends on the pH and ionic strength of the medium. It is usually considered that PE provide a mild environment to proteins, which may help keeping their activity unaffected by surface immobilization. Here, we explore two different approaches to take advantage of the PE-protein interactions for the controlled and tunable surface immobilization of proteins.

STRATEGY: In a first approach, mixed brushes of poly(ethylene oxide) (PEO), a protein-repellent polymer, and of a negatively- or positively-charged PE, respectively poly(acrylic acid) (PAA) and poly(2-(dimethylamine)ethylmethacrylate) (PDMAEMA), were prepared by the “grafting to” approach. These stimuli-responsive mixed brushes were used to selectively adsorb/desorb a given protein from a mixture of several proteins. In a second approach, PE-protein complexes were prepared then immobilized at interfaces within layer-by-layer (LbL) assemblies. This was in particular performed for PE-enzyme complexes, including PE-lysozyme and PE-glucose oxidase complexes. Systems including several enzymes were designed, with a view to further enable enzymatic cascades. Polymer brush formation and protein immobilization were monitored using quartz crystal microbalance, X-ray photoelectron spectroscopy and time-of-flight secondary ions mass spectrometry. Gel electrophoresis was used to determine the nature of proteins collected from the interface. PE-protein complex formation was assessed based on turbidimetry and dynamic light scattering measurements. Enzyme activity was measured based on standard assays.

RESULTS: (i) Mixed polymer brushes-protein interactions: From adsorption experiments with single and mixed solutions of albumin, lysozyme and fibrinogen on PAA/PEO and PDMAEMA/PEO brushes, it was demonstrated that the selective adsorption of one protein could be achieved, as well as the sequential desorption of these proteins when the three of them were adsorbed initially, by means of appropriate pH and I triggers. (ii) PE-enzyme complexes as building blocks for LbL assembly: PE-enzyme complexes were successfully built and characterized, then incorporated into LbL assemblies. The specific activity of lysozyme was higher when immobilized as a complex rather than in its native form.

CONCLUSION:The developed systems may find direct applications in separation technology, on the one hand, and in biocatalysis, on the other hand.