Lysozymes on surfaces with tailored chemistries: Comparison between adsorption from aqueous solution and transfer by gas cluster beams in vacuum

Details

Supervisors

Delcorte, Arnaud ; Dupont-Gillain, Christine C.

Faculty

Faculté des bioingénieurs

Degree label

Master [120] : bioingénieur en chimie et bioindustries, à finalité spécialisée

Abstract

enThis master’s thesis compare two approaches for immobilizing lysozyme onto model surfaces of controlled chemistry: conventional adsorption from aqueous solution and solvent-free transfer by argon gas cluster ion beam (GCIB-Ar) under ultra-high vacuum (UHV). The central question is how surface chemistry shapes the protein-surface interaction in each case, and whether the behavior of functional surfaces in solution still holds with solvent-free deposition. The model surfaces were gold substrates were functionalised with two alkanethiol self-assembled monolayers (SAMs), a 3-MPA-NHS ester SAM intended to covalently anchor the protein through its lysine residues, and a PEG-thiol SAM acting as an antifouling reference. A bare gold wafer served as a nonspecific adsorption control. The formation and lateral homogeneity of all three surfaces were confirmed by Time-of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS). Lysozyme enzyme was then immobilized by the two approaches in parallel. The vacuum transfer with Ar+ 3000 clusters accelerated to 10 keV (approximately 3.3 eV per atom), that enables the deposition of intact molecules while limiting fragmentation. Both pathways were characterized by ToF-SIMS spectra, imaging, depth profiling, and complemented by a fluorometric enzymatic activity assay to estimate the amount of active protein and probe the robustness of attachment through rinsing. In solution, adsorption produced a thin monolayer whose coverage followed surface chemistry, the PEG surface reducing the protein signal by about two orders of magnitude and thereby confirming its antifouling character. The GCIB pathway produced a thick multilayer on every surface. Furthermore, under UHV, the antifouling effect of PEG was bypassed even after rinsing. The transferred protein was found to intermix with the polymer chains rather than being repelled. Activity assays confirmed that a significant fraction of the transferred lysozyme remained bioactive after transfer, with equivalent active masses of roughly 5 to 6 µg on all three chemistries. Rinsing later removed the majority of physisorbed protein film. Overall, GCIB-Ar transfer deposits substantially more protein than solution adsorption, in a dose-controlled and chemistry-independent manner, opening new perspectives for biofunctionalizing surfaces that resist adsorption from solution.