Using Neutron Reflectometry to Characterize Antimicrobial Protein Surface Coatings

Abstract

Understanding the interaction of adsorbed or covalently immobilized proteins with solid substrates at the molecular level guides the successful design of functionalized surfaces used in biomedical applications. In this report, neutron reflectometry (NR) was used to characterize the structure of surface-attached antimicrobial protein films, with antimicrobial activity assessed using an adaption of the Japanese Industrial Standard Test JIS Z 2801. NR allowed parameters influencing bioactivity to be measured at nanometer resolution and for conclusions about structural characteristics relating to bioactivity to be drawn. Hydramacin-1 (HM-1) and lysozyme were covalently attached to poly(methyl methacrylate) (PMMA) and 3-aminopropyltriethoxysilane (APTES) films in the presence and absence of a four-unit poly(ethylene glycol) PEG-based spacer and measured using NR, followed by antimicrobial assays. APTES-PEG-protein films were structurally unique, with a layer of 80% water directly beneath the protein layer, and were the only films that displayed antimicrobial activity against Escherichia coli and Bacillus subtilis. The hydration content of these films combined with the subtle difference in the PEG layer thickness of APTES versus PMMA films played a role in defining antimicrobial activity of the prepared surface coatings.

In-situ Neutron Reflectometry Study on Adsorption of Glucose Oxidase at Mesoporous Aluminum Oxide Film

“…The protein distribution on and in nanoporous materials is thus an important subject to be clarified, and in-situ spectroscopic techniques for protein adsorption have been developed. [4][5][6][7][8][9][10][11][12][13][14][15][16][17] The distribution of adsorbate on a single nanoporous particle has been often observed by microspectroscopy techniques. 4,5 For a nanoporous film on a solid substrate, spectroscopic methods utilizing optical interference [6][7][8][9][10][11][12][13][14] and waveguide mode [15][16][17] are available for the in-situ observation of the molecular adsorption.…”

“…In-situ neutron reflectometry (NR) is one useful technique for structural characterization of an adsorption layer at a solid/ liquid interface. [11][12][13][14] The scattering length densities (SLD) of D2O and H2O are quite different (Table 1), and SLD contrast between materials within the nanoporous film and solvent can be easily tuned by adjusting solvent composition of a D2O/H2O mixture. When NR measurement is performed under a series of D2O/H2O mixtures, simultaneous analysis of reflectivity data obtained for the D2O/H2O mixtures can reduce ambiguities while modeling, and facilitates determination of the structural properties of the adsorption layer.…”

“…When NR measurement is performed under a series of D2O/H2O mixtures, simultaneous analysis of reflectivity data obtained for the D2O/H2O mixtures can reduce ambiguities while modeling, and facilitates determination of the structural properties of the adsorption layer. 18 The in-situ NR has been applied to characterize lipid membranes assembled at a surfactant-templated mesoporous silica film 12,13 and adsorption of polyelectrolyte at an anodized nanoporous aluminum oxide film. 14 The purpose of this study is application of in-situ NR to clarify whether negatively-charged glucose oxidase (GOD) can penetrate into pores in mesoporous aluminum oxide (MAO) film or not.…”

Active antibacterial coating of cotton fabrics with antimicrobial proteins

“…The observation that total cell death was not achieved with HM-1 surface immobilization suggests that tethering reduced protein activity, the amount of HM-1 immobilized to the cotton swatches was too low, or particular coupling reactions gave rise to inactive HM-1, i.e., a tethered orientation that hampered activity. To overcome such possibilities, different linkers, the introduction of a spacer such as polyethylene glycol, or specific attachment should improve the activity profile of these HM-1 functionalized surfaces (Akers et al 2017). Moreover, given the observed aggregation and attachment of cells on the fibers because of HM-1 activity (Figs.…”

“…While direct anchorage onto desired surfaces is the simplest approach, significant loss of activity is often observed because of surface-induced conformational changes and reduced dynamics once proteins are tethered onto the solid support (Faccio 2018;Mukherjee and Gupta 2015;Xiao et al 2018). Therefore, a linker or spacer is often inserted between the protein and the surface to retain activity of the protein once immobilized on the surface (Akers et al 2017;Balamurugan et al 2008;Gunda et al 2014;Wu et al 2018). Previously, Edwards et al demonstrated the use of citric acid and (3-aminopropyl) triethoxysilane (APTES) as linkers for the immobilization of lysozyme onto cotton surfaces (Edwards et al 2011).…”