T. J. Su

Structural conformation of lysozyme layers at the air/water interface studied by neutron reflection

The adsorption of chicken egg white lysozyme at the air/water interface has been studied by specular neutron reflection. The variation of the total thickness of the lysozyme layer at the surface of water under varying solution conditions has been determined. The use of mixed H2O and D2O allowed the determination of the extent of immersion of the layer in water at all concentrations. The measured layer thickness combined with the globular dimensions of lysozyme suggests that the adsorbed lysozyme molecules retain their globular structure with no significant denaturation. Measurements were made over a lysozyme concentration range of 9×10-4 g dm-3 to 4 g dm-3 at pH 7 and at an ionic strength of 0.02 M. The thickness of the layer was determined by measuring neutron reflectivities in null reflecting water (NRW) where the signal is only from the adsorbed protein layer. Below 0.1 g dm-3 the surface coverage increases with bulk concentration but the thickness of the layer is constant at 30±3 A, suggesting that lysozyme is adsorbed sideways-on. As the bulk concentration increases, the layer thickness gradually increases to a value of 47±3 A2 at a bulk concentration of 1 g dm-3, suggesting that the molecules switch from sideways-on to longways-on orientations. The area per molecule at 1 g dm-3 was found to be 950±50 A2 which is close to the limit of 30×30 A2 for a saturated layer of longways-on molecules. The extent of mixing of the layer with water was determined directly by measuring reflectivity profiles in mixed H2O and D2O. A two layer model was found to be appropriate with an upper layer in air and a lower layer fully immersed in water. The thickness of the layer in air was found to vary from 15±5 Aat the lowest bulk concentration to 9±3 Aat the highest concentration studied. The results show that as the total layer thickness increases with bulk concentration the fraction of the layer immersed in water increases from 50 to 85%. At the highest concentration of 4 g dm-3 the adsorbed layer is better described by a two layer model consisting of a close packed top layer of thickness 47±3 Aand a loosely packed sublayer of 30±3 A.

Competitive adsorption of lysozyme and C12E5 at the air/liquid interface

We have studied the adsorption of lysozyme and pentaethylene glycol monododecyl ether (C12E5) at the air/water interface using neutron reflection and surface tension measurements. The effect of C12E5 concentration was examined at three fixed lysozyme concentrations of 0.01, 1 and 4 g dm−3. The surface tension showed little variation with the addition of C12E5 over the low surfactant concentration region, but with the increase of C12E5 concentration, the surface tension gradually became identical to that corresponding to pure C12E5. These results suggest a progressive replacement of lysozyme by C12E5 and that the observed surface event is dominated by competitive adsorption. The parallel neutron measurements showed that, at low surfactant concentration, the surface was predominantly occupied by lysozyme. At intermediate C12E5 concentrations, the surface layer consisted of both lysozyme and C12E5, with the C12E5 eventually completely replacing the adsorbed lysozyme as the surfactant concentration was further increased. While the neutron results confirm the inference from surface tension measurement, structural analysis clearly showed the partial breakdown of the globular structure of lysozyme induced by the nonionic surfactant. Furthermore, neutron data showed that the adsorbed C12E5 molecules are present at the top surface layer only, suggesting no preferential association or binding between the surfactant and any immersed protein fragments at the interface.

Adsorption of proteins from aqueous solutions on hydrophobic surfaces studied by neutron reflection

The two proteins, β-casein (β-CN) and β-lactoglobulin (β-Lg), were adsorbed on hydrophobic silicon substrates from buffer solutions at pH 8, 7, 5 and 3. The structure, in terms of thickness and composition of the adsorbed species, was determined by means of neutron reflectivity. At pH 7 β-CN forms a structure that is described by two layers, a compact layer adjacent to the solid surface and a looser layer protruding into the solution. β-Lg adsorbs as a uniform layer. At lower pH both proteins adsorb more, with thicker layers, and β-Lg also adsorbs as a non-uniform layer. The adsorption of both proteins is irreversible. The merits of contrast variation are discussed and, in particular, the importance for the systems studied of the use of water of scattering length density 4.5×10−6 A−2 is described. Owing to the large size of the proteins, this contrast, intermediate between those of D2O and silicon, allows details masked by the higher critical angle of D2O to be revealed.

Structure of self-assembled layers on silicon: Combined use of spectroscopic variable angle ellipsometry, neutron reflection, and atomic force microscopy

Neutron reflection (NR), spectroscopic ellipsometry (SE), and atomic force microscopy (AFM) have been used to characterize the structure of self-assembled octadecyltrichlorosilane (OTS) layers on silicon. The first two of these techniques rely on modeling of the experimental data and may thus result in the unrealistic representation of the composition and structure at the interface. Ambiguities arise from model-dependent analysis complicated by the lack of sufficient external constraints to converge nonunique solutions to a unique one. We show in this work that AFM measurements provide extra constraints to allow us to obtain a physical description closer to the actual structure of the film. It was found that “the simpler the better” modeling strategy very often employed during the fitting of ellipsometric and neutron reflection data is, therefore, not necessarily the best way to obtain a reliable description of the interfacial structure. Our AFM findings necessitated the refit of both neutron and ellipsom...