We report on a planar poly(acrylic acid) (PAA) brush as a new kind of materials coating showing a variable protein resistance that can be controlled by the ionic strength of the protein solution. Using neutron reflectometry it has been found that a silicon wafer covered with a PAA brush strongly binds bovine serum albumin (BSA) under electrostatic repulsion. However, when adding sodium chloride to the protein solution, the PAA brush appears essentially protein resistant, although the direct repulsive electrostatic forces between BSA and PAA are screened under this condition. This effect of salt is unique and in agreement with that found earlier for the binding of proteins to spherical PAA brush particles. From the analysis of the neutron reflectivities, the protein density profile across the interface has been determined. At low ionic strength in the solution, BSA penetrates deeply into the PAA brush. In this view, the immobilization of proteins at a poly(acrylic acid) brush may be regarded as an entrapment within a confined geometry rather than an adsorption to a twodimensional interface. A simple mean-field argument is given that explains these experimental findings. The model predicts a large gain of free energy associated with the release of BSA counterions on transferring a BSA molecule from the solution into the PAA brush. The free energy of this counterion "evaporation" is entropic in nature and dominates over the electrostatic repulsion between the BSA molecule and the likecharged PAA brush.

The effect of temperature on the adsorption of hen egg white lysozyme at the silica/water interface has been studied. By use of optical reflectometry, the adsorbed mass per surface area has been determined over a large temperature range with protein solution concentrations ranging from 0.03 to 0.09 mg mL -1 . It has been found that the amount of adsorbed protein is strongly increased by an increase in temperature, which indicates an endothermic and thus an entropy-driven adsorption process. This can be explained by an adsorption-induced modification of the protein conformation. At high temperatures, where dissolved lysozyme is mainly unfolded, hydrophobic dehydration effects will play a role as well. The results support the concept that thermodynamically unstable proteins adsorb more strongly at interfaces than stable proteins. The positive enthalpy of adsorption suggests that significant repulsive electrostatic interactions between the protein molecules of the adsorbate are present. Concentration profiles of lysozyme adsorbates have been determined at 23, 63, and 80 °C using neutron reflectometry. At each temperature, three reflectivity curves have been measured applying the contrast variation method. The reflectivities have been fitted globally on the basis of a four-layer model, Si/SiO 2/adsorbate/solution. The obtained concentration profiles clearly show that in the range of 23-63 °C the temperature-induced increase of the degree of lysozyme adsorption at the silica/water interface is caused in part by a denser packing of the lysozyme in the adsorbate. Whereas the neutron reflectivity data are consistent with a monolayer adsorption at 23 and 63 °C, at least two layers of protein molecules are adsorbed at 80 °C.

We used two-photon excitation fluorescence correlation spectroscopy (FCS) and neutron reflectometry to study in situ the effect of salt concentration on the degree of protein binding to polyelectrolyte brushes. The binding of bovine serum albumin (BSA) to poly(acrylic acid) (PAA) brushes was characterized at neutral pH values where both the protein and the brushes carry a negative charge. Spherical PAA brush particles were used in the FCS experiments, whereas a planar PAA brush served as protein substrate in the neutron reflectometry experiments. It has been found that BSA binds strongly to both the spherical and the planar PAA brushes under electrostatic repulsion at low ionic strength. The BSA volume fraction profile, as determined from the neutron reflectivities, indicates a deep penetration of the BSA molecules into the PAA brush. However, the analysis of the FCS data reveals that the protein affinity of the spherical PAA brush particles decreases drastically when increasing the concentration of sodium chloride to a few 100 mM. This observation is in line with the measured neutron reflectivities of the planar PAA brush. The reflectivity curve obtained in the absence of protein is virtually overlapping with that measured when the PAA brush is in contact with a BSA solution but containing 500 mM sodium chloride which suggests protein resistance of the planar PAA brush at this elevated salt concentration. The results of this study provide evidence for a new kind of protein-resistant interfaces. Whereas protein binding to the PAA brush is likely to be dominated by the release of counterions, this driving force vanishes as the ionic strength of the solution is raised and protein molecules are repelled from the interface by steric interactions. In a general view, the ''switching'' of the protein affinity of a PAA brush by varying the ionic strength of the protein solution over a relatively small range may appear to be useful for biotechnological applications.

The change in the secondary and tertiary structure of bovine serum albumin (BSA) induced by the interaction with spherical polyelectrolyte brushes (SPB) has been investigated using fluorescence and circular dichroism (CD) spectroscopy. The SPB consist of poly(acrylic acid) chains grafted to a poly(styrene) core. The colloidal SPB represent a new substrate for protein immobilization because their protein binding capacity can be controlled by the ionic strength of the solution: SPB bind large amounts of BSA at low ionic strength (pH=6.1), but they are largely protein resistant at moderate salt concentrations of 500 mM. The conformation of BSA which was labeled with the environmentally sensitive dansyl fluorophore was studied before adsorption to the SPB, in the adsorbed state, and after desorption from the SPB. In the adsorbed state the obtained fluorescence spectrum is red-shifted which indicates a hydration of the dansyl fluorophores due to a distortion of the tertiary structure of BSA. Fluorescence and CD spectroscopic analysis of BSA that was desorbed from the SPB shows that the adsorption-induced conformational changes are largely reversible. Convex constraint analysis of the observed CD spectra of BSA yield α-helix fractions of 68% and 57% before adsorption to and after desorption from the SPB, respectively. In a general view, the results of this study demonstrate that spherical polyelectrolyte brushes are suitable for a controlled immobilization and release of proteins without major conformational changes.

The spatial distribution of protein molecules interacting with a planar polyelectrolyte multilayer was determined using neutron reflectometry. Staphylococcal nuclease (SNase) was used as model protein that was adsorbed to the multilayer at 22 degrees C and 42 degrees C. At each temperature, the protein solution was adjusted to pD -values of 4.9 and 7.5 to vary the net charge of the protein molecules. The multilayer was built up on a silicon wafer by the deposition of poly(ethylene imine) (PEI), poly(styrene sulfonate) (PSS), and poly(allylamine hydrochloride) (PAH) in the order Si-PEI-PSS- (PAH-PSS)(5). Applying the contrast variation technique, two different neutron reflectivity curves were measured at each condition of temperature and pD -value. From the analysis of the curves, protein density profiles normal to the interface were recovered. Remarkably, it has been found that SNase is partially penetrating into the polyelectrolyte multilayer after adsorption at all conditions studied. The measured neutron reflectivities are consistent with a penetration depth of 50 A at pD=4.9 and 25 A at pD=7.5. Since SNase has an isoelectric point of pH=9.5, it carries a net positive charge at both pD -values and interacts with the PSS final layer under electrostatic attraction conditions. However, when increasing the temperature, the amount of adsorbed protein is increasing at both pD -values indicating the dominance of entropic driving forces for the protein adsorption. Interestingly, at pD=4.9 where the protein charge is relatively high, this temperature-induced mass increase of immobilized protein is more pronounced within the polyelectrolyte multilayer, whereas at pD=7.5, closer to the isoelectric point of SNase, raising the temperature has mainly the effect to accumulate protein molecules outside the polyelectrolyte multilayer at the water interface. It is suggested that the penetration of SNase into the polyelectrolyte multilayer is related to a complexation mechanism. The complexation is essentially entropic in nature due to the release of counterions.