Spatial distribution of protein molecules adsorbed at a polyelectrolyte multilayer

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 $22°\mathrm{C}$ and $42°\mathrm{C}$. At each temperature, the protein solution was adjusted to $p\mathrm{D}$-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-${\left(\mathrm{PAH}\text{−}\mathrm{PSS}\right)}_5$. Applying the contrast variation technique, two different neutron reflectivity curves were measured at each condition of temperature and $p\mathrm{D}$-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 $50\mathrm{\AA }$ at $p\mathrm{D}=4.9$ and $25\mathrm{\AA }$ at $p\mathrm{D}=7.5$. Since SNase has an isoelectric point of $p\mathrm{H}=9.5$, it carries a net positive charge at both $p\mathrm{D}$-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 $p\mathrm{D}$-values indicating the dominance of entropic driving forces for the protein adsorption. Interestingly, at $p\mathrm{D}=4.9$ where the protein charge is relatively high, this temperature-induced mass increase of immobilized protein is more pronounced within the polyelectrolyte multilayer, whereas at $p\mathrm{D}=7.5$, 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.

Figure 1

Schematic representation of the interfacial structure model used to analyze the measured neutron reflectivity data. (A) Structure in the absence of protein. An upper polyelectrolyte layer of $50\mathrm{\AA }$ thickness is assumed into which protein molecules can penetrate after adsorption. (B) Structure in the presence of proteins. Protein molecules are incorporated into the upper polyelectrolyte layer and form an additional adsorbate layer. (C) Protein molecules are allowed to penetrate into the upper $25\mathrm{\AA }$ of the polyelectrolyte multilayer only.

Figure 2

Neutron reflectivity curves of a polyelectrolyte multilayer at the interface between a Si wafer and a buffer solution without protein $(p\mathrm{D}=7.5,T=22°\mathrm{C})$. The symbols represent the experimental data with ${\mathrm{D}}_2\mathrm{O}$ as the solvent (circles) and HDO as the solvent (triangles, shifted by 0.1 on the reflectivity axis), whereas the solid lines show a global fit to both curves.

Figure 3

Neutron reflectivity curves of a Si-PEI-PSS-${\left(\mathrm{PAH}\text{−}\mathrm{PSS}\right)}_5$-solution interface with ${\mathrm{D}}_2\mathrm{O}$ as the solvent at $p\mathrm{D}=4.9$. In (A), measured reflectivities are shown over a limited $Q$-range to illustrate the changes when SNase binds to the polyelectrolyte multilayer at different temperatures. Data are given for the buffer solution at $22°\mathrm{C}$ (circles), the buffer solution containing $0.05\mathrm{mg}∕\mathrm{mL}$ SNase at $22°\mathrm{C}$ (triangles), and the buffer solution containing $0.05\mathrm{mg}∕\mathrm{mL}$ SNase at $42°\mathrm{C}$ (crosses). In (B), the same measured data are shown as symbols together with fitted neutron reflectivity curves represented as solid lines. Data in (B) are shifted on the reflectivity axis for clarity.

Figure 4

Quality of the data fitting as a function of the protein volume fraction in the upper $50\mathrm{\AA }$ of the polyelectrolyte multilayer [solid symbols, see Fig. 1b] and in the upper $25\mathrm{\AA }$ of the polyelectrolyte multilayer [open symbols, see Fig. 1c]. Neutron reflectivity curves at $p\mathrm{D}=4.9$ and $22°\mathrm{C}$ (solid and open circles) and at $p\mathrm{D}=7.5$ and $22°\mathrm{C}$ (solid and open triangles) were analyzed. The ${\chi }^2$-values are normalized to those where no SNase is incorporated into the polyelectrolyte multilayer. Physically reasonable minima are indicated by arrows.

Figure 5

Volume fraction profiles of a polyelectrolyte multilayer (solid lines), SNase at $22°\mathrm{C}$ (dashed lines), and SNase at $42°\mathrm{C}$ (dotted lines). Data are shown at $p\mathrm{D}=4.9$ (A) and $p\mathrm{D}=7.5$ (B). The surface of the silicon wafer is located at $z=0$.