Multivalent-Ion-Activated Protein Adsorption Reflecting Bulk Reentrant Behavior

Protein adsorption at the solid-liquid interface is an important phenomenon that often can be observed as a first step in biological processes. Despite its inherent importance, still relatively little is known about the underlying microscopic mechanisms. Here, using multivalent ions, we demonstrate the control of the interactions and the corresponding adsorption of net-negatively charged proteins (bovine serum albumin) at a solid-liquid interface. This is demonstrated by ellipsometry and corroborated by neutron reflectivity and quartz-crystal microbalance experiments. We show that the reentrant condensation observed within the rich bulk phase behavior of the system featuring a nonmonotonic dependence of the second virial coefficient on salt concentration $c_s$ is reflected in an intriguing way in the protein adsorption $d(c_s)$ at the interface. Our findings are successfully described and understood by a model of ion-activated patchy interactions within the framework of the classical density functional theory. In addition to the general challenge of connecting bulk and interface behavior, our work has implications for, inter alia, nucleation at interfaces.

Figure 1

(a) Charge inversion of BSA as a consequence of adding trivalent yttrium ions. (b) Schematic of the bulk phase diagram of BSA and ${\mathrm{YCl}}_3$ (modified from Ref. [18]) showing a liquid-liquid phase separation (LLPS) and reentrant condensation. The dashed red arrow indicates the path taken in the experiments.

Figure 2

Individual symbols: Adsorbed protein layer thickness $d$ extracted from ellipsometry as a function of $c_s/c_p$. $c^{*}$ and $c^{**}$ denote the phase transitions of the bulk solution [16] [Fig. 1]. Note that, around $c^{**}$, there is an experimental difference between the data in regime III vs regime II. The centrifuged samples in regime II reflect the adsorption trend for overall lower adsorption values due to the removal of big clusters in bulk solution but still follow the same adsorption trend. In addition, the top $c_s$ axis is included showing the absolute $c_s$ in the system (at $c_p=20\mathrm{mg}/\mathrm{ml}$). The blue shaded area shows the approximate range of the bulk turbidity. Solid and dashed lines: Protein adsorption based on DFT calculations as bore out by the ion-activated attractive patch model, while neglecting long-range forces, as a function of $c_s/c_p$ for two different values of $\beta ϵ$. Inset: $B_2/B_2^{\mathrm{HS}}$ is the reduced second virial coefficient obtained via SAXS measurements.

Figure 3

(a) Illustration of the different interaction mechanisms of the proteins, salt, and interface. (b) Sketch of protein adsorption on the attractive surface by increasing $c_s/c_p$.