Under many circumstances proteins adsorb at air-water or oil-water interfaces, and at sufficient concentration such interfacial proteins can form layers with pronounced elasticity. The mechanical behavior of such layers is often key to their utility in current and developing technologies, particularly those related to the food, biomedical, and pharmaceutical industries. Interfacial microrheology, which uses colloidal probe particles to interrogate the mechanical properties of films at fluid interfaces, is emerging as a powerful approach to investigate interfacial layers. This proposal describes a set of experiments that will advance this approach significantly by providing new insights into the nature of colloid mobility at interfaces and will then exploit these insights to gain new understanding of the interfacial rheology of protein layers.
Intellectual Merit:
Knowledge of the rheological properties of protein layers is crucial both for understanding fundamental aspects of their formation and stability, as well as for enabling their adoption for technological applications. The study of protein layers can further provide unique perspectives on issues of protein denaturation, protein-protein interactions, and gel transitions. The proposed research program will be a joint effort involving three Investigators with a strong record of collaboration and with the complementary expertise needed to make substantial progress in this area. A central element of the proposed research will be the use of colloidal probes with different geometries that are employed both in active and passive interfacial microrheology measurements. As previous work by the Investigators has shown, this combined approach provides sets of complementary information that, when treated self consistently, can resolve ambiguities in the interpretation of any single measurement. The result is insight into both the nature of particle motion at interfaces and the interfacial rheology of the host layers implied by that motion. A key approximation in the analysis of the hydrodynamic forces on a particle confined to an interfacial layer is the incompressibility of the layer. Determining experimentally the range of validity of this approximation and the impact on particle mobility when it breaks down would have far reaching implications, and would help solidify the connection between particle motion and interfacial rheology that is at the center of the microrheology approach. Hence, the research will begin with experiments that investigate the limits of the incompressibility approximation. Informed by the resulting insights into the drag on colloids in interfacial layers, the proposed experiments will then address three key topics in protein layer rheology: (i) correlations in the evolution of surface viscosity and dynamic surface tension of protein solutions, (ii) the interfacial rheology of mixed solutions of proteins and small molecule surfactants, and (iii) the comparative mechanical properties of spread versus adsorbed protein layers.
Broader Impacts:
Through systematic investigation of the limits and consequences of a key property of surfactant layers, their incompressibility, the research will bring to the fore an issue relevant to a range of problems, such as multiphase flows at interfaces. By advancing a new, high sensitivity approach to interfacial rheology, the work will help expand the tools of interfacial science, potentially impacting a range of fields within materials and chemical engineering and the biosciences. For example, as microrheological techniques, the measurement approaches being developed require far smaller samples than conventional rheological methods. Thus, they could make feasible mechanical characterization of interfacial systems where large sample sizes are impossible or prohibitively expensive to synthesize. As part of this program, graduate students and undergraduates will receive research training in a highly interdisciplinary field that will prepare them for careers in academia and industry. Collaboration with a local science magnet high school will provide Baltimore City students with opportunities for research internships. To help promote the advancement of women in science and engineering, female high school students will also be recruited through the Women in Science and Engineering Program, a Johns Hopkins University outreach initiative, to participate in the research.
Proteins are long chain molecules that play key roles in virtually all biological processes. When dissolved in water, they often become stuck at the interface between the water and the air above it, thereby altering the properties of the air-water interface and leading eventually to the creation of a mechanically rigid protein layer at the interface. (A similar effect can also occur at the interface between the water and oil.) The formation and mechanical properties of protein layers are central to numerous current and developing technologies, particularly those in the food, biomedical, and pharmaceutical industries. For example, the tendency of proteins to form rigid layers at air-water interfaces creates a serious bottleneck in many protein-based drug-development efforts. Consequently, work to understand the formation of protein layers and their properties is a major focus of research. However, progress is hampered by difficulties inherent in reliably characterizing such molecularly thin films, making improved methods to study protein layers vitally important. This NSF grant has funded graduate-student and undergraduate researchers who have developed and applied "microrheology" methods to investigate the mechanical properties of protein layers as they are forming. In the microrheology experiments, we track the positions of micrometer-sized particles confined to the interface and infer the mechanical properties of the interface from the particles’ motion. Due to the particles’ small size, their motion is strongly influenced by the mechanical behavior of the interface, enabling unusually sensitive interrogation of layer properties. The experiments employ two complementary measurements of particle motion -- one in which small, wire-shaped magnets at the interface are rotated by an external magnet, and one in which the random, thermally excited motion of particles is tracked. Taking advantage of the sensitivity of this approach, we have focused our studies on the early stages of layer formation, when the protein molecules first begin adsorbing at the interface to create an incipient, monolayer film. During these early stages, the layers behave as fluids with a viscosity that steadily increases. Eventually, the layers undergo a transformation from fluid to solid that is reflected in a qualitative change in the mobility of the probe particles. Comparing this process for different proteins and different interfaces (e.g., oil-water or air-water), we have found that the nature of the fluid-solid transition varies depending on the specific system. In some cases, the transition is characteristic of a process known as gelation, wherein the protein molecules bind together to form a network that spans the interface. In other cases, the transition is more akin to a process termed "jamming," in which the interface becomes crowded with protein to the point that mobility is arrested. Additional experiments employing variants of the same protein that assume different conformations in the water (folded versus unfolded) have illustrated a strong dependence of the layer-formation process on the protein’s shape. Overall, this unique experimental approach has enabled our group to obtain precise, microscopic information needed for understanding and controlling the mechanical properties of interfacial protein layers.
