Liat Rosenfeld

Fluorinated Pickering Emulsions Impede Interfacial Transport and Form Rigid Interface for the Growth of Anchorage-Dependent Cells

This study describes the design and synthesis of amphiphilic silica nanoparticles for the stabilization of aqueous drops in fluorinated oils for applications in droplet microfluidics. The success of droplet microfluidics has thus far relied on one type of surfactant for the stabilization of drops. However, surfactants are known to have two key limitations: (1) interdrop molecular transport leads to cross-contamination of droplet contents, and (2) the incompatibility with the growth of adherent mammalian cells as the liquid-liquid interface is too soft for cell adhesion. The use of nanoparticles as emulsifiers overcomes these two limitations. Particles are effective in mitigating undesirable interdrop molecular transport as they are irreversibly adsorbed to the liquid-liquid interface. They do not form micelles as surfactants do, and thus, a major pathway for interdrop transport is eliminated. In addition, particles at the droplet interface provide a rigid solid-like interface to which cells could adhere and spread, and are thus compatible with the proliferation of adherent mammalian cells such as fibroblasts and breast cancer cells. The particles described in this work can enable new applications for high-fidelity assays and for the culture of anchorage-dependent cells in droplet microfluidics, and they have the potential to become a competitive alternative to current surfactant systems for the stabilization of drops critical for the success of the technology.

Structural and Rheological Properties of Meibomian Lipid

PURPOSE We explore the unique rheological and structural properties of human and bovine meibomian lipids to provide insight into the physical behavior of the human tear-film lipid layer (TFLL). METHODS Bulk rheological properties of pooled meibomian lipids were measured by a commercial stress-controlled rheometer; a home-built interfacial stress rheometer (ISR) probed the interfacial viscoelasticity of spread layers of meibomian lipids. Small- and wide-angle x-ray scattering detected the presence and melting of dispersed crystal structures. Microscope examination under cross polarizers provided confirmation of ordered crystals. A differential scanning calorimeter (DSC) analyzed phase transitions in bulk samples of bovine meibum. RESULTS Bulk and interfacial rheology measurements show that meibum is extremely viscous and highly elastic. It is also a non-Newtonian, shear-thinning fluid. Small- and wide-angle x-ray diffraction (SAXS and WAXS), as well as differential scanning calorimetry (DSC) and polarizing microscopy, confirm the presence of suspended lamellar-crystal structures at physiologic temperature. CONCLUSIONS We studied meibum architecture and its relation to bulk and interfacial rheology. Bovine and human meibomian lipids exhibit similar physical properties. From all structural probes utilized, we find a melt transition near eye temperature at which lamellar crystals liquefy. Our proposed structure for the tear-film lipid layer at physiologic temperature is a highly viscoelastic, shear-thinning liquid suspension consisting of lipid lamellar-crystallite particulates immersed in a continuous liquid phase with no long-range order. When spread over on-eye tear, the TFLL is a duplex film that exhibits bulk liquid properties and two separate interfaces, air/lipid and water/lipid, with aqueous protein and surfactantlike lipids adsorbed at the water/lipid surface.

Molecular Structure of Interfacial Human Meibum Films

Meibum is the primary component of the tear film lipid layer. Thought to play a role in tear film stabilization, understanding the physical properties of meibum and how they change with disease will be valuable in identifying dry eye treatment targets. Grazing incidence X-ray diffraction and X-ray reflectivity were applied to meibum films at an air-water interface to identify molecular organization. At room temperature, interfacial meibum films formed two coexisting scattering phases with rectangular lattices and next-nearest neighbor tilts, similar to the Ov phase previously identified in fatty acids. The intensity of the diffraction peaks increased with compression, although the lattice spacing and molecular tilt angle remained constant. Reflectivity measurements at surface pressures of 18 mN/m and above revealed multilayers with d-spacings of 50 Å, suggesting that vertical organization rather than lateral was predominantly affected by meibum-film compression.

Consequences of Interfacial Viscoelasticity on Thin Film Stability

The phenomenon of dewetting is frequently observed in our everyday life. It is of central importance in many technological applications as well as in a variety of physical and biological systems. The presence of nonsoluble surfactants at an air/liquid interface may affect the dewetting properties of the aqueous layer. An important example is the tear film, which comprises an aqueous layer covered with a ∼100-nm-thick blanket of lipids, known as the meibomian lipids. Interfacial rheological measurements of meibomian lipids reveal that these films are remarkably viscoelastic. Tear film dewetting is of central importance to understanding tear film stability. To better understand the role of surface viscoelasticity in tear film stability, we have developed a methodology to systematically control interfacial rheology of thin aqueous layers at the onset of dewetting events. The apparatus allows control over the surface pressure of the monolayer, which is a key feature since this variable controls the surface viscoelasticity. Three insoluble monolayer materials were used: newtonian arachidyl alcohol (AA), DPPC, a phospholipid that is slightly viscoelastic, and meibum, which produces a strongly viscoelastic monolayer. It is reported that monolayers of viscoelastic surfactants are able to stabilize thin films against spontaneous dewetting. As the surface pressure of these layers is increased, their effectiveness is enhanced. Moreover, these surfactants are able to reduce the critical film thickness for dewetting. Meibum is particularly effective in stabilizing thin films. Our results suggest that the meibomian lipids play a vital role in maintaining tear film stability in addition to suppressing evaporation.

Rapid detection of tuberculosis using droplet-based microfluidics

Tuberculosis is one of the most deadly diseases that kills over one million people each year and infects one-third of the world’s population. The disease is spread by infection with Mycobacterium tuberculosis (Mtb). Owing to its airborne transmission, early diagnosis is critical to the prevention and control of TB. Standard diagnostic methods, acid-fast smear from sputum, often do not become positive until after transmission occurs, which allows the spread of the disease. Culture-based techniques are more sensitive, but take weeks to obtain results because of the extremely slow growth rate of Mtb. In this study a new method to detect indicator enzyme based on the isolation of tubercle bacillus in a large number of picoliter droplets combined with a fluorescent probe has been developed. We use BlaC (an enzyme naturally expressed/secreted by tubercle bacilli) as a marker and a designed BlaC-specific fluorogenic substrates as probes for Mtb detection. We present here a new method to detect the indicator enzyme based on the isolation, digitization and concentration of bacteria samples in a large number of picoliter drops. We show that by controlling the size of the droplets we can control the rate of conversion. Hence rapid increase in signal has been observed as the size of the drops has been decreased. Our vision is that this tool will be able to detect tubercle bacilli in a sensitive, rapid, specific and quantitative manner in vitro at a low cost, particularly in resource limited settings where TB is the most prevalent.

3-Hydroxybutyric acid interacts with lipid monolayers at concentrations that impair consciousness.

3-Hydroxybutyric acid (also referred to as β-hydroxybutyric acid or BHB), a small molecule metabolite whose concentration is elevated in type I diabetes and diabetic coma, was found to modulate the properties of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) monolayers when added to the subphase at clinical concentrations. This is a key piece of evidence supporting the hypothesis that the anesthetic actions of BHB are due to the metabolites abilities to alter physical properties of cell membranes, leading to indirect effects on membrane protein function. Pressure-area isotherms show that BHB changes the compressibility of the monolayer and decrease the size of the two-phase coexistence region. Epi-fluorescent microscopy further reveals that the reduction of the coexistence region is due to the significant reduction in morphology of the liquid condensed domains in the two-phase coexistence region. These changes in monolayer morphology are associated with the diminished interfacial viscosity of the monolayers (measured using an interfacial stress rheometer), which gives insight as to how changes in phase and structure may contribute to membrane function.