Experimental Soft Condensed Matter Group

Experimental Soft Condensed
Matter Group
Prof. David A. Weitz
School of Engineering and Applied Sciences / Department of Physics
Harvard University, Cambridge, MA 02138

RESEARCH AREAS

COLLOID PHYSICS
FLOW IN MICROCHANNELS

PARTICLE SYNTHESIS ENCAPSULATION

MECHANICS OF BIOLOGICAL NETWORKS BIOPHYSICS OF CELLS

RHEOLOGY AND MICRORHEOLOGY BIOLOGY IN MICROFLUIDIC DEVICES

MOLECULAR AGGREGATION & X-RAY IMAGING OLDER RESEARCH

COLLOID PHYSICS
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Understanding the dynamics of closely packed microgels: This research focuses on the study of colloidal suspension of soft spherical particles, specifically microgels. Microgels are interesting because of their numerous applications in industry, where they are used, for example, as drug carriers and to modify the mechanical properties of cosmetic products and car paints. They are interesting also because of their potential to serve as a model to study fundamental physics such as the dynamics of close packing and glass formation.

Instability in a droplet: We study instability in a drying colloidal suspension droplet.

Reverse coffee-ring effect: We explore a tendency for solute to segregate at the center of water droplets - named here the "reverse coffee-ring effect".

Delayed collapse of gels: Many industrial and consumer products including food products, such as yogurt and mayonnaise, plant fertilizer, cosmetics products, or fabric enhancers consist of particles or emulsions dispersed in a continuous phase. The stability of many dispersed particle systems is determined by the formation of a weak space-filling gel. For an initial period of time (the delay time) this solid-like particulate gel can support its own weight, and thus slows gravitational collapse and phase separation of microscopic constituents. After this time, however, the gel catastrophically collapses into a consolidated sediment – the sample phase separates, and the product is likely unusable. Understanding the fundamental parameters that govern this process of delayed collapse, as well as connecting these to control parameters – such as sample composition and preparation - would be a significant step towards rational control of the behavior of these gels.

Crystallization of charged colloids in nonpolar solvents: Charged colloidal crystals are typically formed in polar solvents. In nonpolar solvents (e ~ 2) where the low dielectric constant inhibits charges from dissociating, crystallization by charge can not occur. However, it is known that adding surfactants or micelles gives an electrokinetic response and enhanced stability to particles, e.g., electrophoretic ink in flexible electronic displays and studies on the stability of soot particles in oil. Furthermore, recent studies about such charging of colloids by micelles have demonstrated that the electrostatic interaction have the same functional form as those predicted from DLVO theory, and particle surface potentials are remarkably large, comparable to those of highly charged aqueous colloids.

Seeing nucleation by eye: Crystal nucleation from melt is poorly understood, despite of being among the oldest problems in physics. For many systems, theoretical and experimental nucleation rates differ by many orders of mangitude. A suspension of hard spheres is a very simple system, and nucleation in this system has to be fully understood. We use confocal microscopy to visualize early nuclei in a system of hard spheres, in three dimensions, in vivo. The morphology of early nuclei is different from the assumptions of classical nucleation theory, and plays important role in the process of nucleation.

PLuTARC: Target-Locking Microscopy: In any typical data gathering process, objects are observed from a fixed viewpoint (think of a camera on a tripod). If the objects are moving, this limits the observation time, as the objects move out of the field of view. In a microscope, this is a particularly severe problem when studying moving objects like swimming cells, or freely-diffusing cluster of colloidal beads. What we've done with the PLuTARC (Peter Lu Target-Locking Acquisition in Real-time Confocal) system is to implement target-locking. Images from the microscope are analyzed in real-time, allowing determination of the largest object's center.

Drying of a colloidal suspension: How does a colloidal suspension dry? Is evaporation the only factor we need to consider in this process? By watching the drying of an index matched colloidal suspension with a confocal microscope, we found that drying is a two-stage process: in the first stage, due to faster evaporation rate at edge, colloidal particles accumulate and compact at the edge, forming a porous medium filled by solvent; after all the particles are packed, the second stage starts: air invades this porous medium and solvent retreats, eventually all liquid is replaced by air.

The Physics of Attractive Colloids: We use confocal microscopy to determine the three-dimensional positions of thousands of particles as a function of time. We control the interactions between the particles to make them attractive, and can control the range and strength of this attraction potential. We are able to create and observe a number of phases, including equilibrium fluids, a kinetically-arrested gels, and large clusters that persist. Our ultimate aim is to establish a general framework for understanding the behavior of attractive colloid systems, and which physics drives their formation and properties. One particular limit, equilibrium phase separation near the critical, takes a very long time to observe, and we therefore conduct experiments in space, where we don't have to worry about long-time issues of sedimentation.

Investigation of Grain Boundaries in Colloidal Crystals: Almost all engineering applications of metals involve their use in polycrystalline form. Recently the emphasis in the study of mechanical properties has moved away from the processes which occur inside the individual grains to those which are governed by the boundaries between the grains. Diverse phenomena such as high temperature creep, superplasticity, recrystallization, yielding and embrittlement all depend strongly on effects at grain boundaries. Grain boundaries are also important for diffusion phenomena as they provide pathways for diffusions into or within a material that are orders of magnitude faster than through crystalline regions. Interactions of grain boundaries and defects are also a topic of current research. Recent studies emphasize the role of grain boundaries for premelting of a crystal. Despite the important role of grain boundaries in material properties our knowledge at the microscopic level is limited. The direct observation of grain boundary structure is limited by the lack of resolution of experimental techniques such as high resolution transmission electron microscopy. Thus colloidal crystals can serve as a model system to study grain boundary characteristics as they are much larger and show a much slower dynamics which makes them accesible to experimental techniques like confocal microscopy.

FLOW IN MICROCHANNELS
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Two-phase flow in porous media: Using confocal microscopy we monitor the evolution of fluid configurations during two-phase flow through a porous material under a variety of controlled conditions.

Photoreactive sol-gel coating for high contrasts spatial patterning of microfluidic device wettability: We introduce a photoreactive sol-gel coating that can be used to spatially pattern high contrasts in the wettability of microfluidic channels.

Droplet Micro-Fluidics for High Throughput Screening: Droplet-based microfluidics can produce monodisperse picoliter size microreactors at a speed of 10 kHz. This technology can be utilized to enable biochemical screening at astonishing rates. We can prepare two droplet trains where one contains a unique biochemical probe and the other train contains a probe target and other biochemical reagents that are necessary to mimic the cellular environment.

Droplet break-up and pressure measurement in a model 2D porous medium: We use PDMS microfluidic devices as a model system for two-dimensional porous media, and study drop break-up in a two-phase flow.

Valve-based flow focusing for drop formation: We have demonstrated modulation of the size and formation frequency of drops in a microfluidic device, using single-layer membrane valves to provide local control of the channel geometry. This control is independent of the flow rates of the fluids and can be modulated from a steady state up to hundreds of Hertz. We have also made double emulsions with a controllable number of inner drops, adjustable independently of volume fraction.

These valves may find application for prototyping devices, allowing many geometry and flow rate combinations to be explored on just one fabricated chip. We expect they will also be useful for synchronizing drop trains, which can then be merged or sorted, and for improving drop uniformity over time despite flow rate drift. The valves are compatible with our sol-gel coating.

Valve-based flow focusing for microfluidic drop formation

Dropspots: a picoliter array of drops on chip: Studying temporal processes on small scales is essential for understanding phenomena ranging from nucleation to single enzyme kinetics to cellular heterogeneity. Drops of water-in-oil emulsions uniquely provide femtoliter to picoliter-volume reaction vessels, however, it is challenging to follow reactions encapsulated in drops over time. We have developed a device to immobilize drops, allowing us to track single cell growth and to monitor levels of secreted enzymes that rapidly attain high concentrations due to the small drop volume. This picoliter array can be imaged by microscopy or a microarray scanner.

Using microfluidics to encapsulate cells in drops: Encapsulation of single cells and beads within picolitre-size monodisperse drops provides new means to perform quantitative studies for individual members of large populations. Microfluidic devices made using soft lithography techniques are used produce to monodisperse drops containing single cells at rates of up to thousands of drops per second, allowing for screening of large libraries. We're particularly interested in using drop-based microfluidics for monoclonal antibody screening and directed evolution.

Glass coating for PDMS channels by sol-gel methods: Polydimethylsiloxane (PDMS) is widely used for fabrication of microfluidic devices: it is inexpensive and it can be fashioned to have complex channel structures. However, PDMS channels have several drawbacks. Even when cured, PDMS remains permeable to liquids and gases, which can affect reactions that occur in the channels. Organic solvents can swell PDMS significantly, seriously degrading device performance. The limited chemical compatibility of PDMS is, therefore, a major problem that can limit the wider application of PDMS to microfluidic technology. We coat PDMS microfluidic devices with a glass-like layer using sol-gel chemistry. The coating greatly increases the chemical resistance of the channels, enabling the use of organic solvents. In addition, the coating can be functionalized with a wide range of silanes, for example, to make the channels either hydrophilic or hydrophobic.

Fabrication of micron-scaled monodisperse oil-in-water emulsions in glass microcapillaries: We work on generating monodisperse oil-in-water emulsions below conventional droplet sizes using droplet breakup in glass microcapillaries. Smaller droplets necessitates narrower channels. In the micron range, typical pressure required is too high for conventional microfluidic devices. In this work, we show that micron-sized monodisperse emulsions can be prepared in a modified microcapillary device. Moreover, we present potential applications for these emulsion droplets in both materials engineering and fundamental research.

Geometrically Controlled Jet-Like Instabilities in Microfluidic Two-Phase Flows: We are interested in the effects of confinement in two phase co-flows in microfluidic devices. When the flow rate of the inner fluid is small compared to the flow rate of the outer fluid, and the resulting width of the inner fluid is smaller than the height of the channel, the inner fluid breaks into droplets, as expected for a three-dimensional system. On the other hand, when the width of the second phase becomes comparable to the height of the microfluidic device, Rayleigh capillary instabilities are suppressed, and the inner fluid forms a jet that does not break, as might be expected for a purely two-dimensional system. We show that by changing the dimensions of the microfluidic channel we can transition from a stable co-flow to drop break-up. These results can be explained with a model of this two phase flow.

Giant Phospholipid Vesicles from Double Emulsions: Vesicles made from phospholipids (also known as liposomes) provide excellent model systems for studying the biophysics of plasma membranes. Also these liposomes hold much promise in the areas of encapsulation and delivery of active ingredients. In this project, we aim to fabricate phospholipid vesicles from double emulsions (water-in-oil-in-water droplets) which are prepared by using a glass microcapillary device. Using our technique, it is possible to continuously generate monodisperse liposomes and at the same time achieve high encapsulation efficiency.

Microfluidics as a Formulation Tool: We investigate the usefulness of microfluidic devices as a low energy-input formulation tool for producing emulsions in a controlled fashion.

PARTICLE SYNTHESIS
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Microfluidic synthesis of Janus Particles from TEOS and NIPMAM: An interesting phase separation phenomenon of TEOS aqueous emulsions in hexadecane was found, and based on this phenomenon we want to produce the Janus particles using capillary tube-based microfluidics producing single emulsions of TEOS and NIPMAM. Polymerization of NIPMAM will be done during the phase separation process, and the Janus particles should contain one part of silica particle and the other part of PNIPMAM gel.

Synthesis of Biocompatible Polymer Nanoparticles and Their Applications in Drug Delivery: 1 in 10 marketed drugs have solubility problems; over a third of pipeline drugs are poorly soluble. However, loading drug into environmentally responsive polymer nanoparticles is an effective way to enhance its biocompatibility. First, size reduction to nanometer order increases drug’s dissolution rate in human body. Second, drug release kinetics can be controlled by applying external stimuli to polymer nanoparticles. The purpose of this study is to synthesize "smart" polymer nanoparticles, and then use them as drug carrier.

Combining colloid science and microfluidics:Research efforts in this area are focused on the fabrication of novel monodisperse "supraparticles" by the directed assembly of sub-micron sized colloidal particles in droplets. These include smart particles and capsules that are responsive to external fields and anisotropic particles that exhibit biphasic characteristics. Depending upon their tailorable properties, such particles can be used as vehicles for transporting and releasing materials, and as visual probes for chemical, biological, and rheological phenomena. We employ capillary-based microfluidic techniques to ensure the monodispersity of these supra-structures.

Monodisperse Biodegradable Polymersomes with Microfluidics:Vesicles are compartments surrounded by bilayered membranes of amphiphilic molecules such as diblock copolymers and phospholipids. To minimize the exposure of their hydrophobic part to water, amphiphilic molecules self-assemble into aggregates of different structures. When the hydrophobic to hydrophilic ratio is close to unity, amphiphiles self assemble into bilayers, which tend to fold themselves into vesicles. These vesicles are useful for encapsulating and transporting actives such as drugs, flavor, and fragrance. To solve the problems of low encapsulation efficiency and large vesicle size distributions afforded by traditional techniques to create vesicles, we engineer a novel route to generate vesicles using monodisperse double emulsions prepared in microfluidics as templates.

ENCAPSULATION
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Antibody Dectection: PDMS microfluidic devices are used to produce monodisperse aqueous emulsion droplets in a continuous oil phase. The drops serve as individual, picoliter sized compartments for cells and enable us to detect and screen cells which secrete specific molecules, as they can easily reach detectable concentrations within the droplets. We are currently working on a high-throughput assay for monoclonal antibody screening. We encapsulate hybridoma cells in drops and incubate them for several hours to allow for antibody production. Co-encapsulated beads bind the particular target antibody. A fluorescent secondary antibody is used to visualize the binding of the primary antibody. In this case, the fluorescence is localized on the bead surface, whereas it is diffuse in the droplet if the desired primary antibody is not present. We are able to detect this fluorescent signal in real time, sort the drops containing cells that produce the desired antibody, and collect those cells for further studies.

Using microfluidics to encapsulate cells in drops: Encapsulation of single cells and beads within picolitre-size monodisperse drops provides new means to perform quantitative studies for individual members of large populations. Microfluidic devices made using soft lithography techniques are used produce to monodisperse drops containing single cells at rates of up to thousands of drops per second, allowing for screening of large libraries. We're particularly interested in using drop-based microfluidics for monoclonal antibody screening and directed evolution.

Giant Phospholipid Vesicles from Double Emulsions: Vesicles made from phospholipids (also known as liposomes) provide excellent model systems for studying the biophysics of plasma membranes. Also these liposomes hold much promise in the areas of encapsulation and delivery of active ingredients. In this project, we aim to fabricate phospholipid vesicles from double emulsions (water-in-oil-in-water droplets) which are prepared by using a glass microcapillary device. Using our technique, it is possible to continuously generate monodisperse liposomes and at the same time achieve high encapsulation efficiency.

Directed evolution of enzymes in microfluidic devices: We are recreating the mechanisms of natural selection in the laboratory at the single enzyme level.One goal is to engineer new enzyme functions using this method. These may have interesting industrial, synthetic, or therapeutic uses. In addition, In vitro evolution also gives us the ability to observe evolutionary intermediates, control selective pressure, mutation rate, and population sizes in ways that aren't possible using other methods, and this allows us to ask new questions about the evolutionary process itself.

Directed evolution of enzymes in microfluidic devices

MECHANICS OF BIOLOGICAL NETWORKS
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AFM investigations of Actin networks: Actin networks have been widely studied by confocal imaging, which provides an uncomplicated technique to study their structural features and reaction to mechanical forces in water. Although a lot of valuable information can be extracted from these scans, however the resolution of the confocal is limited to several hundred nanometers and cannot reveal the fine structure of the network. Thus it is desirable to study the networks in parallel with a high-resolution technique that can provide complementary information. The AFM can operate in liquid environment and does not require any sophisticated sample preparation protocol while providing a resolution at the scale of a few nm. Apart from this it also allows to study the mechanical properties of the different actin aggregates found in an actin network.

Intermediate Filament Proteins: Intermediate filament (IF) proteins are an essential structural element of metazoan cells; they form a dense network and provide critical structural support in the cytoplasm and nucleus. Impaired assembly of IF networks results in cell and nuclear shape change, destabilization of actin and microtubule networks, cell fragility in response to mechanical stress, as well as multiple diseases. To understand the mechanical properties of the cell as a whole, it is thus essential to understand the structure and mechanical properties of IF protein networks. However, IF proteins are difficult to isolate and purify, and thus have not been widely studied.

Cell and cytoskeletal mechanics: The cell is a material that has both elastic and viscous properties, arising from a composite and highly dynamic intracellular biopolymer network called the cytoskeleton. The material properties of a cell determine how mechanical forces are transmitted through and sensed by that cell. The cellular mechanical response is quite unusual compared to that of more common materials. For instance, the stiffness of a cell is highly nonlinear; some types of cells stiffen passively under large external forces, but they can also alter their own stiffness in response to the local mechanical microenvironment or biochemical cues. This active stiffening is thought to arise from internal prestress generated in the actin cytoskeleton by molecular motors. We want to understand the physical origins of this nonlinear elasticity, determine the molecular mechanisms of this behavior within cells, and understand the biological relevance of these remarkable material properties.

Mechanotransduction: Cells within an organism must not only generate forces to move, but they must also be able to react to forces in their environment. This conversion of physical forces into biological responses is known as mechanotransduction. In this study, we are investigating the ability of an actin crosslinking protein, Filamin A, to function as a biological force sensor.

Quantifying three-dimensional traction forces exerted by cells in a collagen matrix: Cells in our body are strongly influenced by the mechanical properties of the matrix, which surrounds them (the extracellular matrix or ECM); in turn, the mechanical forces generated by cells are correlated with other aspects of their behavior, such as tissue morphogenesis, cell motility, wound healing and metastasis in cancer.

Visualization and Analysis of Biopolymer Networks under Shear: When you impose a small strain on a sample its response will be roughly linear. In other words, the amount of force necessary to deform the sample a given amount is roughly constant. As you increase the strain most materials will start to weaken and eventually break (strain weaken). Interestingly, many biopolymer networks exhibit the opposite behavior. Namely, as you increasingly strain them, they stiffen before they break. (strain stiffening).

There has been a lot of theoretical work trying to predict and model how individual filament dynamics give rise to strain stiffening. We are taking an experimental approach and directly visualize the individual filaments using confocal microscopy as the networks undergo shear.

Rheology of Intermediate Filament Solutions: Cells interact mechanically with their environment through their cytoskeleton, a network consisting largely of filamentous protein polymers. Reconstituted solutions and networks of these biopolymers have rich rheological and elastic properties that arise from their semi-flexibility, with thermal persistence lengths comparable to their contour length. While the viscoelastic properties of reconstituted models of other cytoskeletal filaments, most notably F-actin, have been widely studied in vitro, relatively little is known about the network properties of intermediate filaments (IF). Such knowledge is urgently needed for understanding how disease-causing inherited mutations in human IF proteins yield an increase in cell fragility in response to mechanical stresses. We study both the vimentin and neurofilament networks in vitro by using the multiple particle tracking technique and the conventional rheometry.

Rheology of Microtubule Solutions: In eukaryotic cells, microtubules form a network that guides active intracellular transport and supports the overall cell structure. Microtubules also play an important role in the organization of cell locomotion, morphogenesis, and reproduction. By studying the mechanical properties of microtubules we hope to understand more about the mechanism of these fundamental cellular processes. We study the microtubule networks in vitro by using the multiple particle tracking technique that has previously been successfully utilized with F-actin networks in our lab.

BIOPHYSICS OF CELLS
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Self generated surfactant gradient for collective force generation in biofilms: Many species of bacteria are known to excrete surfactants. It is widely believed that the physical role of these surfactants is to lubricate surfaces so that bacteria can slide around. However, a more directed type of motion could be generated if surfactant gradients were produced in bacterial colonies. To explore this possibility, we study Bacillus Subtilis biofilms grown as pellicles at fluid-air interfaces. We find that the biofilms rapidly climb the walls of their vessel, and that this kind of wall climbing requires the production of a surfactant called Surfactin, but does not require flagellar motility. Furthermore, the nutrient medium surface tension changes in direct proportion to biofilm climbing height.

Physical Aspects of Biofilm Formation: Biofilms are colonies of bacteria that switch from a swimming, motile stage to a sessile state, upon which the bacteria form an extracellular matrix (ECM) of excreted polymeric compounds that serves to protect the colony and transport nutrients. Our goals is to study the physical aspects of biofilm formation.

Cell and cytoskeletal mechanics: The cell is a material that has both elastic and viscous properties, arising from a composite and highly dynamic intracellular biopolymer network called the cytoskeleton. The material properties of a cell determine how mechanical forces are transmitted through and sensed by that cell. The cellular mechanical response is quite unusual compared to that of more common materials. For instance, the stiffness of a cell is highly nonlinear; some types of cells stiffen passively under large external forces, but they can also alter their own stiffness in response to the local mechanical microenvironment or biochemical cues. This active stiffening is thought to arise from internal prestress generated in the actin cytoskeleton by molecular motors. We want to understand the physical origins of this nonlinear elasticity, determine the molecular mechanisms of this behavior within cells, and understand the biological relevance of these remarkable material properties.

Quantifying three-dimensional traction forces exerted by cells in a collagen matrix: Cells in our body are strongly influenced by the mechanical properties of the matrix, which surrounds them (the extracellular matrix or ECM); in turn, the mechanical forces generated by cells are correlated with other aspects of their behavior, such as tissue morphogenesis, cell motility, wound healing and metastasis in cancer.

Beads in cells: What are the differences in the forces of molecular motor in vitro and in vivo? Vesicles containing magnetic beads are transported by molecular motors along microtubules within cells. Transportation is observed and forces are interpreted through the use of magnetic tweezers.

Rheology of Microtubule Solutions: In eukaryotic cells, microtubules form a network that guides active intracellular transport and supports the overall cell structure. Microtubules also play an important role in the organization of cell locomotion, morphogenesis, and reproduction. By studying the mechanical properties of microtubules we hope to understand more about the mechanism of these fundamental cellular processes. We study the microtubule networks in vitro by using the multiple particle tracking technique that has previously been successfully utilized with F-actin networks in our lab.

Cell Prestress, Stiffness, and Density: To explore the prestress mechanism of cell elasticity we have cultured adherent cos-7 cells on PDMS substrates with periodic patterns of varying stiffness and constant chemical environment. We have varied the moduli and the wavelengths of the patterned regions, and have observed preferred growth of cells on stiffer regions.

Collective Contractile Dynamics in Confluent Cell Layers: Many types of cells are sensitive to the mechanical properties of their environment. In cultures of fibroblasts and endothelial cells, for example, it has been shown that cell morphology and motility are sensitive to substrate elasticity and are related to the traction forces exerted by cells. It was shown that in human airway soft muscle cells, the stiffness of the cell itself increases in proportion to the traction forces the cell exerts on the substrate. Taken together, these results suggest that, at confluence, cells will exhibit different mechanical behavior than at sparse densities because in a confluent layer the ?mechanical environment? is active; it is composed of a substrate and neighboring cells. To explore collective mechanical behavior of cells at confluent densities, we cultured cos-7 epithelial cells on PDMS (Young?s modulus ~10 kPa), and monitored the motion of beads embedded below the elastomer surface.

RHEOLOGY AND MICRORHEOLOGY
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Jamming of Solid-Stabilized Emulsions: A significant amount of theoretical and experimental work for over a decade has focused on understanding jamming, the non-equilibrium process by which a densely-packed disordered system abruptly transitions from a fluidlike to a solidlike state - that is, it develops a yield stress. Emulsions - stable suspensions of droplets of one fluid dispersed within another - are particularly interesting jammed systems because they are deformable. In particular, highly concentrated emulsions, while mostly composed of fluid, can be solid-like with a high elastic modulus. This is because droplets, packed together, further deform upon shear; the energy to densely pack these droplets is fully stored in their interfaces. The nature of this elasticity is well understood for the case of surfactant-stabilized emulsions. But what if surfactants were solid particles? How would that affect the rheology?

Rheology of Intermediate Filament Solutions: Cells interact mechanically with their environment through their cytoskeleton, a network consisting largely of filamentous protein polymers. Reconstituted solutions and networks of these biopolymers have rich rheological and elastic properties that arise from their semi-flexibility, with thermal persistence lengths comparable to their contour length. While the viscoelastic properties of reconstituted models of other cytoskeletal filaments, most notably F-actin, have been widely studied in vitro, relatively little is known about the network properties of intermediate filaments (IF). Such knowledge is urgently needed for understanding how disease-causing inherited mutations in human IF proteins yield an increase in cell fragility in response to mechanical stresses. We study both the vimentin and neurofilament networks in vitro by using the multiple particle tracking technique and the conventional rheometry.

Rheology of Microtubule Solutions: In eukaryotic cells, microtubules form a network that guides active intracellular transport and supports the overall cell structure. Microtubules also play an important role in the organization of cell locomotion, morphogenesis, and reproduction. By studying the mechanical properties of microtubules we hope to understand more about the mechanism of these fundamental cellular processes. We study the microtubule networks in vitro by using the multiple particle tracking technique that has previously been successfully utilized with F-actin networks in our lab.

BIOLOGY IN MICROFLUIDIC DEVICES
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Droplet Micro-Fluidics for High Throughput Screening: Droplet-based microfluidics can produce monodisperse picoliter size microreactors at a speed of 10 kHz. This technology can be utilized to enable biochemical screening at astonishing rates. We can prepare two droplet trains where one contains a unique biochemical probe and the other train contains a probe target and other biochemical reagents that are necessary to mimic the cellular environment.

Droplet Compartmentalization to select non-immortalized cells that secrete antibodies with desired binding characteristics: We are developing a new microfluidics-based method (Droplet Compartmentalized Selection - DCS) to isolate cells that produce antibodies with desired binding characteristics. In DCS, individual cells are encapsulated (together with components to assay binding activity) in pico-liter scale droplets, which can then be screened for binding activity at rates of about 1,000 droplets per second. Individual cells will fill the droplets with antibody to μM concentrations within a few hours. A fluorescent read-out can then be used to identify and collect droplets that contain the desired antibody-producing cells. Crucially, there is no need to immortalize the antibody-producing cells. Instead, the antibody-encoding genes can be isolated from individual selected cells by reverse-transcriptase PCR and then cloned into appropriate expression vectors.

Antibody Dectection: PDMS microfluidic devices are used to produce monodisperse aqueous emulsion droplets in a continuous oil phase. The drops serve as individual, picoliter sized compartments for cells and enable us to detect and screen cells which secrete specific molecules, as they can easily reach detectable concentrations within the droplets. We are currently working on a high-throughput assay for monoclonal antibody screening. We encapsulate hybridoma cells in drops and incubate them for several hours to allow for antibody production. Co-encapsulated beads bind the particular target antibody. A fluorescent secondary antibody is used to visualize the binding of the primary antibody. In this case, the fluorescence is localized on the bead surface, whereas it is diffuse in the droplet if the desired primary antibody is not present. We are able to detect this fluorescent signal in real time, sort the drops containing cells that produce the desired antibody, and collect those cells for further studies.

Using microfluidics to encapsulate cells in drops: Encapsulation of single cells and beads within picolitre-size monodisperse drops provides new means to perform quantitative studies for individual members of large populations. Microfluidic devices made using soft lithography techniques are used produce to monodisperse drops containing single cells at rates of up to thousands of drops per second, allowing for screening of large libraries. We're particularly interested in using drop-based microfluidics for monoclonal antibody screening and directed evolution.

Glass coating for PDMS channels by sol-gel methods: Polydimethylsiloxane (PDMS) is widely used for fabrication of microfluidic devices: it is inexpensive and it can be fashioned to have complex channel structures. However, PDMS channels have several drawbacks. Even when cured, PDMS remains permeable to liquids and gases, which can affect reactions that occur in the channels. Organic solvents can swell PDMS significantly, seriously degrading device performance. The limited chemical compatibility of PDMS is, therefore, a major problem that can limit the wider application of PDMS to microfluidic technology. We coat PDMS microfluidic devices with a glass-like layer using sol-gel chemistry. The coating greatly increases the chemical resistance of the channels, enabling the use of organic solvents. In addition, the coating can be functionalized with a wide range of silanes, for example, to make the channels either hydrophilic or hydrophobic.

Directed evolution of enzymes in microfluidic devices: We are recreating the mechanisms of natural selection in the laboratory at the single enzyme level. One goal is to engineer new enzyme functions using this method. These may have interesting industrial, synthetic, or therapeutic uses. In addition, In vitro evolution also gives us the ability to observe evolutionary intermediates, control selective pressure, mutation rate, and population sizes in ways that arent possible using other methods, and this allows us to ask new questions about the evolutionary process itself.

Directed evolution of enzymes in microfluidic devices

Gene expression changes in response to environmental stimuli: When genetically identical cells are exposed to the same environmental conditions, they exhibit phenotypic variation. To study the mechanisms that give rise to this variation, w**e have developed microfluidic devices to encapsulate populations of single cells. Using these devices, we can continuously flow media past the cells, vary environmental conditions, and monitor gene expression in individual cells by fluorescence microscopy. We design the chambers so that cells are constrained to grow in a single line. This makes it easy to study gene expression in single cells and their progeny. Ultimately these experiments provide insights into how patterns in gene expression are passed on to progeny cells.

MOLECULAR AGGREGATION & X-RAY IMAGING
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X-ray imaging for soft matter dynamics: We exploit synchrotron real-time phase-contrast X-ray microscopy to study a variety of soft matter dynamics.

Molecular Aggregation: The heavy oil reserves in northern Canada may be the largest hydrocarbon reserves on the planet. However, these oils are loaded with asphaltenes: an ambiguous chemical definition based on a solubility class. These asphaltenes are aromatic molecules with a complex phase behavior and play a crucial role in the economics of oil production all the way from extraction to transport and refining. From the stand point of physical chemistry, we would like to know more about this phase behavior, and about their interfacial and chemical properties. In order to achieve this, the first step is elemental and compositional analysis. Determination of molecular sizes or diffusivities is the second step. The next step is to understand their thermodynamics: in particular their aggregation thermodynamics and kinetics. The first condition was addressed decades ago. The second condition has been studied for roughly the same amount of time, but the results are still a matter of debate. Using Fluorescence Correlation Spectroscopy we have been able to determine an average molecular diffusivity for asphaltenes dissolved in toluene.

NMR Pulse Shaping

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