Thomas Pfohl

Droplet based microfluidics

Droplet based microfluidics is a rapidly growing interdisciplinary field of research combining soft matter physics, biochemistry and microsystems engineering. Its applications range from fast analytical systems or the synthesis of advanced materials to protein crystallization and biological assays for living cells. Precise control of droplet volumes and reliable manipulation of individual droplets such as coalescence, mixing of their contents, and sorting in combination with fast analysis tools allow us to perform chemical reactions inside the droplets under defined conditions. In this paper, we will review available drop generation and manipulation techniques. The main focus of this review is not to be comprehensive and explain all techniques in great detail but to identify and shed light on similarities and underlying physical principles. Since geometry and wetting properties of the microfluidic channels are crucial factors for droplet generation, we also briefly describe typical device fabrication methods in droplet based microfluidics. Examples of applications and reaction schemes which rely on the discussed manipulation techniques are also presented, such as the fabrication of special materials and biophysical experiments.

Hydrodynamic Flow-Mediated Protein Sorting on the Cell Surface of Trypanosomes

The unicellular parasite Trypanosoma brucei rapidly removes host-derived immunoglobulin (Ig) from its cell surface, which is dominated by a single type of glycosylphosphatidylinositol-anchored variant surface glycoprotein (VSG). We have determined the mechanism of antibody clearance and found that Ig-VSG immune complexes are passively sorted to the posterior cell pole, where they are endocytosed. The backward movement of immune complexes requires forward cellular motility but is independent of endocytosis and of actin function. We suggest that the hydrodynamic flow acting on swimming trypanosomes causes directional movement of Ig-VSG immune complexes in the plane of the plasma membrane, that is, immunoglobulins attached to VSG function as molecular sails. Protein sorting by hydrodynamic forces helps to protect trypanosomes against complement-mediated immune destruction in culture and possibly in infected mammals but likewise may be of functional significance at the surface of other cell types such as epithelial cells lining blood vessels.

Tunable Silk: Using Microfluidics to Fabricate Silk Fibers with Controllable Properties

Despite widespread use of silk, it remains a significant challenge to fabricate fibers with properties similar to native silk. It has recently been recognized that the key to tuning silk fiber properties lies in controlling internal structure of assembled β-sheets. We report an advance in the precise control of silk fiber formation with control of properties via microfluidic solution spinning. We use an experimental approach combined with modeling to accurately predict and independently tune fiber properties including Youngs modulus and diameter to customize fibers. This is the first reported microfluidic approach capable of fabricating functional fibers with predictable properties and provides new insight into the structural transformations responsible for the unique properties of silk. Unlike bulk processes, our method facilitates the rapid and inexpensive fabrication of fibers from small volumes (50 μL) that can be characterized to investigate sequence-structure-property relationships to optimize recombinant silk technology to match and exceed natural silk properties.

Preparation of Monodisperse Block Copolymer Vesicles via Flow Focusing in Microfluidics

We demonstrate that microfluidic flow devices enable a rapid, continuous, well-reproducible and size-controlled preparation of unilamellar block copolymer vesicles. The PDMS-based microfluidic device consists of perpendicularly crossed channels allowing hydrodynamic flow focusing of an ethanolic block copolymer solution in a stream of water. By altering the flow rate ratio in the water and ethanolic inlet channels, the vesicle size can be tuned over a large size range from 40 nm to 2 microm without subsequent processing steps manipulating size and shell characteristics. The ability of tuning the vesicle mean size over a range of several orders of magnitude with the possibility of in situ encapsulation of active ingredients creates new opportunities for the preparation of tailored drug delivery systems in science, medicine and industry.

Trypanosome Motion Represents an Adaptation to the Crowded Environment of the Vertebrate Bloodstream

Blood is a remarkable habitat: it is highly viscous, contains a dense packaging of cells and perpetually flows at velocities varying over three orders of magnitude. Only few pathogens endure the harsh physical conditions within the vertebrate bloodstream and prosper despite being constantly attacked by host antibodies. African trypanosomes are strictly extracellular blood parasites, which evade the immune response through a system of antigenic variation and incessant motility. How the flagellates actually swim in blood remains to be elucidated. Here, we show that the mode and dynamics of trypanosome locomotion are a trait of life within a crowded environment. Using high-speed fluorescence microscopy and ordered micro-pillar arrays we show that the parasites mode of motility is adapted to the density of cells in blood. Trypanosomes are pulled forward by the planar beat of the single flagellum. Hydrodynamic flow across the asymmetrically shaped cell body translates into its rotational movement. Importantly, the presence of particles with the shape, size and spacing of blood cells is required and sufficient for trypanosomes to reach maximum forward velocity. If the density of obstacles, however, is further increased to resemble collagen networks or tissue spaces, the parasites reverse their flagellar beat and consequently swim backwards, in this way avoiding getting trapped. In the absence of obstacles, this flagellar beat reversal occurs randomly resulting in irregular waveforms and apparent cell tumbling. Thus, the swimming behavior of trypanosomes is a surprising example of micro-adaptation to life at low Reynolds numbers. For a precise physical interpretation, we compare our high-resolution microscopic data to results from a simulation technique that combines the method of multi-particle collision dynamics with a triangulated surface model. The simulation produces a rotating cell body and a helical swimming path, providing a functioning simulation method for a microorganism with a complex swimming strategy.

Dynamics of intermediate filament assembly followed in micro-flow by small angle X-ray scattering

The assembly of intermediate filaments (IFs) is a complex process that can be recapitulated through a series of distinct steps in vitro. The combination of microfluidics and small angle X-ray scattering (SAXS) provides a powerful tool to investigate the kinetics of this process on the relevant timescales. Microfluidic mixers based on the principle of hydrodynamic focusing allow for precise control of the mixing of proteins and smaller reagents like ions. Here, we present a multi-layer device that prevents proteins from adsorbing to the channel walls by engulfing the protein jet with a fluid layer of buffer. To ensure compatibility with SAXS, the device is fabricated from UV-curable adhesive (NOA 81). To demonstrate the successful prevention of contact between the protein jet and the channel walls we measure the distribution of a fluorescent dye in the device by confocal microscopy at various flow speeds and compare the results to finite element method (FEM) simulations. The prevention of contact enables the investigation of the assembly of IFs in flow by gradually increasing the salt concentration in the protein jet. The diffusion of salt into the jet can be determined by FEM simulations. SAXS data are collected at different positions in the jet, corresponding to different salt concentrations, and they reveal distinct differences between the earliest assembly states. We find that the mean square radius of gyration perpendicular to the filament axis increases from 13 nm(2) to 58 nm(2) upon assembly. Thereby we provide dynamic structural data of a complex assembly process that was amenable up to now only by microscopic techniques.

Microfluidics of soft matter investigated by small-angle X-ray scattering.

The combination of X-ray microdiffraction and microfluidics is used to investigate the dynamic behaviour of soft materials. A microfocused X-ray beam enables the observation of the influence of droplet formation on the nanostructure of a smectic liquid crystal in water. Using a hydrodynamic focusing device, the evolution of the intercalation of DNA into multilamellar membranes can be studied. Owing to the elongational flow at the centre of this device, alignment of the material is induced which allows for an improved structural characterization. Furthermore, the influence of strain applied to these materials can be tested.

Impact of Microscopic Motility on the Swimming Behavior of Parasites: Straighter Trypanosomes are More Directional

Microorganisms, particularly parasites, have developed sophisticated swimming mechanisms to cope with a varied range of environments. African Trypanosomes, causative agents of fatal illness in humans and animals, use an insect vector (the Tsetse fly) to infect mammals, involving many developmental changes in which cell motility is of prime importance. Our studies reveal that differences in cell body shape are correlated with a diverse range of cell behaviors contributing to the directional motion of the cell. Straighter cells swim more directionally while cells that exhibit little net displacement appear to be more bent. Initiation of cell division, beginning with the emergence of a second flagellum at the base, correlates to directional persistence. Cell trajectory and rapid body fluctuation correlation analysis uncovers two characteristic relaxation times: a short relaxation time due to strong body distortions in the range of 20 to 80 ms and a longer time associated with the persistence in average swimming direction in the order of 15 seconds. Different motility modes, possibly resulting from varying body stiffness, could be of consequence for host invasion during distinct infective stages.

Polymer Nanoreactors with Dual Functionality: Simultaneous Detoxification of Peroxynitrite and Oxygen Transport

The design of multifunctional systems is in focus today as a key strategy for coping with complex challenges in various domains that include chemistry, medicine, environmental sciences, and technology. Herein, we introduce protein-containing polymer nanoreactors with dual functionality: peroxynitrite degradation and oxygen transport. Vesicles made of poly-(2-methyloxazoline)-poly(dimethylsiloxane)-poly(2-methyloxazoline) successfully encapsulated hemoglobin (Hb), which serves as a model protein because of its dual function in oxygen transport and peroxynitrite degradation. By inserting channel proteins, the polymer membranes of vesicles permitted passage of various compounds that served for the assessment of in situ Hb activity. The requisite conformational changes in the protein structure and the change in oxidation states that took place within the confined space of the vesicle cavity demonstrated that Hb preserved its dual functionality: peroxynitrite degradation and oxygen transport. The functionality of our nanoreactor, combined with its simple procedure of production and extensive stability over several months, supports it as a promising system for further medical applications.

Gold nanoparticles stabilized by thioether dendrimers

Ligand-stabilized gold nanoparticles (Au NPs) are promising materials for nanotechnology with applications in electronics, catalysis, and sensors. These applications depend on the ability to synthesize stable and monodisperse NPs. Herein, the design and synthesis of two series of dendritic thioether ligands and their ability to stabilize Au NPs is presented. The dendrimers have 1,3,5-trisubstituted benzene branching units bridged by either meta-xylene or ethylene moieties. A comparison between the two ligands shows how both size control and the stability of the NPs are influenced by the nature of the ligand-NP wrapping interaction. The meta-xylene-bridged ligands provided NPs with a narrow size distribution centered around a diameter of 1.2 nm, whereas the NPs formed with ethylene-bridged dendrimers lack long-term stability with NP aggregation detected by UV/Vis spectroscopy and transmission electron microscopy. The bulkier tert-butyl-functionalized meta-xylene bridges form larger ligand shells that inhibit further growth of the NPs and thus provide a simple route to stable and monodisperse Au NPs that may find use as functional components in nanoelectronic devices.