Jason P. Beech

Sorting cells by size, shape and deformability

While size has been widely used as a parameter in cellular separations, in this communication we show how shape and deformability, a mainly untapped source of specificity in preparative and analytical microfluidic devices can be measured and used to separate cells.

Separation of parasites from human blood using deterministic lateral displacement.

We present the use of a simple microfluidic technique to separate living parasites from human blood. Parasitic trypanosomatids cause a range of human and animal diseases. African trypanosomes, responsible for human African trypanosomiasis (sleeping sickness), live free in the blood and other tissue fluids. Diagnosis relies on detection and due to their often low numbers against an overwhelming background of predominantly red blood cells it is crucial to separate the parasites from the blood. By modifying the method of deterministic lateral displacement, confining parasites and red blood cells in channels of optimized depth which accentuates morphological differences, we were able to achieve separation thus offering a potential route to diagnostics.

Tipping the balance of deterministic lateral displacement devices using dielectrophoresis.

We report the use of dielectrophoresis (DEP) to achieve tunability, improve dynamic range and open up for the separation of particles with regard to parameters other than hydrodynamic size in deterministic lateral displacement (DLD) devices. DLD devices have been shown capable of rapidly and continuously separating micrometer sized plastic spheres by size with a resolution of 20 nm in diameter and of being able to handle the separation of biological samples as wide ranging as bacterial artificial chromosomes and blood cells. DEP, while not exhibiting the same resolution in size separation as DLD, has the benefit of being easy to tune and can, by choosing the frequency, be used to probe a variety of particle properties. By combining DLD and DEP we open up for the advantages, while avoiding the drawbacks, of the two techniques. We present a proof of principle in which the critical size for separation of polystyrene beads is tuned in the range 2-6 microm in a single device by the application of moderate (100 V cm(-1)), low frequency (100 Hz) AC electric fields. The behaviour of the device was further investigated by performing simulations of particle trajectories, the results of which were in good qualitative agreement with experiments, indicating the potential of the method for tunable, high-resolution separations with respect to both size and polarisability.

Tuneable separation in elastomeric microfluidics devices.

We describe how the elastomeric properties of PDMS (polydimethylsiloxane) can be utilised to achieve tuneable particle separation in Deterministic Lateral Displacement devices via strain controlled alteration of inter-obstacle distances, a development that opens up new avenues toward more effective separation of particles in microfluidics devices.

Shear-Driven Motion of Supported Lipid Bilayers in Microfluidic Channels.

In this work, we demonstrate how a lateral motion of a supported lipid bilayer (SLB) and its constituents can be created without relying on self-spreading forces. The force driving the SLB is instead a viscous shear force arising from a pressure-driven bulk flow acting on the SLB that is formed on a glass wall inside a microfluidic channel. In contrast to self-spreading bilayers, this method allows for accurate control of the bilayer motion by altering the bulk flow in the channel. Experiments showed that an egg yolk phosphatidylcholine SLB formed on a glass support moved in a rolling motion under these shear forces, with the lipids in the upper leaflet of the bilayer moving at twice the velocity of the bilayer front. The drift velocity of different lipid probes in the SLB was observed to be sensitive to the interactions between the lipid probe and the surrounding molecules, resulting in drift velocities that varied by up to 1 order of magnitude for the different lipid probes in our experiments. Since the method provides a so far unattainable control of the motion of all molecules in an SLB, we foresee great potential for this technique, alone or in combination with other methods, for studies of lipid bilayers and different membrane-associated molecules.

Multidirectional sorting modes in deterministic lateral displacement devices.

Deterministic lateral displacement (DLD) devices separate micrometer-scale particles in solution based on their size using a laminar microfluidic flow in an array of obstacles. We investigate array geometries with rational row-shift fractions in DLD devices by use of a simple model including both advection and diffusion. Our model predicts multidirectional sorting modes that could be experimentally tested in high-throughput DLD devices containing obstacles that are much smaller than the separation between obstacles.

Mechanical Behavior of a Supported Lipid Bilayer under External Shear Forces

Shear forces from a pressure-driven bulk flow in a microfluidic channel can be used to induce and control the motion of a supported lipid bilayer (SLB) formed on the walls of the channel. We here present a theoretical model that relates the experimentally observed drift velocities of an egg yolk phosphatidylcholine (egg PC) SLB to the hydrodynamic drag force from the bulk flow, the intermonolayer friction coefficient, b, of the bilayer, and the friction coefficient, bls, between the lower leaflet of the bilayer and the supporting substrate. The drift velocity and diffusivity of the lipids in the SLB were obtained by photobleaching a delimited area of fluorescently labeled lipids and subsequently monitoring the recovery and convective motion of the bleached spot. A striking observation was that the drift velocity of the lipids was observed to be nearly 6 orders of magnitude smaller than the bulk velocity at the center of the channel. This predicts a value for bls that is at least 25 times as high as predicted by the traditional model with the SLB and the support spaced by a homogeneous 1 nm thick film of water. In addition, the intermonolayer friction coefficient was estimated to 2x10(7) Pa s/m, a value that increased after addition of glycerol to the bulk solution. This increase was accompanied by an equal decrease in the lipid diffusivity, with both observations indicating an increased viscous drag within the bilayer when glycerol was added to the bulk solution.

Vertical oxide nanotubes connected by subsurface microchannels

AbstractWe describe the fabrication of arrays of oxide nanotubes using a combination of bottom up and top down nanofabrication. The nanotubes are made from epitaxially grown semiconductor nanowires that are covered with an oxide layer using atomic layer deposition. The tips of the oxide-covered nanowires are removed by argon sputtering and the exposed semiconductor core is then selectively etched, leaving a hollow oxide tube. We show that it is possible to create fluidic connections to the nanotubes by a combination of electron beam lithography to precisely define the nanotube positions and controlled wet under-etching. DNA transport is demonstrated in the microchannel. Cells were successfully cultured on the nanotube arrays, demonstrating compatibility with cell-biological applications. Our device opens up the possibility of injecting molecules into cells with both spatial and temporal control.

Sorting cells by their dynamical properties

Recent advances in cell sorting aim at the development of novel methods that are sensitive to various mechanical properties of cells. Microfluidic technologies have a great potential for cell sorting; however, the design of many micro-devices is based on theories developed for rigid spherical particles with size as a separation parameter. Clearly, most bioparticles are non-spherical and deformable and therefore exhibit a much more intricate behavior in fluid flow than rigid spheres. Here, we demonstrate the use of cells’ mechanical and dynamical properties as biomarkers for separation by employing a combination of mesoscale hydrodynamic simulations and microfluidic experiments. The dynamic behavior of red blood cells (RBCs) within deterministic lateral displacement (DLD) devices is investigated for different device geometries and viscosity contrasts between the intra-cellular fluid and suspending medium. We find that the viscosity contrast and associated cell dynamics clearly determine the RBC trajectory through a DLD device. Simulation results compare well to experiments and provide new insights into the physical mechanisms which govern the sorting of non-spherical and deformable cells in DLD devices. Finally, we discuss the implications of cell dynamics for sorting schemes based on properties other than cell size, such as mechanics and morphology.

Solute transport on the sub 100 ms scale across the lipid bilayer membrane of individual proteoliposomes

Screening assays designed to probe ligand and drug-candidate regulation of membrane proteins responsible for ion-translocation across the cell membrane are wide spread, while efficient means to screen membrane-protein facilitated transport of uncharged solutes are sparse. We report on a microfluidic-based system to monitor transport of uncharged solutes across the membrane of multiple (>100) individually resolved surface-immobilized liposomes. This was accomplished by rapidly switching (<10 ms) the solution above dye-containing liposomes immobilized on the floor of a microfluidic channel. With liposomes encapsulating the pH-sensitive dye carboxyfluorescein (CF), internal changes in pH induced by transport of a weak acid (acetic acid) could be measured at time scales down to 25 ms. The applicability of the set up to study biological transport reactions was demonstrated by examining the osmotic water permeability of human aquaporin (AQP5) reconstituted in proteoliposomes. In this case, the rate of osmotic-induced volume changes of individual proteoliposomes was time resolved by imaging the self quenching of encapsulated calcein in response to an osmotic gradient. Single-liposome analysis of both pure and AQP5-containing liposomes revealed a relatively large heterogeneity in osmotic permeability. Still, in the case of AQP5-containing liposomes, the single liposome data suggest that the membrane-protein incorporation efficiency depends on liposome size, with higher incorporation efficiency for larger liposomes. The benefit of low sample consumption and automated liquid handling is discussed in terms of pharmaceutical screening applications.