MicrofluidicsStella Foršček

Mentor: prof. Simon Širca

24.5.2016

Abstract:

In this seminar I am going to present the concept of microfluidics, some basic facts, relavantto it, and present the meaning of the Reynolds and Peclet numbers. Further on, I presenttypes of cell manipulation. At the end I am going to present an experiment involving ourknowledge of microfluidics, called PARTCELL.

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Table of contents2

1 Introduction 2

2 What is microfluidics? 3

3 Cell manipulation 53.1 Magnetic manipulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53.2 Optical manipulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63.3 Electrical manipulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73.4 Mechanical manipulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

4 Droplet formation 9

5 PARTCELL 10

6 Conclusion 12

1 IntroductionMicrofluidics is a quite new multidisciplinary field combining engineering, physics, chem-istry, biochemistry, nanotechnology and biotechnology. As its name itself suggests, it isdealing with systems with low volumes of fluids. The behaviour of those fluids can differfrom systems on a macro scale and can exhibit some unintuitive properties [1]. Knowledgeof microfluidics helps us provide some new devices and techniques for cell manipulationwhich is important especially for medical research (like lab-on-a-chip). Field of microflu-idics is very wide so in this seminar, I will focus more in biological part of it. But beforewe discuss real problems and experiments, let us introduce some basic properties of thisscientific discipline.

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2 What is microfluidics?In microfluidic experiments we usually have a channel in which we inject different streamsand observe their behaviour. To understand microfluidics we should first learn somethingabout dimensionless numbers which will help us understand some basic processes. Thefirst one we are going to discuss is the Reynolds number (Re) which represents the relationbetween inertial and viscous forces [1]. It is defined as

Re = ρU0L0

η, (2.1)

where ρ represents the density, U0 is the flow velocity, L0 is the length of the channel andη is the viscosity [1]. In microfluidics the Reynolds numbers are always very low, usuallymuch below 1. This means that our flows are not turbulent, as they are in the "everydayworld", but rather laminar. Low Reynolds numbers imply interesting phenomena, likewhen we have two fluids side-by-side they do not necessarily mix in the traditional sense.Mixing occurs only through diffusion, which means that it is much slower than on themacro scale [1].

The next important number we are going to observe is the Peclet number. Let us say thatwe have a so called T junction (Fig. 1) in which two fluids are injected from either side. Weobserve how long the fluids need to flow channel to become homogenized. The time neededfor the molecules to diffuse is given by τD = w2/D, where w is the width of the channeland D is diffusion. During this time, the stripe will move distance z = U0τD = U0w

2/D.The Peclet number is the ratio of length z and width w, so we get

Pe = z

w= U0w

D. (2.2)

The Peclet expresses the relative importance of convection with respect to diffusion [1].We can use this knowledge to separate flow ingredients.

The so called rotary Peclet number for active swimmers (like sperm cells) is defined as aratio between time scale D−1

Ω , corresponding to the reorientation of the swimmer due torotary diffusion and the other time scale G−1, which is characteristic time scale for thelaminar flow [2].

Pe = G/4DΩ. (2.3)

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Fig 1: Left: the T-junction. Two streams are brought together down a channel andthe streams diffuse into each other, forming an interdiffusion zone whose boundary wecan measure with a fluorescent marker. This device is used to measure concentrations,diffusivities and reaction kinetics. Right: the H-junction. It is a simple device that filtersparticles by size without a membrane. We have two different streams that are broughttogether to flow alongside each other down a channel. We have a solution with different-sized particles with different diffusivities and Peclet numbers. As we saw before, the Pecletnumber determines the length of the channel required for particles to diffuse across thechannel width. The H-filter works when we choose the length and flow velocity so thatfor one type of particles Pe is small and for the others Pe is large. Particles with smallPe diffuse and fill the channel before exiting it, while particles with large Pe stay on theirside of the channel [1]. Real experiment that shows us the use of H-filters is presented inFigure 2.

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Fig 2: A variant of the H filter that separates motile sperm cells from the nonmotile.Common H-filters rely on particle diffusivity differences and different Peclet number forseparation. But this device exploits the fact that motile sperm cells disperses across andhomogenizes along the channel much more rapidly than the nonmotile one, which spreadvia diffusion alone [1]. Peclet number for active swimmers (like sperm cells are) is definedas ratio between charateristic time scale for the linear flow and time scale correspondingto the orientation of the swimmer due to rotary diffusion [2].

3 Cell manipulationLet us introduce some methods that can be used to manipulate cells by means of magnetic,optical, mechanical and electrical effects.

3.1 Magnetic manipulationMagnetic manipulation is a method where we have magnetic particles attached to cells.This method is used for cell separation or purification in microfluidic devices [3]. The sizeof super-paramagnetic (ferromagnetic nano-particles) beads is 10-100 nm which makesthem small enough to be gentle on the cells and with no bigger effect on the cellularfunction and viability [3]. If we impose magnetic field gradients we can capture the beadand thus the cell attached to it. That method is very useful in handling, for exapmle, redblood cells (their size is about 6-8 µm). In Fig. 3 we see the schematic of the microdevice.At the bottom of the chamber we have ferromagnetic wires at a 45 angle with respect to

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the flow of the cells. Unlabelled cells will flow in the central outlet and those which areattached to super-paramagnetic beads, will feel the magnetic force and their path will bedifferent [3].

Fig 3: A: the schematic of the microfabricated cell fractionation chip; inset: magneticwires embedded in the floor of the central chamber (SEM image). B: the idea of magneticforce separation. High magnetic field gradients provide forces at an angle to the flow ofcells and change the path of the cell which would otherwise follow hydrodynamic flow (inimage called hydrodynamic force) [3].

3.2 Optical manipulationOptical manipulation is useful due to its non-contact and contamination-free manipulationcapability. The most commonly used optical manipulation tool is the optical tweezer whichcaptures cells in the focal point of an electric field gradient. Optical tweezers offer highresolution for single cells trapping, but have limited manipulation area owing to tightfocusing requirements [3]. Flynn et al. developed a so called VCSEL (vertical cavitysurface emitting laser), where each laser in the array is focused and acts as an individualtrapping and manipulation source (Fig. 4).

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Fig 4: VCSEL array optical tweezers for the parallel transport of samples on a chip. Everytweezer is used to manipulate single particle [3].

3.3 Electrical manipulationDielectrophoresis (DEP) is the electric analog of optical tweezers. The standard way tomake a DEP trap is to create an electric field gradient with an arrangement of planarmetallic electrodes (Fig. 5) either directly, connected to a voltage source, or free-floatingin presence of an AC field. The force pulls particles with higher permittivity than sur-roundings in the direction of increasing electrical filed (so called positive dielectrophore-sis) and the other way round for particles with smaller permittivity than surroundings(so called negative dielectrophoresis). Advantage of DEP is that we can manipulate verysmall objects, such as cells, DNA, proteins and nanoparticles [3].

Fig 5: (A) A metallic DEP trap made of microfabricated wires (red rectangle) on asubstrate. The wires may be either free-floating or connected to a voltage source. Blackarrows show the electric field lines E [3].

3.4 Mechanical manipulationWe can also separate cells within microchannels by fabricating constriction structures suchas microfilters, microwells, microgripper and dam structure (Fig. 6). Microfilters can beused, for example, to separate cultured neuroblastoma cells when mixed with blood, toseparate the blood cells from serum in real-time. A microgripper can be used to manipulate

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single cells in physiological ionic solutions and hold a cell without exerting any forces inthe closed position. A dam structure is composed of two parallel channels separated by adam, where the height of the dam is smaller than the vertical height of the channels. Thestructure allows the cells to move in microfluidic channels and dock in desired locations.Cells docked on the parallel dam structure are exposed to minimal stress caused by fluidicpressure [3].

Fig 6: (A) SEM image of microfilters showing the transition from the 15 µm wide channelsto the 10 µm wide channels. (B) SEM image of a microwell structure. (C) SEM image ofa microgripper. (D) Dam structure and channels [3].

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4 Droplet formationMicrofluidic devices can be used to create controllable droplet emulsions in immisciblefluids, by injecting water into a stream of oil at a T-junction [5],[6]. If there would be nointerfacial tension between oil and water, the streams would flow alongside one anotheras we described before for T-sensors. But for water and oil the surface-tension stressestend to reduce the interface area, and viscous stresses act to extend and drag the interfacedownstream. These stresses destabilize streams and form droplets of radius R [5] (Fig. 7).

Fig 7: Schematic presentation of a T-junction (left). From one inlet we inject oil andfrom another we inject water. The interface between the fluids is driven by two stresses.Viscous shear stresses tend to extend and drag the interface and surface tension tends toreduce the interfacial area. The competition between these two types of stresses leads toformation of droplets of radius R [5].

If we want to know the size of the droplets formed in this manner, we can estimate itby balancing the two stresses at the interface [5]. Capillary stresses of magnitude γ/Rbalance the viscous stresses ηU0/h (where γ is the surface tension, η is the viscosity andh is the channel hight) and give us characteristic droplet size

R ∼ γ/ηU0h = h/Ca,

where Ca is the capillary number Ca = ηU0/γ. It is a dimensionless number used wheneverwe wish to quantify the relative importance of viscous and interfacial stresses [5].

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5 PARTCELLThere are many experiment in wich it would be useful to treat a part of the cell with abiochemical reagent. Takayama et al. developed a technique called PARTCELL. PART-CELL is a method using laminar flows for partial treatment of cells. Flows can deliver andremove small-molecule reagents to cells with subcellular resolution inside closed channelsystem [7]. In PARTCELL we combine flows from multiple inlets. Because of small chan-nels and slow flow (U0 = 0.6cm/s) the Re is small (Re<1) so there is no mixing amongthose flows but diffusion. In PARTCELL, the interface between the liquid streams is po-sitioned over a single living cell (tipical size of cell about 5-100 µm) to expose differentregions of the cell to different streams [7]. Such localized exposure allows treatment ofsubcellular domains of the cell.

In experiment they have used a capillary system with three inlet channels that convergeinto a single main channel (Fig. 8). From the three inlets we get parallel streams ofdifferent liquids. The width and the shape of the interface depends on flow velocity,channel geometry, wall interactions and diffusion coefficient. In the presented experimentthe interface between the liquid streams is typically 1-10 µm in width in the region wherethe cells are treated (50-300 µm from the point of convergence) [7]. By adjusting therelative amounts of fluid flowing from each inlet we control the widths of the streamsand the position of the interface between adjacent streams. In the experiment we have amammalian cell over which flow two different streams (we have two inlets with mediumand one inlet with MGFM solution, which is membrane-permeable) (Fig. 8). By streamingdifferent solutions containing fluorescent tags over an endothelial cell, for example, theywere able to maintain selective labelling of subpopulations of mitochondria in differentregions of the cell. Mitotracker Green FM was injected in the solution over extendedperiods of time to study mitochondria labelling in more detail [7]. Pictures show usdifferent extent of labelling after 5, 11 and 35 min of runing PARTCELL (Fig. 9).

Fig 8: Three inlets used in an experiment involving parallel flow over a mammalian cell.In one inlet we have Mitotracker Green FM solution (green arrow), while the other twoinlets are filled with medium [7].

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Fig 9: The results of PARTCELL. Mitotracker Green FM solution flowing over the leftpart of the cell and medium flow over the right part of the cell for 5, 11 and 35 min ofrunning PARTCELL. The white dotted lines indicate the position of the interface betweenthe flows with and without Mitotracker Green. Mitotracker diffused to different extentsinto the cell beyond the boundary defined by the fluid streams with time [7].

Another experiment using PARTCELL is seen on Figure 10. In one part of inlet we havetrypsin-mediated stream over a cell (trypsin is a protease which hydrolyses proteins). Wecan see that the cell detached from the substrate in the part where trypsin was presentand the untreated region was not affected. Because of cell reorganisation the nucleus andmitochondria moved to the right side of the cell (Fig. 10).

Fig 10: The result of PARTCELL in trypsin treatment. Left image is the cell beforetrypsin stream was injected. White dotted line indicates position of the interface betweendifferent streams (left side with trypsin and right side without trypsin). Second imageshow cell organisation after 1 min of trypsin treatment and third one after 3.5 min. Whitearrow shows position of nucleus. We can see that nucleus shifted to the right as the leftpart of the cell detached [7].

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6 ConclusionIn this seminar I could only explain few basic themes presenting such a wide field asmicrofluidic. I focused on some basic processes which make the field different and specialand tried to show how wide the impact of microfluidics is not only on manipulating cellsand understanding biological processes in cells, DNA, etc. but also on fabricating toolsfor their manipulation. To show the width of field, the seminar lost some more focusedfeatures, so I also presented experiment-PARTCELL-to touch one theme in more detailand to show where can we use knowledge of microfluidics.

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