CONTINUOUS SIZE-BASED SEPARATION OF MICROPARTICLES IN STRAIGHT CHANNELS

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2 CONTINUOUS SIZE-BASED SEPARATION OF MICROPARTICLES IN STRAIGHT CHANNELS A thesis submitted to the Division of Research and Advanced Studies of the University of Cincinnati in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE in the School of Electronics and Computing Systems of the College of Engineering and Applied Science 2011 By Taher Kagalwala B.E. (Hons), University of Mumbai, India, 2007 Committee Chair: Ian Papautsky, Ph.D.

3 ABSTRACT This work describes the use of a rectangular straight microchannel for separation of microparticles with a diameter difference of as little as 2 μm. Inertial microfluidics is exploited to achieve separation of particles. Inertial lift forces acting on particles in a straight channel causes them to equilibrate at a position near the channel sidewall. The inertial force is dependent on the particle size which facilitates in the formation of separate streams of particles. With a proper design for the outlet system, these particle streams can be collected to achieve a complete separation. This principle was demonstrated by fabricating straight channels in PDMS using soft lithography. The focusing region was 15 μm x 50 μm (W x H) with an opening angle of 28º connecting to the separation region. The separation region was a low aspect ratio segment with the dimension of 1200 μm x 50 μm which connected to a 5 outlet collection region. This device was used to demonstrate the separation of 2 μm and 4.16 μm diameter particles from a homogeneous mixture. Separation efficiency of 75~85% was achieved. This simple, planar device can be easily integrated with any lab-on-a-chip system for particle separation.

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5 ACKNOWLEDGEMENTS I would like to express my sincere gratitude and appreciation towards my advisor, Dr. Ian Papautsky, for his guidance for the duration of this work. His patience and constant motivation has made it an enjoyable and memorable experience. I would like to thank Jeff Simkins and Ron Flenniken for their help with the clean room processing. I would also like to extend my sincere regards to the members of the BioMicroSystems Lab for their continuous support and friendship. I would like to specially thank Dr. Ali Asgar Bhagat for mentoring me in different aspects of the project. It was a great pleasure working with my fellow lab members, especially Preetha Jothimuthu, Arpita Chatterjee, Nivedita, Jian Zhou, and Srinidi Kuntaegowdanahalli. Many thanks go to all my friends in Cincinnati, Neha, Nikhil, Bala, Ghazal, Kovid, Aseem, Suraush, Bhargava, Madhu for being supportive of my efforts during this work. Last but not the least, I would like to thank my family, parents Aziza and Enayat Kagalwala, brothers Murtuza, Mohammed and Husein for their unconditional love, for being supportive and encouraging and for always being there for me. I wish to dedicate this work to them.

6 TABLE OF CONTENTS LIST OF FIGURES...vi LIST OF TABLES...viii CHAPTER 1 INTRODUCTION...1 Design principle...4 Chapter summaries...7 CHAPTER 2 EXPERIMENTAL METHODS...8 CHAPTER 3 RESULTS AND DISCUSSION...14 Validation of particle focusing in straight microchannels...14 Effects of channel expansion...16 Separation of 2 μm and 4.16 μm diameter particles...18 Design criteria for particle separation in straight microchannels...26 CHAPTER 4 CONCLUSIONS...31 REFERENCES...33 v

7 LIST OF FIGURES Figure Page 1. (a) Inertial lift forces acting on particles in straight channel. (b) Cross-section view of the inlet and outlet of a rectangular microchannel illustrating particle focusing. (c) Schematic of a straight channel microfluidic device illustrating downstream progression of particle focusing Schematic of the soft lithography fabrication process Cross-section view of the focusing channel region. The dimension of the focusing region is 15 μm x 50 μm (W x H) Photograph of a straight separator fabricated in PDMS Setup used to perform the experiments Close up of the straight channel device with the inlet and outlet tubes attached (a) Illustration showing that as the particle size increases, the distance from sidewall also increases. (b) Normalized position from channel sidewall as a function of particle size. In the y-axis, x/w indicates the ratio of distance of the focused stream from the sidewall (x) to the total width (W) of the separation region channel Normalized position from channel sidewall as a function of particle size for various opening angles. Inset depicts the opening angle which was varied (a) Composite florescent images illustrating randomly distributed particles at the inlet and focused streams at outlet of a 15μm x 50μm rectangular microchannel. (b) Intensity line scan shows distinct peaks for 2μm and 4.16μm diameter particles (a) Composite florescent images illustrating focused positions of 2 μm and 4.16 μm particles for different outlet openings. (b) Graph indicating that as the outlet width increases, the difference between the focusing positions of different particles also increases (a) Separation of particles in a curved outlet design. (b) Separation of particles in trifurcating outlet design. (c) Separation of particles in a 5-outlet design. The 2 μm diameter particles are plum purple and the 4.16 μm diameter particles are dragon green in color Photograph of the outlet section of the selected design. Fabrication using SU becomes difficult when the outlet is 800 μm in width Graph illustrating the ratio of focused width to particle size v/s the particle size. When the y-axis value equals to 1 then the particle stream is completely focused vi

8 14. (a) Composite image of the inlet with 2 μm and 4.16 μm diameter particles. (b) Composite image of the outlet section showing separation of 2 and 4.16 μm diameter particles. Image was stitched after taking images of the upper and lower section of the outlet. Difference in the symmetry of focused streams, in the upper and lower section, is attributed to flow variations that may have occurred while collecting individual images (which were stitched later) at different time Results of particle counting using hemocytometer. (a) Majority of 2 μm diameter particles tend to flow through Outlet1. (b) Majority of 4.16 μm diameter particles tend to flow through Outlet Normalized position of particles ranging from 2 μm to 10 μm in diameter, in a 1000 μm wide separation region vii

9 LIST OF TABLES Table Page 1. Summary of particles used in this work Design rules for particle separation...29 viii

10 CHAPTER 1 INTRODUCTION Microfluidic particle separation and filtration techniques have proven their benefits in the biomedical, environmental and pharmaceutical fields [1-5]. Amongst the various modules in a lab-on-a-chip (LOC) system, the separation modules are usually located after synthesis and before analysis processes [1]. The applications of separation techniques are many which includes preparation of biological microassays and microchemical processing [1]. Microfluidic separators can be used in water purification for prevention of health and environmental issues. Biochemical analysis requires separation of particles, cell or DNA molecules, proteins and smaller molecules [2]. Microfluidic separation techniques have been recently reviewed by a number of investigators [1-5]. At a glance, separation techniques can be broadly classified as active and passive techniques. Active techniques make use of external forces like electric or magnetic field to manipulate the sample. These techniques include dielectrophoresis [7, 8], acoustophoresis [9], flow induced electro-kinetic trapping (FIET) [10], magnetophoresis [11] and SPLITTfractionation [12]. Most of these techniques work in batch mode where only a finite volume of the sample undergoes separation at a given time. This yields low throughput for applications which require a large volume processing or a continuous operation. Also, the throughput for particle manipulation faces limitation as the reaction time of the external forces will reduce with increase in flow rate [13]. Macroscale techniques like fluorescence-activated cell sorting 1

11 (FACS) and magnetic-activated cell sorting (MACS) have been proven as effective techniques for separation with high throughput and high efficiency, but require skilled operators and require fluorescent or magnetic labeling, and thus can be cost prohibitive. Also, the use of sheath flows may exert significant sheer stress on cells under concern [5]. On the other hand, the passive techniques make use of particle properties, channel geometry or obstacles in the flow path to achieve separation. These techniques include pinched flow fractionation [14, 15], deterministic lateral displacement [16], crossflow filtration [17] and hydrophoretic separation [18]. Passive techniques have continuous operation and hence usually have higher throughput as compared to active methods. Also these techniques do not use external force fields, thus reducing the device cost. Continuous flow separation has many advantages compared to batch processing techniques. These advantages include 1) continuous processing of sample, 2) continuous readout of separation efficiency, 3) lateral separation of sample components, which enables collection of different samples in various outlets to facilitate further processing/analysis downstream, 4) potential for integration, in lab-on-a-chip (LOC) systems, and 5) in most cases, label-free, which makes the system more cost effective [2]. Some of the disadvantages of continuous flow separators are that they may be less accurate as compared to their counterparts. Also, most of the times the sample is diluted by orders of magnitude and hence there is a lot of waste fluid generated at the output. The sample might also be required to be re-concentrated. Inertial microfluidics has gained a lot of interest recently for filtration and separation applications. These devices make use of hydrodynamic forces acting on particles or cells to achieve positioning within the fluid flow necessary for separation [13, 19-26]. Important parameters which these forces are dependent upon are channel dimensions and aspect ratio, 2

12 diameter of the particle or cell, and the flow rate. It is advantageous to use inertial migration for particle and cell separation and filtration because 1) inertial lift forces are directly proportional to the power of particle size, and 2) higher flow rates can be applied which proves advantageous in most separation and filtration applications. Such microfluidic devices can find applications in separation/filtration without membranes in industrial and chemical processes, waste-water treatment and emulsion processing [27]. Recently it was reported that particles can be filtered (concentrated) from a mixture using inertial microfluidics in straight microchannels [20, 21]. Spiral [19, 22] and asymmetric serpentine [24] channels can be used to modulate Dean forces for complete separation. This approach can also be used for high-throughput cell separation and counting (sheathless flow cytometry) [23]. Kuntaegowdanahalli et al. [19] showed that spiral channels can be used to simultaneously and continuously separate 10 μm, 15 μm, and 20 μm diameter polystyrene particles as well as 10 μm and 20 μm cells. The device achieved 80% separation efficiency and a throughput of ~1 million cells/min. Wu et al. [28] demonstrated separation of bacteria from blood using soft inertial microfluidics. But this system has low throughput due to the low (18 μl/min) flow rate. Also, it is difficult to parallelize these devices due to their system design. Alternatively, straight channels have been used for high throughput filtration of bacteria from blood [25]. With 40 single microchannels placed radially to achieve a throughput of 400 million cells/min. In this work, separation of microparticles is demonstrated using the concept of inertial microfluidics in a straight rectangular microchannel. Previously, straight channels have only been used in filtration of particles [20, 21, 25], but not for complete separation. Particle/cell separation using inertial microfluidics have been demonstrated using various designs [19, 28], 3

13 but the size difference between the particles/cells have been 5 μm. Here, for the first time is shown that particles with size difference as little as 2 µm can be separated by taking advantage of inertial focusing and diverging outlets. Particle sizes of 2 μm and 4.16 μm were used for demonstrating separation because typical bacteria size is approximately 2 μm. This technique can be used to separate particles of any size difference, but to demonstrate the minimum possible size difference between two particles mixture, 4.16 μm particles were chosen as the other primary size under consideration. Such size-based separators are label free and can be integrated in LOC systems. Such LOCs can find many applications in removal of pathogenic bacteria or fungi from blood and other fluids, or in cell sorting and cell engineering. Design principle The basic concept was first described by former group members, Bhagat et al. [20, 21]. Particles or cells flowing in rectangular microchannel experience viscous drag force and inertial lift forces. The viscous drag force helps in the movement of the particles along the flow stream. Inertial lift forces are responsible for the lateral motion of the particles across the flow direction. Particles experience two types to lift forces, namely the shear induced inertial lift force (F SIL ) and the wall induced inertial lift force (F WIL ), as shown in Figure 1(a). The shear induced lift force is produced due to the parabolic nature of Poiseuille flow. The particles are forced towards the channel sidewalls as though they are rolling down the parabola. The wall induced lift force is produced due to the asymmetric wake around the particle when it approaches the channel side wall. The particles will equilibrate at a position near the channel side wall when F SIL and F WIL balance each other [21]. This principle was first shown by Segre and Silberberg [29] in circular tube where uniformly distributed particles migrated to form bands near channel sidewall. 4

14 Figure 1. (a) Inertial lift forces acting on particles in straight channel. (b) Cross-section view of the inlet and outlet of a rectangular microchannel illustrating particle focusing. (c) Schematic of a straight channel microfluidic device illustrating downstream progression of particle focusing. 5

15 The viscous drag force (F D ) acting on a particle is expressed using Stoke s law, where, is the fluid viscosity, a p is the particle diameter and U f is the average flow velocity. The net lift force acting on a particle was given by Asmolov [30] and is written as where, is the fluid density, G is the fluid shear rate and is given by G = 2U f /D h in which D h is the hydraulic diameter of a channel (D h = 4 x Area/Perimeter) and C L is the lift coefficient which is approximately equal to 0.5 when the Reynolds number (Re) <100 [30]. The simplified net lift can be stated as, In the case of a square channel, the characteristic length scale is its channel width or height which is same as the hydraulic diameter. However, in case of a rectangular channel, the characteristics length scale is the narrowest channel dimension (L C ). The lift force in a high aspect ratio channel is larger in magnitude along L C due to varying shear rates which leads to faster lateral migration. Thus by using high aspect ratio channels, focusing of particles can be achieved at low Re and shorter channel lengths [21]. Particles equilibrate only when the particle diameter (a p ) and the critical channel dimension (L C ) satisfy the condition of a p /L C 0.07 [21]. The required channel length is related to the a p /L C as, This relation shows that with a minor change in the particle diameter, the required channel length for particle focusing will change drastically. Figure 1(b) illustrates cross-section view of the 6

16 particle focusing after travelling through a rectangular microchannel. The particles will equilibrate along the longer side walls when 20 Re 100 [20]. Figure 1(c) illustrates downstream particle focusing in straight-channel separator. The focusing region is 15μm x 50μm (W x H) and the separation region is 1200μm x 50μm. The particles flow through the focusing region, where they experience inertial forces and equilibrate at stable positions near the channel sidewalls. Particles of different size will focus at different planes with respect to the sidewalls. Smaller particles focus closer to the sidewall as compared to the larger particles [31]. After the focusing region, the particles flow through the separation region, where the distance between the 2 particle streams is enlarged due to the expansion in width. With properly designed outlet system, the different particle streams can be collected in separate outlets to achieve separation. Separation between particles with the diameter difference of only 2 μm is possible with this design. Chapter summaries Following this introduction, a detailed description of the experimental methods and the characterization techniques has been presented in chapter 2. Chapter 3 presents the experimental results and discussion. A brief summary of the work and final conclusions are presented in chapter 4. 7

17 CHAPTER 2 EXPERIMENTAL METHODS In this work, rectangular microchannels were fabricated in polydimethylsiloxane (PDMS, Sylgard 184, Dow Corning) using standard soft lithography methods. Figure 2 shows a schematic of the fabrication process for soft lithography. Briefly, a negative silicon master was fabricated using SU-8 photoresist (2075, Microchem Corp.). The required height was attained by following the conventional photolithography process [32]. A 3 inch silicon wafer was first cleaned with acetone, methanol and IPA, and then rinsed with DI water. This was followed by dipping the wafer for 30 s in buffered oxide etchant (BOE, 3:3:1 (v/v/v), H 2 O:NH 4 F:HF) to remove any native oxide from the surface. The wafer was once again rinsed with DI water for approximately 1 min, and blown dry using N 2. The wafers were dehydrated on a hot plate at 150ºC for 20 min. After cooling down to room temperature, the wafers were spin coated with SU at 2500rpm, held for 45 s. After spin coating they were kept on a leveled surface to settle and relax for 15 min. Next, wafers were kept on leveled hot plate for soft bake at 65ºC for 5 min and then ramped to 95ºC at the rate of ~5ºC/min for 15 min. Again, the wafers were allowed to cool down to room temperature. Using an I-line (365nm) high pass filter, the soft baked resist was patterned by exposure energy of 175 mj/cm 2 (5mW/cm 2 for 35 s). Glycerin was used between the wafer and the mask to reduce any diffraction effects due to surface roughness. The wafers were rinsed with DI water to remove the glycerin and then kept on the 8

18 Figure 2. Schematic of the soft lithography fabrication process. leveled hot plate for post exposure bake. The hot plate was held at 65ºC for 5 min and then ramped to 95ºC (~5ºC/min) and held for 15 min. After the wafers cooled down to room temperature, they were placed in the SU-8 developer (MicroChem Corp.) for 2 hours with continuous stirring. Following development, wafers were rinsed with DI water and blown dry with N 2. This was followed by de-scumming in O 2 plasma (13.56 MHz, 20 sccm, 300W, 3 min) to clean the surface of any residual photoresist. After the silicon master was ready, it was coated with Sigmacote (Sigma-Aldrich Co.) for a single time, to facilitate easy removal of PDMS mold from the master. The PDMS polymer was mixed with the curing agent in the ratio of 10:1 by weight. This mixture was cast over the master and was kept for de-gassing followed by curing at 80 C for 2 h. The cured PDMS mold 9

19 was peeled off and the input-output ports were made using a 14 gauge syringe needle. This PDMS mold was then bonded with a 1mm thick microscope glass slide by using oxygen plasma produced by corona wand (BD-20AC, Electro-Technic Products Inc.). The fabricated devices consisted of straight rectangular microchannels with single inlet and different outlet systems. The entire channel was divided in two parts, namely the focusing region and the separation region. The focusing region consisted of a high aspect ratio rectangular channel of dimensions 15 μm x 50 μm (W x H). The cross-section image of the focusing region is shown in Figure 3. The peeled PDMS layer from the SU-8 substrate was sliced in the z-direction and the image was captured using the inverted microscope at 40x magnification. The channel width of 15 μm was selected to facilitate the focusing of 2 μm and 4.16 μm diameter particles. Also, high aspect ratio channels cause the focusing of particles along the channel side walls. Hence a height of 50 μm was selected which would give an aspect ratio > 3. The separation region was primarily the outlet section which expanded from the focusing region. The expansion magnified the distance between the particle streams and eventually helped in separation via properly designed outlet system (Figure 1(c)). Various experiments were performed for outlet widths varying from 100 μm to 1500 μm, with the height constant at 50 μm. This was done to verify the eventual position of the particle stream in the separation region so that the outlet section could be appropriately designed for the respective separation section width. Different outlet designs were tested to determine the best geometry for collecting individual particle streams. These experiments were performed to determine the best channel configuration to separate 2 μm and 4.16 μm particles. The reason of selection of these particle 10

20 Figure 3. Cross-section view of the focusing channel region. The dimension of the focusing region is 15 μm x 50 μm (W x H). sizes is explained in chapter 3. The channel ended with a 5-outlet system as shown in Figure 4. Separation experiments were performed using polystyrene particles ranging from 1.0 μm to 7.32 μm in diameter (Bangs Labs Inc. and Polysciences Inc.), labeled with DAPI, FITC, and TRITC fluorophores, as shown in Table 1. Particles were diluted in DI water (~0.05% volume fraction) and introduced using a 5cc syringe driven with a syringe pump (KDS101, KD Scientific Inc.). Tween-20 (Fisher Scientific) was used (0.1% volume fraction) to alleviate particle sticking to the sidewalls. Flow rates in the range of μl/min were used. Particles can easily focus in this range of flow rates. If the flow rate is increased any further, the pressure drop across the channel will increase which could cause leakages. Fluorescent images were captured with a high-speed 12-bit CCD camera (RetigaEXi, QImaging) attached to an inverted epi-flourescence microscope (Olympus IX71). The captured images were stacked using ImageJ, to form a composite (100 images each) indicating particle stream position within the microchannel. Figure 5 shows the setup used to perform the experiments, while Figure 6 illustrates a close-up of the device under test. 11

21 Table 1. Summary of particles used in this work. Particle diameter (µm) Std Dev (µm) Excitation (nm) Emission (nm) Fluorophore 1 NA FITC DAPI FITC FITC FITC FITC TRITC Outlet Inlet Separation region Focusing region Figure 4. Photograph of a straight separator fabricated in PDMS. 12

22 Figure 5. Setup used to perform the experiments. Figure 6. Close up of the straight channel device with the inlet and outlet tubes attached. 13

23 CHAPTER 3 RESULTS AND DISCUSSION This chapter presents the experimental results of this work. The first section deals with the validation of particle focusing in straight microchannels. In the second section, the effects of channel expansion will be discussed. The third section will present the result for separation of 2 μm and 4.16 μm particles. The final section will summarize the overall results and present the design criteria for particle separation using straight microchannels. Validation of particle focusing in straight microchannels Inertial lift forces acting on different sized particles were exploited to demonstarte separation of 2 μm and 4.16 μm diameter particles. The particles of size varying from 1 μm to 7.32 μm were used in the experiments. Individual particle experiments were performed before performing particle mixture experiments. Each particle size was tested in each outlet system to determine the relationship of the focusing distance from the channel sidewalls. Figure 7(a) shows that as the particle size increases, in an 800 μm wide outlet section, the distance of the focused stream from the channel sidewall increases. Figure 7(b) contains the corresponding graph showing the normalized position of the various particles in the 800 μm outlet width. It is observed that the various sized particles focus at approximately around the 0.2W distance as reported previously [33]. As seen in Figure 7(a), 1 μm particles are not completely focused 14

24 x/w Particle size (μm) Figure 7. (a) Illustration showing that as the particle size increases, the distance from sidewall also increases. (b) Normalized position from channel sidewall as a function of particle size. In the y-axis, x/w indicates the ratio of distance of the focused stream from the sidewall (x) to the total width (W) of the separation region channel. 15

25 whereas the 2 μm particles are focused. To get a focused stream for 1 μm particles, either the flow velocity is required to be increased (F L U 2 f ) which would increase the lift force experienced by the particles or the channel length is required to be increased (L a -3 p ) to focus smaller particles. Increasing the flow velocity would increase the pressure drop for the channel and increasing the channel length would increase the risk of channel clogging due to larger particles. Hence the smallest particle size, which could be considered for separation using a 15 μm wide focusing section, was chosen to be 2 μm. These sized particles are of interest because typically the size of many bacteria species is ~2 μm. The aim of this work was to demonstrate the separation of 2 μm and 4.16 μm particles. The channel length of the focusing region was chosen as 1 cm. This would prove advantageous in saving real estate on a Lab-on-a-chip system. Flow rate of 80 μl/min was selected to ensure the focusing of the smallest particle in the set of experiments. Effects of channel expansion Experiments were performed to check the relevance of the opening angle for the channel when the particles transition from the focusing region to the separation region. The channel dimensions of the focusing region were kept constant at 15 μm x 50 μm for all experiments. The channel dimensions for the separation region were also kept constant at 400 μm x 50 μm. Experiments were performed for the opening angle ( º) having values of 20º, 28º and 60º. The opening angle is depicted in the inset of Figure 8. As seen in Figure 8 there is not much variation in the particle position with change in opening angle. It can be seen that as the opening angle increases the particles tend to equilibrate at positions much further away from the channel side walls. 16

26 x/w Particle Size (μm) 400um 20degrees 400um 28 degrees 400um 60degrees Figure 8. Normalized position from channel sidewall as a function of particle size for various opening angles. Inset depicts the opening angle which was varied. The particles travel from a high aspect ratio channel (H/W > 1) to a low aspect ratio channel (H/W < 1). The particles are completely focused in two-single streams, near the channel sidewalls, while entering the low aspect ratio channel. For a low aspect ratio channel, randomly distributed particles would ideally equilibrate along the top and bottom of the channel [20, 21]. But since they are already focused they equilibrate at the positions near the channel sidewall. With a gradually opening outlet the particles tend to keep close to the sidewall as the velocity profile is slightly blunted at the center. This creates a higher shear induced lift force near the sidewalls which in turn moves the particles closer to the sidewalls. Comparatively for an abrupt opening outlet, the particles tend to equilibrate closer to the center as the velocity profile remains parabolic and also due to the reduction in the lateral migration velocity of the particles. This 17

27 effect can be exploited to conveniently filter out two-single streams of particles through a trifurcating outlet system [20, 21]. But in this work however, this effect is not important as the aim is to separate two particle streams from each other. The distance between the two particle streams will remain constant whether the opening is gradual or abrupt. Hence the opening angle of 28º was selected primarily for the reason of convenient geometry for the channel design. Separation of 2 µm and 4.16 µm diameter particles Figure 9 illustrates that a homogeneous mixture of randomly distributed particles at an inlet can be focused in two distinct streams at the 400 μm wide outlet using a 2 cm long straight channel. Florescent intensity line scan across channel shows sharply derived particle stream (Figure 9(b)). The lift force experienced by particles varies with particle diameter (F L a 4 p ). It can be seen that as the particle diameter increases, the particles focus at a distance further from the sidewalls. This variation in the distance is negligible in a 15 μm wide channel. But the effect gets magnified when the focusing section is expanded to a wider outlet. This can be clearly seen in Figure 9(a) where 2 μm and 4.16 μm particles form visibly separate streams. Figure 10(a) shows composite images indicating positions of 2 μm and 4.16 μm diameter particles in channels of varying outlet widths from 100 μm to 1500 μm. It is observed that as the width of the outlet section increases the distance between the two particle streams also increases. From a difference of 2 μm between the two particle streams in a 100 μm outlet, there is an increase in the difference to 35 μm in a 1500 μm outlet. An approximate linear relationship is observed between the separation distance and the outlet section width. This phenomenon is important to be taken under consideration while designing the outlet system. Using a 100 μm 18

28 Intensity (a.u.) Microchannel width (µm) Figure 9. (a) Composite florescent images illustrating randomly distributed particles at the inlet and focused streams at outlet of a 15μm x 50μm rectangular microchannel. (b) Intensity line scan shows distinct peaks for 2μm and 4.16μm diameter particles. 19

29 wide outlet system is not feasible as the separation distance between the two particle streams is negligible. Ideally an outlet system with very large width is preferred in which the separation distance between the two particle streams would be large. Figure 10(b) illustrates the graph showing the relationship of the particle positions relative to the channel sidewall and the outlet width. 100μm μm μm 1200μm 800μm Position (μm) Outlet width (μm) Figure 10. (a) Composite florescent images illustrating focused positions of 2 μm and 4.16 μm particles for different outlet openings. (b) Graph indicating that as the outlet width increases, the difference between the focusing positions of different particles also increases. Various channel outlet designs were explored using a 1500 μm wide separation region. This was done once the individual particle focusing positions were determined. Using the previously collected data the outlet system was designed in such a way that an obstruction was placed at a position between the two particle streams. Due to the obstruction the individual particle streams could be collected at various outlets to achieve separation. 20

30 Figure 11. (a) Separation of particles in a curved outlet design. (b) Separation of particles in trifurcating outlet design. (c) Separation of particles in a 5-outlet design. The 2 μm diameter particles are plum purple and the 4.16 μm diameter particles are dragon green in color. 21

31 Figure 11 shows the final 3 designs, including the curved and the trifurcating outlet designs. These designs worked well for separation of 2 μm and 4.16 μm diameter particles, but due to particle defocusing, the streams widen. Due to this the outlet needs to be precisely designed. Figure 11(c) shows a similar outlet system as used by Kuntaegowdanahalli et al. [19]. In this design the outlet is divided into 5 segments. The segments near the channel side wall will collect the particles which focus closer to the sidewall i.e. 2 μm diameter particles. Subsequently the 2 nd and 4 th segment are used to collect the particles which are further away from the channel sidewall i.e μm diameter particles. The center segment is present to have equal pressure drops at individual segments so that the particle flow is not biased. It was seen that this design gave the best results for separation and hence it was chosen as the final design for the outlet system. Selection of proper dimensions for the separation region was an important criterion to get complete separation between 2 μm and 4.16 μm diameter particles. Using a 400 μm wide outlet system is not advisable. This is because the separation distance between the two particle streams is approximately 8 μm. This distance is too small to create a proper outlet system using SU The minimum feature size recommended for SU is around 15 μm when channel aspect ratio is high (H/W > 3). When using an 800 μm wide outlet the separation between 2 μm and 4.16 μm diameter particle streams is ~16 μm. To allow minor variations in the particle stream positions, a separating segment of 10 μm was placed in between the predicted stream positions. As shown in Figure 12, the SU structures collapsed and thus will cause mixing of the two particle streams at the outlet (not shown in the figure). Particle separation was achieved by using the 1500 μm wide outlet. But the 2 μm diameter particles get defocused due to the reduction of Reynolds number when entering the big outlet region. Now since Re U f 22

32 and F L U 2 f, a reduction in Re will decrease the inertial lift force acting on the particle. Thus, even though having a 1500 μm outlet region would make the design and fabrication process much simpler, it will not achieve complete separation between the 2 μm and 4.16 μm diameter particles. Figure 12. Photograph of the outlet section of the selected design. Fabrication using SU becomes difficult when the outlet is 800 μm in width. The amount of defocusing can be judged by looking at the graph in Figure 13. The y-axis is represented by the ratio of focused width to particle size and the x-axis is representing the particle size. Particles are considered to be perfectly focused when the stream width equals the particle size, i.e. y-axis value is 1. Thus it can be seen that 1 μm particles are never fully focused in any of the outlet systems, whereas the 7.32 μm particles are always focused. It can also be seen that the particles ranging from 1 μm to 4.16 μm lose focusing when the outlet is 1500 μm. When working with 1200 μm outlet 2 μm and 4.16 μm particles are reasonably focused and can be separated from each other. Thus 1200 μm was chosen as the final outlet width which was a compromise between ease in fabrication and not much lose in focusing of particles. Figure 14(a) shows a homogeneous mixture of 2 μm and 4.16 μm diameter particles at the inlet before they enter the 15 μm x 50 μm focusing region. A flow rate of 35 μl/min was 23

33 Focused width to particle size Particle size (μm) used to achieve separation of the particles. The 2 μm particles are labeled with DAPI flurophore, hence the channel produce auto fluorescence which can be seen in Figure 14(a). Separation of these particles using a 1200 μm wide outlet and the 5 segment outlet system is shown in Figure 14(b). Individual particles were passed through the straight channel and photographs were taken at the outlet section. Image of the top section and the bottom section of the channel were taken separately and later stitched. It can be seen that two streams of particles can be separated at the strategically placed outlet obstruction. Once the two particle streams are separated, they are collected in different outlets for further analysis. To quantify separation efficiency, particle streams from each of the five outlets were collected and analyzed with a hemocytometer. Figure 15 illustrates the results of particle counting using hemocytometer. A hemocytometer is typically used in counting of blood cells [34] but it can be easily implemented in particle counting. Different particles can be easily distinguished depending on their um 800um 1200um 1500um Figure 13. Graph illustrating the ratio of focused width to particle size v/s the particle size. When the y-axis value equals to 1 then the particle stream is completely focused.

34 Figure 14. (a) Composite image of the inlet with 2 μm and 4.16 μm diameter particles. (b) Composite image of the outlet section showing separation of 2 and 4.16 μm diameter particles. Image was stitched after taking images of the upper and lower section of the outlet. Difference in the symmetry of focused streams, in the upper and lower section, is attributed to flow variations that may have occurred while collecting individual images (which were stitched later) at different time. 25

35 characteristic fluorophore. Only half of the outlet section was used for evaluation since the flow used to get disrupted by the movement of the outlet tubes used for collection of the samples. The outlet closest to the channel sidewall is labeled as Outlet1. The outlet which is second from the channel sidewall is labeled as Outlet2. The center channel is labeled as Outlet3. As shown in Figure 15(a) majority of the 2 μm diameter particles flow through the Outlet1, while the rest flow through Outlets 2 and 3. Approximately 75% of the 2 μm diameter particles are calculated to be passing through the outlets close to the sidewalls. This is because the 2 μm diameter particles loose focusing when they enter the 1200 μm wide separation region. Figure 15(b) shows that majority of the 4.16 μm diameter particles flow through Outlet2 while the rest flow through Outlet1. There were negligible 4.16 μm diameter particles detected in Outlet3. Approximately 85% of the 4.16 μm diameter particles were calculated to flow through outlets second farthest from the sidewall μm diameter particles were tightly focused but some of these particles passed through Outlet1 because of flow fluctuations. The lower separation efficiency can be attributed to the variation in the individual particle sizes. Improvements in the design of the outlet section will also lead to higher separation efficiency. The length of the segments in which particles travel after getting split can be reduced further to reduce pressure drop variations and ease the particles into the respective outlets. Design criteria for particle separation in straight microchannels Thus it is seen that when the microchannel is properly designed, separation of two particles can be easily achieved. The various channel sections need to be designed individually. These sections are namely the focusing region, the separation region and the outlet system. The focusing region channel dimensions are limited by the larger particle diameter. The width of the 26

36 Number of particles 2.00E E E E E+05 2μm 0.00E+00 Outlet1 Outlet2 Outlet3 Channel Outlet Number of particles 2.50E E E E E E+00 Outlet1 Outlet2 Outlet3 Channel Outlet 4.16μm Figure 15. Results of particle counting using hemocytometer. (a) Majority of 2 μm diameter particles tend to flow through Outlet1. (b) Majority of 4.16 μm diameter particles tend to flow through Outlet2. 27

37 focusing region needs to be at least 4-5 times the largest particle diameter under consideration. The focusing region needs to be rectangular with high aspect ratio (H/W > 3). After the focusing region, it is important to select a proper opening angle from focusing region to the separation region. The opening angle does not affect the distance between the two particle streams. But it is important to know where the two streams will equilibrate in the separation region so that the outlet obstruction can be placed exactly between the particle streams. Hence prior experiments are required with individual particles to determine the location of the particle positions in the separation region. Next, it is required to select the width of the separation region. A very wide separation region will help in increasing the distance between the particle streams but at the same time will cause de-focusing which may lead to particles mixing with each other. On the other hand a narrow separation region has the advantage that the particles do no loose focusing but then the distance between the particle streams is also reduced. This increases fabrication issues due to the limitations of the critical size while using SU-8. Hence there needs to be a compromise while selecting the width of the separation region. In the end a proper outlet system needs to be used which has obstructions placed between the particle streams. The design should not produce pressure gradients in which the motion of the particles will become biased towards a single segment. The individual particle streams can be collected after they are separated at the obstruction. Table 2 summarizes the design principle required to separate two particles, of size less than 10 μm, from a homogeneous mixture. These rules can be used in separation of particles with greater diameters. Figure 16 shows the focusing positions of particles ranging from 2 μm to 10 μm in diameter. The 28

38 Table 2. Design rules for particle separation. Channel Section Focusing region Rule W 4(a p max) Opening angle 28º Separation region Outlet system 800 μm < W 1200 μm 5 segments x/w Particle size (μm) Figure 16. Normalized position of particles ranging from 2 μm to 10 μm in diameter, in a 1000 μm wide separation region. 29

39 dimensions of the focusing region are 30 μm x 50 μm whereas the dimensions of the separation region are 1000 μm x 50 μm. The opening angle is at 28º. It can be seen that the behavior of the particles is similar but now separation of two particle streams of larger diameters is possible. Thus this technique of separation is scalable. It should also be noted that the scope of this work is limited to the separation of two particles from a homogeneous mixture. Separation of 3 particles can also be achieved by designing the appropriate outlet system to accommodate another particle stream. 30

40 CHAPTER 4 CONCLUSIONS In this work, it was demonstrated that microparticles with a diameter difference of as little as 2 µm can be separated using a rectangular straight microchannel. Inertial lift forces have been exploited in straight channels to focus particles of different size at different positions. Particles with larger diameter are observed to equilibrate at a distance further away from the sidewall as compared to smaller particles. This has proven to be against the conventional belief that any particle flowing through a rectangular straight channel will only focus at a distance of 0.2W away from the channel sidewall, where W is the width of the microchannel. It was demonstrated that by using a 15 μm x 50 μm rectangular straight channel which opened out to a 1200 μm wide outlet, separation of 2 μm and 4.16 μm diameter particles from a homogeneous mixture is possible. A separation efficiency of 75~85% was achieved, based on hemocytometry measurements. The various design parameters have been explained to facilitate separation of particles of any size. Focusing region should be kept at a minimum width which is approximately 4(a p max), to avoid clogging. Opening angle, from focusing region to separation region, is not critical for separation of 2 particles in straight channels. But it is necessary to know the final position of the focused stream, once it is in the separation region, to efficiently design the outlet section. The width of the separation region is also an important factor in the design of the microfluidic device. A wide separation region is required to increase the distance 31

41 between the 2 particle streams for easier separation. But this may cause the smaller particles to lose its focusing and reduce the separation efficiency. A narrow separation region has limitations in fabrication for soft lithography. Hence a compromise needs to be achieved for a proper width for the separation region. A 5-outlet section was used to collect the different particle streams. When the channel dimensions are scaled up, larger particles having small size difference can also be separated. For instance, white blood cells (10-20 μm) can be separated from red blood cells (6-8 μm). This system can be used for high throughput operation by parallelization of the device to simultaneously separate particles/cells. This simple, planar device can be easily integrated on a LOC system for precise microparticle/cell separation. 32

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