Active Control of Inertial Focusing Positions and Particle Separations

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Active control of inertial focusing positions and particle separations enabled by velocity profile tuning with co-flow systems Dongwoo Lee, Sung Min Nam, Jeong-ah Kim, Dino Di Carlo, and Wonhee Lee Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b05143 • Publication Date (Web): 27 Jan 2018 Downloaded from http://pubs.acs.org on January 31, 2018

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Analytical Chemistry

Active control of inertial focusing positions and particle separations enabled by velocity profile tuning with co-flow systems Dongwoo Leea, Sung Min Nama, Jeong-ah Kima, Dino Di Carlob and Wonhee Leea, c *

a

Graduate school of Nanoscience and Technology, Korea Advanced Institute of Science and

Technology (KAIST), Daejeon 34141, Republic of Korea b

Department of Bioengineering, Mechanical and Aerospace Engineering, and California

NanoSystems Institute, University of California, Los Angeles 90095, USA c

Departiment of Physics, Korea Advanced Institute of Science and Technology (KAIST),

Daejeon 34141, Republic of Korea

*

To whom correspondence should be addressed

E-mail: [email protected]

Keywords: inertial microfluidics, inflection point focusing, co-flow, tunable inertial focusing, cell separation. 1 ACS Paragon Plus Environment

Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Abstract Inertial microfluidics has drawn much attention not only for its diverse applications but also for counterintuitive new fluid dynamic behaviors. Inertial focusing positions are determined by two lift forces, i.e., shear gradient and wall-induced lift forces, that are generally known to be opposite in direction in the flow through a channel. However, the direction of shear gradient lift force can be reversed if velocity profiles are shaped properly. We used coflows of two liquids with different viscosities to produce complex velocity profiles which lead to inflection point focusing and alteration of inertial focusing positions; the number and the locations of focusing positions could be actively controlled by tuning flow rates and viscosities of the liquids. Interestingly, 3-inlet co-flow systems showed focusing mode switching between inflection point focusing and channel face focusing depending on Reynolds number and particle size. The focusing mode switching occurred at a specific size threshold, which was easily adjustable with the viscosity ratio of the co-flows. This property led to different-sized particles focusing at completely different focusing positions and resulted in highly efficient particle separation of which the separation threshold was tunable. Passive separation techniques including inertial microfluidics generally have a limitation in the control of separation parameters. Co-flow systems can provide a simple and versatile platform for active tuning of velocity profiles and subsequent inertial focusing characteristics, which was demonstrated by active control of the focusing mode using viscosity ratio tuning and temperature changes of the co-flows.

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Microfluidic manipulation of micro-size objects, especially cells, is not only an important problem in a variety of biomedical applications including biological and chemical analysis, therapeutics, and cell biology research1-8 but also an interesting topic in fluid dynamics. Various microfluidic techniques such as dielctrophoresis9,10, acoustophoresis11-13, deterministic lateral displacement (DLD)14,15, hydrophoresis16, pinch flow fractionation17, and inertial microfluidics18-21 have been developed and extensively studied for manipulation of microparticles within microfluidic flows4,22,23. Among these techniques, inertial microfluidics has drawn much attentions due to its advantages including extreme high-throughput and simplicity of device fabrication and operation, which enabled many applications including particle separations, single cell analysis, single cell encapsulations, sample preparation for flow cytometry, and fluid exchange18,20,24. Inertial migration and focusing of particles can be explained by two major lift forces: wall effect lift force and shear gradient lift force25,26. The shear gradient lift force is a function of fluid velocity and particle position in the flow, and the direction of shear gradient lift force can be determined by the sign of shear gradient lift shape, αβ, where α is shear rate and β is gradient of shear rate25. With a simple parabolic velocity profile, the shear gradient lift shape decreases or increases monotonically and change its sign at the channel center where the velocity maximum of parabolic velocity profiles lies. Therefore, shear gradient lift force directs particles away from the channel center in most microfluidic channel flows. Equilibrium positions, i.e., focusing positions, are formed near the channel walls by the balance between shear gradient lift force and opposing wall effect lift force that is always directed away from the wall. However, the shear gradient lift shape can have a complex shape and the direction of shear gradient lift force can also change in such a way that stable focusing positions can be formed by the shear gradient lift force alone26. Such conditions can be satisfied by a velocity profile that has inflection points, shown in Figure 1A. This inflection point focusing can be an 3 ACS Paragon Plus Environment


interesting inertial focusing mode that can be easily adjustable. Here we show active control of inertial focusing positions can be achieved by creating and tuning complex velocity profiles using co-flow systems of two liquids having different viscosities. The inertial focusing positions for a straight channel changes with particle size and Reynolds number (Re); the focusing positions shift slightly towards the channel center with increasing particle size or decreasing Re27. The Reynolds number is defined as Re = ρUH/μ (Particle Reynolds number, Rep = Re(a2/H2) = ρUa2/μH), where U is the average fluid velocity, H is the characteristic dimension of the microfluidic channel and ρ and μ are the fluid density and the dynamic viscosity, respectively. Curved channels such as spiral and serpentine channels21,28 have been devised to utilize Dean flows and increase the changes in focusing positions, which resulted in highly efficient size-based particle separation. Recent reports showed that focusing positions shift along channel faces in triangular channels with increasing Re29 and focusing positions in rectangular channels can split into multiple stable focusing positions along long channel faces at high Re (> ~ 300)30. However, inertial focusing positions in straight microfluidic channels are generally known to be fixed and difficult to adjust. With a co-flow system, it was found that not only the number and locations of inertial focusing positions can be adjusted but also the focusing mode can be switched between inflection point focusing and channel center focusing. In addition, we investigated focusing mode switching phenomena and developed methods to control it. Especially interesting mechanism of particlesize-dependent focusing mode switching was revealed and applied to particle separation applications. Due to easy controllability of the velocity profile with co-flows, ‘active control’ of inertial focusing and separation became possible; with the separation threshold was shown to be actively adjustable over time.


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Experimental Section Preparation of particles and cells We used 10 μm, 15 μm and 20 μm polystyrene particles (PS particles, micromer®) for inertial focusing in co-flow systems and particle separation experiments. 1.0 μm, 9.9 μm (Thermo fluoro-maxTM, excitation 468 nm and emission 508 nm) and 15.45 μm (Bangs Laboratories, Inc., excitation 480 nm and emission 520 nm) green fluorescent particles (GFPs) were also used for the fluorescent images. A particle densities are 1.03 g/cm3 and 1.05 g/cm3 for plain PS particles and GFPs, respectively. NaCl was added to adjust the densities of the solution to prevent precipitation in the solution. In addition, all particles were dispersed in the solutions with 1 % tween 20 to avoid particle aggregation. The particle concentration was in the range of 0.05 – 0.4 w/v%. Polydisperse PDMS particles were synthesized by mixing uncured PDMS solution and DI water with 1 % tween 20. The ratio of base and cross-linker for uncured PDMS solution was 10:1. The volume ratio of uncured PDMS solution and DI water was 1:9 and the mixture was mixed with a vortex mixer for 2 minutes. The resulting mixed solution was cured for 2 hours. After curing, the solution was centrifuged for 10 seconds and the supernatant was eliminated to remove particles smaller than 5 μm. The resultant was re-dispersed in DI water and filtered using a membrane filter of 30 μm pore to eliminate particles larger than 30 μm. Human blood and MCF-7 cells (breast cancer cells, ~ 15-27 μm in diameter) were used for cell experiments. Blood was taken from a healthy donor. The MCF-7 cells were cultured in a CO2 incubator (SANYO) with Dulbecco’s Modified Eagle’s Medium (DMEM, WELGENE) containing 10 v/v% Fetal Bovine Serum (FBS, WELGENE) and 1 v/v% PenicillinStreptomycin Solutions (WELGENE). For fluorescent labeling of MCF-7 cells, a cell-labeling solution (Vybrant® DiO Cell-labeling solution, absorption 484 nm and emission 501 nm) was used. Blood was diluted 500-fold and blood and fluorescent-labeled MCF-7 cells were 5 ACS Paragon Plus Environment


dispersed in DPBS with the ratio of red blood cells to fluorescent-labeled MCF-7 cells of 5000:1. For cell viability test of cells after cell separation, separated MCF-7 cells were fluorescentlabeled with 40 μM Calcein-AM solution (Sigma-Aldrich, absorption 495 nm and emission 517 nm after hydrolysis).

Experimental setup and measurement All microfluidic channels were fabricated by conventional photolithography and soft lithography using PDMS. The length of focusing channels was 4 cm for all experiments except the channels used for the particle and cell separation, and the real-time control of focusing position where the length of the channel was shorten to 3 cm to reduce the fluidic resistance. Particle and cell solutions were injected using a syringe pump (Harvard Apparatus PHD ULTRA CP syringe pump) with controlling volumetric flow rates. For uniform particle and cell concentration, a small magnetic stirring bar was placed in syringes and continuously stirred. For the cell experiments, the cells collected from the outlets were immediately dispersed in DPBS with 1:4 (Sample:DPBS) volume ratio to avoid damage to the cells by glycerol. Dynamics of particles and cells was observed using an optical microscope (Nikon Eclipse Ti-U), a digital high speed camera (Phantom v7.3), a fluorescent illuminator (Nikon Intenslight C-HGFIE) and a non-contact confocal optical profiler (Nanofocus AG). Particle positions were detected using MATLAB code that detects the change in the image gradient from the particle center (Circular Hough Transform algorithm). All images were taken at the consistent position at ~100 μm upstream of the expansion channel that connecting the main channel to the outlets.

Simulation of velocity profiles


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Flow velocity profiles were computed with a FEM simulator (COMSOL Multiphysics V5.2). A no-slip on the channel wall was set as a boundary condition and the flow velocity of each inlet was given by normal velocity from the calculated volumetric flow rates. The effect of the solution mixing by diffusion was neglected. The velocity profile was obtained from 1 mm downstream from the inlet, where the velocity profile is fully-developed.

Results and Discussion Co-flow generation and inertial focusing to inflection points A co-flow of liquids with dissimilar viscosities provides a convenient method for controlling velocity profiles of microfluidic flows. When two liquids with different viscosities form a laminar co-flow, the liquid having a lower viscosity flows faster than the liquid with a higher viscosity in a low aspect ratio microchannel and a complex velocity profile can be created as shown in Figure 1A. The velocity profile along the channel-width direction (ydirection) has an inflection point, which can lead to a reversal of the direction of shear gradient lift force and formation of additional focusing positions. Figure 1B summarizes the sign changes of α, β and the directions of shear gradient lift force in a 2-inlet co-flow system. Unlike a simple parabolic velocity profile, the sign of β changes from minus to plus near the interface of liquids, which allows shear gradient lift force directed towards the inflection point. We created velocity profile as described in Figure 1A and 1B, and confirmed the ‘inflection point focusing’ experimentally. Particles were focused to an inflection point and the location of the inflection point could be adjusted in a 2-inlet co-flow system by adjusting the flow rates, shown in Figure 1C. The particle diameter, a, was 10 μm. The co-flows were formed using two miscible liquids with different viscosities. Water was used for the lower viscosity 7 ACS Paragon Plus Environment


Figure 1. Control of inertial focusing with co-flow systems. (A) Co-flow of two liquids with different viscosities and resulting velocity profile (μ1 20 μl/min. The value of D used here was ~ 1×10-9 m2/s for glycerol in water. The number of focusing positions also can be changed by increasing the number of inflection points, shown in Figure 1D. We constructed 3- and 5-inlet co-flow systems where water and water/glycerol mixtures were flowed through each inlet in alternating order and observed four and six focusing positions respectively. The two focusing positions near the side channel walls were formed by the balance between the shear gradient lift force and wall effect 9 ACS Paragon Plus Environment


lift force, and the other focusing positions were formed by inflection point focusing. The dimensions (width : height) of the main channel in 3- and 5-inlet co-flow systems were 95 µm : 43 µm and 200 µm : 50 µm, respectively.

Inertial focusing in a 2-inlet co-flow system After confirming inflection point focusing in a co-flow system, we further investigated the inertial focusing in detail with a 2-inlet co-flow system, shown in Figure 2. Figure 2A shows that the co-flow in the 2-inlet co-flow system was formed stably. The flow rate (Q1:Q2) and viscosity ratio (μ2/μ1) of liquid A and B were kept constant at 1:1 and 3.5 respectively for all conditions. Microparticles with diameters of 10 μm were dispersed in both liquids and the positions of the particles were measured near the outlet using a microscope equipped with high speed camera, shown in Figure 2B, while varying the total flow rate (Qtot) from 20 μl/min to 180 μl/min, shown in Figure 2C. With a channel width of 95 μm and height of 43 μm, the Re for liquid A was 105 (Rep = 1.2) and liquid B was 16 (Rep = 0.02) when Qtot =180 μl/min. Under these conditions, two focusing positions were found: one located near the wall in the pure water phase and the other near the channel center. If the flow was a single phase, the focusing position near the short channel face would be unstable, which is typically known for low aspect ratio channels. In a 2-inlet co-flow system, the velocity profile in the pure water region resembles that of high aspect ratio channel (inset of Figure 2D). The side focusing position that is near the short channel face becomes stable as the velocity profile changes rapidly in the y-direction. The other focusing position was located near the interface between the fluids, which was the inflection point as in Figure 1. Note this focusing position is on the lower viscosity side. Particles dispensed in glycerol/water mixture migrated towards the pure water side and actually moved across the interface between the co-flowing fluids. Under closer 10 ACS Paragon Plus Environment

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Figure 2. Inertial focusing in 2-inlet co-flow systems. (A) Bright-field image of a co-flow system containing randomly distributed particles at inlet. (B) Inertially focused particles at the downstream near the outlet (10 high speed images are overlapped). (C) Distribution of particle positions with varying flow rate. Confocal image shows focusing positions in cross-sectional view (Qtot = 180 µl/min) (D) Comparison of the particle position distribution (Qtot=140 µl/min) and the focusing positions expected from the shear gradient lift shape. The focusing positions are indicated in a cross-sectional view (inset). The white dotted line indicates the basins of attraction. inspection, the focusing position near the inflection point is actually composed of two focusing positions: top and bottom. Three focusing positions were identified with confocal microscopy (inset of Figure 2C). The focusing position at the inflection point is split into two by the parabolic velocity profile along the z-axis. As the flow rate increases, the peaks become sharper due to the increase of shear gradient lift forces towards the inflection point. We calculated the values of αβ along the y-axis from a simulated velocity profile (COMSOL Multiphysics 5.2), and compared the resulting shear gradient lift shape graph to particle position distribution when Qtot was 140 μl/min, shown in Figure 2D. The point where the sign of αβ changes from plus to minus matches well with the second peak of the particle position distribution.



Inertial focusing in a 3-inlet co-flow system Figure 3 shows the results for inflection point focusing in a 3-inlet co-flow system. All inlet flow rates were the same (Q1::Q2:Q3=1:1:1) and the viscosity ratio between liquid A and B was 3.5. The particle position distribution was measured while varying Qtot from 30 μl/min to 270 μl/min with all other variables kept constant, shown in Figure 3C. Unlike the 2-inlet system, the focusing positions of the 3-inlet system displayed different characteristics depending on flow rate, or Re. At low flow rates (Qtot ≤ 60 μl/min), the particles dispersed in liquid B tended to focus near the channel center, which would be the focusing positions occupied in the case of a single phase flow. As the flow rate increased, the center peak divided into two peaks, which corresponds to the inflection point focusing. The two peaks became sharper as the flow rates increased as expected. Confocal microscopy image shows the actual 6 focusing positions for high flow rates (inset of Figure 3C). The shear gradient lift force increases faster than wall effect lift force as Re increases, which leads to the focusing positions shifting closer to the channel wall26. The focusing mode changes in a 3-inlet co-flow system may be explained by this effect. Near the top and bottom channel walls, wall effect lift force pushes particles towards the center focusing positions while the shear gradient lift force pushes towards the inflection points. That is, the strength of the competing two lift forces is the Re dependent, such way that the wall effect lift force dominates at the lower Re (top of Figure 3D) and the shear gradient lift force dominates at the higher Re (bottom of Figure 3D). The lift-force directions in the 3-inlet co-flow system could be somewhat complicated and shear gradient lift shapes were plotted for both y-direction and z-direction at 9 different positions in Figure 3E and 3F. In the z-direction, all of the shear gradient lift shapes change


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Figure 3. Inertial focusing in 3-inlet co-flow systems. (A) Bright-field image at inlet. (B) Inertially focused particles at the downstream near the outlet (10 high speed images are overlapped). There exist two distinct regime with different focusing behaviors. (C) Distribution of particle positions with varying flow rates. Confocal image shows focusing positions in a cross-sectional view (Qtot =270 µl/min). (D) Inertial focusing positions for different flow rate changes. The direction and the relative magnitude of the lift forces are indicated with arrows (red: shear gradient lift forces. green: wall effect lift force). The white dotted line indicates the basins of attraction. (E and F) Shear gradient lift shapes (αβ) in the direction of z-axis and yaxis. (G) Comparison of the particle position distribution (Qtot=210 µl/min) and the focusing position expected from the shear gradient lift shape. monotonically as expected for parabolic velocity profiles, shown in Figure 3E. The side regions that are occupied by the lower viscosity liquid (|𝑦𝑦⁄𝐻𝐻 | ≥ 0.3) have larger shear rates compared

to the middle region and display shear gradient lift shapes with larger magnitudes. In y-

directions, shear gradient lift shapes change in such a way that there exist two stable 13 ACS Paragon Plus Environment


equilibrium positions, shown in Figure 3F. The equilibrium positions are located at the inflection points, where 𝜕𝜕𝑣𝑣 2 ⁄𝜕𝜕 2 𝑦𝑦 = 0. The locations of inflection points do not change with varying z positions. On the other hand the magnitude of the shear gradient lift shape near the

inflection points changes significantly; shear gradient lift forces towards the infection points are larger near channel center (|𝑧𝑧⁄𝐻𝐻 |~0) due to a smaller flow speed near the top and bottom

channel walls. Similar to the 2-inlet system, the inflection points are located further away from

the center than the interfaces of the co-flowing fluids, shown in Figure 3G. The observed peak positions are slightly closer to the channel center than the calculated equilibrium positions in the particle position distribution. This small mismatch makes sense due to the added wall effect lift force towards the channel centerline.

Switching inertial focusing mode based on particle size and viscosity ratio We investigated the particle size dependence of inertial focusing positions in a 3-inlet co-flow system. Here, the particles were dispersed only in liquid B to observe the inflection point focusing and remove unnecessary particle focusing at the side focusing positions. Liquid A and B were injected into the side inlets and center inlet, respectively. The viscosity ratio was 3.5. Figure 4A show that the inertial focusing positions of particles with three different sizes (10, 15 and 20 μm) were compared at the Qtot of 180 μl/min (Q1:Q2:Q3=1:1:1). Larger, 15 and 20 μm, particles experience larger inertial lift forces and were expected to show inflection point focusing at the smaller Re than 10 μm particles. Contrary to this expectation, larger particles were found to be focused at the center line under the conditions that 10 μm particles were focused to the inflection points. In addition, the focusing position peak of the 20 μm particles is sharper than that of 15 μm particles, which indicates that the attraction to the center focusing position is stronger for larger particles. This observation seems conflicting with the results in 14 ACS Paragon Plus Environment

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the Figure 3 because the Figure 3C shows the inflection point focusing became strengthened with increasing Re.



Figure 4. Particle size dependent focusing position changes and its control with viscosity ratio. (a) Distributions of particle positions of different size particles (Qtot=180 µl/min). Schematic for focusing positions of smaller and larger particles in a cress-section (inset). (b) High-speed images showing flow disturbances due to particle-induced mixing for different size particles (top). Fluorescent images of the co-flows to quantify the particle-induced mixing (middle) and their intensity profiles for flows containing different size particles (bottom). (c) Particle size vs. particle positions at different viscosity ratio conditions (Qtot=210 µl/min). Two different focusing modes can be observed and the threshold size increases along with viscosity ratio. There were flow disturbances caused by particles near the interfaces of liquids found from high-speed imaging, shown in Figure 4B and Supplementary video 1, 2 and 3. It is known that inertially focused particles can induce secondary flows around the particle and enhance fluid mixing32. As the size of particles became larger, the disturbance also became more significant. In the case of 20 μm particles, the disturbance was clearly noticed on both interfaces when a particle flows near one of the interfaces. The amount of the flow disturbance was quantified by measuring fluid mixing (Florescent images, Figure 4B). The number density of each particle type was matched for comparison. Liquid A was dispersed with fluorescent particles (a=1 μm) and other conditions were kept the same as the conditions for Figure 4A. Fluorescent particles were used instead of fluorescent dye because fluorescent dye can diffuse into PDMS and affect the fluorescent intensity measurements. The fluorescent intensities at the outlet show an increasing tendency of mixing with increasing particle size. The slope of fluorescence intensity near the interfaces become smaller and the intensity near the channel center also increased with increasing particle size. These results show that the velocity profiles can be disturbed by particle-induced mixing. We believe this particle-induced mixing reduces 16 ACS Paragon Plus Environment

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the strength of the inflection point in velocity and shear-gradient lift leads to the center focusing mode beyond a certain threshold size. In the 3-inlet co-flow system, the strength of shear gradient lift force towards the inflection points can be easily adjusted with little change in the location of the inflection points by adjusting the viscosity of the co-flow. A larger viscosity ratio can result in a larger gradient in velocity profile, which is expected to provide stronger shear gradient lift force and to result in changes to the onset of the focusing mode switching. Liquid B was prepared at different volumetric mixing ratios of water and glycerol and the viscosity ratios were adjusted to a range of 2 to 6.5. Polydisperse PDMS particles with diameters ranging from 5 μm to 35 μm were prepared and individual particle sizes versus particle positions were measured near the outlet. The results in Figure 4C clearly show that particle focusing mode changes from inflection point focusing to center focusing if the particle size is larger than a certain threshold. The threshold size for focusing mode switching changes with the viscosity ratio; the threshold size became larger with larger viscosity ratio as intended. For example, 15 μm particles could not be focused near the inflection points with a smaller viscosity ratio (μ2/μ1~3.5), while they could be focused near the inflection points with a larger viscosity ratio (μ2/μ1~5.3 or 6.3). We defined switching of focusing mode when more than 80 % of particles were preferentially focused either at the inflection points or channel center. In between the focusing modes, there are ranges of particle size that show broad distribution or focused at both inflection points and center. The size median of the unfocused particles was defined as a threshold size. Medians of each condition were 11.3 (± 1.1), 14.0 (± 1.1), 17.6 (± 0.8) and 24.8 (± 1.6) μm for μ2/μ1 = 2, 3.5, 5.3,and 6.5, respectively. The adjustment of shear gradient lift force with viscosity ratio provides a powerful tool for controlling inertial focusing of microparticles. In the case of passive inertial microfluidic systems, a new device with a different design would be required each time to change the separation parameters. A simple adjustment of the viscosity ratio in this co-flowing 17 ACS Paragon Plus Environment


system enables the adjustment of separation parameters (i.e. size threshold) without device redesign and replacement.

Particle and rare cell separation in a 3-inlet co-flow system The size-dependent focusing mode change can allows highly efficient size-dependent particle separation because the focusing position of particles with different sizes are completely at different locations. We demonstrated separation of particles and enrichment of rare cells using size-dependent focusing in a 3-inlet co-flow system. First, we conducted separation of micro-particles based on the results of Figure 4A. For efficient separation, we modified 3-inlet co-flow system as Figure 5A. A 5-inlet channel was used to achieve the same velocity profile. Liquid A (low viscosity) was flowed through the side inlets (inlet 1, 2 and 4, 5) and liquid B (high viscosity) was flowed through center inlet (inlet 3) only. Particles were dispersed in liquid A and flowed through inlet 2 and 4 only. This configuration helps particles to focus to inflection points faster. Smaller particles (10 μm particle) are focused to inflection points that are close to original stream-lines while larger particles (15 and 20 μm) migrate further away and are focused to the center focusing position. As a result, smaller particles and larger particles can be focused to completely different positions that are separated with a distance several times larger than the particle diameter and can be efficiently separated into different outlets after expansion, shown in Figure 5B. For the particle separation, particle suspensions were prepared and mixed in such way that the number of the particles of different size are the same. The suspensions of each particle are prepared to have concentrations of 0.05, 0.17 and 0.40 w/v% for 10, 15 and 20 μm particles respectively. The 10 μm particle suspension was mixed with each of the 15 and 20 μm suspension with 1:1 volume ratio. After the particle separation, the separation efficiency for each particle size was calculated. High separation efficiency was 18 ACS Paragon Plus Environment

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Figure 5. Particle and cell separations using a co-flow system. (A) Schematic of co-flow system for particle and cell separations. (B) Particle separation results. Stacked images of particles at the beginning of expansion channel in case of 10 and 15 µm particle mixture (left top), and 10 and 20 µm (left bottom) particle mixture. An overall image of the expansion channel and outlets for separation of 10 µm and 15 µm particle (right). (C) Cell separation results. Collected samples from the outlets and fluorescent microscopy images of the samples showing most RBCs are collected from outlet 1&3 while fluorescent labeled MCF-7 are collected from outlet 2. achieved for larger particles: the efficiency for 15 μm particles was 99.3 ± 0.411 % (standard deviation, N=500) and the efficiency for 20 μm particles was 100 ± 0.000 % at the center outlet 19 ACS Paragon Plus Environment


(outlet 2). And, the efficiency for 10 μm particles were 95.7 ± 0.189 % with 15 μm mixture and 96.9 ± 1.11 % with 20 μm mixture at the side outlets (outlet 1 and 3). Here the efficiency is calculated by the number of target particles collected from the designated outlet divided by the number of target particles collected from all outlets. Some portion of 10 μm particles was collected from the center outlet and the efficiency for 10 μm at the side outlets were slightly lower (~96 %) compared to 15 and 20 μm particles. The major source lowering the efficiency is the fact that the smaller particles can also be affected by the disturbed velocity profile due to the rotation and fluid transport induced by larger particles and smaller particles adjacent to larger particles also migrated to center focusing positions. Smaller particles flowed into the side outlets without disturbance when there were no adjacent larger particles. However, smaller particle adjacent to the larger particle was flowed near the center of the channel and separated into the center outlet due to the influence of disturbed velocity profile induced by the larger particle as shown in Figure S-3. Recently, an inertial particle separation method by deflecting the larger particles within a co-flow system has been reported33. Despite the similarity of the velocity profile, there was no observation of inflection point focusing in this study. Furthermore, the physical mechanism of inertial particle migration was based on Saffman lift force34, while inflection point focusing is caused by a shear gradient lift force. In this study, smaller particles (1 μm) remained in streamline without migration by lift force, however, larger particles (9.9 μm) were deflected from the original streamline to the side of the higher streamline by Saffman lift force33, which is the opposite direction of our particle migration results, migrating toward the inflection points by the shear gradient lift forces. Saffman lift force is weaker inertial lift force by three orders of magnitude in a/H than shear gradient lift. Whereas particle manipulation of our co-flow


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system was based on equilibrium focusing, which provided a flexibility and controllability on particle manipulation. The same system used for the solid particle separation experiment was also applied cell separation to demonstrate feasibility in rare cell enrichment. Red blood cells (~ 7-8 μm diameter) and MCF-7 cells (~ 15-27 μm diameter) correspond to small and large particles, respectively. The viscosity of DPBS is similar with that of water, and DPBS was used to prepare liquid A and B instead of water. The whole blood was diluted 500-fold with DPBS and spiked with MCF-7 at a ratio of 5000:1 (RBCs:MCF-7 cells). This ratio was selected for effective cell counting by increasing cell count after cell collection, which helps the data to be statistically reliable. The cell suspension was flowed through inlet 2 and 4. As expected, most of the RBCs migrated toward the inflection points due to their size, causing the RBCs collected at the side outlets. Similar to 15 and 20 μm particles, Figure 5C shows that the MCF-7 cells were focused at the center and collected at the center outlet. The separation efficiency of MCF7 at the center outlet was 73 % with a standard deviation of 9.5 % (N=3). Because of the wide range of cell size and cell deformability, the efficiency of cell separation was low compared to the solid microparticle separations. In fact, MCF-7 cells that were collected from the side outlets were similar in size with RBCs. The enrichment ratio was ~3400 ± 163 (N=3). The enrichment ratio was calculated by counting cells from samples before separation and after the separation. The fluid velocities used were ~ 0.1-1 m/s and the time that cells stayed in the higher viscosity solution is on the order of tens of milliseconds. Therefore, the cells are hardly affected at all by glycerol (e.g., osmotic pressure) if the cells are immediately dispensed in DPBS at the collection outlet. The cell viability of separated MCF-7 cells was examined and the resultant viability was 92 % ± 2.2 % (N=3, Figure S-4). There are increasing demands to use whole blood for rare cell separations. Here, we used 500 diluted blood for demonstration purpose. We expect the high feasibility of using higher concentration or even whole blood with 21 ACS Paragon Plus Environment


further optimization of channel size and experimental conditions due to the large separation distance of our separation technique.

Real-time control of focusing positions Tuning of the velocity profile in co-flow systems allows active control of focusing positions for passive inertial microfluidic systems. Tuning of velocity profile can be done by changing flow rate or changing viscosity. Feasibility in the real-time tuning of the velocity profile in a 3-inlet co-flow system is demonstrated with two methods: real-time adjustment of the mid-stream viscosity with an integrated passive mixer and adjustment of the viscosity ratio by controlling the fluid temperature. The particles were dispersed in the liquid B only. First, we controlled the focusing mode in a 3-inlet co-flow system by changing the viscosity of liquid B on-chip as shown in Figure 6A. Liquid B used for the middle-stream was formulated within a passive mixing channel and the viscosity of liquid B could be changed by adjusting the flow rate of water and glycerol. Passive microfluidic mixing techniques are available for wide range of Re35,36. A connected-groove design was applied to achieve efficient mixing for a wide Re range and low fluidic resistance37. A schematic for the mixing channel and fluorescent images before and after mixing are shown in Figure 6A (Overall design and dimension of mixing channel can be found in Figure S-5). Viscosities of liquids A and liquid B injected into the mixing channel were 1 cp (μ1) and 6.5 cp (μ2), respectively, and the final viscosity of the mixture (μ3) was controlled by varying the flow rates (Q1 and Q2). For example, when Q1 and Q2 were 56 μl/min and 44 μl/min respectively, the resultant flow rate (Q4) and viscosity (μ3) became 100 μl/min and 2.0 cp. The Q1 and Q2 were changed to 10 μl/min and 90 μl/min respectively, and Q4 was same, but μ3 was changed to 5.0 cp. The 15 μm fluorescent particles were focused and their focusing positions were controlled in the system in real-time. 22 ACS Paragon Plus Environment

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Figure 6. Real-time tuning of focusing positions in the 3-inlet co-flow system. (A) Schematic for real time viscosity ratio control of co-flow using on-chip mixer and the results of mixing in the mixing channel (fluorescent images). (B) Real-time tuning of focusing mode of 15 μm particles by adjusting the viscosity ratio. (C) Particle position distribution changes as a result of active viscosity tuning. (D) Particle position distribution changes as a result of active fluid temperature tuning. The particles were dispersed in liquid B only. The position of fluorescent streaks, i.e. the focusing position, changed responding to the changing flow rates Q1 and Q2, shown in Figure 6B and supplementary movie 4. The flow rates were adjusted slowly because sudden changes in flow rates caused unstable co-flow. The switching time between the focusing modes was less than 10 seconds. The statistics of particle positions as a result of real-time adjustment of focusing positions is shown in Figure 6C. Temperature is another interesting control parameter for inertial focusing in co-flow systems. Generally, the viscosity of a fluid is a function of temperature, and the viscosity ratio increases with temperature decrease for the water and glycerol pair, which enables the tuning of the velocity profile and focusing position changes. The temperature of the liquid was controlled by a circulating temperature controlled water around the tubing and the microfluidic device as shown in Figure S-6. The temperature was set to 5 °C and 37 °C and focusing of 15 μm particles was observed (Figure 6D). Particles were suspended in liquid B only. The 23 ACS Paragon Plus Environment


calculated μ2/μ1 for cold (5 °C) and hot (37 °C) conditions were ~ 5.9 and 4.0, respectively. As intended, the focusing position could be switched between inflection points and the center of the channel. The temperature change of the whole device requires a rather long time in the current setup. Integration of on-chip thermoelectric heater/coolers can allow very fast temperature changes, especially with thin film microfluidic structures.

Conclusion Inertial focusing and particle separation were studied with co-flow systems of two miscible liquids with different viscosities. Co-flow systems allow easy manipulation of velocity profiles to create inflection points, by which inflection point focusing was confirmed experimentally and its properties were investigated. The shear gradient lift force switches sign near the inflection points and stable equilibrium positions can be formed by the shear gradient lift force alone, not by a balance with the wall effect lift force. The shear gradient lift shape predicts the inflection point focusing precisely and it is expected that the shear gradient lift shape can give guidelines for the design of microfluidic devices and experimental conditions and help deeper understanding of inertial focusing. The location and number of focusing positions can be easily controlled by creating and adjusting inflection points with velocity profile tuning using co-flow systems. Particularly, 3-inlet co-flow systems showed interesting complexity in inertial focusing modes; both inflection point focusing and channel center focusing modes were observed and these competing modes of inertial focusing were determined by the Re, the velocity gradient, and particle size. When shear gradient lift force was not sufficiently strong compared to the wall effect lift force, the focusing positions were located at the center of long channel faces. Increasing the Re and increasing the velocity 24 ACS Paragon Plus Environment

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gradient by changing the viscosity ratio or flow rates led to an increase in the strength of shear gradient lift force, which stabilized the inflection point focusing and the focusing mode switched from the channel center focusing mode. Interestingly, the focusing mode change due to particle size dependency showed contradicting trend with the Re changes; larger particle sizes which would lead to larger particle Re were actually found to result in destabilization of inflection point focusing. Larger particles induced larger particle-induced mixing effects, which disturbs the velocity profile near the inflection point and likely resulted in focusing of the larger particles near the center of the channel walls. We have achieved highly efficient particle separation using these size-dependent focusing mode changes. The focusing position changes are not a continuous shift but complete switching from one focusing mode to another. Therefore, the distance between focusing position is relatively large, which allows high separation efficiency and purity. The size threshold of focusing mode switching, which defines the separation threshold, can be easily adjustable with the viscosity ratio. Although it was not demonstrated, tuning of the velocity profile with other methods such as changing the flow rate ratio of different liquids could also be used to control the threshold size. Device designs directly manifest into device functions for passive particle manipulation techniques, which includes inertial microfluidics. Devices for passive techniques need to be designed and optimized for each application independently while the same device can perform multiple operations for active techniques. The possibility for active control of device function in passive techniques, like our approach, eliminate such difficulty while taking advantages of passive techniques. Especially, optimization of separation efficiency could be easily achieved without going through searching for the optimal operation condition in a large parameter space, which may involve tedious redesign and feedback processes. Alteration of device function can be achieved during device operation; switching of focusing modes in real-time, which can be used to change separation size thresholds, as was 25 ACS Paragon Plus Environment


demonstrated by adjusting the viscosity ratio by fluid mixing near inlet region and controlling the temperature of the fluid. Velocity profile control and subsequent manipulation of focusing positions using coflows can provide a high degree of freedom for particle controllability and adjustability of focusing positions in passive microfluidics. Particularly, particle manipulation in real-time can be helpful to compensate for fabrication defects in pre-built devices. In addition, sophisticated control of the focusing position can suggest a new platform for observation or measurement of fluid properties such as viscosity. In conclusion, particle manipulation using co-flow systems can offer flexibility and wide applicability for microfluidic fields including particle manipulation and fluid exchange.

Acknowledgments This work was supported by Radiation Technology R&D program through the National Research Foundation of Korea funded by the Ministry of Science, ICT & Future Planning (NRF-2015M2A2A4A02044826) and the Technology Innovation Program (10054488) funded by the Ministry of Trade, Industry & Energy (MOTIE, Korea).

Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: Figure S-1: Control of the position of the interface between liquid A and B by varying flow rates. 26 ACS Paragon Plus Environment

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Figure S-2: Side view images of inertial focusing in 3-inlet co-flow system. Figure S-3: Influence of larger particles (15 μm) to focusing of adjacent smaller particles (10 μm). Figure S-4: Cell viability after separation. Figure S-5: Schematic and dimensions of the mixing channel for the viscosity control in 3-inlet co-flow system. Figure S-6: Schematic for fluid temperature control setup. Video S-1, S-2 and S-3: Flow disturbance by particle-induced mixing. Video S-4: Real-time switching of focusing mode of fluorescent particles (15 μm) by adjusting the viscosity ratio.

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Active Control of Inertial Focusing Positions and Particle Separations

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