Multiorifice Flow Fractionation: Continuous Size-Based Separation of

Sep 23, 2009 - To whom correspondence should be addressed. Phone: +82-(0)2-2123-5814. Fax: +82-(0)2-312-2159. E-mail: [email protected]...
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Anal. Chem. 2009, 81, 8280–8288

Multiorifice Flow Fractionation: Continuous Size-Based Separation of Microspheres Using a Series of Contraction/Expansion Microchannels Jae-Sung Park and Hyo-Il Jung* School of Mechanical Engineering, Yonsei University, 262 Seongsan-no Seodaemun-gu, Seoul 120-749, Republic of Korea We designed a new microfluidic method, multiorifice flow fractionation (MOFF), for continuous size-based separation of spherical microparticles. This method utilizes inertial lift force and momentum-change-induced inertial force generated in a series of contraction/expansion microchannels. The particles were concentrated gradually along the walls of microchannels by those inertial forces as they passed through the channels. The particle trajectory was deflected from the carrier fluid by means of numerous sudden turns formed in a multiorifice channel. The inertial force was induced by this trajectory mismatching between fluid and particle that resulted from the momentum change. The trajectory mismatching induces the lateral drift of the equilibrium position of particle distribution, and its extent is variable according to particle size and flow rate. In the case of polydispersion, the sizebased particle separation could be achieved in the specific range of the channel Reynolds number (Rec). It was found that, at Rec of 63-91, large polymer particles (∼15 µm) were aligned along the centerline of outlet channel, whereas small particles (∼7 µm) remained near both sidewalls. This method has potential in continuous separation without using a sheath flow. The purification or separation of microparticles such as polymer beads, cells, emulsions, colloids, and airborne dust has become one of the main priorities of recent chemical, biomedical, and environmental studies. A diverse array of miniaturized devices, so-called lab-on-a-chip (LOC) or micro-total-analysis systems (µTAS), have been contrived to realize particle separation using a microfluidic system. At present, many separation methods for minute particles in a channel flow have originated from two fundamental techniques: hydrodynamic chromatography (HDC)1,2 and field flow fractionation (FFF).3-5 Both techniques have been developed to achieve enrichment or depletion of target particles which are normally in the range of 1 nm to 100 µm. * To whom correspondence should be addressed. Phone: +82-(0)2-2123-5814. Fax: +82-(0)2-312-2159. E-mail: [email protected]. (1) Small, H. J. Colloid Interface Sci. 1974, 48, 147–161. (2) Blom, M. T.; Chmela, E.; Oosterbroek, R. E.; Tijssen, R.; vandenBerg, A. Anal. Chem. 2003, 75, 6761–6768. (3) Giddings, J. C. Sep. Sci. Technol. 1978, 13, 241–254. (4) Giddings, J. C. Science 1993, 260, 1456–1465. (5) Schimpf, M. E., Caldwell, K., Giddings, J. C., Eds. Field-Flow Fractionation Handbook; Wiley-Interscience: New York, 2000.

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In general, separation methods can be divided into two categories, active and passive. Active separation methods normally utilize an external energy source to manipulate the behavior of particles. Over the past few decades, the basic scheme of FFF has been successfully integrated with manifold energy fields in applications including electrophoresis,6,7 dielectrophoresis,8-11 magnetophoresis,12-14 ultrasound,15,16 centrifugation,17 gravitation,18,19 and optical manipulation.20-22 These active methods provide good separation accuracy but are normally limited to low throughput and require a precision instrument for the additional energy source. Passive separation methods induce changes in particle behavior using hydrodynamic effects that are driven by the geometrical features of microchannels. Passive methods have received significant attention in the past decade due to the discoveries of many interesting fluid dynamic phenomena in microfluidics. These discoveries allowed the development of methods incorporating particle size-based lateral displacement or filtration performed using a patterned array of micropillars,23 a particle-pinched effect (6) (7) (8) (9) (10) (11) (12) (13) (14) (15) (16) (17) (18) (19) (20) (21) (22)

(23)

Caldwell, K. D.; Gao, Y. S. Anal. Chem. 1993, 65, 1764–1772. Radko, S. P.; Chrambach, A. Electrophoresis 2002, 23, 1957–1972. Pethig, R. Crit. Rev. Biotechnol. 1996, 16, 331–348. Morgan, H.; Hughes, M. P.; Green, N. G. Biophys. J. 1999, 77, 516–525. Wang, X. B.; Yang, J.; Huang, Y.; Vykoukal, J.; Becker, F. F.; Gascoyne, P. R. C. Anal. Chem. 2000, 72, 832–839. Gascoyne, P. R. C.; Vykoukal, J. Electrophoresis 2002, 23, 1973–1983. Ahn, C. H.; Allen, M. G.; Trimmer, W.; Jun, Y. N.; Erramilli, S. J. Microelectromech. Syst. 1996, 5, 151–158. Watarai, H.; Suwa, M.; Iiguni, Y. Anal. Bioanal. Chem. 2004, 378, 1693– 1699. Pamme, N.; Wilhelm, C. Lab Chip 2006, 6, 974–980. Hawkes, J. J.; Coakley, W. T. Sens. Actuators, B 2001, 75, 213–222. Petersson, F.; Aberg, L.; Sward-Nilsson, A.-M.; Laurell, T. Anal. Chem. 2007, 79, 5117–5123. Madou, M.; Zoval, J.; Jia, G.; Kido, H.; Kim, J.; Kim, N. Annu. Rev. Biomed. Eng. 2006, 8, 601–628. Moon, M. H.; Kim, H.-J.; Kwon, S.-Y.; Lee, S.-J.; Chang, Y.-S.; Lim, H. Anal. Chem. 2004, 76, 3236–3243. Huh, D.; Bahng, J. H.; Ling, Y.; Wei, H. H.; Kripfgans, O. D.; Fowlkes, J. B.; Grotberg, J. B.; Takayama, S. Anal. Chem. 2007, 79, 1369–1376. Fu, A. Y.; Spence, C.; Scherer, A.; Arnold, F. H.; Quake, S. R. Nat. Biotechnol. 1999, 17, 1109–1111. MacDonald, M. P.; Spalding, G. C.; Dholakia, K. Nature 2003, 426, 421– 424. Wang, M. M.; Tu, E.; Raymond, D. E.; Yang, J. M.; Zhang, H.; Hagen, N.; Dees, B.; Mercer, E. M.; Forster, A. H.; Kariv, I.; Marchand, P. J.; Butler, W. F. Nat. Biotechnol. 2005, 23, 83–87. Huang, L. R.; Cox, E. C.; Austin, R. H.; Sturm, J. C. Science 2004, 304, 987–990. 10.1021/ac9005765 CCC: $40.75  2009 American Chemical Society Published on Web 09/23/2009

Table 1. Comparison of Throughput Performance for Various Particle Separation Methods method ultrasound optical manipulation pinched flow splitting flow slanted obstacles Dean flow momentum loss

concentration (particles/µL) 3.6 × 104 to ∼1.1 × 105 3.0 × 104 to ∼5.0 × 106 ∼5 × 102 1.8 × 103 to ∼7.0 × 103 3.0 × 103 to ∼4.0 × 104 6.8 × 103 to ∼1.8 × 104 1.7 × 102 to ∼7.8 × 104 1.5 × 102 to ∼1.0 × 105 2.3 × 103 to ∼1.3 × 105 ∼2.3 × 105

with a sheath flow,24,25 and a splitting flow to a side channel.26-28 Such approaches provide the advantage of preserving sample condition since they do not require labeling processes that are essential to several active methods. However, they require a laminar flow associated with low Reynolds numbers, which has the effect of prolonging operation time. New approaches in hydrodynamic separation using nonlaminar flow have recently been introduced. In these methods, lateral particle migration is induced by secondary flow generated on a cross section by specific geometric properties of microchannels: for example, lateral flow by three-dimensional (3-D) slanted obstacles29,30 and Dean flow in curved rectangular channels.31-33 These methods attain lateral particle displacement with differential inertial effects according to particle size. With these techniques, biological and polymer particles could be separated in a continuous flow with a moderate flow rate and a low particle concentration. Also, another passive-type separation method was recently introduced on the basis of a trajectory mismatch between particle and fluid by momentum loss, which was controlled by asymmetric sheath flows.34 In a previous study, we introduced a novel hydrodynamic method for particle focusing using a multiorifice microchannel.35 In this study, we explore the feasibility of our hydrodynamic method for size-based particle separation. This new separation method is named multiorifice flow fractionation (MOFF), since the microparticle is moved laterally by hydrodynamic inertial forces driven by a multiorifice structure. The extent of lateral movement varies according to particle size. We demonstrated that polymer microspheres can be concentrated separately at different lateral positions in a microchannel. We examined the separation efficiency in terms of the Reynolds number. The throughput of microfluidic particle separation is an important parameter in evaluating the performance of separation device. Table 1 sumYamada, M.; Nakashima, M.; Seki, M. Anal. Chem. 2004, 76, 5465–5471. Takagi, J.; Yamada, M.; Yasuda, M.; Seki, M. Lab Chip 2005, 5, 778–784. Yamada, M.; Seki, M. Lab Chip 2005, 5, 1233–1239. Yamada, M.; Seki, M. Anal. Chem. 2006, 78, 1357–1362. Yang, S.; Undar, A.; Zahn, J. D. Lab Chip 2006, 6, 871–880. Choi, S.; Park, J.-K. Lab Chip 2007, 7, 890–897. Choi, S.; Song, S.; Choi, C.; Park, J.-K. Anal. Chem. 2009, 81, 50–55. Seo, J.; Lean, M. H.; Kole, A. J. Chromatogr., A 2007, 1162, 126–131. Di Carlo, D.; Edd, J. F.; Irimia, D.; Tompkins, R. G.; Toner, M. Anal. Chem. 2008, 80, 2204–2211. (33) Bhagat, A. A. S.; Kuntaegowdanahalli, S. S.; Papautsky, I. Lab Chip 2008, 8, 1906–1914. (34) Wu, Z.; Willing, B.; Bjerketorp, J.; Jansson, J. K.; Hjort, K. Lab Chip 2009, 9, 1193–1199. (35) Park, J. S.; Song, S. H.; Jung, H. I. Lab Chip 2009, 9, 939–948. (24) (25) (26) (27) (28) (29) (30) (31) (32)

flow rate (µL/min) 40 40 1-9 ∼16 20 0.5 20 0.4 1000 10 18

throughput (particles/s) ∼3 × 104 (bead) ∼1.5 × 106 (blood) 20-100 ∼50 ∼100 ∼5.0 × 103 ∼1.0 × 102 ∼90 1.7 × 106 (DNA) ∼106 (blood) ∼104 (bead) 6.9 × 104

ref 16 20-22 24 and 25 26 and 27 29 and 30 32 and 33 34

marizes the throughput ranges of various separation methods reported in previous works. We expect that the throughput performance of our method can grade up with the state of the art in the particle separation. THEORY AND MECHANISM In microfluidics, physical phenomena of fluid and particle behavior are analyzed by using dimensionless numbers. Normally, we can interpret the inertial and viscous effect of fluid flow using the Reynolds number (Re), which is defined as the ratio of inertial forces to viscous forces. When particles are suspended in the working fluid, the particle behavior is affected by the inertial and viscous forces occurring in the interaction with fluid. We can estimate the fluid dynamic phenomenon of particle flowing through a microchannel by using the particle Reynolds number (Rep), which is expressed by the following equation:

Rep ) Rec

Umd2 d2 ) νDh Dh2

(1)

where Rec is the channel Reynolds number, d is the particle diameter, Dh is the hydraulic diameter of the channel, Um is the maximum flow velocity in the channel, and ν is the kinematic viscosity of the fluid. Note that Rec uses the maximum velocity instead of the mean velocity that is normally applied for Re. For Rep . 1, the inertial force becomes a dominant parameter for driving the lateral migration of particles transverse to fluid streamlines. In contrast, in the case of Rep , 1, the particle behavior is strongly promoted to follow the flow pattern by a viscous drag force acting on the particle surface. For analysis of mass transport in the particle suspension, the particle motion can be estimated by the Peclet number (Pe), which is defined as the ratio of advection rate to diffusion rate:

Pe )

UDh D

(2)

where U is the average velocity of the fluid and D is the diffusion coefficient of the particle. Normally, for Pe . 1, the inertial effect of the fluid flow contributes a dominant force for the particle behavior compared with the diffusion effect. When a particle encounters an accelerating flow in a nonlinear channel, the particle behavior can be estimated by the Stokes number, which is defined Analytical Chemistry, Vol. 81, No. 20, October 15, 2009

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as the ratio of the relaxation time (τp) of the particle to the characteristic time (τf) of the flow:

St )

Fpd2 /18µ βd2Um τp β ) ) ) Rep τf Dh /Um 18νDh 18

(3)

where β is the specific gravity of the particle () Fp/Ff) and Fp and Ff are the particle density and the fluid density, respectively. For St . 1, a particle will continue in its original moving direction instead of following the fluid streamline when the flow turns suddenly by the channel geometry. The size-based particle separation in a multiorifice microchannel is driven by a combination of two fluid-mechanical forces: inertial lift force and momentum-change-induced inertial force. The inertial lift force originates from the lateral pressure gradient exerted on a particle migrating in a straight channel with a Poiseuille velocity profile. In 1962, Segre´ and Silberberg36 observed that the lateral migration of particles yields an equilibrium position of around 0.6 radius from the axis of pipe. This is called the tubular pinch effect. According to a number of theoretical analyses, this inertial migration phenomenon can be explained by a shear-gradient-induced lift force that causes particles to migrate away from the axis of pipe and a wall-effect-induced lift force that repels particles away from a pipe wall.37-43 The equilibrium position of the lateral migration is then achieved by a balance of the two lift forces acting bidirectionally on a particle. In the case of square ducts, particles are concentrated at four corners and four walls (top, bottom, left, and right). This seems to reflect the tubular pinch effect.44,45 The channel geometry of multiorifice pattern was designed so as to transform the tubular-pinched particle distribution in a cross section of a straight channel and subsequently concentrate particles close to both side walls of channel. The mechanism of particle focusing is based on the vortex flow (or the separated flow) formed at the expansion chamber, as shown in Figure 1a. Unlike the equilibrium of two lift forces in the contraction channel, the wall-effect-induced lift forces by the side walls are weakened comparably to the shear-gradient-induced lift force in the expansion chamber due to the long distance from the main stream to the side walls. The particles flowing near the centerline are drifted to the sides by the dominance of the shear-gradient-induced lift force. Then, the majority of particles can be focused near sidewalls as long as they pass through a series of contraction/expansion channels. The mechanism of particle migration induced by solely inertial lift forces cannot describe dissimilarity in the behavior of differently sized particles. Notice that the previously proposed theory for the separation mechanism by the authors in 200935 is incomplete and that the mechanism presented in this manuscript (36) (37) (38) (39) (40) (41) (42) (43) (44) (45)

Segre´, G.; Silberberg, A. J. Fluid Mech. 1962, 14, 136–157. Saffman, P. G. J. Fluid Mech. 1965, 22, 385–400. Gotoh, K. Nature 1970, 225, 848–850. Feng, J.; Hu, H. H.; Joseph, D. D. J. Fluid Mech. 1994, 277, 271–301. Asmolov, E. S. J. Fluid Mech. 1999, 381, 63–87. Matas, J. P.; Morris, J. F.; Guazzelli, E. Oil Gas Sci. Technol. 2004, 59, 59–70. Matas, J. P.; Morris, J. F.; Guazzelli, E´. J. Fluid Mech. 2004, 515, 171–195. Zeng, L.; Balachandra, S.; Fischer, P. J. Fluid Mech. 2005, 536, 1–25. Chun, B.; Ladd, A. J. C. Phys. Fluids 2006, 18, 031704. Di Carlo, D.; Irimia, D.; Tompkins, R. G.; Toner, M. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 18892–18897.

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Figure 1. Principle of multiorifice flow fractionation (MOFF). (a) Particles are focused toward side walls by the dominant effect of shear gradient lift force in an expansion chamber. (b) A particle experiences momentum-change-induced inertial force when it passes through an expansion chamber. From point 1 to 2, the particle loses momentum ∆(mv)12, and the direction is toward the centerline. From point 2 to 3, the particle loses momentum ∆(mv)23, and the direction corresponds with the particle trajectory. From point 3 to 4, the particle gains momentum ∆(mv)34, and the direction is toward the side walls. From point 4 to 5, the particle gains momentum ∆(mv)45, and the direction is toward the centerline.

now corresponds better to theoretical considerations. For a principle of the size-based separation, we will apply a theory of momentum change for a particle experiencing the rapidly changing flow by channel geometry. The momentum-change-induced inertial force is based on a mismatch between fluid and particle trajectories, which is induced by a geometric configuration of channel with sudden expansion/ contraction elements.34,46 When a particle experiences a rapid change in momentum with high values of St and Rep, a trajectory mismatch between the fluid and particle can occur. The subsequent inertial force leads the particle to escape from the original driving stream. Figure 1b illustrates the concept of momentum-change-induced inertial force in a multiorifice channel. From a contraction channel to the next one through an expansion chamber, the moving particle gains or loses momentum at four variation points of rate. Here, it is more meaningful to reckon the size and the direction of inertial force induced by the momentum change than to inquire into momentum gain or loss. The first and (46) Squires, T. M.; Quake, S. R. Rev. Mod. Phys. 2005, 77, 977.

second momentum changes may not affect the trajectory mismatch between particle and fluid due to fluent curves of streamlines. However, the third momentum change can induce a certain trajectory mismatch since the sudden turn of fluid stream is introduced by the wall geometry of expansion chamber. From point 3 to 4, a particle directs to a wall by inertial force and is restricted near the wall regardless of particle size. This phenomenon is another concept distinct from the particle focusing described in Figure 1a. The particle focusing is achieved by a long-term effect of the inertial force accumulated by a series of expansion chambers. The last momentum change is a key factor in the size-based particle separation. From point 4 to 5, the particle behavior will be accompanied with an apparent mismatch in trajectories since there is no restriction by side walls as the third momentum change. Note that the mismatch is amplified as the Stokes number increases. In a vector coordinate system, momentum change over a certain time interval is equivalent to inertial force driven from Newton’s the second law. The relation between inertial force Fi and momentum change ∆(mv) can be estimated by

Fi ) ma ) m

∆ν ∆(mν) dν ∼m ) dt ∆t ∆t

(4)

At each turning point in Figure 1b, if assuming that a particle flows with the average fluid velocity U through the characteristic channel dimension Dh, the time period ∆t corresponds to Dh/ U. Then, the inertial force Fi in eq 4 can be transformed to

Fi ∼ m

U2 Dh

(5)

For a spherical particle with the diameter of d, the inertial force Fi can be expressed by the following:

Fi ∼

Fpπd3U2 6Dh

(6)

The lateral migration driven by the inertial force Fi will be balanced by Stokes drag force (Fd ) 3πµdUd). The particle migration velocity Ud is calculated by

Ud )

Fpd2U2 18µDh

Table 2. Specifications of Three-Sized Particle Suspensions particle diameter (µm)

particle size uniformity (%)

volume fraction (%)

particle concentration (× 103 per µL)

interparticle distance (µm)

2 7 15

5 18 14

0.05 0.5 1

98.2 26.5 5.4

19.2 26.0 41.1

then the particle focusing and size-based particle separation will be accomplished at a same time. EXPERIMENTAL SECTION Preparation of Particle Suspension. Fluorescent polystyrene microspheres 2 µm (B0200, blue, 365/445 nm), 7 µm (35-2, green, 468/508 nm), and 15 µm (36-4, red, 542/612 nm) in diameter were used for the particle suspensions (Thermo Fisher Scientific Inc., Massachusetts). The specific gravity of the three microsphere sizes was about 1.05, and the microspheres were prepared in a 0.5 wt % Tween 20 (Sigma-Aldrich Co., Missouri) aqueous solution. Particle size uniformities, volume fractions, initial particle concentrations, and interparticular distances of three-sized particle suspensions are listed in Table 2. Prior to each experiment, the particle suspensions were treated with a well-type sonicator for at least 10 min so as to minimize particle aggregation. After conducting independent flow measurements of the individual particle sizes, the three particle sizes were mixed and applied to the simultaneous flow measurement, where the partial volume fractions of each size were identical with their initial conditions. Microchannel Design. The microchannel design is shown in Figure 2. The microchannel is composed of one inlet, a filter, a multiorifice segment, and a broadened outlet. The designed depth of the microchannel is 50-55 µm. The multiorifice segment is formed through an alternating series of contraction channel and expansion chamber. The total length of the multiorifice segment is about 36 mm and consists of 120 repeated contraction/ expansion elements. The dimensions of the contraction channels are 45-50 µm in width and 100 µm in length, whereas those of the expansion chambers are 200 µm in both width and length. At the outlet of the microchannel, the width expands to 800 µm. Microchannel Fabrication. Microchannels were fabricated using well-known soft-lithography techniques, i.e., an SU-8 master template, PDMS (poly(dimethylsiloxane)) replica molding, and

(7)

From eq 7, the lateral migration velocity of particles is proportional to d2 and U2 when other parameters are consistent independently of the particle size. This means that the separation effect of differently sized particles can be amplified to the extent of a square of particle size ratio at the same flow rate. Also, note that eq 7 is analogous to the Stokes number multiplied by the fluid velocity U. This means that the trajectory mismatching of particle is accelerated exponentially according to increasing flow rate. The competition of inertial and viscous forces lead particles to be aligned on different equilibrium positions according to particle sizes. Consequently, the particle distribution will transform gradually from random to order through a multiorifice channel,

Figure 2. Schematic diagram of the multiorifice microchannel used in this study. Analytical Chemistry, Vol. 81, No. 20, October 15, 2009

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Figure 3. Transition in the behavior of polystyrene microspheres through the multiorifice microchannel according to various Reynolds numbers: (a) 7 and (b) 15 µm.

plasma bonding.47 First, a film mask of the microchannel design was made using a photoplotter (RP-316SST, First EIE SA, Switzerland) at a print resolution of 5000 dpi. Second, negative photoresist (SU-8 2050, MicroChem Corp., Massachusetts) was spin-coated on a 4 in. silicon wafer at 3000 rpm for 30 s with an acceleration of 300 rpm/s. The photoresist film was subsequently treated by several physicochemical processes such as soft baking, UV light exposure, postexposure baking, development, and hard baking. As a result, the SU-8 master template was formed on a silicon wafer with a thickness of approximately 50-55 µm true to the proposed design. Next, for the replica molding, an instant barrier was made by wrapping the master silicon wafer in aluminum foil. Then, the 10:1 volumetric mixture of PDMS (Sylgard 184, Dow Corning Corp., Michigan) and a curing agent was poured on the master wafer. After a degassing process of the polymer mixture, the master wafer overspread with clear PDMS was cured on a hot plate at 90 °C for 30 min. After the curing, the PDMS replica was taken off the master wafer and perforated at the channel inlet and outlet using a punch. Lastly, the PDMS replica was bonded to a clean glass slide using a plasma generator (Cute-B Plasma, FEMTO Science Co., Korea). Operation. A syringe pump (KDS200, KD Scientific Inc., Massachusetts) was used to generate a continuous, stable microflow through a wide flow rate range of 10-190 µL/min. The 2 mL syringe was connected to the inlet hole of the PDMS microchannel using silicone and aluminum tubes. At the early infusion of the sample fluid, it was difficult to wet the corners of the expansion chambers due to the channel geometry and the hydrophobic nature of PDMS. However, the air trapped in the corners could be removed by increasing the pressure inside the channel, since PDMS is a gas-permeable polymer. An inverted optical microscope (I-70, Olympus Co., Japan) was used to observe the moving microspheres beyond the slide glass part of the microchannel facing an objective lens. To achieve color images of blue-, green-, and red-fluorescent particles, the microscope was equipped with a 100 W mercury lamp and three fluorescence mirror units (U-MWU2, U-MWB2, U-MWG2; Olympus Co., Japan). The fluorescent images were captured using a color CCD (47) Duffy, D. C.; McDonald, J. C.; Schueller, O. J. A.; Whitesides, G. M. Anal. Chem. 1998, 70, 4974–4984.

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camera (ProgRes C10, JENOPTIK-Group., Germany) and then analyzed with the image-processing programs Adobe Photoshop (Adobe Systems Inc., California) and ImageJ (NIH, Maryland). RESULTS AND DISCUSSION Visualization and Analysis of Particle Distribution. We first examined the specific ranges of Reynolds number in which the particles would be concentrated laterally according to their sizes by a multiorifice microchannel. We applied a Rec ranging from 7 to 133 that was based on a characteristic length of a contraction channel and a flow rate of 10-190 µL/min. Figure 3 shows the progress of particle migration through the multiorifice microchannel with various Reynolds numbers. Fluorescence images in Figure 3, parts a and b, were obtained with 7 and 15 µm particles, respectively. The measurement positions in the upstream, the midstream, and the downstream corresponded to the first, the 50th, and the 100th orifices, respectively. To compare particle trajectories with fluid streamlines, a CFD simulation work using COMSOL Multiphysics was conducted under given experimental conditions (Figure S1, Supporting Information). Overall, significant variation in the particle distribution was observed in accordance with the measurement position and Reynolds number. We could observe that the particle distribution was stabilized behind around 80th orifice. In the case of 7 µm particles (Figure 3a) at the lowest channel Reynolds number (Rec ) 7), particles were randomly distributed over the entire region of the microchannel. This phenomenon was a natural result of the low particle Reynolds number (Rep ) 0.14). At Rec ) 28 (Rep ) 0.55), the particle distribution set to the equilibrium state and looked two-lined focusing in the downstream. However, the width of the focused distribution was a little thick because the flow rate was not enough to make a fully developed concentration. At Rec ) 49 (Rep ) 0.97), the equilibrium state was almost fully developed and the two focused lines could be observed in the midstream as well as the downstream. As Rec (Rep) increased to 91 (1.79), the focused lines stayed distinct and came up gradually close to the centerline. With conditions higher than Rec ) 112, the particle distribution was changed to a single-lined pattern focused on the channel center in the downstream.

Figure 4. Photographs from independent experiments of three (2, 7, and 15 µm) polystyrene microspheres according to various Reynolds numbers.

In the case of 15 µm particles (Figure 3b), at Rec ) 7, particles were distributed randomly over the expansion chamber in the upstream and an empty space was observed near the side wall in the downstream. This was a result of the strong wall-effectinduced lift force occurred in the contraction channel since the particle diameter was as large as one-third of the channel width. As Rec increased, the particle distribution changed to form a single line concentrated on the centerline. Particularly, in the cases over Rec ) 70, most particles passed through the centerline in the downstream. This phenomenon was also observed at Rec ) 112 of the 7 µm particle distributions (Figure 3a). A major difference in the behavior of two-size particles is that the larger particle can be aligned to the centerline at low Rec compared to smaller particle. The large Stokes number of the 15 µm particle induced the significant trajectory mismatch compared with fluid streamlines (Figure S1, Supporting Information). On the basis of eq 6, the large particle can experience apparent inertial force enough for the lateral migration even at the low Rec conditions. After passing downstream of the multiorifice channel, particles arrived at a broadened outlet segment. Figure 4 shows fluorescence images of three-sized (2, 7, and 15 µm) particle distributions at the broadened outlet of the multiorifice channel with respect to different Reynolds numbers. Each subfigure was collected with various conditions of Rec and Rep. From Figure 4, we can examine precisely characteristics in particle distributions obtained with three different-sized particles. First, a two-line focusing of 7 µm particles was observed at Rec ) 28-70, whereas 15 µm particles show a similar phenomenon at solely Rec ) 7. In view of the particle Reynolds number, the two-line focusing can be explained with Rep close to a unity. Then, the focusing of 15 µm particles was not as clear as in the case of 7 µm particles. This undeveloped focusing was caused by a weak inertial effect on lateral migration due to low flow rate of U ) 0.067 m/s (Rec ) 7). From eq 7, the migration rate of 15 µm particles at Rec ) 7 is calculated as one-fourth of 7 µm particles at Rec ) 28. These two cases involve similar Stokes numbers, but the separation effect was distinct relatively at higher flow rate. Second, in both

the 7 and 15 µm particle cases at Rec ) 133, some particles were dispersed near the side walls as well as the centerline. This phenomenon occurs when the separated flow zones in the corners of the expansion chamber are fully developed (Figure S1, Supporting Information). The fluid streamlines forms into the nearly linear pattern without severe sudden turns as if the fluid and particles pass through a straight channel. In a straight square channel, the lateral migration of particles is influenced by secondary flow and the equilibrium position lies on the center or corner of side walls. Consequently, the three-lined distributions at Rec ) 133 can be described by the particle equilibrium in a straight noncircular channel which has been reported in the previous works by Chun Ladd44 and Di Carlo et al.45 In the case of 2 µm particles, significant concentration was not achieved in the whole range of Rec. A narrow clear space around the centerline was all that could be seen at values of Rec ) 28-70. At higher Rec, the particles were dispersed again over the entire field of the outlet channel. The underdevelopment of particle focusing we observed was mainly caused by the minute size of the particles relative to the dimensions of the contraction channel, which corresponds to low values of Rep ranging from 0.01 to 0.24. That is, the 2 µm particles hardly experienced lift forces that induce lateral movement. As a result, equilibrium of lift forces was not realized in this microchannel. By observing the behavior of other particle sizes, we can estimate that a Rep of at least 0.6 is required for particles to focus near both side walls. We may increase Rep for a 2 µm particle if we make the orifice configuration having a tighter channel constriction. In this study, we selected three particle sizes (2, 7, and 15 µm) to model three biomaterials (platelet, red blood cell, and white blood cell) for a practical application. The presented channel dimension will be possibly fit for the separation of white blood cells from the whole blood sample. We evaluated separation performance for a three-size (2, 7, and 15 µm) particle mixture using a multiorifice microchannel. Figure 5 shows representative fluorescence images obtained from a simultaneous infusion of three particles, specifically at a flow rate of 100 µL/min corresponding to Rec ) 70. More images are Analytical Chemistry, Vol. 81, No. 20, October 15, 2009

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distribution. We estimated how similarly the intensity distribution correlated with the particle population. In this experiment, the accuracy of intensity level could be influenced by a few factors, i.e., the particle retention time in the field of view, the particle concentration, the depth of focus, and the channel depth. First, we reduced the exposure time of CCD camera to prevent the overlapping of particle streaks due to a long retention time. Second, with the applied concentrations, the interparticle distances sustained over 3 times of particle diameters, which was enough to distinguish individual particles. Third, the used microscopy system acquired fluorescence images with the depth of focus, approximately 10 µm, that was smaller than the channel depth, 50 µm. However, in a wide-field microscopy, the off-focused fluorescence particles can be also captured by CCD camera even though their intensities are much dimmer than those of wellfocused particles. Finally, we estimate that the intensity distribution will represent effectively the particle distribution with an acceptable error. From Figure 6a-f, we can compare the distribution variation of the three-sized particle suspensions according to flow rate. The intensity profile of the 7 µm particles exhibits a two-line concentration having peaks at Y ) ±220-260 µm for Rec ) 28-91 and a single-line concentration at the centerline for Rec ) 112-133. The average width of the 7 µm concentrated distribution is about 150 µm. For the 15 µm particles, a two-line concentration is detected at Rec ) 7 (not shown in Figure 6). For Rec ) 70-133, the central concentrated distribution is clearly formed from Y ) -150 µm to Y ) +150 µm. For the 2 µm particles, there is no remarkable peak in the entire range of Rec. However, at Rec ) 112, it can be seen that the intensity of the 2 µm profile is higher than the other particle sizes at the side regions from Y ) ±200 µm to Y ) ±400 µm, where 7 and 15 µm particles scarcely exist. In conclusion, the position of the particle concentration shifts laterally according to the Reynolds number, and the concentration width also tends to vary with the flow rate condition. Of course, the transformation of the

Figure 5. Photographs from a simultaneous experiment using a mixture of three (2, 7, and 15 µm) polystyrene microspheres at Rec ) 70. (a) The 7 and 15 µm particles appeared as green (dark arrow) and yellow (white arrow), respectively, whereas the 2 µm particles were not shown due to a blockage of their fluorescence by the greenfluorescence filter cube (U-MWB2, Olympus). (b) Most of the area of the outlet channel was filled with 2 µm particles. The 7 and 15 µm particles (7 µm, dark arrow, and 15 µm, white arrow) appeared dimly by using a blue fluorescence filter cube (U-MWU2, Olympus).

presented in the Supporting Information (Figure S2), where data are obtained with wide flow rates of Rec ) 7-112. Different particle sizes behaved differently and independently according to flow rate, similar to the individual migrations presented in Figure 4. With this visualization, we can verify the feasibility of size-based separation for solid polymer microspheres. To quantitatively analyze particle distribution, we used fluorescence intensity that indirectly represents particle population. Figure 6 shows fluorescence intensity curves corresponding to the images shown in Figure 4. The intensity data were measured along a detection line that was located at 1.5 mm position of the broadened outlet channel away from the final contraction channel. The data symbols obtained from 41 positions of the detection line present partial values relative to a total intensity, which represents the local fraction of the particle population. We considered that this theoretical extraction from the fluorescence-based image could involve a tolerance from the exact solution of particle

Table 3. Separation Efficiency of Three Polystyrene Microspheres in Terms of the Channel Reynolds Number channel Reynolds number (Rec) collection channel 300 µm center 140 µm center two 220 µm sides

300 µm center 140 µm center two 220 µm sides

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particle diameter (µm)

14

28

42

15 7 2 15 7 2 15 7 2

36.0 17.8 35.1 12.5 3.0 13.4 57.4 75.4 59.4

(a) Recovery 62.9 75.1 6.2 4.1 32.9 27.0 29.1 36.4 1.3 1.1 10.0 5.9 28.7 17.2 87.9 92.6 61.2 66.9

15 7 2 15 7 2 15 7 2

4.7 11.5 83.8 4.6 5.4 90.0 3.8 24.5 71.7

9.1 4.4 86.5 13.3 3.0 83.7 1.8 27.4 70.7

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(b) Purity 12.8 3.4 83.8 24.4 3.6 72.0 1.0 26.9 72.1

56

70

84

98

112

126

140

71.0 3.7 25.6 38.7 1.3 5.1 23.2 93.5 68.4

86.7 4.1 24.9 65.0 1.4 5.9 11.1 93.7 69.2

85.2 6.5 27.1 64.2 2.3 8.1 12.3 88.9 67.3

90.1 41.8 30.3 75.2 15.6 12.3 8.9 49.5 64.5

86.3 71.8 33.8 69.7 44.5 13.3 11.8 22.4 60.6

80.3 76.0 38.4 61.4 58.3 15.9 18.2 21.8 55.9

81.5 62.2 40.8 64.2 51.0 19.9 17.1 34.7 53.7

12.8 3.3 83.9 28.1 4.6 67.3 1.3 26.6 72.1

15.5 3.6 80.9 36.4 3.7 59.9 0.6 26.6 72.8

14.0 5.3 80.8 28.8 5.0 66.2 0.7 26.1 73.2

10.6 24.3 65.1 20.1 20.4 59.5 0.6 17.0 82.3

8.2 33.4 58.4 13.2 41.3 45.6 1.0 9.0 90.1

7.0 32.4 60.6 9.6 44.9 45.5 1.6 9.4 89.1

7.2 27.0 65.7 9.5 37.0 53.5 1.5 14.6 83.9

Figure 6. Intensity distribution curves of three polystyrene microspheres (triangle for 2 µm, square for 7 µm, and circle for 15 µm) at the detection line according to various Reynolds numbers. (a) The 7 µm profile exhibits two peaks at Y ) (240 µm, having a distribution width of ∼220 µm. The 15 µm profile shows that most particles reside in a central area from Y ) -220 µm to Y ) +240 µm. The 2 µm profile shows a particle-free zone of ∼60 µm on the centerline. (b) The 7 µm profile moves slightly to peak positions at Y ) (250 µm, having a width of ∼200 µm. In the 15 µm profile, the central concentration is formed from Y ) -260 µm to Y ) +260 µm. The particle-free zone in the 2 µm profile changes to ∼80 µm. (c) Two peaks of the 7 µm profile shift to Y ) (260 µm, having a width of ∼220 µm. In the 15 µm profile, the width of the central concentration comes to ∼300 µm from Y ) -160 µm to Y ) +140 µm. (d) The 7 µm peak positions (Y ) (220 µm) move slightly to the centerline. The 15 µm central concentration becomes so compressed that it has a width of ∼240 µm from Y ) -140 µm to Y ) +100 µm. In the 2 µm profile, the particle-free zone at Y ) 0 disappears, and a uniform distribution pattern is established up to Rec ) 133. (e) The 7 µm profile changes to a central concentration having a width of ∼340 µm from Y ) -180 µm to Y ) +160 µm. (f) The 7 µm profile is more compressed, as if its pattern overlaps with that of the 15 µm profile. Their concentration widths are ∼280 µm from Y ) -140 µm to Y ) +140 µm.

distribution profile is achieved differently according to the particle size. Separation Efficiency. We investigated the performance efficiency in enrichment or depletion of a specific particle size by using a pseudo-multi-outlet system. Figure 7 shows the accumulated fluorescence intensities of the three particle sizes passing through a central region with a width of 140 (Figure 7a) and 300 µm (Figure 7b). The accumulated fluorescence intensity can be interpreted as the partial fraction relative to the total particle population infused at the inlet, which is defined as a recovery. In addition, a purity of each particle size is estimated to evaluate the cross-contamination of different particle sizes. Table 3 summarizes the recovery and purity of three-sized particles that were concentrated at the designated center and side sections according to the channel Reynolds numbers. For a 140 µm outlet, the highest recovery (75.2%) of 15 µm particle can be achieved at Rec ) 98, and the highest purity (36.4%) of 15 µm particle can be achieved at Rec ) 70. If the width of the central collecting region is

adjusted to 300 µm, the recovery of 15 µm particle is increased to 90.1%, but the purity is decreased to 15.5%. The poor purity of the 15 µm particle is a result from its lower particle concentration relative to other sizes. For the enrichment of 7 µm particle, we also presume additional outlets near both side walls, specifically in a section of Y ) ±180-400 µm. The highest recovery (93.7%) of 7 µm particle can be achieved at Rec ) 70 while its purity is 26.6%. The low purity of 7 µm particles is caused by the highest fraction (69.2%) of 2 µm particle gathered at the side outlets. The 2 µm particle has the highest purity (∼90%) at two positions: 140 µm center at Rec ) 14 and 220 µm sides at Rec ) 112. In conclusion, by adjusting the width and position of a branch of the multioutlet channel, we can accomplish optimal performance according to separation targets, for example, enrichment or depletion, high recovery or high purity, and small particles or large particles. In this theoretical calculation, we must consider the influence of velocity profile on the separation performance. The lateral Analytical Chemistry, Vol. 81, No. 20, October 15, 2009

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population. The actual population in the high-intensity region can be undervalued if intensity is saturated. We adjusted fluorescence intensity to fall into the middle of the full range to prevent intensity saturation. Despite this, it is inevitable that the intensity of overlapped particles is slightly lower compared to the actual population. We estimate that the uncertainty in fluorescence intensity values is about 15%, particularly in the high-intensity region. Last, the recovery and purity data based in Figure 7 may have an error of ∼10%, which can be estimated by the uncertainties mentioned above and an additional uncertainty such as an experimental performance error. Error bars in Figures 6 and 7 indicate standard deviations (σ) obtained from 10 representative experiment data. The experimental performance error is defined as 1.96σ that is equivalent of 95% confidence in the data distribution.

Figure 7. Accumulated fluorescence intensities of three polystyrene microspheres in terms of the Reynolds number. Each intensity bar (gray for 2 µm, black for 7 µm, and white for 15 µm) indicates the particle volume fraction that is accumulated through a specific outlet region: (a) a 140 µm center outlet of -70 µm < Y < +70 µm and (b) a 300 µm center outlet of -150 µm < Y < +150 µm.

velocity profile at the detection line is almost uniform except both edges, and the vertical velocity profile is parabolic over entire region, as shown in the Supporting Information (Figure S3). The uniform velocity profile on the horizontal plane removed the lateral variation of particle flux that can usually occur in a low-aspectratio channel. Also, the parabolic flow profile on the vertical plane induced rarely a horizontal migration of particles since it involved the varying inertia in z-direction. To be honest, it is premature to declare that the performance data calculated by the pseudo-multioutlets will be similarly realized by using a real multioutlet channel. Note that the high performance of recovery and purity in Table 3 was extracted from a theoretical analysis. We need to explore the more experiments for the acceptable performance with revised devices in the future. Experimental Uncertainties. The uncertainty in measurement data depicted in Figures 6 and 7 was analyzed qualitatively and quantitatively according to several experimental parameters. First, the accuracy of the peak positions of the intensity distribution curves in Figure 6 is dependent on particle size and Reynolds number. The precision of distribution curve widths is influenced directly by uncertainties in particle diameter (∼15%) and Rec (∼5%). We estimate that the peak positions and widths of distribution curves have overall errors of ∼10%. Second, there is an uncertainty in the linear correlation between fluorescence intensity and particle

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CONCLUSIONS We described a new method for particle separation according to size using a multiorifice microchannel. This method was demonstrated through confirmation of separation performance using microspheres of three different sizes. The separation throughput of this multiorifice channel was approximately 1-5 × 104 particles/s, which corresponded to an intermediate level of other separation methods introduced in Table 1. This technique is advantageous for continuous processing in that it can be operated by one syringe pump without the use of sheath flow. In addition, the microfluidic channel can be fabricated simply and rapidly by PDMS soft-lithography without supplementing any mechanical or electrical parts. However, the flow rate must be adjusted to a range designated for the separation of a specific size from the mixed populations, and the particle concentration is not high enough to claim a high-throughput separation. If particle fractions are separately collected by using a multioutlet channel, it may also admit the loss of separation efficiency due to unexplored factors. In future work, this multiorifice channel will be further developed to achieve the more acceptable performance in the size-dependent separation of biomaterials. ACKNOWLEDGMENT This work was supported in part by the Basic Research Program (Grant No. 2008-05943), by the National Core Research Center (NCRC) for Nanomedical Technology (Grant No. R15-2004024-00000-0) of the National Research Foundation, and by a Grant from the ICBIN of the Seoul R&BD program (Grant No. 10816). SUPPORTING INFORMATION AVAILABLE Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org. Received for review March 19, 2009. Accepted August 31, 2009. AC9005765