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Controllable trajectory of inertial focusing in microfluidics
MEE 9874
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17 April 2015 Microelectronic Engineering xxx (2015) xxx–xxx 1
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Controllable trajectory of inertial focusing in microfluidics
7 8 9 10 11
12 1 5 4 2 15 16 17 18 19 20 21 22 23 24
Dianshen Yang a, Heng Zou c,d, Weiliang Zhong b,⇑, Tao Xu a,⇑ a
Liaoning Medical University, Jinzhou, Songpo Road, Liaoning Province, PR China Department of Orthopaedics, First Affiliated Hospital of Dalian Medical University, 22 Zhongshan Road, Dalian 116011, PR China c Department of Biomedical Sciences, City University of Hong Kong, 83 Tat Chee Avenue, Kowloon, Hong Kong, China d Key Laboratory of Biochip Technology, Shenzhen Biotech and Health Centre, City University of Hong Kong, Shenzhen, PR China b
a r t i c l e
i n f o
Article history: Received 10 March 2015 Accepted 6 April 2015 Available online xxxx Keywords: Microfluidics Inertial focusing Slant groove Herringbone
a b s t r a c t Inertial focusing in microfluidics has been widely utilized to sort cells based on cell specific properties for clinical assay, the detection of environmental pathogenic microorganisms and cell biology research. Especially, cell inertial focusing in microchannels, containing slant groove structures, has been utilized for cell separation, enrichment and sample preparation. However, cell inertial focusing in slantgroove-structure-based microchannels, including slant-groove-structure (SGS), asymmetric-herringbone-structure (AHS) and integrated-asymmetric-herringbone-structure (IAHS) microchannels, has not been systematically explored. In this report, three types of microfluidic channels, including SGS, AHS and IAHS microchannels, were fabricated to study cell movement and cell inertial focusing. One single trajectory and two trajectories of inertial focusing were observed in SGS and AHS microchannels, respectively. The trajectories of inertial focusing were interpreted by the combination of lift force and Dean force, according to the results from fluid filed simulation and experiments. On the basis of the theoretical analysis, it was found that the number of inertial focusing trajectories was controllable through the design of slant-groove-structure-based microchannels. Furthermore, IAHS microchannels were fabricated and multiple trajectories of inertial focusing were obtained. These results demonstrate the controllability of inertial focusing trajectories in microfluidics. This work contributes to the control of the number of inertial focusing trajectories, provides an opportunity to increase the throughput of cell sorting due to the augment of cell inertial focusing trajectories and make microfluidics more applicable to the research of cell biology, disease diagnosis and clinical testing. Ó 2015 Published by Elsevier B.V.
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Owing to the microscale channels, microfluidics offers some useful capabilities: the ability to exactly control liquid and mass exchange [1], reduce time for analysis [2] and increase the sensitivity of detection [3]. Based on these capabilities, microfluidics has been widely applied to many fields, such as cell biology [4], medicine engineering [5] and analytical chemistry [6]. Especially, utilizing inertial focusing [7], microfluidics has been employed in cell sorting and flow cytometry, which provides huge potential in market, research and clinical diagnosis. So far several microfluidic chips have been developed for cell sorting by using inertial focusing [8–12]. In these chips, the throughput of cell sorting depends on two factors: one is flow rate and the other is the number of cell focusing trajectory. High shear stress exerts on cells leads to cell damage under high flow rate conditions [13]. In contrast, the ⇑ Corresponding authors. Tel.: +86 411 83635963; fax: +86 411 83632383 (W. Zhong). Tel.: +86 18104069106 (T. Xu). E-mail addresses: [email protected] (W. Zhong), xutao23219895 @126.com (T. Xu).
increase of the number of inertial focusing trajectory can enhance the throughput of cell sorting without the loss of cell viability. However, in these chips, only one trajectory of inertial focusing was often formed. Therefore, it is eager to develop new chips, in which there are multiple trajectories of inertial focusing. Recently, there have been several reports about cell sorting based on inertial focusing. Dino Di Carlo utilized asymmetric turns in microfluidics for particle or cell separation with different diameters [9]. In this chip, the particles or cells with the similar diameters could form only single trajectory of inertial focusing. The high throughput was obtained only under a high flow rate condition. Sung Young Choi employed slant-groove-structure (SGS) microchannels for particle separation and cell sorting [14]. In this design of the chip, there was also one trajectory of inertial focusing. The author increased the number of channels to raise the throughput of cell sorting, which leaded to the complexity of the fabrication of microfluidic chips. So far there has been only one report, in which multiple trajectories of inertial focusing were formed. The design of microchannels was based on symmetric herringbone
http://dx.doi.org/10.1016/j.mee.2015.04.051 0167-9317/Ó 2015 Published by Elsevier B.V.
Please cite this article in press as: D. Yang et al., Microelectron. Eng. (2015), http://dx.doi.org/10.1016/j.mee.2015.04.051
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structure (SHS) [15]. The authors suggested that inertial focusing could form only in SHS microchannels. If not, the drag force could not balance and inertial focusing disappeared. Until now there has no report about inertial focusing in asymmetric-herringbonestructure (AHS) microchannels. In this report, we deeply investigated cell movement behavior in SGS microchannels. Utilizing experiments and numerical simulation, we exploited the mechanism of inertial focusing in SGS microchannels. We found that the symmetric structures, which was reported in SHS microchannels [15], were not prerequisite for inertial focusing. Furthermore, AHS microchannels were fabricated to demonstrate our discovery. In AHS microchannels, two trajectories of inertial focusing formed in AHS microchannels, which were also interpreted by numerical simulation and experiments. Finally, we integrated AHS in one channel to construct IAHS, in which there were four AHS, and multiple (2 4 = 8) trajectories of inertial focusing were obtained in one IAHS
microchannel. This work has three novel points: (1) inertial focusing could form in AHS microchannels; (2) inertial focusing in SGS, AHS and IAHS microchannels was interpreted with the combination of lift force and Dean force; (3) the trajectories of inertial focusing could be easily controlled by the integration of AHS in microchannels. The microchannel was fabricated using standard soft lithography methods. SU-8 photoresist (3035, Microchem Corp.) was photolithographically patterned on glass to form two-layer featured masters. The first layers of SU-8 features form the main microfluidic channels and the second layers form SGS, AHS and IAHS (Fig. 1). The heights of the two layers are both 50 lm. The masters were used as molds. PDMS prepolymer mixture (prepolymer:the curing agent is 10:1) was cast on the masters. After curing in a conventional oven at 65 °C for 24 h, the PDMS mold was peeled off from the masters. Through O2 plasma treatment, the PDMS with the feather was irreversibly sealed with a thick PDMS slab.
Fig. 1. The fabricated microfluidic chips for inertial focusing research. (A) The schematic drawing of chips with two types of inlets. The chip with only one inlet (left) is utilized for cell inertial focusing study in slant-groove-structure (SGS), asymmetric-herringbone-structure (AHS) and integrated-asymmetric-herringbone-structure (IAHS) microchannels. Y type inlets (right) is utilized for the study of cell moving behavior in a slant-groove-structure (SGS) chip. (B) I A schematic drawing of a SGS microchannel. The height of the microchannel, h1, is 50 lm. The height of the grooves, h2, is 50 lm. The width of the interval between two grooves, b, is 50 lm. The width of the microchannel, w, is 200 lm. II A schematic drawing of an AHS microchannel. The widths of the grooves, w1 and w2, are 100 lm and 200 lm, respectively. The width of the microchannel, w, is 300 lm. Other factors, including h1, h2, a and b, are the same as I. III A schematic drawing of an IAHS microchannel. IAHS contains four AHS, therefore, the width of the microchannel, w, is 1200 lm (300 lm 4). Other factors are the same as II. (C) The top view of images of slant-groove-structure (SGS), asymmetric-herringbonestructure (AHS) and integrated-asymmetric-herringbone-structure (IAHS) from I to III. Scale bar: 200 lm.
Please cite this article in press as: D. Yang et al., Microelectron. Eng. (2015), http://dx.doi.org/10.1016/j.mee.2015.04.051
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HepG2 cells were obtained from ATCC (American Type Culture Collection) and cultured in DMEM (high glucose) supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 1 unit/ml penicillin, and 100 mg/L of streptomycin (Invitrogen, Carlsbad, CA, USA). Cells were maintained at 37 °C in a 5% CO2 environment before use. The HepG2 cell (diameter 15 lm) suspension (2.5 105/ml), rhodamine 123 (2 106/ml) and PBS buffer were injected into the microfluidic chips by Longer Pump (Baoding Longer Precision Pump Co., Ltd.). Videos were recorded by Leica S8AP0 and photos were taken by Olympus IX71. 488 nm was used to excite rhodamine 123 and 505–530 nm filter was used for emission. To investigate inertial focusing in SGS, AHS and IAHS microchannels, these microfluidic chips were fabricated. Fig. 1A and B shows the schematic drawings of the microfluidic chips and Fig. 1C shows the top view images of the microfluidic chips. SGS, AHS and IAHS in PDMS were fabricated by the mold masters with two-layer features. The heights of the SGS, AHS, IAHS and main channels are all 50 lm. The width of SGS is 200 lm. AHS can be considered as the combination of two types of SGS with different widths 200 lm and 100 lm. AHS is asymmetric. Four AHS are integrated to form IAHS. Therefore, the widths of IAHS is 1200 lm (4 300 lm) (Fig. 2B and C I–III). At first, the movement of cells was studied in SGS microchannels with Y type inlets. When the flow rate of Inlet 1 and Inlet 2 were 10 ll/h and 70 ll/h, respectively, cells were injected through Inlet 1 and flowed along Line 1 (Fig. 2A) due to laminar flow in the microchannel. The cells lifted into the grooves, slipped along the grooves, descended into the microchannels and traversed the microchannel (Fig. 2B along Line 1 and Video S1). When the flow rates of Inlet 1 and Inlet 2 were 70 ll/h and 10 ll/h, respectively, cells were injected through Inlet 2 and then flowed along Line 2 (Fig. 2A) due to laminar flow in the microchannel. The cells traversed the microchannel (Fig. 2B alone Line 2 and Video S2), lifted into the grooves, slipped along the grooves and descended into the microchannels. When the total flow rates were 160 ll/h, 240 ll/h and 320 ll/h, respectively, the movement of cells was similar with those at 80 ll/h. Abraham D. Stroock found that slant groove structures on the top of the microchannel made flows traverse and circulate back in the grooves, so that helical streamlines were generated in a SGS microchannel (Fig. 2C) [16]. Therefore, the movement of cells in the SGS microchannel was caused by helical streamlines. The vortex motion as we observed could enhance the contact of cells with the microchannels, which could promote cell capture. However, cell inertial focusing appeared at downstream. Inertial focusing was observed in the SGS microchannel (about 2.5 mm away from inlets). Fig. 3B shows cell focusing occurred at 80 ll/h, 160 ll/h, 240 ll/h and 320 ll/h in SGS microchannels. Although some papers report the utility of SGS microchannels for cell separation, the inertial focusing and its principle in SGS microchannels have not been observed and explored. Particles in a channel flow could experience shear gradient force and wall effect force [7]. The balance of the two forces generates equilibrium positions, where particle focusing occurs. For example, in square channels, the symmetry of the system, there are four equilibrium regions, where shear gradient force are equal to wall effect force (Fig. 3D I). In a curved channel, two counter-rotating vortices perpendicular to the primary flow direction, called Dean flow (Fig. 3D II), were created due to inertia1. The two vortices generate drag force, namely Dean force, exerted on particles. Therefore, in a spiral channel, inertial focusing was the result of the interaction of shear gradient force, wall effect force and Dean force. In SGS microchannels, the helical flow, generating a vortice in the cross section of SGS microchannels, exerts drag force on particles. Therefore, it is reasonable to speculate that cell focusing in SGS microchannels was the result of the equilibrium of shear
3
Fig. 2. Cell movement and flows in a SGS microchannel with Y type inlets. (A) When the flow rate of Inlet 1 and Inlet 2 were 10 ll/h and 70 ll/h, respectively, cells were injected through Inlet 1 and flowed along Line 1 (indicated by Line 1). When the flow rates of Inlet 1 and Inlet 2 were 70 ll/h and 10 ll/h, respectively, cells were injected through Inlet 2 and then flowed along Line 2 (indicated by Line 2). (B) When cells flowed into the microchannel along Line 1, they lifted into slant grooves on the top of the microchannel, slipped along the grooves, indicated by black arrows (up), descended into the microchannel and traversed the microchannel. When cells flowed through Inlet 2 into the SGS microchannel, they flowed along Line 2, they traversed the microchannel, indicated by the black arrow (down), lifted into slant grooves, slipped along the grooves and descended into the microchannel. (C) Indicates a helical streamline in the SGS microchannels. Scale bar: 200 lm.
gradient force, wall effect force and drag force, exerted by a vortice in SGS microchannels (Fig. 3D III). To explore the principle of cell focusing in SGS microchannels, a commercial computational fluid dynamics software FLUENT was used to simulate 3D velocity field in SGS microchannels [17]. To verify the simulation, the average velocities of the lateral flow obtained from experiments were compared with those from simulation (Table S1). The results from the simulation and the experiment were similar to each other. Therefore, the simulation was reliable.
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position across the channel cross-section assuming an average value of 0.5 and ap is the average diameter of cells [18]. Fd is the drag force exerted on cells due to a vortice in SGS microchannels, which can be obtained by assuming the Strokes drag [19].
F d ¼ 3plU D ap
Fig. 3. Cell focusing in SGS and AHS microchannels. (A) Bright field images of SGS (Left) and AHS (Right) microchannels. (B) I–IV HepG2 cells were stained by Celltracker (Green fluorescence, Invitrogen). Cell focusing, indicated by green fluorescence, occur in SGS microchannels at 80, 160, 240 and 320 ll/h, respectively. (C) I–IV Cell focusing, indicated by green fluorescence, occur in AHS microchannels at 120, 240, 360 and 480 ll/h. (D) I In a square channel, there are four equilibrium positions, where shear gradient force are equal to wall effect force. II In a curved channel, due to inertia, two counter-rotating vortices are generated. The two vortices exert drag force on particles. III Due to the equilibrium of shear gradient force, shear gradient force and drag force, cell focusing occurs in SGS microchannels. Because flow fields were symmetrical in SGS microchannels, there would be two equilibrium positions. However, the equilibrium position (indicated by dashed line) disappears due to the obstacle of the slant grooves to cell movement. Scale bar: 100 lm.
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In SGS microchannels, cells experienced Fs+w and Fd. Fs+w is the combination of shear gradient force and wall effect force. Asomolov derived a function describing the magnitude of Fs+w.
194 2
C L a4p
196
F sþw ¼ qG
197
where q is the density of fluid medium (kg/m3), G is the shear rate of the fluid, CL is a coefficient, which is a function of the particle
198
ð1Þ
ð2Þ
where p is the fluid viscosity (kg/ms) and Uy is the average velocity of lateral flow (Fig. S1A). According to the data from the simulation of the flow fields, the forces exerted on cells were calculated and list in Table 1. Fs+w and Fd had the similar magnitude. Therefore, in SGS microchannels, cells could be focused at the flow rates due to the combination of the two forces. Because of the symmetrical distribution of flow fields in SGS microchannels, there would be another equilibrium position (indicated by dashed line in Fig. 3D III). However, in our experiment, only one equilibrium position was observed in SGS microchannels. The reason is that cells changed their velocity and slipped in the grooves due to the obstacle of the grooves (Fig. 3A left and B). This caused the change of Fs+w and Fd and broke the equilibrium of Fs+w and Fd. Chia-Hsien Hsu fabricated symmetric herringbone structure (SHS) microchannels for particle focusing. In the work, focusing largely depended on the symmetry of microchannels [15]. However, according to our analysis, the symmetry of microchannels was not requisite. Therefore, asymmetric-herringbone-structure (AHS) microchannel was fabricated to demonstrate that inertial focusing was possible in an asymmetric structures (Fig. 1B II and C II). Fig. 3A (right) and C show that there are two focusing lines at four flow rates, although AHS microchannel is not symmetrical. According to the data from the simulation of the flow fields, Fs+w and Fd had the same magnitude (Table 2). This explained why focusing occurred in AHS. This finding has not reported yet. Multiple focusing can increase the throughput of cell sorting and save time. Therefore, integrated-asymmetric-herringbonestructure (IAHS) microchannels (Fig. 1B III and C III) were fabricated to demonstrate that it is easy to obtain multiple focusing in IAHS microchannels. At 480, 960, 1440 and 1920 ll/h, 2.5 mm away from the inlets, multiple focusing was observed (Video S3 480, 960, 1440 and 1920). The number of focusing positions is the same as the number of integrated SGS. 8 SGS were integrated to construct IAHS, so there were 8 focusing positions in IAHS microchannels.
Table 1 Summary of Fs+w and Fd in SGS microchannels. Fluid rate (ll/h)
Fs+w (1011 N)
Fd (1011 N)
80 160 240 320
1.54 6.17 13.89 24.69
1.90 3.38 5.76 7.67
Table 2 Summary of Fs+w and Fd in AHS microchannels. Fluid rate (ll/h)
Fd (w2) (1011 N)
Fd (w1) (1011 N)
Fs+w (1011 N)
120 240 360 480
1.99 3.98 5.97 7.76
1.69 3.39 5.07 6.78
0.339 1.34 3.01 5.43
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In this work, we found that cell focusing occurred in SGS and ASHS microchannels. The flow fields were simulated. Fs+w and Fd were both calculated. These results explained why focusing occurred in these microchannels. These results have not reported yet. Furthermore, using IAHS microchannels, we found that multiple focusing could be easily achieved by the integration of slant groove structures in microchannels. In future, with the combination of antibody modification on the surface of microchannels, these microchannels could be applied for cell separation, such as cancer stem cell separation and circulating tumor cell separation.
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Acknowledgements
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We thank Professor Michalael M. Yang for paper writing and organizing. This work was supported by grants from the National Key Scientific Research Program (973 Program No. 2012 CB933302), the National Natural Science Foundation of China (Grant No. 81201689), Knowledge Innovation Program of Shenzhen Municiple Government (JCYJ20140419115507575), Doctor Startup Fund of Liaoning Province (Grant No. 20121053), Program Funded by Liaoning Province Education Administration (Grant No. L2014360), the Research Grants Council of the Hong Kong Special Administrative Region, China (Project No. CityU_ 103312, CRF Project No. CityU9/CRF/13G), State Key Laboratory of Environmental and Biological Analysis and Strategic Development Fund of HKBU (SKLP_14-15_P008) and Aohongboze Medicine-Pharmacy Innovation Foundation, the President Fund of Liaoning Medical University.
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Appendix A. Supplementary data
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Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.mee.2015.04.051.
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No. of Pages 5, Model 5G
17 April 2015 Microelectronic Engineering xxx (2015) xxx–xxx 1
Contents lists available at ScienceDirect
Microelectronic Engineering journal homepage: www.elsevier.com/locate/mee
2
Accelerated Publication
5 4 6
Controllable trajectory of inertial focusing in microfluidics
7 8 9 10 11
12 1 5 4 2 15 16 17 18 19 20 21 22 23 24
Dianshen Yang a, Heng Zou c,d, Weiliang Zhong b,⇑, Tao Xu a,⇑ a
Liaoning Medical University, Jinzhou, Songpo Road, Liaoning Province, PR China Department of Orthopaedics, First Affiliated Hospital of Dalian Medical University, 22 Zhongshan Road, Dalian 116011, PR China c Department of Biomedical Sciences, City University of Hong Kong, 83 Tat Chee Avenue, Kowloon, Hong Kong, China d Key Laboratory of Biochip Technology, Shenzhen Biotech and Health Centre, City University of Hong Kong, Shenzhen, PR China b
a r t i c l e
i n f o
Article history: Received 10 March 2015 Accepted 6 April 2015 Available online xxxx Keywords: Microfluidics Inertial focusing Slant groove Herringbone
a b s t r a c t Inertial focusing in microfluidics has been widely utilized to sort cells based on cell specific properties for clinical assay, the detection of environmental pathogenic microorganisms and cell biology research. Especially, cell inertial focusing in microchannels, containing slant groove structures, has been utilized for cell separation, enrichment and sample preparation. However, cell inertial focusing in slantgroove-structure-based microchannels, including slant-groove-structure (SGS), asymmetric-herringbone-structure (AHS) and integrated-asymmetric-herringbone-structure (IAHS) microchannels, has not been systematically explored. In this report, three types of microfluidic channels, including SGS, AHS and IAHS microchannels, were fabricated to study cell movement and cell inertial focusing. One single trajectory and two trajectories of inertial focusing were observed in SGS and AHS microchannels, respectively. The trajectories of inertial focusing were interpreted by the combination of lift force and Dean force, according to the results from fluid filed simulation and experiments. On the basis of the theoretical analysis, it was found that the number of inertial focusing trajectories was controllable through the design of slant-groove-structure-based microchannels. Furthermore, IAHS microchannels were fabricated and multiple trajectories of inertial focusing were obtained. These results demonstrate the controllability of inertial focusing trajectories in microfluidics. This work contributes to the control of the number of inertial focusing trajectories, provides an opportunity to increase the throughput of cell sorting due to the augment of cell inertial focusing trajectories and make microfluidics more applicable to the research of cell biology, disease diagnosis and clinical testing. Ó 2015 Published by Elsevier B.V.
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 61
Owing to the microscale channels, microfluidics offers some useful capabilities: the ability to exactly control liquid and mass exchange [1], reduce time for analysis [2] and increase the sensitivity of detection [3]. Based on these capabilities, microfluidics has been widely applied to many fields, such as cell biology [4], medicine engineering [5] and analytical chemistry [6]. Especially, utilizing inertial focusing [7], microfluidics has been employed in cell sorting and flow cytometry, which provides huge potential in market, research and clinical diagnosis. So far several microfluidic chips have been developed for cell sorting by using inertial focusing [8–12]. In these chips, the throughput of cell sorting depends on two factors: one is flow rate and the other is the number of cell focusing trajectory. High shear stress exerts on cells leads to cell damage under high flow rate conditions [13]. In contrast, the ⇑ Corresponding authors. Tel.: +86 411 83635963; fax: +86 411 83632383 (W. Zhong). Tel.: +86 18104069106 (T. Xu). E-mail addresses: [email protected] (W. Zhong), xutao23219895 @126.com (T. Xu).
increase of the number of inertial focusing trajectory can enhance the throughput of cell sorting without the loss of cell viability. However, in these chips, only one trajectory of inertial focusing was often formed. Therefore, it is eager to develop new chips, in which there are multiple trajectories of inertial focusing. Recently, there have been several reports about cell sorting based on inertial focusing. Dino Di Carlo utilized asymmetric turns in microfluidics for particle or cell separation with different diameters [9]. In this chip, the particles or cells with the similar diameters could form only single trajectory of inertial focusing. The high throughput was obtained only under a high flow rate condition. Sung Young Choi employed slant-groove-structure (SGS) microchannels for particle separation and cell sorting [14]. In this design of the chip, there was also one trajectory of inertial focusing. The author increased the number of channels to raise the throughput of cell sorting, which leaded to the complexity of the fabrication of microfluidic chips. So far there has been only one report, in which multiple trajectories of inertial focusing were formed. The design of microchannels was based on symmetric herringbone
http://dx.doi.org/10.1016/j.mee.2015.04.051 0167-9317/Ó 2015 Published by Elsevier B.V.
Please cite this article in press as: D. Yang et al., Microelectron. Eng. (2015), http://dx.doi.org/10.1016/j.mee.2015.04.051
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structure (SHS) [15]. The authors suggested that inertial focusing could form only in SHS microchannels. If not, the drag force could not balance and inertial focusing disappeared. Until now there has no report about inertial focusing in asymmetric-herringbonestructure (AHS) microchannels. In this report, we deeply investigated cell movement behavior in SGS microchannels. Utilizing experiments and numerical simulation, we exploited the mechanism of inertial focusing in SGS microchannels. We found that the symmetric structures, which was reported in SHS microchannels [15], were not prerequisite for inertial focusing. Furthermore, AHS microchannels were fabricated to demonstrate our discovery. In AHS microchannels, two trajectories of inertial focusing formed in AHS microchannels, which were also interpreted by numerical simulation and experiments. Finally, we integrated AHS in one channel to construct IAHS, in which there were four AHS, and multiple (2 4 = 8) trajectories of inertial focusing were obtained in one IAHS
microchannel. This work has three novel points: (1) inertial focusing could form in AHS microchannels; (2) inertial focusing in SGS, AHS and IAHS microchannels was interpreted with the combination of lift force and Dean force; (3) the trajectories of inertial focusing could be easily controlled by the integration of AHS in microchannels. The microchannel was fabricated using standard soft lithography methods. SU-8 photoresist (3035, Microchem Corp.) was photolithographically patterned on glass to form two-layer featured masters. The first layers of SU-8 features form the main microfluidic channels and the second layers form SGS, AHS and IAHS (Fig. 1). The heights of the two layers are both 50 lm. The masters were used as molds. PDMS prepolymer mixture (prepolymer:the curing agent is 10:1) was cast on the masters. After curing in a conventional oven at 65 °C for 24 h, the PDMS mold was peeled off from the masters. Through O2 plasma treatment, the PDMS with the feather was irreversibly sealed with a thick PDMS slab.
Fig. 1. The fabricated microfluidic chips for inertial focusing research. (A) The schematic drawing of chips with two types of inlets. The chip with only one inlet (left) is utilized for cell inertial focusing study in slant-groove-structure (SGS), asymmetric-herringbone-structure (AHS) and integrated-asymmetric-herringbone-structure (IAHS) microchannels. Y type inlets (right) is utilized for the study of cell moving behavior in a slant-groove-structure (SGS) chip. (B) I A schematic drawing of a SGS microchannel. The height of the microchannel, h1, is 50 lm. The height of the grooves, h2, is 50 lm. The width of the interval between two grooves, b, is 50 lm. The width of the microchannel, w, is 200 lm. II A schematic drawing of an AHS microchannel. The widths of the grooves, w1 and w2, are 100 lm and 200 lm, respectively. The width of the microchannel, w, is 300 lm. Other factors, including h1, h2, a and b, are the same as I. III A schematic drawing of an IAHS microchannel. IAHS contains four AHS, therefore, the width of the microchannel, w, is 1200 lm (300 lm 4). Other factors are the same as II. (C) The top view of images of slant-groove-structure (SGS), asymmetric-herringbonestructure (AHS) and integrated-asymmetric-herringbone-structure (IAHS) from I to III. Scale bar: 200 lm.
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HepG2 cells were obtained from ATCC (American Type Culture Collection) and cultured in DMEM (high glucose) supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 1 unit/ml penicillin, and 100 mg/L of streptomycin (Invitrogen, Carlsbad, CA, USA). Cells were maintained at 37 °C in a 5% CO2 environment before use. The HepG2 cell (diameter 15 lm) suspension (2.5 105/ml), rhodamine 123 (2 106/ml) and PBS buffer were injected into the microfluidic chips by Longer Pump (Baoding Longer Precision Pump Co., Ltd.). Videos were recorded by Leica S8AP0 and photos were taken by Olympus IX71. 488 nm was used to excite rhodamine 123 and 505–530 nm filter was used for emission. To investigate inertial focusing in SGS, AHS and IAHS microchannels, these microfluidic chips were fabricated. Fig. 1A and B shows the schematic drawings of the microfluidic chips and Fig. 1C shows the top view images of the microfluidic chips. SGS, AHS and IAHS in PDMS were fabricated by the mold masters with two-layer features. The heights of the SGS, AHS, IAHS and main channels are all 50 lm. The width of SGS is 200 lm. AHS can be considered as the combination of two types of SGS with different widths 200 lm and 100 lm. AHS is asymmetric. Four AHS are integrated to form IAHS. Therefore, the widths of IAHS is 1200 lm (4 300 lm) (Fig. 2B and C I–III). At first, the movement of cells was studied in SGS microchannels with Y type inlets. When the flow rate of Inlet 1 and Inlet 2 were 10 ll/h and 70 ll/h, respectively, cells were injected through Inlet 1 and flowed along Line 1 (Fig. 2A) due to laminar flow in the microchannel. The cells lifted into the grooves, slipped along the grooves, descended into the microchannels and traversed the microchannel (Fig. 2B along Line 1 and Video S1). When the flow rates of Inlet 1 and Inlet 2 were 70 ll/h and 10 ll/h, respectively, cells were injected through Inlet 2 and then flowed along Line 2 (Fig. 2A) due to laminar flow in the microchannel. The cells traversed the microchannel (Fig. 2B alone Line 2 and Video S2), lifted into the grooves, slipped along the grooves and descended into the microchannels. When the total flow rates were 160 ll/h, 240 ll/h and 320 ll/h, respectively, the movement of cells was similar with those at 80 ll/h. Abraham D. Stroock found that slant groove structures on the top of the microchannel made flows traverse and circulate back in the grooves, so that helical streamlines were generated in a SGS microchannel (Fig. 2C) [16]. Therefore, the movement of cells in the SGS microchannel was caused by helical streamlines. The vortex motion as we observed could enhance the contact of cells with the microchannels, which could promote cell capture. However, cell inertial focusing appeared at downstream. Inertial focusing was observed in the SGS microchannel (about 2.5 mm away from inlets). Fig. 3B shows cell focusing occurred at 80 ll/h, 160 ll/h, 240 ll/h and 320 ll/h in SGS microchannels. Although some papers report the utility of SGS microchannels for cell separation, the inertial focusing and its principle in SGS microchannels have not been observed and explored. Particles in a channel flow could experience shear gradient force and wall effect force [7]. The balance of the two forces generates equilibrium positions, where particle focusing occurs. For example, in square channels, the symmetry of the system, there are four equilibrium regions, where shear gradient force are equal to wall effect force (Fig. 3D I). In a curved channel, two counter-rotating vortices perpendicular to the primary flow direction, called Dean flow (Fig. 3D II), were created due to inertia1. The two vortices generate drag force, namely Dean force, exerted on particles. Therefore, in a spiral channel, inertial focusing was the result of the interaction of shear gradient force, wall effect force and Dean force. In SGS microchannels, the helical flow, generating a vortice in the cross section of SGS microchannels, exerts drag force on particles. Therefore, it is reasonable to speculate that cell focusing in SGS microchannels was the result of the equilibrium of shear
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Fig. 2. Cell movement and flows in a SGS microchannel with Y type inlets. (A) When the flow rate of Inlet 1 and Inlet 2 were 10 ll/h and 70 ll/h, respectively, cells were injected through Inlet 1 and flowed along Line 1 (indicated by Line 1). When the flow rates of Inlet 1 and Inlet 2 were 70 ll/h and 10 ll/h, respectively, cells were injected through Inlet 2 and then flowed along Line 2 (indicated by Line 2). (B) When cells flowed into the microchannel along Line 1, they lifted into slant grooves on the top of the microchannel, slipped along the grooves, indicated by black arrows (up), descended into the microchannel and traversed the microchannel. When cells flowed through Inlet 2 into the SGS microchannel, they flowed along Line 2, they traversed the microchannel, indicated by the black arrow (down), lifted into slant grooves, slipped along the grooves and descended into the microchannel. (C) Indicates a helical streamline in the SGS microchannels. Scale bar: 200 lm.
gradient force, wall effect force and drag force, exerted by a vortice in SGS microchannels (Fig. 3D III). To explore the principle of cell focusing in SGS microchannels, a commercial computational fluid dynamics software FLUENT was used to simulate 3D velocity field in SGS microchannels [17]. To verify the simulation, the average velocities of the lateral flow obtained from experiments were compared with those from simulation (Table S1). The results from the simulation and the experiment were similar to each other. Therefore, the simulation was reliable.
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position across the channel cross-section assuming an average value of 0.5 and ap is the average diameter of cells [18]. Fd is the drag force exerted on cells due to a vortice in SGS microchannels, which can be obtained by assuming the Strokes drag [19].
F d ¼ 3plU D ap
Fig. 3. Cell focusing in SGS and AHS microchannels. (A) Bright field images of SGS (Left) and AHS (Right) microchannels. (B) I–IV HepG2 cells were stained by Celltracker (Green fluorescence, Invitrogen). Cell focusing, indicated by green fluorescence, occur in SGS microchannels at 80, 160, 240 and 320 ll/h, respectively. (C) I–IV Cell focusing, indicated by green fluorescence, occur in AHS microchannels at 120, 240, 360 and 480 ll/h. (D) I In a square channel, there are four equilibrium positions, where shear gradient force are equal to wall effect force. II In a curved channel, due to inertia, two counter-rotating vortices are generated. The two vortices exert drag force on particles. III Due to the equilibrium of shear gradient force, shear gradient force and drag force, cell focusing occurs in SGS microchannels. Because flow fields were symmetrical in SGS microchannels, there would be two equilibrium positions. However, the equilibrium position (indicated by dashed line) disappears due to the obstacle of the slant grooves to cell movement. Scale bar: 100 lm.
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In SGS microchannels, cells experienced Fs+w and Fd. Fs+w is the combination of shear gradient force and wall effect force. Asomolov derived a function describing the magnitude of Fs+w.
194 2
C L a4p
196
F sþw ¼ qG
197
where q is the density of fluid medium (kg/m3), G is the shear rate of the fluid, CL is a coefficient, which is a function of the particle
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ð1Þ
ð2Þ
where p is the fluid viscosity (kg/ms) and Uy is the average velocity of lateral flow (Fig. S1A). According to the data from the simulation of the flow fields, the forces exerted on cells were calculated and list in Table 1. Fs+w and Fd had the similar magnitude. Therefore, in SGS microchannels, cells could be focused at the flow rates due to the combination of the two forces. Because of the symmetrical distribution of flow fields in SGS microchannels, there would be another equilibrium position (indicated by dashed line in Fig. 3D III). However, in our experiment, only one equilibrium position was observed in SGS microchannels. The reason is that cells changed their velocity and slipped in the grooves due to the obstacle of the grooves (Fig. 3A left and B). This caused the change of Fs+w and Fd and broke the equilibrium of Fs+w and Fd. Chia-Hsien Hsu fabricated symmetric herringbone structure (SHS) microchannels for particle focusing. In the work, focusing largely depended on the symmetry of microchannels [15]. However, according to our analysis, the symmetry of microchannels was not requisite. Therefore, asymmetric-herringbone-structure (AHS) microchannel was fabricated to demonstrate that inertial focusing was possible in an asymmetric structures (Fig. 1B II and C II). Fig. 3A (right) and C show that there are two focusing lines at four flow rates, although AHS microchannel is not symmetrical. According to the data from the simulation of the flow fields, Fs+w and Fd had the same magnitude (Table 2). This explained why focusing occurred in AHS. This finding has not reported yet. Multiple focusing can increase the throughput of cell sorting and save time. Therefore, integrated-asymmetric-herringbonestructure (IAHS) microchannels (Fig. 1B III and C III) were fabricated to demonstrate that it is easy to obtain multiple focusing in IAHS microchannels. At 480, 960, 1440 and 1920 ll/h, 2.5 mm away from the inlets, multiple focusing was observed (Video S3 480, 960, 1440 and 1920). The number of focusing positions is the same as the number of integrated SGS. 8 SGS were integrated to construct IAHS, so there were 8 focusing positions in IAHS microchannels.
Table 1 Summary of Fs+w and Fd in SGS microchannels. Fluid rate (ll/h)
Fs+w (1011 N)
Fd (1011 N)
80 160 240 320
1.54 6.17 13.89 24.69
1.90 3.38 5.76 7.67
Table 2 Summary of Fs+w and Fd in AHS microchannels. Fluid rate (ll/h)
Fd (w2) (1011 N)
Fd (w1) (1011 N)
Fs+w (1011 N)
120 240 360 480
1.99 3.98 5.97 7.76
1.69 3.39 5.07 6.78
0.339 1.34 3.01 5.43
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In this work, we found that cell focusing occurred in SGS and ASHS microchannels. The flow fields were simulated. Fs+w and Fd were both calculated. These results explained why focusing occurred in these microchannels. These results have not reported yet. Furthermore, using IAHS microchannels, we found that multiple focusing could be easily achieved by the integration of slant groove structures in microchannels. In future, with the combination of antibody modification on the surface of microchannels, these microchannels could be applied for cell separation, such as cancer stem cell separation and circulating tumor cell separation.
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Acknowledgements
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We thank Professor Michalael M. Yang for paper writing and organizing. This work was supported by grants from the National Key Scientific Research Program (973 Program No. 2012 CB933302), the National Natural Science Foundation of China (Grant No. 81201689), Knowledge Innovation Program of Shenzhen Municiple Government (JCYJ20140419115507575), Doctor Startup Fund of Liaoning Province (Grant No. 20121053), Program Funded by Liaoning Province Education Administration (Grant No. L2014360), the Research Grants Council of the Hong Kong Special Administrative Region, China (Project No. CityU_ 103312, CRF Project No. CityU9/CRF/13G), State Key Laboratory of Environmental and Biological Analysis and Strategic Development Fund of HKBU (SKLP_14-15_P008) and Aohongboze Medicine-Pharmacy Innovation Foundation, the President Fund of Liaoning Medical University.
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Appendix A. Supplementary data
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Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.mee.2015.04.051.
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