- Email: [email protected]
Electrokinetic Separation of Polystyrene Microspheres in Conductive Media on a Microfluidic Chip
CHINESE JOURNAL OF ANALYTICAL CHEMISTRY Volume 43, Issue 2, February 2015 Online English edition of the Chinese language journal
Cite this article as: Chin J Anal Chem, 2015, 43(2), 176–180.
RESEARCH PAPER
Electrokinetic Separation of Polystyrene Microspheres in Conductive Media on a Microfluidic Chip SONG Ning-Ning1, ZHANG Hao1, LI Jin-Bo1, ZHEN Jun-Hui2,*, GAO Jian1,* 1 2
Department of Chemistry, Qilu University of Technology, Jinan 250353, China School of Medicine, Shandong University, Jinan 250012, China
Abstract:
A polydimethylsiloxane (PDMS)/glass microfluidic chip consisting of a three-layer sandwich structure and a
three-parallel micro-electrode system was fabricated for the separation of polystyrene microspheres depending on different the particle size in high conductivity solution by electric field that produced by alternating current. The principle of electrokinetics of microspheres directional movement was investigated in the experiment. The results showed that when the applied voltage was 14 V at 100 kHz, the separation efficiency for the 10-μm and 25-μm polystyrene microspheres was the best. Similarly, with a voltage of 10 V at 2 MHz, the separation efficiency for the 5-μm and 25-μm polystyrene microspheres could achieve the highest. Meanwhile, 11 V at 1 MHz was used for the quick and efficient separation of 5-μm, 10-μm and 25-μm polystyrene microspheres. The separation efficiency of the three groups were all above 90% evenly. At the same time, the formation of the laminar region in the middle of the electrode gap played a key role in the microsphere separation. Key Words:
1
Microfluidic chip; Electrokinetics; High-conductivity; Polystyrene microspheres
Introduction
AC electric field can affect microfluid or charged particles through microelectrode and produce a variety of electrical characteristics[1‒3], such as dielectrophoresis (DEP)[4,5], electrophoresis (EP)[6,7], electroosmosis (EO)[8,9] and AC electrothermal flow (ACEF)[10,11], and so on. Electrokinetics is one of the most powerful approaches for microfluidic manipulation. The past studies on controlling micro- and nano-scale biological targets with electric mainly focused on low conductivity solutions (< 0.01 S m‒1), especially relied on the effect of dielectrophoresis (DEP)[12‒14]. However, most of the physiological fluids had high conductivities (about 1 S m‒1), which required samples pretreatment to adjust the conductivity to a lower level (about 0.01 S m‒1). Such sample treatment may cause the loss of target cell or diluted, reducing the detection sensitivity. In addition, it was difficult to exert the characteristics of other electrokinetics, limiting the use of
the method. In order to operate and detect the targets of biological fluids directly, the study for the laws of electrokinetics manipulation on the targets with different sizes in high conductivity media has important significance for ultimately achieving the efficient electrokinetic manipulation of biological samples. The time-averaged force in the dipole approximation is as follows[2,15]: FDEP = πa3εmRe(fCM)|E|2 (1) * * * * fCM = (εP ‒ εm )/(εP + 2εm ) (2) ε* = ε ‒ jσ/w, j2 = ‒1 (3) where, a is the particle radius, εm is the permittivity of the suspending medium, Re(fCM) is the real part of the Clausius-Mos-sotti (CM) factor, E is the electric field, εp is the permittivity of the particle, σ is the conductivity and ω is the angular frequency of the applied electric field. Re(fCM) is one of the factors in determining the DEP force and represents the dielectric characteristics of the particle and suspending solution in electric field at different frequencies.
________________________ Received 8 September 2014; accepted 25 October 2014 *Corresponding author. Email: [email protected];[email protected] This work is sponsored by the National Natural Science Foundation of China (Nos. 21375068, 81400729), the Scientific Research Foundation for Outstanding Young Scientists of Shandong Provence, China (No. BS2012SW017), and the Basic Research Foundation of Shandong University, China (No. 2014JC033). Copyright © 2015, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences. Published by Elsevier Limited. All rights reserved. DOI: 10.1016/S1872-2040(15)60801-5
SONG Ning-Ning et al. / Chinese Journal of Analytical Chemistry, 2015, 43(2): 176–180
When the Re(fCM) is positive, the particles are more polarizable than the surrounding media, and the DEP force will drive the particles in solution towards the regions with high electric field strength (generally towards the electrodes). This phenomenon is known as positive dielectrophoresis (pDEP). Conversely, it is called negative dielectrophoresis (nDEP)[16,17]. Equations (2) and (3) show that Re(fCM) will always be negative if εm >100 mS m‒1[15], therefore, the particles experience negative DEP in high conductivity solutions. In addition, the previous studies found that multiple electrokinetic phenomena could exist and work together at the same time in conductive biological fluids. Generally, ACEF is long-range hydrodynamic effect, and it can entrain target particles far away from the bulk solution toward the electrode surface. However, EP and DEP are short-range effects, and are more effective in the vicinity of the electrode[18,19]. Theoretically, the electrokinetics have different effects for different solutions and different types of particles[3], thus, the separation and capture of the targets with different sizes can be achieved. Currently, the separation of different size particles in high conductivity solutions has not been researched systematically. Since the density and size of polystyrene (PS) microspheres are similar with cells, PS can be used as the replacement of cells of different kinds and sizes. In this study, the PS microspheres with different sizes were used as the research object. Under the static conditions, the electric conditions and theories of the separation of different size PS microspheres by hybrid electrokinetic technique combined with EP, DEP and ACEF in microfluidic systems was investigated, and a theoretical basis for the separation and capture of physiological samples was provided. Under high conductivity conditions, conventional Au-Cr microelectrode was difficult to adapt to the environment of high conductivity, and easy to be electrolyzed off under the experimental conditions. However, a modified Ti-Au-Ti sandwich electrode[18,19] could work well for a long time in the high conductivity solutions under the experimental conditions (< 20 V, AC). Microfluidic chips based on polydimethylsiloxane (PDMS) have the advantages such as convenient preparation, durable and so on. In this study, the PS microspheres were evenly dispersed into
aa a
the conductive solution (10 × TAE, about 1.59 S m‒1), and the separation of PS microspheres with different sizes was carried out by hybrid electrokinetic technique with PDMS/glass microfluidic chip as the operating platform.
2
Experimental
2.1
Preparation of microfluidic chip
As shown in Fig.1a, the microfluidic chip with 3 sets of 3-parallel-electrodes arranged in series had an inlet and an outlet. The electrode was fabricated by sputtering 30-nm titanium, 30-nm gold and 30-nm titanium on a glass substrate and patterned by lift-off. The length and distance of each electrode were 2.5 mm and 125 μm, the width of the top and bottom electrode were 100 μm and the center electrode were 50 μm. The microfluidic channels were fabricated by molding poly-dimethylsiloxane (PDMS) at 70 °C for 2 h. PDMS channels and the glass substrates with microelectrode arrays were sealed by plasma treatment (PDC-32G, Harrick Plasma). A function generator (SDG1020, Siglent) was used to supply the voltage signals. 2.2
Instruments and reagents
In this research, polystyrene microspheres were used as separating targets. Polystyrene microspheres of 5, 10 and 25 μm were purchased from Micromod (Micromod, GER). Before the experiment, the polystyrene microspheres were diluted with Tris-acetate-EDTA (TAE, Sigma, USA) buffer to reached to the appropriate concentrations. The conductivity of the TAE buffer was 1.59 Sm‒1 approximately. The electrokinetic chip was placed in a digital inverted microscope (XD-101, Nanjing Jiangnan Novel Optics Co., Ltd). A lamp was used to provide illumination for observations. The separation polystyrene microspheres were performed under the microscope with a magnification of 100. The processes of particle manipulation were recorded by a CCD camera and directly digitized into a video capture system for the analysis of the velocity of the particles. b
Fig.1 Schematic diagram of microfluidic chips (a) and schematic diagram of hybrid electrokinetic (b)
SONG Ning-Ning et al. / Chinese Journal of Analytical Chemistry, 2015, 43(2): 176–180
3 3.1
Results and discussion Microspheres separation on chip
As has been noted, if εm >100 m Sm‒1, the Re(fCM) < 0 and the particles experience negative DEP in high conductivity solutions and are pushed away to the regions with weak electricfield strengths; while ACEF will generating annular liquid flow on the top of the electrodes[20]. In the experiments, it also found that after a certain DC bias voltage was applied, the moving speed of the microspheres would increase, demonstrating the involvement of EP in hybrid electrokinetics and the enhance effect of EP on hybrid electrokinetic. Therefore, multiple electrokinetic phenomena can exist and work together in conductive biological fluids. After the electric field was applied, 5 and 10 μm microspheres near the electrode were pushed away from the electrode by ACEF, DEP and EP. Figure 1b shows that the annular liquid flow caused by ACEF exists on top of the parallel and adjacent electrodes, and in the opposite direction. Meanwhile, there was a laminar flow region with a weak ACEF in the gap between the adjacent electrodes. In the region, the ACEF effect was weak or even without effect on the particles. The dashed box in Fig.1b showed the laminar flow region within a certain voltage range. When 5-μm and 10-μm microspheres moved into this area, they were easy to stay in the region due to the smaller size, and could not be entrained out by ACEF. At the same time, the microspheres also experienced the effects of ACEF, DEP and EP that came from the opposite electrode, and the majority of them gathered in the area in the gap of the electrodes. At this area, ACEF, DEP and EP were balanced (Fig.2c and Fig.2f). Under the same conditions, 25-μm microspheres near the electrode were also pushed away from the electrode by ACEF, DEP and EP. However, 25-μm microspheres could not stay in the laminar flow region due to its larger size, and entrained by the long-range hydrodynamic
effect of ACEF toward the surface of the center electrode, where DEP and EP were most effective. With the increase of DEP and EP force, the microspheres were pushed away from the electrode, the rate of 25-μm microspheres gradually slowed down and the microspheres were eventually captured on the top of the center electrode until the DEP, EP and ACEF force achieved a balance (shown in Fig.2c and Fig.2f, it is invisible because of the occlusion of the electrode). For the separation of 10-μm and 25-μm microspheres, Fig.2a‒2c showed that the separation was completed in 15 s after the electric field was applied. Similarly, the same results were obtained for the separation of 5-μm, 10-μm and 25-μm microspheres (Fig.2d and Fig.2f) in 18 s. As shown in Fig.2, except individual 25 μm microspheres could not move due to the adhesion, the vast majority of the microspheres could move directly, and the separation was achieved with an efficiency of around 90%. Since the electrokinetics discussed in the experiment all depended on the size of microspheres (FDEP ~ a3, FACEF ~ 1/a3, FEP ~ a), the size of microspheres was an important factor of the separation. 3.2
Influence of frequency
In the voltage frequency range from 100 kHz to 10 MHz, the effect of frequency on the directional manipulation of PS microspheres was studied. As can be seen from Equation (1), the frequency is an effective parameter for adjusting the strength of DEP. Similarly, it also affects ACEF (FACEF-M(ω,T)). Therefore, at different frequencies, the total force of ACEF, EP and DEP is different, and the greater the force, the faster movement of the microspheres. So the effect of frequency on the microspheres could be observed by investigating the moving speed of the microspheres. As shown in Fig.3a, with 25-μm microspheres, for example, the frequency have the same influence on microspheres in different positions, and in
Fig.2 (a‒c) Time lapse images for visualizing the movement of 10-μm and 25-μm polystyrene microspheres at voltage of 14 Vp-p at 100 kHz with 0.3 V DC offset; (d‒f) Time lapse images for visualizing the movement of 5-μm, 10-μm and 25-μm polystyrene microspheres at voltage of 11 Vp-p at 1 MHz with 1.0 V DC offset
SONG Ning-Ning et al. / Chinese Journal of Analytical Chemistry, 2015, 43(2): 176–180
Fig.3 Frequency dependence of velocity at different locations from the center electrode. Among them, (a) was 25-μm polystyrene microspheres in 10-μm and 25-μm microsphere mixture; (b) was 25-μm polystyrene microspheres in 5-μm and 25-μm microsphere mixture
the same position, the speed of the microspheres reached the maximum when the frequency was set at 100 kHz. The result showed that the resultant force effect on 25-μm microspheres was the largest at 100 kHz. It also could be concluded that the times of 25-μm microspheres moving to the center electrodes at 100 kHz were the shortest. The same analysis could also be applied to 10-μm microspheres in the solution. The experiments also found that the 25-μm microspheres far from the electrode stopped at the gap of the electrode before they reached the center electrode when the microspheres velocity was too small, reducing the separation efficiency. Therefore, the greater the velocity of the microspheres, the greater the resultant force they suffered. As the velocity of the microspheres increased, the 25-μm microspheres far from the electrode were captured on the center electrode, showing an increasing separation efficiency. The results indicated that the voltage frequency of 100 kHz was the optimal conditions for rapid and effective separation. Similarly, for the separation of 5-μm and 25-μm microspheres, as shown in Fig.3b, the separation efficiency was maximized at 2 MHz. Within the same frequency range, 5-μm, 10-μm and 25-μm microspheres could also be separated well, and the separation efficiency was maximized at 1 MHz. These results indicated that the optimum frequency could be
achieved for microspheres separation by optimize the frequency, and the frequency was another important factor for different sizes microspheres separation. 3.3
Influence of voltage
The voltage effect on the PS microspheres separation efficiency was investigated in the experiment. In voltage range of 7–14 Vp-p, it was found that the PS microspheres separation efficiency increased with the increasing voltage. This is because ACEF, DEP and EP force increased with the increasing voltage (FACEF ~ E4, FDEP ~ E2, FEP ~ E), thus the resultant force and its scope expanded, which made ACEF entrain more 25-μm microspheres far from the center electrodes toward the surface of the center electrode and microspheres captured on the center electrode increase, showing an increasing separation efficiency. According to the results in Section 3.2, the microsphere velocity depended on the resultant force and the microspheres separation efficiency. As shown in Fig.4, the optimum voltage was set at 14 V for the separation of 10-μm and 25-μm microspheres, and when the voltage was greater than 14 V, the 10-μm microspheres moved to the electrodes and could not be separated. This was because the increasing effect scope of ACEF caused by the
Fig.4 Voltage dependence of 25 μm polystyrene microspheres velocity. (a) 25 μm polystyrene microspheres in 10 and 25 μm mixture; (b) 25 μm polystyrene microspheres in 5 and 25μm mixture
SONG Ning-Ning et al. / Chinese Journal of Analytical Chemistry, 2015, 43(2): 176–180
increasing voltage made the laminar flow region mentioned described in Section 3.1 fall or even disappear, resulting in 10 μm microspheres were also entrained by ACEF toward the areas near the electrode, and the equilibrium position of 10-μm microspheres was changed, resulting in a bad separation. The same analysis was suitable for 5-μm microspheres. The results also showed that the optimum voltage for the separation of 5-μm and 25-μm microspheres was 10 V, and for 5-μm, 10-μm and 25-μm microspheres were 11 V. The results suggested that the voltage was another important factor affecting the separation efficiency. Although the higher voltage had higher separation efficiency, there was a maximum voltage limit, and exceeded the limit voltage, the equilibrium position of the smaller microspheres would change and the separation efficiency would reduce. These results also illustrated that the separation of the microspheres with different sizes were carried out by a combination effect of ACEF, DEP and EP.
provided a theoretical basis for the research of actual samples.
References [1]
Wong P K, Wang T H, Deval J H, Ho C M. Ieee-asme T. Mech., 2004, 9(2): 366‒376
[2]
Sin M L Y, Shimabukuro Y, Wong P K. Nanotechnology, 2009, 20(16): 165701
[3]
Li D Q. Electrokinetics in Microfluidics, Academic Press, 2004, Chapter 8
[4]
Hardt
S,
Schönfeld
F.
Microfluidic
Technologies
for
Miniaturized Analysis Systems. Springer ,2007: 315‒317 [5]
Sin M L Y, Gau V, Liao J C, Haake D A, Wong P K. J. Phys. Chem. C., 2009, 113: 6561–6565
[6]
Hardt
S,
Schönfeld
F.
Microfluidic
Technologiesfor
[7]
Gao J, Yin X F, Fang Z L. Lab Chip , 2004, 4: 47‒52
[8]
Sun Y, Lim C S, Liu A Q, Ayi T C, Yap P H. Sens. Actuators A:
Miniaturized Analysis Systems. Springer, 2007: 393‒395
Phys., 2007, 133(2): 340‒348
4
Conclusions
[9]
Takamura Y, Onoda H, Inokuchi H, Adachi S, Oki A, Horiike Y. Electrophoresis, 2003, 24: 185‒192
In summary, under the optimum conditions, a hybrid electrokinetic technique combined with DEP, EP and ACEF was used to separate the microspheres with different sizes in conductive solution on the microfluid chip, and its theories of the separation was studied and discussed. Meanwhile the microspheres size, frequency and voltage as the important factors for the separation were also optimized. In particular, under certain conditions, there was a laminar flow region in the gap of the adjacent electrodes, in which the fluid was relatively stationary, and ACEF was weaker and evenly could not affect the movement of microparticles in this region. Thus the microspheres with smaller size (< 10 μm) were easy to stay in the regions, and could not be entrained out by ACEF, in the meantime, the larger size microspheres (25 μm) entrained out by ACEF toward the surface of the center electrode due to its bigger size in the laminar flow region, thus achieved the separation of different sizes microspheres. By separating the PS microspheres of different sizes, the operating rules were found for the hybrid electrokinetic effect on different sizes microspheres and the function area in high conductivity solution. The results
[10] Feldman H C, Sigurdson M, Meinhart C D. Lab Chip, 2007, 7: 1553‒1559 [11] Mandy L Y S, Vincent G, Joseph C L, Pak Kin W. J. Assoc. Lab. Autom., 2010, 15: 426‒432 [12] Li Y, Dalton C, Crabtree H J, Nilsson G, Kaleret K V I S. Lab Chip, 2007, 7(2): 239‒248 [13] Doh I, Cho Y H. Sens. Actuators A: Phys., 2005, 121(1): 59‒65 [14] Cheung K, Gawad S, Renaud P. Cytometry Part A, 2005, 65A(2): 124‒132 [15] Krishnan R, Sullivan B D, Mifflin R L, Esener S C, Heller M J. Electrophoresis, 2008, 29(9): 1765‒1774 [16] Lin R Z, Ho C T, Liu C H, Chang H Y. Biotechnol. J., 2006, 1(9): 949‒957 [17] Xu J, Zhao Z, Fang Z, Liu Y H, Du L D,Geng D Q. Chinese J. Anal. Chem., 2011, 39(3): 295‒299 [18] Gao J, Sin M L Y, Liu T, Gau V, Liao J C, Wong P K. Lab Chip, 2011, 11(10): 1770‒1775 [19] Gao J, Riahi R, Sin M L Y, Zhang S, Wong P K. Analyst, 2012, 137(22): 5215‒5221 [20] Gonzalez A, Ramos A, Morgan H, Green N G, Castellanos A. J. Fluid Mech., 2006, 564: 415‒433
Cite this article as: Chin J Anal Chem, 2015, 43(2), 176–180.
RESEARCH PAPER
Electrokinetic Separation of Polystyrene Microspheres in Conductive Media on a Microfluidic Chip SONG Ning-Ning1, ZHANG Hao1, LI Jin-Bo1, ZHEN Jun-Hui2,*, GAO Jian1,* 1 2
Department of Chemistry, Qilu University of Technology, Jinan 250353, China School of Medicine, Shandong University, Jinan 250012, China
Abstract:
A polydimethylsiloxane (PDMS)/glass microfluidic chip consisting of a three-layer sandwich structure and a
three-parallel micro-electrode system was fabricated for the separation of polystyrene microspheres depending on different the particle size in high conductivity solution by electric field that produced by alternating current. The principle of electrokinetics of microspheres directional movement was investigated in the experiment. The results showed that when the applied voltage was 14 V at 100 kHz, the separation efficiency for the 10-μm and 25-μm polystyrene microspheres was the best. Similarly, with a voltage of 10 V at 2 MHz, the separation efficiency for the 5-μm and 25-μm polystyrene microspheres could achieve the highest. Meanwhile, 11 V at 1 MHz was used for the quick and efficient separation of 5-μm, 10-μm and 25-μm polystyrene microspheres. The separation efficiency of the three groups were all above 90% evenly. At the same time, the formation of the laminar region in the middle of the electrode gap played a key role in the microsphere separation. Key Words:
1
Microfluidic chip; Electrokinetics; High-conductivity; Polystyrene microspheres
Introduction
AC electric field can affect microfluid or charged particles through microelectrode and produce a variety of electrical characteristics[1‒3], such as dielectrophoresis (DEP)[4,5], electrophoresis (EP)[6,7], electroosmosis (EO)[8,9] and AC electrothermal flow (ACEF)[10,11], and so on. Electrokinetics is one of the most powerful approaches for microfluidic manipulation. The past studies on controlling micro- and nano-scale biological targets with electric mainly focused on low conductivity solutions (< 0.01 S m‒1), especially relied on the effect of dielectrophoresis (DEP)[12‒14]. However, most of the physiological fluids had high conductivities (about 1 S m‒1), which required samples pretreatment to adjust the conductivity to a lower level (about 0.01 S m‒1). Such sample treatment may cause the loss of target cell or diluted, reducing the detection sensitivity. In addition, it was difficult to exert the characteristics of other electrokinetics, limiting the use of
the method. In order to operate and detect the targets of biological fluids directly, the study for the laws of electrokinetics manipulation on the targets with different sizes in high conductivity media has important significance for ultimately achieving the efficient electrokinetic manipulation of biological samples. The time-averaged force in the dipole approximation is as follows[2,15]: FDEP = πa3εmRe(fCM)|E|2 (1) * * * * fCM = (εP ‒ εm )/(εP + 2εm ) (2) ε* = ε ‒ jσ/w, j2 = ‒1 (3) where, a is the particle radius, εm is the permittivity of the suspending medium, Re(fCM) is the real part of the Clausius-Mos-sotti (CM) factor, E is the electric field, εp is the permittivity of the particle, σ is the conductivity and ω is the angular frequency of the applied electric field. Re(fCM) is one of the factors in determining the DEP force and represents the dielectric characteristics of the particle and suspending solution in electric field at different frequencies.
________________________ Received 8 September 2014; accepted 25 October 2014 *Corresponding author. Email: [email protected];[email protected] This work is sponsored by the National Natural Science Foundation of China (Nos. 21375068, 81400729), the Scientific Research Foundation for Outstanding Young Scientists of Shandong Provence, China (No. BS2012SW017), and the Basic Research Foundation of Shandong University, China (No. 2014JC033). Copyright © 2015, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences. Published by Elsevier Limited. All rights reserved. DOI: 10.1016/S1872-2040(15)60801-5
SONG Ning-Ning et al. / Chinese Journal of Analytical Chemistry, 2015, 43(2): 176–180
When the Re(fCM) is positive, the particles are more polarizable than the surrounding media, and the DEP force will drive the particles in solution towards the regions with high electric field strength (generally towards the electrodes). This phenomenon is known as positive dielectrophoresis (pDEP). Conversely, it is called negative dielectrophoresis (nDEP)[16,17]. Equations (2) and (3) show that Re(fCM) will always be negative if εm >100 mS m‒1[15], therefore, the particles experience negative DEP in high conductivity solutions. In addition, the previous studies found that multiple electrokinetic phenomena could exist and work together at the same time in conductive biological fluids. Generally, ACEF is long-range hydrodynamic effect, and it can entrain target particles far away from the bulk solution toward the electrode surface. However, EP and DEP are short-range effects, and are more effective in the vicinity of the electrode[18,19]. Theoretically, the electrokinetics have different effects for different solutions and different types of particles[3], thus, the separation and capture of the targets with different sizes can be achieved. Currently, the separation of different size particles in high conductivity solutions has not been researched systematically. Since the density and size of polystyrene (PS) microspheres are similar with cells, PS can be used as the replacement of cells of different kinds and sizes. In this study, the PS microspheres with different sizes were used as the research object. Under the static conditions, the electric conditions and theories of the separation of different size PS microspheres by hybrid electrokinetic technique combined with EP, DEP and ACEF in microfluidic systems was investigated, and a theoretical basis for the separation and capture of physiological samples was provided. Under high conductivity conditions, conventional Au-Cr microelectrode was difficult to adapt to the environment of high conductivity, and easy to be electrolyzed off under the experimental conditions. However, a modified Ti-Au-Ti sandwich electrode[18,19] could work well for a long time in the high conductivity solutions under the experimental conditions (< 20 V, AC). Microfluidic chips based on polydimethylsiloxane (PDMS) have the advantages such as convenient preparation, durable and so on. In this study, the PS microspheres were evenly dispersed into
aa a
the conductive solution (10 × TAE, about 1.59 S m‒1), and the separation of PS microspheres with different sizes was carried out by hybrid electrokinetic technique with PDMS/glass microfluidic chip as the operating platform.
2
Experimental
2.1
Preparation of microfluidic chip
As shown in Fig.1a, the microfluidic chip with 3 sets of 3-parallel-electrodes arranged in series had an inlet and an outlet. The electrode was fabricated by sputtering 30-nm titanium, 30-nm gold and 30-nm titanium on a glass substrate and patterned by lift-off. The length and distance of each electrode were 2.5 mm and 125 μm, the width of the top and bottom electrode were 100 μm and the center electrode were 50 μm. The microfluidic channels were fabricated by molding poly-dimethylsiloxane (PDMS) at 70 °C for 2 h. PDMS channels and the glass substrates with microelectrode arrays were sealed by plasma treatment (PDC-32G, Harrick Plasma). A function generator (SDG1020, Siglent) was used to supply the voltage signals. 2.2
Instruments and reagents
In this research, polystyrene microspheres were used as separating targets. Polystyrene microspheres of 5, 10 and 25 μm were purchased from Micromod (Micromod, GER). Before the experiment, the polystyrene microspheres were diluted with Tris-acetate-EDTA (TAE, Sigma, USA) buffer to reached to the appropriate concentrations. The conductivity of the TAE buffer was 1.59 Sm‒1 approximately. The electrokinetic chip was placed in a digital inverted microscope (XD-101, Nanjing Jiangnan Novel Optics Co., Ltd). A lamp was used to provide illumination for observations. The separation polystyrene microspheres were performed under the microscope with a magnification of 100. The processes of particle manipulation were recorded by a CCD camera and directly digitized into a video capture system for the analysis of the velocity of the particles. b
Fig.1 Schematic diagram of microfluidic chips (a) and schematic diagram of hybrid electrokinetic (b)
SONG Ning-Ning et al. / Chinese Journal of Analytical Chemistry, 2015, 43(2): 176–180
3 3.1
Results and discussion Microspheres separation on chip
As has been noted, if εm >100 m Sm‒1, the Re(fCM) < 0 and the particles experience negative DEP in high conductivity solutions and are pushed away to the regions with weak electricfield strengths; while ACEF will generating annular liquid flow on the top of the electrodes[20]. In the experiments, it also found that after a certain DC bias voltage was applied, the moving speed of the microspheres would increase, demonstrating the involvement of EP in hybrid electrokinetics and the enhance effect of EP on hybrid electrokinetic. Therefore, multiple electrokinetic phenomena can exist and work together in conductive biological fluids. After the electric field was applied, 5 and 10 μm microspheres near the electrode were pushed away from the electrode by ACEF, DEP and EP. Figure 1b shows that the annular liquid flow caused by ACEF exists on top of the parallel and adjacent electrodes, and in the opposite direction. Meanwhile, there was a laminar flow region with a weak ACEF in the gap between the adjacent electrodes. In the region, the ACEF effect was weak or even without effect on the particles. The dashed box in Fig.1b showed the laminar flow region within a certain voltage range. When 5-μm and 10-μm microspheres moved into this area, they were easy to stay in the region due to the smaller size, and could not be entrained out by ACEF. At the same time, the microspheres also experienced the effects of ACEF, DEP and EP that came from the opposite electrode, and the majority of them gathered in the area in the gap of the electrodes. At this area, ACEF, DEP and EP were balanced (Fig.2c and Fig.2f). Under the same conditions, 25-μm microspheres near the electrode were also pushed away from the electrode by ACEF, DEP and EP. However, 25-μm microspheres could not stay in the laminar flow region due to its larger size, and entrained by the long-range hydrodynamic
effect of ACEF toward the surface of the center electrode, where DEP and EP were most effective. With the increase of DEP and EP force, the microspheres were pushed away from the electrode, the rate of 25-μm microspheres gradually slowed down and the microspheres were eventually captured on the top of the center electrode until the DEP, EP and ACEF force achieved a balance (shown in Fig.2c and Fig.2f, it is invisible because of the occlusion of the electrode). For the separation of 10-μm and 25-μm microspheres, Fig.2a‒2c showed that the separation was completed in 15 s after the electric field was applied. Similarly, the same results were obtained for the separation of 5-μm, 10-μm and 25-μm microspheres (Fig.2d and Fig.2f) in 18 s. As shown in Fig.2, except individual 25 μm microspheres could not move due to the adhesion, the vast majority of the microspheres could move directly, and the separation was achieved with an efficiency of around 90%. Since the electrokinetics discussed in the experiment all depended on the size of microspheres (FDEP ~ a3, FACEF ~ 1/a3, FEP ~ a), the size of microspheres was an important factor of the separation. 3.2
Influence of frequency
In the voltage frequency range from 100 kHz to 10 MHz, the effect of frequency on the directional manipulation of PS microspheres was studied. As can be seen from Equation (1), the frequency is an effective parameter for adjusting the strength of DEP. Similarly, it also affects ACEF (FACEF-M(ω,T)). Therefore, at different frequencies, the total force of ACEF, EP and DEP is different, and the greater the force, the faster movement of the microspheres. So the effect of frequency on the microspheres could be observed by investigating the moving speed of the microspheres. As shown in Fig.3a, with 25-μm microspheres, for example, the frequency have the same influence on microspheres in different positions, and in
Fig.2 (a‒c) Time lapse images for visualizing the movement of 10-μm and 25-μm polystyrene microspheres at voltage of 14 Vp-p at 100 kHz with 0.3 V DC offset; (d‒f) Time lapse images for visualizing the movement of 5-μm, 10-μm and 25-μm polystyrene microspheres at voltage of 11 Vp-p at 1 MHz with 1.0 V DC offset
SONG Ning-Ning et al. / Chinese Journal of Analytical Chemistry, 2015, 43(2): 176–180
Fig.3 Frequency dependence of velocity at different locations from the center electrode. Among them, (a) was 25-μm polystyrene microspheres in 10-μm and 25-μm microsphere mixture; (b) was 25-μm polystyrene microspheres in 5-μm and 25-μm microsphere mixture
the same position, the speed of the microspheres reached the maximum when the frequency was set at 100 kHz. The result showed that the resultant force effect on 25-μm microspheres was the largest at 100 kHz. It also could be concluded that the times of 25-μm microspheres moving to the center electrodes at 100 kHz were the shortest. The same analysis could also be applied to 10-μm microspheres in the solution. The experiments also found that the 25-μm microspheres far from the electrode stopped at the gap of the electrode before they reached the center electrode when the microspheres velocity was too small, reducing the separation efficiency. Therefore, the greater the velocity of the microspheres, the greater the resultant force they suffered. As the velocity of the microspheres increased, the 25-μm microspheres far from the electrode were captured on the center electrode, showing an increasing separation efficiency. The results indicated that the voltage frequency of 100 kHz was the optimal conditions for rapid and effective separation. Similarly, for the separation of 5-μm and 25-μm microspheres, as shown in Fig.3b, the separation efficiency was maximized at 2 MHz. Within the same frequency range, 5-μm, 10-μm and 25-μm microspheres could also be separated well, and the separation efficiency was maximized at 1 MHz. These results indicated that the optimum frequency could be
achieved for microspheres separation by optimize the frequency, and the frequency was another important factor for different sizes microspheres separation. 3.3
Influence of voltage
The voltage effect on the PS microspheres separation efficiency was investigated in the experiment. In voltage range of 7–14 Vp-p, it was found that the PS microspheres separation efficiency increased with the increasing voltage. This is because ACEF, DEP and EP force increased with the increasing voltage (FACEF ~ E4, FDEP ~ E2, FEP ~ E), thus the resultant force and its scope expanded, which made ACEF entrain more 25-μm microspheres far from the center electrodes toward the surface of the center electrode and microspheres captured on the center electrode increase, showing an increasing separation efficiency. According to the results in Section 3.2, the microsphere velocity depended on the resultant force and the microspheres separation efficiency. As shown in Fig.4, the optimum voltage was set at 14 V for the separation of 10-μm and 25-μm microspheres, and when the voltage was greater than 14 V, the 10-μm microspheres moved to the electrodes and could not be separated. This was because the increasing effect scope of ACEF caused by the
Fig.4 Voltage dependence of 25 μm polystyrene microspheres velocity. (a) 25 μm polystyrene microspheres in 10 and 25 μm mixture; (b) 25 μm polystyrene microspheres in 5 and 25μm mixture
SONG Ning-Ning et al. / Chinese Journal of Analytical Chemistry, 2015, 43(2): 176–180
increasing voltage made the laminar flow region mentioned described in Section 3.1 fall or even disappear, resulting in 10 μm microspheres were also entrained by ACEF toward the areas near the electrode, and the equilibrium position of 10-μm microspheres was changed, resulting in a bad separation. The same analysis was suitable for 5-μm microspheres. The results also showed that the optimum voltage for the separation of 5-μm and 25-μm microspheres was 10 V, and for 5-μm, 10-μm and 25-μm microspheres were 11 V. The results suggested that the voltage was another important factor affecting the separation efficiency. Although the higher voltage had higher separation efficiency, there was a maximum voltage limit, and exceeded the limit voltage, the equilibrium position of the smaller microspheres would change and the separation efficiency would reduce. These results also illustrated that the separation of the microspheres with different sizes were carried out by a combination effect of ACEF, DEP and EP.
provided a theoretical basis for the research of actual samples.
References [1]
Wong P K, Wang T H, Deval J H, Ho C M. Ieee-asme T. Mech., 2004, 9(2): 366‒376
[2]
Sin M L Y, Shimabukuro Y, Wong P K. Nanotechnology, 2009, 20(16): 165701
[3]
Li D Q. Electrokinetics in Microfluidics, Academic Press, 2004, Chapter 8
[4]
Hardt
S,
Schönfeld
F.
Microfluidic
Technologies
for
Miniaturized Analysis Systems. Springer ,2007: 315‒317 [5]
Sin M L Y, Gau V, Liao J C, Haake D A, Wong P K. J. Phys. Chem. C., 2009, 113: 6561–6565
[6]
Hardt
S,
Schönfeld
F.
Microfluidic
Technologiesfor
[7]
Gao J, Yin X F, Fang Z L. Lab Chip , 2004, 4: 47‒52
[8]
Sun Y, Lim C S, Liu A Q, Ayi T C, Yap P H. Sens. Actuators A:
Miniaturized Analysis Systems. Springer, 2007: 393‒395
Phys., 2007, 133(2): 340‒348
4
Conclusions
[9]
Takamura Y, Onoda H, Inokuchi H, Adachi S, Oki A, Horiike Y. Electrophoresis, 2003, 24: 185‒192
In summary, under the optimum conditions, a hybrid electrokinetic technique combined with DEP, EP and ACEF was used to separate the microspheres with different sizes in conductive solution on the microfluid chip, and its theories of the separation was studied and discussed. Meanwhile the microspheres size, frequency and voltage as the important factors for the separation were also optimized. In particular, under certain conditions, there was a laminar flow region in the gap of the adjacent electrodes, in which the fluid was relatively stationary, and ACEF was weaker and evenly could not affect the movement of microparticles in this region. Thus the microspheres with smaller size (< 10 μm) were easy to stay in the regions, and could not be entrained out by ACEF, in the meantime, the larger size microspheres (25 μm) entrained out by ACEF toward the surface of the center electrode due to its bigger size in the laminar flow region, thus achieved the separation of different sizes microspheres. By separating the PS microspheres of different sizes, the operating rules were found for the hybrid electrokinetic effect on different sizes microspheres and the function area in high conductivity solution. The results
[10] Feldman H C, Sigurdson M, Meinhart C D. Lab Chip, 2007, 7: 1553‒1559 [11] Mandy L Y S, Vincent G, Joseph C L, Pak Kin W. J. Assoc. Lab. Autom., 2010, 15: 426‒432 [12] Li Y, Dalton C, Crabtree H J, Nilsson G, Kaleret K V I S. Lab Chip, 2007, 7(2): 239‒248 [13] Doh I, Cho Y H. Sens. Actuators A: Phys., 2005, 121(1): 59‒65 [14] Cheung K, Gawad S, Renaud P. Cytometry Part A, 2005, 65A(2): 124‒132 [15] Krishnan R, Sullivan B D, Mifflin R L, Esener S C, Heller M J. Electrophoresis, 2008, 29(9): 1765‒1774 [16] Lin R Z, Ho C T, Liu C H, Chang H Y. Biotechnol. J., 2006, 1(9): 949‒957 [17] Xu J, Zhao Z, Fang Z, Liu Y H, Du L D,Geng D Q. Chinese J. Anal. Chem., 2011, 39(3): 295‒299 [18] Gao J, Sin M L Y, Liu T, Gau V, Liao J C, Wong P K. Lab Chip, 2011, 11(10): 1770‒1775 [19] Gao J, Riahi R, Sin M L Y, Zhang S, Wong P K. Analyst, 2012, 137(22): 5215‒5221 [20] Gonzalez A, Ramos A, Morgan H, Green N G, Castellanos A. J. Fluid Mech., 2006, 564: 415‒433