APPLIED PHYSICS LETTERS 96, 193701 共2010兲
Droplet electric separator microfluidic device for cell sorting Feng Guo, Xing-Hu Ji, Kan Liu, Rong-Xiang He, Li-Bo Zhao, Zhi-Xiao Guo, Wei Liu, Shi-Shang Guo, and Xing-Zhong Zhaoa兲 Key Laboratory of Artificial Micro- and Nano-structures of Ministry of Education and School of Physics and Technology, Wuhan University, Wuhan 430072, People’s Republic of China
共Received 7 December 2009; accepted 16 February 2010; published online 13 May 2010兲 A simple and effective droplet electric separator microfluidic device was developed for cell sorting. The aqueous droplet without precharging operation was influenced to move a distance in the channel along the electric field direction by applying dc voltage on the electrodes beside the channel, which made the target droplet flowing to the collector. Single droplet can be isolated in a sorting rate of ⬃100 Hz with microelectrodes under a required pulse. Single or multiple mammalian cell 共HePG2兲 encapsulated in the surfactant free alginate droplet could be sorted out respectively. This method may be used for single cell operation or analysis. © 2010 American Institute of Physics. 关doi:10.1063/1.3360812兴 Droplet-based microfluidics with the advantages of highthroughput, minimal reagents consumption, contaminationfree, fast response and miniaturized space provides a powerful, and necessary platform for the single cell operation and analysis, which is not only highly economical but also unavailable in conventional experiment.1,2 The basic required droplet manipulations including droplet generation, sorting, trapping, and fusion for cell sorting, cell trapping, cell culture, cell lysis, and other applications2–5 have been developed using different methods such as optical,4magnetic,6 acoustic,7 valve-based,8 electric,3,9–12 etc. Basing on the advantages of the electric method as labeling free, strong manipulation, easy to setup and fast response time, dielectrophoresis 共DEP兲,3,9 electrowetting,10 electrostatic manipulation,11,12 etc., have been used for, droplet discretion,10 droplet electrocoalescence,11 highthroughput droplet sorter,9 or a single droplet handling platform. Weitz’s group developed a high-speed droplet sorter for microfluidic device by DEP force.11 Link et al.12 reported the electric droplet handling platform depending on the precharged droplet, while the system needed electrochemical reaction with droplet.9 Griffiths’s group prepared the emulsification of cell suspension with surfactant on a droplet generation chip, then separated the enzyme activation E. coli cells by the fluorescence-activated droplet sorting microfluidic device using DEP method.3 Here, we report the electric manipulation of the droplet generated by the flow-focusing channel without precharging operation under dc voltage. After optimizing the electrodes and the square pulse, single mammalian cell encapsulated alginate droplet can be isolated. The microfluidic device 关Fig. 1共a兲兴 including three parts: droplet generator, electric controller, and droplet collector was fabricated by the standard soft lithography.13 The polydimethylsiloxane bulk with both electrode channels and fluidic channels in same layer was bonded to a glass sides with oxygen plasma, and the electrodes were made by microsolidics.11 The device was cured at 80 ° C for a兲
Author to whom correspondence should be addressed. Electronic mail:
[email protected]. Tel.: ⫹86 27 87642784.
0003-6951/2010/96共19兲/193701/3/$30.00
two weeks to ensure surfaces of microchannels to be hydrophobic. A steady stream of uniform droplets produced by flowfocusing method14 关Fig. 1共a兲兴 with frequency from 5 to 250 Hz can be obtained, using deionization 共DI兲 water or 2.5% sodium alginate 共China National Medicines Co. Ltd., China兲 as the dispersed phase and soybean oil 共Beiya Medical Oil Co. Ltd., China with viscosity = 70 mpa s兲 as the continuous phase driven by three syringe pumps 共TS2–60, Longer, China兲. The distance between each two droplets in the droplet stream was adjusted by the other oil flow. Without electric field, droplets were driven by the oil laminar flow as Fig. 1共b兲. Under the electric field produced by a function generator 共33220A, Agilent, American兲 and a voltage amplifier 共610D, Track, American兲, the target droplet moved to the oil flow with red dye 关Fig. 1共c兲兴. The movement of droplet was observed and recorded by an inverted microscope 共IX71, Olympus, Japan兲 coupled with a high-speed charge coupled device camera 共Evolution VF cooled monochrome camera, Media Cybernetics, Inc.兲. Under the optimized flow rate of continuous phase and dispersed phase, the aqueous droplet with 30– 50 m diameters was generated without prechanging operation. Compar-
FIG. 1. 共Color online兲 共a兲 Schematic of the microfluidic device. 共b兲 and 共c兲 show the droplet movement in the oil laminar flow without or with electric field.
96, 193701-1
© 2010 American Institute of Physics
193701-2
Appl. Phys. Lett. 96, 193701 共2010兲
Guo et al.
FIG. 2. 共Color online兲 共a兲 DI water droplet movement under the electric field. 共b兲 The relationship between dc voltage and terminal velocity of DI water or 2.5% alginate droplet along electric field direction.
ing with 65 m high channels, the aqueous droplet did not touch the surfaces of the channel in the whole flowing process through the channel. However, droplet movement was influenced under the electric field 关Fig. 2共a兲兴, and we called that “self-charged” behavior. That was different from the electrical charging droplet15 which got charges from touching with the electrode surface.12 Similar surface charge phenomena of droplet or colloid got the great interests from colloid chemists and physicists,15–17 but no generally accepted conclusion had been reached yet. The charges may be contributed by induced charge18 or electrons 共or ions兲 transformation between aqueous droplet and oil phase16 or simultaneously. Wherever charges come from, they could be used to direct droplet movement under electric field. The electric force is given by FE = Uq / L, where q is the amount of charge on the droplet. U and L denote dc voltage and the distance between two electrodes, respectively. In order to study the physical mechanisms for the droplet separation, we established a hydrodynamic model of droplet trajectory. The droplet movement in the oil flow influenced by an electric force FE is governed by mdu/dt = FE − ␥共u-v兲,
共1兲
where m denotes the droplet mass, u and v mean the droplet and fluid velocities, respectively, and ␥ the friction factor of the droplet in the fluid, which is expressed as ␥ = 6a for the droplet of radius a with being the fluid viscosity. The velocity and position vector are given by integrations of Eq. 共1兲, u共t兲 = 共FE/␥ + v兲关1 − exp共− ␥t/m兲兴,
共2兲
and r共t兲 = 共FE/␥ + v兲兵t + m关exp共− ␥t/m兲 − 1兴/␥其,
共3兲
respectively. The exponential term in Eq. 共2兲 vanishes rapidly with respect to the observation time so that the charge of droplet is approximately given by q = 6L · a · 共ut/U兲.
共4兲
ut is the terminal velocity of droplet along electric field direction under different voltages and obtained by measuring the displacement of droplet as a function of time from each frame of the movies. As shown in Fig. 2共b兲, ut is almost linear to the voltage so that q can be estimated experimentally. Such as the 48.99 m diameter water droplet flowing in the 60 l / h oil flow and directed by the electric field with the 560 m distance between two electrodes 共L兲, the charge
FIG. 3. 共Color online兲 Boundary conditions for single droplet isolation by adjusting square pulse width and amplitude. 关共a兲–共c兲兴 Single droplet was sorted out within 15 ms with adding a 6 ms width and 800 V amplitude pulse.
can be estimated around 7.44⫻ 10−15C. The distance of droplet influenced by electric force along the electric field direction can get from Eq. 共3兲, y共t兲 = Uq兵␥t + m关exp共− ␥t/m兲 − 1兴其/共L␥2兲.
共5兲
Depending on Eq. 共5兲, single droplet can be isolated instantaneously from the droplet flow by a suitable electric force within a droplet generation period. After optimizing the electrodes integrated into the device and using pulse electric field, the single droplet was isolated such as adding a 1 Hz square pulse with altering amplitude and width on the 100 m width electrodes. All the droplets were driven to the center of main channel and collected in the middle of waste reservoir under a suitable flow rate of two oil phases without electric field. Single droplet was sorted out within 15 ms by a pulse with 6 ms width and 800 V amplitude as Figs. 3共a兲–3共c兲 described. Whether single droplet can be isolated or not depended on the critical condition: y共t兲 ⱖ d, where d is the distance of droplet away from the center of the oil flow into the collecting channel. The relationship between the square pulse amplitude U and minimal separation width t became the boundary condition for single droplet isolation, then the simulation of critical condition curve 共by MATLAB兲 and the experiment data were obtained as Fig. 3 shown to demonstrate the droplet separation ability of our system at 10–100 Hz. The differences of the simulation curve and experiment data came from the fluidic dynamic fluctuation and the neglect of the distance of the decelerate process after a very short signal. This present method was used for cell sorting. The mixture of 2 ⫻ 106/mL HePG2 cells suspending in PBS solution and 2.5% sodium alginate solution in PBS buffer was used as the dispersed phase with 1:1 volume ratio, which avoided cell subsiding. The droplet containing with cells was formed by the droplet generator of our device. By premonitoring of the droplet with a low flowing velocity in the channel under the microscopy, programmed electric field was applied. The single cell alginate droplet was sorted out into one collecting channel by a square pulse as Fig. 4共a兲 shown, while two cells alginate droplet was separated to the other collecting channel by a reversed pulse 关Fig. 4共b兲兴. In fact, the droplet containing with three, four or multiple cells could be isolated respectively. Then the cells separated by the electric force were all collected into the prepared tube, washed three times to get rid of the oil. The viability of the cells sorted out in several
193701-3
Appl. Phys. Lett. 96, 193701 共2010兲
Guo et al.
This work was supported by China National Funds for Distinguished Young Scientists 共Grant No. 50125309兲 and National Natural Science Foundation of China 共Grant Nos. 10904117 and 10804087兲. We would like to thank Dr. Xu Yu’s kind help and cell supply. Y. Schaerli and F. Hollfelder, Mol. Biosyst. 5, 1392 共2009兲. C. E. Sims and N. L. Allbritton, Lab Chip 7, 423 共2007兲. 3 J. C. Baret, O. J. Miller, V. Taly, M. Ryckelynck, A. El-Harrak, L. Frenz, C. Rick, M. L. Samuels, J. B. Hutchison, J. J. Agresti, D. R. Link, D. A. Weitz, and A. D. Griffiths, Lab Chip 9, 1850 共2009兲. 4 M. Y. He, J. S. Edgar, G. D. M. Jeffries, R. M. Lorenz, J. P. Shelby, and D. T. Chiu, Anal. Chem. 77, 1539 共2005兲. 5 M. A. McClain, C. T. Culbertson, S. C. Jacobson, N. L. Allbritton, C. E. Sims, and J. M. Ramsey, Anal. Chem. 75, 5646 共2003兲. 6 L. B. Zhao, L. Pan, K. Zhang, S. S. Guo, W. Liu, Y. Wang, Y. Chen, X. Z. Zhao, and H. L. W. Chan, Lab Chip 9, 2981 共2009兲. 7 T. Franke, A. R. Abate, D. A. Weitz, and A. Wixforth, Lab Chip 9, 2625 共2009兲. 8 S. J. Zeng, B. W. Li, X. O. Su, J. H. Qin, and B. C. Lin, Lab Chip 9, 1340 共2009兲. 9 K. Ahn, C. Kerbage, T. P. Hunt, R. M. Westervelt, D. R. Link, and D. A. Weitz, Appl. Phys. Lett. 88, 024104 共2006兲. 10 M. G. Pollack, R. B. Fair, and A. D. Shenderov, Appl. Phys. Lett. 77, 1725 共2000兲. 11 L. M. Fidalgo, G. Whyte, D. Bratton, C. F. Kaminski, C. Abell, and W. T. S. Huck, Angew. Chem., Int. Ed. 47, 2042 共2008兲. 12 D. R. Link, E. Grasland-Mongrain, A. Duri, F. Sarrazin, Z. D. Cheng, G. Cristobal, M. Marquez, and D. A. Weitz, Angew. Chem., Int. Ed. 45, 2556 共2006兲. 13 K. Liu, H. J. Ding, J. Liu, Y. Chen, and X. Z. Zhao, Langmuir 22, 9453 共2006兲. 14 S. L. Anna, N. Bontoux, and H. A. Stone, Appl. Phys. Lett. 82, 364 共2003兲. 15 Y. M. Jung, H. C. Oh, and I. S. Kang, J. Colloid Interface Sci. 322, 617 共2008兲. 16 M. E. Leunissen, A. van Blaaderen, A. D. Hollingsworth, M. T. Sullivan, and P. M. Chaikin, Proc. Natl. Acad. Sci. U.S.A. 104, 2585 共2007兲. 17 W. D. Ristenpart, J. C. Bird, A. Belmonte, F. Dollar, and H. A. Stone, Nature 共London兲 461, 377 共2009兲. 18 M. Z. Bazant and T. M. Squires, Phys. Rev. Lett. 92, 066101 共2004兲. 1 2
FIG. 4. 共Color online兲 共a兲 Single cell droplet was sorted out to left collecting channel under square pulse. 共b兲 Two cells encapsulated in the alginate droplet were driven to right collecting channel by a reversed pulse. 共c兲 The viability of HePG2 cells separated under different voltages in 4 h was shown.
hours was investigated by 0.4% trypan blue buffer as Fig. 4共c兲 shown and the selected cells were suitable for cell lysis4,5 or other biochemistry assay. In conclusion, we have demonstrated the potential of this electric droplet manipulation microfluidic device for cell sorting and operation. Single droplet can be sorted out in several milliseconds under the optimized separation condition. This method will be an easy and effective platform for single cell operation and analysis such as single cell sorting, handling, lysis, and so on.
