ABSTRACT. This paper presents a novel method for continuous particle sorting and collection by using of cascade squeeze-jumping effect under microfluidic.
CONTINUOUS PARTICLE SORTING UTILIZING CASCADE SQUEEZE-JUMPING EFFECT UNDER MICROFLUIDIC CONFIGURATION Che-Hsin Lin1, Cheng-Yan Lee1, Lung-Ming Fu2 Department of Mechanical and Electro-mechanical Engineering, National Sun Yat-Sen University, Taiwan, ROC 2 Graduate Institute of Materials Engineering, National Pingtung University of Science and Technology, Taiwan, ROC 1
sorting devices have been successfully demonstrated using dielectrophoresic forces[3, 4], optical tweezing forces[5, 6], magnetic forces[7] and even ultrasonic forces[8]. In general, delicate control systems and chip fabrication processes are required to achieve reasonable separation efficiency while using these technologies to separate cell samples. In addition, the through-put and sorting efficiency of using the optical or magnetic approaches are also problematic for practical applications. Separating cell samples using micro-dam structures with difference geometries is also a common approach [9] but clogging of the microchannel is an issue need to be overcame. Mood et. al. reported a cell sorter utilizing an upstream ultrafiltration method by gravitational separation.[10] However, microparticles tend not sensitive to the gravity force such that a long flow channel was used to separate particles with enough distance apart. Takagi et. al. reported a micro device for continuous particle separation using asymmetric pinched flow fractionation. A mixture of 1.0 ~ 5.0 µm particles was successfully separated.[11] This paper presents a novel method for continuous particle sorting utilizing cascade squeezejumping effect under microfluidic configuration. A simple and reliable electrokinetic flow system is used for generating cascade sheath flows in microfluidic channel. Microparticles with different size can be separated and sorted at different stages of the cascade flow in the microchannel with this approach. Microchip devices are designed, fabricated and evaluated using numerical simulation and experimental operation. Experimental results indicate the proposed microfluidic device is capable of continuous particle sorting and collection.
ABSTRACT This paper presents a novel method for continuous particle sorting and collection by using of cascade squeeze-jumping effect under microfluidic configuration. Microparticles with different sizes can be successfully separated at different stages of squeezed sheath flows. Separated microparticles then flow into expansion channel blocks to enlarge the distance of the separated flow streams and are collected at different side channels. Only one high voltage is required for generating all the sheath flows for particle separation by applying a series of variable resister to create different electric field levels such that the system is simple and reliable. This study also adopts numerical simulation to analyze and predict the flow fields and stream lines within the microchannel. Experimental results show that the microchip is capable of continuously separating microparticles with the sizes of 5, 10 and 20 Pm using the proposed squeeze-jumping effect. The method proposed in this study provides a simple way to continuously separate microparticles/cells with different sizes. More importantly, no delicate control system and fabrication process are required to achieve the sorting and collection.
1.
INTRODUCTION
Flow cytometry techniques have been widely used for clinical diagnostics, environmental monitoring, food pathogen screening, drug discovery and fundamental biomedical researches by researchers who working in the field of medical diagnosis [1]. In this operation, optical detection is a common and well-established method for large-scale flow cytometers. Cell samples are firstly labeled with fluorescence for optically identification, counting and finally sorting.[2] One of the important issues in developing flow cytometers is to sort and collect functional cell samples with a simple and highthroughput method. In the past years, miniaturized cell sorting devices have been attracted lots of interest for researchers who work on Bio-MEMS. Many cell
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DESIGN AND FABRICATION
The working principle of the proposed microfluidic device is shown in figure 1. The main concept of the proposed method is based on that microparticles are not able to flow within a flow stream of the stream width smaller than their diameters. Therefore, electrokinetically induced neighboring sheath flows can simply be used to
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MEMS 2006, Istanbul, Turkey, 22-26 January 2006.
squeeze the sample flow into a narrow stream for particle separation purpose. Big particles will jump from their original flow stream into the neighboring sheath flow around the squeezed region while small particles remain to stable flow in the squeezed flow stream. A cascade channel for generating multi-stage sheath flows with different widths is utilized to sort microparticles of the size from small to large. (a)
Figure 3a shows a picture of the proposed microfluidic chip after bonding. The dimension of the microfluidic chip is 3 cm in length and 2.6 cm in width. Two cascade structures for separation of microparticles with 3 different sizes were designed. Figure 3b and 3c show close-up OM images of the sealed microchannel. The width of the main channel, sheath flow channels and particle collection channels are 150, 120 and 100 Pm, respectively. The 300-Pm wide expansion channel blocks are designed for enlarging the distance of two separated cell streams in a passive and efficient way.
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Fig. 1 (a) Schematic diagram of the working principle for the proposed cell sorter. (b) An enlarged picture for explaining the squeezing effect.
Fig. 3 (a) A photo picture of the chip after fabrication, Close up views of the cascade microfluidic channels (b) and a sheath flow channel(c).
Figure 2 shows a simplified schematic of the chip fabrication process. The chip was fabricated in low cost glass slides using a fast fabrication process. [12] Briefly, a thin layer of AZ4620 positive photoresist was firstly spin-coated on the glass slide for the pattern definition and was use as the etching mask for glass etching. The patterned substrate was then immersed into BOE solution for 40 min to generate 36-Pm deep microchannels. Note that the substrate was immersed into a 1 M HCl solution for 10 s every 5 min during the etching process for removing the precipitations. The photoresist layer was then removed using a 10% KOH solution. Fluid via-holes were drilled using a diamond drill bit on another bare upper substrate. The microchip was then sealed in a sintering oven at 580qC for 10 min.
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EXPERIEMNTAL
Figure 4 presents a simplified schematic for the experimental setup in this study. The experiment was operated under a fluorescence stereo microscope (SZX-9, Olympus, Japan) with a CCD moldule (DXC-190, Sony, Japan). A high voltage power supply (MP3500, Major Science, Taiwan) was used to electrokinetically drive the running fluids. A series of high-ohm variable resisters (30 M:) were utilized to generate various electric field levels for different reservoirs. Multi-stage sheath flows with different widths were then induced for squeezing the sample particles. Polystyrene beads with the size of 5, 10 and 20 Pm (Duke Scientific, USA) were used for the continuous cascade separation tests.
Fig. 4 Schematic illustration of the experimental setup for the proposed cell sorter. Note that only one power supply is required for the system.
Fig. 2 A simplified schematic for the fabrication process of the proposed cell sorter.
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Figure 5 presents the experimental and numerical flow fields of the squeeze phenomena at the first stage of the cascade channel. The electric field applied at the sample channel and side channel were 100 and 200 V/cm, respectively. Results show that the sample channel can be squeezed into the width of 6 Pm which is suitable for collecting microparticles of the size smaller than 5 Pm.(Fig. 5a and 5b) A smaller squeezed stream can also be obtained by applying a higher electric field on the side channel. Figure 5c, 5d and 5e present the calculated electrical potential contours, velocity vectors and the streamline plots under the same operation conditions. It is clear that the expansion channel increases the flow stream significantly. Figure 6a shows the calculated flow field of the fluids at the first stage of the cascade channel. The corresponding flow velocity and acceleration profiles of the flows were presented in Fig. 6b and 6c. The positive acceleration values at the squeezed region provide the jumping forces for big particles to move into the neighboring sheath flow stream. Experiment
Figure 7 shows the continuous images of 20-Pm microparticles jumping from the original flow stream into the neighboring sheath flow by the squeeze-jump effect. The width of the squeezed sample flow is around 10 Pm. This result confirms that the squeezed flow can force big particles to move into the wider neighboring flow stream. Figure 8 presents continuous images showing that the successful separation of 5 Pm microparticles out of a mixture composed of 5, 10 and 20 Pm microparticles at the first stage of the cascade channel utilizing the proposed method. As shown in the continuous images, the 5-Pm microspheres flow stable in the squeezed sample flow along the channel wall while the 10 and 20-Pm microspheres switch their flow paths into the sheath fluids and flow farther from the channel wall. The separated particles then flow into the expansion section to increase the distance of the separated particles as shown in Fig. 5e. This design will enhance the sorting efficiency significantly.
Numerical Model
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Fig. 5 Experimental (a,b) flow contours and numerical results of (c) electrical potential contours, (d) velocity vectors and (e) the streamline plots.
Fig. 7 Continuous images of 20-Pm microparticles jump into the neighboring sheath flow by the squeezejump effect.
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Fig. 6 Calculated (a) flow field (b) velocity profile and (c) acceleration profile at the first-stage squeeze region. Note that the values for the velocity and acceleration were estimated at the interface of the sample and sheath flows.
Fig. 8 Continuous images showing the successful separation of 5 Pm microspheres (red arrow) out of 10 Pm (blue arrow) and 20 Pm (black arrow) particle mixtures using the squeeze-jumping effect.
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A side channel placed at the middle of the expansion section is used to collect the sorted 5-Pm microspheres. Figure 9 presents the continuous images of sorting the 5 Pm microparticles (red arrow) into the first collection side channel. The 10 Pm (blue arrow) and 20 Pm (black arrow) microparticles keep flowing toward downstream and will be finally separated and sorted at the second stage of the cascade channel. Therefore, microparticles with different size can be sorted continuously in the microchannel utilizing the proposed cascade squeezejumping effect. The proposed method provides a simple, low-cost yet high efficient way to sort and collect mixed cell samples in microfluidic channel.
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REFERENCE [1] M. Brown and C. Wittwer, "Flow cytometry: Principles and clinical applications in hematology", Clin Chem, vol. 46, pp. 1221-1229, 2000. [2] A. Y. Fu, C. Spence, A. Scherer, F. H. Arnold, and S. R. Quake, "A microfabricated fluorescence-activated cell sorter", Nat Biotechnol, vol. 17, pp. 1109-1111, 1999. [3] L. Cui, T. Zhang, and H. Morgan, "Optical particle detection integrated in a dielectrophoretic lab-on-a-chip", J Micromech Microeng, vol. 12, pp. 7-12, 2002. [4] M. Durr, J. Kentsch, T. Muller, T. Schnelle, and M. Stelzle, "Microdevices for manipulation and accumulation of micro- and nanoparticles by dielectrophoresis", Electrophoresis, vol. 24, pp. 722-731, 2003. [5] J. Prikulis, F. Svedberg, M. Kall, J. Enger, K. Ramser, M. Goksor, and D. Hanstorp, "Optical spectroscopy of single trapped metal nanoparticles in solution", Nano Lett, vol. 4, pp. 115-118, 2004. [6] J. Enger, M. Goksor, K. Ramser, P. Hagberg, and D. Hanstorp, "Optical tweezers applied to a microfluidic system", Lab Chip, vol. 4, pp. 196200, 2004. [7] R. Rong, J. W. Choi, and C. H. Ahn, "A Functional Magnetic Bead/Biocell Sorter Using Fully Integrated Magnetic Micro/Nano Tips", 16th IEEE International Conference on Micro Electro Mechanical Systems 2003. [8] H. Jagannatha, G. G. Yaralioglu, A. S. Ergun, and B. T. Khuri-Yakub, "An Implementation of A Microfluidic Mixer and Switch Using Micromachined Acoustic Transducers", 16th IEEE International Conference on Micro Electro Mechanical Systems pp. 104-107, 2003. [9] S. Metz, C. Trautmann, A. Bertsch, and P. Renaud, "Flexible Microchannels with Integrated Nanoporous Membranes for Filtration and Separation of Molecules and Particles", 15th IEEE International Conference on Micro Electro Mechanical Systems pp. 81-84, 2002. [10] M. H. Moon, D. J. Kang, D. W. Lee, and Y. S. Chang, "On-line Particle Concentrator with Upstream Ultrafiltration in Continuous SPLITT Fractionation", Anal Chem, vol. 73, pp. 693-697, 2001. [11] J. Takagi, M. Yamada, M. Yasuda, and M. Seki, "Continuous Particle Separation in A Microchannel Having Asymmetrically Arranged Multiple Branches", Lab Chip, vol. 5, pp. 778-784, 2005. [12] C. H. Lin, G. B. Lee, Y. H. Lin, and G. L. Chang, "A Fast Prototyping Process for Fabrication of Microfluidic Systems on Soda-Lime Glass", J Micromech Microeng, vol. 11, pp. 726-732, 2001.
Fig. 9 Continuous images showing the successful collection of the 5 Pm microparticles (red arrow) from the mixed sample.
4.
CONCLUSIONS
This paper reports an innovative method for continuous particle sorting using a microfluidic phenomenon of squeeze-jumping effect. A cascade microfluidic channel was designed and fabricated in a low cost soda-lime glass substrate for continuous cell sorting. No delicate transducer such as embedded microelectrode, ultrasonic generator or external optical system was required for constructing this system. Experimental results confirm the proposed microdevice can successfully separate microparticles with different sizes. Similar concept can be adopted to sort biological cells such as stem cells, embryos, oocytes out of a mixed cell sample. It is the authors’ believe that this simple approach will give substantial impacts on cell separation technology.
ACKNOWLODGEMENTS Financial supports from National Science Council in Taiwan are great acknowledged. (NSC 942320-B-110-003 and NSC 94-2320-B-020-001).
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