SEPARATING PLASMA AND BLOOD CELLS BY DIELECTROPHORESIS IN MICROFLUIDIC CHIPS
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1 Fourth International Symposium on Physics of Fluids (ISPF4) International Journal of Modern Physics: Conference Series Vol. 19 (2012) World Scientific Publishing Company DOI: /S SEPARATING PLASMA AND BLOOD CELLS BY DIELECTROPHORESIS IN MICROFLUIDIC CHIPS TZONG-SHYNG LEU AND ZHI-FENG LIAO Department of Aeronautics and Astranautics, National Cheng Kung University, No. 1 University Road, Tainan 701, Taiwan tsleu@mail.ncku.edu.tw In this paper, a dielectrophoretic (DEP) micro separator is studied for plasma-blood separation. DEP forces created by non-uniform electric fields are used as deflected forces to deplete blood cells from side walls at a given inlet flow rate (Q in). Then one can extract plasma through a microchannel on side wall at certain extraction flow rate (Q p). In this experiment, saline isotonic solution is chosen as dilute solution for whole blood. The minimum dilute ratio (whole blood: saline dilute) is found to be 1:3 for DEP to substantially deplete blood cells from side walls. Exraction of plasma from whole blood sample by DEP force is also investigated. Experimetnal results show blood cells do not enter side channel by DEP force at inlet flow rate Q in=0.5μl/min when plasma extract flow rates is Q p 0.3 µl/min. By calculating pure plasma extraction volume fraction, the efficiency in current experiment can reach as high as 20% if dilute ratio 1:3 of whole blood sample is considered. Keywords: Dielectrophoresis; whole blood separation; plasma. 1. Introduction Modern methods in many biomedical applications often require the analysis of bio-fluid of interest from mixtures of various kinds of bio-particles. The conventional methods commonly used in laboratories for manipulation, concentration, and separation of bioparticles include dilution, dispersion and filtration. These approaches are disadvantageous especially for valuable or rare samples. It has been widely acknowledged that many microfluidic devices had great potential for single cell experiments 1. Over the years, many methods have been developed. In this paper, a dielectrophoretic (DEP) micro separator is studied for plasma-blood separation. The DEP separator approaches are particularly suitable for operating at the micrometer scale. Such separators operate through the interaction of induced polarization charge with non-uniform electric fields that can induce DEP forces acting on particles. With proper design of DEP micro separators, particles of interest can be trapped 2, sorted 3, manipulated 4, rotated 5 and focused 6 to a predestined point. Since there is no sheath flow like conventional hydrodynamic focusing, the current DEP micro separator design can result in substantial reduction of dilute fluid consumption. DEP separation technique is also label-free, i.e., a unique advantage that cells are unaltered during the measurement process. Meanwhile, the flow in the present DEP micro separator is smooth and without any dead spaces. In 185
2 186 T.-S. Leu and Z.-F. Liao addition, DEP micro separator can be easily integrated with other microfluidic devices for cellomics. These features obtained in the design of this paper are, in fact, required by many micro-fluidic systems. 2. Design and Fabrication of DEP Micro Separator Figure 1 shows the schematic design of the DEP micro separator. The width of main channel is W in =750µm. In DEP micro separator, pairs of electrodes with angle α=30 o, width w e =50µm and gap w g =50µm are used to generate non-uniform electric fields when applying an ac signal between them. The whole blood sample is injected into the micro separator by using a syring pump at inlet flow rate Q in. It has been verified that blood cells are negative-dep-type particles at current operation frequency f=1.0 MHz. Blood cells flowing through the non-uniform electric fields are expected to experience DEP forces in Y direction. The DEP force in Y direction of micro separator chip is designed to act as a confining force. The blood cells are expected to be focused into a narrower particle stream near the centerline of the micro separator, as shown in Figure 1. On one side of main channel, a side channel is used to extract plasma from whole blood sample with a flow rate Q p. The width of side channel is W p =50µm. Fig. 1. Principle and design of DEP micro separator when applying a sinusoidal ac signal with frequency f and peak-to-peak voltage V pp. (Win=750µm, We=50µm, Wg=50µm, Wp=50µm, α=30 o ) The experimental studies are implemented to demonstration the feasibility and efficacy of the DEP micro separator for plasma extraction. The DEP micro separator are fabricated in-house. Au/Cr electrodes are first deposited on a glass substrate by using E- beam evaporator and the electrodes are patterned by using standard photolithography techniques. The microchannel is fabricated by using polydimethylsiloxane (PDMS). For PDMS microchannel, a master mold is fabricated by spinning SU8-50 (MicroChem Corp. Newton, MA, USA) on the silicon wafer to define the micrchannels. PDMS prepolymer mixture (Sylgard-184 Silicone Elastomer Kit, Dow Corning, Midland, MI, USA) is poured and cured on the master mold to replicate the patterned structures. It was bonded with the glass substrate after treatment of the oxygen plasma in the O 2 plasma cleaner. Fig. 2 depicted the picture of microfabricated DEP micro separator.
3 Separating Plasma and Blood Cells by Dielectrophoresis in Microfluidic Chips 187 Fig. 2. Picture of DEP micro separator. 3. Results and Discussions Depletion of blood cells by DEP force is first investigated in the present work. Figure 3 shows the depletion of whole blood sample by DEP force near one side wall at different dilute ratios when inlet flow rate (Q in ) is 0.1µl/min and plasma outlet flow rate (Q p ) is 0µl/min. The isotonic dilute medium in current experiment is using 0.9% NaCl saline. The ac signal for DEP micro separator is sinudoidal wave signal with frequency f=1mhz and peak-to-peak voltage V pp =5V. Figure 3 indicates that blood cells are negative-deptype cells which move away from side wall within DEP micro separator. (a) (c) (b) (d) Fig. 3. The depletion of blood cells within the DEP micro separator when input ac signal is V pp=5 volt, frequency f=1 MHz, the inlet flow rate Q in=0.1 l/min and plasma outlet flow rate Q p=0µl/min for whole blood sample at different dilute ratio (whole blood: saline) (a) 1:0 (b) 1:1 (c) 1:2 (d) 1:3. One can find that cells in whole blood sample without dilution are so dense that blood cells do not have enough space to deplete by DEP force (Fig. 3(a)). When dilute ratio of whole blood is set as 1:1, and 1:2, the depletion region of blood cells increases (Figs. 3(b) and 3(c)). As shown in Fig. 3(d), dilute ratio 1:3 of whole blood sample allows one to
4 188 T.-S. Leu and Z.-F. Liao extract cell-free plasma from side wall channel suscessfully. The depletion thickness (δ p ), as shown and defined in Fig. 1, equals about 150 µm in Fig. 3(d). Exraction of plasma from whole blood sample by DEP force is also investigated. The snapshots of blood cell image in DEP micro separator at different plasma extraction rates (Q p ) are illustrated in Fig. 4 when inlet flow rate is Q in =0.5µl/min. Figs. 4(a)~4(c) show no blood cells enter side channel at plasma extract flow rate Q p 0.3µl/min. The depletion thickness (δ p ) is greater than 50 µm, as shown in Figs. 4(a)~4(c). Experimental results of Fig. 4(d) depicts clearly that blood cells enter the side channel when plasma extraction rate is 0.4 µl/min. Therefore, the blood cells are excluded from side channel successfully if Q p 0.3µl/min when Q in =0.5µl/min. The effiency of plasma extraction can reach as high as 60% in the present study. The actual plasma extraction efficiecy is about 20% if dilute ratio 1:3 of whole blood sample is considered. (a) (b) Fig. 4. The depletion of blood sample with dilute ratio 1:3 at differnt plasma extraction flow rate (Q p) (a) Q p=0.1µl/min (b) Q p=0.2 µl/min, (c) Q p=0.3 µl/min (d) Q p=0.4 µl/min in DEP micro separator when ac signal is V pp=5 volt, frequency f=1 MHz and the inlet flow rate Q in=0.5µl/min, (c) (d) 4. Conclusions The present studies involvs the use of DEP micro separator with different dilute ratios and flow rates conditions, to extract the cell-free plasma from whole blood samples. Experimetal studies are performed to analyze the effects of dilute ratio and extraction flow rate of plasma in DEP micro separator, as well as the efficiency of plasma extraction. The minimum dilute ratio of whole blood sample that can be depleted by DEP force is obtained to be 1:3. Real plasma extraction from whole blood sample is presented in this
5 Separating Plasma and Blood Cells by Dielectrophoresis in Microfluidic Chips 189 work. The plasma efficiency of extraction by using DEP micro separator is demonstrated as high as 20% of whole blood in this experiment. Acknowledgments Funding for this project is provided by National Science Council, R.O.C under the contract of NSC E MY2. References 1. P. Telleman, U. Larsen, J. Kutter, and et al., Proc. SPIE Conf. Microfluidic Dev. Sys. 4177, 1 (2000) 2. T.Schnelle, R. Hagedorn, G. Fuhr, and et al., Biochem. Biophys. Acta, 1157, 127 (1993). 3. K. Takahashi, A.Hattori, I. Suzuki, and et al., J. Nanobiotechn. 2, 5 (2004). 4. G. Fuhr, T. Müller, T. Schnelle, and et al., Naturwiss. 81, 528 (1994). 5. C. Reichle,T. Müller, T. Schnelle and G., J. Phys. D: Appl. Phys.32, 2128 (1999). 6. T. Muller, A. Pfennig, P. Klien, and et al., IEEE Eng. Medical Bio. Mag. 51 (2003).
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