對於生物微粒的分離，非但要求高純度的分離效率外，經分離後細胞本體不會受破壞更是重要的考量。本研究設計了一介電泳晶片，主要根據細胞與分離溶液間不同的介電特性，且利用非均勻性交流電場對細胞具非破壞特性的介電誘發能力，產生非對稱的電極化程度，使細胞受電場力作用後往高或低電場強度的方向移動而分離。此介電泳力之大小和方向依存於細胞和分離溶液的介電特性、外加交流電場強度等參數。
　　此介電泳分離晶片藉光微影蝕刻法製作的微小電極組晶片，其上對準接合以複製翻模製成的polydimethylsiloxane (PDMS)分離反應槽共組合而成本研究的晶片系統。實驗中以信號產生器作為交流電場源，並利用城垛形交指狀之微小電極組(interdigitated castellated microelectrodes)提供非均勻性電場，藉頻率掃瞄方式(1 kHz ~ 20 MHz)或調控不同懸浮溶液之導電率，尋求出最佳分離生物微粒的條件。
　　本研究中我們藉此晶片進行了兩項應用；(1)在乳膠微粒(latex)與修飾小牛血清蛋白的乳膠微粒(BSA-latex)的分離測試結果，發現可在10.2 µS/cm 的KCl分離溶液下，以10 Vp-p的正弦信號300 kHz，將latex受正介電泳力吸附於具高電場密度的電極組上，然此時BSA-latex則無受明顯介電泳力而懸浮於溶液中。(2)在活酵母菌與經沸煮30分鐘後之死酵母菌的分離實驗中，發現在5 µS/cm 之KCl溶液中，以10 Vp-p的正弦信號20 MHz為最佳分離條件，亦即可將活酵母菌細胞吸附於電極組上，相對地死酵母菌細胞則受負介電泳力，集中於低電場強度的電極區間中。特別的是我們以活酵母菌與死酵母菌進行分離系統的重複實測性探討，結果顯示出微流體以1 µl/min的注入流速，可先將聚集在電極區間之死酵母菌沖出，而不影響受正介電泳力吸附於電極組上之活酵母菌。其次，若將交流電場關閉，則活酵母菌會開始脫離電極，而終究被沖流至分離槽匯集取出。

　　High separation purity and maintenance of cell viability in cell separation procedure was required. In this study, a dielectrophoretic(DEP) chip was fabricated to test the separating effect for bioparticles. Dielectrophoresis is the lateral movement of cells caused by electrical polarization effects in nonuniform AC electric fields. Movement of the cells to regions of high electric field strength is called positive dielectrophoresis, whereas movement to regions of low electric field strength is called negative dielectrophoresis. The different cells may be induced opposite polarity through a suitable electrical condition with positive or negative DEP forces, and then be separated. Actually, the force responsible for this motion is also governed by the dielectric properties both of the suspending medium and of the particles, as well as the geometry of the electric field.
　　The DEP chip was constructed by a top layer of poly-dimethylsiloxane replica containing the separation chamber, and a bottom microelectrode chip manufactured by standard photolithography. The resulting microelectrodes of interdigitated castellated geometry were energized by a 10 V peak-to-peak sinusoidal signal from the function generator. The frequency was swept from 1 kHz to 20 MHz and the conductivity of the suspending medium was changed in order to obtain the best separation conditions.
　　First application on separations showed that latex could be separated from a mixture of latex and latex coated with bovine serum albumin (BSA-latex).The latex was attracted to the electrode tips by positive DEP force at frequency of 300 kHz in a 10.2 µS/cm diluted KCl medium, however, BSA-latex was not affected by DEP force. On the other hand, viable and nonviable yeast cells(heated at 100℃ for 30 minutes) were successfully separated under the conditions of the applied voltage of 5 Vp-p at frequency of 20 MHz in 5 µS/cm diluted KCl medium. The viable yeast cells were attracted at the electrode tips by positive DEP force while the nonviable yeast cells experienced a negative DEP force and were collected into a triangular pattern. In this study, a DEP separation system was constructed to remove nonviable yeast cells at a flow rate of 1 µl/min by a syringe pump while viable yeast cells were retained at the electrode tips. The separated viable yeast cells could then be collected by turning off the field.

[1]魏正舒、宋金丹, “醫學細胞生物學”, 合記圖書出版社, 87年, 31-34.

[2]A. Radbruch, “Flow cytometry and cell sorting”, Springer-Verlag Inc., 1992, 7-11.

[3]R. Pethig, “Dieletrophoresis: using inhomogeneous AC electrical fields to separate and manipulate cells”, Critical Review in Biotechnology, 1996, 16, 331.

[4]M. S. Talary, J. P. H. Burt, P. Pethig, “Future trends in diagnosis using
laboratory-on-a-chip technologies”, Parasitology, 1998, 117, S191-S203.

[5]R. Pethig, J. P. H. Burt, A. Partonz, N Rizvix, M. S. Talary and J. A. Tamey, “Development of biofactory-on-a-chip technology using excimer laser micro- machining”, Journal of Micromechanical and Microengeering, 1993, 8, 57–63.

[6]R. Pethig, Y. Huang, X. B. Wang, J. P. H. Burt, “Positive and negative dielectrophoretic collection of colloidal particles using interdigitated castellated microelectrodes”, Journal of Physics D: Applied Physics, 1992, 24, 881.

[7]K. Asami, E. Gheorghiu, T. Yonezawa, “Dielectric behavior of budding yeast in cell separaion”, Biochimica et Biophysica Acta, 1998, 1381, 234-239.

[8]P. R. C. Gascoyne, X. B. Wang, Y. Huang, F. F. Becker, “Dielectrophoretic separation of cancer cells from blood”, IEEE Transactions on Industry Applications, 1997, 33, 670-678.

[9]F. F. Becker, X. B. Wang, Y. Huang, R. Pethig, J. Vykoukal, P. R. C. Gascoyne, “The removal of human leukaemia cells from blood using interdigitated microelectrodes”, Journal of Physics D: Applied Physics, 1994, 27, 2659-2662.

[10]J. Cheng, E. L. Sheldon, L. Wu, M. J. Heller, J. P. O’Connell, “Isolation of cultured cervical carcinoma cells mixed with peripheral blood cells on a bioelectronic chip”, Analytical Chemistry, 1998, 70, 2321-2326.

[11]P. R. C. Gascoyne, J. Noshari, F. F. Becker, R. Pethig, “Use of dielectrophoretic collection spectra for characterizing difference between normal and cancerous cells”, IEEE Transactions on Industry Applications , 1994, 30, 829-834

[12] Paul T Sharpe, “Methods of cell separation”, Elsevier, 1988, 23-26.

[13]C. L. Colyer, S. D. Mangru, D. J. Harrison, “Microchip-based capillary electrophoresis of human serum proteins”, Journal of Chromatography A, 1997, 781, 271-276.

[14]A. T. Woolley, K. Q. Lao, A. N. Glazer, R. A. Mathies, “Capillary electrophoresis chips with integrated electrochemical detection”, Analytical Chemistry, 1998, 70, 684-688.

[15]S. F. Y. Li, “Capillary electrophoresis: principles, practice and applications”, Elsevier, 1993.

[16]M. Berger, J. Castelino, R. Huang, M. Shah, R. H. Austin, “Design of a microfabricated magnetic cell separator”, Electrophoresis, 2001, 22, 383-3892.

[17]A. J. Cunningham, “Introduction to bioanalytical sensors”, Wiley Interscience, 1998.

[18]A. E. G. Cass, “Biosensor : a practical approach”, IRL Press, 1990.

[19]M. Bengtsson, S. Ekström, J. Drott, A. Collins, E. Csöregi, G. Marko-Varga, T. Laurall, “Application of microstructured porous silicon as a biocatalytical surface”, Physica Status Solidi, 2000, 182, 495-504.

[20]P. C. Wang, D. L. DeVoe, C. S. Lee, “Integration of polymeric membranes with microfluildic networks for bioanalytical applications”, Electrophoresis, 2001, 22, 3857-3867.

[21]L. P. Lee, S. A. Berger, D. Liepmann, L. Pruitt, “High aspect ratio polymer microstructures and cantilevers for bioMEMS using low energy ion beam and photolithography”, Sensors and Actuators A, 1998, 71, 144-149.

[22]I. Moser, G. Jobst, G. A. Urban, “Biosensor arrays for simultaneous measurement of glucose, lactate, glutamate, and glutamine”, Biosensors & Bioelectronics, 2002, 17, 297-302.

[23]L. Lauer, S. Ingebrandt, M. Scholl, A. Offenhausser, “Aligned microcontact printing of biomolecules on microelectronic device surfaces”, IEEE Transaction on Biomedical Engineering, 2002, 48, 838-842.

[24]K. K. Jain, “Applications of biochip and microarray systems in pharmacogenomics”, Pharmacogenomics, 2000, 3, 289-307.

[25]L.W. van H. Nicole, V. Oscar, M. M. L. van H. Adele, J. K. Esther, P. Ad, A. Aharoni, J. van T. Arjen, K. Jaap, “The application of DNA microarrays in gene expression analysis”, Journal of Biotechnology, 2000, 78, 271–280.

[26]I. Erill, R. Villa , P. Goudignon, L. Fonseca, J.A. Plaza, “Silicon microsystem passivation for high-voltage applications in DNA chips”, Microelectronics and Reliability, 2000, 40, 787-789.

[27] F. Vinet , P. Chaton, Y. Fouillet, “Microarrays and microfluidic devices: miniaturized systems for biological analysis”, Microelectronic Engineering, 2002, 61–62, 41–47.

[28]D. Beebe, M. Wheeler, H. Zeringue, E. Walters, S. Raty, “Microfluidic technology for assisted reproduction”, Theriogenology, 2002, 57,125-135.

[29]T. Richter, L. S. L. Loranelle, O. D. Richard,U. Bilitewski, D. J. Harrison, “Bi-enzymatic and capillary electrophoretic analysis of non-fluorescent compounds in microfluidic devices: Determination of xanthine”, Sensors and Actuators B, 2002, 81, 369-376.

[30]G. R. Fuhr, C. Reichle, “Living cells in opto-electrical cages”, Analytical Chemistry, 2000, 19, 6, 402-409

[31]B. Malyan, W. Balachandran, “Sub-micron sized biological particle manipulation and characterization”, Journal of Electrostatic, 2001, 51-52, 15-19

[32]T. Schnelle, T. Muller, G. Gradl, S. G. Shirley, G. fuhr, “Dielectrophoretic manipulation of suspended submicron particles”, Electrophoresis, 2001, 21, 66-73.

[33]S. Fiedler, S. G. Shirley, T. Schnelle, G. Fuhr, “Dielectrophoretic sorting of particles and cells in a microsystem”, Analytical Chemistry, 1998, 70, 1909-1915.

[34]M. S. Talary, J. P. H. Burt, J. A. Tame, R. Pethig, “Electromanipulation and separation of cells using traveling electric fields”, Journal of Physics D: Applied Physics, 1996, 29, 2198–2203.

[35]F. F. Becker, X. B. Wang, Y. Huang, R. Pethig, J. Vykoukal, P. R. C. Gascoyne, “The removal of human leukaemia cells from blood using interdigitated microelectrodes”, Journal of Physics D: Applied Physics, 1994, 27, 2659-2662.

[36]N. G. Green, H. Morgan, “Separation of submicrometre particles using a combination of dielectrophoretic and electrohydrodynamic forces”, Journal of Physics D: Applied Physics, 1998, 31, L25–L30.

[37]A. D. Goater and R. Pethig, “Electrorotation and dielectrophoresis”, Parasitology, 1998,117, S177-S189.

[38]J. Suehiro, R. Pethig, “The dielectrophoretic movement and positioning of a biological cell using a three-dimensional grid electrode system”, Journal of Physics D: Applied Physics, 1998, 31, 3298-3305.

[39]N. G. Green, A. Ramos, H. Morgan, “Ac electrokinetics: a survey of
sub-micrometre particle dynamics”, Journal of Physics D: Applied Physics, 2000, 33, 632-641.

[40]H. Morgan, A. G. Izquierdo, D. Bakewell, N. G. Green, A. Ramos, “The dielectrophoretic and travelling wave forces generated by interdigitated
electrode arrays: analytical solution using Fourier series”, Journal of Physics D: Applied Physics, 2001, 34, 1553-1561.

[41]T. B. Jones, “Electromechanics of particles”, Cambridge University Press, 1995.

[42]J. P. H. Burt, K. L. Chan, D. Dawson, A. Parton, R. Pethig, “Assay for microbial contamination and DNA analysis based on electrorotation”, Annales Biologie Clinique, 1996, 54, 253-257.

[43]D. Walz, H. Berg, G. Milazzo, “Bioelectrochemistry of cells and tissues”, Birkhauser Verlag, 1995.

[44]X. Chuanxiang, Z. Lisheng, M. Hong, “Frequency characteristics of dielectrophoresis in cell suspension solution”, Proceedings of the 3rd International Conference on Properties and Applications of Dielectric Materials, 1991, 1112-1115.

[45]D. W. E. Allsopp, K. R. Milner, A. P. Brown, W. B. Betts, “Impedance technique for measuring dielectrophoretic collection of microbiological particles”, Journal of Physics D: Applied Physics,1999, 32, 1066–1074.

[46]J. Sanny, W. Moebs, ”University physics”, Wm. C. Brown Publisher, 1996, 444-446.

[47] http://www.ibmm.informatics.bangor.ac.uk/pages/people/pethig.htm/

[48] http://www.dielectrophoresis.org/

[49] http://www.ibmt.fhg.de/ibmt3ambtbiotechnol_e.html/

[50] http://www.ee.rochester.edu:8080/users/jones/

[51] http://www.nanogen.com/

[52]T. Fujii, “PDMS-based microfluidic devices for biomedical applications”, Microelectronic Engineering, 2002, 61–62, 907–914.

[53]B. H. Jo, L. M. V. Lerberghe, K. M. Motsegood, D. J. Beebe, “Three-Dimensional Micro-Channel Fabrication in Polydimethylsiloxane (PDMS) Elastomer”, Journal of Microelectromechanical Systems, 2000, 9, 76-81.

[54]N. G. Green, H. Morgan, “Dlelectrophoretic separation of nano-particles”,Journal of Physics D: Applied Physics, 1997, 30, L41-L44.

[55]A. Ramos, H. Morgan, N. G. Green, A. Castellanos, “AC electrokinetics : a review of forces in microelectrode structures”, Journal of Physics D: Applied Physics, 1998, 31, 2338-2353.