臺灣博碩士論文加值系統

由於細胞相較於工作溶液易受到電場誘導產生偏極化，在不均勻電場作用下會產生正介電泳力，進而趨向強電場方向移動。本研究利用COMSOL有限元素分析模擬軟體進行電場模擬做為探討細胞受電場影響變化與最佳化電極設計之依據，再以微製程技術製作出不規則形狀電極，藉此產生不均勻強度之電場來驅動細胞移動，進而脫離基材，藉此方式量測培養於PDMS與PLA兩種不同生醫材料之細胞貼附力。
從介電泳量測結果發現培養在PDMS之細胞無論培養時間長短仍未有明顯移動現象；而培養在PLA之細胞於培養時間4、6小時，在低電壓(約6V)操作下細胞即產生移動；當培養時間至8小時，需要施加較高電壓(約16V)才可驅動細胞移動；培養24小時之細胞已不見移動現象。當施加越高電壓，表示驅動細胞之作用力越大，細胞貼附越佳，故隨著培養時間增加，細胞培養於PLA之貼附力增加。而培養於PDMS之細胞貼附已達一定程度，持續施加電壓仍無法造成細胞移動，反而造成細胞被裂解。
由材料之親疏水性量測結果可知，PDMS之接觸角為92.21度；PLA之接觸角為83.11度，顯示PLA比PDMS具有較佳的親水性，理論上PLA會有較佳細胞貼附情形。但由細胞培養之外觀與介電泳量測結果均顯示PDMS有較佳之貼附效果，推測材料之其他特性較親水性更能影響細胞之貼附。

Compared with the working medium, cell is easily to encounter an electric field induced polarization. Hence a positive dielectrophoretic force, which is generated due to an unbalanced electric field, will drive the cell toward the area of high electric field density. In this study, the finite element analysis software COMSOL is used to investigate the influence of an electric field on cell behavior for a better design of the dielectrophoretic electrode. Accordingly, a dielectrophoretic device that contains several micro electrodes for producing unbalanced electric field is fabricated using microelectromechanical fabrication technique. The proposed device is then used for the detection of cell adhesive force on PDMS and PLA.
It is observed that those cells cultured on PDMS firmly stick on the material without any movement under the actions of different dielectrophoretic force. However, the dielectrophoretic force due to a 6 V applied potential is enough to move those cells having cultured on the PLA for 4 and 6 hr. When the culture period is increased to 8 hr, a higher applied potential of 16V is required to move the cells. The cells cultured on the PLA for 24 hr are not relaxed by the dielectrophoretic force even a higher potential is applied. Theoretically, a higher applied voltage can induce a larger driving force to move the cultured cells. For the PLA scaffold, a higher potential is required to move those cells having longer culture time. While for the PDMA scaffold, the cells cultured on it have a much higher adhesive force such that the dielectrophoretic force generated by the proposed device cannot lift the cells from the scaffold. A higher applied potential results in the unwanted cell lysis.
The contact angles of PDMS and PLA are measured to be 92.21and 83.11, respectively. It indicates that the PLA surface is more hydrophilic than PDMS. In general, cells should have a better adhesion on PLA scaffold. However, the experimental results in this study reveal that cells have better adhesion on the PDMS scaffold. It is presumed that other properties of the scaffold influence the cell adhesion more than its hydrophilic-hydrophobic property.

致謝 I
摘要 II
Abstract III
圖目錄 VI
表目錄 VIII
第一章 緒論 1
1.1研究背景與動機 1
1.2 研究目的 8
1.3論文大綱 9
第二章 研究方法與材料 10
2.1 研究方法 10
2.1.1介電泳(Dielectrophoresis) 10
2.1.2誘導偶極矩 10
2.1.3電中性微粒之介電泳現象 12
2.1.4細胞之介電泳力 15
2.1.5 介電泳相關文獻與介電泳應用 16
2.2 實驗材料 20
2.2.1聚二甲基矽氧烷(Polydimethylsiloxane，PDMS) 20
2.2.2聚乳酸(Polylactic acid，PLA) 21
2.3 實驗材料製備 21
2.3.1聚二甲基矽氧烷(Polydimethylsiloxane，PDMS) 21
2.3.2聚乳酸(Polylactic acid，PLA) 22
第三章 電極設計分析與製作 23
3.1電極設計 23
3.2電場模擬 24
3.2.1 COMSOL模擬步驟 24
3.2.2 參數設定 24
3.2.3 模擬結果與分析 27
3.3電極製作 31
3.3.1 黃光微影製程 31
3.3.2 蒸鍍(熱阻式蒸鍍機) 32
3.3.3 Lift off製程 32
第四章 實驗方法與步驟 33
4.1 親水性量測 33
4.2 細胞培養 33
4.2.1 實驗儀器與使用藥品 33
4.2.2 細胞植覆 34
4.3 介電泳量測 35
4.3.1實驗儀器與使用藥品 35
4.3.2 實驗條件設置 35
4.3.3實驗方法 37
4.3.4 實驗架構 37
第五章 實驗結果與討論 38
5.1 實驗結果 38
5.1.1 親水性量測 38
5.1.2細胞培養 38
5.1.3介電泳量測 40
5.2 結果討論 44
第六章 結論與未來展望 46
6.1 結論 46
6.2 未來展望 47
參考文獻 48

[1]宋信文、梁晃千，“建立人類的人體工房—組織工程，”科學發展
　Vol.362,6-11,2003。
[2]Kovacs GTA, Maluf NI and Petersen KE, “Bulk micromachining
　of silicon, ”Proceedings of the IEEE,86(8):1536-1551,1998.
[3]Pan L. C. , Liang Y. C. , Tseng F. G. , Leou K. C. , Chen L. D. and
　Lai Y. Y. , “A novel application of acoustic plate mode sensor in
　tissue regeneration. Proceedings of the IEEE-EMBS Special Topic
Conference on Microtechnologies, ”Medicine & Biology,143-144,
2002.
[4]Han A. , Rooij N. F. D. and Staufer U. , “Design and fabrication of
nanofluidic devices by surface micromachining, ” Nanotechnology,
17:2498–2503,2006.
[5]Chang H. K. and Kim Y. K. , “UV-LIGA process for high aspect ratio
structure using stress barrier and C-shaped etch hole, ”Sensors and
Actuators,84(3):342-350,2000.
[6]Xia Y. and Whitesides G. M. , “Soft lithography, ”Annu.Rev.Mater.
Sci,28:153-184,1998.
[7]Rogers J. A. and Nuzzo R. G. , “Recent progress in soft lithography, ”
Materials today,8:50-56,2005.
[8]李世光、胡毓忠，“微機電系統與奈米科技，”科學發展Vol.378
,57-61,2004。
[9]Madou M. J. , Lee L. J. , Daunert S. , Lai S. and Shih C. H. ,“Design
and fabrication of CD-like microfluidic platforms for diagnostics:
microfluidic functions, ”Biomedical Microdevices,3(3):245-254
,2003.
[10] Martin R. S. , Gawron A. J. , Lunte S. M. and Henry C. S. , “Dual-
electrode electrochemical detection for poly(dimethylsiloxane)
-fabricated capillary electrophoresis microchips, ”Anal.Chem.
,72:3196-3202,2000.
[11]Jiang G. , Attiya S. , Ocvirk G. , Lee W. E. and Harrison D. J. ,
“Red diode laser induced fluorescence detection with a confocal
microscope on a microchip for capillary electrophoresis, ”
Biosensors and Bioelectronics,14(10-11):861-869,2000.
[12] 潘吉祥，“微機電系統技術於生醫的應用，”國立勤益技術學院
機械系，勤益學報第十九期。
[13] 生醫材料的特性－ http://mmrl.cgu.edu.tw/rehab/mme/rehab/organize/chap3/material/no2.htm
[14]黃世偉，”高分子材料與醫療器材，”科學發展Vol.455,14-19,
2010。
[15] 黃何雄、何俊德、游惠婷、潘思蓉、李天翎、楊明鈴、廖保鑫、
周明勇、徐啟智，“Ti及Ti-6Al-4V合金表面粗糙度對類造骨
U-2OS細胞初期貼附及增殖之影響，”中華牙醫學雜誌Vol.23
,3,2004。
[16]丁彥中，“奈米級直溝地形對骨母細胞貼附形態之影響，”國立
台灣大學化學工程學研究所碩士學位論文，2006。
[17]湯為淳，“羊膜培養環境中內皮細胞可提升整合素表現與附著
度，”東海大學生命科學系碩士班碩士論文，2007。
[18] Becker W. M. , Kleinsmith L. J. , Hardin J. and Bertoni G. P. , The
world of the cell,7th edition.PEARSON,2009.
[19] Integrin regulation of cell migration and focal adhesions－
http://www.google.com.tw/imgres?imgurl=http://www.nature.com/nrc/journal/v8/n8/images/nrc2353-i2.jpg&imgrefurl=http://www.na
ture.com/nrc/journal/v8/n8/box/nrc2353_BX2.html&usg=__XPg0j
Gi7zrgQQw5-fozBQkkGCkI=&h=579&w=305&sz=87&hl=zh-T
W&start=4&sig2=Ym7MezCjc0rNZ4YSaklx8w&zoom=1&tbnid=
XzFzILVNluUOuM:&tbnh=134&tbnw=71&ei=u1OsTb2BA46Su
APHntmECQ&prev=/search%3Fq%3Dfocal%2Badhesion%26um
%3D1%26hl%3Dzh-TW%26sa%3DN%26rlz%3D1R2PPST_zh-T
WTW418%26biw%3D1659%26bih%3D814%26tbm%3Disch&u
m=1&itbs=1
[20]謝夙惠，“Galectin-1透過neuropilin-1調控人類內皮細胞的貼附
和移動，”國立成功大學口腔醫學研究所碩士論文，2006。
[21]Boveri T. , “The origin of malignant tumours, ” Baillière, tindall
&Cox ,1914.
[22]Benoit M. , Gabriel D. , Gerisch G. and Gaub H. E. , “Discrete
interactions in cell adhesion measured by single-molecule forces
pectroscopy, ” Nature Cell Biology, 2:313-317, 2000.
[23]Chu Y. S. , Thomas W. A. , Eder O. , Pincet F. , Perez E. , Thiery
J. P. and Dufour S. , “Force measurements in E-cadherin–mediated
cell doublets reveal rapid adhesion strengthened by actin cytoske-
leton remodeling through Rac and Cdc42 , ” The Journal of cell
biology,167(6):1183-1194,2004.
[24] Ko T. M. , Lin J. C. and Cooper S. L. ,“Surface characterization
and platelet adhesion studies of plasma-sulphonated polyethylene, ”
Biomaterials,14(9):657-64,1993.
[25] Tamada Y. and Ikada Y. , “Fibroblast growth on polymer surfaces
and biosynthesis of collagen, ” J Biomed Mater Res,28(7):783-789,
1994.
[26] Lotz M. M. , Burdsal C. A. , Erickson H. P. , and McClay D. R. ,
“Cell adhesion to fibronectin and tenascin：quantitative measu-
rements of initial binding and subsequent strengthening respo-
nse, ” The Journal of Cell Biology,109:1795-1805,1989.
[27]Maheshwari G. , Brown G. , Lauffenburger D. A. , Wells A. and
Griffith L. G. ,“Cell adhesion and motility depend on nanoscale
RGD clustering, ” Journal of Cell Science,113:1677-1686,2000.
[28] Furukawa K. S. , Ushida T. , Nagase T. , Nakamigawa H. ,Noguchi
T. ,Tamaki T. , Tanaka J. and Tateishi T. ,“Quantitative analysis of
cell detachment by shear stress, ” Materials Science and Engineeri-
ng C17: 55–58, 2001.
[29] Moussya F. , Linb F. Y. H. , Lahooti S. , Policova Z. , Zinggd W.
and Neumannc A. W. , “A micropipette aspiration technique to
investigate the adhesion of endothelial cells, ”Elsevter Scrence
B.V.,2:493-503,1994.
[30] Song G. B. , Qin J. , Luo Q. , Shen X. D. , Yan R. B. and Cai
S. X. ,“Adhesion of different cell cycle human hepatoma cells to
endothelial cells and roles of integrin β1, ” World J Gastroenterol
,11(2):212-215, 2005.
[31] Yamamoto A. , Mishima S. , Maruyama N. and Sumita M. ,“ A
new technique for direct measurement of the shear force necessary
to detach a cell from a material, ” Biomaterials, 19:871-879, 1998.
[32] Lee C. C. , Wu C. C. and Su F. C. ,“The technique for measurem-
ent of cell adhesion force, ”Taiwan R.O.C. , National Cheng
Kung University, 2004.
[33] Kwon K. W. , Choi S. S. , Kim B. , Lee S. N. , Park M. C. , Kim P. , Lee S. H. ,Park S. H. and Suh K. Y.,“ A microfluidic flow sensor for measuring cell adhesion , ” IEEE Sensors, 2006.
[34] Lu H. , Koo L. Y. , Wang W. M. , Lauffenburger D. A. , Griffith L.
G. , and Jensen K. F. ,“Microfluidic shear devices for quantitative
analysis of cell adhesion, ” Analytical Chemistry,76:5257-5264
,2004.
[35]Hung M. S. , Chen Y. W. , Lin C. J. , Ay C. and Chiou C. P. ,“
Measurement of Human Endothelial Cell adhesion using dielectrophoresis, ” Journal of The Chinese Society of Mechanical Engineers,30(5):393-400,2009.
[36]Pohl H. A. ,“The motion and precipitation of suspensoids in
divergent electric fields,”J. Appl. Phys. 22:869-871,1951.
[37]粘正勳、邱聞鋒，“介電泳動─承先啟後的奈米操縱術，”物理
雙月刊，廿三卷六期，2004。
[38]林彥亨，“利用介電泳力操控細胞之生物晶片研究，”國立成功
大學工程科學系碩士班碩士論文，2002。
[39] Goater A. D. and Pethig R. ,”Electrorotation and dielectrophoresis,
” Parasitology,117,S177-S189, 1998.
[40]林育德，“平面式微介電泳系統之研發與其在生物微粒分離上
之應用，”國立成功大學醫學工程研究所碩士論文，2002。
[41]鄭傑輿，“利用介電泳力評估人類臍靜脈內皮細胞貼附力最適化
量測模式之研究，”國立嘉義大學生物機電工程學系碩士論文，
2007。
[42] Morgan H. and Green N. G. ,“AC electrokinetics: colloids and
nanoparticles , ”Microtechnologies and microsystems series.
Research Studies Press,2003.
[43]Heller J. H. , Teixeira-Pinto A. A. , Nejelski L. L. and CutlerJ.
L. ,“The behavior of unicellular organisms in an electromagnetic
field, ”Cell Res 20,548-564 ,1960.
[44] Pohl H. A. and Crane J. S. ,“Dielectrophoresis of cells, ”Biophysic-
al Journal,11(9)1971.
[45]Masuda S. , Washizu M. and Kawabatai I. , “ Movement of Blood
Cells in liquid by Nonuniform Traveling Field, ” IEEE Transactio-
ns On Industry Applications,24(2),1988.
[46] Pethig R. and Markx G. H. , “Applications of dielectrophoresis in
biotechnology, ”Elsevier Science,15,1997.
[47] Iliescu C. , Xu G. L. , Samper V. and Tay F. E. H. ,“Fabrication of
a dielectrophoretic chip with 3D silicon electrodes, ” Journal of
Micromechanics and Microengineering,2005.
[48] Tay F. E. H. , Yu L. , Pang A. J. and Iliescu C. ,“Electrical and
thermal characterization of a dielectrophoretic chip with 3D
electrodes for cells manipulation, ” Electrochimica Acta 52:2862
–2868,2007.
[49] Lapizco-Encinas B. H. , Simmons B. A. , Cummings E. B. and
Fintschenko Y. ,“Dielectrophoretic Concentration and
Separation of Live and Dead Bacteria in an Array of Insulators, ”
Analytical Chemistry, 76(6):1571-1579, 2004.
[50] Lin Y. H. and Lee G. B. , “ An optically induced cell lysis device
using dielectrophoresis, ” Applied Physics Letters,94(3),2009.
[51] 聚二甲基矽氧烷－ http://zh.wikipedia.org/zh-tw/%E8%81%9A%E4%BA%8C%E7%94%B2%E5%9F%BA%E7%9F%BD%E6%B0%A7%E7%83%B7
[52] Eddings M. A. , Johnson M. A. and Gale B. K. ,“ Determining the
optimal PDMS–PDMS bonding technique for microfluidic devices
, ” Journal Of Micromechanics And Microengineering,18,2008.
[53] Jo B. H. , Lerberghe L. M. V. , Motsegood K. M. and Beebe D. J. ,
“Three-Dimensional Micro-Channel Fabrication in Polydimethyl-
siloxane(PDMS) Elastomer, ” Journal Of Microelectromechanical
Systems,9(1),2000.
[54] Fuard D. , Tzvetkova-Chevolleau T., Decossas S., Tracqui P. and
Schiavone P. ,“Optimization of poly-di-methyl-siloxane (PDMS)
substrates for studying cellular adhesion and motility, ”
Microelectronic Engineering,85:1289–1293, 2008.
[55]聚乳酸－http://zh.wikipedia.org/wiki/%E8%81%9A%E4%B9%B3%E9%85%B8
[56]柯文昌、楊正昌、王敦正、馮琮涵、陳立典、李憲坤、李勝揚，
“聚乳酸骨釘骨板與骨斷裂面間於癒合過程中之交互作用，”中
華牙醫學雜誌Vol.23,3,2004。
[57] 聚乳酸(PLA)的應用－
http://www.weimon.com.tw/files/bulletin/f7e1a821949a4e9f86d2d
5aeb2ceba64.doc
[58]吳岳桐， “微粒子導電度量測與在其介電泳晶片上的應用，”
國立成功大學醫學工程研究所碩士論文， 2004。
[59] 在高頻交流電場下藉非對稱誘導電荷極化所造成之非線性電滲拉伸流－http://conf.ncku.edu.tw/research/articles/c/20071019/7.html
[60]李靜雯，“奈米結構與微水平震動平台對細胞生長之影響研究，”
國立中興大學生醫工程研究所碩士學位論文，2009。