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研究生:林育德
研究生(外文):Yu-De Lin
論文名稱: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
論文名稱(外文):Development of a Planar Dielectrophoretic System and Its Applications on Bioparticles
指導教授:張憲彰
指導教授(外文):Hsien-Chang Chang
學位類別:碩士
校院名稱:國立成功大學
系所名稱:醫學工程研究所碩博士班
學門:工程學門
學類:綜合工程學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:中文
論文頁數:78
中文關鍵詞:酵母菌介電泳晶片非均勻交流電場分離乳膠微粒
外文關鍵詞:dielectrophoresisyeastseparationlatexnonuniform AC electric field
相關次數:
  • 被引用被引用:17
  • 點閱點閱:434
  • 評分評分:
  • 下載下載:88
  • 收藏至我的研究室書目清單書目收藏:2
  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.
中文摘要………………………………………………………………I
英文摘要………………………………………………………………II
致謝……………………………………………………………………III
目錄……………………………………………………………………IV
表目錄…………………………………………………………………VI
圖目錄…………………………………………………………………VII

第一章 緒論…………………………………………………………1
  1.1 研究背景……………………………………………………1
  1.2 細胞分離技術………………………………………………2
    1.2.1 離心法………………………………………………3
    1.2.2 親和性分離法………………………………………5
    1.2.3 電泳法………………………………………………6
    1.2.4 流式細胞分選儀……………………………………7
    1.2.5 磁場細胞分選儀……………………………………8
  1.3 生物技術的發展……………………………………………9
    1.3.1 生醫微機電系統之發展……………………………10     1.3.2 生物晶片之發展……………………………………10
  1.4 介電泳分離原理……………………………………………12
    1.4.1 介電泳效應之產生…………………………………12
    1.4.2 介電泳應用於生物分離系統………………………17
  1.5 介電泳於國內外生物科技研究之應用……………………29
  1.6 研究架構……………………………………………………33
第二章 設備與方法…………………………………………………34
  2.1 實驗設備……………………………………………………34
  2.2 分離樣本配製………………………………………………35
  2.3 分離晶片製作………………………………………………37
    2.3.1 微小電極陣列晶片的製作…………………………37
    2.3.2 分離反應槽之製作…………………………………39
    2.3.3 分離參數測試晶片…………………………………40
    2.3.4 介電泳式細胞分離晶片……………………………41
  2.4 系統架構……………………………………………………42
    2.4.1 分離參數測試平台…………………………………42
    2.4.2 介電泳分離系統硬體架構…………………………43

第三章 結果與討論…………………………………………………44
  3.1 介電泳分離晶片製作之探討………………………………44
    3.1.1 微小電極陣列設計…………………………………44
    3.1.2 PDMS微型分離通道設計與製作……………………44
    3.1.3 電極晶片與PDMS分離通道之接合技術……………45
  3.2 Latex 之介電泳效應探討…………………………………49
    3.2.1 Latex 於不同電導率溶液之介電泳探討…………49
    3.2.2 BSA-latex 於不同電導率溶液之介電泳探討……53
    3.2.3 Latex與BSA-latex之介電泳分離效果……………56
  3.3 酵母菌細胞之介電泳晶片分離實驗結果…………………57
    3.3.1 活酵母菌之介電泳探討……………………………57
    3.3.2 死酵母菌之之介電泳探討…………………………60
    3.3.3 酵母菌細胞之介電泳測試…………………………62
    3.3.4 ANSYS 電場模擬及實驗結果預測…………………65
    3.3.5 酵母菌細胞之介電泳晶片分離系統測試…………67

第四章 結論…………………………………………………………69
  4.1 最佳分離條件之選擇………………………………………69
  4.2 介電泳晶片式分離系統之探討……………………………70
  4.3 未來發展方向………………………………………………70

附錄……………………………………………………………………71
參考文獻………………………………………………………………73


表目錄
表1.1 現今國際上介電泳研究群的發展近況………………………32
表3.1 頻率與溶液電導率對latex 介電泳力變化整理……………54
表3.2 頻率與溶液電導率對BSA-latex 介電泳力變化整理………55
表3.3 頻率與溶液電導率與活酵母菌之介電泳力變化整理………62
表3.4 頻率與溶液電導率與死酵母菌之介電泳力變化整理………64
表3.5 活與死酵母菌細胞之最佳分離條件…………………………64


圖目錄
圖1.1 Rate-zonal離心法流程圖……………………………………3
圖1.2 Isopycnic 離心法流程圖……………………………………4
圖1.3 親和性分離法流程圖…………………………………………5
圖1.4 電泳分離原理…………………………………………………6
圖1.5 流式細胞分選儀………………………………………………7
圖1.6 磁場細胞分選儀………………………………………………8
圖1.7 生物感測分析系統之概念圖…………………………………9
圖1.8 球體於平行電板作用下產生電極化…………………………14
圖1.9 電中性球體於非均勻電場之介電泳現象……………………15
圖1.10球體於非均勻交變電場作用之結果…………………………15
圖1.11水中之氣泡於非均勻電場作用………………………………16
圖1.12不同介電特性的微粒造成正和負介電泳現象………………16
圖1.13細胞懸浮於中性溶液之電荷分佈……………………………19
圖1.14細胞產生極化及表面電雙層之扭曲現象……………………19
圖1.15溶液與細胞之電極化現象……………………………………21
圖1.16溶液與細胞之電極化現象的簡化圖…………………………21
圖1.17誘發偶極距與電場之關係……………………………………22
圖1.18偶極距在不同頻率之變化……………………………………23
圖1.19相互介電泳……………………………………………………25
圖1.20球體於兩線狀電極之交變電場移動吸引現象………………26
圖1.21細胞互相吸引呈串珠狀………………………………………27
圖1.22研究架構圖……………………………………………………33
圖2.1 真空蒸鍍機基本結構圖………………………………………40
圖2.2 分離參數測試晶片實體圖……………………………………40
圖2.3 介電泳分離晶片實體圖………………………………………41
圖2.4 分離參數測試平台架構圖……………………………………42
圖2.5 介電泳分離細胞系統架構圖…………………………………43
圖3.1 不同形式之微小電極設計……………………………………48
圖3.2 蝕刻時間過長而造成電極失真情形…………………………48
圖3.3 Latex 懸浮於純水中在不同頻率之介電泳效應……………49
圖3.4 SU-8母模與PDMS微分離槽製作流程…………………………50
圖3.5 立體式電極晶片接合…………………………………………74
圖3.6 利用水玻璃之晶片接合………………………………………74
圖3.7 Latex於10.2 µS/cm之分離溶液中之介電泳現象…………50
圖3.8 BSA-latex於10.2 µS/cm之分離溶液之介電泳現象………54
圖3.9 Latex 與BSA-latex之分離結果……………………………56
圖3.10活酵母菌於不同頻率之介電泳現象…………………………58
圖3.11死酵母菌於不同頻率之介電泳現象…………………………59
圖3.12活與死酵母菌細胞最佳分離結果……………………………63
圖3.13以crystal violet染劑染死酵母菌之結果…………………63
圖3.14正介電泳及負電泳力分離圖…………………………………64
圖3.15 ANSYS電場模擬分析圖………………………………………65
圖3.16分離實驗結果預測圖…………………………………………66
圖3.17晶片式流動分離系統實測結果………………………………68
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