成功大學博碩士論文系統

簡易檢索 / 詳目顯示

回結果列表

研究生：	鄭宜肪 Cheng, I-Fang
論文名稱：	可連續分選、捕捉、濃縮與偵測生物粒子之三維介電泳晶片的發展與其應用 A Bio-chip for Continuous Bio-particle Sorting, Trapping, Concentrating and Detecting Based on 3D Dielectrophoretic Field and Its Application
指導教授：	張憲彰 Chang, Hsien-Chang
學位類別：	碩士 Master
系所名稱：	工學院 - 醫學工程研究所 Institute of Biomedical Engineering
論文出版年：	2007
畢業學年度：	95
語文別：	英文
論文頁數：	78
中文關鍵詞：	分離、定量、濃縮、捕捉、拉曼光譜、介電泳
外文關鍵詞：	Raman spectrum, trapping, dual-layers dielectrophoresis, sorting, concentrating
相關次數：	點閱：91 下載：4
分享至:	分享至facebook 分享至twitter

查詢本校圖書館目錄查詢臺灣博碩士論文知識加值系統勘誤回報

將特定細菌從檢體中快速且精確的分離收集出來在臨床診斷與檢測是非常重要的，然目前傳統的微生物檢測方法其操作過程，依然相當繁雜且耗時。基於此，本研究想利用介電泳設計之微流道中來進行分離，藉各種生物微粒與其所處在懸浮液之間，存在有某程度介電特性的不同，會造成在交流電場下產生電極化程度的差異，經由微流道設計及即時性的拉曼光譜法，希望能對生物樣本進行連續式的分離、濃縮、鑑定與收集。
本研究製作並整合了平行對稱、傾斜、曲線漸縮等三區的雙層電極於流道的頂蓋與下板處，以期能達過濾、聚焦、分選與捕捉濃縮之功效。其間以20 m厚的SU 8光阻層相隔與UV膠的銜接構裝，形成一種整個具非均一的三維電場分佈之穩固微流道，非但可改善傳統平面電極介電泳力隨著離電極表面高度衰減的現象，且隨適當的流體條件之導入，可對生物微粒形成連續式分離濃縮與捕捉。我們成功的將大小不同的乳膠微粒分選至其各自獨立的子流道，也將腸球菌(E. faecium)從稀釋的全血中分離並於其各自的子流道中捕捉與濃縮，並印證箭頭漸縮之曲線形電極呈現最佳的捕捉濃縮效果。而針對尺寸接近的大腸桿菌(E. coli Nissle 1917)與乳酸桿菌(Lactobacillus)，亦成功的分離與捕捉濃縮，其後在細菌濃縮處以表面增顯拉曼光譜法偵測，得到可供辨識的訊號。最後，此技術也被應用於牛乳中含生物微粒之檢測，結果可將牛乳中之體細胞與細菌分選至各自的子流道中，並於其各自的濃縮區，以數位影像定義出該捕捉面積，經與單顆體細菌與捕捉細菌體積回推，可估算出各生物微粒的濃度。如此，可顯示本研究在對於生物檢體中所的含微粒，具鑑定與定量的功能。
透過微機電製程技術發展出快速、簡便、小型化、經濟與低破壞性之多功能性之生物處理系統，對細菌為非接觸且不需生化標定之分離操控方式，將不同的微生物群分離並量化收集出來，以利後續研究使用，其成果亦朝向開發新式lab-on-a-chip晶片邁進一大歩。

Separation of the microorganism and sorting their individual population from mixture sample was very important in the biomedical application. In this research, we develop an AC dielectrophoretic (DEP) bio-chip that integrated high throughput bacteria sorting, trapping, concentrating and detecting in continuous flow. A dual-layers dielectrophoresis (DEP) chip with these functions can be fabricated by standard lithography technique and UV bonding method. The 3D chip was constructed by two face to face electrode chips at top and bottom of microfluidic channel. The specific dielectrophoretic force can be induced by different dielectric properties of particles. We have separated different sizes of latex particles successfully based on their different magnitudes of negative DEP (nDEP) force and we also have sorted E. faecium from diluted whole blood cells and trapped E. faecium. Mixture of E. coli Nissle 1917 and Lactobacillus can be sorted and concentrated in this chip based on their different dielectric properties, and detected the fingerprints by surface enhanced Raman scattering (SERS). We have got well Raman signal during our DEP concentrator procedure. Consequently, About 80 % sorting and trapping efficiency was calculated. When the bacteria concentration was 107 CFU/ml, about 500 particles per second can be sorted and trapped. The particle concentration of the milk sample also can be roughly calculated through trapping area of the captured image. According to the results, this chip can be promoted to be a lab-on-a-chip in the future.

摘要                       I
Abstract                   II
致謝                       III
List of Figures            VI
Chapter 1  Introduction	1
1 Background and Motivation	1
2 Conventional Separation of Microorganism	4
2.1 Centrifuge Method	4
2.2 Flow cytometry	5
2.3 Biochemical methods	6
3	Micro-Electro-Mechanical Systems for Bio-Chip	7
3.1 Active micro-sorter	8
3.2 Passive micro sorter	9
4 Electrokinetics-based Cell Separation	11
4.1 Electrophoresis	11
4.2 Dielectrophoresis	13
5 Theory of Dielectrophoresis	14
5.1 Dielectric polarization	15
5.2 Field induced dielectrophoresis	19
6 Paper Review for Dielectrophoresis	22
6.1 Conventional dielectrophoresis	22
6.2 Traveling wave dielectrophoresis	22
6.3 Electro-rotation	23
6.4 Flow through dielectrophoretic chip	25
7 Research configuration	28
Chapter 2  Materials and Methods	29
1 Chip Design and Construction	29
2 Instrument and System Configuration	30
2.1 Experiment instrument	30
2.2 System configuration	32
3 Force calculation and related physical effect	33
4 Micro Fabrication	36
4.1 Fabrication of electrode chip	36
4.2 Fabrication of micro fluidic channel	38
4.3 Alignment and bonding	39
5 Sample preparation	41
Chapter 3  Results and Discussion	43
1 DEP Filtering and Focusing	43
1.1 Field simulation for electrode designs	43
1.2 Particle filtering	45
1.3 Particle focusing and alignment	46
2.1 Electrode design and theory	50
2.2 Particle guiding	53
2.3 Particle sorting	53
4 DEP Gating for Particle Trapping	56
4.1 Electrode design and theory	56
4.2 DEP trapping and concentrating	58
4.3 Trapping efficiency	60
4.5 DEP trapping combined with Raman detection	62
5 Particle Sorting and Trapping for Blood Sample	63
5.1 Blood sample and bacteremia	63
5.2 DEP parameter and buffer condition	63
5.3 Sorting and Trapping bacteria from blood	64
6 Particle Sorting and Trapping for Milk Sample	66
6.1 Total cell concentration for milk sample	66
6.2 Size separation	67
6.3 Detection of total cell concentration	69
Chapter 4  Conclusion	72
1 The advantages of continuously flow 3D DEP Chip	73
2 Future application and recommendation	74
References	75
                                    

[1] A. Minerick, P. Takhistov, R. Zhou and H.-C. Chang, “Manipulation and characterization of red blood cells with alternating current fields in microdevices”, Electrophoresis, 24(2003)3703-3717.
[2] M. P. Hughes, “Strategies for dielectrophoretic separation in laboratory-on-a-chip systems”, Electrophoresis, 23(2002)2569-2582.
[4] A. Radbruch, “Flow cytometry and cell sorting”, Springer-Verlag Inc., (1992)7-11.
[5] Daniel C. Harris,” Exploring chemical analysis”, second edition.
[6] 陳建人, 微機電系統技術與應用, 國科會精儀中心出版, (2004).
[7] G. B. Lee, C. I. Hung et. al, “Micromachined pre-focused 1×N flow switches
for continuous sample injection”, J. Micromech. Microeng., 11(2001)567-573.
[8] A. Y. Fu1, C. Spence, A. Scherer, F. H. Arnold, and S. R. Quake, “A
microfabricated fluorescence-activated cell sorter”, Nature Biotech.,
17(1999)1109-1111.
[9] James P. B. and Paul Y., “Diffusion-based extraction in a microfabricated device”, Sensor. Actuat. A-Phys., 58(1997)13-18.
[10] M. Yamada, M. Nakashima and M. Seki, “Pinched flow fractionation: Continuous size separation of particles utilizing laminar flow profile in a pinched microchannel”, Anal. Chem., 76(2004)5465-5471.
[11] P. D. Grossman, J. C. Colburn, “Capillary electrophoresis: theory and practice”, Academic press, 1992.
[12] H. A. Pohl, “Dielectrophoresis”, Cambridge Uni. Press, 1978.
[13] N.G. Green, H. Morgan, “Sub-micrometre ac Electrokinetics”, Proc IEEE Conference on Engineering in Medicine and Biology, 20(1998)2974-2977.
[14] H. Li, R. Bashir, “Dielectrophoretic separation and manipulation of live and heat-treated cells of Listeria on microfabricated devices with interdigitated electrodes”, Sensor. Actuat. B-Chem., 86(2002)215-221.
[15] M. P. Hughes, R. Pethig, X-B Wang, “Dielectrophoretic forces on particles in traveling electric fields”, J. Phys. D: Appl. Phys., 29(1996)474-482.
[16] R. Pethig, M. S. Talary and R. S. Lee, “Enhanceing Traveling-Wave Dielectrophoresis with signal Superposition”, IEEE Eng. Med. Biol. Mag., 22(2003)43-50.
[17] Jones T. B., “Electromechanics of Particles”, Cambridge: Cambridge University Press, 1995.
[18] D. Mietchen, T. Schnelle, T. Müller, R. Hagedorn and G. Fuhr, “Automated dielectric single cell spectroscopy-temperature dependence of electrorotation”, J. Phys. D: Appl. Phys., 35(2002)1258-1270
[19] X. B Wang, J. Yang, Y. Huang and J. Vykoukal, Frederick F. Becker, and Peter R. C. Gascoyne*,“ Cell Separation by Dielectrophoretic Field-Flow-Fractionation.”, Anal. Chem., 72(2000)832-839.
[20] Sungyoung Choi and Je-Kyun Park, “Microfluidic system for dielectrophoretic separation based on a trapezoidal electrode array”, Lab Chip, 5(2005)1161-1167.
[21] D. Holmes,N. G. Green and H. Morgan, “Microdevices for dielectrophoretic flow through cell separation”, IEEE Eng. Med. Biol. Mag., 22(2003)85-90.
[22] Fiedler, S., Shirley, S.G., Schnelle, T., Fuhr, G., “Dielectrophoretic sorting of particles and cells in a microsystem“, Anal. Chem., 70(1998)1909-1915.
[23] T. Schnelle, T. Muller, S. Fiedler1, G. Fuhr, “The influence of higher moments on particle behaviour in dielectrophoretic Þeld cages”, J. Electrostat., 46(1999)13-28.
[24] Th. Schnelle, T. Muller and G. Fuhr, “Trapping in AC octode field cages”, J. Electrostat, 50(2000)17-29.
[25] M. Durr, J. Kentsch, T. Muller, T. Schnelle and M. Stelzle, “Microdevices for manipulation and accumulation of micro- and nanoparticles by dielectrophoresis”, Electrophoresis, 24(2003)722-731.
[26] X. Hu, P. H. Bessette, J. Qian, C. D. Meinhart, P. S. Daugherty and H. T. Soh, “Marker-specific sorting of rare cells using dielectrophoresis”, PNAS, 102(2005)15757-15761
[27] P. C. Lee, and D. Meisel, “Adsorption and surface-enhanced Raman of dyes on silver and gold sols”, J. Phys. Chem. 86(1982)3391.
[28] T. Schnelle, T. Müller, G. Gradl, S. G. Shirley, G. Fuhr, “Paired microelectrode system: dielectrophoretic particle sorting and force calibration”, J. Electrostat., 47(1999)121-132.
[29] M. P. Hughes, Nanoelectromechanics in Engineering and Biology, Research Studies Press, 2003.
[30] N. G. Green, et al., “Electrothermally induced ﬂuid ﬂow on Microelectrodes”, J. Electrostat, 53(2001)71-87.
[31] A. Castellanos, A. Ramos, A. Gonzalez, N. G. Green and H. Morgan, “Electrohydrodynamics and dielectophoresis in microsystems: saling laws”, J. Phys. D: Appl. Phys., 36(2003)2584-2579.
[32] X. Wang, J. Yang, and P. R. C. Gascoyne, “Role of peroxide in AC electrical field exposure effects on Friend murine erythroleukemia cells during dielectrophoretic manipulations,” Biochim. Biophys. Acta, 1426(1999)53-68.
[33] J. Wu, Y. Ben, D. Battigelli and H.-C. Chang, ”Long-range AC electroosmotic trapping and detection of bioparticles”, Ind. Eng. Chem. Res., 44(2005)2815-2822.
[34] H. Morgan, N. G. Green, “Ac electrokinetics: colloids and nanoparticles”, Research Studies Press, 2003.
[35] 蕭孟芳, 圖解醫學微生物學與感染症, 合記圖書, (2002).
[36] 曾岐元, 最新病理學, 匯華圖書,(2000)
[37] S. Gawad, L. Schild and Ph. Renaud, “Micromachined impedance spectroscopy flow cytometer for cell analysis and particle sizing”, Lab Chip, 1(2001)76-82.

校內：2008-07-28公開
校外：2010-07-28公開

簡易檢索 / 詳目顯示

相關論文