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研究生： 吳姿賢
Tzu-Hsien Wu
論文名稱： 兩性離子微珠抗阻塞過濾裝置應用於分離大量循環腫瘤細胞
Anti-Clogging Filtration Device with Zwitterionized Microspheres for Mass Isolation of Circulating Tumor Cells
指導教授： 黃俊仁
Chun-Jen Huang
口試委員:
學位類別： 碩士
Master
系所名稱： 生醫理工學院 - 生醫科學與工程學系
Department of Biomedical Sciences and Engineering
論文出版年： 2018
畢業學年度： 106
語文別： 英文
論文頁數： 98
中文關鍵詞： 雙離子磺甜菜鹼 、抗阻塞特性 、循環腫瘤細胞 、無抗體捕捉系統 、血液接觸之醫療器材
外文關鍵詞： Zwitterionic sulfobetaine silane, Anti-clogging property, Circulating tumor cells, Ligand-free capture system, Blood-contacting medical device
相關次數： 點閱：13  下載：0 
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    • 現今，循環腫瘤細胞 (CTCs) 在癌症的診斷及預後中扮演著極為關鍵的角色，因其可應用於臨床上即時監測腫瘤復發及制定新的治療標靶藥物，同時也可透過循環腫瘤細胞的數量變化去評估癌症轉移的情形。目前許多微過濾裝置已被開發應用於分離極稀少循環腫瘤細胞，然而仍有許多缺點存在，像是: 低效率，低純度，壽命短，選擇性低。而造成上述缺點的原因在於血液中的纖維蛋白原以非特異性吸附於材料表面而引起一連串的連鎖效應並形成血栓導致過濾裝置堵塞。因此，本研究利用兩性雙離子 (Sulfobetaine silane, SBSi) 在二氧化矽微珠材料上進行自組裝單層膜修飾。利用SBSi超親水與抗粘黏特性加速過濾效果與避免血栓。此外，將SBSi修飾在二氧化矽微珠上，並將之填充管柱內，利用微珠間空隙尺寸大小選擇性地分離血液中CTCs。爾後透過傅立葉轉換紅外線光譜 (FT-IR) 及固態核磁共振光譜儀 (C13 NMR) 進行定性分析；熱重分析儀（TGA）及元素分析（EA）進行定量分析。透過蛋白質定量 (BCA Kit)證實SBSi修飾後的微珠具有抗蛋白質吸附之能力。同時，有鑑於應用端為生物醫學領域，利用溶血試驗、血小板貼附試驗及細胞存活率試驗進行安全性評估。而試驗顯示修飾之微珠除了具優異之血液相容性及可大幅降低血小板貼附外，同時也具良好之生物相容性。 此後，將各種不同直徑之二氧化矽微珠填充入管住中以控制微珠間孔洞尺寸，利用循環腫瘤細胞與血球細胞大小差異來捕獲血液中稀少之循環腫瘤細胞。經修飾之微珠相較於未修飾之微珠裝填之微過濾裝置相比具有更高之體積通量，表明具親水性表面之微珠能抵抗非特異性吸附。此外，使用自動化細胞計數器 (Cell counter) 來驗證血球細胞的滯留管柱情形及優化最佳微珠直徑以用於有效分離循環腫瘤細胞及血球細胞。最後，選用大腸癌細胞(CRCs) 為分離癌細胞實驗之細胞株，將含有大量大腸癌細胞之血清緩衝液通過不同直徑微珠裝填之過濾裝置以鑑別其細胞選擇性。不同直徑微珠裝填之過濾裝置皆表現高捕獲性能，細胞捕獲效率高達100％。由上述結果得知，功能性二氧化矽微珠將具有潛力進一步發展為抗堵塞分離大量循環腫瘤細胞之過濾系統。

    Isolation and enumeration of circulating tumor cells (CTCs) from periphery blood are of significance in diagnosis and prognosis of cancers. A key challenge to the clinical utility of CTCs is the mass collection of viable rare cells. Although many microfiltration-based cell separation devices were developed to isolate individual circulating tumor cells from blood, the drawbacks, such as low efficiency, high impurity, short utility time, low selectivity, and fail in the collection of CTC clusters remain. Herein, the aim of the thesis is to develop an efficient filtration system to isolate CTCs and clusters using zwitterionized silica microspheres. Specifically, the silica microspheres were modified with zwitterionic sulfobetaine silane (SBSi), to provide the excellent resistance against clogging due to the contact of blood. The microspheres of various diameters were packed in a column in order to control the pore sizes between microspheres for selectively capturing CTCs with the larger size than blood cells. In this study, the surface characterization of the coatings on microspheres was conducted by Fourier Transform Infrared Spectroscopy (FT-IR) and Solid State Nuclear Magnetic Resonance Spectrometer (C13 NMR) for qualitative analysis, and Thermogravimetric Analyzer (TGA) and Elemental analysis (EA) for quantitative analysis. The tests for protein adsorption revealed the excellent antifouling property of SBSi coating prepared from a controlled condition. For the biomedical application, the biocompatibility of SBSi-modified microspheres were confirmed by the hemolysis test, platelet adhesion test, and cell viability assay. The results indicated that SBSi-modified silica microspheres possess good biocompatibility, superior blood cell compatibility and great resistance against the platelet adhesion in comparison with the un-modified microspheres. Moreover, the anti-clogging effect of SBSi-modified microspheres has been further proven by the volumetric flux of blood and retention of blood cells in columns. The cell retention of white blood cells (WBCs) and red blood cells (RBCs) after passing through the filtration devices was characterized using an automated cell counter for optimization the pore size of the column with microspheres. More importantly, the colorectal cancer cells (CRCs) were effectively and selectively captured by the column packed with the SBSi-modified silica microspheres. Consequently, the anti-clogging filtration prototype device was demonstrated to have the high entrapped performance, up to 100%. The potential implementation of the anti-clogging filtration system for isolating mass CTCs and clusters in clinical application is expected.

    中文摘要 I Abstract III Acknowledgment V Table of Contents VII List of Figures XI List of Tables XVI Lists of Abbreviations XVII CHAPTER 1: Introduction 1 1.1 Introduction to Circulating Tumor Cells (CTCs) 1 1.1.1 Metastatic Cancer 1 1.1.2 Circulating Tumor Cells (CTCs) 3 1.1.3 Clinical Utility of CTC Detection 3 1.1.4 Challenges in Capturing Rare Circulating Tumor Cells 4 1.2 CTC Detection Technology 5 1.2.1 Physical Enrichment Method 7 1.2.1.1 Cell-Size Based CTC Enrichment Method 7 1.3 Challenges of Cell-Size Based Method 15 1.3.1 Size Overlap 15 1.3.1.1 Size of the Circulating Tumor Cells (CTCs) 16 1.3.2 Blood Clot 17 1.3.2.1 Blood Coagulation Process 17 1.3.2.2 Biointerface and Blood Coagulation Cascade 18 1.4 Surface Modification Technology 20 1.4.1 Adsorption of Biomolecules on Surfaces 20 1.4.2 Antifouling Materials 22 1.4.2.1 Zwitterionic Materials 22 1.4.3 Self-Assembling Monolayers (SAMs) 23 1.4.3.1 Silane Functional Group 24 1.4.4 Silicon-Based Inorganic Materials 27 1.4.4.1 Introduction of Silica Microspheres 27 CHAPTER 2: Research Objective 28 CHAPTER 3: Materials and Methods 29 3.1 Materials 29 3.2 Methods 29 3.2.1 Silica Microspheres Characterization 29 3.2.1.1 Low Vacuum Scanning Electron Microscope (LV-SEM) 29 3.2.1.2 Sieving Method 29 3.2.1.3 Laser Diffraction Particle Size Analyzer 30 3.2.2 Synthesis Sulfobetainesilane (SBSi) 30 3.2.2.1 Liquid State Nuclear Magnetic Resonance Spectrometer (1H NMR) 31 3.2.3 Surface Modification of Silica Microspheres with Sulfobetainesilane (SBSi) 31 3.2.4 Characterization of SBSi-modified Silica Microspheres 32 3.2.4.1 Fourier Transform Infrared Spectroscopy (FTIR) 32 3.2.4.2 Solid State Nuclear Magnetic Resonance Spectrometer (13C NMR) 32 3.2.4.3 Elemental Analyzer (EA) 32 3.2.4.4 Thermogravimetric Analyzer (TGA) 32 3.2.5 Protein Adsorption Test 32 3.2.6 Cell Viability Assay 33 3.2.7 Hemocompatibility 34 3.2.7.1 Hemolysis Test 34 3.2.7.2 Platelet Adhesion 35 3.2.8 Design of the Silica Microspheres Filtration Device 36 3.2.9 Cell Retention Test 36 3.2.10 Collection of Colorectal Cancer Cells 37 CHAPTER 4: Results and Discussions 39 4.1 Characterization of Silica Microspheres 39 4.1.1 Morphology of Silica Microspheres 39 4.1.2 Particle Size Distribution of Silica Microspheres 40 4.1.2.1 Sieving Method for Size Distribution 40 4.1.2.2 Laser Diffraction Particle Size Analyzer 42 4.1.2.3 Characterization of the Pore Space 43 4.2 SBSi Analysis (NMR Spectrum Analysis) 48 4.3 Characterization of SBSi-modified Silica Microspheres 48 4.3.1 Qualitative Analysis of SBSi-modified Silica Microspheres 49 4.3.1.1 Fourier Transform Infrared Spectroscopy (FTIR) 49 4.3.1.2 Solid State Nuclear Magnetic Resonance Spectrometer (13C NMR) 50 4.3.2 Quantitative Analysis of SBSi-modified Silica Microspheres 51 4.3.2.1 Elemental Analysis (EA) 51 4.3.2.2 Thermogravimetric Analyzer (TGA) 53 4.4 Protein Adsorption Test 54 4.4.1 Effect of the various coating concentration of SBSi-modified Silica Microspheres on Protein Adsorption 54 4.4.2 Effect of the various size of SBSi-modified Silica Microspheres on Protein Adsorption 56 4.5 Biocompatibility Test 57 4.6 Hemocompatibility 59 4.6.1 Hemolysis Test 59 4.6.2 Platelet Adhesion 61 4.7 Isolating Mass Colorectal Cancer cells (CRCs) from the Serum-Containing Buffer using SBSi-modified Silica Microspheres Filtration Device 62 4.7.1 Volumetric Flux 63 4.7.2 Cell Retention of whole blood pass through the Filtration Devices 64 4.7.3 Capture Efficiency of SBSi-modified Silica Microspheres Filtration Device 66 CHAPTER 5: Conclusions 68 Bibliographies 71

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