微流體晶片選擇性抓取CD14+骨髓細胞應用於全血敗血症檢驗

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研究生：	周重羽 Chou, Chung-Yu
論文名稱：	微流體晶片選擇性抓取CD14+骨髓細胞應用於全血敗血症檢驗 Selectively Capturing CD14+ Myeloid Cell from Whole Blood on Microfluidic Biochip for Sepsis Diagnosis
指導教授：	劉承賢 Liu, Cheng-Hsien
口試委員:	邵耀華 Shau, Yio-Wha 盧向成 Lu, Shiang-Cheng
學位類別：	碩士 Master
系所名稱：	工學院 - 動力機械工程學系 Department of Power Mechanical Engineering
論文出版年：	2018
畢業學年度：	106
語文別：	中文
論文頁數：	85
中文關鍵詞：	敗血症檢驗、確定性側向位移、CD14+ 、微坑洞陣列、人類白血球抗原
外文關鍵詞：	Sepsis diagnosis, Deterministic lateral displacement, CD14+, Micro-hole matrix, Human leukocyte antigen
相關次數：	點閱：2 下載：0
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敗血症是一種由感染引起的全身性發炎反應的嚴重疾病，常好發於年幼、年老以及免疫系統受損的族群中。在臨床檢驗上，可以透過人類白血球抗原在白血球上的表現量來檢驗該敗血症患者之免疫系統狀況，在進行這類檢驗時，往往需要收集病人的血液樣本來分析並且將白血球分離以減少紅血球、血小板造成的誤差，這類的檢驗往往耗時、需要依賴專業檢驗人員以及昂貴的檢測儀器。本研究提出並製作微流體生物晶片，建立確定性側向位移將白血球從少量的全血樣品中分離，並且利用預先附著CD14抗體的微孔陣列來獨立抓取白血球中的CD14+骨髓細胞並透過螢光抗體檢測其表面人類白血球抗原的表現量。實驗結果顯示，確定性側向位移結構能夠在一分鐘內將10微升的全血樣本內86.28%的白血球分離，並透過後端的附著抗體的微孔陣列選擇性地抓取掉落於孔洞內白血球當中的CD14+骨髓細胞，實驗顯示在使用全血樣品的操作下，微孔陣列平均在每10微升能抓取5689~6790(6129±378 in average)個骨髓細胞於孔洞內以利於進行後續的疾病檢測。我們期望當敗血症患者在留院觀察的不同時段內，藉由這類診斷測試的系統能改善敗血症的診斷並且給予最佳的照護。

Sepsis, a clinical syndrome caused by infection, has occurred frequently in the patient who are young, old, and with a weakened immune system. The low expression of HLA (Human leukocyte antigen) on the surface of leukocyte is reported to reliably examine the sepsis patient’s immune system. Medical technologist usually needs leukocyte from whole blood to examine sepsis which needs long time to process, professional technicians and expensive machine. In this master study, a microfluidic biochip, which quickly separates leukocytes from minute whole blood sample, is developed and reported by using deterministic lateral displacement to capture CD14+ myeloid cells from leukocytes to examine the expression of HLA-DR on its surface by antibody-coating micro-hole matrix. In experimental results, our biochip can separate leukocytes from 10μl whole blood with 86.28% recovery rate averagely in one minute. After that, the micro-hole matrix selectively captures CD14+ myeloid cells from separated leukocytes by CD14 antibody when leukocytes drop into holes. By using whole blood sample in experiments, our device can capture 5689~6790(6129±378 in average) CD14+ myeloid cells from separated leukocytes for disease diagnosis. By establishing this POC (Point of care) system, we anticipate to improve sepsis diagnosis in the different stage when patients stay in the hospital.

Abstract    I
摘要    II
致謝    III
目錄    1
圖目錄    4
表目錄    8
第一章 緒論    9
1    前言    9
2    研究動機與目的    11
3    研究背景    12
3.1    生醫微機電    12
3.2    實驗室晶片(Lab on a chip)    12
3.3    敗血症    13
3.3.1    敗血症簡介     13
3.3.2    敗血症檢驗     14
3.3.3    檢驗方法爭議     15
3.4    Point of care testing    16
3.5    血液分析    16
4    文獻回顧    20
4.1    白血球分離技術    20
4.1.1    物理性白血球分離     20
4.1.2    化學性白血球分離     23
4.2    單細胞分析    27
第二章 系統理論與晶片設計    32
1    系統理論    32
1.1    微流體晶片設計理論    32
1.1.1    雷諾數 (Reynolds number)     32
1.1.2    微流體流阻分析     33
1.2    確定性側向位移理論(Deterministic lateral         displacement)    35
1.2.1    簡介     35
1.2.2    參數設計     36
1.3    免疫抑制    37
1.4    螢光檢測分析    39
2    微流體晶片設計    41
2.1    設計概念    41
2.1.1    晶片設計細節     41
2.1.2    晶片前處理─抗體選擇性附著技術     44
2.1.3    實驗操作流程     46
第三章 微流體晶片製程    48
1    製作流程    48
1.1    微流體母模製程    48
1.2    系統晶片製程    51
2    製程結果    54
第四章 實驗材料與方法    55
1    實驗材料    55
1.1    人類單核球細胞(Human acute monocyte leukemia cell line, THP-1)培養    55
1.2    Purified anti-human CD14 抗體    56
1.3    Anti-HLA-DR PE Clone L243 抗體    56
1.4    Anti-Human CD66b APC Clone G10F5 抗體    57
1.5    螢光免疫分析法(Immunofluorescence)    57
2    實驗架設    58
第五章 實驗結果與討論    59
1    確定性側向位移參數設計    59
1.1    初步參數設計    59
1.2    優化DLD陣列參數    60
2    全血樣本應用於DLD陣列    62
2.1    DLD陣列應用於全血分選    62
2.2    DLD陣列分離全血效率值    63
3    選擇性抗體附著測試    64
4    微孔陣列    67
4.1    微孔陣列抓取細胞測試    67
4.2    微孔陣列專一性抓取測試    69
4.3    微孔陣列效率測試    73
4.3.1    THP-1效率測試     73
4.3.2    全血樣本效率測試     74
4.4    自動化螢光免疫分析系統建立    76
第六章 結論    81
參考文獻    83

                                

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