Impedance Measurements of Single Basophils Stimulated by Pollen Allergen

本研究探討花粉引起的過敏反應，搭配阻抗分析儀達到無標記與非侵入式的量測結果，並藉由阻抗頻譜的變化了解過敏機制。本實驗使用黃光微影技術製作捕捉及量測單一細胞的微流道電極晶片。針對過敏反應，本實驗以單一嗜鹼性細胞經被動傳輸的方式與樺樹花粉免疫球蛋白E(IgE)結合，再以樺樹花粉過敏原刺激，使嗜鹼性細胞產生脫顆粒現象。實驗結果發現細胞捕獲前後阻抗增加約1倍，在濃度1 μg/ml的樺樹花粉刺激後阻抗下降23%，嗜鹼性細胞的外觀有逐漸膨脹的趨勢，在15分鐘內膨脹的幅度較明顯；在後續至45分鐘的反應趨於平緩，細胞直徑從原本的13.3μm增加至16.1μm；然而以濃度10 μg/ml的樺樹花粉刺激後阻抗下降24%，變化並不明顯。本實驗成功的使用液動力捕獲單一嗜鹼性細胞，並透過阻抗與相位的量測分辨細胞的捕捉及受過敏原刺激後的變化，經顯微鏡觀察到刺激後細胞的外型明顯改變。This study investigates the allergic reaction caused by birch pollen based on impedance response as a label-free and non-invasive analysis. In the present experimental study, light lithography techniques for microelectromechanical systems (MEMS) were employed to produce the bio-chip for capturing and measuring single cells. In this study, a basophilic cell line KU812 were cultured with specific birch pollen (Betula verrucosa, Bet v 1) IgE through passive transfer as the sample cells. Then specific antigen (anti-Bet v 1 IgE) were used to stimulate the sensitized single KU812 cells that were captured in the bio-chip for investigating the degranulation phenomenon. Experimental results show that the impedance magnitude for capture of single KU812 cell increases to nearly twice compared with the one before the capturing. As the single sensitized cell was stimulated with birch pollen allergens at a concentration of 1 μg/ml, the impedance was found to decrease about 23%. Time-varying images of the stimulated single KU812 cell also shows a rapid expansion in size in about 15 min and then followed by a slower growth until 45 min, resulting in a total increase in cell diameter from 13.3 to 16.1 μm. Using a much higher birch pollen concentration of 10 μg/ml for stimulation also demonstrated a decrease in impedance by about 24%, indicating indiscernible change as compared with the lower concentration of 1 μg/ml. The bio-chip developed in the present experimental study allows one to measure and observe the allergic reaction of IgE-mediated single basophils stimulated by the specific allergens and may serve as a platform for allergy testing.致謝 i 摘要 ii Abstract iii 目錄 iv 圖目錄 vi 表目錄 vii 第一章緒論 1 1.1研究背景與動機 1 1.2文獻回顧 2 1.2.1實驗室晶片 2 1.2.2細胞的捕捉 3 1.2.3被動傳輸引起的I型過敏反應 6 1.2.4單一細胞阻抗量測 9 1.3研究目標 12 第二章 實驗方法 13 細胞捕捉原理 13 2.2被動傳輸 14 2.3等效電路 15 2.4 實驗流程 18 第三章微流體電極晶片設計與製作 19 3.1流道與電極設計 19 3.2微流道製作 20 3.2.1微流道母模製作步驟與相關參數 20 3.2.2軟微影技術 22 3.3微電極製作 23 3.4微流道與微電極之對準貼合 26 第四章 細胞與抗體抗原配置 28 4.1被動傳輸的原理與實驗配置 28 4.2嗜鹼性細胞KU812介紹 28 4.3細胞培養 28 4.3.1細胞培養液配置 29 4.3.2初代細胞解凍 29 4.3.3細胞繼代 29 4.3.4細胞凍存 30 4.3.5細胞計數 30 4.4抗體抗原配置 31 第五章 結果與討論 33 5.1細胞阻抗量測儀器配置 33 5.2儀器校正 34 5.3背景溶液量測 35 5.4不同頻率下細胞的響應 36 5.5不同流道深度對阻抗的影響 36 5.6不同濃度抗體抗原對KU812的影響 39 5.7不同過敏階段KU812的阻抗變化 42 第六章結論與建議 46 6.1結論 46 6.2建議 46 參考文獻 4