系統識別號 U0026-2208201116082200
論文名稱(中文) 以交流電驅動促進親和性反應之電化學式免疫晶片
論文名稱(英文) AC Electrokinetics Enhanced Sensitivity and Reaction Rate in an Electrochemical Impedance-Based Immuno-chip
校院名稱 成功大學
系所名稱(中) 醫學工程研究所碩博士班
系所名稱(英) Institute of Biomedical Engineering
學年度 99
學期 2
出版年 100
研究生(中文) 楊筱嵐
研究生(英文) Hsiao-Lan Yang
學號 p86981083
學位類別 碩士
語文別 英文
論文頁數 49頁
口試委員 指導教授-張憲彰
中文關鍵字 交流電滲流  電化學阻抗分析  免疫分析法  蛋白質 A 
英文關鍵字 AC electroosmosis (ACEO)  electrochemical impedance spectroscopy (EIS)  Immunoassay  Protein A 
中文摘要 近年來,免疫分析法常應用於食物、動物和臨床疾病的檢測上,因為免疫分析法是具有高靈敏度和高精確性的診斷工具可以確定早期感染而給予藥物治療。傳統的免疫分析技術皆需使用螢光的標定與酵素呈色反應並以擴散作用的方式與待測物結合,使得檢測分析需耗費冗長的時間並須搭配高成本的大型光學儀器,故如何建立快速、準確、低成本的免疫平檢測台為當前值得重視的課題。本研究提出,以結合交流電滲流與電化學阻抗量測應用於操控生物粒子及檢測親和性反應之微流體晶片,以達到快速檢測、靈敏度高且不須生物標定的免疫感測器。在操控方法上,藉由使用交流電滲流的技術使電極表面產生的渦流來促進免疫分析上的反應效率,並利用電化學阻抗量測法針對抗原與抗體(IgG-Protein A)間反應完成後的阻抗變化量(ΔRet)進行量測。做為生物探針的抗體(IgG)會藉由自組性單層膜物質11-MUA經過EDC/NHS活化尾端的羧基而固定在基材上,並使用1%小牛血清蛋白阻隔空位。藉由微小電極的電化學檢測技術,來分析Protein A在免疫感測器(BSA/IgG/MUA/Au)上的阻抗變化。在低交流電壓操作下所引發的電滲流現象已有效的濃縮在低濃度的樣本中粒子/分子,且均能提高檢測的靈敏度及反應速率。因此藉由交流電滲流所誘發的擾動流場所設計的同心圓電極將可達到遠程之生物分子的濃縮。在實驗中也顯示了在數分鐘內,奈米等級之粒子及生物分子會快速的濃縮在電化學式的量測電極上,並且完成電化學阻抗量測。在交流電滲流之最佳參數的作用下(15 Vpp, 8 Vpp, 0.5 V, 800 Hz),非但可於90秒內測得其已達到飽和(免疫反應達到飽和)的阻抗值,而且使檢測極限降低至0.2 ng/ml。本研究結合電化學抗分析法與交流電滲流的技術,可改善檢測靈敏度與反應速率,將來再搭配其他技術開發出快速、高靈敏度、自動化的免疫分析平台。
英文摘要 In recent years, immunoassay is usually used to specifically attract biomolecules as biomarkers for detection of food, animal, and clinical diseases to determine the early stage infection and medical treatments. Commonly used ELISA kits include the lacks of low sensitivity (~ 2 ng/ml) and long reaction time (~2 hr) due to its diffusion dominated transport-limiting kinetics. Moreover, conventional techniques of immunoassay typically use fluorescent labeling or enzyme-chromogenic reaction for enabling detection and analysis. Two issues described above that could be the major challenges for field useable applications due to huge and expensive optical instruments are required. In this research, we reported a rapid, sensitive and label-free immunosensor that combines AC electroosmosis (ACEO) and electro- chemical impedance spectroscopy (EIS) to manipulate biomolecules and detect its affinity reaction respectively in a microfluidic chip. Enhancement of immunoassay by ACEO that produces the vortices on the electrode surface can promote efficiency of affinity reaction, and the varied resistance (ΔRet) between the antibody-antigen (IgG-Protein A) binding can be measured in real-time by the EIS method. Concentric electrodes were designed for long-range biomolecular concentration by ACEO-induced convection. Low AC voltage induced ACEO enables effective collection of particles/molecules from very dilute samples, which can promote the sensitivity and reaction rate. The experimental results show that nanoparticles/molecules can be rapidly concentrated into the electrochemically-measured electrode within a few minutes. The antibody immobilized substrate as an immunosensor (BSA/IgG/MUA/Au) showed the varied ΔRet that responded to the behavior of antibody-antigen binding for quantification of the Protein A by microelectrode-based electrochemical impedance techniques. The measured impedance was saturated (complete reaction) after ACEO concentrating (at the optimal frequency of 800 Hz) for 90 sec and the detection limit can reach to 0.2 ng/ml. EIS integrated with ACEO could improve the sensitivity and the reaction rate of immunoassay. In the future, this system could be integrated with other techniques for developing a rapid, high sensitivity and automatic detection immuno-platform.
論文目次 Abstract I
摘要 II
誌謝 III
Contents IV
List of Figures VI
List of Tables IX
Chapter 1 Introduction 1
1.1 Background and Motivation 2
1.2 Micro-electro-mechanical system technology 3
1.3 Theory of electro-osmosis 5
1.3.1 Electric double layer 5
1.3.2 DC electroosmosis flow 7
1.3.3 AC electro-osmosis flow (ACEO) 8
1.4 Electrochemical impedance spectroscopy (EIS) 9
1.5 Immunoassay technique 14
1.5.1 The different detection methods for immunoassay 14
1.6 Electrokinetic-based Immunosensor 16
1.6.1 Dielectrophoretic Force 16
1.6.2 Electrowetting on Dielectric 18
1.7 Research Configuration 20
Chapter 2 Materials and Methods 22
2.1. The working principle of immuno-chip 22
2.2. Chip design and micro-fabrication 22
2.3. Sample preparation 24
2.4. Protein immobilization and immunoassay procedure 26
2.5. System configuration 27
Chapter 3 Results and Discussions 29
3.1. EIS and CV respond to each immobilized layers 29
3.2. Optimal frequency of ACEO concentration for EIS detection 33
3.3. Ion concentration affected antibody-antigen docking 37
3.4. Saturation time versus different ACEO frequencies 39
3.5. Specificity and sensitivity 41
Chapter 4 Conclusion 45
References 46
Personal Information 49
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