||Electrochemical Detector Prepared by Electro-deposition on a Capillary Electrophoresis Microchip for Histamine
||Department of BioMedical Engineering
過敏是最常見的疾病之一，而過敏人口逐年增加，因此快速檢測過敏有其必要性。目前的過敏檢測方法，主要檢測免疫球蛋白IgE來判斷患者過敏原因，然而並不是所有過敏反應都與IgE有關，因為過敏本質上主要原因為嗜鹼性白血球細胞或肥大細胞受到活化或刺激後分泌組織胺，因此直接針對造成過敏反應的物質進行檢測是極為重要的。本研究開發毛細管電泳晶片，檢測過敏反應中主要分泌物-組織胺。毛細管電泳利用電泳力及電滲流力將樣本分離及輸送至偵測槽中，並搭配電化學做即時性檢測。晶片由電極和聚甲基矽氧烷 (PDMS) 微管道所組成，PDMS微管道利用熱處理的方式來改善氧電漿修飾後的不穩定性，而最佳的熱處理時間為12 h，最後製作完畢的PDMS微管道與電極藉由氧電漿處理將其接合，最後在參考及接地電極上電鍍白金，而工作電極上電鍍金即可完成。組織胺在10 mM MES溶液中最佳的電泳實驗檢測條件為注入電場+500 V/cm、注入時間35 sec、分離電場+180 V/cm以及+120 V/cm之推回電壓，並搭配上電鍍金的方式，增加工作電極表面積，降低組織胺檢測極限，其線性範圍為2 μM ～ 30 μM(R2=0.999)，檢測極限為1 μM (S/N ≧ 3)。最後，初步測得KU-812細胞 (1×108 cells/mL) 經1 μM鈣離子載體 (A23187) 刺激20 min後取上清液檢測，電流響應為0.4 nM，回推組織胺濃度約為3 μM，未來本系統期望能應用在臨床上細胞過敏反應的評估。
An allergy is a hypersensitivity disorder of the immune system that affects many people around the world and the number is still increasing every year; thus immediate diagnosis and prevention of allergies is very important. The conventional detection of allergic reaction does focus on the amount of IgE in the serum to diagnose the reason of allergy. However, the allergic reaction is not entirely depending on IgE. Because major factor for allergic reactions is histamine released from basophiles or mast cells; therefore, detection of substance causing an allergic reaction is important. In the study, we aim to develop a capillary electrophoresis (CE) chip to detect histamine causing an allergic reaction. CE chip used electroosmotic and electrophoretic forces to separate and transport the sample toward the sample reservoir, and then real time detected the sample based on electrochemical method. The CE chip consisted electrode chip and polydimethylsiloxane (PDMS) microchannel, and the PDMS microchannel treated by thermal aging and oxygen plasma to bind with electrode chip. The reference and decoupler electrodes were deposited by Pt and the working electrode was deposited by Au. We obtain the optimum conditions for histamine detection in MES buffer in the injection field of 500 V/cm for 35 sec, separation field of 180 V/cm, and pushback field of 120 V/cm. To decrease the detection limit of histamine, we deposited Au nanoparticle on the working electrode for increasing the surface area. A regression line of histamine concentration showed at 2 and 30 μM and the correlation coefficient was 0.999 (n=2). The detection limit of histamine was 1 μM. Lastly, we stimulated KU-812 cells (1×108 cells/mL) by calcium ionophore (A23187) for 20 min, and then extracted the supernatant to detection. We have successfully detected histamine in supernatant, the current and concentration of histamine was 0.4 nA and 3 μM. In the future, the system could be applied for clinical investigation of allergic reaction.
Table List VI
Figures List VII
Chapter 1 Introduction 1
1.1 Allergic Reaction 1
1.2 Metabolic Pathway of Histamine 3
1.2 The Methods of Histamine Detection 4
1.3 The Theory of Capillary Electrophoresis 5
1.3.1 Electrophoretic Force 5
1.3.2 Electroosmotic Force 6
1.4 The Properties of Polydimethylsiloxane 7
1.5 The Method of PDMS Modification 8
1.6 The Method of Increasing the Area of Detection Electrode 10
1.7 Allergy Diagnosis in Cellular Level 13
1.8 Motivation and Objective 16
Chapter 2 Materials and Experiment 18
2.1 Equipments 18
2.2 Chemicals for Microfabrication 18
2.3 Chemical for Analysis 18
2.4 Test Solution 19
2.5 Fabrication of the Microchannel 20
2.6 Fabrication of the Au Electrode and Chip Bonding 21
2.7 Electroosmotic Measurement 24
2.8 Electrophoregrams Measurement 25
2.9 Detection of histamine released from KU-812 Cells 25
2.10 Optical Detection of Histamine 27
Chapter 3 Results and Discussion 29
3.1 Evaluation of Hydrophobic Recovery of PDMS 29
3.1.1 Contact Angle Measurements of PDMS 29
3.1.2 Electroosmotic Measurements of Chip 30
3.1.3 Electrophoresis Current Measurements of Chip 31
3.2 The Choose of Detection Potential of Histamine 32
3.3 CE-EC System Evaluation 33
3.3.1 Optimum Parameters for Decoupling 33
3.3.2 Effect of Injection Time, Separation Voltage 39
3.4 Optimum Parameters for Deposition of Au on Working Electrode 41
3.4 Calibration Curve for the Electrochemical Detection of Histamine 43
3.5 Detection of Histamine Released from KU-812 Cells 45
Chapter 4 Conclusions 48
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