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研究生:薛世峰
研究生(外文):Shih-Feng Hsueh
論文名稱:電漿和接枝聚合處理對無機基材表面固定酵素於生物感測器之研究
論文名稱(外文):The Study of Immobilization of Glucose Oxidase on Inorganic Surface for Biosensor
指導教授:陳克紹陳克紹引用關係
指導教授(外文):Ko-Shao Chen
學位類別:碩士
校院名稱:大同大學
系所名稱:材料工程學系(所)
學門:工程學門
學類:綜合工程學類
論文種類:學術論文
論文出版年:2006
畢業學年度:94
語文別:英文
論文頁數:119
中文關鍵詞:葡萄糖氧化酵素生物感測器梳型電極
外文關鍵詞:biosensorinterdigital electrodeglucose oxidase
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對於一些阻抗型或是質量型的感測器,必須要固定一層感測材料於電極或是陶瓷的基材上,但因為感測材料一般為有機化合物,所以就必須利用一層中間層使感測材料對基材有良好的附著性。本研究利用HMDSZ電漿沉積一層含矽有機薄膜於無機基材表面,使其表面具有過氧化基和自由基,並利用光接枝聚合的方式,將AAm固定於無機基材上,然後以PEI-GA和Chitosan-GA的方式來固定酵素GOD,以PEI和Chitosan來擴增胺基,GA當作交聯劑來連接基材和酵素。當酵素被固定於QCM的表面時,可以由QCM的頻率下降量來確認固定酵素於無機基材上,並將此技術應用於無機基材的梳型電極上,當GOD催化葡萄糖產生氧化還原反應時,會放出電子增加梳型電極的導電度,使阻抗值下降,來測量葡萄糖溶液的濃度。由結果可知,PEI-GA法來固定酵素,其葡萄糖濃度量測範圍為2-10mM,反應時間約需要70秒,較一般的檢測方法快速許多,而Chitosan-GA法則偏向於較高濃度的檢測範圍10-500mM,檢測時間只有30秒,而且經過10天的保存後,依然保有90%的準確性。
Many research workers attempt to fabricate sensitive, selective, reliable and low cost glucose sensors. Among these sensors, amperometric electrodes based on the immobilized glucose oxidase (GOD) have attracted considerable interest due to their simple alternative analytical systems. Because sensitive layer always is coating in conductive electrode. In this study, we used the hexamethyldisilazane (HMDSZ) to plasma deposit organic silicon-containing thin films on the surface of interdigital electrode substrates in order to make an interface layer between the inorganic materials and organic molecules. The UV-light induced surface grafting polymerization of acrylamide (AAm) can provide useful functional NH2-groups sites for binding glucose oxidase (GOD). Hence glutaraldehyde (GA) as cross–linking agent can connect the grafting polymer and the GOD enzyme. In the FTIR spectra, Si-O-C, Si-O-Si Si-CH2-Si, Si-N-Si, and Si-CH3 groups were found after plasma deposition. XPS analysis shows PEI treatment could raise amount of GOD immobilization with GA. Experiment results indicated that GOD can be immobilization effectively on interdigital electrode substrate.
Contents Chinese Abstract……………………………………….…...I
English Abstract………………….……………………..…………….II
Acknowledgement……………………………..……..........................III
Contents …………………………………………………………….....IV
List of Figures …………………………………..………………...…VIII
List of Tables………………………………………………………….XI
Chapter 1 Introduction.......................................1
1.1 Introduction..................................................2
1.2 What is plasma.............................................4
1.3 Reactions between plasma and surface........................7
1.4 Surface grafting method.....................................11
1.5 Quartz crystal microbalance...............................13
1.6 Enzyme biosensor..............................................17
1.7 Electrochemical transducers ...............................18
1.8 Thermal transducers……………………………………………...20
1.9 Optical transducer………………………………………………...20
1.10 Enzyme immobilization method……………..………………….21
Chapter 2 Experiment........................................30
2.1 Materials and reagents........................................31
2.2 Sample surface cleaning........................................31
2.3 Plasma deposition process ................................32
2.4 UV light-induced graft polymerization .....................33
2.5 PEI-GA method for immobilization glucose oxidase .......33
2.6 Chitosan-GA method for immobilization glucose oxidase ...34
2.7 Property analysis and test .............................34
Flow chart of experiments........................................44
Chapter 3 Result and discussion……………………………………45
3.1 Effect of plasma deposition time ..............................46
3.2 Effect of the HMDSZ vapor pressure in plasma deposition process.53
3.3 Effect of plasma deposition power.........................58
3.4 Wet-ability change on the substrate surface after each treatment…..61
3.5 Film thickness………………………….. .............................65
3.6 SEM morphology……………………………69
3.7 Micro FTIR analyses .........................................76
3.8 EXCA analysis of every step treatment on the surface of QCM.......82
3.9 Impendence measurement……...............................94
Chapter 4 Conclusion...............................................99
4.1 Conclusion……………………. ........................100
Reference……………………………………………………….……..101

LIST OF FIGURES
Fig. 1-1The reaction between temperature and pressure in glow discharge ……………………………………………………….23
Fig.1-2 The plasma treatment system with Radio Frequency 13.56MHz generator………………………...…………………………..…24
Fig.1-3 Various commonly used grafting methods………..……...……25
Fig. 1-4Schematic of a typical piezoelectric crystal…………………...26
Fig. 1-5(a) The assignment of axes to a quartz crystal. (b) Each of cut models. (c) AT-cut (in general use for mass sensors), (d) BT-cut quartz crystal (in general use for temperature sensors)……….27
Fig. 2-1Chemical structure of hexamethyldisilazane…….………..…..38
Fig. 2-2Chemical structure of acrylamide………….…….………..…..38
Fig. 2-3Chemical structure of PEI………………….…….………..…..39
Fig. 2-4Chemical structure of Chitosan…………….…….………..…..39
Fig. 2-5Chemical structure of Glutaraldehyde……..…….………..…..40
Fig. 2-6Schematic diagram of plasma CVD system equipment with a bell-jar reaction chamber…………………………………..…..41
Fig. 2-7Schematic diagram of UV-light graft polymerization apparatus……………………………………………………….42
Fig. 2-8Schematic illustration of the preparation of the surface modified and GOD immobilization procedures………………………….43
Fig. 3-1QCM frequency shift (-Hz) of HMDSZ deposition at different deposition time…………………………………………………49
Fig. 3-2QCM frequency shift (-Hz) of grafting AAm at different deposition time…………………………………………….…..51
Fig. 3-3Effect of plasma deposition time on wet-ability……………….52
Fig. 3-4Frequency shift quantity (-Hz) of QCM that HMDSZ deposition at different vapor pressure……………………………………..55
Fig. 3-5Frequency shift quantity (-Hz) of QCM that grafting polymerization AAm on the surface after HMDSZ deposition at different vapor pressure…………………………………..…....57
Fig. 3-6The frequency shift with different plasma deposition power on QCM…………………………………………………..……….59
Fig. 3-7OM of water (10μl) droplet on the sample surface (takes from CCD) measured water contact angle after each treatment. (a) Glass, (b) plasma deposition (30W, 30mtorr, 5min), (c) grafted polymerization (AAm 10wt %, 1000W, 20min)………………63
Fig. 3-8OM of water droplet on (takes from CCD) measured water contact angle that after each treatment. The plasma deposition time is 5min, (d) immersed PEI solution (2.5 wt%, 15min), (e) immersed GA solution (1 wt%, 10min), (f) immobilize glucose oxidase (GOD, 1060U/ml, 60min)…………………………….64
Fig 3-9 The thickness of plasma deposition film treated at different plasma deposition time………………………………………..67
Fig. 3-10 The SEM morphology of QCM device……………………70
Fig. 3-11 The SEM morphology of plasma deposition (30W, 30mtorr, 5min) on QCM device surface…………………………………71
Fig. 3-12 The SEM morphology of plasma deposition (30W, 30mtorr, 5min) on QCM device surface then grafting polymerization AAm……...................................................72
Fig. 3-13 The SEM morphology of PDHMDSZ/grafting-AAm QCM device immersed PEI solution……………………………..…73
Fig. 3-14 The SEM morphology of PDHMDSZ/grafting-AAm/PEI QCM device immersed GA solution.………………………….....….74
Fig. 3-15 The SEM morphology of PD/AAm/PEI-GA QCM device with immobilization GOD………………………………………….75
Fig. 3-16 Micro-IR spectra of silicon wafer with (A)plasma deposition and (B)grafting polymerization AAm treatment……………….78
Fig. 3-17 Micro-IR spectra of silicon wafer with (B)grafting polymerization AAm and immersed (C)PEI solution treatment.79
Fig. 3-18 Micro-IR spectra of silicon wafer with immersed (C)PEI solution and (D)GA solution treatment.……..………………...80
Fig. 3-19 Micro-IR spectra of silicon wafer with immersed (D)GA solution and immobilized (E)glucose oxidase treatment..……..81
Fig. 3-20 The (a) C1s and (b) N1s spectrums of plasma modified specimens.……………………………………………………...87
Fig. 3-21 The (C) O1s (D) Si2p spectrums of plasma modified specimens…………………………………………………………88
Fig. 3-22 The (a) C1s and (b) N1s spectrums of acrylamide grafted onto PD-HMDSZ silicon wafer..………………..…………………...89
Fig. 3-23 The (c) O1s and (d) Si2p spectrums of acrylamide grafted onto PD-HMDSZ silicon wafer..…………………………………...90
Fig. 3-24 The (a) C1s and (b) N1s (c) O1s spectrums of immersed PEI solution after surface-grafting process.….……………………...91
Fig. 3-25 The (a) C1s and (b) N1s (c) O1s spectrums of immersed GA solution after PEI step..…………………………………............92
Fig. 3-26 The (a) C1s and (b) N1s (c) O1s spectrums of immobilization glucose oxidase..……………...………………………………...93
Fig. 3-27 Calibration curve for the glucose biosensor in pH 7.0 PBS. Impedance change of glucose electrodes operated in buffered glucose solutions of increasing concentration..………………...96
Fig. 3-28 Calibration curve for the glucose biosensor in pH 7.0 PBS. Impedance change of glucose electrodes operated in buffered glucose solutions of increasing concentration..………………...97
Fig. 3-29 Impedance change of glucose electrodes operated in constant glucose concentration...……………...……….………………...98

LIST OF TABLES
Table 1-1Different transducers have been used in enzyme biosensor construction………………………………………………..28
Table 1-2 Enzymes are immobilized on transducer or support matrices by physical and chemical method…………………………29
Table 3-1QCM frequency shift (-Hz) and deposition mass (ng) measured from plasma deposition process………………..50
Table 3-2Effect of vapor pressure of HMDSZ plasma on wet-ability…………………………………………………56
Table 3-3The amount of HMDSZ deposition and grafting polymerization with different deposited power observes from QCM frequency shift (-Hz)………………………….60
Table 3-4Wet-ability (water contact angle) changes after different treatment on the surface of glass…………………………62
Table 3-5Density of thin film measured by deposition mass and thickness of film…………………………………………68
Table 3-6XPS analysis of surface modified on silicon wafer……86
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