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研究生:蔡立志
研究生(外文):Li-Chih Tsai
論文名稱:應用介電泳固定奈米碳材與葡萄糖氧化酵素混合物之血糖感測器的開發
論文名稱(外文):Study of Applying Dielectrophoresis to Immobilize Carbon Nanomaterial/Glucose Oxidase Composite for Construction of Glucose Biosensors
指導教授:吳靖宙
指導教授(外文):Ching-Chou Wu
口試委員:孫嘉良曾志明
口試委員(外文):Chia-Liang SunJyh-Myng Zen
口試日期:2015-07-22
學位類別:碩士
校院名稱:國立中興大學
系所名稱:生醫工程研究所
學門:工程學門
學類:生醫工程學類
論文種類:學術論文
論文出版年:2015
畢業學年度:103
語文別:英文
論文頁數:84
中文關鍵詞:多壁奈米碳管多壁奈米碳管與氧化石墨稀帶異構體葡萄糖氧化酵素介電泳葡萄糖生物感測器
外文關鍵詞:Multi-wall carbon nanotubeMulti-wall carbon nanotube@graphene oxide nanoribbonglucose oxidedielectrophoresisglucose biosensor
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相較於傳統結合多壁奈米碳管與葡萄糖氧化酵素的複合式感測器,本研究引進一新型奈米材料「多壁奈米碳管與氧化石墨稀帶異構體」(Multi-wall carbon nanotube@graphene oxide nanoribbon,MWCNT@GONR),從而提供了對葡萄糖更靈敏的檢測能力。多壁奈米碳管與氧化石墨稀帶異構物和葡萄糖氧化酵素(Glucose oxidase, GOx)在混合比例1:1 時,可以達到最佳的電子傳遞效果。另一方面,我們開發一種運用介電泳力操控多壁奈米碳管與葡萄糖氧化酵素於薄膜金電極。透過介電泳操控可具有讓多壁奈米碳管與葡萄糖氧化酵素的複合物吸附於電極表面的潛力,從而提供更靈敏的第三代葡萄糖感測器。

Compared to traditional multi-wall carbon nanotube (MWCNT)/glucose oxidase (GOx) composite sensors, a novel carbon heterostructure nanomaterial of multi-wall carbon nanotube@graphene oxide nanoribbon (MWCNT@GONR)presented the better sensing properties for glucose detection. The optimal ratio of MWCNT@GONR to GOx was 1:1 with the largest electron transfer rate. Furthermore, we developed a dielectrophoretic (DEP) technique to fix the MWCNT/GOx composite on the thin-film gold electrodes. The method has promising potential to absorb the MWCNT/GOx composite on the surface of electrodes for constructing a more effective third-generation glucose biosensor.

Content
Abstract ………………………………………………………………………….... I
摘要 ………………………………………………………………………............. II
Special thanks …………………………………………………………………… III
誌謝 ………………………………………………………………………........... IV
Content ……………………………………………………………………….........V
List of Figures …………………………………………………………………. VIII
List of Tables …………………………………………………………………….. X
List of Abbreviations and Denotations ………………………………………….. XI
Chapter 1 Introduction …………………………………………………………. 1
1.1 Glucose biosensor …………………………………………………………… 1
1.1.1 Biosensor ………………………………………………………………... 2
1.1.2 Development of enzyme-based biosensor ………………………………. 4
1.1.3 Three generations of glucose biosensor ……………………………….... 5
1.2 Carbon nanomaterials …………………………………………………….... 12
1.2.1 Development of carbon nanomaterials ……………………………...…. 12
1.2.2 Functionality of carbon nanomaterials ……………………………….... 13
1.2.3 Carbon nanotube (CNT)-modified sensors …………………………..... 14
1.2.4 Graphene-modified sensors …………………………………………..... 15
1.3 Dielectrophoresis (DEP) ……………………………………………...……. 16
1.3.1 The effect of alignment …………………………………………..……. 17
1.3.2 DEP on the manipulation of CNT ……………………………...……… 17
1.4 Research construction ……………………………………………...………. 20

Chapter 2 Materials and Method …………………………………...……….... 22
2.1 Reagents and Instruments ……………………………………………….. 22
2.1.1 Reagents and Chemicals ………………………………………….... 22
2.1.2 Instruments ………………………………………………...………. 31
2.2 Material analysis ………………………………………………...……… 35
2.2.1 X-ray photoelectron spectrum (XPS) ………………………...……. 35
2.2.2 Raman spectroscopy …………………………………………...…... 35
2.3 Preparation of buffer and solutions …………………………………...… 37
2.4 Preparation of screen printed carbon electrodes (SPCEs) ………………. 39
2.4.1 Pretreatment on SPCEs ………………………………………...… 39
2.4.2 Preparation of MWCNT@GONR/GOx/PO-SPCE …………...….. 39
2.4.3 Preparation of MWCNT/GOx/PO-SPCE ………………………… 40
2.4.4 Preparation of AP-MWCNT@GONR/GOx/PO-SPCE ….............. 40
2.5 Preparation of Au electrodes ………………………………………...…... 41
2.5.1 Fabrication of Au electrodes (Au) ……………………………...… 41
2.5.2 Gold nanoparticle sediment (GNS) on Au ……………………..… 42
2.5.3 Pretreatment on Au ……………………………………………...... 43
2.5.4 Preparation of MWCNT/GOx/CA/GNS/Au …………………...… 43
2.6 Dielectrophoresis (DEP) manipulation ………………………………..... 44
2.6.1 DEP theory ……………………………………………………...... 44
2.6.2 Chip design for DEP …………………………………………..…. 45
2.6.3 Parameters used for DEP …………………………………………. 46
Chapter 3 Results and Discussion …………………………………………...... 48
3.1 Characterization of carbon nanomaterials …………………………...…. 48
3.1.1 XPS of MWCNT and MWCNT@GONR ……………………..… 48
3.1.2 Raman spectroscopy of MWCNT and MWCNT@GONR …...…. 49
3.2 Electrochemical behavior on SPCE …………………………………….. 51
3.2.1 Ratio and concentration effect of MWCNT@GONR/GOx ……… 51
3.2.2 Direct electron transfer (DET) between GOx and SPCE ………… 54
3.2.3 DET of MWCNT@GONR/GOx with different preparation …..… 59
3.2.4 pH shift of MWCNT@GONR/GOx/PO-SPCE ……………...…... 60
3.2.5 Glucose detection on MWCNT@GONR/GOx/PO-SPCE …..…... 63
3.3 Electrochemical behavior on Au electrode ………………………...…… 66
3.3.1 Pretreatment effect on Au electrode ………………………..……. 66
3.3.2 MWCNT/GOx absorption using DEP force …………………..…. 67
3.3.3 Test of GOx absorption on CA/Au …………………………...….. 70
Chapter 4 Conclusion and Future Prospective ………………………….....… 74
Reference ……………………………………………………………………...… 75







List of Figures
Fig. 1-1 Construction of biosensor ……………..………………………………… 4
Fig. 1-2 Clark-type oxygen electrode …………………………………………….. 6
Fig. 1-3 Reaction pathway of glucose biosensor ………………………………..... 8
Fig. 1-4 Shape of SWCNT, DWCNT and MWCNT ……………………………. 13
Fig. 1-5 Construction of this research …………………………………………... 21
Fig. 2-1 Shape and component of glucose oxidase ……………………………... 24
Fig. 2-2 Functionality of cysteamine ……………………………………………. 25
Fig. 2-3 Fabrication pathway of Au electrodes …………………………………. 42
Fig. 2-4 The theory and relational expressions of DEP …………………………. 45
Fig. 2-5 Chip design (ITO/Au) of DEP …………………………………………. 46
Fig. 3-1 The XPS spectra of MWCNT and MWCNT@GONR ………………… 48
Fig. 3-2 Raman spectra of MWCNT and MWCNT@GONR …………………... 50
Fig. 3-3 Cyclic voltammograms at PO-SPCEs modified by MWCNT@GONR and GOx composite with the ratio of 2:1, 1:1, 0.5:1 and 0:1 ……..…………………. 52
Fig. 3-4 Cyclic voltammograms at PO-SPCEs modified by MWCNT@GONR and GOx composite with the concentration of 1, 2.5, 5 mg/mL ….…………………. 54
Fig. 3-5 Cyclic voltammograms at PO-SPCEs modified by MWCNT@GONR and GOx composite with the concentration of 2.5 mg/mL and different scan rate range from 0.05 to 0.5 V/s ….………………………………………………………..… 56
Fig. 3-6 Linear dependence of Ipa and Ipc versus scan rates ………………........... 56
Fig. 3-7 Linear dependence of Epa and Epc versus logarithm of scan rates …........ 57
Fig. 3-8 Cyclic voltammograms at PO-SPCEs modified by MWCNT@GONR and GOx composite with the concentration of 2.5 mg/mL in air-saturated PBS with different pH values at the scan rate of 0.35 V/s …………………………………. 61
Fig. 3-9 pH value versus cathodic peak potential (Epc) ……………………......... 62
Fig. 3-10 Cathodic peak current versus varied pH values ………………………. 63
Fig. 3-11 Glucose detection of MWCNT@GONR/GOx/PO-SPCE in air-saturated PBS. Inset: the linear range of glucose detection …………………………….…. 65
Fig. 3-12 Comparison of pretreatments on Au electrode ……………………….. 67
Fig. 3-13 Principle of DEP applied ITO/Au chip ……………………………….. 68
Fig. 3-14 The SEM image before the injection of MWCNT/GOx solution and DEP applied to CA/GNS/Au ………….………………………………………………. 69
Fig. 3-15 The SEM image after the injection of MWCNT/GOx solution with DEP applied to CA/GNS/Au ………………………………………………………….. 70
Fig. 3-16 Glucose absorption of before injection, 1st injection and 2nd injection of MWCNT/GOx with DEP applied …….…………………………………………. 72
Fig. 3-17 Test of absorption before and after the injection of MWCNT/GOx solution during DEP applied …………………………………………………….. 73

List of Tables
Table 1-1 Mediator and oxidation potential in different enzymes ……………….. 9
Table 1-2 Comparison of DET between GOx and different electrodes ………… 11
Table 1-3 Comparison of optimal parameters of DEP to manipulate carbon nanotubes in different researches ……………………………………………...... 19
Table 3-1 The effect of ratio of MWCNT@GONR to GOx on cathodic peak current (Ipc), electron transfer rate constant (ks), surface coverage (Γ) and charge transfer coefficient (α) …………………………………………………………... 58
Table 3-2 The effect of concentration of MWCNT@GONR/GOx on the cathodic peak current (Ipc), electron transfer rate constant (ks), surface coverage (Γ) and the charge transfer coefficient (α) …………………………………………………... 59
Table 3-3 The effect of different preparation to DET of MWCNT@GONR/GOx/ PO-SPCE ………………………………………………………………………... 60


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