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研究生:曾達峯
研究生(外文):Ta-Feng Tseng
論文名稱:植入應用之無氧葡萄糖感測技術探討
論文名稱(外文):Studies of oxygen independent glucose sensing technologies used for implant applications
指導教授:婁世亮
指導教授(外文):Shyh-Liang Lou
學位類別:博士
校院名稱:中原大學
系所名稱:生物醫學工程研究所
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:2011
畢業學年度:99
語文別:中文
論文頁數:186
中文關鍵詞:葡萄糖去氫酶含鉻黃血鹽無氧式葡萄糖感測器電位處理
外文關鍵詞:Chromium hexacyanoferrateOxygen-independent glucose sensorGlucose dehydrogenasePotential treatment
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本研究旨在發展植入式無氧葡萄糖感測器之關鍵技術,藉無氧/無酵素電極和無氧/酵素電極做為研發平台,所探討的技術則以電極介質修飾、電極加熱和電極電位處理(potential treatment, PT)為主,其中電極電位處理是本研究之獨特創意技術。細胞實驗是用以探討電位處理及電極材料對於肌肉細胞(C2C12)生長的影響,以資植入生物體可行性之考量。無氧/無酵素電極的探討,是以開路電位法分析各種電極材料如白金(Pt)、黃金(Au)、玻璃碳(GC)及石墨(GP)於電位處理後之效應,結果顯示,電位處理有增益電極氧化或還原反應之進行。修飾有含鉻黃血鹽(CrHCF)之白金電極(CrHCF/Pt)是以安培法獲得其最佳操作電位及電位處理時間,並比較電位處理及電極加熱前後對於葡萄糖的反應訊號,結果顯示,電位處理及電極加熱後之葡萄糖訊號可有518%的增益。無氧/酵素電極的探討是以CrHCF/Pt為基底,再修飾以Nafion®和葡萄糖去氫酶修飾於白金電極上。由於本研究使用含鉻黃血鹽為中間介質,其價態原為還原態,吾人以X-射線光電子能譜(XPS)確認該電極在經電位處理後之價態是由還原態轉變為氧化態。最佳參數探討則是著重於線性濃度範圍、氧氣的干擾及穩定度,其結果顯示,電極線性範圍可達20 mM (R = 0.9923)、Km = 25.9 mM,並且於氧氣飽和狀況下無任何氧氣干擾情況;電極在保存272天後,酵素仍保有88 %的活性。細胞實驗是以裸白金電極、修飾白金電極及修飾白金生物電極三種材料探討電位處理前、後對C2C12細胞生長的影響,除了MTT測試細胞存活率,並以LDH含量試驗、TUNEL試驗及細胞週期分析佐證細胞死亡方式,結果顯示,裸白金電極、修飾白金電極及修飾白金生物電極三種材料不具有毒性,但是,由AFM分析結果得知,修飾白金電極及修飾白金生物電極之電極表面粗糙度會造成細胞死亡,再由LDH含量試驗之結果顯示細胞是經壞死的途徑死亡。總言之,運用電位處理、中間介質修飾及電極加熱可有效提高偵測靈敏度,具有應用於植入式微電極系統之潛能。

This dissertation aims to develop key technologies specifically for implantable oxygen-independent glucose sensors. Electrodes modified with and without enzyme were used in the studies. Intermediate coating on electrodes, heating on electrodes, and potential treatment on electrodes were the main technologies studied. In particular, the potential treatment technology is an innovation of this work. C2C12 cells were used to determine the possible negative effects that potential treatment and electrode substrates would have on cell growth. Future usage of potential treatment and electrode substrates in implant applications is contingent upon the results of the in vitro studies mentioned above. The open circuit potentiometry technique was used in the oxygen-independent non-enzyme electrode study to investigate potential treatment effects of various electrode substrates, including platinum (Pt), gold (Au), glassy carbon (GC), and graphite (GP). Results indicate that the proposed potential treatment method on the eletrode can enhance redox reactions. The study of mediator effect was based on a modified chromium hexacyanoferrate (CrHCF) Pt electrode. The optimal operational potential and potential treatment time length of the CrHCF/Pt electrode were determined via amperometry. Amperometric measurements of the electrode before and after the potential treatment and electrode heating assessed the glucose response enhancement of the electrodes. Results indicate that the potential treatment and electrode heating increased the glucose response by 518 %. The studies of the oxygen-independent enzyme glucose electrode were based on the CrHCF/Pt electrode modified with Nafion® and pyrroloquinoline quinone-glucose dehydrogenase (PQQ-GDH). To assure that CrHCF will be able to accept the electrons from PQQ-GDH, CrHCF must be in oxidation stage. Because the initial form of CrHCF was in the reduction stage, the potential treatment effect of converting CrHCF from the reduction stage to the oxidation stage was confirmed by X-ray photoelectron spectroscopy (XPS). The characteristics of the bio-electrode (Nafion/PQQ-GDH/CrHCF/Pt), such as linearity, oxygen interference, and stability, were studied. Results show that the linearity feature of the bio-electrode was 20 mM (R = 0.9923). There was no signal response detected from the bio-electrode even when saturated oxygen was applied in the tested solution, and the residual activity of the bio-electrode did not fall below 88% after 272 days. In vitro study was to evaluate cell damage effect by cisplatin, which may be produced when a power voltage was given to platinum in a solution containing chloride and NH3. In this study, a bare Pt electrode, a CrHCF/Pt electrode, and a Nafion/PQQ-GDH/CrHCF/Pt bio-electrode were individually co-cultured with a cell line of C2C12 and then followed by potential treatment. The MTT assay was used to test the viability of the C2C12 cells. In addition, LDH assay, TUNEL assay, and cell cyclic assay were used to analyze cell death mechanism. Results show that the electrode materials of bare Pt, CrHCF/Pt and Nafion/PQQ-GDH/Nafion/CrHCF/Pt are not cytotoxic. It is the surface roughness, in particular the CrHCF/Pt and Nafion/PQQ-GDH/Nafion/CrHCF/Pt, from the AFM analysis leading C2C12 cells die. The LDH releases from the C2C12 cells co-cultured with the electrode materials are high enough to conclude that the cells death is through necrosis mode. In conclusion, the techniques of OPT, mediation and electrode heating can enhance glucose response sensitivity, hence have potential to be implemented in implantable micro-electrode systems.

中文摘要 I
Abstract II
謝 誌 IV
Content VV
List of figures VIII
List of tables XIII
Chapter 1 Introduction 1
1.1 Diabetes Mellitus 1
1.2 Electrochemical biosensor 3
1.2.1 Potentiometry 5
1.2.2 Voltammetry 6
1.2.3 Amperometry 8
1.3 Oxygen independent based implantable glucose sensor 10
1.3.1 Enzymatic approaches 12
1.3.2 Non-enzymatic approaches 14
1.4 Study Aims 15
1.5 Dissertation framework 16
Chapter 2 Effects of electric potential treatment of a CrHCF modified biosensor based on PQQ-GDH 17
2.1 Introduction 17
2.2 Experimental 20
2.2.1 Materials 20
2.2.2 Apparatus 20
2.2.3 Preparation of chromium hexacyanoferrate modified platinum discs 21
2.2.4 Fabrication of chromium hexacyanoferrate modified glucose biosensor 22
2.2.5 Electrochemical measurement 23
2.3 Results and discussions 24
2.3.1 Confirmation of presence of chromium hexacyanoferrate on the Pt electrode 24
2.3.2 Electrochemical impedance spectroscopy 27
2.3.3 Effects of potential pre-treatment and potential boosting 29
2.3.4 XPS spectral analysis 31
2.3.5 Effect of glucose concentration 35
2.3.6 Effect of pH value 37
2.3.7 Effect of temperature 40
2.3.8 Characteristics of the CrHCF/PQQ-GDH glucose biosensor 42
2.3.9 Oxygen interference 43
2.3.10 Stability performance 45
2.4 Conclusions 48
Chapter 3 Thermally stable improved glucose biosensors based on heated electreodes 49
3.1 Introduction 49
3.2 Experimental 51
3.2.1 Materials 51
3.2.2 Apparatus 51
3.2.3 Temperature calibration 52
3.2.4 Sensor fabrication 52
3.3 Results and discussions 53
3.3.1 Effect of heating upon the oxidation of H2O2 53
3.3.2 Temperature effect upon amperometric signals 54
3.3.3 Calibration of glucose concentration 56
3.3.4 Stability of the amperometric signals 58
3.4 Conclusions 59
Chapter 4 Techniques exploring for platinum based non-enzymatic glucose sensor. 60
4.1 Introduction 60
4.2 Experimental 62
4.2.1 Materials 62
4.2.2 Apparatus 63
4.2.3 Pretreatment of bare working electrodes 64
4.3 Results and discussions 66
4.3.1 Oxidation potential treatment effect of bare platinum electrodes 66
4.3.2 Open circuit potentiometry analysis 68
4.3.2.1 OCP analyses of OPT working electrodes 70
4.3.2.2 OCP analyses of RPT working electrodes 73
4.3.2.3 Summary of OCP analysis studies of RPT and OPT effects 76
4.3.3 Effective duration of potential treatment 78
4.3.4 Quantitative studies of potential treatment effect 81
4.3.4.1 The OPT effect studies with amperometry 82
4.3.4.2 The RPT effect studies with amperometry 87
4.3.5 Enhancment effect in combination with potential treatment and CrHCF 94
4.3.6 Selection of the operation potential of the CrHCF/Pt electrode 97
4.3.7 Determination of the OPT processing time 100
4.3.8 Stability performance of CrHCF/Pt electrode 102
4.3.9 Heating electrode effect 108
4.4 Conclusions 116
Chapter 5 In vitro studies of platinum-based electrodes 117
5.1 Introduction 117
5.2 Experiment 118
5.2.1 Materials 118
5.2.2 Cell culture and electrode material preparation 119
5.2.3 MTT cell viability assay 120
5.2.4 In vitro cytotoxicity test: Indirect contact (Agar diffusion test) 121
5.2.5 Flow cytometry assay 122
5.2.6 TUNEL assay and LDH content assay 123
5.2.7 LDH content assay 124
5.3 Results and Discussions 125
5.3.1 MTT cell viability assay 125
5.3.1.1 MTT viability assay of pre-OPT effect 128
5.3.1.2 MTT cell viability assay of OPT effect 131
5.3.2 Indirect contact test 134
5.3.3 Atomic force microscope analysis 136
5.3.4 Heat effect study 138
5.3.5 X-ray photoelectron spectroscopy analysis 140
5.3.6 Energy-dispersive X-ray spectroscopy 142
5.3.7 Flow cytometry assay 144
5.3.8 TUNEL assay 150
5.3.9 LDH content assay 152
5.4 Conclusions 154
Chapter 6 Suggestions for future work 155
References 157
Appendix I Chemical reagents used in this work 168
Appendix II Apparatus used in this work 170
Appendix III Datasheet of C2C12 cells 171
Publication list 172

List of figures
Figure 1 1 A cyclic voltammetry diagram with switching potentials at +1.0 V and -1.0 V vs. Ag/AgCl. 7
Figure 2 1 Typical cyclic voltammograms of compounds electrochemically deposited on surface of Pt foils; (a) bare Pt foil, (b) Cr(NO3)3, (c) K3Fe(CN)6, and (d) mix of Cr(NO3)3 and K3Fe(CN)6 in 0.1 M KCl electrolyte, pH = 3. Sweep cycles, 250 cycles. 25
Figure 2 2 Scanning electron micrographs of compounds deposited on Pt foil surface; (a) bare, (b) electrochemically deposited with Cr(NO3)3, (c) K3Fe(CN)6 and (d) mixture of Cr(NO3)3 and K3Fe(CN)6 in 0.1 M KCl electrolyte, pH = 3. 26
Figure 2 3 Fourier transform infrared spectra of Pt electrode surface: (a) bare, and (b) coated with chromium hexacyanoferrate. Other conditions are as given in figure 2-1. 26
Figure 2 4 Electrochemical impedance spectra of (a) bare Pt, (b) CrHCF/Pt, (c) Nafion/CrHCF/Pt, (d) PQQ-GDH/CrHCF/Pt, (e) PQQ-GDH/Nafion/CrHCF/Pt and (f) Nafion/PQQ-GDH/Nafion/CrHCF/Pt electrodes in the solution containing K3Fe(CN)6 and K4Fe(CN)6 (1 mM each) and 0.1M KCl. 28
Figure 2 5 Amperometric responses of CrHCF/PQQ-GDH glucose biosensor in scenarios when it was (a) newly completed, (b) potentially treated, (c) unused overnight, and (d) potentially boosted in detecting 5 mM glucose in 0.1 M phosphate buffer (pH 7). Arrow indicates time of spiking with 5 mM glucose. 30
Figure 2 6 XPS analysis of CrHCF-modified Pt discs when they were: (a) newly modified, (b) potentially treated, (c) unused overnight, and (d) potentially boosted. 34
Figure 2 7 (A) Typical calibration curve of CrHCF/PQQ-GDH biosensor in determination of concentration of glucose. Conditions are as given in Figure 2-5. (B) A Lineweaver-Burke plot derived from the calibration curve in (A). 36
Figure 2 8 Effect of pH on the CrHCF/PQQ-GDH biosensor in sensing 5 mM glucose. Except for the pH setting, other conditions are as given in Figure 2-5. 39
Figure 2 9 Effect of temperature on the CrHCF/PQQ-GDH biosensor in sensing 5 mM glucose. Except for the temperature setting, other conditions are as given in Figure 2-5. 41
Figure 2 10 (A) Amperometric responses of CrHCF/PQQ-GDH glucose biosensor in determination of concentration of glucose in 0.1 M phosphate buffer (pH 7) at the conditions of ambient air (○), deaeration with nitrogen gas (●), and saturation with oxygen gas (). (B) The glucose responses of the biosensor along the time when the glucose buffers at the conditions of (a) ambient air, (b) deaeration with nitrogen gas, and (c) saturation with oxygen gas. Stirring speed: 300 rpm; Operating temperature: 25 oC; Applied potential: +1.0 V 44
Figure 2 11 Stability performance of PQQ-GDH/CrHCF/Pt electrode. Conditions are as given in Figure 2-5. 46
Figure 3 1 Effect of heating upon the oxidation of 1 mM H2O2: (a) non-heating, (b) heating, 36.8 oC, at LTCC gold electrode (A) and at gold bioelectrode (B) in 0.1 M PBS (pH 7.0); scan rate, 50 mV s-1. 53
Figure 3 2 Amperometric response recorded at gold bioelectrode for various applied temperature pulses: (A) 29.4 oC, (B) 36.8 oC, (C) 45.8 oC, (D) 56.0 oC, (E) 67.5 oC in 0.1 M PBS (pH 7.0), (A) [Glucose] = 1 mM and (B) [H2O2] = 1 mM. Inset shows the effect of applied heating current for (a) blank and (b) 1 mM glucose/1 mM H2O2. Working potential, +0.6 V. 55
Figure 3 3 Amperometric response recorded at +0.6 V using the heated glucose sensor (at 50 oC) for various glucose concentrations: (A) blank, (B) 3 mM (C) 6 mM (D) 9 mM and (E) 12 mM in 0.1 M PBS (pH 7.0). Inset shows the calibration curves at (a) 30 oC, (b) 40 oC, and (c) 50 oC. 57
Figure 3 4 Stability of the amperometric response for 1 mM glucose recorded at heated GOx–Nafion-gold electrode (37 oC) in 0.1 M PBS (pH 7.0). Inset shows 20 repetitive measurements at two different temperatures. 58
Figure 4 1 The cyclic voltammetric analysis of the bare Pt electrode in 0.1 M PBS (pH 7.0). (a) pre-OPT, glucose free; (b) pre-OPT, 30 mM glucose; (c) post-OPT, 30 mM glucose. All cyclic scans were performed from -0.1 to 1.0 V; scan rate, 50 mV•s-1. 67
Figure 4 2 OCP analyses of GC, GPE, Au and Pt electrodes in DDW or PBS. (a) The electrodes were at the pre-OPT stage and analyzed in DDW; (b) the electrodes were undergone the OPT process and analyzed in DDW; (c) the electrodes were at the pre-OPT stage and analyzed in PBS; and (d) the electrodes were undergone the OPT process and analyzed in PBS 72
Figure 4 3 OCP analyses of the GC, GPE, Au and Pt electrodes in DDW and PBS. (a) The electrodes were at the pre-RPT stage and analyzed in DDW; (b) The electrodes were undergone the RPT process and analyzed in DDW; (c) The electrodes were at the pre-RPT stage and analyzed in PBS; and (d) The electrodes were undergone the RPT process and analyzed in PBS. 75
Figure 4 4 Effective duration of potential treatment study; (A) The OCP potential diminishing response of a Pt working electrode receiving the OPT, (B) the amperometric response (●) versus the OCP potential (○) along the time. The OCP potential diminishing response study was performed in 0.1 M PBS (pH 7.0). The amperometric response study measured 0.1 mM H2O2 in 0.1 M PBS (pH 7.0) with +0.6 V, 200 rpm, at room temperature. 80
Figure 4 5 Amperometric responses of the working electrodes in detecting 0.1 mM H2O2 in 0.1 M phosphate buffer (pH 7) at the stages of pre-OPT and post-OPT. (A) Pt, (B) Au, (C) GC, and (D) GPE. 85
Figure 4 6 Amperometric responses of the working electrodes in detecting 0.1 mM H2O2 in 0.1 M phosphate buffer (pH 7) at the stages of pre-RPT and post-RPT. (A) Pt, oxygen, (B) Pt, oxygen free, (C) Au, oxygen, (D) Au, oxygen free, (E) GC, oxygen, (F) GC, oxygen free, (G) GPE, oxygen, and (H) GPE, oxygen free. 92
Figure 4 7 The cyclic voltammetry analysis of the electrodes without the OPT process in 0.1 M PBS (pH 7.0); 30 mM glucose, scan rate, 50 mV s-1. (a) the bare Pt electrode, and (b) the CrHCF/Pt electrode. 95
Figure 4 8 The cyclic voltammetry analysis of the electrodes with the OPT process in 0.1 M PBS (pH 7.0); 30 mM glucose, scan rate, 50 mV s-1. (a) the bare Pt electrode, and (b) the CrHCF/Pt electrode. 96
Figure 4 9 The cyclic voltammetry analysis of the CrHCF/Pt electrode detecting 30 mM glucose in 0.1 M PBS (pH 7.0); scan rate, 50 mV s-1. (a) at the pre-OPT stage, and (b) at the post-OPT stage. 96
Figure 4 10 Amperometric currents versus operation potentials at (a) pre-OPT, (b) post-OPT, determining 5 mM glucose in 0.1 M pH 7.0 phosphate buffer solution. Operation condition is stirring speed 150 rpm; operating temperature at 25 oC. 99
Figure 4 11 Amperometric glucose oxidation currents of CrHCF/Pt electrode at difference post-OPT time. Measurement potential was +0.5 V in 0.1 M phosphate buffer (pH 7.0) solution. The measurement condition was determining 5 mM glucose; stirring speed, 150 rpm; operating temperature at 25 oC. 101
Figure 4 12 Stability performance of the OPT processed CrHCF/Pt electrode at room temperature. the electrode (○) stored in 0.1 M PBS (pH 7.0), (●) stored in 0.1 mM hydrogen peroxide with an operation potential of +0.5 V to detect 0.1 mM hydrogen peroxide. 107
Figure 4 13 Stability performance of the OPT processed CrHCF/Pt electrode at 37oC. the electrode stored in 0.1 M PBS (pH 7.0) with an operation potential of +0.5 V to detect 0.1 mM hydrogen peroxide. 107
Figure 4 14 The electrode heating apertures 113
Figure 4 15 The calibration function to be used to map the OCP response of the Pt electrode for a given solution temperature. The solution was a mixture of 0.1 M KCl and 5 mM ferro-/ferricyanide. 113
Figure 4 16 The calibration curve of temperature by regulating various heating potential and plotting the temperature difference in 0.1 M phosphate buffer (pH 7.0) solution. 114
Figure 4 17 The amperometric glucose responses of the heating electrodes for the five scenarios: (A) the bare Pt electrode, glucose free, pre-OPT; (B) the bare Pt electrode, pre-OPT; (C) the bare Pt, post-OPT; (D) the modified CrHCF/Pt electrode, pre-OPT; (E) the modified CrHCF/Pt electrode, post-OPT in detecting 30 mM glucose in 0.1 M PBS. The time between 60 and 90 seconds was the period of heating voltage applied. 114
Figure 5 1 MTT viability test flowchart 126
Figure 5 2 The apparatus used to perform the OPT in the MTT cell viability test 127
Figure 5 3 Time course of performing the OPT process and the MTT cell viability test 127
Figure 5 4 C2C12 cell viability (Left) and OD values (Right) of the four groups: control, bare Pt, CrHCF/Pt, and Nafion/PQQ-GDH/Nafion/CrHCF/Pt in the pre-OPT stage at the 24-, 48-, and 72-hr checkpoints. 130
Figure 5 5 C2C12 cell viability (Left) and OD values (Right) of the six groups: control (-), control (+), cisplatin, bare Pt, CrHCF/Pt, and Nafion/PQQ-GDH/Nafion/CrHCF/Pt in the post-OPT stage at the checkpoints 24-, 48-, and 72-hr. 133
Figure 5 6 Microscope images of agar diffusion test from (A) positive control, (B) negative control, (C) bare Pt, (D) CrHCF/Pt and (E) Nafion/PQQ-GDH/Nafion/CrHCF/Pt substrates at the pre-OPT stage. 135
Figure 5 7 AFM spectra of (A) dish, (B) bare Pt, (C) CrHCF/Pt, and (D) Nafion/PQQ-GDH/Nafion/CrHCF/Pt at the pre-OPT stage. 137
Figure 5 8 Temperature plotting for the recordings of ambient environment, OPT and pre-OPT. 139
Figure 5 9 One XPS overall survey and three XPS elemental survey scans, Pt4f, Cl2p, and N1s. (A) the bare Pt foil in the medium culturing C2C12 cells at the pre-OPT stage and (B) the bare Pt foil in the medium with the OPT process 3 times in a period of 72 hours. 141
Figure 5 10 EDS study of the two bare Pt foils, (A) at the pre-OPT stage, and (B) at the post-OPT stage. 143
Figure 5 11 Number of C2C12 cell histograms from the control group and the three test groups in the cell cycle phases of G0/G1, S, and G2/M at (A) the pre-OPT stage, and (B) the post-OPT stage. 146
Figure 5 12 DNA content frequency histograms of C2C12 cells cultured with (A) culturing plate (control), (B) bare Pt, (C) CrHCF/Pt, (D) Nafion/PQQ-GDH/Nafion/CrHCF/Pt at the pre-OPT stage by flow cytometry. 147
Figure 5 13 DNA content frequency histograms of C2C12 cells cultured with (A) culturing plate (control), (B) bare Pt, (C) CrHCF/Pt, (D) Nafion/PQQ-GDH/Nafion/CrHCF/Pt at the post-OPT stage by flow cytometry. 148
Figure 5 14 Folds of cell apoptosis resulted from the TUNEL assay for the control groups and the test groups of bare Pt, CrHCF/Pt, and Nafion/ PQQ-GDH/Nafion/CrHCF/Pt. 151
Figure 5 15 LDH releases resulted from the LDH assay for the control groups and the test groups of bare Pt, CrHCF/Pt, and Nafion/ PQQ-GDH/Nafion/CrHCF/Pt. 153

List of tables
Table 2 1 Comparison of the performance of different PQQ-GDH based glucose biosensors. 47
Table 4 1 The difference of potential values summaries of four working electrode substrates in DDW and PBS electrolyte by OPT and RPT. 77
Table 4 2 The H2O2 response enhancement of the OPT effect 86
Table 4 3 The H2O2 response enhancement of the RPT effect 93
Table 4 4 The difference glucose response current density by operation potential. 99
Table 4 5 The glucose response enhancement of the techniques: the OPT, the mediation, and the heating voltage. 115
Table 5 1 C2C12 cells viability rates and corresponding OD values of the control group and the test groups in the pre-OPT stage at the 24-, 48-, and 72-hr checkpoints. 130
Table 5 2 C2C12 cells viability rates and corresponding OD values of the control groups and the test groups in the post-OPT stage at the 24-, 48-, and 72-hr checkpoints. 133
Table 5 3 Cell population distributions in cell cycle phases of the test samples at the stages of post-OPT and pre-OPT. 149



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