本研究利用多樣化印刷方式，採逐層堆積(Layer-by-layer)的方式，製成生物感測器工作電極，利用網刷技術製作碳膠導電層，接著利用點膠機沉積普魯士藍中介層，隨後在電極表面利用噴墨技術噴塗葡萄糖氧化酶酵素層，最後再利用浸製塗佈在電極表面修飾石蠟層，以便固定葡萄糖氧化酶。應用噴墨技術噴塗各式圖樣便利性，將此技術發展至生物感測器的製造用途上。

研究證實噴墨製程應用在生物感測器的可行性，探討系統最佳的電化學環境，發現若將操作電位越往負電位移動，可得到越大的電流訊號，然而操作電位的設定不僅要考慮電流訊號大小，仍要考慮操作電位過大會驅動其他干擾物質反應；探討電解質酸鹼值pH值6.53與7.4對電流訊號的影響，發現其兩者對於感測器的訊號大小影響不大；最後確認干擾測試中，系統是否能避開干擾物質的反應雜訊。

進一步探討改變噴墨參數、沉積不同葡萄糖氧化酶圖樣與靈敏度的關係，發現噴塗速度需與液滴、表面性質及乾燥情況配合，藉由調整溫度，以便得到完整、均勻沉積的表面；藉由噴墨技術在電極表面沉積快速，且可噴塗多種圖樣修飾的優勢，探討葡萄糖氧化酶圖樣是否會影響工作電極電流訊號，發現在使用同樣量的葡萄糖氧化酶時，若葡萄糖氧化酶與普魯士藍層接觸點越多，則訊號越高。除了改變葡萄糖氧化酶圖樣，增加接觸點外，本研究也藉由改變普魯士藍層的粗糙度，嘗試增加葡萄糖氧化酶與普魯士藍接觸點，以便增加反應生成的過氧化氫與普魯士藍接觸的機會，發現藉由碳紙吸附普魯士藍，增加其粗糙度，可確實增強過氧化氫感測訊號，然而增強葡萄糖感測訊號，則需均勻混合葡萄糖氧化酶與普魯士藍墨水，使其沉積在電極表面時，兩者界面均勻接觸，以期能增強訊號。

By using versatile printing technique and layer-by-layer depositing method, the working electrode of biosensors are manufactured in this research. The manufacturing process is as following: screen-printing carbon paste conductive layer, depositing Prussian blue (PB) mediator layer by fluid dispenser, inkjet printing glucose oxidase (GOx) enzyme layer, and dip coating paraffin wax for enzyme immobilization. This research applies the inkjet printing method on biosensors fabrication for the convenience of printing patterns.

The feasibility of fabricating biosensors by inkjet printing process has been proved. Discussing the best electrochemical environment in this system, the result was found that the more negative applied voltage, the bigger response current. However, the setting of proper applied voltage has to take many factors into consideration not only the bigger amount of current signals but also the less interference reaction that the bigger applied voltage would trigger the interference substance reacts. The results from different pH values of electrolyte at pH 6.53 and pH 7.4 have no difference on current response signals. Furthermore, by the interference test, the research needs to make sure the selectivity of this system.

Then, this study discusses the relationship between the printing parameters, the printing patterns of glucose oxidase on the biosensor surface, and sensitivity. The printing velocity is adjusted by droplet and surface properties, and printing temperature is controlled to print uniform deposited surface. Due to the advantage of inkjet printing technique to print various patterns flexibly and fast, this study shows that the glucose oxidase patterns could effect on current signals, and the result shows that in the same amount of glucose oxidase, the more contacting points between PB and GOx are, the bigger electrochemical signals are. Besides, by changing the glucose oxidase patterns, this study also shows increasing the roughness of PB layer could increase the contacting points between GOx and PB, and the reacting area between PB and hydrogen peroxide which is produced by glucose and GOx. The results show that the biosensor electrochemical performance of hydrogen peroxide detection gets better by depositing PB adsorbed on carbon paper, which increases the roughness of PB surface. Then, it is expected to increase the glucose detection signals by mixing glucose oxidase and Prussian blue ink in order to make the homogeneous connecting surface while depositing on the electrode surface.

誌謝 I
摘要 II
Abstract III
目錄 V
圖目錄 VIII
表目錄 XIV
第一章 緒論 1
1. 1 前言 1
1. 2 生物感測器簡介 3
1. 3 塗佈技術介紹 8
1. 3. 1 噴墨技術 9
1. 3. 2 網印技術 12
1. 3. 3 針筆繪製技術 14
1. 3. 4 點膠塗佈技術 16
1. 4 葡萄糖簡介 18
1. 5 葡萄糖氧化酶概述 20
1. 5. 1 葡萄糖氧化酶(Glucose Oxidase)簡介 20
1. 5. 2 酵素動力學 22
1. 5. 3 固定化酵素 25
1. 6 過氧化氫簡介 31
1. 7 普魯士藍概述 34
1. 8 電化學原理 41
1. 8. 1 電極界面－電雙層現象 43
1. 8. 2 循環伏安法 46
1. 8. 3 計時安培法 49
1. 9 研究動機與目的 51
1. 10 論文架構 52
第二章 實驗用品與流程 53
2. 1 實驗藥品 53
2. 2 實驗儀器 55
2. 3 墨水製作 57
2. 3. 1 普魯士藍墨水－噴墨製程(Inkjet Printing)用 57
2. 3. 2 普魯士藍墨水－針筆繪製(Pen-writing)用 57
2. 3. 3 普魯士藍墨水－點膠噴塗製程用 57
2. 3. 4 葡萄糖氧化酶墨水－噴墨製程(Inkjet Printing)用 58
2. 3. 5 石蠟墨水－浸製塗佈(Dip Coating)用 58
2. 4 工作電極製作 59
2. 4. 1 碳膠層 59
2. 4. 2 普魯士藍層 61
2. 4. 3 葡萄糖氧化酶層 63
2. 4. 4 石蠟層 64
2. 4. 5 噴墨技術介紹 66
2. 5 自製工作電極電化學表現測試 73
2. 5. 1 磷酸緩衝水溶液 73
2. 5. 2 葡萄糖水溶液 73
2. 5. 3 過氧化氫水溶液 73
2. 5. 4 尿酸水溶液 74
2. 5. 5 維生素C溶液 74
2. 5. 6 電化學分析 75
第三章 利用噴墨製程之葡萄糖感測器表現 77
3. 1 感測器結構介紹 77
3. 1. 1 感測器表面結構 77
3. 1. 2 利用明膠與戊二醛的交聯性質固定葡萄糖氧化酶 81
3. 1. 3 利用石蠟固定葡萄糖氧化酶 85
3. 1. 4 利用醋酸纖維素固定葡萄糖氧化酶 86
3. 2 操作電壓對於葡萄糖氧化酶感測器靈敏度影響探討 87
3. 3 電解液酸鹼值對於葡萄糖氧化酶感測器靈敏度影響探討 89
3. 4 干擾測試 91
第四章 改變噴墨參數對葡萄糖感測器靈敏度之探討 95
4. 1 噴塗參數對於沉積葡萄糖氧化酶於感測器表面之探討 95
4. 1. 1 噴墨速度對感測器表面樣態之影響 95
4. 1. 2 噴墨基板溫度對感測器表面樣態與電化學表現之影響 97
4. 1. 3 材料噴塗層數對感測器電化學表現之影響 99
4. 2 塗佈形狀對於葡萄糖感測器靈敏度影響探討 104
4. 3 表面粗糙度對葡萄糖感測器靈敏度之影響 111
第五章 結論 121
第六章 未來展望 123
參考文獻 125

1.S.P. Mohanty, and E. Kougianos, Biosensors: a tutorial review. Potentials, IEEE, 2006. 25(2): p. 35-40.
2.D.R. Thevenot, et al., Electrochemical biosensors: Recommended definitions and classification - (Technical Report). Pure and Applied Chemistry, 1999. 71(12): p. 2333-2348.
3.F.W. Scheller, et al., Research and development in biosensors. Current Opinion in Biotechnology, 2001. 12(1): p. 35-40.
4.Wenju Wang, et al., Amperometric bienzyme glucose biosensor based on carbon nanotube modified electrode with electropolymerized poly(toluidine blue O) film. Electrochimica Acta, 2010. 55(23): p. 7055-7060.
5.Joseph Wang., Electrochemical glucose biosensors. Chemical Reviews, 2008. 108(2): p. 814-825.
6.Xin Che, et al., A glucose biosensor based on chitosan-Prussian blue-multiwall carbon nanotubes-hollow PtCo nanochains formed by one-step electrodeposition. Colloids and Surfaces B-Biointerfaces, 2011. 84(2): p. 454-461.
7.O.D. Renedo, et al., Recent developments in the field of screen-printed electrodes and their related applications. Talanta, 2007. 73(2): p. 202-219.
8.A.A. Karyakin, et al., Prussian Blue-Based First-Generation Biosensor. A Sensitive Amperometric Electrode for Glucose. Analytical Chemistry, 1995. 67(14): p. 2419-2423.
9.S.A. Jaffari, and J.C. Pickup, Novel hexacyanoferrate (III)-modified carbon electrodes: Application in miniaturized biosensors with potential for in vivo glucose sensing. Biosensors & Bioelectronics, 1996. 11(11): p. 1167-1175.
10.Chunyan Deng, et al., Direct electrochemistry of glucose oxidase and biosensing for glucose based on boron-doped carbon nanotubes modified electrode. Biosensors & Bioelectronics, 2008. 23(8): p. 1272-1277.
11.Yu Liu, et al., Amperometric glucose biosensor with high sensitivity based on self-assembled Prussian Blue modified electrode. Electrochimica Acta, 2009. 54(28): p. 7490-7494.
12.Min-Hua Xue, et al., In situ immobilization of glucose oxidase in chitosan-gold nanoparticle hybrid film on Prussian Blue modified electrode for high-sensitivity glucose detection. Electrochemistry Communications, 2006. 8(9): p. 1468-1474.
13.F. Ricci, et al., Prussian Blue based screen printed biosensors with improved characteristics of long-term lifetime and pH stability. Biosensors & Bioelectronics, 2003. 18(2-3): p. 165-174.
14.A.A. Karyakin, et al., Optimal environment for glucose oxidase in perfluorosulfonated ionomer membranes: Improvement of first-generation biosensors. Analytical Chemistry, 2002. 74(7): p. 1597-1603.
15.Yeon Hee Yun, et al., A Glucose Sensor Fabricated by Piezoelectric Inkjet Printing of Conducting Polymers and Bienzymes. Analytical Sciences, 2011. 27(4): p. 375-379.
16.Po-Chin Nien, et al., Amperometric glucose biosensor based on entrapment of glucose oxidase in a poly(3,4-ethylenedioxythiophene) film. Electroanalysis, 2006. 18(13-14): p. 1408-1415.
17.Lei Li, et al., Facile and controllable preparation of glucose biosensor based on Prussian blue nanoparticles hybrid composites. Bioelectrochemistry, 2008. 74(1): p. 170-175.
18.C.P. McMahon, et al., Design variations of a polymer-enzyme composite biosensor for glucose: Enhanced analyte sensitivity without increased oxygen dependence. Journal of Electroanalytical Chemistry, 2005. 580(2): p. 193-202.
19.D. Moscone, et al., Construction and analytical characterization of Prussian Blue-based carbon paste electrodes and their assembly as oxidase enzyme sensors. Analytical Chemistry, 2001. 73(11): p. 2529-2535.
20.Tong Li, et al., Development of an amperometric biosensor based on glucose oxidase immobilized through silica sol-gel film onto Prussian Blue modified electrode. Sensors and Actuators B-Chemical, 2004. 101(1-2): p. 155-160.
21.M.S. Lin, and W.C. Shih, Chromium hexacyanoferrate based glucose biosensor. Analytica Chimica Acta, 1999. 381(2-3): p. 183-189.
22.Laura Gonzalez-Macia, et al., Advanced printing and deposition methodologies for the fabrication of biosensors and biodevices. Analyst, 2010. 135(5): p. 845-867.
23.Joseph T. Delaney, Jr, et al., Inkjet printing of proteins. Soft Matter, 2009. 5(24): p. 4866-4877.
24.M. Singh, et al., Inkjet Printing—Process and Its Applications. Advanced Materials, 2010. 22(6): p. 673-685.
25.P.C. Duineveld, The stability of ink-jet printed lines of liquid with zero receding contact angle on a homogeneous substrate. Journal of Fluid Mechanics, 2003. 477: p. 175-200.
26.M. Tudorache, and C. Bala, Biosensors based on screen-printing technology, and their applications in environmental and food analysis. Analytical and Bioanalytical Chemistry, 2007. 388(3): p. 565-578.
27.A.L. Hart, et al., On the use of screen- and ink-jet printing to produce amperometric enzyme electrodes for lactate. Biosensors and Bioelectronics, 1996. 11(3): p. 263-270.
28.B. Nguon, and M. Jouaneh, Design and characterization of a precision fluid dispensing valve. International Journal of Advanced Manufacturing Technology, 2004. 24(3-4): p. 251-260.
29.American Diabetes Association: http://www.diabetes.org/.
30.Liu Chao, et al., Progress on Glucose Oxidase. Food and Drug, 2010. 12(7): p. 285-288.
31.Gabriel Zoldak, et al., Irreversible thermal denaturation of glucose oxidase from Aspergillus niger is the transition to the denatured state with residual structure. Journal of Biological Chemistry, 2004. 279(46): p. 47601-47609.
32.Hao Tang, et al., Amperometric glucose biosensor based on adsorption of glucose oxidase at platinum nanoparticle-modified carbon nanotube electrode. Analytical Biochemistry, 2004. 331(1): p. 89-97.
33.A.J. Cunningham, Introduction to Bioanalytical Sensors, 1998. p. 167-192.
34.Michael L. Shuler, and F. Kargi, Bioprocess engineering. 2002: Prentice Hall New York.
35.J.F. Kennedy, et al., Immobilized Enzymes and Cells. Chemical Engineering Progress, 1990. 86(7): p. 81-89.
36.S. Rauf, et al., Glucose oxidase immobilization on a novel cellulose acetate-polymethylmethacrylate membrane. Journal of Biotechnology, 2006. 121(3): p. 351-360.
37.F. Ricci, et al., Prussian Blue and enzyme bulk-modified screen-printed electrodes for hydrogen peroxide and glucose determination with improved storage and operational stability. Analytica Chimica Acta, 2003. 485(1): p. 111-120.
38.Brain A. Gregg, and Adam Heller, Cross-Linked Redox Gels Containing Glucose-Oxidase for Amperometric Biosensor Applications. Analytical Chemistry, 1990. 62(3): p. 258-263.
39.Asha Chaubey, et al., Co-immobilization of lactate oxidase and lactate dehydrogenase on conducting polyaniline films. Analytica Chimica Acta, 2000. 407(1–2): p. 97-103.
40.Nobuo Kageyama, A direct colorimetric determination of uric acid in serum and urine with uricase-catalase system. Clinica Chimica Acta, 1971. 31(2): p. 421-426.
41.A.M. Azevedo, et al., Ethanol biosensors based on alcohol oxidase. Biosensors and Bioelectronics, 2005. 21(2): p. 235-247.
42.Xiaocui Zhao, et al., Redox reactions of reactive oxygen species in aqueous solutions as the probe for scanning electrochemical microscopy of single live T24 cells. Canadian Journal of Chemistry-Revue Canadienne De Chimie, 2010. 88(6): p. 569-576.
43.R. Garjonyte, et al., Prussian Blue- and lactate oxidase-based amperometric biosensor for lactic acid. Sensors and Actuators B: Chemical, 2001. 79(1): p. 33-38.
44.F. Ricci, and G. Palleschi, Sensor and biosensor preparation, optimisation and applications of Prussian Blue modified electrodes. Biosensors & Bioelectronics, 2005. 21(3): p. 389-407.
45.Chia-hsiang Peng, Photochemical synthesis of Prussian blue film and its application for H2O2 sensor. Master Thesis in Department of Chemical Engineering(2007), Feng Chia University, Taiwan.
46.J.F. Keggin, and F.D. Miles, Structures and formulae of the Prussian blues and related compounds. Nature, 1936. 137(7): p. 577-578.
47.Jacek C. Wojdeł, First principles calculations on the influence of water-filled cavities on the electronic structure of Prussian Blue. Journal of Molecular Modeling, 2009. 15(6): p. 567-572.
48.H.J. Buser, et al., The crystal structure of Prussian Blue: Fe4[Fe(CN)6]3．xH2O. Inorganic Chemistry, 1977. 16(11): p. 2704-2710.
49.Yu-Ping Lin, Operational Stability Enhancement of a Hydrogen Peroxide Sensor Based on Prussian Blue Fabricated through Inkjet Process. Master Thesis in Department of Chemical Engineering(2012), National Taiwan University, Taiwan.
50.R.A. Huggins, Mixed-conducting host structures into which either cations or anions can be inserted. Solid State Ionics, 1998. 113–115(0): p. 533-544.
51.V.D. Neff, Electrochemical Oxidation and Reduction of Thin-Films of Prussian Blue. Journal of the Electrochemical Society, 1978. 125(6): p. 886-887.
52.A.A. Karyakin, et al., The electrocatalytic activity of Prussian blue in hydrogen peroxide reduction studied using a wall-jet electrode with continuous flow. Journal of Electroanalytical Chemistry, 1998. 456(1-2): p. 97-104.
53.R. Garjonyte, and A. Malinauskas, Operational stability of amperometric hydrogen peroxide sensors, based on ferrous and copper hexacyanoferrates. Sensors and Actuators B-Chemical, 1999. 56(1-2): p. 93-97.
54.J.Y. Hu, et al., Inkjet Printed Prussian Blue Films for Hydrogen Peroxide Detection. Analytical Sciences, 2012. 28(2): p. 135-140.
55.Yu Liu, et al., A sensitivity-controlled hydrogen peroxide sensor based on self-assembled Prussian Blue modified electrode. Electrochemistry Communications, 2009. 11(2): p. 484-487.
56.Jun-Yu Hu, Applications and Process Development of Biosensors by Inkjet Printing Methods. Master Thesis in Department of Chemical Engineering(2011), National Taiwan University, Taiwan.
57.A.J. Bard, and L.R. Faulkner, Electrochemical Methods: Fundamentals and Applications 2nd ed. 2001: New Work: John Wiley.
58.L.L. Zhang, and X.S. Zhao, Carbon-based materials as supercapacitor electrodes. Chemical Society Reviews, 2009. 38(9): p. 2520-2531.
59.S. Matsuda, et al., Bioadhesion of gelatin films crosslinked with glutaraldehyde. Journal of Biomedical Materials Research, 1999. 45(1): p. 20-27.
60.Kespi Pithawala, et al., Immobilization of urease in alginate, paraffin and lac. Journal of the Serbian Chemical Society, 2010. 75(2): p. 175-183.
61.Chengyan Wang, et al., Glucose biosensor based on the highly efficient immobilization of glucose oxidase on Prussian blue-gold nanocomposite films. Journal of Molecular Catalysis B-Enzymatic, 2011. 69(1-2): p. 1-7.
62.A.A. Karyakin, et al., On the mechanism of H2O2 reduction at Prussian Blue modified electrodes. Electrochemistry Communications, 1999. 1(2): p. 78-82.
63.I. Willner, and E. Katz, Integration of layered redox proteins and conductive supports for bioelectronic applications. Angewandte Chemie-International Edition, 2000. 39(7): p. 1180-1218.