本文研究中主要將研究之方向分為二部分: (I) 合成調控不同原子數比例之鉑鈀/多層壁奈米碳管(PtxPd1-x/MWCNTs)之電化學觸媒應用於雙氧水氧化反應感測分析 (II) 應用電化學沉積法沉積甲殼素以固定酵素製備葡萄糖生物感測器。
(I) 探討不同強氧化劑進行改質反應對於多層壁奈米碳管表面官能基之影響，發現硝酸與硫酸(1:1)混合之溶液對於改質多層壁奈米碳管具有較佳之氧化反應效果，以修飾Watanabe膠體化還原法在液相環境中調整適當之pH值皆具有接近100 %之還原率，最佳活性觸媒Pt3Pd1/MWCNTs其顆粒粒徑大小分布介於2~4 nm之間，其於調控之不同比例自製觸媒分別為2~4 nm與3~5 nm，由此可知，觸媒在相同金屬負載量下，其觸媒粒徑大小與雙氧水氧化反應之間無明顯直接之關係。利用實驗室所建立觸媒表面組成鑑定關係式搭配新竹同步輻射中心EXAFS所計算出原子總配位數，由結果可知，Pt3Pd1/MWCNTs觸媒表面的原子組成接近1:1，代表觸媒表面有足夠的鉑原子能夠吸附雙氧水分子，且有適量的鈀原子能提供OH基，來幫助雙氧水進行電化學氧化反應，進而提高奈米金屬觸媒的活性。
(II) 建立最適化電極感測層之結構，利用添加微量交聯劑幫助酵素與甲殼素溶液間行交聯反應，找尋電沉積溶液配方與電化學沉積參數之關係，由酵素動力學反應Michaelics-Menten constant=5.3 mM與穩定性測試探討(電極保存七天後效能約減少10 %)等結果得知，此酵素固定化條件應用於電化學沉積法所製備之酵素感測器不僅能提供溫和環境固定化酵素於電極表面，所形成之三維空間網狀薄膜幫助酵素與待測物質間進行生物反應，使酵素具良好之親合性與穩定性。
本研究所製備之葡萄糖感測器於0.6 V v.s SCE之施加電位下，具有感測葡萄糖線性範圍為0.031mM ~14.07 mM (R2=0.99)，且其偵測靈敏度為104 μA/mM cm2，將所建立的最適化修飾感測結構層轉移至微型感測試片，其偵測葡萄糖感測之線性範圍涵蓋至10 mM (R2=0.99，N=3)，偵測之靈敏度為344.57 μA/mM cm2，本研究結果顯示，藉由合成之雙元金屬電化學觸媒搭配電化學沉積法應用於生物感測器之製程系統，成功製備具優良再現性與量測準確度、結構穩定之高靈敏度微型葡萄糖感測試片。

This study is comprised of two parts: (I) the synthesis of novel Pt-Pd/ multi-wall carbon nanotubes (MWCNTs) catalysts for the applications of electrochemical sensing of hydrogen peroxide. (II) Immobilization of glucose oxidase by electrochemi
cal deposition of chitosan.
(I) Oxidation of Multi-wall carbon nanotube was attempted by various acids, such as sulfuric acid, nitric acid.The oxidation who achieved effectively by mixing sulfuric acid and nitric acid in 1:1 ratios. The syntheses of (PtxPd1-x/MWCNTs) catalysts were carried out at different atomic ratios of metal precursor by modified Watanabe colloidal synthesis method in liquid phase at pH 6. The Pt3Pd1/MWCNTs catalyst was found to show the best performance toward hydrogen peroxide quantification. TEM analyses showed the particle size of the catalysts was approximately same (2-5 nm). From this observation, it was found that particle size and hydrogen peroxide oxidation reaction are independent at the same metal loading on the carbon nanotube.
(II) The optimum structure of the sensing layers who developed. The additions of cross-linking agent enhanced the cross-linking reaction between enzyme and chitosan. The solution recipe and parameters for the electrochemical deposition were optimized. The enzyme kinetics (Michaelics-Menten constant = 5.3 mM) and stability tests have been demonstrated that the sensitivity decay of the assembled electrodes is around 10 % after 7 days. It shows that the assembled sensor prepared under this condition provide mild environment for enzyme immobilization and 3D network membrane facilitate the bio-reaction between enzyme and bio-species, which makes enzyme exhibiting good affinity and stability.
The prepared glucose sensor exhibits a sensitivity of 104 μA/mMcm2 in detection range between 0.031 mM and 14.07 mM (R2 =0.990) at 0.6 V (vs. SCE). The optimum mini-strip shows 344.57 μA/mM cm2 in detection range up to 10 mM (R2 =0.99, N=3). The results the glucose strips with a high sensitivity, reproducibility and stability are fabricated via the combination of synthesized bimetallic nanocatalyst and electrochemical deposition.

1.Wang, J., Electrochemical glucose biosensors. Chemical Reviews, 2008. 108(2): p. 814-825.
2.CLARK Jr., L.C., LYONS, C. , Electrode systems for continuous monitoring in cardiovascular surgery. Annals of the New York Academy of Sciences 1962. 102.
3.Lubrano, G.G.G.a.G.J., An enzyme electrode for the amperometric determination of glucose Analytica Chimica Acta, 1973. 64(3): p. 439-455.
4.Shen, J., L. Dudik, and C.C. Liu, An iridium nanoparticles dispersed carbon based thick film electrochemical biosensor and its application for a single use, disposable glucose biosensor. Sensors and Actuators B-Chemical, 2007. 125(1): p. 106-113.
5.Lu, J., et al., Simple fabrication of a highly sensitive glucose biosensor using enzymes immobilized in exfoliated graphite nanoplatelets nafion membrane. Chemistry of Materials, 2007. 19(25): p. 6240-6246.
6.Moussy, F., et al., In vitro and in vivo performance and lifetime of perfluorinated ionomer-coated glucose sensors after high-temperature curing. Analytical Chemistry, 1994. 66(22): p. 3882-3888.
7.Wang, J. and H. Wu, Permselective lipid-poly(o-phenylenediamine) coatings for amperometric biosensing of glucose. Analytica Chimica Acta, 1993. 283(2): p. 683-688.
8.Li, J. and X. Lin, Glucose biosensor based on immobilization of glucose oxidase in poly(o-aminophenol) film on polypyrrole-Pt nanocomposite modified glassy carbon electrode. Biosensors and Bioelectronics, 2007. 22(12): p. 2898-2905.
9.Sternberg, R., et al., Covalent enzyme coupling on cellulose acetate membranes for glucose sensor development. Analytical Chemistry, 1988. 60(24): p. 2781-2786.
10.Zhilei, W., et al., Synergistic contributions of fullerene, ferrocene, chitosan and ionic liquid towards improved performance for a glucose sensor. Biosensors and Bioelectronics, 2010. 25(6): p. 1434-1438.
11.Wang, C., 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.
12.Ming, L., X. Xi, and J. Liu, Electrochemically platinized carbon paste enzyme electrodes: A new design of amperometric glucose biosensors. Biotechnology Letters, 2006. 28(17): p. 1341-1345.
13.Wang, J., et al., Highly Selective and Sensitive Amperometric Biosensing of Glucose at Ruthenium-Dispersed Carbon Paste Enzyme Electrodes. Analytical Letters, 1993. 26(9): p. 1819 - 1830.
14.Wang, J., G. Rivas, and M. Chicharro, Iridium-Dispersed Carbon Paste Enzyme Electrodes. Electroanalysis, 1996. 8(5): p. 434-437.
15.Huang, J., et al., Electrospun Palladium Nanoparticle-Loaded Carbon Nanofibers and Their Electrocatalytic Activities towards Hydrogen Peroxide and NADH. Advanced Functional Materials, 2008. 18(3): p. 441-448.
16.Chaubey, A. and B.D. Malhotra, Mediated biosensors. Biosensors and Bioelectronics, 2002. 17(6-7): p. 441-456.
17.李坤易, 高感度葡萄糖生物感測器之研究 95.
18.D.H., A.W.J.a.C., Amperometric enzyme electrodes: theory and experiment. Biosensors:Fundaments and Applications: p. Chap 12 pp 180-210.
19.Ghindilis, A.L., P. Atanasov, and E. Wilkins, Enzyme-Catalyzed Direct Electron Transfer: Fundamentals and Analytical Applications. Electroanalysis, 1997. 9(9): p. 661-674.
20.He, F., Tang, Y., Yu, M., Wang, S., Li, Y., Zhu, D. , Fluorescence-amplifying detection of hydrogen peroxide with cationic conjugated polymers, and its application to glucose sensing.Advanced Functional Materials 2006. 16: p. 91-94.
21.Soichi Hanaoka, J.-M.L., Chemiluminescent flow sensor for H2O2 based on the decomposition of H2O2 catalyzed by cobalt(II)-ethanolamine complex immobilized on resin. Analytica Chimica Acta, 2011. 426: p. 57-64
22.Hrapovic, S., Liu, Y., Male, K.B., Luong, J.H.T. , Electrochemical Biosensing Platforms Using Platinum Nanoparticles and Carbon Nanotubes.Analytical Chemistry 2004. 76: p. 1083-1088
23.Miscoria, S.A., G.D. Barrera, and G.A. Rivas, Analytical Performance of a Glucose Biosensor Prepared by Immobilization of Glucose Oxidase and Different Metals into a Carbon Paste Electrode. Electroanalysis, 2002. 14(14): p. 981-987.
24.Lu, J., et al., Nanometal-Decorated Exfoliated Graphite Nanoplatelet Based Glucose Biosensors with High Sensitivity and Fast Response. ACS Nano, 2008. 2(9): p. 1825-1832.
25.Zou, Y., et al., Glucose biosensor based on electrodeposition of platinum nanoparticles onto carbon nanotubes and immobilizing enzyme with chitosan-SiO2 sol–gel. Biosensors and Bioelectronics, 2008. 23(7): p. 1010-1016.
26.Che, X., 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.
27.Immobilization of enzymes at high loadactivity by aqueous electrodeposition of enzyme-tethered chitosan for highly sensitive amperometric biosensing.
28.Zhou, Q., et al., Electrodeposition of carbon nanotubes-chitosan-glucose oxidase biosensing composite films triggered by reduction of p-benzoquinone or H2O2. CHEM BR, 2007. 111(38): p. 11276-11284.
29.黃炳照、莊睦賢, 電化學感測器. 化工技術, 1999. 第七卷(第二期).
30.Hage, D.S., Analytical Chemistry. 1999.
31.Castillo, J., et al., Biosensors for life quality: Design, development and applications. Sensors and Actuators B: Chemical, 2004. 102(2): p. 179-194.
32.D.Diamond, Chemical Analysis. Vol. 1.150. 1998, New York.
33.Mohanty, S.P. and E. Kougianos, Biosensors: a tutorial review. Potentials, IEEE, 2006. 25(2): p. 35-40.
34.Shen, J., Development & Characterization of thick-film printed electrochemical biosensor., in Department of Chemical Engineering. 2007, Case Western Reserve University: Cleveland,Ohio.
35.陳冠榮, 以奈米金修飾電極製備電流式免疫型感測器, in 化學工程研究所. 2008, 國立台灣科技大學.
36.田蔚城, 生物技術發展與應用. 1997: 九州圖書文物.
37.陳春吉, 自主性單層薄膜電極之阻抗分析與其在內毒素檢測上之應用, in 醫學工程研究所. 2002, 國立成功大學.
38.謝振傑, 光纖生物感測器. Vol. 廿八卷四期. 2006: 物理雙月刊.
39.許峰碩, 奈米碳黑在免疫層析檢測上的應用, in 工程研究所. 2002, 國立中興大學化學.
40.Spichiger-Keller, U.E., Chemical Sensors and Biosensors for Medical and Biological Applications. 1998: Wiley-VCH.
41.A.J.Bard, L.F., Electrochemical Methods Fundaments and applications. 2001, New York: Wiley.
42.汪玉銘, 定電流聚合導電性高分子法製備葡萄糖生物感測器之研究, in 化學工程學系碩博士班. 2003, 國立成功大學　
43.洪珮珍, 奈米結構葡萄糖感測器的製備與活性分析, in 化學工程系. 2004, 南台科技大學　
44.蘇宏基編著, 化學生物感測器. 東華大學化學系.
45.Yalcinkaya, F. and E.T. Powner, Intelligent structures. Sensor Review, 1996. 16(2): p. 32-37.
46.D'Orazio, P., Biosensors in clinical chemistry. Clinica Chimica Acta, 2003. 334(1-2): p. 41-69.
47.Heineman, P.T.K.W.R., Laboratory Techniques in Electroanalytical Chemistry. 2 ed. 1996, New York: Marcel Dekker.
48.Mehrvar, M. and M. Abdi, Recent developments, characteristics, and potential applications of electrochemical biosensors. Analytical Sciences, 2004. 20(8): p. 1113-1126.
49.劉英俊, 酵素工程. 1995: 中央圖書出版社.
50.林哲瑩, 高敏度表面與高解析技術診斷具疾病表現知單一核苷酸多
51.葉旻鑫, 均勻結構之複合式鉑銥奈米金屬觸媒/葡萄糖氧化酵素電極及其製備與應用.
52.Gamati, S., J.H.T. Luong, and A. Mulchandani, A microbial biosensor for trimethylamine using Pseudomonas aminovorans cells. Biosensors and Bioelectronics, 1991. 6(2): p. 125-131.
53.Jones, T.P. and M.D. Porter, Optical pH sensor based on the chemical modification of a porous polymer film. Analytical Chemistry, 1988. 60(5): p. 404-406.
54.Zhujun, Z. and W.R. Seitz, Optical sensor for oxygen based on immobilized hemoglobin. Analytical Chemistry, 1986. 58(1): p. 220-222.
55.B.D.Ratner, biomaterials science an introduction to materials in medicine academic press, 1996: p. 124-130.
56.Gill, I. and A. Ballesteros, Encapsulation of biologicals within silicate, siloxane, and hybrid sol- gel polymers: An efficient and generic approach. Journal of the American Chemical Society, 1998. 120(34): p. 8587-8598.
57.Cabral, J.M.S., Kennedy, J.F., Covalent and coordination immobilization of proteins. In: Taylor, R.F. (Ed.), Protein Immobilization; Fundamentals and Applications. Marcel Dekker. 1991, New York. 73–138.
58.Taylor, R.F., Development and applications of antibody- and receptor-based biosensors Bioinstrumentation: Research, Developments and Applications, ed D. L. Wise, Boston,MA;Butterworths.
59.洪爭坊、郭肇凱、張正英, 淺談酵素. Vol. 84. 2007: 台中區農情月刊.
60.呂鋒洲，林仁混, 基礎酵素學. 1991: 聯經出版社.
61.王世敏，許祖勛，傅晶, 奈米材料原理與製備. 2004: 五南出版社.
62.Yonezawa, Y., et al., Photochemical formation of colloidal silver: Peptizing action of acetone ketyl radical. Journal of the Chemical Society, Faraday Transactions, 1991. 87(12): p. 1905-1910.
63.Okitsu, K., et al., Sonochemical preparation of ultrafine palladium particles. Chemistry of Materials, 1996. 8(2): p. 315-317.
64.Antolini, E. and F. Cardellini, Formation of carbon supported PtRu alloys: An XRD analysis. Journal of Alloys and Compounds, 2001. 315(1-2): p. 118-122.
65.Takasu, Y., et al., Effect of structure of carbon-supported PtRu electrocatalysts on the electrochemical oxidation of methanol. Journal of the Electrochemical Society, 2000. 147(12): p. 4421-4427.
66.Watanabe, M., M. Uchida, and S. Motoo, Preparation of highly dispersed Pt + Ru alloy clusters and the activity for the electrooxidation of methanol. Journal of Electroanalytical Chemistry, 1987. 229(1-2): p. 395-406.
67.Hamnett, A., B.J. Kennedy, and F.E. Wagner, PtRu anodes for methanol electrooxidation: A ruthenium-99 Mo?ssbauer study. Journal of Catalysis, 1990. 124(1): p. 30-40.
68.Wang, X. and I.M. Hsing, Surfactant stabilized Pt and Pt alloy electrocatalyst for polymer electrolyte fuel cells. Electrochimica Acta, 2002. 47(18): p. 2981-2987.
69.Liu, Z., et al., Physical and Electrochemical Characterizations of Microwave-Assisted Polyol Preparation of Carbon-Supported PtRu Nanoparticles. Langmuir, 2004. 20(1): p. 181-187.
70.Zhang, X. and K.Y. Chan, Water-in-oil microemulsion synthesis of platinum-ruthenium nanoparticles, their characterization and electrocatalytic properties. Chemistry of Materials, 2003. 15(2): p. 451-459.
71.Nalwa, H.S., Academic press. 2000.
72.張義泉, 具界面活性擬樹枝狀聚乙烯亞胺之合成與製備燃料電池觸媒之應用, in 化學工程研究所. 2005, 國立成功大學.
73.Reetz, M.T., W. Helbig, and S.A. Quaiser, Electrochemical Preparation of Nanostructural Bimetallic Clusters. Chemistry of Materials, 1995. 7(12): p. 2227-2228.
74.Reetz, M.T. and S.A. Quaiser, A new method for the preparation of nanostructured metal clusters. Angewandte Chemie (International Edition in English), 1995. 34(20): p. 2240-2241.
75.Wang, J., et al., Highly selective membrane-free, mediator-free glucose biosensor. Analytical Chemistry, 1994. 66(21): p. 3600-3603.
76.Huang, J.S., et al., Electrospun palladium nanoparticle-loaded carbon nanofibers and their electrocatalytic activities towards hydrogen peroxide and NADH. Advanced Functional Materials, 2008. 18(3): p. 441-448.
77.Shimazu, K., D. Weisshaar, and T. Kuwana, Electrochemical dispersion of Pt microparticles on glassy carbon electrodes. Journal of Electroanalytical Chemistry, 1987. 223(1-2): p. 223-234.
78.S. Iijima, Helical microtubules of graphitic carbon. Nature (London), , 1991, . 354: p. 56-58.
79.D. S. Bethune, C.H.K., M. S. de Vries, G. Gorman, R. Savoy, J.vazquez, R. Beyers,, Nature, 1993. 363: p. 605-609.
80.A. C. Dillon, P.A.P., J. L. Alleman, J. D. Perkins and M. J. Heben, “Controlling single-wall nanotube diameters with variation in laser pulse power”. Chemical Physics Letters, 2000: p. 13-18.
81.Z. W. Pan, S.S.X., B. H. Chang, L. F. Sun, W. Y. Zhou and G. Wang, “Direct growth of aligned
open carbon nanotubes by chemical vapor deposition”,. ,Chemical Physics Letters, 1999. 97-102.
82.Tsang, S.C., et al., A simple chemical method of opening and filling carbon nanotubes. Nature, 1994. 372(6502): p. 159-162.
83.Lago, R.M.T., S. C.; Lu, K. L.; Chen, Y. K.; Green M. L. H. J. , chem .Soc ., chem. Commu, 1995: p. 1355.
84.Hyunmin Yi, ‡ Li-Qun Wu,† William E. Bentley,†,§ Reza Ghodssi,|,^ Gary W. Rubloff,‡,| and a.G.F.P. James N. Culver, †, Biofabrication with Chitosan. American Chemical Society, 2005.
85.Begin, A. and M.-R. Van Calsteren, Antimicrobial films produced from chitosan. International Journal of Biological Macromolecules, 1999. 26(1): p. 63-67.
86.al, Q.S.e., Direct electrochemistry of glucose oxidase immobilized on NdPO4 nanoparticles-chitosan composite film on glassy carbon electrodes and its biosensing application. bioelectrochemistry2009.
87.胡啟章, 電化學原理與方法. 2002: 五南圖書出版公司.
88.彭文權, 以沈積法製備甲醇燃料電池用之Pt-Ru雙金屬觸媒, in 化學工程學系. 1997, 元智大學.
89.R.Brian , E., Chemical Sensors and Biosensors. 2005.
90.J. B. Allen, R.F.L., JOHN WILEY ＆ SONS. INC, 2001.
91.Michael L. Bishop, E.P.F., and Larry Schoeff. Baltimore, MD, Clinical Chemistry: Principles, Procedures, Correlations. 2005: Lippincott Williams & Wilkins.
92.Z.Fan,a.D.H.,Permeability of glucose and other neutral species through through recast perfluorosulfonated ionomer films. Analytical-Chemistry.199
2. 64: p. 1304-1311.
93.Lim, S.H., et al., A glucose biosensor based on electrodeposition of palladium nanoparticles and glucose oxidase onto Nafion-solubilized carbon nanotube electrode. Biosensors and Bioelectronics, 2005. 20(11): p. 2341-2346.
94.Tsai, Y.C., S.C. Li, and J.M. Chen, Cast thin film biosensor design based on a nafion backbone, a multiwalled carbon nanotube conduit, and a glucose oxidase function. Langmuir, 2005. 21(8): p. 3653-3658.
95.Chu, X., et al., A new amperometric glucose biosensor based on platinum nanoparticles/polymerized ionic liquid-carbon nanotubes nanocomposites. Electrochimica Acta, 2010. 55(8): p. 2848-2852.
96.Zou,Y., Glucose biosensor based on electrodeposition of platinum nanoparticles onto carbon nanotubes and immobilizing enzyme with chitosan-SiO2 sol-gel. Biosensors and Bioelectronics, 2008. 23(7): p. 1010-1016.
97.Zhou, H., Glucose biosensor based on platinum microparticles dispersed in nano-fibrous polyaniline. Biosensors and Bioelectronics, 2005. 20(7): p. 1305-1311.
98.Zeng,Q.,Palladiumnanoparticle/chitosan-grafted graphene nanocomposites for construction of a glucose biosensor. Biosensors and Bioelectronics, 2011. 26(8): p. 3456-3463.
99.Yang,H.andY.Zhu, Glucose biosensor based on nano-SiO2 and “unprotected” Pt nanoclusters. Biosensors and Bioelectronics, 2007. 22(12): p. 2989-2993.
100.He, J.-L.,β-Cyclodextrin incorporated carbon nanotube-modified electrode as an electrochemical sensor for rutin. Sensors and Actuators B: Chemical, 2006. 114(1): p. 94-100.