本研究利用電沉積之幾丁聚醣作為酵素固定之基材，使穀氨酸氧化酵素可選擇性地固定於陣列微電極表面，用以製備可植入式穀氨酸感測器探針。研究中使用兩種不同的酵素固定方式製備穀氨酸感測器並比較其效能，方法一：以幾丁聚醣選擇性沉積法固定酵素所製備之穀氨酸感測器(利用電化學方法將幾丁聚醣選擇性沉積在目標微電極上，用以吸附固定穀氨酸氧化酵素)；方法二：以人工塗覆法固定酵素所製備之穀氨酸感測器(利用戊二醛交聯劑和牛血清蛋白穩定劑與穀氨酸氧化酵素進行交聯，在顯微鏡下以人工徒手將酵素塗覆固定在目標微電極上)；以此二種酵素固定法製備感測器時，皆利用過氧化聚吡咯與全氟磺酸這兩種滲透選擇性高分子薄膜作為感測器的抗干擾層。找出幾丁聚醣電沉積之最適化條件參數後，以方法一製備出的穀氨酸感測器其線性範圍為20-217 μM，響應時間約1秒，靈敏度為38.1 ± 5.4 nA/μM∙cm2 (n = 9)，感測極限為2.5 ± 1.2 μM，儲存穩定性為70% (存放9天後其靈敏度與其初始靈敏度比值之百分比)；由方法二所製備出的穀氨酸感測器其線性範圍為20-352 μM，響應時間約2-3秒，靈敏度為95.0 ± 8.8 nA/μM∙cm2 (n = 12)，感測極限為6.5 ± 1.7 μM，儲存穩定性為50% (存放9天後其靈敏度與其初始靈敏度比值之百分比)。以此二種方法所製備出的穀氨酸感測器皆能有效阻擋多數具電化學活性之干擾物(如: 抗壞血酸與多巴胺)。由方法一所製備出的穀氨酸感測器，擁有較佳的儲存穩定性，且此感測器在連續重複操作下其靈敏度標準差相較之下非常小，可說明此感測器有較好的感測結果再現性，且在系統中加入穀氨酸後，陣列中鄰近的對照感測器(其電極表面沒有沉積幾丁聚醣)並沒有產生任何穀氨酸的感測電流，成功證明在緊密排列的微電極陣列中，穀氨酸氧化酵素能有效地選擇性固定在微電極表面上，此製備方法簡單、穩定、快速又經濟，有潛力成為量產此感測器之建議方法；而由方法二製備出的穀氨酸感測器，則有較高的感測靈敏度及線性範圍，其較長的響應時間可能由於其較厚的酵素層所導致。進行白鼠動物實驗時，將由方法二所製備出的穀氨酸感測器植入白鼠大腦視丘中，用以瞬時偵測由痛覺刺激所產生的穀氨酸釋放。

In this study, the electrodeposited chitosan is used as the substrate for the enzyme adsorption for the selective immobilization of glutamate oxidase on the arrayed microelectrode surface in order to prepare implantable glutamate sensor probe. In the thesis, we compare figures of merit of glutamate sensors on the micromachined probes prepared by two different enzyme immobilization methods – adsorption with electrodeposited chitosan (Method 1) and crosslinking with glutaraldehyde crosslinker and the stabilizing agent bovine serum albumin (Method 2). The permselective films overoxidized polypyrrole and Nafion® were used for rejecting common interferents. Glutamate sensors prepared by Method 1 have response time ~1 sec, linear detection range 20-217 μM, and sensitivity 38.1 ± 5.4 nA/μM• cm2 (n = 9), detection limit 2.5 ± 1.2 μM, and storage stability that the sensor remains 70% of its initial activity after 9 days of storage and that prepared by Method 2 have response time 2~3 sec, linear detection range 20-352 μM, and sensitivity 95.0 ± 8.8 nA/μM• cm2 (n = 12), detection limit 6.5 ± 1.7 μM, and storage stability that the sensor remains 50% of its initial activity after 9 days of storage. Sensors prepared by both methods can reject common interferents (ascorbic acid and dopamine) very effectively; little interferent current was observed. In general, glutamate sensors prepared by Method 1 have higher storage stability and their performance is more reproducible due to smaller standard deviations of sensor sensitivities after continuous repeating operations; on the other hand, closely arrayed control sensors without the chitosan film showed no signal upon the addition of glutamate. Successful selective glutamate oxidase immobilization on closely packed microelectrodes is demonstrated. Glutamate sensors prepared by Method 2 have higher sensor sensitivity and selectivity; however, its slower response time may due to the thicker enzyme layer prepared by Method 2. This glutamate sensor (prepared by Method 2) was implanted into the rat’s brain for real-time monitoring the release of glutamate after giving the pain stimulation (preliminary data will be shown).

1. McEntee, W.J. and T.H. Crook, Glutamate: its role in learning, memory, and the aging brain. Psychopharmacology (Berl), 1993. 111(4): p. 391-401.
2. Cosnier, S., et al., An electrochemical method for making enzyme microsensors. Application to the detection of dopamine and glutamate. Anal Chem, 1997. 69(5): p. 968-71.
3. Cooper, J.M. and D.J. Pritchard, Biomolecular sensors for neurotransmitter determination: electrochemical immobilization of glutamate oxidase at microelectrodes in a poly(o-phenylenediamine) film. Journal of Materials Science: Materials in Electronics, 1994. 5(2): p. 111-116.
4. Ryan, M.R., J.P. Lowry, and R.D. O'Neill, Biosensor for neurotransmitter L-glutamic acid designed for efficient use of L-glutamate oxidase and effective rejection of interference. Analyst, 1997. 122(11): p. 1419-24.
5. Kulagina, N.V., L. Shankar, and A.C. Michael, Monitoring glutamate and ascorbate in the extracellular space of brain tissue with electrochemical microsensors. Anal Chem, 1999. 71(22): p. 5093-100.
6. GLUTAMATE BIOSENSOR. 2013; Available from: http://www.pinnaclet.com/glutamate.html.
7. Wassum, K., et al., Silicon Wafer-Based Platinum Microelectrode Array Biosensor for Near Real-Time Measurement of Glutamate in Vivo. Sensors, 2008. 8(8): p. 5023-5036.
8. Tseng, T.T.C. and H.G. Monbouquette, Implantable microprobe with arrayed microsensors for combined amperometric monitoring of the neurotransmitters, glutamate and dopamine. Journal of Electroanalytical Chemistry, 2012. 682(0): p. 141-146.
9. Castillo, J., et al., Biosensors for life quality - Design, development and applications. Sensors and Actuators B-Chemical, 2004. 102(2): p. 179-194.
10. Cunningham, A.J., Introduction to Bioanalytical Sensors. 1998, New York: John Wiley & Sons, INC.
11. Li, W., et al., A MEMS Thermal Biosensor for Metabolic Monitoring Applications. Microelectromechanical Systems, Journal of, 2008. 17(2): p. 318-327.
12. Hao, R.Z., et al., DNA probe functionalized QCM biosensor based on gold nanoparticle amplification for Bacillus anthracis detection. Biosens Bioelectron, 2011. 26(8): p. 3398-404.
13. Spichiger-Keller, U.E., Frontmatter, in Chemical Sensors and Biosensors for Medical and Biological Applications. 2007, Wiley-VCH Verlag GmbH. p. I-XII.
14. F. Yalcinkaya, E.T.P., Intelligent structures. Sensor Review, 1996. 16 (2): p. 32 - 37.
15. Allen J. Bard, L.R.F., Electrochemical Methods: Fundamentals and Applications. 2001, New York: Wiley.
16. D.), J.S.P., Development and Characterization of Thick-film Printed Electrochemical Biosensors. 2007: Case Western Reserve University.
17. Spichiger-Keller, U.E., Chemical Sensors and Biosensors for Medical and Biological Applications. 1998: Wiley-VCH.
18. Grieshaber, D., et al., Electrochemical Biosensors - Sensor Principles and Architectures. Sensors, 2008. 8(3): p. 1400-1458.
19. 田蔚城, 生物技術的發展與應用. 1998, 台灣: 九州.
20. Peter Kissinger, W.R.H., Laboratory Techniques in Electroanalytical Chemistry. 2 ed. 1996, New York: Marcel Dekker.
21. Mehrvar, M. and M. Abdi, Recent developments, characteristics, and potential applications of electrochemical biosensors. Anal Sci, 2004. 20(8): p. 1113-26.
22. Stulik, K., et al., Microelectrodes. Definitions, characterization, and applications (Technical Report). Pure and Applied Chemistry, 2000. 72(8): p. 1483-1492.
23. Dzyadevych, S.V., et al., Amperometric enzyme biosensors: Past, present and future. Irbm, 2008. 29(2-3): p. 171-180.
24. Clark Jr LC, L.C., Electrode systems for continuous monitoring in cardiovascular surgery. Annals of the New York Academy of Sciences. 1962.
25. Guilbault, G.G. and G.J. Lubrano, An enzyme electrode for the amperometric determination of glucose. Analytica Chimica Acta, 1973. 64(3): p. 439-455.
26. 胡啟章, 電化學原理與方法. 2002: 五南圖書.
27. 伏安法分析. Available from: http://222.91.187.3/jpkhx/jiaoan5.htm.
28. Carlsson, A., The occurrence, distribution and physiological role of catecholamines in the nervous system. Pharmacol Rev, 1959. 11(2, Part 2): p. 490-3.
29. Chaurasia, C.S., et al., AAPS-FDA workshop white paper: microdialysis principles, application and regulatory perspectives. Pharm Res, 2007. 24(5): p. 1014-25.
30. Chefer, V.I., et al., Overview of brain microdialysis. Curr Protoc Neurosci, 2009. Chapter 7: p. Unit7 1.
31. 張峻豪, 應用微透析技術探討未結合態咖啡因在大白鼠之藥物動力學及其與吳茱萸之交互作用, in 藥理學研究所2004, 國立陽明大學: 台北市. p. 120.
32. Burmeister, J.J., et al., Improved ceramic-based multisite microelectrode for rapid measurements of L-glutamate in the CNS. J Neurosci Methods, 2002. 119(2): p. 163-71.
33. Pomerleau, F., et al., Real time in vivo measures of L-glutamate in the rat central nervous system using ceramic-based multisite microelectrode arrays. Ann N Y Acad Sci, 2003. 1003: p. 454-7.
34. Walker, E., et al., Selective detection of extracellular glutamate in brain tissue using microelectrode arrays coated with over-oxidized polypyrrole. Analyst, 2007. 132(11): p. 1107-1111.
35. Wassum, K.M., et al., Transient extracellular glutamate events in the basolateral amygdala track reward-seeking actions. J Neurosci, 2012. 32(8): p. 2734-46.
36. Qin, S., et al., Microsensors for in vivo Measurement of Glutamate in Brain Tissue. Sensors, 2008. 8(11): p. 6860-6884.
37. P. Lowry, J., M. R. Ryan, and R. D. O'Neill, Behaviourally induced changes in extracellular levels of brain glutamate monitored at 1 s resolution with an implanted biosensor. Analytical Communications, 1998. 35(3): p. 87-89.
38. Hamdi, N., et al., An electroenzymatic l-glutamate microbiosensor selective against dopamine. Journal of Electroanalytical Chemistry, 2006. 591(1): p. 33-40.
39. Espey, M.G., et al., Extracellular glutamate levels are chronically elevated in the brains of LP-BM5-infected mice: a mechanism of retrovirus-induced encephalopathy. J Neurochem, 1998. 71(5): p. 2079-87.
40. Spector, R., Vitamin homeostasis in the central nervous system. N Engl J Med, 1977. 296(24): p. 1393-8.
41. Hallström, Å., et al., Simultaneous determination of lactate, pyruvate, and ascorbate in microdialysis samples from rat brain, blood, fat, and muscle using high-performance liquid chromatography. Journal of Pharmacological Methods, 1989. 22(2): p. 113-124.
42. Iniouchine, M.Y., et al., Blockers of monoamine transporters influence high dopamine concentration uptake in rat brain slices. Doklady Biological Sciences, 2008. 419(1): p. 80-82.
43. Chung, Y.C., et al., Effect of abiotic factors on the antibacterial activity of chitosan against waterborne pathogens. Bioresour Technol, 2003. 88(3): p. 179-84.
44. Yi, H., et al., Biofabrication with chitosan. Biomacromolecules, 2005. 6(6): p. 2881-94.
45. Kaminski, K., et al., pH-sensitive genipin-cross-linked chitosan microspheres for heparin removal. Biomacromolecules, 2008. 9(11): p. 3127-32.
46. Baxter, A., et al., Improved method for i.r. determination of the degree of N-acetylation of chitosan. Int J Biol Macromol, 1992. 14(3): p. 166-9.
47. Sabnis, S. and L. Block, Improved infrared spectroscopic method for the analysis of degree of N-deacetylation of chitosan. Polymer Bulletin, 1997. 39(1): p. 67-71.
48. 劉英俊, 酵素工程. 1987: 中央圖書出版社.
49. Rusmini, F., Z. Zhong, and J. Feijen, Protein Immobilization Strategies for Protein Biochips. Biomacromolecules, 2007. 8(6): p. 1775-1789.
50. Cretich, M., et al., Protein and peptide arrays: recent trends and new directions. Biomol Eng, 2006. 23(2-3): p. 77-88.
51. 翟祐暄, 以二十四面奈米金屬觸媒修飾製備電流式雙氧水感測器與其應用, in 化學工程系2012, 國立臺灣科技大學: 台北市. p. 131.
52. Cabral, J.M. and J.F. Kennedy, Covalent and coordination immobilization of proteins. Bioprocess Technol, 1991. 14: p. 73-138.
53. Tominaga, J., et al., An enzymatic strategy for site-specific immobilization of functional proteins using microbial transglutaminase. Enzyme and Microbial Technology, 2004. 35(6–7): p. 613-618.
54. 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.
55. Zhujun, Z. and W.R. Seitz, Optical sensor for oxygen based on immobilized hemoglobin. Analytical Chemistry, 1986. 58(1): p. 220-222.
56. Gamati, S., J.H. Luong, and A. Mulchandani, A microbial biosensor for trimethylamine using Pseudomonas aminovorans cells. Biosens Bioelectron, 1991. 6(2): p. 125-31.
57. Arenkov, P., et al., Protein Microchips: Use for Immunoassay and Enzymatic Reactions. Analytical Biochemistry, 2000. 278(2): p. 123-131.
58. 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.
59. Bohmer, A., et al., A novel L-glutamate oxidase from Streptomyces endus. Purification and properties. Eur J Biochem, 1989. 182(2): p. 327-32.
60. Chuah, L.H., et al., Curcumin-containing chitosan nanoparticles as a potential mucoadhesive delivery system to the colon. Pharm Dev Technol, 2013. 18(3): p. 591-9.
61. O'Neill, R.D., et al., Comparisons of platinum, gold, palladium and glassy carbon as electrode materials in the design of biosensors for glutamate. Biosens Bioelectron, 2004. 19(11): p. 1521-8.
62. Chen, X., et al., Multilayer assembly of positively charged polyelectrolyte and negatively charged glucose oxidase on a 3D Nafion network for detecting glucose. Biosensors and Bioelectronics, 2007. 22(12): p. 3256-3260.
63. 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.
64. 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.
65. Malitesta, C., et al., Glucose fast-response amperometric sensor based on glucose oxidase immobilized in an electropolymerized poly(o-phenylenediamine) film. Analytical Chemistry, 1990. 62(24): p. 2735-2740.
66. Eremenko, A., et al., Biosensor based on an enzyme modified electrode for highly-sensitive measurement of polyphenols. Biosensors and Bioelectronics, 1995. 10(8): p. 717-722.
67. Sternberg, R., et al., Covalent enzyme coupling on cellulose acetate membranes for glucose sensor development. Analytical Chemistry, 1988. 60(24): p. 2781-2786.
68. Debiemme-Chouvy, C., A very thin overoxidized polypyrrole membrane as coating for fast time response and selective H2O2 amperometric sensor. Biosensors and Bioelectronics, 2010. 25(11): p. 2454-2457.
69. Tseng, T.-C., Bioanalytical Techniques: Homogeneous Immunoassays for Estradiol & Arrayed Microsensor for Neurotransmitters. 2011, University of California.