本研究利用半導體微機械加工製程技術製備植入型微電極陣列感測探針，其中包含光罩設計與半導體加工製程兩大重點方向，在光罩設計方面，我們以繪圖軟體Auto CAD設計繪製光罩上元件圖樣，本製程包含三道光罩，分別用以電極金屬沉積、絕緣層蝕刻及探針形狀定義，每片4吋晶圓上共設有164隻探針，其中有(1)四極式、(2)四+二長極式和(3)六極式共3種探針設計，分別占有53隻、74隻與37隻，每根探針由電極端(electrode sites)、導線端(channels)、封裝端(bonding pads)組成，四+二長極式與六極式微電極陣列探針為同一尺寸，全長18 mm (針軸部長11 mm)、寬度3 mm、探針尖端寬度150 μm、厚度200 μm，電極大小為200 μm × 50 μm；而四極式全長13 mm (針軸部長9 mm)、寬度3 mm，探針尖端寬132 μm，電極大小為140 μm × 30 μm，尺寸較小。可根據未來的應用選擇所需之探針；在製程方面，我們於微影、擴散、蝕刻、薄膜等加工製程中，將製程參數進行最佳化試驗，製作出精確、高產量、低成本的微型電極陣列探針，良率高達95%。不同於傳統的三極式電化學感測系統，我們所設計的探針具有多效合一的概念，以本研究為例，將銀/氯化銀(Ag/AgCl)沉積於探針上所設置的長型微電極後，可形成自身參考電極，再選擇另一長型微電極作為輔助電極，而探針上的微工作電極陣列於修飾後則可用以感測不同待測分析物質，形成工作電極、輔助電極、參考電極三極合一的生物感測器，可應用於植入型生物感測以及大幅提升其使用上與商品化的可行性及便利性。
另一方面，為驗證所製備之微電極陣列探針的感測效能，我們將經過封裝、表面修飾與酵素塗佈後製備而成穀氨酸感測探針，進行一系列的效能測試，包含靈敏度(190±7.5 nA·μM-1·cm-2)、感測極限(1.15±0.01 μM)、感測範圍(20-500 μM)、響應時間(3±2 s)，以及干擾物測試，驗證結果顯示所製備的微電極陣列探針具有良好的感測能力。未來可於所製備的探針上進行不同酵素的修飾，以應用在不同研究領域進行分析量測，包含植入型的動物實驗。

In this research, we used the semiconductor manufacturing technique to fabricate implantable multi-electrode array microprobes and AutoCAD software to design masks. The process to prepare microprobes was divided into three parts, including formation of the specific pattern of platinum metal layer on the silicon wafer, etching of the specific insulation layer and defining the outline of microprobes. We have three kinds of microprobes designed on a 4-inch wafer, including 53 probes with 4 microelectrodes, 74 probes with 4 microelectrodes and 2 long electrodes, 37 probes with 6 microelectrodes. Each probe consists of electrode sites, channels, bonding pads. Additionally, the 4-electrode microprobes were smaller than the others with full length 13 mm, width 3 mm, probe tip width 132 μm, and platinum area size 140 μm × 30 μm; while the others were of the same size with full length 18 mm, width 3 mm, thickness 200 μm, probe tip width 150 μm, and platinum area size 200 μm × 50 μm. The appropriate pattern can be selected according to future applications.
Moreover, we optimized parameters in each processing step (i.e. thermal oxidation, photolithography, thin film deposition, and etching) to miniaturize the size, reduce cost, and improve the production rate of multi-electrode array microprobes. Different from the traditional three-electrode system, we designed our microprobes as all-in-one biosensor probes. For example, silver/silver chloride can be deposited onto one of electrode sites to make a self-reference electrode, while others are responsible for the counter and working electrode. An all-in-one biosensor can be applied in implantable biological sensing and greatly enhance its applications, feasibility, and convenience.
In the second part of this thesis, sensors abilities were tested. In detail, we modified the electrode surface with permselective polymer layers and glutamate oxidase layers to construct glutamate biosensors. For this study, our glutamate sensors have fast response time 3±2 s, wider linear detection range 20-500 μM, low detection limit 1.15±0.01 μM, and high sensitivity 190±7.5 nA·μM-1·cm-2. We have proposed a multi-electrode array glutmate biosensor probe with good sensing ability. The proposed all-in-one glutmate sensor microprobes could be applied for different kinds in vivo experiment in live rodents in the future.

1. Clark Jr L. C., Lyons C., “Electrode systems for continuous monitoring in cardiovascular surgery”, Annals of the New York Academy of Sciences, Vol. 102, pp. 29-45 (1962).
2. J. Castillo., Gáspár. S., S. Leth., M. Niculescu., A. Mortari., I. Bontidean., V. Soukharev., S.A. Dorneanu., A.D. Ryabov., E. Csöregi., “Biosensors for life quality： Design, development and applications”, Sensors and Actuators B： Chemical, Vol. 102, No. 2, pp.179-194 (2004).
3. Walter H Brattain, Bardeen John,“Three-electrode circuit element utilizing semiconductive materials”, Bell Telphone Lab Inc., U.S. Pat.,2524035 (1950).
4. N.C. Danbolt, “Glutamate uptake”, Progress in Neurobiology, Vol. 65, pp.1-105 (2001).
5. R.J. Weinberg, “Glutamate： an excitatory neurotransmitter in the mammalian CNS”, Brain Research Bulletin, Vol. 50, pp. 353-354 (1999).
6. M. Fillenz, “Physiological release of excitatory amino acids”, Behav. Brain Res., Vol. 71, pp. 51-67 (1995).
7. T.P. Obrenovitch, J. Urenjak ,“ALTERED GLUTAMATERGIC TRANSMISSION IN NEUROLOGICAL DISORDERS： FROM HIGH EXTRACELLULAR GLUTAMATE TO EXCESSIVE SYNAPTIC EFFICACY”, Progress in Neurobiology, Vol. 51, pp. 39-87 (1997).
8. P.J. Conn, J.P. Pin, “Pharmacology and functions of metabotropic glutamate receptors”, Annu Rev Pharmacol Toxicol, Vol. 37, pp. 205-237 (1997).
9. J.W. Olney, N.B. Farber, “Glutamate receptor dysfunction and schizophrenia”, Arch. Gen. Psychiatry, Vol. 52, pp. 998-1007 (1995).
10. J. Smythies, “Redox mechanisms at the glutamate synapse and their significance： a review”, Eur J Pharmacol., Vol. 370, pp. 1-7 (1999).
11. Sitges et al., “Effects of carbamazepine, phenytoin, lamotrigine, oxcarbazepine, to piramate and vinpocetine on Na+ channel-mediated release of [H] glutamate in hippocampal nerve endings”, Neuropharmacology, Vol. 52, pp. 598-605 (2007).
12. G. Szénási, J.L.G. Hársing, “Pharmacology and prospective therapeutic usefulness of negative allosteric modulators of AMPA receptors”, Drug Discovery Today： Therapeutic Strategies, Vol. 521, pp. 69-76 (2004).
13. Ghobadi S, Csöregi E, Marko-Varga G, “Bienzyme carbon paste electrodes for L-glutamate determination”, Current Separations, Vol. 14, pp. 94-102 (1996).
14. E. Valero, F. Garcia-Carmona, “A Continuous Spectrophotometric Method Based on Enzymatic Cycling for Determining L-Glutamate”, ANALYTICAL BIOCHEMISTRY, Vol. 259, pp. 265-271 (1998).
15. J. Chapman, M. Zhou, “Microplate-based fluorometric methods for the enzymatic determination of L-glutamate： application in measuring l-glutamate in food samples”, Analytica Chimica Acta, Vol. 402, pp. 47-52 (1999).
16. T.P. Piepponen, A. Skujins, “Rapid and sensitive step gradient assays of glutamate, glycine, taurine and g-aminobutyric acid by high-performance liquid chromatography–fluorescence detection with o-phthalaldehyde–mercaptoethanol derivatization with an emphasis on microdialysis”, Journal of Chromatography B, Vol. 757, pp. 277-283 (2001).
17. A. Tivesten, A. Lundqvist, S. Folestad, “Selective chiral determination of aspartic and glutamic acid in biological samples by capillary electrophoresis”, Chromatographia, Vol. 44, pp. 623-633 (1997).
18. Iniouchine, M.Y., et al., “Blockers of monoamine transporters influence high dopamine concentration uptake in rat brain slices”, Doklady Biological Sciences, Vol. 419, No. 1, pp. 80-82 (2008).
19. Wei WJ, Song YL, Shi WT et al., “A high sensitivity MEA probe for measuring real time rat brain glucose flux”, Biosensors and Bioelectronics, Vol. 55, pp. 66-71(2014).
20. M.R. Ryan, J.P. Lowry, R.D. O‘Neill, “Biosensor for neurotransmitter l-glutamic acid designed for efficient use of l-glutamate oxidase and effective rejection of interference, Analyst, Vol. 122, pp. 1419-1424 (1997).
21. Å. Hallström, A. Carlsson, L. Hillered, U. Uncerstedt, “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, Vol. 22, No. 2, pp.113-124 (1989).
22. C. Debiemme-Chouvy, “A Very Thin Overoxidized Polypyrrole Membrane as Coating for Fast Time Response and Selective H2o2 Amperometric Sensor”, Biosensors & Bioelectronics, Vol. 25, pp. 2454-57 (2010).
23. S. T. Pan, and M. A. Arnold, “Selectivity Enhancement for Glutamate with a Nafion/Glutamate Oxidase Biosensor”, Talanta, Vol. 43, pp. 1157-62 (1996).
24. R. D. O'Neill, S. C. Chang, J. P. Lowry et al, “Comparisons of Platinum, Gold, Palladium and Glassy Carbon as Electrode Materials in the Design of Biosensors for Glutamate”, Biosensors & Bioelectronics, Vol. 19, pp. 1521-28 (2004).
25. M. Ammam, and J. Fransaer, “Hghly Sensitive and Selective Glutamate Microbiosensor Based on Cast Polyurethane/Ac-Electrophoresis Deposited M ultiwalled Carbon Nanotubes and Then Glutamate Oxidase/Electrosynthesized Polypyrrole/Pt Electrode”, Biosensors & Bioelectronics, Vol. 25, pp. 1597-602 (2010).
26. S. Govindarajan, C. J. McNeil, J. P. Lowry, C. P. McMahon, R. D. O'Neill ,“Highly selective and stable microdisc biosensors for l-glutamate monitoring”, Sensors and Actuators B： Chemical, Vol. 178, pp. 606-614 (2013).
27. M. Zhang, C. Mullens, W. Gorski, “Amperometric glutamate biosensor based on chitosan enzyme film”, Electrochimica Acta, Vol. 51, No. 21, pp. 4528-4532(2006).
28. O. V. Soldatkina, O. O. Soldatkin, B. Ozansoy Kasap et al., “A Novel Amperometric Glutamate Biosensor Based on Glutamate Oxidase Adsorbed on Silicalite”, Nanoscale Research Letters, Vol. 12, No. 1, pp. 2026-8 (2017).
29. M. Bäcker, L. Delle, A. Poghossian, M.Biselli, W. Zang et al., “Electrochemical sensor array for bioprocess monitoring”, Electrochimica Acta, Vol. 56, No. 26, pp. 9673-9678 (2011).
30. V. M.Tolosa, K. M. Wassum, N. T. Maidment et al., “Electrochemically deposited iridium oxide reference electrode integrated with an electroenzymatic glutamate sensor on a multi-electrode arraymicroprobe”, Biosensors and Bioelectronics, Vol. 42, pp. 256-260 (2013).
31. O. Frey, T. Holtzman, R. M. McNamara et al., “Enzyme-based choline and l-glutamate biosensor electrodes on silicon microprobe arrays”, Biosensors and Bioelectronics, Vol. 26, No. 2, pp. 477-484 (2010).
32. Wenjing Wei, Yilin Song, Li Wang et al., “An implantable microelectrode array for simultaneous L-glutamate and electrophysiological recordings in vivo”, Microsystems & Nanoengineering,15002 (2015).
33. Eric Walker, Jianjun Wang, Naser Hamdi et al., “Selective detection of extracellular glutamate in brain tissue using microelectrode arrays coated with over-oxidized polypyrrole”, The Analyst, Vol. 132, pp. 1107-1111 (2007).
34. Michael W. Varney, Dean M. Aslam, Abed Janoudi et al., “Polycrystalline-Diamond MEMS Biosensors Including Neural Microelectrode-Arrays”, Biosensors (Basel), Vol. 1, No. 3, pp. 118-133 (2011).
35. Matthew D.Johnson, Robert K.Franklin, Matthew D.Gibson et al., “Implantable microelectrode arrays for simultaneous electrophysiological and neurochemical recordings”, Journal of Neuroscience Methods, Vol. 174, No. 1, pp. 62-70 (2008).
36. KarlSchügerl, “Progress in monitoring, modeling and control of bioprocesses during the last 20 years”, Journal of Biotechnology, Vol. 85, No. 2, pp. 149-173 (2001).
37. Huicheng Yu, Zhenzhen Ma, Zhaoyang Wu, “Immobilization of Ni–Pd/core–shell nanoparticles through thermal polymerization of acrylamide on glassy carbon electrode for highly stable and sensitive glutamate detection”, Analytica Chimica Acta, Vol. 896, pp. 137-142 (2015).
38. Qi Zeng, Kai Xia, Bin Sun et al., “Electrodeposited Iridium Oxide on Platinum Nanocones for Improving Neural Stimulation Microelectrodes”, Electrochimica Acta, Vol. 237, pp. 152-159 (2017).
39. D. Carelli, D. Centonze, A. De Giglio, M. Quinto, and P. G. Zambonin, “An Interference-Free First Generation Alcohol Biosensor Based on a Gold Electrode Modified by an Overoxidised Non-Conducting Polypyrrole Film”, Analytica Chimica Acta, Vol. 565, pp. 27-35 (2006).
40. M. Q. Liu, J. H. Jiang, Y. L. Feng et al., “Glucose Biosensor Based on Immobilization of Glucose Oxidase in Electrochemically Polymerized Polytyramine Film and Overoxidised Polypyrrole Film on Platinized Carbon Paste Electrode”, Chinese Journal of Analytical Chemistry, Vol. 35 , pp. 1435-38 (2007).
41. N. Wahono, S. Qin, P. Oomen et al., “Evaluation of Permselective Membranes for Optimization of Intracerebral Amperometric Glutamate Biosensors”, Biosensors & Bioelectronics, Vol. 33, pp. 260-66 (2012).
42. Xiao, H. 著，羅正忠、張鼎張 譯，台灣培生教育出版：學銘圖書發行，台北，半導體製程技術概論(Introduction to Semiconductor Manufacturing Technology)， (2002)。
43. 張鴻彥，「利用微電極陣列感測探針探討痛覺刺激與穀氨酸於脊隨丘腦逕之神經傳導的關聯」，碩士論文，國立台灣科技大學，台北(2015)。
44. 盧偉凡，「微機械加工技術製備微電極陣列探針與測量分析物絕對濃度的方法」，碩士論文，國立台灣科技大學，台北(2014)。
45. 余政霖，「應用於生物感測器之植入型微電極陣列探針製備與微影光罩改良」，碩士論文，國立台灣科技大學，台北(2014)。
46. 詹文進，「選擇性酵素固定法製備電流式微電極陣列穀氨酸感測器」，碩士論文，國立台灣科技大學，台北(2013)。