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研究生:林雅琴
研究生(外文):Lin, Ya-Chin
論文名稱:魚類皮質醇(壓力荷爾蒙)電化學感測裝置之開發與應用
論文名稱(外文):Development of a cortisol (stress hormone) electrochemical biosensor for a living fish
指導教授:黃士豪黃士豪引用關係
指導教授(外文):Huang, Shih-Hao
口試委員:莊昀儒吳志偉
口試委員(外文):Chuang, Yun-JuWu, Chih-Wei
口試日期:2019-01-10
學位類別:碩士
校院名稱:國立臺灣海洋大學
系所名稱:機械與機電工程學系
學門:工程學門
學類:機械工程學類
論文種類:學術論文
論文出版年:2019
畢業學年度:107
語文別:中文
論文頁數:69
中文關鍵詞:吳郭魚皮質醇植入式皮質醇電化學感測裝置免標定式電化學阻抗免疫感測晶片11β -羥基類固醇脫氫酶氧化石墨烯葡萄糖氧化酶安培法
外文關鍵詞:TilapiaCortisolImplantable cortisol electrochemical sensing deviceLabel-free electrochemical impedance immunosensing chip11β -hydroxysteroid dehydrogenasegrephene oxideglucose oxidaseAmperometry
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吳郭魚為台灣養殖漁經濟魚種,但每年受病毒及寒害重創造成魚群死亡率高,除了血糖、乳酸與膽固醇外,壓力賀爾蒙-皮質(Cortisol)亦是生理異常的重要指標。正常狀況下魚類皮質醇濃度40~200ng/ml,當分泌過多時易造成死亡,因此監控魚的皮質醇濃度尤其重要。本研究提出兩種生醫感測裝置以監測皮質醇,分別為1.植入式皮質醇電化學感測裝置:可即時量測、但靈敏度低。2.免標定式電化學阻抗免疫感測晶片:以免疫法進行鍵結,具高專一性,但須抽取血液量測。
植入式皮質醇電化學感測裝置:於工作電極塗佈11β-羥基類固醇脫氫酶(11β-Hydroxysteroid dehydrogenase,11β-HSD1),催化皮質醇,另塗佈黃遞激素(Diaphorase,DP)催化NADP+ 、加入維生素K3 (Vitamin K3¸VK3 作為電子媒介物、添加琴黑(Ketjenblack¸KB)提升電子傳導率。將皮質醇標準液進行校正,靈敏度為8.4μA/μM。採用三種類固醇賀爾蒙進行專一性測試,分別為皮質素(cortisone)、雌激素(11β-estradiol,E2)、排卵素(17α,20β-dihydroxy-4-pregnen-3-one,DHP),其中只有皮質醇有顯著變化。將植入式皮質醇電化學感測裝置植入魚體進行量測,於27℃水溫下量測出皮質醇濃度為0.357μM,15℃ 濃度為2.176μM,濃度上升6倍,回溫至27℃下降為1.434μM。
免標定式電化學阻抗免疫感測晶片:利用還原氧化石墨烯沉積於工作電極提升導電率,並將抗體共價鍵結後塗佈葡萄糖氧化酶,利用氧化酶催化葡萄糖訊號,間接量測皮質醇濃度。當皮質醇抗原與抗體共價鍵結後,電子傳導率降低,再以葡萄糖溶液作為導電液,使葡萄糖氧化酶催化葡萄糖,利用安培法量測電流訊號,相較於不採用葡萄糖氧化酶間接量測的方式,其靈敏度大約為0.26μA/μM,檢測極限0.075μM。取上述三種類固醇進行專一性測試,只有皮質醇電流值明顯下降4倍,得知晶片具有專一性。將晶片量測不同水溫下抽出之魚血,27℃水溫下魚血皮質醇濃度為0.762μM,水溫15℃為0.978μM,回溫至27℃為0.798μM。
綜合以上結果,推論當魚所處的環境水溫產生急遽變化時,亦會對魚的生理狀態造成影響,如代謝降低、活動力降低,因此會提升體內皮質醇濃度,促使肌肉收縮增加代謝,對抗外界惡劣環境,當水溫回復至正常狀態(27℃)時皮質醇濃度會逐漸降低直至水平狀態。因此可以將此量測結果用於魚的生理監控,當進行監控時若發現皮質醇有急遽上升之狀態,即時給予治療,提升產值。
Tilapia is an important economic species of fish farming in Taiwan. However, mortality rate of fish caused by viruses and cold damage is high every year. In addition to blood sugar, lactic acid and cholesterol, stress hormone-cortisol is also an important indicator of physiological abnormalities. The cortisol concentration in normal conditions is 1pM~100μM, when excessive secretion which caused death. Therefore, monitoring the cortisol concentration of fish is very important. This study propose two biomedical sensing devices to monitor cortisol. 1. Implantable cortisol electrochemical sensing device : can real-time measure but low sensitivity. 2. Label-free electrochemical impedance immunosensing chip : the method is bonded with immunoassay have high specificity, but have to blood test for measure.
Implantable cortisol electrochemical sensing device : Coating 11β-hydroxysteroid dehydrogenase at the working electrode to catalyze cortisol, also coated with Diaphorase to catalyze NADP+, add Vitamin K3 as an electron carrier, and add Ketjenblack to increase the electron conductivity. The cortisol standard was calibrated to a sensitivity of 8.4μA/μM. Testing with three types of steroid hormones, respectively cortisol, 11β-estradiol, 17α,20β-dihydroxy-4-pregnen-3-one. Only cortisol has a significant change. The implantable cortisol electrochemical sensing device was implanted into the fish body for measurement. The cortisol concentration was 0.357 uM at a water temperature of 27°C, the concentration at 15°C was 2.176uM, the concentration riased six times and when the temperature was returned to 27°C the concentration was 1.344uM.
Label-free electrochemical impedance immunosensing chip: Using reduced graphene oxide deposited on the working electrode to increase conductivity, Coating glucose oxidase after the antibody is covalently bonded. Using glucose oxidase catalyzed the signal indirectly measuring the concentration of cortisol. When the cortisol antigen is covalently bonded to the antibody, the electron conductivity decreases. Therefore, the glucose solution is used as a conductive liquid catalyze by the glucose oxidase . Using Amperometry to mearure current signal, compare with the method of without glucose oxidase, the sensitivity approximately 0.26μA/μM, limit of detection is 0.075μM. Take the above three types of steroid specificity for testing, only cortisol current value obviously decreased four times, thus the chip that has specificity. The chip is measured for fish blood drawn at different water temperatures, The fish cortisol concentration was 0.762 uM at 27°C water temperature, 0.978uM at 15°C water temperature, and when the water temperature was returned to 27°C the fish cortisol concentration was 0.798uM.
Based on the above results, When the ambient water temperature changes rapidly, it will also affect the physiological state of the fish. For example, lower metabolism and lower activity, Therefore, it will increase the concentration of cortisol in the body, promote muscle contraction and increase metabolism to resist the harsh environment of the outside world. When the temperature returned to normal conditions (27℃) cortisol concentration will gradually decrease until the horizontal state. Therefore, the measurement results can be used for physiological monitoring of fish. When the monitoring has found that cortisol state rising sharply, gave treatment immediately to increase production value
目錄
摘要..............................................I
Abstract.........................................II
圖目錄...........................................VII
表目錄............................................X
第一章 緒論......................................1
1.1前言...........................................1
1.2研究背景.......................................2
1.2.1糖皮質素.....................................2
1.2.2酵素.........................................2
1.2.3生物型感測器..................................3
1.2.4氧化石墨烯....................................4
1.2.5抗體.........................................4
1.2.6吳郭魚(Tilapia)..............................4
第二章 文獻回顧....................................6
2.1皮質醇免標定晶片於生物感測之應用.................6
2.2針狀酵素感測針之相關研究........................18
2.3研究動機與目的.................................24
第三章 實驗原理...................................25
3.1酵素催化反應...................................25
3.2抗原抗體結合原理................................25
3.3電化學反應.....................................26
3.3.1循環伏安法(cyclic voltammetry, CV)...........26
3.3.2線性掃描伏安法(Linear Sweep Voltammetry, LSV).26
3.3.3安培法(Amperometry ,IT)......................26
3.3.4電化學阻抗圖譜(Electrochemical impedance spectroscopy,EIS)..................................27
4.1感測系統之機台-電化學CHI系列分析儀.................28
4.1.1機台軟硬體介紹..................................28
4.1.2電化學技術.....................................29
4.2植入式皮質醇電化學感測裝置.........................30
4.2.1植入式皮質醇電化學感測裝置結構...................30
4.2.2植入式皮質醇電化學感測裝置工作電極修飾............30
4.3免標定式電化學阻抗免疫感測晶片製作..................32
4.3.1免標定式電化學阻抗免疫感測晶片結構................33
4.3.2電化學清洗感測晶片...............................34
4.3.3氧化石墨烯於工作電極上之層積與還原................34
4.3.4抗體於工作電極之修飾.............................35
4.3.5皮質醇抗原鍵結於工作電極之上.....................36
4.4感測裝置量測魚體與感測晶片量測魚血..................36
4.4.1植入式皮質醇電化學感測裝置於魚體量測..............37
4.4.2免標定式電化學阻抗免疫感測晶片量測魚血............39
第五章 結果與討論....................................40
5.1植入式皮質醇電化學感測裝置之效能分析................40
5.1.1植入式皮質醇電化學感測裝置於安培法中量測皮質醇校正液40
5.1.2植入式皮質醇電化學感測裝置專一性測試..............42
5.2免標定式電化學阻抗免疫感測晶片效能分析..............44
5.2.1免標定式電化學阻抗免疫感測晶片氧化石墨烯電化學法還原44
5.2.2免標定式電化學阻抗免疫感測晶片工作電極表面修飾之測試46
5.2.3免標定式電化學阻抗免疫感測晶片檢測皮質醇濃度........47
5.2.4免標定式電化學阻抗免疫感測晶片結合葡萄糖氧化酶於皮質醇之量...................................................50
5.2.5免標定式電化學阻抗免疫感測晶片專一性測試............56
5.3量測吳郭魚於應激環境下的生理變化.....................58
5.3.1植入式皮質醇電化學感測裝置植入魚體量測.............58
5.3.2植入式皮質醇電化學感測裝置量測抽出之魚血...........60
5.3.3免標定式電化學阻抗免疫感測晶片量測抽出之魚血........61
第六章 結論與未來展望..................................65
6.1結論...............................................65
6.2未來展望...........................................66
第七章 參考文獻.......................................67
[1] 維基百科,Cortisol
Available: https://en.wikipedia.org/wiki/Cortisol.
[2] Kim;, S.L.H.-S.L.H.-J.S.S.-A., J.P.H.-C.H.H.J. Kim;, and Y.-T.K.-R.L.a.Y.-J. Kim, Simultaneous Determination of Cortisol and Cortisone from Human Serum by Liquid Chromatography-Tandem Mass Spectrometry. Analytical Methods in Chemistry, 2014.
[3] Pottinger, A.D.P.a.T.G., Stress responses and disease resistance in salmonid fish: Effects of chronic elevation of plasma cortisol. Fish Physiology and Biochemistry, 1989. 7: p. 253-258.
[4] MARCEL MARTÍNEZ-PORCHAS, L.R.M.-C., ROGELIO and RAMOS-ENRIQUEZ, Cortisol and Glucose: Reliable indicators of fish stress? Pan-American, 2009. 4: p. 158-178.
[5] 維基百科,Glucocorticoid
Available:https://en.wikipedia.org/wiki/Glucocorticoid.
[6] 維基百科,Cortisone
Available:https://en.wikipedia.org/wiki/Cortisone.
[7] 維基百科,Enzyme
Available::https://en.wikipedia.org/wiki/Enzyme.
[8] 生物型感測器-醫療機電設計與整合研發室-長庚大學
Available:http://mmrl.cgu.edu.tw/data/mme/rehab/organize/chap2/sensor/no2.htm.
[9] Pei, S. and H.-M. Cheng, The reduction of graphene oxide. Carbon, 2012. 50(9): p. 3210-3228.
[10] 維基百科,Antibody
Available:https://en.wikipedia.org/wiki/Antibody.
[11] 國立臺灣海洋大學-水生動物實驗中心Available:http://aac.ntou.edu.tw/touchmaker/front/bin/ptdetail.phtml?Part=267&Category=100037.
[12] Wen-Hsiung Chen, L.-T.S., Ching-Lin Tsai,Yen-Lin Song, and Ching-Fong Chang, Cold-Stress Induced the Modulation of Catecholamines, Cortisol, Immunoglobulin M, and Leukocyte Phagocytosis in Tilapia. General and Comparative Endocrinolog, 2002. 126: p. 90-100.
[13] 林峰右‧吳育甄‧沈子耘‧許晉榮‧葉信利, 短鰭黃鱲鰺對低溫緊迫耐受性研究. Taiwan Fisheries Research, 2011. 19: p. 37-43.
[14] Mohammad Nabi Adloo, S.S., Mahmoud Hafeziyeh, Nastaran Ghadimi1, Cortisol and Glucose Responses in Juvenile Striped Catfish Subjected to a Cold Shock. Veterinary Science Development 2015. 5: p. 78-81.
[15] Sunil K. Arya, G.C., Manju Venugopal and Shekhar Bhansali, Antibody functionalized interdigitated μ-electrode (IDμE) based impedimetric cortisol biosensor. Analyst, 2010. 135: p. 1941–1946
[16] Hirai, M., et al., Carbon nanotube enhanced label-free immunosensor for amperometric determination of oocyte maturation-inducing hormone in fish. Fish Physiol Biochem, 2013. 39: p. 299-308.
[17] Yamaguchi, M., et al., Immunosensor with fluid control mechanism for salivary cortisol analysis. Biosens Bioelectron, 2013. 41: p. 186-91.
[18] Syed Khalid Pasha, A.K., Abhay Vasudev,Shedra Amy Snipes, and Shekhar Bhansalia, Electrochemical Immunosensing of Saliva Cortisol. The Electrochemical Society, 2014. 161: p. 3077-3082.
[19] Kaushik, A., et al., Electrochemical sensing method for point-of-care cortisol detection in human immunodeficiency virus-infected patients. Dovepress, 2015. 10: p. 677–685.
[20] Wu, H., et al., Carbon-Nanotube-Enhanced Label-Free Immunosensor for Highly Sensitive Detection of Plasma Cortisol Level in Fish. Sensors and Materials, 2015. 27: p. 793-803.
[21] Wu, H., et al., New approach for monitoring fish stress: A novel enzyme-functionalized label-free immunosensor system for detecting cortisol levels in fish. Biosens Bioelectron, 2017. 93: p. 57-64.
[22] Wu, H., et al., Flow immunosensor system with an electrode replacement unit for continuous cortisol monitoring for fish. Sensing and Bio-Sensing Research, 2017. 13: p. 122-127.
[23] Omar, A., et al., Development of Cortisol Immunosensor Based Reduced Graphene Oxide (rGO) for Future Application in Monitoring Stress Levels Among Military Personnel. Vol. 10. 2017. 142-148.
[24] Kwang Su Kim, S.R.L., , Sung-Eun Kim, Jun Young Lee, Chan-Hwa Chung, and P.J.Y. Woo-Seok Choe, Highly sensitive and selective electrochemical cortisol sensor using bifunctional protein interlayer-modified graphene electrodes. Sensors and Actuators, 2017. 242: p. 1121-1128.
[25] Neethirajan, S.K.T.C.O.S., Noninvasive Label-Free Detection of Cortisol and Lactate Using Graphene Embedded Screen-Printed Electrode. Nano-Micro 23 January 2018. 10.
[26] Gordicb;, A.K.S.A.M. and S.B.S.S. Mohapatraa;, Ultrasensitive detection of cortisol with enzyme fragment complementation technology using functionalized nanowire. Biosensors and Bioelectronics 2007. 22: p. 2138–2144.
[27] Takase, M., et al., Mediator-type biosensor for real-time wireless monitoring of blood glucose concentrations in fish. Chemistry and Biochemistry, 2012. 78: p. 691–698.
[28] Kyoko Hibi, K.H., Mai Takase, Huifeng Ren and Hideaki Endo, Wireless Biosensor System for Real-Time L-Lactic Acid Monitoring in Fish. sensors, 2012. 12,: p. 6269-6281.
[29] Mai Takase , R.H., Masataka Murata , Kyoko Hibi • Hideaki Endo, Development of mediator-type biosensor to wirelessly monitor whole cholesterol concentration in fish. Fish Physiol Biochem, 2014. 40: p. 385–394.
[30] Haiyun Wu, A.A., Takafumi Arimoto, Toshiki Nakano, Hitoshi Ohnuki, and H.R. Masataka Murata, Hideaki Endo,, Fish stress become visible: A new attempt to use biosensor for real-time monitoring fish stress. Biosensors and Bioelectronics, 2015. 67: p. 503-510.
[31] Wei-Hung Chen, S.-H.H., 植入式無線酵素感測裝置於受鏈球菌感染之吳郭魚生理狀態量測. 2018.
[32] 胡啟章, 電化學原理與方法. 2011.
[33] 禪譜科技-電化學技術Available:http://www.zensor.com.tw/assets/download/Zensor%20R&D%20Technology-1.2%20Electrochemical%20Method-%E4%B8%AD%E6%96%87.pdf.
[34] Takeo Miyake, K.H., Nobuhiro Nagai, Yohei Yatagawa,Hideyuki Onami,Syuhei Yoshino, and M.N. Toshiaki Abe, Enzymatic biofuel cells designed for direct power generation from biofluids in living organisms. Energy &Environmental Science, 2011. 4: p. 5008–5012.
[35] Chih Chio Yang, A.S.K., Jyh Myng Zen, Precise blood lead analysis using a combined internal standard and standard addition approach with disposable screen-printed electrodes. ANALYTICAL BIOCHEMISTRY, 2005. 338: p. 278–283.
[36] K. Sudhakara Prasad, G.M., Jyh-Myng Zen, The role of oxygen functionalities and edge plane sites on screen-printed carbon electrodes for simultaneous determination of dopamine, uric acid and ascorbic acid. Electrochemistry Communications, 2008. 10: p. 559–563.
[37] Binesh Unnikrishnan, S.P., Shen-Ming Chen, A simple electrochemical approach to fabricate a glucose biosensor based on grapheme-glucose oxidase biocomposite. Biosensors and Bioelectronics, 2013. 39: p. 70-75.
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