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系統識別號 U0026-1908202016404700
論文名稱(中文) 以氧化石墨烯建立適體傳感器檢測汞與鉛離子的微流體裝置
論文名稱(英文) A Microfluidic Device for Detecting Mercury (II) and Lead (II) Ions Based on Graphene Oxide by Aptasensor
校院名稱 成功大學
系所名稱(中) 工程科學系
系所名稱(英) Department of Engineering Science
學年度 108
學期 2
出版年 109
研究生(中文) 吳若茵
研究生(英文) Ruo-Yin Wu
學號 N96074421
學位類別 碩士
語文別 中文
論文頁數 64頁
口試委員 指導教授-楊瑞珍
口試委員-傅龍明
口試委員-楊鏡堂
口試委員-張建成
口試委員-楊煥成
中文關鍵字 適體  螢光共振能量轉移  汞二價離子  鉛二價離子  氧化石墨烯  微流體晶片裝置 
英文關鍵字 Aptamer  FRET  Mercury and Lead ions  Graphene oxide  Microfluidic device 
學科別分類
中文摘要 本研究利用適體結合螢光標記製作可同時檢測汞與鉛離子的檢測裝置,並利用微流體晶片裝置僅使用少量樣品即可進行檢測,且還能有效縮短反應與分析時間的特性,藉此製作低成本檢測裝置的雛形。近年來,在生物傳感器領域上,螢光標記適體已廣泛被應用於各種領域,包含目標物追蹤以及環境的即時檢測,使用特定的序列設計,適體即可與多種目標物結合,且具有可以大量生產、低成本以及高穩定性的優點。另外,氧化石墨烯作為優秀的兩親二維材料,可輕易地分散在水溶液中並與適體的鹼基做結合,再藉由螢光共振能量轉移(FRET)的機制,將適體上的螢光標記淬滅。本實驗利用不同序列及螢光標記物(FAM及HEX)的適體,其分別可與汞及鉛二價離子做專一性結合,在與氧化石墨烯結合後螢光消失,當與汞及鉛離子的反應後,適體分子的構型將由原本的鏈狀轉變為髮夾型及四鏈體結構,同時脫離了氧化石墨烯表面致使螢光恢復。在微流體晶片裝置中同時檢測汞與鉛離子,結果表明本實驗裝置在汞與鉛離子的檢測上分別具有10 nM至250 nM及10 nM至100 nM的線性檢測範圍,其最低檢測濃度分別約為 2.01 ppb 及 2.07 ppb,皆比世界衛生組織(WHO)分別規範的6 ppb 及 15 ppb 還低,並具有良好的選擇性。
英文摘要 One of the key environmental problems for population health is the heavy metal pollution such as Mercury (II) and Lead (II). These metals could react with organic compounds in the food chain of biological world resulting in toxic substances, which is harmful to human health. This study proposed a method to detect the metal ions using graphene-based two-dimensional materials Graphene Oxide, and reagent aptamers mixed with sample solutions in a microfluidic device. The advantages of the microfluidic device are fast-reaction time, stable mixing reaction and good fluorescence recovery. The different sequence of the specific aptamers are labeled by FAM and HEX fluorescent that is used to capture the fluorescence resonance energy transfer process of the mercury Hg(II) and lead Pb(II) ions. The experimental calibration curves show that the linear range of the aptamer sensors is in the range of 10 nM-250 nM for Hg(II) ion and 10 nM-100 nM for Pb(II) ion. The lowest detection limit is about 2 ppb for both metals, which are much lower than the standard of 6 and 10 ppb for Hg(II) and Pb(II), respectively, provided by World Health Organization (WHO) for a drinkable water.
論文目次 證明書 I
摘要 II
致謝 X
目錄 XI
圖目錄 XIV
表目錄 XVII
縮寫表 XVIII
第一章 緒論 1
1.1 研究背景與動機 1
1.1.1 環境汙染 1
1.1.2 汞與鉛檢測的意義 2
1.1.3 重金屬檢測技術 4
1.1.4 微流體晶片的簡介 4
1.2 研究目的與架構 6
第二章 文獻討論 7
2.1 微流體晶片 7
2.1.1 微流道的製作技術 7
2.1.2 微混合器之簡介 9
2.2 螢光共振能量轉移原理 10
2.3 生物傳感器 12
2.3.1 石墨烯及其衍生物 13
2.3.2 適體傳感器 16
2.3.3 重金屬與適體傳感器的研究 18
第三章 實驗材料與方法 23
3.1 化學品 23
3.2 微流道設計 24
3.2.1 微流道之結構 24
3.2.2 微流道系統裝置 25
3.3 儀器設備 26
3.3.1 雕刻機 26
3.3.2 氧電漿機 27
3.3.3 酸鹼度計 28
3.3.4 電子天秤 29
3.3.5 真空幫浦 30
3.3.6 試管震盪器 31
3.3.7 離心機 32
3.3.8 倒立式螢光顯微鏡 33
3.3.9 掃描式雷射共軛焦顯微鏡 34
3.3.10 超音波震盪器 35
3.3.11 雙通道注射幫浦 36
3.3.12 Image J 37
3.3.13 Zeiss ZEN 37
3.5 溶液配置 38
3.5.1 緩衝溶液 38
3.5.2 氧化石墨烯懸浮溶液 38
3.5.3 適體溶液 39
3.5.4 金屬離子溶液 39
第四章 結果與討論 40
4.1 微流體裝置的混合 40
4.1.1 微流體的混合時間 40
4.1.2 混合時間的實驗設置 43
4.1.3 混合效率的評估原理與結果 45
4.1.4 微流體裝置之檢測區域 47
4.3 氧化石墨烯的淬滅效率(Quenching efficiency) 48
4.3.1 氧化石墨烯對於不同適體的淬滅效率 49
4.3.2 時間對於氧化石墨烯淬滅效率的影響 52
4.4 在微流體裝置內汞與鉛離子的檢測 54
4.4.1 通過微流道檢測汞離子 54
4.4.2 通過微流道檢測鉛離子 56
4.4.3 通過微流道同時檢測汞與鉛離子 56
4.4.4 通過微流道檢測裝置之特異性 57
第五章 結論 59
參考文獻 60
參考文獻 1. Colborn T., vom F.S., Soto A.M. Developmental effects of endocrine-disrupting chemicals in wildlife and humans, Environmental health perspectives, 101(5), 378-384. (1993)
2. Duruibe J.O., Ogwuegbu M.O.C., Egwurugwu J.N. Heavy metal pollution and human biotoxic effects, International Journal of Physical Sciences, 2(5), 112-118. (2007)
3. Clarkson T.W. The toxicology of mercury, Critical Reviews in Clinical Laboratory Sciences, 34(4), 369-403. (1997)
4. Campbell L., Dixon D., Hecky R. A review of mercury in Lake Victoria, East Africa: implications for human and ecosystem health, Journal of Toxicology and Environmental Health, Part B, 6(4), 325-356. (2003)
5. Mason R.P., Choi A.L., Fitzgerald W.F., Hammerschmidt C.R., Lamborg C.H., Soerensen A.L., Sunderland E.M. Mercury biogeochemical cycling in the ocean and policy implications, Environmental Research, 119, 101-117. (2012)
6. Zalups R.K. Molecular interactions with mercury in the kidney, Pharmacological reviews, 52(1), 113-144. (2000)
7. Zahir F., Rizwi S.J., Haq S.K., Khan R.H. Low dose mercury toxicity and human health, Environmental toxicology and pharmacology, 20(2), 351-360. (2005)
8. Lin C.C., Yee N., Barkay T. Microbial transformations in the mercury cycle, Environmental Chemistry and Toxicology of Mercury, 155-191. (2012)
9. Harvey P., Handley H., Taylor M. Widespread copper and lead contamination of household drinking water, New South Wales, Australia, Environmental Research, 151, 275-285. (2016)
10. Subramanian K.S., Connor J.W. Lead contamination of drinking water: Metals leaching from soldered pipes may pose health hazard, Journal of Environmental Health, 29-32. (1991)
11. Papanikolaou N.C., Hatzidaki E.G., Belivanis S., Tzanakakis G.N., Tsatsakis A.M. Lead toxicity update. A brief review, Medical Science Monitor, 11(10), RA329-RA336. (2005)
12. Liu C.W., Lin K.H., Kuo Y.M. Application of factor analysis in the assessment of groundwater quality in a blackfoot disease area in Taiwan, Science of the Total Environment, 313(1-3), 77-89. (2003)
13. MacLehose R., Pitt G., Will S., Jones A., Duane L., Flaherty S., Hannant D., Stuttard B., Silverwood A., Snee K., Murray V., Syed Q., House I., Bellis M.A. Mercury contamination incident., Journal of Public Health, 23(1), 18-22. (2001)
14. Dai B., Cao M., Fang G., Liu B., Dong X., Pan M., Wang S. Schiff base-chitosan grafted multiwalled carbon nanotubes as a novel solid-phase extraction adsorbent for determination of heavy metal by ICP-MS, Journal of Hazardous Materials, 219-220, 103-110. (2012)
15. Aranda P.R., Gil R.A., Moyano S., De Vito I., Martinez L.D. Slurry sampling in serum blood for mercury determination by CV-AFS, Journal of Hazardous Materials, 161(2-3), 1399-1403. (2009)
16. . (!!! INVALID CITATION !!! [18-21])
17. Andersson H., Van den Berg A. Microfluidic devices for cellomics: a review, Sensors and Actuators B: Chemical, 92(3), 315-325. (2003)
18. Abgrall P., Gué A.M. Lab-on-chip technologies: making a microfluidic network and coupling it into a complete microsystem—a review, Journal of Micromechanics and Microengineering, 17(5), R15-R49. (2007)
19. Zhang C., Xing D. Single-Molecule DNA Amplification and Analysis Using Microfluidics, Chemical Reviews, 110(8), 4910-4947. (2010)
20. Streets A.M., Huang Y. Chip in a lab: Microfluidics for next generation life science research, Biomicrofluidics, 7(1), 011302. (2013)
21. Liu J., Enzelberger M., Quake S. A nanoliter rotary device for polymerase chain reaction, Electrophoresis, 23(10), 1531-1536. (2002)
22. Gawad S., Schild L., Renaud P. Micromachined impedance spectroscopy flow cytometer for cell analysis and particle sizing, Lab Chip, 1(1), 76-82. (2001)
23. Terry S.C., Jerman J.H., Angell J.B. A gas chromatographic air analyzer fabricated on a silicon wafer, IEEE transactions on electron devices, 26(12), 1880-1886. (1979)
24. Lin C.H., Lee G.B., Lin Y.H., Chang G.L. A fast prototyping process for fabrication of microfluidic systems on soda-lime glass, Journal of Micromechanics and Microengineering, 11(6), 726. (2001)
25. McDonald J.C., Whitesides G.M. Poly (dimethylsiloxane) as a material for fabricating microfluidic devices, Accounts of Chemical Research, 35(7), 491-499. (2002)
26. Xia Y., Si J., Li Z. Fabrication techniques for microfluidic paper-based analytical devices and their applications for biological testing: A review, Biosensors and Bioelectronics, 77, 774-789. (2016)
27. Ren K., Zhou J., Wu H. Materials for Microfluidic Chip Fabrication, Accounts of Chemical Research, 46(11). (2013)
28. Carrilho E., Martinez A.W., Whitesides G.M. Understanding Wax Printing A Simple Micropatterning Process for Paper-Based Microfluidics, Analytical Chemistry, 81(16), 7091-7095. (2009)
29. Li X., Tian J., Garnier G., Shen W. Fabrication of paper-based microfluidic sensors by printing, Colloids and Surfaces B: Biointerfaces, 76(2), 564-570. (2010)
30. Cate D.M., Adkins J.A., Mettakoonpitak J., Henry C.S. Recent developments in paper-based microfluidic devices, Analytical Chemistry, 87(1), 19-41. (2015)
31. Gaal G., Mendes M., de Almeida T.P., Piazzetta M.H., Gobbi Â.L., Riul Jr A., Rodrigues V. Simplified fabrication of integrated microfluidic devices using fused deposition modeling 3D printing, Sensors Actuators B: Chemical, 242, 35-40. (2017)
32. Romoli L., Tantussi G., Dini G. Experimental approach to the laser machining of PMMA substrates for the fabrication of microfluidic devices, Optics Lasers in Engineering, 49(3), 419-427. (2011)
33. Chen P.C., Pan C.W., Lee W.C., Li K.M. Optimization of micromilling microchannels on a polycarbonate substrate, International Journal of Precision Engineering, 15(1), 149-154. (2014)
34. Chen P.C., Pan C.W., Lee W.C., Li K.M. An experimental study of micromilling parameters to manufacture microchannels on a PMMA substrate, The International Journal of Advanced Manufacturing Technology, 71(9-12), 1623-1630. (2014)
35. Yasuda K. Non-destructive, non-contact handling method for biomaterials in micro-chamber by ultrasound, Sensors Actuators B: Chemical, 64(1-3), 128-135. (2000)
36. Vivek V., Kim E.S., editors. Novel acoustic-wave micromixer. Proceedings IEEE Thirteenth Annual International Conference on Micro Electro Mechanical Systems (Cat No 00CH36308); 2000: IEEE.
37. Lee Y.-K., Deval J., Tabeling P., Ho C.-M. Chaotic mixing in electrokinetically and pressure driven micro flows. Microreaction Technology: Springer; 2001. 185-191.
38. Mengeaud V., Josserand J., Girault H.H. Mixing processes in a zigzag microchannel: finite element simulations and optical study, Analytical Chemistry, 74(16), 4279-4286. (2002)
39. Stroock A.D., Dertinger S.K., Ajdari A., Mezić I., Stone H.A., Whitesides G.M. Chaotic mixer for microchannels, Science, 295(5555), 647-651. (2002)
40. Gobby D., Angeli P., Gavriilidis A. Mixing characteristics of T-type microfluidic mixers, Journal of Micromechanics and microengineering, 11(2), 126. (2001)
41. Das S.S., Tilekar S.D., Wangikar S.S., Patowari P.K. Numerical and experimental study of passive fluids mixing in micro-channels of different configurations, Microsystem Technologies, 23(12), 5977-5988. (2017)
42. Hussain S.A. An introduction to fluorescence resonance energy transfer (FRET), arXiv preprint arXiv:09081815. (2009)
43. Hochreiter B., Pardo-Garcia A., Schmid J.A. Fluorescent proteins as genetically encoded FRET biosensors in life sciences, Sensors, 15(10), 26281-26314. (2015)
44. Lovell J.F., Chen J., Jarvi M.T., Cao W.-G., Allen A.D., Liu Y., Tidwell T.T., Wilson B.C., Zheng G. FRET quenching of photosensitizer singlet oxygen generation, The Journal of Physical Chemistry B, 113(10), 3203-3211. (2009)
45. Bhalla N., Jolly P., Formisano N., Estrela P. Introduction to biosensors, Essays in Biochemistry, 60(1), 1-8. (2016)
46. Goode J.A., Rushworth J.V., Millner P.A. Biosensor Regeneration: A Review of Common Techniques and Outcomes, Langmuir, 31(23), 6267-6276. (2015)
47. K.S. N., A.K. G., S.V. M., Jiang D., Zhang Y., S.V. D., I.V. G., A.A. F. Electric field effect in atomically thin carbon films, Science, 306(5696). (2004)
48. Geim A.K., Novoselov K.S. The rise of graphene. Nanoscience and technology: a collection of reviews from nature journals: World Scientific; 2010. 11-19.
49. Zhu Y., James D.K., Tour J.M. New routes to graphene, graphene oxide and their related applications, Advanced Materials, 24(36), 4924-4955. (2012)
50. C.N. R., A.K. S., K.S. S., A. G. Graphene: the new two-dimensional nanomaterial, Angewandte Chemie International Edition, 48(42), 7752-7777. (2009)
51. Hummers Jr W.S., Offeman R.E. Preparation of graphitic oxide, Journal of the american chemical society, 80(6), 1339-1339. (1958)
52. Chen J., Yao B., Li C., Shi G. An improved Hummers method for eco-friendly synthesis of graphene oxide, Carbon, 64, 225-229. (2013)
53. Gao W. The chemistry of graphene oxide. Graphene Oxide: Reduction Recipes, Spectroscopy, and Applications: Springer International Publishing; 2015. 61-95.
54. Wang Y., Li Z., Wang J., Li J., Lin Y. Graphene and graphene oxide: biofunctionalization and applications in biotechnology, Trends in Biotechnology, 29(5), 205-212. (2011)
55. Ramakrishna Matte H.S.S., Subrahmanyam K.S., Venkata Rao K., George S.J., Rao C.N.R. Quenching of fluorescence of aromatic molecules by graphene due to electron transfer, Chemical Physics Letters, 506(4-6), 260-264. (2011)
56. Li F., Pei H., Wang L., Lu J., Gao J., Jiang B., Zhao X., Fan C. Nanomaterial-Based Fluorescent DNA Analysis: A Comparative Study of the Quenching Effects of Graphene Oxide, Carbon Nanotubes, and Gold Nanoparticles, Advanced Functional Materials, 23(33), 4140-4148. (2013)
57. Biotechnology D.C.f. The advantages of Aptamer and Antibody, Development Center for Biotechnology. (2017)
58. Feng C., Dai S., Wang L. Optical aptasensors for quantitative detection of small biomolecules: a review, Biosensors Bioelectronics, 59, 64-74. (2014)
59. Lu L.M., Zhang X.B., Kong R.M., Yang B., Tan W.H. A ligation-triggered DNAzyme cascade for amplified fluorescence detection of biological small molecules with zero-background signal, Journal of the American Chemical Society, 133(30), 11686-11691. (2011)
60. Du H., Disney M.D., Miller B.L., Krauss T.D. Hybridization-based unquenching of DNA hairpins on Au surfaces: prototypical “molecular beacon” biosensors, Journal of the American Chemical Society, 125(14), 4012-4013. (2003)
61. He S., Song B., Li D., Zhu C., Qi W., Wen Y., Wang L., Song S., Fang H., Fan C. A Graphene Nanoprobe for Rapid, Sensitive, and Multicolor Fluorescent DNA Analysis, Advanced Functional Materials, 20(3), 453-459. (2010)
62. Ono A., Togashi H. Highly selective oligonucleotide‐based sensor for mercury (II) in aqueous solutions, Angewandte Chemie International Edition, 43(33), 4300-4302. (2004)
63. De Acha N., Elosua C., Corres J.M., Arregui F.J. Fluorescent Sensors for the Detection of Heavy Metal Ions in Aqueous Media, Sensors, 19(3). (2019)
64. Cui X., Zhu L., Wu J., Hou Y., Wang P., Wang Z., Yang M. A fluorescent biosensor based on carbon dots-labeled oligodeoxyribonucleotide and graphene oxide for mercury (II) detection, Biosensors Bioelectronics, 63, 506-512. (2015)
65. Li X., Wang G., Ding X., Chen Y., Gou Y., Lu Y. A “turn-on” fluorescent sensor for detection of Pb 2+ based on graphene oxide and G-quadruplex DNA, Physical Chemistry Chemical Physics, 15(31), 12800-12804. (2013)
66. Zhan S., Wu Y., Luo Y., Liu L., He L., Xing H., Zhou P. Label-free fluorescent sensor for lead ion detection based on lead(II)-stabilized G-quadruplex formation, Analytical Biochemistry, 462, 19-25. (2014)
67. Zhang M., Yin B.C., Tan W., Ye B.C. A versatile graphene-based fluorescence "on/off" switch for multiplex detection of various targets, Biosens Bioelectronics, 26(7), 3260-3265. (2011)
68. Li Y., Xu Y., Feng X., Liu B.-F. A rapid microfluidic mixer for high-viscosity fluids to track ultrafast early folding kinetics of G-quadruplex under molecular crowding conditions, Analytical chemistry, 84(21), 9025-9032. (2012)
69. Wu X., Xing Y., Zeng K., Huber K., Zhao J.X. Study of Fluorescence Quenching Ability of Graphene Oxide with a Layer of Rigid and Tunable Silica Spacer, Langmuir, 34(2), 603-611. (2018)
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