臺灣博碩士論文加值系統

在本篇論文當中，我利用了實驗室許純淵博士所開發出的微波電漿加熱法，成功地在短時間之內製作出20片金奈米粒子薄膜基板，並使用成本便宜的anti-IgG來測試基板，藉以改善先前的研究方法。此優化後的方法最大優點在於讓實驗時間大大縮短，以加快後續的研究步驟。為了將我們研究出的方法利用於臨床醫學上，並了解到修飾含有GA分子與IgG之金奈米粒子薄膜基板的保存期限，我將實驗分成五種不同的階段以測試基板的保存期限，經過六個禮拜後，發現到當金奈米粒子薄膜基板上修飾GA分子，並利用1X PBS buffer進行保存後，只能在0℃底下保存三天；而帶有IgG之金奈米粒子薄膜基板以30ppm IgG溶液來進行保存後，所得到的結果證明IgG分子可以穩定地長時間存放在0℃中。利用我所找到的最佳實驗條件，對於偵測10ng/mL anti-IgG (即anti human-IgG)仍然具有選擇性，同時anti-IgG的偵測極限為5ng/mL(33pM)。

The thesis provides a method to make Au NPs substrates at the short time by using homemade-microwave plasma bombardment, and optimize the previous detection method to improve the detecting sensitivity. The method not only decrease the experimental time but also speed up the follow-up experimental steps. For application to clinical medicine development and understand the expiration dates of GA-immobilized and IgG-immobilized substrates, we divided the expiration date into five stages to test how long theses substrates could preserve. After 6 weeks, we found that GA-immobilized substrates preserved in 1X PBS buffer only can preserve in 0℃for three days. On the other hand, IgG-immobilized substrates can be preserved in 30 ppm IgG solution in 0℃stably for a long time. This method is not only selectivity to anti-IgG (i.e. anti human-IgG), but the limit of detection of anti-IgG is 5ng/mL(33pM).

中文摘要 Ⅰ
英文摘要 Ⅱ
目錄 Ⅲ
圖目錄 Ⅶ
表目錄 XVΙ

第1章、緒論 1
1.1、源起 1
1.2、奈米簡介 1
1.3、奈米材料製備與應用 2
第2章、基礎理論與文獻回顧 4
2.1、生物感測器 4
2.1.1、生物感測器簡介 4
2.1.2、生物感測器之歷史發展 5
2.1.3、免疫感測器之類型 7
2.1.3.1、電化學免疫感測器(electrochemical immunosensors) 7
2.1.3.2、壓電式免疫感測器(piezoelectric immunosensors) 8
2.1.3.3、熱學免疫感測器(thermometric immunosensors) 8
2.1.3.4、光學免疫感測器(optical immunosensors) 8
2.2、表面電漿共振效應 8
2.2.1、表面電漿共振 9
2.2.2、表面電漿共振應用於生物感測器 10
2.2.2.1、直接法 12
2.2.2.2、競爭法 12
2.2.2.3、三明治法 12
2.2.2.4、抑制法 12
2.3.2、局域性表面電漿共振 13
2.3.2.1、粒子形狀對LSPR吸收峰的影響 14
2.3.2.2、粒子大小對LSPR吸收峰的影響 15
2.3.2.3、金屬奈米粒子之間距對LSPR吸收峰的影響 16
2.3.2.4、周圍環境介電常數(或折射率)對LSPR吸收峰的影響 18
2.3.3、比較SPR與LSPR 19
2.3.4、局域性表面電漿共振應用於生物感測器 21
2.3、手持式儀器 30
2.3.1、手持式儀器的調控 30
2.3.2、SPR與LSPR在手持式儀器的應用 31
2.4、人體免疫球蛋白G (Immunoglobulin G , IgG) 34
2-5、研究動機 37
第3章、實驗 40
3.1、實驗儀器 40
3.2、藥品與器材 41
3.3、實驗流程 42
3.3.1、金奈米粒子薄膜基板的製作 42
3.3.2、金奈米粒子薄膜基板的表面修飾 42
3.3.2.1、APTMS分子修飾於金奈米粒子之間 42
3.3.2.2、戊二醛(glutaraldehyde，GA)修飾於APTMS分子上 43
3.3.2.3、Immunoglobulin G (IgG)修飾於GA分子上 44
3.3.2.4、利用帶有IgG分子的金奈米粒子薄膜基板偵測anti-human Immunoglobilin G (anti-IgG) 44
第4章、實驗結果與討論 46
4.1、金奈米粒子薄膜感測器之條件優化探討 46
4.1.1、金奈米粒子薄膜基板修飾APTMS 46
4.1.1.1、基板放置12小時後之差異性 46
4.1.1.2、基板在不同濃度之APTMS溶液中的差異性 47
4.1.1.3、不同浸泡時間下所得之訊號差異性 49
4.1.1.4、熱處理造成的差異性 51
4.1.2、金奈米粒子薄膜基板修飾GA分子 55
4.1.2.1、不同溫度下所得之基板差異性 55
4.1.3、金奈米粒子薄膜基板偵測抗體分子anti-IgG 58
4.1.3.1、非專一性(non-specific)測試 58
4.1.3.2、anti-IgG之檢量線及偵測極限 60
4.2、金奈米粒子薄膜感測器之保存期限測試 63
4.2.1、金奈米粒子薄膜基板上修飾GA分子之保存期限測試 63
4.2.1.1、第一階段：三天後 64
4.2.1.2、第二階段：一個禮拜後 65
4.2.1.3、第三階段：兩個禮拜後 67
4.2.1.4、第四階段：四個禮拜後 68
4.2.1.5、第五階段：六個禮拜後 69
4.2.2、金奈米粒子薄膜基板修飾IgG分子之保存期限測試 71
4.2.2.1、第一階段：三天後 71
4.2.2.2、第二階段：一個禮拜後 73
4.2.2.3、第三階段：兩個禮拜後 75
4.2.2.4、第四階段：四個禮拜後 76
4.2.2.5、第五階段：六個禮拜後 78
第5章、結論 80
第6章、未來展望 81
附錄Ⅰ 82
◆ 金奈米粒子薄膜基板修飾GA分子之保存期限測試 82
I-1、第一階段：三天後 82
I-1-1、基板保存於0℃環境中 82
I-1-2、基板保存於室溫環境中 84
I-2、第二階段：一個禮拜後 86
I-2-1、基板保存於0℃環境中 86
I-2-2、基板保存於室溫環境中 88
I-3、第三階段：兩個禮拜後 90
I-3-1、基板保存於0℃環境中 90
I-3-2、基板保存於室溫環境中 92
I-4、第四階段：四個禮拜後 94
I-4-1、基板保存於0℃環境中 94
I-4-2、基板保存於室溫環境中 96
I-5、第五階段：六個禮拜後 98
I-5-1、基板保存於0℃環境中 98
I-5-2、基板保存於室溫環境中 100
附錄Ⅱ 102
◆ 金奈米粒子薄膜基板修飾IgG分子之保存期限測試 102
Ⅱ-1、第一階段：三天後 102
Ⅱ-1-1、基板保存於0℃環境中 102
Ⅱ-1-2、基板保存於室溫環境中 104
Ⅱ-2、第二階段：一個禮拜後 106
Ⅱ-2-1、基板保存於0℃環境中 106
Ⅱ-2-2、基板保存於室溫環境中 108
Ⅱ-3、第三階段：兩個禮拜後 110
Ⅱ-3-1、基板保存於0℃環境中 110
Ⅱ-3-2、基板保存於室溫環境中 112
Ⅱ-4、第四階段：四個禮拜後 114
Ⅱ-4-1、基板保存於0℃環境中 114
Ⅱ-4-2、基板保存於室溫環境中 116
Ⅱ-5、第五階段：六個禮拜後 118
Ⅱ-5-1、基板保存於0℃環境中 118
Ⅱ-5-2、基板保存於室溫環境中 120
附錄Ⅲ 122
◆ 手持式儀器初步成果 122
參考資料 124

1.Shankaran, D.; Gobi, K.; Miura, N., Recent advancements in surface plasmon resonance immunosensors for detection of small molecules of biomedical, food and environmental interest. Sensor Actuat. B : Chem. 2007, 121 (1), 158-177.
2.胡淑芬, 奈米科技發展之介紹. 毫微米通訊 第8卷 (第4期).
3.馬遠榮, 低維奈米材料. 科學發展 2004, (第382期), 72-75.
4.Stewart, M. E.; Anderton, C. R.; Thompson, L. B.; Maria, J.; Gray, S. K.; Rogers, J. A.; Nuzzo, R. G., Nanostructured plasmonic sensors. Chem. Rev. 2008, 108, 494-521.
5.陳世明, 簡明的科技：重點照護檢驗. 福島醫療器材有限公司 2001.
6.Heeren, H. v., Fabrication for nanotechnology.
7.Luppa, P. B.; Sokoll, L. J.; Chan, D. W., Immunosensors-principles and applications to clinical chemistry. Clin. Chim. Acta 2001, 314, 1-26.
8.Jr., L. C.; Lyons, C., Electrode systems for continuous monitoring in gradiovascular surgery. Ann. NY Acad. Sci. 1962, 102, 29-45.
9.林宇聲; 簡正邦; 張正義, 生物感測器.
10.黃磁婷, 以離子感測場效電晶體用於新型乳酸生醫感測器之研究.
11.Michelle Duval Malinsky; K. Lance Kelly; George C. Schatz; Duyne*, R. P. V., Chain length dependence and sensing capabilities of the localized surface plasmon resonance of silver nanoparticles chemically modified with alkanethiol self-assembled monolayers. J. Am. Chem. Soc. 2001, 123, 1471-1482.
12.Resnick, G., Biosensors overview. 2010.
13.Ricci, F.; Volpe, G.; Micheli, L.; Palleschi, G., A review on novel developments and applications of immunosensors in food analysis. Anal. Chim. Acta 2007, 605 (2), 111-129.
14.E. Mallat, D. B.; C. Barzen, G. G.; Abuknesha, R., Immunosensors for pesticide determination in natural waters. Trends Anal. Chem. 2001, 20, 124-132.
15.Pedersen, L. H., Biosensor technology-an overview. 2005.
16.W.Wood, R., On a remarkable case of uneven distribution of light in a diffraction grating spectrum Philos. Mag. Ser. 6 1902, 4, 396-402.
17.張勝雄; 戴朝義, 奈米電漿子波導元件於積體光學之應用. 物理雙月刊 2008, 第30卷 (第6期).
18.Ritchie, R., Plasma losses by fast electrons in thin films. Phys. Rev. 1957, 106 (5), 874-881.
19.Pitarke, J. M.; Silkin, V. M.; Chulkov, E. V.; Echenique, P. M., Theory of surface plasmons and surface-plasmon polaritons. Rep. Prog. Phys. 2007, 70 (1), 1-87.
20.吳民耀; 劉威志, 表面電漿子理論與模擬. 物理雙月刊 2006, 第28卷 (第2期).
21.Otto, A., Excitation of nonradiative surface plasma waves in silver by the method of frustated total reflection. Z. Phys. 1968, 216, 398-410.
22.Seshadri, S. R., Attenuated total reflection method of excitation of the surface polarition in te Kretschmann configuration. J. Appl. Phys. 1991, 70 (7), 3647-3654.
23.Homola, J. ı.; Yee, S. S.; Gauglitz, G. n., Surface plasmon resonance sensors─review. Sens. Actuators, B 1999, 54, 3-15.
24.Abdulhalim, I.; Zourob, M.; Lakhtakia, A., Surface plasmon resonance for biosensing : a mini-review. Electromagnetics 2008, 28 (3), 214-242.
25.Marquette, C. A.; Blum, L. J., State of the art and recent advances in immunoanalytical systems. Biosens. Bioelectron. 2006, 21 (8), 1424-1433.
26.Homola, J. ı.; Dosta’lek, J.; Chen, S.; Rasooly, A.; Jiang, S.; Yee, S. S., Spectral surface plasmon resonance biosensor for detection of staphylococcal entertoxin B in milk. Int. J. Food Microbiol. 2002, 75, 61-69.
27.Sapsford, K. E.; Charles, P. T.; Jr., C. H. P.; Ligler, F. S., Demonstration of four immunoassay formats using the array biosensor. Anal. Chem. 2002, 74, 1061-1068.
28.Mayer, K. M.; Hafner, J. H., Localized surface plasmon resonance sensors. Chem. Rev. 2011, 111 (6), 3828-3857.
29.曾賢德, 金奈米粒子的表面電漿共振特性：耦合、應用與樣品製作. 物理雙月刊 2010, 第32卷 (第2期), 126-135.
30.K. Lance Kelly; Coronado, E.; Zhao, L. L.; Schatz, G. C., The optical properties of metal nanoparticles : the influence of size, shape, and dielectric environment J. Phys. Chem. B 2003, 107, 668-677.
31.Ghosh, S. K.; Pal, T., Interparticle coupling effect on the surface plasmon resonance of gold Nnanoparticles : from theory to applications. Chem. Rev. 2007, 107, 4797-4862.
32.Anker, J. N.; Hall, W. P.; Lyandres, O.; Shah, N. C.; Zhao, J.; Duyne, R. P. V., Biosensing with plasmonic nanosensors. Nature materials 2008, 7, 442-453.
33.Kuwata, H.; Tamaru, H.; Esumi, K.; Miyano, K., Resonant light scattering from metal nanoparticles : practical analysis beyond Rayleigh approximation. Appl. Phys. Lett. 2003, 83 (22), 4625-4627.
34.Jain, P. K.; El-Sayed, M. A., Noble metal nanoparticle pairs : effect of medium for enhanced nanosensing. Nano Lett. 2008, 8 (12), 4347-4352.
35.Chen, C.-F.; Tzeng, S.-D.; Chen, H.-Y.; Lin, K.-J.; Gwo, S., Tunable plasmonic response from alkanethiolate-stabilized gold nanoparticle superlattices : evidence of near-field coupling. J. Am. Chem. Soc. 2007, 130 (3), 824-826.
36.Talley, C. E.; Jackson, J. B.; Oubre, C.; Grady, N. K.; Hollars, C. W.; Lane, S. M.; Huser, T. R.; Nordlander, P.; Halas, N. J., Surface-enhanced Raman scattering from individual Au nanoparticles and nanoparticle dimer substrates. Nano Lett. 2005, 5 (8), 1569-1574.
37.Garcı’a-Vidal, F. J.; Pendry, J. B., Collective theory for surface enhanced Raman scattering. Phys. Rev. Lett. 1996, 77 (6), 1163-1166.
38.Shalaev, V.; Botet, R.; Jullien, R., Resonant light scattering by fractal clusters. Phys. Rev. B 1991, 44 (22), 12216-12225.
39.Wark, A. W.; Lee, H. J.; Corn, R. M., Long-range surface plasmon resonance imaging for bioaffinity sensors. Anal. Chem. 2005, 77 (13), 3904-3907.
40.Akiyama, T.; Nakada, M.; Terasaki, N.; Yamada, S., Photocurrent enhancement in a porphyrin-gold nanoparticle nanostructure assisted by localized plasmon excitation. Chem. Comm. 2006, (4), 395-397.
41.Kadir Aslan; Zhang, J.; Lakowicz, J. R.; Geddes, C. D., Saccharide sensing using gold and silver nanoparticles─a review. J. Fluoresc. 2004, 14 (4), 391-400.
42.Radhakumary, C.; Sreenivasan, K., Naked eye detection of glucose in urine using glucose oxidase immobilized gold nanoparticles. Anal. Chem. 2011, 83 (7), 2829-2833.
43.Endo, T.; Kerman, K.; Nagatani, N.; Hiepa, H. M.; Kim, D.-K.; Yonezawa, Y.; Nakano, K.; Tamiya, E., Multiple label-free detection of antigen−antibody reaction using localized surface plasmon resonance-based core−shell structured nanoparticle layer nanochip. Anal. Chem. 2006, 78 (18), 6465-6475.
44.Vestergaard, M. d.; Kerman, K.; Kim, D.-K.; Hiep, H. M.; Tamiya, E., Detection of Alzheimer''s tau protein using localised surface plasmon resonance-based immunochip. Talanta 2008, 74 (4), 1038-1042.
45.Nath, N.; Chilkoti, A., A colorimetric gold nanoparticle sensor to interrogate biomolecular interactions in real time on a surface. Anal. Chem. 2002, 74 (504-509).
46.Nath, N.; Chilkoti, A., Label-free biosensing by surface plasmon resonance of nanoparticles on glass: optimization of nanoparticle size. Anal. Chem. 2004, 76 (18), 5370-5378.
47.Marinakos, S. M.; Chen, S.; Chilkoti, A., Plasmonic detection of a model analyte in serum by a gold nanorod sensor. Anal. Chem. 2007, 79 (14), 5278-5283.
48.Karakouz, T.; Tesler, A. B.; Bendikov, T. A.; Vaskevich, A.; Rubinstein, I., Highly stable localized plasmon transducers obtained by thermal embedding of gold island films on glass. Adv. Mater. 2008, 20 (20), 3893-3899.
49.Karakouz, T.; Maoz, B. M.; Lando, G.; Vaskevich, A.; Rubinstein, I., Stabilization of gold nanoparticle films on glass by thermal embedding. ACS Appl. Mater. Inter. 2011, 3 (4), 978-987.
50.Hsu, C.-Y.; Huang, J.-W.; Gwo, S.; Lin, K.-J., The facile fabrication of tunable plasmonic gold nanostructure arrays using microwave plasma. Nanotechnology 2010, 21 (3), 035302.
51.Naimushin, A., A portable surface plasmon resonance (SPR) sensor system with temperature regulation. Sensor Actuat. B : Chem. 2003, 96 (1-2), 253-260.
52.Neuzil, P.; Reboud, J., Palm-sized biodetection system based on localized surface plasmon resonance. Anal. Chem. 2008, 80 (15), 6100-6103.
53.Shin, Y.-B.; Kim, H. M.; Jung, Y.; Chung, B. H., A new palm-sized surface plasmon resonance (SPR) biosensor based on modulation of a light source by a rotating mirror. Sensor Actuat. B : Chem. 2010, 150 (1), 1-6.
54.Stevens, R. C.; Soelberg, S. D.; Near, S.; Furlong, C. E.,Detection of cortisol in saliva with a flow-filtered, portable surface plasmon resonance biosensor system. Anal. Chem. 2008, 80, 6747-6751.
55.Stevens, R.; Soelberg, S.; Eberhart, B.; Spencer, S.; Wekell, J.; Chinowsky, T.; Trainer, V.; Furlong, C., Detection of the toxin domoic acid from clam extracts using a portable surface plasmon resonance biosensor. Harmful Algae 2007, 6 (2), 166-174.
56.Nakamoto, K.; Kurita, R.; Niwa, O., One-chip biosensor for simultaneous disease marker/calibration substance measurement in human urine by electrochemical surface plasmon resonance method. Biosens. Bioelectron. 2010, 26 (4), 1536-1542.
57.台北榮民總醫院病理檢驗部, Creatiniee(Cr)肌酸酐. 2003.
58.Nelson, D. L.; Cox, M. M., Lehninger principles of biochemistry, fifth edition. fifth ed.; 2006; Vol. chaptor 5.
59.Goldsby, R. A.; Kindt, T. J.; Osborne, B. A., Kuby Immunology 免疫學, fourth edition. 2004; Vol. chaptor4.
60.Silverton, E. W.; Navia, M. A.; Davies, D. R., Three-dimensional structure of an intact human immunoglobulin. P. Natl. Acad. Sci. 1977, 74 (11), 5140-5144.
61.Hsu, C.-Y.; Huang, J.-W.; Lin, K.-J., High sensitivity and selectivity of human antibody attachment at the interstices between substrate-bound gold nanoparticles. Chem. Comm. 2011, 47 (3), 872-874.
62.Beeram, S. R.; Zamborini, F. P., Selective attachment of antibodies to the edges of gold nanostructures for enhanced localized surface plasmon resonance biosensing. J. Am. Chem. Soc. 2009, 131 (33), 11689-11691.
63.Lee, S.; Mayer, K. M.; Hafner, J. H., Improved localized surface plasmon resonance immunoassay with gold bipyramid substrates. Anal. Chem. 2009, 81 (11), 4450-4455.
64.Choi, S.; Chae, J., A microfluidic biosensor based on competitive protein adsorption for thyroglobulin detection. Biosens. Bioelectron. 2009, 25 (1), 118-123.
65.Billakanti, J. M.; Fee, C. J.; Lane, F. R.; Kash, A. S.; Fredericks, R., Simultaneous, quantitative detection of five whey proteins in multiple samples by surface plasmon resonance. Int. Dairy J. 2010, 20 (2), 96-105.
66.Rakesh Singh Moirangthem; Mohammad Tariq Yaseen; Pei-Kuen Wei; Ji-Yen Cheng; Chang, Y.-C., Enhanced localized plasmonic detections using partially-embedded gold nanoparticles and ellipsometric measurments. Biomed. Opt. Express 2012, 3, 900-910.