免疫色層分析試紙（Immunochromatographic test strip, ICTS）由於其便宜、快速且方便檢驗而受到重視，而能依據檢測材料的不同，使用吸收或者螢光來判定結果。若利用吸收來判定結果，會由於變化不明顯而不利於定量；利用螢光判定則必須有激發光源才能判讀結果，在定性上較不方便。而雙顯色試紙則是同時具有吸收及螢光，能夠利於肉眼直接定性，以及用螢光強度變化增加定量的靈敏度。本篇研究將具有螢光的半導體高分子奈米顆粒（Semiconducting Polymer Dots, Pdots）結合金奈米粒子作為材料，放入免疫色層分析試紙中進行應用。利用金奈米粒子表面電漿共振（Surface Plasmon Resonance, SPR）的特性，使試紙上有明顯的顏色有助於肉眼直接辨識結果，且能增強高分子奈米顆粒的螢光，以降低偵測極限，期望此種雙顯色的試紙能夠做到癌症檢測以及食品安全檢驗。
（1）以電漿子增強高分子點之螢光結合雙顯色試紙對攝護腺特異抗原檢測
癌症又稱惡性腫瘤，十幾年來蟬聯國人十大死因之首，隨著健康意識的提高，人們開始注重自己的身體健康，希望遠離癌症，由於癌症的檢測都需要專業人員的幫助以及使用貴重的儀器，必須花費大量的時間和金錢且場地也受到限制，為了能夠更便捷的檢測癌症，科學家們開始開發檢驗癌症標誌物（Tumor marker）的免疫色層分析試紙，期望能夠快速且有效的達到定性及定量的效果，利用體積小、操作簡易及花費時間短的優點，將癌症檢測變得容易且普及化。
在此題目中，我們結合了高分子奈米顆粒和金奈米粒子做為檢測材料，修飾上對攝護腺特定抗原（Prostate Specific Antigen, PSA）有專一性的抗體，另外在試紙上修飾不同的抗體作為測試線和控制線，利用三明治法進行檢測。當血液透過毛細現象在試紙上流動經過測試線和控制線時，會因為抗原抗體的專一性作用，只要血液中含有攝護腺特定抗原，偵測材料就會和測試線上的抗體結合，透過金奈米粒子的顏色可以用肉眼初步辨識線條的數量，達到定性的效果；而透過高分子奈米顆粒的螢光，比較測試線和控制線的螢光亮度就能夠做到定量的效果，利用金奈米粒子具有表面電漿共振的特性，能使半導體高分子奈米顆粒的螢光增強，降低定量的偵測極限。

關鍵字： 半導體高分子奈米顆粒、金奈米粒子、免疫色層分析試紙、表面電漿共振增強螢光、癌症標記物、攝護腺特定抗原

（2）多色螢光高分子點結合雙顯色試紙於多種真菌毒素檢測
民以食為天，食品的挑選是每日都會遇到的問題，近日食安問題越來越嚴重，使得人心惶惶，我們希望能將雙顯色的免疫色層分析試紙應用到食品安全檢驗上。
真菌毒素的含量，也是食品安全的一大課題。黃麴毒素（Aflatoxin, AF）、玉米赤烯銅（Zearalenone, ZEA）、脫氧雪腐鐮刀菌烯醇（Deoxynivalenol, DON），是三種在穀物中常見的真菌毒素。在此題目中我們利用三種螢光顏色不同的半導體高分子顆粒搭配不同吸收的金奈米粒子。要利用金奈米粒子的表面電漿共振增強螢光，必須要讓金奈米粒子的吸收與高分子奈米顆粒的螢光重疊，因此我們選用具有紅色、黃色、藍色三種螢光的高分子奈米顆粒，分別包覆在吸收650、550、518 nm的金奈米粒子上，再分別修飾上黃麴毒素、玉米赤烯銅、脫氧雪腐鐮刀菌烯醇，期望能做出能同時偵測上述三種真菌毒素的雙顯色免疫色層分析試紙。

關鍵字： 半導體高分子奈米顆粒、金奈米粒子、免疫色層分析試紙、表面電漿共振、真菌毒素

Immunochromatographic test strip (ICTS) has been valued as an important subject because of it features like cost-effective, fast-responsive, easy-to-use. By using different methods to detect different kind signal reporting reagents, like colorimetric method and fluorescent method, signal reporting reagents play an important role in ICTS. Take ICTS is based on the colorimetric method in which colloidal gold nanoparticles for example, the qualitative detection results can be directly observed by naked eyes, but its sensitivity ability weaker. Compare with ICTS is based on the fluorescent method, the signal will have a high detection sensitivity, but need extra light source to read out. In our study, we will combine the advantages of colorimetric ICTS and fluorometric ICTS to create a bimodal readout ICTS. We use semiconducting polymer dots (Pdots) and gold nanomaterial as signal reporting reagents, because gold nanomaterial can plasmon enhanced fluorescence of polymer dots. We hope this kind of bimodal readout ICTS can be used in cancer detection and food safety detection.






(1) Colorimetric and Fluorescent Bimodal Readout Immunochromatography Test Strip with Plasmon Enhanced Fluorescence of Polymer Dots for Detection of PSA

Cancer leading cause of death in Taiwan for decades, which causes cancer detection become a very important subject. Currently detection of cancer take too much time and cost too much money, required professional staff and specific location as well. Under this precondition, scientists began to develop ICTS as a tumor markers detector. ICTS is a rapid method to detect tumor markers, which can reduce time and money during the detection, make cancer detection more convenient and popular.

In this study, different gold nanomaterial were coated on three different types and three different distinct emission Pdots, then functionalized the product with different antibodies. This bimodal readout ICTS was based on a sandwich assay, we fabricated a control line (modified by IgG antibody) and a test line (modified by capture antibody) on a single test strip, while the PSA monoclonal antibody was conjugated to reporting reagents as the detection antibody. After applying sample to the bimodal readout ICTS, based on the colorimetric method could determine the result is positive or negative, and the concentration of PSA could be measured by the fluorescence ratio of test line to control line.
Keyword: Semiconducting polymer dots, Gold nanorod, Immunochromatogrphy test strip, Plasmon enhanced fluorescence, Tumor marker, PSA

(2) Bimodal Signal Readout Immunochromatogrphy Test Strip with Multicolor Pdots for Multiplexed Mycotoxin Deretmination

Since many food safety issues happened recently, the public seen to lose their faith of our food safety and get panic of their daily eating. Therefore, we hope our bimodal signal readout ICTS can be used in solving this food safety problem. Mycotoxins are a significant hidden worry in food safety concern, we choose three different mycotoxins, Aflatoxin (AF) , Zearalenone (ZEA) and Deoxynivalenol (DON) , which were used as models to demonstrate the performance of the bimodal signal readout ICTS. In this study, we used three different types of Pdots of three distinct emission were coated on different gold nanomaterial and functionalized with different antibodies. When absorption of gold nanomaterial overlaps with emission of Pdot, gold nanomaterial will plasmon enhanced the fluorescence of Pdots. This design allows the simultaneous detection of multiple mycotoxins.
Keyword: Semiconducting polymer dots, Gold nanomaterial, Immunochromatogrphy test strip, Plasmon enhanced fluorescence, Mycotoxins

論文審定書 i
致謝 ii
摘要 iii
Abstract v
目錄 viii
圖目錄 xi
表目錄 xiii
縮寫表 xiii
第1章 緒論 1
一、 前言 1
二、 重點照護 1
I. 電化學檢測法 2
II. 磁性物質檢測法 2
III. 微流體 3
IV. 免疫色層分析試紙 4
三、 免疫色層分析試紙的歷史 5
四、 免疫色層分析試紙原理 9
I. 組件 9
II. 機制 10
五、 偵測材料 13
I. 吸收、比色 13
II. 螢光 14
III. 電化學 17
IV. 磁力 17
V. 熱度 19
VI. 拉曼增強 19
VII. 複合材料 20
六、 電漿子增強螢光 21
七、 研究動機 22
第2章 以電漿子增強高分子點之螢光結合雙顯色試紙對攝護腺特異抗原檢測 24
一、 前言 24
二、 實驗藥品及器材 25
I. 藥品與試劑 25
II. 化學結構式 27
III. 儀器 27
三、 實驗步驟 29
I. 製作金奈米棒 29
II. 製作Pdots 30
III. 將Pdots包覆在金奈米棒上 30
IV. 修飾diamine PEG 30
V. 修飾抗體 31
VI. 製作試紙 31
VII. 製備TEM樣品 32
四、 實驗設計原理 32
五、 實驗結果與討論 35
I. 偵測材料的選擇 35
II. 材料製作方法優化 35
III. 以電漿子增強Pdots螢光 38
IV. 時間條件優化 40
V. 抗原選擇性測試 41
VI. 抗原濃度定量 42
VII. 真實樣品檢測 44
六、 結論 45
第3章 多色螢光高分子點結合雙顯色試紙於多種真菌毒素檢測 46
一、 前言 46
二、 實驗藥品及器材 47
I. 藥品與試劑 47
II. 化學結構式 50
III. 儀器 50
三、 實驗步驟 52
I. 製作金奈米粒子 52
II. 製作Pdots 53
III. 將Pdots包覆在3.5 nm大小之金奈米粒子上 54
IV. 將Pdots包覆在650、550nm吸收之金奈米棒上 55
V. 修飾抗體 55
VI. 製作試紙 56
VII. 製備TEM樣品 56
四、 實驗設計原理 57
五、 實驗結果與討論 60
I. 偵測材料的選擇 60
II. 材料包覆方法優化 61
III. 將偵測材料應用至試紙 63
IV. 抗原濃度測試 64
V. 未來方向 65
第4章 參考資料 66

1. Qin, Z.; Chan, W. C.; Boulware, D. R.; Akkin, T.; Butler, E. K.; Bischof, J. C., Significantly improved analytical sensitivity of lateral flow immunoassays by using thermal contrast. Angew. Chem. Int. Ed. 2012, 51 (18), 4358-61.
2. Cazacu, A. C.; Demmler, G. J.; Neuman, M. A.; Forbes, B. A.; Chung, S.; Greer, J.; Alvarez, A. E.; Williams, R.; Bartholoma, N. Y., Comparison of a new lateral-flow chromatographic membrane immunoassay to viral culture for rapid detection and differentiation of influenza A and B viruses in respiratory specimens. J. Clin. Microbiol. 2004, 42 (8), 3661-4.
3. Yao, Y.; Guo, W.; Zhang, J.; Wu, Y.; Fu, W.; Liu, T.; Wu, X.; Wang, H.; Gong, X.; Liang, X. J.; Chang, J., Reverse Fluorescence Enhancement and Colorimetric Bimodal Signal Readout Immunochromatography Test Strip for Ultrasensitive Large-Scale Screening and Postoperative Monitoring. ACS. Appl. Mater. Inter. 2016, 8 (35), 22963-70.
4. Koets, M.; van der Wijk, T.; van Eemeren, J. T.; van Amerongen, A.; Prins, M. W., Rapid DNA multi-analyte immunoassay on a magneto-resistance biosensor. Biosens. Bioelectron. 2009, 24 (7), 1893-8.
5. Wang, J.; Cao, F.; He, S.; Xia, Y.; Liu, X.; Jiang, W.; Yu, Y.; Zhang, H.; Chen, W., FRET on lateral flow test strip to enhance sensitivity for detecting cancer biomarker. Talanta 2018, 176, 444-449.
6. Chen, Y.; Sun, J.; Xianyu, Y.; Yin, B.; Niu, Y.; Wang, S.; Cao, F.; Zhang, X.; Wang, Y.; Jiang, X., A dual-readout chemiluminescent-gold lateral flow test for multiplex and ultrasensitive detection of disease biomarkers in real samples. Nanoscale 2016, 8 (33), 15205-12.
7. Bai, Y.; Tian, C.; Wei, X.; Wang, Y.; Wang, D.; Shi, X., A sensitive lateral flow test strip based on silica nanoparticle/CdTe quantum dot composite reporter probes. RSC. Adv. 2012, 2 (5), 1778.
8. Lou, S. C.; Patel, C.; Ching, S.; Gordon, J., One-step competitive immunochromatographic assay for semiquantitative determination of lipoprotein(a) in plasma. Clin. Chem. 1993, 39 (4), 619-624.
9. Feng, S.; Caire, R.; Cortazar, B.; Turan, M.; Wong, A.; Ozcan, A., Immunochromatographic Diagnostic Test Analysis Using Google Glass. ACS Nano 2014, 8 (3), 3069-3079.
10. Cooper, J.; Yazvenko, N.; Peyvan, K.; Maurer, K.; Taitt, C. R.; Lyon, W.; Danley, D. L., Targeted Deposition of Antibodies on a Multiplex CMOS Microarray and Optimization of a Sensitive Immunoassay Using Electrochemical Detection. PLoS One 2010, 5 (3), e9781.
11. Xu, H.; Mao, X.; Zeng, Q.; Wang, S.; Kawde, A.-N.; Liu, G., Aptamer-Functionalized Gold Nanoparticles as Probes in a Dry-Reagent Strip Biosensor for Protein Analysis. Anal. Chem. 2009, 81 (2), 669-675.
12. Hwang, J.; Lee, S.; Choo, J., Application of a SERS-based lateral flow immunoassay strip for the rapid and sensitive detection of staphylococcal enterotoxin B. Nanoscale 2016, 8 (22), 11418-25.
13. Li, Z.; Wang, Y.; Wang, J.; Tang, Z.; Pounds, J. G.; Lin, Y., Rapid and Sensitive Detection of Protein Biomarker Using a Portable Fluorescence Biosensor Based on Quantum Dots and a Lateral Flow Test Strip. Anal. Chem. 2010, 82 (16), 7008-7014.
14. Shyu, R.-H.; Shyu, H.-F.; Liu, H.-W.; Tang, S.-S., Colloidal gold-based immunochromatographic assay for detection of ricin. Toxicon 2002, 40 (3), 255-258.
15. Li, M.; Cushing, S. K.; Wu, N., Plasmon-enhanced optical sensors: a review. Analyst 2015, 140 (2), 386-406.
16. Ren, M.; Xu, H.; Huang, X.; Kuang, M.; Xiong, Y.; Xu, H.; Xu, Y.; Chen, H.; Wang, A., Immunochromatographic assay for ultrasensitive detection of aflatoxin B(1) in maize by highly luminescent quantum dot beads. ACS. Appl. Mater. Inter. 2014, 6 (16), 14215-22.
17. Lee, M.; Lee, K.; Kim, K. H.; Oh, K. W.; Choo, J., SERS-based immunoassay using a gold array-embedded gradient microfluidic chip. Lab. Chip. 2012, 12 (19), 3720-7.
18. Fang, C. C.; Chou, C. C.; Yang, Y. Q.; Wei-Kai, T.; Wang, Y. T.; Chan, Y. H., Multiplexed Detection of Tumor Markers with Multicolor Polymer Dot-Based Immunochromatography Test Strip. Anal. Chem. 2018, 90 (3), 2134-2140.
19. Edwards, K. A.; Baeumner, A. J., Optimization of DNA-tagged dye-encapsulating liposomes for lateral-flow assays based on sandwich hybridization. Anal. Bioanal. Chem. 2006, 386 (5), 1335-43.
20. Gubala, V.; Harris, L. F.; Ricco, A. J.; Tan, M. X.; Williams, D. E., Point of care diagnostics: status and future. Anal. Chem. 2012, 84 (2), 487-515.
21. Sun, J.; Xianyu, Y.; Jiang, X., Point-of-care biochemical assays using gold nanoparticle-implemented microfluidics. Chem. Soc. Rev. 2014, 43 (17), 6239-53.
22. Hoegger, D.; Morier, P.; Vollet, C.; Heini, D.; Reymond, F.; Rossier, J. S., Disposable microfluidic ELISA for the rapid determination of folic acid content in food products. Anal. Bioanal. Chem. 2007, 387 (1), 267-75.
23. Ma, Y.; Mao, Y.; Huang, D.; He, Z.; Yan, J.; Tian, T.; Shi, Y.; Song, Y.; Li, X.; Zhu, Z.; Zhou, L.; Yang, C. J., Portable visual quantitative detection of aflatoxin B1 using a target-responsive hydrogel and a distance-readout microfluidic chip. Lab. Chip. 2016, 16 (16), 3097-104.
24. Ngom, B.; Guo, Y.; Wang, X.; Bi, D., Development and application of lateral flow test strip technology for detection of infectious agents and chemical contaminants: a review. Anal. Bioanal. Chem. 2010, 397 (3), 1113-35.
25. Mao, X.; Ma, Y.; Zhang, A.; Zhang, L.; Zeng, L.; Liu, G., Disposable Nucleic Acid Biosensors Based on Gold Nanoparticle Probes and Lateral Flow Strip. Anal. Chem. 2009, 81 (4), 1660-1668.
26. Posthuma-Trumpie, G. A.; Korf, J.; van Amerongen, A., Lateral flow (immuno)assay: its strengths, weaknesses, opportunities and threats. A literature survey. Anal. Bioanal. Chem. 2009, 393 (2), 569-582.
27. Sajid, M.; Kawde, A.-N.; Daud, M., Designs, formats and applications of lateral flow assay: A literature review. J. Saudi. Chem. Soc. 2015, 19 (6), 689-705.
28. Quesada-Gonzalez, D.; Merkoci, A., Nanoparticle-based lateral flow biosensors. Biosens. Bioelectron. 2015, 73, 47-63.
29. Qiu, W.; Xu, H.; Takalkar, S.; Gurung, A. S.; Liu, B.; Zheng, Y.; Guo, Z.; Baloda, M.; Baryeh, K.; Liu, G., Carbon nanotube-based lateral flow biosensor for sensitive and rapid detection of DNA sequence. Biosens. Bioelectron. 2015, 64, 367-72.
30. Gong, X.; Cai, J.; Zhang, B.; Zhao, Q.; Piao, J.; Peng, W.; Gao, W.; Zhou, D.; Zhao, M.; Chang, J., A review of fluorescent signal-based lateral flow immunochromatographic strips. J. Mater. Chem. B. 2017, 5 (26), 5079-5091.
31. Akanda, M. R.; Joung, H. A.; Tamilavan, V.; Park, S.; Kim, S.; Hyun, M. H.; Kim, M. G.; Yang, H., An interference-free and rapid electrochemical lateral-flow immunoassay for one-step ultrasensitive detection with serum. Analyst 2014, 139 (6), 1420-5.
32. Wang, D. B.; Tian, B.; Zhang, Z. P.; Wang, X. Y.; Fleming, J.; Bi, L. J.; Yang, R. F.; Zhang, X. E., Detection of Bacillus anthracis spores by super-paramagnetic lateral-flow immunoassays based on "Road Closure". Biosens. Bioelectron. 2015, 67, 608-14.
33. Marks, H.; Schechinger, M.; Garza, J.; Locke, A.; Coté, G., Surface enhanced Raman spectroscopy (SERS) for in vitro diagnostic testing at the point of care. Nanophotonics 2017, 6 (4).
34. Amendola, V.; Pilot, R.; Frasconi, M.; Marago, O. M.; Iati, M. A., Surface plasmon resonance in gold nanoparticles: a review. J. Phys.: Condens. Matter 2017, 29 (20), 203002.
35. Faraday, M., The Bakerian Lecture: Experimental Relations of Gold (and Other Metals) to Light. Philos. Trans. R. Soc. London 1857, 147 (0), 145-181.
36. Lakowicz, J. R.; Ray, K.; Chowdhury, M.; Szmacinski, H.; Fu, Y.; Zhang, J.; Nowaczyk, K., Plasmon-controlled fluorescence: a new paradigm in fluorescence spectroscopy. Analyst 2008, 133 (10), 1308-46.
37. Zhao, Y.; Brun, E.; Coan, P.; Huang, Z.; Sztrokay, A.; Diemoz, P. C.; Liebhardt, S.; Mittone, A.; Gasilov, S.; Miao, J.; Bravin, A., High-resolution, low-dose phase contrast X-ray tomography for 3D diagnosis of human breast cancers. Proc. Natl. Acad. Sci. USA 2012, 109 (45), 18290-4.
38. Hernandez, J.; Thompson, I. M., Prostate-specific antigen: a review of the validation of the most commonly used cancer biomarker. Cancer 2004, 101 (5), 894-904.
39. Wang, M. C.; Valenzuela, L. A.; Murphy, G. P.; Chu, T. M., Purification of a human prostate specific antigen. Invest. Urol. 1979, 17 (2), 159-163.
40. Oesterling, J. E., Prostate Specific Antigen: A Critical Assessment of the Most Useful Tumor Marker for Adenocarcinoma of the Prostate. J. Urol. 1991, 145 (5), 907-923.
41. Ni, W.; Kou, X.; Yang, Z.; Wang, J., Tailoring Longitudinal Surface Plasmon Wavelengths, Scattering and Absorption Cross Sections of Gold Nanorods. ACS Nano 2008, 2 (4), 677-686.
42. Ke, C. S.; Fang, C. C.; Yan, J. Y.; Tseng, P. J.; Pyle, J. R.; Chen, C. P.; Lin, S. Y.; Chen, J.; Zhang, X.; Chan, Y. H., Molecular Engineering and Design of Semiconducting Polymer Dots with Narrow-Band, Near-Infrared Emission for in Vivo Biological Imaging. ACS Nano 2017, 11 (3), 3166-3177.
43. Liu, H. Y.; Wu, P. J.; Kuo, S. Y.; Chen, C. P.; Chang, E. H.; Wu, C. Y.; Chan, Y. H., Quinoxaline-Based Polymer Dots with Ultrabright Red to Near-Infrared Fluorescence for In Vivo Biological Imaging. J. Am. Chem. Soc. 2015, 137 (32), 10420-9.
44. Chan, Y.-H.; Wu, P.-J., Semiconducting Polymer Nanoparticles as Fluorescent Probes for Biological Imaging and Sensing. Part. Part. Syst. Char. 2015, 32 (1), 11-28.
45. Luo, W.; Wu, M.; Li, S.; Xu, Y.; Ye, Z.; Wei, L.; Chen, B.; Xu, Q. H.; Xiao, L., Nanoprecipitation of Fluorescent Conjugated Polymer onto the Surface of Plasmonic Nanoparticle for Fluorescence/Dark-Field Dual-Modality Single Particle Imaging. Anal. Chem. 2016, 88 (13), 6827-35.
46. Yezhelyev, M. V.; Gao, X.; Xing, Y.; Al-Hajj, A.; Nie, S.; O'Regan, R. M., Emerging use of nanoparticles in diagnosis and treatment of breast cancer. Lancet Oncol. 2006, 7 (8), 657-667.
47. Song, S.; Liu, N.; Zhao, Z.; Njumbe Ediage, E.; Wu, S.; Sun, C.; De Saeger, S.; Wu, A., Multiplex lateral flow immunoassay for mycotoxin determination. Anal. Chem. 2014, 86 (10), 4995-5001.
48. Khlangwiset, P.; Shephard, G. S.; Wu, F., Aflatoxins and growth impairment: A review. Crit. Rev. Toxicol. 2011, 41 (9), 740-755.
49. Kuiper-Goodman, T.; Scott, P. M.; Watanabe, H., Risk assessment of the mycotoxin zearalenone. Regul. Toxicol. Pharm. 1987, 7 (3), 253-306.
50. Pestka, J. J., Deoxynivalenol: Toxicity, mechanisms and animal health risks. Anim. Feed Sci. Technol. 2007, 137 (3-4), 283-298.