利用以羧甲基亞磷酸修飾之氧化鋅奈米線進行癌症細胞辨識

Cancer Cell Identification by Using ZnO Nanowires Modified by Carboxyalkylphosphonic Acids

黃柏豪(Po-Hao Huang)

指導教授 ： 黃建璋

國立臺灣大學/電機資訊學院/光電工程學研究所/碩士(2015年)

https://doi.org/10.6342/NTU.2015.00458

摘要

在現今癌症檢測技術中，最常見為螢光染色法，然而螢光染色法存在許多缺點，例如螢光衰退、螢光顏色的限制與細胞自體螢光分辨上的困難，這些都使得醫師在手術的過程中難以達成癌症細胞之即時檢測。
為了解決上述困難，氧化鋅奈米柱 ─ 一種半導體奈米尺度材料，有能力傳統螢光染色之有機螢光物質。氧化鋅擁有優異的光學特性並常應用於生醫相關研究及產品，且氧化鋅對於蛋白質皆有很高的親和力，因此我們利用具有特異性的生物抗體與奈米線的接合形成了特殊的生物標記，可成功識別癌症細胞。
在這篇論文中，我們利用另一種半導體材料 ─ 二氧化鈦奈米線，來達到雙色的癌症細胞檢測。二氧化鈦亦擁有優異的光學特性、對於蛋白質也有很高的親和力。氧化鋅/抗體的生物標記將用來標記癌細胞，而二氧化鈦/抗體的生物標記則用於標記正常細胞。由光致發光頻譜與顯微影像的結果，可證明此技術能成功辨別癌細胞與正常細胞。我們也做了一系列的量化實驗，將生物標記的濃度、有效檢測細胞數量範圍與取得之光學頻譜分析比較並找出規律，最後更模擬真實細胞分布狀況將正常細胞與癌症細胞共同培養並且按照不同比例做癌症細胞檢測實驗。
另外，雖然奈米線與抗體間的接合可藉由簡單的吸收來完成，但利用有機連結物在兩者間形成共價鍵結是較好的方法。一方面能使奈米線與抗體結合更為堅固，另一方面也能增加抗體的吸附量、並避免非專一性接合。在此篇論文中，我們利用羧甲基亞磷酸在奈米線表面形成自組單層；利用EDC與NHS活化此自組單層後，抗體便能藉由新生成的共價鍵固定於奈米線上。藉由抗體接合測試、光致發光頻譜與顯微影像的結果，我們證實經修飾的奈米線與抗體的接合將更為堅固，因此，對氧化鋅奈米線的修飾能成功提升癌症細胞檢測的效率。

關鍵字

癌症細胞檢測 ； 氧化鋅 ； 二氧化鈦 ； 奈米線 ； 自組單層 ； EDC ； NHS

並列摘要

Among cancer cell detection methods, fluorescence microscopy cancer detection is one of the most common methods. However, there are many disadvantages for fluorescence cancer detection such as the phototoxicity, the limited number of available fluorescent channels, and the overlap of the excitation and emission spectra of the stains. Furthermore, under a constant light illumination, it possesses the issue of photobleaching, making real time surgery difficult.
ZnO nanowires, one of the semiconductor materials in nano-scale, have the ability to replace the organic fluorescent substances for those drawbacks mentioned above. ZnO nanowires have exceptional optical properties, and they are often applied to biomedical research and commercial products. In addition, due to great affinities between ZnO nanowires and many proteins, ZnO bounded to specific antibodies are regarded as biomarkers to identify the cancer cells.
In this thesis, we applied another kind of the semiconductor materials, TiO2 nanowires, to achieve the bi-color cancer cell identification. TiO2 nanowires also have great optical properties, and they have great affinities to many proteins, too. The ZnO/antibody biomarkers were applied to mark the cancer cells, while the TiO2/antibody biomarkers were applied to mark the normal cells. From PL spectra and bio-images, our idea for cancer cell detection by semiconductor nanowires was confirmed. A series of quantitative analyses, including biomarker concentration limit, the valid range of cell numbers, and co-culture case to simulation the real situation were conducted to examine the relationship between the optical response from biomarkers and cell numbers.
Though the attachment of antibodies to the surface of ZnO can be achieved through simple adsorption, formation of a covalent bond between the antibody and the oxide surface via a chemical crosslinker is the preferred method. The covalent bonds makes the conjugation more robust, and prevents low coverage of antibodies or non-specific binding. In this thesis, carboxyalkylphosphonic acid would form the self-assemble monolayers (SAMs) on the nanowires. After the activation of the SAMs by EDC/NHS, the antibodies would be immobilized on the nanowires by the newly-formed covalent bonds. Regarding the results of binding tests of antibodies, Photoluminescence spectra, and the bio-images, we proved that the conjugation between nanowires and antibodies would be stronger during the modification process, so the modification on the ZnO nanowires successfully enhances the sensitivity of the cancer cell detection.

並列關鍵字

Cancer cell detection ； ZnO ； TiO2 ； Nanowires ； Self-assemble monolayers ； EDC ； NHS

參考文獻

[1-2] S. C. Yang, Y. C. Shen, T. C. Lu, T. L. Yang, and J. J. Huang, "Tumor detection strategy using ZnO light-emitting nanoprobes," Nanotechnology, vol. 23, p. 055202, 2012.

[1-3] T. C. Lu, M. Y. Ke, S. C. Yang, Y. W. Cheng, L. Y. Chen, G. J. Lin, et al., "Characterizations of low-temperature electroluminescence from ZnO nanowire light-emitting arrays on the p-GaN layer," Optics letters, vol. 35, pp. 4109-4111, 2010..

[1-4] N. X. Jun Zhou, and Zhong L. Wang, "Dissolving Behavior and Stability of ZnO Wires in Biofluids: A Study on Biodegradability and Biocompatibility of ZnO Nanostructures," Advanced Materials, vol. 18, pp. 2432-2435, 2006.

[1-6] Q.S. Wei, K. Hirota, K. Tajima, K. Hashimoto 2006 Design and Synthesis of TiO2 Nanorod Assemblies and Their Application for Photovoltaic Devices Chemistry of Materials 18 5080-5087

[1-8] J. Homola, S. S. Yee, and G. Gauglitz, "Surface plasmon resonance sensors: review," Sensors and Actuators B: Chemical, vol. 54, pp. 3-15, 1999.