以金奈米棒及金奈米球為表面增強拉曼 散射基材之生物檢測應用

Gold Nanorods and Gold Nanospheres as Surface-Enhanced Raman Scattering Substrates for Biodetection

黃家琪(Chia-Chi Huang)

指導教授 ： 楊子萱 ； 周禮君

國立中正大學/理學院/化學暨生物化學系/博士(2012年)

摘要

生物感測器以表面增強拉曼光譜 (surface enhanced Raman spectrum, SERS) 形式讀出，大致可區分為表面增強拉曼標籤 (SERS tags) 及免標記法 (label-free method) 兩種不同型態之應用。本研究發展這兩項不同型態的生物檢測並分三個部分介紹如下：
第一部分研究為調控金奈米棒上的十六烷基三甲基溴化铵 (cetyl- trimethylammonium bromide, CTAB) 保護層的結構，成功地發展出以單顆金奈米棒為 SERS 基材之表面增強拉曼核殼標籤。其拉曼增強效應可與金奈米球聚集體相較。核殼結構提高拉曼標籤分子之穩定性並提供生物分子修飾的額外空間。將金奈米棒拉曼核殼標籤接上探針，以修飾biotin的聚苯乙烯球作為模擬細菌，兩者鍵結後，得到拉曼標籤訊號強度約為金奈米球聚集標籤 (Nanoaggregate-embedded beads, NAEBs) 的 1/3 倍；且 6 次實驗測得訊號強度變化量為6％內。該項標籤除了既有的拉曼特徵指紋外，還兼具螢光特色，可作為二元檢測。
第二部分研究為比較多種形狀之金奈米粒子直接偵測蛋白質的能力。利用免標記法進行 20 種胺基酸、類 RGD (Arg-Gly-Asp) 胜肽與蛇毒蛋白之表面增強拉曼光譜偵測。發現其中狗骨頭形之金奈米棒具最強的拉曼增效因子。本研究首次以該 SERS 基材獲得雨傘節 (Bungarus multicinctus) 和青竹絲 (Trimeresurus stejnegeri) 蛇毒蛋白之二級結構資訊。因此以狗骨頭形之金奈米棒作為 SERS 基材可提高生物巨分子檢測靈敏度。
第三部分研究以金奈米棒表面之CTAB 在不同濃度下的不同組態的模型進一步應用於大分子 DNA 序列之檢測。利用 CTAB 之正電離子與帶負電的 Hum23 序列 (TAGGG(TTAGGG)3) 庫倫吸引力及 CTAB 組態改變，成功地偵測到濃度為2 × 10-7 M Hum23序列之訊號。此外抗癌藥物 3,6-bis(1-methyl-4-vinylpyrazinium)carbazole diiodide (BMVC-4) 與 Hum23 形成的複合物可直接吸附於金表面。比較 BMVC-4 分子與 Hum23作用前後的 SERS 光譜差異，得到BMVC-4分子的pyrazine 環會與 DNA 骨架作用。本研究建立的 CTAB 於金奈米棒表面之不同組態的模型可作為操控分析物吸附於金奈米棒之原則，使得金奈米棒更適於作為生物感測元件。

關鍵字

金奈米棒 ； 金奈米球 ； 狗骨頭形金奈米棒 ； 表面增強拉曼光譜 ； 表面增強拉曼核殼標籤 ； RGD 胜肽 ； 青竹絲蛇毒蛋白 ； 雨傘節蛇毒蛋白 ； 咔唑衍生物 ； 生物檢測 ； 基材

並列摘要

There are two biosensing approaches based on surface enhanced Raman spectroscopy (SERS): surface enhanced Raman tags (SERS tags) and label-free methods. In this study, the biosensor fabrication methodology and the signal amplification technique can be described as the following three parts:
First, the status of protective layer of cetyltrimethylammonium bromide (CTAB) on gold nanorod (AuNR) surface can be modulated by controlling the concentration of CTAB. Thus, the monodispersive SERS core-shell tag based on an AuNR was fabricated successfully. Because of the core-shell structure, the SERS tags were more stable and the outer shell can provide a larger surface area for biomoleculear immobilization. AuNR SERS core-shell tags attached to polystyrene bead which mimics bacteria via biotin–streptavidin labeling protocol were used in SERS detection. These results show that the SERS signal intensity of AuNR core-shell SERS tags is about 1/3 fold compared to that of the nanoaggregate-embedded beads (NAEBs), suggesting a strong enhancement effect from the AuNR substrate. Moreover, the SERS signal variation among six repeated measurements of the detected AuNR core-shell beads was within 6%. Besides, the AuNR core-shell SERS tag encoded with both fluorescence characteristic and Raman signatures may lead to a kind of powerful dual-mode biosensor.
Second, 5 different types of SERS substrates were fabricated and applied to detection of 20 kinds of amino acids, mimicked RGD (Arg-Gly-Asp) peptides and snake venom. The SERS efficiency of 5 different SERS substrates was compared. Among these SERS substrates, the dog-bone shaped gold nanorod exhibited excellent SERS enhancement. Furthermore, this substrate can successfully distinguish the secondary protein structure between Bungarus multicinctus and Trimeresurus stejnegeri from their collected SERS spectra. According to the SERS results of dog-bone shaped AuNRs, the present work has developed a SERS substrate suitable for biological macromolecules detection.
Third, the model of the effect of CTAB morphology on the surface properties of AuNR accompanied by varying the solution CTAB concentration was established, and this model applied to DNA sequence detection was further demonstrated. The SERS signal of Hum23 at 2 × 10-7 M resulting from the coulombic interactions between the positive charge of CTAB and negative charge of Hum23 sequence (TAGGG(TTAGGG)3) was detected successfully. Furthermore, the anticancer drug 3,6-bis(1-methyl-4-vinylpyrazinium)carbazole diiodide (BMVC-4) and Hum23 sequence can form a complex and directly absorb on AuNR surface. According to the difference SERS spectra of BMVC-4 after interacting with Hum23, the results indicate that the pyrazine ring interacts with the backbone of DNA. Thus, this model could be a protocol for controlling the analyte absorbed on the Au surface, indicating the potential of AuNRs as a substrate for SERS detection.

並列關鍵字

gold nanosphere ； dog-bone shaped gold nanorod ； gold nanorod (AuNR) ； surface enhanced Raman spectroscopy (SERS) ； AuNR SERS core-shell tags ； RGD (Arg-Gly-Asp) peptides ； Trimeresurus stejnegeri ； Bungarus multicinctus ； snake venom ； 3,6-bis(1-methyl-4-vinylpyrazinium)carbazole diiodide (BMVC-4) ； biodetection ； SERS substrate

參考文獻

245. 方宏志，"利用拉曼探針分辨不同去氧核糖核酸結構"，碩士論文，國立臺灣大學化學研究所，( 2009)。

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