以二氧化矽包覆含有標記分子的金奈米棒做為生物檢測之表面增強拉曼標籤

Silica-coated Marker-embedded Gold Nanorods as Surface-enhanced Raman Scattering Tags for Biochemical Detection

劉盈君(Liou, Ying Jyun)

指導教授 ： 楊子萱

國立中正大學/理學院/化學暨生物化學系/碩士(2014年)

摘要

表面增強拉曼散射(SERS)核殼標籤具有高靈敏度、使用單一波長雷射激發、適用於多重性檢測(multiplex detection)等優點。在本實驗室先前的研究中，使用二氧化矽包覆金奈米球與標記分子(marker molecule)，製成奈米聚集團粒子(nanoaggregate-embedded bead, NAEBs)。此粒子訊號強大並且穩定，但缺點為不易控制核殼標籤中金奈米球的聚集程度，因此沒有強度的再現性。使用金奈米棒為SERS基材可以改善上述缺點:單顆金奈米棒產生的SERS效應較金奈米球強，因此以二氧化矽包覆金奈米棒作為SERS核殼標籤為一種有較好強度再現性的辦法。先前的研究已經確定SERS核殼標籤的訊號約為NAEBs的1 /3倍，顯示金奈米棒可提供SERS核殼標籤足夠的訊號強度。
本實驗使用4-nitrothiophenol (4-NTP)作為標記分子、金奈米棒為SERS基材，並使用二氧化矽作為殼層製成SERS核殼標籤。在合成過程中，金奈米棒須要二次離心，但導致金棒不穩定且容易聚集，因此需要額外加入保護劑十六烷三甲基銨溴(cetyl-trimethylammonium
bromide, CTAB)。我們根據金奈米棒之介達電位(zeta potential)來判斷需要加入之保護劑最終濃度。當金棒的介達電位為48 ~ 50 mV、51 ~ 54 mV及54 ~ 57 mV時，需要額外添加的CTAB最終濃度為28.8 ~ 29.3 mM、28 ~ 28.8 mM及27.3 ~ 28 mM。
第二部分的實驗為檢測SERS核殼標籤訊號強度之再現性。首先測量水溶液中的SERS核殼標籤，發現在15 nM ~ 45 nM濃度範圍SERS訊號強度與濃度呈現線性關係。測量3次不同批的樣品其訊號強度偏差為10 %內。將SERS核殼標籤連結模擬細菌的聚苯乙烯球(polystyrene beads, PS球)進行SERS強度檢測，SERS訊號強度會隨著雷射光的聚焦位置不同而改變。訊號強度再現性最佳之聚焦位置在PS球表面。測量四顆PS球之訊號強度差異在9 %以內。最後，利用二股互補的DNA分別修飾在矽基板及SERS核殼標籤表面，並透過DNA的雜交將SERS核殼標籤固定於矽基板上。偵測不同位置之SERS訊號，初步得到SERS強度的偏差值為21 %。

關鍵字

拉曼散射 ； 標籤

並列摘要

The surface-enhanced Raman Scattering (SERS) core-shell tag has several advantages on multiplex detection of biological species in one sample simultaneously. For example, one can in principle prepare numerous Raman barcodes, they have very high sensitivity on detection, and all of them can be read out by a single laser excitation. In previous research, the nanoaggregate-embedded bead (NAEB) were synthesized as the Raman tag, which was modified by an antibody to specifically combine to the antigen that was modified on a polystyrene bead (PS bead); the SERS signals of the NAEBs were successfully detected on a single PS bead. However, the SERS intensity of the NAEB is not reproducible because it is difficult to control the aggregation number of nanoparticles.
Therefore, the first part of our research is the synthesis of silica-coated marker-embedded single gold nanorod (AuNR) as a SER tag. 4-nitrothiophenol (4-NTP) is used as the marker molecule. The SERS intensity of such SERS tags was measured as one-third of the intensity of NAEBs. It showed a strong enhancement effect from the AuNR substrate. Compared with previous research, we adjusted the concentration of cetyl-trimethylammonium bromide (CTAB) before silica coating. The concentration of CTAB is determined by the zeta potential of the AuNRs. When the zeta potential of the AuNR are48 ~50 mV, 51 ~54 mV, or 54~57 mV, the additional concentration of CTAB is 28.8 ~ 29.3 mM, 28 ~28.8 mM, or 27.3 ~ 28 mM, respectively.
In the second part of this thesis, we demonstrated the reproducibility of SERS intensity on the AuNR SERS tags. Firstly, we measured the SERS signals of the SERS tags in different concentrations to confirm that there is a linear correlation between the SERS intensity of 4-NTP and the concentration of AuNR tags. The percentage of the standard deviation (STD) of three samples is about 10 %.Then we measured the SERS signals of the Raman tags on the mimic bacterium PS beads at different focal positions to determine the experimental condition that can achieve high reproducibility. The result shows that the focal position on the top of the PS bead gave the best reproducibility of SERS intensity. At that position, the average value, the STD, and the percentage of the STD to the peak intensity are 3115, 280, and 9%, respectively. Finally, we immobilized the SERS tags on the silicon wafer by DNA hybridization and detected the SERS signals at different position. The preliminary result showed that the percentage STD of SERS intensity is about 21 %.

並列關鍵字

Raman scattering ； SERS tag

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