毛細管電泳雷射激發螢光偵測系統在適體量測上的應用

本篇論文研究的目的，是建立出一套、快速、多樣性、高靈敏度與高信賴度的蛋白質偵測法。採用具辨識功能的嶄新適體，來取代量少而昂貴的抗體偵測技術，並且結合奈米科技，將適體與帶有螢光的去氧核醣核酸同時鍵結在奈米等級的金粒子上，形成一個可將訊號放大的生物探針。這種生物探針，希望可藉由不同長度的去氧核醣核酸，來當作各個不同偵測物的代號，在同一時間與樣品下，做多樣性的分析。實驗中並架設出一台化學分析常用的毛細管電泳儀器，以雷射激發螢光方式偵測，在這種最高靈敏且安全偵測下，偵測範圍可接近毫微微莫爾濃度。並可以運用平台掃瞄裝置，在一次實驗之中，進行多毛細管電泳的分析。使用此系統另一個好處，可以快速在半個小時內完成約100鹼基長度分析。最後在類似酵素免疫分析法的三明治法，藉由兩個不同的適體對同一種蛋白質做雙重確認，達到高專一性的檢驗。此論文研究，先有系統一步步地證明毛細管電泳的高解析度與高靈敏度。利用密閉壓力注射方法，可將自由溶液或半黏稠膠質溶液，灌入細小的毛細管中。實驗證明出在較細微的毛細管徑中，於長時間強鹼作用之下，可以建立更好的介面電位，並大幅地提升分離解析度的理論板數。在雷射激發螢光的光學偵測系統中，藉由高倍率的物鏡，光學帶通濾鏡及偵測器的組成，利用空間解析的方法，降低背景的雜訊，得到較好的訊噪比，可以得到接近微微莫爾等級的偵測。辨識標的蛋白質的適體，分別固定在奈米金粒子與奈米磁珠上，待測樣中的標的蛋白質與這兩種奈米粒子上的適體作用後，形成三明治的複合結構。利用磁座吸附帶有磁性的複合結構後，洗滌掉未進行反應的物質，並利用DTT將奈米金粒子表面上的螢光長度條碼切下，以毛細管電泳辨別長度。樣品中多樣標的蛋白質的檢測與鑑定，可經由電泳尖銳而完整的生物條碼訊號辨別。

關鍵字

適體；凝血酵素；奈米金粒子；生物探針

並列摘要

This study establishes a new protein detection method, which is for fast, high sensitive, and accurate. The method uses a new class of biomolecules for molecular recognition to replace antibody which is expensive and bulky. Combining aptamer with nanoparticle constitutes a special probe. In this study, gold nanoparticles are conjugated with aptamers and fluorescently labeled oligonucleotides to detect the presence of target protein and to amplify the signals. This method can be extended to simultaneously detect multiple protein targets by incorporating barcode oligonucleotides of different lengths on the gold nanoparticles. A capillary electrophoresis (CE) instrument with laser-induced fluorescence (LIF) was developed. LIF is a very sensitive method enabling femto-molr detection limit. I also establish a scanning system for detection of multiple capillaries. Comparing to other separation methods, CE is efficient and has high resolution to separate 50 mer DNA. This new model system works like enzyme-link immunosorbent assay (ELISA) in liquid phase. By using two different aptamers recognizing different eitpopes of a target protein, this method identifies the protein target with high specificity. Parametric studies revealed that the established capillary electrophoresis system had high sensitivity and resolution. The studies concluded that smaller diameter of capillary yielded high zeta potential and the number of theoretical plates can be increased by longer alkaline treatment. The signal-to-noise ratio of the detection system can be optimized by using objective lens of higher numerical aperture and spatial filter as well as bandpass filter. With optimization, the system could reach detection limit in femto-molar concentration. To detect the target protein, aptamer probes recognizing the first epitope of the target protein were immobilized on gold nanoparticles, Apt-GNP. The Apt-GNP nanoparticles were also treated to have fluorescently labeled beacon oligonucleotides in the GNP. The aptamer molecules recognizing the second epitope of the target protein were immobilized on magnetic nanoparticles, Apt-MNP. In the presence of target protein, the Apt-GNP an Apt-MNP form a sandwich complex with the target protein. After they were captured by a magnet, the beacon oligonucleotides on the GNP were cleaved by dithiothreitol (DTT) and detected by the CE system with LIF detector to reveal the presence of target proteins in the sample.

並列關鍵字

aptamer ； thrombin ； gold nanoparticle ； bio-probe

參考文獻

[4]黃志清, 2004, 毛細管電泳與奈米感應器應用於DNA及蛋白質分析, 國立台灣大學化研所博士論文

[3]黃冠銘, 2005, 甘安酸溶液中金銀汞三金屬奈米棒子的合成與鑑定,國立台灣大學化研所碩士論文

[5]楊正義, 陳吉峰, 葉怡均, 陳正龍, 陳家俊,2001 ,金屬、半導體奈米晶體在生物檢測及分析上的應用

[6]S. Klussmann, 2006, The aptamer handbook

[7]R. Kuhn; S. Hoffstetter-kuhn, 1993, Capillary electrophoresis: principles and practice, Springer-Verlag

國際替代計量

毛細管電泳雷射激發螢光偵測系統在適體量測上的應用

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