- 學位論文
金屬奈米粒子標記於 DNA 微陣列晶片應用
Application of Metal Nanoparticles (MNPs) Labeling to DNA Microarray
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摘要
目前在微陣列晶片( microarray )分析中，對於基因表現( gene expression )的測量與定量越來越為重要，目前最常使用的便是螢光表現量的偵測方式為架構，然而使用螢光標記時我們需要將目標物放大( target amplification )以期得到較為足夠的信號源，再者螢光偵測所需的儀器系統也較為複雜，而偵測上容易受到螢光本身及螢光背景值的影響，因此我們希望可以發展一套更為簡單、低成本且快速的偵測方式。 本研究目標主要為發展金屬奈米粒子 DNA 檢測平台，利用各種不同金屬奈米粒子散射光譜的不同來達到多樣信號源的目的。在本論文中主要著重在金奈米粒子於 DNA 晶片的應用，由金奈米粒子區域表面電漿共振的特性，我們可以得到其散射光譜及粒子影像。論文中提出了金奈米粒子應用於 DNA 晶片的製備流程：包括晶片表面修飾、金奈米粒子與 DNA 的三明治雜交反應流程、估算金奈米粒子表面 DNA 分子數、金奈米粒子應用的偵測極限以及不同奈米粒子的初步實驗結果。 與 Cy3 螢光標記方法互相比較，本論文提出的金屬奈米粒子光學標記方法，大約有 1000 倍靈敏度的增加。利用本實驗室所發展的傅立葉光譜儀系統掃描，可達到大面積的散射光譜掃描，與商用的微陣列晶片掃描儀器比較，也較為簡便且成本為低。 本研究所發展的金屬奈米粒子 DNA 檢測平台，利用實驗室所建構的傅立葉光譜儀光譜掃描，可以讓我們達到低成本、快速掃描且高靈敏的偵測方式。
並列摘要
DNA microarray is a powerful tool allowing simultaneous detection of many different target molecules present in a sample. By using these techniques, large-scale parallel analyses can be performed in short time periods. Fluorescence technology has been the gold standard for detection of DNA on microarrays due to its high sensitivity, dynamic range, and multiplexing capabilities. Despite these attributes, fluorescence labeling typically requires target amplification to obtain sufficient amounts of target and complex instrumentation is needed for detection, ultimately limiting its utility in the applications described above. The development of a metal nanoparticle-based detection methodology for sensitive and specific DNA microarray is described. The technology utilizes gold and silver nanoparticles derivatives with thiol modified oligonucleotides that are designed to bind complementary DNA targets. Scattered light from nanoparticle-tagged biomolecules can be imaged and quantified extremely sensitively. It allows for detection and quantization by measuring evanescent wave induced light scatter with low-cost optical detection systems. Compared to Cy3-based fluorescence, gold nanoparticle probes provide for a 1000-fold increase in sensitivity.
參考文獻
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