應用多層自組裝金奈米粒子晶片於分析方法之開發與應用

Abstract

形狀不同的金奈米粒子會有各自獨特的侷域性表面電漿共振 (Local surface Plasmon resonance, LSPR) 吸收峰，由於這些金奈米粒子的LSPR對於外在環境的改變很靈敏，常被使用來感測微量樣品，隨著越多金奈米粒子被應用在分析領域中，可以從中得知當LSPR吸收峰位於近紅外光範圍會有較好的靈敏度，因此本論文第一部份將單層金奈米粒子晶片逐層自組裝修飾上金奈米粒子，利用金奈米粒子之間的交互作用產生出位於近紅外光波長的吸收峰，將此擁有近紅外光吸收波長的雙層金奈米粒子晶片結合上可對磷酸根有很好親和能力的二氧化鈦薄膜，來對複雜環境中的磷酸化胜□進行辨認，並使用吸收光譜為偵測方式來判斷樣品是否含有磷酸化胜□，並經由質譜確認後可達到快速檢測磷酸化胜□的目的。

傳統酵素消化反應的過程十分漫長，許多研究團隊致力於加速酵素消化的反應，例如使用微波、超音波以及紅外光等技術，可以將原本冗長的酵素消化反應時間給縮短到5分鐘甚至是30秒左右，因此本論文第二部份則是利用金奈米粒子具有的光熱轉換效應，將具有近紅外光吸收的多層金奈米粒子晶片，在經由紅外光雷射的照射下，金奈米粒子會吸收雷射能量轉換成熱能，提高胰蛋白□的活性來加快蛋白質酵素消化反應縮短所需的反應時間，經由快速酵素消化後的蛋白質的序列涵蓋率可和傳統酵素消化反應所得到的序列涵蓋率有一樣的效果，進一步將此方法應用在電泳膠內酵素消化上，可成功地應用在血清中蛋白質的分析，此種具有近紅外光吸收的多層金奈米粒子晶片結合紅外光雷射加速酵素消化反應的方法可將膠內酵素消化所需的時間大幅縮短。

Localized surface plasmon resonance (LSPR) is the unique optical feature of gold nanoparticles. The LSPR shifts of gold nanoparticles depend on the dielectric constants of surrounding environment. This LSPR property of gold nanoparticles has been applied in the development of analytical methods. On the basis this feature, two novel analytical methods were explored in this study. In the first part of this thesis, a two-layer-gold nanoparticle-based glass chip, which has a LSPR band appearing in the NIR region of the electromagnetic spectrum arising from dipole-dipole interactions between nanoparticles, was fabricated from layer-by-layer self-assembly. The surface of the glass chip was then spin-coated with titania, which is capable of interacting with phosphopeptides from complex samples. The LSPR shift resulting from binding specific analytes is greater when the LSPR band is in the infrared (NIR) region than that in ultraviolet-visible region. Absorption spectrophotometry was employed for the detection and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) was used for further confirmation of the results. In the second part of the thesis, a novel method for accelerating protein enzymatic digestion was proposed using multilayer of gold nanoparticle-based chip as the sample substrate. Conventional protein enzymatic protein digestion is quite time-consuming, which may generally take several hours. On the basis of the photothermal effect of the multilayer of gold nanoparticle-based chip which has the absorption capacity in the NIR region of the electromagnetic spectrum, protein enzymatic digestion was conducted in the glass chip under illumination of a NIR laser (808 nm) within 5 min. The digestion efficiency of the protein enzymatic digestion obtained using this approach is similar or even better than those obtained from conventional methods. The feasibility of using this approach for proteins-in-gel was also demonstrated.