全反射螢光顯微技術應用於蛋白質分子之即時偵測與操控

Real-Time Protein Monitoring and Manipulation Using Total Internal Reflection Fluorescence Microscopy

顏毅廣(Yi-Kuang Yen)

指導教授 ： 黃榮山

國立臺灣大學/工學院/應用力學研究所/碩士(2004年)

https://doi.org/10.6342/NTU.2004.01844

摘要

自從90 年代後期人類基因計劃解出大量基因體資料，以及電腦處理資訊速度大為發展，使得蛋白質可以被快速測定。蛋白質結構、功能性與其分子間的作用，都是蛋白質體研究裡相當重要的課題。
本文首次成功地觀測與分析抗原分子產生的動態行為和其與抗體分子間作用，並量測到其動態運動三度空間的位置與速度表現，此乃是藉著整合全反射螢光顯微術與微機電製程技術，進行蛋白質在物性環境改變時，。利用全反射光場以漸逝波激發螢光，使全反射螢光顯微術具有離玻璃基材150至700奈米尺寸等級以內的觀測功能，並且其影像的訊號背景比遠高於其他的顯微技術；而在橫向尺寸的功能也藉著量測1微米的螢光粒子得到了驗證。另外，配合速度每秒30張畫面的影像擷取系統，可對標定螢光之抗原分子Anti-IgG，進行即時偵測了解其從運動至與抗體鍵結時之狀態，並加以分析。
利用微機電製程技術，設計並製作整合氧化銦錫 (ITO)透明電極與PDMS微流道之生物晶片，為了達到尺寸微小化(1.6與0.8微升)，且能與符合目前商用蛋白質生物晶片之規格，與蛋白質分子IgG和Anti-IgG相容。藉此在單分子偵測上，本文首次利用全反射螢光顯微鏡即時觀察到在固液相單一抗原分子Anti-IgG結合於抗體的狀態(1張/30秒)；同時也觀察到分子在流體邊界層運動行為，並成功地追蹤並分析抗原分子在流體邊界層的三度空間運動軌跡(空間範圍間距：X軸 14.7微米；Y軸 1.3微米；Z軸 0.24微米)與速度表現，與流體理論作驗證，發現反應物分子(抗原)擴散受到流體邊界層所影響。此外，藉著生物晶片以施加直流電的方式操控奈米粒子，實際達到操控粒子的運動，觀測粒子的運動狀態，並發現電壓與粒子運動速度呈線性增長，將有助於未來在蛋白質分子間結合與解離作用的研究。
由於生物分子領域上仍有許多未知的現象，全反射螢光顯微術與微機電製程技術在單分子影像之觀測將扮演重要的角色。以了解生物分子在生物晶片上之表面特性與流體邊界層在晶片表面對生物分子之擴散的影響，以利未來在研究上或是奈米生物技術上之發展潛力。

關鍵字

全反射螢光顯微鏡 ； 漸逝波 ； 微機電技術

並列摘要

The study of biomolecular recognition has been becoming crucial to provide insights into molecular genetics, design of biosensor devices, drug design, and development of targeted drug delivery systems. The in-depth understanding of biomolecular recogntion involves adsorption, interaction and desorption as well as associated manipulation between biomolecules. In this present study, we have successfully demonstrated a single biomolecular detection and real-time tracking of anti-IgG in a microchannel using total internal reflection flurorescence (TIRF) microscopy for illustration of protein adsorption and recognition.
TIRF microscopy is a well-suited technique for real-time imaging and monitoring of a single protein molecule in nano-layer fluidics due to its unique evanencent wave at the optically index-mismatch interaface that may excites fluorescences at the transparent near-wall region. Recent advances in charge coupled device (CCD) camera detection efficiency and speed have enabled the microscopy of temporal and spatial resolutions to be far-reaching 0.033 ms and 0.3 μm, respectively. A modified inverted TIRF microscopy was newly established, which allows a directed laser beam underneath through the inverted microscopy to be incident in a critical angle into the surface inside the microchannel. In this microchannel, the conductive ITO film was deposited to be electrically feedthrough for electrical manipulation. Based on the fluorescent beads analysis, which are 1.1 μm in size, the capability of TIRFM for single molecules monitoring and tracking, even the measurement in lateral size of molecules was demonstrated.
In this study, the TIRFM incorporates a MEMS-based electrical control biochip to monitor and track the motion of a single antigen molecule in which the real-time position and velocity of each frames were tracked and measured. The motion of an antigen molecule was founded to be dominated in a hydrodynamic boundary layer. A further comparison of experimental results with theoretics appears to be deviated unexpectedly, which provokes a further thought that a nano-layer fluidics exhibits a novel non-classical fluidic characteristics.
At last, the motion of fluorescence nanobeads manipulated by the application of DC voltage was real-time monitored. The velocities of beads were shown to be in a linear accordance with the applied voltages. As a result of the accordance, the manipulation of nonobeads with electrical control was demonstrated and verified.

並列關鍵字

MEMS ； TIRFM ； evanescent wave

參考文獻

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