單分子光譜技術在生物物理和化學分析上的應用: I.微小脂質體的脂質動力學 II. 微流道內氧氣濃度梯度的量測和分析

Biophysical and chemical investigation using single molecule spectroscopy: I. Lipid dynamics in lipid bilayer of small unilamellar vesicles II. Probing oxygen gradient across micro-fluidic channel

10.6342/NTU201602829

趙孟軒

共聚焦顯微鏡 ； DMPC ； DPPC ； 微脂粒 ； 小型單層微脂粒 ； 異構化 ； 氧氣淬息 ； 側向擴散 ； 氧氣梯度 ； 微流道 ； 馬可夫分析 ； confocal microscopy ； DMPC ； DPPC ； liposome ； small unilamellar vesicles ； isomerization ； intersystem crossing ； oxygen quenching ； lateral diffusion ； oxygen tension ； Birth-death Markov process ； micro-fluidic device

國立臺灣大學化學系學位論文

2016年

博士

林金全

英文

過去的一二十年，顯微鏡的解析度大幅提升使單一分子的動態行為偵測變得可能，進而找出隱藏在原本大量粒子的平均量測中所看不到的細微變化。單分子技術已被廣泛應用於生物學、物理學至材料科學等眾多領域。此論文即以單分子技術為核心，進行二大主軸的研究。主軸一為利用單分子技術研究仿生膜的脂質動力學。我們製備了四種不同的微小脂質體，直徑大約一百奈米，其成分組成為DMPC、DMPC/膽固醇、DPPC、DPPC/膽固醇，並在膜內嵌入染料(DiD)好進行偵測。DiD在膜內的光動力學和擴散行為會受到周圍環境的影響，表現在其螢光放射隨時間的變化。首先，透過訊號的自相關(autocorrelation)分析，我們發現脂雙層的極化區域有可能是氧氣穿透的障礙。接著，藉由三重態(triplet-state)生命期與擴散常數(diffusion constant)的交叉比較圖，我們可推斷仿生膜內除了過去所認定的相位(phase)外，應該還有極小的脂質簇合物(ultra-small lipids cluster)產生，仿生膜的相位也有可能呈現不均勻分布。在主軸二，我們結合單分子和微流道技術，希望量測數十微米流道內氧氣濃度隨空間的變化。此實驗中，藉由靈敏高速的攝影機(EM-CCD)，我們可同時拍到散佈在流道內的染料分子的螢光表現，進而得到橫跨流道的氧氣梯度。因染料分子極小，空間上的解析度只取決於顯微鏡的繞射極限(~400nm)。相比於溶氧電極的大小(~3mm)，單分子技術偵測的策略大幅提升了空間解析度，相信能成為觀察血管中或著膜內外溶氧變化的主要工具。

In the last decades, single molecule spectroscopy has been highly developed to visualize exactly one molecule in complex environments. The well-established method allows the investigation of heterogeneity which is hidden in ensemble observation and help to enlarge our knowledge raging from biology to material science. In topic I, we utilized the technique to explore lipid dynamics in the membrane of four types small unilamellar vesicles (SUV): DMPC, DMPC/cholesterol (55:45 molar ratio), DPPC, and DPPC/cholesterol (55:45 molar ratio). We observed that the polar region of membrane might become oxygen transport barrier where lipid polar heads and cholesterols reduce oxygen diffusivity. Moreover, we found that lateral translational diffusion of DiD in lipids membrane is not only highly dependent on the lipids membrane phase, but also sensitive to the microenvironment discrepancy. Through the diffusion coefficient and triplet-state 2D correlation spectrum, we found DiD may locate at huge lipids domain, the lipids-packing defects or DiD induced ‘bouquet’ ultra-small clusters. In topic II, we probed the oxygen gradient across microfluidic channels using single molecule spectroscopy. Dye molecule is served as a probe. Due to the oxygen sensitivity to the triplet-state lifetime, the random-distributed dyes could sense the oxygen concentration at specific place. Each probe was measured under wide-field mode so that the gas variation could be visualized simultaneously. The detection limit in space ~400nm originates from diffraction limit which is largely lower than commercial oxygen elctrode ~3mm. The small, highly-respond technique might be an inspiration to the detection of dissolved oxygen (DO) distribution in vessel and gas gradient across membrane.

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