發展零差量測之奈秒解析螢光各向異性技術以進行蛋白質聚集狀態之即時定量檢測

Abstract

本論文提出零差量測法的奈秒解析螢光各向異性技術，並將此技術應用於蛋白質聚集相關疾病的即時定量檢測。現有量測方法只能量測溶液中的蛋白質聚集現象，無法量測活細胞或是活體中的蛋白質聚集狀態，更無法進行即時定量之量測。為了突破現有技術的限制，本論文完成了兩部分的儀器架設與驗證。第一部分是改裝螢光顯微鏡之光路及光學元件，以進行螢光各向異性的顯微影像量測，並應用此新型螢光各向異性顯微鏡，針對癌症及免疫疾病有重大相關之鈣離子通道進行研究，本論文量測非興奮性細胞的膜蛋白中，調控鈣離子通道之鈣池調控鈣離子流入（SOCE）機制相關的基質交互分子（STIM1）與鈣離子通道蛋白（Orai1）聚集現象的動態變化。由實驗結果中，驗證本論文所架設的新型螢光各向異性顯微鏡能夠觀測蛋白質聚集狀態之即時變化。論文的第二部分中，發展零差量測之奈秒解析螢光各向異性技術，並且應用免疫球蛋白G（IgG）進行系統驗證與量測。本論文利用不同螢光標定方式，比較免疫球蛋白G（IgG）之分子運動行為與分子聚集數量。奈秒解析螢光各向異性實驗結果之中，對於不同螢光分子標定方式，會有以往文獻中不曾探討的差異存在，而此差異無法以穩態螢光各向異性進行解釋。則此部分實驗結果，驗證零差量測之奈秒解析螢光各向異性技術之優勢：對於蛋白質的聚集狀態具有更高度準確性，以及具有即時定量檢測的便利性。未來將可以藉由本論文之二部分實驗結果，進行零差量測之奈秒解析螢光各向異性顯微鏡系統之開發，以結合顯微影像以及奈秒解析技術，將可對蛋白質聚集現象有更精準快速的量測。

In this thesis, I developed a homodyne-based nanosecond-resolved fluorescence anisotropy technique, in order to achieve quantitative detection of protein aggregations in real time. Conventional methods can only measure protein aggregation in solution, which cannot be applied for living cells measurement in real time. In addition, conventional methods cannot provide quantitative information. Therefore, two different fluorescence anisotropy systems were built here, in order to demonstrate the potential of fluorescence anisotropy technique for protein aggregation detection. The first part of my thesis is on building fluorescence anisotropy microscope. This fluorescence anisotropy microscope was applied to measure formation of a calcium ion channel called store-operated calcium entry (SOCE), which is related to protein aggregation of the stromal interaction molecule 1(STIM1) and the calcium channel 1(Orai1) in non-excitable cells. My result showed the setup of fluorescence anisotropy microscope successfully captured in vivo protein aggregation process in real time. The second part of my work is to develop a homodyne-based nanosecond-resolved fluorescence anisotropy technique. The instrument I built was applied to measure immunoglobulin G (IgG) aggregation. Different fluorescent labelling methods were used to monitor and compare molecular motion of IgG aggregates, and interesting molecular motion was observed from nanosecond-resolved fluorescence anisotropy data. In conclusion, I have demonstrate the great potential of both fluorescence anisotropy microscopy and nanosecond-resolved fluorescence anisotropy technique for quantitative protein aggregation measurement. Future development of homodyne-based nanosecond-resolved fluorescence anisotropy microscope technique will certainly be useful for in vivo protein aggregation in real time.