應用螢光各向異性檢測蛋白質聚集狀態之研究

Abstract

本論文整合螢光各向異性技術與適體生物晶片於手機式全內反射螢光顯微鏡，進行免疫球蛋白 G 聚集狀態之即時量測，目的是應用於相關疾病的檢測。傳統儀器大部分利用分子量或等效粒徑對蛋白質聚集進行定量，但常受限於本身的解析度。由於分子偏振狀態與分子型態之間密切相關，因此在本研究中利用螢光各向異性作為定量的依據，藉此光學方式達到即時性與非侵入式的量測。螢光各向異性產生機制包含分子的旋轉擴散與同分子間的螢光共振能量轉移。大多數相關研究中，僅考慮單一機制的影響，然而這兩種機制同時存在且無法忽略其中一方，則釐清在不同分子尺寸下兩者對於螢光各向異性的影響程度與彼此之間的差異，有助於提升定量的準確性。在實驗中我們透過有標定及沒有標定螢光的免疫球蛋白G進行特定莫耳濃度比例的配製，來達到旋轉擴散、同分子間螢光共振能量轉移與同於包含兩種機制等三種情況，產生分子聚集後再分離為單體、二聚體及低聚體，並利用穩態與奈秒解析方式進行量測。實驗結果中我們成功區分出兩種機制，並且證實旋轉擴散的確對於螢光各向異性有一定程度上的影響，同時驗證在奈秒解析量測下，不同分子尺寸之間螢光各向異性的差異更加明顯。為了提升對於不同分子尺寸穩態螢光各向異性的差異，我們利用全內反射螢光顯微術的表面激發與高訊噪比以及去氧核醣核酸適體的專一性等優點，將螢光各向異性技術與其整合在一起，並且設計出基於適體生物晶片並以螢光各向異性進行分析的手機式全內反射螢光顯微鏡。實驗結果中我們驗證全內反射式量測的優勢，同時也提高穩態螢光各向異性的差異性。本論文除了達到蛋白質聚集狀態的快速篩檢，並且利用結合手機的生物晶片量測裝置進行通訊醫療的研究與發展。

In this thesis, I integrated fluorescence anisotropy technique and aptamer biochip into a cellphone-based TIRF microscope, called TIRF Box, to analyze IgG aggregations in real time. Conventional apparatuses classify protein aggregations according to their molecular weight or equivalent diameter, but such analyses usually have limited resolution. Fluorescence polarization state is a direction measurement of molecular conformation, therefore fluorescence anisotropy is utilized here. Fluorescence anisotropy is decided by either rotational diffusion or homo-FRET on the molecules. Most studies discuss about homo-FRET effect only, but two mechanisms happen at the same time and none of them can be ignored. Hence, to distinguish the two different mechanisms will help the accuracy of fluorescence anisotropy measurements. In my experiments, samples having each of the two mechanisms were designed and prepared using specific molar ratio of FITC-IgG to IgG; they were measured by both steady-state and nanosecond time-resolved methods. In my results, the two mechanisms values in large proteins like IgG. My results also show that better resolution of fluorescence anisotropy data is obtained using time-resolved measurement. Furthermore, I combined DNA aptamer biochips with fluorescence anisotropy to build a compact TIRF microscope. In results, measurements under TIRF mode had better performance, and difference on fluorescence anisotropy in the steady-state way was successfully resolved by a cellphone camera. In conclusion, I’ve accomplished a method for rapid characterization of protein aggregations to realize the promise for telemedicine.