功能化螢光奈米鑽石的開發及其在生物標定與影像之應用

Abstract

以氮-空缺（nitrogen-vacancy center, NV center）中心為發光中心的螢光奈米鑽石為碳原子組成的新穎材料。因具有獨特且穩定的螢光性及高度的生物相容性，其已被廣泛的運用為生物影像應用中重要的顯影材料之一。然而，因其易於在生理緩衝溶液系統中產生凝集以及缺乏辨識專一蛋白的能力，致其難以運用於專一膜蛋白的顯影。因此，本論文首先致力於於開發具有專辨識功能的螢光奈米鑽石，隨後將開發之功能化螢光奈米鑽石做為顯影試劑用以解決現有顯影技術之瓶頸。 此篇論文共開發了兩種修飾螢光奈米鑽石使其具專一辨識功能的方法。其一為脂肪包覆法（lipid encapsulation method），此方法具備簡單及快速兩優點且其用於專一辨識功能用的官能機團能隨其特殊需求而加至奈米鑽石表面上。另一種方法為炔化超支化聚合甘油酯接枝包覆法（alkyne modified hyperbranched polyglycerol method），此方法較前述方法耗時，但由於此方法以共價鍵結方式逐一將甘油酯覆蓋於奈米鑽石表面，因此在奈米鑽石表面所形成的修飾層較前述脂肪包覆所形成的修飾層較為穩定。但此二種方法都能有效的使螢光奈米鑽石在生理緩衝溶液中依能保有良好分散性且具有專一辨識生物分子的功能。 在成功改良螢光奈米鑽石後，本論文接著利用此改良後螢光奈米鑽石應用於標定、定量及顯影細胞表面之膜蛋白。首先，藉由螢光奈米鑽石之螢光不易受外在環境干擾的獨特特性，我們成功的將生物素修飾之脂肪包覆螢光奈米鑽石做為整合式光學電子顯微鏡（correlative light electron microscopy）之顯影試劑來定位細胞表面的膜蛋白。再者，利用氮-空缺中心的磁光特性，生物素修飾之脂肪包覆螢光奈米鑽石能成功地被用於高靈敏度及高準確的測量細胞表面抗原的數目。最後，我們成功地將炔化超支化聚合甘油酯接枝包覆之螢光奈米鑽石用於長時間且連續的觀測細胞表面膜蛋白的動態。此應用是藉由螢光奈米鑽石螢光永不滅的特性，因此在目前其他現有的螢光染劑中（如：螢光蛋白或量子點），很難找到能達成相同的目的的試劑。 總括來說，此論文成功的提供兩種改進螢光奈米鑽石增加其實用性（如：使其具有辨識功能）方法。螢光奈米鑽石在經過此改良後，能成功的標定細胞上膜蛋白做為顯影試劑供長時間顯影使用，並能用於簡化現有整合式光學電子顯微鏡在樣品製備中所遇到的難題。

Fluorescent nanodiamonds (FNDs) containing nitrogen-vacancy (NV) defects as light emitters have been extensively used as contrast agents for bioimaging due to their superior optical properties, such as high photostability. Forming aggregates in biofluids and lacking specific targeting ability, however, have significantly impeded their applications in specific protein labelling and imaging. In this thesis, two strategies were developed to modify and functionalize FNDs to overcome existing limitations. The first approach, lipid encapsulation method has the advantages of simple manipulation and time-effective. Desired functional groups can be added onto FND surface through minor changes in the lipid composition. Alkyne-modified hyperbranched polyglycerol (alkyne-HPG) grafting by ring opening reaction is the other approach for functionalization of FNDs. Although this approach has longer processing time, the coating layer is much more stable because of the formation of covalent bonds between the coating layer and particles. Both coatings endow FNDs with not only high dispersity in physiological medium but also specific targeting ability of cell membrane proteins. By combining the exclusive optical features (e.g., chemical inert), biotinylated lipid coated FNDs (bL-FNDs) successfully simplified the complicated protocol to localize CD44 antigens on cell surface by correlative light electron microscopy (CLEM). A thorough literature search reveal that FND is to date, the only carbon nanomaterial having the ability to act as a dual contrast agent for CLEM. Moreover, taking advantage of magneto-optical property of NV- centers, highly sensitive and accurate quantification of CD44 antigens on cell surface with 35 nm of bL-FNDs was accomplished. Finally, high temporal and spatial resolution of continuous long-term observation of integrins α5 was achieved with alkyne-HPGFNDs. The superb photostability (no photo-bleaching and -blinking) of FNDs allows for the detailed transportation route of integrins α5 to be studied through short- and long-term observation, which cannot be viewed by any other dye molecules or quantum dots. To sum up, two reliable surface functionalization methods for FNDs was successfully demonstrated in this thesis. These novel FNDs shorten the gap between light and electron microscopy and serve as a platform for continuous long-term imaging of membrane protein tracking with high temporal and spatial resolution. In the future, the applicability of other kinds of biohybrid FNDs (e.g., antibody modified HPGFNDs or L-FNDs) may be conducted to further simplify the protocol of FNDs for biolabeling.