Surface Modification and Functionalization of Fluorescent Nanodiamonds for Membrane Protein Targeting and Imaging

• Main Authors: Feng-Jen Hsieh, 謝豐任
• Other Authors: Chun-Hung Lin
• Format: Others
• Language: en_US
• Published: 2019
• Online Access: http://ndltd.ncl.edu.tw/handle/j53esv

博士 === 國立臺灣大學 === 生化科學研究所 === 107 === 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.