Detection and Characterization of Protein Aggregates by Fluorescence Anisotropy

• Main Authors: Feng, Ya-Lan, 馮雅蘭
• Other Authors: Chen, Yi-Chun
• Format: Others
• Language: en_US
• Published: 2016
• Online Access: http://ndltd.ncl.edu.tw/handle/33066040771199748512

碩士 === 國立交通大學 === 光電系統研究所 === 105 === 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.