Detection of Protein-Protein Interactions and Imaging MCF-7 Cells with (3-(2,2,2-Trifluoroethyl)phenyl)me- thanethiol-ConA Capped Gold Nanoparticles
Date Issued
2008
Date
2008
Author(s)
Yang, Cheng-Han
Abstract
The objective of this thesis is to develop a chemical method to immobilize sugar binding proteins onto gold nanoparticles with different sizes as well as to investigate the possibilities of using the resultant proteins-capped gold nanoparticles in screening protein/protein interactions and imaging surface tomography of breast cancer cells. The cognate substrate of ConA appended with 3-(trifluoroethyl)-3- phenyldiazirine was synthesized and the resultant photoaffinity ligand 8 was introduced to the surface of ConA based on the characteristics of ConA’s specific substrate binding and photo-activated labeling. By removing 8, ConA not only displaying the surface thiol groups (denoted as SH-ConA) but also possessing the free binding site for the mannose are thus generated. The structural features of linkers play some roles in the dispersion of gold nanoparticles. Thus, we design and synthesize some functional (namely Linker MMUA and Linker MTA) linkers with maleimide functionality and nonfunctional (Linker DO, Linker DOEG and Linker DA) linkers. SH-ConA is chemically modified to 32-nm gold nanoparticles by reacting with maleimide of functional linkers via 1,4-addition yielding ConA-GNP-MTA. Nonfunctional linkers not only dilute the concentration of functional linkers on the gold nanoparticle surface, but also increase the repulsion among gold nanoparticle by hydrophilicity and charge density. Based on experimental observation, the best linker to serve our purposes is Linker DA. By monitoring the changes of plasmon resonance absorption bands of gold nanoparticles, we successfully find the optimal concentration of sodium chloride solution, which can neutralize surface charge of ConA-GNP-MTA, for ConA-GNP-MTA to interact with BS-I in the protein-protein interaction experiment. However, the selectivity of ConA-GNP-MTA to other proteins is not as good as what we expected. Applying the same immobilization strategy, SH-ConA successfully encapsulates 1.2-nm gold nanodots, which fluoresce strongly in aqueous solution. The resultant ConA-Audot-MTA is thus evaluated in the application of MCF-7 breast cancer cells imaging. However, ConA-Audot-MTA aggregates under the cell imaging experiment condition. The experiment results could be possibly improved by changing the signal transduction unit or characteristic features of linkers.
Subjects
Gold Nanoparticles
Concanavalin A
Protein-Protein Interactions
SDGs
Type
thesis
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