Capillary Electrophoresis for Protein Analysis and Bioapplications of Gold-based Nanoparticles
Date Issued
2006
Date
2006
Author(s)
Huang, Yu-Fen
DOI
en-US
Abstract
In order to develop novel analytical techniques for the analysis of biomolecules such as proteins and aminothiol compounds, we take the advantages of high efficiency, rapidity of capillary electrophoresis (CE) and unique optical and large surface properties of nanomaterials. CE in conjunction with laser-induced fluorescence has been separately applied to study the differential activity of enzymes in free solution and those are bound to gold nanoparticles (AuNPs) and improved sensitivity and efficiency for protein analysis. Differently shaped and sized Au-based nanoparticles (NPs) have been prepared and some of them have been tested to be efficient selectors and matrices for aminothiols compounds in surface assisted laser desorption ionization-mass spectrometry (SALDI-MS).
The specificity and activity of trypsin that is conjugated to gold NPs (AuNP-trypsin) for proteins have been investigated by micellar electrokinetic chromatography (MEKC) and capillary zone electrophoresis (CZE) with laser-induced-fluorescence (LIF) detection. In the presence of sodium dodecyl sulfate (SDS), adsorption of the tryptic fragments on AuNP-trypsin and on the capillary wall is reduced. Thus, the sensitivity and resolution of the tryptic fragments from bovine serum albumin (BSA) is improved. According to the electropherograms, the activity of AuNP-trypsin is lower than that of free trypsin. It is suggested that changes in the conformations and steric effects contribute to the loss of activity and changes in specificity of trypsin adsorbed on AuNPs.
An on-line concentration and separation method for analyzing large-volume protein samples by CE-LIF is described. After the injection of 1.0-μL samples, proteins migrate against the electroosmotic flow (EOF) and enter the poly(ethylene oxide) (PEO) zone; this process causes them to slow down and stack at the boundary between the PEO. Either 0.01% SDS or 0.01% PEO was used as sample additives to improve the stacking and separation efficiencies. By applying a short plug of 0.2% SDS prior to sample injection, the microheterogeneity of the proteins can be resolved. 12 peaks are detected when injecting 1.0-μL of sample containing six model proteins (0.1 μM). The limit of detection (LOD) for α-lactalbumin is 0.5 nM, which is an 84-fold sensitivity enhancement over the traditional method.
Preparation of Au core–Au–Ag shell NPs in different morphologies can be easily achieved by controlling both the pH and the glycine concentration. Using a seed–growth method, we prepared high-quality Au–Ag NPs from a glycine solution under alkaline conditions (pH > 8.5). Dumbbell- and peanut-shaped Au core–Au–Ag shell NPs were prepared in aqueous solution at the concentrations of glycine greater than 0.5 M and greater than 0.2 M at pH 9.5 and 10.5, respectively. In addition, we were able to affect the shapes and sizes of the Au–Ag NPs by controlling the reaction time. At pH 9.7, we observed the changes in the morphologies of the Au core–Au–Ag shell NPs—from regular (rectangular) to peanut- and dumbbell-shaped, and finally to jewel-, diamond-, and/or sphere-shaped—that occurred during the course of a 60-min reaction.
We further prepared differently shaped and sized Au–Ag nanocomposites from gold nanorod (AuNR) seeds in various amino acid solutions at the pH values ranging over 8.0–11.5. Our study shows that the pH as well as the concentration and species of amino acids have great impacts on the preparation of I-liked, dumbbell-liked, sphere-liked, peanut-liked, and corn-liked NPs as well as nanowires (NWs), mainly through the control of the reducing ability of ascorbate (or amino acids), oxidizing abilities of Au and Ag ions, and/or recognition capability as well as surface charges of the amino acids on the AuNRs. Depending on the value of pH, we were able to prepare I-shaped, dumbbell-shaped, and/or sphere-shaped Au–Ag nanocomposites in 0.1 M solutions of Arg, Gly, Glu, Gln, Lys, and Met. In His solutions at pH 8.0 and 9.0, we obtained peanut-shaped Au–Ag nanocomposites. Corn-shaped Au–Ag nanocomposites were prepared in 0.1 M Met solutions (pH 9.0 and 10.0). By controlling the Lys concentration at pH 10.0, we synthesized pearl-necklace-shaped Au–Ag nanoparticles and Au–Ag wires.
Nile Red-adsorbed AuNPs (NRAuNPs) has been demonstrated as selective probes and matrices for the determination of aminothiols through SALDI-MS. Due to the high specificity of NRAuNPs toward thiol-containing compounds as well as the aggregation induced by the binding of these molecules to the surfaces of NRAuNPs, NRAuNPs are capable to selectively concentrate three aminothiols—glutathione (GSH), cysteine (Cys), and homocysteine (HCys) in the precipitate—from a mixtures containing four amino acids. The preconcentration approach also provides an LOD of 25, 54, and 34 nM, for the determinations of GSH, Cys, and HCys, respectively. This method was validated in the analyses of GSH in red blood cells and of Cys in plasma and has great potential for diagnosis.
Subjects
毛細管電泳
金奈米粒子
金-銀奈米粒子
表面輔助雷射脫附游離化質譜
Capillary electrophoresis
gold nanoparticles
gold-silver nanocomposites
SALDI-MS
Type
thesis
File(s)![Thumbnail Image]()
Loading...
Name
ntu-95-F91223005-1.pdf
Size
23.31 KB
Format
Adobe PDF
Checksum
(MD5):a2361402549a6bd7203aac47b4242e0b