Biomedical Engineering Reference
In-Depth Information
anticoagulant (sodium citrate, lithium heparin, EDTA) which is used for the
collection process [
8
]. In order to increase the accuracy, researchers have employed
N-terminal sequencing on individual spots to better identify the proteins in the
2D-PAGE pattern. For identification of individual proteins in each spot of 2D
pattern, other techniques can also be employed such as mass spectrometry followed
by peptide sequencing or immunoblotting and Western blotting [
8
].
In addition to 2D-PAGE, other techniques which can be employed for protein
separation are gel filtration such as size-exclusion chromatography or affinity
chromatography. The proteins collected from chromatography (which in most
cases do not need more separation) can be identified by mass spectroscopy and
peptide sequencing or N-terminal sequencing [
8
].
4.5 UV-Visible Spectroscopy
The UV-Visible Spectroscopy (UV-Vis) is based on the measurement of ratio of
the passed light (
I
1
) with respect to the incident light (
I
0
) in the wavelengths ranging
from UV to the visible. The UV-Vis spectra can be shown as absorbance or as
transmission. The adsorption of protein on the surface on NPs induces some
changes on the absorption spectrum such as broadening or shift of the absorption
peak. In the case of metallic NPs, the plasmonic peak can be monitored during the
protein adsorption on the surface of NPs. The plasmonic absorption (which is due to
collective oscillation of surface electron in metals) is highly sensitive to the surface
and environmental condition of the NP. In a study on the adsorption of BSA on gold
NPs, by increasing the time of incubation, the plasmonic peak showed a redshift
accompanied by peak widening (Fig.
4.4
)[
21
]. Casals et al. [
22
] also reported the
redshift of plasmonic peak of gold NPs incubated in cell culture medium due to
formation of a dense dielectric layer onto the surface of NPs.
The UV-Vis spectroscopy method is fast and simple and does not require
sophisticated sample preparation; however, it is not very accurate for quantitative
analysis. The dependence of absorption spectra on different parameters such as
size, aggregation, concentration, pH, and dielectric constant of environment makes
quantitative analysis difficult. As an example of the role of environment on absorp-
tion spectra, it has been shown that by increasing the concentration of azurin in the
solution, the absorption spectra of gold NP incubated in azurin change [
23
]. It
should be mentioned that although the quantitative application of UV-Vis spectra is
challenging, however, for those samples which follow the Beer-Lambert law,
especially when concentrations are low, by overlapping the spectra of single
components, it is possible to find the concentration of adsorbed protein. This
method has been employed to study BSA adsorption on carbon nanotubes [
24
].
