Biology Reference
In-Depth Information
To determine the sensitivity of the colloidal probe method of mapping cell
surface proteins, Kim
estimated the density of speciic molecules
over a glass surface.
44
Since direct counting of the protein molecules after
immobilization on the glass surface was not feasible, they measured the
number density of cross-linkers that were ixed on the substrate surface. First
the amino-silanized glass was reacted with the covalent cross-linker, Sulfo-LC-
SPDP (Sulfosuccinimidyl 6-(3'-[2-pyridyldithio]-propionamido) hexanoate),
in a buffer solution. The amount of Sulfo-LC-SPDP that reacted with amino
groups on the glass surface was determined by measuring the optical density
of the buffer that contained the chromogenic by-product, pyridine-2-thione,
at 343 nm. The results showed that the surface-immobilized SPDP showed a
clear saturation behaviour. They chose a condition where the number density
of the cross-linker on the surface was about 1 × 10
4
/μm
2
. They then reacted
the activated glass surface with synthetic peptide named VN7 and coated
the glass bead on an AFM cantilever with anti-VN7 antibody. By changing
the concentration of antibody in the modifying solution, they prepared
AFM probes with different relative densities of antibody and measured the
adhesion property of each of the probe with the VN7-coated glass surface.
They concluded that it is easy to differentiate the peptide density of 100
times difference and possibility of differentiating 10 times difference under
favourable conditions. To estimate the number density of protein molecules
subsequently immobilized on the substrate surface by reacting with SPDP,
they assumed that the reaction eficiency of this step is 100%. This assumption
may not be correct, and a more direct way of counting the immobilized
peptides and protein on the surface should be tried.
Hinterdorfer
et al.
irst
developed a new method of imaging by AFM, named
“recognition imaging”, based on a speciic interactions between the ligand
immobilized on an AFM probe and speciic receptors on solid or biological
surfaces (see
Chapter 7
).
45-47
The method is based on the detection of
decreasing amplitude of cantilever oscillation where ligand-receptor
interaction sets in. The AFM probe is coated with ligand molecules with
a long tether of polyethylene glycol spacer and raster scans the sample
surface in a similar manner as in the tapping mode imaging of AFM.
48,49
By
using recognition imaging, Chtcheglova
et al.
et al.
50,51
mapped the localization
of vascular endothelial-cadherin on gently ixed microvascular endothelial
cells and ergtoxin-1 receptors on hERG HEK 293 cells within 2 μm square
regions. The method is capable of giving a simultaneous topographic image
of the ixed cell surface. Their method has a iner resolution compared with
Kim's method, but the area for mapping was conined to a narrower one of
approximately 2 μm
2 μm. It is interesting to observe that the high density
interaction areas in their mapping, when overlapped on the topography map,
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