Biomedical Engineering Reference
In-Depth Information
antibodies binding to latex colloids from the resulting increase in
DI
[
38
].
An investigation with sub-micrometer pores by Uram et al
.
expanded on this
concept to detect immune complexes formed from the potential biowarfare agent
staphylococcal enterotoxin B (SEB) with polyclonal antibodies against SEB. Anal-
ysis of values for
DI
made it possible to estimate the number of proteins in these
immune complexes; this number ranged from 610 to 17,300 proteins [
49
].
An important point to note about (
9.1
) is that the net charge of proteins can alter
DI
<
if the ionic strength in the recording buffer is low (i.e.
300 mM). This effect occurs
because ions with an opposite polarity of a charged protein associate with the protein
in solution [
5
,
17
,
44
]. At low ionic strength, these counterions can affect the
conductance of the solution within a pore. In practice, however, low ionic strength
solutions are rarely used due to the decreased conductivity of the solution, which
reduces the magnitude of
DI
(
9.1
). The charge of a protein also has a significant effect
on the translocation time of proteins as discussed in the subsequent section.
9.2.2 Determining the Charge of Proteins with Nanopores
In addition to volume, the charge of a protein affects the translocation time,
t
d
,
of proteins passing through a nanopore. In general, translocation times are in the
range of tens to hundreds of microseconds, though for very large molecules, translo-
cation times have been observed in the range of milliseconds [
45
,
46
]. In particular,
the translocation time can be characteristic for certain proteins and thus useful
for protein identification. Measurements of translocation time are, however, chal-
lenging to predict accurately due to the effect of electroosmotic flow and the
possibility of interactions between the pore walls and a protein. Recent research by
Talaga and Li, described the distribution of
t
d
by the distribution function,
P
(
t
d
):
2
"
#
2
ð
d
p
v
t
d
Þ
"
#
exp
ðv t
d
þd
p
Þ
4
D t
d
l
p
l
m
< l
p
l
p
þ l
m
l
m
l
p
p
4
with
d
p
¼
Pðt
d
Þ¼
;
(9.2)
2
t
d
D t
d
p
where
v
(m s
1
) is the electrophoretic drift velocity and
D
(m
2
s
1
) is the diffusion
constant of the protein
within the nanopore
[
46
]. The electrophoretic drift velocity
is given by:
v ¼u
e
e;
(9.3)
2
The factor of 2 in the denominator of equation (9.2) is not present in the cited work by Talaga and
Li. Working with Talaga and Li, we determined that the factor of 2 in the denominator is required
for correct normalization such that the area of this probability density function equals 1.
Search WWH ::
Custom Search