Biomedical Engineering Reference
In-Depth Information
blocking of the remaining free functional groups, were car-
ried out as
per
the i rst method. If BrCN was used, the bio-
logical material was bound 1.8 times more ef ectively. In this
case, the signal was higher and consequently the method
became more precise. Nitrocellulose (NC) was used for the
membrane strips, which were immersed in the biomaterial
solution (concentration of 0.1 μg/ml, 1 h at room tempera-
ture). Alter that they were desiccated and the remaining free
functional groups were blocked. h is procedure was similar
to that used in the “dot”-ELISA-method.
Careful examination disclosed that the biomaterial may be either
directly immobilized on the surface of the optrode, or tightly connected
with the special membrane. One of the components of the immunochemi-
cal reaction was conjugated with HRP. h e latter catalysis breaks up H
2
O
2
into oxygen radicals which are oxidized by luminol to yield aminophthalic
acid. h e excited part of the oxidized molecules of luminol emits quanta
of light. h e number of l ashes may be drastically increased in the pres-
ence of some chemical compounds. An example of such an enhancer is
used
p
-iodophenol. h e level of ChL depends upon a number of factors:
concentration of H
2
O
2
,
p
-iodophenol, luminol, pH media etc. h e optimal
conditions were: 2 mM H
2
O
2
, 0.06 mM
p
-iodophenol, 0.06 mM luminol
and 50 mM TB, pH 8.1.
Immunochemical reactions were carried out in two dif erent regimes:
(i) in stationary conditions, when the optrode was placed in the measur-
ing cell or in a capillary into which the analyzing solution was sucked and
on the wall of which the immunochemical interaction would occur; (ii) in
l owing regimes, where the analyzing solution was pumped through the
cell. h e optrode alone or placed in a capillary was immersed in a special
non-transparent cuvette. h e value of the luminescent signal was mea-
sured for a few minutes.
13.2.3.2
Determination of the Individual Indexes which are
Important for Diagnostics
On the surface of the optrode or the NC membrane, IgG was immobilized.
h e measuring cell was i lled with a solution of Ab to IgG, which were con-
jugated with HRP, and a sample of diluted serum. h e titer of Ab labeled
by HRP was 1:500. All dilutions were made in PBS, pH 7.2, containing
0.05% twin-20 and 1% BSA. At er 10 minutes, the cell was washed and
rei lled with a solution of the components needed to carry out the reaction
