Biomedical Engineering Reference
In-Depth Information
of enhanced ChL. Two-fold dilutions of IgG starting from 300 ng/ml
were used as standard solutions. h e signal reaches maximum value at er
17 min of exposure in the solution to be analyzed. h e response period
was half as much at er the addition of PEG in 0.01% i nal concentration.
h e sensitivity of analysis by the biosensors was similar with the ELISA
method and achieved about several ng/ml. In case of the determination
of the chorionic honadotropine (ChH) level the specii c monoclonal Ab
to its α-subunit were immobilized on the surface of the optrodes. h en
they were immersed into cell which contained 5 μl of blood serum diluted
in PBS (1:3) in the presence of 0.05% twin-20 and 1% BSA as well as 5 μl
of specii c Ab conjugated with HRP (dilution 1:100). For the calibration
curve the sample contained 5 μl of the above mentioned mixture and 5
μl of consecutive two-fold ChH dilutions (starting from 150 ng/ml) were
used. h e sensitivity of the analysis reached 10 ng/ml and the response
time was 5-7 min. h ese results were coni rmed by the ELISA-method in
the range of concentration of 10-100 ng/ml.
At the control of the estradiol-17 concentration the specii c monoclonal
Ab were covalently bound to the optrode surface. h en it was immersed
(on 10 min) in a cell containing 0.01 ml of human blood serum diluted
with PBS (1:3) in the presence of 0.05% of twin-20. Measurements were
carried out by two dif erent ways. In the i rst one at er washing in the above
mentioned buf er the optrodes were placed in a cell containing 0.01 ml
of estradiol-isoluminol conjugate (50 ng/ml). h en they were placed in
a non-transparent cell i lled with a mixture of
p
-iodophenol (0.06 mM),
HRP (4 mM), H
2
O
2
(20 mM) and TB (10 mM), pH 8.2. For calibartion
of optrodes the samples containing 5 μl of consecutive two-fold estra-
diol-17 dilutions starting from 50 ng/ml and 5 μl (50 ng/ml) of estradiol-
isoluminol conjugate were used. According to the second way (sandwich
variant) the estradiol-isoluminol conjugate was replaced by specii c Ab
linked with HRP. h e controlled concentration was the same as in case
for ChH. In both cases the sensitivity was about 6 ng/ml. h e total time of
analysis in the second case was approximately in two times less than in the
i rst one (~20 min). A strong correlation (r=0.95) was found between these
results and obtained by the ELISA- method.
In the next experiments, the developed method was examined at the con-
trol of α-2-interferon in the serum blood. h e monoclonal Ab to recombi-
nant α-2-mterferon were immobilized on the inside wall of a capillary (inner
diameter 1.0, length 6 mm). h e quartz optical i ber (diameter 0.8, length 75
mm) was put into the capillary. h e sample (2 μl) of purii ed α-2-interferon
linked with HRP (1:200 dilutions with 0.05% twin-20 and 1% BSA) and the
analyzing sample (of the same volume) were sucked into the capillary. At er
