Biomedical Engineering Reference
In-Depth Information
1.6
Negative
1.4
Positive
1.2
1.0
0.8
0.6
0.4
0.2
0.0
1 - 1,000
1- 2,000
1-5,000
1-10,000
1-20,000
Sample dilution
FIGURE 22.6
Amperometric signal obtained for control hemolyzed mouse blood sample using AP-labeled conjugate and
nafion-coated electrodes at various sample dilutions. Error bars represent standard deviation from the mean of
four runs, on a single control sample.
22.4.2 Effect of H
2
O
2
Used in the Substrate
Approximately 10
L of hemolyzed blood samples and nonhemolyzed samples were
treated with 30 µL of 1 M H
2
O
2
on a glass plate. Evolution of gas was observed suggest-
ing decomposition of H
2
O
2
in the case of hemolyzed samples while there is no reaction in
the other case. This effect is observed in reactions involving hydrogen peroxide/peroxi-
dase because of premature decomposition of hydrogen peroxide by hemoglobulin [22].
The plot of various background signals obtained can be seen in the Figure 22.6. The
response of the device obtained for case 4 is very high compared to other background sig-
nals. This is due to the absorbance of contents of the hemolyzed blood on to the electrode
surfaces interfering with the H
2
O
2
in the substrate solution. Therefore, hydrogen peroxide
is considered to be interfering with contents of the hemolyzed blood that can react or yield
overlapping signals. Hence, we studied the possibility of using other enzyme markers
such as AP instead of HRP. This was used to avoid the use of H
2
O
2
thereby eliminating the
interference of the contents of hemolyzed blood with the H
2
O
2
used in the substrate.
22.4.3
Alkaline Phosphatase vs. Horse-Radish Peroxidase
Electrochemical enzyme analysis requires electrochemically active reaction products.
-
Naphthyl phosphate was chosen as the suitable substrate for the electrochemical meas-
urement of AP activity. It is electrochemically inactive while the reaction product
-naphthol (Equation 22.1) can be readily oxidized at a potential of
100 mV vs. Ag/AgCl.
AP
2
- Naphthyl Phosphate
H O
→
- Naphthol
HPO
(22.1)
2
4
The experimentl procedure performed with the use of AP-labeled conjugate is the same as
described before, apart from the change of conjugate and substrate. The results attained
with the use of AP-labeled conjugate are presented in Figure 22.7. It shows a summary of
