Biomedical Engineering Reference
In-Depth Information
rhodium-graphite electrodes. Thin films of gold nanoparticles were deposited onto the
rhodium-graphite electrodes by drop casting. Cytochrome P450scc was deposited onto
both gold nanoparticle-modified and bare rhodium-graphite electrodes. Cyclic voltam-
metry indicated enhanced activity of the enzyme at the gold nanoparticle-modified
surface. The role of the nanoparticles in mediating electron transfer to the cytochrome
P450scc was verified using ac impedance spectroscopy (69).
The amperometric responses were obtained by adding aliquots of clozapine (40 µM in
methanol) in a working stirred system of 10 mM K-phosphate buffer, pH 7.4 (2 ml).
Equilibration time was 5 min for each measurement. The cytochrome was electrochemi-
cally reduced at the potential of -600 mV vs. Ag/AgCl.
Protein studies utilizing electrochemical techniques such as cyclic voltammetry have
provided insight into the functional properties of redox-active centers.
In several studies, we performed an electrochemical study of the cytochrome P450scc
(15) and defined the best electrochemical parameters for this enzyme, which are also used
for cytochrome P4501A2 and 2B4.
Electrodes with gold nanoparticles and P450scc were used for measurements of choles-
terol concentration in solution.
Aliquots of a 100
M cholesterol stock solution in 0.3% sodium cholate were added to
the analyzed solution and the steady-state current was recorded. The steady-state current
as a function of cholesterol concentration is shown in Figure 9.5. The plot shows a sensi-
tivity of the biosensor of 0.13
M of
cholesterol. This indicates that gold nanoparticles mediate electron transfer to the P450scc
better than riboflavin (sensitivity 13.8 nA
A
M
1
and a detection range between 10 and 70
M). The current
response of the cytochrome P4501A2 electrode, in a function of clozapine concentration,
was measured by chronoamperometry (6). This analysis was performed using the screen-
printed thick film electrodes: a three-electrode standard configuration was used, where
M
1
and detection limit 300
8
7
6
5
4
3
2
1
0
0
10
20
30
40
50
60
70
80
Concentration of cholestrol (
µ
m)
FIGURE 9.5
The amperometric response of screen-printed rhodium-graphite Au-P450scc electrode to increasing cholesterol
concentration. About 100
M stock solution in 0.3% sodium cholate, measured when fractions of 10
M of cho-
lesterol has been repeatedly added. The total volume of electrolyte was 60
l. The current was measured at the
potential
400 mV (vs. Ag/AgCl).
