Biomedical Engineering Reference
In-Depth Information
17.3.1.1 Biosensors based on direct electron transfer of proteins
cytochrome c
It has been said above that cyt c was one of the most important and extensively stud-
ied electron-transfer proteins with active heme centers. Thus, cyt c was widely used in
enzyme-based biosensors and to study the mechanism of the catalytic process between
redox enzyme and substrate.
The determination of H 2 O 2 is very important in many different fi elds, such as in
clinical, food, pharmaceutical, and environmental analyses [202]. Many techniques
such as spectrophotometry, chemiluminesence, fl uorimetry, acoustic emission, and
electrochemistry methods have been employed to determine H 2 O 2 . Electrochemical
methods are often used because of their advantages. Among these electrochemical
methods, the construction of the mediator-free enzyme-based biosensors based on the
direct electrochemistry of redox proteins has been reported over the past decade [203-
204]. The enzyme-based biosensors, which use cyt c as biocatalyzer to catalyze H 2 O 2 ,
were widely studied.
Many materials were used to immobilize cyt c , and multi-walled carbon nanotubes
(MWNTs) were one of these materials. CNTs consist of cylindrical graphitic sheets
with nanometer diameters, superb electrical conductivity, high chemical stability, and
extremely remarkable mechanical strength and modulus [22], as described above. Zhao
[195] developed a hydrogen peroxide biosensor based on the direct electrochemistry of
cyt c on an MWNTs-modifi ed GC electrode to study the electrochemical reduction of
hydrogen peroxide. As shown in Fig. 17.1, no redox response can be observed in the
potential range from 0.4 to
1.0 V at the bare MWNTs-modifi ed GC electrode. However,
at the cyt c /MWNTs-modifi ed glass carbon electrode, an obvious catalytic reduction peak
appears at the potential of
0.277 V. The cathodic peak current of cyt c increased but its
anodic peak current decreased with an increase in the concentration of H 2 O 2 , indicating
the typical electrocatalytic reduction process. Cyt c immobilized with other materials has
the same behavior when it catalyzes H 2 O 2 [205]. The catalytic currents increased linearly
with the concentration of H 2 O 2 in different ranges with different immobilized materials,
and this is the base of the determination of hydrogen peroxide. The calculated apparent
Michaelis-Menten constant ( K m app ), which can indicate the catalytic activity of enzyme to
its substrate, can be obtained from the Lineweaver-Burk equation:
K app / I max C
1/ I ss
1/ I max
(12)
where I ss is the steady-state current after the addition of a substrate, which can be
obtained from amperometric experiments. I max is the maximum current under saturated
substrate condition and C is the bulk concentrate of the substrate. The value of the
apparent Michaelis-Menten constant ( K app ) can be calculated from the slope ( K app / I max )
and the intercept (1/ I max ) of the plot of the reciprocals of the I ss vs C H 2 O 2 . The con-
centration of H 2 O 2 can also be determined by amperometric method. The amperomet-
ric response of the cyt c /MWNTs-modifi ed electrode to H 2 O 2 was recorded through
successively adding H 2 O 2 to a continuous stirring PBS solution. The amperometric
response has linear relationship with the concentration of H 2 O 2 (Fig. 17.2).
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