Biosensors based on direct electron transfer of protein - Electrochemical Sensors, Biosensors and Their Biomedical Applications - page 553

passively adsorb cyt c onto the electrode surface. They employed this modifi ed electrode
in vivo to measure O 2 production in rat brain during hypoxia, focal ischemia, reper-
fusion, and fl uid percussion brain injury. Despite the inherently high background noise
detected by the large surface area of this porous electrode material, some O 2 generation
was seen following brain injury in rats.
NO has been employed as a parameter to evaluate environmental air pollution. A
most keen interest is currently being paid to NO function in living systems. The detec-
tion of NO has been widely studied by many methods. One of these methods is an
electrochemical method based on the direct electron transfer of redox proteins on elec-
trode. Haruyama [217] studied the nitric oxide biosensor based on the electrochemical
properties of cyt c deposited on a modifi ed electrode. Heat-denatured cyt c deposited
on a 4-mercaptopyridine-modifi ed gold electrode responded to NO with an increase of
cathodic current through electrochemical reduction of cyt c (Fe 3 ), when the electrode
potential was controlled at 0 mV vs Ag/AgCl. The cathodic current response is linearly
correlated with NO concentration in the range from 0.5 to 4.0

M in aqueous solution.
The NO sensing, which used heat-denatured cyt c , was performed at a lower potential
of 0 mV, where no serious interference might be associated in living systems. A nota-
ble NO sensing system has been accomplished by implementing heat-denatured cyt c
on a mercaptopyridine-modifi ed gold electrode. Besides these normal small molecules,
many other molecules like phenol, chromate, and histamine can also be detected by cyt
c -based biosensors.

µ

17.3.1.2 Biosensors based on direct electron transfer of proteins
cytochrome p450 [218]
Cytochrome P450 (CYP) is a large family of enzymes containing heme as the active
site. It forms a large family of heme enzymes that catalyze a diversity of chemical
reactions such as epoxidation, hydroxylation, and heteroatom oxidation. The enzymes
are involved in the metabolism of many drugs and xenobiotics. They are also responsi-
ble for bioactivation. Many of these compounds are even inducers for CYP expression
in different organs [219]. The catalytic abilities of the CYP family attracted the interest
of enzyme engineers in the 1970s [220]. However, these studies had to face complica-
tions due to the limited stability of the labile multi-enzyme system and the need of
the regeneration of the cofactor NADPH or NADH. Many biosensers are based on the
direct electrochemistry of CYP [221].
The electrochemistry of CYP has been investigated using a variety of metal elec-
trodes such as Au, Pt, and Tin oxide, as well as non-metal electrodes such as glassy
carbon, pyrolytic graphite, edge-plane graphite, and carbon cloth. Although direct
electron transfer has been observed on bare solid electrodes, modifying the electrode
with an appropriate medium like a polymer or a polyelectrolyte has been very popular
in recent years. These modifying materials can attain native structure and appropriate
orientation of enzyme, which increases electron transfer between the enzyme and the
electrode. In the fi rst biosensor based on the direct electron transfer of CYP [221],
solubilized CYP from rabbit liver showed a polarographic reduction step at a mercury