Biomedical Engineering Reference
In-Depth Information
17.2.3 Direct electron transfer of enzymes
Study of electron-transfer reactions of enzymes at the electrode-solution interface
is a convenient and informative means for understanding the kinetics and thermody-
namics of biological redox processes [121-123]. It can also establish a foundation for
fabricating the third-generation biosensors without using redox mediators. Work on
direct electrochemistry of biomolecules has largely focused on relatively small pro-
teins [121]. However, few studies have been reported on the direct electrochemistry of
redox enzymes. Part of the diffi culty may have stemmed from the large spatial separa-
tion between the prosthetic group(s) of catalytically active proteins/enzymes and the
electrode surface. Many types of metal electrodes or chemically modifi ed electrodes
may not be suitable for aligning the redox center(s) of the enzymes close to the surface
or could even lead to denaturation of the enzymes upon adsorption [124].
17.2.3.1 Direct electron transfer of HRP
Horseradish peroxidase (HRP) is a member of the large class of peroxidases, which
are enzymes defi ned as oxidoreductases using hydroperoxide as electron acceptor. Due
to its commercial availability in high purity, HRP has long been a representative sys-
tem for investigating the structure, dynamic, and thermodynamic properties of peroxi-
dases, especially for understanding their biological behaviors of catalyzing oxidation
of substrates by H
2
O
2
[125]. HRP can react with H
2
O
2
to form a powerful enzymatic
oxidizing agent known as Compound I, which is a two-equivalent oxidized form con-
taining an oxyferryl heme (Fe
4
¨
O) and a porphyrin
cation radical. Compound I is
catalytically active and can abstract one electron from the substrate to form a second
intermediate, called Compound II, which is subsequently reduced to the resting state
of the native enzyme, HRP-Fe(III), by accepting an additional electron from the sub-
strate. HRP-Fe(III) can also be further reduced to HRP-Fe(II). Effi cient electron trans-
fer between HRP and electrodes has been reported for many years [126]. However, in
most cases, direct electrochemistry was proven in the presence of H
2
O
2
or other per-
oxides by amperometry, and attributed to electrochemical reduction of Compound I or
Compound II. Only a few examples of independent quasi-reversible CVs of HRP were
reported in the absence of peroxides, probably because of the large molecular mass
and extended structure of HRP, and the inaccessibility of its redox centers [127-130].
Ferri [127-128] reported a pair of quasi-reversible CV peaks for HRP Fe(III)/Fe(II)
redox couple by entrapping HRP in the fi lm of tributylmethyl phosphonium chlo-
ride (TBMPC) polymer bound to an anionic exchange resin at PG electrodes. Chen
[129-130] then explored the direct electrochemistry of HRP Fe(III)/Fe(II) couple
by CV in DDAB and DNA fi lms at PG electrodes. These fi lms provided a suitable
microenvironment for HRP, which greatly facilitated the electron exchange between
HRP and electrodes. Electrochemical catalytic reduction of H
2
O
2
by HRP in these
fi lms was also described. Recently, a novel Nafi on-cysteine functional membrane was
constructed [131]. Rapid and direct electron transfer of HRP was carried out on the
functional membrane-modifi ed gold electrode with good stability and repeatability.
π
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