Biosensors based on direct electron transfer of protein - Electrochemical Sensors, Biosensors and Their Biomedical Applications - page 540

proton transfer. The immobilized Mb displayed good electrocatalytic responses to the
reduction of both H 2 O 2 and nitrite (NO 2 ), which were used to develop novel sensors
for H 2 O 2 and NO 2 . The HMS provided a novel matrix for protein immobilization and
the construction of biosensors via the direct electron transfer of immobilized protein.

17.2.2.3 Direct electron transfer of hemoglobin
Hemoglobin (Hb) is a heme protein that can store and transport oxygen in blood in
vertebrates. It has a molar mass of approximately 67 000 g mol 1 and comprises four
polypeptide subunits (two

subunits). The electron-transfer reactivity of
Hb is physiologically hampered, though it contains four iron-bearing hemes within
molecularly accessible crevices, which are known to act as electron-transfer centers
in other proteins. Although Hb does not function biologically or physiologically as
an electron carrier, it can be used as an ideal model molecule for study of electron
transfer of heme enzymes due to its commercial availability and a known structure.
Unlike some other small heme proteins such as cyt c , it, however, is diffi cult for Hb to
exhibit heterogeneous electron-transfer process in most cases, which means that the
rate of electron transfer of Hb is very slow. As a result, no useful currents appear at
the conventional electrodes, even when rather large overvoltages are applied, due to
its extended three-dimensional structure and resulting inaccessibility of the electroac-
tive centers as well as its strong adsorption onto the electrode surface for subsequent
passivation. Great efforts have been made to enhance the electron transfer of Hb by
using mediators and promoters [110-111] and some interesting results were obtained.
Recently, Rusling [112], Lu [113], and Wang [114] developed a technique of protein-
fi lm to incorporate Hb into fi lms, such as composite fi lms, protein-polyion layer-by-
layer assembly fi lms-polymer and natural lipid fi lms on PG electrodes. For example,
Wang [114] observed the direct electrochemistry of Hb in stable thin fi lm composed
of a natural lipid (egg-phosphatidylcholine) and Hb on PG electrode. Hb in lipid fi lms
showed thin layer electrochemistry behavior and exhibited elegant catalytic activity for
electrochemical reduction of H 2 O 2 , based on which an unmediated biosensor for H 2 O 2
was developed.
Some efforts have been taken to obtain the electrochemical response of Hb at solid
electrode surfaces. Fan's electrochemical researches revealed that the electron-transfer
reactivity of Hb could be greatly enhanced, simply by treating it with an organic sol-
vent, dimethyl sulfoxide (DMSO) [115]. Hb can also achieve its direct electron trans-
fer in N , N -dimethylformamide (DMF) fi lm, as Xu [116] reported. These, therefore,
suggested that there are many different factors that regulate electron-transfer reactivity
of proteins. It also pointed out the complicated and precise regulation mechanisms of
proteins in vivo .
Nanotechnology has provided a novel way to enhance the electron-transfer rates
between Hb and the electrode. As in the case of cyt c and Mb, nanocrystalline TiO 2
fi lm has been proposed to be a promising interface for the immobilization of Hb.
GNPs are renowned for their good biocompatibility. With the help of these GNPs, Hb
can exhibit a direct electron-transfer reaction without being denatured. To improve the

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