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Journal of
Electroanalytical
Chemistry
Journal of Electroanalytical Chemistry 597 (2006) 51–59
www.elsevier.com/locate/jelechem
Direct electrochemistry and electrocatalysis of hybrid film
assembled by polyelectrolyte–surfactant polymer, carbon
nanotubes and hemoglobin
Liang Chen
a
a,b
, Gongxuan Lu
a,*
State Key Laboratory for Oxo Synthesis and Selective Oxidation, Lanzhou Institute of Chemical Physics, Chinese Academy of Sciences,
Lanzhou 730000, PR China
b
The Graduate School of the Chinese Academy of Sciences, Chinese Academy of Sciences, Beijing 100039, PR China
Received 24 March 2006; received in revised form 24 July 2006; accepted 9 August 2006
Available online 22 September 2006
Abstract
Functional hybrid film based on the incorporation of multiwalled carbon nanotubes modified with hemoglobin into polyelectrolyte–
surfactant polymer was fabricated. Such assembled films are found to possess facilitated electron transfer of hemoglobin. Cyclic voltammetric results showed a pair of well-defined redox peaks for the Hb heme Fe(III)/Fe(II) redox couple at about 0.273 (versus SCE) in a
pH 7.0 phosphate buffer solution. The formal potential of the Hb heme Fe(III)/Fe(II) couple shifted linearly with pH with a slope of
52.8 mV pH1, indicating that an electron transfer is coupled with a one-proton transportation. An FT-IR and UV–vis spectroscopy
study confirms that the secondary structure of Hb entrapped in the hybrid film still maintains the original arrangement. It is suggested
that the achieved faradic response of the hemoglobin be due to the ability of carbon nanotubes to promote the electron transfer of
enzymes and the biomembrane properties of the polyelectrolyte–surfactant polymer. And the entrapped Hb exhibits the features of a
peroxidase and acts in an electrocatalytic manner in the reduction of hydrogen peroxide, oxygen, and nitrite. The properties of the functional hybrid films, combined with the bioelectrochemical catalytic activity, could make them useful for the development of bioelectronic
devices and investigation of protein electrochemistry at functional interface.
2006 Elsevier B.V. All rights reserved.
Keywords: Hybrid film; Polyelectrolyte–surfactant; Carbon nanotubes; Hemoglobin; Direct electrochemistry; Electrochemical catalysis
1. Introduction
Among the best-known synthetic self-assembling polymeric systems, polyelectrolyte–surfactant polymer is a
new kind of complexes of charged polymeric chain (polyelectrolytes) and appositely charged small amphiphilic
molecules (surfactant), consisting of a polar ‘‘headgroup’’
and a nonpolar ‘‘tail’’ [1,2]. The complexation process is
an ion-exchange reaction mainly driven by electrostatic
attraction between the polymer chain units and ionic surfactants. Depending on the polyelectrolytes to surfactant
*
Corresponding author. Fax: +86 931 4968178.
E-mail address: [email protected] (G. Lu).
0022-0728/$ - see front matter 2006 Elsevier B.V. All rights reserved.
doi:10.1016/j.jelechem.2006.08.002
ratio in aqueous solution, the complexes formed are as
either stoichiometric or nonstoichiometric. Nonstoichiometric complexes containing an excess of either charged
polymer chain units or surfactant molecules are generally
soluble in water. If equimolar amounts of charged polymer
chain units and surfactant molecules are mixed in water,
stoichiometric complexes are formed. Stoichiometric polyelectrolyte–surfactant complexes can be viewed as a new
type of comb-shaped polymer, in which every polymer
chain unit has an electrostatically bound ‘‘side chain’’.
Such complexes combine in unique ways the properties of
polymers with those of low molecular weight amphiphiles.
The polymeric components can provide, for instance,
mechanical strength and thermal stability, while the
surfactants retain their tendency to assemble in layered
52
L. Chen, G. Lu / Journal of Electroanalytical Chemistry 597 (2006) 51–59
structures. This may be particularly useful for fabrication
of multifunctional materials for technological application,
e.g., electronic devices, microsensors, separation membrane, and biomembranes, etc. [2–5].
Carbon nanotubes (CNTs) represents a new kind of carbon-based nanomaterials and is superior to other carbon
material mainly in special structure feature, unique electronic properties and mechanical strength [6]. The unique
properties of carbon nanotubes make them extremely
attractive for the construction of ultrasensitive electrochemical sensors [7]. Recently, electrochemical studies have
revealed that the ability of CNTs to promote electron
transfer of some important molecules, such as catecholamine neurotransmitters [8], ascorbic acid [9], dopamine
[10], nitric oxide [11] and enzymes (proteins) [12–16], etc.
The striking properties of the CNTs coupled with the
advantages of polyelectrolyte–surfactant polymer essentially suggest a new platform for facilitate the investigation
of the protein electrochemistry.
This article reports on a new and simple avenue for
preparing bioelectrochemically hybrid film based on polyelectrolyte–surfactant polymer, carbon nanotubes and
hemoglobin. The direct electrochemistry of hemoglobin
entrapped in the hybrid film was studied and used to catalyze reduction of hydrogen peroxide, oxygen and nitrite.
This demonstration may pave a new route to CNTs-based
bioelectrochemical devices and would be useful for the
investigation of protein electrochemistry at functional
interface.
2. Experimental
2.1. Chemicals and solutions
Hemoglobin (Hb, Mw 66,000) was purchased from
Shanghai Lizhu Dong Feng Biotechnology Company
(China). Poly(sodium-p-styrene-sulfonate) (PSS, Mw
70,000) was obtained from Acros Organics, USA. Cetyltrimethylammonium bromide (CTAB) was purchased from
Shanghai Reagent Co. MWNTs (95%) purchased from
Shenzhen Nanotech Port. Co., Ltd. (Shenzhen, China)
were used as received. The 0.1 mol L1 phosphate buffers
of various pHs were prepared by mixing the stock solutions
of NaH2PO4 and Na2HPO4 and adjusted by 0.1 mM
NaOH and 0.1 mM H3PO4 solutions. All solutions were
made up with double-distilled water. All other chemicals
were of analytical grade and used without further
purification.
After the solution was centrifuged, washed, and dried,
the dry yellow CTAB–PSS solids were obtained. The polymer can be dissolved in chloroform. Prior coating, the
glassy carbon electrode (GCE, diameter 3 mm) was polished sequentially with slurries of 0.3 and 0.05 lm alumina
to mirror finish. After rinsing with doubly distilled water, it
was cleaned ultrasonically in water for 10 min. CNTs
(10 mg) can be well dispersed in 10 ml aqueous CTAB solution (1% by weight) with aid of ultrasonication to give a
1 mg mL stable black suspension. CNTs suspensions
(10 lL) were mixed well with 10 lL Hb solution (20 mg Hb
dissolved in 1 mL pH 7.0 phosphate buffer) thoroughly.
Then 6 lL of the mixture was deposited onto the surface
of a GCE with a microsyringe and allowed to dry at ambient temperature. Finally, a total of 3 lL of 15 mg mL
CTAB–PSS in chloroform was cast and used as binder to
hold the Hb–MWNTs on the electrode surface stably.
Then a small bottle was fit tightly over the electrode to
serve as a closed evaporation chamber so that chloroform
was evaporated slowly to give uniform film.
2.3. Instrumentation
For all electrochemical experiments, a CHI660A electrochemical workstation (CH Instruments, Shanghai, China)
was employed. The electrochemical cells consisted of a
three-electrode arrangement with a Pt wire counter
electrode, a saturated calomel electrode and a working
electrode (hybrid film modified GC electrode). The phosphate buffer solution (PBS) was purged with high-purity
nitrogen for 15 min prior to experiments and a nitrogen
environment was then kept over the solution in the cell.
All experiments were performed at room temperature.
Scanning electron microscopy (SEM) was carried out using
a JSM-5600LV microscopy (Japan) at an acceleration voltage of 20 kV. MWNTs, Hb–MWNTs, polymer–Hb–
MWNTs films for SEM characterization were coated on
copper slice and dried in the same way as the fabrication
of the hybrid film modified GC electrode. FT-IR spectra
were obtained on an IFS120HRFTIR spectrometer (Bruker). Hb, Hb–MWNTs, surfactant polymer–Hb–MWNTs
films were coated on KBr wafer and dried for characterization. UV–vis spectra were obtained on a HP8453 UV–vis
spectrometer (HP).
3. Results and discussion
3.1. FT-IR spectra of the polyelectrolyte–surfactant
polymer
2.2. Film assembly on glassy carbon electrode
Polyelectrolyte–surfactant polymer CTAB–PSS was prepared by a similar method described by Kunitake et al.
[17]. A total of 50 mL of 10 mg mL1 PSS solutions and
50 mL of 15 mg mL1 CTAB solutions were mixed well
with stirring for 4 h at about 65 C until reaction was completed. A white precipitate of CTAB–PSS was formed.
A comparison of CTAB, PSS and comb-shaped surfactant polymer CTAB–PSS FT-IR spectra is shown in Fig. 1.
As shown in Fig. 1 (curve a), the symmetric and antisymmetric CH2 stretching vibrations modes of CTAB appear
at 2849 and 2919 cm1, respectively, and the CH3–(N+)
asymmetric deformation modes are at 1481 cm1. The
characteristic peaks of PSS samples were found at about
L. Chen, G. Lu / Journal of Electroanalytical Chemistry 597 (2006) 51–59
53
trolytes) and CTAB (surfactants) in the polymer. The
details of peak-assignments of three samples are given in
Table 1.
Transmittance %
a
3.2. Film characterization
b
c
3000
2500
2000
1500
1000
500
Wavenumber / cm -1
Fig. 1. FT-IR spectra of the three samples: CTAB (curve a), PSS (curve b)
and polymer CTAB–PSS (curve c).
1195, 1128, 1039, 1011 and 836 cm1 [18]. The SO
3 group
antisymmetric and symmetric vibrational adsorption peaks
can be assigned to the peaks at 1195 and 1039 cm1,
respectively. Peaks at 1128 and 1011 cm1 can be attributed to the in-plane skeleton vibration of benzene ring
and in-plane bending vibration of benzene ring. Peak at
835 cm1 is assigned to CH out-of-plane vibration for
papa-disubstituted benzene. In the FT-IR spectrum of
polymer, the CH3(N+) asymmetric deformation modes
of CTA+ blue-shifted at 1486 cm1 , and the SO
3 group
antisymmetric and symmetric vibration modes red-shifted
at 1191 and 1037 cm1. These spectral changes clearly demonstrated their strong interaction between PSS (polyelecTable 1
Infrared frequencies and assignments of the three samples (CTAB, PSS
and CTAB–PSS)
CTAB
(cm1)
PSS
(cm1)
CTAB–PSS
(cm1)
3063
3015
2919
2849
2923
2850
1603
1570
1497
1481
1469
1486
1473
1450
1195
1128
1191
1127
1039
1011
1037
1009
832
963
911
833
720
720
962
910
725
3017
2919
2850
1601
1571
Assignment
Aromatic C–H stretch
N+–CH3 C–H asym stretch
CH2 C–H asym stretch
CH2 C–H sym stretch
Benzene ring [email protected] stretch
Benzene ring [email protected] stretch
Benzene ring [email protected] stretch
N+–CH3 C–H sym bend
CH2 scissoring
Benzene ring [email protected] stretch
SO
3 [email protected] asym stretch
In-plane skeleton vibration of
benzene ring
SO
3 [email protected] sym stretch
In-plane bending vibration of
benzene ring
C–N+ stretch
C–N+ stretch
Aromatic C–H out-of-plane
bend
CH2 rocking
Macroscopic characterization of films was studied using
scanning electron microscopy. Fig. 2 illustrates the SEM
photographs of the MWNTs film (a), the Hb–MWNTs film
(b) and the polymer–Hb–MWNTs hybrid film ((c) and (d)).
As shown in photograph (a), many twisted MWNTs bundles can be observed. In the photograph (b), MWNTs bundles cannot be clearly observed. It could be due to the
adsorption of hemoglobin molecules on the MWNTs.
However, the surfactant film shows significant differences
from above two films. It is observed that this film displays
porous structure. The diameter of the holes varied from
100 nm to 1 lm. This unique porous structure could be
propitious to the small molecule transfer between the electrode and the solution.
Positions of the Soret absorption band of iron heme
which is located at 406 nm may provide information about
possible denaturation of heme proteins, especially that of
conformational change in the heme group region. Previous
studies showed that the band will diminish if the protein is
fully denatured [19]. Our experimental results revealed that
the corresponding Soret bands for adsorbed Hb on
MWNTs or entrapped Hb in the composite film are
located at 410 nm and have nice shapes. It is suggested that
there exists slight structure variations in the vicinity of the
heme group, and no significant denaturation occurs
(Fig. 3).
FT-IR spectroscopy is a sensitive technique to probe
into the secondary structure of proteins. Our FT-IR studies
also show Hb is not denatured after incorporated into the
hybrid film. The profiles of the amide I and II infrared
bands of Hb, especially, provide detailed information on
the polypeptide chain [20]. The amide I band (1700–
1600 cm1) is attributed to the [email protected] stretching vibration
of peptide linkage in the backbone of protein, while the
amide II band (1620–1500 cm1) is assignable to the combination of NAH bending and CAN stretching. Although
the band of Hb from 1500 to 1600 cm1 overlaps with that
of the polymer, the intensities of peaks at about 1653 and
1541 cm1 are mainly attributed to the absorbance of the
amide I and II bands of Hb (see Fig. 4). It was shown that
the amide I and II bands of Hb adsorbed on the MWNTs
or entrapped in the hybrid film (1653 and 1541 cm1), are
very similar to those obtained in the protein spectrum
(1658 and 1541 cm1). It is suggested that Hb retains the
essential features of its original structure in the hybrid film.
Also five characteristic peaks of the surfactant polymer
(1190, 1124, 1035, 1009 and 833 cm1) were observed in
the spectrum of the hybrid film. Our FT-IR spectroscopy
study indicated that the functional hybrid film could be
successfully assembled by carbon nanotube, hemoglobin
and polyelectrolyte–surfactant polymer.
54
L. Chen, G. Lu / Journal of Electroanalytical Chemistry 597 (2006) 51–59
Fig. 2. SEM images of the MWNTs film (a), the Hb–MWNTs film (b), the polymer–Hb–MWNTs hybrid film 10,000· magnification (c) and 20,000·
magnification (d).
a
a
Hb
Transmittance %
Absorbance
1.0
b
0.5
c
b
Hb-MWNTs
d
c
polymer-Hb-MWNTs
300
400
/ nm
500
Fig. 3. UV–vis spectra of (a) 0.8 mg mL1 Hb solution, (b) 0.8 mg L1
Hb + 1 mg mL1 MWNT solution, (c) dry polymer–Hb–MWNTs hybrid
film, (d) 1 mg mL1 MWNT solution.
3.3. Direct electron transfer of Hb entrapped
in the hybrid film
Fig. 5 depicts typical cyclic voltammograms obtained
with the polymer–Hb-MWNTs hybrid film modified electrode into pH 7.0 PBS buffer. For comparison, voltammograms of the Hb and Hb–MWNTs and polymer–Hb films
modified GC electrode were also given. As shown, no
4000
3500
3000
2500
2000
1500
1000
500
Wavenumber / cm-1
Fig. 4. FT-IR spectra of the Hb film (curve a), Hb–MWNTs film (curve b)
and polymer–Hb–MWNTs hybrid film (curve c).
redox peaks were observed at the above three films in the
potential range of interest, but a pair of well defined and
nearly symmetric redox peaks located at 0.213 V and
0.333 V was obtained at the polymer–Hb–MWNTs film.
And the potential difference between the two peak current,
DEp, is 125 mV at a scan rate of 150 mV s1, which indicates a fast heterogeneous electron transfer process on
the surface of the hybrid film modified GC electrode. The
0
formal potential (E0 ) calculated by averaging the cathodic
L. Chen, G. Lu / Journal of Electroanalytical Chemistry 597 (2006) 51–59
Current / 1e-6A
2
b
a
d
c
0
-2
-0.8
-0.4
0.0
Potential / V vs SCE
0.4
Fig. 5. Typical cyclic voltammograms of Hb–MWNTs (curve a), polymer–Hb–MWNTs film (curve b), Hb film (curve c) and polymer–Hb film
(curve d) modified GC electrodes in 0.1 M phosphate buffer (pH 7.0) at a
scan rate of 100 mV s1.
and anodic peak potentials, is 0.273 V versus SCE, which
agrees well with that obtained at a Hb film at the CNT
powder microelectrodes electrode [21]. Table 2 lists the val0
ues of E0 of Hb at different electrodes for comparison.
According to those reported previously, the nearly revers-
Table 2
0
Formal potentials ðE0 Þ and heterogeneous electron transfer rate constants
(Ks) for hemoglobin on different electrodes
0
Electrode
pH
E0 (V) versus
SCE
Ksa (s1)
References
Polymer–Hb–CNTs/GC
Nafion–Hb–CNTs/GC
Hb–AQ/PG
Hb–PAM/PG
Hb–DDAB/GC
Hb–DDAB–PSS/PG
Hb–DDAB–clay/PG
Hb–Au colloid–
cystamine/Au
Hb–Au nanoparticle/GC
7.0
6.8
7.0
7.0
7.0
7.0
7.0
7.0
0.273
0.343
0.341
0.320
0.200
0.220
0.309
0.051
0.90
1.25
62
45
2.6
33
75
0.49
This work
[22]
[23]
[24]
[25]
[3]
[26]
[27]
7.0
0.257
[28]
Hb–sol–gel/PG
(Hb–PDDA)n/PG
Hb–DHP–PDDA/PG
Hb/CNTPMEs
Hb–SDS–CNTs/GC
7.0
7.0
7.0
7.0
7.0
0.312
0.340
0.340
0.278
0.300
Hb–DMPC/PG
Hb–SP Sephadex/PG
Hb–PHB/PG
7.0
7.0
5.0
0.329
0.290
0.254
Not
shown
1.58
60
30
0.062
Not
shown
70
102.12
10.33
55
ible CV peaks is a characteristic of the Hb heme Fe(III)/
Fe(II) redox couple.
Hb is a kind of biological macromolecules containing
four subunits. Several factors make it difficult to achieve
their direct electrochemistry at conventional electrodes
such as GC, Au and Pt electrodes [35]. These include (I)
electroactive prosthetic group deep within the protein
structure, (II) adsorptive denaturation of proteins onto
electrodes, (III) unfavorable orientations at electrodes. In
our experimental result, no redox peaks obtained at the
Hb–MWNTs film. It is probably ascribed to adsorbed
Hb molecules in unfavorable orientations. The unique
porous structure of surfactant polymer film could yield
biomembrane-like microenvironment for Hb. So Hb
entrapped in the hybrid film may has suitable orientations
and exhibits well-behaved electrochemistry and good catalytic properties towards a variety of substrate.
The CV reduction and oxidation peaks currents for
entrapped Hb are found to increase linearly with potential
scan rates from 20 to 400 mV s1 (see Figs. 6a and 6b). The
linear regression equations are: y = 0.118 + 0.1260x,
r = 0.9998 (anodic peak), and y = 0.3103 0.1240x,
r = 0.9999 (cathodic peak). Fig. 7 shows the logarithm plot
of anodic peak current versus logarithm of scan rate gives a
linear relationship, with a correlation coefficient of 0.9998
and a slope of 0.92, which is very close to the theoretical
slope of 1 for thin layer voltammetry. These results suggest
that all electroactive Hb Fe(III) in the film has been converted to Hb Fe(II) on the reverse scan. Integration of
reduction or oxidation peaks at different scan rates gave
nearly constant charge (Q) values. The surface concentration of the electroactive Hb entrapped in the hybrid film,
C (in mol/cm2), can be estimated using the Laviron’s
equation [36]:
[29]
[30]
[4]
[21]
[31]
[32]
[33]
[34]
GC, glassy carbon; PG, pyrolytic graphite; CNT, carbon nanotube;
CNTPMEs, CNT powder microelectrode electrodes; AQ, Eastman AQ;
PAM, polyacrylamide; DDAB, didodecyldimethylammonium bromide;
PSS, poly(styrene sulfonate), PDDA, poly(diallyldimethylammonium);
DHP, dihexadecylphosphate; SDS, sodium dodecylsulfate; DMPC,
dimyristoyl phosphatidylcholine; PHB, poly-3-hydroxybutyrate.
a
Ks for hemoglobin on different electrodes were estimated by different
methods.
Fig. 6a. Cyclic voltammograms of the polymer–Hb–MWNTs hybrid film
modified GC electrode in a 0.1 MPBS buffer (pH 7.0) at various scan rates.
Scan rate (from inner to outer): 20, 40, 60, 80, 100, 150, 200, 250, 300, 350,
400 mV s1.
56
L. Chen, G. Lu / Journal of Electroanalytical Chemistry 597 (2006) 51–59
5
4
Peak current / 10-6A
3
2
1
0
-1
-2
-3
-4
-5
-6
0.0
0.1
0.2
0.3
0.4
V / vs -1
Fig. 6b. Plot of peak current versus scan rate in a 0.1 M pH 7.0 phosphate
buffer.
0.8
log(peak current)
0.6
0.4
0.2
0.0
-0.2
-0.4
-0.6
1.2
1.4
1.6
1.8
2.0
2.2
2.4
2.6
2.8
log(scan rate)
Fig. 7. Linear logarithm plot of anodic peak current versus scan rate in
0.1 M phosphate buffer solution.
Fig. 8. Cyclic voltammograms of the polymer–Hb–MWNTs film modified
GC electrode in 0.1 M phosphate buffer at different pH values. Scan rate:
150 mV s1.
that the electron transfer of Hb entrapped in the hybrid
film is a fast process.
It is known that solution pH modulates the accessibility
of water to the heme pocket of Hb and the protonation of
the heme iron-bound proximal histidine and/or the distal
histidine in the heme pocket and accordingly influences
the redox potential of Hb [38]. So the dependence of Epa,
0
Epc, and E0 of the hybrid film modified electrode on the
solution pH was examined (see Fig. 8). It is observed that
the increase of the value of pH in buffers led to a negative
0
0
shift of Epa, Epc, and E0 . Epa, Epc, and E0 had linear relationships with solution pH with slopes of 45.8, 49.4, and
52.8 mV pH1 unit, respectively, in the range of 5–11.
0
The E0 values is close to the 58 mV pH1 unit expected
for a reversible, one-electron coupled one-proton reaction
process at 20 C, which indicates that a single protonation
accompanies a single electron transfer between the electrode and those of the four heme Fe(III) of Hb.
I p ¼ n2 F 2 ACv=4RT ¼ nFQv=4RT
3.4. Catalytic reduction of H2O2
Here, n is the number of electron transferred, F, Faraday’s
constant, and A, the electrode area. According to the
C Q relationship, the average surface concentration of
electroactive Hb was estimated to be 1.1 · 1010 mol cm2,
which accounted for only about 2.5% of the total amount
of Hb deposited on the electrode. This may suggest that
only those Hb molecules in the inner layers of the films
closest to the electrodes and with a suitable orientation
can exchange electrons with the electrode and contribute
to the observed redox reaction. From the dependence of
DEp on the various scan rates, the apparent heterogeneous
electron transfer rate constant, Ks, can be estimated to be
0.9 s1 using the method developed by Laviron [37] for a
surface-controlled electrochemical system. It was indicated
Having demonstrated the redox properties of the hybrid
film, we next investigated its bioelectrochemically catalytic
activity. It has been reported that proteins containing the
heme group had the ability to reduce H2O2 electrocatalytically. To check the bioelectrocatalytic activity of the polymer–Hb–MWNTs hybrid film modified electrode, cyclic
voltammetric experiments were performed (Fig. 9). When
H2O2 was added to a pH 7.0 buffer, an increase in reduction peak at about 0.34 V was seen with the disappearance of the oxidation peak for HbFe(II) [30]. Controlled
experimental results indicated that no catalytic current
was observed at the bare GC electrode and only a very
small cathodic current was observed at the MWNT/GC
electrode in the presence of H2O2, which was also observed
in previous publication [22]. Fig. 10 showed the steady-
L. Chen, G. Lu / Journal of Electroanalytical Chemistry 597 (2006) 51–59
57
Here, Iss is the steady-state current after the addition of
substrate, c is the bulk concentration of the substrate and
Imax is the maximum current measured under saturated
substrate conditions. The Km value of the hybrid film was
found to be 1.4 · 104 M. The low value of Km implied that
entrapped Hb exhibits a high biological affinity for H2O2.
3.5. Catalytic reduction of O2 and NO
2
Fig. 9. Cyclic voltammograms obtained at the hybrid film modified GC
electrode in 0.1 M phosphate buffer with pH 7.0 before (curve a) and after
the addition of 0.06 (curve b), 0.21 mM (curve c) H2O2. Scan rate:
150 mV s1.
Catalytic CV behavior was also observed for oxygen at
the hybrid film modified electrodes (Fig. 11), which is very
similar to that of a hydrogen peroxide system. When a certain volume of air was injected into a previously deaerated
buffer at pH 7.0 in a sealed cell by a syringe, a significant
increase in reduction peak at 0.25 V was observed,
accompanied by the disappearance of the oxidation peak
for HbFe(II), the reduction peak current increased with
the amount of oxygen in solution. Compared with MWNT
film modified electrode, the hybrid film modified electrode
decreased the reduction overpotential of oxygen about
200 mV and the peak current for the oxygen reduction is
larger (see Fig. 12). So it was concluded that the active
Hb entrapped in the hybrid film play an important role
in the electroreduction of oxygen. The catalytic efficiency
expressed as the ratio of the reduction peak current of
HbFe(II) in the presence (Ic) and absence of oxygen (Id),
i.e., Ic/Id, decreases within the increase of scan rate, which
is also a characteristic of electrochemical catalytic reduction of oxygen by Hb entrapped in the composite film.
The potential of the reduction peak is about 100 mV more
0
positive than the E0 of the Hb heme Fe(III)/Fe(II) redox
couple, which could be explained by the fact that the reduction peak belonged to the reduction of HbFe(II)–O2, rather
than reduction of HbFe(III). When the solution was deoxygenated and oxygenated in turn, a reproducible reduction
Fig. 10. Current–time response curve of the hybrid films with successive
additions of 0.06 mM H2O2, in a 0.1 M phosphate buffer (pH 7.0) at the
applied potential 0.2 V. Inset is the calibration curve.
state current response of H2O2. The response was linearly
within the concentration range from 8 · 106 M to
2.4 · 104 M, the linearly regression equation was I
(lA) = 0.3752 + 2.236 · 103[H2O2] (M), with a correlation
coefficient of 0.997. The detection limit was 4 · 106 M
(three times the ratio of signal to noise). When the concentration of H2O2 was higher than 3 · 104 M, a response
plateau is observed, showing the characteristic of the
Michaelis–Menten kinetic mechanism. The apparent
Michaelis–Menten constant (Km), which is an indication
of the enzyme–substrate kinetics, can be calculated using
the Lineweaver–Burk equation [39]:
1=I ss ¼ 1=I max þ K m =I max c
Fig. 11. Cyclic voltammograms of the hybrid film modified GC electrode
in 15 mL 0.1 MPBS (pH 7.0): (a) with no oxygen present; (b) after
injection of 6 mL air; (c) after injection of 12 mL air; (d) after injection of
18 mL air; (e) after injection of 24 mL air. Scan rate: 100 mV s1.
58
L. Chen, G. Lu / Journal of Electroanalytical Chemistry 597 (2006) 51–59
0.8
Current/1e-5A
0.4
0.0
-0.4
-0.8
a
b
c
-1.2
-1.0
-0.8
-0.6
-0.4
-0.2
0.0
0.2
0.4
0.6
Potential / V vs SCE
Fig. 12. Cyclic voltammograms of the MWNTs film modified GC
electrode in 15 mL 0.1 MPBS (pH 7.0): (a) with no oxygen present; (b)
after injection of 12 mL air; (c) after injection of 24 mL air. Scan rate:
100 mV s1.
peak current could be obtained. In other words, the reduction activity of Hb to oxygen did not decrease in the processes. The exact mechanism of catalytic reduction of
oxygen on the hybrid film is not yet clear but probably
similar to that reported by Hu [30] and Rusling [40]. For
example, direct electrochemical reduction of HbFe(III) to
HbFe(III) + e-
HbFe(II)
at electrode
HbFe(II) + O 2 → HbFe(II)-O2
fast
HbFe(II)-O2 + 2e + 2H → HbFe(II) + H2O2
at electrode
-
+
Scheme 1.
HbFe(II) occurred at the electrode, followed by a fast
reduction of HbFe(II) with oxygen. The product of
HbFe(II)–O2 could then undergo electrochemical reduction at the potential of HbFe(II) reduction, producing
hydrogen peroxide and HbFe(II) again. Thus, the simplified mechanism for the electrochemical catalytic reduction
of O2 on the hybrids modified electrode maybe expressed as
Scheme 1.
The hybrid film modified electrode can be also used to
catalyze reduction of nitrite (see Fig. 13). When a modified
electrode was placed in pH 6.0 PBS buffer containing NO
2,
a new reduction peak appeared at about 0.82 V. The
direct reduction of NO
2 at blank MWNTs film electrode
was hardly observed in the studied potential window. The
reduction peak currents of NO
2 at the hybrid film varied
approximately linearly with the concentration of NO
2.
Although the mechanism of NO
2 reduction at on the polymer–Hb–MWNTs hybrid film is yet not clear, probably it
is similar to that on Mb–DDAB film reported by Farmer
et al. [41]. On Mb–DDAB film modified electrodes, the
conformed catalytic reduction product was N2O, which
was detected by mass spectroscopy on electrocatalysis at
0.895 V in pH 7.0 buffer.
4. Conclusions
The properties of Hb entrapped in the hybrid film were
characterized by both spectroscopic and electrochemical
methods. It was observed that Hb could facilely exchange
electrons with the electrode at the same time and achieve
a high peroxidase activity. It was also demonstrated that
carbon nanotubes combined with polyelectrolyte–surfactant polymer could yield a new platform for facilitate direct
electron transfer of proteins. This demonstration may pave
a new way to CNT-based biosensors, catalytic bioreactors
and biomedical devices, etc.
Acknowledgements
The financial supports by Chinese Academy of Sciences,
Department of Sciences and Technology of China
(2003CB214503) are gratefully acknowledged.
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Fig. 13. Cyclic voltammograms of the hybrid film modified GC electrode
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addition of (b) 0.13, (c) 0.40, (d) 0.65 and (e) 0.93 mM NaNO2. Scan rate:
100 mV s1.
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