Ionic liquid-hemoglobin-carbon paste composite bioelectrode and its electrocatalytic activity

Article abstract of DOI:10.1002/jccs.201000187

Anew carbon ionic liquid paste bioelectrode was fabricated by mixing hemoglobin (Hb) with graphite powder, ionic liquid 1-ethyl-3-methylimidazolium tetrafluoroborate (EMIMBF4) and liquid paraffin homogeneously. Nafion film was cast on the electrode surface to improve the stability of bioelectrode. Direct electrochemistry of Hb in the bioelectrode was carefully investigated. Cyclic voltammetric results indicated that a pair of well-defined and quasi-reversible electrochemical responses appeared in pH 7.0 phosphate buffer solution (PBS), indicating that direct electron transfer of Hb was realized in themodified electrode. The formal potential (E^0') was calculated as -0.316 V (vs. SCE), which was the typical characteristic of the electrochemical reaction of heme Fe(III)/Fe(II) redox couple. Based on the cyclic voltammetric results the electrochemical parameters of the electrode reaction were calculated. This bioelectrode showed high electrocatalytic activity towards the reduction of trichloroacetic acid (TCA) with good stability and reproducibility.

Full text of DOI:10.1002/jccs.201000187

1262
Journal of the Chinese Chemical Society, 2010, 57, 1262-1267
Ionic Liquid-hemoglobin-carbon Paste Composite Bioelectrode and Its
Electrocatalytic Activity
Zong-wei Xuana (
Zi-qin Zhaib (
), Xiao-ying Sunb (
), Wei Sunb* (
), Guo-song Jiaoc (
) and Lu-de Lua (
),
)
aKey Laboratory for Soft Chemistry and Functional Materials of Ministry Education,
College of Chemical Engineering, Nanjing University of Science and Technology,
Nanjing 210094, P. R. China
bCollege of Chemistry and Molecular Engineering, Qingdao University of Science and Technology,
Qingdao 266042, P. R. China
cQingdao Environmental Monitoring Central Station, Qingdao 266003, P. R. China
A new carbon ionic liquid paste bioelectrode was fabricated by mixing hemoglobin (Hb) with graph-
ite powder, ionic liquid 1-ethyl-3-methylimidazolium tetrafluoroborate (EMIMBF4) and liquid paraffin
homogeneously. Nafion film was cast on the electrode surface to improve the stability of bioelectrode. Di-
rect electrochemistry of Hb in the bioelectrode was carefully investigated. Cyclic voltammetric results in-
dicated that a pair of well-defined and quasi-reversible electrochemical responses appeared in pH 7.0
phosphate buffer solution (PBS), indicating that direct electron transfer of Hb was realized in the modified
electrode. The formal potential (E0¢) was calculated as -0.316 V (vs. SCE), which was the typical charac-
teristic of the electrochemical reaction of heme Fe(III)/Fe(II) redox couple. Based on the cyclic
voltammetric results the electrochemical parameters of the electrode reaction were calculated. This
bioelectrode showed high electrocatalytic activity towards the reduction of trichloroacetic acid (TCA)
with good stability and reproducibility.
Keywords: Hemoglobin; Nafion; Ionic liquid; Carbon paste electrode; Direct electrochemistry.
INTRODUCTION
Direct electron transfer between redox proteins and
ent kinds of modifier incorporated in the carbon paste,
chemically modified carbon paste electrodes show many
specific functions and applications. Various modifiers such
as organic, inorganic and biomolecules have been used in
the fabrication processes. For example Matuszewski et al.18
firstly mixed glucose oxidase with graphite powder and sil-
icon oil to get a carbon paste bioelectrode. Gorton19 re-
viewed the progress of carbon paste bioelectrodes with var-
ious biological substances including enzyme, tissues and
cells. The carbon paste enzyme electrode had been demon-
strated to show excellent acidic and thermal ability, which
was attributed to the conformational rigidity of the bio-
catalyst within the highly hydrophobic pasting liquid.20,21
In recent years ionic liquids (ILs) had been used in the
field of electrochemical biosensors due to the higher ionic
conductivity and wider electrochemical windows. ILs can
be used as not only the modifier but also the binder in the
preparation of the chemically modified electrodes. Wei et
electrodes has established the basis for the development of
biosensors and bioreactors, which can provide the mecha-
nistic investigation on the electron transfer in the biological
system.1 But the electron exchange rate of protein with bare
electrode is often slow due to the deep burying of the elec-
troactive center and the unfavorable orientation of protein
on the electrode surface.2 This problem can be solved by
using the redox mediators or fabricating a specific interface
to fasten the electron transfer rate.3-5 In recent years differ-
ent kinds of modified electrodes had been devised for the
third-generation biosensors with different methods or ma-
terials such as sol-gel,6,7 surfactant,8,9 hydrogel,10,11 bio-
polymer,12,13 composite film14,15 or nanoparticles.16,17
Carbon paste electrode had been widely studied in the
field of electroanalytical chemistry with the advantages
such as low cost and easy preparation. By applying differ-
* Corresponding author. E-mail: sunwei@qust.edu.cn

Ionic Liquid-hemoglobin-carbon Paste Bioelectrode
J. Chin. Chem. Soc., Vol. 57, No. 6, 2010 1263
al.22 reviewed the application of ILs in the electrochemical
sensors. Maleki et al.23 applied N-octylpyridinum hexa-
fluorophosphate as a binder for the construction of carbon
ionic liquid electrode (CILE) and investigated its electro-
chemical behaviors. Qiao24 investigated a gellan and IL
modified CILE for the direct electrochemistry of hemoglo-
bin (Hb). Our groups also fabricated different kinds of ILs
modified carbon paste electrode and applied to the detec-
tion of some electroactive substances.25,26
to the embedment of high viscosity EMIMBF4 between the
layers of graphite particles. On Hb-CILE (Fig. 1b) many
spots were found homogeneously distributed on the sur-
face, which indicated that Hb molecules were dispersed in
the carbon ionic liquid paste. After Nafion was cast on the
carbon ionic liquid bioelectrode, a layer of film was formed
(Fig. 1c). Nafion is a commonly used ion-exchange poly-
mer with the characteristics such as good film forming abil-
ity, excellent biocompatibility and chemical resistance.28,29
So the presence of Nafion can form a stable film on the
electrode surface, which can prevent the leakage of Hb
from dissolving into the buffer solution.
1-Ethyl-3-methylimidazolium
tetrafluoroborate
(EMIMBF4) is a hydrophilic ionic liquid with higher con-
ductivity and smaller viscosity, also the hydrophilic solvent
is benefit for the maintenance of structure and activity of
protein. EMIMBF4 had been used as the modifier for the
preparation of ionic liquid modified carbon paste electrode
and used as the substrate electrode for the furthur modifica-
tion by multi-walled carbon nanotube (MWCNT). Direct
electrochemistry of Hb on MWCNT/CILE was realized
and carefully investigated.27 In this paper Hb and hydro-
philic IL of EMIMBF4 were mixed with traditional carbon
paste to get a carbon ionic liquid paste bioelectrode. A
Nafion film was cast on the bioelectrode surface to improve
the stability of electrode. Electrochemical behaviors of Hb
in the bioelectrode were carefully investigated with a pair
of well-defined redox peaks appeared, indicating the real-
ization of direct electron transfer process. The fabricated
bioelectrode showed good electrocatalytic ability to tri-
chloroacetic acid (TCA), which provided a potential ap-
proach to the fabrication of the third-generation mediator-
free biosensors.
Electrochemical impedance spectroscopy
Electrochemical impedance spectroscopy (EIS) is of-
ten used to investigate the impedance changes during the
modification process, which can reflect the interface changes
of electrode surface. The semicircular part at higher fre-
quencies corresponds to electron transfer limited process
and its diameter is equal to electron transfer resistance
(Ret), which controls the electron-transfer kinetics of the
redox probe at the electrode interface. In the frequencies
range from 0.05 to 105 Hz with [Fe(CN)6]3-/4- redox couples
as the electrochemical probe, the Nyquist plots of (a) Nafion/
Hb-CPE, (b) Nafion/Hb-CILE and (c) Nafion/CILE were
shown in Fig. 2. The Ret value (191.4 W) of Nafion/Hb-
CILE (curve b) was bigger than that of Nafion/CILE (52.94
W) (curve c), indicating the existance of Hb in the CILE.
The Ret value (595.9 W) of Nafion/Hb-CPE (curve a) was
RESULTS AND DISCUSSION
SEM images of modified electrodes
Scanning electron microscopic images of different
modified electrodes were recorded and shown in Fig. 1. On
the surface of CILE, an uniform surface could be observed
without separated graphite layers (Fig. 1a), which was due
Fig. 2. Electrochemical impedance spectra for (a)
Nafion/Hb-CPE, (b) Nafion/Hb-CILE, (c)
Nafion/CILE in the presence of 10.0 mmol L-1
[Fe(CN)6]3-/4- and 0.1 mol L-1 KCl with the fre-
quencies swept from 105 to 0.05 Hz.
Fig. 1. SEM images of (a) CILE, (b) Hb-CILE and (c)
Nafion/Hb-CILE.

1264 J. Chin. Chem. Soc., Vol. 57, No. 6, 2010
Xuan et al.
greatly larger than that of Nafion/Hb-CILE and Nafion/
CILE. This might be due to the presence of non-conductive
liquid paraffin in the carbon paste, which hindered the elec-
tron transfer rate and made it more difficult for the electron
transfer reaction to take place.
ment with the typical characteristics of heme Fe(III)/Fe(II)
redox couples. The peak-to-peak separation (DEp) was cal-
culated as 125 mV, indicating that Hb immobilized in the
carbon ionic liquid paste bioelectrode displayed a quasi-re-
versible electron transfer reaction. According to the refer-
ences,20, 21 the mixture of enzyme with carbon paste was an
attractive method for the preparation of versatile and re-
newable reagentless biosensors, which showed the im-
provement to the stability of enzyme in the carbon paste
microenvironment. The enzyme can also retain the biocat-
alytic activity in the hostile paste environment, which is re-
lated to the enzymology in the nonaqueous media such as
the organic-phase environments. ILs are also green media
for enzymes to retain their bioactivity, so the presence of
ILs in the carbon paste can also provide a biocompatible
microenvironment for enzymes. On the other hand, the
presence of Nafion on the electrode surface exhibited good
ability to retain Hb on the electrode surface and prevent in-
terfering substances in the solution from reaching the elec-
trode interface. So the direct electron transfer of Hb was
successfully realized in the fabricated carbon ionic liquid
paste bioelectrode.
Direct electrochemistry of Nafion/Hb-CILE
Fig. 3 showed the typical cyclic voltammograms of
different modified electrodes at the scan rate of 100 mV s-1
in pH 7.0 PBS. No electrochemical responses appeared on
the Nafion/CILE (curve b), which indicated no electro-
active substances existed on the electrode. On the Nafion/
Hb-CPE a pair of redox peaks appeared with small electro-
chemical response (curve c), indicating the direct electron
transfer of Hb in the CPE could be realized but with slow
rate. While on the Nafion/Hb-CILE (curve a) a pair of well-
defined and stable redox peaks appeared, and the electro-
chemcial responses were bigger than that of Nafion/Hb-
CPE. The results indicated that the presence of IL in the
carbon paste greatly improved the electron transfer rate of
Hb. Since ILs exhibit the characteristics including high
ionic conductivity, wide electrochemical windows and
good biocompatibility. Proteins and enzymes are usually
-
active in IL that containing tetrafluoroborate (BF4 ) or
hexafluorophosphate (BF6 ) anions.30 Since the Hb mole-
Influence of scan rate
-
The influence of scan rate on the electrochemical re-
sponse of Nafion/Hb-CILE was investigated with the re-
sults shown in Fig. 4. A pair of well-defined redox peaks
appeared with almost equal height of redox peak current,
which indicated that all the electroactive ferric hemoglobin
[Hb Fe(III)] was reduced to ferrous hemoglobin [Hb
Fe(II)] on the forward scan and then the Hb Fe(II) produced
cules and ILs were co-existed in the carbon paste, then the
direct electron transfer of Hb was greatly improved within
the IL matrix. From curve a it can be seen that the cathodic
(Epc) and anodic (Epa) peak potentials appeared at -0.253
V and -0.378 V (vs. SCE), respectively, and the formal po-
tential (E0¢) was calculated as -0.316 V, which was in agree-
Fig. 4. Cyclic voltammograms of Nafion/Hb-CILE
with different scan rates (from a to j: 50, 100,
150, 200, 250, 300, 350, 400, 450, 500 mV s-1)
in pH 7.0 PBS.
Fig. 3. Cyclic voltammograms of (a) Nafion/Hb-
CILE, (b) Nafion/CILE and (c) Nafion/Hb-CPE
in pH 7.0 PBS at the scan rate of 100 mV s-1.

Ionic Liquid-hemoglobin-carbon Paste Bioelectrode
J. Chin. Chem. Soc., Vol. 57, No. 6, 2010 1265
was reoxidized to Hb Fe(III) on the reverse scan. Both the
redox peak currents were proportional to scan rate in the
range from 50.0 to 500.0 mV s-1, which indicated that the
electrode reaction was a surface-controlled process. The
linear regression equations between the redox peak cur-
rents and scan rate were calculated as Ipa(mA) = -663.31 u
(V s-1) -53.97 (g = 0.997) and Ipc(mA) = 629.33 u (V s-1)
+60.17 (g = 0.999), respectively.
The surface concentration (G*) of electroactive Hb
can be further calculated by the integration of the reduction
peak on the cyclic voltammograms based on the equation
of G* = Q/nAF. The integration of the reduction peak at dif-
ferent scan rates from 50.0 to 500.0 mV s-1 gave a nearly
constant value of charge (Q), so the average value of G* was
got as 1.332 × 10-8 mol cm-2, which was much higher than
the reported value.31 The result indicated that Hb molecules
in the carbon ionic liquid paste matrix had closer and effec-
tive contact to graphite powder with ILs as the medium.
With the increase of scan rate the redox peak poten-
tials were also shifted with the increment of the peak-to-
peak separation (DEp). Based on the Laviron’s equa-
tions:32,33
electrode, the Nafion/Hb-CILE showed good electrocata-
lytic ability to the reduction of trichloroacetic acid (TCA).
The catalytic activity of Nafion/Hb-CILE toward different
amounts of TCA was investigated with the typical cyclic
voltammograms shown in Fig. 5. With the increase of the
TCA concentrations, the reduction peak currents increased
gradually at -0.30 V (vs. SCE) with the simultaneously dis-
appearance of the oxidation peak (curve a-g), which was a
typical electrocatalytic reduction on the Hb modified elec-
trode. The results suggested that the Hb Fe(II) had reacted
with TCA to produce Hb Fe(III), which was further re-
duced on the electrode to form a catalytic cycle. The cata-
lytic reduction mechanism can be expressed as follows:
Hb heme Fe(III) + e «
Hb heme Fe(II) at electrode
(1)
(2)
Cl3CCOOH « Cl3CCOO- + H+ pKa = 0.89
2Hb heme Fe(II) + Cl3CCOOH + H+ «
2Hb heme Fe(III) + Cl2HCCOOH + Cl-
(3)
The linear relationship between the reduction peak
current and the TCA concentration was constructed in the
range of 33.0~154.0 mmol L-1 with the linear regression
equation as Ip(mA) = 4.17C (mmol L-1) + 218.84 (g =
0.998) and the detection limit was calculated as 8.5 mmol
L-1 (3s). At a higher concentration of TCA in the buffer so-
lution, the calibration curve tended to level off and reached
a constant value, which indicated a typical Michaelis-
Menten kinetic process. Based on the electrochemical ver-
sion of the Lineweaver-Burk equation,36
RT
Epa = E0¢
Epc = E0¢
+
ln u
(1-a)nF
RT
-
ln u
anF
RTK S
RT
RT
Ep = E0¢
+
ln
-
anF anF
ln u
anF
RT
logkS = a log(1-a)+ (1-a)log a -log
nFn
nFDEpa(1-a)
-
2.3RT
The electrochemical parameters can be calculated
based on the relationship of the redox peak potentials with
lnu. The linear regression equations were got as Epa(V) =
0.046lnu - 0.147 (g = 0.990) and Epc(V) = -0.072lnu -
0.551 (g = 0.997), respectively. From the slope and the in-
tercept, the values of electron transfer coefficient (a) and
electrode reaction standard rate constant (ks) were calcu-
lated as 0.390 and 0.568 s-1, respectively. The ks value was
bigger than some other reported values,34,35 which indi-
cated a fast electron transfer rate of Hb in the carbon ionic
liquid bioelectrode.
Fig. 5. Cyclic voltammograms of Nafion/Hb-CILE in
pH 7.0 PBS before (a) and after the addition of
38.0, 48.0, 72.0, 100.0, 124.0, 128.0 mmol L-1
of TCA (curve b-g), respectively, at the scan
rate of 100 mV s-1.
Electrocatalysis towards trichloroacetic acid
Due to the presence of Hb molecules in the modified

1266 J. Chin. Chem. Soc., Vol. 57, No. 6, 2010
Xuan et al.
K Mapp
BMIMPF6. The fabricated bioelectrode showed good
electrocatalytic ability to the reduction of TCA, which pro-
vided a new way for fabricating third generation biosensor.
1
1
=
ISS Imax Imax
+
C
the apparent Michaelis-Menten constant (KMapp) can be cal-
culated, where C is the concentration of TCA in the solu-
tion, Iss is the steady current after the addition of TCA, and
EXPERIMENTAL
Apparatus and reagents
I
max is the maximum current measured under saturated sub-
Bovine hemoglobin (Hb, MW. 64 500, Tianjin Chuanye
Biochemical Company, China), 1-ethyl-3-methylimida-
zolium tetrafluoroborate (EMIMBF4, Hangzhou Kemer
Chemical Company, China), graphite powder (average par-
ticle size 30 mm, Shanghai Colloid Chemical Company,
China), liquid paraffin (Laiyang Shuangshuang Chemical
Limited Company, China) and Nafion (0.5% ethanol solu-
tion, Sigma) were used as received without further purifi-
cation. All the electrochemical measurements were carried
out with 0.1 mol L-1 phosphate buffer solution (PBS) as
supporting electrolyte. Other chemicals used were of ana-
lytical grade and doubly distilled water was used through-
out.
app
strate condition. According to this equation, the KM
value for Nafion/Hb-CILE based TCA biosensor was cal-
culated as 0.0969 mol L-1, which was similar to a previous
reported value.37 It is known that the smaller KMapp value in-
dicates the higher catalytic activity, so the Hb molecules in
the Nafion/Hb-CILE show good bioactivity.
Stability of the modified electrode
The stability of the Nafion/Hb-CILE was firstly tested
by comparing the cyclic voltammetric peak currents of Hb
with time intervals of 2 h. Only slight changes happened in
cyclic voltammograms after 2 days, which indicated the
presence of Nafion on the electrode surface could form a
stable film. Nafion can be served as an effective membrane
to prevent the leakage of Hb and protect the electrode sur-
face from interference. After two weeks storage of the
modified electrode at 4 ºC, 97.3% of its initial current re-
sponse was retained, demonstrating the Nafion/Hb-CILE
had good long-term storage stability.
All the electrochemical experiments including cyclic
voltammetry (CV) and electrochemical impedance spec-
troscopy (EIS) were performed on a CHI 750 B electro-
chemical workstation (Shanghai CH Instrument, China). A
conventional three-electrode system was employed with a
Hb modified electrode as working electrode, a platinum
wire as counter electrode and a saturated calomel electrode
(SCE) as reference electrode. Scanning electron micros-
copy (SEM) was performed with a JSM-6700F scanning
electron microscope (Japan Electron Company, Japan).
Preparation of ionic liquid-graphite powder-Hb com-
posite paste electrode
CONCLUSION
In this paper, a new Hb modified carbon ionic liquid
paste bioelectrode was fabricated by mixing hemoglobin,
ionic liquid EMIMBF4, graphite powder and liquid paraf-
fin together in an agate mortar. Nafion was used as a stable
film material to retain the Hb molecules on the surface of
electrode. Direct electron transfer of Hb was realized and
enhanced on the electrode with a pair of well-defined redox
peaks appeared. The results can be attributed to the pres-
ence of biocompatibility and higher ionic conductivity of
EMIMBF4, which provided a suitable microenviroment for
Hb to mix homogeneously with graphite powder. Com-
pared with another ionic liquid 1-butyl-3-methylimida-
zolium hexafluorophosphate (BMIMPF6) used for the fab-
rication of the paste bioelectrode,38 the smaller value of
DEp with the bigger values of G* and ks appeared in this pa-
per showed the faster electron transfer rate. The results
were attributed to the different physicochemcial properities
of EMIMBF4, which was a hydrophilic solvent with high
conductivity and small viscosity than the hydrophobic
Carbon paste (CP) was prepared by simply mixing
graphite powder and liquid paraffin (3 g: 1 mL) thoroughly
in an agate mortar. Carbon ionic liquid paste (CIL) was ob-
tained by mixing graphite powder with liquid paraffin and
EMIMBF4 (3 g: 0.25 mL: 0.75 mL). The Hb modified CIL
(Hb-CIL) was prepared by directly mixing Hb with the car-
bon ionic liquid paste at the weight ratio of 3:5 and ground
homogeneously in an agate mortar. As for comparison, the
Hb modified CP (Hb-CP) was prepared by the similar pro-
cedure with the same weight ratio. Portions of the modified
carbon paste was packed into one end of glass tube (F = 4.2
mm) and copper wire was inserted through the opposite end
to establish an electrical contact. Then different kinds of
chemically modified carbon paste electrodes (CILE, Hb-
CPE, Hb-CILE) were fabricated and these electrodes were
stored at 4 ºC when not in use. Prior to use the surfaces of

Ionic Liquid-hemoglobin-carbon Paste Bioelectrode
J. Chin. Chem. Soc., Vol. 57, No. 6, 2010 1267
2006, 18, 379.
the prepared electrodes were gently polished on a piece of
weighing paper and a 8 mL volume of 0.5% Nafion solution
was cast on the electrode surface to form a stable film,
which could prevent the leakage of Hb from electrode sur-
face and increase the stability of the modified electrode.
Then these modified electrodes were demonstrated as
Nafion/CILE, Nafion/Hb-CPE and Nafion/Hb-CILE, re-
spectively.
12. Sun, W.; Wang, D.-D.; Gao, R.-F.; Jiao, K. Electrochem.
Commun. 2007, 9, 1159.
13. Li, X.-Q.; Wang, Y.; Sun, X.-Y.; Zhan, T.-R.; Sun, W. J.
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Electrochemical procedure
The three-electrode system was placed in a 10 mL
electrochemical cell containing 0.1 mol L-1 pH 7.0 PBS and
cyclic voltammetric experiments were performed in the po-
tential range from +0.2 to –0.9 V (vs. SCE) at the scan rate
of 100 mV s-1. Electrochemical impedance spectroscopy
(EIS) was preformed in a 0.1 mol L-1 KCl solution contain-
ing 10.0 mmol L-1 [Fe(CN)6]3-/4- with the applied AC volt-
age amplitude as 5 mV and the voltage frequencies swept
from 105 to 0.05 Hz. All the buffers were purged with
high-purity nitrogen for 15 min before the experiments and
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the electrochemical experiments were carried out at 25
ºC.
2
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Received July 12, 2010
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