Direct electrochemistry and electrocatalysis of myoglobin with ionic liquid through multilayers film on carbon ionic liquid electrode

Article abstract of DOI:10.1002/jccs.201190147

Multilayers of myoglobin (Mb) with ionic liquid 1-ethyl-3-methylimidazolium tetrafluoroborate ([EMIM]BF₄) was assembled on carbon ionic liquid electrode (CILE) based on the electrostatic attraction between the negatively charged Mb and the positively charged imidazolium ion of IL. The CILE was fabricated with 1-ethyl-3-methylimidazolium ethylsulfate ([EMIM]EtOSO ₃) as the modifier, which exhibited imidazolium ion on the electrode surface. Then Mb molecules were assembled on the surface of CILE step-by-step to get a {IL/Mb}_n multilayer film modified electrode. UV-Vis adsorption and FT-IR spectra indicated that Mb remained its native structure in the IL matrix. In deaerated phosphate buffer solution (pH 7.0) a pair of well-defined quasi-reversible redox peaks appeared with the apparent formal potential (E ⁰') as -0.212 V (vs. SCE), which was the characteristic ofMb heme Fe(III)/Fe(II) redox couples. The results indicated that the direct electron transfer of Mb was realized on the modified electrode. The {IL/Mb} _n/CILE displayed excellent electrocatalytic ability to the trichloroacetic acid reduction in the concentration range from 2.0 to 22.0 mmol/L with the detection limit of 0.6 mmol/L (3σ). The proposed method provides a new platform to fabricate the third generation biosensor based on the self-assembly of redox protein with ILs.

Full text of DOI:10.1002/jccs.201190147

9
30
Journal of the Chinese Chemical Society, 2011, 58, 930-936
Direct Electrochemistry and Electrocatalysis of Myoglobin with Ionic
Liquid through Multilayers Film on Carbon Ionic Liquid Electrode
a
a
b
b
a
b
Chengxiang Ruan, * Jin Lou, Yan Wang, Yaqing Guo, Weimin Gao and Wei Sun *
Jiangxi Key Laboratory of Surface Engineering, Jiangxi Science and Technology Normal University,
Jiangxi 330013, P. R. China
a
b
College of Chemistry and Molecular Engineering, Qingdao University of Science and Technology,
Qingdao 266042, P. R. China
Received January 20, 2011; Accepted May 23, 2011; Published Online June 13, 2011
Multilayers of myoglobin (Mb) with ionic liquid 1-ethyl-3-methylimidazolium tetrafluoroborate
[EMIM]BF ) was assembled on carbon ionic liquid electrode (CILE) based on the electrostatic attraction
between the negatively charged Mb and the positively charged imidazolium ion of IL. The CILE was fab-
ricated with 1-ethyl-3-methylimidazolium ethylsulfate ([EMIM]EtOSO ) as the modifier, which exhib-
ited imidazolium ion on the electrode surface. Then Mb molecules were assembled on the surface of CILE
step-by-step to get a {IL/Mb} multilayer film modified electrode. UV-Vis adsorption and FT-IR spectra
indicated that Mb remained its native structure in the IL matrix. In deaerated phosphate buffer solution
(
4
3
n
(
(
pH 7.0) a pair of well-defined quasi-reversible redox peaks appeared with the apparent formal potential
0
E ) as -0.212 V (vs. SCE), which was the characteristic of Mb heme Fe(III)/Fe(II) redox couples. The re-
¢
sults indicated that the direct electron transfer of Mb was realized on the modified electrode. The
IL/Mb} /CILE displayed excellent electrocatalytic ability to the trichloroacetic acid reduction in the
{
n
concentration range from 2.0 to 22.0 mmol/L with the detection limit of 0.6 mmol/L (3s). The proposed
method provides a new platform to fabricate the third generation biosensor based on the self-assembly of
redox protein with ILs.
Keywords: Myoglobin; Direct electrochemistry; Carbon ionic liquid electrode; 1-Ethyl-3-methyl-
imidazolium tetrafluoroborate.
INTRODUCTION
Investigations on the direct electrochemistry of pro-
surface and then enhance the electron transfer rate of redox
proteins.
teins or enzymes can provide a good model for the mecha-
nistic studies on electron transfer in biological systems,
which can help us understand the energy transform and ma-
terial metaboly in life processes. However, it is difficult for
the redox proteins in solutions or on the surface of conven-
tional electrode to exchange electrons directly with the na-
ked solid electrodes because of the embedment of the redox
center of proteins in polypeptide chain structures. In these
circumstances, an important object for biochemists is to
achieve efficient electrical contact between the protein and
Ionic liquids (ILs) are molten salts that composed of
organic cations and either organic or inorganic anions.
They have exhibited many specific characteristics such as
high chemical and thermal stability, negligible vapor pres-
sure and high conductivity, which make them environmen-
tal friendly solvents used in various electrochemical pro-
cesses. In recent years the applications of ILs in the fields
of electrochemistry and electroanalysis have received
6
much attention. ILs can be used as a novel electrode ma-
,7
terial for catalysis or sensing, or as the supporting electro-
1
8
9
the electrode surface. Various methods including sol-gel,
lyte for the electrochemical reaction. Maleki et al. re-
ported the simultaneous determination of dopamine, ascor-
bic acid and uric acid using carbon ionic liquid electrode
2
electropolymerization, covalent-binding, layer-by-layer
4
assembly and direct embedded in biocompatible mem-
3
5
brane were used to immobilize the protein on the electrode
10
(CILE). Sun et al. also applied CILE to the simultaneous
*
Corresponding author. Tel. & Fax: +86-532-84023927; E-mail: cxruan@126.com and sunwei@qust.edu.cn

Direct Electrochemistry of Myoglobin
J. Chin. Chem. Soc., Vol. 58, No. 7, 2011 931
detection of adenine and guanine. Recently the direct elec-
trochemistry of redox proteins has also been achieved with
different types of ILs. Due to the biocompatibility of the
ILs, the activity of protein shows similar or even higher ef-
fect in ILs than in the conventional organic solvents. For
4
tetrafluoroborate ([EMIM]BF ) were purchased from
Hangzhou Kemer Chemical Limited Company. Horseheart
myoglobin (Mb, MW.17800, Sigma), graphite powder (av-
erage particle size £ 30 mm, Shanghai Colloid Chemical
Plant) and trichloroacetic acid (TCA, Tianjin Kermel
Chemical Reagent Co. Ltd.) were used as received. 0.1
mmol/L phosphate buffer solutions (PBS) were used as
supporting electrolyte, which was deaerated for 30 min
with high-purity nitrogen and kept under the nitrogen at-
mosphere during the experiments. Other chemicals were of
analytical reagent grade and the solutions were prepared
with doubly distilled water.
11
example Wang et al. investigated the direct electrochem-
istry and electrocatalysis of heme proteins with IL as elec-
1
trolyte. Gao et al. reported direct electrochemistry of
2
1
3
myoglobin (Mb) on DNA modified CILE. Sun et al. elec-
trodeposited Co nanoparticles on CILE and further used it
as the platform to construct a Mb electrochemical biosen-
1
sor. Chen et al. investigated the direct electrochemistry of
4
horseradish peroxidase (HRP) in the Nafion-IL composite
Apparatus
1
film. Zhang et al. used IL-hyaluronic acid biocomposite
5
Electrochemical experiments were performed on a
CHI 750B electrochemical workstation (Shanghai CH In-
strument, China) with a conventional three-electrode sys-
tem, where a Mb modified electrode was used as the work-
ing electrode, a platinum wire as auxiliary electrode and a
saturated calomel electrode (SCE) as reference electrode.
UV-Vis spectroscopy was recorded on a Cary 50 probe
spectrophotometer (Varian Company, Australia) and FT-IR
spectra were performed with a Tensor 27 FT-IR spectro-
photometer (Bruker Company, Germany).
material modified electrode to realize the direct electro-
1
6
chemistry of Mb. Safavi et al. investigated the direct elec-
trochemistry and electrocatalytic of hemoglobin (Hb) on
CILE with octylpyridinium chloride ([OcPy][Cl]). Sun et
1
7
6
al. fixed Mb in Nafion-BMIMPF composite film on
surface of CILE and studied the direct electrochemical
behaviors.
Trichloroacetic acid (TCA) is an important organo-
halide pollutant present in the environment. It is widely
used in biochemistry for the precipitation of macromole-
cules such as proteins, DNA and RNA. Also TCA can be
found in the drinking water due to the chlorine disinfection
and is the major metabolite of the trichloroethylene and
Fabrication of carbon ionic liquid electrode
The following procedure was used to prepare the
3
CILE. Typically 200 µL of [EMIM]EtOSO , 800 µL of liq-
uid paraffin and 3.2 g of graphite powder were put into an
agate mortar and hand-mixed carefully. Then a portion of
carbon paste was packed firmly into a glass tube with the
diameter of 4 mm. A copper wire was inserted into the end
of the paste in the inner hole of the tube to make the electri-
cal contact and the CILE surface was polished on a weigh-
ing paper to get a smooth interface.
1
8
tetrachloroethylene. So it is necessary to establish sensi-
tive methods for the TCA detection.
In this paper a carbon ionic liquid electrode (CILE)
was fabricated by using 1-ethyl-3-methylimidazolium
ethylsulfate ([EMIM]EtOSO
and ionic liquid 1-ethyl-3-methylimidazolium tetrafluoro-
borate ([EMIM]BF ) was alternatively adsorbed on the
electrode surface to get a {IL/Mb} /CILE. Experimental
3
) as the binder. Then the Mb
4
n
Construction of {IL/Mb} multilayer film
n
The newly prepared CILE was used as the basal elec-
trode for the multilayer film assembly. Firstly, the CILE
was immersed in 10.0 mg/mL Mb solution (in pH 8.0 PBS)
for 40 min to adsorb a layer of Mb on the surface of CILE.
The obtained electrode was rinsed with water and dried in
results indicated that the direct electron transfer of Mb was
realized in the LBL film with a pair of well-defined redox
peaks appeared. The {IL/Mb}
catalytic activity to the reduction of trichloroacetic acid
TCA) with high sensitivity.
n
/CILE showed good electro-
(
air. Then Mb/CILE was alternately immersed in [EMIM]BF
4
and Mb solution (10.0 mg/mL in pH 8.0 PBS) for 40 min,
with intermediate water washing and air drying. This pro-
cedure was repeated for the desired number of bilayers (n)
EXPERIMENTAL
Chemical
Ionic liquids 1-ethyl-3-methylimidazolium ethylsul-
fate ([EMIM]EtOSO ) and 1-ethyl-3-methylimidazolium
to get the {IL/Mb}
CILE.
n
multilayer films on the surface of
3

9
32 J. Chin. Chem. Soc., Vol. 58, No. 7, 2011
Ruan et al.
RESULTS AND DISCUSSION
uted to the combination of N–H bending and C–N stretch-
ing. Fig. 2 showed the typical FT-IR spectra of Mb film and
IL-Mb film, the amide I and amide II bands of Mb in the IL
UV-Vis spectroscopy
-
matrix (1639 and 1573 cm ) are very similar to those ob-
1
UV-Vis spectroscopy is a conventional tool for iden-
tifying the secondary structure of redox protein in different
matrix and it is used to investigate the influence of IL on the
structure of Mb. Due to the hydrophilic properties of IL
-
1
tained in Mb film (1655 and 1539 cm ), suggested that Mb
retained its native structure in the IL matrix.
[
EMIM]BF
4
, Mb solution (in pH 8.0 PBS) can easily mixed
. And the Uv-Vis spectra were recorded
n
Cyclic voltammograms of {IL/Mb} /CILE
2
2
with [EMIM]BF
4
According to the reference a layer of IL film was
present on the surface of CILE. Due to the specific of IL
structure, the positive charges existed on the surface of
CILE with the presence of the organic cationic ions. While
the isoelectric point (pI) of Mb molecule is at 6.8, so the Mb
molecules have the net negative charges in pH 8.0 PBS,
which can interact with the positive charged imidazolium
ions on CILE surface by the electrostatic force. Then Mb
by a transmission model. The position of the Soret band of
the Mb molecules can provide the information about the
conformational change in the heme protein, which is sensi-
tive to the changes of microenvironments around the heme
1
9
site. Fig. 1 showed the UV-Vis adsorption spectra of Mb
and its mixture with IL. The Soret absorption band of na-
tive Mb molecules appeared at 409.1 nm in water (curve a).
After mixed with [EMIM]BF
4
, the Soret absorption band
and IL [EMIM]BF
4
were assembled on the surface of CILE
/CILE.
appeared at 410.3 nm (curve b), which was about 1.2 nm
shift to the red direction. The results suggested that Mb re-
mained its native structure in the IL matrix, which indicated
that the biocompatible IL had the ability to provide a favor-
by a step-by-step procedure to make a {IL/Mb}
n
Firstly the Mb molecules were adsorbed on the CILE to get
an electrode denoted as Mb/CILE, which was further im-
mersed in [EMIM]BF
on the surface of Mb/CILE. After several repetition in IL
and Mb solution, a {IL/Mb} multilayer film was formed
4
to adsorb a layer of IM cationic ions
2
0
able microenvironment to stabilize enzyme.
n
FT-IR spectroscopy
on the surface of CILE. The multilayer assembly process
was characterized by electrochemical method and Fig. 3
FT-IR spectroscopy is very sensitive to the confor-
2
1
mational changes of the protein, and the conformational
changes will affect the electron transfer reactivity. The am-
ide I and amide II bands of proteins provide detailed infor-
mation on the secondary structure of polypeptide chain.
n
showed the cyclic voltammetric results of {IL/Mb} /CILE
with different assembly cycles (n). It can be seen that a pair
of well-defined quasi-reversible redox peaks appeared
with almost equal height of peak currents at different n
value from 1 to 4, indicating the direct electron transfer of
Mb was realized on the multilayer film modified electrode.
-
The amide I band (1700–1600 cm ) is caused by C=O
1
stretching vibrations of peptide linkages in the protein’s
-
backbone and the amide II band (1620–1500 cm ) is attrib-
1
n
So the {IL/Mb} film was favorable to realize the electro-
Fig. 1. UV-Vis absorption spectra of Mb (a) in aque-
ous solution, (b) in the IL matrix.
Fig. 2. FT-IR spectra of (a) Mb and (b) IL-Mb film.

Direct Electrochemistry of Myoglobin
J. Chin. Chem. Soc., Vol. 58, No. 7, 2011 933
chemical reaction of Mb. IL is a biocompatible green sol-
vent with high ionic conductivity, and it can provide a suit-
able microenvironment for Mb to retain its native structure
and enhance the electron transfer rate with the underlying
electrode. From Fig. 3 it can be seen that the redox peak
current increased with the assembly cycles of IL/Mb ad-
sorption bilayers up to 3 and then began to decrease slowly
with the further increase of the assembly number. The re-
sults was similar to a recent report about the sol-gel pro-
cessed ionic liquid-hydrophilic carbon nanoparticles layer-
appeared at the number of 3, which also indicated that 3
layers of multifilm gave the maximum electrochemical re-
*
sponses. All the G values calculated were larger than the
-
11
2 11
theoretical monolayer value of 1.89 × 10 mol/cm ,
which indicated that several layers of Mb in the multilayer
films took part in the electron transfer with the underlying
electrode.
3
Direct electrochemistry of {IL/Mb} /CILE
From the curve c of Fig. 3, it can be seen that a pair of
2
3
by-layer film modified electrode, which may be attrib-
uted to the neutralization of the imidazolium groups that re-
sulted in the less adsorption of Mb molecules in the multi-
layer film. While no redox peaks appeared in the selected
potential range when Mb was not present on the electrode
surface. To get the biggest voltammetric response the as-
sembly cycles were fixed at three in the following experi-
ments. The surface concentration of electroactive Mb in the
well-defined and stable symmetric redox peaks appeared
on the {IL/Mb}
3
/CILE with the anodic (Epa) and cathodic
(Epc) peak potential located at -0.283 and -0.141 V (vs.
0
¢
SCE). The apparent formal potential (E ), which is calcu-
lated form the midpoint of Epa and Epc, was got as -0.212
V. The result was the typical characteristic of Mb heme
2
Fe(III)/Fe(II) redox couples, which indicated that the di-
4
rect electron transfer of Mb with electrode was achieved in
{
IL/Mb}
n
/CILE was further calculated to explain the corre-
4
the layer-by-layer film with [EMIM]BF . It has been re-
sponding electrochemical results. According to the Fara-
*
ported that the refolding of protein is improved with the ac-
tivity remained in the IL that containing tetrafluoroborate
*
day’s law (Q = nFAG ), where F is Faraday constant and G
-
-
25
) anions. Typical
is the surface concentration of electroactive Mb, Q can be
calculated from the integration charge of the reduction
peak, A is the area of CILE, and n stands for the number of
electron transferred. Based on the cyclic voltammetric re-
duction peak area of different {IL/Mb} multilayer films,
(BF
4
) and hexafluorophosphate (PF
6
electrochemical responses of the {IL/Mb}
3
/CILE at differ-
ent scan rates were further investigated with the cyclic
voltammograms shown in Fig. 4. In the scan rate range
from 30 to 300 mV/s a pair of well-defined redox peaks ap-
*
the value of G was calculated as 2.63 × 10 mol/cm for the
-9
2
3
peared on the {IL/Mb} /CILE. The oxidation and reduc-
first layer. With the increase of the assembly cycle number
*
from 2 to 4, the values of G were calculated to be 8.67 ×
tion peak currents increased linearly with the scan rate, and
two linear regression equations were plotted as Ipc(µA) =
28.918 + 1.023u(mV/s) (g = 0.995) and Ipa(µA) = -44.288
- 0.722u(mV/s) (g = 0.992), indicating the electrode pro-
-
0 mol/cm , 1.45 × 10 mol/cm and 1.20 × 10 mol/cm ,
9
2
-8
2
-8
2
1
-
respectively. The maximum value of 1.45 × 10 mol/cm
8
2
Fig. 4. (A) Cyclic voltammograms of {IL/Mb}
with different scan rates (from a to k as 30, 50,
0, 100, 120, 150, 180, 200, 220, 250, 300
3
/CILE
8
mV/s) in pH 7.0 PBS; (B) Plot of the cathodic
and anodic peak current with scan rate (u); (C)
The relationship of the cathodic and anodic
peak potential against lnu.
n
Fig. 3. Cyclic voltammograms of the {IL/Mb} film
electrode in pH 7.0 PBS with assembly number
as 1,2,3,4 (a-d) at the scan rate of 100 mV/s.

9
34 J. Chin. Chem. Soc., Vol. 58, No. 7, 2011
Ruan et al.
cess was a typical diffusionless surface-confined electro-
chemical process. With the increase of scan rate the oxida-
tion peak moved to more positive direction and the reduc-
tion peak moved to the negative direction. The peak-to-
peak separation (DEp) was increased gradually, indicating
a quasi-reversible electrode process. So the Laviron’s
of electrocatalytic reaction. So the presence of Mb mole-
cules can great enhance the electrocatalytic of TCA with
the reduction overpotential decreased for more than 0.8 V,
which indicated the large decrease of the activation energy
for the reduction reaction. The results indicated that the Mb
Fe(II) produced in the electrode reaction was chemically
oxidized by TCA and returned back to Mb Fe(III) with a
catalytic cycle. When the concentration of TCA is higher
than 4.0 mmol/L, a new reduction peak appeared at -0.54 V,
which was attributed to the formation of highly reduced
form of Mb that could dechlorinate di- and monochloro-
actetic acid after the dechlorination of TCA with the Mb
2
6,27
model
eters such as the charge transfer coefficient (a) and the
electron transfer rate constant (k ). By exploring the rela-
was used to calculate the electrochemical param-
s
tionship of the redox peak potential with the value of lnu,
two straight lines were got with the linear regression equa-
tions as Epc(V) = -0.0274 - 0.063lnu (mV/s) (g = 0.994)
and Epa(V) = -0.300 - 0.0352lnu (mV/s) (g = 0.994), then
3
0
31
Fe(II). According to the reference, a highly reduced
form of Mb could be produced in a didodecyldimethyl-
ammonium bromide (DDAB) film modified electrodes,
which was proved by the spectroelectrochemical results.
This highly reduced Mb Fe(I) acted as an active reductant
during the dechlorination of trichloroethylenes. So the sec-
ond reduction peak on the cyclic voltammograms with high
concentration of TCA can be tentatively assign to the
highly reduced form of Mb Fe(I), which can dechlorinate
di- and monochloroacetic acid after the dechlorination of
TCA by Mb Fe(II). Then the overall electrocatalytic reac-
tion by the Mb molecules in the modified film may be
deduced with the following equations.
-
1
the values were calculated with a as 0.36 and k
s
as 0.45 s ,
respectively. The k value was in accordance with the previ-
s
2
8,29
ous reported values,
kinetic process.
indicating a fast electrochemical
Electrocatalytic to TCA
It has been reported that redox proteins with heme
groups can catalyze different kinds of substrates. So the
3
fabricated {IL/Mb} /CILE was used to test the electrocata-
lytic activity to the reduction of TCA. Fig. 5 showed the cy-
clic voltammograms obtained at above modified electrode
in the presence of different concentrations TCA added in
the oxygen free PBS. On the CILE without Mb modifica-
tion, the direct reduction of TCAwas found to be more than
Mb Fe(III) + e ® Mb Fe(II)
2Mb Fe(II) + Cl CCOOH + H ®
2Mb Fe(III) + Cl CHCOOH + Cl
Mb Fe(II) + e ® Mb Fe(I)
(1)
+
-
1.1 V (curves a and b). While on the {IL/Mb}
3
/CILE the
3
-
reduction peak appeared at -0.31 V with the disappearances
of the oxidation peak, which was the typical characteristic
2
(2)
(3)
+
2
Mb Fe(I) + Cl
Mb Fe(II) + ClCH
Mb Fe(I) + ClCH COOH + H ®
Mb Fe(II) + CH
2
CHCOOH + H ®
-
2
2
COOH + Cl
(4)
(5)
+
2
2
-
2
3
COOH + Cl
The total reaction included the electrochemical re-
duction of Mb Fe(III) to Mb Fe(II) on the electrode, the re-
duction of TCA with Mb Fe(II) in the film, the further re-
duction of Mb Fe(II) to Mb Fe(I) in the modified film, the
reduction of di- and monochloroactetic acid with formed
Mb Fe(I), and the reoxidation of Mb Fe(I) back to Mb
Fe(II). All the above results indicated that the Mb mole-
cules in the LBL multilayer films retained its bioelectro-
catalytic activity and exhibited excellent electrocatalytic
ability to the reduction of TCA.
Fig. 5. (A) Cyclic voltammograms of CILE in the pres-
ence of (a) 0, (b) 1.0 mmol/L TCA in 0.1 mol/L
pH 7.0 PBS and cyclic voltammograms of
{
IL/Mb}
ferent concentrations of TCA (from c to h: 0,
.0, 6.0, 8.0, 10.0, 12.0 mmol/L TCA) with the
3
/CILE in pH 7.0 PBS containing dif-
4
scan rate of 100 mV/s. (B) The relationship of
the catalytic reduction peak current with the
TCA concentration.
The reduction peak current increased with the TCA

Direct Electrochemistry of Myoglobin
J. Chin. Chem. Soc., Vol. 58, No. 7, 2011 935
-
concentration in the range from 2.0 to 22.0 mmol L and a
1
was realized. The result can be attributed to the specific
characteristics of IL such as high ionic conductivity and
good biocompatibility that provide a suitable microenvi-
ronment for Mb. The electrochemical behavior of Mb in
the multilayer film was carefully investigated with the elec-
trochemical parameters calculated. The Mb bioelectrode
showed excellent electrocatalytic ability to the reduction of
TCA. So the Mb and IL LBL multilayer film modified elec-
trode provided an efficient platform for the preparation of a
new third generation electrochemical biosensor.
linear regression equation was obtained as Iss(mA) =
4
7.01C (mmol/L) + 3.87 (g = 0.997) with the detection
limit as 0.6 mmol/L (3s), which was smaller than some pre-
3
2,33
vious results,
indicating the proposed Mb bioelectrode
exhibited higher sensitivity to TCAdetection. The apparent
app
Michaelis-Menten constant (K
M
) can be calculated from
the electrochemical version of the Lineweaver-Burk equa-
3
tion:
4
app
1
1
K M
=
Iss Imax Imax
+
c
ACKNOWLEDGEMENTS
where Iss is the steady current after the addition of sub-
strate, c is the bulk concentration of the substrate, and Imax
We are grateful to the financial support of the Natural
Science Foundation of Jiangxi Province (2009GZC0031)
and the S&T plan projects of Jiangxi Provincial Education
Department (GJJ10590).
is the maximum current measured under the saturated sub-
app
strate condition. Based on the above equation the K
M
value was calculated as 21.36 mmol/L, which was smaller
7,35
than some reported values.
1
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1
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