Direct Electrochemistry and Electrocatalysis of …carbon nanotube (CNT), graphene, metal,...

Electrochemistry of Myoglobin with CoMoO4 Nanorods Bull. Korean Chem. Soc. 2013, Vol. 34, No. 2 475

http://dx.doi.org/10.5012/bkcs.2013.34.2.475

Direct Electrochemistry and Electrocatalysis of Myoglobin with CoMoO4

Nanorods Modified Carbon Ionic Liquid Electrode

Zengying Zhao, Lili Cao,† Anhui Hu,† Weili Zhang,† Xiaomei Ju,† Yuanyuan Zhang,† and Wei Sun†,*

School of Science, National Laboratory of Mineral Materials, China University of Geosciences, Beijing 100083, P.R. China†College of Chemistry and Molecular Engineering, Qingdao University of Science and Technology, Qingdao 266042, P.R. China

*E-mail: [email protected]

Received October 19, 2012, Accepted November 19, 2012

By using ionic liquid 1-hexylpyridinium hexafluorophosphate (HPPF6) based carbon ionic liquid electrode

(CILE) as the substrate electrode, a CoMoO4 nanorods and myoglobin (Mb) composite was casted on the

surface of CILE with chitosan (CTS) as the film forming material to obtain the modified electrode (CTS/

CoMoO4-Mb/CILE). Spectroscopic results indicated that Mb retained its native structures without any

conformational changes after mixed with CoMoO4 nanorods and CTS. Electrochemical behaviors of Mb on the

electrode were carefully investigated by cyclic voltammetry with a pair of well-defined redox peaks from the

heme Fe(III)/Fe(II) redox center of Mb appeared, which indicated that direct electron transfer between Mb and

CILE was realized. Electrochemical parameters such as the electron transfer number (n), charge transfer

coefficient (α) and electron transfer rate constant (ks) were estimated by cyclic voltammetry with the results as

1.09, 0.53 and 1.16 s−1, respectively. The Mb modified electrode showed good electrocatalytic ability toward

the reduction of trichloroacetic acid in the concentration range from 0.1 to 32.0 mmol L−1 with the detection

limit as 0.036 mmol L−1 (3σ), and the reduction of H2O2 in the concentration range from 0.12 to 397.0 μmol L−1

with the detection limit as 0.0426 μmol L−1 (3σ).

Key Words : Carbon ionic liquid electrode, Electrochemistry, CoMoO4 nanorods, Myoglobin

Introduction

Direct electrochemistry of redox proteins and electrode is

currently attracting widespread attentions due to its impor-

tance to understand the structure and function of proteins.

The research can also provide the basic information for the

development of electrochemical biosensors. Myoglobin (Mb)

is an important oxygen transporting protein in the biological

system, which is consisted of a polypeptide chain and a

heme cofacter.1-3 However, due to the deep burying of electro-

active center in the complicated structure of protein and the

denaturation of proteins or the unfavorable orientation on the

electrode interface, direct electron transfer of proteins on the

bare electrode is difficult to be realized.4 Great efforts have

been made to increase the electron transfer kinetics of pro-

teins with electrodes by using mediators or promoters such

as polymer films,5 clay,6 nanoparticles7 and biomembranes.8

By using these functionalized materials direct electrochemistry

of proteins could be greatly enhanced on the modified elec-

trodes with a pair of redox peaks appeared.

With the development of nanotechnology different kinds

of nanoparticles had been applied to direct electrochemistry

of protein. Nanostructure materials have exhibited unique

properties such as large surface area, exceptional chemical

stability, tunable porosity, good electrical conductivity, high

mechanical strength, biocompatibility and catalytic activities.9

The presence of nanomaterials on the electrode can provide

a novel way to enhance electron transfer rate between

proteins and electrode due to the quantum size effect and

surface effect.10 Different kinds of nanoparticles such as

carbon nanotube (CNT), graphene, metal, semiconductor

with various morphologies had been used in the protein

electrochemistry. For example, Wang et al.11 investigated the

direct electrochemistry and electrocatalysis of Mb adsorbed

in gold nanoshells. Ruan et al.12 studied the direct electro-

chemistry and electrocatalysis of Mb based on graphene-

ionic liquid (IL)-chitosan bionanocomposites. Sun et al.13

prepared graphene-TiO2-IL nanocomposite film modified

electrode and applied it to investigate the direct electro-

chemistry of hemoglobin (Hb). Ma et al.14 developed a

biocompatible nano-platform for immobilization of Hb

based on ZrO2 nanotubes-IL for the electrochemical sensing

of NaNO2. But there are seldom reports about the appli-

cation of molybdate compounds in the field of protein

electrochemistry. Metal molybdates are important inorganic

materials with many applications in photoluminescence,

electrochemistry and catalyst. As for CoMoO4, there are

three basic types existing under atmospheric pressure, that is

the low temperature α-CoMoO4, the high temperature β-

CoMoO4 and the hydrate CoMoO4·nH2O. Kichambare et al.15

applied CoMoO4 as a photo anode in the polycrystalline

pellet form for photovoltaic electrochemical cell. Rodriguez

et al.16 investigated the electronic properties and phase trans-

formations of CoMoO4 and NiMoO4 by X-ray absorption

near-edge spectroscopy and XRD studies. Pandey et al.17

studied the structural, optical, electrical and photovoltaic

electrochemical properties of CoMoO4 thin film. Peng et

al.18 proposed a large scale synthesis method of nanostruc-

476 Bull. Korean Chem. Soc. 2013, Vol. 34, No. 2 Zengying Zhao et al.

tured CoMoO4 by precipitation strategy. By decreasing the

dimensionality, nanostratured CoMoO4 exhibits many novel

properties such as high surface area, optical transparency,

good antibacterial activities, good biocompatibility, relative-

ly good conductivity, which are of great importance in

heterogeneous catalysis and industrial application. Ding et

al.19 synthesized molybdate hydrates nano/microcrystals by

a hydrothermal approach and investigated the magnetic,

photocatalytic and electrochemical properties. Pei et al.20

prepared nanostructured CoMoO4 catalysts supported on

mesoporous silica SBA-15 with different loadings and

investigated the catalytic activity of these catalysts in the

oxidative dehydrogenation of propane. Xiao et al.21 synthe-

sized a one-dimensional CoMoO4 nanorods by hydrothermal

methods and investigated the lithium storage capability. Mai

et al.22 fabricated asymmetric supercapacitor based on

hierarchical MnMoO4/CoMoO4 heterostructured nanowires,

which showed a specific capacitance and good reversibility.

Due to the specific electronic properties, CoMoO4 nano-

materials have the potential application to direct electro-

chemistry of redox proteins.

In recent years carbon ionic liquid electrode (CILE) has

been widely used in the field of electrochemical sensors.23

CILE was prepared by using IL as the binder and the

modifier in the traditional carbon paste electrode (CPE). ILs

are compounds consisted entirely of ions that exist in liquid

state around room temperature, which exhibit the properties

such as extraordinarily high chemical and thermal stability,

good conductivity, wide electrochemical windows and good

dissolving capability.24 Due to the present of IL in CPE,

CILE has been proven to exhibit excellent performances

such as resistivity towards electrode fouling, high rates of

electron transfer and the inherent catalytic activity. It has

been reported that CILE combines the advantages of carbon

electrode such as glassy carbon electrode (GCE), carbon

ceramic electrode and pyrolytic graphite electrode.25 So

CILE has been used as the working electrode in the field of

electrochemical sensors. Maleki et al.26 made a CILE with

noctylpyridinum hexafluorophosphate and carbon powder to

investigate the electrochemical behaviors of different electro-

active compounds. Sun et al.27,28 fabricated an N-butylpyri-

dinium hexafluorophasphate based CILE for the electro-

chemical applications. Safavi et al.29 fabricated a glucose

sensor based on nanoscale nickel hydroxide modified CILE.

In this work, CoMoO4 nanorods were synthesized and

used for the investigation on the direct electrochemistry of

Mb with CILE as the substrate electrode. Mb modified

electrode was fabricated with the addition of CoMoO4

nanorods and chitosan (CTS) was used as the film to fix the

composite on the electrode surface, which could increase the

stability of the modified electrode. Direct electrochemistry

of Mb was realized and enhanced with a pair of well-defined

redox peaks appeared, which could be attributed to the

specific properties of CoMoO4 nanorods. Electrochemical

behaviors of Mb were carefully investigated with the electro-

chemical parameters calculated. The modified electrode

exhibited excellent catalytic activity to the electrochemical

reduction of trichloroacetic acid (TCA) and hydrogen per-

oxide (H2O2) with wider linear range and lower detection

limit.

Experimental

Reagents. Myoglobin (Mb, MW. 17800, Sigma), 1-hexyl-

pyridinium hexafluorophosphate (HPPF6, Lanzhou Green-

chem ILS. LICP. CAS., China), chitosan (CTS, minimum

95% deacetylated, Dalian Xindie Chemical Reagents Ltd.

Co., China), graphite powder (average particle size 30 µm,

Shanghai Colloid Chemical Plant, China), trichloroacetic

acid (TCA, Tianjin Kemiou Chemical Ltd. Co., China) and

hydrogen peroxide (H2O2, Tianjin Bodi Chemical Holding

Ltd. Co., China) were used as received. CoMoO4 nanorods

(nano-CoMoO4) were synthesized based on the reported

procedure.21 0.1 mol L−1 phosphate buffer solutions (PBS)

with various pH values were prepared and used as the sup-

porting electrolyte. All the other chemicals were of analy-

tical reagent grade and doubly distilled water was used to

prepare all solutions.

Apparatus. All the electrochemical measurements were

executed on a CHI 750 B electrochemical workstation

(Shanghai CH Instrument, China) with a conventional three-

electrode system, which was composed of a modified CILE

as working electrode, a saturated calomel electrode (SCE) as

reference electrode and a platinum wire as counter electrode.

UV-Visible absorption spectrum and FT-IR spectrum were

operated with a Cary 50 probe spectrophotometer (Varian

Company, Australia) and a Tensor 27 FT-IR spectrophoto-

meter (Bruker, Germany), respectively. Scanning electron

microscopy (SEM) was conducted on a JSM-6700F scann-

ing electron microscope (Japan Electron Company, Japan).

Electrode Preparation. CILE was prepared with the

following procedure: 0.8 g of HPPF6 and 1.6 g of graphite

powder were ground carefully in a mortar to get an IL modi-

fied carbon paste. Then a portion of homogeneous paste was

packed firmly into a glass tube (Φ = 4.2 mm) with the elec-

trical contact established through a copper wire to the end of

the paste. Before use the surface of CILE was polished on a

weighing paper to get a mirror-like interface for the further

modification.

The modifier was prepared by mixing Mb and nano-

CoMoO4 homogeneously to obtain a suspension solution

with the final concentrations of 15.0 mg mL−1 and 0.3 mg

mL−1, respectively. Then 8 μL of mixture was evenly spread

onto the CILE surface and the electrode was left in the air to

allow the water evaporated gradually. Finally, 5.0 µL of 1.0

mg mL−1 CTS (in 1.0% HAC) solution was dropped on the

electrode surface and dried to get a uniform film. The fabri-

cated electrode was denoted as CTS/CoMoO4-Mb/CILE and

kept at 4 oC refrigerator when not use. Other modified elec-

trodes such as CTS/CILE, CTS/Mb/CILE etc. were fabri-

cated with the similar procedures for comparison.

Electrochemical Measurement. The three-electrode system

was immersed in a 10 mL cell containing 0.1 mol L−1 pH 3.0

PBS and scanned in the potential range from 0.2 to –0.6 V


(vs SCE) at the scan rate of 100 mV s−1. Before the measure-

ment the buffer solutions were deoxygenated by bubbling

high pure nitrogen thoroughly for 30 min and the nitrogen

atmosphere environment was kept in the electrochemical

cell during the procedure.

Results and Discussion

SEM Images. SEM was used to record the image of the

synthesized CoMoO4 nanomaterial with the results shown in

Figure 1 and the inset was the enlarged magnitude of the

image. It can be seen that CoMoO4 appeared as rodlike

nanostructures with the average diameter of 200 nm. Also

some of the nanorods aggregated together to form bundles

like structure. The disorder distribution of CoMoO4 nano-

rods can form a porous structure on the electrode surface

with the increase of the effective area, which is suitable for

the immobilization of Mb molecules.

FT-IR and UV-Vis Absorption Spectroscopy. FT-IR spectro-

scopy is a sensitive tool for studying the secondary structure

of heme proteins. The characteristic amide I (1700-1600

cm−1) and amide II (1620-1500 cm−1) bands of proteins can

provide the detailed information on the secondary structure

of polypeptide chain. The amide I band is caused by the

C=O stretching vibration of peptide linkages in the back-

bone of the protein, and the amide II band is assigned to a

combination of N-H bending and C-N stretching vibrations.30

FT-IR spectroscopy was recorded with the results shown in

Figure 2(A). It can be seen that the amide I and II bands of

native Mb were located at 1651 and 1528 cm−1 (curve a).

While the mixture of CTS, CoMoO4 and Mb also gave the

similar positions with the amide I and II bands at 1649 and

1525 cm−1 (curve b). The almost same position of the wave-

number in FT-IR spectra indicated that Mb still retained its

original structure after immobilized with CTS and CoMoO4

nanorods.

The secondary structure of proteins in the composite can

be further confirmed by UV-Vis absorption spectroscopy. In

UV-Vis absorption spectrum the Soret absorption band from

the four iron heme groups of heme proteins is sensitive to

the variation of the microenvironment around the heme

group and the band shift may provide the information about

the denaturation on the tertiary structure in heme proteins.31

Figure 2(B) presented the typical UV-Vis absorption spectro-

scopy of Mb in the composite material. It can be seen that

the native Mb gave a Soret absorption band at 409 nm in pH

3.0 PBS (curve a). While the mixture solution of CTS-

CoMoO4-Mb also had the Soret band at 409 nm without

changes (curve b), suggested that the secondary structure of

proteins did not change after mixing with CTS and CoMoO4

nanorods. CTS is a biodegradable and nontoxic natural

biopolymer originated from the exoskeleton of crustaceans,

which has been widely reported as an immobilization matrix

for the preparation of biosensor and bioreactor with ex-

cellent film-forming ability.32,33 So the results indicated that

CoMoO4 nanorods were also biocompatible nanomaterials

without the change of Mb structure.

Direct Electrochemistry of Mb Modified Electrode.

Cyclic voltammograms of different modified electrodes in

pH 3.0 PBS at the scan rate of 100 mV s−1 were recorded and

demonstrated in Figure 3. It was observed that no voltam-

metric responses appeared on both CILE (curve a) and CTS/

CILE (curve b), indicating no electroactive substance present

on the electrode. On CTS/Mb/CILE (curve c) a pair of un-

symmetric redox peaks appeared on cyclic voltammogram

with the cathodic peak current (Ipc) as 6.709 μA and the

anodic peak current (Ipa) as 6.057 μA. The redox peak

potential was got with the values of Epc as −0.284 V and

Epa as −0.171 V, and the peak-to-peak separation (ΔEp) as

113 mV. The results indicated that direct electron transfer

between Mb and CILE was realized with slow electron

Figure 1. SEM images of CoMoO4 nanorods. Inset is the highmagnication of SEM image.

Figure 2. (A) FT-IR spectra of (a) Mb film; (b) CTS/CoMoO4-Mb composite film; (B) UV-Vis absorption spectra of native Mb (curve a)and CTS-CoMoO4-Mb mixture (curve b) solution with pH 3.0 PBS, respectively.


transfer rate, which was in accordance with the former

reported result.34 CILE has been proven to exhibit some

specific characteristics such as good conductivity, high rates

of electron transfer and the inherent catalytic activity. Also a

layer of IL film is present on the electrode surface, which

could provide a specific interface for the immobilization of

Mb and the realization of the electron transfer. With the

addition of CoMoO4 nanorods on the electrode surface, a

pair of well-defined and quasi-reversible redox peaks ap-

peared with the increase of redox peak currents (curve d),

indicating the presence of CoMoO4 nanorods enhanced the

electron transfer between Mb and CILE. The redox peak

current was obtained as 18.92 μA (Ipc) and 17.08 μA (Ipa),

respectively, which was about 2.8 times higher than that of

CTS/Mb/CILE. The peak potentials were located at −0.179

V (Epa) and −0.260 V (Epc), respectively, with the ΔEp

value of 81 mV. The increase of the redox peak currents and

the decrease of the ΔEp value indicated that the presence of

CoMoO4 nanorods on the electrode surface exhibited ex-

cellent electrocatalytic ability and improved the electron

exchange rate. The formal potential, which was calculated

from the midpoint of Epa and Epc, was obtained as −0.220

V (vs. SCE), and the result was the typical characteristics of

heme Fe(III)/Fe(II) redox couples. So Mb molecules on the

electrode underwent a quasi-reversible electrochemical reac-

tion due to the presence of CoMoO4 nanorods on the CILE

surface.

Effect of Scan Rate. To further investigate electrochemical

characteristics of the immobilized Mb on the electrode

surface, the effect of scan rate on the cyclic voltammetric

response was investigated in the scan rate range from 80 to

800 mV s−1. As shown in Figure 4, the cathodic and anodic

peak currents of Mb increased gradually with the increase of

scan rate. Two linear regression equations between the redox

peak current and scan rate were calculated as Ipc(μA) =

104.2 ν(V s-1) + 8.221 (γ = 0.997) and Ipa(μA) = −101.6 ν

(V s−1) − 6.454 (γ = 0.993), respectively. The results indicat-

ed that the electron transfer process of Mb in CTS/CoMoO4/

CILE was a typical surface-confined electrochemical behavior.

The surface concentration (Γ*) of the electroactive Mb on

the electrode can be further calculated by integration of the

cyclic voltammetric reduction peaks based on the formula

of Q = nFAΓ*,35 where Q is the integration charge of the

reduction peak, n is the electrons transfer number, F is the

Faraday constant, A is the area of the modified electrode.

The charge value (Q) was nearly constant at different scan

rates and the average value of Γ* was obtained as 7.13 ×

10−9 mol cm−2. While the theoretical monolayer concentration

of protein on the electrode surface was 2.0 × 10−11 mol cm−2,

so multilayers of Mb on the electrode surface took place the

electrochemical reaction. Also the total surface concent-

ration of Mb in the composite film on the electrode surface

was calculated as 5.36 × 10−8 mol cm−2, then 13.28% of the

immobilized Mb molecules took part in the reaction. The

results could be attributed to the presence of CoMoO4

nanorods resulted in a porous structure with higher surface

area. The interaction of CoMoO4 nanorods with Mb also

happened in the film, then the several layers of Mb mole-

cules near the electrode surface underwent the electron

transfer with the help of CoMoO4 nanorods.

From Figure 4 it can also be seen that the redox peak

potentials moved gradually with the increase of scan rate.

The oxidation peak shifted to the positive direction and the

reduction peak shifted to the negative direction with the

peak-to-peak separation (ΔEp) increased, indicating a quasi-

reversible process. The relationship of redox peak potentials

and the natural logarithm of scan rate (ln ν) were calculated,

which was linearly dependant in the range from 80 to 800

mV s−1 with the linear regression equations as Epc(V) =

−0.0445 lnν − 0.285 (γ = 0.993) and Epa(V) = 0.0509 lnν −

0.128 (γ = 0.996), respectively. Then the electrochemical

parameters of Mb in the composite film could be calculated

based on the Laviron's equations:36,37

log ks=αlog(1−α)+(1−α)logα−log −

Epc = E0′

−RT

αnF----------lnν

Epa = E0′

+ RT

1 α–( )nF----------------------lnν

RTnFν---------- 1 α–( )αnFΔ Ep

2.3RT--------------------------------------

Figure 3. Cyclic voltammograms of (a) CILE, (b) CTS/CILE, (c)CTS/Mb/CILE and (d) CTS/CoMoO4-Mb/CILE in pH 3.0 PBS atthe scan rate of 100 mV s−1.

Figure 4. Cyclic voltammograms of CTS/CoMoO4-Mb/CILE inpH 3.0 PBS with different scan rates (a-k: 80, 100, 160, 200, 280,340, 400, 500, 600, 700, 800 mV s−1).


where α is the electron transfer coefficient, n is the number

of electron transferred, ν is the scan rate, and E0' is the

formal potential, ks is the electron transfer rate constant and

ΔEp is the peak-to-peak potential separation, R, T and F

have their conventional meanings. Based on these equations

the value of n was estimated as 1.09, suggesting that totally

one electron was involved in the electrode reaction. The

values of α and ks were further calculated as 0.53 and 1.16 s−1.

The value of ks is bigger than that of 0.34 s−1 on Mb/NiO/

GCE38 and 0.41 s−1 on Mb-HSG-SN-CNTs/GCE,39 indicating

that CoMoO4 nanorod acted as an excellent promoter to the

direct electron transfer between Mb and the electrode.

It is well-known that most of the heme proteins exhibit a

pH-dependent conformational equilibrium and the pH value

of the buffer solution influences the electrochemical reaction

of the heme proteins. The effect of buffer pH on the response

of CTS/CoMoO4-Mb/CILE was investigated in the pH range

from 1.0 to 7.0. The increase of buffer pH led to a negative

shift of both reduction and oxidation peak potentials, which

indicated that protons involved in the electrode reaction. The

maximum redox peak currents were obtained at pH 3.0

buffer solution, which was selected for the furthur investi-

gation.

Electrocatalysis of CTS/CoMoO4-Mb/CILE. Because

of the presence of similar active center of Mb with per-

oxidase, Mb exhibits intrinsic peroxidase activity in the cata-

lytic reaction. Due to the presence of Mb on the electrode

surface, the modified electrodes often showed good electro-

catalytic ability toward the reduction of different substrates

with a large decrease of activation energy. The electro-

catalytic activity of CTS/CoMoO4-Mb/CILE to TCA and

H2O2 was investigated to probe its potential applications.

Figure 5 showed the cyclic voltammograms of the modified

electrode with different concentrations of TCA. It can be

seen that a new reduction peak appeared at −0.258 V (vs

SCE) with the addition of TCA, and the reduction peak

currents increased gradually with the disappearance of the

oxidation peak current, which was a typical electrocatalytic

reduction process. The linear relationship of the electro-

catalytic reduction peak current and TCA concentration was

constructed in the range from 0.1 to 32.0 mmol L−1 with the

linear regression equation as Iss (μA) =8.443C (mmol L−1) +

34.30 (n = 17, γ = 0.998). The detection limit, which was

calculated based on a signal-to-noise ratio of 3, was got as

0.036 mmol L−1 (3σ). When the TCA concentration was

more than 32.0 mmol L−1 the catalytic reduction peak currents

began to level off with a plateau appeared, which was a

typical Michaelis-Menten kinetic mechanism. So the apparent

Michaelis-Menten constant (KMapp), which gives an indi-

cation of the enzyme-substrate kinetics, can be obtained

from the following Lineweaver-Burk equation:40

where c is the bulk concentration of the substrate, Iss is the

steady current after the addition of substrate, and Imax is the

maximum current measured under saturated substrate condi-

tion. Then the KMap value was calculated as 0.547 mmol L−1

with the above equation. So the Mb molecules immobilized

on the electrode surface exhibited a high affinity for TCA. In

order to compare the electrochemical performance of Mb

modified electrodes, the electrochemical data were sum-

marized in Table 1. It can be seen that the CTS/CoMoO4-

Mb/CILE exhibited wider linear range and lower detection

limits for TCA detection.

Electrocatalysis of CTS/CoMoO4-Mb/CILE towards the

reduction of H2O2 was further investigated with the cyclic

voltammograms shown in Figure 6. With the addition of

different amounts of H2O2 into PBS, a new reduction peak

appeared at −0.255 V, indicating the electrocatalytic reduc-

tion. So the electrocatalytic reduction of H2O2 by Mb on the

modified electrode can be explained with the following

1

Iss

----- = 1

Imax

-------- + KM

app

Imaxc-----------

Figure 5. Cyclic voltammograms of CTS/CoMoO4-Mb/CILE inthe presence of 2.0, 4.0, 6.0, 8.0, 10.0, 12.0, 16.0, 22.0, 26.0, 30.0mmol L−1 TCA (a-j) in pH 3.0 PBS at the scan rate of 100 mV s−1.

Table 1. Electrochemical data of different Mb modified electrodes towards the reduction of TCA

Modified ElectrodesLinear Range

(mmol L−1)

Detection Limit

(mmol L−1)

Sensitivity

(µA mM−1)

KMapp

(mmol L−1)Refs

Mb/DNA/CILE 0.5-40.0 0.083 10.6 0.82 41

Nafion/Mb/Co/CILE 0.4-12.0 0.2 – 4.11 42

Nafion–BMIMPF6/Mb/CPE 0.2-11.0 0.016 48 – 43

Mb/ZrPNS/GCE 0.1-2.2 0.025 – – 44

Nafion/Mb/MWCNTs/CILE 1.57-12.0 0.1 58.2 3.396 45

CTS/CoMoO4-Mb/CILE 0.1-32.0 0.036 8.443 0.547 This work

BMIMPF6: 1-Butyl-3-methylimidazolium hexafluorophosphate; ZrPNS: zirconium phosphate nanosheets; MWCNTs: multi-walled carbon nanotubes


equations:

Mb heme Fe(III) + H+ + e− → Mb heme Fe(II)

2 Mb heme Fe(II) + H2O2 + 2H+ → 2 Mb heme Fe(III) + 2H2O

The reduction peak current increased linearly with H2O2

concentration in the range from 0.12 to 397.0 μmol L−1 with

the linear regression equation as Iss(μA) = 0.0686C (μmol

L−1) + 16.62 (n = 19, γ = 0.998) and the detection limit was

calculated as 0.0426 μmol L−1 (3σ).

Based on the same method the apparent Michaelis-Menten

constant (KMapp) was further calculated as 0.016 mmol L−1.

A comparison of the results for the determination of H2O2

with different modified electrodes was also summarized

with the results listed in Table 2. Compared with other H2O2

biosensors, the fabricated CTS/CoMoO4-Mb/CILE had

wider linear range, lower detection limit, higher sensitivity

and smaller KMapp.

Interference. The influence of some possible interferents

on the voltammetric responses of 4.0 μmol/L H2O2 was

investigated by using the tolerable limit with the relative

deviation of ± 5%. No obvious interferences could be

observed with 1000 fold concentrations of K+, NH4+, Zn2+,

Mg2+, SO42−, Cl−, and 500 fold of ascorbate, uric acid,

acetaminophenol. The results indicated that this modified

electrode exhibited good selectivity.

Stability and Reproducibility. The stability and repro-

ducibility of CTS/CoMoO4-Mb/CILE was also studied. The

electrode was stored at room temperature when not in use, it

could remain 97.8% and 94.1% of its initial responses after

storage of 14 days and 30 days, respectively, which indicated

that the Mb modified electrode had good stability. Four

modified electrodes were fabricated with the same proce-

dure and further applied to the detection of 20.0 mmol L−1

TCA with a relative standard deviation (RSD) value of

2.37%. The results indicated that CTS/CoMoO4-Mb/CILE

exhibited good stability and reproducibility in the electro-

chemical application.

Conclusion

CoMoO4 nanorods were incorporated with Mb and further

modified on the CILE surface to get the Mb modified

electrode. The presence of CoMoO4 nanorods acted as an

efficient promoter to accelerate the direct electron transfer of

Mb with CILE, which may be due to the specific properties

of nanostructured CoMoO4. A pair of well-defined redox

peaks appeared on CTS/CoMoO4-Mb/CILE, which was

attributed to the direct electron transfer of Fe(III)/Fe(II) in

the redox protein. The electrocatalysis of Mb in the modified

electrode to the reduction of TCA and H2O2 was further

investigated with wider linear range and lower detection

limit. The CTS/CoMoO4-Mb/CILE electrode exhibited the

advantages such as long-term stability, high electrocatalytic

ability, good reproducibility and simply preparation proce-

dure. The results indicated the CoMoO4 nanorods had

potential applications in the field of the electrochemical

enzyme biosensor.

Acknowledgments. This work was supported by “the

Fundamental Research Funds for the Central Universities of

China, No. 2010ZY38” and the Laboratory Open Funds of

China University of Geosciences (Beijing).

Figure 6. Cyclic voltammograms of CTS/CoMoO4-Mb/CILE inthe presence of 0.0, 30.0, 100.0, 180.0, 280.0 µmol L−1 H2O2 (a-e)in pH 3.0 PBS at the scan rate of 100 mV s−1.

Table 2. Electrochemical data of different Mb modified electrodes towards the detection of H2O2

Modified ElectrodesLinear Range

(mmol L−1)

Detection Limit

(µmol L−1)

Sensitivity

(µA mM−1)

KMapp

(mmol L−1)Refs

Mb-HSG-SN-CNTs/GCE 2.0-1200 0.36 – 1.62 39

Mb/ZrPNS/GCE 0.8-12.8 0.14 – 0.034 44

{SG-Al2O3/Mb}9/PGE 1.0-125 0.6 0.265 – 46

Mb/ZrO2/MWCNT/GCE 1.0-116.0 0.53 17 0.085 47

Mb/HMS/GCE 4.0-124 0.062 424 0.065 ± 0.005 48

Mb/clay-IL/GCE 3.9-259 0.733 – 0.0173 49

Mb–TiO2/MWCNT/GCE 1-160 0.41 16.4 0.0831 50

PDDA/{ZrO2}3-Mb/PGE 0.02-3.0 0.01 2970 – 51

CTS/CoMoO4-Mb/CILE 0.12-397.0 0.0426 68.6 0.016 This work

PDDA: poly(diallyldimethylammonium); PGE: pyrolytic graphite electrode; GCE: glassy carbon electrode; SG: sol-gel; MWCNT: multi-walled carbonnanotube; HMS: hexago-nal mesoporous silica; IL: ionic liquid; HSG: hybrid silica sol-gel; SN-CNTs: Ag nanoparticle doped carbon nano-tubs;ZrPNS: zirconium phosphate nanosheets.


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