Anal. Chem. 2010, 82, 3588–3596
Electrochemical Approach for Detection of
Extracellular Oxygen Released from Erythrocytes
Based on Graphene Film Integrated with Laccase
and 2,2-Azino-bis(3-ethylbenzothiazoline-6-sulfonic
acid)
Xiuming Wu,†,‡ Yaojuan Hu,† Juan Jin,† Ninglin Zhou,† Ping Wu,† Hui Zhang,*,† and Chenxin Cai*,†
Jiangsu Key Laboratory of Biofunctional Materials, Laboratory of Electrochemistry, College of Chemistry and
Environmental Science, Nanjing Normal University, Nanjing 210097, P. R. China, and School of Chemical and
Material Engineering, Jiangnan University, Wuxi 214122, P. R. China
This work develops a novel electrochemical approach for
detection of the extracellular oxygen released from human
erythrocytes. The sensing is based on the bioelectrocatalytic system of graphene integrated with laccase (Lac) and
2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS)
toward the reduction of oxygen. ABTS and laccase are
assembled on the surface of graphene, which is synthesized by a chemistry route, utilizing the π-π and electrostatic interactions of these components. Transmission
electron microscopy (TEM), atomic force microscopy
(AFM), and FT-IR spectroscopy demonstrate that graphene
has been successfully synthesized, and ABTS and laccase
have been effectively assembled on a graphene surface
with the formation of Lac-ABTS-graphene hybrid. The
voltammetric results indicate that ABTS can be used as a
redox mediator when it is in immobilized form. The hybrid
deposited on the glassy carbon (GC) electrode is demonstrated to be a good bioelectrocatalyst for the reduction
of oxygen with inherent enzyme activity, accepted stability,
high half-wave potential (ca.670 mV vs NHE), and unimpeded electrical communication to the copper redox sites
of laccase. Therefore, this study has not only established
a novel approach of detection of extracellular oxygen but
also provided a general route for fabricating a graphenebased biosensing platform via assembling enzymes/
proteins on a graphene surface.
All primary animal cells are aerobic; i.e., oxygen is involved in
their essential cellular functions. For example, they constantly
consume oxygen in their energy production and use oxygen as
an electron acceptor at the end of the aerobic pathway of glucose
oxidation.1 The stepwise reduction of oxygen in a biological
system generates a number of potentially harmful oxygen metabolites, such as the superoxide anion radical, hydrogen peroxide,
* Corresponding author. E-mail: [email protected] (H.Z.); cxcai@
njnu.edu.cn (C.C.).
†
Nanjing Normal University.
‡
Jiangnan University.
(1) Garrett, R. H.; Grisham, C. M. Biochemistry; Saunders College Publishing:
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Analytical Chemistry, Vol. 82, No. 9, May 1, 2010
and hydroxyl radical.2,3 This sequence can take place in various
compartments of the cell4 and may be involved in a number of
pathological events connected with lipid peroxidation,5 organ
injury,6 DNA damage,7 tumor promotion,7 etc. Obviously, the
concentration of oxygen in cells and cell compartments as well
as cell metabolic activity determines the intensity of cell oxidative
processes and intracellular radical generation. There is much data
concerning the dependence of tissue oxidative injury on oxygen
level.8 In addition, evidence has demonstrated that oxygen also
functions as second messengers for several growth factors,
cytokines, signal transduction, and modulators of specific regulation of the expression of various genes.9,10
Regarding the importance of oxygen in cellular biology and
pathophysiology, its concentration measurements can be used to
study biochemical events in which some part of the aerobic
pathway is affected.11,12 Therefore, a selective and sensitive
method for reliable determination of cellular oxygen is very useful
for gaining a full understanding of the role that oxygen plays in
pathology and physiology. Until now, a variety of assay methods
have been developed for measuring oxygen levels of cultured cells.
These methods include the amperometric Clark electrode,13,14
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10.1021/ac100621r  2010 American Chemical Society
Published on Web 04/12/2010
spectrophotometry,15 HPLC-MS analysis,16 chemiluminescence,2,17,18
fluorescence,3,19,20 phosphoresence,12,21,22 electron spin resonance,11 etc. Recently, biosensors based on electrochemical
transduction have received considerable attention and have
become an attractive method for in vivo and ex vivo biological
application, including continuous in vivo monitoring, measurement
of analytes in extremely small volumes, monitoring of localized
events, or biosensing resistive media because of their high
sensitivity, low cost, rapid response, compatibility for miniaturization, low manpower requirements, and compatibility with microfabrication technology.23-27 Although there were much works
published on the investigation of a tissue oxygen microenvironment using a tissue electrode (with a diameter of 0.1-0.5 µm),28
to our best knowledge, the works on the determination of cellular
oxygen with the use of the biosensors have not been reported so
far. In this study, such an electrochemical approach is developed
to detect extracellular O2 released from erythrocytes based on
the bioelectrocatalytic system of graphene integrated with
laccase (Lac) and 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic
acid) (ABTS).
Graphene, a new form of carbon material with carbon atoms
parked in a two-dimensional honeycomb lattice,29 has attracted
great attention of scientists in recent years.30-35 One of the factors
that makes graphene so attractive is its low energy dynamics of
electrons with atomic thickness.36 It is a semiconductor with zero
band gap and high carrier mobilities and concentrations and
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shows nearly ballistic transport at room temperature.37 These
unusually electronic properties make graphene one of the most
promising candidate materials for future nanoelectronic applications, including graphene-based field-effect transistors,38 gas
sensors,39 nanoelectromechanical switch,35c supercapacitors,31b
integrated ballistic carrier devices,40 and so forth. Of particular
interest for us is to explore its application in the field of
electrochemical research. To fully exploit the electrochemical
properties of graphene, it is important to understand the electrochemical characteristics of the graphene surface, including the
electron transfer kinetics of the redox system, adsorption, and
electrocatalysis. Recently, several groups have demonstrated that
graphene sheets show fast electron transfer (ET) kinetics and
excellent electrocatalytic characteristics compared with graphite
and GC.30a,31a,b However, the immobilization and electrochemistry
of proteins and/or enzymes on the surface of graphene have not
been widely studied so far.33a Here, we describe the fabrication
and characterization of a novel mediating system in which laccase
(Lac) and ABTS are assembled onto a graphene surface. The
resulting hybrid (Lac-ABTS-graphene) has been characterized
by transmission electron microscopy (TEM), FT-IR, and cyclic
voltammetry. The bioelectrocatalytic activity of the hybrid toward
the reduction of oxygen was studied and utilized to effectively
detect the extracellular oxygen released from human erythrocytes.
Our goal is not only to design a novel biosensing platform but
also to present a new approach for preparation of a graphenebased hybrid, which has potential utility to bioelectroanalytical
chemistry, cellular biology, pathophysiology, etc.
EXPERIMENTAL SECTION
Chemicals. Laccase (EC 1.10.3.2, from Rhus vernicifera, g50
U mg-1) and ABTS (diammonium salt) were purchased from
Sigma and used as received. Graphite powder (99.99995%, 325
mesh) was obtained from Alfa Aesar. All other chemicals were
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Adv. Funct. Mater. 2009, 19, 2297–2302. (b) Park, S.; An, J.; Piner, R. D.;
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of analytical grade or better. All solutions were prepared with
double distilled water. Phosphate buffer solution (PBS) was
made from Na2HPO4 and NaH2PO4.
Synthesis of Graphene. Graphene was synthesized according
to a published route involving the steps of graphite oxidation,
exfoliation, and chemical reduction.41 In brief, graphite oxide (GO)
was prepared by a modified Hummers method, starting from
graphite powder. Graphite was oxidized by concentrated H2SO4,
K2S2O8, and P2O5 to produce preoxidized graphite, which was
then subjected to reoxidization by concentrated H2SO4 and
KMnO4. Exfoliation was carried out by sonicating GO dispersion (0.1 mg mL-1) under ambient temperature for ca 20 min.
The resulting homogeneous yellow-brown dispersion was
reduced by hydrazine. The reduction reaction was carried out
by adding hydrazine (1.2 mL) into the dispersion of GO (60
mg of GO in 50 mL of water). After being sonicated for 1 h
and kept stirring for 24 h at 50 °C, graphene sheets were
obtained by filtration of the product and drying in vacuum. The
details of synthesizing are described in the Supporting Information.
Assembly of ABTS and Laccase on Graphene. Several
experimental parameters were optimized to obtain the best
voltammetric response. Typically, graphene sheets (6 mg) were
dispersed into ABTS solution (1 mL, 5 mM in PBS) and were
stirred for ca. 15 min. Then, the ABTS-graphene was collected
by centrifugation for 10 min. The supernatant was removed, and
ABTS-graphene was thoroughly washed at least thrice with water
to remove the loosely adsorbed ABTS. Laccase was assembled
on the surface of negatively charged ABTS-graphene via the
electrostatic interaction by stirring PBS (1 mL, pH 7.4) containing
ABTS-graphene (2 mg) and laccase (10 mg) at 4 °C for 2 h. After
that, the mixture was centrifuged and the Lac-ABTS-graphene
hybrid was collected by removing the supernatant. The hybrid
was washed with PBS for removing the loosely assembled enzyme
molecules. Each step of the above was characterized by FT-IR
spectroscopy.
Fabrication of the Lac-ABTS-Graphene/GC Electrode.
To fabricate the modified electrode, the Lac-ABTS-graphene
hybrid (2 mg) was dispersed into PBS (1 mL, pH 7.4) to form a
homogeneous suspension (2 mg mL-1). Then, the suspension
(5 µL) was cast onto the surface of the pretreated GC
electrode42 (3 mm in diameter, CH Instruments) with a
microsyringe, and solvent was allowed to be evaporated at
ambient temperature before use. The electrode (denoted as
the Lac-ABTS-graphene/GC electrode) was stored at 4 °C
when not in use.
Using the similar procedures, the ABTS-graphene/GC,
Lac-graphene/GC, graphene/GC, ABTS/GC, and Lac-ABTS/
GC electrodes were fabricated, and their electrocatalytic characteristics toward the reduction of O2 were compared with that of
the Lac-ABTS-graphene/GC electrode. Due to its hydrophobic characteristic, graphene (2 mg) was dispersed by sonication
in N,N-dimethylformamide (DMF, 1 mL) for fabrication of the
graphene/GC electrode.
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Instruments and Procedures. The FT-IR spectrum was
recorded on a Nexus 670 FT-IR spectrophotometer (Nicolet
Instruments) using a KBr disk at a resolution of 4 cm-1.
Transmission electron microscopy (TEM) images were obtained with a JEOL-2010 transmission electron microscope
operating at an accelerating voltage of 120 kV. Atomic force
microscopic (AFM) images were recorded with a Nanoscope
IIIa scanning probe microscope (Digital Instruments, USA)
using a tapping mode. The sample used for measurements was
prepared by casting the suspension of graphene (0.1 mg mL-1)
or Lac-ABTS-graphene (0.1 mg mL-1) on the surface of a
mica sheet. The solvent was allowed to evaporate before
measurements. The electrochemical experiments were performed with a CHI 660B electrochemical workstation (CH
Instruments). A coiled Pt wire and a saturated calomel
electrode (SCE) were used as the counter electrode and the
reference electrode, respectively. The voltammetric characteristics of ABTS-graphene/GC electrode were studied in an O2free buffer (0.1 M PBS, pH 7.4). The electrocatalytic characteristics of the Lac-ABTS-graphene/GC electrode were
recorded in O2-saturated buffer, which was obtained by bubbling O2 for 15 min and also by maintaining an O2 environment
over the solution during the experiments.26,27 All electrochemical experiments were performed at room temperature (22 ± 1 °C).
The apparent surface area (A) of the electrodes including the
bare GC and graphene/GC electrodes was estimated using cyclic
voltammetry. The cyclic voltammetric experiments of 1 mM
Ru(NH3)6Cl3 in 0.5 M KNO3 solution were performed at
different electrodes and at various scan rates. A straight line
of peak currents (ip) versus scan rates (v1/2) could be obtained
according to the equation of ip ) 2.69 × 105n2/3AD1/2v1/2c*, and
the values of A for the bare GC and graphene/GC were
estimated to be 0.083 ± 0.008 and 0.121 ± 0.026 cm2, respectively, from the slope of the lines with the use of the diffusion
coefficient of (7.74 ± 0.23) × 10-6 cm2 s-1 for Ru(NH3)63+ in
0.5 M KNO3 solution. The value of the diffusion coefficient of
Ru(NH3)63+ was estimated using a Pt microelectrode (5 µm in
diameter, BAS, West Lafayette, IN).
Detection of Extracellular Oxygen. Erythrocytes were
obtained by removing plasma and the leukocyte layer from human
red blood cells and were washed thrice with a cold PBS buffer
(0.145 M NaCl, 1.9 mM NaH2PO4, 8.1 mM Na2HPO4, pH 7.4).
Erythrocytes were centrifuged to obtain a cell-packed pellet
with the cell density of ca. (4 to 5) × 105 cells cm-2, which was
estimated with a hemocytometer, for the electrochemical
experiments. Before measurements, the buffer was deoxygenated by gently shaking the cells under a humidified nitrogen
stream. The Lac-ABTS-graphene/GC electrode, which was
carefully adjusted to near the cell pellet under a microscope
(AxioObserver A1, Carl Zeiss), was biased at 0.3 V (vs SCE).
After a steady state background was attained, NaNO2 solution
(the final concentration is 2 mM) was injected into buffer, and
response current corresponding to the electrocatalytic reduction of O2 released from erythrocyte was recorded. The
measurements were performed under the physiological pH and
temperature (pH 7.4 and 37 °C).
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Figure 2. Typical tapping mode AFM images and the cross-sectional
analysis of graphene sheets with (b) and without (a) assembly of
ABTS and laccase.
Figure 1. Typical TEM image of a graphene sheet.
RESULTS AND DISCUSSION
Characterization of the Lac-ABTS-Graphene Hybrid.
Laccase, which belongs to the multicopper group of oxidases and
drives effectively the four-electron reduction of oxygen to water,43
is one of the most commonly considered enzymes for bioelectrocatalytic cathodes in biofuel cells for the reduction of oxygen.44
To ensure the effective ET communication between the redox
centers of the enzyme and electrode, ABTS is commonly introduced as a mediator.44,45 ABTS can be easily assembled onto the
surface of graphene through π-π electronic interactions forming
a new kind of nanocomposites (ABTS-graphene). After the
assembly of ABTS, a layer of negative charges is created on the
surface of graphene. Therefore, the positively charged laccase (at
pH 7.4, the isoelectric point of laccase from Rhus vernicifera is
ca. 8.246) could be assembled on the surface of ABTS-graphene
based on electrostatic interaction with the formation of the
Lac-ABTS-graphene hybrid. These assembly processes were
characterized by AFM and FT-IR spectroscopy.
Figure 1 shows a typical TEM image of graphene. Large
graphene sheets with a length up to ca. 4 µm and a width of ca.
3 µm are observed. The graphene sheets are rippled and resemble
crumpled silk veil waves. However, when its size is down to ca.
1 × 1 µm, the surface is flat as shown by an AFM image in Figure
2a. The cross-sectional analysis indicates that the thickness of
graphene is ca. 0.7 nm, which matches well with the reported
apparent thickness of graphene sheets,30,47 suggesting the single(43) (a) Solomon, E. I.; Sundaram, U. M.; Machonkin, T. E. Chem. Rev. 1996,
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sheet nature of graphene is obtained in this work. The AFM image
reveals that no sheets either thicker or thinner than 0.7 nm are
observed, demonstrating that complete exfoliation of graphene
oxide down to an individual graphene sheet is indeed achieved
under our experimental conditions.
The image in Figure 2a also shows that graphene sheets
distribute separately on the surface of the electrode without
forming aggregates or entangling each other. The uniform
nanostructure can significantly increase the effective surface of
the electrode for loading of biomolecules and accelerating ET
kinetics. After the assembly of laccase, the morphology of
graphene changes significantly. Some small and uniformly distributed island-like nanostructures appear (Figure 2b). These
nanostructures are the aggregates of laccase molecules, indicating
that the enzyme has been effectively loaded on the graphene.
Moreover, the thickness of graphene has a significant increase
with the assembly of laccase (5 to 6 nm for laccase-assembled
graphene, please refer to the corresponding cross-sectional
analysis), further indicating the formation of Lac-ABTS-graphene
hybrid. The morphology of the hybrid on the electrode surface
possesses a similar structure to that of graphene. Such a structure
is expected to be very attractive for the detection of substrates
because each of the hybrids is fully and easily accessible to
substrates and, therefore, can be used as an electrochemical
sensing unit, yielding a high ratio of signal-to-noise for electrochemical determination.
The assembly of ABTS and laccase were also examined by
FT-IR spectroscopy. In an IR spectrum of graphene oxide (Figure
3b), the characteristic vibration modes of the O-H groups
(∼3410 cm-1), CdO groups (∼1730 cm-1), the deformation
peak of O-H groups (1410 cm-1), the stretching peak of
C-OH (1225 cm-1), and the stretching peak of C-O (1050
cm-1) are obvious, demonstrating the successful oxidation of
graphite.34d The peak at 1620 cm-1 is assigned to the vibrations
of the adsorbed water molecules and the skeletal vibrations of
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3591
Figure 3. FT-IR spectra of the graphene (a), graphite oxide (b),
ABTS-graphene (c), and Lac-ABTS-graphene hybrid (d).
unoxidized graphitic domains.34d After reduction with hydrazine, the residual oxygen functionalities are still present on the
graphene surface; however, the IR intensities of those oxygencontaining groups decrease significantly (Figure 3a), indicating
that graphene oxide has been well deoxygened and the graphene
is successfully prepared. The assembly of ABTS on graphene
results in the characteristic vibration of -CH3 and -CH2(∼2852 and ∼2927 cm-1) groups in the IR spectrum (Figure
3c). The peaks at ∼1180 and 1020 cm-1 are assigned to the -SO3
group antisymmetric and symmetric vibration adsorption,
respectively.48 Peaks at ∼1120 and 1010 cm-1 can be assigned
to the in-plane skeleton vibration and in-plane bending vibration
of the benzene ring, respectively. These results indicate that
ABTS has been effectively assembled on the surface of
graphene. The demonstration of amide I (1642 cm-1) and amide
II (1540 cm-1)49 in the IR spectrum of Lac-ABTSgraphene hybrid (Figure 3d) indicates that laccase has been
successfully assembled on graphene via ABTS. This result is in
good agreement with that obtained from the AFM image.
Voltammetric Characteristics of the Lac-ABTS-Graphene
Hybrid. The use of laccase combined with ABTS as a diffusion
mediator for the reduction of oxygen in a biofuel cell has already
been well-established.44a More recently, attempts have been made
to immobilize ABTS together with laccase on the electrode
surface.45a,50,51 In the present work, the cyclic voltammetric
responses of ABTS-graphene and Lac-ABTS-graphene hybrid
deposited on GC electrode (denoted as the ABTS-graphene/GC
and Lac-ABTS-graphene/GC electrode, respectively) are studied. For comparison, cyclic voltammogram of ABTS adsorbed on
the bare GC electrode (ABTS/GC) is also presented.
The cyclic voltammogram of the graphene/GC electrode does
not show any observable redox peaks (Figure 4a, 0.1 M PBS, pH
7.4) in the potential range of interest, indicating that graphene
cannot undergo the redox reaction in the experimental potential
ranges. However, the cyclic voltammetric response of the ABTSgraphene/GC electrode is characterized by a pair of well-defined
(48) Yang, J. C.; Jablonsky, M. J.; Mays, J. W. Polymer 2002, 43, 5125–5132.
(49) (a) Niwa, K.; Furukawa, M.; Niki, K. J. Electroanal. Chem. 1988, 245, 275–
285. (b) Irace, G.; Bismuto, E.; Savy, F.; Colonna, G. Arch. Biochem. Biophys.
1986, 244, 459–469.
(50) Nogala, W.; Rozniecka, E.; Zawisza, I.; Rogalski, J.; Opallo, M. Electrochem.
Commun. 2006, 8, 1850–1854.
(51) Karnicka, K.; Miecznikowski, K.; Kowalewska, B.; Skunik, M.; Opallo, M.;
Rogalski, J.; Schuhmann, W.; Kulesza, P. J. Anal. Chem. 2008, 80, 7643–
7648.
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redox peaks with the cathodic (Epc) and anodic (Epa) peak
potential of ca. 415 and 425 mV (at 10 mV s-1), respectively
(Figure 4c). These peak potentials are similar to those for the
oxidation and reduction of ABTS2-/ABTS•- couple adsorbed on
the surface of carbon nanotubes.51 This result indicates the
formation of ABTS-graphene hybrid, which is in agreement
with that obtained from the spectroscopic results. The cathodic
and anodic peak currents are almost equal at all scan rates (v)
and increase linearly with v up to at least 1000 mV s-1 (panel
B, Figure 4), indicating the presence of surface-confined redox
processes. Although Epc and Epa shift slightly with v increasing
up to 1000 mV s-1, the value of E0′, defined as the average
value of the Epc and Epa, is independent of v, demonstrating
the fast ET kinetics of the ABTS2-/ABTS•- couple on the
surface of graphene, implying ABTS can be utilized as a redox
mediator when it is in immobilized form. Another important
feature of the cyclic voltammetric response of ABTS-graphene
is that the redox peaks are symmetrical with small separation
(∆Ep) between the cathodic and anodic peak (10 mV at 10 mV
s-1, and 30 mV at 1000 mV s-1), although the value of ∆Ep is
not zero. The nonzero separation parameter most likely reflects
differences in the nature and relative strengths of adsorption
of the oxidized and reduced forms of ABTS on the graphene
surface. The full width at half-maximum of the peaks for ABTS
oxidation is equal to ca. 130 mV (Figure 4c), and it is somewhat
higher than the theoretical value of 90 mV expected for an ideal
one-electron surface-confined voltammetric peak.52 The above
observation may originate from strong repulsive interactions
between the oxidized (ABTS•-) or reduced (ABTS2-) states of
the mediator, or both.51
To illustrate the special effects of graphene in assembling
ABTS, the cyclic voltammetric responses of the ABTS/GC
electrode were also recorded and compared with that of the
ABTS-graphene/GC electrode. As shown in Figure 4b, ABTS at
the bare GC electrode also produces a well-defined redox peak
with Epc and Epa of 390 and 450 mV, respectively. While the
formal potential (420 mV) is comparable to that of the
ABTS-graphene/GC electrode, a higher value of ∆Ep (60 mV)
is observed at the ABTS/GC electrode. Moreover, the peak
current density of the electrode is only ca. 1/7 of that obtained
at the ABTS-graphene/GC electrode. Please note that the
cyclic voltammograms in panel A of Figure 4 are plotted as
current density (j, in µA cm-2) versus potential in order to obtain
more information on the relative ET characteristic itself,
compare the results better, and eliminate the effects of the
apparent surface area (A) of the bare GC and graphene/GC
electrodes on the peak current. The above results demonstrate
that graphene can remarkably enhance ET kinetics and peak
current of the ABTS2-/ABTS•- couple with respect to the bare
GC electrode, similarly to what has been reported in the carbon
nanotube.51,53 The superior ET kinetics of graphene can be
ascribed to its unique electronic structure.31a It may also be due
to graphene having more surface defects than a polished GC
(52) Bard, A. J.; Faulkner, L. R. Electrochemical Methods, 2nd ed.; John Wiley &
Sons: New York, 2001, p 591.
(53) (a) Meng, L.; Wu, P.; Chen, G.; Sun, Y.; Yuan, Z. Biosens. Bioelectron. 2009,
24, 1751–1756. (b) Meng, L.; Wu, P.; Chen, G.; Cai, C.-X. J. Electrochem.
Soc. 2008, 155, F231–F236. (c) Meng, L.; Jin, J.; Yang, G.; Lu, T.; Zhang,
H.; Cai, C.-X. Anal. Chem. 2009, 81, 7271–7280.
Figure 4. (A) Voltammetric responses of the graphene/GC (a), ABTS/GC (b), ABTS-graphene/GC (c), and Lac-ABTS-graphene/GC electrode
(d) at a scan rate of 10 mV s-1 in O2-free PBS (0.1 M, pH 7.4). (B) Cyclic voltammograms of the Lac-ABTS-graphene/GC electrode at scan
rates of (from curve e to n) 100, 200, 300, 400, 500, 600, 700, 800, 900, and 1000 mV s-1 in oxygen-free PBS (0.1 M, pH 7.4). The inset of
panel B shows dependence of peak currents on scan rates for the Lac-ABTS-graphene/GC electrode.
electrode because the graphene sheets were synthesized via a
chemical method. These defects result in the high density of
electronic states near the Fermi level leading to the enhancement
of ET kinetics. Moreover, the high current obtained at the
graphene/GC electrode may also be due to graphene having a
higher adsorption capacity and an interaction much stronger with
ABTS than that of the bare GC, and therefore, a large amount of
ABTS can be assembled. The value of surface concentration
(Γ, in mol cm-2) corresponding to the saturated adsorption of
ABTS on the graphene is estimated to be ca. (4.3 ± 0.2) × 10-10
mol cm-2 using Γ ) Q/nAF (where Q is the charge consumed
in coulombs, obtained from integrating the anodic or cathodic
peak area in cyclic voltammograms under the background
correction, n is the number of electron transfer for the reaction
of ABTS (n ) 1), F is the Faraday constant, and A is the
apparent area of the electrode). The value obtained at the bare
GC electrode is only ca. (1.8 ± 0.3) × 10-11 mol cm-2.
Cyclic voltammogram of the Lac-ABTS-graphene/GC electrode (Figure 4d) also shows a pair of redox peaks with the peak
potential and currents almost identical with those presented in
Figure 4c, demonstrating that assembly of the enzyme does not
affect the electrochemical characteristics of ABTS. This feature
is important for ABTS being utilized as a useful mediator after it
was immobilized on the electrode surface with an enzyme.
The ABTS-graphene and Lac-ABTS-graphene on the GC
electrode surface are fairly stable because the voltammetric
characteristics of the ABTS-graphene/GC electrode or LacABTS-graphene/GC electrode remain unchangeable even after
the electrode was scanned continuously for a long time in the
potential range of interest (ca. 100 cycles at a 10 mV s-1). This
result is consistent with the persistence of the attachment of
ABTS and laccase on graphene. Therefore, the hybrid is
expected to act as a stable mediating system of potential utility
to bioelectrocatalytic applications.
Bioelectrocatalytic Reduction of Oxygen. Since ABTS dissolved in solution or immobilized on suitable substrates50,51 is a
good mediator for the reduction of oxygen catalyzed by laccase,
Lac-ABTS-graphene is also expected to be a good catalyst for
the reduction of oxygen. The electrocatalytic reduction of oxygen
at the Lac-ABTS-graphene/GC (panel A in Figure 5), ABTSgraphene/GC (panel B in Figure 5), graphene/GC (panel C in
Figure 5), Lac-graphene/GC (panel D in Figure 5), ABTS/GC
(panel E in Figure 5), and Lac-ABTS/GC electrode (panel F in
Figure 5. Cyclic voltammograms of the Lac-ABTS-graphene/GC
(A), ABTS-graphene/GC (B), graphene/GC (C), Lac-graphene/GC
(D), ABTS/GC (E), and Lac-ABTS/GC electrode (F) in oxygen-free
(curve a) and oxygen-saturated (curve b) PBS (0.1 M, pH 7.4). Scan
rate: 10 mV s-1.
Figure 5) was studied in PBS (pH 7.4), and their electrocatalytic
characteristics were compared. Upon the presence of O2 in
solution, the voltammetric features of the Lac-ABTS-graphene/
GC electrode change significantly, being characterized with a
large cathodic peak at ca. 330 mV and complete disappearance
of the anodic peak (panel A, Figure 5). The height of cathodic
peak is sensitive to the change of the O2 concentration in solution
(not show here). The reduction currents in air-saturated
solution are less than 1/5 of that obtained in an oxygensaturated one. These characteristics are the typical features of
electrocatalytic reactions. The reduction of O2 starts at a very
positive potential of ca. 580 mV, and the observed reduction
current increases quite rapidly during negative potential scanning to reach the current density of more than 120 µA cm-2 at
ca. 330 mV. The onset potential is 250 mV more positive than
the reduction peak actually appears. The half-wave potential
of the catalytic wave is ca. 430 mV (vs SCE; i.e., ca. 670 mV vs
NHE). Such positive reduction potentials are usually observed
Analytical Chemistry, Vol. 82, No. 9, May 1, 2010
3593
only for ABTS mediator dissolved in the solution, in gel phase,
or on the surface of carbon nanotubes.50,51 The above results
clearly imply that the ABTS-graphene hybrid functions as an
effective mediating system in the laccase-catalyzed electroreduction of O2. It is reasonable to depict the electrocatalytic
processes as electrons are transferred from the electrode
surface along the conductive graphene to reduce ABTS•- to
ABTS2- at ca. 430 mV (as shown in Figure 4c). ABTS2- should
donate effectively four electrons to four Cu(II) ions in the active
site of laccase. This step also results in regeneration of ABTS•-.
Finally, O2 molecules are activated at two of four Cu(I) ions,
and they are subsequently reduced to water at 670 mV (vs
NHE).44b
The results of panels B, C, D, and E in Figure 5 show that the
presence of O2 in PBS does not affect the voltammetric features
of the ABTS-graphene/GC, graphene/GC, Lac-graphene/
GC, and ABTS/GC electrode, demonstrating that graphene,
ABTS-graphene, Lac-graphene, or ABTS does not have any
appreciable electrocatalytic activity toward the reduction of O2.
The electrocatalytic reduction of O2 by Lac-ABTS deposited
on the GC electrode (denoted as the Lac-ABTS/GC electrode)
was also studied and compared with those obtained at the
Lac-ABTS-graphene/GC electrode. The cyclic voltammograms (panel F of Figure 5) show that the electrocatalytic
features of the Lac-ABTS/GC electrode are similar to those of
the Lac-ABTS-graphene/GC electrode in terms of the onset and
cathodic peak potentials. However, by carefully analyzing the curve
b in panels A and F, it is obvious that the electrocatalytic current
density at the Lac-ABTS-graphene/GC electrode is more than
6 times higher than that obtained at the Lac-ABTS/GC electrode
(please note the different current density scale in panels A and
F). Moreover, the Lac-ABTS/GC electrode loses its electrocatalytic activity quickly due to the enzyme denaturation. However, it
should be stressed that the electrocatalytic characteristics of
Lac-ABTS-graphene hybrid are fairly stable since the features
of the cyclic voltammograms remain almost invariable after the
electrode was scanned continuously for a long time (ca. 50 cycles,
at 10 mV s-1) in O2-saturated PBS. In addition, no obvious
change is detected after the electrode has been stored in PBS
at 4 °C for 1 week. Therefore, the hybrid has acceptable stability
in the potential range where it is used as a bioelectrocatalyst
for the reduction of oxygen.
Optimizing the Performance Parameters of the LacABTS-Graphene/GC Electrode and Detection of O2. The
electrocatalytic activity of the Lac-ABTS-graphene/GC electrode
toward the reduction of oxygen can be affected by a variety of
parameters, such as the amount of laccase loading, detection
potential, pH of the buffer solution, and temperature of the
detection system. In this work, these conditions are optimized.
The concentration of laccase in PBS during the Lac-ABTSgraphene hybrid preparation procedure may affect the loading
amount of the enzyme (see Experimental Section). Therefore, the
hybrid was prepared in various concentrations of laccase, and the
response of the prepared electrode toward the reduction of O2
(in O2-saturated PBS) was recorded. The results were presented in panel A of Figure 6. Increasing the enzyme loading
leads to an enhancement in the response current for the reduction
O2, and the response reaches a maximum at ca. 10 mg mL-1
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Analytical Chemistry, Vol. 82, No. 9, May 1, 2010
Figure 6. Dependence of the electrocatalytic responses of the
Lac-ABTS-graphene/GC electrode toward the reduction of oxygen
in oxygen-saturated PBS (0.1 M) on the amount of laccase loading
(A), the detection potential (B), the pH of the buffer (C), and the
temperature of the system (D). Every value is an average value of
five independent measurements.
for the concentration of laccase. The response has a slight
decrease at a higher concentration of laccase (for example, 12
mg mL-1) probably due to the blockage created by laccase
overloading, similarly to what has been reported for glucose
oxidase assembled into polyaniline nanotube.54 The electron
relaying capacity of the graphene may also be disturbed
because of the presence of the large amount of the insulating
and bulky enzyme molecules. Therefore, a concentration of 10
mg mL-1 of laccase is chosen as the optimal concentration for
construction of the electrode.
The choice of the detection potential is necessary to achieve
the highest sensitivity and avoid the electrochemical interfering
species. The response increases with the detection potential from
0.5 to 0.3 V (vs SCE) and then reaches a plateau at higher
potentials (panel B in Figure 6). Thus, a detection potential of 0.3
V is selected.
A further optimized experimental parameter is the solution pH.
In the range of pH variant from 5 to 9, an optimal response is
found at approximately pH 8 (panel C in Figure 6), which is in
good agreement with that reported for the laccase dissolved in
solution.55 This result indicates that the assembly procedure can
keep the native activity of laccase. The response decreases
significantly at a higher pH value. This may be caused by the
leakage of the enzyme molecules from the electrode surface due
to the electrostatic repulsion between the negatively charged
ABTS and laccase molecules. (The enzyme molecule should be
negatively charged when the solution pH is higher than its
isoelectric point, which is ca. 8.2 for laccase from Rhus vernicifera.)46
The temperature effect of the electrode response in O2saturated PBS was evaluated between 20 and 60 °C. The
response increases when the temperature rises from 20 to 30
°C, and then, it keeps almost constant in the range of 30 to 50
°C. At a higher temperature, the response decreases due to
(54) Wang, Z.; Liu, S.; Wu, P.; Cai, C.-X. Anal. Chem. 2009, 81, 1638–1645.
(55) Yang, W. Y.; Min, D. Y.; Wen, S. X.; Jin, L.; Rong, L.; Tetsuo, M.; Bo, C.
Process Biochem. 2006, 41, 1378–1382.
Figure 7. Typical steady state current response of the Lac-ABTSgraphene/GC electrode on successive increase of O2 concentration
in solution (PBS, pH 7.4) at an applied potential of 0.3 V. Insets show
the calibration curve of the electrocatalytic current on the concentration of O2 (inset A) and the Lineweaver-Burk plot (inset B).
the denaturation of the enzyme (panel D in Figure 6). The
optimum temperature for laccase dissolved in solution is usually
in the range of ca. 30-40 °C.55 The present results indicate that
the immobilized laccase can tolerate the temperature higher than
the dissolved one does. Therefore, the physiological pH and
temperature (pH 7.4 and 37 °C) are chosen as the optimal
condition for the Lac-ABTS-graphene/GC electrode sensing
extracellular O2 level released from cell by considering the
above results and the environments of the cell survival.
The determination of O2 concentration has been realized with
benzoquinone as a probe at a monoolein cubic phases-laccase
modified electrode26 and with ABTS as a probe (dissolved in
soultion) at a carbon nanotubes-chitosan-laccase modified
electrode.27 Here, the developed Lac-ABTS-graphene/GC
electrode can also be used to detect O2 quantitatively. The
concentration of O2 in a saturated supporting electrolyte was
1.1 mM;26,27 the solution with a different concentration of O2 was
prepared by diluting the O2-saturated solution with the deaerated buffer. Figure 7 displays the typical steady state amperometric response of the electrode to a stepwise increase of O2
concentration (0.05 mM for each addition) at an optimum
condition. Immediately after the addition, the response increases and reaches 95% of the steady state value within 5 s,
suggesting that the electrode responds rapidly to the change
of the O2 concentration. The electrode linearly responds to O2
concentration at lower concentrations and attains saturation
levels at higher concentrations (inset A, Figure 7). The response
displays a good linear range from 0.05 to 0.4 mM with a correlation
coefficient of 0.995 and a slope of (10.55 ± 0.35) µA mM-1.
Therefore, the sensitivity is evaluated to be ca. 90 µA mM-1
cm-2. The detection limit is estimated to be ca. (10 ± 2) µM
(at a signal/noise of 3), which is comparable to that obtained
at the carbon nanotubes-chitosan-laccase modified electrode
app
(7.8 µM).27 The apparent Michaelis-Menten constant (KM
)
is estimated to be (0.32 ± 0.05) mM from the Lineweaverapp
Burk plot (inset B of Figure 7). The KM
and the sensitivity
are superior to those reported at monoolein cubic phaseslaccase (5.5 mM and 0.23 µA mM-1 cm-2, data was estimated
from the given plot)26 and at a carbon nanotubes-chitosanlaccase modified electrode (3.22 mM and 27.31 µA mM-1
cm-2).27 The sensitivity is also higher than that obtained at a
carbon nanotubes-laccase modified boron-doped diamond
electrode (15-37 µA mM-1 cm-2, the value was calculated from
Figure 8. Typical amperometric responses obtained at the ABTSgraphene/GC (a) and Lac-ABTS-graphene/GC electrode (b) for the
electrocatalytic reduction of oxygen released from human erythrocytes. The release of oxygen from erythrocytes is induced by addition
of 2 mM NaNO2. The measurements were performed in PBS (10 mM)
under the physiological pH and temperature (pH 7.4 and 37 °C). The
electrodes were biased at a potential of 0.3 V (vs SCE).
the reported data).56 The linear range is much wider than that
obtained at a poly(nile blue) modified electrode (1.3-26 µM).57
These results suggest that graphene can be used as a
biocompatible platform for development of laccase-based amperometric biosensors for O2 determination.
Detection of Extracellular Oxygen Released from Human
Erythrocytes. The erythrocyte, lacking protein synthesis machinery and living in the presence of permanently high concentrations of oxygen and hemoglobin iron, is a good model to
investigate the processes of O2 release and consumption.58
Figure 8 depicts amperometric response obtained at the LacABTS-graphene/GC electrode located near the cell in PBS (pH
7.4) at a detection potential of 0.3 V (vs SCE). After a steady state
background was reached, 2 mM NaNO2 is injected into PBS. A
reduction current of ca. (3 ± 0.3) µA (this value is an average
of five independent measurements) is observed within ca.
10-20 s after the addition of NaNO2 (Figure 8b), which has
been reported to induce the release of oxygen from human
erythrocytes.14 This measurement was repeated up to five times
to establish the observation; Figure 8b represents a typical
response curve. However, no response is attained at the
ABTS-graphene/GC electrode (Figure 8a). Therefore, the observed cathodic current is ascribed to the electrocatalytic reduction of the released O2.
Due to a relative high detection potential (0.3 V vs SCE) being
used in this work, some coexisting oxidizable species in cells,
such as ascorbic acid (AA), uric acid (UA), etc., might be oxidized
at the electrode and their responses inevitably affect the electrocatalytic responses of the reduction of O2 provided these
substances can also be released from the cells after addition
of NaNO2. To further verify the response depicted in Figure
8b is only due to the electrocatalytic reduction of the released O2
from erythrocyte, a commercial O2 meter (CyberScan DO600,
Eutech Cyberscan) was connected to the system and used for
monitoring the amount of O2 release induced by NaNO2. The
measurements were also repeated up to five times, and the
(56) Stolarczyk, K.; Nazaruk, E.; Rogalski, J.; Bilewicz, R. Electrochim. Acta
2008, 53, 3983–3990.
(57) Ju, H.-X.; Shen, C. Electroanalysis 2001, 13, 789–793.
(58) van der Zee, J.; Dubbelman, T. M.; Van Stereninck, J. Biochim. Biophys.
Acta 1985, 818, 38–44.
Analytical Chemistry, Vol. 82, No. 9, May 1, 2010
3595
average O2 concentration released from the cells was ca. (0.31
± 0.05) mM. This value is in good agreement with that obtained
at the developed electrode (0.28 ± 0.08 mM), which is
calculated based on the average response currents (3 ± 0.3 µA)
and the calibration curve of the electrocatalytic current on the
concentration of O2 (inset A in Figure 7). These results show
that there is a good concordance between the responses of the
developed sensor and the O2 meter, suggesting the addition of
NaNO2 can only induce the release of O2 from human
erythrocytes and cannot lead to the release of the other
oxidizable species including AA, UA, etc. These results also
demonstrate that the observed signal at the Lac-ABTSgraphene/GC electrode (Figure 8b) is actually due to the
electrocatalytic reduction of O2 released from the cells. Therefore, the determination of O2 release by the developed method
is not interfered with the oxidizable species in the cells.
Though it is difficult to estimate precisely the concentration
of O2 in erythrocyte using the developed method since a little
of O2 is inevitably released before NaNO2 was injected into PBS
(this can be evidenced from the steady state background
presented in Figure 8b that is not level (slightly inclined) before
addition of NaNO2), the above observation substantially demonstrates that the developed Lac-ABTS-graphene/GC electrode represents a new biosensing platform for determination
of extracellular O2 released from cell. Therefore, the method
should be extremely helpful for studying the kinetics of O2
release from the cell and could be potentially useful for further
physiological and pathological studies.
In comparison with the classical Clark electrode, the developed
sensor still suffers a bit of slow response. This deficiency, however,
can be improved by properly modifying the electrode design; for
example, the device can be fabricated at a micrometer sized
electrode. The ultrasmall electrode can be used for measuring
the O2 content release from the single cell. More importantly,
the small electrode can also be used for monitoring the rate of
O2 release or uptake by a single cell. The work on modifying
the electrode design and measurements on the single cell is
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Analytical Chemistry, Vol. 82, No. 9, May 1, 2010
currently being carried out and will be reported in another
manuscript.
CONCLUSIONS
In summary, a novel efficient biosensing platform based on
graphene sheets integrated with laccase and ABTS has been
proposed. The hybrid has been demonstrated to be a good
electrocatalyst for the reduction of oxygen with not only inherent
enzyme activity, accepted stability, and a high half-wave potential
(ca. 670 mV vs NHE) but also unimpeded charge transport and
electrical communication to the copper redox sites of laccase. An
electrochemical approach for detection of the extracellular oxygen
released from human erythrocytes based on the bioelectrocatalytic
reduction of oxygen by bioelectrocatalyst has been developed.
This study has not only established a general route for fabricating
graphene-based hybrid via assembling enzymes/proteins on
graphene but also expanded the scope of graphene applications
to the field of bioelectroanalytical chemistry and cellular biology,
which may open up a new challenge and approach to explore the
electrochemical features of graphene or its hybrid materials for
the potential utilizations.
ACKNOWLEDGMENT
This work is supported by the National Natural Science
Foundation of China (20673057, 20773067, 20833006, and 20905036),
the Program for New Century Excellent Talents in University
(NET-06-0508), and the Foundation of the Jiangsu Education
Committee (09KJA150001 and 09KJB150006).
SUPPORTING INFORMATION AVAILABLE
Additional information as noted in text. This material is
available free of charge via the Internet at http://pubs.acs.org.
Received for review November 25, 2009. Accepted April 1,
2010.
AC100621R