Study of direct electron transfer and enzyme activity of glucose oxidase on graphene surface

Article abstract of DOI:10.1016/j.elecom.2014.10.016

In recent years, graphene has been widely used as a high performance two-dimensional material in the development of biosensors and biofuel cells for facilitating direct electron transfer (DET) of glucose oxidase (GOx). However, almost all of these reports perform experiments in the presence of oxygen (a natural mediator of oxidase) and whether the GOx with DET property retained their catalytic activity in the absence of mediators has not been studied in detail so far. In this paper, we investigated the DET property and enzyme activity of GOx on graphene surface without and with mediators. Experimental results showed that the biosensor had no response to glucose in mediator-free solutions, even though the DET of GOx was observed, indicating that the GOx with DET property lacked enzymatically catalytic activity. However, in the presence of mediators, the biosensor showed sensitive response to glucose, illustrating that the mediated enzymatic oxidation of glucose occurred, which can be attributed to the catalytically active GOx without DET capability. These results suggest that DET property and enzyme catalytic activity cannot occur on the same GOx simultaneously. Therefore, keeping enzyme activity and DET of GOx at the same time is still a major challenge for biosensor and biofuel cell researches.

Full text of DOI:10.1016/j.elecom.2014.10.016

Electrochemistry Communications 50 (2015) 1–5
Contents lists available at ScienceDirect
Electrochemistry Communications
journal homepage: www.elsevier.com/locate/elecom
Study of direct electron transfer and enzyme activity
of glucose oxidase on graphene surface
Bo Liang a,1, Xishan Guo b,1, Lu Fang , Yichuan Hu , Guang Yang , Qin Zhu , Jinwei Wei , Xuesong Ye a,c,⁎
a
a
a
a
a
a
Biosensor National Special Laboratory, College of Biomedical Engineering and Instrument Science, Zhejiang University, PR China
College of Biosystems Engineering and Food Science, Zhejiang University, PR China
Cyrus Tang Center for Sensor Materials and Applications, Zhejiang University, Hangzhou 310027, PR China
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 9 October 2014
Received in revised form 28 October 2014
Accepted 28 October 2014
Available online 3 November 2014
In recent years, graphene has been widely used as a high performance two-dimensional material in the develop-
ment of biosensors and biofuel cells for facilitating direct electron transfer (DET) of glucose oxidase (GOx). How-
ever, almost all of these reports perform experiments in the presence of oxygen (a natural mediator of oxidase)
and whether the GOx with DET property retained their catalytic activity in the absence of mediators has not been
studied in detail so far. In this paper, we investigated the DET property and enzyme activity of GOx on graphene
surface without and with mediators. Experimental results showed that the biosensor had no response to glucose
in mediator-free solutions, even though the DET of GOx was observed, indicating that the GOx with DET property
lacked enzymatically catalytic activity. However, in the presence of mediators, the biosensor showed sensitive re-
sponse to glucose, illustrating that the mediated enzymatic oxidation of glucose occurred, which can be attribut-
ed to the catalytically active GOx without DET capability. These results suggest that DET property and enzyme
catalytic activity cannot occur on the same GOx simultaneously. Therefore, keeping enzyme activity and DET of
GOx at the same time is still a major challenge for biosensor and biofuel cell researches.
Keywords:
Direct electron transfer
Enzyme activity
Graphene
Glucose oxidase
©
2014 Elsevier B.V. All rights reserved.
1
. Introduction
Graphene has attracted considerable attention from both the exper-
imental and theoretical scientific communities in recent years [28,29].
Owing to the excellent electrical, chemical and mechanical properties,
graphene has stimulated exploding interest in sensor applications
[30–32]. Numerous studies of DET behavior of GOx on graphene deco-
rated electrodes have been reported [33–38]. In our previous research,
we also studied the DET of GOx self-assembled on graphene surface
[39]. However, almost all of these reports examined glucose detection
performances in the presence of oxygen, indicating that those glucose
biosensors were not the real mediator-less DET-based biosensors. In
fact, the real meaning of DET is between enzymatically active GOx and
electrode. Up to now, whether the GOx on graphene surface with DET
performance retained their enzyme activity and whether the graphene
based biosensor can be used for glucose detection without mediators
have not been studied detailedly.
The direct electron transfer (DET) between enzymatically active
glucose oxidase (GOx) and conductive electrode surface is crucial for
the development of mediator-less biosensors [1–4] and high perfor-
mance biofuel cells [5–7]. The DET-based enzymatic biosensor is an
ideal detection technique, possessing several advantages such as
reagent-less sensing, low operation potential, and high selectivity
[8–10]. The DET-based enzymatic biofuel cells can work in a potential
range close to the redox potential of the enzyme itself and give the
maximum potential difference between the anode and cathode,
resulting in a higher power output [11–13].
The DET of enzymatically active GOx is extremely difficult, since the
active site of GOx, flavin adenine dinucleotide (FAD), is deeply embed-
ded within
nanomaterial, such as gold nanoparticles [16,17], carbon nanotubes
18,19], graphene [20,21] and their composites [22–24] have been
a protective protein shell [14,15]. Thus, various
Here, we investigated the DET property and enzyme activity of GOx
on graphene modified electrode in mediator-free and mediator contain-
ing solutions. Experimental results show that GOx with DET property
lost their enzyme activity when adsorbed onto graphene surface while
GOx with enzyme activity couldn't direct transfer electrons to electrode.
[
used to promote the electron transfer of redox proteins. However,
recent studies of DET and enzyme activity of GOx on carbon nanotubes
showed that DET did not occur between enzymatically active GOx and
carbon nanotubes [25–27].
2. Experimental
⁎
Corresponding author. Tel.: +86 571 87952756; fax: +86 571 87951676.
E-mail address: yexuesong@zju.edu.cn (X. Ye).
These authors contributed equally to this work.
Graphene modified electrode was prepared by electrochemical
reduction of graphene oxide (GO) as previously reported [39–41].
1
http://dx.doi.org/10.1016/j.elecom.2014.10.016
388-2481/© 2014 Elsevier B.V. All rights reserved.
1

2
B. Liang et al. / Electrochemistry Communications 50 (2015) 1–5
Briefly, 6 μL GO water dispersion (1 mg/mL) was cast on the polished
glassy carbon electrode (GCE) surface and dried in the ambient condi-
tion. 5 cycles of cyclic voltammetric scanning from 0.7 to −0.9 V at a
on GCE is necessary to facilitate the DET of GOx. The observed redox is
due to the electron transfer of the cofactor FAD of GOx via reaction (1):
−
1
GOxðFADÞ þ 2Hþ þ 2e ↔GOxðFADH2Þ
−
scan rate of 50 mV s in N
2
-saturated 0.5 M NaCl solution were per-
ð1Þ
formed. The electrode was denoted as ERGO/GCE. Then 6 μL GOx/PBS
solution (10 mg/mL GOx, pH 7.4) was cast on the ERGO/GCE surface,
dried in the ambient condition. Afterwards, 6 μL 1% Nafion was cast on
the electrode surface to maintain the stability of the modified electrode.
The final electrode denoted as GOx/ERGO/GCE was rinsed thoroughly
with deionized water and dried in air. For comparison, bare GCE,
GOx/GCE and ERGO/GCE were prepared respectively, and were
casted with 6 μL 1% Nafion finally.
According to reaction (1) the DET of GOx is a two-electron and two-
proton coupled reaction. The cathodic peak current is attributed to the
reduction of GOx(FAD), while the anodic peak current is attributed to
the oxidation of GOx(FADH ). The pH value of the solution will influ-
2
ence the direct electron transfer of GOx. As shown in Fig. 1B, both the
anodic and cathodic peak potentials shift to negative direction with
the increase of the solution pH. The formal potential exhibits a linear de-
pendence on the pH ranging from 5 to 9 with a slope of −59 mV/pH
(r = 0.999).
3
. Results and discussion
As we know, the DET-based third generation glucose biosensor
doesn't need any mediators, including oxygen. Glucose is oxidized by
3
2
.1. DET between GOx and electrode in N -saturated solution
2
GOx into gluconolactone while GOx(FAD) is reduced into GOx(FADH ),
DET of GOx adsorbed on ERGO modified GCE was studied by cyclic
voltammetry in N -saturated PBS. Fig. 1A shows the cyclic voltammo-
and then the GOx is directly recycled at the electrode surface by electro-
2
chemical oxidation of GOx(FADH ). Both reactions are shown as
2
grams (CVs) of GCE with or without ERGO and GOx modification.
A pair of well-defined and quasi-reversible redox peaks was observed
at the CVs of GOx/ERGO/GCE (Fig. 1A line 4). The formal potential of
the redox peaks was −0.48 V, and the separation of peak potentials
was 40 mV, which is close to redox peaks of GOx and its cofactor FAD
on modified electrode surfaces in previous reports [20,42]. However,
no redox peaks were observed at the CVs of bare GCE (Fig. 1A line
follows:
GOxðFADÞ þ glu cose→GOxðFADH Þ þ gluconolactone
ð2Þ
ð3Þ
2
GOxðFAD2Þ→GOxðFADÞ þ 2Hþ þ 2e−
1
) and ERGO/GCE (Fig. 1A line 3), indicating that the redox peaks of
However, when glucose was added into the solution, the CVs of
GOx/ERGO/GCE had no variation (Fig. 1C). The lack of glucose response
indicated that no enzymatic reaction happened to GOx with DET
the GOx/ERGO/GCE should be ascribed only to GOx. What's more,
when GOx was directly adsorbed onto a bare GCE surface, no redox
peaks were observed (Fig. 1A line 2), suggesting that ERGO modification
2
capability; otherwise GOx(FAD) could be reduced into GOx(FADH )
Fig. 1. (A) CVs of bare GCE (1), GOx/GCE (2) ERGO/GCE (3), and GOx/ERGO/GCE (4) in N
and 9. The inset is the plot of formal potential vs. pH. (C) CVs of GOx/ERGO/GCE in N -saturated PBS with 0 mM, 1 mM, 5 mM, and 10 mM glucose respectively. Insets: enlargement of the
redox peak areas (dashed box). (D) CVs of GOx/ERGO/GCE in air-saturated PBS in various concentrations of glucose. Scan rates, panel A: 50 mV s , panel B, C, and D: 25 mV s
2 2
-saturated PBS. (B) CVs of GOx/ERGO/GCE in N -saturated PBS with different pH values of 5, 6, 7, 8
2
−
1
−1
.

B. Liang et al. / Electrochemistry Communications 50 (2015) 1–5
3
by glucose according to reaction (2) and the anodic oxidation peak of
GOx should be increased according to reaction (3). Unfortunately, no
such peak current change was observed. This result indicates that the
DET active GOx has no glucose catalytic activity.
3
.2. Enzyme activity of GOx in air-saturated solution
In the presence of oxygen (O
glucose is oxidized by GOx into gluconolactone while O
GOx into H (reaction 4). Besides, O can be electrochemically
2
), the natural mediator of oxidase,
2
is reduced by
2
O
2
2
reduced on electrode surfaces (reaction 5), forming a cathodic current.
GOx
glucose þ O2 → gluconolactone þ H O
ð4Þ
ð5Þ
2
2
þ
−
O þ 4H þ 4e →2H O
2
2
As shown in Fig. 1D, a remarkable enhancement of the cathodic peak
current was obtained in the presence of O , accompanied by a decrease
of anodic peak current, which was caused by the overlapping of the
current of the reduction of O (reaction 5) with the DET current of
GOx (reaction 1) [33,34]. The reduction peak of O could be observed
2
2
2
at about −0.38 V. When glucose was added into the air-saturated solu-
tion, the cathodic peak current decreased. This result should be ascribed
to the decrease of O
consumption of dissolved O
oxidation (reaction 4). This indicates that some GOx immobilized on the
electrode retain their enzyme activity in the presence of O . However,
2
reduction current (reaction 5) due to the
2
according to the enzyme catalytic glucose
2
these glucose catalytically active GOx shouldn't be the GOx with DET
property; otherwise the anodic peak current of the CVs in Fig. 1C should
be increased when glucose was added into the O
ing to reactions (2) and (3).
2
-free solution accord-
Fig. 2. (A) CVs of GOx/ERGO/GCE in N
concentrations increased from 0 mM to 8 mM. (B) CVs of DET-GOx/ERGO/GCE in
2
-saturated PBS containing 0.5 mM FCA with glucose
N
2
-saturated PBS containing 0.5 mM FCA with 0 mM, 1 mM, 4 mM, and 8 mM
3
2
.3. Enzyme activity of GOx in N -saturated solution with
−1
glucose respectively. Scan rates, 25 mV s
.
artificial mediator
To further evaluate the electroactivity and catalytic activity of GOx
on ERGO/GCE surface, we performed cyclic voltammetry in the presence
These results suggest that there are two types of GOx adsorbed on
ERGO/GCE surface. As shown in Fig. 3, one type GOx with DET property
was denoted as DET-GOx, while the other type GOx with enzyme
2
of ferrocene (an artificial mediator of oxidase) in O -free solutions.
Fig. 2A shows the CVs of GOx/ERGO/GCE in O -free solutions with
2
FCA as the mediator. As shown, there are two pairs of redox peaks in
CVs. The first pair at the formal potential of −0.48 V was due to the
DET of GOx, while the second pair at the formal potential of 0.3 V was
due to the redox of FCA. When glucose was added into the solution,
the anodic peak current of FCA increased significantly. The peak current
enhancements attributed to the enzyme catalytic reaction of glucose,
which can be represented by reactions (2), (6) and (7). Firstly,
GOx(FAD) is reduced into GOx(FADH
2
) by glucose according to reaction
+
(
2). Then, the reduced GOx(FADH ) is oxidized by FCA to GOx(FAD)
2
+
and FCA is reduced to FCA according to reaction (6). Lastly, the
increased peak current comes from the electrochemical oxidation of
+
FCA to FCA according to reaction (7). This result indicates that some
GOx on ERGO/GCE surface maintain their catalytic enzyme activity,
and the electron transfer between these GOx and ERGO/GCE was
mediated by FCA.
þ
þ
GOxðFADH Þ þ FCA →GOxðFADÞ þ FCA þ 2H
ð6Þ
ð7Þ
2
þ
−
2
FCA→2FCA þ 2e
However, no change was observed on the first pair of redox peak in
CVs in Fig. 2A in the absence and presence of glucose, giving more
evidence that the DET active GOx didn't remain their enzyme activity.
Fig. 3. Schematic illustration of GOx adsorbed on the surface of ERGO modified GCE.

4
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