Direct electrochemistry of glucose oxidase and sensing of glucose at a glassy carbon electrode modified with a reduced graphene oxide/fullerene-C60 composite

Article abstract of DOI:10.1039/c5ra12018g

In the present work, a glucose biosensor was fabricated based on the direct electrochemistry of glucose oxidase at glassy carbon modified with a reduced graphene oxide (RGO) and fullerene-C60 (C60) composite. The reduced graphene oxide/fullerene (RGO-C60) composite was prepared by electrochemical reduction of a graphene oxide (GO) and C60 composite at -1.4 V for 200 s in pH 5 solution; while the GO-C60 composite was prepared by a simple sonication of C60 in GO solution for 6 hours at 45°C. A well-defined and enhanced reversible redox peak of GOx was observed at RGO-C60 composite compared with other modified electrodes. The heterogeneous electron transfer rate constant (K_s) and the surface coverage concentration of GOx at RGO-C60/GOx modified electrode were calculated to be 2.92 s^-1 and 1.19 × 10^-10 mol cm^-2, respectively. Under optimum conditions, the amperometry response of the biosensor was linear against the concentration of glucose from 0.1 to 12.5 mM with a response time of 3 s. The limit of detection was estimated to be 35 μM based on S/N = 3 with a high sensitivity of 55.97 μA mM^-1 cm^-2. In addition, the fabricated biosensor showed a good practical ability for the detection of glucose in human blood serum samples.

Full text of DOI:10.1039/c5ra12018g

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Schematic representation for the sensing of glucose at RGO–C60/GOx composite.

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Journal Name
RSCPublishing
ARTICLE
Direct electrochemistry of glucose oxidase and
sensing of glucose at glassy carbon electrode modified
Cite this: DOI: 10.1039/x0xx00000x
with
reduced
graphene
oxide/fullerene-C60
nanocomposite
Received 00th January 2012,
Accepted 00th January 2012
a‡
a‡
a*
Balamurugan Thirumalraj,
Selvakumar Palanisamy,
ShenꢀMing Chen
and BihꢀShow Lou c***
DOI: 10.1039/x0xx00000x
ChengꢀYu Yang, a Prakash Periakaruppan
b**
www.rsc.org/
In the present work, the glucose biosensor was fabricated based on the direct electrochemistry
of glucose oxidase at glassy carbon modified with reduced graphene oxide (RGO) and fullereneꢀC60
(C60) composite. Reduced graphene oxide/fullerene (RGO–C60) composite was prepared by
electrochemical reduction of graphene oxide (GO) and C60 composite at ꢀ1.4 V for 200 s in pH 5
solution; while the GO–C60 composite was prepared by a simple sonication of C60 in GO solution for 6
hours at 45˚C. A wellꢀdefined and enhanced reversible redox peak of GOx was observed at RGO–C60
composite compared to that of other modified electrodes. The heterogeneous electron transfer rate
constant (Ks) and surface coverage concentration of GOx at RGO–C60/GOx modified electrode were
calculated to be 2.92 sꢀ1 and 1.19 x 10–10 mol cm–2, respectively. Under optimum conditions, the
amperometry response of the biosensor was linear against the concentration of glucose from 0.1 to 12.5
mM with a response time of 3 s. The limit of detection was estimated to be 35 µM based on S/N=3 with
a high sensitivity of 55.97 µAmM–1cm–2. In addition, the fabricated biosensor showed good practical
ability
for
the
detection
of
glucose
in
the
human
blood
serum
samples.
(GOx) is an ideal redox active enzyme for studying the electron
transfer properties and has been widely used for construction glucose
biosensors owing to its high specificity toward glucose.4 The
determination of glucose is always more important in the case of
diabetes mellitus which is increasing rapidly.5 As of 2013, 382
million people have been affected by diabetes worldwide as reported
by International Diabetes Federation.5 Hence, the fabrication of
simple and sensitive glucose biosensors is necessary even though
1. Introduction
The direct electron transfer (DET) of redox active enzymes and
proteins are continuously receiving much attention in different
disciplines owing to their unique redox active electron transfer
properties that are associated with redox active centre.1, 2 The unique
DET properties of the enzymes are more helpful for studying the key
biological reactions and construction of biosensors and biofuel
cells.3 Among different redox active enzymes, glucose oxidase
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tons of glucose biosensors already are available in the literature. The it is reported earlier that C60 can be easily dispersed into the
high specificity of GOx is more helpful for the construction of graphene oxide (GO) aqueous solution via covalent and nonꢀ
selective glucose biosensors.6 However, the DET of GOx is often covalent interactions.20, 21 In addition, GO has been widely used for
complicated or quite difficult at conventional electrodes due to the the preparation of hybrid carbon materials including carbon
complex structure of GOx scaffold which directly prohibit the redox nanotubes and C60 owing to the presence of abundant oxygen
properties of the catalytic sites of GOx.3 Over the past decades, the functional groups.21 In the present study, we have dispersed the C60
DET of GOx have successfully been realized and studied with the into the GO aqueous solution by simple sonication and the resulting
help of micro and/or nanomaterials modified electrodes, including GO–C60 composite has been electrochemically reduced to RGO–
the carbon materials, metal nanoparticles, metal oxides, conducting C60 composite by electrochemical method. Compared with other
polymers, ionic liquids, etc.,7–11; Since the composites of nano and reduction methods, electrochemical method is more helpful to retain
micro materials have shown an enhanced DET of GOx compared to some of the oxygen functionalities on the carbon basal plane and the
individual one.
properties of RGO and C60. The reasons why we have chosen
Reduced graphene oxide (RGO) is a carbon allotrope and the RGO¬C60 composite for biosensor are: i) the electrochemical
single atomic layer of carbon atoms that are arranged in a behaviour of C60 is greatly improved in the presence of RGO, since
honeycomb lattice.2 Since the discovery of RGO in 2004, it has been pristine C60 has poor electrochemical behavior in aqueous solutions;
regularly used for different potential applications including energy ii) The electron transfer rate of enzyme towards the electrode surface
conversion and storage, electronic devices, electrocatalysis, sensors is much improved at RGO–C60 composite when compared with
13
and biosensors.12,
However, the DET of GOx at pristine RGO pristine C60 and RGO, the amino terminal group of enzyme can be
surface is difficult due to the van der Waals force of attraction easily attracted by oxygen functional group of RGO and electronꢀ
between each RGO sheets. Hence, the different approaches or micro releasing nature of enzymes and proteins can be easily attracted by
or nanomaterials have been used with RGO to further enhance the conjugate π electron structure of C60 molecules.
electron transfer properties of GOx thus leading to the high
Herein, we report the electrochemical preparation of the RGO–
sensitivity for the detection of glucose.7 Recently, the composites of C60 composite by electrochemical reduction of GO–C60 composite
RGO with carbon nanomaterials, metal nanoparticles and metal at –1.2 V for 200 s in pH 5 solution. The GO–C60 composite was
oxides have been used as an electrode material for immobilization of prepared by a simple sonication of C60 in GO aqueous solution for 6
GO, and they have shown an enhanced DET with better catalytic hours at 45˚C. The fabricated RGO–C60 composite was further used
performance of GOx towards glucose compared to RGO.14–16
as an immobilization matrix for GOx. The DET of GOx is greatly
Fullerene C60 (C60) is one of the carbon allotropes and is made improved in presence of C60 with RGO. The GOx immobilized
up of 60 closely packed carbon atoms into fused hexagons and RGO–C60 composite electrode was subsequently used for the
pentagons without any edges.17 Furthermore, it has been used as a construction of glucose biosensor. The practicality of the biosensor
promising electron transfer mediator for biomolecules and oxidase in human blood serum samples have also been studied in detail.
based amperometric biosensors and ECL biosensors.19–21 In addition,
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Preparation of RGO–C60 composite
2. Experimental
GO was synthesized from natural graphite by using the
Hummers’ method as reported previously.23 The GO aqueous
solution was prepared by dispersing the GO (2 mg mL–1) in water
with the help of ultrasonication. In order to prepare the GO–C60
composite, the C60 in toluene solution was added into the GO
aqueous solution (1:2, V: V %) and ultrasonicated for 6 hours at
45˚C. After 6 hours of ultrasonication at 45˚C, the colour of GO
solution changes from brown to dark brown indicates the formation
of GO–C60 composite. Ultrasonicator DC150H (Taiwan Delta New
Instrument Co. Ltd.) with operating frequency of 40 kHz and
ultrasonic power output of 150 W has been used for sonication.
Materials and methods
FullereneꢀC60 (99.5% purity) and graphite powder (98.0% purity)
were obtained from Sigma–Aldrich. Glucose oxidase from
Aspergillus niger was obtained from Sigma–Aldrich. Glucose,
dopamine, ascorbic acid and uric acid were obtained from Aldrich.
The supporting electrolyte 0.05 M pH 7 solution (PBS) was used for
all electrochemical experiments and was prepared by using 0.05 M
Na2HPO4 and NaH2PO4 solutions in doubly distilled water. All other
chemicals used in this work were of analytical grade and all the
solutions were prepared using doubly distilled water without any
further purification. Human blood serum sample was collected from
valley biomedical, Taiwan product & services, Inc. This study was
reviewed and approved by the ethics committee of ChangꢀGung
memorial hospital through the contract no. IRB101ꢀ5042A3.
Cyclic voltammetry (CV) and amperometry iꢀt measurements
were performed using the CHI 1205b and CHI 750a electrochemical
stations. The surface morphology studies were carried out JSMꢀ
6500F scanning electron microscope. Raman spectrum was recorded
using a Raman spectrometer (Dong Woo 500i, Korea) equipped with
a 50 × objective and a chargeꢀcoupled detector. Ultraviolet–visible
(UVꢀVis) absorption spectra were obtained using a Hitachi Uꢀ3300
UV spectrophotometer. A conventional threeꢀelectrode system
consisting of modified glassy carbon electrode (electrochemically
active surface area = 0.12 cm2) as a working electrode, an external
saturated Ag/AgCl as a reference electrode and a platinum wire as
the auxiliary electrode was used. Amperometric i–t measurements
were performed using a PRDEꢀ3A (AS distributed by BAS Inc.
Japan) rotating ring disc electrode apparatus with an
electrochemically working surface area of 0.033 cm2. The
electrochemically active surface area (EASA) of the GCE and RDE
was calculated by CV according to our previous reported method.22
Fig. 1 A schematic representation for the preparation of RGO–C60
composite.
The effect of loading amount of GO and C60 on the biosensor
response was optimized and the optimized conditions were used for
the fabrication of the biosensor. The prepared GO–C60 composite
was centrifuged at 2000 rpm to remove the unattached C60. About 8
ꢁL of GO–C60 composite (optimum) was drop casted onto the preꢀ
cleaned GCE and dried at room temperature. The GO–C60
composite modified GCE was transferred to N2 saturated pH 5
solution and was electrochemically reduced to RGO–C60 composite
by applying ꢀ1.4 V for 200. For comparison, the RGO modified
GCE was prepared by electrochemical reduction of GO modified
electrode without C60 by applying ꢀ1.4 V for 200 s. A schematic
representation for the preparation of RGO–C60 composite is shown
in Fig. 1.
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composite. The findings confirm the formation of RO–C60
For the fabrication of biosensor, about 6 µL of GOx solution (5 composite.
Fabrication of RGO–C60/GOx biosensor
mg mL–1 in PBS) was drop coated onto the RGO–C60 composite
The formation of RGO–C60 composite was further confirmed
modified GCE. The fabricated biosensor was allowed to dry at room by Raman spectroscopy and UVꢀVis spectroscopy. Raman
temperature and rinsed with deionized water before the spectroscopy is widely used for the confirmation of the electronic
electrochemical experiments. All electrochemical measurements and lattice structures of carbon materials. Fig. 3A shows the Raman
were carried out at room temperature.
spectra of GO (a), C60 (b) and RGO–C60. It can be seen that C60
exhibits two distinct peaks at 1460 and 1680 cm−1, which ascribed
to the Ag and Hg mode of C60. While, the Raman spectrum of
RGO–C60 composite shows D and G band at 1342 and 1596 cm–1,
which is related to the vibrations of sp3 carbon atoms of disordered
3. Results and Discussion
Characterization of RGO–C60 composite
Fig. 2 shows the SEM image of (A) GO and GO sonicated for 6
hr at 45˚C (inset), GO–C60 (B), C60 (inset of B), RGO (C) and
RGO–C60 (D). The GO shows thin sheet morphology and RGO graphene nanosheets and vibrations of sp2 carbon atom domains of
exhibits its typical crumble morphology associated with bundle of graphite.18 It should be noted that the Hg mode of C60 is not
nanosheets. Fig. 2A inset clearly reveal that the surface morphology obviously visible on RGO–C60 composite, which maybe
of GO did not changed upon sonication of GO for 6 hr at 45˚C.
overlapping of Hg mode of C60 with the Gꢀband of RGO. Similar
results has been reported before for the presence of C60 with carbon
nanomaterials.18 In addition, the ID/IG ratio increase (ID/IG = 1.12) in
RGO–C60 composite compared to GO (ID/IG = 0.91) which is due
to the removal of oxygen functional groups in GO and formation of
conjugated graphene network (sp2 carbon) by electrochemical
reduction process. The results confirm the formation of RGO–C60
composite.
The formation of GO–C60 composite is confirmed by
UVꢀVis spectroscopy and the results are shown in Fig. 3B. The C60
(curve a) shows a shoulder peak at 332 nm, which is assigned to πꢀ
π* electronic transitions of C60 molecules. The GO (curve b)
exhibits an absorption maximum at 310 nm corresponding to n →
π* transitions of C=O bonds.25 On the other hand, RGO did not
show the absorption peak at 310 nm, is due to the deoxygenation of
the GO by electrochemical reduction processes. The GO–C60
composite shows absorption peaks at 331 nm and is related to the πꢀ
π stacking of C60 and GO. Since, the peak of C60 at 335 nm is a
Fig. 2 SEM images of (A) GO and GO sonicated for 6 hr at 45˚C
(inset), GO/C60 (B), RGO (C) and RGO/C60 (D). The inset of B
shows the SEM image of C60. Scale bar = 1.5 µm.
On the other hand, the C60 reveals the typical rodꢀshape
morphology of C60. The SEM image of GO–C60 composite clearly
shows that the C60 rods are enfolded by GO sheets which indicate
the formation of GO–C60 composite. While, the C60 rods are
clearly seen on the RGO nanosheets and the morphology of RGO
does not affect upon the electrochemical reduction of GO–C60
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vital indicator for the π–π interaction between the C60 and other
organic compounds.20 The RGO–C60 exhibits absorption peak at
The direct electrochemistry of GOx immobilized different
modified electrodes was investigated using CV. Fig. 4 shows the CV
332 nm and the peak of C60 at 310 nm disappear, which indicates response of GOx immobilized at C60 (a), GO–C60 (b), RGO (c) and
that C60 molecule is assembled on graphene sheets through πꢀπ RGO–C60 (d) modified GCEs in N2 saturated PBS at a scan rate of
interaction. The observed results are much similar to the previously 50 mV s–1 in the potential scanning from –0.7 to ꢀ0.1 V. A weak
reported RGO–C60 composite.20, 26
redox couple is observed at C60/GOx and the anodic peak potential
(Epa) and cathodic peak potential (Epc) of redox couple is located at –
0.396 and –0.432 V with a peak to peak separation (∆Ep) of 36 mV,
which is attributed to the direct electrochemical behavior of redox
couple (FAD/FADH2) in GOx.27 The GO–C60/GOx modified GCE
shows a pair of enhanced redox couple for GOx compared to that of
C60. The Epa and Epc of redox couple is located at –0.405 and –0.396
V with a peak to peak separation (∆Ep) of 31 mV, which is 5 mV
lower than those observed at C60/GOx modified GCE. The GOx
immobilized bare and GO modified GCEs does not show the redox
couple at the same potential window (figure not shown), which
indicates that C60 plays an important role in the DET of GOx
towards the electrode surface.
Fig. 3 A) Raman spectra of GO (a), C60 (b) and RGO–C60. B) UVꢀ
Vis spectra of C60 (a), GO (b), GO–C60 (c), RGO–C60 (d) and
RGO (e).
Fig. 4 A) CV response of C60/GOx (a), GO–C60/GOx (b), RGO (c)
and RGO–C60/GOx modified electrode in N2 saturated PBS at a
scan rate of 50 mV s–1.
Electrochemistry of GOx at different modified electrodes
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On the other hand, RGO–C60/GOx modified electrode Where ꢂEp = peakꢀtoꢀpeak separation of the redox couple, n = 2, α is
shows a wellꢀdefined redox peak for GOx and Epa and Epc of redox assumed to be 0.5 and all other symbols have usual meanings. The
couple is observed at –0.439 and –0.405 V with a peak to peak surface coverage concentration (Γ) of GOx at the modified electrode
separation (∆Ep) of 34 mV. Furthermore, the Ipa and Ipc of redox is calculated as 1.19 x 10–10 mol cm–2 using the following equation
couple is 5.6 folds higher than those observed at C60 and GO–C60 as reported early.22
modified GCEs. RGO/GOx modified GCE shows less intense redox
couple for GOx compared with other GOx immobilized modified
GCEs. The results clearly indicate that RGO–C60 composite is
favourable for DET between the electrode surface and the redox
centers of GOx compared to other modified electrodes. The
enhanced DET of GOx at RGO–C60 modified electrode is due to the
combined unique properties of RGO and C60. Hence, RGO–
C60/GOx modified electrode can be used as a sensitive biosensor for
the detection of glucose.
Γ = Q/nFA
Where Q = total charge (5.7925 × 10−6 C), n = number of electrons
involved in electrochemical reaction (n = 2), A= electrochemically
active surface area of the electrode (0.12 cm2) and all other symbols
have usual meanings. The calculated value of Ks is a bit higher than
that of previously reported GOx immobilized composite modified
electrodes indicating that the fast DET of GOx towards the electrode
surface. In addition, the higher value of Γ indicates the high loading
of GOx on RGO–C60 composite.19, 29, 30
Fig. S1A shows the effect of scan rate on the DET of GOx
The pH is an essential parameter to evaluate the DET of
GOx at RGO–C60 modified electrode, since it greatly affects the
DET behavior of GOx. Fig. S2A shows the CV response of GOx
immobilized RGO–C60 modified electrode in N2 saturated different
pH solutions from 3 to 9 at a scan rate of 50 mV s–1. It can be seen
that a wellꢀdefined reversible redox peaks of GOx observed in each
pH solution; the Epa and Epc of redox couple of GOx shift towards
positive and negative direction upon decreasing and increasing the
pH of the solution. As shown in Fig. S2B, the formal potential (Eo’,
immobilized RGO–C60 modified electrode in PBS. It can be seen
that the redox peak current of GOx increases with increasing the
scan rates; while the Epa and Epc of redox couple is slightly shifted
towards positive and negative direction upon increasing the scan
rates from 10 to 200 mV s–1. As shown in Fig. S1B, the anodic (Ipa)
and cathodic peak current (Ipc) of redox couple have a linear
dependence over the scan rates from 10 to 120 mV s–1 with the
correlation coefficient of 0.9978 and 0.9983, respectively. The result
suggests that the redox electrochemical behavior of GOx at RGO–
ꢒꢓꢘꢙ ꢒꢓꢚ
ꢗ′
ꢖ
=
) of GOx has a linear dependence over the pH range
ꢔ
C60 modified electrode is controlled by
a surfaceꢀconfined
from 3 to 9 with a slope of –51.9 mV/pH and a correlation
coefficient of 0.9939. The observed slope value (−51.9 mV/pH) is
close to the theoretical value of the Nernst equation for an equal
number of proton and electron transfer reversible electrochemical
process and it confirms that the DET of GOx involves equal number
of protons (H+) and electrons (e−) transferred reaction. The DET of
GOx can be written by the following Eqn. as reported previously.31
electrochemical process. The heterogeneous electron transfer rate
constant (Ks) of GOx at RGO–C60/GOx modified electrode is
calculated when the ꢂEp > 100 mV; since the number of electrons
transferred in electrochemical reaction of GOx is 2. The value of Ks
is calculated as 2.92 sꢀ1 by using the following Laviron equation.28
ꢉꢊ
ꢆ
ꢇ
ꢆ
ꢇ
ꢀꢁꢂꢃꢄ = ꢅꢀꢁꢂ 1 − ꢅ + 1 − ꢅ ꢀꢁꢂꢅ − ꢀꢁꢂ ꢈꢋꢌꢍꢎ −
–
ꢙ
ꢇ
ꢅ ꢆ
ꢏꢐꢑ ꢋꢌꢂꢒꢓ
ꢆ
ꢇ
ꢆ
ꢇ
ꢛꢜꢝ– ꢞꢟꢠ + 2ꢡ + 2ꢢ ↔ ꢛꢜꢝ– ꢞꢟꢠꢢꢔ
ꢔ.ꢕꢉꢊ
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biosensor for addition of 1 mM glucose into the PBS. C) The
Amperometric determination of glucose
The electrocatalytic activity of the proposed biosensor calibration plot of [glucose] vs. amperometric current response.
towards glucose was evaluated by CV in oxygen saturated PBS at a Fig. S3B shows the CV response of RGO–C60/GOx modified
scan rate of 50 mV s–1. As shown in Fig. S3A, in absence of oxygen electrode in the absence (a) and presence of different concentrations
(curve a), a wellꢀdefined reversible redox couple of GOx is of glucose (b–g) in oxygen saturated PBS at a scan rate of 50 mV s–
observed; while increase in cathodic peak current and decrease in 1. It can be seen that the cathodic peak current decreases with
anodic peak current is observed in the presence of oxygen (curve b). increasing the concentration of glucose into the oxygen saturated
The result confirms that GOx is highly active in the presence of PBS. The decrease in the cathodic peak current is due to the
molecular oxygen. The reduction of molecular oxygen by of GOx consumption of molecular oxygen and formation of H2O2. The high
can be written by following reaction.
activity of GOx with molecular oxygen is resulting to the decrease of
oxidized form of GOx on electrode surface. This is the reason why
the addition of glucose restrained the electrocatalytic reaction and
lead to the decrease of reduction current in the presence of oxygen.
The similar phenomenon has been reported early in the literatures for
oxidation of glucose by GOx in presence of oxygen.31–33 The
mechanism of glucose oxidation by GOx in presence of oxygen can
be expressed by following equations, and the schematic
representation of electroꢀoxidation of glucose in the presence of
oxygen at RGO–C60/GOx composite is shown in Fig. 5A.
–
ꢙ
ꢆ
ꢇ
ꢆ
ꢇ
ꢛꢜꢝ– ꢞꢟꢠ + 2ꢡ + 2ꢢ ↔ ꢛꢜꢝ– ꢞꢟꢠꢢꢔ ꢆꢠꢖꢣ ꢁꢤ ꢛꢜꢝꢇ
ꢆ
ꢇ
ꢛꢜꢝ– ꢞꢟꢠꢢꢔ + ꢜꢔ
ꢆ
ꢇ
→ ꢛꢜꢝ– ꢞꢟꢠ + ꢢꢔꢜꢔ ꢆꢜꢔꢥꢡꢦꢧꢨꢩꢪꢁꢫꢇ
ꢆ
ꢇ
ꢆ
ꢇ
ꢛꢜꢝ– ꢞꢟꢠ + ꢂꢬꢧꢨꢁꢭꢡ → ꢛꢬꢧꢨꢁꢫꢁꢬꢮꢨꢩꢁꢫꢡ + ꢛꢜꢝ– ꢞꢟꢠꢢꢔ
ꢆ
ꢇ
ꢆ
ꢇ
ꢛꢜꢝ– ꢞꢟꢠꢢꢔ + ꢜꢔ → ꢛꢜꢝ– ꢞꢟꢠ + ꢢ2ꢜ2
Amperometric iꢀt method was used for the determination of
glucose using the proposed biosensor, since it is more sensitive
method than that of other voltammetric methods. This is the reason
why amperometric iꢀt method has been widely used for enzymatic
biosensors. Fig. 5B shows the amperometric iꢀt response of RGO–
C60/GOx modified electrode for the addition of different
concentrations of glucose into the constantly stirred oxygen saturated
PBS with the working potential of –0.42 V. A wellꢀdefined
amperometric response is obtained for the each addition of 500 µM
glucose into the PBS (inset). The amperometric reduction current
decreases upon addition of further concentrations of glucose in to the
Fig. 5 A) Schematic representation for the sensing of glucose at
RGO–C60/GOx composite. B) Amperometric iꢀt response obtained
at RGO–C60/GOx biosensor for the addition of 0.1 mM (a), 0.5 mM
(b) and 1 mM of glucose (c and d) up to 14.6 mM glucose into the
constantly stirred oxygen saturated PBS; working potential is –0.42
V. Inset shows the enlarged view of amperometric response of the
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PBS and shows the response up to the addition of 14.6 mM glucose.
The selectivity of the proposed biosensor is always an
The biosensor reaches its steady state current in 3 s. As shown in important one especially for amperometric enzymatic biosensors;
Fig. 5C, the amperometric iꢀt response current of the biosensor is hence, the selectivity of the biosensor was investigated in the
linear against the concentration of glucose in the range of 0.1–12.5 presence of potentially interfering compounds such as dopamine,
mM. The limit of detection (LOD) was calculated as 35 µM based ascorbic acid and uric acid. The selectivity of the biosensor was
on S/N=3. The sensitivity is calculated from the slope of the investigated using amperometry and shown in Fig. S4. The
calibration plot as 55.97 µAmM–1 cm–2. The obtained linear response experimental conditions are similar as of Fig. 5B. It can be seen that
range of glucose is more suitable for the detection of glucose in the 5 mM addition of interfering compounds causes less interference
human blood samples, since the normal glucose range in human effect on the modified electrode; while a sharp response for the
blood in the range of 4.4 ꢀ 6.6 mM. The Michaelis–Menten constant addition of 1 mM glucose. The negative working potential is more
(Km) is calculated as 4.44 mM using a following Lineweaver–Burk beneficial for the less interference effect on the detection of glucose.
equation.34 The obtained Km value is lower than those previously The result confirms the appreciable selectivity of the biosensor
reported glucose biosensors, 33–36 which indicates the higher affinity towards the detection of glucose and can be used for the real time
of glucose towards biosensor electrode surface.
sensing of glucose in human serum samples without any interference
effect.
1
1
1
ꢆꢯꢃ ꢇ ꢆꢳ
ꢇ
ꢰ
=
+
ꢯꢄꢄ ꢯꢰꢱꢲ
ꢰꢱꢲ
ꢴꢵꢶꢷꢸꢄꢹ
The practical ability of the biosensor was evaluated for the
detection of glucose in human blood serum samples using
amperometry. The concentration of glucose was predetermined by a
commercial detector and the appropriate concentration of blood
serum was diluted with PBS for the real sample analysis. The
experimental conditions are similar as of Fig. 5B. The recovery of
glucose in human serum samples was calculated using the standard
addition method31, and the recovery of glucose is listed in Table ST2.
It can be seen that the proposed sensor shows a satisfactory recovery
towards glucose in human serum samples and the recovery was in
the range of 97.5%. The satisfactory recovery of glucose in human
serum samples authenticates the good practicality of the proposed
biosensor.
Where C = concentration of glucose in solution, Iss = steadyꢀstate
current, Imax = maximum current. Km/Imax = slope of the plot of the
reciprocals of Iss vs. C and 1/Imax = intercept of the plot of the
reciprocals of Iss vs. C. It is necessary to compare the analytical
performance of the proposed sensor with similar state of the art of
other glucose biosensors. Hence, the comparison table is shown for
the comparison of the analytical performance of the proposed
glucose biosensor with previously reported glucose biosensors in
Table ST1. It can be seen that the proposed biosensor shows higher
sensitivity, low LOD along with wider linear response range to
glucose compared to that of previously reported graphene based
glucose biosensors and thus it can be used for electrochemical
determination of glucose. 14, 25, 29, 31–33, 36, 37 The result confirms that
the proposed biosensor has wider linear response range, better
sensitivity with lower LOD. In addition, the high Km value of the
biosensor further confirms that it has high affinity towards glucose.
The storage stability is of the biosensor was evaluated by CV
and the cathodic response current of biosensor towards 1 mM
glucose containing PBS was measured periodically and the results
are shown in Fig. S5; the biosensor was stored in the refrigerator
8 | J. Name., 2012, 00, 1-3
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DOI: 10.1039/C5RA12018G
bDepartment of Chemistry, Thiagarajar College, Maduraiꢀ625009,
Tamilnadu, India.
when not in use. The biosensor retains its 90.9 % of its initial
response towards glucose after stored in six days, which indicates
the good storage stability of the proposed biosensor. The strong
immobilization of the GOx in the composite matrix thus leads to the
high activity of the biosensor. The repeatability and reproducibility
of the sensor was evaluated using CV for the detection of 1 mM
glucose. The three biosensors were prepared and used for the
detection of glucose in the PBS and the relative standard deviation
(RSD) of 3.9 % was observed. The RSD of 4.4 % was observed for
the 8 measurements of the detection of 1 mM glucose containing
PBS using a single biosensor. These results indicate a good precision
and accuracy of the proposed sensor.
cChemistry Division, Center for General Education, Chang Gung University,
TaoꢀYuan, Taiwan.
‡ These authors contributed equally.
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This project was supported by the National Science Council and the
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aElectroanalysis and Bioelectrochemistry Lab, Department of Chemical
Engineering and Biotechnology, National Taipei University of
Technology, No. 1, Section 3, ChungꢀHsiao East Road, Taipei 106,
Taiwan, ROC. Eꢀmail: smchen78@ms15.hinet.net;
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Fax: +886ꢀ2ꢀ27025238; Tel: +886ꢀ2ꢀ27017147.
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