Sensors and Actuators B 194 (2014) 71–78

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Sensors and Actuators B: Chemical

journal homepage: www.elsevier.com/locate/snb

A method for determination of glucose by an amperometric bienzyme

biosensor based on silver nanocubes modified Au electrode
Penghao Yang, Lisha Wang, Qi Wu ∗ , Zhichun Chen, Xianfu Lin ∗
Department of Chemistry, Zhejiang University, Hangzhou 310027, PR China

a r t i c l e i n f o a b s t r a c t

Article history: This paper reported the fabrication of an amperometric glucose biosensor based on silver nanocubes
Received 28 September 2013 (AgNCs) and horseradish peroxidase (HRP)–chitosan (CS)–glucose oxidase (Gox) bienzymatic film. The
Received in revised form fabrication process was characterized by scanning electron microscopy (SEM), transmission electron
17 December 2013
microscopy (TEM), and atomic force microscopy (AFM). Cyclic voltammetry (CV) and amperometry mea-
Accepted 19 December 2013
surements were used to study and optimize the performance of the resulting peroxide biosensor. Under
Available online 27 December 2013
the optimal conditions, the sensor exhibits good electrocatalytic activity toward the oxidation of glucose,
and this enables the determination of glucose in the 10 ␮M to 1.5 mM concentration range, with a detec-
Keywords:
Silver nanocubes
tion limit at 0.6977 ␮M (at an S/N of 3). The response time is less than 5 s. The bienzymatic electrode
Chitosan presented a number of attractive features such as high sensitivity, low detection limit, fast response,
Horseradish peroxidase and good stability, making this Gox–CS–HRP/AgNCs–CS nanocomposite film a promising candidate for
Glucose oxidase glucose sensors.
Biosensor © 2014 Elsevier B.V. All rights reserved.

1. Introduction active site of Gox and electrode, various nanomaterials, especially

Glucose played a crucial role in life processes, and the con-
centration of it in extracellular fluid of central nervous system
controlled the brain activity, which was closely linked to brain
energy metabolism and synaptic transmission. Moreover, diabetes
could be caused by high level of glucose in blood [1]. Since Clark
and Lyons proposed the first initial concept of glucose enzyme elec-
trodes in 1962 [2], tremendous efforts have been made to develop
reliable devices for diabetes control. During the last decades
many approaches including SPR [3], electrochemiluminescence [4],
fluorescence [5], colorimetric [6], flow injection with spectropho-
tometry [7], and electrochemical [8], have been developed for the
detection of glucose. They are mostly based on the analysis of
hydrogen peroxide generated by reaction between glucose and Gox
in most techniques. Among all of the methods, electrochemical
sensors are considered as one of the most convenient methods,
because of their high sensitivity, simplicity and rapidity and no
sample preparation.
However, the active site of Gox, flavin adenine dinucleotide
(FAD), which is of vital importance to the direct electron transfer for
Gox, is deeply embedded within a protective protein shell [9,10].
Thus, in order to promote the direct electron transfer between
∗ Corresponding authors. Tel.: +86 571 87951588; fax: +86 571 87951895.
E-mail address: llc123@zju.edu.cn (X. Lin).
noblemetal nanoparticles, such as gold [11], silver [12], platinum
[13], palladium [14], iridium [15], and their hybrids [15–17], have
attracted considerable attention due to their unique electronic and
catalytic properties.
The synthesis of silver nanomaterials has undergone extensive
research due to their low cost and high efficiency in catalysis of
the redox reaction of some analytes. Various silver nanostructures
have been achieved using different techniques: Ag nanowires [18],
nanograins [19], nanobelts [20], nanorods [21], and hybrids of Ag
nanoparticles [17,22,23]. They have many excellent properties such
as large surface-to-volume ratio, good electrical properties, strong
adsorption ability, high surface reaction activity, small particle size
and good surface properties. These excellent properties are help-
ful for the immobilization of biomolecules. Our research group
have reported the 1-D nanomaterials AgNWs in the use of glu-
cose [18] and H2 O2 [24] sensors. It is known that the shape of the
nanocrystals and the number of surface atoms (corner and edge)
are important parameters that contribute to the active catalytic
sites profoundly. Thus, shape control may efficiently lead to cat-
alytic selectivity in chemical reactions. Recently, nanocubes have
drawn more and more attention in the reason that they exhib-
ited extremely high electrocatalytic activity than truncated cube
or commercial spherical catalysts due to their rich (1 0 0) faces.
For example, Jiang Yang et al. developed an amperometric non-
enzymatic glucose sensor by electrodepositing copper nanocubes
onto vertically well-aligned multi-walled carbon nanotube arrays

0925-4005/$ – see front matter © 2014 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.snb.2013.12.074
72 P. Yang et al. / Sensors and Actuators B 194 (2014) 71–78
[25]; Juan Ren et al. reported an amperometric glucose biosen-
sor based on platinum nanocubes to enhance the electrocatalytic
activity of the reduction and oxidation of H2 O2 [13]. Jonathan
C. Claussen et al. employed the networks of single-walled car-
bon nanotubes (SWCNTs) decorated with Au-coated Pd (Au/Pd)
nanocubes as electrochemical biosensors that exhibited excellent
sensitivity [26]. However, in the case of Ag nanocubes, much of the
research has focused on using them as templates in the produc-
tion of Au nanocages [27], Au nanoframes [28] and the plasmonic
substrate [29], and relatively little has directed toward the sensor
applications [30–32].
Since the first report on amperometric glucose biosensors [33],
monolayer and multilayer competition between oxygen and an
oxidized mediator for the regeneration of the oxidase [34]. Bien-
zyme biosensor systems showed “unusual amperometric response”
[35–37]. In our research, hydrogen peroxide produced by the Gox
is subsequently reduced by the HRP which was then reduced
through media AgNCs at the electrode at low applied potentials.
The main advantage of the Gox-HRP bienzyme glucose biosensor
is that cascade schemes, where an enzyme is catalytically linked
to another enzyme, may produce signal amplification and there-
fore enhance the sensitivity of the biosensor [38]. In addition, the
removal of H2 O2 could reduce peroxide-induced degradation of the
Gox enzyme [39,40].
In the present work, a bienzyme amperometric glucose biosen-
sor was developed with both Gox and HRP enzymes coimmobilized
in CS film coated on AgNCs modified Au electrode. The bienzyme
electrode has been characterized by electrochemical methods and
offered highly sensitive amperometric response to glucose at low
potential. By a combination of AgNCs, Gox, HRP and CS, an electro-
analytical biocomposite electrode was produced by simple casting.
CS immobilized AgNCs acts as an efficient conduit for electron
transfer, while Gox and HRP act as effective biological catalysts.
A wider linear range, a lower detection limit and fast response of
the Gox–CS–HRP/AgNCs–CS modified Au implied that the proposed
method provided an excellent platform for sensitive electrochem-
ical sensing.
2. Experimental
2.1. Reagents
HRP (EC 1.11.1.7, 250 U mg−1 ) was purchased from Shanghai
Sanjie Biotechnology. Gox (EC 1.1.3.4, 200 U mg−1 ) was purchased
from Sigma. Sliver nitrate (AgNO3 ), poly(vinyl phrrolidone) (PVP,
K value: 30) and HCl were purchased from Sinopharm Chemi-
cal Reagent Co., Ltd. (Shanghai, China). Anhydrous ethylene glycol
(EG) was obtained from Wuxi Haishuo Biology Co., Ltd. (Wuxi,
China). Chitosan (CS, MW 480000, 92% deacetylation) was pur-
chased from Golden-shell Biochemical Co., Ltd. (Zhejiang, China).
Phosphate buffered saline (PBS, 0.02 M) at various pHs were pre-
pared with 0.2 M NaH2 PO4 and 0.2 M Na2 HPO4 . All other reagents
were of analytical grade and used without further purification.
2.2. Instruments
Electrochemical measurements were performed on a CHI
650 electrochemical analyzer (Shanghai CH Instrument). A con-
ventional three-electrode system was used with modified Au
electrode as the working electrode, saturated calomel electrode
as the reference electrode, and platinum (Pt) disk electrode as the
auxiliary electrode. Scanning electron microscopy (SEM) images
were obtained on a JEOL (TSM-5510 < V) field emission scanning
electron microscope at 5.0 kV. Transmission electron microscopy
(TEM) images were performed using an interface high-resolution
transmission electron microscopy. The TEM images were taken
using a HT-7700 transmission electron microscope at an accelerat-
ing voltage of 120 kV. Atomic force microscopy (AFM) images were
obtained using Nanascopy IV A system (Digital Instruments, Inc.)
in the tapping mode, and the samples were prepared on the silicon
substrate. The X-ray diffraction (XRD) measurement was per-
formed on an X-ray powder diffractometer (Bruker D8 Advance)
with Cu K␣ radiation. The sample for XRD characterization was
prepared by placing 20 ␮L of the suspension on a glass slide.
2.3. Preparation of AgNCs
AgNCs were synthesized based on a previous method [41]. In a
typical synthesis, 5 mL EG was added into a 20 mL round-bottom
flask equipped with a water-cooling condenser, followed by heat-
ing in an oil bath at 140 ◦ C under vigorous magnetic stirring for
1 h. 1 mL of 3 mM HCl solution in EG was then quickly added. After
10 min, 3 mL EG containing 94 mM AgNO3 solution and 147 mM
poly(vinyl pyrrolidone) (PVP) was added at a rate of 45 mL per hour
to the stirring solution simultaneously. Upon injection of the solu-
tion, the reaction mixture went through a series of color changes
that included milky white, light yellow, transparent, red, and ocher.
Then the solution was maintained at 140 ◦ C for another 26 h. The
resulting dispersion was cooled in an ice-water bath and then
washed by acetone. After collecting by centrifugation and washing
with ethanol three times, the AgNCs were dispersed in 5 ml ethanol
for further use.
2.4. Construction of AgNCs-bienzyme electrodes
Before modification, bare Au electrode was successively pol-
ished with emery paper and 0.05 ␮m ␣-Al2 O3 slurry. After being
sonicated in deionized water for 5 min, the electrode was immersed
in freshly prepared Piranha solution (30% H2 O2 and concentrated
H2 SO4 , 3:1, v/v) for 20 min. After ultrasonic rinsing, the elec-
trode was electrochemically pretreated by cyclic potential scanning
between 1.4 and −0.2 V in 0.1 M H2 SO4 until a cyclic voltam-
mogram of clean Au electrode was obtained, and then dried in
air.
AgNCs ethanol dispersion liquid was centrifuged for 5 min at
4000 rpm with the supernatant discarded, followed by addition of
5 mL CS solution (0.0625 wt%, pH 5.0) and sonicated for 20 min to
obtain a homogeneous dispersion. 3 ␮L of AgNCs suspension was
dropped onto the surface of the electrode film. The CS–AgNCs/Au
was obtained after drying at room temperature. Then we prepared
Gox–CS–HRP bienzyme solution as following: mix Gox solution
(8 mg mL−1 in 0.02 M pH 7.4 phosphate buffer), the chitosan solu-
tion (0.5 wt%, pH 5.0) and HRP solution (168 mg mL−1 in 0.02 M pH
7.4 phosphate buffer) at a 2:1:1 (v/v) ratio, namely, the mixture
consisted of 4 mg mL−1 Gox, 0.125 0.25 wt% CS and 4 mg mL−1 HRP.
Finally the HRP–CS–Gox/CS–AgNCs/Au was prepared with 5 ␮L of
the bienzyme mixture casted onto the pretreated CS–AgNCs/Au
electrode successively.
2.5. Electrochemical measurements
The electrochemical measurements were performed in an
electrochemical cell at the room temperature of 25 ◦ C. Cyclic
voltammograms (CVs) were recorded in 0.02 M PBS (pH 7.5) from 0
to −0.6 V (versus SCE) at a scan rate of 100 mV/s. For amperometric
detection, all measurements were performed at −0.15 V versus sat-
urated calomel electrode (SCE), allowing the transient background
current to decay to a steady-state value before the addition of the
glucose. A stirred solution was employed to provide convective
transport.
 P. Yang et al. / Sensors and Actuators B 194 (2014) 71–78
Fig. 1. (A) TEM and SEM (the inset) image of AgNCs (B) XRD pattern of the AgNCs.
3. Results and discussion
3.1. Characterization of bienzyme–AgNCs biocomposite
The formation of the AgNCs was firstly confirmed by SEM and
TEM observations. Fig. 1A and the inset of it showed the represen-
tative TEM and SEM images of the as-prepared AgNCs, respectively.
We could clearly observe that Ag products mainly contained small
nanocubes with an average edge length of about 100 nm. Moreover,
Fig. 2. AFM images of (A) AgNCs, (B) CS/AgNCs film, (C) Gox–CS–HRP/AgNCs–CS film.
73
all these particles are well separated from each other, suggesting
good dispersibility. Fig. 1B shows the X-ray diffraction pattern of
the prepared AgNCs. All peaks can be indexed to diffraction from
the (1 1 1), (2 0 0), and (2 2 0) planes of face-centered cubic sil-
ver. The ratio of the intensity of the (2 0 0) to (1 1 1) diffraction
peaks was higher than the conventional value (1.09 versus 0.4,
respectively), indicating that as-prepared silver nanocubes were
abundant in {1 0 0} facets, and thus their {1 0 0} planes tended to
be preferentially oriented parallel to the supporting substrate [41].
AFM were used to monitor the procedure of modified elec-
trode. The surface roughness of AgNCs film, AgNCs–CS film, and
GOx–CS–HRP/AgNCs film were visualized by AFM in tapping mode
as shown in Fig. 2. Each AFM image was obtained using an area of
5 ␮m × 5 ␮m. The data of the root mean square roughness (RMS)
were 25.078, 69.934 and 40.564 nm, respectively. They are much
more than the RMS of the silicon substrate (about 0.617 nm).
Cubic structures with an ordered array can be found in Fig. 2A,
indicating the existence of AgNCs. After CS was mixed with the
AgNCs as shown in Fig. 2B, a rougher configuration was presented.
Cube-like nanostructures became mistiness and the surface had
many cavities. This might the reason that AgNCs were surrounded
by CS, forming larger particles, and producing larger cavities. In
Fig. 2C, cavities disappeared and the surface morphology became
smoother, which might be ascribed to Gox–HRP filling the inter-
stitial places between CS–AgNC and CS–AgNC, suggesting that the
Gox–HRP was effectively immobilized on the surface of AgNCs–CS
film.
3.2. Cyclic voltammetric characterization of the glucose biosensor
The working mechanism of the Gox–HRP bienzyme electrodes
studied in this work is depicted in Scheme 1. The biocatalytic
scheme used for the determination of glucose involved the reduc-
tion of H2 O2 , generated in the enzyme reaction with Gox, and
regeneration of HRP with AgNCs which is a mediator. With Gox
and HRP coimmobilized with AgNCs, the reaction is given below.
Gox
Glucose + O2 −→Gluconic acid + H2 O2
HRP
H2 O2 + 2H+ + Ag−→2H2 O2 + Ag+
Ag+ + e− → Ag
Cyclic voltammetric (CV) experiment was used to evaluate
the electrochemical performance of the electrodes. The cyclic
voltammograms (CVs) of different modified electrodes were
shown in Fig. 3. Fig. 3A compared the cyclic voltammograms of
HRP–CS/AgNCs–CS/Au without participation of Gox in the absence
(a) and presence (b) of 0.4 mM glucose, no obvious difference was
observed. It clearly confirmed the important catalytic role of Gox
74 P. Yang et al. / Sensors and Actuators B 194 (2014) 71–78

Scheme 1. Reaction occurring at the Gox–CS–HRP/AgNCs–CS/Au biosensor.

in the oxidation process of modified electrode and HRP had no
catalysis for glucose.
As shown in Fig. 3B, no obvious peak was observed at bare Au
electrode (curve a), CS/Au (curve b) and Gox–CS–HRP/Au (curve
c), indicating no faraday responses at bare Au electrode, CS/Au
and Gox–CS–HRP/Au in the investigated potential window. When
AgNCs were embedded into the CS membrane, an obvious peak
was observed (curve e and f). The presence of peak is an oxidation
peak of Ag(0)/Ag(I) oxide transitions [31]. The reduction peak of
H2 O2 is so approximate to the oxide peak of AgNCs that they are
overlapped. Compared curve f with curve e, the reduction peak cur-
rent increased with the addition of 0.4 mM glucose. The increased
current (from curve e to f) is higher than the current (from curve
c to d) with the same concentration of glucose. It suggested that
AgNCs could improve the charge transport of the composite film
and lead to an increasing direct electron transfer signal of Gox.
The phenomenon indicated that a catalytic reaction occurred on
the biosensor. These results demonstrated that the response of the
biosensor to glucose only resulted from the catalytic activity of
bienzymes immobilized in the biocomposite film. Therefore, this

15

A b
12

a
9
Current/µA

-3

-0.6 -0.5 -0.4 -0.3 -0.2 -0.1 0.0

Potential/V
15

B
12

9 f

6
Current/µA

3 a

-3

-6

-0.7 -0.6 -0.5 -0.4 -0.3 -0.2 -0.1 0.0

Time/s

Fig. 3. (A) Cyclic voltalmmograms of HRP–CS/AgNCs–CS/Au in the 0.02 M pH 7.5 PBS at 100 mV/s in the (a) absence and (b) presence of glucose. (B) Cyclic voltalmmograms
in 0.02 M PBS (pH 7.5) at 100 mV/s of (a) bare Au, (b) CS/Au, (c) Gox–CS–HRP/Au without glucose, (d) Gox–CS–HRP/Au with 0.4 mM glucose, (e) Gox–CS–HRP/AgNCs–CS/Au
without glucose and (f) Gox–CS–HRP/AgNCs–CS/Au containing 0.4 mM glucose.
 P. Yang et al. / Sensors and Actuators B 194 (2014) 71–78 75

0.6
A 0.6
C
0.5

0.4 0.5
Currrent/mA

Current/µA
0.3

0.4
0.2

0.1
0.3

0.0
6.0 6.5 7.0 7.5 8.0 8.5 1 2 3 4 5
pH VAgNCs/µL

0.6
B 0.6
D

0.4
0.5
Current/µA

Current/µA

0.4 0.2

0.3 0.0
-0.20 -0.15 -0.10 -0.05 0 2 4 6 8
Potential/V CGox/mg mL
-1

Fig. 4. Influence of (A) applied potential, (B) pH of PBS, (C) amount of AgNCs and (D) concentration of Gox on the current response to successive addition of 0.1 mM glucose.

modified electrode could be employed to determine the glucose
concentration.
3.3. Optimization of experimental conditions
The influence of the buffer pH was very essential to the sensi-
tivity of the biosensors. It was well known that a strong acidic or
basic solution would decrease the bioactivity of Gox, leading to an
obvious decrease in response current. Thus, a range of pH values
from 6.0 to 8.5 was studied (Fig. 4A). The result revealed that the
current response for 0.1 mM glucose reached the maximum when
the pH of PBS was 7.5.
It is well known that the applied potential can affect the sensi-
tivity and selectivity of electrochemical sensors. Fig. 4B displayed
the effect of applied potential on the electrocatalytic oxidation of
glucose on the Gox–CS–HRP/AgNCs–CS/Au. The current responses
were recorded after successive addition of 0.1 mM glucose in a
stirred PBS (0.02 M, pH 7.4) with different applied potentials, and
the results showed that the current response was highest when the
potential was −0.15 V. Thus −0.15 V is selected in the subsequent
experiments as working potential.
The effect of the amount of AgNCs on the current response was
demonstrated in Fig. 4C. It could be found that the current response
increased initially with the amount of AgNCs and then tended to
level off. The AgNCs would more easily fall off from the electrode
surface when more suspension was cast. So 3 ␮L of AgNCs suspen-
sion was selected for the fabrication of the sensor.
Further studies were performed to investigate the depend-
ence of the biosensor response on the concentration of Gox. As
expected, with the increase of the concentration from 0.5 mg mL−1
to 4 mg mL−1 , the electrochemical response was increased as
shown in Fig. 4D. Further increase of the concentration of Gox can-
not improve the electrochemical response continuously. Thus an
optimal concentration of 4 mg mL−1 was selected. According to a
previous report that the optimum ratio of HRP and Gox on the bien-
zyme electrode was 1:1 [38], the concentration of HRP was selected
as 4 mg mL−1 .
3.4. Calibration curve of the modified electrode
The amperometric response of the Gox–CS–HRP/AgNCs–CS/Au
modified electrode was given in Fig. 5A on successive injection of
0.01 mM and 0.05 mM glucose into the stirring 0.02 M PBS (pH 7.5)
at an applied potential of −0.15 V. An obvious increase in the reduc-
tion current upon successive addition of glucose occurred, and
a well-defined amperometric response was obtained within the
response time less than 5 s. These results implied that the amper-
ometric sensor had a rapid and sensitive response to the change of
glucose concentration. The amperometric response showed a lin-
ear relation with glucose concentration from 10 ␮M to 1.5 mM with
a correlation coefficient of 0.9997 (curve a in Fig. 5C). The detection
limit was estimated to be 0.6977 ␮M (S/N = 3). The glucose biosen-
sor showed high sensitivity, low detection limit, fast response,
which could be attributed to good electrocatalytic activity, high
76 P. Yang et al. / Sensors and Actuators B 194 (2014) 71–78

5
10
A B

8
4

Current/µA
Current/µA

50µM
3
4 10µM 50µM

2
2
200 400 600 800 100 200 300 400 500

Time/s Time/s

12

C
10

a
8
Current/µA

6
b

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4

Concentration/mM

Fig. 5. Typical amperometric response of the fabricated sensor (A) Gox–CS–HRP/AgNCs–CS/Au and (B) Gox–CS/AgNCs–CS/Au to successive addition of 0.4 mM glucose in a
stirred PBS (0.02 M, pH 7.5) at the applied potential of −0.15 V. (C) Calibration curve of (a) Gox–CS–HRP/AgNCs–CS/Au and (b) Gox–CS/AgNCs–CS/Au between the current
response and the concentration of 0.4 mM glucose.

conductivity, and large surface-to-volume ratio of AgNCs. In order
to demonstrate the better behavior of the bienzyme biosensor, the
amperometric response of Gox–CS/AgNCs–CS/Au modified elec-
trode was also researched on successive injection of 0.05 mM
glucose at same condition, which was showed in the Fig. 5B. By con-
trast, the bienzyme biosensor had a higher current value and wider
detection range. Moreover, compared with some reported glucose
sensors (Table 1), Gox–CS–HRP/AgNCs–CS/Au was more sensitive
with wider linear range simultaneously.
interfering species were examined. Herein, the current responses of
the Gox–CS–HRP/AgNCs–CS/Au to successive addition of 0.42 ␮M
urea (UA), 0.2 mM galactose (Gal), 4 ␮M ascorbic acid (AA), 0.1 mM
phenylalanine (Phe),6.6 ␮M tyrosine (Tyr), and 0.1 mM glucose
(Glu) were displayed in Fig. 6. Compared with well-defined
responses obtained upon the addition of Glu, there was a slightly
signal observed toward Gal and AA and no obvious signal toward
other interferences. The results indicated that the proposed sensor
might be used for glucose determination in serum or uric samples.

3.5. Interference study of the modified electrode 3.6. Glucose determination in serum samples

Anti-interference ability is one of the important indicators To demonstrate the practical use of the glucose biosen-
whether an electrochemical sensor is practical, so some common sor, human serum samples were assayed. The recovery was

Table 1
Comparison of performances of different electrochemical biosensors for determination of glucose.

Electrode Linear range (mM) Detection limit (␮M) Refs.

GOD/In2 O3 /GCE 0.005–1.3 1.9 [42]

GOD/SnS2 /Nafion/GCE 0.025–1.1 10.0 [43]
Nafion/GOD/Ag-Pdopa @CNT/GCE 0.05–1.1 17.0 [44]
GOD–CS/AgNWs/GCE 0.01–0.8 2.83 [18]
GOD/AuNPs–SnS2 –CS/GCE 0.02–1.32 1.0 [45]
GOD–CS–HRP/AgNTs/GCE 0.01–1.5 0.6977 This work
a
Polydopamine.
 P. Yang et al. / Sensors and Actuators B 194 (2014) 71–78 77

3.5 Furthermore, the glucose biosensor revealed excellent selectivity

as no interference from various amino acid and AA were detected,
Glu and the biosenser could be used for the determination of glucose
3.0 in serum.

Glu Acknowledgement
Current/µA

2.5

This research was supported by the National Natural Science

Glu Foundation of China (Nos. 30800247 and 20805043).
2.0

UA Gal AA Phe Tyr

1.5
50 100 150 200
Time/s
Fig. 6. Interference test protocols of the glucose biosensor to the interfering species
in the presence of glucose.
Table 2
Recovery studies of glucose in real samples.
Sample Added (mM) Founded (mM)
1 0.1 0.109
2 0.3 0.319
3 0.6 0.548
4 0.8 0.791
5 1.2 1.27
6 1.3 1.281
investigated by standard additions of glucose to the serum sam-
ple in the concentration range from 0.1 to 1.3 mM (Table 2). The
samples were diluted 1000 times before determination. The val-
ues of the recovery were in the range of 91.3–109.0%. These results
indicate that the biosensor can be used to determine glucose in
serum.
3.7. Stability of developed bienzyme biosensor
The stability of the bienzyme biosensor was evaluated by mon-
itoring the response currents in the presence of 0.1 mM glucose
over 8 days. The electrode was stored at 4 ◦ C in a refrigerator when
not used. The biosensor retained approximately 93% of its origi-
nal response after storage for 9 days, and about 70% response after
two weeks. The reproducibility and storage stability of the pro-
posed biosensor also were studied. The relative standard deviation
(RSD) of the biosensor response to 0.1 mM glucose was 1.27% for
six successive measurements. The fabrication reproducibility was
investigated by preparing four biosensors independently. Repro-
ducibility with an RSD of 2.14% for the detection of 0.1 mM glucose
was obtained.
4. Conclusion
In this study, an amperometric bienzyme glucose biosensor
was developed by co-immobilization HRP and Gox on the AgNCs
with CS as a linker. Electrochemical and amperometric responses
of this bienzyme electrode for glucose detection were examined
in detail. The results indicated that AgNCs played an important
role in the enhanced electron transfer between the immobilized
Gox–CS–HRP and the surface of electrode, which are attributed to
large surface-to-volume ratio and high conductivity of AgNCs. The
bienzyme biosensor had a higher current value and wider detec-
tion range because the catalytic cycle of HRP was driven forward
and gave an amperometric transducer for the detection of glucose.
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Biographies
Penghao Yang is currently a M.Sc. student of Zhejiang University, China, majoring
in analytical chemistry. Her interested fields mainly surround the electrochemical
activation on biosensors and their applications in sensing of bio-active molecules.
Lisha Wang is currently a M.Sc. student of Zhejiang University, China, majoring in
analytical chemistry. Her interests include construction and application of enzyme-
sensor.
Qi Wu is an associate professor of chemistry, Department of Chemistry, Zhejiang
University. He obtained his Ph.D. from Zhejiang University in 2003. His research
interests cover organic synthesis and bioelectrochemistry.
Zhichun Chen received his Ph.D. in Department of Chemistry from Zhejiang Uni-
versity, China in 2008. His research interests cover biosensors and Raman spectrum.
Xianfu Lin was born in 1963. He received his Ph.D. in chemistry from Zhejiang
University, China, in 1997. He has been working as a professor since 1998 and
vice-chairman of Department of Chemistry, Zhejiang University since 2006. He has
published 200 papers and two scientific books. His research interests cover enzy-
matic synthesis, chemical biology, bioelectrochemistry and sensors.