In situ growth of metallic silver on glucose oxidase for a highly sensitive glucose sensor

Article abstract of DOI:10.1039/c5ra00134j

This work presents a new idea to fabricate an enzyme glucose sensor. A bio-composite was assembled by in situ reducing Ag⁺ to Ag in glucose oxidase (GOD) solution. In this way, metallic silver can directly deposit onto the GOD surface and induce tight contact between Ag and GOD. The obtained composites were characterized by FESEM, EDS mapping, FTIR, CD, and electrochemical measurements. The Ag-GOD composite formed by an in situ reducing process exhibits facile, direct electrochemistry and good electrocatalytic performance without any electron mediator. The designed glucose biosensor shows high sensitivity, high selectivity, long life and accurate measurement in real serum samples, which is contributed to by a fast, direct electron transfer due to close proximity between Ag and GOD.

Full text of DOI:10.1039/c5ra00134j

RSC Advances
View Article Online
View Journal | View Issue
PAPER
In situ growth of metallic silver on glucose oxidase
for a highly sensitive glucose sensor
Cite this: RSC Adv., 2015, 5, 34486
*
Qiang-Qiang Sun, Ya-Nan Yu, Jin-Jin Li and Shu-Juan Bao
This work presents a new idea to fabricate an enzyme glucose sensor. A bio-composite was assembled by
in situ reducing Ag+ to Ag in glucose oxidase (GOD) solution. In this way, metallic silver can directly deposit
onto the GOD surface and induce tight contact between Ag and GOD. The obtained composites were
characterized by FESEM, EDS mapping, FTIR, CD, and electrochemical measurements. The Ag–GOD
composite formed by an in situ reducing process exhibits facile, direct electrochemistry and good
electrocatalytic performance without any electron mediator. The designed glucose biosensor shows
high sensitivity, high selectivity, long life and accurate measurement in real serum samples, which is
contributed to by a fast, direct electron transfer due to close proximity between Ag and GOD.
Received 5th January 2015
Accepted 9th April 2015
DOI: 10.1039/c5ra00134j
www.rsc.org/advances
cross-linking method of an immobilized enzyme is easy to
operate and has good stability. However, this method leads to
Introduction
reduced electrocatalytic activity of the enzyme and long
response time. Encapsulation is commonly used to immobilize
an enzyme while also improving its stability. This method is
easy to control and can be used repeatedly, but the enzyme has a
long response. The enzyme immobilized by covalent-bond
combination method has a quick response and nice stability;
unfortunately, this method weakens the enzyme's activity and
has a complex operation. Although some of these nano-
materials and immobilized method could help enzyme-
induced, direct electrochemistry reactions, there is still great
challenge in developing new ways for better activity, specicity,
and stability for super bioelectrocatalysis.
Here, we report on a new, simple in situ growth method to
realize effective immobilization of GOD. In our work, silver ions
are reduced to metallic silver and in situ deposited on the
surface of GOD to form Ag–GOD composite. For comparison,
the Ag and GOD composite was also prepared by the conven-
tional direct adsorption method. The results indicate that the
sensor prepared based on the former composite has a higher
sensitivity, quicker response, and better stability. More impor-
tantly, this approach reported here is a new method to immo-
bilize bio-enzyme for sensing. The nanomaterial which could be
reduced not only silver but other metals on the bio-enzyme. It
should be noted that the reaction condition must be moderate
enough to ensure enzymes active.
Glucose detection is very important in many types of studies
from food processing and fermentation to clinical, biological,
and chemical studies.1–4 Techniques including uorescence,
colorimetric, ow injection, and electrochemistry analysis have
been developed for this purpose.5–8 Among these detection
techniques, amperometric biosensors, which are based on
direct electron transfer (DET) between the electrode and glucose
oxidase (GOD), have attracted much attention due to their high
sensitivity, excellent selectivity, wide test range, and easy oper-
ation.9 Unfortunately, deeply buried redox centers in enzymes
and instability of the biological matrix upon interaction with
electrode surfaces make direct electrochemistry of most redox
enzymes on bare or “naked” solid electrodes very difficult. The
key for designing a high performance biosensor is to under-
stand how to efficiently transfer an electron directly between the
electrode and enzyme.
In the past few decades, many works on producing such a
biosensor have been performed. Nanomaterials like gold,
various carbon materials, titanium dioxide, and other conduc-
tors have been introduced to initiate direct electron transfer.10–13
Various morphologies of these materials, including nanowire,
nanorods, hollow nanospheres, nanolm, and nanoparticles
were used to enhance the electro-catalytic performance of
enzymes, which were restricted due to their small size, high
specic area, and appropriate pore size distribution.14–18
However, since GOD is freely soluble in water, enzymes cannot
be immobilized on nanomaterial simply by an adsorption
process. Hence, various methods have been developed to
effectively immobilize GOD on test electrodes. For example, the
Experimental
Reagents
The glucose oxidase, ascorbic acid (AA), dopamine (DA) and uric
acid (UA) were purchased from Sigma Chemical Co. Fetal
bovine serum as a real sample is obtained from Gibco,
Institute for Clean Energy & Advanced Materials, Faculty of Material & Energy,
Southwest University, Chongqing, 400715, P. R. China. E-mail: baoshj@swu.edu.cn
34486 | RSC Adv., 2015, 5, 34486–34490
This journal is © The Royal Society of Chemistry 2015

View Article Online
Paper
RSC Advances
Invitrogen, USA. AgNO3 is from Fluka Co., D-maltose and
D-glucose were purchased from KeLong Chemical Reagent Co.,
(Chengdu, China). Ammonium hydroxide purchased from
ChuanDong Chemical Reagent Co., (Chongqing, China). They
were used as received, without further purication. 0.1 M
phosphate buffer solutions (PBS) consisted of Na2HPO4 and
NaH2PO4, were employed as supporting electrolyte. The desired
solution pH (7.4) was adjusted by different amount of 0.2 M
Na2HPO4 and NaH2PO4 solutions. Deionized water was used
throughout.
Fig. 1 FSEM images of bio-composites obtained by in situ growth
(A) and conventional direct adsorption method (B).
The preparation of the materials
Initially, 0.01 M AgNO3 and maltose solution were prepared
followed by dissolving 2 mg of GOD into 100 mL of maltose
solution containing 3 mL of 3% ammonium hydroxide. Aer
mixing uniformly, 10 mL of prepared AgNO3 was added into the
miscible liquids above. Aer that, the mixture was shaken a few
minutes and put in 4 ꢀC refrigerator for 24 h. This sample is
named as Ag–GOD. For comparison, metallic silver was rst
synthesized by the same method but without GOD at room
temperature. Next, the prepared silver was mixed with GOD and
stored in 4 ꢀC refrigerator for another 24 h. The mixture is
abbreviated as Ag/GOD. Ag–GOD composites with different
amount of Ag were synthesized by change the amount of AgNO3.
is about 1 mm. Ag/GOD composites (B) consist of nanoparticles
and some ocs. The diameter of the nanoparticles is far smaller
than that of Ag–GOD, around 100 nm. There are several reasons
why the differences between these two samples are so distinc-
tive. It is well known that GOD is a dimeric protein, which is
covered with carbohydrate chains.19 In the reduction process,
positive silver ions can rst be adsorbed onto GOD molecules
and then are in situ reduced by maltose. Thus, Ag deposits
directly on the surface of GOD and tightly bond with GOD to
form Ag–GOD aggregations, in which Ag particles can contact
with GOD closely. For Ag/GOD, GOD is simply mixed with Ag
nanoparticles.
Characterization of the materials
In order to prove silver has in situ grown onto GOD
successfully in the synthesis process, Ag, C, and N element
distributions have been investigated by EDS mapping. As seen
in Fig. 2, Ag–GOD samples (le part) have a similar graphic
The morphologies of bioconjugates were characterized by
FESEM (S-4800, Japan). The element distribution is measured
by EDS Mapping. FTIR was used for analyzing the change of
functional groups. The Circular dichroism (CD) spectroscopy
was taken using a biologic MOS500 spectropolarimeter. Quartz
cells of 0.5 mm optical path length were used for all CD
measurements of GOD (GOD concentration ¼ 1 mg mLꢁ1) and
Ag–GOD solutions. Deionized water was used to prepare these
solutions.
Fabrication of working electrode
Glass carbon electrodes (GCEs) with a diameter of 3 mm were
polished with 0.3 and 0.05 mm alumina powder, followed by
thorough rinsing with deionized water. The electrodes were
then dried at room temperature, and 5 mL of bioconjugates were
dropped onto the center of GCEs. The latter was le to dry at
room temperature, aer which 5 mL of 2.5% Naon was
subsequently placed on the electrode surface to form a Naon
membrane, which was used to x the GOD-impregnated Ag
materials. The electrode fabricated with Ag–GOD is expressed as
Ag–GOD/Naon, while the electrode made by sample Ag/GOD is
marked as Ag/GOD/Naon.
Results and discussion
The surface morphologies of the Ag and GOD composites
obtained by different method have been investigated by FESEM.
It is clear that they have different morphologies as shown in
Fig. 1. Samples of Ag–GOD (A) contain some aggregations of
Fig. 2 Electronic images and elements distributions of the obtained
nanoplates and nanoparticles. The diameter of the aggregations sample. (Ag–GOD (left) and Ag/GOD (right)).
This journal is © The Royal Society of Chemistry 2015
RSC Adv., 2015, 5, 34486–34490 | 34487

View Article Online
RSC Advances
Paper
pattern just like the electronic image; that is to say, Ag, C and N
The fabricated electrodes are employed as biosensor for the
have a same distribution. Because GOD contains C and N glucose sensing in order to compare the bioactivity of the GOD
elements, we can infer that Ag has successful in situ growth on immobilized by different ways. The cyclic voltammograms (CVs)
the surface of GOD. While only Ag distribution is similar with of the Ag–GOD electrode (a) and Ag/GOD electrode (b) are
the electronic image in the right side, we can conclude that GOD shown in Fig. 4A. It is apparent that the Ag–GOD electrode
does not adsorb on silver well in the conventional direct exhibits a pair of obvious symmetric peaks located at about
adsorption process.
ꢁ0.45 V and ꢁ0.5 V. This indicates that the electro-catalytic
The in situ growth method can ensure that the enzyme and performance of GOD had been improved with help of Ag,
material are combined closely. FTIR and electrochemical while the Ag/GOD electrode displays similar behaviour but has
measurements are further performed to estimate the effective- weak redox peaks. It is concluded that direct electron transfer of
ness of GOD immobilization in different methods. In Fig. 3A, GOD on the Ag–GOD electrode is more effective than that of the
two IR absorption bands from a are centered at 1650 and 1550 Ag/GOD electrode. In the in situ growth process, silver deposits
cmꢁ1 that are ascribed to the typical amide I and amide II on the surface of GOD directly and tightly bonds, which is not
adsorption bands of GOD, respectively.20 In sample Ag/GOD (c), only benecial for direct electron transfer but could protect
the relative intensity of IR absorption of amide I and amide II
band remains constant, while in the sample (b), a slight red
shi with respect to the amide peak positions of GOD is
observed. This shi may come from the force change among
amines, OH groups and silver, which caused by electrostatic
interaction in the reaction process due to their different elec-
trical property. Further red shi also means the system has
lower energy,21 which indicate that the combination of Ag and
GOD in situ reaction is more stable than that of simple
adsorption process. CD spectroscopy was further used to char-
acterize the fresh GOD and Ag–GOD colloid. The CD spectrum
in Far-UV region (200–250 nm) is usually used to determine the
secondary structure change of protein. As shown in Fig. 3B, the
CD spectra exhibited two negative bands at 208 and 222 nm,
which is representative of the a-helix structure of protein. The
band at 208 nm corresponds to p / p* transition of the a-helix,
whereas the band at 222 nm corresponds to p / p* transition
for both the a-helix and random coil. The results displayed in
Fig. 3B indicated that the structure of native GOD predomi-
nately contain a-helix conformation.22 From 200 to 250 nm, the
CD spectra of the fresh GOD and Ag–GOD colloids are almost
similar, but he peak has slightly changed at about 205 nm. It
indicated that the GOD have been adsorbed on the surface of
silver nanoparticles.23 Only when the state of GOD have a little
transformed this change can happen. And the small trans-
formation should be caused by the combination with Ag
nanoparticle. Actually, the similar secondary structure content
of GOD and Ag–GOD suggested that the secondary structure of
GOD was preserved in the presence of Ag nanoparticles.23,24
Fig. 4 (A) CV comparison of Ag–GOD/Nafion (a) and Ag/GOD/Nafion
(b) electrode in N2-saturated pH 7.4 PBS. (B) CV comparison of Ag–
GOD/Nafion electrode in N2-saturated (a) and air-saturated (b) pH 7.4
PBS. (C) The CV of Ag–GOD/Nafion electrode in air-saturated PBS
without glucose (a) and with 2 mM glucose (b). (D) The CVs with an
increase of 0.5 mM glucose for each time. (E) Amperometric response
of Ag–GOD/Nafion electrode with successive addition 0.5 mM
glucose at the potential of ꢁ0.45 V, inset is the corresponding linear
relation. (F) Amperometric response of Ag/GOD/Nafion electrode with
Fig. 3 (A) Infrared spectrograms comparison of GOD (a), sample successive addition 0.5 mM glucose at the potential of ꢁ0.45 V. (G) (H)
Ag–GOD (b) and sample Ag/GOD (c). (B) Circular dichroism (CD) Amperometric response of Ag–GOD/Nafion and Ag/GOD/Nafion
spectroscopy of Ag–GOD nano-composites (b) and fresh pure electrode with successive addition 0.5 mM glucose, 0.05 mM AA, DA
GOD (a).
and UA at the potential of ꢁ0.45 V.
34488 | RSC Adv., 2015, 5, 34486–34490
This journal is © The Royal Society of Chemistry 2015

View Article Online
Paper
RSC Advances
GOD from dissolving into an electrolyte during the test process. the surface of GOD. In the in situ preparation process, more
Cyclic voltammograms of the Ag–GOD electrode in N2-saturated GOD was covered by Ag nanoparticles effectively and greatly
(a) and air-saturated (b) PBS are presented in Fig. 4B. It is increased stability. In simple mixing of Ag and GOD, more GOD
evident that the oxidation current increases and reduction were absorbed on the surface of Ag particles, which let the
current decreases in air-saturated PBS compared to the results modied electrode deliver higher sensitivity and lead to GOD
of N2-saturated PBS, suggesting that the Ag–GOD xed on the gradually dissolving into PBS solution and decreased stability.
electrode can electrocatalyze the reduction of dissolved oxygen.
The selectivity of Ag–GOD and Ag/GOD electrodes are eval-
The electrode modied with GOD and nanomaterials can elec- uated in the presence of common interfering species such as
trocatalyze the reduction of dissolved oxygen according to ascorbic acid, dopamine, and uric acid in air-saturated PBS. As
following equations.25 The direct electrochemistry of GOD on shown in Fig. 4G and H, for each addition of 0.5 mM glucose the
the electrode involves a two-proton redox reaction as eqn (1):
biosensors show quick response. However, no noteworthy
response was observed for the addition of 0.05 mM of ascorbic
acid, dopamine, and uric acid. Thus, Ag–GOD and Ag/GOD
electrodes are highly selective for glucose due to the low test
potential.
The long-term stability of the two kinds of electrodes is
investigated by recording the current response to 0.5 mM
glucose. The current response of the Ag–GOD electrode
remained about 94% of the initial response current aer two
weeks, while the Ag/GOD electrode only kept about 72%. The
good stability of the Ag–GOD based biosensor may be attributed
to the unique in situ immobilization method.
GOD(FAD) + 2eꢁ + 2H+ # GOD(FADH2)
(1)
GOD (FADH2) further electro-catalyze the reduction of dis-
solved oxygen according to eqn (2) as shown in Fig. 4B.
GOD(FADH2) + O2 # GOD(FAD) + H2O2
(2)
In the presence of glucose, the electrocatalytic reaction is
restrained due to the enzyme-catalyzed reaction:
The effect of silver amount in Ag–GOD composites on their
electro-catalytic performance has also been observed. As shown
Glucose + GOD(FAD) / gluconolactone + GOD(FADH2) (3)
Hence, upon addition of glucose to an air-saturated PBS in Fig. 5, with the increasing of Ag content in Ag–GOD
solution, the cathodic peak current decreases because of the composites, the intensity of redox peaks increased. But when
enzymatic reaction between the oxidized form of GOD and the amount of Ag in Ag–GOD composites further increased, its
glucose.
redox peaks current cut down obviously, and its reversibility
Fig. 4E and F show the typical current–time responses of also become worse. These results indicated that a certain
different electrodes for successive addition of 0.5 mM glucose to amount of silver can help GOD be used fully, and then when the
air-saturated and stirred 0.2 M pH 7.4 PBS at ꢁ0.45 V. Each amount of silver is too high, it will reduce the activity of GOD
successive addition of 0.5 mM glucose results in a decrease in due to a high concentration of silver ions may induce a big
the steady state current, and the Ag–GOD electrode shows a change of the secondary structure of protein. In addition, the as-
sensitivity of 13.8 mA mMꢁ1 and fast response time of less than prepared glucose biosensor has been used to detect glucose in
5 seconds. The calibration curve of the electrode (Fig. 4E inset) the serum samples. The glucose concentration in raw serum is
shows a linear response range from 0.014 mM to 3 mM with a 3 mM, detected by commercialized glucose meter. Adding 1 mL
correlation coefficient of 0.998. The plot of amperometric of serum sample into 5 mL of pH 7.4 PBS, hence the glucose
response vs. the detected glucose concentration illustrates a concentration of the dilute serum is 0.5 mM based on glucose
well-dened, typical behavior of an enzymatic kinetic reaction, meter. The current response of the dilute serum was performed
while the Ag/GOD electrode has a far narrower linear range at ꢁ0.45 V under stirring, as shown in Fig. 5B. The concentra-
within 1 mM and higher sensitivity of 21.2 mA mMꢁ1. This may tion of glucose in the serum sample was calibrated using
be caused by the different combination modes between silver standard glucose solution. We can know DI is 0.487 mA from
and GOD leading to various adsorption quantity and stability on Fig. 5B. Based on Fig. 4E, y ¼ ꢁ0.0131 + 0.9569x, the glucose
concentration of the diluted serum is 0.523 mM. That means
the relative standard deviations is 4.6% compared to the stan-
dard method. So the results from the biosensor are similar to
that tested by the glucose meter, indicating the as-prepared
biosensor may have potential in real sample analysis.
Conclusions
A new and effective enzyme immobilization method, in situ
growth metal on enzyme, is presented in our work. The exper-
Fig. 5 (A) The cyclic voltammograms of the electrode modified with
imental results indicate that Ag–GOD has good sensitivity of
Ag–GOD composites synthesized by different amount of AgNO3.
(5 mL of 0.01 M AgNO3 (a), 10 mL of 0.01 M AgNO3 (b), 20 mL of 0.01 M
AgNO3 (c)). (B) Amperometric response of the diluted serum on
13.8 mA mMꢁ1, wide detection range from 0.014 mM to 3 mM,
fast response of less than 5 s, and long life. These properties
Ag–GOD/Nafion electrode at ꢁ0.45 V under stirring.
demonstrate that in situ growth method is an attractive way to
This journal is © The Royal Society of Chemistry 2015
RSC Adv., 2015, 5, 34486–34490 | 34489

View Article Online
RSC Advances
Paper
immobilize enzymes and exhibits facile, direct electrochemistry
of GOD without any electron mediator. Moreover, this approach
presents a new platform for us to develop other biosensor.
9 E. Turkmen, S. Z. Bas, H. Gulce and S. Yildiz, Electrochim.
Acta, 2014, 123, 93–102.
10 N. German, A. Ramanavicius, J. Voronovic and
A. Ramanaviciene, Colloids Surf., A, 2012, 413, 224–230.
11 K. Y. Hwa and B. Subramani, Biosens. Bioelectron., 2014, 62,
127–133.
Acknowledgements
This work is nancially supported by National Natural Science
Foundation of China (21163021, 21375108); Chongqing Key
Laboratory for Advanced Materials and Technologies of Clean
Energies, Start-up grant under SWU113077 from Southwest
University; Natural Science Foundation of Chongqing
(cstc2013jcyjA5004); Fundamental Research Funds for the
Central Universities (XDJK2013B031) and Program for Excellent
Talents in Chongqing (102060-20600218).
12 Y. Y. Shao, J. Wang, H. Wu, J. Liu, I. A. Aksay and Y. H. Lin,
Electroanalysis, 2010, 22, 1027–1036.
13 J. Tang, Y. C. Wang, J. Li, P. M. Da, J. Geng and G. F. Zheng, J.
Mater. Chem. A, 2014, 2, 6153–6157.
14 E. Murphy-Perez, S. K. Arya and S. Bhansali, Analyst, 2011,
136, 1686–1689.
15 A. Wei, X. W. Sun, J. X. Wang, Y. Lei, X. P. Cai, C. M. Li,
Z. L. Dong and W. Huang, Appl. Phys. Lett., 2006, 89,
123902–123905.
16 X. Zhong, Y. Q. Chai and R. Yuan, Talanta, 2014, 128, 9–14.
17 J. Li, D. Kuang, Y. Feng, F. Zhang and M. Liu, Microchim.
Acta, 2012, 176, 73–80.
18 Y. Piao, D. J. Han and T. S. Seo, Sens. Actuators, B, 2014, 194,
454–459.
19 S. B. Bankar, M. V. Bule, R. S. Singhal and
L. Ananthanarayan, Biotechnol. Adv., 2009, 27, 489–501.
20 J. N. Hui, J. W. Cui, G. Q. Xu, S. B. Adeloju and Y. C. Wu,
Mater. Lett., 2013, 108, 88–91.
Notes and references
1 M. Mathew and N. Sandhyarani, Anal. Biochem., 2014, 459,
31–38.
2 S.-R. Lee, Y.-T. Lee, K. Sawada, H. Takao and M. Ishida,
Biosens. Bioelectron., 2008, 24, 410–414.
3 S. Vaddiraju, I. Tomazos, D. J. Burgess, F. C. Jain and
F. Papadimitrakopoulos, Biosens. Bioelectron., 2010, 25,
1553–1565.
4 P. Raghu, T. M. Reddy, K. Reddaiah, B. E. K. Swamy and
M. Sreedhar, Food Chem., 2014, 142, 188–196.
5 H. Zhang, H. Huang, Z. Lin and X. Su, Anal. Bioanal. Chem.,
2014, 406, 6925–6932.
21 S. Giri, C. H. Moore, J. T. McLeskey and P. Jena, J. Phys.
Chem. C, 2014, 118, 13444–13450.
22 S. Karimi, H. Ghourchian and A. Banaei, Int. J. Biol.
Macromol., 2013, 56, 169–174.
6 L. Su, W. Qin, H. Zhang, Z. U. Rahman, C. Ren, S. Ma and
X. Chen, Biosens. Bioelectron., 2015, 63, 384–391.
7 M. Yang, K. G. Lee, J. W. Kim, S. J. Lee, Y. S. Huh and
B. G. Choi, RSC Adv., 2014, 4, 40286–40291.
23 X. Ren, D. Chen, X. Meng, F. Tang, A. Du and L. Zhang,
Colloids Surf., B, 2009, 72, 188–192.
24 L.-M. Liu, J. Wen, L. Liu, D. He, R.-y. Kuang and T. Shi, Anal.
Biochem., 2014, 445, 24–29.
8 Y. F. Gao, T. Yang, X. L. Yang, Y. S. Zhang, B. L. Xiao, J. Hong,
N. Sheibani, H. Ghourchian, T. Hong and A. A. Moosavi-
Movahedi, Biosens. Bioelectron., 2014, 60, 30–34.
25 S. Q. Liu and H. X. Ju, Biosens. Bioelectron., 2003, 19, 177–
183.
34490 | RSC Adv., 2015, 5, 34486–34490
This journal is © The Royal Society of Chemistry 2015