Mercury(ii)-stimulated oxidase mimetic activity of silver nanoparticles as a sensitive and selective mercury(ii) sensor

Article abstract of DOI:10.1039/c3ra45226c

Research on oxidase mimics is challenging but important. In this article, mercury(ii) ion enabled citrate-capped silver nanoparticles (Cit-AgNPs) exhibit catalytic activity toward the oxidation of typical chromogenic substrate 3,3′,5,5′-tetramethylbenzidi

Full text of DOI:10.1039/c3ra45226c

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Mercury(II)-stimulated oxidase mimetic activity of
silver nanoparticles as a sensitive and selective
mercury(II) sensor†
Cite this: RSC Adv., 2014, 4, 5867
a
a
b
b
a
Guang-Li Wang,* Xiu-Fang Xu, Li-Hua Cao, Chong-Hui He, Zai-Jun Li
and Chi Zhang
a
Research on oxidase mimics is challenging but important. In this article, mercury(II) ion enabled citrate-
capped silver nanoparticles (Cit–AgNPs) exhibit catalytic activity toward the oxidation of typical
0
0
chromogenic substrate 3,3 ,5,5 -tetramethylbenzidine (TMB) by dissolved oxygen under mild conditions,
suggesting a new type of oxidase mimic. In addition, the oxidase-like activity of Cit–AgNPs was sensitive
to the concentration of mercury(II) ions and selective towards mercury(II) ions among other metal ions.
Received 19th September 2013
Accepted 5th November 2013
2+
Based on this, a facile colorimetric mercury(II) ion sensor was developed. Hg was reduced on the
surface of Cit–AgNPs to form Hg–Ag alloys. The Hg–Ag alloys activated oxygen and generated
superoxide anions, which oxidized TMB. This discovery indicates the potential of nanoparticles for
efficient enzyme mimetics.
DOI: 10.1039/c3ra45226c
www.rsc.org/advances
possess several advantages including controlled synthesis at a
low-cost, high catalytic activity, and high stability under harsh
1
. Introduction
It was well known that natural enzymes have been found conditions. Therefore, nanomaterials are promising candidates
extensive applications in various elds because they can cata- as enzyme mimics.
lyze chemical reactions specically and efficiently under mild
Oxidation reactions are of fundamental importance in
conditions. Unfortunately, the catalytic activity of natural nature. In addition, catalytic oxidations that use oxygen as a
9
enzymes is sensitive to environmental conditions and they can green oxidant in organic synthesis are of high signicance.
easily be denatured and digested. Furthermore, the prepara- Therefore, research on oxidase mimics is challenging but
tion, purication and storage of natural enzymes are usually important. Compared to peroxides mimics, there is less
time-consuming and expensive. Therefore, articial enzyme research on oxidase mimics based on nanomaterials. In 2008,
10
2
mimics are of great interest. Enzyme mimics show similar Asati et al. found that CeO nanoparticles possessed oxidase-
catalytic activities (for example, to oxidize typical substrates to like activity. However, it was found that instead of being an
their corresponding oxidation products) as that of natural oxidase mimic, nanoceria is a plain nanoparticulate oxidant
11
enzymes and the catalytic reaction process follows typical and dissolves completely aer the redox reaction. Inspired by
Michaelis–Menten curves. Since the pioneering work of Yan the fact that Pt is the dominant anode catalyst in fuel cells for
1
et al. using Fe O nanocrystals as peroxidase-mimics, different the reduction of oxygen to water, Pt-based bimetallic nano-
3
4
2
12
nanostructures including graphene oxide, single-wall carbon structures including AuPt and PdPt supported on gold nano-
nanotubes, CoFe O , FeTe, metal oxides and bimetallic rods were explored as oxidase mimics of horseradish
nanostructures have also been found to possess peroxidase-like peroxidase (HRP) to catalyze the reaction of the substrates
activity which can catalyze the oxidation of typical chromogenic o-phenylenediamine (OPD) and 3,3 ,5,5 -tetramethylbenzidine
substrates by H
. It was found that the enzyme-like activity of (TMB) in the presence of molecular oxygen. These reports are
3
4
5
6,7
13
2
4
8
0
0
2 2
O
nanomaterials was dependant on their structure, which could interesting in the development of novel oxidase mimetic
be controlled by the synthesis conditions. In comparison with nanomaterials. The oxidase-like activity of the Pt-based nano-
natural enzymes, enzyme mimics based on nanostructures structures was obviously inhibited by some metal ions such as
12
iron(II) ions, copper(II) ions and mercuric(II) ions.
In contrast to the idea that the mercuric(II) ions could inhibit
the oxidase-like catalytic activity of Au@Pt nanorods, in this
contribution, a new kind of mercury(II)-stimulated oxidase
a
12
The Key Laboratory of Food Colloids and Biotechnology, Ministry of Education, School
of Chemical and Material Engineering, Jiangnan University, Wuxi 214122, P. R. China.
E-mail: glwang@jiangnan.edu.cn; Fax: +86 510 85917763; Tel: +86 510 85917090
mimetic activity of citrate-capped silver nanoparticles (Cit–
b
Nanjing Entry-Exit Inspection and Quarantine Bureau, Nanjing 211106, P. R. China
2
+
AgNPs) was found. The reduction of Hg on the surface of Cit–
AgNPs resulted in the formation of a Hg–Ag alloy, which
†
Electronic supplementary information (ESI) available. See DOI:
0.1039/c3ra45226c
1
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demonstrated high catalytic effect for typical chromogenic and then the solution become golden yellow aer several drops,
0
0
substrates, such as 3,3 ,5,5 -tetramethylbenzidine (TMB), in the which was considered to be indication of the presence of iso-
presence of dissolved oxygen. The oxidase-like activity of mer- lated colloidal AgNPs. Then the mixture was allowed to react at
cury(II)-stimulated Cit–AgNPs could result in the oxidation of ambient temperature for 48 h before further applications.
TMB very quickly (within two minutes). The oxidase-like activity
2
+
of Cit–AgNPs in the presence of Hg was highly selective among
2
+
2
.4. Hg stimulated oxidase-like activity of Cit–AgNPs for
other metal ions. Therefore, the system could selectively detect
2
+
the detection of Hg
2
+
Hg . This is the rst report of oxidase mimics based on non
Pt-based nanostructures. In addition, compared to peroxidase,
oxidase mimetics could oxidize substrates using oxygen as a
green oxidant, which can avoid the use of the destructive and
unstable H O . Our work is not only helpful to develop efficient
First, 500 mL Cit–AgNPs, different amounts of HgSO and 300 mL
4
ꢀ
3
ꢀ1
5
.0 ꢁ 10 mol L TMB were sequentially added to a 5 mL
calibrated test tube. Then, 1 mL acetate buffer (HAc–NaAc, pH ¼
.0) was added to the above solution. Finally, the mixed solution
was dissolved to the required volume with ultrapure water.
4
2
2
nanomaterial-based oxidase mimetics, but also to nd new
applications of nanomaterial-based catalysts.
3
. Results and discussion
2
. Experimental
The Cit–AgNPs were prepared from AgNO
ment with NaBH . The HRTEM image revealed the spherical
shape of the Cit–AgNPs with an average diameter of about 7–
1 nm (Fig. 1A). The hydrodynamic size and zeta potential of the
3
by reductive treat-
2.1. Chemicals and materials
4
Silver nitrate, trisodium citrate dihydrate (Na C H O $2H O),
3
6
5
7
2
sodium borohydride (NaBH
4
, 96%), t-butanol, p-benzoquinone,
,3 ,5,5 -tetrametylbenzidine (TMB), AgNO and HgSO were all
obtained from Sinopharm Chemical Reagent Co., Ltd
Shanghai, China). All other chemicals used were of analytical
grade. All solutions were prepared with ultrapure water
1
0
0
3
3
4
Cit–AgNPs were 30.9 nm and ꢀ33.2 mV, respectively, measured
by dynamic light scattering measurements. The negative
charges of Cit on the surface of the AgNPs resulted in an elec-
trostatic double layer, which provided a repulsive force between
separated AgNPs and enabled the Cit–AgNPs to be dispersed
(
ꢀ
1
(18.2 MU cm ) obtained from a Healforce water purication
system.
15
stably in an aqueous solution. The nal Cit–AgNPs suspension
showed a typical intense absorption peak (around 400 nm)
1
6
2
.2. Instrumentation
(Fig 1B) due to the surface plasmon excitation.
2
+
It was found that neither Cit–AgNPs or Hg alone showed
any catalytic oxidation effect for typical chromogenic substrates
High resolution transmission electron microscopy (HRTEM)
images of Cit–AgNPs were obtained on a JEOL JEM-2100
transmission electron microscope (Hitachi, Japan). The zeta
potential and size distribution of the Cit–AgNPs were measured
using a ZetaPALS zeta potential and particle size analyzer
0
0
such as 3,3 ,5,5 -tetramethylbenzidine (TMB), in the presence of
dissolved oxygen under ambient conditions (Fig. 2 curve a and
b). Aer the addition of Hg , the catalytic activity of the Cit–
AgNPs was stimulated immediately with the appearance of the
characteristic absorbance peaks of the oxidized TMB (oxTMB) at
2
+
(
Brookhaven, USA). UV-Vis absorption spectroscopic measure-
ments were carried out using a TU-1901 spectrophotometer
Beijing Purkinje General Instrument Co., Ltd, China). Cyclic
3
70 nm and 652 nm and the simultaneous appearance of a
bright blue color of the oxTMB (Fig. 2 curve c). In addition, the
(
voltammetry was performed with a CHI 800C electrochemical
workstation (Shanghai, China). A conventional three-electrode
cell was used, including a Pt wire counter electrode, and a
saturated Ag/AgCl reference electrode. An indium tin oxide
2
+
Cit–AgNPs–Hg could also oxidize two other typical substrates
such as o-phenylenediamine (OPD) and 2,2-azinobis-(3-ethyl-
benzothizoline-6-sulfonic acid) (ABTS) to produce their corre-
sponding oxidation products with typical colors. However, the
maximum absorption peaks of oxOPD and oxABTS were 450 nm
and 417 nm, respectively, which overlapped with the peak of the
(ITO) electrode was used as the working electrode. The pH of the
HAc–NaAc buffer solution was measured with a glass electrode
connected to a PHS-3C pH meter (Shanghai, China).
2.3. Preparation of citrate-capped AgNPs
Citrate-capped AgNPs (Cit–AgNPs) were prepared using a
14
previously reported method with modications. First, 5 mL 1.0
ꢀ
2
ꢀ1
ꢀ3
ꢀ1
ꢁ
10 mol L trisodium citrate and 20 mL 1.0 ꢁ 10 mol L
AgNO were mixed in a 100 mL beaker. Then, 50 mL H O was
was
3
2
ꢀ3
ꢀ1
added to the beaker, and 10 mL 2.7 ꢁ 10 mol L NaBH
4
added drop by drop with stirring. In this method, the sodium
+
borohydride was a reducing agent capable of reducing Ag to
Ag and forming AgNPs in the presence of trisodium citrate as a
0
stabilizing agent. This was a very fast process at room temper-
ature. The colorless reactant mixture immediately turned to
Fig. 1 HRTEM image (A) and UV-vis absorption spectrum (B) of Cit–
dark yellow aer about ve drops of NaBH
4
had been added, AgNPs.
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Fig. 4 TMB oxidation was conducted in air with dissolved oxygen (a)
and after bubbling the solution with high purity nitrogen for two hours
Fig. 2 UV-vis spectra of (a) Hg + TMB; (b) Cit–AgNPs + TMB; (c) Cit– (b).
2
+
2+
2+
AgNPs + Hg + TMB; (d) the mixture of Cit–AgNPs and Hg was
added 500 mL 10 mol L NaBH and was allowed to react for 24 h,
4
ꢀ2
ꢀ1
and finally TMB solution was added. The inset image shows the cor-
responding colors of the above solutions. All the above solutions were
prepared in acetic buffer with pH 4.0.
2+
Fig. 5 The catalytic activity of Hg stimulated oxidase-like activity of
Cit–AgNPs is pH (A) and temperature (B) dependent.
Scheme 1 The corresponding reaction equation of the oxidization of
2+
TMB by dissolved oxygen using Cit–AgNPs and Hg
.
Fig. 6 Michaelis–Menten curve fit (A) and Lineweaver–Burk plot of the
double reciprocal of the Michaelis–Menten equation (B).
Fig. 3 Time dependent absorbance evolution at 652 nm of the TMB
2+
2+
oxidation system (a) Cit–AgNPs + Hg + TMB; (b) Hg + TMB; (c)
Cit–AgNPs + TMB.
AgNPs. Therefore, we chose TMB as a typical substrate to
investigate the enzyme-like activity. The oxidase-like activity of
2
+
Hg stimulated Cit–AgNPs for the oxidation of typical organic
substrates (TMB) can be described by the equation in Scheme 1.
It should be emphasized that the catalytic course of the reaction
system was very fast (within 2 min) when using TMB as the
model substrate (Fig. 3), though Hg or Cit–AgNPs alone hardly
showed any catalytic effect at the same time intervals. It was
2+
Fig. 7 Linear calibration plot for Hg detection.
2
+
2
+
found that the catalytic reaction activity of TMB oxidation by the indicating the nature of the oxidase-like activity of AgNPs–Hg
2
+
Cit–AgNPs in the presence of Hg decreased obviously aer using oxygen as a green oxidant. That is, the TMB oxidation
bubbling the solution with high purity nitrogen (Fig. 4), originated from the catalytic ability of the catalyst to activate
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Fig. 8 (A) Catalytic activity of Cit–AgNPs for TMB stimulated by
various metal ions; (B) absorption change of TMB at l ¼ 652 nm in the
presence of Cit–AgNPs and various metal ions. All the metal ions are at
ꢀ5
ꢀ1
.
concentrations of 1.0 ꢁ 10 mol L
Fig. 10 Oxidation of TMB over the Ag–Hg alloy under different
conditions: no scavenger (black line), 10 mM t-butanol (blue line), and
ꢀ1
5
mol L p-benzoquinone (red line).
Additionally, the concentration of TMB is a key factor because it
determined the absorbance and color level of the reaction
system. Therefore, the inuence of the concentration of TMB
was investigated from 50 to 400 mM. For all the concentrations
2
+
investigated, the Cit–AgNPs–Hg system could catalyze TMB
oxidation in two minutes (Fig. S2†). The absorption intensity of
oxTMB (652 nm) increased gradually with TMB concentration at
lower levels until reaching a maximum at high concentrations
(about 300 mM, Fig. S3†).
Under the optimum conditions, the steady-state kinetics for
2
+
the catalytic activity of Hg stimulated Cit–AgNPs towards the
oxidization of TMB with dissolved oxygen were determined and
typical Michaelis–Menten curves were obtained (Fig. 6A). The
2+
Fig. 9 UV-vis spectra of (a) Cit–AgNPs, and (b) Cit–AgNPs + Hg at
pH ¼ 4.0.
m
Michaelis–Menten constant (K ), which is an indicator of
enzyme affinity for its substrate, was obtained by using a
1
n
Km
1
1
Lineweaver–Burk plot:
¼
ꢁ
þ
, where n, nmax, [S],
nmax ½Sꢃ nmax
and K
m
are the reaction rate, maximum reaction velocity,
concentration of the substrate, and Michaelis constant,
respectively. For the TMB oxidation, K is 230 mM, and nmax is
80 nM s . The much lower apparent Km and higher maximum
m
ꢀ
1
3
2
+
reaction velocity of this system demonstrated that Hg stimu-
lated Cit–AgNPs had much higher intrinsic catalytic activity
2 2
towards oxygen than the natural HRP using H O as a
17,18
substrate.
The stimulating effect of Hg on the Cit–AgNPs catalytic
2
+
Scheme 2 The mercury(0) stimulated oxidase-like activity of Cit–
2+
AgNPs. Hg ions are reduced by citrate and form the Ag–Hg alloy, activity was concentration dependent and could be observed
2
+
changing the surface properties of the AgNPs and stimulating the with some sensitivity. With an increasing concentration of Hg ,
AgNPs-based catalytic oxidation of TMB with dissolved oxygen.
there was an increase in the absorbance of oxTMB at 652 nm. In
fact, under optimal experimental conditions, the calibration
2
+
curve of the absorbance at 652 nm against Hg concentration
ꢀ
7
ꢀ5
ꢀ1
ꢀ1
dissolved oxygen but not the direct reaction between Cit–AgNPs was linear in a range from 1.0 ꢁ 10 to 1.0 ꢁ 10 mol L
2
+
ꢀ8
or Hg with TMB.
(Fig. 7) with a detection limit of 2.8 ꢁ 10 mol L (S/N ¼ 3).
Similar to natural enzymes, the catalytic activity of the Cit– The detection limit was lower than the maximum mercury level
2
+
AgNPs–Hg system was dependent on pH and temperature. dened for drinking water by the World Health Organization
ꢀ1
19
The experimental results indicated that the optimized pH and (WHO) (ꢄ30 nmol L ). This approach holds great potential
ꢂ
2+
temperature for the proposed system were 4.0 and 40 C (Fig. 5). for monitoring Hg in environmental samples.
It should be pointed out that the Cit–AgNPs showed improved It was found that the oxidase-like catalytic effect of Cit–
stability compared to HRP at lower/higher pH solutions and at AgNPs was stimulated by Hg selectively (Fig. 8). Other metal
2
+
2
+
2+
2+
2+
3+
3+
2+
2+
2+
higher temperatures (Fig. S1†), indicating that Cit–AgNPs as ions including Pb , Cu , Ni , Cd , Fe , Al , Fe , Co , Zn ,
enzyme mimics showed better stability under harsh conditions. Mn and Ag did not display the same stimulating catalytic
2
+
+
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2
+
effects for TMB oxidation and no absorption at 652 nm of fast colorimetric assay for Hg . Owing to the facile preparation
oxTMB could be distinguished owing to the catalysis of Cit– of AgNPs and their potential for the chemical reaction activity,
AgNPs in the presence of other metal ions even at a concen- our work could help to develop a variety of applications of
ꢀ
5
ꢀ1
tration of 1.0 ꢁ 10 mol L
.
oxidase-based, simple, cost-effective, and easy-to-make sensors
It was found that citrate-capped noble metal nanoparticles in biotechnology, medicine and environmental chemistry.
2
+
0
20
could reduce Hg to elemental mercury (Hg ). A slight blue
shi in the absorption peak from 400 to 397 nm was observed
2
+
Acknowledgements
aer the addition of Hg to Cit–AgNPs (Fig. 9). This phenom-
2
+
enon may be ascribed to the reduction of Hg by Cit–AgNPs
and subsequent deposition of elemental Hg on the surface of
AgNPs, yielding the Ag–Hg alloy due to the high affinity between
This work was supported by the National Natural Science
Foundation of China (no. 21275065, 21005031), the Funda-
mental Research Funds for the Central Universities
2
1,22
0
Ag and Hg.
enhancement of the catalytic activity of AgNPs, Ag–Hg alloy
synthesized by NaBH (a stronger reductant than citrate)
reduction of Hg was used for TMB oxidation. The Ag–Hg alloy
obtained by NaBH reduction showed even higher oxidase-like
catalytic activity for TMB oxidation than that obtained by Cit–
In order to conrm that Hg results in the
(JUSRP51314B), the 111 Project (B13025) and Project of Jiangsu
23,24
Inspection and Quarantine Bureau (2011KJ17).
4
2
+
4
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+
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2
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4
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2
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(
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27
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ꢀ
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