Copper nanoparticles modified graphitic carbon nitride nanosheets as a peroxidase mimetic for glucose detection

Article abstract of DOI:10.1039/c5ra18957h

In this study, copper nanoparticles modified graphitic carbon nitride nanosheets (Cu NPs/g-C₃N₄) as a novel peroxidase mimetic were successfully prepared by the calcination of dicyandiamide-Cu²⁺ complex with the assistance of humic acid. The morphology and structure of the product were characterized by X-ray powder diffractometry (XRD), Fourier transform infrared spectroscopy (FTIR) and transmission electron microscopy (TEM). The prepared Cu NPs/g-C₃N₄ was found to have highly peroxidase-like activity, which can rapidly catalyze the oxidation of peroxidase substance 3,3′,5,5′-tetramethylbenzidine (TMB) to produce a blue color in the presence of H₂O₂. Accordingly, a simple, selective and fast colorimetric method was developed for H₂O₂ and glucose detection. The prepared Cu NPs/g-C₃N₄ exhibited low detection limits of 3.2 × 10^-8 M and 3.7 × 10^-7 M for H₂O₂ and glucose, respectively, due to the excellent peroxidase-like activity of Cu NPs/g-C₃N₄ originating from the synergistic effect of Cu NPs and g-C₃N₄. In this work, we utilized the easily forming complex ability of dicyandiamide and Cu²⁺ ions to form a homogeneous precursor solution, and then obtained the CuNPs/g-C₃N₄ product; such a method may have wide applications in novel composite nanomaterial preparation. The product may have promising applications in medical diagnostics and biotechnology fields.

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Graphical Abstract
CuNPs/g-C3N4 was successfully prepared by the calcination of dicyandiamide-Cu2+
complex as peroxidase mimic for sensitive detection of glucose.

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Copper nanoparticles modified graphitic carbon nitride nanosheet
as peroxidase mimetic for glucose detection
Received 00th January 20xx,
Accepted 00th January 20xx
Nan Wang, Zhenwei Han, Hai Fan*, Shiyun Ai*
In this study, copper nanoparticles modified graphitic carbon nitride nanosheet (CuNPs/g-C3N4) as a novel peroxidase
mimetic was successfully prepared by the calcination of dicyandiamide-Cu2+ complex with the assistance of humic acid.
The morphology and structure of the product was characterized by X-ray powder diffractmeter (XRD), Fourier transform
infrared spectroscopy (FTIR) and transmission electron microscopy (TEM). The prepared CuNPs/g-C3N4 was found to have
highly peroxidase-like activity, which can rapidly catalyze the oxidation of peroxidase substance 3, 3′, 5, 5′-
tetramethylbenzidine (TMB) to produce a blue color reaction in the presence of H2O2. Accordingly, a simple, selective and
fast colorimetric method was developed for H2O2 and glucose detection. The prepared CuNPs/g-C3N4 exhibited low
detection limits with 3.2 × 10-8 M and 3.7 × 10-7 M for H2O2 and glucose, respectively, due to the excellent peroxidase-like
activity of Cu NPs/g-C3N4 originated from the synergistic effect of Cu NPs and g-C3N4. In this work, we utilized the easily
forming complex ability of dicyandiamide and Cu2+ ions to form a homogeneous precursor solution, and then obtained
CuNPs/g-C3N4 product, such method may have wide applications in novel composite nanomaterials preparation. The
DOI: 10.1039/x0xx00000x
www.rsc.org/
product
may
have
promising
applications
in
medical
diagnostics
and
biotechnology
fields.
oxidized to form copper oxides. To solve this problem,
stabilizing agents are necessary during the synthesized
process. For example, Nirmal Goswami et al. reported
extremely stable, water-soluble Cu quantum clusters by
capped with bovine serum albumin 18. However, the Cu NPs
obtained by these methods are easily aggregated, which
hindered their further applications. Therefore, it is necessary
to provide facile synthetic route for synthesis of Cu NPs with
good dispersity and stability.
1. Introduction
Since Fe3O4 magnetic nanoparticles were discovered to exhibit
1
an intrinsic peroxidase-like activity for the first time , great
interest has been focused on the nanoparticles as enzyme
mimetics. Consequently, a variety of nanoparticles were found
to have peroxidase- or oxidase-like activity 2, 3. Among them,
metal nanoparticles have attracted much attention due to
4
their low toxicity, ultrafine size and high enzyme activity .
5
Several metal nanomaterials such as Au nanoparticles and Pt
6
nanoparticles were discovered to exhibit a high intrinsic
Graphitic carbon nitride (g-C3N4) has become a hot
research topic recently due to many advantages such as high
thermal and chemical stability, tunable electronic structure,
abundant and inexpensive raw materials 19. Several potential
applications have been developed, such as photocatalyst,
bioimaging and biomedical applications 20-27. Recently, g-C3N4
has also been found to have peroxidase-like activity. g-C3N4
has a stacked two-dimensional (2D) structure, which can be
easily exfoliated into nanosheets. Nanosheets are regarded as
ideal supports for the formation of functional composites. As
we all know that the precursors for the preparation of g-C3N4
are usually amino groups contained compounds, such as
dicyandiamide. These amino groups can interact with copper
ions to form stable complex due to the strong coordination
force between amino and copper ions. This inspired us to take
advantage of dicyandiamide-Cu2+ complex as the precursor for
the formation of carbon nitride and Cu composite with
functional properties. During the formation of carbon nitride
nanosheets, Cu NPs would be dispersed on nanosheets
uniformly, which could largely enhance the catalytic activity
and reduce the cost at the same time.
peroxidase-like enzyme mimetic activity. Consequently,
bimetallic or hybrid nanomaterials such as Au@Pt
7
8
9
nanostructures , CuPt nanorods , Bi-Au nanoparticles and
Au@Pd nanoparticles 10, have also been reported to have
excellent peroxidase-like activity. However, these noble metals
usually have high cost, which limited their practical
applications.
Recently, Cu based nanomaterials have brought increasing
attention due to their much lower cost compared to noble
metals such as Au, Pt, Pd. Several methods, such as vapor
depositions 11, 12, reverse micellar system 13, 14, soft and hard
15, 16
17
template processes
and electrochemical reaction have
been developed for the synthesis of Cu nanocrystals. However,
pure Cu nanocrystals are not stable in air, which is easily
a.College of Chemistry and Material Science, Shandong Agricultural University,
Taian, 271018, Shandong, P. R. China. E-mail: fanhai@sdau.edu.cn;
ashy@sdau.edu.cn; Fax: +86 538 8242251; Tel: +86 538 8247660
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Herein, we report the formation of Cu NPs modified g-C3N4 Colorimetic detection of glucose based on Cu NPs/g-C3N4 as
nanosheet by calcination of dicyandiamide-Cu2+ complex with peroxidase mimetic was investigated. Michaelis-Menten
the reduction of humic acid. The prepared Cu NPs/g-C3N4 constant (Km) was obtained under the optimal conditions by
possesses intrinsic peroxidase-like activity, which have much varying the concentrations of H2O2 and TMB. Km was
better peroxidase-like activity than Cu NPs and g-C3N4 under calculated using Lineweaver-Burk plots of the double
the reaction of H2O2 and TMB. Accordingly,
a novel reciprocal of the Michaelis-Menten equation, 1/v = (Km/Vmax)
colorimetric method for the detection of H2O2 and glucose was (1/[S]) + 1/Vmax. In this equation, v is the initial velocity, Vmax is
developed. Most importantly, this rapid, sensitive, and the maximal reaction velocity, and [S] is the concentration of
convenient approach could be applied for glucose detection in the substrate.
real serum samples, which can be potentially applied in
biotechnology and medical diagnostics.
H2O2 detection was carried out as follows: 80 μL Cu NPs/g-
C3N4 (0.5 mg mL-1), 40 μL TMB (8 mM) and 200 μL phosphate
buffer (25 mM, pH 3.0) were mixed and incubated in 500 μL
centrifugal tube. Then 80 μL H2O2 solutions with different
concentrations were added. The mixed solution was further
incubated at room temperature for 10 min. The absorbance of
supernatant was recorded.
2. Experimental Section
2.1 Chemicals and reagents
Dicyandiamide, copper acetate, glucose oxidase and TMB were
analytically pure and purchased from Aladdin (Shanghai,
China). Humic acid, H2O2 and other chemical reagents were
purchased from Kay Tong Chemical Reagents Co., Ltd (Tianjin,
China). The blood samples were provided by the university
hospital. All reagents were of analytical grade and used
without further purification. Phosphate buffer solution (PBS,
pH from 2.0 to 7.0) was used in this work and double distilled
water was applied throughout the experiment.
Glucose detection was realized as follows: 5 μL GOx (40 mg
mL-1) was added into 100 μL phosphate buffer (25 mM)
containing different concentrations of glucose. Then the mixed
solution was incubated at 37 °C for 1 h to produce H2O2. The
other detection procedure was the same as that of H2O2.
The specificity of the Cu NPs/g-C3N4 for glucose was tested
using 10 mM maltose, 10 mM fructose, 10 mM lactose, and 5
mM glucose. The detection procedure was the same as that of
H2O2. Before the detection of glucose in blood samples, the
blood samples were pretreated by salting-out and
centrifugation to remove proteins. The supernatant solution
was taken and diluted with phosphate buffer (25 mM). 100 μL
of above solution was mixed with 5 μL GOx (40 mg mL-1), and
then the mixture was incubated at 37 °C for 1 h to produce
H2O2. The other detection procedure was the same as that of
H2O2.
2.2 Synthesis of the Cu NPs/g-C3N4
Firstly, dicyandiamide and copper acetate were mixed in the
aqueous solution with stirring. Cu2+ ions were coordinated
with dicyandiamide to form a dicyandiamide-Cu2+ complex due
to the strong coordination interaction between Cu2+ ions and
the amino groups on the dicyandiamide. Then the mixture was
filtered and dried. Secondly, the obtained product and humic
acid were grinded on the agate mortar and put into a porcelain
crucible covered with a lid in a muffle fumace at 3 °C/min up to
550 °C for 2 hours in nitrogen atmosphere.
3. Results and discussion
2.3 Characterization
3.1 Characterization of the products
The crystal structure and phase purities of as-synthesized
products were characterized by X-ray powder diffractmeter
(XRD). The XRD data was collected using a Rigaku DLMAX-
2550V diffractmeter. Transmission electron microscopy (TEM)
was performed on
a JEM-2010 transmission electron
microscope (Japan). Kinetic measurements and UV-Vis
absorption spectra were carried out using a UV-2450 Shimadzu
Vis-spectrometer. Photographs were taken using a Canon G11
digital camera.
2.4 The catalytic activity of Cu NPs/g-C3N4
Fig. 1 (A) XRD patterns and (B) FTIR spectra of g-C3N4 and Cu NPs/g-C3N4.
The catalytic activity measurements of Cu NPs/g-C3N4 were
performed in PBS buffer (25 mM, pH 4.0) containing Cu NPs/g-
C3N4 samples (80 μL), in the presence of H2O2 (100 mM) and
TMB (8 mM) at room temperature, unless otherwise stated.
The assays were monitored in wavelength-scan mode after
reacted for 10 min or time-course mode at 652 nm under the
optimal conditions as described above, unless otherwise
stated.
The phase and structure of as-synthesized products were
characterized by XRD and FTIR. In the XRD pattern (Fig. 1A),
the apparent peak of 27.7° is assigned to the characteristic
peak of g-C3N4 28, 29. To the red XRD pattern of the Cu NPs/g-
C3N4, besides the C3N4 diffraction peak at 27.7°, all the other
three peaks at 43.4°, 50.5° and 74.2° can be assigned to the
(111), (200) and (220) crystal face of Cu (JCPDS No. 04-0836).
No other diffraction peaks of impurities can be observed. It
confirmed the successful preparation of pure Cu NPs/g-C3N4
2.5 Detection of H2O2 and glucose using Cu NPs/g-C3N4 as
peroxidase mimetic
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nanocomposite. FTIR was further used to characterize the samples shows an ignorable absorbance. However, the H2O2 +
product, as shown in Fig. 1B. The peak at 807 cm-1 belongs to TMB + g-C3N4 system has an absorbance at 652 nm indicating
triazine ring mode, one typical peak in carbon nitride. The the production of oxidized TMB. And thus proves that the g-
absorption bands near 1572 and 1632 cm-1 are attributed to C3N4 owns intrinsic peroxidase-like activity. The H2O2 + TMB +
C=N stretching, while the bands at 1250, 1325, 1420 cm-1 are Cu NPs/g-C3N4 system has a strongest absorbance at 652 nm,
consistent with aromatic C-N stretching. The broad band at demonstrating the high catalytic efficiency and the excellent
3000-3500 cm-1 appears for uncondensed terminal amino peroxidase-like activity of Cu NPs/g-C3N4. The excellent activity
groups (-NH2 or
= NH groups). To Cu NPs/g-C3N4 is due to the Cu nanoparticles dispersed on the C3N4
nanocomposite, all absorption peak positions in the case of Cu nanosheets, which produces the synergistic enhanced catalytic
NPs/g-C3N4 are almost similar to those in the case of g-C3N4, activity effect.
which indicates that the structure of g-C3N4 keeps almost the 3.3 Optimization of reaction conditions
same with that after addition of Cu NPs 29
.
Fig. 2 TEM images: (A) g-C3N4, (B) and (C) Cu NPs/g-C3N4 with different magnification.
Fig.
4 Dependence of peroxidase-like activity of Cu NPs/g-C3N4 on (A) pH, (B)
temperature and (C) concentration of Cu NPs/g-C3N4. Experiments were carried out
using Cu NPs/g-C3N4 in 25 mM PBS (pH 3.0) solution with 10 mM H2O2 and 800 μM TMB
as substrates.
The morphology of the Cu NPs/g-C3N4 nanocomposite is
investigated by TEM. In Fig. 2A, g-C3N4 nanosheet can be
obviously observed. Fig. 2B showed that Cu NPs are uniformly
dispersed on the surface of g-C3N4 nanosheets, forming the
assembly of g-C3N4 nanosheets and Cu spherical
nanostructures. From the magnification image in Fig. 2C, it can
be clearly observed that each Cu spherical nanostructure is
composed of many little Cu nanoparticles. Such special
morphology may be due to the viscosity of humic acid besides
its reduction property. The characterization results suggest
that Cu NPs/g-C3N4 has been successfully synthesized.
To explore the optimal reaction conditions, influence of pH
and temperature on the catalytic activity of Cu NPs/g-C3N4 was
studied. When the pH of phosphate buffer varied from 2.0 to
7.0, the activity of Cu NPs/g-C3N4 was investigated as shown in
Fig. 4A. It was found that the peak intensity increases first and
then decrease with the pH varying from 2.0 to 7.0. At pH 3.0,
the absorption intensity becomes the maximum. Therefore,
the optimal pH value is pH 3.0. The temperature ranging from
20 °C to 45 °C was also performed in this experiment. As
shown in Fig. 4B, the absorbance at 652 nm increased
obviously initially but much more slowly after 35 °C. Thus, 35
°C is selected during the experiment. In addition, the
concentration of Cu NPs/g-C3N4 also has an influence on the
catalytic reaction. Fig. 4C shows the time-dependent
absorbance changes of TMB solution with addition different
concentration of Cu NPs/g-C3N4 in 25 mM PBS (pH 3.0) at room
temperature. With the concentration of Cu NPs/g-C3N4
increased, the reaction rate increased correspondingly. 50
μg·mL-1 was used in our following experiment.
3.2 Peroxidase-like activity of Cu NPs/g-C3N4
In order to study the catalytic mechanism, steady-state
kinetics with H2O2 or TMB as substrate was measured by
changing the concentration of one substrate while the other
conditions kept constant. Typical Michaelis-Menten curves
could be acquired for both TMB and H2O2 in a range of
concentrations of one substrate as shown in Fig. 5A and B. The
double reciprocal plots were obtained according to the
calculated series of the initial reaction rates. In Fig. 5C, every
straight line was obtained by changing reciprocal initial
velocity with reciprocal TMB concentration keeping H2O2
concentration constant. Three approximate parallel lines were
obtained by varying the concentration of H2O2. Similarly,
reciprocal initial velocity versus reciprocal H2O2 concentration
was collected by varying concentration of TMB as shown in Fig.
Fig. 3 Absorbance spectra of TMB (800 μM) in different reaction systems. Solutions in
25 mM PBS (pH 3.0) incubated at the room temperature for 10 min.
In this study, catalytic oxidation of the typical peroxidase
substrate TMB in the different systems was conducted to
demonstrate the peroxidase-like activity of as-synthesized Cu
NPs/g-C3N4. In Fig. 3, the TMB + Cu NPs/g-C3N4 system without
H2O2 results in a very slight absorbance at 652 nm. And the
H2O2 + TMB system without any g-C3N4 or Cu NPs/g-C3N4
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5D. The results show that the slopes of the plots are almost response curve exhibits that glucose can be visually
parallel, which should be the characteristic of a ping-pong determined. In Fig. 6D, the linear range of glucose detection is
mechanism. Moreover, Lineweaver-Burk plot was used to from 1.0 × 10-6 to 1.0 × 10-4 M with a detection limit of 3.7 ×
calculate Michaelis-Menten constant (Km) and maximal 10-7 M. The detection limits of our prepared Cu NPs/g-C3N4 are
reaction velocity (Vmax) in this system. The calculated values much lower than those in most of the reported literatures, as
are recorded in table 1. The Km values indicate the affinity for shown in table 2.
the structure. The Km value for the Cu NPs/g-C3N4 was 0.389
mM, which is much lower than that of AuNPs/PVP-GNs (2.63
mM) 30. The result indicated that the Cu NPs/g-C3N4 have a
significantly high affinity.
Fig. 6 (A) Absorbance changes at 652 nm of TMB solutions catalyzed by Cu NPs/g-C3N4
in the presence of different concentrations of H2O2 in 25 mM PBS (pH 3.0) at room
temperature. (B) A dose-response curve for H2O2 detection. (C) Absorbance changes at
652 nm of TMB reaction solutions catalyzed by Cu NPs/g-C3N4 in the presence of
different concentrations of glucose in 25 mM PBS (pH 3.0) at room temperature. (D) A
dose-response curve for glucose detection. Insert: photos of the solution during and
Fig. 5 Steady-state kinetic assay and catalytic mechanism of Cu NPs/g-C3N4. (A) The
after the detection of H2O2 and glucose.
concentration of H2O2 was 4 mM and TMB concentration was varied. (B) The
concentration of TMB was 8 mM and H2O2 concentration was varied.（C and D）
Table
2 The comparisons of detection limits using different materials as
Double reciprocal plots of activity of Cu NPs/g-C3N4 with the concentration of one
substrate (H2O2 or TMB) fixed and the other varied. The velocity (v) of the reaction was
measured using 0.15 mg·mL-1 Cu NPs/g-C3N4 in 400 μL of 25 mM PBS (pH 3.0) at room
temperature.
peroxidase mimetics for H2O2/glucose detection.
Materials
Methods
H2O2/gluc
ose
Detection
limit
Reference
Cu NPs/g-
C3N4
colorimetric
H2O2
0.032 μM
0.37 μM
/
Table 1 The Michaelis-Menten constant (Km) and maximum reaction rate (Vmax) of
glucose
Cu NPs/g-C3N4.
31
32
6
g-C3N4
colorimetric
colorimetric
colorimetric
colorimetric
H2O2
5 μM
1 μM
catalyst
substance
TMB
Km [mM]
0.389
Vmax (10-7 M s-1)
5.84
glucose
Cu NPs/g-C3N4
Cu NPs/g-C3N4
Cu NCs
H2O2
10 μM
H2O2
9.27
3.84
glucose
100 μM
Pt NTs
H2O2
18.6 μM
/
3.4 Detection of H2O2 and glucose
glucose
According to the excellent peroxidase-like activity of our
prepared Cu NPs/g-C3N4, the colorimetric method for
detection of H2O2 and glucose using Cu NPs/g-C3N4-catalyzed
blue color reaction was developed. The catalytic activity of Cu
NPs/g-C3N4 is H2O2-concentration-dependent and thus can be
used to detect H2O2. Fig. 6A shows the typical H2O2
concentration-response curve under the optimal conditions
(pH 3.0, 35 °C). In Fig. 6B, the linear calibration plot ranges
from 1.0 × 10-7 to 2.0 × 10-6 M. The detection limit is calculated
to be H2O2 is 3.2 × 10-8 M. Because, H2O2 is the main product
of glucose oxidation in the presence of O2 while catalyzed by
glucose oxidase, therefore, the detection of glucose can also
be realized according the quantitative relationship between
glucose and H2O2. In Fig. 6C, the typical glucose concentration-
33
Fe-g-C3N4
H2O2
0.05 μM
0.5 μM
glucose
It is interesting to note that the detection sensitivity of our
prepared Cu NPs/g-C3N4 for the detection of H2O2 and glucose
31
is not only much higher than those of g-C3N4 and Cu NCs
32
,
but higher than those of many reported peroxidase mimetics.
Such unusual high detection performance probably originated
from the synergistic effect of Cu NPs and g-C3N4, as shown in
scheme 1. Firstly, g-C3N4 nanaosheets provide an ideal
platform for the high dispersion of Cu NPs. Secondly, g-C3N4
nanosheets have π-conjugated electronic structure. The
electrons would transfer from g-C3N4 nanosheets to Cu NPs
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due to the low Fermi level of metals 34. More electrons would
catalylize more H2O2 to ·OH by giving electrons to H2O2.
Herein, the existence of Cu NPs accelerates the electron
transfer rate from C3N4 to H2O2. Therefore, our prepared Cu
NPs/g-C3N4 demonstrates much higher catalytic activity.
4. Conclusion
In summary, we successfully prepared Cu NPs/g-C3N4 via the
calcination of dicyandiamide-Cu2+ complex with the assistance
of humic acid. The prepared Cu NPs/g-C3N4 was found to have
highly intrinsic peroxidase-like activity. Cu NPs/g-C3N4 could
catalyze the oxidation of TMB by H2O2 to produce the typical
blue color reaction. The catalytic activity was sensitive to
variations of pH, temperature, catalyst amount, and substrate
concentration. The Cu NPs/g-C3N4 catalyzed the reaction of
H2O2 and TMB which showed typical Michaelis-Menton
kinetics. A sensitive colorimetric method to detect H2O2 and
glucose was developed by using Cu NPs/g-C3N4 peroxidase-
like catalytic activity. The detection limits of H2O2 and glucose
can be reached to 3.2 × 10-8 M and 3.7 × 10-7 M, respectively.
This approach could be applied for glucose detection in real
serum samples. As a novel peroxidase mimetic with highly
sensitive property, Cu NPs/g-C3N4 would have potential
applications in biocatalysts and clinical diagnosis.
Scheme 1 The enhanced catalytic mechanism of Cu NPs/g-C3N4.
In order to test the selectivity of the colorimetric method
for glucose, 10 mM fructose, 10 mM lactose, and 10 mM
maltose were selected as the control samples. Fig. 7A shows
the absorption intensity of solution in the presence of glucose
or other analogues. To glucose, the absorption intensity is
much higher than that of other analogues, even though the
concentrations of analogues were 2 times higher than that of
glucose. The main reason is that glucose oxidase has a certain
degree of specificity to glucose, so the absorbance hardly
increased for analogues as shown in Fig. 7A. Therefore, the
colorimetric method proposed in our experiment is a simple,
speed, and selective colorimetric method for the detection of
glucose. To test the application of this method on real
samples, blood sample was selected to detect glucose. Fig. 7B
shows the time-dependent absorbance changes of solutions in
the absence or presence of samples. According to the linear
calibration curve, glucose in the blood sample can be
calculated to be 7.12 mM. The error is minimal for the
provided value of 6.85 mM from the hospital. In addition, the
normal range of blood glucose concentration in healthy is
about 3-8 mM. Therefore, this colorimetric method can be
conveniently applied to glucose detection.
Acknowledgements
This work was supported by the National Natural Science
Foundation of China (Nos. 21375079, 51402175) and Project of
Development of Science and Technology of Shandong
Province, China (No. 2013GZX20109).
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Fig. 7 (A) Selectivity analysis for glucose detection by monitoring the relative
absorption intensity. The analyte concentrations were as follows: 10 mM
maltose, 10 mM fructose, 10 mM lactose, and 5 mM glucose. The error bars
represent the standard deviation of three measurements. (B) The time-
dependent absorbance changes at 652 nm for different samples (buffer solution
or diluted blood samples) after incubation with GOx. Diluted blood was diluted
100-fold.
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