Pd nanoparticles supported on nitrogen, sulfur-doped three-dimensional hierarchical nanostructures as peroxidase-like catalysts for colorimetric detection of xanthine

Article abstract of DOI:10.1039/c5ra02312b

Pd nanoparticles supported on nitrogen, sulfur-doped three-dimensional hierarchical nanostructures (Pd/N-S-CS) were successfully prepared by glycol reduction of Pd on nitrogen, sulfur-doped three-dimensional hierarchical nanostructures (N-S-CS) using humic acid (HA) and thiourea as the precursors. Transmission electron microscopy (TEM), X-ray diffraction (XRD), energy-dispersive X-ray spectroscopy (EDX) and X-ray photoelectron spectroscopy (XPS) were employed to confirm the characterization of Pd/N-S-CS. The results showed that the obtained Pd/N-S-CS had interesting three-dimensional hierarchical nanostructures which were composed of nanosheets, and Pd nanoparticles with an average diameter of 6 nm were distributed on the surface of the nanosheets. Furthermore, the Pd/N-S-CS exhibited excellent peroxidase-like activity and it can effectively catalyze the classical peroxidase substrate 3,3′,5,5′-tetramethylbenzidine (TMB) by H₂O₂ to produce a color reaction. Based on this finding, a simple, sensitive, and selective colorimetric method for H₂O₂ and xanthine detection was developed and applied to detect xanthine in urine samples. The detection limit of H₂O₂ and xanthine were 3.3 × 10^-6 M and 2.9 × 10^-7 M, respectively. This work is expected to provide a novel and efficient method for the detection of xanthine in the human body. This journal is

Full text of DOI:10.1039/c5ra02312b

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Pd nanoparticles supported on nitrogen, sulfur-doped three-dimentional
hierarchical nanostructures as peroxidase-like catalyst for colorimetric
detection of xanthine
Weijie Shia,b, Hai Fana, Shiyun Ai*a, Lusheng Zhu*b
5
Received (in XXX, XXX) Xth XXXXXXXXX 20XX, Accepted Xth XXXXXXXXX 20XX
DOI: 10.1039/b000000x
Pd nanoparticles supported on nitrogen, sulfur-doped three-dimentional hierarchical nanostructures
(Pd/N-S-CS) was successfully prepared by glycol reduction of Pd on nitrogen, sulfur-doped three-
dimentional hierarchical nanostructures (N-S-CS) using humic acid (HA) and thiourea as the precursors.
10 Transmission electron microscopy (TEM), X-ray diffraction (XRD), energy-dispersive X-ray spectra
(EDX) and X-ray photoelectron spectra (XPS) were employed to confirm the characterizations of Pd/N-S-
CS. The results showed that the obtained Pd/N-S-CS had interesting three-dimensional hierarchical
nanostructures which were composed of nanosheets, and Pd nanoparticles with average diameter of 6 nm
were distributed on the surface of nanosheets. Furthermore, the Pd/N-S-CS exhibited excellent
15 peroxidase-like activity and it can effectively catalyze the classical peroxidase substrate 3,3′,5,5′-
tetramethylbenzidine (TMB) by H2O2 to produce a color reaction. Based on this finding, a simple,
sensitive, and selective colorimetric method for H2O2 and xanthine detection was developed and applied
to detect xanthine in urine samples. The detection limit of H2O2 and xanthine were 3.3 × 10−6 M and 2.9 ×
10−7 M, respectively. This work is expected to provide a novel and efficient method for the detection of
20 xanthine in human body.
nanoparticles. One is the loss of rare and expensive metal, which
brings economic concerns; the other is that Pd nanoparticles are
usually instable and easily aggregated quickly without proper
surface modification. Therefore, in order to improve their
50 dispersion and stability as well as alleviate the cost of Pd
catalysts, it is helpful to load them on a proper support.
1. Introduction
Colorimetric detection as a simple, cheap, practical and easily
controlled method has attracted much attention in portable and
inexpensive daily life applications.1 In recent years, enzyme-
25 mimetic nanoscale materials have emerged as a new type of
important and excellent tools for colorimetric detection because
of their low cost, easy preparation, high stability, and tunability in
catalytic activities in comparison with natural enzymes.2 Since
Fe3O4 nanoparticles were reported to have intrinsic peroxidase-
30 like activity similar to that of horseradish peroxidase (HRP),3
various nanomaterials have been successfully used as peroxidase
mimics, including CuO,4 CeO2,5 MFe2O4 (M = Co, Mn, Zn),6-8
V2O5 nanowires,9 Au,10 Pt nanoparticles,11 and carbon-based
nanomaterials (carbon nanotubes,12 carbon nanodots13 and
35 graphene oxide14), etc. With the catalysis of these reported
nanomaterials, some chromogenic substrates can be oxidized by
H2O2 and yield the colored products. Hence these colorimetric
detection have been employed for the detection of H2O2 and the
various H2O2 related materials, such as glucose,15 cholesterol16
40 and thrombin.17 As a famous catalyst, noble metal palladium (Pd)
at nanoscales exhibit very high catalytic activity due to their
unique size, shape and high specific surface area. Recent studies
have shown that Pd nanoparticles can present peroxidase-like
catalysis activity for the detection of H2O2.18 However, there are
45 two main problems limit the catalytic performance of Pd
Heteroatom-doped nanostructured carbon materials acted as
excellent supports have received intensive attentions because of
their outstanding catalytic activity, high specific surface area,
55 excellent reliability and relatively low cost.19 Among the
available heteroatoms that can be suitable for doping in carbon
materials, nitrogen has an extra electron that can improve the
electron donation and in turn enhance catalyst performance, while
the electronegativity of sulfur is close to that of carbon.20
60 Therefore, doping carbon materials with dual heteroatoms is
believed to further improve the catalytic activity, since this can be
controlled not only by the increased number of dopant
heteroatoms but also by the synergistic effect arising from co-
doping of heteroatoms.21 In addition, heteroatom-doped
65 nanostructured carbon materials can provide the main initial
nucleation sites for the deposition of nanoparticles resulting in a
very high loading, dispersion, and stability of nanoparticles. Up
to now, some applications of nitrogen, sulfur-doped
nanostructured carbon materials have been developed, such as
70 catalyst,22 energy storage,23 and sensing.24 However, few
investigations have been systematically conducted on the
peroxidase-like catalysis activity of nitrogen, sulfur-doped
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2.2 Characterizations
nanostructured carbon materials as support to load nanoparticles.
The synthesis of nitrogen, sulfur-doped nanostructured carbon
materials have been studied in the literature. A series of materials
including α-lipoic acid,9 pyrrole25 and glutathione24 as carbon
precursors were used in their preparation. However, the raw
materials are expensive and non-renewable, so it is necessary to
develop the low-cost carbon materials from renewable raw
materials. Humic acid (HA) is one of the major components of
humic substances, which is commonly present in soils, brown and
10 brown-black coals, natural waters, river, lake and sea sediments
and other natural materials as a product of degradation of plant,
algal, and microbial material.26 HA consists of carbon, oxygen,
hydrogen and sometimes small amounts of nitrogen and
occasionally phosphorous and sulphur, and contains many
15 chemical functional groups such as carboxylic, carbonyl,
phenolic, and hydroxyl groups connected with the aliphatic or
aromatic carbons in the macromolecules structures.27 Hence, HA
can be used as carbon source to prepare all kinds of new
functional materials.28, 29 It is worth mentioning that the methods
20 to extract HA from straw and industrial waste liquid (such as
pulping waste liquor and fermentation waste water) have made a
great progress in recent years, which provides the new renewable,
easily available and very cheap resources for the production of
HA. Therefore, fully utilizing of inexpensive and earth-abundant
25 material of HA is consistent with the major goals of sustainable
chemistry.
Based on the mentioned above, we firstly used HA and
thiourea as the precursors to prepare the nitrogen, sulfur-doped
three-dimentional hierarchical nanostructures (N-S-CS) and
30 further deposited Pd nanoparticles on it. As expected, the
prepared Pd nanoparticles supported on N-S-CS (Pd/N-S-CS)
possessed intrinsic peroxidase-like catalytic activity, which could
catalyze the peroxidase substrate 3,3′,5,5′-tetramethylbenzidine
(TMB) to produce a blue-colored reaction in the presence of
35 H2O2. Using Pd/N-S-CS peroxidase-like catalytic activity and
xanthine oxidase (XOD), a simple, sensitive and selective
colorimetric method for the detection of xanthine was developed,
and successfully applied for the xanthine detection in urine
samples. This work would pave the way for the usage of
40 renewable HA and promote the further potential application of
Pd/N-S-CS as peroxidase mimics in medical and biochemistry
fields.
Scanning electron microscopy (SEM) images were obVtaieiwneAdrticwleiOthnline
a field emission scanning electron microscopDeO(FI:E10S.E10M39,/SCU5R8A001203,12B
60 Hitachi, Japan). High-resolution transmission electron
microscopy (HRTEM) images were recorded on a high resolution
transmission electron microscopy (Tecnai G2 F20 S-TWIN, FEI
Company, USA) with an accelerating voltage of 200 kV. Powder
X-ray diffraction (XRD) measurements were carried out using a
65 Bruker AXSD8 advanced powder X-ray diffraction system with
Cu Kα-radiation source (Bruker Co., Germany). The X-ray
photoelectron spectroscopy (XPS) analysis was carried out on an
ESCALAB 250 X-ray photoelectron spectrometer (Thermo, USA)
using Mg as the exciting source. The elemental content of the
70 Pd/PCNS material was analyzed with Energy-dispersive X-ray
spectroscope (EDX) (Oxford, England) at an operating voltage of
5 kV. The Brunauer-Emmett-Teller (BET) method was used to
calculate the specific surface areas. UV-Vis absorption spectra
and kinetic measurements were scanned with a Shimadzu UV-
75 2450PC spectrometer (Shimadzu, Japan).
5
2.3 Preparation of N-S-CS
In a typical process, humic acid, thiourea and K2CO3 (the mass
ratio was 8:1:8) were mixed together and ground to
a
homogeneous mixture. The mixture was heated to 600 °C for 2 h
80 in a tube furnace under a dry N2 flow conditions (heating rate:
5 °C·min-1), and then cooled to room temperature. The obtained
product was rinsed with double-distilled water till to constant pH
and finally dried at 60 °C overnight. In addition, carbon
nanosheets (CS) were also prepared under the same experimental
85 conditions without addition of thiourea.
2.4 Preparation of Pd/N-S-CS
The preparation process of Pd/N-S-CS was as follows: 0.2 g N-S-
CS, 5 ml PdCl2 solution (10 mg∙mL-1) and 50 ml glycol solution
were put into three-necked flask. After ultrasonic dispersing for
90 30 min, the reactants were heated at 150 °C for 2min under
microwave irradiation (700W) and cooled down to room
temperature. After that, the dark solution was centrifuged at 3000
rpm for 15 min, and the supernatant was discarded. The
precipitation was rinsed with double-distilled water till to pH = 7,
95 and dialyzed against pure water in a dialysis bag (molecular-
weight cutoff = 3500) for 48h to remove small molecules and
then continue to centrifuge at 3000 rpm for 15 min. Finally, the
product was vacuum-dried at 60 °C for 12 h.
2. Experimental section
2.1 Materials
2.5 Peroxidase-like activity assay
45 Humic
acid
(HA)
(90%
fulvic
acid),
3,3′,5,5′-
100 In order to study the peroxidase-like activity of Pd/N-S-CS, a
typical experiment was performed with 0.1 mg∙mL-1 Pd/N-S-CS
in 400 μL of reaction solution (50 mM PBS, pH 3.5) in the
presence of 10 mM H2O2, using 0.8 mM TMB as the substrate.
The absorbance spectrum of the reaction mixture was measured
105 from 500 to 800 nm. Similar procedures were also performed for
the CS, N-S-CS and Pd nanoparticles. To test the stability of
Pd/N-S-CS, we measured the activities of Pd/N-S-CS at a range
of pH (2.0 ~ 7.0) or a range of temperatures (25 ~ 45 °C) under
the standard conditions mentioned above.
tetramethylbenzidine (TMB), xanthine and ascorbic acid were
purchased from Aladin Ltd. (Shanghai, China). Xanthine oxidase
(XOD), palladium chloride, thiourea and uric acid were received
from Sigma-Aldrich (St. Louis, Missouri, USA). Cholesterol,
50 glucose, glycol, H2O2, NaH2PO4·2H2O and Na2HPO412H2O
were got from Kay Tong Chemical Reagents Co., Ltd. (Tianjin,
China). Dialysis bag (molecular-weight cut off = 3500) was
obtained from Shanghai Sangon biotechnology Development Co.,
Ltd. (Shanghai, China). All chemicals were analytical grade and
55 used as received without further purification. Double-distilled
water was used throughout in all experiments.
110
Furthermore, to investigate the catalytic mechanism, a series of
assays were carried out under standard reaction conditions as
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described above by varying concentrations of H2O2 at a fixed
successful preparation of Pd/N-S-CS.
concentration of TMB or vice versa. The reactants were measured
in time course mode by monitoring the absorbance change at 652
nm for 300 s on a UV-visible spectrophotometer at 30 °C. The
Michaelis–Menten constant was calculated using the
View Article Online
DOI: 10.1039/C5RA02312B
5
Lineweaver–Burk plots of the double reciprocal of the Michaelis–
30
Menten equation, 1/v = (Km/Vmax)(1/[S]) + 1/Vmax
.
In this
equation, v is the initial velocity, Vmax is the maximal reaction
velocity, [S] is the substrate concentration and Km is the
10 Michaelis constant.
45 Fig. 2 (A) XRD patterns of N-S-CS and Pd/N-S-CS. (B) The EDX spectra of
Pd/N-S-CS (inset: the relative weight and atomic percentages of Pd/N-S-
CS.).
2.6 Xanthine detection
Xanthine detection was performed as follows: 15 L of 1 U∙mL-1
XOD and 80 L of xanthine with different concentrations were
incubated for 1 h at 37 °C. Then, 40 L of 8 mM TMB, 80 L of
15 0.5 mg∙mL-1 Pd/N-S-CS, and 185 L of 100 mM PBS (pH 3.5)
were added into the above xanthine reaction solution. The
resulting reaction solution was measured by
spectrophotometer.
For xanthine determination in urine samples, urine samples
20 were firstly treated by centrifugation at 3000 rpm for 10 min, and
then the supernatants were detected as described above.
Fig. 2A shows the typical XRD profiles of N-S-CS and Pd/N-
S-CS. The XRD pattern of N-S-CS has two broader diffraction
50 peaks. The peak at about 2θ = 23.8° corresponds to the (002)
plane reflection, revealing an amorphous carbon phase, which is
attributed to the introduction of nitrogen-, sulfur-, and oxygen-
a UV-Vis
containing groups, and the peak at 43.0° points to the formation
32
of interlayer condensation for the material.31,
In the XRD
55 pattern of Pd/N-S-CS, the sharp peaks at 40.2°, 46.8° and 68.3°
can be referred to the (111), (200) and (222) planes of the cubic
structure of metallic Pd, respectively, suggesting that Pd
nanoparticles are loaded on the N-S-CS material successfully.
EDX analysis was employed to measure the elemental
60 distribution of the Pd/N-S-CS (Fig. 2B). The mass content of C,
N, O, S, and Pd is determined to be 60.82 wt%, 21.48 wt%, 17.51
wt%, 0.11 wt%, and 0.08 wt%, respectively, and the atomic ratio
of N/C is about 0.303 (the inset in Fig. 2B). The large nitrogen
and sulfur doped percentages in carbon texture are suitable for
65 stabilizing highly dispersed Pd NPs and preventing the
reoxidation of Pd0.32
In control experiments, 80 L of 0.25 mM glucose, 0.25 mM
ascorbate, 0.25 mM cholesterol and 0.25 mM uric acid were used
instead of 80 L of 0.25 mM xanthine in a similar way.
25 3. Results and discussion
3.1 Characterization of Pd/N-S-CS
Fig. 1 TEM images of N-S-CS (A, B) and Pd/N-S-CS (C, D).
The TEM images of the prepared N-S-CS are shown in Fig. 1.
30 The product shows interesting three-dimensional hierarchical
nanostructures (Fig. 1A). These nanostructures are composed of
nanosheets with depth gradually becoming thinner to the margin.
From the magnified image in Fig. 1B, the nanosheets morphology
without obvious nanoparticles on it can be clearly observed. Fig.
35 1C shows the image of Pd/N-S-CS. We can clearly see that Pd/N-
S-CS has similar three-dimensional hierarchical nanostructures
with that in Fig. 1A. Furthermore, small nanoparticles can be
observed to be deposited on the surface of three-dimensional
hierarchical nanostructures. As shown in Fig. 1D, nanoparticles
40 with average diameter of 6 nm are uniformly distributed on the
surface of nanosheets (based on the statistical analysis of more
than 50 nanoparticles). These results further indicate the
Fig. 3 XPS spectra of Pd/N-S-CS: (A) XPS survey, (B) C 1s, (C) N 1s, (D) O 1s,
(E) S 2p and (F) Pd 3d.
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The elemental composition and chemical state of Pd/N-S-CS
resulting H2O2 is quantitatively used to oxidize peroxidase
were further confirmed by XPS analysis. Figure 3A shows the
XPS survey spectra of Pd/N-S-CS, which reveals five prominent
peaks associated with C 1s (around 284.4 eV), N 1s (around
400.1 eV), O 1s (around 533.2 eV), S 2p (around 163.1 eV) and
Pd 3d (around 335.7 eV) electrons. This result indicates that both
N and S species have been successfully incorporated into carbon
framework. The high-resolution C 1s XPS spectrum (Fig. 3b)
exhibits five distinct peaks at 284.7, 285.2, 286.0, 286.5, and
substrate TMB to oxTMB in the presence of Pd/N-S-CS. Based
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on the above theories, a sensitive and selective colorimetric
DOI: 10.1039/C5RA02312B
sensor for xanthine detection is established.
5
10 288.3 eV, which confirms the presence of C–C/C＝C, C–S, C–N,
C–O and C＝O/C＝N bonds, respectively.33 The N1s spectrum
(Fig. 3C) can be further deconvoluted into three different peaks at
398.3, 400.5 and 401.2 eV, owing to pyridinic–N, pyrrolic–N and
quaternary–N.34 The O 1s spectrum (Fig. 3D) shows the oxygen
15 signals, including C＝O and C–O, corresponding to the peaks at
531.4 and 532.9 eV, respectively. The S 2p spectrum (Fig. 3E)
confirms two main bands at 163.8 and 168.3 eV, which suggests
sulfur in two forms of –C–S– and –C–SOx–, respectively. The
former can be resolved into two different peaks at 163.5 and
20 164.6 eV, which are assigned to the 2p3/2 and 2p1/2 positions of
thiophene–S owing to their spin-orbit coupling. The latter can be
deconvoluted into three peaks at 167.6, 168.5 and 169.3 eV,
corresponding to some oxidized sulfur such as sulfonate.35 The
Pd 3d XPS spectrum in Fig. 3F consists of two asymmetric peaks
25 attributed to Pd 3d5/2 and Pd 3d3/2 core levels, which can be
fitted using two doublets. The peaks at the binding energies of
335.9 and 341.1 eV are assigned to metallic Pd0, while the peaks
at 337.6 and 342.5 eV correspond to Pd2+ species.36 The
integration of peak areas indicates that most Pd species exist as
30 metallic Pd for Pd/N-S-CS material.
50
55
Fig. 4 Absorbance spectra of TMB in various reaction systems: (a) TMB +
H2O2, (b) TMB + Pd/N-S-CS, and (c) TMB + H2O2 +Pd/N-S-CS. Inset shows
the photographs of the color changes in different reaction systems. The
reaction was performed with 10 mM H2O2 and 0.8 mM TMB in PBS buffer
(50 mM, pH 3.5) for 5 min.
Fig. 4 presents the UV-vis absorption spectra for the different
reaction systems. It is observed that the absorbance at 652 nm
displays negligible change only with the addition of H2O2 (a) or
Pd/N-S-CS (b) to the TMB solution, suggesting that no oxidation
60 reaction is occurred. However, upon addition of H2O2 and Pd/N-
S-CS to the TMB solution (c), there is a great absorbance at 652
nm. This indicates that Pd/N-S-CS can catalyze the oxidation of
TMB by H2O2 to produce the oxTMB, and the solution exhibited
intense characteristic absorbance at 652 nm. Similarly, from the
65 photographs, it is seen that TMB–H2O2 solution in the absence of
Pd/N-S-CS (photograph a) and the TMB–Pd/N-S-CS solution
without H2O2 (photograph b) exhibit no or negligible color
change, while the TMB–H2O2–Pd/N-S-CS system (photograph c)
presents a typical deep blue color. The results indicate that Pd/N-
70 S-CS exhibits excellent peroxidase catalytic ability toward typical
peroxidase substrate TMB. Therefore, a sensitive colorimetric
TMB–H2O2–Pd/N-S-CS sensing platform for H2O2 can be
established due to the extremely low background.
3.2 Peroxidase-like activity of Pd/N-S-CS
Scheme 1 Schematic illustration of peroxidase-like activity of Pd/N-S-CS
and colorimetric detection of xanthine by using XOD and Pd/N-S-CS.
35 Peroxidase-like activity of the synthesized Pd/N-S-CS was
investigated using TMB as the peroxidase substrate and the
xanthine was detected subsequently. The reaction scheme is
described in scheme 1 as follows: upon the addition of Pd/N-S-
CS into TMB–H2O2 solution, a blue-colored reaction is occured,
40 indicating that Pd/N-S-CS has peroxidase-like catalytic behavior,
which can catalyze the colorless TMB to blue oxidized product
(oxTMB) in the presence of H2O2. The catalytic reaction can be
detected by monitoring the oxTMB absorbance change at 652 nm.
The oxidation of xanthine can be catalyzed by XOD in the
45 presence of oxygen to produce uric acid and H2O2, and the
75 Fig. 5 Absorbance spectra of TMB in various reaction systems. Image B is
the magnification of image A. The reaction was performed with 10 mM
H2O2 and 0.8 mM TMB in PBS buffer (50 mM, pH 3.5) for 5 min.
4
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In order to further investigate the catalytic performance of
system containing different concentrations of Pd/N-S-CS. It is
Pd/N-S-CS, we compared the activities of different materials 45 suggested that the catalytic activity of Pd/N-S-CS increases with
under the same conditions. Fig. 5 shows the absorbance spectra of
TMB in various reaction systems. From the image A and its
magnified image (image B), it is clear that there is no obvious
absorption at 652 nm upon the addition of CS (0.1 mg∙mL-1) into
the increasing concentrations of Pd/N-S-CS, whichViieswcAortnicsleisOtnelninte
DOI: 10.1039/C5RA02312B
with the color changes of TMB with different concentrations of
Pd/N-S-CS (Fig. 6A inset). Furthermore, as shown in Fig. 6B, the
intensity of the absorption at 652 nm sharply increases with
5
TMB–H2O2 solution in comparison with blank solution (TMB– 50 increasing Pd/N-S-CS concentration from 0.002 to 0.05 mg∙mL-1,
H2O2), suggesting that TMB is hardly oxidized by H2O2 with CS
as the catalyst. However, the absorbance of the TMB–H2O2–N-S-
and then it gradually slows down when the concentration is
beyond 0.1 mg∙mL-1. Therefore, the concentration of 0.1 mg∙mL-1
is used in our experiment.
10 CS (0.1 mg∙mL-1) system is much higher, indicating that the
catalytic activity of N-S-CS is remarkably enhanced as compared
The catalytic activity of Pd/N-S-CS is also dependent on pH
with CS. The reason is that the doping of N and S into the carbon 55 and temperature. In our experiment, phosphate buffer was chosen
framework makes a great contribution to the enhancement of
catalytic activity, and this synergistic enhancement is considered
15 to be from the redistribution of spin and charge densities after the
dual doping of N and S atoms.37 Pd nanoparticles have been
for the reaction system and the activity of Pd/N-S-CS was
investigated from pH 2.0 to 7.0. As shown in Fig. 6C, the
absorbance at 652 nm increases in intensity with pH in the range
of 2.0-3.5, and then declines on increasing the pH further.
found to exhibit outstanding peroxidase-like activities, so an 60 Similarly, in Fig. 6C inset, the solution exhibits the deep blue
evident absorbance at 652 nm is observed upon the addition of Pd
nanoparticles (0.03 mg∙mL-1) into TMB–H2O2 solution. However,
20 there is the highest absorbance of TMB at 652 nm after adding
Pd/N-S-CS (0.1 mg∙mL-1) into TMB–H2O2 solution, which
color at pH 3.5 but a light blue at lower or higher pH. The
influence of temperature upon the catalytic activity of Pd/N-S-CS
has been investigated and the results are shown in Fig. 6D. We
can see that the absorbance at 652 nm firstly increase and then
suggests that Pd/N-S-CS exhibits higher peroxidase-like activity 65 decrease with temperature ranging from 20 °C to 45 °C, and
than the other catalysts. Furthermore, it is observed that the
catalytic activity of Pd/ N-S-CS is not a simple addition of the
25 activities of N-S-CS and Pd, but exhibits a much stronger
catalytic activity. This interesting phenomena may be that Pd
nanoparticles is highly dispersed on the surface of N, S-doped
carbon nanosheets, and the synergistic effect between Pd and N-
S-CS can create new active reaction sites, which make Pd/ N-S-
30 CS show a superb peroxidase-like activity. In addition, the high
surface area (BET surface area was measured to be 263.330 m2 g-
1) of Pd/N-S-CS may be another reason for the higher peroxidase-
like activity.
giving a maximum value at 30 °C. The experiments results show
that the optimal pH and reaction temperature are approximately
3.5 and 30 °C, respectively.
3.4 Steady-state kinetic assay of Pd/N-S-CS
3.3 Optimization of experimental conditions
70
Fig. 7 Steady-state kinetic assay and catalytic mechanism of Pd/N-S-CS. (A)
The concentration of H2O2 was 90 mM and the TMB concentration was
varied. (B) The concentration of TMB was 1.5 mM and the H2O2
concentration was varied. (C and D) Double reciprocal plots of activity of
75 Pd/N-S-CS with the concentration of one substrate (H2O2 or TMB) fixed
and the other varied. The velocity (V) of the reaction was measured using
0.1 mg∙mL-1 Pd/N-S-CS in 400μL of 50 mM PBS (pH 3.5) for 5 min. The
error bars represent the standard deviation of three measurements.
To investigate the kinetic mechanism of the peroxidase-like
80 activity of Pd/N-S-CS, the steady-state kinetic parameters were
determined using TMB and H2O2 as substrates. A series of
experiments were performed by varying the concentration of one
substrate while keeping the other one constant. Over a suitable
range of TMB and H2O2 concentrations, the plots of initial
85 reaction rates vs TMB or H2O2 concentration show typical
Michaelis−Menten behavior as shown in Fig. 7A and B,
respectively. Furthermore, a series of the initial velocity were
35
Fig. 6 Dependence of the peroxidase-like activity of Pd/N-S-CS on (A, B)
Pd/N-S-CS concentration (C) pH and (D) temperature in PBS buffer (50
mM) with 10 mM H2O2 and 0.8 mM TMB for 5 min. Inset: typical
photographs of different reaction conditions.
40 To find the optimum experimental conditions, the effects of
various concentrations of Pd/N-S-CS, the pH of the reaction
buffer and the incubation temperature were investigated in detail.
Fig. 6A shows the absorbance spectra of the TMB–H2O2
This journal is © The Royal Society of Chemistry [year]
Journal Name, [year], [vol], 00–00 | 5

RSC Advances
Page 6 of 7
calculated and the double reciprocal plots were obtained. From
the double reciprocal plots of the initial velocity against one of
the substrate concentrations when the other substrate is fixed at
xanthine is from 0.001 to 0.05 mM with a detection limit of 2.9×
10−7 M. The limit of detection for xanthine using Pd/N-S-CS is
View Article Online
lower than that reported in the previous report,41 suggesting that
DOI: 10.1039/C5RA02312B
three concentration levels, we demonstrates that the catalytic 50 the prepared Pd/N-S-CS may play an important role in clinical
5
reaction of Pd/N-S-CS follows a ping-pong mechanism due to the
slopes of the lines are almost parallel (Fig. 7C and D).38 This
result indicates that similar to HRP, Pd/N-S-CS binds and reacts
with the first substrate and then releases the first product before
reacting with the second substrate, no tertiary intermediate of an
medicine.
3.6 Detection of xanthine in real urine samples
10 enzyme.3 The Michaelis-Menten constant (Km) and maximal
reaction velocity (Vmax) of Pd/N-S-CS were obtained from the
Lineweaver-Burk plot and the Michaelis-Menten equation, and
were listed in Table 1. Km is often associated with the affinity of
the enzyme for the substrates, and the lower value means the
15 higher affinity.39 In our experiment, the Km value of Pd/N-S-CS
with H2O2 as the substrate is significantly lower than that of
Fe3O4 MNPs (42.7 mM for Pd/N-S-CS vs. 154 mM for Fe3O4
MNPs), suggesting that Pd/N-S-CS has a higher affinity for H2O2
than Fe3O4 MNPs. However, the Km value of Pd/N-S-CS with
20 TMB as the substrate is higher than that of Fe3O4 MNPs (1.44
mM for Pd/N-S-CS vs. 0.098 mM for Fe3O4 MNPs),40 revealing
that Pd/N-S-CS has a lower affinity for TMB than Fe3O4 MNPs,
corresponding to a relative higher TMB concentration used in
experiments.
Fig. 9 (A) Time-dependent absorbance changes at 652 nm for urine
55 sample after incubation with xanthine oxidase. (B) Selectivity analysis of
this system for xanthine detection by measuring the absorbance at 652
nm. The error bars represent the standard deviation of three
measurements.
To validate that the proposed determination method could have
60 practical application for xanthine analysis in real samples, we
applied the TMB–H2O2–Pd/N-S-CS sensor to determine the
levels of xanthine in urine sample of a healthy human volunteer.
Fig. 9A shows the time-dependent absorbance changes at 652 nm
of oxidized TMB for urine sample after incubation with xanthine
65 oxidase. According to the calibration curve and the absorbance of
oxidized TMB in urine sample system, the concentration of
xanthine in the normal human urine sample is found to be 1.09
μM, which is very close to the reported values in urine samples.42
In addition, the xanthine detection with the prosed colorimetric
70 method is in good agreement with the result obtained from high
performance liquid chromatographic (HPLC) method (1.1±0.18
μM),43 indicating that the proposed colorimetric method can be
used for the xanthine detection in real samples.
25 Table 1 The Michaelis-Menten constant (Km) and maximum reaction rate
(Vmax) of Pd/ N-S-CS.
catalyst
substance
TMB
Km [mM]
1.44
Vmax (10-6 M s-1)
0.241
Pd/N-S-CS
Pd/N-S-CS
H2O2
42.7
0.389
3.5 Detection of H2O2 and xanthine
To investigate the selectivity for xanthine detection, control
75 experiments were carried out using glucose, ascorbate,
cholesterol and uric acid. The results (Fig. 9B) demonstrate that
there is no obvious response for the control samples in
comparison with the buffer, indicating that the colorimetric
detection method shows a high selectivity for xanthine detection.
Fig. 8 (A) The linear calibration plots for H2O2. (B) The linear calibration
30 plots for xanthine. Experiments were carried out in PBS buffer (50 mM)
with 10 mM H2O2 and 0.8 mM TMB for 5 min. The error bars represent
the standard deviation of three measurements.
80 4. Conclusion
In summary, a facile and green method had been established to
fabricate N-S-CS by carbonization of HA with the assistance of
thiourea, and then Pd nanoparticles successfully deposited on N-
S-CS by a glycol reduction method. The obtained Pd/N-S-CS
85 showed interesting three-dimensional hierarchical nanostructures
which were composed of nanosheets, and Pd nanoparticles with
average diameter of 6 nm were distributed on the surface of
nanosheets. Furthermore, the as-prepared Pd/N-S-CS possessed
intrinsic peroxidase-like activity, which could catalyze the
90 oxidation of TMB by H2O2 to produce a typical blue color
reaction. Kinetic experiments indicated that the catalysis was in
accord with the typical Michaelis-Menten kinetics and followed a
ping-pong mechanism. Based on this finding, colorimetric
methods for the detection of H2O2 and xanthine were developed,
On the basis of the peroxidase-like activity of Pd/N-S-CS, a
colorimetric method for the detection of H2O2 was developed by
35 using the TMB−H2O2−Pd/N-S-CS system. Fig. 8A presents the
H2O2 calibration curve over H2O2 concentrations ranging from
0.01 to 0.6 mM. As a result, the absorbance at 652 nm increases
with the increasing of H2O2 concentration. The curve is linear in
the range from 0.01 to 0.6 mM, and the linear regression equation
40 is A652nm = 0.1054 + 0.3753x (x: mM, R2 = 0.9986), with a
detection limit for H2O2 of 3.3 × 10−6 M. H2O2 is the main
product of XOD-catalyzed reaction. Therefore, combined with
XOD, the proposed colorimetric method can be used for the
determination of xanthine. Fig. 8B exhibits the linear calibration
45 plots for xanthine. The linear regression equation is A652nm
=
0.0336 + 2.4758x (x: mM, R2 = 0.9967), and the linear range for
6
| Journal Name, [year], [vol], 00–00
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Page 7 of 7
RSC Advances
65 25 Y. Meng, D. Voiry, A. Goswami, X. Zou, X. Huang, M. Chhowalla,
and the practical application for xanthine analysis in urine
Z. Liu and T. Asefa, J. Am. Chem. Soc., 2014, 136, 13554-13557.
samples was performed. Because the method is easily accessible,
low cost, high sensitivity and selectivity, the Pd/N-S-CS material
as enzymatic mimics has great potential for practical use in
biotechnology and biochemistry.
26 J. r. Novák, J. Kozler, P. Janoš, J. Čežıková, V. Tokarová and L.
View Article Online
DOI: 10.1039/C5RA02312B
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This work was supported by the National Natural Science
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15 b College of Resources and Environment, Shandong Agricultural
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