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

Abstract

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.

---

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-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-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 graphene oxide¹⁴), etc. With the catalysis of these reported nanomaterials, some chromogenic substrates can be oxidized by H₂O₂ and yield colored products. Hence this colorimetric detection has been employed for the detection of H₂O₂ and various H₂O₂ related materials, such as glucose,¹⁵ cholesterol¹⁶ and thrombin.¹⁷ As a famous catalyst, noble metal palladium (Pd) at nanoscale 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 two main problems limiting the catalytic performance of Pd 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 dispersion and stability as well as alleviate the cost of Pd catalysts, it is helpful to load them on a proper support.

Heteroatom-doped nanostructured carbon materials acted as excellent supports have received intensive attentions because of their outstanding catalytic activity, high specific surface area, 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 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 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 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 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 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 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 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 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-dimensional hierarchical nanostructures (N-S-CS) and 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 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 renewable HA and promote the further potential application of Pd/N-S-CS as peroxidase mimics in medical and biochemistry fields.

2. Experimental section

2.1 Materials

Humic acid (HA) (90% fulvic acid), 3,3′,5,5′-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, 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 used as received without further purification. Double-distilled water was used throughout in all experiments.

2.2 Characterizations

Scanning electron microscopy (SEM) images were obtained with a field emission scanning electron microscope (FESEM, SU8010, 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 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 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-2450PC spectrometer (Shimadzu, Japan).

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 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 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 30 min, the reactants were heated at 150 °C for 2 min under microwave irradiation (700 W) 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, and dialyzed against pure water in a dialysis bag (molecular-weight cutoff = 3500) for 48 h 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.

2.5 Peroxidase-like activity assay

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 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.

Furthermore, to investigate the catalytic mechanism, a series of assays were carried out under standard reaction conditions as described above by varying concentrations of H₂O₂ at a fixed 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 Lineweaver–Burk plots of the double reciprocal of the Michaelis–Menten equation, 1/v = (K_m/V_max)(1/[S]) + 1/V_max.³⁰ In this equation, v is the initial velocity, V_max is the maximal reaction velocity, [S] is the substrate concentration and K_m is the Michaelis constant.

2.6 Xanthine detection

Xanthine detection was performed as follows: 15 μL of 1 U mL⁻¹ XOD and 80 μL of xanthine with different concentrations were incubated for 1 h at 37 °C. Then, 40 μL of 8 mM TMB, 80 μL of 0.5 mg mL⁻¹ Pd/N-S-CS, and 185 μL of 100 mM PBS (pH 3.5) were added into the above xanthine reaction solution. The resulting reaction solution was measured by a UV-Vis spectrophotometer.

For xanthine determination in urine samples, urine samples were firstly treated by centrifugation at 3000 rpm for 10 min, and then the supernatants were detected as described above.

In control experiments, 80 μL of 0.25 mM glucose, 0.25 mM ascorbate, 0.25 mM cholesterol and 0.25 mM uric acid were used instead of 80 μL of 0.25 mM xanthine in a similar way.

3. Results and discussion

3.1 Characterization of Pd/N-S-CS

The TEM images of the prepared N-S-CS are shown in Fig. 1. 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. 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 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 successful preparation of Pd/N-S-CS.

Fig. 1 TEM images of N-S-CS (A and B) and Pd/N-S-CS (C and D).

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 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-containing groups, and the peak at 43.0° points to the formation of interlayer condensation for the material.^31,32 In the XRD 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 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 stabilizing highly dispersed Pd NPs and preventing the reoxidation of Pd⁰.³²

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.).

The elemental composition and chemical state of Pd/N-S-CS were further confirmed by XPS analysis. Fig. 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 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 N 1s 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 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–SO_x–, respectively. The former can be resolved into two different peaks at 163.5 and 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 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 metallic Pd for Pd/N-S-CS material.

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.

3.2 Peroxidase-like activity of Pd/N-S-CS

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, 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 presence of oxygen to produce uric acid and H₂O₂, and the resulting H₂O₂ is quantitatively used to oxidize peroxidase substrate TMB to oxTMB in the presence of Pd/N-S-CS. Based on the above theories, a sensitive and selective colorimetric sensor for xanthine detection is established.

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.

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 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 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-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.

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.

In order to further investigate the catalytic performance of Pd/N-S-CS, we compared the activities of different materials 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 TMB–H₂O₂ solution in comparison with blank solution (TMB–H₂O₂), suggesting that TMB is hardly oxidized by H₂O₂ with CS as the catalyst. However, the absorbance of the TMB–H₂O₂–N-S-CS (0.1 mg mL⁻¹) system is much higher, indicating that the catalytic activity of N-S-CS is remarkably enhanced as compared with CS. The reason is that the doping of N and S into the carbon framework makes a great contribution to the enhancement of catalytic activity, and this synergistic enhancement is considered to be from the redistribution of spin and charge densities after the dual doping of N and S atoms.³⁷ Pd nanoparticles have been found to exhibit outstanding peroxidase-like activities, so an evident absorbance at 652 nm is observed upon the addition of Pd nanoparticles (0.03 mg mL⁻¹) into TMB–H₂O₂ solution. However, 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 suggests that Pd/N-S-CS exhibits higher peroxidase-like activity than the other catalysts. Furthermore, it is observed that the catalytic activity of Pd/N-S-CS is not a simple addition of the 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-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.

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.

3.3 Optimization of experimental conditions

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 system containing different concentrations of Pd/N-S-CS. It is suggested that the catalytic activity of Pd/N-S-CS increases with the increasing concentrations of Pd/N-S-CS, which is consistent 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 increasing Pd/N-S-CS concentration from 0.002 to 0.05 mg mL⁻¹, 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.

Fig. 6 Dependence of the peroxidase-like activity of Pd/N-S-CS on (A and 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.

The catalytic activity of Pd/N-S-CS is also dependent on pH and temperature. In our experiment, phosphate buffer was chosen 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. Similarly, in Fig. 6C inset, the solution exhibits the deep blue 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 decrease with temperature ranging from 20 °C to 45 °C, and 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

To investigate the kinetic mechanism of the peroxidase-like 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 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 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 three concentration levels, we demonstrates that the catalytic 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 enzyme.³ The Michaelis–Menten constant (K_m) and maximal reaction velocity (V_max) of Pd/N-S-CS were obtained from the Lineweaver–Burk plot and the Michaelis–Menten equation, and were listed in Table 1. K_m is often associated with the affinity of the enzyme for the substrates, and the lower value means the higher affinity.³⁹ In our experiment, the K_m 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 K_m value of Pd/N-S-CS with 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. 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 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 400 μL of 50 mM PBS (pH 3.5) for 5 min. The error bars represent the standard deviation of three measurements.

Table 1 The Michaelis–Menten constant (K_m) and maximum reaction rate (V_max) of Pd/N-S-CS

Catalyst	Substance	Km [mM]	Vmax (10^−6 M s^−1)
Pd/N-S-CS	TMB	1.44	0.241
Pd/N-S-CS	H2O2	42.7	0.389

---

3.5 Detection of H₂O₂ and xanthine

On the basis of the peroxidase-like activity of Pd/N-S-CS, a colorimetric method for the detection of H₂O₂ was developed by 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 is A_{652 nm} = 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 plots for xanthine. The linear regression equation is A_{652 nm} = 0.0336 + 2.4758x (x: mM, R² = 0.9967), and the linear range for 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 lower than that reported in the previous report,⁴¹ suggesting that the prepared Pd/N-S-CS may play an important role in clinical medicine.

Fig. 8 (A) The linear calibration plots for H₂O₂. (B) The linear calibration 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.

3.6 Detection of xanthine in real urine samples

To validate that the proposed determination method could have 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 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 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.

Fig. 9 (A) Time-dependent absorbance changes at 652 nm for urine 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 investigate the selectivity for xanthine detection, control 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.

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 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 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, and the practical application for xanthine analysis in urine 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.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (no. 21375079, no.51402175), and Project of Development of Science and Technology of Shandong Province, China (no. 2013GZX20109).

Notes and references

1. T. Lin, L. Zhong, Z. Song, L. Guo, H. Wu, Q. Guo, Y. Chen, F. Fu and G. Chen, Biosens. Bioelectron., 2014, 62, 302–307.
2. F. Qiao, L. Chen, X. Li, L. Li and S. Ai, Sens. Actuators, B, 2014, 193, 255–262.
3. L. Gao, J. Zhuang, L. Nie, J. Zhang, Y. Zhang, N. Gu, T. Wang, J. Feng, D. Yang, S. Perrett and X. Yan, Nat. Nano, 2007, 2, 577–583.
4. W. Chen, J. Chen, Y.-B. Feng, L. Hong, Q.-Y. Chen, L.-F. Wu, X.-H. Lin and X.-H. Xia, Analyst, 2012, 137, 1706–1712.
5. I. Celardo, J. Z. Pedersen, E. Traversa and L. Ghibelli, Nanoscale, 2011, 3, 1411–1420.
6. Y. Fan and Y. Huang, Analyst, 2012, 137, 1225–1231.
7. T. Zhang, Y. Lu and G. Luo, ACS Appl. Mater. Interfaces, 2014, 6, 14433–14438.
8. L. Su, J. Feng, X. Zhou, C. Ren, H. Li and X. Chen, Anal. Chem., 2012, 84, 5753–5758.
9. R. André, F. Natálio, M. Humanes, J. Leppin, K. Heinze, R. Wever, H. C. Schröder, W. E. G. Müller and W. Tremel, Adv. Funct. Mater., 2011, 21, 501–509.
10. Y. Jv, B. Li and R. Cao, Chem. Commun., 2010, 46, 8017–8019.
11. M. Ma, Y. Zhang and N. Gu, Colloids Surf., A, 2011, 373, 6–10.
12. Y. Song, X. Wang, C. Zhao, K. Qu, J. Ren and X. Qu, Chem.–Eur. J., 2010, 16, 3617–3621.
13. W. Shi, Q. Wang, Y. Long, Z. Cheng, S. Chen, H. Zheng and Y. Huang, Chem. Commun., 2011, 47, 6695–6697.
14. Y. Song, K. Qu, C. Zhao, J. Ren and X. Qu, Adv. Mater., 2010, 22, 2206–2210.
15. T. Lin, L. Zhong, J. Wang, L. Guo, H. Wu, Q. Guo, F. Fu and G. Chen, Biosens. Bioelectron., 2014, 59, 89–93.
16. R. Li, C. Xiong, Z. Xiao and L. Ling, Anal. Chim. Acta, 2012, 724, 80–85.
17. C. Zheng, A.-X. Zheng, B. Liu, X.-L. Zhang, Y. He, J. Li, H.-H. Yang and G. Chen, Chem. Commun., 2014, 50, 13103–13106.
18. J. Lan, W. Xu, Q. Wan, X. Zhang, J. Lin and J. Chen, Anal. Chim. Acta, 2014, 825, 63–68.
19. F. Su, Z. Tian, C. K. Poh, Z. Wang, S. H. Lim, Z. Liu and J. Lin, Chem. Mater., 2010, 22, 832–839.
20. H. Wang, X. Bo, Y. Zhang and L. Guo, Electrochim. Acta, 2013, 108, 404–411.
21. J. Liang, Y. Jiao, M. Jaroniec and S. Z. Qiao, Angew. Chem., Int. Ed., 2012, 51, 11496–11500.
22. J. Shi, X. Zhou, P. Xu, J. Qiao, Z. Chen and Y. Liu, Electrochim. Acta, 2014, 145, 259–269.
23. W. Si, J. Zhou, S. Zhang, S. Li, W. Xing and S. Zhuo, Electrochim. Acta, 2013, 107, 397–405.
24. W. Wang, Y.-C. Lu, H. Huang, A.-J. Wang, J.-R. Chen and J.-J. Feng, Sens. Actuators, B, 2014, 202, 741–747.
25. Y. Meng, D. Voiry, A. Goswami, X. Zou, X. Huang, M. Chhowalla, Z. Liu and T. Asefa, J. Am. Chem. Soc., 2014, 136, 13554–13557.
26. J. R. Novák, J. Kozler, P. Janoš, J. Čežıíková, V. Tokarová and L. Madronová, React. Funct. Polym., 2001, 47, 101–109.
27. S. Maghsoodloo, B. Noroozi, A. K. Haghi and G. A. Sorial, J. Hazard. Mater., 2011, 191, 380–387.
28. S. Salati, G. Papa and F. Adani, Biotechnol. Adv., 2011, 29, 913–922.
29. F. N. Crespilho, V. Zucolotto, J. R. Siqueira, C. J. L. Constantino, F. C. Nart and O. N. Oliveira, Environ. Sci. Technol., 2005, 39, 5385–5389.
30. L. Chen, K. Sun, P. Li, X. Fan, J. Sun and S. Ai, Nanoscale, 2013, 5, 10982–10988.
31. D. Sun, R. Ban, P.-H. Zhang, G.-H. Wu, J.-R. Zhang and J.-J. Zhu, Carbon, 2013, 64, 424–434.
32. X. Xu, Y. Li, Y. Gong, P. Zhang, H. Li and Y. Wang, J. Am. Chem. Soc., 2012, 134, 16987–16990.
33. H. Ding, J.-S. Wei and H.-M. Xiong, Nanoscale, 2014, 6, 13817–13823.
34. W. Si, J. Zhou, S. Zhang, S. Li, W. Xing and S. Zhuo, Electrochim. Acta, 2013, 107, 397–405.
35. Y. Hu, J. Yang, J. Tian, L. Jia and J.-S. Yu, Carbon, 2014, 77, 775–782.
36. P. Zhang, Y. Gong, H. Li, Z. Chen and Y. Wang, Nat. Commun., 2013, 4, 1593–1603.
37. H. Ding, J. S. Wei and H. M. Xiong, Nanoscale, 2014, 6, 13817–13823.
38. K.-I. Hsu, C.-W. Lien, C.-H. Lin, H.-T. Chang and C.-C. Huang, RSC Adv., 2014, 4, 37705–37713.
39. T. Zhang, Y. Lu and G. Luo, ACS Appl. Mater. Interfaces, 2014, 6, 14433–14438.
40. L. Gao, J. Zhuang, L. Nie, J. Zhang, Y. Zhang, N. Gu, T. Wang, J. Feng, D. Yang, S. Perrett and X. Yan, Nat. Nanotechnol., 2007, 2, 577–583.
41. R. Devi, S. Yadav and C. S. Pundir, Colloids Surf., A, 2012, 394, 38–45.
42. X. X. Wang, Q. Wu, Z. Shan and Q. M. Huang, Biosens. Bioelectron., 2011, 26, 3614–3619.
43. R. A. Harkness, J. Chromatogr., Biomed. Appl., 1988, 429, 255–278.

---