Enhanced peroxidase-like activity of Mo-doped ceria nanoparticles for sensitive colorimetric detection of glucose

Abstract

The development of highly efficient nanomaterial-based mimetic enzymes is much needed to replace natural enzymes due to their low cost, high stability and efficient catalytic ability. Herein, multiple Mo-doped CeO₂ nanoparticles (Mo/CeO₂ NPs) with different molar ratios of Mo were synthesized using a facile microwave-assisted method. For characterizing the as-prepared Mo/CeO₂ NPs, XRD, TEM, HRTEM, BET and XPS have been performed systematically. The peroxidase-like activity of Mo/CeO₂ NPs was investigated, and they exhibited enhanced peroxidase-like activity in comparison to that of pure CeO₂ NPs, the best peroxidase-like activity was obtained for the Mo/CeO₂ NPs when the molar ratio of Mo was 9%. On the basis of the high activity of Mo/CeO₂ NPs, the reaction provides a simple, sensitive and selective method for colorimetric detection of glucose with the detection limit of 4 × 10⁻⁷ mol L⁻¹ and in a dynamic range from 5 × 10⁻⁷ to 5 × 10⁻⁵ mol L⁻¹. This work will pave a way for high throughput synthesis of other ultra-small doped metal oxide nanoparticles, which have bright prospects in biomedical analysis.

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Introduction

Natural enzymes with high substrate specificity and efficiency under mild reaction conditions have been widely studied and applied in biotechnology, clinical diagnosis, biosensing, environmental science, the food industry, etc.^1–3 In particular, horseradish peroxidase (HRP) is one of the most important natural enzymes, which can be used in the detection of H₂O₂ and glucose, and thus HRP has enormous potential in biomedicine or environmental monitoring.⁴ However, the intrinsic drawbacks of natural enzymes, such as easy denaturation and high costs in preparation and purification, have hampered their widespread applications.^5,6 In order to overcome the limitations, great efforts have been devoted to exploring various artificial enzyme mimetics based on nanomaterials. Compared with natural enzymes, peroxidase-like nanomaterials exhibit many advantages such as good stability, easy preparation, low cost, great resistance to harsh chemical environments and tunable catalytic activities. Various nanomaterials have been evaluated as peroxidase-like mimetics, including ferromagnetic-based nanomaterials,^7–10 noble metal nano-particles,^11–13 carbon-based nanomaterials,^14–17 graphene dots¹⁸ and metal oxide nanomaterials.^19–26

Among various peroxidase-like nanomaterials, nanoceria has been attracting particular attention due to its unique physical and chemical properties.^27–32 The catalytic activity of CeO₂ is mainly ascribed to the reversible switch between Ce⁴⁺ ↔ Ce³⁺ and the presence of associated oxygen vacancies. Therefore, there has been increasing interest focused on the wide usage of CeO₂ as peroxidase mimetics and subsequent application in bioanalysis.^30,33–35 In order to improve the catalytic activity of CeO₂, obtaining more surface defects and adjusting the Ce⁴⁺/Ce³⁺ ratio via homogeneous doping with suitable heteroatoms has been developed as a feasible strategy;^36–38 the improvement in catalytic activity is due to the exchange of oxygen in the oxide network by decreasing the energy barrier for oxygen migration and the existence of more surface defects.³⁹ Multiple metal elements, such as Co,³⁸ Mn,⁴⁰ Cu,⁴¹ Fe³⁶ and Ti⁴², have been used as dopants for CeO₂ to change the structure and surface, which can efficiently increase the concentration of oxygen vacancies, as well as enhance peroxidase mimic activity. Particularly, several reports have proved that Mo could promote the catalytic activity and thermal stability of the catalyst.^37,43 Wang et al.³⁷ reported that Mo-doped CeO₂ solid solution modified Pt catalysts exhibited remarkable catalytic activity and stability, showing superior performance compared to the commercial Pt/C-JM catalyst. Zhan et al.⁴³ reported that CeMo (0.3) hollow microspheres exhibited much better NH₃-SCR catalytic activity, stability and H₂O resistance in the low-temperature region than pure CeO₂ microspheres. To the best of our knowledge, there were few studies about the peroxidase mimetic catalytic properties of Mo-doped CeO₂ NPs (Mo/CeO₂ NPs).

Herein, Mo/CeO₂ NPs with high yield, a narrow size range and high catalytic activity are synthesized via a facile microwave-assisted synthesis route. Compared with other preparation methods, microwave-assisted methods can obtain Mo/CeO₂ NP catalysts with a higher surface area and higher crystallinity^44,45 at lower temperature. Besides, it was found that the Mo/CeO₂ NPs possess enhanced intrinsic peroxidase-like activity in contrast to the pure CeO₂ NPs without being doped with Mo. The effects of doping with Mo on the material structure, properties and catalytic performance as peroxidase mimics were discussed in detail, and reaction kinetics and mechanisms of obtained Mo/CeO₂ NPs were also investigated. Based on the greatly enhanced peroxidase-like activity, a novel facile colorimetric method with a wide linear range and low detection limit for glucose is further developed (Scheme 1).

Scheme 1 The procedures for the preparation of CeO₂ and Mo/CeO₂ NPs and glucose detection.

Experimental

Chemicals and materials

Cerium nitrate (Ce(NO₃)₃·6H₂O, 99.9%), ammonium molybdate tetrahydrate (H₂₄Mo₇N₆O₂₄·4H₂O, 99.9%), glucose, glucose oxidase (GOx, ≥180 U mg⁻¹), 3,3,5,5-tetramethylbiphenyl dihydrochloride (TMB·2HCl) and horseradish peroxidase (HRP, ≥300 U mg⁻¹) were purchased from Aladdin Chemical Reagent Co., China. Ammonium hydroxide (25–28 wt%, NH₃ H₂O), hydrogen peroxide (30 wt%, H₂O₂), acetic acid (HAc), sodium acetate (NaAc), disodium hydrogen phosphate (Na₂HPO₄), and sodium dihydrogen phosphate (NaH₂PO₄) were purchased from Kelong Reagent Co. (Chengdu, China). All chemicals were of analytical grade and used as received without further purification. Deionized water (18.25 MΩ cm) was obtained using a ULUPURE Water Purification System (NEX UP 1000) and was used to make all aqueous solutions. Human serum samples were obtained from the Affiliated Hospital of Zunyi Medical College (Zunyi, China).

Synthesis of Mo/CeO₂ NPs

Mo/CeO₂ NPs with various contents of Mo (3, 6, 9, 12, 15 and 18%) were prepared via a microwave-assisted synthesis route; the obtained products were labeled as 3Mo/CeO₂, 6Mo/CeO₂, 9Mo/CeO₂, 12Mo/CeO₂, 15Mo/CeO₂ and 18Mo/CeO₂. In each experiment, the desired Mo doping atomic percentage (at%) was calculated according to the following equation:

In a typical synthesis, for 6% Mo-doped CeO₂ NPs, 0.7903 g of Ce(NO₃)₃·6H₂O and 0.2224 g of H₂₄Mo₇N₆O₂₄·4H₂O were dissolved in 20 mL distilled water and kept stirring for 10 minutes. Then, 0.1 mol L⁻¹ NaOH solution was slowly added to the above solution until the pH value reached 12 and aged for 0.5 h under vigorous stirring. Afterwards, the resultant mixture was transferred into a 100 mL microwave-reactor for 4 h at 100 °C. The precipitate was collected by centrifugation, washed with distilled water and ethanol several times until neutrality. The sample was dried at 80 °C for 12 h in a vacuum oven for the following experiments and characterizations. The synthesis conditions were optimized through adjusting the reaction temperature, reaction time and reaction pH respectively. CeO₂ was prepared by the same method without doping with Mo.

The dried nanoparticle powders were dispersed in ultra-pure water and then the mixture was homogenized by ultrasonication (80 W) for 30 min. The concentration of CeO₂ NPs was about 0.6 mg mL⁻¹ for the following experiments.

Characterization and instruments

The micro-wave reactor was Discover SP Installation from GEM Company. The UV-vis absorption spectra were obtained on aqueous dispersions at room temperature using a TU-1901 spectrophotometer (Persee). Powder X-ray diffraction (XRD) measurements were performed using an X'Pert Pro X-ray diffractometer (Philips) with Cu K_α radiation (λ = 1.5406 Å). The TEM, HRTEM images were obtained from a Tecnai G2F20 S-TWIN (FEI). X-ray photoelectron spectroscopy (XPS) was performed with an XSAM 800 Electron Spectrometer (Kratos) using monochromatic Mg K_α radiation. The Brunauer–Emmett–Teller (BET) surface area and the pore size distribution of the materials were determined using N₂ adsorption and desorption (QUADRASORB SI, Quantachrome, USA) at 77 K over a relative pressure ranging from 0.050 to 0.99.

Peroxidase-like activity of Mo/CeO₂ NPs

The peroxidase-like activity of the CeO₂ NPs, Mo/CeO₂ NPs and HRP were evaluated in the catalysis of peroxidase substrate TMB·2HCl in the presence of H₂O₂. The detailed process: the mixtures including the catalyst (10 μL Mo/CeO₂ NPs, 10 μL CeO₂ NPs or 1.0 ng HRP) in 2.0 mL 0.2 mol L⁻¹ acetate buffer, with 5 mM TMB·2HCl as the substrate in the presence of H₂O₂ with a concentration of 10 mM were incubated in a water bath for 30 min at 45 °C. The UV absorbance was recorded at 652 nm after reaction for 30 min to reach the stable state. The influences of pH or temperature on the catalytic activity of the catalyst (Mo/CeO₂ NPs, CeO₂ NPs, and HRP) were measured according to the above assay, except that the pH (2.0–12.0) or temperature (25–80 °C) of the reaction system was changed, respectively. The Mo/CeO₂ NPs were incubated with different concentrations of H₂O₂ at 45 °C to determine their H₂O₂ concentration-related catalytic activity.

The reaction kinetics measurements were carried out in time course mode by monitoring the absorbance change at 652 nm with one minute intervals using a Perkin Elmer UV spectrophotometer. The experiments were carried out at 45 °C with 10 mL 0.6 mg mL⁻¹ Mo/CeO₂ solution or 0.6 mg mL⁻¹ CeO₂ in 2.0 mL reaction buffer (0.2 mol L⁻¹ NaAc, pH 4.0) with 5 mM TMB·2HCl as the substrate, the H₂O₂ concentration was 50 mM for Mo/CeO₂ and CeO₂ NPs. The Michaelis–Menten constant was calculated from the Lineweaver–Burk double reciprocal plot based on the equation 1/v = (K_m/V_max) × (1/[S]) + 1/V_max, where v is the initial reaction velocity, K_m is the Michaelis–Menten constant, V_max is the maximum reaction velocity, and [S] is the concentration of the substrate.

Glucose detection using Mo/CeO₂ NPs as peroxidase mimetics

Firstly, 10 μL of GOx (10 mg mL⁻¹) and 200 μL glucose of different concentrations in 10 mM Na₂HPO₄ buffer (pH = 7.0) were incubated at 37 °C for 30 min; then, 10 μL of Mo/CeO₂ NP stock solution (0.6 mg mL⁻¹), 500 μL of TMB·2HCl (5 mM), and 1680 μL of acetate buffer (0.2 M, pH 4.0) were successively added to the above glucose reaction solution. Finally, the mixed solution was incubated in a 55 °C water bath for 30 min, and the absorbance was measured at 652 nm. 5 mM maltose, 5 mM D-galactose, 5 mM sucrose, 5 mM sorbose, 5 mM fructose, 5 mM dopamine and 5 mM uric acid were used instead of glucose for the selectivity experiment.

Results and discussion

Characterization of the as-synthesized Mo/CeO₂ NPs

The formation of the special structure of the catalyst is strongly dependent on the preparation method. Mo/CeO₂ NPs were fabricated using a simple single-step microwave method that did not employ any surfactant or stabilizer. Microwave irradiation, which could achieve rapid and homogeneous heating energy, was an excellent strategy to fabricate metal nanoparticles with narrow size distribution. Nanoparticles with high catalytic activity can be obtained by adjusting the reaction pH value and the mixing ratio of Mo (Fig. S1a and b†). The microwave temperature and time played important roles in the Mo/CeO₂ NPs synthesis, which were demonstrated to optimize experiments (Fig. S1c and d†). Certainly, Mo/CeO₂ NPs with high catalytic activity were synthesized under the optimum reaction conditions (pH 12, doping ratio 9%, the temperature is 100 °C for 4 h).

Fig. 1 shows the XRD patterns of CeO₂ and Mo-doped CeO₂ samples with different Mo doping ratios (3%, 6%, 9% and 15%). Pure CeO₂ NPs exhibited diffraction peaks at about 28.76°, 33.16°, 47.56°, 56.59° and 59.08° corresponding to the (111), (200), (220), (311) and (222) planes, which can be attributed to the cubic fluorite structured ceria [JCPDS file, no. 34-0394].³⁷ All characteristic diffraction peaks of each Mo-doped CeO₂ sample are indexed to the fluorite cubic structure of CeO₂ which has no diffraction peaks corresponding to oxidized molybdenum species. The XRD patterns of Mo-doped CeO₂ proved that Mo species were dispersed on the surface of cubic CeO₂ in the form of relatively small crystallites or existed as amorphous species. The peak intensity decreased gradually and the slight increase in the peak width with increasing the Mo doping ratio, indicating that the Mo species can inhibit the particle growth of CeO₂. The average diameters of the five CeO₂ samples prepared, evaluated by means of the Scherrer formula,⁴⁶ are about 9.3, 5.4, 5.1, 5.7 and 7.3 nm for the Mo-doped CeO₂ NPs obtained with an increase in the Mo/Ce ratio from 0% to 15%, respectively. The calculated size of 15Mo/CeO₂ NPs increased, which can be attributed to the agglomeration. Then we can conclude that the optimum doping rate of Mo is 9%. The 9Mo/CeO₂ NPs contain smaller CeO₂ crystallites than others products. All the Mo/CeO₂ NPs mentioned in the following experiment are Mo/CeO₂ NPs with a doping ratio of 9%, abbreviated as 9Mo/CeO₂ NPs.

Fig. 1 Powder XRD patterns of CeO₂, 3Mo/CeO₂, 6Mo/CeO₂, 9Mo/CeO₂, and 15Mo/CeO₂ NP catalysts.

Moreover, the N₂ adsorption–desorption performance of CeO₂ NPs and 9Mo/CeO₂ NPs was analyzed to observe their surface area and the pore structures (Fig. 2). The BET surface areas of CeO₂ NPs and 9Mo/CeO₂ NPs can be obtained as 98.95 and 89.73 m² g⁻¹, respectively. Doping with Mo is responsible for the slight reduction of the specific surface area of this sample. However, the average pore radius estimated from the Barrett–Joyner–Halenda (BJH) method was about 18.07 and 29.92 nm, increasing the wide pore radius of the 9Mo/CeO₂ NP sample is indicative of the role played by cerium–molybdate and to its partial deposition inside the micropores creating their own pore system and thus enhancing the pore radius.⁴⁷ However, the BET surface area of the Mo/CeO₂ NPs is smaller than those of the CeO₂ NPs. This result suggests that the surface area is not the only factor that affects the activity of Mo/CeO₂ NP based catalysts for enzyme-mimic catalytic reactions; pore size and active component dispersion could also influence the enzyme-mimic catalytic activity.

Fig. 2 Nitrogen adsorption–desorption isotherms and pore size distributions (inset) of 9Mo/CeO₂ NPs (black) and CeO₂ NPs (red).

The oxidation states and compositions of the as-prepared 9Mo/CeO₂ NP catalysts can be obtained using XPS technology. Fig. 3 shows the survey scan for representative 9Mo/CeO₂ NPs. Ce, Mo, and O peaks are observed in Fig. 3a with the absence of any other elements. Fig. 3b is used to identify Ce 3d XPS peaks, where v and u indicate the spin–orbit coupling 3d_5/2 and 3d_3/2 respectively. The three pairs of peaks (v, u; v2, u2; and v3, u3) arise from the Ce 4p oxidation state. The couple (v1, u1) corresponds to the Ce 3p oxidation state. Therefore, a mixture of Ce 3p/Ce 4p oxidation states exists on the surface of the sample,⁴⁸ which is crucial to the formation of oxygen vacancies, unsaturated chemical bonds and a catalysis cycle. The O 1s XPS spectra are also investigated as shown in Fig. 3c, which can be decomposed into two distinct peaks. The binding energies located at 529.5 eV can be assigned to lattice oxygen O²⁻; the binding energies at 531.7 eV belonging to surface active oxygen species like defect-oxide O₂²⁻ or hydroxyl-like group OH⁻. The strong peak of the sample at about 531.7 eV manifests that they are expected to have better catalytic activity in oxidation reactions. On the other hand, more information can be obtained from the Mo 3d XPS spectra in Fig. 3d. The peak of Mo 3d for the representative 9Mo/CeO₂ NPs can be decomposed into two distinct peaks, suggesting that Ce and Mo have chemical interactions.

Fig. 3 XPS spectrum (a), Ce 3d XPS spectrum (b), Mo 3d XPS (c) and O 1s (d) of 9Mo/CeO₂ NPs.

The morphology and structural features of CeO₂ NPs and 9Mo/CeO₂ NPs were elucidated by TEM and HRTEM analyses. A typical TEM image of CeO₂ NPs shown in Fig. 4a clearly illustrates the homogeneous, well-dispersed nanoparticles, which might result from the uniform heating in the microwave synthesis method. The crystallite size of CeO₂ NPs is distributed in a narrow range between 8 and 12 nm, as elucidated in the high magnification (the inset in Fig. 4b) of the approximate area part outlined in the white rectangle. When Mo is doped in CeO₂ NPs, the average diameter of nanoparticles is decreased and the corresponding values are 5 nm for the 9Mo/CeO₂ NPs. As can be seen from the observed TEM images (Fig. 4c), a porous structure formed by the agglomeration of the NPs. In the HRTEM image (Fig. 4d), most of the lattice fringes of 0.31 nm can be clearly observed, which was in good agreement with the crystal plane (111) of CeO₂. Several lattice fringes of 0.27 nm corresponding to its (200) lattice were also marked in the HRTEM image.

Fig. 4 (a) Low-magnification TEM image of CeO₂ NPs. (b) High-magnification TEM of the approximate area outlined with the white rectangle in (a). (c) Low-magnification TEM image of 9Mo/CeO₂ NPs. (d) The cross-sectional high magnification TEM image of 9Mo/CeO₂ NPs.

Peroxidase-like catalytic activity of Mo/CeO₂ NPs

The peroxidase-like activity of CeO₂ NP catalysts with different Mo doping ratios was primarily evaluated for continuous oxidation of the TMB·HCl substrate in the presence of H₂O₂ as shown in Fig. S1a.† The observed relative peroxidase-like activity of 9Mo/CeO₂ NP catalysts was the highest of all materials, which are selected as the artificial enzyme in the next experiment. It is obvious that the catalytic activity of CeO₂ NPs doped with Mo was much higher than that of pure CeO₂ NPs alone, revealing that an activated sample of Mo/CeO₂ NPs exhibited enhanced peroxidase-like catalytic activity under identical conditions (Fig. 5). Meanwhile the catalytic activity increases gradually as the Mo/CeO₂ NP concentration increases from 0.01 mg mL⁻¹ to 1.50 mg mL⁻¹ (Fig. S2†). When the concentration of Mo/CeO₂ NPs increases to 0.6 mg mL⁻¹, the absorbance intensity reaches a plateau, suggesting that the optimal concentration was 0.6 mg mL⁻¹ in following experiments.

Fig. 5 UV-vis spectra at a reaction time of 30 min in different systems; inset is the photograph of the solution at a reaction time of 30 min in different reaction systems. (a) TMB + 9Mo/CeO₂ NPs, (b) TMB + H₂O₂, (c) TMB + H₂O₂ + CeO₂ NPs, and (d) TMB + H₂O₂ + 9Mo/CeO₂ NPs in an acetate buffer with a pH of 4.0 at 45 °C for 30 min.

The catalytic activity of CeO₂ NPs and Mo/CeO₂ NPs was analogous to that of natural enzyme HRP, depending on pH and temperature. The effects of pH on the peroxidase-like activity of HRP, CeO₂ NPs and Mo/CeO₂ NPs were measured by varying the pH from 2.0 to 12.0 (Fig. 6a). The catalysts exhibited good catalytic activity at pH 4.0, which is consistent with the previously reported results.³⁶ In addition, the peroxidase-like activity of CeO₂ NPs, 9Mo/CeO₂ NPs and HRP was investigated while varying the temperature from 25 °C to 80 °C (Fig. 6b). It can be found that the activity remains stable between 40 °C and 50 °C, and decreases dramatically when the temperature is higher than 55 °C. Finally, the optimal pH and temperature for subsequent analysis were approximately pH 4.0 and 45 °C for 9Mo/CeO₂ NPs, pH 4.0 and 50 °C for CeO₂ NPs, and pH 4.0 and 45 °C for HRP respectively.

Fig. 6 The optimization of pH (a) and temperature (b) for the peroxidase-like activity of the HRP, CeO₂ and 9Mo/CeO₂ NP catalyst, respectively. The maximum point in each curve was set as 100%. Error bars shown represent the standard error derived from three repeated measurements.

Robustness of Mo/CeO₂ NPs

The peroxidase-like activity of CeO₂ NPs, Mo/CeO₂ NPs and HRP after treatment in a wide range of pH from 2.0 to 12.0 for 2 h is presented in Fig. 7a. The pH stability was also proved by observing 90% of relative catalytic enzyme activity over 9Mo/CeO₂ NPs in a wide range of pH from 2.0 to 12.0. In contrast, the relative catalytic enzyme activity of HRP was indeed decreased greatly after incubating at pH lower than 4.0 or larger than 10.0. As shown in Fig. 7b, the relative catalytic enzyme activity of Mo/CeO₂ NPs was indeed maintained at nearly identical values over the temperature from 10 to 80 °C. The peroxidase-like activity of HRP was decreased at treatment temperatures higher than 50 °C. Thus, it was clear that the Mo/CeO₂ NPs exhibited higher relative enzyme catalytic activity and were stable at higher temperatures and wide pH.

Fig. 7 (a) The pH stability of the HRP, CeO₂ and 9Mo/CeO₂ NP catalyst. The enzyme activity of HRP was defined as 100% at pH 8.0. (b) The temperature stability of the HRP, CeO₂ and 9Mo/CeO₂ NP catalyst. The enzyme activity of HRP was defined as 100% at 10 °C.

Catalytic kinetics and the proposed mechanism of Mo/CeO₂ NPs

To further understand the mechanism of peroxidase-like activity, the steady-state kinetics of 9Mo/CeO₂ NPs and CeO₂ NPs were investigated under optimal conditions, respectively. Typical Michaelis–Menten curves were observed for both Mo/CeO₂ NPs and CeO₂ NPs within a suitable range of H₂O₂ concentrations and TMB concentrations. As shown in Fig. S3a–d,† in a certain substrate concentration, the plots of the initial rate versus TMB and H₂O₂ concentrations both follow typical Michaelis–Menten behavior. The Michaelis–Menten constant (K_m) and the maximum initial velocity (V_max) were obtained by using Lineweaver–Burk plots (Fig. S3e–g†) and are listed in Table 1. The apparent K_m value of Mo/CeO₂ NPs with H₂O₂ as the substrate was higher than that of CeO₂ NPs, agreeing with the observation that a higher H₂O₂ concentration was required to achieve maximal activity for Mo/CeO₂ NPs. In contrast with CeO₂ NPs, 9Mo/CeO₂ NPs exhibited a significantly lower K_m value with TMB as the substrate, indicating that the Mo/CeO₂ NPs have a higher affinity for TMB than CeO₂ NPs.

Table 1 Comparison of K_m and V_m of 9Mo/CeO₂ NPs and CeO₂ NPs

Catalysts	V max [10^−8 m s^−1]		K m (mM)
	TMB	H2O2	TMB	H2O2
9Mo/CeO2 NPs	9.64 × 10^−9	8.01 × 10^−8	0.001	8.85
CeO2 NPs	1.95 × 10^−8	6.88 × 10^−9	0.623	0.067

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It was found that 9Mo/CeO₂ NPs show enhanced peroxidase-like catalytic activity, and the possible reaction mechanism is hypothesized. As is known, the ˙OH and ˙O₂⁻ radicals could be easily produced in the H₂O₂ containing system and play primary roles in the catalytic reaction.^36,49 Fig. S2† shows that the relative activity of the peroxidase mimic was switched on upon the addition of H₂O₂ and 9Mo/CeO₂ NPs, which enhanced with the increase of 9Mo/CeO₂ NP amounts. Therefore, the mechanism of 9Mo/CeO₂-catalyzed oxidation of TMB can be ascribed to the generation of ˙OH from the decomposition of H₂O₂. 9Mo/CeO₂ NPs showed a high amount of surface defects such as Ce³⁺ ions and oxygen vacancies due to Mo doping that was observed from the XPS results (Fig. 3d). The highly reactive oxygen species were produced when Ce³⁺ ions reacted with H₂O₂. Thus, TMB was absorbed on the surface of 9Mo/CeO₂ NPs on account of its high affinity and donates lone-pair electrons in the amino groups to 9Mo/CeO₂ NPs, leading to an increase in electron density and mobility on 9Mo/CeO₂ NPs, further enhancing the reaction efficiency and catalytic activity.⁵⁰ In addition, as the catalytic activity of nanomaterials for many reactions is often dependent on the morphology, size, surface area and pore structures, the peroxidase-like activity of 9Mo/CeO₂ NPs also exhibited a similar phenomenon compared with CeO₂ NPs. It is not a surprise to observe the best performance with 9Mo/CeO₂ NPs as the artificial peroxidase, which have a smaller size and a bigger pore radius (shown in the Characterization part). Furthermore, the addition of Mo can lead to the aggregation of CeO₂ NPs, which may accelerate decomposition of H₂O₂ that triggers the oxidation of TMB. Therefore, the peroxidase-like activity enhancement of 9Mo/CeO₂ NPs may be due to the possible reasons above.

Glucose detection using Mo/CeO₂ NPs

A simple colorimetric method to detect glucose contents was developed based on the efficient catalytic effect of 9Mo/CeO₂ NPs under optimized conditions. Generally, glucose oxidase (GOx) is used as an oxidant to oxidise glucose to produce gluconic acid and H₂O₂ in the presence of oxygen.^36,51 Therefore, the colorimetric detection of the converted TMB can be exploited to indirectly measure glucose content. As shown in Fig. 8, the absorbance at 652 nm was proportional to glucose concentrations, the linear detection range of glucose was from 5 × 10⁻⁷ to 5 × 10⁻⁵ mol L⁻¹, and the detection limit was estimated to be 4 × 10⁻⁷ mol L⁻¹. The present method has a very low detection limit compared with other colorimetric methods based on enzyme mimics for glucose detection.^14,36,38 Furthermore, this method was used to detect serum with complicated components, and the selectivity of the colorimetric detection method was verified. In this work, substances including common sugars and other substances were selected as interferents. As shown in Fig. 9, the method had less response to other tested sugars such as maltose, D-galactose, sucrose and sorbose in the experiment and found that the Mo/CeO₂ NP catalyst showed excellent selectivity towards glucose detection.

Fig. 8 Glucose concentration response curve for glucose detection using GOx and the 9Mo/CeO₂ NP catalyst (inset: linear calibration plot for glucose).

Fig. 9 The selectivity of glucose detection with 1 mM glucose, 5 mM maltose, 5 mM D-galactose, 5 mM sucrose, 5 mM sorbose, 5 mM fructose, 5 mM dopamine and 5 mM uric acid (inset: images of the corresponding colour changes). Error bars shown represent the standard error derived from three repeated measurements.

In addition, the practical application of the present method was evaluated by determining the concentration of glucose in real serum samples under the optimal experimental conditions. The serum was obtained from the Affiliated hospital of Zunyi Medical College, and the results obtainedusing this method are summarized in Table 2. The concentrations of glucose examined by this colorimetric method were similar to the observed concentrations based on automatic biochemical instruments in hospitals, which indicates that this simple method can be potentially applied to the determination of glucose content in real biological samples.

Table 2 Determination of glucose in human serum samples

Samples	Content (10^−3 mol L^−1)	Found^a (10^−3 mol L^−1)
a Value = mean ± S.D. (n = 3).
1	5.36	5.57 ± 0.016
2	6.81	7.01 ± 0.025
3	10.56	10.21 ± 0.028

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Conclusions

In summary, CeO₂ NPs with different Mo doping concentrations were prepared using a facile microwave-assisted synthesis method and possessed enhanced peroxidase-like activity. The TEM and XRD results confirmed that the Mo doping significantly reduced the average diameter of CeO₂ NPs. The 9Mo/CeO₂ NPs had a higher enzyme activity toward the H₂O₂-mediated TMB reaction than those of un-doped and Mo/CeO₂ NPs with other molar ratios of Mo. Furthermore, the prepared 9Mo/CeO₂ NPs were proven to possess peroxidase-like catalytic activity over a wide range of pH values and temperatures compared with natural enzymes and un-doped CeO₂ NPs. In view of the excellent peroxidase-like catalytic performance, a simple, cheap and high sensitive method for colorimetric detection of glucose was successfully established, and the detection limit of glucose could reach as low as 0.4 μM. This study would be significant to accelerate the development of novel peroxidase mimic materials with enhanced catalytic activities.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

The authors acknowledge financial support from the Natural Science Foundation of China (No. 21405107) and the United Foundation of Guizhou Province (LH20147558).

Notes and references

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c7ay02459b

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