Ultrasonic synthesis of nano-PrO1.8 as nanozyme for colorimetric determination of trans-resveratrol

In this study, nano-PrO1.8 were synthesized successfully in ionic liquids (ILs) as template assisted ultrasonic irradiation method. Various precipitating agents and different types of ILs were investigated to determine their respective effects on the morphology of the end products. Using hydrazine hydrate as a precipitating agent and 1-carboxymethyl-3-methylimidazolium chloride as a template, spherical structure with an average diameter of 250 nm was obtained. It is worth noting that the prepared material exhibits high peroxidase-like activity and weak oxidase activity. Then, the catalytic oxidation capacity of the nano-PrO1.8 was evaluated by the peroxidase substrate 3,3′,5,5′-tetramethylbenzidine (TMB). The colorless of TMB can be converted into blue oxidized TMB (oxTMB) in the presence of nano-PrO1.8, but trans-resveratrol inhibited its peroxidase-like activity and weakened the blue color. Hence, we developed a sensitive, selective and simple colorimetric method for trans-resveratrol detection using nano-PrO1.8 as peroxidase-like enzyme. A linear relationship was found in the range of 0.30 µM–16 µM trans-resveratrol with the detection limit of 0.29 µM. Satisfactory results were achieved when the method was submitted to the determination of trans-resveratrol in white wine samples.

they eat or drink may have positive or negative effects on their heath. There is a general trend for the healthy life style in order to slow down the aging process. Trans-resveratrol as a star molecule, the level in food or wine is attracted people's attention naturally. Therefore, it's determination and quantification are of very important. Many detection methods are reported for determination of trans-resveratrol, including high-performance liquid chromatography (HPLC) 21 , capillary electrophoresis (CE) 22 , and gas chromatography (GC) 23 . Hence, developing a more simple and fast analysis method for trans-resveratrol is important for clinical analysis. In general, the colorimetric analysis methods have many merits including the simple and easy procedures, mild reaction conditions and low cost. Besides, the color changes can be visually observed by naked eye on-site. In the present paper, we reported a facile strategy to prepare nano-PrO 1.8 by sonochemical synthesis in an ILs, and found the synthesized nano-PrO 1.8 had a good peroxidase-like activity and weak oxidase-like activity. As a result, the developed PrO 1.8 was able to catalyze the peroxidase substrate TMB into blue oxidized oxTMB, and trans-resveratrol inhibited this catalytic effect and weakened the blue color. Hence, a simple and sensitive colorimetric sensing platform was established to determine the concentrations of trans-resveratrol on the color change. Furthermore, the analytical method was also successfully employed in white wine sample (Fig. 1).   Synthesis of nano-pro 1.8 nanocrystal. In a typical synthesis, 0.20 g of Pr(NO 3 ) 3 and 0.1624 g of 1-carboxymethyl-3-methylimidazolium chloride ([HO 2 CMMIm]Cl) were dissolved in 40 mL water. Then, 500 μL of N 2 H 4 ·H 2 O was added with a magnetic stirrer, and a slurry-like green suspension was formed in the glass beaker. Then, the glass beaker was placed in an ultrasonic homogenizer at 45% power (427.5 W) for 30 minutes, and next the beaker was cooled naturally to room temperature until a dark green powder was formed at the bottom of the beaker. The powder was obtained after the green powder was centrifuged, filtered, rinsed several times with water and ethanol, and calcinated at 800 °C for 4 hours in a muffle furnace finally. The control experiments were performed by adjusting the types of ILs ( peroxidase-like activity and enzyme kinetic analysis of nano-pro 1.8 . The peroxidase-like activity of the nano-PrO 1.8 was performed at varied temperatures and pH using 1 mg/mL PrO 1.8 with 4 mM TMB and 100 mM H 2 O 2 as substrate. Ultrapure water was used in this experiment to avoid the influence of other ions on the activity of the nano-PrO 1.8 , and the pH of the reaction system was adjusted with a 0.2 M sodium acetate buffer solution. Then the enzyme kinetic analysis of the reaction was carried out by recording the absorption spectra in

Materials and instruments. All
Where V is the initial velocity, V max is the maximal reaction velocity, [S] is the substrate concentration and Km is a Michaelis constant.
To study the influence of reaction buffer pH on the catalytic activity of nano-PrO 1.8 , the pH of 0.1 M acetic acid buffer solution varying from pH 2.6 to 7.9 was investigated at 40 °C. To compare the influence of temperature on the nano-PrO 1.8 catalytic activity, the catalytic reactions were incubated in different temperature water bath from 25 to 80 °C under pH 3.5. The relative activity was defined as: Relative activity (%) = A 1 /A 2 × 100, where A 2 was the maximum absorbance, and A 1 was the sample absorbance measured at the same conditions. colorimetric detection of trans-resveratrol. Under the optimum conditions, 150 μL of nano-PrO 1.8 (dispersed with 30% ethanol, the concentration was 1 mg/mL), 3.33 mM H 2 O 2 and 1.03 mM TMB were added  (420) planes. This observation is in good agreement with the standard JCPDS 06-0639. To further confirm the oxygen content in praseodymium oxide, the prepared powder was analyzed by EMGA-920 Oxygen Elemental Analyzer. The sample in an inert atmosphere was heated to gas, which passes by the carbon dioxide converter where oxygen reacts with carbon, forming CO 2 and is measured by infrared absorption. The result of oxygen concentration was 16.95 ± 0.43%, which proved the oxidation state of praseodymium is PrO 1.8 .
The impacts of the ILs types and precipitating agents on final products morphology were compared in the experiment. Three types of precipitating agents (N 2 H 4 .H 2 O, NaOH and C 2 H 8 N 2 ) and two types of ILs ([HOOCMMIm]Cl and [HMIm]I) were used to study the effect on morphology of the products. The molar ratio of Pr(NO 3 ) 3 to the ILs was 1:2 and the calcination temperature was 800 °C for 4 hours. Figure 3 shows the SEM images of the products prepared with different precipitating agents and ILs. The results demonstrated that the morphology of the products was a well-crystallized spherical or hexagonal shape and had little dependence on the precipitating agents and ILs types. The morphology of the material prepared from N 2 H 4 .H 2 O and [HOOCMMIm]Cl was also characterized by HRTEM. As shown in Fig. 4A, the material has a hexahedral structure, and the lattice spacing of the material is 0.12 nm (Fig. 4B). In addition, the element distribution was analyzed by mapping images. The distribution of Pr and O elements in materials is uniform (Fig. 4C,D). The EDX (Energy Dispersive X-ray) spectrum also shows that the main element in the prepared material is Pr and O (Fig. 4E).
In order to determine the valence of the various elements in nano-PrO 1.8 , XPS study was carried out. The survey spectra of nano-PrO 1.8 confirmed that the presence of Pr and O. The binding energies (BEs) were calibrated using the C 1 s energy of 284.6 eV. The single C 1 s peak (Fig. 5A) is attributed to adventitious carbon that seems to exhibit an unavoidable presence on all air exposed materials. The binding energies for Pr3d at 932.91 eV (Fig. 5B), are in good agreement with the literature 24 . The O1s spectrum (Fig. 5C) is broad and asymmetric and can be  Fig. 5D. The results show that the particle size of the material is approximately normal distribution, the particle size of the material is between 100 and 550 nm, and the range of particle size distribution at 292.7 nm is the largest.

Peroxidase-like activity and oxidase-like activity of the material. The peroxidase-like and
oxidase-like activity of nano-PrO 1.8 were tested by oxidizing its enzyme substrate TMB and o-phenylenediamine in the presence and absence of H 2 O 2 at room temperature, respectively. The experimental results show that when the precipitating agent is hydrazine hydrate, the product maintains the highest catalytic activity under the same experimental conditions. Figure 6 shows the typical absorption curves of the different reaction systems. The nano-PrO 1.8 can catalyze the oxidation of colorless TMB in the absence of H 2 O 2 to produce bright blue color, indicating that the nano-PrO 1.8 has oxidase-like activity (Fig. 6A). However, when trans-resveratrol (t-res.) was  www.nature.com/scientificreports www.nature.com/scientificreports/ added into the solution, the catalytic activity of the nano-PrO 1.8 was rapidly lowered due to the suppression of oxidase-like activity, and the blue color was faded. Similar experimental results can also be observed in the presence of H 2 O 2 , indicating that the nano-PrO 1.8 have peroxidase-like activity (Fig. 6A). Simultaneously, the prepared nano-PrO 1.8 can catalyze the oxidation of o-diaminobenzene in the presence of H 2 O 2 or in the absence of H 2 O 2 (Fig. 6B). The absorbance value in the presence of H 2 O 2 is higher than in the absence of H 2 O 2 at the same conditions, this indicates that the activity of peroxidase-like is higher than oxidase-like under the same conditions. Thus, we only studied the peroxidase-like activity of nano-PrO 1.8 and analytical application in the next experiments.
Then, the peroxidase-like catalytic activity of the nano-PrO 1.8 using TMB as the substrate was proved to be dependent on the pH values, temperature, concentrations of H 2 O 2 and concentrations of TMB (Fig. 7). These results demonstrated that the nano-PrO 1.8 have similar catalytic activities with nature enzyme, with maximum activity in a mildly acidic (pH at 3.5), negligible catalytic activity lost over a range of temperature (25-55 °C), and  www.nature.com/scientificreports www.nature.com/scientificreports/ maximum activity at a 6.5 mM concentration of H 2 O 2 . Therefore, nano-PrO 1.8 with peroxidase-like activity can be used in broad ranges of pH and temperature. the kinetic assay of nano-pro 1.8 . In order to study the kinetics of the peroxidase-like catalytic activity of nano-PrO 1.8 , the catalytic oxidation of the oxidase substrate TMB was carried out in the presence of H 2 O 2 . Like some natural enzymes, the catalytic activity of nano-PrO 1.8 was further investigated based on the enzyme kinetics theory, in which H 2 O 2 and TMB were the substrates under the optimal conditions, as shown in Fig. 8. The evident steady-state kinetic parameters V max and K m by applying a reciprocal plot 26 Table 1. The results show that the K m of nano-PrO 1.8 for TMB is 0.0367 mM, and the k m of nano-PrO 1.8 with H 2 O 2 is 0.1502 mM, indicating that the Km of the nano-PrO 1.8 with TMB was smaller than that of HRP and the nano-PrO 1.8 possess better affinity for TMB than horseradish peroxidase (HRP) studied previously 8 . Furthermore, the Km of the nano-PrO 1.8 with H 2 O 2 is also smaller than that of the HRP, implying that nano-PrO 1.8 possess higher affinity for H 2 O 2 than HRP. Similarly, we also compared the K m and V max of other reported nanozyme. The results showed that the mimic enzyme activity of nano-PrO 1.8 was stronger than these reported materials.
Trans-resveratrol detection using tMB-H 2 o 2 -nano-pro 1.8 system. Based on the previous experimental results that trans-resveratrol can effectively inhibit the activity of peroxidase-like of nano-PrO 1.8 , trans-resveratrol was detected in white wine with TMB-H 2 O 2 -nano-PrO 1.8 system. The results showed a linear relationship between trans-resveratrol concentration and ΔA, as shown in Fig. 9A. The corresponding linear regression equation is Y = 0.05797X + 0.0021, (R 2 = 0.996, n = 5), linear range is 0.3-16 μM and the detection limit is 0.29 μM. In Table 2, the comparison is made with different electrochemical methods. In order to study the anti-interference of this method, other metal ions and antioxidants possibly existed in white wine, including Ca 2+ , Mg 2+ , Zn 2+ , Cu 2+ , Mn 2+ , Na + , K + , glucose, fructose, citric acid, tannic acid, 4-methylpyrocatechol and cyanidin   Table 3. Detection of trans-resveratrol in white wine and champagne samples.

conclusions
In this study, the nano-PrO 1.8 were prepared successfully by ultrasonic as the power, ILs as template and hydrazine hydrate as precipitating agent. The prepared nano-PrO 1.8 possess high peroxidase-like activity and oxidase-like activity, and the peroxidase-like K m values of the nano-PrO 1.8 with TMB and H 2 O 2 were smaller than that of HRP, indicating that the nano-PrO 1.8 possess better affinity for TMB and H 2 O 2 than HRP. In addition, trans-resveratrol can inhibit the peroxidase-like activity of nano-PrO 1.8 significantly. On this basis, a reliable and low-cost colorimetric quantitative detection method for trans-resveratrol was proposed. The linear range was from 0.3 to 16 μM with a detection limit of 0.29 μM. This work provides a novel method for rapid preparation of peroxidase-like nano-PrO 1.8 and extends the sensing application of trans-resveratrol to the fields of wine samples.