Influence of VO2 Nanoparticle Morphology on the Colorimetric Assay of H2O2 and Glucose

Nanozyme-based colorimetric sensors have received considerable attention due to their unique properties. The size, shape, and surface chemistry of these nanozymes could dramatically influence their sensing behaviors. Herein, a comparative study of VO2 nanoparticles with different morphologies (nanofibers, nanosheets, and nanorods) was conducted and applied to the sensitive colorimetric detection of H2O2 and glucose. The peroxidase-like activities and mechanisms of VO2 nanoparticles were analyzed. Among the VO2 nanoparticles, VO2 nanofibers exhibited the best peroxidase-like activity. Finally, a comparative quantitative detections of H2O2 and glucose were done on fiber, sheet, and rod nanoparticles. Under the optimal reaction conditions, the lower limit of detection (LOD) of the VO2 nanofibers, nanosheets, and nanorods for H2O2 are found to be 0.018, 0.266, and 0.41 mM, respectively. The VO2 nanofibers, nanosheets, and nanorods show the linear response for H2O2 from 0.025–10, 0.488–62.5, and 0.488–15.625 mM, respectively. The lower limit of detection (LOD) of the VO2 nanofibers, nanosheets, and nanorods for glucose are found to be 0.009, 0.348, and 0.437 mM, respectively. The VO2 nanofibers, nanosheets, and nanorods show the linear response for glucose from 0.01–10, 0.625–15, and 0.625–10 mM, respectively. The proposed work will contribute to the nanozyme-based colorimetric assay.

Vanadium dioxide (VO 2 ) have received considerable attention for their redox activity and layered structures, which can serve as very good intercalation materials and smart sensors [33]. The VO 2 exists in multiple morphologies, such as fibers, nanorods, nanosheets, spheres, and hollow spheres [34,35]. The shape of the nanoparticle has attracted growing interest due to its effect on the catalytic, optical, electronic, and magnetic properties [9,[36][37][38][39][40][41]. For example, one-dimensional (1D) nanostructures-such as nanotubes, nanorods, and nanowires-exhibit higher activity and durability, compared with zero-dimensional (0D) nanostructures, due to possessing fewer lattice boundaries, fewer defect sites, and longer segments of surface crystalline planes [36]. Therefore, we focused on the effect of different morphology on the catalytic activities of VO 2 nanoparticles in order to obtain more information for their potential applications in biosensor and biocatalysts.
The catalysis activities and kinetic mechanic of various VO 2 nanoparticles were investigated upon the reaction of hydrogen peroxide with its reducing substrates 3,3 ,5,5 -tetramethybenzidine (TMB). The hydrogen peroxide and glucose colorimetric sensors were developed based on VO 2 nanoparticles with different shapes. In this colorimetric assay, different analytical parameters-such as concentrations of nanoparticles, buffer solution, and pH of the analyte medium-were determined. Under optimal reaction conditions, the detection system of fiber-like VO 2 nanoparticles shows the most sensitive response to H 2 O 2 and glucose than the other two VO 2 nanoparticles.

Characterization of VO 2 Nanoparticles
The structural characterizations of the VO 2 nanoparticles were done by transmission electron microscopy (TEM) and X-ray powder Diffraction (XRD). TEM images indicate the VO 2 nanoparticles of different morphology, fibers, rods, and sheets ( Figure 1). The formation of VO 2 nanoparticles is confirmed from the X-ray diffraction pattern ( Figure 2). The VO 2 nanoparticles with fiber, sheet, and rod shapes have the same crystal structures as those reported in the literature [34,35], and are monoclinic VO 2 (Joint Committee on Powder Diffraction Standards card No. 31-1438 and No. 65-7960: see Figure 2). Nanomaterials 2017, 7, 347 2 of 10 nanostructures, due to possessing fewer lattice boundaries, fewer defect sites, and longer segments of surface crystalline planes [36]. Therefore, we focused on the effect of different morphology on the catalytic activities of VO2 nanoparticles in order to obtain more information for their potential applications in biosensor and biocatalysts.
Herein, different morphologies VO2 nanoparticles-including fibers, sheets, and rods-were synthesized. The catalysis activities and kinetic mechanic of various VO2 nanoparticles were investigated upon the reaction of hydrogen peroxide with its reducing substrates 3,3′,5,5′-tetramethybenzidine (TMB). The hydrogen peroxide and glucose colorimetric sensors were developed based on VO2 nanoparticles with different shapes. In this colorimetric assay, different analytical parameters-such as concentrations of nanoparticles, buffer solution, and pH of the analyte medium-were determined. Under optimal reaction conditions, the detection system of fiber-like VO2 nanoparticles shows the most sensitive response to H2O2 and glucose than the other two VO2 nanoparticles.

Characterization of VO2 Nanoparticles
The structural characterizations of the VO2 nanoparticles were done by transmission electron microscopy (TEM) and X-ray powder Diffraction (XRD). TEM images indicate the VO2 nanoparticles of different morphology, fibers, rods, and sheets ( Figure 1). The formation of VO2 nanoparticles is confirmed from the X-ray diffraction pattern ( Figure 2). The VO2 nanoparticles with fiber, sheet, and rod shapes have the same crystal structures as those reported in the literature [34,35], and are monoclinic VO2 (Joint Committee on Powder Diffraction Standards card No. 31-1438 and No. 65-7960: see Figure 2).   nanostructures, due to possessing fewer lattice boundaries, fewer defect sites, and longer segments of surface crystalline planes [36]. Therefore, we focused on the effect of different morphology on the catalytic activities of VO2 nanoparticles in order to obtain more information for their potential applications in biosensor and biocatalysts.
Herein, different morphologies VO2 nanoparticles-including fibers, sheets, and rods-were synthesized. The catalysis activities and kinetic mechanic of various VO2 nanoparticles were investigated upon the reaction of hydrogen peroxide with its reducing substrates 3,3′,5,5′-tetramethybenzidine (TMB). The hydrogen peroxide and glucose colorimetric sensors were developed based on VO2 nanoparticles with different shapes. In this colorimetric assay, different analytical parameters-such as concentrations of nanoparticles, buffer solution, and pH of the analyte medium-were determined. Under optimal reaction conditions, the detection system of fiber-like VO2 nanoparticles shows the most sensitive response to H2O2 and glucose than the other two VO2 nanoparticles.

Characterization of VO2 Nanoparticles
The structural characterizations of the VO2 nanoparticles were done by transmission electron microscopy (TEM) and X-ray powder Diffraction (XRD). TEM images indicate the VO2 nanoparticles of different morphology, fibers, rods, and sheets ( Figure 1). The formation of VO2 nanoparticles is confirmed from the X-ray diffraction pattern ( Figure 2). The VO2 nanoparticles with fiber, sheet, and rod shapes have the same crystal structures as those reported in the literature [34,35], and are monoclinic VO2 (Joint Committee on Powder Diffraction Standards card No. 31-1438 and No. 65-7960: see Figure 2).

Principle
In pH 4 citrate buffer solution at room temperature, VO 2 nanoparticles with different morphologies catalyzed the oxidation of a peroxidase substrate 3,3 ,5,5 -tetramethylbenzidine (TMB) in the presence of H 2 O 2 to obtain the TMB oxidized product with blue color. As shown in Figure 3, when various VO 2 nanoparticles were added into the TMB/H 2 O 2 solution, the strong absorption peaks were obtained at 656 nm. However, there were no strong absorption peaks when the solution did not contain H 2 O 2 or VO 2 nanoparticles. The absorbance becomes stronger due to more TMB being oxidized with the increasing of the concentration of H 2 O 2 . The absorbance also showed a linear trend depending on the concentration of H 2 O 2 .

Principle
In pH 4 citrate buffer solution at room temperature, VO2 nanoparticles with different morphologies catalyzed the oxidation of a peroxidase substrate 3,3′,5,5′-tetramethylbenzidine (TMB) in the presence of H2O2 to obtain the TMB oxidized product with blue color. As shown in Figure 3, when various VO2 nanoparticles were added into the TMB/H2O2 solution, the strong absorption peaks were obtained at 656 nm. However, there were no strong absorption peaks when the solution did not contain H2O2 or VO2 nanoparticles. The absorbance becomes stronger due to more TMB being oxidized with the increasing of the concentration of H2O2. The absorbance also showed a linear trend depending on the concentration of H2O2.

Effect of pH
The effect of pH value (pH 3.0-8.0) on absorption value with TMB was investigated in the citrate buffer system, as shown in Figure 4. Each of the VO2 nanofibers, nanosheets, and nanorods of the system reached their maximum peaks when the pH value was 4.0. Therefore, pH 4.0 was selected to detect H2O2 and glucose with various VO2 nanoparticles.

Effect of Buffers
The effect of buffers on absorption value of TMB oxide product was examined. The time response curves of TMB with H2O2 catalyzed by VO2 with different morphologies, in pH 4.0, 0.2 M acetate, phosphate, and citrate buffers. The results were shown in Figure 5. Up to 300 s, the VO2 nanoparticles were more active in the citrate buffer solution. Thus, the citrate buffer solution (pH = 4.0, 0.2 M), was chosen as the optimal reaction solution for the H2O2 and glucose colorimetric assay.

Effect of pH
The effect of pH value (pH 3.0-8.0) on absorption value with TMB was investigated in the citrate buffer system, as shown in Figure 4. Each of the VO 2 nanofibers, nanosheets, and nanorods of the system reached their maximum peaks when the pH value was 4.0. Therefore, pH 4.0 was selected to detect H 2 O 2 and glucose with various VO 2 nanoparticles. Nanomaterials 2017, 7, 347 3 of 10

Principle
In pH 4 citrate buffer solution at room temperature, VO2 nanoparticles with different morphologies catalyzed the oxidation of a peroxidase substrate 3,3′,5,5′-tetramethylbenzidine (TMB) in the presence of H2O2 to obtain the TMB oxidized product with blue color. As shown in Figure 3, when various VO2 nanoparticles were added into the TMB/H2O2 solution, the strong absorption peaks were obtained at 656 nm. However, there were no strong absorption peaks when the solution did not contain H2O2 or VO2 nanoparticles. The absorbance becomes stronger due to more TMB being oxidized with the increasing of the concentration of H2O2. The absorbance also showed a linear trend depending on the concentration of H2O2.

Effect of pH
The effect of pH value (pH 3.0-8.0) on absorption value with TMB was investigated in the citrate buffer system, as shown in Figure 4. Each of the VO2 nanofibers, nanosheets, and nanorods of the system reached their maximum peaks when the pH value was 4.0. Therefore, pH 4.0 was selected to detect H2O2 and glucose with various VO2 nanoparticles.

Effect of Buffers
The effect of buffers on absorption value of TMB oxide product was examined. The time response curves of TMB with H2O2 catalyzed by VO2 with different morphologies, in pH 4.0, 0.2 M acetate, phosphate, and citrate buffers. The results were shown in Figure 5. Up to 300 s, the VO2 nanoparticles were more active in the citrate buffer solution. Thus, the citrate buffer solution (pH = 4.0, 0.2 M), was chosen as the optimal reaction solution for the H2O2 and glucose colorimetric assay.

Effect of Buffers
The effect of buffers on absorption value of TMB oxide product was examined. The time response curves of TMB with H 2 O 2 catalyzed by VO 2 with different morphologies, in pH 4.0, 0.2 M acetate, phosphate, and citrate buffers. The results were shown in Figure 5. Up to 300 s, the VO 2 nanoparticles were more active in the citrate buffer solution. Thus, the citrate buffer solution (pH = 4.0, 0.2 M), was chosen as the optimal reaction solution for the H 2 O 2 and glucose colorimetric assay.

Effect of VO2 Nanoparticle Morphologies and Concentrations
As shown in Figure 6, the absorption values at OD656nm of TMB oxide product increased gradually with the concentration of VO2 nanoparticles. The system reached its maximum absorption value when the concentrations of VO2 nanofibers, nanosheets, and nanorods were 10, 10, and 2 mM, respectively. The results show that the catalytic activity of VO2 nanofibers is stronger than the other two, shown in the Figure 6.

Effect of VO 2 Nanoparticle Morphologies and Concentrations
As shown in Figure 6, the absorption values at OD 656nm of TMB oxide product increased gradually with the concentration of VO 2 nanoparticles. The system reached its maximum absorption value when the concentrations of VO 2 nanofibers, nanosheets, and nanorods were 10, 10, and 2 mM, respectively. The results show that the catalytic activity of VO 2 nanofibers is stronger than the other two, shown in the Figure 6.

Effect of VO2 Nanoparticle Morphologies and Concentrations
As shown in Figure 6, the absorption values at OD656nm of TMB oxide product increased gradually with the concentration of VO2 nanoparticles. The system reached its maximum absorption value when the concentrations of VO2 nanofibers, nanosheets, and nanorods were 10, 10, and 2 mM, respectively. The results show that the catalytic activity of VO2 nanofibers is stronger than the other two, shown in the Figure 6.

Steady-State Kinetic Assay
For further understanding the influence of particle morphology on the catalytic mechanism of VO 2 nanoparticles, the steady-state kinetic assay for VO 2 nanoparticles were determined in detail. As shown in Figure 7, the typical Michaelis-Menten curve were obtained for VO 2 nanozymes. Michaelis-Menten constant (K M ) and maximum initial velocity (V max ) were known from Michaelis-Menten curve use a Lineweaver-Burk plot. A comparison of the kinetic parameters of VO 2 nanozymes, V 2 O 5 nanozymes, Fe 3 O 4 magnetic nanoparticle (MNP S ), and HRP was given in

Calibration Curve for H 2 O 2 and Glucose Detection
Under the optimal conditions (pH 4.0 citrate buffer, the concentrations of VO 2 nanofibers, nanosheets and nanorods were 2, 10, and 10mM, respectively.) the calibration curves of H 2 O 2 were obtained with VO 2 nanoparticles different morphologies (Figure 8). The correlation between the absorbance values and H 2 O 2 concentration are linear over the range of 0-100 mM (nanofibers), 0-500 mM (nanosheets) and 0-500 mM (nanorods) with correlation coefficients 0.99981, 0.99364, and 0.99222, respectively. The lower limit of detection (LOD) of the VO 2 nanofibers, nanosheets, and nanorods for H 2 O 2 are found to be 0.018, 0.266, and 0.41 mM, respectively.

Calibration Curve for H2O2 and Glucose Detection
Under the optimal conditions (pH 4.0 citrate buffer, the concentrations of VO2 nanofibers, nanosheets and nanorods were 2, 10, and 10mM, respectively.) the calibration curves of H2O2 were obtained with VO2 nanoparticles different morphologies (Figure 8). The correlation between the absorbance values and H2O2 concentration are linear over the range of 0-100 mM (nanofibers), 0-500 mM (nanosheets) and 0-500 mM (nanorods) with correlation coefficients 0.99981, 0.99364, and 0.99222, respectively. The lower limit of detection (LOD) of the VO2 nanofibers, nanosheets, and nanorods for H2O2 are found to be 0.018, 0.266, and 0.41 mM, respectively. As glucose oxidase (GOx) can catalyze the oxidation of glucose and produce H2O2, the absorption value with TMB was changing by H2O2 in presence of VO2 nanoparticles. Because the GOx would be denatured in pH 4.0 buffer, the glucose detection was produced in two steps: first, H2O2 was induced by GOx oxidation of glucose and then the reaction solutions were detected by TMB/different VO2 nanoparticles system. As shown in Figure 9, the absorbtion increases gradually with the increasing of glucose concentration. The correlation between the absorbance at 656 nm and glucose concentration are linear over the range of 0-30 mM (nanofibers), 0-40 mM (nanosheets) and 0-40 mM (nanorods) with the correlation coefficient of 0.98557, 0.98919, and 0.99502, respectively. The lower limit of detection (LOD) of the VO2 nanofibers, nanosheets, and nanorods for glucose are found to be 0.009, 0.348, and 0.437 mM, respectively.
The VO2 nanofibers showed the highest peroxidase activity in the H2O2 and glucose colorimetric assay, followed by VO2 nanosheets, and finally VO2 nanorods. Additionally, it was reported that the specific surface area of VO2 nanoparticles greatly influences their catalytic activities. The specific surface area of VO2 nanofibers (185 to 122 m 2 g −1 ) [34] is also much larger than that of other VO2 micro/nanoparticles, such as hollow microspheres (22.3 m 2 g −1 ), nanowires (12.3 m 2 g −1 ) [42], nanobelts (18.6 m 2 g −1 ) [43], nanorods (42 m 2 g −1 ) As glucose oxidase (GOx) can catalyze the oxidation of glucose and produce H 2 O 2 , the absorption value with TMB was changing by H 2 O 2 in presence of VO 2 nanoparticles. Because the GOx would be denatured in pH 4.0 buffer, the glucose detection was produced in two steps: first, H 2 O 2 was induced by GOx oxidation of glucose and then the reaction solutions were detected by TMB/different VO 2 nanoparticles system. As shown in Figure 9, the absorbtion increases gradually with the increasing of glucose concentration. The correlation between the absorbance at 656 nm and glucose concentration are linear over the range of 0-30 mM (nanofibers), 0-40 mM (nanosheets) and 0-40 mM (nanorods) with the correlation coefficient of 0.98557, 0.98919, and 0.99502, respectively. The lower limit of detection (LOD) of the VO 2 nanofibers, nanosheets, and nanorods for glucose are found to be 0.009, 0.348, and 0.437 mM, respectively.

Synthesis of VO2 Nanoparticles
The synthesis of VO2 nanofiber contains two steps: synthesis of VO2 hollow sphere and the supernatant collecting and drying. According to the literature procedure [35] synthesis of VO2 hollow sphere, with minor adjustment. Briefly, V2O5 and oxalic acid (the ratio of molar is 1:3) were first dissolved in 7 mL distilled water and stirred for 10 min at room temperature. Then the 23 mL methanol was added in the solution and stirred for another 10 min. The mix solution was transferred to a Teflon-lined autoclave with stainless steel, and heated at 200 °C for 24 h. The sample was cooled down naturally. The black precipitates were filtered off and washed with distilled water and ethanol, and then dried at 80 °C overnight, and finally the VO2 hollow spheres were dissolved, the supernatant was collected and dried. Similar procedures were adopted to prepare nanorods and nanosheets: when the water content is 10 mL, the product is nanorods, and when the solution is completely water, the product is just nanosheets (with water and methanol measures maintained at 30 mL).

Synthesis of VO 2 Nanoparticles
The synthesis of VO 2 nanofiber contains two steps: synthesis of VO 2 hollow sphere and the supernatant collecting and drying. According to the literature procedure [35] synthesis of VO 2 hollow sphere, with minor adjustment. Briefly, V 2 O 5 and oxalic acid (the ratio of molar is 1:3) were first dissolved in 7 mL distilled water and stirred for 10 min at room temperature. Then the 23 mL methanol was added in the solution and stirred for another 10 min. The mix solution was transferred to a Teflon-lined autoclave with stainless steel, and heated at 200 • C for 24 h. The sample was cooled down naturally. The black precipitates were filtered off and washed with distilled water and ethanol, and then dried at 80 • C overnight, and finally the VO 2 hollow spheres were dissolved, the supernatant was collected and dried. Similar procedures were adopted to prepare nanorods and nanosheets: when the water content is 10 mL, the product is nanorods, and when the solution is completely water, the product is just nanosheets (with water and methanol measures maintained at 30 mL).

Physical Characterization
The morphology and size of the VO 2 nanoparticles were acquired using a transmission electron microscopy (TEM) by JEM-1011 transmission electron microscopy (JEOL, Tokyo, Japan) with a working voltage at 100 kV. The X-ray powder diffraction method was carried out in a D/max-rα power diffractometer (Rigaku, Tokyo, Japan) using Cu-Kα monochromatic radiation (λ = 1.5418 Å).

H 2 O 2 Detection Using VO 2 Nanoparticles as Peroxidase Mimetics
To discover the peroxidase-like character of VO 2 nanoparticles, the experiments were performed as follows: 60 µL VO 2 nanoparticles solution (the concentrations of nanofibers, nanosheets, and nanorods are 2, 10, and 10 mM, respectively) in a reaction volume of 2400 µL citrate buffer solution (pH = 4.0) and 480 µL TMB solution (1.5 mM in ethanol), followed by the addition of 60 µL H 2 O 2 (30%). The mixed solution was reacted for 5 min at room temperature. Then used for the UV-Vis spectrophotometer (Metash Instruments Inc., Shanghai, China) record the spectra at 656 nm for TMB.
To investigate the influence of buffer solution on the VO 2 nanoparticle characteristics, the pH-ranging from 3.0 to 8.0 of the buffer solution-was examined, under conditions identical to these used above.
To investigate the influence of different reaction buffers on the VO 2 nanoparticles characteristics, catalytic reactions incubated in difference buffer solution-including citrate, phosphate, and acetate-were examined, under conditions identical to these used in above. For a blank, only substrate solution was used. All experiments were conducted at room temperature (25 • C).

Glucose Detection Using VO 2 Nanoparticles
Glucose detection was examined as follows: (a) 200 µL of GOx (1 mg/mL) and 200 µL of glucose of different concentrations in 400 µL of phosphate buffered saline (PBS, pH = 7.0) were incubated at 37 • C for 60 min; (b) 400 µL of TMB (1.5 mM in ethanol) and 50 µL of VO 2 nanoparticles solution (the concentrations of nanofibers, nanosheets, and nanorods are 2, 10, and 10 mM, respectively) in 1750 µL of citrate buffer solution (pH = 4.0) were added into the above glucose reaction solution; (c) The mixed solutions with different concentrations of glucose were incubated for 5 min; the (d) the UV-Vis spectrophotometer was used to record the spectra.

Conclusions
VO 2 nanoparticles with different structures-nanofibers, nanosheets, and nanorods-have been successfully fabricated and show peroxidase-like activities. The catalytic behaviors of VO 2 nanoparticles show Michaelis-Menten kinetics and good affinity to both H 2 O 2 and TMB. The VO 2 nanoparticle-based colorimetric assay provides fast, sensitive, and low-cost H 2 O 2 and glucose sensors. Compared with VO 2 nanorods and VO 2 nanosheets, the VO 2 nanofibers demonstrated the most sensitive response during the H 2 O 2 and glucose sensing. This investigation is significant for vanadium-based nanozyme application in biosensor and biocatalysis.