Preparation and application of magnetic zinc pyridinedicarboxylic acid nanocomposite (Zn-(PDC)₂@Fe₃O₄)

2.1 Experimental reagents and apparatuses

2,6-pyridinedicarboxylic acid, ferric chloride hexahydrate (AR, Xiya Chemical Reagent Factory); zinc sulfate heptahydrat (AR, Xilong Chemical Co., Ltd.); anhydrous sodium acetate, hydrochloric acid, acetic acid, ethanol, 30% H₂O₂, anhydrous sodium sulfite (AR, Guangzhou Chemical Reagent Factory); polyethylene glycol 6000 (AR, Tianjin Kermel Chemical Reagent Co., Ltd.); polyvinylpyrrolidone (PVP), 3-aminopropyl triethoxy silane (APTES), 3,3′,5,5′-tetramethyl benzidine (TMB) (AR, Aladdin Industrial Corporation); glycol (AR, Sinopharm Group Chemical Reagent Co., Ltd.); pure nitrogen (≥99.99%, Gas Plant, Sanshui District, Foshan City); deionized water; water from Xijiang River (collected from Zhaoqing section, stored at 4 °C); lake water (collected from the Calligraphy Pond of Zhaoqing University, stored at 4 °C); hylocereus undatus 1, hylocereus undatus 2, tremella fuciformis and Chinese wolfberry (purchased from school supermarket).

AUY120 type electronic balance (SHIMADZU), HH-S2 thermostatic water bath (Universal Scientific Instruments Factory, Jintan City, Jiangsu Province), PHS-3C pH meter (Shanghai Rex), vortex mixing (Haimen Qilinbeier Instrument Manufacturing Co., Ltd.), 149A0279 type ultrasonic cleaner (Shanghai Kudos Ultrasonic Instrument Co., Ltd.), vacuum drying oven (Shanghai Yiheng Technical Co., Ltd.), electric constant temperature drying oven (Shanghai Sumsung Laboratory Instrument Co., Ltd.), PTEF autoclave (Shanghai Sumsung Laboratory Instrument Co., Ltd.), pipette and UV-2600 UV–VIS Spectrophotometer (SHIMADZU), APEX ⅡQUAZAR X-ray single crystal diffractometer (Bruker, German), IRT racer-100 Fourier transform infrared spectrometer (SHIMADZU), D/max 2000 X-ray powder diffractometer (XRD) (Rigaku), SUPRA 55 SAPPHIRE Fs-SEM (Carl Zeiss Jena), etc.

2.2 Synthesis and activation of nano-scaled Fe₃O₄

Synthesis of nano-scaled Fe₃O₄ using the hydrothermal and solvothermal method (Hu and Lou, 2013): 2.70 g FeCl₃·6H₂O (iron source) was weighed, and dissolved in 60 mL ethylene glycol solvent, and the mixture solution was stirred for 30 min to form a clear bright yellow solution. Then, 7.20 g sodium acetate (electrostatic stabilizer) and 1 g (polyethylene glycol stabilizer) were added, and the mixture solution was stirred for 30 min. The mixture solution finally formed was put into two 50 mL PTEF lined reaction kettles to react (solvothermal reduction) at 200 °C for 9 h, and then cooled down to room temperature, separated with magnet, washed with water and ethanol to obtain nano-scaled Fe₃O₄ by vacuum drying at 50 °C.

Activation of the surface hydroxyl groups of nano-scaled Fe₃O₄: 0.5 g nano-scaled Fe₃O₄ was added to 50 mL deionized water, going through ultrasonic dispersion for 10 min, then heated in a water bath at a constant temperature of 60 °C, and mechanically stirred. The pH of the reaction solution was adjusted to 4 with a certain amount of dilute hydrochloric acid (0.001 mol/L), and then measured every 30 min to guarantee pH = 4 at any time with dilute hydrochloric acid until no more changes occurred (N₂ protection for the whole reaction).

2.3 Amino-modification of the surface of nano-scaled Fe₃O₄

With 3-aminopropyl triethoxy silane (APTES) as the silane coupling agent, after hydrolysis and condensation under acid conditions, the APTES was adhered to the surface of nano-scaled Fe₃O₄ by using blending surface package embedding method so as to achieve the amino-modification of the surface of nano-scaled Fe₃O₄: 0.5 g activated nano-scaled Fe₃O₄ was added to 100 mL ethanol water solution (50%), and the pH of the mixture solution was adjusted to 4 with glacial acetic acid, then went through ultrasonic dispersion at room temperature for 10 min. Then 1 mL APTES (the mass ratio of Fe₃O₄ to APTES was 3:8) was added, and the mixture went through ultrasonic dispersion (53 kHz) with N₂ protection, at room temperature for 30 min, aging for 3 h. After reaction, magnetic separation, and being washed with deionized water and absolute ethyl alcohol many times until no oily suspension appeared, it then went through vacuum drying at 40 °C for 12 h.

2.4 Preparation of Zn-(PDC)₂

Preparation of Zn-(PDC)₂: the traditional methods for preparing metal organic frameworks and even nano-metal organic frameworks include: the solvent diffusion method, the low pressure steam method, the traditional hydrothermal method and the solvothermal method, which have disadvantages such as long reaction time, slow crystallization rate and low crystallization ratio (Zhang, 2014). The commonly used new methods for preparing MOFs crystals include the ultrasonic synthesis method and the microwave method, which have advantages such as fast speed, simple operation and high efficiency.

Syntheses of MOFs with the ultrasonic-assisted method: 2.70 g (2.00 × 10⁻³ mol/L) H₂PDC was weighed, dissolved in 20 mL absolute ethanol solution, in which 10 mL water solution containing 0.30 g (1.00 × 10⁻³ mol/L) ZnSO₄·7H₂O was dropped slowly, and then went through ultrasonic dispersion (53 kHz) at room temperature for 1 h. After the reaction, the mixture solution was cooled down and filtered, and the filtrate volatilized under natural conditions to obtain almost colorless and transparent bulk MOFs crystals.

2.5 Preparation of Zn-(PDC)₂@Fe₃O₄

In this paper, Zn-(PDC)₂@Fe₃O₄ was prepared using the ultrasonic-assisted adhesion method, which can reduce the crystallization time and form more uniform nuclei compared with the traditional preparation method. With the ultrasonic-assisted adhesion method, the magnetic nano-scaled Fe₃O₄ was added to the MOFs precursor solution, where the magnetic nano-scaled Fe₃O₄ adhered to the surfaces of MOFs to form MMOFs (Wang et al., 2015). With the ultrasonic-assisted method, the magnetic nano-scaled Fe₃O₄ was embedded in MOFs: 0.5 g Fe₃O₄-NH₂ was added to 30 mL absolute ethanol solution containing ZnSO₄·7H₂O 0.862 g (3 mmol) for ultrasonic dispersion at room temperature for 30 min to obtain the mixture solution A; 1.50 g (9 mmol) H₂PDC and 100 mg PVP were dissolved in 30 mL absolute ethanol solution for uniform ultrasonic dispersion to obtain the mixture solution B; the mixture solution B was slowly added to the mixture solution A, went through ultrasonic dispersion at room temperature for 1 h, and aged at 60 °C for 3 h; after magnetic separation, the mixture solution was washed with anhydrous ethanol and deionized water, and went through vacuum drying at 50 °C for 24 h to obtain the Zn-(PDC)₂@Fe₃O₄.

2.6 Study of the activity of mimetic peroxidase of Zn-(PDC)₂@Fe₃O₄

5 test tubes were taken, and added with 3 mL 0.2 mol/L NaAc-HAc (pH = 4.0) buffer solution. No. 1 test tube was added with 300 μL 200 μM H₂O₂; No. 2 test tube was added with 300 μL 200 μM H₂O₂ and 10 mg Zn-(P-DC)₂ @ Fe₃O₄; No. 3 test tube was added with 300 μL 200 μM H₂O₂ and 200 μL 2 mg/mL TMB; No. 4 test tube was added with 200 μL 2 mg/mL TMB and 10 mg Zn-(PDC)₂@Fe₃O₄; No. 5 test tube was added with 300 μL 200 μM H₂O₂, 10 mg Zn-(PDC)₂@Fe₃O₄ and 200 μL 2 mg/mL TMB. After reaction at room temperature for 10 min, the absorbance of the mixture solution in the 5 test tubes was determined with a UV–VIS spectrophotometer at the wavelength of 400–800 nm.

2.7 Experiment proving that the catalytic activity of Zn-(PDC)₂@Fe₃O₄ was not caused by metal impurities

To prove that the catalytic activity of mimetic peroxidase derived from the hydroxyl radical (OH) produced by catalyzation of hydrogen peroxide was caused by the magnetic Zn-(PDC)₂@Fe₃O₄ rather than the iron ions dissolved from magnetic nano-scaled Zn-(PDC)₂@Fe₃O₄, 10 mg magnetic nano-scaled Zn-(PDC)₂@Fe₃O₄ was dissolved in 3 mL 0.2 mol/L NaAc-HAc (pH = 4.0) buffer solution for 2 h. After magnetic separation, the supernatant was collected and added with 200 μL 2 mg/mL TMB solution and 300 μL 200 μM H₂O₂ solution to react under the best color reaction condition for 10 min, then the absorbance of the reaction solution was determined. Then, 300 μL deionized water and 300 μL 200 μM H₂O₂ solution were prepared for a control experiment, with other conditions being the same: 10 mg magnetic nano-scaled Zn-(PDC)₂@Fe₃O₄, 200 μL 2.0 mg/mL TMB and 3 mL 0.2 M NaAc-HAc (pH = 4.0) buffer solution. If the results showed that the absorbance of the supernatant remained unchanged, it would prove that the catalytic activity of mimetic peroxidase derived from the hydroxyl radical (OH) produced by the catalyzation of hydrogen peroxide was caused by magnetic nano-scaled Zn-(PDC)₂@Fe₃O₄ rather than metal impurities.

2.8 Catalytic steady-state kinetics of Zn-(PDC)₂@Fe₃O₄

3 mL 0.2 M NaAc-HAc (pH = 4.0) buffer solution was respectively added to No. 1 and No. 2 test tubes with a pipette, relative to different concentrations of H₂O₂ solutions. After the amount of TMB solution (c = 2.0 mg/mL) was maintained at 200 μL, 200 μL TMB solution (c = 2.0 mg/mL) was respectively added to No. 1 and No. 2 test tubes, then 10 mg Zn-(PDC)₂@Fe₃O₄ was respectively added to No. 1 and No. 2 test tubes for insulation at 40 °C for 5 min, and then 300 μL 400 μM H₂O₂ solution was added to No. 1 and 2 test tubes. After even vortex mixing, the mixture solution in No. 1 and No. 2 test tubes adopted insulation and coloration at 40 °C for 2 min and 4 min, respectively. After the reaction stopped through magnetic separation of Zn-(PDC)₂@Fe₃O₄ and freezing of reaction solution, a certain volume of reaction solution was added to a cuvette, and its absorbance was determined at the wavelength of 652 nm. With the same method, the absorbances of 20, 40, 80, 200 μM H₂O₂ reaction solutions at 2 min and 4 min were determined, respectively; similarly, after the amount of H₂O₂ solution was maintained at 300 μL 200 μM, the absorbances of 4, 6, 8, 10, 12 mM TMB reaction solutions at 2 min and 4 min were determined, respectively too. With the same method, the Michaelis constants of TMB and H₂O₂ by nano-scaled Fe₃O₄ were determined.

2.9 Drawing of the standard curve of SO₃²⁻

3 mL 0.2 M NaAc-HAc (pH = 4.0) buffer solution was respectively added to a test tube, and 100 μL 400 μM SO₃²⁻ solution and 100 μL 200 μM H₂O₂ solution were added to the test tube, after even vortex mixing, the mixture solution in the test tube went through insulation at 40 °C for 15 min. Then, 200 μL TMB solution (c = 2.0 mg/mL) and 10 mg Zn-(PDC)₂@Fe₃O₄ were added to the test tube for insulation and coloration at 40 °C for 10 min. After the reaction stopped due to the magnetic separation of Zn-(PDC)₂@Fe₃O₄, a certain volume of the reaction solution was added to a cuvette, and its absorbance was determined at the wavelength of 652 nm. With the same method, the absorbance of 0.08, 0.4, 0.8, 4, 8, 40, 80 μM SO₃²⁻ reaction solutions was determined, respectively. Thus, the absorbance of different concentrations of SO₃²⁻ solutions was obtained, and the linear regression of data was conducted to obtain the equation that can represent the relationship between the absorbance of SO₃²⁻ reaction solution and H₂O₂ reaction solution.

2.10 Pretreatment of samples

The method of GB/T 5009.34-2003 with appropriate modifications was adopted. For the solid sample:, 5 g uniform solid samples were ground into powder with a knife (or a pair of scissors), mixed and then weighed. For the liquid sample: 10.0 mL liquid sample was directly absorbed and then dropped into a 500 mL round-bottom distillation flask, in which 150 mL water was added. Then, a condenser tube was installed, whose lower end was inserted into a conical flask containing 20 mL sodium acetate (20 g/L). Then, 5 mL hydrochloric acid (1 + 1) was added to the round-bottom distillation flask. After the stopper was covered quickly, the mixture was heated for distillation. When the distillation solution was about 100 mL, the lower end of the condenser tube was removed from the liquid surface, and the mixture was distilled again for 1 min, and the condenser tube inserting into the conical flask containing 20 mL sodium acetate (20 g/L) was washed with a small amount of distilled water. Meanwhile, a blank experiment was conducted simultaneously.

2.11 Determination of the content of SO₃²⁻ in the samples

3 mL 0.2 M NaAc-HAc (pH = 4.0) buffer solution, 100 μL 100 μL sample distillate and 100 μL 200 μM H₂O₂ solution were added to the test tube. After even vortex mixing, the mixture in the test tube adopted insulation at 40 °C for 15 min. Then, 200 μL TMB solution (c = 2.0 mg/mL) and 10 mg Zn-(PDC)₂@Fe₃O₄ were added to the test tube for insulation and coloration at 40 °C for 10 min. After the reaction stopped through magnetic separation of Zn-(PDC)₂@Fe₃O₄, a certain volume of the reaction solution was added to a cuvette, and its absorbance was determined at the wavelength of 652 nm, which then was substituted into the linear regression equation of work curve to obtain the content of SO₃²⁻. With the same method, the content of SO₃²⁻ in the water sample collected from Xijiang River, the lake water sample from the Calligraphy Pond, the hylocereus undatus 1 sample, the hylocereus undatus 2 sample, the tremella fuciformis sample and the Chinese wolfberry sample was determined, respectively. A blank experiment with deionized water was conducted simultaneously.

3.1 X-ray single crystal diffraction characterization of Zn-(PDC)₂

The analysis of the results of X-ray single crystal diffraction showed that: the relative molecular mass of the complex was: 431.66, among which the number of C was 14.5 (14 theoretically), the number of H was 10 (10 theoretically), the number of N was 2 (2 theoretically), the number of O was 10.5 (10 theoretically), and the number of Zn was 1 (1 theoretically). This suggests that the molecular formula of Zn-2,6-pyridinedicarboxylic acid should be Zn(C₇H₃NO₄)₂·2H₂O. With the References (Gao et al., 2012; Kjell et al., 1993), its structure is inferred to be what is shown in Fig. 1. The complex was formed by the self-assembling of Zn and 2,6-pyridinedicarboxylic acid, the three teeth chelate coordination of Zn with carboxyl groups O₂ and O₄ on H₂PDC ligand of a molecule and N1 on pyridine ring, as well as with carboxyl groups O₆ and O₇ on H₂PDC ligand of a molecule and N2 on pyridine ring to form a distorted octahedral geometry.

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Fig. 1

Molecular structure diagram of the complex and coordination polyhedron diagram of the metal complex.

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3.2 Infrared spectrum characterization of nano-scaled Fe₃O₄ and Zn-(PDC)₂@Fe₃O₄

References (Zhang, 2007; Su et al., 2011; Zhang, 2008), from Fig. 2 we can see that: the absorption peak of the complex at 3118 cm⁻¹ was the C—H absorption peak of ν_C—H on pyridine ring, and there was no characteristic peak of the carboxyl group at 1750–1700 cm⁻¹, suggesting that all the carboxyl groups in 2,6-pyridinedicarboxylic acid were ionized and involved in coordination. The absorption peaks of the complex at 1679, 1620 and 1471 cm⁻¹ were the infrared absorption peaks of ν_C⚌C and ν_C—N on pyridine ring, and the infrared absorption peaks of the complex at 1580, 1402 and 1302 cm⁻¹ were the absorption peaks of asymmetric stretching vibration ν_as(COO⁻) and symmetric stretching vibration ν_s(COO⁻) of carboxyl groups on pyridine ring. In addition, in the low frequency region, the absorption peaks at 440 cm⁻¹ and 688 cm⁻¹ were the stretching vibration peaks of ν_Zn-N and ν_Zn-0, the absorption peak at 836 cm⁻¹ was the symmetric stretching vibration peak of Si-O-Si, the absorption peak at 1075 cm⁻¹ was the asymmetric stretching vibration peak of Si-O-Si, the stretching vibration peak of Fe—O bond was at 588 cm⁻¹, and the absorption peak wavenumber of Fe—O bond moved 10 cm⁻¹ compared with that of the unmodified Fe₃O₄. The above results showed that the Zn-(PDC)₂@Fe₃O₄ was composed of Fe₃O₄-NH₂ and Zn-(PDC)₂ (see Fig. 3).

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Fig. 2

Infra-red spectrogram of nano-scaled Fe₃O₄ and Zn-(PDC)₂@Fe₃O₄.

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Fig. 3

XRD spectrogram of nano-scaled Fe₃O₄ and Zn-(PDC)₂@Fe₃O₄.

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3.3 X-ray powder diffraction characterization of nano-scaled Fe₃O₄ and Zn-(PDC)₂@Fe₃O₄

References (Zhang, 2008; Zhang and Li, 2009), from the XRD spectrogram and the data of nano-scaled Fe₃O₄ and Zn-(PDC)₂@Fe₃O₄, we can see that the appearance positions of main diffraction peaks of the two were basically the same. 2θ was around 18°, 30°, 35°, 37°, 43°, 53°, 57°, 62°, 71° and 74°, suggesting that both of them had a face-centered cubic structure; but the peak of Zn-(PDC)₂@Fe₃O₄ was greater than that of nano-scaled Fe₃O₄, and the Zn-(PDC)₂@Fe₃O₄ had obvious diffraction absorption peaks at around 10°, 11° and 18°, namely crystal peaks of Zn-(PDC)₂, which proved that the Zn-(PDC)₂@Fe₃O₄ prepared by using the ultrasonic-assisted method contained Zn-(PDC)₂ and nano-scaled Fe₃O₄, and had a good crystallinity and perfect crystal structure (see Fig. 4).

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Fig. 4

SEM image of Zn-H₂PDC@Fe₃O₄. Fig. A was the SEM image of Zn-H₂PDC@Fe₃O₄ below 2 μm, and Fig. B was the SEM image of Zn-H₂PDC@Fe₃O₄ below 100 nm, and Figs. C and D were the SEM images of Zn-H₂PDC@Fe₃O₄ below 20 nm.

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3.4 SEM characterization of Zn-(PDC)₂@Fe₃O₄

From the SEM image of Zn-(PDC)₂@Fe₃O₄ we can see that: the Zn-(PDC)₂@Fe₃O₄ bascially looked like a spherical nanoflower, with a rough surface and a particle diameter of about 270 nm.

3.5 Study of the activity of mimetic peroxidase of Zn-H₂PDC@Fe₃O4

From Fig. 5 we can see that the absorbance of Curve a at 652 nm was 0.515, suggesting that the Zn-(PDC)₂@Fe₃O₄ could catalyze H₂O₂ to produce hydroxyl radical (OH), which could turn the chromogenic substrate TMB blue, and the absorbance of Curves b, c, d, e at 652 nm was almost 0, namely the Zn-(PDC)₂@Fe₃O₄ could not catalyze the H₂O₂ to produce hydroxyl radical (OH) and could not turn the chromogenic substrate TMB blue. The above results proved that the Zn-(PDC)₂@Fe₃O₄ had the good property of mimetic peroxidase, and could reach an obvious absorption peak at the wavelength of 652 nm. Therefore, 652 nm was selected as the maximum absorption wavelength of the reaction system.

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Fig. 5

Proving of the mimetic peroxidase activity of Zn-(PDC)₂@Fe₃O₄ and selection of maximum absorption wavelength of Zn-(PDC)₂@Fe₃O₄. a was H₂O₂ + TMB + catalyst; b was H₂O₂ + TMB; c was H₂O₂; d was H₂O₂ + catalyst; e was TMB + catalyst.

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3.6 Experiment proving that the catalytic activity of Zn-(PDC)₂@Fe₃O₄ was not caused by metal impurities

Fig. 6 showed that the absorbance of the supernatant remained unchanged, which proved that the activity of mimetic peroxidase derived from the hydroxyl radical (OH) produced by the catalyzation of hydrogen peroxide was caused by magnetic nano-scaled Zn-(PDC)₂@Fe₃O₄ rather than by metal impurities.

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Fig. 6

Proving of the catalytic activity of Zn-(PDC)₂@Fe₃O₄ not being caused by metal impurities. a was catalyst; b was supernatant; c was blank group.

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3.7 Optimization of coloration conditions

With the NaAc-HAc (pH = 4.0) buffer solution as the buffer medium, the pH was adjusted from 3 to 8. As shown in Fig. 7(A), the absorbance was large when the pH was 4. Under strong acid conditions, the reaction solution may become pale yellow due to the generation of diimine. Under neutral or alkaline conditions, the absorbance decreased because the number of available hydroxyl radicals produced by the catalytic decomposition of hydrogen peroxide by catalyst decreased sharply due to the poor solubility of diimine and the accelerated decomposition of hydrogen peroxide with the pH greater than 4. Therefore, pH = 4.0 was selected as the acidity of the reaction solution. Fig. 7B showed the influence of the reaction temperature of 20–80 °C on the absorbance, and the optimum reaction temperature was about 40 °C. As the reaction temperature rose, the catalytic activity of Zn-(PDC)₂@Fe₃O₄ decreased, and the decomposition of hydrogen peroxide accelerated. Fig. 7C showed that within 5–10 min, the absorbance of the reaction solution gradually increased, and the reaction rate gradually accelerated. 10 min later, the absorbance of the reaction solution hardly changed as time goes by. Therefore, it can be concluded that the reaction basically completed at 10 min. Fig. 7D showed that the best dosage of Zn-(PDC)₂@Fe₃O₄ was 10 mg, and the absorbance was basically unchanged with the mass of Zn-(PDC)₂@Fe₃O₄ greater than 10 mg. Fig. 7(E), (F) showed the effects of H₂O₂ concentration on the catalytic activity Zn-(PDC)₂@Fe₃O₄, and the absorbance increased gradually as the H₂O₂ concentration increased. There was a good linear relationship between the H₂O₂ concentration and the absorbance at 2 × 10⁻⁶–4×10⁻⁴ mol/L. The linear equation was: y = 0.0025x + 0.038, R² = 0.9995 and the detection limit was: 2 × 10⁻⁷ mol/L. 200 μM H₂O₂ solution was determined 6 times, and the result was shown in Fig. 8. The relative standard deviation RSD = 1.5%, showing that the method for determining H₂O₂ concentration had a good accuracy, and the established colorimetric method could be used for the determination of H₂O₂ concentration.

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Fig. 7

Optimization of the coloration conditions of the mimetic peroxidase. A, B, C, D, E and F were the influence of pH, temperature, reaction time, catalyst dosage and H₂O₂ concentration, respectively.

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Fig. 8

Determination of the accuracy of H₂O₂ by the mimetic peroxidase. A and B were the UV–VIS spectrogram and line chart determining 200 μM H₂O₂ solution 6 times, respectively.

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3.8 Comparative experiment of the catalytic activity of Zn-(PDC)₂@Fe₃O₄, Fe₃O₄ and Zn-(PDC)₂

Under the optimum conditions, the absorbance of 200 μM H₂O₂ reaction solution was determined with the Zn-(PDC)₂@Fe₃O₄, Fe₃O₄ and Zn-(PDC)₂ as the catalysts, and a comparison experiment without catalysts was conducted simultaneously. The results, as shown in Fig. 9, suggestedthat the catalytic activity of Zn-(PDC)₂@Fe₃O₄ was the highest, indicating that the Zn-(PDC)₂@Fe₃O₄ synthesized by Fe₃O₄ and Zn-(PDC)₂ had a synergistic catalytic effect (see Figs. 10–14).

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Fig. 9

Comparative experiment of the catalytic activity of Zn-(PDC)₂@Fe₃O₄, Fe₃O₄ and Zn-(PDC)₂. a, b, c and d were the influence of Zn-(PDC)₂@Fe₃O₄, Fe₃O₄ and Zn-(PDC)₂ respectively as well as without catalyst.

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Fig. 10

Double-reciprocal plot of the K_m and v_max of H₂O₂ by Zn-(PDC)₂@Fe₃O₄.

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Fig. 11

Double-reciprocal plot of the K_m and v_max of TMB by Zn-(PDC)₂@Fe₃O₄.

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Fig. 12

Double-reciprocal plot of the K_m and v_max of H₂O₂ by Fe₃O₄.

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Fig. 13

Double-reciprocal plot of the K_m and v_max of TMB by Fe₃O₄.

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Fig. 14

Schematic diagram of the colorimetric sensor based on the mimetic peroxidase.

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3.9 Catalytic steady-state kinetics of Zn-(PDC)₂@Fe₃O₄

The following equation (Li, 2014): c = A/εb, where c wass the concentration of H₂O₂ oxidation products, A was the absorbance value, ε = 39,000 M⁻¹ S⁻¹, b = 1.0 cm was used. The Michaelis constant K_m was calculated by the Lineweaver-Burk (Xie, 2012) equation 1/v = (1/v_max)(1 + K_m/c), and a straight line was obtained by drawing 1/v against 1/c. The horizontal intercept of the straight line was −1/K_m, and the longitudinal intercept of the straight line was 1/v_max. In this way, the K_m and v_max could be calculated, where v was the initial reaction rate, v_max was themaximum reaction rate, and c was the substrate concentration. With the data from Tables 1–4, the following could be calculated: the Michaelis constant of H₂O₂ by Fe₃O₄ was: K_m = 1.116 mM, and the maximum reaction rate: v_max = 4.394 × 10⁻⁸ M s⁻¹; the Michaelis constant of TMB by Fe₃O₄ was: K_m = 0.599 mM, and the maximum reaction rate: v_max = 2.155 × 10⁻⁸ M s⁻¹; The Michaelis constant of H₂O₂ by Zn-(PDC)₂@Fe₃O₄ was: K_m = 0.457 mM, and the maximum reaction rate: v_max = 3.946 × 10⁻⁸ M s⁻¹; the Michaelis constant of TMB by Zn-(PDC)₂@Fe₃O₄ was: K_m = 0.207 mM, and the maximum reaction rate: v_max = 2.424 × 10⁻⁸ M s⁻¹. The Michaelis constant of H₂O₂ by Fe₃O₄ was lower than that of H₂O₂ by HRP, and the Michaelis constant of TMB by Fe₃O₄ was close to that of TMB by HRP, suggesting that the affinity for H₂O₂ by synthesized Fe₃O₄ was higher than that for H₂O₂ by HRP; the Michaelis constants of H₂O₂ and TMB by Zn-(PDC)₂@Fe₃O₄ were lower than those of H₂O₂ and TMB by HRP, suggesting that the affinities for H₂O₂ by Zn-(PDC)₂@Fe₃O₄ were higher than those for H₂O₂ and TMB by Fe₃O₄, and the Zn-(PDC)₂ @Fe₃O₄ had a better catalytic activity than Fe₃O₄ (see Tables 5 and 6).

4.1 Optimization of the reaction time for determining the content of SO₃²⁻

The effects of the 1, 5, 10, 15, 20, 25 and 30 min reaction times were studied, and other conditions adopted Zn-(PDC)₂@Fe₃O₄ as the best coloration conditions of mimetic peroxidase; Fig. 15 showed that the optimum reaction time for determining the content of SO₃²⁻ was 15 min (see Fig. 16).

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Fig. 15

Optimization of the reaction time for determining the content of SO₃²⁻.

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Fig. 16

Standard curve of SO₃²⁻.

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4.2 Drawing of the standard curve of SO₃²⁻

The experimental results showed that: the linearity was good when the content of SO₃²⁻ was determined to be 8 × 10⁻⁷–8×10⁻⁵ mol/L by using the Zn-(PDC)₂@Fe₃O₄ mimetic peroxidase colorimetric method, and the linear equation was: y = −0.00098x + 0.18391(R² = 0.998), and the detection limit of SO₃²⁻ was 8 × 10⁻⁸ mol/L.

4.3 Experiment of interference by metal ions

Fig. 17 showed that: the interference index of metal ions was less than 5.41%, so it can be considered that the above metal ions did not interfere with the method for determining the content of SO₃²⁻.

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Fig. 17

Experiment of interference by metal ions.

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4.4 Selectivity of the method for determining the content of SO₃²⁻

Fig. 18 showed that: only SO₃²⁻ led to the decreased absorbance, indicating that the method for determining the content of SO₃²⁻ had a good selectivity (see Figs. 19 and 20).

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Fig. 18

Selectivity of the method for determining the content of SO₃²⁻.

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Fig. 19

Reusability of Zn-(PDC)₂@Fe₃O₄.

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Fig. 20

Magnetic separation of Zn-(PDC)₂@Fe₃O₄.

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4.5 Determination of the content of SO₃²⁻ in samples and its precision and accuracy

The content of SO₃²⁻ in the samples was determined using the method in Experiment 2.11, and the accuracy of the method for determining the content of SO₃²⁻ was determined using the standard addition recovery method, and the results are shown in Table 6. The recovery rate of standard addition was 88–110% and the RSD was 2.7–9.2%, suggesting that the method for determining the content of SO₃²⁻ had a good precision and accuracy. Among the 6 samples to be determined, SO₃²⁻ was not detected in the hylocereus undatus, the tremella fuciformis orthe Chinese wolfberry, while 36.1 μM (2.9 μg/mL) SO₃²⁻ and 17.2 μM (1.4 μg/mL) SO₃²⁻ were detected in Xijiang River and the lake water, respectively.

4.6 Reusability of catalyst

After being used 8 times, the absorbance of the Zn-(PDC)₂@Fe₃O₄ basically remained unchanged; after being used 9–12 times, the absorbance of the Zn-(PDC)₂@Fe₃O₄ decreased slightly; after being used 13–15 times, the absorbance of the Zn-(PDC)₂@Fe₃O₄ decreased significantly. The above results showed that the Zn-(PDC)₂@Fe₃O₄ had a good reusability.