Cobalt-based nano-enzyme with yolk shell structure and application of cobalt-based nano-enzyme in mercury ion detection

Abstract

The invention belongs to the technical field of pollutant detection, and relates to a cobalt-based nano-enzyme with an egg yolk shell structure and application of the cobalt-based nano-enzyme in mercury ion detection. The preparation method comprises the following steps: adding cobalt nitrate hexahydrate and hexadecyl trimethyl ammonium bromide into water, and uniformly mixing; mixing with a 2-methylimidazole solution, stirring and reacting to prepare a ZIF-67 cube; and adding the ZIF-67 / PTA into ethanol, uniformly mixing, uniformly mixing with an aqueous solution of polybasic oxyacid, and then carrying out stirring reaction to prepare the ZIF-67 / PTA. The preparation method comprises the following steps: adding PTA into an ethanol solution, uniformly mixing, then adding a chloroauric acid solution and a sodium borohydride solution, and carrying out a stirring reaction to prepare ZIF-67 / PTA / Au. The synthesized material has a yolk shell nanocube mesoporous structure, and is low in raw material price, stable in performance and suitable for large-scale synthesis. The visual detection on Hg can be realized.

Description

Technical Field

[0001] The present invention belongs to the technical field of pollutant detection, and relates to a cobalt-based nanozyme with a yolk-shell structure and its application in the detection of mercury ions. Background Art

[0002] With the rapid development of modern industry, the problem of heavy metal ion pollution has attracted great attention to its negative impacts on water environment and organisms. As a typical toxic heavy metal, Hg²⁺ comes from natural emissions and anthropogenic pollution, and can enter the human body through skin contact, inhalation, diet intake, etc. The maximum allowable concentration of Hg²⁺ in water is set at 6 μg / L. The toxicity of Hg²⁺ is mainly reflected in its damage to the nervous system, kidneys and immune system. Long-term exposure to a low-concentration Hg²⁺ environment may lead to chronic poisoning, manifested as symptoms such as memory loss, mood instability, muscle tremors, etc. In addition, Hg²⁺ can also accumulate in organisms through the biomagnification effect of the food chain, causing serious damage to the ecosystem.

[0003] Therefore, people have made a lot of efforts in the detection technology of Hg²⁺, such as atomic absorption spectrometry (AAS), atomic fluorescence spectrometry (AFS), electrochemical analysis and inductively coupled plasma mass spectrometry (ICP-MS). Although these traditional methods have high sensitivity and accuracy, they have some obvious limitations. First of all, these methods usually require complex sample pretreatment processes, including digestion, extraction and concentration steps, which not only increase the detection time, but also may lead to sample loss and contamination. Secondly, these technologies rely on large-scale instrument equipment and require professional technical personnel for operation and maintenance, making it difficult to achieve on-site rapid detection. For example, although ICP-MS has extremely high sensitivity and the ability to detect multiple elements simultaneously, its equipment is expensive, the operation is complex, and the purity requirements for samples are relatively high. Therefore, in practical applications, especially in the fields of environmental monitoring and food safety, there is an urgent need for a rapid, simple and on-site Hg²⁺ detection method.

[0004] In recent years, with the rapid development of nanotechnology and biosensor technology, some new Hg²⁺ detection methods have gradually emerged. For example, the sensor technology based on surface plasmon resonance (SPR) shows great application potential due to its advantages of high sensitivity, rapid response and label-free. The SPR sensor realizes the detection of target ions by detecting the resonance change of the metal surface plasmon, and has the ability of real-time monitoring and high-throughput screening. In addition, significant progress has also been made in fluorescence probes and electrochemical sensors based on nanomaterials. These new detection technologies not only improve the detection efficiency, but also reduce the detection cost, providing the possibility for on-site rapid detection of Hg²⁺. However, these technologies are still in the development stage, and there is still much room for improvement in their application prospects and detection effects. Summary of the Invention

[0005] The present invention proposes a novel cobalt-based nanozyme with a yolk-shell structure and its application in mercury ion detection in view of the problems existing in traditional Hg²⁺ detection.

[0006] In order to achieve the above object, the present invention is implemented by the following technical solutions: A preparation method of a cobalt-based nanozyme with a yolk-shell structure, the steps are as follows.

[0007] (1) Cobalt nitrate hexahydrate and cetyltrimethylammonium bromide are added to water and mixed evenly to obtain a mixed solution A; the mixed solution A is mixed evenly with a 2-methylimidazole solution, and stirred and reacted to prepare ZIF-67 cubes.

[0008] (2) The ZIF-67 cubes are added to ethanol and mixed evenly to obtain a mixed solution B; the mixed solution B is mixed evenly with an aqueous solution of a polyoxoacid, and then stirred and reacted to prepare ZIF-67 / PTA.

[0009] (3) ZIF-67 / PTA is added to an ethanol solution and mixed evenly, then chloroauric acid solution and sodium borohydride solution are added, and then stirred and reacted to prepare ZIF-67 / PTA / Au.

[0010] Preferably, the polyoxoacid in step (2) is phosphotungstic acid, the volume fraction of ethanol in the ethanol solution in step (3) is 50%, and the chloroauric acid solution and the sodium borohydride solution need to be added sequentially.

[0011] Preferably, the stirring reaction time in step (1) is 10-40 min; the stirring reaction time in step (2) is 4-6 h; the total stirring reaction time in step (3) is 4-6 h.

[0012] The present invention proposes the application of the cobalt-based nanozyme with a yolk-shell structure prepared by the above method in mercury ion detection.

[0013] The application method is to mix the cobalt-based nanozyme, TMB and NaAc-HAc buffer solution evenly to obtain a reaction system, let it stand naturally for at least 5 min, then add the test solution, mix evenly and let it stand naturally for at least 5 min, filter, and record the absorbance value at 652 nm with a UV-visible spectrophotometer.

[0014] Preferably, the pH of the NaAc-Hac buffer solution is 5.0, and the volume fraction of the NaAc-HAc buffer solution in the reaction system is 75-80%.

[0015] Compared with the prior art, the advantages and positive effects of the present invention are as follows: 1. The synthesized ZIF-67 / PTA / Au material of the present invention has a mesoporous structure of yolk-shell nanocubes, effectively increasing the contact area with the substrate and providing more active sites. By adding PTA to compete for coordination with 2-methylimidazole in ZIF-67 to capture Co²⁺, it can also act as a local reducing agent to effectively improve the charge transfer rate. In addition, the rapid etching effect of PTA assisted by NaBH4 is also beneficial to the catalytic oxidation reaction of the nanozyme.

[0016] 2. The raw materials are cheap, rich in resources, with low synthesis cost, and the product performance is stable, suitable for large-scale synthesis.

[0017] 3. Based on the principle of enhancing the activity of ZIF-67 / PTA / Au by Hg²⁺, the visual detection of Hg²⁺ can be realized, with high sensitivity, low detection limit (0.26 μM), wide linear response range (1 - 2000 μM), and good selectivity and stability. Description of the Drawings

[0018] Figure 1 are the X-ray diffraction patterns (XRD) of ZIF-67, ZIF-67 / PTA, and ZIF-67 / PTA / Au.

[0019] Figure 2 A is the scanning electron microscope image (SEM) of ZIF-67, Figure 2 B is the SEM image of ZIF-67 / PTA, Figure 2 C is the SEM image of ZIF-67 / PTA / Au, Figure 2 D is the transmission electron microscope image (TEM) of ZIF-67, Figure 2 E is the TEM image of ZIF-67 / PTA, Figure 2 F is the TEM image of ZIF-67 / PTA / Au.

[0020] Figure 3 is the high-resolution transmission electron microscope (HRTEM) image of ZIF-67 / PTA / Au.

[0021] Figure 4 A is the N2 adsorption - desorption isotherm of ZIF-67 / PTA / Au, Figure 4 B is the corresponding pore size distribution curve.

[0022] Figure 5 is the comparison chart of the OXD-like activities of different nanozymes.

[0023] Figure 6 is the specific detection chart of ZIF-67 / PTA / Au nanozyme.

[0024] Figure 7Figure showing the effect of the pH value of the buffer solution on the activity of ZIF-67 / PTA / Au nanozyme. Detailed implementation mode

[0025] In order to more clearly understand the above-mentioned objects, features and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments. It should be noted that, without conflict, the embodiments of the present application and the features in the embodiments can be combined with each other.

[0026] In the following description, many specific details are set forth in order to fully understand the present invention. However, the present invention can also be implemented in other ways different from those described herein. Therefore, the present invention is not limited by the limitations of the specific embodiments disclosed in the following specification.

[0027] Example 1 Add 0.6 g of Co(NO3)2·6H2O and 10 mg of cetyltrimethylammonium bromide (CTAB) to 20 mL of deionized water. Then, quickly add a solution containing 140 mL of ultrapure water and 9.03 g of 2-methylimidazole during manual stirring with a glass rod, and stir at room temperature for 20 min for the reaction. The stirring rate is 800 rpm. After the reaction, wash by centrifugation alternately with ethanol and deionized water 3 times, with each amount about 20 mL. The centrifugation rate is 8000 rmp, and the centrifugation time for each time is 8 min. Then scrape the solid and dry it at 60 °C for 12 h to obtain a purple solid, denoted as ZIF-67.

[0028] Disperse 50 mg of the prepared ZIF-67 nanocubes in 40 mL of ethanol. Dissolve phosphotungstic acid (PTA) in ultrapure water to prepare 20 mL of a solution with a concentration of 0.125 g / L. Subsequently, slowly add the PTA solution to the ZIF-67 dispersion, and stir and react at room temperature for 6 h. The stirring rate is 500 rpm. After the reaction, wash by centrifugation alternately with ethanol and deionized water 3 times, with each amount about 20 mL. The centrifugation rate is 8000 rmp, and the centrifugation time for each time is 8 min. Then scrape the solid and dry it at 60 °C for 12 h to obtain a dark purple solid, denoted as ZIF-67 / PTA.

[0029] Disperse 20 mg of the prepared ZIF-67 / PTA in 4 mL of an ethanol solution with a volume fraction of 50%, sonicate for 2 min, and then add 0.5 mL of a chloroauric acid solution with a concentration of 25 mM. After stirring at 1000 rpm at room temperature for 4 h, quickly add 2.4 mL of a sodium borohydride solution with a concentration of 0.05 M, and continue to stir at 1000 rpm for 1 h for the reaction. After the reaction, wash by centrifugation alternately with ethanol and deionized water 3 times, with about 20 mL used each time, the centrifugation rate is 8000 rmp, and the centrifugation time for each time is 8 min. Then scrape the solid, dry it at 60 °C for 12 h to obtain a black solid, denoted as ZIF-67 / PTA / Au.

[0030] Perform XRD characterization on the prepared ZIF-67, ZIF-67 / PTA, and ZIF-67 / PTA / Au, and the results are as Figure 1 shown. It can be Figure 1 seen that the crystal form of the ZIF-67 material does not change significantly after coating with PTA; ZIF-67 / PTA / Au can correspond to the standard colorimetric card of Au.

[0031] Perform SEM and TEM characterization on the prepared ZIF-67, ZIF-67 / PTA, and ZIF-67 / PTA / Au, and the results are as Figure 2 shown in A-2F. From Figure 2 A and 2D, it can be seen that ZIF-67 is a uniform cubic structure with a smooth surface; from Figure 2 B and 2E, it can be seen that after coating with PTA, an obvious yolk-shell structure is formed on the surface of ZIF-67, and the shell layer presents a flaky structure, and the surface roughness of the material increases; from Figure 2 C and 2F, it can be seen that on the basis of ZIF-67 / PTA, ZIF-67 / PTA / Au successfully introduces gold nanoparticles (Au NPs), forming an obvious yolk-shell structure, and the Au NPs are evenly distributed in the shell layer.

[0032] The lattice state of the ZIF-67 / PTA / Au material is as Figure 3 , Figure 3 showing two sets of lattice fringes of about 2.36 Å and 2.04 Å, corresponding to the (111) and (200) crystal planes of Au respectively.

[0033] The N2 adsorption-desorption isotherm of the ZIF-67 / PTA / Au material is as Figure 4As shown, it is of the typical type IV and has an obvious hysteresis loop, indicating that ZIF-67 / PTA / Au has a mesoporous structure. The specific surface area of the material was detected to be 683.33 m² / g, and the total pore volume was 0.85 cm³ / g. The mesoporous property of the nanozyme is beneficial to the contact between the substrate and the material. The large contact area between the two materials can provide more active sites, thus facilitating the enhancement of the catalytic oxidation performance of the material.

[0034] Example 2 Unless otherwise specified, this example is the same as Example 1. Add 0.3 g of Co(NO3)2·6H2O and 10 mg of CTAB to 20 mL of deionized water. Then, quickly add a solution containing 140 mL of ultrapure water and 9.03 g of 2-methylimidazole under stirring, and stir at room temperature for 20 min at a stirring rate of 800 rpm. After the reaction, wash by centrifugation with ethanol and deionized water alternately 3 times, with a centrifugation rate of 8000 rmp and a centrifugation time of 8 min each time. Then, dry at 60 °C for 12 h to obtain purple solid, ZIF-67 nanocubes.

[0035] Disperse 50 mg of the prepared ZIF-67 nanocubes in 40 mL of ethanol. Dissolve phosphotungstic acid (PTA) in ultrapure water to prepare 20 mL of a solution with a concentration of 0.125 g / L. Subsequently, slowly add the PTA solution to the ZIF-67 dispersion, and stir and react at room temperature for 10 h at a stirring rate of 500 rpm. After the reaction, wash by centrifugation with ethanol and deionized water alternately 3 times, with a centrifugation rate of 8000 rmp and a centrifugation time of 8 min each time. Then, dry at 60 °C for 12 h to obtain dark purple solid, ZIF-67 / PTA.

[0036] Disperse 20 mg of the prepared ZIF-67 / PTA in 4 mL of ethanol solution. After ultrasonic treatment, add 0.5 mL of chloroauric acid solution with a concentration of 25 mM. Stir at room temperature for 4 h, then quickly add 2.4 mL of freshly prepared sodium borohydride solution with a concentration of 0.05 M, and continue to stir for 1 h at a stirring rate of 1000 rpm. After the reaction, wash by centrifugation with ethanol and deionized water alternately 3 times, with a centrifugation rate of 8000 rmp and a centrifugation time of 8 min each time. Then, dry at 60 °C for 12 h to obtain black solid, ZIF-67 / PTA / Au.

[0037] Example 3 Unless otherwise specified, this example is the same as Example 1. Add 0.6 g of Co(NO3)2·6H2O and 10 mg of CTAB to 20 mL of deionized water. Then, quickly add a solution containing 50 mL of ultrapure water and 9.03 g of 2-methylimidazole during stirring, and stir at room temperature for 40 min at a stirring rate of 800 rpm. After the reaction, centrifuge and wash alternately with ethanol and deionized water 3 times, with a centrifuge rate of 8000 rmp and a centrifugation time of 8 min each time. Then, dry at 60 °C for 12 h to obtain a purple solid, ZIF-67.

[0038] Disperse 50 mg of the prepared ZIF-67 nanocubes in 40 mL of ethanol. Dissolve phosphotungstic acid (PTA) in ultrapure water to prepare a 20 mL solution with a concentration of 0.125 g / L. Subsequently, slowly add the PTA solution to the ZIF-67 dispersion, react at room temperature for 6 h with a stirring rate of 500 rpm. After the reaction, centrifuge and wash alternately with ethanol and deionized water 3 times, with a centrifuge rate of 8000 rmp and a centrifugation time of 8 min each time. Then, dry at 60 °C for 12 h to obtain a dark purple solid, ZIF-67 / PTA.

[0039] Disperse 20 mg of the prepared ZIF-67 / PTA in 4 mL of ethanol. After ultrasonic treatment, add 0.5 mL of a chloroauric acid solution with a concentration of 25 mM. Stir at room temperature for 2 h, then quickly add 2.4 mL of a newly prepared sodium borohydride solution with a concentration of 0.05 M, and continue to stir for 1 h with a stirring rate of 1000 rpm. After the reaction, centrifuge and wash alternately with ethanol and deionized water 3 times, with a centrifuge rate of 8000 rmp and a centrifugation time of 8 min each time. Then, dry at 60 °C for 12 h to obtain a black solid, ZIF-67 / PTA / Au.

[0040] Perform performance tests on the cobalt-based nanozyme prepared in Example 1.

[0041] 1. Determination of the OXD-like activity of the cobalt-based nanozyme Add the ZIF-67 / PTA / Au nanozyme prepared in Example 1 to ethanol to make a dispersion with a concentration of 500 μg / mL. Add 50 μL of 3,3',5,5'-tetramethylbenzidine (TMB, purchased from Sangon Biotech (Shanghai) Co., Ltd.) to 850 μL of NaAc-HAc buffer solution (pH 5.0), and then take 100 μL of the dispersion and add it thereto. React naturally at room temperature for 5 min, then filter, and detect the filtrate with a UV-visible spectrophotometer, and record the UV-visible absorption spectrum at 652 nm. The results are as Figure 5 , from Figure 5It can be seen that the ZIF-67 / PTA / Au nanozyme has excellent OXD-like activity.

[0042] 2. Standard curve determination The detection was carried out by changing the concentration and volume of the Hg²⁺ solution respectively. During the detection, the added volume of TMB was kept at 50 μL, and the system was made up to a total volume of 1 mL with NaAc-HAc buffer solution at pH 5.0. After reaction and filtration, the ultraviolet-visible absorption spectrum at 652 nm was measured to test the detection linear range of the ZIF-67 / PTA / Au nanozyme. After detection, there was a good linear relationship between the concentration of the Hg²⁺ solution and the absorbance in the range of 1-2000 μM, and the detection limit was 0.26 μM. Among them, in the concentration range of 1-2000 μM, y = 0.00119x + 0.49326, R 2 = 0.99, where x is the concentration of the Hg²⁺ solution and y is the absorbance.

[0043] 3. Specificity test Detect Fe 2+ , Sn 2+ , Cu 2+ , Fe 3+ , Cr 3+ , Pb 2+ , Ni 2+ , Co 2+ , Zn 2+ , Sn 4+ , Mn 2+ and Cd 2+ for the interference of the detection results. (The interfering ions were provided by the following substances in turn: ferrous chloride, stannous chloride dihydrate, copper chloride, ferric chloride, chromium(III) nitrate nonahydrate, lead nitrate, nickel(II) acetate tetrahydrate, cobalt(II) nitrate hexahydrate, zinc chloride, tin(IV) chloride pentahydrate, manganese chloride and chromium chloride) The specific operation is as follows: Weigh the substances providing the interfering ions mentioned above and add them to 10 mL of deionized water respectively to prepare interference solutions with the concentration of each interfering ion reaching 1.5 mmol / L. The ZIF-67 / PTA / Au nanozyme prepared in Example 1 was added to ethanol to make a dispersion with a concentration of 500 μg / mL. Then, 100 μL of the dispersion was taken and added to 750 μL of NaAc-HAc buffer solution (pH 5.0) containing 50 μL of TMB and 100 μL of the interference solution. React naturally at room temperature for 5 min, then filter, and the filtrate was detected with a UV-visible spectrophotometer, and the ultraviolet-visible absorption spectra at 652 nm were recorded respectively. The results are shown in Figure 6 , and only after adding the Hg²⁺ solution, there is an obvious enhancing effect on the OXD-like activity of ZIF-67 / PTA / Au.

[0044] 4. Real sample test The standard addition method was used to detect the detection of Hg²⁺ by ZIF-67 / PTA / Au in actual water samples. The specific operation is as follows: Tap water from the Chemistry Building of Nanjing Tech University and lake water from Zhengxin Lake were taken for real sample detection respectively. In the experiment, Hg²⁺ solutions with different concentrations were added to each water sample by the standard addition method, and 100 μL of the water sample was taken for detection respectively. The actual contents of Hg²⁺ in the water samples of each detection group are shown in Table 1. The ZIF-67 / PTA / Au nanozyme prepared in Example 1 was added to ethanol to make a dispersion with a concentration of 500 μg / mL. Then, 100 μL of the dispersion was added to 750 μL of NaAc-HAc buffer solution (pH 5.0) containing 50 μL of TMB and 100 μL of the water sample, and the reaction was allowed to proceed naturally at room temperature for 5 min. Then, it was filtered, and the filtrate was detected by an ultraviolet-visible spectrophotometer, and the ultraviolet-visible absorption spectrum at 652 nm was recorded. This was repeated three times. Substituting into the working curve, the detected amount was calculated. The results are shown in Table 1. From the results in Table 1, it can be seen that the relative standard deviation of the detection values ≤ 3.7% (n = 3), indicating that this colorimetric sensor has good repeatability in the rapid detection of Hg²⁺ content in water. In addition, the recovery rate is between 97.0% - 107.5%, proving that this method has good accuracy and reliability for the determination of Hg²⁺ in actual water samples.

[0045] Table 1 Real sample test results Comparative Example 1 Phosphotungstic acid was not added, and the rest of the preparation process was the same as that in Example 1. The prepared nanozyme was denoted as ZIF-67 / Au. It was added to ethanol to make a dispersion with a concentration of 500 μg / mL. Then, 100 μL of the dispersion was added to 800 μL of NaAc-HAc buffer solution (pH 5.0) containing 50 μL of TMB, and the reaction was allowed to proceed naturally at room temperature for 5 min. Then, it was filtered, and the filtrate was detected by an ultraviolet-visible spectrophotometer, and the ultraviolet-visible absorption spectrum at 652 nm was recorded. The results are as Figure 5 (ZIF-67 / Au). It can be Figure 5 seen that the OXD activity of the ZIF-67 / Au nanozyme prepared in this comparative example is not as high as that of the ZIF-67 / PTA / Au nanozyme prepared in Example 1.

[0046] Comparative Example 2 The difference between this comparative example and Example 1 lies in the different pH values of the buffer solution. The NaAc-HAc buffer solution with pH values of 2, 4, 5, 6, 7, and 8 was sequentially substituted. The remaining conditions were the same as those in Example 1. After the samples were processed using the same steps for the determination of the OXD-like activity of the cobalt-based nanozyme, the ultraviolet-visible absorption spectrum at 652 nm was recorded. The results are as Figure 7 shown. It can be seen from Figure 7 that the absorbance value is the highest when the pH of the buffer solution is 5.0.

[0047] As mentioned above, the above are only the preferred embodiments of the present invention, and it is not intended to limit the present invention in other forms. Any person skilled in the art may use the technical content disclosed above to make changes or modifications into equivalent embodiments with equivalent changes and apply them to other fields. However, any simple modification, equivalent change, and modification made to the above embodiments based on the technical essence of the present invention without departing from the technical solution content of the present invention still fall within the protection scope of the technical solution of the present invention.

Claims

1. A preparation method of a cobalt-based nanozyme with a yolk-shell structure, characterized in that, The steps are as follows: (1) Cobalt nitrate hexahydrate and cetyltrimethylammonium bromide are added to water and mixed evenly to obtain a mixed solution A; the mixed solution A is mixed evenly with a 2-methylimidazole solution, and stirred and reacted to prepare ZIF-67 cubes; (2) The ZIF-67 cubes are added to ethanol and mixed evenly to obtain a mixed solution B; the mixed solution B is mixed evenly with an aqueous solution of a polyoxoacid, and then stirred and reacted to prepare ZIF-67 / PTA; (3) The ZIF-67 / PTA is added to an ethanol solution and mixed evenly, then chloroauric acid solution and sodium borohydride solution are added, and then stirred and reacted to prepare ZIF-67 / PTA / Au.

2. The preparation method of the cobalt-based nanozyme with a yolk-shell structure according to claim 1, wherein, The polyoxoacid in step (2) is phosphotungstic acid, and the volume fraction of ethanol in the ethanol solution in step (3) is 50%.

3. The preparation method of the cobalt-based nanozyme with an egg yolk shell structure according to claim 1, characterized in that, The stirring reaction time in step (1) is 10 - 40 min; the stirring reaction time in step (2) is 4 - 6 h; the total stirring reaction time in step (3) is 4 - 6 h.

4. Application of the cobalt-based nanozyme with a yolk-shell structure prepared by the method according to any one of claims 1 - 3 in the detection of mercury ions.

5. The application according to claim 4, characterized in that, The application method is to mix the cobalt-based nanozyme, TMB and NaAc-HAc buffer solution evenly to obtain a reaction system, let it stand and react naturally for at least 5 min, then add the solution to be measured, mix evenly and let it stand and react naturally for at least 5 min, filter, and record the absorbance value at 652 nm with a UV-visible spectrophotometer.

6. The application according to claim 5, characterized in that The pH of the NaAc-Hac buffer solution is 5.0, and the volume fraction of the NaAc-HAc buffer solution in the reaction system is 75 - 80%.

Images