Zirconium phosphate-based composite material, and preparation method and application thereof

Abstract

The application belongs to the technical field of adsorbents, and particularly relates to a zirconium phosphate-based composite material, a preparation method and application thereof. The application provides a preparation method of a zirconium phosphate-based composite material, which comprises the following steps: mixing a soluble zirconium source, a hydrated phosphate and a mineralizer, and obtaining the zirconium phosphate-based composite material through thermal crystallization; and the number of crystal water in the hydrated phosphate is greater than or equal to 2. In the application, the hydrated phosphate containing more than 2 crystal waters is used as a raw material, the obtained composite material has high removal efficiency for ammonium in a water system or a cell culture medium; and the obtained composite material has good biocompatibility, has a slight influence on the pH of a system, and can be used repeatedly.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of adsorbents, and particularly relates to a zirconium phosphate-based composite material and a preparation method and application thereof. BACKGROUND

[0002] In the process of high-density cell culture, cells quickly consume nutrients such as glucose and glutamine, and produce metabolic byproducts such as ammonia, lactic acid and carbonic acid. Among them, ammonium is considered to be a toxic substance that inhibits cell growth, the accumulation of ammonia will affect cell growth and the production of recombinant proteins, change the electrochemical gradient of the cell chamber, damage the enzyme activity in the cell body, and trigger cell apoptosis. When the concentration of ammonia accumulates to a certain extent, the cell culture medium must be replaced in time to maintain the normal life activities of the cells. The cost of the cell culture medium is high, especially fetal bovine serum, growth factors and nutrients. Therefore, in the future operation process of large-scale bioreactors, it is necessary to minimize the inhibitory effect of metabolic byproducts on cells and achieve efficient recycling of the culture medium as much as possible to ensure the sustainable development of cell agriculture.

[0003] Layered materials are a new type of material, especially zirconium phosphate and its derivatives, which have many applications such as flame retardation, ion exchange, catalysis, antibiosis, etc. Research on zirconium phosphate and its derivatives in removing heavy metals in wastewater, fixing radioactive elements and rare earth elements in nuclear pollution is increasingly rich. Some previous studies have confirmed that zirconium phosphate can adsorb ammonia gas and can achieve multiple cycles; at the same time, in the field of medical kidney dialysis, it can be used as an ion exchanger for cations.

[0004] Due to the complexity of the components in the cell culture medium and the large number of competing cations, the existing zirconium phosphate and its derivatives have poor effect on removing ammonium in the culture medium. SUMMARY

[0005] The purpose of the present application is to provide a zirconium phosphate-based composite material and a preparation method and application thereof. The method provided by the present application can prepare a zirconium phosphate-based composite material that has a high removal effect on ammonium in a water system or a cell culture medium.

[0006] In order to achieve the above-mentioned purpose, the present application provides the following technical scheme:

[0007] The present application provides a preparation method of a zirconium phosphate-based composite material, comprising the following steps:

[0008] Mixing a soluble zirconium source, a hydrated phosphate and a mineralizer, and hydrothermally crystallizing to obtain the zirconium phosphate-based composite material;

[0009] The number of crystal water in the hydrated phosphate is ≥2.

[0010] Preferably, the soluble zirconium source includes one or more of zirconium oxychloride octahydrate, zirconium nitrate, and zirconium sulfate.

[0011] Preferably, the hydrated phosphate includes one or more of dihydrogen phosphate, hydrogen phosphate, metaphosphate, and orthophosphate;

[0012] The cations in the hydrated phosphate include sodium ions, potassium ions, calcium ions, or magnesium ions.

[0013] Preferably, the mineralizing agent includes one or more of sodium fluoride, hydrofluoric acid, and ammonium fluoride.

[0014] Preferably, the molar ratio of the soluble zirconium source to the hydrated phosphate is 1:1 to 5, respectively, based on the molar amounts of Zr and P.

[0015] Preferably, the molar ratio of the hydrated phosphate to the mineralizing agent is 5 to 15:1.

[0016] Preferably, the hydrothermal crystallization temperature is 100–120°C, and the holding time is 12–36 h.

[0017] The present invention also provides a zirconium phosphate-based composite material prepared by the preparation method described above, wherein the zirconium phosphate-based composite material has a two-dimensional layered structure;

[0018] The zirconium phosphate-based composite material has a particle size of 300–1400 nm; the interlayer spacing of the two-dimensional lamellar structure is…

[0019] The crystallinity of the zirconium phosphate-based composite material is 68-75%.

[0020] The present invention also provides the application of the zirconium phosphate-based composite material described in the above technical solution in the adsorption of ammonium.

[0021] Preferably, the ammonium comprises ammonium in an aqueous solution or ammonium in a cell culture medium.

[0022] This invention provides a method for preparing a zirconium phosphate-based composite material, comprising the following steps: mixing a soluble zirconium source, hydrated phosphate, and a mineralizing agent, followed by hydrothermal crystallization to obtain the zirconium phosphate-based composite material; wherein the hydrated phosphate contains ≥2 water molecules of crystallization. In this invention, using hydrated phosphate containing more than two water molecules of crystallization as a raw material can improve the crystallinity of the crystals, forming wider and more regular ion channels, and increasing the ion adsorption capacity; simultaneously, the water molecules of crystallization in the raw material can increase the hydrophilicity of the crystals, accelerate the ion exchange rate, and thus enable the obtained composite material to have high removal efficiency for ammonium in aqueous systems or cell culture media; furthermore, the obtained composite material has good biocompatibility, minimal impact on the pH of the system, and can be recycled multiple times. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 SEM image of the zirconium phosphate sodium obtained in Example 1;

[0024] Figure 2 SEM image of the zirconium phosphate sodium obtained in Comparative Example 1;

[0025] Figure 3 Particle size distribution graph of the zirconium phosphate sodium obtained in Example 1 and Comparative Example 1;

[0026] Figure 4 Contact angle test graph of the zirconium phosphate sodium obtained in Example 1 and Comparative Example 1;

[0027] Figure 5 Biological toxicity test result graph of the zirconium phosphate sodium obtained in Example 1 and Comparative Example 1;

[0028] Figure 6 Ammonium removal effect graph of the zirconium phosphate sodium obtained in Example 1 in cell culture medium;

[0029] Figure 7 Actual image of the mixed slurry obtained after artificial grinding in Example 1;

[0030] Figure 8 Actual image of the mixed slurry obtained after artificial grinding in Comparative Example 1;

[0031] Figure 9 Removal effect graph of the zirconium phosphate sodium obtained in Example 1 in aqueous solution for repeated use. DETAILED DESCRIPTION

[0032] The present application provides a preparation method of a zirconium phosphate-based composite material, comprising the following steps:

[0033] Mixing a soluble zirconium source, a hydrated phosphate and a mineralizer, and obtaining the zirconium phosphate-based composite material through hydrothermal crystallization;

[0034] The number of crystal water in the hydrated phosphate is ≥2.

[0035] In the present application, all the preparation raw materials are commercially available products well known to those skilled in the art, unless otherwise specified.

[0036] In the present application, the number of crystal water is ≥2, and is further preferably 2, 7 or 12.

[0037] In the present application, the soluble zirconium source preferably includes one or more of zirconium oxychloride octahydrate, zirconium nitrate and zirconium sulfate.

[0038] In the present application, the hydrated phosphate salt preferably comprises one or more of dihydrogen phosphate, hydrogen phosphate, metaphosphate and orthophosphate. In the present application, the cation in the hydrated phosphate salt preferably comprises sodium ion, potassium ion, calcium ion or magnesium ion. In a specific embodiment of the present application, the hydrated phosphate salt is specifically disodium hydrogen phosphate dodecahydrate.

[0039] In the present application, the molar ratio of the soluble zirconium source and the hydrated phosphate salt, respectively in terms of the molar amount of Zr and P, is preferably 1:1-5, further preferably 1:2-4, and more preferably 1:3.

[0040] In the present application, the mineralizer preferably comprises one or more of sodium fluoride, hydrofluoric acid and ammonium fluoride. In the present application, the molar ratio of the hydrated phosphate salt and the mineralizer is preferably 5-15:1, further preferably 8-12:1, and more preferably 10-11:1. In the present application, the mineralizer can increase the stability of the crystal.

[0041] The present application does not have a special limitation on the process of mixing, which can be known to those skilled in the art. In a specific embodiment of the present application, the mixing mode is preferably manual grinding; and the time of manual grinding is preferably 10-20 min. The present application can release the crystal water in the raw material through grinding, as a solvent liquid for the hydrothermal crystallization process, so as to make the crystallization uniform.

[0042] In the present application, the temperature of the hydrothermal crystallization is preferably 100-120℃, further preferably 105-110℃; and the holding time is preferably 12-36 h, further preferably 18-30 h, and more preferably 24 h.

[0043] In the present application, the process of the hydrothermal crystallization preferably comprises:

[0044] The mixture obtained by mixing is placed in a polypropylene reaction bottle, the bottle cap is tightly covered, and then the polypropylene reaction bottle is placed in an oven for heating to perform hydrothermal crystallization.

[0045] After the hydrothermal crystallization, the present application further preferably comprises sequentially performing water washing, filtering, drying and grinding on the obtained reaction liquid.

[0046] The present application does not have a special limitation on the process of water washing and filtering, which can be known to those skilled in the art.

[0047] In the present application, the drying mode is preferably drying in an oven at 60℃ overnight.

[0048] The present application does not have a special limitation on the process of grinding, which can be known to those skilled in the art.

[0049] The preparation method is simple, and can realize scale-up production.

[0050] The application further provides the zirconium phosphate-based composite prepared by the preparation method. Further preferably, the interlayer distance of the two-dimensional sheet structure is 0.7-1.2 nm. In the application, the crystallinity of the zirconium phosphate-based composite is preferably 68-75%, and further preferably 73.7%.

[0051] The application further provides the application of the zirconium phosphate-based composite in adsorbing ammonium.

[0052] In the application, the ammonium preferably includes ammonium in an aqueous solution or ammonium in a cell culture medium.

[0053] In the application, when the zirconium phosphate-based composite is applied to adsorb ammonium in an aqueous solution, the adsorption process preferably includes:

[0054] The zirconium phosphate-based composite and the aqueous solution containing ammonium are mixed, and the obtained mixed solution is placed in a conical flask, and the conical flask is placed in a constant-temperature shaker for oscillation.

[0055] In the application, the concentration of ammonium in the aqueous solution containing ammonium is preferably 0.1-20 mmol / L.

[0056] In the application, the temperature of the constant-temperature shaker is preferably 37℃.

[0057] After the oscillation, the application further preferably includes detecting the mixed system after oscillation, and the detection process preferably includes:

[0058] The sample is taken by using a disposable syringe, filtered through a filter membrane with a pore size of 0.22 μm, and the filtrate is collected to detect the change of ammonium concentration and the change of pH value in the filtrate.

[0059] The specific detection method of the ammonium concentration change and the pH value change is not particularly limited in the present application, and any method known to those skilled in the art can be used.

[0060] In the present application, when the zirconium phosphate-based composite is applied to adsorb ammonium in a cell culture medium, the adsorption process preferably comprises:

[0061] The zirconium phosphate-based composite and the cell culture medium containing ammonium are mixed, and the obtained mixed solution is placed in a conical flask, which is placed in a constant-temperature shaker for oscillation.

[0062] The source of the cell culture medium containing ammonium is not particularly limited in the present application, and any kind of cell culture medium containing ammonium can be used.

[0063] In the present application, the concentration of ammonium in the cell culture medium containing ammonium is preferably 0.1-20 mmol / L. In the present application, the pH value of the cell culture medium containing ammonium is preferably 7.0-7.4, and further preferably 7.2-7.4. In the present application, the ratio of the amount of the zirconium phosphate-based composite to the cell culture medium containing ammonium is preferably 2.5-20 g:1 L.

[0064] In the present application, the temperature of the constant-temperature shaker is preferably 37℃. In the present application, the rotation speed of the constant-temperature shaker is preferably 150 rpm, and the oscillation time is preferably 30 min.

[0065] After the oscillation, the present application further preferably comprises detecting the mixed system after oscillation, and the detection process preferably comprises:

[0066] The sample is taken by a disposable syringe, filtered through a 0.22 μm filter membrane, and the filtrate is collected to detect the ammonium concentration change and the pH value change.

[0067] The specific detection method of the ammonium concentration change and the pH value change is not particularly limited in the present application, and any method known to those skilled in the art can be used.

[0068] The zirconium phosphate-based composite provided by the present application can be used as an adsorbent for ammonium in a cell culture medium, which can meet the application requirements for reducing the toxic effect caused by ammonium accumulation in the process of animal cell culture, and is helpful for the recycling of the cell culture medium in the process of high-density cell culture.

[0069] In order to further illustrate the present application, a zirconium phosphate-based composite, a preparation method and application thereof provided by the present application are described in detail below in conjunction with the drawings and examples, but they should not be understood as limiting the scope of protection of the present application.

[0070] Example 1

[0071] After 3.222 g of zirconium oxychloride octahydrate, 10.744 g of disodium hydrogen phosphate dodecahydrate and 0.126 g of sodium fluoride were mixed, they were put into a mortar for manual grinding for 15 min, and then the obtained mixture (the actual physical picture is shown in Figure 7 ) was transferred into a 30 mL brown polypropylene reaction bottle, the bottle cap was tightly covered, and the bottle was put into an oven with a temperature of 105°C for hydrothermal crystallization for 24 h; the obtained reaction solution was sequentially subjected to water washing, filtration, and drying at 60°C overnight, and then grinding was performed to obtain zirconium sodium phosphate with an average particle size of 1400 nm (labeled as α-Na2ZrP A, with a crystallinity of 73.7% and an interlayer spacing of ).

[0072] Comparative Example 1

[0073] After 3.222 g of zirconium oxychloride octahydrate, 4.258 g of disodium hydrogen phosphate anhydrous and 0.126 g of sodium fluoride were mixed, they were put into a mortar for manual grinding for 15 min, and then the obtained mixture (the actual physical picture is shown in Figure 8 ) was transferred into a 30 mL brown polypropylene reaction bottle, the bottle cap was tightly covered, and the bottle was put into an oven with a temperature of 105°C for hydrothermal crystallization for 24 h; the obtained reaction solution was sequentially subjected to water washing, filtration, and drying at 60°C overnight, and then grinding was performed to obtain zirconium sodium phosphate with an average particle size of 1400 nm (labeled as α-Na2ZrP A, with a crystallinity of 73.7% and an interlayer spacing of ).

[0074] Application Example 1

[0075] 50 mg of zirconium sodium phosphate obtained in Example 1 and Comparative Example 1 was respectively weighed and added into a conical flask containing 20 mL of an NH4Cl aqueous solution with a concentration of 50 mmol / L and a pH of 7.3, a rubber stopper was covered, and the conical flask was put into a constant-temperature shaker with a temperature of 37°C and a rotation speed of 150 rpm for oscillation for 30 min.

[0076] After the oscillation was completed, a disposable syringe was used for sampling, the sample was filtered through a filter membrane with a pore size of 0.22 μm, the filtrate was collected, and the change in the ammonium concentration in the solution and the change in the pH of the solution were detected.

[0077] The detection showed that the ammonium adsorption capacity of α-Na2ZrP A and α-Na2ZrP B was 5.61±0.09 mmol / g and 5.33±0.45 mmol / g respectively, and the pH of the solution was increased by 0.58±0.01 and 0.65±0.02 respectively.

[0078] Application Example 2

[0079] 26.87 mg of NH4Cl was dissolved in 100 mL of DMEM high-sugar culture medium (Wuhan Punsai Life Science and Technology Co., Ltd., China) without phenol red and without sodium pyruvate to configure an NH4+ The culture medium with a concentration of 50 mM was diluted tenfold to prepare the test solution.

[0080] Weigh 50 mg, 100 mg, and 200 mg of the adsorbent obtained in Example 1 into 30 mL glass sample bottles, add 10 mL of test solution, tighten the cap, and place the bottles in a shaker at 37°C and 150 rpm for 30 min.

[0081] After completion, samples were taken using a disposable syringe, filtered through an organic filter membrane with a pore size of 0.22 μm, and the filtrate was collected for NH4 testing. + Concentration and pH changes in culture medium; the pH of the culture medium was measured using a pH meter (Leici PHSJ-4F, Shanghai Instrument & Electronics Scientific Instrument Co., Ltd., China); NH4 in DMEM was determined using a urea nitrogen kit (catalog number C013-2, Nanjing Jiancheng Biotechnology Co., Ltd., China). + The concentration of NH4 before and after adsorption was determined by constructing a standard curve. + concentration;

[0082] Test results are as follows Figure 6 As shown, from Figure 6 It can be seen that the sodium zirconium phosphate α-Na2ZrPA obtained in Example 1 has a positive effect on 5mM NH4 under three dosage conditions of 5 g / L, 10 g / L and 20 g / L. + The removal efficiencies were 19.88%, 28.25%, and 38.09%, respectively. Furthermore, the pH of the culture medium system only fluctuated slightly, because with increasing dosage, Na... + With NH4 + The exchange capacity increases, and the amount of NH4 in the solution increases. + The molecules gradually transfer to the adsorbent, causing a slight increase in the pH of the solution.

[0083] In addition, the amount of adsorbent precipitated during the adsorption process was tested, as was the amount of Zr4. + The amount of Zr4 released increases with increasing adsorbent dosage, but at a dosage of 20 g / L, the amount of Zr4 released is relatively small. + The amount precipitated was only 4.23 mg / L.

[0084] Application Example 3

[0085] To increase the ammonium concentration in the culture medium, a complete culture medium that had been stored in a 4°C refrigerator for a period of time was selected. This culture medium was DMEM (gibco, ThermoFisher Biochemical Products (Beijing) Co., Ltd.) containing 10% fetal bovine serum.

[0086] Mouse myoblasts (C2C12) were seeded in 90×15 mm cell culture dishes at a seeding density of 9.25 x 10⁻⁶.4 cells / mL, 10 mL complete medium was added, and the cells were cultured in a 37°C, 5% CO2 and 95% humidity incubator for three days; the ammonium concentration in the cell culture medium was 15.99 mM;

[0087] 80 mg of the prepared sodium zirconium phosphate α-Na2ZrPA adsorbent in Example 1 was added to a 20 mL glass bottle, 4 mL of the cell culture medium after the cells were cultured was added, and the mixture was shaken at 37°C, 150 rpm for 30 min; the test results showed that the ammonium concentration in the culture medium decreased by 25.94 ± 8.39% after the addition of the adsorbent.

[0088] Performance test

[0089] Test Example 1

[0090] The sodium zirconium phosphate obtained in Example 1 and Comparative Example 1 was subjected to scanning electron microscope test and particle size distribution test, and the SEM images obtained are shown in Figures 1-2 , wherein Figure 1 is Example 1, Figure 2 is Example 2, and the particle size distribution chart is shown in Figure 3 , and it can be seen from Figures 1-3 that the two adsorbents are uniformly dispersed and present a flaky structure;

[0091] The nanoparticle size of α-Na2ZrPA is relatively large, about 1.4 μm, while the particle size of α-Na2ZrPB is relatively small, with an average particle size of about 300 nm; this is related to the surface water content during the synthesis of the material. There is more surface-bound water in α-Na2ZrPA, and when the tiny crystals grow to a certain size, the crystals are connected by hydrogen bonds in the water molecules to form crystal adhesion, increasing the crystal size and resulting in an average particle size much larger than that of α-Na2ZrPB.

[0092] Test Example 2

[0093] The hydrophilic and hydrophobic properties of the sodium zirconium phosphate obtained in Example 1 and Comparative Example 1 were detected, and the contact angles obtained are shown in Figure 4 , and it can be seen from Figure 4 that the contact angle of α-Na2ZrPA is smaller than that of α-Na2ZrPB, indicating that α-Na2ZrPA is more easily hydrophilic than α-Na2ZrPB.

[0094] Test Example 3

[0095] The biocompatibility of the sodium zirconium phosphate obtained in Example 1 was detected:

[0096] The cytotoxicity test of the adsorbent was carried out in accordance with the relevant standards of GB / T 16886.5-2017 "Biological Evaluation of Medical Devices Part 5: In Vitro Cytotoxicity Test".

[0097] Weigh 5-25 mg of the adsorbent prepared in step 1 and place it in a glass bottle for moist heat sterilization. Add 10 mL of culture medium containing 10% fetal bovine serum and let it stand in a refrigerator at 4°C for 24±2 h. After sterile filtration, inoculate the cells.

[0098] 50 μL of passaged C2C12 cells (passaged 2-3 times) were seeded into 24-well plates at a density of approximately 1 x 10⁻⁶ cells per well. 5 Each cell was placed in a 96-well plate with 450 μL of the soaked culture medium added. After 24 h of culture, the cell morphology was observed under a microscope. Then, 10% of the volume of Cell Counting Kit-8 (CCK8) reagent was added and incubated for 1 h. 100 μL of the liquid was added to each well and placed in a 96-well plate. The CCK-8 reagent solution was used as a blank control. The OD value was measured at 450 nm wavelength using an ELISA reader to indirectly reflect the number of viable cells.

[0099] Each experiment was performed in triplicate, with two OD values ​​measured each time. After the test, 500 μL of the corresponding culture medium was added to each well for further incubation. Cell viability was determined by calculating the relative cell viability; the test results are shown below. Figure 5 As shown; from Figure 5 The results showed that after 24 hours of culture, the cell viability in all experimental groups with different soaking concentrations was above 80%, and the microstructure was no different from the blank control group, indicating that the cells were in a healthy state. This demonstrates that the zirconium phosphate-based composite material provided by this invention has good biocompatibility and potential for application in the fields of biomedicine and cell food.

[0100] Test Example 4

[0101] The reusability of sodium zirconium phosphate obtained in Example 1 for adsorbing 5 mM ammonium in aqueous solution was determined. The used adsorbent was rinsed with distilled water, dried at 60–80°C, and reused. Figure 9 It can be seen that sodium zirconium phosphate maintains a similar adsorption effect after being used three times.

[0102] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. Other embodiments can be obtained based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Use of a zirconium phosphate-based composite material for adsorbing ammonium, characterized in that, The adsorbed ammonium is ammonium adsorbed in the cell culture medium; The zirconium phosphate-based composite material is α-sodium zirconium phosphate; The preparation method of the zirconium phosphate-based composite material comprises the following steps: The soluble zirconium source, the hydrated phosphate and the mineralizer are mixed, and the hydrothermal crystallization is performed to obtain the zirconium phosphate-based composite material; the hydrated phosphate is disodium hydrogen phosphate; and the mineralizer is sodium fluoride. The number of crystal water in the hydrated phosphate is ≥2.

2. Use according to claim 1, characterized in that, The soluble zirconium source comprises one or more of zirconium oxychloride octahydrate, zirconium nitrate and zirconium sulfate.

3. Use according to claim 1, characterized in that, The molar ratio of the soluble zirconium source and the hydrated phosphate is 1:1-5, respectively in terms of the molar amount of Zr and P.

4. Use according to claim 3, characterized in that, The molar ratio of the hydrated phosphate and the mineralizer is 5-15:

1.

5. The use according to any one of claims 1 to 4, characterized in that, The temperature of the hydrothermal crystallization is 100-120 ℃, and the holding time is 12-36 h.

6. Use according to claim 1, characterized in that, The zirconium phosphate-based composite material has a two-dimensional sheet structure. The particle size of the zirconium phosphate-based composite material is 300-1400 nm; and the interlayer spacing of the two-dimensional sheet structure is 8.4-8.6 Å. The crystallinity of the zirconium phosphate-based composite material is 68-75%.

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