A method for preparing polyvinyl phosphonic acid grafted enzyme immobilized magnetic nanochain adsorbent by droplet confinement and its application in uranium extraction

Abstract

The application belongs to the technical field of preparation of adsorption separation functional materials, and relates to a method for preparing a polyvinyl phosphonic acid grafted enzyme fixed magnetic nanochain adsorbent by droplet confinement, and high-efficiency uranium adsorption application of the adsorbent. The application can construct the effective uranium ion trap in the continuous water phase of the high internal phase emulsion through the space limited conjugation of the enzyme fixed magnetic nanochain and the polyvinyl phosphonic acid polymer. The polyvinyl phosphonic acid polymer is grown from the surface of a model enzyme, i.e. alpha-chymotrypsin, by using a novel water-soluble atom transfer radical polymerization (ATRP) initiator, and provides high-density accessible phosphonic groups for capturing uranium ions, so that the polyvinyl phosphonic acid polymer and the enzyme fixed magnetic nanochain adsorbent with uniform surface functional sites are prepared, and are applied to high-efficiency adsorption of uranium cation.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of preparation of adsorption separation functional materials, and relates to preparation of a magnetic nanochain adsorbent, in particular to a method for preparing a polyvinyl phosphonic acid grafted enzyme fixed magnetic nanochain adsorbent by liquid drop confinement, and application of the adsorbent to efficient uranium extraction. BACKGROUND

[0002] Uranium is mainly distributed in the ocean, with a total amount of about 4.5 billion tons, which is 1000 times the uranium content of land ores. At the same time, with the development of the nuclear industry, a large amount of uranium-containing radioactive waste is also generated. Uranium is considered one of the main pollutants due to its radioactivity and chemical toxicity, which is harmful to the environment and humans. Inhalation of uranium compounds can cause them to deposit in the lungs and reach the kidneys through the bloodstream, leading to progressive or irreversible kidney damage, and in acute cases, leading to kidney failure and death. Therefore, it is very necessary to develop effective technology to achieve efficient uranium extraction.

[0003] Spatially confined synthesis strategies can provide a mild approach to confine nucleation and growth of nanoparticles without the need for vigorous stirring, and compared with other rigid confined spaces, emulsion droplets with water / oil / air as frame boundaries are strong reactors for realizing confined synthesis and assembly due to their simple preparation and easy regeneration. Magnetic nanocomposites have been found to be one of the most attractive tools in the sensing field due to their excellent superparamagnetism, photothermal properties and biocompatibility, combined with fast separation, catalytic reactions, switchable nanomotors, site-specific delivery and multimodal imaging. The unique demand for uranium extraction from seawater or radioactive wastewater has generated magnetic nanocomposites with abundant specific sites, strong interfacial stability and high cost-effectiveness. In previous reports, many magnetic nanocomposites were synthesized according to different methods, which are usually affected by harsh reaction conditions, including fast stirring or stirring to fully mix and disperse. In particular, low interfacial stability and weak interaction between the magnetic core and the modified chemical moiety lead to irreversible denaturation of the affinity sites, thereby shortening the service life of the nanocomposites.

[0004] Enzymes are multifunctional active biomolecules that exhibit good chemical and spatial region selectivity in the surrounding environment. In addition, enzyme immobilization on the surface of magnetic nanoparticles is considered to be a site-directed reaction platform for post-modification or interfacial growth. At the same time, this strategy can establish a stable intermediate layer between the organic-inorganic structures in a magnetic complex, thereby providing a compatible microenvironment around the enzyme molecules for the introduction of functionality and available sites. Limited space can increase the ordered collision of reactants and rapid and uniform mixing, thereby achieving the purpose of synthesizing functional nanoparticles with adjustable size and structure. The typical "flexible closed space" of the thin film of the continuous phase of the high internal phase emulsion has the advantages of simple preparation and reusability, and also has good application prospects in the field of preparation of nanofunctional materials (catalysts, adsorbents, etc.). SUMMARY

[0005] In order to solve the problems of the nanometer adsorbent not easy to recover, the nanometer adsorbent preparation is complicated and the controllable uniform growth of the nanometer particles in the prior art, a method for preparing a polyvinyl phosphonic acid grafted enzyme immobilized magnetic nanochain adsorbent based on a droplet limited space is established. Through the spatial limited conjugation of the enzyme immobilized magnetic nanochain and the polyvinyl phosphonic acid polymer, the effective uranium ion trap can be constructed in the continuous aqueous phase of the high internal phase emulsion. The polyvinyl phosphonic acid polymer is grown from the surface of the model enzyme alpha-chymotrypsin by using a new type of water-soluble atom transfer radical polymerization ATRP initiator, which provides a high density of accessible phosphonic groups for capturing uranium ions, and the application of the adsorbent in uranium extraction is discussed.

[0006] A method for preparing a polyvinyl phosphonic acid grafted enzyme immobilized magnetic nanochain adsorbent based on a droplet limited space, comprising the following steps:

[0007] Step one, preparation of magnetic nanochain MNPs-GMA

[0008] The magnetic Fe3O4 nanoparticles MNPs were dispersed in a solution of ethanol and deionized water, ultrasonically dispersed uniformly, NH3·H2O and 3-glycidyloxypropyltrimethoxysilane were continuously added, the mixture was stirred again, collected by a magnet, washed with deionized water and ethanol, and dried to obtain the product MNPs-MPS.

[0009] Then the MNPs-MPS were uniformly dispersed in acetonitrile, glycidyl methacrylate, N,N'-methylenebisacrylamide and azobisisobutyronitrile were added under ultrasonication, and a distillation precipitation polymerization reaction was carried out in an oil bath at 110°C, collected, washed with ethanol and deionized water to remove excess monomers and oligomers, and then dried at 45°C to obtain the product MNPs-GMA.

[0010] Step two, preparation of enzyme linked magnetic nanochain EMC

[0011] The MNPs-GMA was added into the α-chymotrypsin aqueous solution and phosphate buffer solution, stirred at room temperature to complete the chemical coupling process, collected, washed with deionized water, and then freeze-dried to obtain the product EMC.

[0012] Step three, preparation of ATRP reaction precursor emulsion HIPEs-1

[0013] EMC and ATRP initiator N-2-bromo-2-methylpropionyl-β-alanine N'-oxysuccinimidyl ester were dissolved in PBS buffer solution at 0°C to synthesize EMC-Br;

[0014] Then dimethylaminoethyl methacrylate, vinylphosphonic acid VPA, EMC-Br and sodium dodecyl sulfate were dispersed in deionized water as a continuous phase, and the continuous phase was sealed and continuously blown with N2, followed by adding the alkane dispersed phase into the continuous phase at 4°C, and stirring to form HIPEs-1.

[0015] Step four, preparation of deoxygenated catalyst high internal phase emulsion HIPEs-2

[0016] At 4°C, 1,1,4,7,10,10-hexamethyltrivinyltetramine, CuBr(I) and deionized water were mixed as a continuous phase, and then the alkane dispersed phase was added into the continuous phase, and stirred to form HIPEs-2.

[0017] Step five, preparation of polyvinylphosphonic acid grafted enzyme immobilized magnetic nanochain adsorbent E-EMC-VPA

[0018] HIPEs-1 and HIPEs-2 were mixed to form a uniform HIPEs-3 droplet reactor without stirring, and the spatially confined conjugation was initiated by ATRP reaction. After the reaction was completed, the product was washed with ethanol and deionized water, and freeze-dried to obtain the product E-EMC-VPA.

[0019] In step one, the mass volume ratio of MNPs, ethanol, deionized water, NH3·H2O and 3-glycidyloxypropyl trimethoxysilane was 100-300 mg: 30-60 mL: 1.0-20 mL: 1.0-3.0 mL: 0.1-0.3 mL;

[0020] The mass volume ratio of MNPs-MPS, acetonitrile, glycidyl methacrylate, N,N'-methylene bisacrylamide and azobisisobutyronitrile was 30-60 mg: 30-50 mL: 100-200 mg: 100-200 mg: 5-10 mg;

[0021] Stirring conditions: temperature 50-80°C, rotation speed 500-1000 rpm, time 12-36 h.

[0022] Preferably, the mass volume ratio of MNPs, ethanol, deionized water, NH3H2O and 3-glycidyloxypropyltrimethoxysilane is 100 mg:40 mL:10 mL:1.5 mL:0.2 mL;

[0023] The mass volume ratio of MNPs-MPS, acetonitrile, glycidyl methacrylate, N,N'-methylenebisacrylamide and azobisisobutyronitrile is 50 mg:40 mL:150 mg:150 mg:6 mg;

[0024] The stirring condition is that the temperature is 70℃, the rotating speed is 800 rpm and the time is 24 h.

[0025] In the second step, the mass volume ratio of MNPs-GMA, α-chymotrypsin aqueous solution and phosphate buffer solution is 100-300 mg:1.0-3.0 mL:10-30 mL. The stirring time at room temperature is 3.0-6.0 h and the mass percentage concentration of α-chymotrypsin aqueous solution is 1.0-10 wt%.

[0026] Preferably, the phosphate buffer solution is 50 mM, pH=8, the mass volume ratio of MNPs-GMA, α-chymotrypsin aqueous solution and phosphate buffer solution is 100 mg:2.0 mL:20 mL. The stirring time at room temperature is 5.0 h and the mass percentage concentration of α-chymotrypsin aqueous solution is 6.0 wt%.

[0027] In the third step, the mass volume ratio of EMC, ATRP initiator and PBS buffer solution is 40-60 mg:40-60 mg:10-30 mL;

[0028] Secondly, the mass volume ratio of dimethylaminoethyl methacrylate, vinyl phosphoric acid, EMC-Br, sodium dodecyl sulfate and deionized water is 100-200 μL:50-200 μL:50-200 mg:50-100 mg:20-50 mL,

[0029] The alkane is n-decane, dodecane or n-tridecane;

[0030] The internal phase ratio of the formed HIPEs-1 is 75%-90%.

[0031] Preferably, the mass volume ratio of EMC and ATRP initiator and PBS buffer solution is 50 mg: 58 mg: 20 mL, the phosphate buffer solution is 50 mM, pH = 8, the mass volume ratio of dimethylaminoethyl methacrylate, vinyl phosphate, EMC-Br, sodium dodecyl sulfate and deionized water is 169 μL: 100 μL: 100 mg: 75 mg: 30 mL, the alkane is n-decane, and the prepared HIPEs-1 internal phase ratio is 75%;

[0032] The stirring speed is 1200 rpm, and the stirring time is 5.0 min.

[0033] In step four, the mass volume ratio of 1,1,4,7,10,10-hexamethyltrivinyltetramine, CuBr(I) and deionized water is 50-100 μL: 20-40 mg: 5-15 mL, and the alkane is n-decane, dodecane or n-tridecane;

[0034] The formed HIPEs-2 internal phase ratio is 75%-90%.

[0035] Preferably, the mass volume ratio of 1,1,4,7,10,10-hexamethyltrivinyltetramine, CuBr(I) and deionized water is 55 μL: 29 mg: 10 mL, the alkane is n-decane, the formed HIPEs-2 internal phase ratio is 75%, and the stirring reaction condition is: temperature is 4℃, stirring speed is 1200 rpm, and stirring time is 5.0 min.

[0036] In step five, the time for starting the spatially confined conjugation is 2.0-5.0 h, and the temperature is 4℃.

[0037] The volume ratio of HIPEs-1 to HIPEs-2 is 1:1.

[0038] Preferably, the time for starting the spatially confined conjugation is 3.0 h.

[0039] The polyvinyl phosphonic acid grafted enzyme immobilized magnetic nanochain adsorbent prepared based on the droplet confined reactor of the application is applied to the study of uranium extraction, the environment is 25℃, the prepared adsorbent E-EMC-EVA 3.0 mg is placed in 8.0 mL of 20 mg / L fluoride ion solution for adsorption, and after 1.0 h, the adsorbed solution is taken out and tested.

[0040] The adsorption capacity calculation formula is:

[0041] Wherein C0(mg L -1 ) and C e (mg / L) are the initial concentration and equilibrium concentration of fluoride ions respectively. V(mL) is the volume of the test solution, and m(g) is the mass of the adsorbent.

[0042] In step three, the ATRP initiator N-2-bromo-2-methylpropionyl-β-alanine N'-oxysuccinimidyl ester was synthesized according to the prior art and commissioned to Shanghai Landis Biomedicine Technology Co., Ltd. The reference is:

[0043] Hironobu Murata, Chad S. Cummings, Richard R. Koepsel, and Alan J. Russell, Polymer-Based Protein Engineering Can Rationally Tune Enzyme Activity, pH-Dependence, and Stability, Biomacromolecules, 2013, 14, 6, 1919-1926. The synthesis method is: a mixture of 2-bromo-2-methylpropionyl bromide (12.4 mL, 100 mmol) and dichloromethane (50 mL) is slowly added to a solution of β-alanine (8.9 g, 100 mmol) and sodium bicarbonate (21 g, 250 mmol). Deionized water (200 mL) is added at 0°C, and then the mixture is stirred at room temperature for 2 hours. The aqueous phase is washed with dichloromethane (100 mL x 3) and adjusted to pH = 2 with 1.0 M aqueous HCl. At 0°C, the product is extracted with ethyl acetate (150 mL x 6). The organic phase is dried with MgSO4 and evaporated to remove the solvent. N-2-bromo-methylpropionyl-β-alanine is isolated by recrystallization from a mixture of ethyl ether and n-hexane (1 / 9 volume ratio). N,N'-diisopropylcarbodiimide (2.8 g, 22 mmol) is slowly added to a solution of N-2-bromo-2-methylpropionyl-β-alanine (4.8 g, 20 mmol) and N-hydroxysuccinimide (2.5 g, 22 mmol) in dichloromethane (200 mL) at 0°C. The mixture is stirred at room temperature for 4 hours. After filtering out the precipitated urea, the solution is evaporated to remove the solvent. Finally, N-2-bromo-2-methylpropionyl-β-alanine N'-oxysuccinimidyl ester is purified by recrystallization from 2-propanol.

[0044] The beneficial effects of the present application are:

[0045] The confined space can increase the ordered collision and rapid and uniform mixing of reactants, so as to achieve the purpose of synthesizing functional nanoparticles with adjustable size and structure. The confined space is constructed by the thin film of the continuous phase of the high internal phase emulsion, and the space-enclosed conjugation is realized. The polyvinyl phosphonic acid polymer with uniform surface functional sites and the enzyme immobilized magnetic nanochain adsorbent are prepared. The adsorbent is applied to the efficient adsorption of uranyl ion. BRIEF DESCRIPTION OF DRAWINGS

[0046] Figure 1 TEM images of each substance prepared in Example 1;

[0047] Figure 2 Magnetic nanochain and magnetic nanochain adsorbent saturation magnetization images prepared in step (1) and step (5) of Example 1

[0048] Figure 3 Effect of pH conditions on the adsorption capacity of E-EMC-VPA in Example 1 in performance test;

[0049] Figure 4 Comparison of adsorption effects of Comparative Example W-EMC-VPA and Example 1 E-EMC-VPA in performance test;

[0050] Figure 5 Adsorption kinetics and model fitting curve of E-EMC-VPA of Example 1 in performance test;

[0051] Figure 6 Adsorption thermodynamics and model fitting curve of E-EMC-VPA of Example 1 in performance test;

[0052] Figure 7 Regeneration capacity verification results of E-EMC-VPA of Example 1 in performance test. DETAILED DESCRIPTION

[0053] The present application will be described in detail below with reference to the accompanying drawings and specific examples, so that those skilled in the art can better understand the present application, but the present application is not limited to the following examples.

[0054] Comparative Example

[0055] (1) Preparation of magnetic nanochain (MNPs-GMA)

[0056] First, monodisperse magnetic Fe3O4nanoparticles MNPs with a diameter of about 120 nm were prepared by hydrothermal method. Second, 100 mg of MNPs were dispersed in a solution of ethanol (40 mL) and deionized water (10 mL). After ultrasonic dispersion for 5.0 min, NH3·H2O (1.5 mL) and 3-glycidyloxypropyltrimethoxysilane (0.2 mL) were continuously added at 70°C and 800 rpm, and then the mixture was stirred for another 24 hours. Collected by magnet, washed with deionized water and ethanol for several times, and then dried to obtain the product MNPs-MPS.

[0057] Subsequently, MNPs-MPS (50 mg) was uniformly dispersed in 40 mL acetonitrile in a round bottom flask (100 mL), and then glycidyl methacrylate (150 mg), N,N'-methylenebisacrylamide (150 mg) and azobisisobutyronitrile (6.0 mg) were added by ultrasonic bath. The flask was immersed in an oil bath at 110 °C, and 20 mL acetonitrile was distilled out within 30 min. The product was collected, washed with ethanol and deionized water to remove excess monomers and oligomers, and then dried at 45 °C overnight to obtain the product MNPs-GMA.

[0058] (2) Preparation of enzyme-linked magnetic nanochain (EMC)

[0059] After 100 mg MNPs-GMA was added to 2.0 mL aqueous solution of α-chymotrypsin (6.0 wt%) and 20 mL PBS buffer solution (50 mM, pH = 8.0), it was stirred at room temperature for 5.0 h to complete the chemical coupling process. The product was collected, washed with deionized water several times, and then freeze-dried to obtain the product EMC.

[0060] (3) First, a water-soluble ATRP initiator (N-2-bromo-2-methylpropionyl-β-alanine N'-oxysuccinimidyl ester) was synthesized. Subsequently, 50 mg EMC and 58 mg ATRP initiator were dissolved in 20 mL PBS buffer solution at low temperature to synthesize EMC-Br.

[0061] Second, 169 μL dimethylaminoethyl methacrylate, 100 μL vinyl phosphoric acid, 100 mg EMC-Br and 75 mg sodium dodecyl sulfate were dispersed in 30 mL deionized water as a continuous phase under continuous stirring at 4 °C, and the mixture was sealed and blown with N2 for 30 min. No dispersed phase was added.

[0062] (4) A deoxygenated catalyst solution was prepared by adding 55 μL 1,1,4,7,10,10-hexamethyltriethylenetetramine and 29 mg CuBr(I) to 10 mL deionized water. No dispersed phase was added.

[0063] (5) Preparation of non-confined polyvinyl phosphonic acid grafted enzyme immobilized magnetic nanochain adsorbent (W-EMC-VPA): The two solutions obtained in steps (3) and (4) were mixed and stirred for 10 h, and the product prepared under non-confinement was called W-EMC-VPA. Figure 1 The TEM image of W-EMC-VPA is shown in FIG. 6, and it can be seen that the chain-like structure is wrapped with less polymer on the surface.

[0064] Example 1

[0065] (1) Preparation of magnetic nanochain MNPs-GMA

[0066] First, monodisperse magnetic Fe3O4 nanoparticles (MNPs) with a diameter of approximately 120 nm were prepared using a hydrothermal method. Next, 100 mg of MNPs were dispersed in a solution of ethanol (40 mL) and deionized water (10 mL). After ultrasonic dispersion for 5.0 min, NH3·H2O (1.5 mL) and 3-glycidyloxypropyltrimethoxysilane (0.2 mL) were continuously added at 70 °C and 800 rpm, and the mixture was then stirred for another 24 hours. The mixture was collected using a magnet, washed several times with deionized water and ethanol, and then dried to obtain the product MNPs-MPS.

[0067] MNPs-MPS (50 mg) was then uniformly dispersed in 40 mL of acetonitrile in a 100 mL round-bottom flask. Glycidyl methacrylate (150 mg), N,N'-methylenebisacrylamide (150 mg), and azobisisobutyronitrile (6.0 mg) were then added via ultrasonic bath. The flask was immersed in an oil bath at 110 °C, and 20 mL of acetonitrile was distilled off within 30 min. The collected product was washed with ethanol and deionized water to remove excess monomers and oligomers, and then dried overnight at 45 °C to obtain the product MNPs-GMA.

[0068] Figure 1 The TEM image of MNPs in section a shows that the particle size is uniform. Figure 1 In the image b, which is a TEM image of MNPs-GMA, it can be seen that the MNPs particles have become chain-like structures.

[0069] from Figure 2 As can be seen from Figure a, the saturation magnetization of MNPs indicates that MNPs have good magnetism.

[0070] (2) Preparation of enzyme-linked magnetic nanochains (EMC)

[0071] 100 mg of MNPs-GMA was added to 2.0 mL of α-chymotrypsin aqueous solution (6.0 wt%) and 20 mL of PBS buffer solution (50 mM, pH 8.0), and stirred at room temperature for 5.0 hours to complete the chemical coupling process. The product was collected, washed several times with deionized water, and then freeze-dried to obtain EMC.

[0072] Figure 1 In the image c, which is a TEM image of EMC, it can be seen that the surface shell of the chain-like products is thicker.

[0073] (3) Preparation of ATRP reaction precursor emulsion HIPEs-1

[0074] Synthesis of water-soluble ATRP initiator (N-2-bromo-2-methylpropionyl-β- alanine N'-oxysuccinimidyl ester), then, 50 mg EMC and 58 mg ATRP initiator were dissolved in 20 mL of PBS buffer solution (50 mM, pH = 8.0) at 0 °C to synthesize EMC-Br.

[0075] Secondly, dimethylaminoethyl methacrylate (169 μL, 1.0 mmol), vinyl phosphoric acid (100 μL, 0.1 mmol), EMC-Br (100 mg) and 75 mg sodium dodecyl sulfate were dispersed in deionized water (30 mL) as a continuous phase, and the mixture was sealed and blown with N2for 30 minutes. The dispersed phase n-decane was added to the continuous phase to form HIPEs-1 with an internal volume fraction of 75% under stirring at 1200 rpm at 4.0 °C.

[0076] (4) Preparation of deoxygenated catalyst high internal phase emulsion HIPEs-2

[0077] A deoxygenated catalyst solution of 1,1,4,7,10,10-hexamethyltrivinyltetramine (55 μL) and CuBr(I) (29 mg) and deionized water (10 mL) as a continuous phase, the dispersed phase n-decane was added to the continuous phase to obtain another HIPEs-2 with an internal volume fraction of 75%, the detailed conditions were the same as those of HIPEs-1.

[0078] (5) Preparation of polyvinyl phosphonic acid grafted enzyme immobilized magnetic nanochain adsorbent E-EMC-VPA

[0079] HIPEs-1 and HIPEs-2 were mixed in a volume ratio of 1:1 to form a uniform HIPEs droplet reactor HIPEs-3, without stirring, after the spatially confined conjugation was initiated by ATRP reaction for 3.0 hours, they were washed with ethanol and deionized water respectively, and then they were subjected to freeze-drying treatment to obtain the product E-EMC-VPA.

[0080] Figure 1 Fig. 3 is a TEM image of EMC-Br, wherein d is a TEM image of EMC-Br, Figure 1 Fig. 5 is a TEM image of E-EMC-VPA, wherein it can be seen that the surface has many particles.

[0081] Figure 2 Fig. 6 shows the saturation magnetization of E-EMC-VPA, compared with MNPs, the magnetization of E-EMC-VPA is weaker, but still has good magnetism, as shown in Fig. 6b. Figure 2 Fig. 6b shows the saturation magnetization of E-EMC-VPA.

[0082] Example 2

[0083] (1) Preparation of magnetic nanochain MNPs-GMA

[0084] Monodispersed magnetic Fe3O4nanoparticles MNPs with diameter of about 120 nm were first prepared by hydrothermal method. Second, 100 mg of MNPs were dispersed in a solution of ethanol (40 mL) and deionized water (10 mL). After ultrasonic dispersion for 5.0 min, NH3H2O (3.0 mL) and 3-glycidyloxypropyltrimethoxysilane (0.3 mL) were continuously added at 70 °C and 800 rpm, then the mixture was stirred for another 24 h, collected by a magnet, washed with deionized water and ethanol for several times, and then dried to obtain the product MNPs-MPS.

[0085] Then MNPs-MPS (50 mg) were uniformly dispersed in 40 mL acetonitrile in a round bottom flask (100 mL), and glycidyl methacrylate (150 mg), N,N'-methylenebisacrylamide (150 mg) and azobisisobutyronitrile (6.0 mg) were added by ultrasonic bath. The flask was immersed in an oil bath at 110 °C, and 20 mL acetonitrile was distilled out within 30 min. After collection, washed with ethanol and deionized water to remove excess monomers and oligomers, and then dried at 45 °C overnight to obtain the product MNPs-GMA.

[0086] Steps (2)-(5) were operated as steps (2)-(5) of Example 1.

[0087] Example 3

[0088] Step (1) was operated as step (1) of Example 1.

[0089] (2) Preparation of enzyme-linked magnetic nanochain (EMC)

[0090] After 100 mg of MNPs-GMA were added to 2.0 mL of an aqueous solution of α-chymotrypsin (3.0 wt%) and 20 mL of PBS buffer solution (50 mM, pH = 8.0), stirring was performed at room temperature for 5.0 h to complete the chemical coupling process. After collection, washed with deionized water for several times, and then freeze-dried to obtain the product EMC.

[0091] Steps (3)-(5) were operated as steps (3)-(5) of Example 1.

[0092] Example 4

[0093] Step (1) was operated as step (1) of Example 1.

[0094] (2) Preparation of enzyme-linked magnetic nanochain (EMC)

[0095] After 100 mg of MNPs-GMA was added into 2.0 mL of aqueous α-chymotrypsin (1.0 wt%) and 20 mL of PBS buffer solution (50 mM, pH = 8.0), the chemical coupling process was completed by stirring at room temperature for 5.0 h. The product EMC was collected, washed with deionized water for several times, and then freeze-dried.

[0096] Steps (3)-(5) were operated as steps (3)-(5) of Example 1.

[0097] Example 5

[0098] Steps (1) and (2) were operated as steps (1) and (2) of Example 1.

[0099] (3) Preparation of ATRP reaction precursor emulsion HIPEs-1

[0100] The water-soluble ATRP initiator (N-2-bromo-2-methylpropionyl-β-alanine N'-oxysuccinimidyl ester) was synthesized, and then 50 mg of EMC and 58 mg of ATRP initiator were dissolved in 20 mL of PBS buffer solution (50 mM, pH = 8.0) at 0°C to synthesize EMC-Br.

[0101] Secondly, dimethylaminoethyl methacrylate (169 μL, 1.0 mmol), vinyl phosphoric acid (100 μL, 0.1 mmol), EMC-Br (100 mg), and 75 mg of sodium dodecyl sulfate were dispersed in deionized water (30 mL) as a continuous phase, and the mixture was sealed and blown with N2for 30 min. Next, the dispersed phase of dodecane was added to the continuous phase at 4.0°C under stirring at 1200 rpm to form HIPEs-1 with an internal volume fraction of 75%.

[0102] (4) Preparation of deoxygenated catalyst high internal phase emulsion HIPEs-2

[0103] A deoxygenated catalyst solution of 1,1,4,7,10,10-hexamethyltriethylenetetramine (55 μL) and CuBr(I) (29 mg) and deionized water (10 mL) as a continuous phase, and the dispersed phase of dodecane was added to the continuous phase to obtain another HIPEs-2 with an internal volume fraction of 75%, and the detailed conditions were the same as those of HIPEs-1.

[0104] (5) Preparation of polyvinyl phosphonic acid grafted enzyme immobilized magnetic nanochain adsorbent (E-EMC-VPA)

[0105] The HIPEs-1 was mixed with the HIPEs-2 to form a uniform HIPEs droplet reactor HIPEs-3, without stirring, after 3.0 hours ATRP reaction initiated the spatially confined conjugation, they were washed with ethanol and deionized water respectively, then they were freeze-dried to obtain the product E-EMC-VPA.

[0106] Example 6

[0107] Steps (1) and (2) were operated as steps (1) and (2) of Example 1.

[0108] (3) Preparation of ATRP reaction precursor emulsion HIPEs-1

[0109] Secondly, dimethylaminoethyl methacrylate (169 μL, 1.0 mmol), vinyl phosphoric acid (100 μL, 0.1 mmol), EMC-Br (100 mg) and 75 mg sodium dodecyl sulfate were dispersed in deionized water (30 mL) as the continuous phase, and the mixture was sealed and blown with N2for 30 minutes. Next, the dispersed phase n-tridecane was added to the continuous phase at 4.0 °C under stirring at 1200 rpm to form HIPEs-1 with an internal volume fraction of 75%.

[0110] (4) Preparation of deoxygenated catalyst high internal phase emulsion HIPEs-2

[0111] A deoxygenated catalyst solution of 1,1,4,7,10,10-hexamethyltriethylenetetramine (55 μL) and CuBr(I) (29 mg) and deionized water (10 mL) as the continuous phase, the dispersed phase n-tridecane was added to the continuous phase to obtain another HIPEs-2 with an internal volume fraction of 75%, the detailed conditions were the same as HIPEs-1.

[0112] (5) Preparation of polyvinyl phosphonic acid grafted enzyme immobilized magnetic nanochain adsorbent (E-EMC-VPA)

[0113] The HIPEs-1 was mixed with the HIPEs-2 to form a uniform HIPEs droplet reactor HIPEs-3, without stirring, after 3.0 hours ATRP reaction initiated the spatially confined conjugation, they were washed with ethanol and deionized water respectively, then they were freeze-dried to obtain the product E-EMC-VPA.

[0114] Example 7

[0115] Steps (1) and (2) were operated as steps (1) and (2) of Example 1.

[0116] (3) Preparation of ATRP reaction precursor emulsion HIPEs-1

[0117] The water-soluble ATRP initiator (N-2-bromo-2-methylpropionyl-β-alanine N'-oxysuccinimidyl ester) was synthesized, and then 50 mg of EMC and 58 mg of ATRP initiator were dissolved in 20 mL of a PBS buffer solution (50 mM, pH = 8.0) at 0°C to synthesize EMC-Br.

[0118] Secondly, dimethylaminoethyl methacrylate (169 μL, 1.0 mmol), vinyl phosphoric acid (100 μL, 0.1 mmol), EMC-Br (100 mg), and 75 mg of sodium dodecyl sulfate were dispersed in deionized water (30 mL) as a continuous phase, and the mixture was sealed and blown with N2for 30 minutes. Next, the dispersed phase n-decane was added to the continuous phase at 4.0°C under stirring at 1200 rpm to form HIPEs-1 with an internal volume fraction of 90%.

[0119] (4) Preparation of deoxygenated catalyst high internal phase emulsion HIPEs-2

[0120] A deoxygenated catalyst solution of 1,1,4,7,10,10-hexamethyltriethylenetetramine (55 μL) and CuBr(I) (29 mg) and deionized water (10 mL) as a continuous phase, and the dispersed phase n-decane was added to the continuous phase to obtain another HIPEs-2 with an internal volume fraction of 90%, and the detailed conditions were the same as those of HIPEs-1.

[0121] (5) Preparation of polyvinyl phosphonic acid grafted enzyme immobilized magnetic nanochain adsorbent (E-EMC-VPA)

[0122] HIPEs-1 and HIPEs-2 were mixed to form a uniform HIPEs droplet reactor HIPEs-3, without stirring, after the spatially confined conjugation was initiated by a 3.0 hour ATRP reaction, they were washed with ethanol and deionized water, respectively, and then subjected to freeze-drying treatment to obtain the product E-EMC-VPA.

[0123] Example 8

[0124] Steps (1) and (2) were operated as steps (1) and (2) of Example 1.

[0125] (3) Preparation of ATRP reaction precursor emulsion HIPEs-1

[0126] Secondly, dimethylaminoethyl methacrylate (169 μL, 1.0 mmol), vinyl phosphoric acid (100 μL, 0.1 mmol), EMC-Br (100 mg) and 75 mg sodium dodecyl sulfate were dispersed in deionized water (30 mL) as the continuous phase, and the mixture was sealed and blown with N2for 30 minutes. Next, the dispersed phase n-decane was added to the continuous phase at 4.0 °C under stirring at 1200 rpm to form HIPEs-1 with an internal volume fraction of 80%.

[0127] (4) Preparation of deoxygenated catalyst high internal phase emulsion HIPEs-2

[0128] A deoxygenated catalyst solution of 1,1,4,7,10,10-hexamethyltriethylene tetramine (55 μL) and CuBr(I) (29 mg) and deionized water (10 mL) as the continuous phase, the dispersed phase n-decane was added to the continuous phase to obtain another HIPEs-2 with an internal volume fraction of 80%, and the detailed conditions were the same as those of HIPEs-1.

[0129] (5) Preparation of polyvinyl phosphonic acid grafted enzyme immobilized magnetic nanochain adsorbent (E-EMC-VPA)

[0130] Afterwards, HIPEs-1 and HIPEs-2 were mixed to form a uniform HIPEs droplet reactor HIPEs-3, without stirring, after 3.0 hours of ATRP reaction initiated space-enclosed conjugation, they were washed with ethanol and deionized water respectively, and then they were subjected to freeze-drying treatment to obtain the product E-EMC-VPA.

[0131] Example 9

[0132] Steps (1) and (2) were operated in the same way as steps (1) and (2) of Example 1.

[0133] (3) Preparation of ATRP reaction precursor emulsion HIPEs-1

[0134] Synthesis of water-soluble ATRP initiator (N-2-bromo-2-methylpropionyl-β- alanine N'-oxysuccinimidyl ester), then, 50 mg EMC and 58 mg ATRP initiator were dissolved in 20 mL of PBS buffer solution (50 mM, pH = 8.0) at 0 °C to synthesize EMC-Br.

[0135] Secondly, dimethylaminoethyl methacrylate (169 μL, 1.0 mmol), vinyl phosphoric acid (100 μL, 0.1 mmol), EMC-Br (100 mg) and 75 mg sodium dodecyl sulfate were dispersed in deionized water (30 mL) as a continuous phase, and the mixture was sealed and blown with N2for 30 minutes. Next, the dispersed phase n-decane was added to the continuous phase at 4.0 °C under stirring at 1200 rpm to form HIPEs-1 with an internal volume fraction of 85%.

[0136] (4) Preparation of deoxygenated catalyst high internal phase emulsion HIPEs-2

[0137] A deoxygenated catalyst solution of 1,1,4,7,10,10-hexamethyltriethylenetetramine (55 μL) and CuBr(I) (29 mg) and deionized water (10 mL) as a continuous phase, the dispersed phase n-decane was added to the continuous phase to obtain another HIPEs-2 with an internal volume fraction of 85%, the detailed conditions were the same as those of HIPEs-1.

[0138] (5) Preparation of polyvinyl phosphonic acid grafted enzyme immobilized magnetic nanochain adsorbent (E-EMC-VPA)

[0139] HIPEs-1 and HIPEs-2 were mixed to form a uniform HIPEs droplet reactor (HIPEs-3), without stirring, after 3.0 hours of ATRP reaction to initiate spatially confined conjugation, they were washed with ethanol and deionized water respectively, and then subjected to freeze-drying treatment to obtain the product E-EMC-VPA.

[0140] Performance test:

[0141] 3.0 mg of polyvinyl phosphonic acid polymer prepared by the method described in Example 1 and enzyme immobilized magnetic nanochain adsorbent (E-EMC-VPA) were added to 8.0 mL of prepared uranium ion test solution (concentration value of 20 mg / L) with pH of 3-9, and the final concentration of uranium ion was determined by inductively coupled plasma spectrometer (ICP) to calculate the remaining concentration. The experiment was carried out three times, and the equilibrium adsorption capacity was calculated.

[0142] The results show that the adsorption solution with pH from 3-9 was studied. From Figure 3It can be seen that the adsorption capacity increased as the pH increased from 3.0 to 8.0. Then, an interesting phenomenon was found that the adsorption capacity remained at a high value as the pH continuously increased from 7.0 to 9.0. This characteristic makes E-EMC-VPA have good adsorption performance in seawater environment (pH = 8.3). This advantage may be related to the amino group of EMC and the phosphate of VPA.

[0143] From the comparison of adsorption experiments, Figure 4 It can be seen that the adsorption effect of E-EMC-VPA prepared in the droplet confined space of HIPEs (Example 1) is better, and increasing the amount of VPA does not have a significant change on the adsorption effect, which indicates that the maximum loading capacity on the surface of the particles has been reached.

[0144] The adsorption capacity of E-EMC-VPA of Example 1 at different time nodes was investigated, the effect of contact time between equal mass of adsorbent and three volumes (7.0, 15, 30 mL) of uranyl ion solution on adsorption kinetics was studied, and the data were fitted by using pseudo-first-order and pseudo-second-order kinetic models, and the kinetic and fitting curves (as shown in Figure 5 It can be seen from Figure 5 that the adsorption capacity of U(VI) increases sharply with time until the equilibrium stage, and the adsorption rate of U(VI) ion on E-EMC-VPA is fast within the first 80 minutes, and then gradually reaches equilibrium at about 150 minutes. In order to ensure that the adsorption process reaches equilibrium, the subsequent experiment was carried out for 300 minutes. In order to achieve the best adsorption capacity, it is necessary to study the effect of solution volume on the separation test of uranium ion containing solution in water sample. Through the study of the adsorption results of three volumes of solution, E-EMC-VPA shows fast adsorption kinetics, indicating that there is strong chelation (oxygen, nitrogen, phosphorus) between U(VI) ion and organic functional groups on the surface edge of the material.

[0145] Figure 6 For the results of thermodynamic and adsorption model fitting curves, respectively at 298 K and 308 K, the adsorption capacity of E-EMC-VPA of Example 1 increases with the increase of uranyl ion concentration, and the adsorption process is an endothermic process.

[0146] Figure 7 The adsorption capacity of E-EMC-VPA of Example 1 in five adsorption and desorption cycles is shown. After five cycles, the adsorption capacity of E-EMC-VPA decreases slightly compared with the original value, and decreases by 12.1% in the fifth cycle. The decrease of adsorption capacity in the fifth cycle may be due to the degradation of VPA functionalization, which still shows good reusability.

[0147] The above merely illustrates the embodiments of the present application, and does not limit the patent scope of the present application, and any equivalent structure or equivalent process transformation, or direct or indirect application in other related technical fields, are also included in the patent protection scope of the present application.

Claims

1. A method for preparing a polyvinylphosphonic acid grafted enzyme-immobilized magnetic nanochain adsorbent using droplet confinement, characterized in that, Includes the following steps: (1) Preparation of magnetic nanochains MNPs-GMA Magnetic Fe3O4 nanoparticles (MNPs) were dispersed in a solution of ethanol and deionized water and ultrasonically dispersed until uniform. NH3·H2O and 3-glycidyloxypropyltrimethoxysilane were continuously added, and the mixture was stirred. The mixture was collected with a magnet, washed with deionized water and ethanol, and dried to obtain the product MNPs-MPS. MNPs-MPS were uniformly dispersed in acetonitrile. Under ultrasonication, glycidyl methacrylate, N,N'-methylenebisacrylamide and azobisisobutyronitrile were added. The mixture was subjected to distillation precipitation polymerization in an oil bath. The product was collected, washed with ethanol and deionized water to remove excess monomers and oligomers, and then dried to obtain the product MNPs-GMA. (2) Preparation of enzyme-linked magnetic nanochains (EMC) MNPs-GMA were added to an aqueous solution of α-chymotrypsin and a phosphate buffer solution. The mixture was stirred at room temperature to complete the chemical coupling process. The mixture was collected, washed with deionized water, and then freeze-dried to obtain the product EMC. (3) Preparation of ATRP reaction precursor emulsion HIPEs-1 EMC-Br was synthesized by dissolving EMC and the ATRP initiator N′-oxosuccinimide ester of N-2-bromo-2-methylpropionyl-β-alanine in PBS buffer solution. Then, dimethylaminoethyl methacrylate, vinyl phosphate VPA, EMC-Br and sodium dodecyl sulfate were dispersed in deionized water as a continuous phase, and the continuous phase was sealed and continuously blown with N2. The alkane dispersion phase was added to the continuous phase and stirred to form HIPEs-1. (4) Preparation of deoxygenation catalyst high internal phase emulsion HIPEs-2 1,1,4,7,10,10-hexamethyltriethylenetetramine, CuBr(I) and deionized water were mixed as a continuous phase, and then an alkane dispersion phase was added to the continuous phase and stirred to form HIPEs-2. (5) Preparation of E-EMC-VPA, a magnetic nanochain adsorbent immobilized by polyvinylphosphonic acid grafting enzyme. HIPEs-1 and HIPEs-2 were mixed to form a homogeneous HIPEs-3 droplet reactor. Without stirring, the reaction was initiated by ATRP with a closed conjugate structure. After the reaction was completed, the products were washed with ethanol and deionized water, respectively, and then freeze-dried to obtain the product E-EMC-VPA.

2. The preparation method according to claim 1, characterized in that, In step (1), the mass-to-volume ratio of MNPs, ethanol, deionized water, NH3·H2O and 3-glycidyloxypropyltrimethoxysilane is 100–300 mg: 30–60 mL: 1.0–20 mL: 1.0–3.0 mL: 0.1–0.3 mL; Stirring conditions: temperature 50-80℃, speed 500-1000rpm, time 12-36h; The mass-to-volume ratio of MNPs-MPS, acetonitrile, glycidyl methacrylate, N,N'-methylenebisacrylamide, and azobisisobutyronitrile is 30–60 mg: 30–50 mL: 100–200 mg: 100–200 mg: 5–10 mg; The oil bath temperature is 110℃; the drying temperature is 45℃.

3. The preparation method according to claim 2, characterized in that, The mass-to-volume ratio of MNPs, ethanol, deionized water, NH3·H2O and 3-glycidyloxypropyltrimethoxysilane was 100 mg: 40 mL: 10 mL: 1.5 mL: 0.2 mL. The mass-to-volume ratio of MNPs-MPS, acetonitrile, glycidyl methacrylate, N,N'-methylenebisacrylamide, and azobisisobutyronitrile was 50 mg:40 mL:150 mg:150 mg:6 mg. Stirring conditions: temperature 70℃, speed 800rpm, time 24h.

4. The preparation method according to claim 1, characterized in that, In step (2), the mass-to-volume ratio of MNPs-GMA, α-chymotrypsin aqueous solution, and phosphate buffer solution is 100–300 mg: 1.0–3.0 mL: 10–30 mL; the stirring time at room temperature is 3.0–6.0 h; and the mass percentage concentration of α-chymotrypsin aqueous solution is 1.0–10 wt%.

5. The preparation method according to claim 4, characterized in that, The mass-to-volume ratio of MNPs-GMA, α-chymotrypsin aqueous solution, and phosphate buffer solution was 100 mg: 2.0 mL: 20 mL. The mass percentage concentration of the α-chymotrypsin aqueous solution was 6.0 wt%, and the phosphate buffer solution was 50 mM with pH = 8. The stirring time at room temperature was 5.0 h.

6. The preparation method according to claim 1, characterized in that, In step (3), the mass-to-volume ratio of EMC, ATRP initiator and PBS buffer solution is 40-60 mg: 40-60 mg: 10-30 mL; the reaction temperature for synthesizing EMC-Br is 0 °C. The mass-to-volume ratio of dimethylaminoethyl methacrylate, vinyl phosphoric acid, EMC-Br, sodium dodecyl sulfate, and deionized water was 100–200 μL: 50–200 μL: 50–200 mg: 50–100 mg: 20–50 mL; the temperature at which HIPEs-1 was formed was 4 °C. The alkane is n-decane, dodecane, or n-tridecane; the internal proportion of the formed HIPEs-1 is 75%–90%; The stirring speed was 1200 rpm, and the stirring time was 5.0 min.

7. The preparation method according to claim 6, characterized in that, The mass-to-volume ratio of EMC and ATRP initiator to PBS buffer solution was 50 mg: 58 mg: 20 mL, and the PBS buffer solution was 50 mM with pH = 8. The mass-to-volume ratio of dimethylaminoethyl methacrylate, vinyl phosphoric acid, EMC-Br, sodium dodecyl sulfate, and deionized water was 169 μL:100 μL:100 mg:75 mg:30 mL; the alkane was n-decane, and the internal proportion of the prepared HIPEs-1 was 75%.

8. The preparation method according to claim 1, characterized in that, In step (4), the mass-to-volume ratio of 1,1,4,7,10,10-hexamethyltriethylenetetramine, CuBr(I), and deionized water is 50–100 μL: 20–40 mg: 5–15 mL; the alkane is n-decane, dodecane, or n-tridecane; the internal proportion of the formed HIPEs-2 is 75%–90%; and the temperature at which the substances are mixed is 4 °C. In step (5), the volume ratio of HIPEs-1 to HIPEs-2 is 1:1; the start-up space is closed and conjugate for 2.0 to 5.0 hours and the temperature is 4°C.

9. The preparation method according to claim 8, characterized in that, In step (4), the mass-to-volume ratio of 1,1,4,7,10,10-hexamethyltriethylenetetramine, CuBr(I) and deionized water is 55 μL: 29 mg: 10 mL; the alkane is n-decane, and the internal proportion of HIPEs-2 formed is 75%; the stirring time is 5.0 min, and the stirring speed is 1200 rpm.

10. The use of the polyvinylphosphonic acid grafted enzyme immobilized magnetic nanochain adsorbent prepared by any one of claims 1 to 9 for the adsorption and separation of uranyl ions.

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