Preparation method and application of brucein D molecularly imprinted polymer functionalized magnetic nanoparticles

Abstract

The invention discloses a preparation method and application of brucein D molecular imprinting polymer functionalized magnetic nanoparticles, including the steps of preparing Fe3O4 nanoparticles, surface silanization of Fe3O4 nanoparticles, amino terminal modification of Fe3O4@SiO2, and preparation of brucein D molecular imprinting polymer functionalized magnetic nanoparticles. The invention uses magnetic nanoparticles as carriers and brucein D molecular imprinting polymers as targeting head groups, and optimizes process conditions to prepare brucein D molecular imprinting polymer functionalized magnetic nanoparticles, which have high adsorption rate and high selectivity for brucein D, are reusable, and can achieve targeted separation of different samples containing brucein D.

Description

Technical Field

[0001] The invention relates to the field of medical technology, and in particular to a preparation method and application of brucein D molecular imprinted polymer functionalized magnetic nanoparticles. Background Art

[0002] Brucea javanica is the dried mature fruit of Brucea javanica, a plant of the genus Brucea in the family Simulaceae. It has the effects of stopping malaria, stopping dysentery, treating warts, and clearing away heat and detoxifying. At present, the application of Brucea javanica is mainly to extract Brucea javanica oil, which is prepared into emulsions or soft capsules and widely used in the adjuvant treatment of various cancers. Quassin compounds are the highest content and best active ingredients in Brucea javanica, especially anti-tumor and anti-malarial effects, and representative compounds include brucein D. However, due to the complex chemical structure and multiple chiral carbons of brucein D, it is difficult to chemically synthesize, and the current source still depends on separation from plants. At present, the traditional extraction methods include solvent method, steam distillation and sublimation method, but the traditional extraction and purification has the disadvantages of complicated process, time-consuming, and the need to consume a variety of organic toxic solvents.

[0003] Molecular imprinting technology refers to the technology of preparing polymers that can selectively recognize target molecules. The preparation principle is that the template molecule and the functional monomer are first prepolymerized, and then a high molecular polymer containing the template molecule is formed under the action of a porogen, a cross-linking agent, and an initiator. Finally, the interaction between the prepolymers is destroyed to wash away the template molecules to obtain molecularly imprinted polymers. This polymer contains three-dimensional holes that match the size and shape of the template molecules. At the same time, the holes contain precisely arranged functional groups provided by functional monomers that are complementary to the functional groups of the template molecules. Such holes have selective recognition characteristics for the template molecules. The recognition performance of this artificial synthetic material is similar to that of natural substances such as enzymes and antibodies. It also has the advantages of synergistic recognition ability, stability and low preparation cost. It is widely used in chiral separation, solid phase extraction, enzyme simulation, chemical sensors and other fields.

[0004] Depending on the preparation principle, the traditional preparation method of molecular imprinted polymers has disadvantages such as fewer binding sites, low sensitivity and low binding efficiency due to the excessive embedding of imprinted sites. Therefore, the selectivity for template molecules is weak and efficient selective adsorption of template molecules cannot be achieved. Summary of the invention

[0005] To solve the above problems, the primary purpose of the present invention is to provide a method for preparing brucein D molecular imprinted polymer functionalized magnetic nanoparticles. The brucein D molecular imprinted polymer functionalized magnetic nanoparticles prepared by the method can achieve efficient and selective adsorption of template molecules.

[0006] The present invention is achieved through the following technical solutions:

[0007] A method for preparing brucein D molecular imprinted polymer functionalized magnetic nanoparticles comprises the following steps:

[0008] (1) Preparation of Fe3O4 nanoparticles:

[0009] FeCl3·6H2O and FeCl2·4H2O were added to deionized water, and ultrasonic mixing was performed until uniform. The mixture was stirred under nitrogen protection, and concentrated ammonia was then added dropwise. The mixture was stirred continuously, and the precipitate was separated by a magnet. The precipitate was washed with deionized water and methanol in turn to obtain Fe3O4 nanoparticles.

[0010] (2) Surface silanization of Fe3O4 nanoparticles:

[0011] Fe3O4 nanoparticles were dispersed in methanol, and ultrasonicated until uniformly mixed. Under nitrogen protection, tetraethyl orthosilicate and ammonia water were added dropwise in sequence. Ultrasonic reaction was carried out at room temperature. The precipitate was separated by a magnet. The precipitate was washed with deionized water and methanol in sequence, and vacuum dried to obtain Fe3O4@SiO2 nanoparticles.

[0012] (3) Amino-terminal modification of Fe3O4@SiO2:

[0013] The Fe3O4@SiO2 nanoparticles were dispersed in methanol with a volume concentration of 50-75%, 3-aminopropyltrimethoxysilane was added dropwise, heated to reflux reaction, washed with deionized water and ethanol in turn until the eluent was neutral, and vacuum dried to obtain amino-terminally modified nanoparticles Fe3O4@SiO2-NH2;

[0014] (4) Preparation of Brucein D Molecularly Imprinted Polymer Functionalized Magnetic Nanoparticles:

[0015] The brucein D and the functional monomer are dissolved and dispersed in acetonitrile, and prepolymerized to obtain a prepolymer; the amino-terminus-modified nanoparticles Fe3O4@SiO2-NH2 are ultrasonically dispersed in a porogen, and a cross-linking agent, a prepolymer, and an initiator are added in sequence, and an ultrasonic reaction is performed. The precipitate is separated by a magnet and ultrasonically cleaned with an eluent. When brucein D is not detected in the eluent, vacuum drying is performed to obtain brucein D molecularly imprinted polymer functionalized magnetic nanoparticles.

[0016] Preferably, in step (4), the functional monomer is one or more of methacrylic acid (MAA), acrylamide (AM) or 2-vinylpyridine (2-VP), preferably methacrylic acid (MAA).

[0017] Preferably, in step (4), the prepolymerization temperature is 4-50° C. and the prepolymerization time is 3-7 hours.

[0018] Preferably, in step (4), the molar ratio of brucein D to the functional monomer is 1:2-1:8, preferably 1:4.

[0019] Preferably, in step (4), the porogen is one or more of methanol, acetonitrile or dichloromethane; the crosslinking agent is one or more of ethylene glycol dimethacrylate (EGDMA), trimethylolpropane trimethacrylate (TRIM) or divinylbenzene (DVB); and the initiator is azobisisobutyronitrile (AIBN).

[0020] Preferably, in step (4), the molar ratio of the initiator to the functional monomer is 1:5-1:40, preferably 1:20.

[0021] Preferably, in step (4), the temperature of the ultrasonic reaction is 50-70° C., and the reaction time is 5-15 hours.

[0022] Preferably, in step (4), the eluent is a methanol / glacial acetic acid solution with a volume ratio of 9:1, a methanol / formic acid solution with a volume ratio of 8:2, or a divinylbenzene solution, preferably a methanol / glacial acetic acid solution with a volume ratio of 9:1.

[0023] The present invention also provides brucein D molecular imprinting polymer functionalized magnetic nanoparticles, which are prepared by the above-mentioned preparation method.

[0024] The present invention also provides the use of the above-mentioned brucein D molecular imprinted polymer functionalized magnetic nanoparticles in the specific recognition, adsorption and separation of brucein D.

[0025] Preferably, the adsorption temperature of the brucein D molecularly imprinted polymer functionalized magnetic nanoparticles is 4-50°C.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] The present invention utilizes magnetic nanoparticles as carriers and brucein D molecular imprinted polymers as targeting head groups, optimizes process conditions, and adopts a surface imprinting method to prepare the imprinted polymers, so that binding sites are evenly distributed on the polymer surface, effectively solving the shortcomings of the traditional preparation method, such as few binding sites, low sensitivity, and low efficiency during binding, caused by over-deep embedding of imprinted sites. The prepared brucein D molecular imprinted polymer functionalized magnetic nanoparticles have high adsorption rate and selectivity for brucein D, are reusable, and can achieve targeted separation of different samples containing brucein D. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The preparation process of brucein D molecularly imprinted polymer functionalized magnetic nanoparticles;

[0029] Figure 2 Transmission electron microscopy (TEM) was used to observe the particle size distribution and particle morphology of the samples; (A-C: Fe3O4@SiO2-NH2; D-E: MIPs; F-G: NIPs);

[0030] Figure 3 The electron diffraction structure of the sample was observed by selected area electron diffraction (SAED); (A, C: Fe3O4@SiO2-NH2; B, D: MIPs);

[0031] Figure 4 Scanning electron microscope (SEM) images; (A, B: MIPs; C, D: NIPs);

[0032] Figure 5 The infrared spectrum of the sample is measured by Fourier transform infrared spectrometer (FTIR);

[0033] Figure 6 The static adsorption results of MIPs and NIPs on brucein D reference solution;

[0034] Figure 7 The dynamic adsorption results of MIPs and NIPs on brucein D reference solution;

[0035] Figure 8 The pseudo-first-order equation fitting results of dynamic adsorption of brucein D reference solution by MIPs and NIPs;

[0036] Fig. 9 The pseudo-second-order equation fitting results of dynamic adsorption of brucein D reference solution by MIPs and NIPs;

[0037] Fig.10 is the adsorption selectivity of MIPs and NIPs to brucein D;

[0038] Fig.11 It is the optimization result of prepolymer synthesis conditions;

[0039] Fig.12 The results are the optimization results of the preparation conditions of molecular imprinted polymers;

[0040] Fig.13 This is the optimization result of adsorption conditions. DETAILED DESCRIPTION

[0041] The present invention is further described below by means of specific implementation methods. The following examples are preferred implementation methods of the present invention, but the implementation methods of the present invention are not limited to the following examples.

[0042] Embodiment 1:

[0043] Figure 1The preparation process of brucein D molecular imprinted polymer functionalized magnetic nanoparticles is shown, and the specific preparation method thereof comprises the following steps:

[0044] (1) Preparation of Fe3O4 nanoparticles:

[0045] 0.02 mol FeCl3·6H2O and 0.01 mol FeCl2·4H2O were added to 50 mL deionized water, and the mixture was thoroughly mixed by ultrasonication for 15 minutes. The mixture was stirred at 70 °C for 30 minutes under nitrogen protection, and then 7 mL concentrated ammonia was added dropwise. The mixture was stirred overnight. After the reaction was completed, the mixture was cooled to room temperature, and the black precipitate was separated by a magnet. The precipitate was washed with deionized water and methanol for 3 times each, and dried in vacuo at 50 °C to obtain Fe3O4 nanoparticles (Fe3O4 MNPs).

[0046] (2) Surface silanization of Fe3O4 nanoparticles:

[0047] 200 mg of Fe3O4 nanoparticles were dispersed in 50 mL of methanol and mixed thoroughly by ultrasonication for 15 minutes. 1.5 mL of tetraethyl orthosilicate (TEOS) and 3 mL of ammonia water were added dropwise under nitrogen protection. The mixture was ultrasonicated at room temperature for 24 hours. After the reaction was completed, the precipitate was separated by a magnet, washed with deionized water and methanol for 3 times each, and dried in vacuum at 50 °C to obtain Fe3O4@SiO2 nanoparticles (Fe3O4@SiO2 MNPs).

[0048] (3) Amino-terminal modification of Fe3O4@SiO2:

[0049] 200 mg of Fe3O4@SiO2 nanoparticles were dispersed in 45 mL of 50% methanol, and the mixture was thoroughly mixed by ultrasonication for 15 minutes. 0.5 mL of 3-aminopropyltrimethoxysilane (APTMS) was added dropwise, and the mixture was refluxed at 90°C overnight. After the reaction was completed, the mixture was cooled to room temperature, and the APTMS residue was washed with deionized water and ethanol in turn until the eluent was neutral. The mixture was dried under vacuum at 50°C to obtain amino-terminally modified nanoparticles Fe3O4@SiO2-NH2.

[0050] (4) Preparation of Brucein D Molecularly Imprinted Polymer Functionalized Magnetic Nanoparticles:

[0051] 0.01 mmol brucein D and 0.04 mmol methacrylic acid (MAA) were dissolved and dispersed in 80 mL acetonitrile, and stirred at 4°C in the dark for 5 hours to perform prepolymerization to obtain a prepolymer;

[0052] 100 mg of amino-terminally modified Fe3O4@SiO2-NH2 nanoparticles were ultrasonically dispersed in 10 mL of methanol, and 2 mmol of ethylene glycol dimethacrylate (EGDMA), prepolymer, and 0.2 mmol of azobisisobutyronitrile (AIBN) were slowly added in sequence. The reaction was ultrasonically reacted at 70 ° C for 10 hours under nitrogen protection. After the reaction was completed, it was cooled to room temperature and the precipitate was separated by magnet. 1 mg of the precipitate was vacuum dried at 50 ° C to make a molecular imprinting polymer (MIPs-Bruceine D) before the template molecule was eluted. At this time, the template molecule recognition site was in a saturated state; this sample was used for the subsequent identification of the functional groups of the molecular imprinting polymer to determine whether bruceine D was successfully imprinted. The remaining precipitate was ultrasonically cleaned with methanol / glacial acetic acid solution (9:1, v / v). When bruceine D was not detected in the eluent, it was vacuum dried at 50 ° C to obtain bruceine D molecular imprinting polymer functionalized magnetic nanoparticles (MIPs).

[0053] Comparative Example 1:

[0054] The difference from Example 1 is that in step (4), the template molecule brucein D is not added when preparing the prepolymer, and the remaining steps are the same as Example 1, so as to obtain non-imprinted polymer functionalized magnetic nanoparticles (NIPs).

[0055] (A) The Fe3O4@SiO2-NH2, MIPs and NIPs prepared in Example 1 and Comparative Example 1 were physically and chemically characterized.

[0056] The particle size distribution and particle morphology were observed using transmission electron microscopy (TEM). Figure 2 As shown in the figure, the particles of Fe3O4@SiO2-NH2 are relatively loose spheres with a relatively dispersed distribution and an average particle size of 10-20nm. After the addition of surface polymers, the particles of MIPs and NIPs are spherical and approximately spherical in an aggregated state, and the average particle sizes of MIPs and NIPs increase to 17-30nm and 13-26nm, respectively. The obvious increase in particle size indicates that the polymer is successfully polymerized on the surface of Fe3O4@SiO2, and the polymerization process successfully increases the active surface area of ​​MIPs, thereby improving the adsorption capacity of MIPs. Compared with MIPs, NIPs have a slightly smaller average particle size than MIPs because there is no participation of template molecules in the polymerization process.

[0057] The electron diffraction structure of the sample was observed by selected area electron diffraction (SAED), and the crystallographic properties of the sample were analyzed. Figure 3 The transmittance of MIPs particles (shown in A) is very strong and has obvious pores; Figure 3The electron diffraction transmittance of the sample (shown in B) is low and the aperture is not obvious. The difference between the two indicates that the polymer is formed on the Fe3O4 surface.

[0058] In addition, the distance from the diffraction ring to the diffraction center was measured. By comparing the standard card value (JCPDSCard79-0417), Figure 3 In Figures C and D, six common characteristic peaks (214), (220), (311), (400), (440), and (511) were compared, belonging to Si and mixed-valence iron oxide. The results show that Fe3O4@SiO2-NH2 and MIPs have the same or very similar crystal structures.

[0059] The pores on the particle surface were observed using a scanning electron microscope (SEM). Figure 4 It can be seen that the morphology of MIPs and NIPs is similar to irregular spheres, but the MIPs particles ( Figure 4 The size of A and B) is obviously larger than that of NIPs particles ( Figure 4 (C, D) are large, indicating that the template molecules may affect the size of the spherical particles during surface polymerization.

[0060] The enlarged morphology shows that the surface structure of MIPs is three-dimensional and the holes are obvious, indicating that the surface of MIPs after the polymerization reaction contains the imprinted position of the template molecule, and the template molecule can enter the imprinted hole during adsorption to complete the recognition. The surface morphology of NIPs is relatively smooth, and the wrinkles and hole structure are not obvious, so low adsorption or non-specific adsorption will occur during adsorption.

[0061] The infrared spectrum of the sample was measured by Fourier transform infrared spectrometer (FTIR) to obtain relevant characteristic functional group information, such as Figure 5 As shown, each sample has a peak at 570 cm -1 The nearby Fe-O-Fe absorption peak is related to the Fe3O4 core.

[0062] Fe3O4@SiO2, Fe3O4@SiO2-NH2, NIPs, MIPs-Bruceine D and MIPs: at 1000cm -1 -1200cm -1 The Si-O-Si characteristic absorption peak appears in the interval, indicating the silanization of the surface of Fe3O4 nanoparticles.

[0063] NIPs, MIPs-Bruceine D and MIPs: about 1300 cm -1 and 1700cm -1 The peaks at about 1460 cm are the stretching vibration characteristic peaks of CO and C=O of EGDMA (crosslinker) and MAA (functional monomer);-1 The CN peaks are characteristic peaks of AIBN (initiator); about 2850cm -1 、3450cm -1 The characteristic peaks of CH and OH are the stretching vibrations of long-chain alkyl groups, indicating the interaction between MIPs / NIPs and APTMS. The appearance of these peaks indicates that the polymer is successfully coated on the Fe3O4@SiO2-NH2 surface.

[0064] MIPs-Bruceine D and MIPs: MIPs at about 3450cm -1 The vibration amplitude of the OH characteristic peak at is larger than that of MIPs-Bruceine D, which may be related to the release of the hydroxyl functional group after the template molecule is removed, which can prove that the template molecule is successfully imprinted on the molecular imprinting polymer.

[0065] (II) Adsorption capacity test of MIPs and NIPs prepared in Example 1 and Comparative Example 1

[0066] Take 1 mg of brucein D powder, add 1 mL of methanol to fully dissolve it, prepare brucein D mother solution, and store it in a refrigerator at 4°C.

[0067] Static adsorption: Weigh 5 mg of MIPs and NIPs respectively and immerse them in 5 mL of brucein D solution with different concentrations (1, 2, 3, 4, 5 μg / mL), shake at room temperature for 24 hours, extract the supernatant, filter it with a 0.45-μm filter membrane, and detect the brucein D concentration in the supernatant by mass spectrometry. The adsorption amount of MIPs and NIPs was calculated based on the change in the concentration of brucein D in the solution before and after adsorption. The adsorption results are shown in Figure 2. Figure 6 As shown, from Figure 6 It can be seen that: with the increase of the initial concentration of brucein D solution, the equilibrium adsorption capacity of MIPs increases, while the equilibrium adsorption capacity of NIPs decreases. When the initial concentration is 4 μg / mL, the adsorption capacity of MIPs is about 20 times that of NIPs.

[0068] Dynamic adsorption: Weigh 5 mg of MIPs and NIPs respectively and immerse them in 5 mL of 4 μg / mL brucein D solution. Oscillate at room temperature for different time periods (5, 10, 15, 20, 35, and 60 min). Draw out the supernatant, filter it with a 0.45-μm filter membrane, and detect the brucein D concentration in the supernatant by mass spectrometry. The adsorption amount of MIPs and NIPs was calculated based on the change in the concentration of brucein D in the solution before and after adsorption. The adsorption results are shown in Figure 2. Figure 7 As shown, from Figure 7The results showed that the adsorption trend of NIPs was similar to that of MIPs, but the adsorption amount of NIPs was lower than that of MIPs, and the equilibrium time was longer than that of MIPs. The reason may be that the recognition sites on MIPs make it easier for brucein D to enter and exit MIPs, making the mass transfer rate of MIPs faster and the binding ability stronger.

[0069] For further study, classical kinetic modeling was performed on the kinetic data, and the results were as follows: Figure 8 , 9 As shown, the pseudo first-order and pseudo second-order model equations are as follows:

[0070]

[0071]

[0072] Where Q e (mg / g) is the equilibrium adsorption capacity, Q cal is the theoretical adsorption capacity, Q t is the adsorption amount at time t, K1 and K2 are the first-order and second-order rate constants, respectively.

[0073] (III) Repeatability study

[0074] 5 mg of MIPs and NIPs were weighed and immersed in 5 mL of 3 μg / mL brucein D solution, and shaken at room temperature for 20 minutes. MIPs / NIPs were separated by magnet, and 10 mL of methanol / glacial acetic acid solution (9:1, v / v) was added to the nanoparticles, and shaken at room temperature for 12 hours to elute the adsorbed compounds. The concentration of brucein D in the eluate was detected by mass spectrometry, and the concentration of the original solution was compared to calculate the adsorption capacity and recovery rate. The above steps were repeated, and the adsorption capacity was compared each time. The reuse of MIPs / NIPs was investigated. The results showed that the adsorption capacity of the three tests was 0.30, 0.25, and 0.28 mg / g, respectively, and the average adsorption capacity was 0.28 mg / g. The RSD of the three repeated results was 9%. The three adsorption capacities and recovery rates were not much different, and repeated use would not affect the adsorption efficiency, proving that MIPs have good repeatability and stability for the adsorption of brucein D.

[0075] Example 2: Optimization of process conditions

[0076] (1) Optimization process of preparing prepolymer

[0077] When preparing prepolymers, many factors will affect the selectivity and binding ability of the final MIPs product. The main factors include: 1) Selection of functional monomers: The complementary effect between the template molecule and the functional monomer determines the binding ability of the binding site of MIPs; 2) Ratio of functional monomers: The stoichiometric relationship and concentration between the template molecule and the functional monomer affect the surface morphology and binding selectivity of MIPs; 3) Prepolymerization temperature: The temperature of the polymerization reaction affects the binding equilibrium speed of the functional monomer and the template molecule, that is, it affects the phase separation time; 4) Prepolymerization time: The choice of phase separation time determines the accessibility of the imprinted polymer binding site. The present invention optimizes and screens the above factors, and the specific process is as follows:

[0078] Optimize the selection of functional monomers:

[0079] In 80 mL of acetonitrile solution containing 0.01 mmol of brucein D, 0.04 mmol of methacrylic acid (MAA), acrylamide (AM), and 2-vinylpyridine (2-VP) were added respectively, and stirred at 4°C for 5 hours to prepare a prepolymer. Then, MIPs were prepared under the same conditions and adsorbed on the brucein D reference solution. The ion peak area of ​​the brucein D finally eluted was determined by LC-MS. The results are as follows: Fig.11 As shown in A in the figure, MAA interacts best with brucein D, so methacrylic acid (MAA) is preferred as the functional monomer.

[0080] Optimize the ratio of functional monomers:

[0081] In 80 mL of acetonitrile solution containing 0.01 mmol of brucein D, 0.01, 0.02, 0.04, and 0.08 mmol of MAA were added, respectively, and stirred at 4°C for 5 hours to prepare prepolymers. MIPs were then prepared under the same conditions and adsorbed on brucein D reference solution. The ion peak area of ​​brucein D finally eluted was determined by LC-MS. The results are as follows: Fig.11 As shown in Figure B, within a certain range, with the increase of functional monomers, the adsorption amount of brucein D by MIPs also increases. When MAA increases to 0.4 mmol, the adsorption amount reaches the highest. However, when the amount of MAA exceeds 0.4 mmol, the adsorption amount decreases significantly. Therefore, the optimal ratio of brucein D to MAA is 1:4.

[0082] Optimizing prepolymerization temperature:

[0083] 0.04 mmol MAA was added to 80 mL acetonitrile solution containing 0.01 mmol brucein D, and stirred at 4°C, 25°C, 50°C, and 70°C for 5 hours to prepare a prepolymer. MIPs were then prepared under the same conditions and adsorbed on the brucein D reference solution. The ion peak area of ​​the brucein D finally eluted was determined by LC-MS. The results are as follows: Fig.11 As shown in Figure C, with the increase of prepolymerization temperature, the adsorption amount of brucein D by MIPs gradually decreased. Therefore, the optimal stirring temperature is 4 °C.

[0084] Optimize pre-polymerization time:

[0085] 0.04 mmol MAA was added to 80 mL acetonitrile solution containing 0.01 mmol brucein D, and stirred at 4°C for 1, 3, 5, and 7 hours to prepare prepolymer. MIPs were prepared under the same conditions and adsorbed on brucein D reference solution. The ion peak area of ​​brucein D finally eluted was determined by LC-MS. The results are as follows: Fig.11 As shown in D, with the increase of stirring time, the adsorption amount of brucein D by MIPs gradually increased and reached the highest at 5 hours. If the stirring time exceeds 5 hours, the adsorption amount does not increase significantly. Therefore, the optimal stirring time is preferably 5 hours.

[0086] (2) Optimization of conditions for preparing molecularly imprinted polymers

[0087] In a certain solvent (porogen), the initiator azobisisobutyronitrile (AIBN) can initiate thermal polymerization, so that the prepolymer and the crosslinker form a highly linked rigid polymer; then the template molecule in the polymer is dissociated by the eluent to obtain a molecularly imprinted polymer. The above optimized method is used to prepare the prepolymer (0.01mmol brucein D and 0.04mmol MAA are dissolved and dispersed in 80mL acetonitrile, stirred at 4℃ in the dark for 5 hours), and the main factors affecting the performance of MIPs are investigated: 1) the selection of porogen, 2) the selection of crosslinker, 3) the amount of initiator, 4) the reaction temperature, 5) the reaction time, 6) the selection of eluent, and the specific optimization process is as follows:

[0088] Optimizing the choice of porogen:

[0089] 100 mg Fe3O4@SiO2-NH2 was ultrasonically dispersed in 10 mL methanol, acetonitrile, and dichloromethane, and 2 mmol ethylene glycol dimethacrylate (EGDMA), prepolymer, and 0.2 mmol AIBN were slowly added in sequence. The mixture was ultrasonically reacted at 70°C for 10 hours under nitrogen protection. After cooling to room temperature, the precipitate was obtained by magnetic separation, ultrasonically cleaned with methanol / glacial acetic acid solution (9:1, v / v), and vacuum dried at 50°C to obtain MIPs of brucein D. The brucein D reference solution was then adsorbed under the same conditions, and the ion peak area of ​​the brucein D finally eluted was determined by liquid chromatography-mass spectrometry. The results are shown in Figure 2. Fig.12 As shown in Figure A, when other conditions are the same, three different porogens are used to synthesize MIPs, and the adsorption amounts of brucein D are different, from high to low, methanol>acetonitrile>dichloromethane. Therefore, methanol is the preferred best porogen.

[0090] Optimizing crosslinker selection:

[0091] 100 mg Fe3O4@SiO2-NH2 was ultrasonically dispersed in 10 mL methanol, and 2 mmol ethylene glycol dimethacrylate (EGDMA), trimethylolpropane trimethacrylate (TRIM), and divinylbenzene (DVB) were slowly added, and then prepolymer and 0.2 mmol AIBN were slowly added in sequence. Ultrasonic reaction was carried out at 70 ° C for 10 hours under nitrogen protection; after cooling to room temperature, the precipitate was obtained by magnetic separation, ultrasonically cleaned with methanol / glacial acetic acid solution (9:1, v / v), and vacuum dried at 50 ° C to obtain MIPs of brucein D. The brucein D reference solution was then adsorbed under the same conditions, and the ion peak area of ​​the final eluted brucein D was determined by liquid chromatography-mass spectrometry. The results are as follows: Fig.12 As shown in B, when other conditions were the same, three different cross-linking agents were used to synthesize MIPs, and the adsorption amounts of brucein D were different, from high to low, EGDMA>TRIM>DVB. Therefore, ethylene glycol dimethacrylate (EGDMA) was the preferred optimal cross-linking agent.

[0092] Optimize the amount of initiator:

[0093] 100 mg Fe3O4@SiO2-NH2 was ultrasonically dispersed in 10 mL methanol, 2 mmol EGDMA and prepolymer were slowly added in sequence, and then 0.05, 0.1, 0.2, and 0.4 mmol AIBN were added respectively. The mixture was ultrasonically reacted at 70°C for 10 hours under nitrogen protection. After cooling to room temperature, the precipitate was obtained by magnetic separation, ultrasonically cleaned with methanol / glacial acetic acid solution (9:1, v / v), and vacuum dried at 50°C to obtain MIPs of brucein D. The brucein D reference solution was then adsorbed under the same conditions, and the ion peak area of ​​the brucein D finally eluted was determined by liquid chromatography-mass spectrometry. The results are shown in Figure 2. Fig.12 As shown in Figure C, when the amount of initiator is less than or equal to 0.2 mmol, the adsorption of brucein D by MIPs gradually increases with the increase of the amount of initiator. However, when the amount exceeds 0.2 mmol, the adsorption of brucein D by MIPs decreases significantly. Therefore, the optimal initiator amount is 0.2 mmol.

[0094] Optimize reaction temperature:

[0095] 100 mg Fe3O4@SiO2-NH2 was ultrasonically dispersed in 10 mL methanol, and 2 mmol EGDMA, prepolymer, and 0.2 mmol AIBN were slowly added in sequence. The reaction was ultrasonically reacted at 4°C, 25°C, 50°C, and 70°C for 10 hours under nitrogen protection. After cooling to room temperature, the precipitate was obtained by magnetic separation, ultrasonically cleaned with methanol / glacial acetic acid solution (9:1, v / v), and vacuum dried at 50°C to obtain MIPs of brucein D. The brucein D reference solution was then adsorbed under the same conditions, and the ion peak area of ​​the brucein D finally eluted was determined by liquid chromatography-mass spectrometry. The results are as follows: Fig.12 As shown in D, when other conditions were the same, four different reaction temperatures were used to synthesize MIPs, and the adsorption amount of brucein D was different, and the adsorption amount increased with the increase of reaction temperature. Therefore, the optimal reaction temperature was preferably 70°C.

[0096] Optimize reaction time:

[0097] 100 mg Fe3O4@SiO2-NH2 was ultrasonically dispersed in 10 mL methanol, and 2 mmol EGDMA, prepolymer, and 0.2 mmol AIBN were slowly added in sequence. The reaction was ultrasonically reacted at 70 °C for 1, 5, 10, and 15 hours under nitrogen protection. After cooling to room temperature, the precipitate was obtained by magnetic separation, ultrasonically cleaned with methanol / glacial acetic acid solution (9:1, v / v), and vacuum dried at 50 °C to obtain MIPs of brucein D. The brucein D reference solution was then adsorbed under the same conditions, and the ion peak area of ​​the brucein D finally eluted was determined by liquid chromatography-mass spectrometry. The results are as follows: Fig.12 As shown in Figure E, within 10 hours, as the molecular imprinting reaction time increases, the adsorption amount of brucein D by MIPs increases significantly. After more than 10 hours, the adsorption amount decreases. Therefore, the optimal reaction time is preferably 10 hours.

[0098] Optimizing eluent selection:

[0099] 100 mg Fe3O4@SiO2-NH2 was ultrasonically dispersed in 10 mL methanol, and 2 mmol EGDMA, prepolymer, and 0.2 mmol AIBN were slowly added in sequence. The mixture was ultrasonically reacted at 70 °C for 10 hours under nitrogen protection. After cooling to room temperature, the precipitate was obtained by magnetic separation, and ultrasonically cleaned with methanol / glacial acetic acid solution (9:1, v / v), methanol / formic acid solution (8:2, v / v), and divinylbenzene (DVB), and vacuum dried at 50 °C to obtain MIPs of brucein D. The brucein D reference solution was then adsorbed under the same conditions, and the ion peak area of ​​the brucein D finally eluted was determined by liquid chromatography-mass spectrometry. The results are as follows: Fig.12 As shown in Figure F, four different eluents were used to elute brucein D from MIPs, and the elution amounts were different, from large to small: methanol / glacial acetic acid solution (9:1, v / v) > methanol / formic acid solution (8:2, v / v) > divinylbenzene (DVB). Therefore, the best eluent was preferably methanol / glacial acetic acid solution (9:1, v / v).

[0100] (3) Optimization of adsorption conditions

[0101] The main factors affecting the adsorption results of MIPs on samples include adsorption temperature, adsorption time, and eluent selection. Since the effect of adsorption time has been investigated in the dynamic adsorption experiment and the eluent selection has been investigated above, only the optimal adsorption temperature is investigated.

[0102] The prepolymer and MIPs of brucein D were prepared using the above optimized method, and the brucein D reference solution was adsorbed. Weigh 5 mg of MIPs and immerse them in 5 mL of 5 μg / mL brucein D solution, and shake at 4°C, 25°C, 50°C, and 70°C for 20 minutes. MIPs were obtained by magnetic separation, and 5 mL of methanol / glacial acetic acid solution (9:1, v / v) was added, and eluted by ultrasonic shaking at room temperature for 1 hour. The ion peak area of ​​brucein D in the eluate was determined by liquid chromatography-mass spectrometry, and the results are as follows: Fig.13 As shown in the figure, within the temperature range of 25°C and below, as the oscillation adsorption temperature increases, the adsorption amount of brucein D by MIPs increases significantly. When the temperature is greater than 25°C, the adsorption amount of brucein D by MIPs decreases significantly. Therefore, the optimal oscillation temperature is preferably 25°C.

[0103] Application Example 1:

[0104] Take 1.0 g of Brucea javanica powder, place it in a 50-mL centrifuge tube, add 20 mL of 50% methanol, ultrasonicate for 30 minutes, and centrifuge at 2800g for 5 minutes. Transfer the supernatant to a 50-mL volumetric flask, repeat the extraction once, and wash the residue with 10 mL of 50% methanol. Combine the extracts, add 50% methanol to the scale, and filter with a 0.45-μm microporous filter membrane to obtain the Brucea javanica extract solution.

[0105] Weigh 5 mg of MIPs and NIPs respectively and immerse them in 5 mL of Brucea brucea extract and shake at room temperature for 30 minutes. Use a magnet to separate MIPs / NIPs, add 10 mL of methanol / glacial acetic acid solution (9:1, v / v) to the nanoparticles, and shake at room temperature for 12 hours to elute the adsorbed compounds. Use mass spectrometry to detect the concentration of brucea brucea D in the eluate, compare it with the concentration of the original solution, and calculate the adsorption capacity and recovery rate. Fig.10 As shown in the figure, in the extract of Brucea brucea, the original concentration of brucein D was 12.3 μg / mL. The adsorption capacities of MIPs and NIPs for brucein D were 261.54 μg / g and 82.53 μg / g, respectively, and the recovery rates of brucein D were 89% and 54%, respectively.

[0106] Weigh 5 mg of MIPs and NIPs respectively and immerse them in 5 mL of Brucea brucea extract and shake at room temperature for 30 minutes. Use a magnet to separate MIPs / NIPs, add 10 mL of methanol / glacial acetic acid solution (9:1, v / v) to the nanoparticles, and shake at room temperature for 12 hours to elute the adsorbed compounds. Use mass spectrometry to detect the concentrations of brucein D, brucein G, and brucein lactone A in the eluate, compare the concentrations of the original solution, and analyze the adsorption selectivity. Fig.10As shown, in the extract of Brucea brucea medicinal material, for the three structural analogs (brucein D, brucein lactone A, and brucein G), the adsorption amount of brucein D on MIPs is the highest, indicating that MIPs still maintain the highest affinity for the template molecule between the template molecule and multiple analogs, and therefore has adsorption selectivity. The adsorption capacities of MIPs for brucein D, brucein lactone A, and brucein G in the extract of Brucea brucea medicinal material are 261.54μg / g, 18μg / g, and 63μg / g, respectively. The adsorption capacities of NIPs for brucein D, brucein lactone A, and brucein G in the extract of Brucea brucea medicinal material are 82.53μg / g, 10μg / g, and 27μg / g, respectively. The results show that the MIPs in the present invention have specific recognition sites for the template molecule brucein D, and can be used to selectively separate and determine brucein D in complex systems (such as Chinese medicine extracts).

Claims

1. A method for preparing brucein D molecularly imprinted polymer functionalized magnetic nanoparticles, characterized in that: The steps include: (1) Preparation of Fe3O4 nanoparticles: Add FeCl3⋅6H2O and FeCl2⋅4H2O to deionized water, mix by ultrasound, stir to react under nitrogen protection, then add concentrated ammonia water dropwise, continue to stir to react, separate the precipitate with a magnet, wash the precipitate with deionized water and methanol in turn, and obtain Fe3O4 nanoparticles; (2) Surface silanization of Fe3O4 nanoparticles: Fe3O4 nanoparticles were dispersed in methanol, and ultrasonicated until uniformly mixed. Under nitrogen protection, tetraethyl orthosilicate and ammonia water were added dropwise in sequence. Ultrasonic reaction was carried out at room temperature. The precipitate was separated by a magnet. The precipitate was washed with deionized water and methanol in sequence, and vacuum dried to obtain Fe3O4@SiO2 nanoparticles. (3) Amino-terminal modification of Fe3O4@SiO2: The Fe3O4@SiO2 nanoparticles were dispersed in methanol with a volume concentration of 50-75%, 3-aminopropyltrimethoxysilane was added dropwise, heated to reflux reaction, washed with deionized water and ethanol in turn until the eluent was neutral, and vacuum dried to obtain amino-terminally modified nanoparticles Fe3O4@SiO2-NH2; (4) Preparation of Brucein D Molecularly Imprinted Polymer Functionalized Magnetic Nanoparticles: The brucein D and the functional monomer are dissolved and dispersed in acetonitrile, and prepolymerized to obtain a prepolymer; the amino-terminus-modified nanoparticles Fe3O4@SiO2-NH2 are ultrasonically dispersed in a porogen, and a cross-linking agent, a prepolymer, and an initiator are added in sequence, and an ultrasonic reaction is performed. The precipitate is separated by a magnet and ultrasonically cleaned with an eluent. When brucein D is not detected in the eluent, vacuum drying is performed to obtain brucein D molecularly imprinted polymer functionalized magnetic nanoparticles.

2. The method for preparing brucein D molecularly imprinted polymer functionalized magnetic nanoparticles according to claim 1, characterized in that: In step (4), the functional monomer is one or more of methacrylic acid MAA, acrylamide AM or 2-vinylpyridine 2-VP.

3. The method for preparing brucein D molecularly imprinted polymer functionalized magnetic nanoparticles according to claim 2, characterized in that: In step (4), the functional monomer is methyl methacrylate (MAA).

4. The method for preparing brucein D molecularly imprinted polymer functionalized magnetic nanoparticles according to claim 1, characterized in that: In step (4), the prepolymerization temperature is 4-50° C. and the prepolymerization time is 3-7 hours.

5. The method for preparing brucein D molecularly imprinted polymer functionalized magnetic nanoparticles according to claim 1, characterized in that: In step (4), the molar ratio of brucein D to the functional monomer is 1:2-1:

8.

6. The method for preparing brucein D molecularly imprinted polymer functionalized magnetic nanoparticles according to claim 5, characterized in that: In step (4), the molar ratio of brucein D to the functional monomer is 1:

4.

7. The method for preparing brucein D molecularly imprinted polymer functionalized magnetic nanoparticles according to claim 1, characterized in that: In step (4), the porogen is one or more of methanol, acetonitrile or dichloromethane; the crosslinking agent is one or more of ethylene glycol dimethacrylate EGDMA, trimethylolpropane trimethacrylate TRIM or divinylbenzene DVB; and the initiator is azobisisobutyronitrile AIBN.

8. The method for preparing brucein D molecularly imprinted polymer functionalized magnetic nanoparticles according to claim 1, characterized in that: In step (4), the molar ratio of the initiator to the functional monomer is 1:5-1:

40.

9. The method for preparing brucein D molecularly imprinted polymer functionalized magnetic nanoparticles according to claim 8, characterized in that: In step (4), the molar ratio of the initiator to the functional monomer is 1:

20.

10. The method for preparing brucein D molecularly imprinted polymer functionalized magnetic nanoparticles according to claim 1, characterized in that: In step (4), the temperature of the ultrasonic reaction is 50-70°C, and the reaction time is 5-15 hours.

11. The method for preparing brucein D molecularly imprinted polymer functionalized magnetic nanoparticles according to claim 1, characterized in that: In step (4), the eluent is a methanol / glacial acetic acid solution with a volume ratio of 9:1, a methanol / formic acid solution with a volume ratio of 8:2, or a divinylbenzene solution.

12. The method for preparing brucein D molecularly imprinted polymer functionalized magnetic nanoparticles according to claim 11, characterized in that: In step (4), the eluent is a methanol / glacial acetic acid solution with a volume ratio of 9:

1.

13. A brucein D molecular imprinted polymer functionalized magnetic nanoparticle prepared by the preparation method according to any one of claims 1 to 12.

14. Use of the brucein D molecularly imprinted polymer functionalized magnetic nanoparticles according to claim 13 in specific recognition, adsorption and separation of brucein D.

Images