CN110871055B - Preparation method and application of molecular engram polymer silica gel microsphere with glutamic acid as end group and fresh polypeptide surface - Google Patents
Preparation method and application of molecular engram polymer silica gel microsphere with glutamic acid as end group and fresh polypeptide surface Download PDFInfo
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- CN110871055B CN110871055B CN201810994201.6A CN201810994201A CN110871055B CN 110871055 B CN110871055 B CN 110871055B CN 201810994201 A CN201810994201 A CN 201810994201A CN 110871055 B CN110871055 B CN 110871055B
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Abstract
The invention relates to a preparation method of a surface molecularly imprinted polymer silica gel microsphere with glutamic acid as a terminal group and fresh polypeptide, which comprises the following steps: adopting a dilute hydrochloric acid soaking method to remove impurities on the surfaces of the silica gel microspheres to obtain activated silica gel microspheres; performing surface silanization modification on the activated silica gel microspheres to obtain silanized silica gel microspheres; carrying out RAFT (reversible addition-fragmentation chain transfer) functionalization treatment on the silanized silica gel microspheres to obtain RAFT functionalized silica gel microspheres with surface grafted with disulfide bonds; after the self-assembly of the substitution template molecules, the metal ions and the functional monomers, adding a cross-linking agent, an initiator and the RAFT functional silica gel microspheres, and obtaining the molecular imprinting coating microspheres with the surfaces coated with the substitution template molecules through RAFT polymerization reaction; eluting to remove surface-coated surrogate template molecules; wherein the substitute template molecule is L-glutamic acid, and the metal ion is Cu 2+ The functional monomer is 1- (N, N-dicarboxymethyl) amino-3-allylglycerol.
Description
Technical Field
The invention belongs to the field of chemical analysis and test, and particularly relates to a preparation method and application of a molecular engram polymer silica gel microsphere with a glutamic acid end group and a fresh polypeptide surface.
Background
Soy sauce is a traditional brewing product in China, and is an indispensable seasoning in daily life of common people. The unique color, fragrance and taste make the seasoning become a traditional seasoning which is deeply favored by people in China. The delicate flavor is the most important evaluation index in soy sauce. The discovery that the brewed soy sauce contains small molecular polypeptides with molecular weight below 500Da, the small molecular polypeptides can obviously improve the overall flavor intensity of foods, and are flavor substances in the brewed soy sauce, and the small molecular polypeptides are called as fresh polypeptides. It has been found that most of the fresh polypeptides are dipeptides and tripeptides with terminal amino acids glutamic acid or aspartic acid. Therefore, people can further reveal and grasp the freshness mechanism of the soy sauce by detecting and analyzing the kind and the content of the freshness-developing polypeptide in the brewed soy sauce.
Currently, the analytical methods for fresh polypeptides are mainly liquid chromatography and liquid chromatography/mass spectrometry. Both methods require a pretreatment of the sample solution, i.e. soy sauce. In the sample pretreatment technology for separating and detecting amino acids, small molecule polypeptides and proteins, the surface blotting technology is receiving more and more attention as a novel sample pretreatment technology. The principle of action of the surface imprinting technology is similar to that of the key-lock interaction of an enzyme-substrate, materials such as silica gel are used as a supporting solid phase, target molecules are used as template molecules, corresponding chemical bonds are generated through chemical reaction to bond the template molecules on the supporting solid phase, and then the template molecules are removed through means such as elution, so that a vacancy is reserved on the surface of the supporting solid phase for bonding the target molecules. Because the template molecules are the same as the target molecules, the binding vacancies reserved on the surface of the support solid phase can only specifically adsorb and bind the target molecules, thereby greatly improving the selectivity of the target molecules in the sample solution, reducing the probability of binding with other molecules in the sample solution, and greatly improving the detection accuracy. In this technique, we refer to the structure that has eluted the template molecule, leaving a supporting solid phase and surface target molecule binding vacancies, as a surface molecularly imprinted polymer. It can be seen that the surface blotting technology can complete the binding and separation of target molecules in the sample solution by only contacting the sample solution with the surface molecularly imprinted polymer, and the operation is very simple and takes less time.
However, in the prior art, the surface molecularly imprinted polymer for detecting amino acid and polypeptide has the defects of uneven coating, uneven thickness, unstable performance and the like. In addition, in the existing preparation method of the surface molecularly imprinted polymer, the template molecules are mainly combined onto the support solid phase through intermolecular force, and the quantity of the template molecules combined onto the support solid phase is limited because of weak intermolecular force, and correspondingly, the quantity of formed combined vacancies is also limited, so that the prepared surface molecularly imprinted polymer can not absorb and combine target molecules in a sample solution as much as possible or completely, and the detection accuracy is affected. In addition, surface blotting techniques have not been applied in the prior art to the detection and analysis of fresh polypeptides.
Disclosure of Invention
Based on the method, the invention provides a preparation method of the molecular engram polymer silica gel microsphere with the end group of glutamic acid and the surface of the fresh polypeptide, so as to prepare the surface molecular engram polymer which has uniform coating, uniform thickness and stable performance, can simultaneously and specifically adsorb a plurality of fresh polypeptide molecules with the end group of L-glutamic acid and has more firm chemical bonds between the silica gel microsphere and a template molecule.
The invention relates to a preparation method of a surface molecularly imprinted polymer silica gel microsphere with glutamic acid as a terminal group and fresh polypeptide, which comprises the following steps:
s1: adopting a dilute hydrochloric acid soaking method to remove impurities on the surfaces of the silica gel microspheres to obtain activated silica gel microspheres;
s2: carrying out surface silanization modification on the activated silica gel microspheres by adopting a silanization reagent to obtain silanized silica gel microspheres;
s3: carrying out RAFT functional treatment on the silanized silica gel microspheres by adopting a RAFT reagent to obtain RAFT functional silica gel microspheres with surface grafted with disulfide bonds;
s4: fully and uniformly mixing the substitution template molecules, the metal ions and the functional monomers in a mixed solvent to enable the substitution template molecules, the metal ions and the functional monomers to self-assemble, then adding a cross-linking agent, an initiator and the RAFT functional silica gel microspheres, and obtaining the molecularly imprinted coating microspheres coated with the substitution template molecules on the surfaces through RAFT polymerization reaction;
s5: eluting the silica gel microsphere obtained in the step S4, removing the surface-coated substitute template molecules, and drying to obtain the surface molecularly imprinted polymer silica gel microsphere;
wherein the substitute template molecule is L-glutamic acid, and the metal ion is Cu 2+ The functional monomer is 1- (N, N-dicarboxymethyl) amino-3-allylglycerol.
Compared with the prior art, the surface molecularly imprinted polymer silica gel microsphere prepared by the method has the advantages of uniform coating, narrow molecular weight distribution of the imprinted polymer silica gel microsphere and stable performance. In addition, as the end groups of the plurality of the fresh polypeptides are L-glutamic acid, the selective adsorption of the plurality of the fresh polypeptides in the water environment is realized at one time, and the workload is greatly reduced. In addition, in the traditional surface molecular imprinting technology, functional monomers are combined with alternative template molecules through intermolecular force, and in the preparation method, metal ions Cu are prepared 2+ And the functional monomer 1- (N, N-dicarboxymethyl) amino-3-allylglycerol is connected to replace template molecule L-glutamic acid through metal chelating force, and the L-glutamic acid and Cu are opposite to intermolecular force 2+ The binding force of the compound is stronger, the surface of the compound can be more stable, and more substituted template molecules can be combined, so that more L-glutamic acid binding vacancies are obtained, and the fresh polypeptide with the end group of glutamic acid in the brewed soy sauce is combined as much as possible or even completely, so that the detection accuracy is improved.
Further, the molar ratio of the substituted template molecule to the functional monomer is 7:1-1:15; the molar ratio of the functional monomer to the metal ion is 9:1-1:12.
Further, the cross-linking agent is N, N-methylene bisacrylamide; the initiator is azodiisobutyronitrile; the dosage of the initiator is 0.1-5% of the mass of the cross-linking agent.
Further, the silylating agent is 4-chloromethylphenyl trimethoxysilane; the mol ratio of the silanization reagent to the activated silica gel microspheres is 8:1-1:12.
Further, in the step S1, the silica gel microspheres are placed in hydrochloric acid with the mass fraction of 5-20%, and are subjected to ultrasonic treatment for 3-20 min, and are heated to 50-200 ℃ and kept for 12-48 h; and washing with deionized water, suction filtering to neutrality, drying, and activating at 50-200 deg.c for 12-48 hr to obtain activated silica gel microsphere. Activating the surface of silica gel microsphere by using dilute hydrochloric acid to enable SiO to be prepared 2 The microsphere surface contains abundant hydroxyl groups, which is beneficial to subsequent surface modification.
Further, in the step S2, activated silica gel microspheres are dispersed in anhydrous toluene by ultrasonic, nitrogen is introduced to remove oxygen, and then 4-chloromethyl phenyl trimethoxy silane is added to react for 12-48 hours at the temperature of 30-150 ℃; washing with toluene and ethanol respectively, and drying to obtain silanized silica gel microspheres. SiO using silylating reagent 2 The microsphere is subjected to silanization treatment, so that the surface of the microsphere is connected with active silane groups, and the subsequent replacement of self-assembled structures of RAFT reagent and functional monomer-metal ion-substitution template molecules is realized by connecting the active silane groups to solid phase SiO 2 The surface of the microsphere.
Further, in step S3, after drying tetrahydrofuran and introducing nitrogen to remove oxygen, phenylmagnesium bromide is added to the pretreated tetrahydrofuran, and CS is then added dropwise 2 Reacting for 0.2-3 h at 20-80 ℃; adding silanized silica gel microspheres, uniformly dispersing by ultrasonic, introducing nitrogen to deoxidize for 3-30 min, heating to 30-80 ℃ and reacting for 24-60 h; and after the reaction is finished, washing the mixture with tetrahydrofuran, methanol and acetone for multiple times, and drying the mixture to obtain the RAFT functional silica gel microspheres. Synthesizing a RAFT reagent with active free radicals by adopting phenylmagnesium bromide and carbon disulfide under an anaerobic condition, wherein the RAFT reagent can be combined with active silane groups, so that the autonomous growth of the polymer on the surface of the microsphere is realized; the phenylmagnesium bromide and the carbon disulfide are small molecules, and RAFT reagent generated by combining the phenylmagnesium bromide and the carbon disulfide is also small molecules, so that the thickness of the formed imprinting film layer is thinner; the size and the length of the imprinting membrane are small, so that the occupied space is small, and the space for combining the RAFT reagent with adjacent active silane groups is not extruded, so that the thickness of the imprinting membrane is uniform; thereby overcoming the common conditionsThe MIP film layer prepared by the free radical polymerization method has the problems of thinner thickness and thickness.
Further, in step S4, L-glutamic acid is ultrasonically dissolved in a mixed solvent, and 1- (N, N-dicarboxymethyl) amino-3-allylglycerol and Cu are added 2+ Then vibrating for 4-14 h at the temperature of 10-80 ℃; adding RAFT functional silica gel microspheres and a cross-linking agent into the mixture, adding an initiator after ultrasonic dispersion, introducing nitrogen for deoxidization after shaking and ultrasonic mixing uniformly, and reacting for 4-12 hours at the temperature of 30-90 ℃ to obtain silica gel microspheres coated with alternative template molecules; the mixed solvent is a mixed solvent of ethanol and water, and the volume ratio of the ethanol to the water is 3:1-1:6. Incubating the substituted template molecule, the metal ion and the functional monomer in a mixed solvent to form a functional monomer-metal ion-substituted template molecule complex. Wherein the functional monomer replaces the RAFT reagent, the functional monomer-metal ion-substituted template molecule complex is combined to the active silane group, and the functional monomer-metal ion-substituted template molecule complex and the metal ion form a combining site of L-glutamic acid together; the substitution of the template molecule L-glutamic acid provides a basis for the formation of a cavity that recognizes and binds the nascent polypeptide whose end group is glutamic acid. Compared with the prior art, the functional monomer and the alternative template molecule are combined through weaker intermolecular force, and the functional monomer is used in the invention. The metal ions and the substitute template molecules act through stronger metal chelating force. In particular, due to Cu 2+ Electron-deficient, in aqueous solution, which is bound to a water molecule or anion, however, the donor atom of L-glutamic acid is capable of reacting with electron-deficient Cu under the action of 1- (N, N-dicarboxymethyl) amino-3-allylglycerol 2+ To form a complex, and replace the original water molecules or anions, thus the L-glutamic acid which is a substitute template molecule can be firmly combined with 1- (N, N-dicarboxymethyl) amino-3-allylglycerol and Cu 2+ And (3) upper part. Because the binding force is strong and the influence of the water phase is smaller, the nonspecific adsorption between the functional monomer and the substitute template molecules is greatly reduced, so that more substitute template molecules are bound to the functional monomer and the metal ions, thereby providing more L-glutamic acid binding vacancies and extracting and binding more in the sample solutionThe end group is glutamic acid, so that the accuracy of detecting the fresh polypeptide in the brewed soy sauce is ensured. In addition, the cross-linking agent and the initiator are added to perform free radical polymerization reaction with the functional monomer-metal ion-substituted template molecule complex, so that a firm cross-linked framework structure is formed, and the stability of the functional monomer-metal ion-substituted template molecule complex can be improved.
Further, in step S5, washing the silica gel microspheres coated with the substituted template molecules with deionized water and ethanol respectively, and drying; then placing the mixture in an ethylene diamine tetraacetic acid disodium eluent with the concentration of 0.1-0.5 mol/L for continuous and repeated elution, and then drying the mixture to obtain the surface molecularly imprinted polymer silica gel microsphere.
The invention also protects the application of the surface molecularly imprinted polymer silica gel microsphere with the end group of glutamic acid and the fresh polypeptide prepared by the preparation method in separation and detection of the fresh polypeptide.
For a better understanding and implementation, the present invention is described in detail below with reference to the drawings.
Drawings
FIG. 1 is a schematic diagram of the preparation process of the molecular engram polymer silica gel microsphere with glutamic acid end group and fresh polypeptide surface.
FIG. 2 is a scanning electron microscope photograph (magnification: 30000) of a blank silica gel microsphere (a) and a molecular engram polymer silica gel microsphere (b) with glutamic acid as a terminal group and a fresh polypeptide surface.
FIG. 3 is a blank silica gel microsphere (a), RAFT-functionalized silica gel microsphere (b), NIP/SiO of the present invention 2 (c) And molecular engram polymer silica gel microsphere with end group of glutamic acid and fresh polypeptide surface (d: MIP/SiO) 2 ) Is a thermogravimetric graph and a primary differential graph (wherein NIP/SiO 2 Except that no template molecule is added in the preparation process, the rest steps are completely the same as the preparation of the molecular engram polymer silica gel microsphere with the end group of glutamic acid and the fresh polypeptide surface.
FIG. 4 is a MIP/SiO of the present invention 2 Schematic diagram of the fresh polypeptide with glutamic acid as end group in the adsorption extraction sample solution.
FIG. 5 is a NIP/SiO of the present invention 2 With MIP/SiO 2 And (5) respectively carrying out selective extraction capacity graphs on different fresh polypeptides.
FIG. 6 is a graph showing the extraction capacity of L-glutamic acid by the functional monomer of the present invention, metal ions and functional monomers of the prior art; wherein the functional monomer in the invention is 1- (N, N-dicarboxymethyl) amino-3-allylglycerol (AGE/IDA), and the metal ion is Cu 2+ The functional monomer in the prior art is 2-acrylamide-2 methacrylic Acid (AMPS).
FIG. 7 is a liquid chromatogram of a fresh polypeptide surface molecularly imprinted polymer silica gel microsphere with glutamic acid end group prepared by the invention for pretreatment of soy sauce and a liquid chromatogram of a fresh polypeptide surface molecularly imprinted polymer silica gel microsphere without pretreatment of soy sauce by the microsphere.
Detailed Description
A preparation method of a molecular engram polymer silica gel microsphere with a glutamic acid end group and a fresh polypeptide surface comprises the following steps:
s1: adopting a dilute hydrochloric acid soaking method to remove impurities on the surfaces of the silica gel microspheres to obtain activated silica gel microspheres;
s2: carrying out surface silanization modification on the activated silica gel microspheres by adopting a silanization reagent to obtain silanized silica gel microspheres;
s3: carrying out RAFT functional treatment on the silanized silica gel microspheres by adopting a RAFT reagent to obtain RAFT functional silica gel microspheres with surface grafted with disulfide bonds;
s4: fully and uniformly mixing the substitution template molecules, the metal ions and the functional monomers in a mixed solvent to enable the substitution template molecules, the metal ions and the functional monomers to self-assemble, then adding a cross-linking agent, an initiator and the RAFT functional silica gel microspheres, and obtaining the molecularly imprinted coating microspheres coated with the substitution template molecules on the surfaces through RAFT polymerization reaction;
s5: eluting the silica gel microsphere obtained in the step S4, removing the surface-coated substitute template molecules, and drying to obtain the surface molecularly imprinted polymer silica gel microsphere;
wherein the substitute template molecule is L-glutamic acid, and the metal ion is Cu 2+ The functional monomer is 1- (N, N-dicarboxymethyl) amino-3-allylglycerol.
The preparation process of the molecular engram polymer silica gel microsphere with the end group of glutamic acid and the fresh polypeptide surface is detailed below. Referring to FIG. 1, the present invention relates to a molecular engram polymer silica gel microsphere (MIP/SiO) with glutamic acid end group and fresh polypeptide surface 2 ) Is a schematic of the preparation process. The preparation method comprises the following steps:
s1 activation of silica gel microspheres
Accurately weighing 5g of silica gel microspheres, adding the silica gel microspheres into a 250mL round-bottom flask, adding 150mL of hydrochloric acid solution with the mass fraction of 5% -20%, performing ultrasonic treatment for 3-20 min, and then placing the round-bottom flask into an oil bath pot with the temperature of 50-200 ℃ and keeping for 12-48 h. And washing with deionized water after the completion, and carrying out suction filtration until the solution is neutral. And then placing the silica gel microspheres in a blast drying oven, and activating for 12-48 hours at 50-200 ℃ to obtain activated silica gel microspheres.
Preparation of S2 silanized silica gel microspheres
2g of activated silica gel microspheres are added into a 50mL conical flask, 20mL of anhydrous toluene is added, the activated silica gel microspheres are uniformly dispersed in the toluene under the ultrasonic action, and nitrogen is introduced for 10min. Then 2mL of 4-chloromethylphenyl trimethoxysilane was added into the conical flask and reacted at 30-150 ℃ for 12-48 h. After the reaction is finished, respectively washing with toluene and ethanol for 5 times, and then drying in a vacuum drying oven at room temperature for 12-48 hours to obtain the silanized silica gel microspheres.
Preparation of S3RAFT functional silica gel microsphere
40mL of dry pretreated tetrahydrofuran is taken and added into a 100mL conical flask, nitrogen is introduced to deoxidize for 10min, and 8-30 mL of phenylmagnesium bromide is injected. Then 1-5 mL CS is added dropwise by a syringe 2 After the dripping is finished, reacting for 0.2 to 3 hours at the temperature of 20 to 80 ℃. After the reaction is finished, 300-1000 mg of silanized silica gel microspheres (namely, the mole ratio of the RAFT reagent to the activated silica gel microspheres is 8:1-1:12) are added, the ultrasonic action is carried out for 5min, nitrogen is introduced for 3-30 min, and the mixture is placed in an oil bath pot at 30-80 ℃ for reaction for 24-60 h after deoxidization. After the reaction is finished, washing with tetrahydrofuran, methanol and acetone for three times in sequence, and finally drying in a vacuum drying oven for 12-48 hours at room temperature to obtain RAFTFunctionalized silica gel microspheres.
Preparation of S4 silica gel microsphere coated with template molecules on surface
Weighing 3-15 mg of L-glutamic acid, ultrasonically dissolving the L-glutamic acid in 10mL of mixed solvent of ethanol and water, adding 300-600 mg of 1- (N, N-dicarboxymethyl) amino-3-allylglycerol and 50-200 mg of CuSO 4 ·5H 2 O (namely, the molar ratio of the substituted template molecule to the functional monomer is 7:1-1:15, and the molar ratio of the functional monomer to the metal ion is 9:1-1:12), and oscillating for 4-14 h in a shaking table at 10-80 ℃ to ensure that L-glutamic acid and Cu are obtained 2+ Self-assembly with 1- (N, N-dicarboxymethyl) amino-3-allylglycerol occurs by metal chelation. Subsequently, 80mg of RAFT functionalized silica gel microspheres, 370mg of N, N-methylenebisacrylamine, were added thereto, and sonicated for 10min. Then adding 500 mu L of Azobisisobutyronitrile (AIBN) solution with the concentration of 0.6mg/mL (the dosage of the initiator is 0.1% -5% of the mass of the cross-linking agent), oscillating for 30min, performing ultrasonic action for 10min, introducing nitrogen for 15min, and sealing a reaction system. And (3) carrying out oscillation reaction for 4-12 h in a shaking table at the temperature of 30-90 ℃ to obtain the silica gel microsphere coated with the template molecule L-glutamic acid.
S5 eluting template molecules
Washing and suction-filtering the silica gel microspheres coated with the template molecules by deionized water and ethanol respectively, and then drying the silica gel microspheres in a vacuum drying oven at 80 ℃ for 24 hours. Placing the mixture into eluent for eluting, and centrifuging the mixture after continuous repeated times to obtain silica gel microspheres from which template molecules are removed; and drying the silica gel microsphere with the template molecules removed to obtain the molecular engram polymer silica gel microsphere with specific recognition cavities. And then washing with deionized water to neutrality, and drying in a vacuum drying oven at 60 ℃ for 10 hours to obtain the L-glutamic acid surface molecularly imprinted polymer silica gel microsphere.
Following the above preparation procedure, the following specific examples were carried out.
The molecular engram polymer silica gel microsphere with the end group of glutamic acid and fresh polypeptide surface prepared by the method is analyzed as follows:
for convenience of comparison and explanation, the non-imprinted polymer silica gel microsphere (NIP/SiO) is prepared by the same method except that the substitution template molecule L-glutamic acid is not added in the preparation process 2 ) Molecular engram polymer silica gel microsphere (MIP/SiO) with end group of glutamic acid prepared in this example 2 ) And (5) comparing and analyzing.
Referring to fig. 2, a scanning electron microscope photograph of a blank silica gel microsphere (a) and a molecular engram polymer silica gel microsphere (b) with glutamic acid as a terminal group and a fresh polypeptide surface is shown. As can be seen from the figure, before the polymerization, the silica gel microsphere is in a regular sphere shape, and the surface roughness is larger (a); MIP/SiO 2 The surface of the silica gel microsphere is in a regular sphere shape, a layer of obvious molecularly imprinted polymer is arranged on the surface of the silica gel microsphere, and the surface roughness of the silica gel microsphere is smaller than that of a blank silica gel microsphere.
Please refer to fig. 3, which shows the blank silica gel microsphere (a), RAFT functionalized silica gel microsphere (b), NIP/SiO of the present invention 2 (c) With MIP/SiO 2 (d) A thermogravimetric plot and a primary differential plot. From the figure, the mass loss of the blank silica gel is about 4.2%, and the mass loss is mainly the weight loss of water and part of impurities adsorbed on the surface of the silica gel microsphere. The weight loss of the RAFT functionalized silica gel microsphere is about 8.3%, and the weight loss rate is maximum at about 450 ℃ and 650 ℃, because the silanized substance grafted on the surface of the functionalized silica gel microsphere and the phenylmagnesium bromide are degraded along with the temperature rise, which shows that the modification treatment of the silica gel microsphere is successful. NIP/SiO compared with RAFT functionalized silica gel microsphere 2 And MIP/SiO 2 The weight loss is larger and is respectively 9.6 percent and 11.2 percent, and the weight loss rate is maximum at about 350 ℃, which indicates that the surface of the silica gel microsphere forms a imprinting thin layer, and the imprinting thin layer is decomposed under the high temperature condition of 350 ℃. In addition, MIP/SiO 2 Weight loss ratio NIP/SiO 2 In many cases, described in MIP/SiO 2 Surface to NIP/SiO of (C) 2 Is higher, the percentage of imprinted polymer is higher.
Referring to FIG. 4, the MIP/SiO of the present invention is shown 2 Schematic diagram of the fresh polypeptide with glutamic acid as end group in the adsorption extraction sample solution. The surface of the molecular engram polymer silica gel microsphere with the end group of glutamic acid is provided with a specific binding vacancy of L-glutamic acid, and the molecular engram polymer silica gel microsphere can adsorb the end group of the fresh polypeptide with glutamic acid in the sample solution and insert the glutamic acid on the molecular engram polymer silica gel microsphere into the binding vacancy of the surface of the microsphere, thereby completing the adsorption extraction of the end group of the fresh polypeptide with glutamic acid in the sample solution.
Please refer to fig. 5, which is MIP/SiO 2 With NIP/SiO 2 The selective extraction capacity diagrams of different fresh polypeptides are respectively shown, wherein the selected polypeptides are aspartic acid-glycine (Asp-Gly), glutamic acid-glycine (Glu-Gly), glutamic acid-glutamic acid (Glu-Glu), serine-glycine-serine (Ser-Gly-Ser), glutamic acid-glycine-serine (Glu-Gly-Ser) and glutamic acid-glycine-glutamic acid (Glu-Gly-Glu) in sequence. The six polypeptides are respectively prepared into 10 ≡ -2 mg/mL standard solution. As can be seen from the figure, MIP/SiO 2 The extraction amount of Glu-Gly is 0.0082mmol, and NIP/SiO 2 The extraction amount of Glu-Gly is 0.0026mmol; and MIP/SiO 2 The extraction amount of Glu-Gly, glu-Glu, glu-Gly-Ser and Glu-Gly-Glu is larger than that of Ser-Gly-Ser. Description of MIP/SiO 2 The fresh polypeptide with glutamic acid at the opposite end group has certain selectivity, and the polypeptide without glutamic acid at the opposite end group has poorer selectivity, which shows that compared with other polypeptides, the MIP/SiO prepared by the method of the invention 2 The polypeptide with glutamic acid at the opposite end group has better selectivity.
FIG. 6 is a graph showing the extraction capacity of L-glutamic acid by the functional monomer of the present invention, metal ions and functional monomers of the prior art; wherein the functional monomer in the invention is 1- (N, N-dicarboxymethyl) amino-3-allylglycerol (AGE/IDA), and the metal ion is Cu 2+ The functional monomer in the prior art is 2-acrylamide-2 methacrylic Acid (AMPS). As can be seen from the graph, the AGE/IDA of the present invention has a higher extraction amount of L-glutamic acid than AMPS. This is due to the van der Waals force connection between AMPS and L-glutamic acid between H and OIs very weak; AGE/IDA and Cu 2+ The structure of the catalyst provides electron orbits for two carboxyl H of the L-glutamic acid, and the binding force is stronger, so that more L-glutamic acid can be extracted.
Referring to fig. 7, a liquid chromatogram of a molecular engram polymer silica gel microsphere with glutamic acid end group and fresh polypeptide surface prepared by the method of the present invention for pretreatment of soy sauce (a), pretreatment of soy sauce without the microsphere (b), and pretreatment of reference solution (non-soy sauce) without the microsphere (c) is shown. By comparison, it was found that MIP/SiO 2 After separation, two species of the fresh polypeptides Glu-Gly and Glu-Gly-Ser with glutamic acid end groups can be detected in soy sauce without MIP/SiO 2 The above two polypeptides were isolated and no fresh polypeptides were detected in soy sauce. Therefore, the molecular engram polymer silica gel microsphere with the end group of glutamic acid on the surface of the fresh polypeptide prepared by the invention can effectively adsorb and extract the fresh polypeptide with the end group of glutamic acid in soy, thereby improving the accuracy of detecting the fresh polypeptide in soy.
Compared with the prior art, the surface molecularly imprinted polymer silica gel microsphere prepared by the method has the advantages of uniform coating, narrow molecular weight distribution of the imprinted polymer silica gel microsphere and stable performance. In addition, as the end groups of the plurality of the fresh polypeptides are L-glutamic acid, the selective adsorption of the plurality of the fresh polypeptides in the water environment is realized at one time, and the workload is greatly reduced. In addition, in the traditional surface molecular imprinting technology, functional monomers are combined with alternative template molecules through intermolecular force, and in the preparation method, metal ions Cu are prepared 2+ And the functional monomer 1- (N, N-dicarboxymethyl) amino-3-allylglycerol is connected to replace template molecule L-glutamic acid through metal chelating force, and the L-glutamic acid and Cu are opposite to intermolecular force 2+ The binding force of the compound is stronger, the surface of the compound can be more stable, and more substituted template molecules can be combined, so that more L-glutamic acid binding vacancies are obtained, and the fresh polypeptide with the end group of glutamic acid in the brewed soy sauce is combined as much as possible or even completely, so that the detection accuracy is improved.
The present invention is not limited to the above-described embodiments, but, if various modifications or variations of the present invention are not departing from the spirit and scope of the present invention, the present invention is intended to include such modifications and variations as fall within the scope of the claims and the equivalents thereof.
Claims (9)
1. A preparation method of a surface molecularly imprinted polymer silica gel microsphere with glutamic acid as a terminal group and a fresh polypeptide is characterized by comprising the following steps: the method comprises the following steps:
s1: adopting a dilute hydrochloric acid soaking method to remove impurities on the surfaces of the silica gel microspheres to obtain activated silica gel microspheres;
s2: carrying out surface silanization modification on the activated silica gel microspheres by adopting a silanization reagent to obtain silanized silica gel microspheres;
s3: carrying out RAFT functional treatment on the silanized silica gel microspheres by adopting a RAFT reagent to obtain RAFT functional silica gel microspheres with surface grafted with disulfide bonds;
s4: fully and uniformly mixing the substituted template molecules, metal ions and functional monomers in a mixed solvent to enable the substituted template molecules, the metal ions and the functional monomers to perform self-assembly through metal chelation, then adding a cross-linking agent, an initiator and RAFT functional silica gel microspheres, and obtaining molecularly imprinted coating microspheres coated with the substituted template molecules through RAFT polymerization reaction;
s5: eluting the silica gel microsphere obtained in the step S4, removing the surface-coated substituted template molecules, eluting by using disodium ethylenediamine tetraacetate eluent, and drying to obtain the surface molecularly imprinted polymer silica gel microsphere;
wherein the substitute template molecule is L-glutamic acid, and the metal ion is Cu 2+ The functional monomer is 1- (N, N-dicarboxymethyl) amino-3-allylglycerol,
the molar ratio of the substituted template molecules to the functional monomers is 7:1-1:15; the molar ratio of the functional monomer to the metal ion is 9:1-1:12.
2. The method for preparing the surface molecularly imprinted polymer silica gel microsphere with the end group of glutamic acid and fresh polypeptide, which is characterized in that: the cross-linking agent is N, N-methylene bisacrylamide; the initiator is azodiisobutyronitrile; the dosage of the initiator is 0.1-5% of the mass of the cross-linking agent.
3. The method for preparing the surface molecularly imprinted polymer silica gel microsphere with the end group of glutamic acid and fresh polypeptide according to claim 2, which is characterized in that: the silylating agent is 4-chloromethylphenyl trimethoxysilane; the mol ratio of the silanization reagent to the activated silica gel microspheres is 8:1-1:12.
4. The method for preparing the surface molecularly imprinted polymer silica gel microsphere with the end group of glutamic acid and fresh polypeptide according to any one of claims 1-3, which is characterized in that: in the step S1, the silica gel microspheres are placed in hydrochloric acid with the mass fraction of 5-20%, are ultrasonically treated for 3-20 min, and are heated to 50-200 ℃ and kept for 12-48 h; and washing with deionized water, suction filtering to neutrality, drying, and activating at 50-200 deg.c for 12-48 hr to obtain activated silica gel microsphere.
5. The method for preparing the surface molecularly imprinted polymer silica gel microsphere with the end group of glutamic acid and fresh polypeptide according to any one of claims 1-3, which is characterized in that: in the step S2, activated silica gel microspheres are dispersed in anhydrous toluene by ultrasonic, nitrogen is introduced to deoxidize, and then 4-chloromethyl phenyl trimethoxy silane is added to react for 12 to 48 hours at the temperature of 30 to 150 ℃; washing with toluene and ethanol respectively, and drying to obtain silanized silica gel microspheres.
6. The method for preparing the surface molecularly imprinted polymer silica gel microsphere with the end group of glutamic acid and fresh polypeptide according to any one of claims 1-3, which is characterized in that: in the step S3, after the tetrahydrofuran is dried and pretreated by introducing nitrogen to remove oxygen, the phenylmagnesium bromide is added into the pretreated tetrahydrofuran, and then CS is added dropwise 2 Reacting for 0.2-3 h at 20-80 ℃; adding silanized silica gel microspheres, uniformly dispersing by ultrasonic, introducing nitrogen to deoxidize for 3-30 min, and heating to 30-8Reacting for 24-60 h at 0 ℃; and after the reaction is finished, washing the mixture with tetrahydrofuran, methanol and acetone for multiple times, and drying the mixture to obtain the RAFT functional silica gel microspheres.
7. The method for preparing the surface molecularly imprinted polymer silica gel microsphere with the end group of glutamic acid and fresh polypeptide according to any one of claims 1-3, which is characterized in that: in the step S4, L-glutamic acid is ultrasonically dissolved in a mixed solvent, and 1- (N, N-dicarboxymethyl) amino-3-allylglycerol and Cu are added 2+ Then vibrating for 4-14 h at the temperature of 10-80 ℃; adding RAFT functional silica gel microspheres and a cross-linking agent into the mixture, adding an initiator after ultrasonic dispersion, introducing nitrogen for deoxidization after shaking and ultrasonic mixing, and reacting for 2-18 hours at 50-60 ℃ to obtain silica gel microspheres coated with alternative template molecules; the mixed solvent is a mixed solvent of ethanol and water, and the volume ratio of the ethanol to the water is 3:1-1:6.
8. The method for preparing the surface molecularly imprinted polymer silica gel microsphere with the end group of glutamic acid and fresh polypeptide according to claim 7, which is characterized in that: in the step S5, the silica gel microspheres coated with the substituted template molecules are respectively washed by deionized water and ethanol and dried; then placing the mixture in an ethylene diamine tetraacetic acid disodium eluent with the concentration of 0.1-0.5 mol/L for continuous and repeated elution, and then drying the mixture to obtain the surface molecularly imprinted polymer silica gel microsphere.
9. The use of the surface molecularly imprinted polymer silica gel microsphere with the end group of glutamic acid and the fresh polypeptide prepared by the preparation method of the surface molecularly imprinted polymer silica gel microsphere with the end group of glutamic acid and the fresh polypeptide according to any one of claims 1-8 in separation and detection of the fresh polypeptide.
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| SE0103404D0 (en) * | 2001-10-12 | 2001-10-12 | Protista Internat Ab | Macroporous gel, ITS preparation and its use |
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