CN110871055A - Preparation method and application of fresh polypeptide surface molecularly imprinted polymer silica gel microspheres with glutamic acid as end group - Google Patents

Preparation method and application of fresh polypeptide surface molecularly imprinted polymer silica gel microspheres with glutamic acid as end group Download PDF

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CN110871055A
CN110871055A CN201810994201.6A CN201810994201A CN110871055A CN 110871055 A CN110871055 A CN 110871055A CN 201810994201 A CN201810994201 A CN 201810994201A CN 110871055 A CN110871055 A CN 110871055A
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silica gel
gel microspheres
glutamic acid
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CN110871055B (en
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胡小刚
何佩玲
罗晓燕
郑雪屏
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South China Normal University
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Abstract

The invention relates to a preparation method of surface molecularly imprinted polymer silica gel microspheres with glutamic acid as an end group and fresh polypeptide, which comprises the following steps: removing impurities from the surface of the silica gel microspheres by using a dilute hydrochloric acid soaking method to obtain activated silica gel microspheres; carrying out surface silanization modification on the activated silica gel microspheres to obtain silanized silica gel microspheres; RAFT functional treatment is carried out on the silanized silica gel microspheres to obtain RA with the surface grafted with disulfide bondsFT functional silica gel microspheres; after the substituted template molecules, the metal ions and the functional monomers are self-assembled, adding a cross-linking agent, an initiator and RAFT functional silica gel microspheres, and carrying out RAFT polymerization reaction to obtain the molecularly imprinted coating microspheres coated with the substituted template molecules on the surface; eluting to remove the surface-coated alternative template molecules; wherein the substitutional template molecule is L-glutamic acid, and the metal ion is Cu2+The functional monomer is 1- (N, N-dicarboxymethyl) amino-3-allyl glycerol.

Description

Preparation method and application of fresh polypeptide surface molecularly imprinted polymer silica gel microspheres with glutamic acid as end group
Technical Field
The invention belongs to the field of chemical analysis and test, and particularly relates to a preparation method and application of a fresh polypeptide surface molecularly imprinted polymer silica gel microsphere with a glutamic acid as an end group.
Background
The soy sauce is a traditional brewed product in China and is an indispensable seasoning for common people in daily life. The unique color, fragrance and taste make the seasoning become a traditional seasoning which is well loved by people in China. And delicate flavor is the most important evaluation index in soy sauce. The brewed soy sauce is found to have a small molecular polypeptide with the molecular weight of below 500Da, the small molecular polypeptide can obviously improve the whole delicate flavor intensity of food, is a flavor substance in the brewed soy sauce, and is called as fresh polypeptide. It has been found that most of the present polypeptides are dipeptides and tripeptides with the terminal amino acid glutamic acid or aspartic acid. Therefore, people can further disclose and master the freshness mechanism of the soy sauce by detecting and analyzing the type and the content of the freshness polypeptide in the brewed soy sauce.
At present, the analytical methods for the fresh polypeptide are mainly liquid chromatography and liquid chromatography/mass spectrometry. Both methods require a pre-treatment of the sample solution, i.e. soy sauce, first. In the sample pretreatment technology for separating and detecting amino acids, small molecular polypeptides and proteins, the surface imprinting technology is more and more widely concerned as a novel sample pretreatment technology. The action principle of the surface imprinting technology is similar to the 'key-lock' interaction principle of enzyme-substrate, silica gel and other materials are used as a supporting solid phase, target molecules are used as template molecules, corresponding chemical bonds are generated through chemical reaction to combine the template molecules on the supporting solid phase, and then the template molecules are removed through means of elution and the like, so that a vacancy is reserved on the surface of the supporting solid phase for combining the target molecules. Because the template molecule is the same as the target molecule, the binding vacancy reserved on the surface of the supporting solid phase can only specifically adsorb and bind the target molecule, thereby greatly improving the selectivity of the target molecule in the sample solution, reducing the probability of binding with other molecules in the sample solution and greatly improving the accuracy of detection. In this technique, we call the structure that has eluted the template molecules leaving binding vacancies for the solid phase and surface target molecules as a surface molecularly imprinted polymer. It can be seen that the surface imprinting technology can complete the binding and separation of target molecules in the sample solution only by contacting the sample solution with the surface molecularly imprinted polymer, and the operation is very simple and convenient and consumes little 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, template molecules are mainly bonded to a supporting solid phase through intermolecular force, and the number of the template molecules bonded to the supporting solid phase is limited due to very weak intermolecular force, and accordingly the number of bonding vacancies formed is also limited, so that the prepared surface molecularly imprinted polymer cannot adsorb and bond target molecules in a sample solution as much as possible or completely, and the detection accuracy is influenced. In addition, the prior art has not applied the surface blotting technique to the detection and analysis of fresh polypeptides.
Disclosure of Invention
Based on the above, the invention provides a preparation method of the fresh polypeptide surface molecularly imprinted polymer silica gel microspheres with the terminal group of glutamic acid, so as to prepare the surface molecularly imprinted polymer which is uniform in coating, uniform in thickness, stable in performance, capable of specifically adsorbing multiple fresh polypeptide molecules with the terminal group of L-glutamic acid and firmer in chemical bonds between the silica gel microspheres and template molecules.
The preparation method of the surface molecularly imprinted polymer silica gel microspheres with the terminal group of glutamic acid and the fresh polypeptide comprises the following steps:
s1: removing impurities from the surface of the silica gel microspheres by using a dilute hydrochloric acid soaking method to obtain activated silica gel microspheres;
s2: carrying out surface silanization modification on the activated silica gel microspheres by using a silanization reagent to obtain silanized silica gel microspheres;
s3: RAFT functional treatment is carried out on the silanized silica gel microspheres by using RAFT reagent to obtain RAFT functional silica gel microspheres with disulfide bonds grafted on the surfaces;
s4: fully and uniformly mixing the substituted template molecules, the metal ions and the functional monomers in a mixed solvent to enable the substituted template molecules, the metal ions and the functional monomers to be self-assembled, then adding a cross-linking agent, an initiator and RAFT functional silica gel microspheres, and obtaining the molecularly imprinted coating microspheres with the surface coated with the substituted template molecules through RAFT polymerization reaction;
s5: eluting the silica gel microspheres obtained in the step S4, removing the substituted template molecules coated on the surfaces of the silica gel microspheres, and drying to obtain surface molecularly imprinted polymer silica gel microspheres;
wherein the substitutional template molecule is L-glutamic acid, and the metal ion is Cu2+The functional monomer is 1- (N, N-dicarboxymethyl) amino-3-allyl glycerol.
Compared with the prior art, the surface molecularly imprinted polymer silica gel microspheres prepared by the method have the advantages of uniform coating, narrow molecular weight distribution of imprinted substances and stable performance. In addition, the terminal group of the plurality of fresh polypeptides is L-glutamic acid, so that the selective adsorption of the plurality of 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, the functional monomer and the substitute template molecule are combined through intermolecular force, and in the preparation method, the metal ion Cu is used2+And the functional monomer 1- (N, N-dicarboxymethyl) amino-3-allyl glycerol is connected with the template molecule L-glutamic acid through metal chelation acting force to replace the template molecule L-glutamic acid, and the L-glutamic acid and Cu are opposite to the acting force between molecules2+The binding force of the compound is stronger, the surface of the polymer can be more stabilized, and more alternative template molecules can be bound, so that more L-glutamic acid binding vacancies are obtained, and the brewing soy sauce can be bound as much as possible or even allThe terminal group in the kit is a fresh polypeptide of glutamic acid, so that the detection accuracy is improved.
Further, 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 ions is 9: 1-1: 12.
Further, the cross-linking agent is N, N-methylene bisacrylamide; the initiator is azobisisobutyronitrile; the dosage of the initiator is 0.1-5% of the mass of the cross-linking agent.
Further, the silanization reagent is 4-chloromethyl phenyl trimethoxy silane; the molar ratio of the silanization reagent to the activated silica gel microspheres is 8: 1-1: 12.
Further, in step S1, placing the silica gel microspheres in 5-20% hydrochloric acid by mass, performing ultrasonic treatment for 3-20 min, and heating to 50-200 ℃ for 12-48 h; and then washing with deionized water, carrying out suction filtration until the mixture is neutral, drying, and activating at 50-200 ℃ for 12-48 h to obtain the activated silica gel microspheres. Activating the surface of the silica gel microsphere by using dilute hydrochloric acid to ensure that SiO is generated2The surface of the microsphere contains abundant hydroxyl, which is beneficial to the subsequent surface modification.
Further, in step S2, ultrasonically dispersing the activated silica gel microspheres in anhydrous toluene, introducing nitrogen to remove oxygen, adding 4-chloromethyl phenyl trimethoxysilane, and reacting at 30-150 ℃ for 12-48 h; and respectively washing with toluene and ethanol, and drying to obtain the silanized silica gel microspheres. Use of silylating agents on SiO2The microspheres are subjected to silanization treatment, so that the surfaces of the microspheres are connected with active silane groups, and the subsequent replacement of the RAFT reagent and the functional monomer-metal ion-substituted template molecule self-assembly structure is realized by connecting the active silane groups on the solid SiO phase2The surface of the microspheres.
Further, in step S3, after the tetrahydrofuran is dried and pretreated by introducing nitrogen and removing oxygen, phenylmagnesium bromide is added to the pretreated tetrahydrofuran, and then CS is added dropwise2Reacting for 0.2-3 h at 20-80 ℃; then adding silanized silica gel microspheres, ultrasonically and uniformly dispersing, introducing nitrogen to remove oxygen for 3-30 min, heating to 30-80 ℃, and reacting for 24-60 h; the reaction is finishedAnd washing the microspheres with tetrahydrofuran, methanol and acetone for multiple times in sequence, and drying to obtain the RAFT functional silica gel microspheres. Phenyl magnesium bromide and carbon disulfide are adopted to synthesize an RAFT reagent with active free radicals under an anaerobic condition, and the RAFT reagent can be combined to active silane groups, so that the autonomous growth of the polymer on the surface of the microsphere is realized; the phenyl magnesium bromide and the carbon disulfide are small molecules, and the RAFT reagent generated by combining the phenyl magnesium bromide and the carbon disulfide is also small molecules, so that the formed imprinted membrane layer is thinner; the small molecules have small volume and small length, so that the occupied space is small, and the space for combining the adjacent active silane-based sites with the RAFT reagent cannot be extruded, so that the thickness of the formed imprinted membrane layer is uniform; thereby overcoming the problems of thin and thick MIP film layer prepared by the conventional free radical polymerization method.
Further, in step S4, L-glutamic acid was dissolved in the mixed solvent by sonication, and 1- (N, N-biscarboxymethyl) amino-3-allylglycerol and Cu were added2+Then oscillating for 4-14 h at the temperature of 10-80 ℃; adding the RAFT functional silica gel microspheres and a cross-linking agent into the mixture, adding an initiator after ultrasonic dispersion, introducing nitrogen to remove oxygen after oscillating ultrasonic mixing, and reacting for 4-12 hours at the temperature of 30-90 ℃ to obtain the silica gel microspheres coated with the substituted 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. And incubating the substitutional template molecule, the metal ions and the functional monomer in a mixed solvent to form a functional monomer-metal ion-substitutional template molecule compound. Replacing the RAFT reagent with the functional monomer, combining the functional monomer-metal ion-substituted template molecule compound onto the active silane group, and forming a combination site of L-glutamic acid together with the metal ion; and the substituted template molecule L-glutamic acid provides a basis for forming a cavity for recognizing and combining the fresh polypeptide of which the terminal group of the target molecule is glutamic acid. Compared with the prior art, the functional monomer is combined with the substituted template molecule through weaker intermolecular force, and in the invention, the functional monomer is used. The metal ions and the substituted template molecules act through stronger metal chelation. In particular, since Cu2+Electron deficient in aqueous solutionWhich will bind to water molecules or anions, whereas the electron-donating atom of L-glutamic acid is capable of interacting with electron-deficient Cu under the action of 1- (N, N-biscarboxymethyl) amino-3-allylglycerol2+The combination forms a complex, replaces the originally combined water molecules or anions, and enables the replacement template molecule L-glutamic acid to be firmly combined with the 1- (N, N-dicarboxymethyl) amino-3-allyl glycerol and the Cu2+The above. Because the binding force is strong and the influence of the aqueous phase is small, the nonspecific adsorption between the functional monomer and the substituted template molecules is greatly reduced, more substituted template molecules are bonded to the functional monomer and the metal ions, so that more L-glutamic acid bonding vacant sites are provided, more fresh polypeptide with the terminal group of glutamic acid in the sample solution is extracted and bonded, and the accuracy of detecting the fresh polypeptide in the brewed soy sauce is ensured. In addition, a cross-linking agent and an initiator are added to carry out free radical polymerization reaction with the functional monomer-metal ion-substituted template molecule compound, so that a firm cross-linked framework structure is formed, and the stability of the functional monomer-metal ion-substituted template molecule compound can be improved.
Further, in step S5, washing the silica gel microspheres coated with the substitute template molecules with deionized water and ethanol, respectively, and drying; and then continuously eluting for multiple times in an ethylene diamine tetraacetic acid eluent with the concentration of 0.1-0.5 mol/L, and drying to obtain the surface molecularly imprinted polymer silica gel microspheres.
The invention also protects the application of the surface molecularly imprinted polymer silica gel microspheres of the fresh polypeptide with the terminal group of glutamic acid prepared by the preparation method in separating and detecting the fresh polypeptide.
For a better understanding and practice, the invention is described in detail below with reference to the accompanying drawings.
Drawings
FIG. 1 is a schematic diagram of the preparation process of the fresh polypeptide surface molecularly imprinted polymer silica gel microspheres with glutamic acid as the terminal group.
FIG. 2 is the scanning electron micrographs (both 30000 magnification) of the blank silica gel microspheres (a) and the fresh polypeptide surface molecularly imprinted polymer silica gel microspheres (b) with the terminal group of glutamic acid according to the present invention.
FIG. 3 shows the blank silica gel microspheres (a), RAFT functionalized silica gel microspheres (b), NIP/SiO2(c) And fresh polypeptide surface molecularly imprinted polymer silica gel microspheres with glutamic acid as terminal group (d: MIP/SiO)2) Thermogravimetric and first derivative plots of (wherein, NIP/SiO)2The preparation method is completely the same as the preparation method of the fresh polypeptide surface molecularly imprinted polymer silica gel microspheres with the terminal group of glutamic acid except that no template molecules are added in the preparation process).
FIG. 4 is a MIP/SiO of the present invention2A schematic diagram of fresh polypeptide with glutamic acid as the end group in the adsorption extraction sample solution.
FIG. 5 shows NIP/SiO of the present invention2With MIP/SiO2Respectively for different fresh polypeptide selective extraction volumetric maps.
FIG. 6 is a graph showing the extraction capacity of the functional monomer of the present invention with metal ions and the functional monomer of the prior art for L-glutamic acid; wherein, the functional monomer in the invention is 1- (N, N-dicarboxymethyl) amino-3-allyl glycerol (AGE/IDA), and the metal ion is Cu2+The functional monomer in the prior art is 2-acrylamide-2-methacrylic Acid (AMPS).
FIG. 7 is a liquid chromatogram of pretreatment of soy sauce with fresh polypeptide surface molecularly imprinted polymer silica gel microspheres with glutamic acid as the terminal group prepared by the present invention, and pretreatment of soy sauce without the above microspheres.
Detailed Description
A preparation method of fresh polypeptide surface molecularly imprinted polymer silica gel microspheres with glutamic acid as an end group comprises the following steps:
s1: removing impurities from the surface of the silica gel microspheres by using a dilute hydrochloric acid soaking method to obtain activated silica gel microspheres;
s2: carrying out surface silanization modification on the activated silica gel microspheres by using a silanization reagent to obtain silanized silica gel microspheres;
s3: RAFT functional treatment is carried out on the silanized silica gel microspheres by using RAFT reagent to obtain RAFT functional silica gel microspheres with disulfide bonds grafted on the surfaces;
s4: fully and uniformly mixing the substituted template molecules, the metal ions and the functional monomers in a mixed solvent to enable the substituted template molecules, the metal ions and the functional monomers to be self-assembled, then adding a cross-linking agent, an initiator and RAFT functional silica gel microspheres, and obtaining the molecularly imprinted coating microspheres with the surface coated with the substituted template molecules through RAFT polymerization reaction;
s5: eluting the silica gel microspheres obtained in the step S4, removing the substituted template molecules coated on the surfaces of the silica gel microspheres, and drying to obtain surface molecularly imprinted polymer silica gel microspheres;
wherein the substitutional template molecule is L-glutamic acid, and the metal ion is Cu2+The functional monomer is 1- (N, N-dicarboxymethyl) amino-3-allyl glycerol.
The preparation process of the fresh polypeptide surface molecularly imprinted polymer silica gel microsphere with the terminal group of glutamic acid is detailed below. Please refer to fig. 1, which shows the fresh polypeptide surface molecularly imprinted polymer silica gel microspheres (MIP/SiO) with glutamic acid as the terminal group according to the present invention2) The preparation process is shown schematically. The preparation method comprises the following steps:
activation of S1 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 pan at the temperature of 50-200 ℃ and keeping the temperature for 12-48 h. After the reaction is finished, washing with deionized water and performing suction filtration until the reaction is neutral. And then, placing the silica gel microspheres in a forced air drying oven, and activating for 12-48 hours at the temperature of 50-200 ℃ to obtain the activated silica gel microspheres.
Preparation of S2 silanized silica gel microspheres
Adding 2g of activated silica gel microspheres into a 50mL conical flask, adding 20mL of anhydrous toluene, uniformly dispersing the activated silica gel microspheres in the toluene under the action of ultrasonic waves, and introducing nitrogen for 10 min. Then 2mL of 4-chloromethylphenyltrimethoxysilane is added into the conical flask, and the reaction is carried out for 12-48 h at 30-150 ℃. After the reaction is finished, respectively washing the microspheres with toluene and ethanol for 5 times, and then placing the microspheres in a vacuum drying oven to dry the microspheres for 12-48 hours at room temperature to obtain the silanized silica gel microspheres.
Preparation of S3RAFT functional silica gel microspheres
Adding 40mL of dried and pretreated tetrahydrofuran into a 100mL conical flask, introducing nitrogen to remove oxygen for 10min, and injecting 8-30 mL of phenylmagnesium bromide. Then dropwise adding 1-5 mL of CS by using an injector2And after the dropwise addition is finished, reacting for 0.2-3 h at the temperature of 20-80 ℃. And after the reaction is finished, adding 300-1000 mg of silanized silica gel microspheres (namely the molar ratio of the RAFT reagent to the activated silica gel microspheres is 8: 1-1: 12), performing ultrasonic action for 5min, introducing nitrogen for 3-30 min, deoxidizing, and then placing in an oil bath kettle at the temperature of 30-80 ℃ for reaction for 24-60 h. And after the reaction is finished, washing the mixture by tetrahydrofuran, methanol and acetone for three times respectively, and finally drying the mixture in a vacuum drying oven at room temperature for 12-48 hours to obtain the RAFT functional silica gel microspheres.
Preparation of silica gel microspheres coated with template molecules on S4 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-allyl glycerol and 50-200 mg of CuSO4·5H2O (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 the mixture in a shaker for 4-14 hours at 10-80 ℃ to ensure that the L-glutamic acid and the Cu are mixed2+Self-assemble with 1- (N, N-biscarboxymethyl) amino-3-allylglycerol by metal chelation. Subsequently, 80mg of RAFT functionalized silica gel microspheres and 370mg of N, N-methylenebisacrylamide were added thereto, and ultrasonication was performed for 10 min. Then adding 500 mu L of Azodiisobutyronitrile (AIBN) solution with the concentration of 0.6mg/mL (the dosage of the initiator is 0.1-5 percent of the mass of the cross-linking agent), oscillating for 30min, performing ultrasonic action for 10min, introducing nitrogen for 15min, and sealing the reaction system. And carrying out oscillation reaction in a shaking table for 4-12 h at the temperature of 30-90 ℃ to obtain the silica gel microspheres coated with the template molecule L-glutamic acid.
S5 elution of template molecules
And washing and filtering the silica gel microspheres coated with the template molecules by using deionized water and ethanol respectively, and then placing the silica gel microspheres in a vacuum drying oven to dry for 24 hours at the temperature of 80 ℃. Placing the gel in an eluent for elution, and centrifuging after continuous multiple times to obtain silica gel microspheres with template molecules removed; and drying the silica gel microspheres with the template molecules removed to prepare the molecularly imprinted polymer silica gel microspheres with the specific recognition holes. Then washing the mixture to be neutral by using deionized water, and placing the mixture in a vacuum drying oven to dry the mixture for 10 hours at the temperature of 60 ℃ to prepare the L-glutamic acid surface molecularly imprinted polymer silica gel microspheres.
Following the above preparation procedure, the following specific examples were carried out.
Figure BDA0001781496640000061
Figure BDA0001781496640000071
The analysis of the fresh polypeptide surface molecularly imprinted polymer silica gel microspheres with the terminal group of glutamic acid prepared by the method is as follows:
for the convenience of comparison, the non-imprinted polymer silica gel microspheres (NIP/SiO) are prepared by the same method except that the substitute template molecule L-glutamic acid is not added in the preparation process2) The fresh polypeptide surface molecularly imprinted polymer silica gel microspheres (MIP/SiO) with glutamic acid as the terminal group prepared in the embodiment2) And (5) carrying out comparative analysis.
Please refer to fig. 2, which is a scanning electron microscope photograph of the blank silica gel microspheres (a) and the fresh polypeptide surface molecularly imprinted polymer silica gel microspheres (b) with glutamic acid as the terminal group. As can be seen from the figure, before the polymerization, the silica gel microspheres are regular spherical and have a large surface roughness (a); MIP/SiO2Is in a regular spherical shape, the surface of the microsphere is provided with a layer of obvious molecularly imprinted polymer, and the surface roughness of the microsphere is smaller than that of a blank silica gel microsphere.
Please refer to fig. 3, which shows the blank silica gel microspheres (a), RAFT functionalized silica gel microspheres (b), NIP/SiO of the present invention2(c) With MIP/SiO2(d) Thermogravimetric plots and first derivative plots. As can be seen from the figure, the mass loss of the blank silica gel was about 4.2%, and mainly the weight loss of the water and part of the impurities adsorbed on the surface of the silica gel microspheres. The weight loss of the RAFT functional silica gel microspheres is about 8.3 percent, the weight loss rate is maximum at about 450 ℃ and 650 ℃,the reason is that the silanization substance grafted on the surface of the functionalized silica gel microspheres and the phenylmagnesium bromide are degraded along with the increase of the temperature, which indicates that the silica gel microspheres are successfully modified. Compared with RAFT functional silica gel microspheres, NIP/SiO2And MIP/SiO2The weight loss is greater, namely 9.6% and 11.2%, respectively, and the weight loss rate is maximum at about 350 ℃, which indicates that a print thin layer is formed on the surface of the silica gel microsphere and is decomposed at the high temperature of 350 ℃. Further, MIP/SiO2Loss-of-weight ratio NIP/SiO2More, is described in MIP/SiO2Surface comparison of (2) NIP/SiO2The percentage of imprinted polymer is higher.
Please refer to fig. 4, which shows the MIP/SiO of the present invention2A schematic diagram of fresh polypeptide with glutamic acid as the end group in the adsorption extraction sample solution. The surface of the molecular imprinting polymer silica gel microsphere with the fresh polypeptide surface having the terminal group of glutamic acid is provided with a specific binding vacancy of L-glutamic acid, and the fresh polypeptide with the terminal group of glutamic acid in the sample solution can be adsorbed, and the glutamic acid on the fresh polypeptide is inserted into the binding vacancy on the surface of the microsphere, so that the adsorption and extraction of the fresh polypeptide with the terminal group of glutamic acid in the sample solution are completed.
Please refer to fig. 5, which shows MIP/SiO2With NIP/SiO2The selective extraction capacity maps 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^ s-2mg/mL of standard solution. As can be seen from the figure, MIP/SiO2The extraction amount of Glu-Gly was 0.0082mmol, and NIP/SiO2The extraction amount of Glu-Gly is 0.0026 mmol; and MIP/SiO2Compared with the extraction amount of Ser-Gly-Ser, the extraction amount of Glu-Gly, Glu-Glu, Glu-Gly-Ser and Glu-Gly-Glu is larger. Description of MIP/SiO2The fresh polypeptide with the terminal group containing glutamic acid has certain selectivity, and the polypeptide without the terminal group containing glutamic acid has poor selectivity, which shows that compared with other polypeptides, the polypeptide prepared by the method of the inventionMIP/SiO of2Has better selectivity to the fresh polypeptide with the terminal group containing glutamic acid.
Please refer to fig. 6, which is a graph of the capacity of the functional monomer of the present invention to extract L-glutamic acid with metal ions and the functional monomer of the prior art; wherein, the functional monomer in the invention is 1- (N, N-dicarboxymethyl) amino-3-allyl glycerol (AGE/IDA), and the metal ion is Cu2+The functional monomer in the prior art is 2-acrylamide-2-methacrylic Acid (AMPS). As can be seen from the figure, AGE/IDA of the present invention has a higher extraction amount of L-glutamic acid than AMPS. This is because the connection between AMPS and L-glutamic acid by van der waals' force between H and O is very weak; AGE/IDA and Cu2+The structure of (2) provides electron orbitals for two carboxyl groups H of the L-glutamic acid, and the bonding force is stronger, so that more L-glutamic acid can be extracted.
Please refer to fig. 7, which is a liquid chromatogram of (a) pre-treating soy sauce with the fresh polypeptide surface molecularly imprinted polymer silica gel microspheres with glutamic acid as the terminal group prepared in the present invention, (b) pre-treating soy sauce without the above microspheres and (c) pre-treating reference solution (non-soy sauce) without the above microspheres. Through comparison, the product is found to be MIP/SiO2After separation, two present polypeptides Glu-Gly and Glu-Gly-Ser with terminal group of glutamic acid can be detected in soy sauce without MIP/SiO2The two fresh polypeptides were isolated and not detected in soy sauce. Therefore, the prepared fresh polypeptide surface molecularly imprinted polymer silica gel microspheres with the terminal group of glutamic acid can effectively adsorb and extract the fresh polypeptide with the terminal group of glutamic acid in the soy sauce, so that the accuracy of detecting the fresh polypeptide in the soy sauce is improved.
Compared with the prior art, the surface molecularly imprinted polymer silica gel microspheres prepared by the method have the advantages of uniform coating, narrow molecular weight distribution of imprinted substances and stable performance. In addition, the terminal group of the plurality of fresh polypeptides is L-glutamic acid, so that the selective adsorption of the plurality of 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, the functional monomer and the substitute template molecule are interacted by moleculesForce bonding, in the preparation method of the invention, the metal ion Cu2+And the functional monomer 1- (N, N-dicarboxymethyl) amino-3-allyl glycerol is connected with the template molecule L-glutamic acid through metal chelation acting force to replace the template molecule L-glutamic acid, and the L-glutamic acid and Cu are opposite to the acting force between molecules2+The binding force is stronger, the surface of the polymer can be stabilized more, and more alternative template molecules can be bound, so that more L-glutamic acid binding vacancies are obtained, the fresh polypeptide with the terminal group of glutamic acid in the brewed soy sauce is bound as much as possible or even completely, and the detection accuracy is improved.
The present invention is not limited to the above-described embodiments, and various modifications and variations of the present invention are intended to be included within the scope of the claims and the equivalent technology of the present invention if they do not depart from the spirit and scope of the present invention.

Claims (10)

1. A preparation method of surface molecularly imprinted polymer silica gel microspheres with glutamic acid as an end group and fresh polypeptide is characterized by comprising the following steps: the method comprises the following steps:
s1: removing impurities from the surface of the silica gel microspheres by using a dilute hydrochloric acid soaking method to obtain activated silica gel microspheres;
s2: carrying out surface silanization modification on the activated silica gel microspheres by using a silanization reagent to obtain silanized silica gel microspheres;
s3: RAFT functional treatment is carried out on the silanized silica gel microspheres by using RAFT reagent to obtain RAFT functional silica gel microspheres with disulfide bonds grafted on the surfaces;
s4: fully and uniformly mixing the substituted template molecules, the metal ions and the functional monomers in a mixed solvent to enable the substituted template molecules, the metal ions and the functional monomers to be self-assembled, then adding a cross-linking agent, an initiator and RAFT functional silica gel microspheres, and obtaining the molecularly imprinted coating microspheres with the surface coated with the substituted template molecules through RAFT polymerization reaction;
s5: eluting the silica gel microspheres obtained in the step S4, removing the substituted template molecules coated on the surfaces of the silica gel microspheres, and drying to obtain surface molecularly imprinted polymer silica gel microspheres;
wherein the substitutional template molecule is L-glutamic acid, and the metal ion is Cu2+The functional monomer is 1- (N, N-dicarboxymethyl) amino-3-allyl glycerol.
2. The method for preparing the surface molecularly imprinted polymer silica gel microspheres of fresh polypeptide with glutamic acid as the terminal group according to claim 1, wherein the method comprises the following steps: 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 ions is 9: 1-1: 12.
3. The method for preparing the surface molecularly imprinted polymer silica gel microspheres of fresh polypeptide with glutamic acid as the terminal group according to claim 2, wherein the method comprises the following steps: the cross-linking agent is N, N-methylene bisacrylamide; the initiator is azobisisobutyronitrile; the dosage of the initiator is 0.1-5% of the mass of the cross-linking agent.
4. The method for preparing the surface molecularly imprinted polymer silica gel microspheres of fresh polypeptide with glutamic acid as the terminal group according to claim 3, wherein the method comprises the following steps: the silanization reagent is 4-chloromethyl phenyl trimethoxy silane; the molar ratio of the silanization reagent to the activated silica gel microspheres is 8: 1-1: 12.
5. The method for preparing the surface molecularly imprinted polymer silica gel microspheres of fresh polypeptide with glutamic acid as the terminal group according to any one of claims 1 to 4, wherein the method comprises the following steps: in the step S1, the silica gel microspheres are placed in hydrochloric acid with the mass fraction of 5% -20%, ultrasonic treatment is carried out for 3-20 min, and the mixture is heated to 50-200 ℃ and kept for 12-48 h; and then washing with deionized water, carrying out suction filtration until the mixture is neutral, drying, and activating at 50-200 ℃ for 12-48 h to obtain the activated silica gel microspheres.
6. The method for preparing the surface molecularly imprinted polymer silica gel microspheres of fresh polypeptide with glutamic acid as the terminal group according to any one of claims 1 to 4, wherein the method comprises the following steps: in step S2, ultrasonically dispersing the activated silica gel microspheres in anhydrous toluene, introducing nitrogen to remove oxygen, adding 4-chloromethyl phenyl trimethoxysilane, and reacting at 30-150 ℃ for 12-48 h; and respectively washing with toluene and ethanol, and drying to obtain the silanized silica gel microspheres.
7. The method for preparing the surface molecularly imprinted polymer silica gel microspheres of fresh polypeptide with glutamic acid as the terminal group according to any one of claims 1 to 4, wherein the method comprises the following steps: in step S3, after the tetrahydrofuran is dried and pretreated by introducing nitrogen and removing oxygen, phenylmagnesium bromide is added to the pretreated tetrahydrofuran, and then CS is added dropwise2Reacting for 0.2-3 h at 20-80 ℃; then adding silanized silica gel microspheres, ultrasonically and uniformly dispersing, introducing nitrogen to remove oxygen for 3-30 min, heating to 30-80 ℃, and reacting for 24-60 h; and after the reaction is finished, washing the microspheres with tetrahydrofuran, methanol and acetone for multiple times in sequence, and drying to obtain the RAFT functional silica gel microspheres.
8. The method for preparing the surface molecularly imprinted polymer silica gel microspheres of fresh polypeptide with glutamic acid as the terminal group according to any one of claims 1 to 4, wherein the method comprises the following steps: in step S4, L-glutamic acid is ultrasonically dissolved in a mixed solvent, and 1- (N, N-biscarboxymethyl) amino-3-allylglycerol and Cu are added2+Then oscillating for 4-14 h at the temperature of 10-80 ℃; adding the RAFT functional silica gel microspheres and a cross-linking agent into the mixture, adding an initiator after ultrasonic dispersion, introducing nitrogen to remove oxygen after oscillating ultrasonic mixing, and reacting for 2-18 h at 50-60 ℃ to obtain the silica gel microspheres coated with the substituted 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.
9. The method for preparing the surface molecularly imprinted polymer silica gel microspheres of fresh polypeptide with glutamate as terminal group according to claim 8, wherein the method comprises the following steps: in step S5, washing the silica gel microspheres coated with the substituted template molecules with deionized water and ethanol respectively, and drying; and then continuously eluting for multiple times in an ethylene diamine tetraacetic acid eluent with the concentration of 0.1-0.5 mol/L, and drying to obtain the surface molecularly imprinted polymer silica gel microspheres.
10. The use of the surface molecularly imprinted polymer silica gel microspheres with terminal group of glutamic acid prepared by the method for preparing surface molecularly imprinted polymer silica gel microspheres with terminal group of fresh polypeptide of glutamic acid according to any one of claims 1 to 9 in separating and detecting fresh polypeptide.
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