CN113318480B - Hydrophilic nano core-shell material and preparation method and application thereof - Google Patents
Hydrophilic nano core-shell material and preparation method and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of chromatographic stationary phase materials, and particularly relates to a hydrophilic nano core-shell material as well as a preparation method and application thereof. The invention provides a preparation method of a hydrophilic nano core-shell material, which comprises the steps of firstly adopting tetrapropoxysilane, formaldehyde and resorcinol to generate solid silica gel microspheres, then calcining to obtain nano-scale core-shell silica gel microspheres, acidifying, carrying out vinyl modification to obtain a vinyl functionalized core-shell silica gel material, and finally adopting a photo-initiated mercapto-alkene click reaction to bond glutathione to the microsphere surface of the vinyl functionalized core-shell silica gel material to obtain the hydrophilic nano core-shell material. The preparation method provided by the invention is simple to operate and easy to master, and can be used for large-scale production expansion; the obtained hydrophilic nano core-shell material has excellent glycopeptide enrichment performance.
Description
Technical Field
The invention belongs to the technical field of chromatographic stationary phase materials, and particularly relates to a hydrophilic nano core-shell material as well as a preparation method and application thereof.
Background
Protein glycosylation is a very important post-translational modification of proteins, and the resulting glycoproteins are involved in many important life processes, such as immune response, information transfer, cell migration, etc. Currently, glycosylation analysis of proteins is usually performed by a high-performance liquid chromatography (HPLC) technique in combination with Mass Spectrometry (MS). However, because the glycopeptide abundance in the complex sample is very low, the non-glycopeptide signal has an obvious inhibitory effect on the glycopeptide signal in the mass spectrometry, so that the glycopeptide in the sample needs to be effectively enriched before the mass spectrometry. Currently, common glycopeptide enrichment methods mainly include hydrazine chemical reaction methods, boric acid chemical reaction methods, lectin affinity methods, hydrophilic interaction chromatography and the like, wherein the Hydrophilic interaction chromatography (HILIC) has the advantages of indiscriminate enrichment of glycopeptides, high glycosylation identification coverage rate, easiness in combination with HPLC-MS and the like, and is receiving more and more attention. However, the current hydrophilic interaction chromatography still has the defect of low selectivity on glycopeptides, and non-glycopeptides cannot be effectively removed, so that the mass spectrum response of glycopeptides is influenced. Therefore, the search and preparation of novel glycopeptide enrichment materials remains the focus of researchers (document Chen z., huang j., li l. Trends anal. Chem.2019,118, 880-892).
The core-shell silica gel microspheres, also called thin-shell silica gel microspheres, are assembled from a solid "core" and a porous "shell" as the name implies. The inner solid core can increase the mass transfer rate and the mechanical stability of the matrix to a certain extent; the external shell structure can provide certain porosity, so that the core shell has certain sample loading capacity. The structure can shorten the mass transfer path of solute molecules in the porous shell layer to accelerate the mass transfer rate between solid and liquid phases, thereby realizing fast and efficient chromatographic separation. The material as chromatographic packing has the other advantages of lower back pressure and better compatibility with a high-pressure liquid chromatograph, is an ideal substitute of the traditional full-porous silica gel microspheres, and is favored by scientific researchers. Various strategies have been developed to prepare core-shell silica gel microspheres, such as multilayer self-assembly method (Dong H, brennan j.d. chem. Commun.,2011,47, 1207-1209), template method (Kang Y, shann W, wu J, etc. chem. Mat., 2006,18, 1861-1866), etc., but these preparation methods are cumbersome, require multiple preparation steps, and have long preparation period.
Therefore, a core-shell silica gel material with simple preparation process and excellent glycopeptide enrichment performance is sought to meet the requirement of glycopeptide enrichment materials, and the core-shell silica gel material has important scientific research significance and great industrial value.
Disclosure of Invention
In view of the above, the present invention aims to provide a hydrophilic nano core-shell material and a preparation method thereof. The preparation method provided by the invention has simple process and is easy to master, and the prepared hydrophilic nano core-shell material has the characteristic of excellent glycopeptide enrichment performance; the invention also provides an application of the hydrophilic nano core-shell material.
In order to achieve the purpose of the invention, the invention provides the following technical scheme:
the invention provides a preparation method of a hydrophilic nano core-shell material, which comprises the following steps:
mixing tetrapropoxysilane, ammonia water, absolute ethyl alcohol and water, and carrying out hydrolytic polycondensation reaction to obtain silicon nuclei;
mixing the silicon core, the m-diphenol and the formaldehyde, carrying out catalytic reaction, and then calcining to obtain core-shell silica gel microspheres;
acidifying the core-shell silica gel microspheres to obtain activated core-shell silica gel microspheres;
mixing the activated core-shell silica gel microspheres with toluene, dripping dimethylvinylchlorosilane and triethylamine into the obtained system, and sequentially carrying out grafting reaction and drying to obtain a vinyl functionalized core-shell silica gel material;
mixing a modifying solution with the vinyl functionalized core-shell silica gel material, and carrying out click reaction to obtain a hydrophilic nano core-shell material; the modification liquid contains glutathione, a photoinitiator and a solvent.
Preferably, the volume ratio of the tetrapropoxysilane to the ammonia water to the absolute ethyl alcohol to the water is (2-5): (2-5): (40 to 80): (20 to 50); the mass percentage concentration of the ammonia water is 20-50%;
the temperature of the hydrolytic polycondensation reaction is 18-25 ℃, and the time is 10-30 min.
Preferably, the volume ratio of the tetrapropoxysilane to the mass of the resorcinol to the volume of the formaldehyde is (2-5) mL: (300-500) mg: (0.3-0.8) mL;
the temperature of the catalytic reaction is 18-25 ℃, and the time is 20-24 h;
the calcining temperature is 500-900 ℃ and the time is 2-6 h.
Preferably, the acidifying agent is hydrochloric acid; the ratio of the mass of the core-shell silica gel microspheres to the volume of the hydrochloric acid is (100-300) mg: (20-30) mL; the volume percentage concentration of the hydrochloric acid is 10-20%;
the acidification temperature is 60-120 ℃, and the time is 5-8 h.
Preferably, the ratio of the mass of the activated core-shell silica gel microspheres to the volume of toluene to the volume of dimethylvinylchlorosilane to the volume of triethylamine is (100-300) mg: (30-50) mL: (0.2-0.5) mL: (0.2-0.5) mL;
the temperature of the grafting reaction is 60-120 ℃, and the time is 20-24 h;
the drying temperature is 50-80 ℃ and the drying time is 6-12 h.
Preferably, the ratio of the mass of glutathione to the mass of photoinitiator to the volume of solvent is (100 to 300) mg: (20-60) mg:30mL; the solvent is ethanol water solution, and the volume percentage concentration of ethanol in the ethanol water solution is 30-80%.
Preferably, the click reaction comprises a first click reaction and a second click reaction which are sequentially performed;
the ratio of the mass of the vinyl functionalized core-shell silica gel material to the volume of the modification liquid in the first click reaction is (100-300) mg: (10-20) mL; the wavelength of the ultraviolet light in the first click reaction is 365nm, the intensity is 70-100 lux, and the exposure time is 10-30 min;
the ratio of the mass of the vinyl functionalized core-shell silica gel material to the volume of the modification liquid in the second click reaction is (100-300) mg: (10-20) mL; the wavelength of the ultraviolet light in the second click reaction is 365nm, the intensity is 70-100 lux, and the exposure time is 10-30 min.
The invention also provides the hydrophilic nano core-shell material prepared by the preparation method of the technical scheme, which has a silicon core and a shell formed by bonding glutathione.
The invention also provides application of the hydrophilic nano core-shell material in the technical scheme in the field of protein glycosylation analysis.
Preferably, the application is that the hydrophilic nano core-shell material is used as a glycopeptide enrichment material for separation and enrichment of glycopeptide in protein glycosylation analysis.
The invention provides a preparation method of a hydrophilic nano core-shell material, which comprises the following steps: mixing tetrapropoxysilane, ammonia water, absolute ethyl alcohol and water, and carrying out hydrolytic polycondensation reaction to obtain silicon nuclei; mixing the silicon core, the m-diphenol and the formaldehyde, carrying out catalytic reaction, and calcining to obtain a core-shell silica gel microsphere; acidifying the core-shell silica gel microspheres to obtain activated core-shell silica gel microspheres; mixing the activated core-shell silica gel microspheres with toluene, then dripping dimethylvinylchlorosilane and triethylamine into the obtained reaction system, and sequentially carrying out grafting reaction and drying to obtain a vinyl functionalized core-shell silica gel material; and (2) mixing a modification solution containing glutathione, a photoinitiator and a solvent with the vinyl functionalized core-shell silica gel material, and carrying out click reaction to obtain the hydrophilic nano core-shell material. The method comprises the steps of firstly, generating solid silica gel microspheres by tetrapropoxysilane, formaldehyde and resorcinol, calcining to obtain nano-scale core-shell silica gel microspheres, acidifying, carrying out vinyl modification to obtain a vinyl functionalized core-shell silica gel material, and finally, bonding glutathione to the microsphere surface of the vinyl functionalized core-shell silica gel material by adopting a light-initiated mercapto-alkene click reaction to obtain the hydrophilic nano core-shell material. The preparation method provided by the invention is simple to operate and easy to master, and can be used for large-scale production expansion; the obtained hydrophilic nano core-shell material has excellent glycopeptide enrichment performance.
The test results of the embodiment show that matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS) and standard protein IgG are adopted to detect the glycopeptide enrichment effect of the hydrophilic nano core-shell material obtained by the preparation method, most of signals with higher peak intensity in a spectrogram are non-glycopeptides before enrichment, the glycopeptide signals are almost inhibited, only one glycopeptide is detected, after enrichment, the non-glycopeptides are obviously reduced, 25 typical N-connected glycopeptides can be detected, and the glycopeptide enrichment effect is excellent.
Drawings
FIG. 1 is a schematic diagram of preparation of a core-shell silica gel material and a route diagram of glutathione-modified core-shell silica gel material in the preparation method of the present invention;
FIG. 2 is a schematic structural diagram of a hydrophilic nano core-shell material provided by the present invention;
FIG. 3 is a transmission electron microscope image of the hydrophilic nano core-shell material obtained in example 1;
FIG. 4 is a scanning electron microscope image of the hydrophilic nano core-shell material obtained in example 1;
FIG. 5 is a nitrogen adsorption/desorption curve of the hydrophilic nano core-shell material obtained in example 1;
FIG. 6 is a pore size distribution diagram of the hydrophilic nano core-shell material obtained in example 1;
FIG. 7 is the hydrophilic contact angles before and after bonding of glutathione to the hydrophilic nano core-shell material obtained in example 1 and the commercial all-porous silica gel of comparative example 1, wherein A is the water contact angle of the hydrophilic nano core-shell material, B is the water contact angle of the nano core-shell material to the glutathione, C is the water contact angle of the commercial all-porous silica gel, and D is the water contact angle of the commercial all-porous silica gel to the glutathione;
fig. 8 is a comparison graph of mass spectra of the hydrophilic nano core-shell material obtained in example 1 and the commercial total porous silica gel bonded glutathione in comparative example 1 before and after the immunoglobulin (IgG) enzymatic hydrolysate is enriched, wherein a is a mass spectrum of the immunoglobulin (IgG) enzymatic hydrolysate, B is a mass spectrum of the hydrophilic nano core-shell material silica gel bonded glutathione after the immunoglobulin (IgG) enzymatic hydrolysate is enriched, C is a mass spectrum of the peptide segment of the hydrophilic nano core-shell material silica gel bonded glutathione after the immunoglobulin (IgG) enzymatic hydrolysate is enriched by using PNGase F enzyme for deglycosylation, and D is a mass spectrum of the commercial total porous silica gel bonded glutathione after the immunoglobulin (IgG) enzymatic hydrolysate is enriched;
FIG. 9 is a chromatogram separation chart obtained by analyzing 2 μ L human serum enzymolysis solution on a C18 silica gel analytical column with the hydrophilic nano core-shell material obtained in example 1.
Detailed Description
The invention provides a preparation method of a hydrophilic nano core-shell material, which comprises the following steps:
mixing tetrapropoxysilane, ammonia water, absolute ethyl alcohol and water, and carrying out hydrolytic polycondensation reaction to obtain silicon nuclei;
mixing the silicon core, the m-diphenol and the formaldehyde, carrying out catalytic reaction, and then calcining to obtain core-shell silica gel microspheres;
acidifying the core-shell silica gel microspheres to obtain activated core-shell silica gel microspheres;
mixing the activated core-shell silica gel microspheres with toluene, dripping dimethylvinylchlorosilane and triethylamine into the obtained reaction system, and sequentially carrying out grafting reaction and drying to obtain a vinyl functionalized core-shell silica gel material;
mixing a modifying solution with the vinyl functionalized core-shell silica gel material, and carrying out click reaction to obtain a hydrophilic nano core-shell material; the modification liquid contains glutathione, a photoinitiator and a solvent.
In the present invention, unless otherwise specified, each component in the preparation method is a commercially available product well known to those skilled in the art.
The method mixes tetrapropoxysilane, ammonia water, absolute ethyl alcohol and water, and carries out hydrolytic polycondensation reaction to obtain the silicon nucleus.
In the present invention, the volume ratio of the tetrapropoxysilane, the ammonia water, the anhydrous ethanol, and the water is preferably (2 to 5): (2-5): (40 to 80): (20 to 50), more preferably (2.5 to 4.5): (2.5-4.5): (45-75): (20 to 40). In the present invention, the mass percentage concentration of the ammonia water is preferably 20 to 50%, and more preferably 25 to 45%.
In the present invention, the temperature of the hydrolytic polycondensation reaction is 18 to 25 ℃, more preferably 19 to 24 ℃; the time is preferably 10 to 30min, more preferably 12 to 25min.
After the silicon core is obtained, the silicon core, the m-diphenol and the formaldehyde are mixed, and are calcined after catalytic reaction, so that the core-shell silica gel microsphere is obtained.
In the present invention, the ratio of the volume of the tetrapropoxysilane, the mass of the resorcinol, and the volume of the formaldehyde is preferably (2 to 5) mL: (300-500) mg: (0.3 to 0.8) mL, more preferably (2.5 to 4.5) mL: (350-450) mg: (0.4-0.7) mL.
In the present invention, the temperature of the catalytic reaction is preferably 18 to 25 ℃, more preferably 19 to 24 ℃; the time is preferably 20 to 24 hours, more preferably 21 to 24 hours.
In the present invention, the temperature of the calcination is preferably 500 to 900 ℃, more preferably 550 to 850 ℃; the time is preferably 2 to 6 hours, more preferably 3 to 5.5 hours. In the present invention, the calcination apparatus is preferably a muffle furnace.
The present invention preferably further comprises washing and drying the product of the catalytic reaction in sequence prior to said calcining. In the present invention, the washing liquid is preferably an aqueous solution of ethanol; the volume ratio of ethanol to water in the ethanol aqueous solution is preferably 2: (1 to 4), more preferably 2: (1.5-3.5). In the present invention, the number of washing is preferably 2 to 4. In the present invention, the drying is preferably vacuum drying; the drying temperature is preferably 60-100 ℃, and more preferably 60-90 ℃; the time is preferably 6 to 12 hours, more preferably 7 to 12 hours; the degree of vacuum for drying is not particularly limited in the present invention, and may be any degree of vacuum known to those skilled in the art.
After obtaining the core-shell silica gel microspheres, the invention acidifies the core-shell silica gel microspheres to obtain the activated core-shell silica gel microspheres.
In the present invention, the acidifying agent is preferably hydrochloric acid; the volume percentage content of the hydrochloric acid is preferably 10-20%, and more preferably 12-18%. In the present invention, the ratio of the mass of the core-shell silica gel microspheres to the volume of hydrochloric acid is preferably (100 to 300) mg: (20 to 30) mL, more preferably (150 to 250) mg: (22-28) mL.
In the invention, the acidification temperature is preferably 60-120 ℃, and more preferably 70-110 ℃; the time is preferably 5 to 8 hours, more preferably 5.5 to 7.5 hours.
After the acidification, the invention preferably further comprises washing and drying the acidified product in sequence. The water washing is not particularly limited, and the pH value of the acidified product is washed by water to be 5-9. In the invention, the drying temperature is preferably 60-100 ℃, and more preferably 70-90 ℃; the time is preferably 6 to 12 hours, more preferably 7 to 12 hours. In the present invention, the drying device is preferably an oven.
After the activated core-shell silica gel microspheres are obtained, the activated core-shell silica gel microspheres are mixed with toluene, dimethylvinylchlorosilane and triethylamine are dropped into the obtained reaction system, and grafting reaction and drying are sequentially carried out to obtain the vinyl functionalized core-shell silica gel material. In the present invention, the toluene is preferably used after drying.
In the present invention, the ratio of the mass of the activated core-shell silica gel microspheres to the volume of toluene is preferably (100 to 300) mg: (30 to 50) mL, more preferably (100 to 250) mg: (35-45) mL. In the invention, the mixing mode of the activated core-shell silica gel microspheres and toluene is preferably ultrasonic; the present invention does not specifically limit the ultrasound, and ultrasound known to those skilled in the art may be used; the invention forms a uniform solution system by the activated core-shell silica gel microspheres and toluene through ultrasound.
In the present invention, the volume ratio of the toluene, dimethylvinylchlorosilane, and triethylamine is preferably (30 to 50): (0.2-0.5): (0.2 to 0.5), more preferably (35 to 45): (0.25-0.45): (0.25-0.45). In the present invention, the dimethylvinylchlorosilane and triethylamine are preferably dropped simultaneously. In the present invention, the rate of the dropping is independently preferably (1 to 3) mL/min, more preferably (1.5 to 2.5) mL/min.
In the present invention, the grafting reaction is preferably carried out under the condition of an oil bath. In the present invention, the temperature of the grafting reaction is preferably 60 to 120 ℃, more preferably 70 to 120 ℃; the time is preferably 20 to 24 hours, more preferably 20.5 to 23.5 hours.
In the present invention, the drying temperature is preferably 50 to 80 ℃, more preferably 55 to 75 ℃; the time is preferably 6 to 12 hours, more preferably 7 to 12 hours.
Before the drying, the invention preferably further comprises alcohol washing of the product obtained by the grafting reaction; the alcohol washing reagent is preferably methanol; the alcohol washing method is not particularly limited in the present invention, and a washing method known to those skilled in the art may be used. The invention removes unreacted dimethylvinylchlorosilane and triethylamine attached to the product obtained by the grafting reaction through alcohol washing.
After the vinyl functionalized core-shell silica gel material is obtained, the modification liquid is mixed with the vinyl functionalized core-shell silica gel material to carry out click reaction, so that the hydrophilic nano core-shell material is obtained; the modification liquid contains glutathione, a photoinitiator and a solvent.
In the present invention, the ratio of the mass of glutathione, the mass of photoinitiator, and the volume of solvent in the modification solution is preferably (100 to 300) mg: (20-60) mg:30mL, more preferably (120 to 250) mg: (25-55) mg:30mL. In the present invention, the solvent is preferably an aqueous ethanol solution; the volume percentage content of the ethanol in the ethanol water solution is preferably 30-80%, and more preferably 40-70%. In the present invention, the photoinitiator is preferably benzoin bis methyl ether (DMPA).
In the present invention, the click reaction preferably includes a first click reaction and a second click reaction which are sequentially performed.
In the present invention, the ratio of the mass of the vinyl-functionalized core-shell silica gel material to the volume of the modification liquid in the first click reaction is preferably (100 to 300) mg: (10-20) mL. In the present invention, the wavelength of the ultraviolet light in the first click reaction is preferably 365nm, the intensity is preferably 70 to 100lux, and the exposure time is preferably 10 to 30min.
In the present invention, the ratio of the mass of the vinyl-functionalized core-shell silica gel material to the volume of the modification liquid in the second click reaction is preferably (100 to 300) mg: (10-20) mL. In the present invention, the wavelength of the ultraviolet light in the second click reaction is preferably 365nm, the intensity is preferably 70 to 100lux, and the exposure time is preferably 10 to 30min.
Before the second click reaction, the present invention preferably further comprises removing the modification solution from the first click reaction system and stirring the product obtained after the first click reaction is completed. In the present invention, the device for removing the finishing liquid is preferably a pipette.
After the second click reaction, the present invention preferably further comprises washing and vacuum drying, which are performed sequentially. In the present invention, the cleaning solution is preferably an aqueous solution of ethanol; the volume percentage content of the ethanol in the ethanol water solution is preferably 30-80%, and more preferably 40-70%. In the present invention, the number of washing is preferably 2 to 4. The cleaning method of the present invention is not particularly limited, and a cleaning method known to those skilled in the art may be used. In the present invention, the temperature of the vacuum drying is preferably 60 to 100 ℃, more preferably 60 to 90 ℃; the time is preferably 6 to 12 hours, more preferably 7 to 12 hours.
Fig. 1 is a schematic diagram of preparation of a core-shell silica gel material and a route diagram of a glutathione-modified core-shell silica gel material in the preparation method of the present invention, and as can be seen from the diagram, the preparation method of the present invention comprises the steps of firstly generating solid silica gel microspheres by tetrapropoxysilane, formaldehyde and resorcinol (i.e., "one-pot method"), calcining to obtain nano-scale core-shell silica gel microspheres, acidifying, performing functional modification by dimethylvinylchlorosilane to obtain a vinyl-functionalized core-shell silica gel material, and finally bonding glutathione to the microsphere surface of the vinyl-functionalized core-shell silica gel material by using a light-initiated mercapto-ene click reaction to obtain a hydrophilic nano core-shell material.
The invention also provides a hydrophilic nano core-shell material prepared by the preparation method in the technical scheme, which has a silicon core and a shell formed by bonding glutathione, and is shown in figure 2.
The invention also provides application of the hydrophilic nano core-shell material in the technical scheme in the field of protein glycosylation analysis.
In the invention, the application is preferably that the hydrophilic nano core-shell material is used as a glycopeptide enrichment material for separating and enriching glycopeptides in protein glycosylation analysis.
For further illustration of the present invention, the following will describe in detail a hydrophilic nano core-shell material provided by the present invention, and its preparation method and application in conjunction with the following examples, but they should not be construed as limiting the scope of the present invention. It is to be understood that the disclosed embodiments are merely exemplary of the invention, and are not intended to be exhaustive or exhaustive. All other embodiments, which can be obtained by a person skilled in the art without making any creative effort based on the embodiments in the present invention, belong to the protection scope of the present invention.
The reagents used in the examples are all commercially available.
Example 1
Mixing 3.46mL of tetrapropoxysilane, 3mL of ammonia water with the mass percentage of 40%, 60mL of absolute ethyl alcohol and 20mL of water, and stirring at room temperature for reaction for 15min to obtain silicon nuclei;
mixing the obtained silicon core, 400mg of resorcinol and 0.56mL of formaldehyde, reacting for 24 hours at room temperature, washing with an ethanol aqueous solution for three times, drying in vacuum at 60 ℃ for 12 hours, and calcining the product at 600 ℃ for 5 hours to obtain core-shell silica gel microspheres;
reacting 300mg of core-shell silica gel microspheres with 30mL of hydrochloric acid solution with the volume percentage of 10% at 90 ℃ for 6 hours, washing with water until the pH value is 7 after the reaction is finished, and drying in an oven at 80 ℃ for 12 hours to obtain activated core-shell silica gel microspheres;
ultrasonically dissolving 300mg of activated core-shell silica gel microspheres and 20mL of dry toluene to form a uniform solution, dripping 0.3mL of dimethylvinylchlorosilane and 0.3mL of triethylamine at the dripping rate of 1mL/min, putting the solution into an oil bath at 120 ℃ for reaction for 24 hours, washing the product with a methanol solution for three times, and then drying the product in vacuum at 60 ℃ for 12 hours to obtain a vinyl functionalized core-shell silica gel material;
dissolving 50mg of glutathione and 30mg of photoinitiator DMPA in 10mL of aqueous solution with the ethanol volume percentage content of 50% to obtain modification solution; slowly transferring 100mg of vinyl functionalized core-shell silica gel material into 5mL of modification liquid, completely immersing the vinyl functionalized core-shell silica gel material in the modification liquid, exposing for 20min under the ultraviolet illumination with the wavelength of 365nm, removing the modification liquid by using a liquid transfer gun, slightly stirring the material, adding 5mL of the modification liquid, exposing for 20min under the ultraviolet illumination with the wavelength of 365nm, finally washing the obtained material by using an ethanol aqueous solution with the volume percentage content of 50% for 3 times, and drying for 12h under the condition of 60 ℃ in vacuum to obtain the hydrophilic nano core-shell silica gel material.
The obtained hydrophilic nano core-shell material is subjected to transmission electron microscope test, and the test chart is shown in figure 3; scanning electron microscope test is carried out on the obtained hydrophilic nano core-shell material, and a test chart is shown in figure 4. As can be seen from FIGS. 3 and 4, the hydrophilic nano core-shell material provided by the invention is spherical, the particles are uniformly dispersed, the core and shell structures are clear, the average particle size of the particles is 300nm, and the shell thickness is 50nm.
Performing nitrogen adsorption/desorption test on the obtained hydrophilic nano core-shell material, wherein the obtained nitrogen adsorption/desorption is shown in a figure 5; for the obtained hydrophilic nano core-shell materialThe pore size distribution was measured and the resulting pore size distribution curve is shown in fig. 6. As can be seen from FIGS. 5 and 6, the BET specific surface area of the hydrophilic nano core-shell material provided by the invention is 154cm 2 The catalyst has obvious detention ring and the average pore diameter of mesopores is 7.3nm.
And (3) carrying out water contact angle test on the obtained core-shell silica gel microspheres and the hydrophilic nano core-shell material, wherein the obtained test result is shown in figure 7, A in figure 7 is the water contact angle of the hydrophilic nano core-shell material, and B in figure 7 is the water contact angle of the nano core-shell material bonded with glutathione. As can be seen from A and B in fig. 7, the water contact angle of the core-shell silica gel microsphere provided by the invention is 16.2 degrees, the water contact angle of the hydrophilic nano core-shell material modified by glutathione is 11.1 degrees, which indicates that the hydrophilic nano core-shell material obtained after glutathione is grafted on the surface of the core-shell silica gel microsphere has good hydrophilicity.
Comparative example 1
Ultrasonically dissolving 300mg of commercial full-porous silica gel with the particle size of 2 mu m and 20mL of dry toluene to form a uniform solution, dripping 0.3mL of dimethylvinylchlorosilane and 0.3mL of triethylamine at the dripping rate of 1mL/min, placing the solution in an oil bath at 120 ℃ for reaction for 24 hours, washing the product with a methanol solution for three times, and then drying the product in vacuum at 60 ℃ for 12 hours to obtain a vinyl functionalized porous silica gel material;
dissolving 50mg of glutathione and 30mg of photoinitiator DMPA in 10mL of aqueous solution with the ethanol volume percentage content of 50% to obtain modification solution; slowly transferring 100mg of vinyl functionalized porous silica gel material into 5mL of modification liquid to completely immerse the vinyl functionalized porous silica gel material in the modification liquid, exposing for 20min under the ultraviolet illumination with the wavelength of 365nm, removing the modification liquid by using a liquid transfer gun, slightly stirring the material, adding 5mL of modification liquid, exposing for 20min under the ultraviolet illumination with the wavelength of 365nm, finally washing the obtained material by using an ethanol aqueous solution with the volume percentage content of 50% for 3 times, and drying in vacuum for 12h at the temperature of 60 ℃ to obtain the hydrophilic full-porous silica gel material.
Performing nitrogen adsorption/desorption test on the obtained hydrophilic full-porous silica gel material, wherein the obtained nitrogen adsorption/desorption is shown in a figure 5; for the obtained hydrophilic full porous silica gelThe pore size distribution of the material was measured and the resulting pore size distribution curve is shown in figure 6. As can be seen from FIG. 5, the BET specific surface area of the hydrophilic fully porous silica gel material provided in the comparative example was 185.6cm 2 G, and the average pore diameter of the mesopores is 16.6nm.
And (3) carrying out a water contact angle test on the commercialized porous silica gel and the obtained hydrophilic nano core-shell material, wherein the obtained test result is shown in figure 7, wherein C in figure 7 is the water contact angle of the commercialized porous silica gel, and D in figure 7 is the water contact angle of the commercialized porous silica gel bonded with glutathione. As can be seen from C and D in fig. 7, the contact angle of the commercial fully porous silica gel was 28.3 °, and the water contact angle of the hydrophilic fully porous silica gel material modified with glutathione was 16.8 °, indicating that glutathione was successfully grafted on the surface of the fully porous silica gel. Comparing a and B with C and D in fig. 7, it can be seen that the hydrophilicity of the hydrophilic nano core-shell material provided by the present invention is better than that of the hydrophilic fully porous silica gel material.
Application example 1
Preparation of an IgG enzymatic sample: human serum immunoglobulin G (IgG) 1mg was dissolved in a 100mM ammonium bicarbonate solution containing 8M urea (pH = 8.2), 80 μmol dithiothreitol was added, the temperature was maintained at 60 ℃ for 1 hour, 40 μmol iodoacetamide was added, and the solution was protected from light for 40min, and the urea concentration was diluted to 1M with a 100mM ammonium bicarbonate solution in a mass ratio to trypsin of 1:40, adding trypsin, reacting for 16 hours in a water bath at 37 ℃, desalting the obtained enzymolysis liquid, freeze-drying and storing in a refrigerator at-20 ℃ for later use.
Separation and enrichment of glycosylated peptide fragments: first, 10. Mu.g of IgG enzymolysis solution was applied to 200. Mu.L of a sample solution (ACN/H) 2 O/TFA,85:14.9:0.1, v/v/v), adding into 10mg cysteine modified core-shell silica gel material, and shaking for 15min at room temperature; centrifuging and removing supernatant; then, leaching the mixture by using a sample solution (400 mu L multiplied by 3 times) to remove non-glycopeptide and other impurities; centrifuging to remove supernatant, adding 60 μ L of eluate (ACN/H) 2 O/TFA,30:69.9:0.1, v/v/v), shaking for 10min at room temperature, centrifuging, taking supernatant, and performing MALDI-TOF/MS analysis by using Triple TOF 5600 mass spectrum; alternatively, the supernatant can be lyophilized, and then added60 μ L of 10mmol/L NH containing 1000U PNGase F enzyme 4 HCO 3 Solution (pH = 8.0), incubated at 40 ℃ for 12h to remove sugar-based fragments; and finally, analyzing the deglycosylated peptide section by using a MALDI-TOF/MS or nano-LC-MS/MS method.
Evaluating the glycopeptide enrichment effect of the material by adopting standard protein IgG; and detecting by using matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS), wherein the detection result is shown in FIG. 8, wherein A in FIG. 8 is a mass spectrogram of immunoglobulin (IgG) enzymolysis liquid, B in FIG. 8 is a mass spectrogram obtained by enriching the immunoglobulin (IgG) enzymolysis liquid by hydrophilic nano core-shell material silica gel bonded glutathione, C in FIG. 8 is a mass spectrogram obtained by deglycosylating a peptide segment obtained by enriching the immunoglobulin (IgG) enzymolysis liquid by the hydrophilic nano core-shell material silica gel bonded glutathione by PNGase F enzyme, and D in FIG. 8 is a mass spectrogram obtained by enriching the commercial full-porous silica gel bonded glutathione enzymolysis liquid. As can be seen from a in fig. 8, before enrichment, most of the signals with higher peak intensity in the spectrogram are non-glycopeptides, and the glycopeptide signals are almost all suppressed, and only one glycopeptide is detected; as can be seen from B in fig. 8, after the hydrophilic core-shell nanomaterial in example 1 is enriched, non-glycopeptides are significantly reduced, and 25 typical N-linked glycopeptides can be detected; the molecular weight and glycoform composition of glycopeptide in IgG enzymatic hydrolysate enriched with the hydrophilic nano core-shell material obtained in example 1 are shown in table 1.
As can be seen from C in fig. 8, in order to verify that the peptide fragments enriched in example 1 are all glycopeptides, only two peptide fragments with significant glycopeptide-to-nucleus ratios of 1158 and 1190 can be obtained after deglycosylation treatment is performed on the peptide fragments with PNGase F enzyme, which indicates that the peptide fragments enriched with the hydrophilic nano core-shell material provided by the present invention are all glycopeptides; as can be seen from D in fig. 8, after the hydrophilic fully porous silica gel material in comparative example 1 is used for enrichment, there are more non-glycopeptides, and only 18 typical N-linked glycopeptides are retained, which is lower than the glycopeptide enrichment effect of the hydrophilic nano core-shell material prepared in example 1 under the same conditions.
TABLE 1 molecular weight and glycoform composition of glycopeptides in IgG enzymatic hydrolysate enriched with hydrophilic nano core-shell material obtained in example 1
Description of the drawings: n # in Table 1 represents a glycosylation site; hex: mannose; hexNac: n-acetylglucosamine; fuc: fucose is used as the fucose.
Application example 2
The hydrophilic nano core-shell material obtained in the example 1 is further applied to more complex samples, and the glycopeptide in the human serum enzymolysis liquid is deeply enriched and analyzed and identified by a nano-LC-MS/MS method. The method comprises the following specific steps: 2 mu L of human serum enzymolysis solution is enriched by 20mg of the hydrophilic nano core-shell material obtained in example 1 according to the method, and the chromatographic separation diagram obtained by analyzing on a C18 silica gel analytical column is shown in FIG. 9. As can be seen in fig. 9, a total of 272 glycopeptides from 108 glycoproteins and 204 glycosylation sites were identified. Compared with the magnetic nanoparticles modified by maltose step by step (Li J, wang F, wan H, et al. Magnetic nanoparticles coated with a polysaccharide-functionalized polysaccharide for high affinity reaction of N-glycopeptides [ J ]. Journal of Chromatography A,2015,1425 213-220.) reported in the literature, 219 glycopeptides and 187 glycosylation sites of 134 glycoproteins are identified in the complex serum sample, the hydrophilic nano core-shell material provided by the invention has more excellent enrichment effect on glycopeptides. The hydrophilic nano core-shell material obtained in the embodiment 1 of the invention has excellent enrichment effect when used as HILIC adsorbent.
Example 2
Mixing 3.46mL of tetrapropoxysilane, 3mL of ammonia water with the mass percentage of 40%, 60mL of absolute ethyl alcohol and 20mL of water, and stirring at room temperature for reaction for 15min to obtain silicon nuclei;
mixing the obtained silicon core, 200mg resorcinol and 0.28mL formaldehyde, reacting for 24h at room temperature, washing with an ethanol aqueous solution for three times, vacuum drying for 12h at 60 ℃, and calcining the product for 5h at 600 ℃ to obtain core-shell silica gel microspheres; the rest of the procedure was the same as in example 1.
Scanning electron microscope test and nitrogen adsorption/desorption test are carried out on the hydrophilic nano core-shell material obtained in the example 2, and the test result shows that the average particle size of the particles of the hydrophilic nano core-shell material is 280nm, and the thickness of the shell layer is 25nm; BET specific surface area of 117cm 2 G, and the average pore diameter of the mesopores is 7nm.
The preparation method provided by the invention is simple to operate and easy to master, and can be used for large-scale production expansion; the obtained hydrophilic nano core-shell material has excellent glycopeptide enrichment performance. The test results of the embodiment show that matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS) and standard protein IgG are adopted to detect the glycopeptide enrichment effect of the hydrophilic nano core-shell material obtained by the preparation method, most of signals with higher peak intensity in a spectrogram are non-glycopeptides before enrichment, the glycopeptide signals are almost inhibited, only one glycopeptide is detected, the non-glycopeptides are obviously reduced after enrichment, 25 typical N-connected glycopeptides can be detected, the glycopeptide enrichment effect is excellent, the requirement of the glycopeptide enrichment material is met, and the method has important scientific research significance and great industrial value.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (9)
1. A preparation method of a hydrophilic nano core-shell material is characterized by comprising the following steps:
mixing tetrapropoxysilane, ammonia water, absolute ethyl alcohol and water, and carrying out hydrolytic polycondensation reaction to obtain silicon nuclei;
mixing the silicon core, the m-diphenol and the formaldehyde, carrying out catalytic reaction, and calcining to obtain a core-shell silica gel microsphere;
acidifying the core-shell silica gel microspheres to obtain activated core-shell silica gel microspheres;
mixing the activated core-shell silica gel microspheres with toluene, dripping dimethylvinylchlorosilane and triethylamine into the obtained system, and sequentially carrying out grafting reaction and drying to obtain a vinyl functionalized core-shell silica gel material;
mixing a modifying solution with the vinyl functionalized core-shell silica gel material, and carrying out click reaction to obtain a hydrophilic nano core-shell material; the modification liquid contains glutathione, a photoinitiator and a solvent;
the click reaction comprises a first click reaction and a second click reaction which are sequentially carried out;
the wavelength of ultraviolet light in the first click reaction is 365nm, the intensity is 70-100 lux, and the exposure time is 10-30 min;
the wavelength of the ultraviolet light in the second click reaction is 365nm, the intensity is 70-100 lux, and the exposure time is 10-30 min;
the photoinitiator is benzoin dimethyl ether;
the ratio of the mass of the vinyl functionalized core-shell silica gel material to the volume of the modification liquid in the first click reaction is (100-300) mg: (10-20) mL;
the ratio of the mass of the vinyl functionalized core-shell silica gel material to the volume of the modification liquid in the second click reaction is (100-300) mg: (10-20) mL.
2. The method according to claim 1, wherein the volume ratio of the tetrapropoxysilane, the ammonia water, the absolute ethanol and the water is (2-5): (2-5): (40 to 80): (20-50); the mass percentage concentration of the ammonia water is 20-50%;
the temperature of the hydrolytic polycondensation reaction is 18-25 ℃, and the time is 10-30 min.
3. The method according to claim 1, wherein the ratio of the volume of tetrapropoxysilane, the mass of resorcinol, and the volume of formaldehyde is (2-5) mL: (300-500) mg: (0.3-0.8) mL;
the temperature of the catalytic reaction is 18-25 ℃, and the time is 20-24 h;
the calcining temperature is 500-900 ℃, and the calcining time is 2-6 h.
4. The process according to claim 1, wherein the acidifying agent is hydrochloric acid; the ratio of the mass of the core-shell silica gel microspheres to the volume of the hydrochloric acid is (100-300) mg: (20-30) mL; the volume percentage concentration of the hydrochloric acid is 10-20%;
the acidification temperature is 60-120 ℃, and the time is 5-8 h.
5. The preparation method according to claim 1, wherein the volume ratio of the mass of the activated core-shell silica gel microspheres, the volume of toluene, the volume of dimethylvinylchlorosilane, and the volume of triethylamine is (100-300) mg: (30-50) mL: (0.2-0.5) mL: (0.2-0.5) mL;
the temperature of the grafting reaction is 60-120 ℃, and the time is 20-24 h;
the drying temperature is 50-80 ℃ and the drying time is 6-12 h.
6. The method according to claim 1, wherein the ratio of the mass of glutathione to the mass of the photoinitiator to the volume of the solvent is (100 to 300) mg: (20-60) mg:30mL; the solvent is ethanol water solution, and the volume percentage concentration of ethanol in the ethanol water solution is 30-80%.
7. The hydrophilic nano core-shell material prepared by the preparation method of any one of claims 1 to 6, which has a silicon core and a shell formed by bonding glutathione.
8. The use of the hydrophilic nucleocapsid material of claim 7 in the field of protein glycosylation analysis.
9. The use of claim 8, wherein the use is of the hydrophilic nucleocapsid as a glycopeptide enrichment material for separation and enrichment of glycopeptides in protein glycosylation analysis.
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