CN115028155A - Preparation method of biomass porous carbon material, carbon material prepared by method and application of carbon material - Google Patents
Preparation method of biomass porous carbon material, carbon material prepared by method and application of carbon material Download PDFInfo
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- C01B32/00—Carbon; Compounds thereof
- C01B32/05—Preparation or purification of carbon not covered by groups C01B32/15, C01B32/20, C01B32/25, C01B32/30
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- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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Abstract
The invention provides a preparation method of a biomass porous carbon material, which is characterized in that chitosan with low price and easy availability is used as a precursor for the first time, and the graphitized porous carbon material is prepared by a high-temperature carbonization and post-oxidation modification method. The porous carbon material obtained by the method has the advantages of large specific surface area, high graphitization degree, strong hydrophilicity and proper pore diameter structure. After the material is enriched, the mass spectrum signal of the sugar chain of the target analyte is obviously enhanced, and the material shows good stability, excellent volume exclusion effect and ultrahigh sensitivity in the enrichment of standard sugar chain samples, and is successfully applied to the analysis of the sugar chain in clinical serum samples.
Description
Technical Field
The invention relates to the technical field of biological materials, in particular to a preparation method of a biomass porous carbon material, the biomass porous carbon material prepared by the method and application of the porous carbon material in separation and identification of biological micromolecule sugar chains.
Background
Protein glycosylation is a common, ubiquitous post-translational modification of proteins, and refers to the process of covalently binding carbohydrate compounds (sugar chains) to proteins under the catalytic action of glycosyltransferases. The structure of sugar chains in glycosylated proteins determines the function and metabolic pathways of the proteins, and plays important roles in various life activities, such as cell recognition, signal transduction, immune regulation, and the like. Alterations in sugar chain structure due to abnormal glycosylation of proteins have been found in a variety of diseases. The research shows that abnormal glycosylation of protein, such as more branched sugar chain and increased sialic acid glycosylation, can cause the occurrence of diseases. Therefore, it is important to fully analyze sugar chains for understanding protein functions, and for diagnosing and treating diseases. However, due to low abundance of sugar chains, variable and non-uniform structure, and complicated samples, mass spectrometry technology is still not able to directly detect sugar chains in complicated samples as the most effective means for sugar chain analysis. Therefore, it is important to selectively enrich sugar chains before mass spectrometric detection.
Based on this, researchers have developed various materials, such as porous carbon materials, lectin-based materials, hydrophilic materials, and the like. Although these reported materials have some effects in sugar chain analysis, most of them have problems of low enrichment efficiency, complicated preparation steps, expensive raw materials, and the like.
In view of this, the invention is particularly proposed.
Disclosure of Invention
The first purpose of the invention is to provide a preparation method of a biomass porous carbon material, which adopts cheap and easily available raw materials and is simple in preparation method.
The second purpose of the invention is to provide the biomass porous carbon material prepared by the method, and the material has better stability and biocompatibility.
The third purpose of the invention is to provide the application of the porous carbon material prepared by the method in separation and identification of the biological small molecular sugar chain.
In order to achieve the purpose, the technical scheme of the invention is as follows:
the invention relates to a preparation method of a biomass porous carbon material, which comprises the following steps:
(1) mixing chitosan powder with potassium bicarbonate, and grinding to obtain a mixture I;
(2) calcining the mixture I obtained in the step (1) in an inert gas atmosphere, and recording the obtained material as a product II;
(3) and (3) dispersing the product II obtained in the step (2) in an aqueous solution of ammonium persulfate for oxidation modification, and sequentially cleaning, filtering and drying the reaction product to obtain the porous carbon material.
Preferably, in the step (1), the weight ratio of the chitosan powder to the potassium bicarbonate is 1 (1-5), preferably 1: 2.5.
Preferably, in step (2), the inert gas is nitrogen or argon, preferably argon.
Preferably, in the step (2), the calcination temperature is 600-900 ℃, and the calcination time is 1-5 h; more preferably, the calcination temperature is 800-900 ℃ and the calcination time is 2-4 h.
Preferably, in the step (3), the concentration of the aqueous solution of ammonium persulfate is 0.1-4.0mol/L, preferably 2.0 mol/L.
Preferably, in the step (3), the mass concentration of the product II in the ammonium persulfate aqueous solution is 5-50g/L, and preferably 20 g/L.
Preferably, in the step (3), the temperature of the oxidation modification is 25-80 ℃, and the reaction time is 2-15 h; more preferably, the reaction temperature is 50-70 ℃ and the reaction time is 6-10 h.
Preferably, in step (3), the solid obtained by filtration is dried under vacuum at 40-80 ℃ for 10-24 h.
The invention also relates to the biomass porous carbon material prepared by the method, which has the advantages of large specific surface area, good hydrophilic property and biocompatibility, high graphitization degree, proper pore diameter structure and excellent stability, shows high-efficiency enrichment separation effect on sugar chain biological micromolecules, and ultrahigh sensitivity, and can be used for selective separation and identification of sugar chain molecules in complex samples.
The invention also relates to application of the biomass porous carbon material prepared by the method in selective separation, enrichment and mass spectrum identification of biological micromolecular sugar chains.
Preferably, the application comprises the steps of: fully mixing the biomass porous carbon material with the sugar chain solution, and incubating for 10-60min in an enzymolysis instrument at 35-40 ℃; washing the separated carbon material with deionized water for 3-5 times, and eluting with 30-60% acetonitrile aqueous solution; 0.8-1.2 mu L of eluent is spotted on a target plate of matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS), after natural drying, 0.75-1.5 mu L of 2, 5-dihydroxybenzoic acid (DHB) solution with the concentration of 5-15mg/mL is dripped on the analyzed liquid drop, and mass spectrometry is carried out after drying.
Preferably, the sugar chain solution has a concentration of 1 ng/. mu.L or more.
Specifically, the biomass porous carbon material prepared by the invention can be used for separation and identification of sugar chains in clinical serum samples. The biomass porous carbon material synthesized by the method selectively enriches sugar chains in a sample and then carries out mass spectrum high-throughput detection, so that the originally unidentified sugar chains are greatly improved in signal intensity, and a sugar chain signal peak with high signal-to-noise ratio is obtained. The biomass porous carbon material is applied to multiple experiments such as standard OVA protein enzymolysis liquid, mixed solution of the OVA protein enzymolysis liquid and Bovine Serum Albumin (BSA) and OVA protein, separation and identification of sugar chains in clinical serum samples and the like.
The invention has the beneficial effects that:
the invention provides a preparation method of a biomass porous carbon material, which is characterized in that chitosan with low price and easy availability is used as a precursor for the first time, and the graphitized porous carbon material is prepared by a high-temperature carbonization and post-oxidation modification method. The potassium bicarbonate in the raw materials is used as a dispersing agent, and the ammonium persulfate is used as an oxidizing agent.
The porous carbon material obtained by the method has the advantages of large specific surface area, high graphitization degree, strong hydrophilicity and proper pore diameter structure. After the material is enriched, the mass spectrum signal of the sugar chain of the target analyte is obviously enhanced, the mass spectrum signal has good stability, excellent volume exclusion effect and ultrahigh sensitivity in the enrichment of a standard sugar chain sample, and the mass spectrum signal is successfully applied to the analysis of the sugar chain in a clinical serum sample.
Experimental results fully prove that the porous carbon material prepared by the invention has excellent stability and biocompatibility and the capability of efficiently and selectively separating and identifying sugar chains, and is suitable for separating and identifying the sugar chains in complex biological samples such as human serum, various tissue extracts, various cell proteins and the like. The porous carbon material can be applied to analysis and identification of sugar chains in serum/urine samples of patients with different types and periods of diseases in the future, so that potential biomarkers related to the diseases are found, and certain commercialization potential is realized.
Drawings
FIG. 1 is a schematic diagram of the synthesis of a biomass porous carbon material according to the present invention.
Fig. 2 is SEM and TEM photographs of the biomass porous carbon material in example 1.
FIG. 3(a) is 10 in test example 1 -6 Mass spectrogram of the OVA enzymolysis solution of M before enrichment; FIG. 3(b) is 10 -6 And (3) performing selective enrichment on the OVA enzymolysis liquid of the M through a biomass porous carbon material to obtain a mass spectrogram.
Fig. 4(a) shows the mass ratio of 1: 1000:1000 of sugar chain signal peak mass spectrogram before selective enrichment of OVA protein enzymolysis liquid, OVA protein and BSA protein mixed solution; fig. 4(b) shows the mass ratio of 1: 1000:1000 of protein signal peak mass spectrogram before selective enrichment of OVA protein enzymolysis liquid, OVA protein and BSA protein mixed solution; fig. 4(c) shows the mass ratio of 1: 1000:1000, wherein fig. 4(d) is a mass ratio of 1: 1000:1000 of protein signal peak mass spectrogram after selective enrichment of OVA protein enzymolysis liquid, OVA protein and BSA protein mixed solution.
Fig. 5 is a mass spectrum of the OVA enzymatic hydrolysate with different concentrations in test example 3 after selective enrichment of the biomass porous carbon material: (a)50 ng/. mu.L, (b)10 ng/. mu.L, (c)5 ng/. mu.L and (d)1 ng/. mu.L.
FIG. 6 is an identification map of the biomass porous carbon material in test example 4 after application to sugar chain enrichment in clinical serum samples.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be described in detail below. It should be apparent that the described embodiments are only some embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the examples given herein without any inventive step, are within the scope of the present invention.
Example 1
A biomass porous carbon material, the preparation and the flow of the separation and enrichment and MALDI-TOF-MS detection of the sugar chain are shown in figure 1, and the preparation method comprises the following steps:
(1) mixing 1.5g of chitosan powder with 4.5g of potassium bicarbonate, and fully grinding in a mortar to obtain a mixture I;
(2) calcining the mixture I for 1h at 900 ℃ in argon, and marking the obtained material as a product II;
(3) dispersing 0.2g of the product II in 10mL of aqueous solution of ammonium persulfate with the concentration of 2.0mol/L, reacting for 3h at 60 ℃, cooling to room temperature to obtain a reaction product, fully washing with deionized water, filtering, and drying a filter cake for 10h at 50 ℃ in vacuum to obtain a product III, namely the biomass porous carbon material.
Scanning electron micrographs of product III are shown in FIGS. 2(a) and 2 (b); the transmission electron micrographs of the product III are shown in FIG. 2(c) and FIG. 2 (d). The product was seen to be relatively thin walled with uniformly dense pores on the surface and inside.
Test example 1
The biomass porous carbon material prepared in the example 1 is used for separation and enrichment of sugar chains in standard OVA enzymatic hydrolysate and detection of MALDI-TOF-MS.
(1) Preparation of standard glycoprotein OVA enzymolysis liquid: OVA glycoprotein (2.0mg) was dissolved in 2mL of 50mM NH 4 HCO 3 Decocting in water at 100 deg.C for 10min, cooling to room temperature, adding PNGaseF enzyme 2 μ L, and performing enzyme digestion at 37 deg.C for 16h to obtain OVA hydrolysate, and storing in refrigerator at-20 deg.C.
(2) Enrichment of sugar chains in standard glycoprotein OVA enzymatic hydrolysis: 500 mu g of the porous carbon material prepared in the example 1 is taken and washed for three times by deionized water, then the porous carbon material is dispersed in water, and a sample to be enriched (the protein OVA enzymolysis liquid prepared in the step (1)) is added to be incubated for 30min at 37 ℃. The collected material was then separated and washed three times with deionized water, followed by the addition of 10. mu.L of 50% ACN/H 2 Shaking the O solution at 37 deg.C for 30min to elute the sugar chain enriched on the porous carbon materialAnd collecting the eluent to be detected by mass spectrometry.
(3) Target spotting: spotting 1. mu.L of the eluate collected in step (2) on a MALDI-TOF-MS target plate, naturally drying in air at room temperature, and collecting 1. mu.L of 2, 5-dihydroxybenzoic acid solution (prepared by dissolving 2, 5-dihydroxybenzoic acid in 0.1% TFA and 30% ACN/H 2 O in deionized water, with a concentration of 20mg/mL2, 5-dihydroxybenzoic acid) was dropped as a matrix onto the analyte droplets, and mass spectrometric detection was performed after drying and crystallization, the results of which are shown in fig. 3.
FIG. 3(a) is 10 -6 Mass spectrogram of the OVA enzymolysis solution of M before enrichment can be seen as 10 before enrichment -6 Only 5 sugar chain signal peaks are identified in OVA standard protein enzymolysis liquid of M, and the signal intensity is very low, and the highest signal intensity is not more than 200. FIG. 3(b) is 10 -6 The mass spectrogram of the OVA enzymatic hydrolysate of M after being selectively enriched by the biomass porous carbon material can show that sugar chain peaks of 22 OVA proteins appear in the mass spectrogram after being enriched by the biomass porous carbon material, the target peak signals are obviously improved, and the maximum signal intensity reaches 12000. The biomass porous carbon material can be used for enriching sugar chains in OVA standard protein enzymolysis liquid with high selectivity.
Test example 2
The biomass porous carbon material obtained in the example 1 is used for enriching OVA enzymolysis liquid, OVA protein and Bovine Serum Albumin (BSA) protein mixed solution and detecting MALDI-TOF-MS.
(1) According to the protein mass ratio of 1: 1000:1000, mixing OVA enzymolysis liquid, OVA and BSA protein solution, adding 10 mu L of standard mixed solution into 150 mu L of deionized water, adding 50 mu L of the dispersion (with the concentration of 10mg/mL) of the biomass porous carbon material obtained in the example 1, and incubating for 30 minutes at 37 ℃; the carbon material was separated by centrifugation, washed three times with 200. mu.L of an aqueous solution, then eluted with 10. mu.L of a 50% acetonitrile solution for 15 minutes, and centrifuged to obtain an eluate.
(2) Target spotting: mu.L of the eluate collected in step (1) was spotted onto a MALDI-TOF-MS target plate, and after leaving it to air-dry at room temperature, 1. mu.L of a 2, 5-dihydroxybenzoic acid solution (1. mu.L, 20mg/mL2,5-Dihydroxybenzoic acid, 0.1% TFA, 30% ACN/H 2 O) as a matrix was dropped on the analyte droplets, dried and subjected to mass spectrometry, and the results are shown in fig. 4.
Fig. 4(a) shows the mass ratio of 1: 1000:1000, wherein the sugar chain signal peak is not identified in the mixed solution before enrichment. FIG. 4(b) shows the mass ratio of 1: 1000:1000, wherein the mixed solution of OVA protein enzymolysis liquid, OVA protein and BSA protein can observe a large number of signal peaks of protein in the linear mode of mass spectrum before enrichment. Fig. 4(c) shows the mass ratio of 1: 1000:1000, wherein the mass spectrum of the sugar chain signal peaks after selective enrichment of the OVA protein enzymatic hydrolysate, the OVA protein and BSA protein mixed solution shows that 16 sugar chain signal peaks are clearly observed in the mass spectrum after the mixed solution is enriched by the biomass porous carbon material. FIG. 4(d) shows the mass ratio of 1: 1000:1000, and a protein signal peak mass spectrogram obtained by selectively enriching OVA protein enzymolysis liquid, OVA protein and BSA protein mixed solution shows that no signal peak of any protein is observed in the enriched eluent in a mass spectrum linear mode. The biomass porous carbon material prepared by the method has good anti-interference capability.
Test example 3
The biomass porous carbon material obtained in the example 1 is used for the enrichment and MALDI-TOF-MS detection of ultralow-concentration OVA enzymolysis liquid.
(1) Enriching sugar chains, namely adding 50 mu L of dispersion liquid (with the concentration of 10mg/mL) of the biomass porous carbon material into 50 mu L of deionized water; diluting the standard OVA protein enzymolysis solution by using 25mM ammonium bicarbonate buffer solution to ensure that the final concentration of the protein enzymolysis solution is 1 ng/mu L; incubating at 37 ℃ for 30min, centrifuging, washing with 200. mu.L deionized water for three times, eluting with 10. mu.L 50% acetonitrile buffer for 15 min, and centrifuging to obtain the eluent.
(2) Target spotting: spotting 1 μ L of the eluate obtained in step (1) on a MALDI-TOF-MS target plate, naturally drying in air at room temperature, and dryingmu.L of a 2, 5-dihydroxybenzoic acid solution (1. mu.L, 20mg/mL2, 5-dihydroxybenzoic acid, 0.1% TFA, 30% ACN/H) 2 O) as a matrix was dropped on the analyte droplets, dried and subjected to mass spectrometry, and the results are shown in fig. 5.
Fig. 5 is a mass spectrum of the OVA enzymatic hydrolysate with different concentrations in test example 3 after selective enrichment of the biomass porous carbon material: wherein FIG. 5(a) is 50 ng/. mu.L, FIG. 5(b) is 10 ng/. mu.L, FIG. 5(c) is 5 ng/. mu.L, and FIG. 5(d) is 1 ng/. mu.L. As can be seen from FIG. 5(d), even when the concentration of OVA proteolysis solution is as low as 1 ng/. mu.L, 8 sugar chain peaks derived from OVA protein can still be clearly observed in the mass spectrum after the biomass porous carbon material is enriched.
Test example 4
The biomass porous carbon material obtained in the embodiment 1 is used for enriching sugar chains in a clinical sample serum enzymolysis solution and detecting mass spectrum.
(1) Preparation of human serum sample enzymolysis liquid: human serum samples (5. mu.L) were dissolved in 25mM NH 4 HCO 3 In solution (100. mu.L). Then boiled in water at 100 ℃ for 10min and then cooled to room temperature naturally, then 1. mu.L of PNGaseF enzyme was added, and the solution sample was placed on a 37 ℃ enzymolysis instrument for enzymolysis overnight. Finally, the obtained sample is stored at-80 ℃ for later use.
(2) Enrichment of sugar chains in serum: adding 100 mu L of human serum enzymolysis solution and 50 mu L of biomass porous carbon material dispersion (with concentration of 10mg/mL) into 150 mu L of deionized water, incubating and mixing for 30min at 37 ℃; and (4) performing centrifugal separation, washing the eluent with 200 mu L of deionized water for three times, eluting the eluent with 10 mu L of 50% acetonitrile buffer solution for 30min, and performing centrifugal separation to obtain the eluent.
(3) Target spotting: spotting 1. mu.L of the eluate obtained in step (2) on a MALDI-TOF-MS target plate, allowing to air-dry at room temperature, and collecting 1. mu.L of a 2, 5-dihydroxybenzoic acid solution (1. mu.L, containing 20mg/mL2, 5-dihydroxybenzoic acid, 0.1% TFA, 30% ACN/H) 2 O) was dropped as a matrix on the analyte droplets, dried and subjected to mass spectrometry, and the results are shown in fig. 6.
FIG. 6 is an identification map of the biomass porous carbon material in test example 4 after application to sugar chain enrichment in clinical serum samples. It can be seen that after the biomass porous carbon material is used for enrichment, 49 sugar chain signal peaks can be clearly identified in normal human serum. However, before enrichment, no mass spectrum signal peak of sugar chains is observed, which indicates that the biomass porous carbon material prepared in example 1 can be well applied to separation and detection of sugar chains in clinical practical samples.
Example 2: the specific steps of the synthesis method of the biomass porous carbon material are the same as those of example 1, and the differences are as follows: example 1 the calcination temperature was changed to 800 c in step (2).
The biomass porous carbon material obtained in example 2 was used for enrichment separation and mass spectrometric detection of sugar chains in a standard OVA enzymatic hydrolysate, and the specific steps of enrichment and detection were the same as steps (1), (2) and (3) in test example 1. After the obtained biomass porous carbon material is enriched, only 18 sugar chain signal peaks of OVA protein appear in a mass spectrogram, and the maximum signal intensity is only 8000, which indicates that the low carbonization temperature has adverse effect on the enrichment effect.
Example 3: the specific steps of the synthesis method of the biomass porous carbon material are the same as those of example 1, and the differences are as follows: example 1 the ammonium persulfate concentration in step (3) was changed to 1.0 mol/L.
The biomass porous carbon material obtained in example 3 was used for enrichment separation and mass spectrometric detection of sugar chains in a standard OVA enzymatic hydrolysate, and the specific steps of enrichment and detection were the same as steps (1), (2) and (3) in test example 1. After the obtained biomass porous carbon material is enriched, only 20 sugar chain signal peaks of OVA protein appear in a mass spectrogram, and the highest signal intensity is only 9000 more, which shows that the concentration of the oxidation-modified ammonium persulfate has certain influence on the enrichment effect of the carbon material.
Test example 5: the biomass porous carbon material obtained in example 1 is used for enriching and separating sugar chains in standard OVA enzymolysis liquid and detecting the sugar chains by mass spectrometry, the specific steps of enriching and detecting are the same as the steps (1), (2) and (3) in the test example 1, and only 50% ACN/H of the step (2) in the test example 1 is used 2 The O elution solution was changed to 80% ACN/H 2 And (4) O solution.
After sugar chain enrichment and separation under the above conditions, only 16 sugar chain signal peaks of OVA protein appear in the mass spectrogram, which indicates ACN/H 2 The concentration of the O-eluting solution will also have an effect on the enrichmentAn influence is produced.
The above description is only for the specific embodiments of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art can easily conceive of the changes or substitutions within the technical scope of the present invention, and all the changes or substitutions should be covered within the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the appended claims.
Claims (10)
1. A preparation method of a biomass porous carbon material is characterized by comprising the following steps:
(1) mixing chitosan powder with potassium bicarbonate, and grinding to obtain a mixture I;
(2) calcining the mixture I obtained in the step (1) in an inert gas atmosphere, and recording the obtained material as a product II;
(3) and (3) dispersing the product II obtained in the step (2) in an aqueous solution of ammonium persulfate for oxidation modification, and sequentially cleaning, filtering and drying the reaction product to obtain the porous carbon material.
2. The method according to claim 1, wherein in step (1), the weight ratio of chitosan powder to potassium bicarbonate is 1 (1-5), preferably 1: 2.5.
3. The method as claimed in claim 1, wherein in step (2), the calcination temperature is 600-900 ℃, and the calcination time is 1-5 h; more preferably, the calcination temperature is 800-900 ℃ and the calcination time is 2-4 h.
4. The method according to claim 1, wherein in the step (3), the concentration of the aqueous solution of ammonium persulfate is 0.1-4.0mol/L, preferably 2.0 mol/L.
5. The method according to claim 1, wherein in the step (3), the mass concentration of the product II in the ammonium persulfate aqueous solution is 5-50g/L, preferably 20 g/L.
6. The method according to claim 1, wherein in the step (3), the temperature for the oxidative modification is 25-80 ℃ and the reaction time is 2-15 h; more preferably, the reaction temperature is 50-70 ℃ and the reaction time is 6-10 h.
7. A biomass porous carbon material produced by the method according to any one of claims 1 to 6.
8. The use of the biomass porous carbon material according to claim 7 in selective separation, enrichment and mass spectrometric identification of small biological molecule sugar chains.
9. The application according to claim 8, characterized in that it comprises the following steps: fully mixing the biomass porous carbon material with the sugar chain solution, and incubating for 10-60min in an enzymolysis instrument at 35-40 ℃; washing the separated carbon material with deionized water for 3-5 times, and eluting with 30-60% acetonitrile aqueous solution; spotting 0.8-1.2 μ L of eluate on MALDI-TOF-MS target plate, naturally drying, dripping 0.75-1.5 μ L of 5-15mg/mL 2, 5-dihydroxybenzoic acid solution on the analyzed liquid drop, drying, and performing mass spectrometry;
preferably, the sugar chain solution has a concentration of 1 ng/. mu.L or more.
10. The use according to claim 9, wherein the sugar chain solution is selected from any one of a standard OVA protein enzymatic hydrolysate, a mixed solution of OVA protein enzymatic hydrolysate and BSA and OVA protein, and a clinical serum sample.
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