Surface-modified 2-aminopurine carbon sphere material, preparation method and application thereof

Abstract

This invention discloses a surface-modified 2-aminopurine carbon sphere material, its preparation method, and its application, belonging to the field of new materials. In this invention, the synthesized surface-modified 2-aminopurine carbon sphere material is used for the separation and enrichment of N-glycopeptides and exosomes, possessing the following advantages: 1. The preparation process is simple, with a very short synthesis cycle, saving significant time and labor; 2. The reaction raw materials are common, and no organic reagents are used as solvents during the reaction, conforming to the design principles of green chemistry to reduce or eliminate the use and generation of hazardous chemicals and processes; 3. The microstructure and size of the material can be continuously adjusted by optimizing the reaction time, temperature, and raw material concentration, thereby obtaining the material with optimal performance and greatly improving the separation and enrichment capabilities of glycosylated peptides and exosomes.

Description

Technical Field

[0001] This invention relates to the field of new materials, specifically to surface-modified 2-aminopurine carbon spheres, their preparation methods and applications, and more specifically, to a novel surface-modified 2-aminopurine carbon sphere material, its one-step synthesis method, and its application in glycosylated proteomics and the separation and enrichment of exosomes. Background Technology

[0002] Exosomes, secreted by healthy or diseased cells, can carry various biomolecules, including proteins and RNA, and are present in body fluids and tissues, such as blood. They reflect the physiology and pathology of apoptosis in diseased cells and have the potential to become disease biomarkers. However, traditional isolation methods are insufficient for effectively isolating and enriching exosomes from complex samples, necessitating the development of efficient methods for analyzing and isolating exosomes and their proteins for accurate disease diagnosis. Many exosome components, including proteins, are highly glycosylated.

[0003] Protein glycosylation accounts for more than half of all mammalian proteins and plays a crucial role in many biological events, such as cell adhesion, immune cell transport, protein stability, and intercellular recognition. Meanwhile, abnormal protein glycosylation is closely associated with various diseases, such as cancer. Therefore, it has become an indicator for disease diagnosis and treatment. However, due to the low abundance of glycosylation in highly complex clinical biological samples such as plasma, these targets remain largely unexplored. Therefore, before using techniques such as mass spectrometry (MS) analysis for detection, effectively enriching glycopeptides while simultaneously eliminating a large number of interferences is of great significance.

[0004] Among various glycopeptide enrichment strategies, hydrophilic interaction chromatography (HILIC) separates non-glycopeptides from glycopeptides based on differences in hydrophilicity, as glycopeptides with numerous hydroxyl groups are more hydrophilic than non-glycopeptides. Therefore, hydrophilic materials can selectively enrich glycopeptides with intact structural information without disrupting their structure and reducing non-specific interactions. Many novel HILIC-based hydrophilic materials have been developed for the successful enrichment of glycopeptides, including silica nanoparticles, graphene oxide, and metal-organic frameworks. However, the synthesis of these materials is cumbersome and time-consuming, typically requiring several steps to modify their surfaces. Furthermore, organic solvents are unavoidable during the synthesis process.

[0005] Hydrothermal carbonization (HTC) is a process that transforms renewable materials into functionalized carbonaceous materials with hydrophilic surfaces, resulting in carbonaceous frameworks that exhibit high thermal and chemical stability. Functional groups can be directly modified to tailor the desired carbonaceous material. The HTC process involves neither organic solvents nor complex procedures. It requires only renewable materials such as cellulose, sucrose, and glucose as carbon sources, water as the sole solvent, and a small amount of water-soluble functionalized organic monomers to provide functionality. Therefore, the HTC process offers a simple, low-cost, environmentally friendly, and versatile route for producing multifunctional carbonaceous materials.

[0006] To date, various functional carbonaceous materials have been synthesized using the HTC process. However, HTC materials for glycoproteomics require further exploration. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to provide an HTC material for glycoproteomics.

[0008] To address the above problems, this invention provides a novel method for synthesizing carbon spheres with surface modification of 2-aminopurine, specifically comprising the following steps:

[0009] S1: Dissolve glucose, 2-aminopurine, 1,2-epoxy-5-hexene, and 4,4'-azobis(4-cyanopentanoic acid) in deionized water to prepare a solution;

[0010] S2: Remove the stainless steel reactor lined with polytetrafluoroethylene and pour the solution obtained in step S1 into it;

[0011] S3: Place the reactor from step S2 into an oven for reaction;

[0012] S4: The product obtained in step S3 is thoroughly washed with deionized water and ethanol to remove impurities and raw material monomers from the surface of the product, and then dried in a vacuum drying oven to obtain carbon spheres with surface modification of 2-aminopurine.

[0013] Preferably, the concentration of glucose in step S1 is 0.1 g / mL.

[0014] Further, in step S1, the mass ratio of glucose, 2-aminopurine, 1,2-epoxy-5-hexene, and 4,4'-azobis(4-cyanopentanoic acid) is (7-12):(7-12):(0-3):(0-10).

[0015] Preferably, the reaction temperature in step S3 is 150–200°C, and the reaction time is 6–8 hours.

[0016] This invention also provides a novel carbon sphere material with a surface modified with 2-aminopurine, which is prepared using the above method and has the composition formula G. 10 2AP 10 E (0～3) A (0～10) In this formula, G is glucose, 2AP is 2-aminopurine, E is 1,2-epoxy-5-hexene, and A is 4,4'-azobis(4-cyanopentanoic acid).

[0017] The present invention also provides an application of a method for preparing carbon spheres with surface modification of 2-aminopurine, namely, using the carbon spheres for selective enrichment of N-glycopeptides.

[0018] Further, the selective enrichment of N-glycopeptides includes the following steps: dispersing carbon spheres into centrifuge tubes containing N-glycopeptides, 1% (v / v) trifluoroacetic acid, and 94% (v / v) acetonitrile, enriching at 37°C, then washing thoroughly twice with a buffer of 1% (v / v) trifluoroacetic acid and 94% (v / v) acetonitrile, and then washing once with a buffer of 0.5% (v / v) phosphate and 85% (v / v) acetonitrile; then eluting with an eluent of 0.1% (v / v) trifluoroacetic acid and 30% (v / v) acetonitrile, spotting the eluent onto a target, and performing matrix-assisted laser desorption / ionization time-of-flight mass spectrometry analysis using 2,5-dihydroxybenzoic acid as the matrix.

[0019] In addition, the present invention also provides an application of a carbon sphere material with a surface modified with 2-aminopurine, specifically, the carbon sphere material is used to enrich exosomes in human serum.

[0020] Further enrichment of exosomes in human serum includes the following steps: dispersing carbon sphere material into human serum, enriching at 37°C, followed by thorough washing with phosphate buffer solution; eluting with 0.1% (v / v) trifluoroacetic acid and 30% (v / v) acetonitrile; and performing Western blot analysis on the eluent.

[0021] The beneficial effects of this invention are as follows: This invention provides a one-step synthesis of carbon spheres with surface modification of 2-aminopurine for the separation and enrichment of N-glycopeptides and exosomes, offering the following advantages: 1. The preparation process is simple, with a very short synthesis cycle, saving significant time and labor; 2. The reactants are readily available, and no organic reagents are used as solvents during the reaction, aligning with the design principles of green chemistry, which aim to reduce or eliminate the use and generation of hazardous chemicals and processes; 3. By optimizing reaction time, temperature, and reactant concentration, the microstructure and size of the material can be continuously adjusted to obtain the best-performing material, greatly improving the separation and enrichment capabilities of glycosylated peptides and exosomes. Attached Figure Description

[0022] Figure 1This is a scanning electron microscope image of carbon spheres with surface modification of 2-aminopurine in a specific embodiment of the present invention;

[0023] Figure 2 The infrared spectrum of the carbon sphere material with surface modification of 2-aminopurine is shown in a specific embodiment of the present invention.

[0024] Figure 3 This is an elemental distribution diagram of the carbon sphere material with surface modification of 2-aminopurine in a specific embodiment of the present invention;

[0025] Figure 4 The mass spectra of glycosylated peptides in horseradish peroxidase digests enriched by carbon sphere materials with different raw material concentration ratios modified with 2-aminopurine in specific embodiments of the present invention are shown.

[0026] Figure 5 This is a graph showing the detection limit of glycosylated peptides enriched by carbon spheres with surface modification of 2-aminopurine in a specific embodiment of the present invention.

[0027] Figure 6 This is a mass spectrum of glycosylated peptides in a mixed peptide fragment (glycated protein horseradish peroxidase digest: non-glycosylated protein digest) enriched by carbon sphere material with surface modification of 2-aminopurine in a specific embodiment of the present invention.

[0028] Figure 7 This is a Western blot analysis diagram of exosomes enriched in human serum using carbon spheres modified with 2-aminopurine in a specific embodiment of the present invention. Detailed Implementation

[0029] To make the above-mentioned objects, features, and advantages of the present invention more apparent and understandable, specific embodiments of the present invention are described in detail below. It should be noted that the following embodiments are only used to illustrate the implementation methods and typical parameters of the present invention, and are not intended to limit the parameter range described in the present invention. Reasonable variations derived therefrom are still within the protection scope of the claims of the present invention.

[0030] It should be noted that the endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0031] Unless otherwise defined, all terms, symbols, and other scientific terms used herein are intended to have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In some instances, terms having a conventional meaning are defined herein for clarification or ease of reference, and such definitions should not be construed as indicating a significant difference from conventional understanding in the art. The technical methods described or referenced herein are generally well understood by those skilled in the art and employed by conventional methods. Unless otherwise stated, the use of commercially available kits, reagents, and instruments shall be performed according to the manufacturer's instructions and parameters.

[0032] The specific embodiment of the present invention provides a method for preparing surface-modified 2-aminopurine carbon spheres and their application.

[0033] Example 1

[0034] Synthesis of carbon spheres with surface-modified 2-aminopurine:

[0035] S1: Dissolve 1.5g of glucose (G) in 15mL of deionized water to prepare a solution with a concentration of 0.1g / mL. Then add 1.5g of 2-aminopurine (2AP), 0.45g of 1,2-epoxy-5-hexene (E), and 0.75g of 4,4'-azobis(4-cyanopentanoic acid) (A) in a mass ratio of 10:10:3:5 to glucose.

[0036] S2: Take out a 25mL stainless steel reactor lined with polytetrafluoroethylene and pour the solution obtained in step (1) into it;

[0037] S3: Place the reactor from step S2 into an oven and react at 180°C for 6 hours;

[0038] S4: The product obtained in step S3 is thoroughly washed with deionized water and ethanol to remove impurities and raw material monomers from the product surface. It is then dried overnight at 50°C in a vacuum drying oven to obtain carbon spheres with 2-aminopurine surface modification.

[0039] Scanning electron microscope images of the obtained carbon spheres with 2-aminopurine surface modification (20 kV, Philips XL30 electron microscope, Netherlands) are shown below. Figure 1 As shown, the Fourier transform infrared spectrum (Thermo Fisher Scientific Nicolet iS 10, USA) is as follows. Figure 2 As shown, the element distribution map is as follows: Figure 3 As shown.

[0040] Example 2

[0041] Preparation steps of surface-modified 2-aminopurine carbon spheres synthesized with different raw material concentration ratios:

[0042] (1) Dissolve 1.5g of glucose (G) and 1.5g of 2-aminopurine (2AP) in 15mL of deionized water to prepare a solution with a concentration of 0.1g / mL. Prepare 6 solutions.

[0043] (2) In order to prepare the product named G 10 2AP 10 E0A1 surface-modified 2-aminopurine carbon sphere material was prepared by adding 0 g of 1,2-epoxy-5-hexene (E) and 0.15 g of 4,4'-azobis(4-cyanopentanoic acid) (A) to a solution at a mass ratio of 10:10:0:1 with glucose; in order to prepare the material named G 10 2AP 10 E1A1 surface-modified 2-aminopurine carbon sphere material was prepared by adding 0.15 g of 1,2-epoxy-5-hexene (E) and 0.15 g of 4,4'-azobis(4-cyanopentanoic acid) (A) to a solution in a mass ratio of 10:10:1:1 with glucose; to prepare the material named G... 10 2AP 10 E3A1 surface-modified 2-aminopurine carbon sphere material was prepared by adding 0.45 g of 1,2-epoxy-5-hexene (E) and 0.15 g of 4,4'-azobis(4-cyanopentanoic acid) (A) to a solution in a mass ratio of 10:10:3:1 to glucose; to prepare a material named G... 10 2AP 10 E 10 A1 surface-modified 2-aminopurine carbon sphere material was prepared by adding 1.5 g of 1,2-epoxy-5-hexene (E) and 0.15 g of 4,4'-azobis(4-cyanopentanoic acid) (A) to a solution in a mass ratio of 10:10:10:1 to glucose; for the preparation of a material named G 10 2AP 10 E3A0 surface-modified 2-aminopurine carbon sphere material was prepared by adding 0.45 g of 1,2-epoxy-5-hexene (E) and 0 g of 4,4'-azobis(4-cyanopentanoic acid) (A) to a solution in a mass ratio of 10:10:3:0 with glucose; to prepare the material named G 10 2AP 10 E3A5 surface-modified 2-aminopurine carbon spheres were prepared by adding 0.45 g of 1,2-epoxy-5-hexene (E) and 0.75 g of 4,4'-azobis(4-cyanopentanoic acid) (A) to a solution in a mass ratio of 10:10:3:5 with glucose; to prepare the material named G... 10 2AP 10 E3A 10Surface-modified 2-aminopurine carbon spheres were prepared by adding 0.45 g of 1,2-epoxy-5-hexene (E) and 1.5 g of 4,4'-azobis(4-cyanopentanoic acid) (A) to a solution in a mass ratio of 10:10:3:10 with glucose; wherein glucose is represented by G, 2-aminopurine by 2AP, 1,2-epoxy-5-hexene by E, and 4,4'-azobis(4-cyanopentanoic acid) by A.

[0044] (3) Take out 6 stainless steel reaction vessels with a capacity of 25 mL PTFE lining and pour the solution obtained in step (2) into them;

[0045] (4) Place the reactor from step (3) into an oven and react at 180°C for 6 hours;

[0046] (5) The product obtained in step (4) is thoroughly washed with deionized water and ethanol to remove impurities and raw material monomers from the surface of the product. It is then placed in a vacuum drying oven and dried overnight at 50°C to obtain carbon ball material with surface modified 2-aminopurine.

[0047] Example 3

[0048] Application of surface-modified 2-aminopurine carbon spheres in the enrichment of glycosylated peptides in horseradish peroxidase digests:

[0049] (1) Sample preparation: Horseradish peroxidase (HRP) was enzymatically hydrolyzed in 25 mmol / L NH4HCO3 solution at 37℃ for 16 h;

[0050] (2) Enrichment: 250 μg of carbon sphere material was dispersed into a centrifuge tube containing 200 μL of 1% trifluoroacetic acid and 94% acetonitrile by volume, and then 2 μL of the sample prepared in step (1) was added. The mixture was enriched at 37 °C for 30 min. The mixture was washed twice with a buffer of 1% trifluoroacetic acid and 94% acetonitrile by volume, and then washed once with a buffer of 0.5% phosphoric acid and 85% acetonitrile by volume. The mixture was eluted with an elution buffer of 0.1% trifluoroacetic acid and 30% acetonitrile by volume for 30 min, and the supernatant was obtained by centrifugation.

[0051] (3) Mass spectrometry analysis: Take 1 μL of the supernatant obtained in step (2) and spot it onto the target. Use DHB as the matrix for mass spectrometry analysis. The spectrum is shown in the figure. Figure 4 As shown, (a): before enrichment; (b): after enrichment and named G. 10 2AP 10 After enrichment of carbon sphere material in E0A1; (c): named G 10 2AP 10 After enrichment of carbon spheres with E1A1; (d): named G 10 2AP 10After enrichment of carbon spheres in E3A1; (e): named G 10 2AP 10 E 10 After enrichment of carbon spheres in A1; (f): named G 10 2AP 10 After enrichment of carbon spheres with E3A0 material; (g): named G 10 2AP 10 After enrichment of carbon spheres with E3A5; (h): named G 10 2AP 10 E3A 10 After enrichment of carbon sphere material; where ● represents glycosylated peptides.

[0052] Named G 10 2AP 10 The detection limit for enriching glycosylated peptides in HRP using surface-modified 2-aminopurine carbon spheres of E3A5 is as follows: Figure 5 As shown, (a): 80 fmol / μL; (b): 8 fmol / μL; (c): 0.8 fmol / μL; (d): 0.08 fmol / μL; where ● represents glycosylated peptides.

[0053] Example 4

[0054] Application of surface-modified 2-aminopurine carbon spheres in enriching glycosylated peptides in mixed protein digests:

[0055] (1) Sample preparation: Bovine serum albumin (BSA) was first reduced and alkylated with dithiothreitol and iodoacetamide, and then enzymatically digested at 37°C for 16 h. HRP was enzymatically digested in 25 mM NH4HCO3 solution at 37°C for 16 h. The digests of HRP and BSA were added to centrifuge tubes containing 1% trifluoroacetic acid and 94% acetonitrile at mass ratios of 1:10, 1:100, 1:500, and 1:1000.

[0056] (2) Enrichment: 250 μg of carbon sphere material was dispersed into 200 μL of centrifuge tube containing 1% (v / v) trifluoroacetic acid and 94% (v / v) acetonitrile of the glycosylated peptide from step (1), and enriched at 37 °C for 30 min; washed twice thoroughly with buffer of 1% (v / v) trifluoroacetic acid and 94% (v / v) acetonitrile, and then washed once with buffer of 0.5% (v / v) phosphate and 85% (v / v) acetonitrile; eluted with elution buffer of 0.1% (v / v) trifluoroacetic acid and 30% (v / v) acetonitrile for 30 min, and centrifuged to obtain the supernatant;

[0057] (3) Mass spectrometry analysis: Take 1 μL of the eluent obtained in step (2) and spot it onto the target. Use DHB as the matrix for mass spectrometry analysis. The mass spectrum is shown below. Figure 6As shown, (a): 1:10; (b): 1:100; (c): 1:500; (d): 1:1000; where ● represents glycosylated peptides.

[0058] Example 5

[0059] Application of 2-aminopurine-modified carbon spheres in the enrichment of exosomes from human serum (diabetic patients):

[0060] (1) Enrichment: 5 mg of carbon pellet material was dispersed into a centrifuge tube containing 50 μL of human serum (diabetic patients) and enriched at 37°C for 30 min; it was thoroughly washed with phosphate buffer solution and centrifuged 3 times; it was eluted with 0.1% trifluoroacetic acid and 30% acetonitrile eluent for 30 min and centrifuged to obtain the supernatant.

[0061] (2) Western blot analysis: Protein concentration in the samples was measured using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). 5×SDS-PAGE protein loading buffer was added to the exosome protein samples, and the samples were heated at 100°C for 10 minutes to denature the proteins. Protein samples were separated by molecular weight using polyacrylamide electrophoresis. Then, proteins were separated by SDS-PAGE (WSHTbio, Hepes-Tris gel) and transferred to polyvinylidene fluoride (PVDF) microporous membranes (Merck Milli-pore, Immobilion-P transfer membrane) using a wet membrane transfer device. The PVDF membranes were blocked with 5% skim milk powder at room temperature for 1–2 hours. Next, the PVDF membranes were incubated overnight at 4°C with different antibodies. After washing, the PVDF membranes were incubated with a secondary antibody (anti-rabbit antibody labeled with horseradish peroxidase) for 2 hours. Finally, the blots were visualized using the ChemiDoc Touch Imaging System (Bio-Rad Laboratories), and the protein blot analysis results are shown below. Figure 7 As shown.

[0062] While the disclosure is as stated above, its scope of protection is not limited thereto. Those skilled in the art can make various changes and modifications without departing from the spirit and scope of this disclosure, and all such changes and modifications will fall within the protection scope of this invention.

Claims

1. A method for preparing carbon spheres with surface modification of 2-aminopurine, characterized in that, Includes the following steps: S1: Dissolve glucose, 2-aminopurine, 1,2-epoxy-5-hexene, and 4,4'-azobis(4-cyanopentanoic acid) in deionized water to prepare a solution; wherein the mass ratio of glucose, 2-aminopurine, 1,2-epoxy-5-hexene, and 4,4'-azobis(4-cyanopentanoic acid) is 10:10:(0~3):(1~10); S2: Remove the stainless steel reactor lined with polytetrafluoroethylene and pour the solution obtained in step S1 into it; S3: Place the reactor from step S2 into an oven for reaction; S4: The product obtained in step S3 is thoroughly washed with deionized water and ethanol to remove impurities and raw material monomers from the surface of the product, and then dried in a vacuum drying oven to obtain carbon spheres with surface modification of 2-aminopurine.

2. The method for preparing carbon spheres with surface modification of 2-aminopurine as described in claim 1, characterized in that, The concentration of glucose in step S1 is 0.1 g / mL.

3. The method for preparing carbon spheres with surface modification of 2-aminopurine as described in claim 1, characterized in that, In step S3, the reaction temperature is 150–200°C and the reaction time is 6–8 hours.

4. A carbon sphere material with a surface modified with 2-aminopurine, characterized in that, It is prepared by any one of the preparation methods described in claims 1 to 3.

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