A spherical silica-based composite material, its preparation method, and its application in proteomics.

Abstract

This invention discloses a spherical silica-based composite material, its preparation method, and its application in proteomics. The preparation method of the spherical silica-based composite material includes the following steps: 1) Mixing polyvinyl alcohol (PVA) and distilled water, stirring and dissolving to obtain a transparent and viscous PVA aqueous solution, which is then set aside; 2) Under an inert gas atmosphere, mixing the PVA aqueous solution, acrylic monomers, crosslinking agent, and silica spheres, followed by the addition of an initiator and catalyst, stirring and reacting at a certain temperature for a certain time, removing the silica sphere component, and drying to obtain the spherical silica-based composite material (MSSMC). The obtained MSSMC has the function of quantitatively adsorbing both finger-prick blood samples and proteins, and can quantitatively adsorb 9-12 μL of blood samples, especially suitable for finger-prick blood. Using it, through three steps of sample adsorption-protein adsorption-washing, the proteome can be extracted from a small amount of finger-prick blood sample, and the protein identification rate of finger-prick blood can be increased by about 50%.

Description

Technical Field

[0001] This invention relates to the field of blood proteomics technology, specifically to a spherical silica-based composite material, its preparation method, and its application in proteomics. Background Technology

[0002] With the increasing incidence of malignant tumors (such as head and neck cancer, prostate cancer, and colorectal cancer) and infectious diseases (such as COVID-19 and HIV), and the growing awareness of health, the demand for point-of-care testing (POCT) is increasing daily. Venous blood collection requires trained medical professionals and is difficult to use in resource-constrained environments; however, finger-prick blood collection, due to its simplicity, greatly reduces barriers to its use and is more popular in the era of personalized medicine. Proteomics analysis based on finger-prick blood will also effectively promote the development of protein biomarkers.

[0003] However, in addition to the challenges of high protein abundance, high proteomic complexity, and low detection depth, similar to venous blood, finger-prick blood also faces the problem of small sample size. Therefore, developing a simple and convenient method to achieve comprehensive and in-depth proteomics analysis of trace finger-prick blood samples is of great significance. Summary of the Invention

[0004] Objective: This invention addresses the technical problems existing in finger-prick blood proteomics analysis by proposing a spherical silica-based composite material, its preparation method, and its application in proteomics. This invention introduces a polyacrylic acid polymer shell onto the surface of the silica spheres, endowing them with strong hydrophilic groups and a certain cross-linked network structure, thereby significantly enhancing the water absorption and retention properties of the silica spheres. The innovative millimeter-sized spherical structure proposed in this invention combines the quantitative adsorption of both finger-prick blood samples and proteins; furthermore, the proteomics sample processing method based on the millimeter-sized spherical structure is simple to operate, further amplifying the advantages of real-time finger-prick blood detection. The introduction of the polyacrylic acid shell significantly increases the number of proteins identified in finger-prick blood.

[0005] Technical solution: To achieve the above-mentioned objectives, the present invention adopts the following technical solution:

[0006] In a first aspect, the present invention provides a method for preparing a spherical silicone-based composite material, comprising the following steps:

[0007] 1) Mix polyvinyl alcohol (PVA) and distilled water, stir to dissolve, and obtain a transparent and viscous PVA aqueous solution for later use;

[0008] 2) Under an inert gas atmosphere, PVA aqueous solution, acrylic monomer, crosslinking agent and silica spheres are mixed, and then initiator and catalyst are added. The mixture is stirred and reacted at a certain temperature for a certain time. The silica sphere component is then removed and dried to obtain the spherical silica-based composite material (MSSMC).

[0009] Preferably, in step 1), the stirring temperature is 60–90°C, and the stirring time is 2–3 hours; the mass fraction of the PVA aqueous solution is 6%–8%.

[0010] Preferably, in step 2), the acrylic monomer is one or more of sodium acrylate, N-isopropylacrylamide, acrylamide, glycidyl methacrylate, ethyl acrylate, and methyl methacrylate; the crosslinking agent is N,N′-methylenebisacrylamide; the diameter of the silica gel ball is 3-4 mm; the initiator is one of ammonium persulfate, potassium persulfate, and azobisisobutyronitrile; and the catalyst is tetramethylethylenediamine.

[0011] Preferably, in step 2), the silica balls and PVA aqueous solution are measured in g by weight, and other raw materials are measured in mmol. The ratio of the amount of silica balls, PVA aqueous solution, acrylic monomer, crosslinking agent, initiator and catalyst is 1:2.5-3:200-250:20-25:1-1.25:2:2.5.

[0012] Preferably, in step 2), the PVA aqueous solution, acrylic monomer, crosslinking agent, and silica gel balls are mixed and stirred in a constant temperature water bath at 20-30℃ for 25-35 minutes; after adding the initiator and catalyst, the mixture is stirred in a constant temperature water bath at 20-30℃ for 25-35 minutes; and the reaction is stirred at a certain temperature for a certain time, wherein the temperature is 55-65℃ and the time is 5-7 hours.

[0013] Secondly, the present invention provides a spherical silicone-based composite material, which is prepared by the aforementioned preparation method.

[0014] Thirdly, the present invention provides the application of the spherical silica-based composite material as an adsorbent in proteomics.

[0015] As a preferred embodiment, the present invention provides the application of the spherical silica-based composite material as an adsorbent in fingertip blood proteomics, wherein the adsorbent is capable of adsorbing fingertip blood and the proteins therein.

[0016] Fourthly, the present invention provides a method for blood proteomics detection using the aforementioned spherical silica-based composite material, characterized by comprising the following steps:

[0017] 1) Place the spherical silicone-based composite material into a blood sample and let it stand to allow MSSMC to quantitatively adsorb a certain volume of fingertip blood;

[0018] 2) Take out the spherical silica-based composite material after step 1), put it into the buffer solution, and let it stand to allow it to adsorb the proteins in the fingertip blood.

[0019] 3) Take out the spherical silica-based composite material after step 2) and wash it in a new buffer solution. The final result is a mixture of the spherical silica-based composite material and the protein it adsorbs.

[0020] 4) Detect the target proteome or target protein.

[0021] Preferred:

[0022] In step 1), the blood sample is a finger prick blood sample, and the sample volume is ≥10μL; the settling time is 2min~3min;

[0023] In step 2), the buffer solution comprises one or any combination of Tris, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, potassium phosphate, phosphoric acid, sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium phosphate, potassium chloride, sodium chloride, citric acid, sodium citrate, barbituric acid, sodium barbital, sodium hydroxide, hydrochloric acid, formic acid, acetic acid, EDTA, SDS, NP-40, CHAPS, Tween, Triton, PEG, acetonitrile, and methanol, preferably a combination buffer of Tris and EDTA, and the standing time is 10 min to 30 min.

[0024] In step 3), the new buffer solution is selected from the buffer solution used in step 2) or the corresponding dilution solution;

[0025] In step 4), the detection is performed using one or more of mass spectrometry, IHC, ELISA, Western blot, and chemiluminescence, with mass spectrometry being preferred.

[0026] As a specific implementation, the MSSMC can be fixed to the end of the dropper's aspiration tip, and then the operations in steps 1), 2), and 3) above can be performed. Preferably, the diameter of the dropper's aspiration tip satisfies: √3 times the radius of the MSSMC ≤ the diameter of the dropper's aspiration tip ≤ the diameter of the MSSMC. The process of washing with new buffer solution is as follows: insert the MSSMC end into the new buffer solution, rotate it clockwise and counterclockwise 3 times each, then remove the dropper and insert the MSSMC end into the new buffer solution. Repeat the above process 3 times.

[0027] Beneficial effects:

[0028] 1. The present invention introduces a polyacrylic acid polymer shell layer on the surface of the silicone ball, endowing it with strong hydrophilic groups and a certain cross-linked network structure, thereby significantly enhancing the water absorption and water retention of the silicone ball.

[0029] 2. The millimeter-sized spherical silica-based composite material prepared by the method proposed in this invention has uniform particle size and stable methodology.

[0030] 3. The innovative millimeter-sized spherical structure proposed in this invention has the function of quantitatively adsorbing both fingertip blood samples and proteins; it can stably adsorb 9-12uL of fingertip blood in 2 minutes.

[0031] 4. The finger-prick blood proteomics sample processing method proposed in this invention, based on a millimeter-scale spherical structure, can extract the proteome from a small amount of finger-prick blood sample through three steps: sample adsorption, protein adsorption, and washing. The operation is simple and has good stability, further amplifying the advantages of real-time finger-prick blood detection.

[0032] 5. The millimeter-sized spherical silicone-based composite material proposed in this invention increases the number of proteins identified in fingertip blood by 50% by introducing a polyacrylic shell layer on the surface of the silicone sphere. Attached Figure Description

[0033] Figure 1 Vernier caliper was used to test the particle size of silica gel balls and MSSMC; the left image shows silica gel balls, and the right image shows MSSMC (obtained in Example 3).

[0034] Figure 2 Particle size distribution of 30 silica gel balls and 30 MSSMCs (obtained in Example 3); the testing tool was a vernier caliper, and the unit was mm.

[0035] Figure 3 The differential gravimetric method was used to test the adsorption amount of MSSMC on the sample within 2 minutes. The left figure is a schematic diagram of the differential gravimetric method, where the weight of ③-① is the amount of sample adsorbed; the right figure shows the sample adsorption distribution of 30 silica gel balls and 30 MSSMCs (obtained in Example 3).

[0036] Figure 4 Schematic diagram of the experimental procedure for the application of MSSMC in fingertip blood. Detailed Implementation

[0037] The following provides a comprehensive description of the present invention. The embodiments described are the most preferred embodiments of the present invention, but the present invention is not limited to the following embodiments.

[0038] Example 1

[0039] 1) Add 8g of polyvinyl alcohol (PVA) and 92mL of distilled water to a three-necked flask, and stir at 80℃ for 2h until the PVA is completely dissolved. Then pour it into an Erlenmeyer flask and let it cool naturally to room temperature to remove air bubbles. You will get an aqueous solution of PVA with a mass fraction of 8% for later use.

[0040] 2) Under a nitrogen atmosphere, 7.5g of PVA aqueous solution from step 1), 35.5g of acrylamide, 7.735g of N,N′-methylenebisacrylamide, 2.5g of silica spheres, and 500mL of distilled water were added to a three-necked flask and stirred in a constant temperature water bath at 25℃ for 30min. Subsequently, 0.675g of potassium persulfate and 0.581g of tetramethylethylenediamine were added, and the mixture was stirred under a nitrogen atmosphere for 30min. After stirring at 55℃ for 4h, the silica sphere component was removed and dried at 60℃ overnight to obtain millimeter-sized spherical silica-based composite material (MSSMC).

[0041] Example 2

[0042] 1) Add 8g of polyvinyl alcohol (PVA) and 92mL of distilled water to a three-necked flask, and stir at 80℃ for 2h until the PVA is completely dissolved. Then pour it into an Erlenmeyer flask and let it cool naturally to room temperature to remove air bubbles. You will get an aqueous solution of PVA with a mass fraction of 8% for later use.

[0043] 2) Under a nitrogen atmosphere, 7.5g of the PVA aqueous solution from step 1), 62.57g of methyl methacrylate, 7.735g of N,N′-methylenebisacrylamide, 2.5g of silica gel spheres, and 500mL of distilled water were added to a three-necked flask and stirred in a water bath at 25°C for 30min. Subsequently, 0.41g of azobisisobutyronitrile and 0.581g of tetramethylethylenediamine were added, and the mixture was stirred under a nitrogen atmosphere for 30min. After stirring at 65°C for 4h, the silica gel sphere component was removed and dried overnight at 60°C to obtain millimeter-sized spherical silica gel-based composite material (MSSMC).

[0044] Example 3

[0045] 1) Add 8g of polyvinyl alcohol (PVA) and 92mL of distilled water to a three-necked flask, and stir at 80℃ for 2h until the PVA is completely dissolved. Then pour it into an Erlenmeyer flask and let it cool naturally to room temperature to remove air bubbles. You will get an aqueous solution of PVA with a mass fraction of 8% for later use.

[0046] 2) Under a nitrogen atmosphere, 7.5g of the PVA aqueous solution from step 1), 47g of sodium acrylate, 7.735g of N,N′-methylenebisacrylamide, 2.5g of silica spheres, and 500mL of distilled water were added to a three-necked flask and stirred in a constant temperature water bath at 25℃ for 30min. Subsequently, 0.57g of ammonium persulfate and 0.581g of tetramethylethylenediamine were added, and the mixture was stirred under a nitrogen atmosphere for 30min. After stirring at 60℃ for 4h, the silica sphere component was removed and dried at 60℃ overnight to obtain millimeter-sized spherical silica-based composite material (MSSMC).

[0047] Example 4

[0048] 1) Fix one MSSMC (obtained in Example 3) to the end of the suction end of a 3.5 mm diameter dropper;

[0049] 2) Insert the MSSMC end of the dropper from step 1) into a fingertip blood sample of about 15 μL, let stand for 2 min, so that the MSSMC can quantitatively adsorb a certain volume of fingertip blood.

[0050] 3) Take out the dropper from 2) and insert its MSSMC end into 200 μL of Tris-EDTA buffer. Let it stand for 15 min to allow it to adsorb the proteins in the fingertip blood.

[0051] 4) Take out the dropper from 3) and insert its MSSMC end into 500 μL of Tris-EDTA buffer. Rotate it clockwise and counterclockwise 3 times each. Then take out the dropper and insert the MSSMC end into a new buffer. Repeat the above process 3 times. The final result is a mixture of MSSMC and the proteins it adsorbs.

[0052] 5) As a comparison: an untreated silica ball was fixed to the end of the liquid-absorbing end of a 3.5 mm diameter dropper; and steps 2) to 4) above were repeated; the silica ball and the protein mixture it adsorbed were obtained.

[0053] LC-MS / MS detection

[0054] Take out the MSSMC and silica gel components from steps 4) and 5) above and place them into new centrifuge tubes, and perform the following processing respectively:

[0055] 1) Add a certain volume of buffer containing DTT to the centrifuge tube and react at 95°C for 1 hour; then add a certain volume of IAM and react at room temperature in the dark for 45 minutes.

[0056] 2) Add 10 μL of digestion buffer containing ammonium bicarbonate and 1 μg of trypsin, mix well, and incubate at 37°C for 4 h.

[0057] 3) Add excess formic acid solution, centrifuge at 12,000g for 5 minutes, collect the supernatant, add it to the SDB desalting column, centrifuge, so that the enzymatically digested peptides bind to the SDB column.

[0058] 4) Wash the SDB column several times and desorb to obtain the purified peptide solution.

[0059] 5) Freeze-dry the purified peptide solution and reconstitute the peptide using the loading buffer.

[0060] 6) DIA data acquisition of peptides was performed using nano-level high-performance liquid chromatography (Thermo Scientific UltiMate 3000UHPLC) and tandem mass spectrometry (Thermo Scientific Orbitrap Q Exactive HF mass spectrometer) for 30 minutes.

[0061] 7) Use DIA-NN software (version 1.8.1) to extract data and obtain qualitative and quantitative results of proteins.

[0062] 8) Three personnel simultaneously processed three finger-prick blood samples from different sources, with three parallel replicates for each sample. The number of proteins identified is shown in Table 1.

[0063] Table 1. Number of proteins identified in peripheral blood.

[0064]

[0065] in conclusion:

[0066] 1. Through Figure 1 It can be seen that the method of the present invention successfully prepared millimeter-sized spherical silicone-based composite materials; the particle size of the silicone spheres is 3.6 mm, and the particle size of MSSMC is 3.91 mm, indicating that a polymer shell layer of about 0.3 mm was formed on the surface of the silicone spheres.

[0067] 2. Through Figure 2 It can be seen that the millimeter-sized spherical silica-based composite material prepared by the preparation method proposed in this invention has a larger particle size than silica spheres, and the particle size is uniform, all between 3.8-4.2 mm.

[0068] 3. Through Figure 3 It can be seen that MSSMC can stably adsorb 9-12 mg of fingertip blood within 2 minutes, which is about 9-12 μL; the adsorption capacity of MSSMC is significantly increased by nearly 50% compared with that of silica gel balls, indicating that the preparation method proposed in this invention effectively enhances the adsorption capacity of silica gel balls.

[0069] 4. Through Figure 4It can be seen that the method for applying millimeter-sized spherical silicone-based composite materials in fingertip blood proteomics proposed in this invention is simple to operate.

[0070] 5. As can be seen from Table 1, the millimeter-sized spherical silicone-based composite material proposed in this invention can significantly increase the number of proteins identified in fingertip blood and has good repeatability.

Claims

1. The application of a spherical silica-based composite material as an adsorbent in fingertip blood proteomics, characterized in that, The adsorbent is capable of adsorbing fingertip blood and the proteins therein; the preparation method of the spherical silica-based composite material includes the following steps: 1) Mix polyvinyl alcohol (PVA) and distilled water, stir to dissolve, and obtain a transparent and viscous PVA aqueous solution for later use; 2) Under an inert gas atmosphere, PVA aqueous solution, acrylic monomer, crosslinking agent and silica spheres are mixed, and then initiator and catalyst are added. The mixture is stirred and reacted at a certain temperature for a certain time. The silica sphere component is then removed and dried to obtain the spherical silica-based composite material.

2. The application according to claim 1, characterized in that, In step 1), the stirring temperature is 60~90℃ and the stirring time is 2h~3h; the mass fraction of the PVA aqueous solution is 6%-8%.

3. The application according to claim 1, characterized in that, In step 2), the acrylic monomer is one or more of sodium acrylate, N-isopropylacrylamide, acrylamide, glycidyl methacrylate, ethyl acrylate, and methyl methacrylate; the crosslinking agent is N,N′-methylenebisacrylamide; the diameter of the silica gel ball is 3-4 mm; the initiator is one of ammonium persulfate, potassium persulfate, and azobisisobutyronitrile; and the catalyst is tetramethylethylenediamine.

4. The application according to claim 1, characterized in that, In step 2), the silica balls and PVA aqueous solution are measured in grams by weight, and other raw materials are measured in mmol. The ratio of silica balls, PVA aqueous solution, acrylic monomers, crosslinking agents, initiators and catalysts is 1:2.5~3:200~250:20~25:1~1.25:2:2.

5.

5. The application according to claim 1, characterized in that, In step 2), the PVA aqueous solution, acrylic monomer, crosslinking agent, and silica gel balls are mixed and stirred in a constant temperature water bath at 20-30℃ for 25-35 minutes; after adding the initiator and catalyst, the mixture is stirred in a constant temperature water bath at 20-30℃ for 25-35 minutes; the reaction is stirred at a certain temperature for a certain time, wherein the temperature is 55-65℃ and the time is 5-7 hours.

6. A method for blood proteomics detection using spherical silica-based composite materials, characterized in that, Includes the following steps: 1) Place the spherical silicone-based composite material into a blood sample and allow it to stand; the preparation method of the spherical silicone-based composite material includes the following steps: 1.1) Mix polyvinyl alcohol (PVA) and distilled water, stir to dissolve, and obtain a transparent and viscous PVA aqueous solution for later use; 1.2) Under an inert gas atmosphere, PVA aqueous solution, acrylic monomer, crosslinking agent and silica spheres are mixed, and then initiator and catalyst are added. The mixture is stirred and reacted at a certain temperature for a certain time. The silica sphere component is taken out and dried to obtain the spherical silica-based composite material. 2) Take out the spherical silica-based composite material after step 1), place it in the buffer solution, and let it stand; 3) Take out the spherical silica-based composite material after step 2) and wash it in a new buffer solution. The final result is a mixture of the spherical silica-based composite material and the protein it adsorbs. 4) Detect the target proteome or target protein.

7. The method according to claim 6, characterized in that, In step 1), the blood sample is a fingertip blood sample, and the sample volume is ≥10μL; the settling time is 2min~3min; In step 2), the buffer solution comprises one or any combination of Tris, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, potassium phosphate, phosphoric acid, sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium phosphate, potassium chloride, sodium chloride, citric acid, sodium citrate, barbituric acid, sodium barbital, sodium hydroxide, hydrochloric acid, formic acid, acetic acid, EDTA, SDS, NP-40, CHAPS, Tween, Triton, PEG, acetonitrile, and methanol, and the settling time is 10 min to 30 min. In step 3), the new buffer solution is selected from the buffer solution or corresponding dilution solution used in step 2); In step 4), the detection is performed using one or more of the following: mass spectrometry, IHC, ELISA, Western blot, and chemiluminescence.

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