Porous silicon-based nano material as well as synthesis method and application thereof

Abstract

The invention discloses a porous silicon-based nano material which comprises a silicon-based nano material and a surface functional group modified porous silicon-based nano material, the pore diameter is 2-200nm, the specific surface area is 100-800m < 2 > / g, and the pore volume is 0.5-5.0 cm < 3 > / g. The invention also discloses a preparation method of the porous silicon-based nano material, and an application of the nano material in protein enrichment and preparation of a proteomic sample of a biological sample.

Description

Technical Field

[0001] The invention belongs to the technical field of nano material application, and relates to a type of porous silicon-based nano material and a synthesis method and application thereof. Background Art

[0002] Proteomics is the study of the composition, structure, function and interaction of all proteins in an organism, aiming to systematically and comprehensively understand biological processes at the protein level, including the occurrence and development of diseases and cellular metabolism. There are extremely rich types of proteins in organisms. The diversity and complexity of proteins make proteomics research more challenging, while also providing more information on potential functions and interactions. Compared with genomics, transcriptomics and metabolomics, proteomics research can reveal the relationship between protein structure and function, infer the function of unknown proteins, and predict the interaction between proteins and other molecules, which is of great significance for drug design, disease diagnosis and treatment.

[0003] Mass spectrometry technology has the advantages of rapidity, high sensitivity, high resolution, and high throughput. It can accurately measure the relative molecular mass, amino acid sequence, post-translational modification, and protein-protein interactions of proteins and peptides, and has become the preferred method in proteomics research. MS-based proteomics has become increasingly mature in the past decade. The development of advanced high-resolution mass spectrometry instruments has promoted large-scale analysis of proteomics research and provided a deep understanding of the properties of proteins (such as post-translational modifications, interactions, and biochemical functions). The mass spectrometry-based protein workflow includes (1) sample preparation, (2) peptide separation, (3) mass spectrometry detection, and (4) data analysis. Sample preparation is a key stage that affects the overall efficiency of proteomics research, but the low reproducibility and low throughput of sample preparation have become the main bottlenecks for many cutting-edge biomedical applications.

[0004] Sample preparation based on in-solution digestion is a traditional but popular method for digesting proteins into peptides. Its workflow includes protein extraction from biological samples, protein denaturation / purification, reduction, alkylation, enzymatic digestion, desalting, and peptide separation. The first is the extraction of proteins from biological samples. Since the protein concentration in biological samples spans at least 6 orders of magnitude, it is difficult to effectively extract proteins. For example, there are about 10,000 proteins in plasma, ranging from 35-50 mg / mL albumin to pg / mL low-abundance proteins. Plasma and serum samples usually need to remove high-abundance proteins to identify low-abundance proteins. Immunodepletion (centrifugal columns and LC) and magnetic beads can remove up to 20 of the most abundant plasma proteins, but will result in residual, low reproducibility, low throughput, and loss of proteins bound to albumin. Secondly, protein denaturation requires high concentrations of denaturants, such as 8M urea to dissolve proteins and denature their three-dimensional structures. The addition of high concentrations of denaturants inhibits enzyme digestion activity, so protein samples are diluted to reduce urea concentrations before adding enzymes. At the same time, the sample needs to be incubated for at least 30 minutes after adding reducing agents such as dithiothreitol, tris(2-carboxyethyl)phosphine (TCEP), and tris(3-hydroxypropyl)phosphine to reduce the sample. Further addition of alkylating agents such as iodoacetamide and iodoacetic acid also requires incubation in the dark. Finally, protein enzymatic digestion requires a long enzymatic hydrolysis time (usually overnight) and a large amount of starting material (usually >100 μg), which is extremely unfavorable for the processing of trace or precious biological samples. Due to the multi-step operation in sample preparation, the in-solution digestion method has a large sample loss, which limits its application in proteomic analysis with limited starting material. At the same time, the cumbersome workflow is inefficient and is not suitable for clinical proteomics and in vivo studies that require high sample processing throughput, reproducibility, and sensitivity.

[0005] Sample preparation is the most critical stage in proteomics research, but it is time-consuming, labor-intensive and prone to errors. Researchers have developed many methods to replace in-solution digestion, including on-membrane digestion (MStern, FASP, and fa-SPEED), magnetic bead-based digestion (proteomic reactor, SP3, and C4-tip), and digestion based on immobilized enzyme reactors to reduce time, increase throughput and improve reproducibility. Currently, these methods have also been successfully applied to proteomics research, but there are still certain limitations in sample preparation for clinical research and biomedical applications. Therefore, there is an urgent need for a sample preparation technology that improves the accuracy and reproducibility of the sample preparation process, integrates multiple steps of sample preparation, thereby reducing sample loss caused by manual transfer steps, simplifying the workflow, and improving the efficiency of proteomics research. Summary of the invention

[0006] In response to the above problems, the present invention provides a type of proteomics sample preparation technology based on porous silicon-based nanomaterials. The porous silicon-based nanomaterials proposed in the present invention have a mesoporous (pore size of 2-50nm) and / or macroporous (pore size>50nm) structure and a large specific surface area, which can effectively adsorb and enrich protein molecules of appropriate sizes. At the same time, the material of the present invention can also introduce nanopores of appropriate sizes through different synthesis methods, which can not only expand the specific surface area, modify more functional groups on the surface, enhance specific recognition performance, but also have a volume exclusion effect on molecules of certain sizes, significantly improving the enrichment efficiency of protein molecules. At the same time, the protein denaturation, reduction and alkylation steps are integrated into one step, which greatly shortens the sample preparation time.

[0007] The present invention provides a class of porous silicon-based nanomaterials, including silicon-based nanomaterials with a mesoporous structure (pore size of 2-50 nm) and / or a macroporous structure (pore size>50 nm) and porous silicon-based nanomaterials with surface functional group modification;

[0008] The porous silicon-based nanomaterial has a pore size of 2-200 nm and a specific surface area of ​​100-800 m 2 / g, pore volume 0.5-5.0cm 3 / g porous silica material; preferably, a pore size of 50-200nm and a specific surface area of ​​100-800m 2 / g, pore volume 0.5-5.0cm 3 / g of macroporous ordered silica material.

[0009] The porous silicon-based nanomaterial can be modified with surface functional groups to synthesize a functionalized porous silicon-based nanomaterial.

[0010] Among them, the surface functional groups in the surface functional group modification refer to hydroxyl-OH, thiol-SH, amino-NH2, nitro-NO2, carboxyl-COOH, sulfonic acid group-SO3H, phosphate group-PO3H2, carbamate group-NHCOCH3, azido-N3, charged group, boric acid group, aromatic group, sugar modification, metal oxide modification, halogen modification, silicone modification, aldehyde ketone modification, polyethylene glycol modification, hydrophobic group, hydrophilic group, cationic coating, anionic coating, etc.

[0011] The present invention also provides a method for preparing a porous silicon-based nanomaterial, the specific steps of which are as follows:

[0012] (1) dissolving a surfactant and an inorganic salt in a buffer solution to obtain a uniform solution, and then adding a silicon source to react for a certain period of time to form a uniform solution;

[0013] (2) subjecting the uniform solution obtained in step (1) to hydrothermal treatment;

[0014] (3) The solution obtained in step (2) is filtered, washed, dried and then calcined to obtain a porous silica material.

[0015] In the step (1), the surfactant is selected from one or more of quaternary ammonium salt cationic surfactants, primary amine salt cationic surfactants, long-chain alkyl sulfate, phosphate, carboxylate anionic surfactants, long-chain alkylamine, polyoxyethylene ether and block copolymer nonionic surfactants; preferably, it is a block copolymer nonionic surfactant.

[0016] The inorganic salt is selected from one or more of sulfates, phosphates, carbonates and carboxylates, etc.; preferably, it is phosphate.

[0017] The concentration of the inorganic salt is 0.01-1.0M; preferably, 0.3M.

[0018] The buffer solution is a NaH2PO4-Na2HPO4, H3PO4-Na2HPO4, NaAc-HAc or H2CO3-NaHCO3 buffer system with a pH of 2-7, preferably, NaH2PO4-Na2HPO4, pH=6.

[0019] The silicon source is selected from tetramethyl orthosilicate (TMOS), tetraethyl orthosilicate (TEOS), etc.; preferably, it is TMOS.

[0020] The mass ratio of the surfactant to the silicon source is (0.5-2):1; preferably, it is 0.7:1.

[0021] The reaction temperature is 25-70°C; preferably, 30°C.

[0022] The reaction time is 10-48 hours; preferably, 12 hours.

[0023] In the step (2), the temperature of the hydrothermal treatment is 50-200°C; preferably, 120°C.

[0024] The hydrothermal treatment time is 10-48 hours; preferably, 12 hours.

[0025] In the step (3), the drying temperature is 25-70°C; preferably, 40°C.

[0026] The drying time is 5-12 hours; preferably, 8 hours.

[0027] The calcination temperature is 300-800°C; preferably, 500°C.

[0028] The calcination time is 2-10 hours; preferably, 5 hours.

[0029] The invention also provides porous silicon-based nanomaterials prepared by the method.

[0030] The porous silicon-based nanomaterial has a pore size of 2-200 nm and a specific surface area of ​​100-800 m 2 / g, pore volume 0.5-5.0cm 3 / g of macroporous ordered silica material; preferably, a pore size of 50-200nm and a specific surface area of ​​100-800m 2 / g, pore volume 0.5-5.0cm 3 / g of macroporous ordered silica material.

[0031] The invention also provides application of the porous silicon-based nanomaterial in protein enrichment and proteomics sample preparation in biological samples.

[0032] In a specific embodiment, in the application of the present invention, the protein denaturation, reduction and alkylation steps are integrated into one step.

[0033] Compared with the existing materials used for proteomics sample preparation, the material synthesis of the present invention has the following advantages: low raw material cost, mild and controllable reaction conditions, simple operation and easy synthesis; silicon-based materials with different pore sizes and / or specific surface areas can be synthesized by changing experimental conditions, such as feed ratio, reaction temperature, buffer solution pH, etc.; at the same time, the material surface can be modified with functional groups to increase its specific enrichment of proteins; the synthesized porous silicon-based nanomaterial has a suitable pore size and a large specific surface area, has been successfully used for protein enrichment, and has excellent performance, and has broad application prospects in the field of proteomics sample preparation.

[0034] The present invention also provides a method for preparing a proteomics sample in a biological sample based on a porous silicon-based nanomaterial, the method comprising: first adding the porous silicon-based nanomaterial to the biological sample for protein enrichment, and then performing protein cleavage, reduction and alkylation, protein digestion, protein desalting and other steps to obtain the proteomics sample. In a specific embodiment, the method comprises the following specific steps:

[0035] (1) Protein extraction: add the corresponding lysis buffer to cells, tissues and other samples to homogenize the samples and then extract the protein solution. Biological fluids (serum, plasma, etc.) are directly used without dilution;

[0036] (2) Protein enrichment: Add the porous silicon-based nanomaterial to the protein sample (protein: material (w:w) = 0.2-10:1), place it on a constant temperature mixer at 37°C, incubate at 300-1500 rpm for 5-30 minutes, centrifuge, remove the supernatant, and wash three times;

[0037] (3) Protein lysis, reduction and alkylation: add protein lysis, reduction and alkylation buffer reagents, place on a constant temperature mixer at 70-95°C, 300-1500 rpm and incubate for 5-60 minutes; the final concentration of the protein lysis reagent is 5-50 mM, the final concentration of the reducing reagent is 5-100 mM, the final concentration of the alkylation reagent is 10-100 mM, the buffer: protein = 1-100:1, and the buffer pH is 7-8.5;

[0038] (4) Protein digestion: according to the BCA protein quantification result, add digestive enzyme at a ratio of enzyme: protein = 1:20 to 1:100, place on a constant temperature mixer at 37°C, incubate at 300-1500 rpm for 0.5 to 6 hours, add stop reagent and mix well to stop the enzymatic reaction, centrifuge at 10000-20000 g for 1 to 10 minutes, and aspirate the supernatant;

[0039] (5) Protein desalting: The supernatant was loaded into a C18 desalting column for desalting. The eluate was concentrated by a vacuum refrigerated centrifugal concentrator and stored at -80°C for LC-MS / MS detection.

[0040] The proteomics sample preparation method provided by the present invention has the following advantages over the existing sample preparation methods: the sample preparation technology of the present invention does not need to pre-remove high-abundance proteins, and realizes indiscriminate enrichment of protein samples by surface modification of porous silicon-based nanomaterials and combination of different porous silicon-based nanomaterials, thereby realizing indiscriminate enrichment and concentration normalization of high- and low-abundance proteins in the pores of the nanomaterials; by integrating multiple steps of protein sample preparation, the workflow is simplified, the time is greatly shortened, the work efficiency is improved, and sample preparation can be completed within 1 to 8 hours; the materials used in this technology are inexpensive, with a cost of 0.2 yuan / 100 μl sample, and it is expected to be widely used in clinical testing in the future. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without paying any creative work.

[0042] Figure 1 This is a characterization picture of the porous silicon-based nanomaterial prepared in specific embodiment 1 of the present invention. Figure 1a is the transmission electron microscope TEM characterization picture, Figure 1 b is the SEM characterization picture. Figure 1 c is the characterization diagram of nitrogen adsorption-desorption isotherm.

[0043] Figure 2 The porous silicon-based nanomaterials in specific embodiments 5 and 6 of the present invention are enriched with fetal bovine serum (FBS) ( Figure 2 a) and plasma samples ( Figure 2 b) The number of protein groups identified by LC-MS / MS detection was compared with the protein identification results of samples processed by commercial kits. DETAILED DESCRIPTION

[0044] The present invention is further described in detail with reference to the following specific examples and drawings. The process, conditions, experimental methods, etc. for implementing the present invention, except for the contents specifically mentioned below, are all common knowledge and common common sense in the art and are not particularly limited by the present invention.

[0045] Example 1: Preparation of porous silicon-based nanomaterials

[0046] 0.7 g of block copolymer P123 (polyethylene oxide-polypropylene oxide-polyethylene oxide triblock copolymer PEO-PPO-PEO), 0.3 g of CTAB, and 1.4 g of Na2SO4 were dissolved in 30 g of NaAc-HAc buffer solution with a pH of 4, and reacted at 30°C for 1 h. 1.5 g of TMOS was added to the above homogeneous solution, and the reaction was continued for 12 hours. After that, the mixture was hydrothermaled at 120°C for 12 hours, filtered, washed, and dried. After drying, the mixture was calcined at 500°C for 5 hours to obtain a pore size of 50 nm and a specific surface area of ​​217 m 2 / g, pore volume 3.9cm 3 / g porous silica nanomaterials.

[0047] Figure 1 This is a characterization picture of the porous silicon-based nanomaterial prepared in Example 1. Figure 1 a is the transmission electron microscope TEM characterization picture, Figure 1 b is the SEM characterization picture. Figure 1 c is the characterization diagram of nitrogen adsorption-desorption isotherm.

[0048] Example 2: Preparation of porous silicon-based nanomaterials

[0049] 0.7 g of block copolymer F-127 (poly(propylene glycol)-block-poly(ethylene glycol)-block-poly(propylene glycol) PPG-PEG-PPG), 0.3 g of CTAB and 1.4 g of Na3PO4 were dissolved in 30 g of NaH2PO4-Na2HPO4 buffer solution of pH=6, and reacted at 30°C for 1 h. 1.5 g of TMOS was added to the above homogeneous solution, and the reaction was continued for 12 hours. After that, the solution was hydrothermaled at 120°C for 12 hours, filtered, washed and dried; after drying, the solution was calcined at 500°C for 5 hours to obtain a pore size of 20 nm and a specific surface area of ​​291 m 2 / g, pore volume 2.7cm 3 / g porous silica nanomaterials.

[0050] Example 3: Preparation of porous silicon-based nanomaterials

[0051] 1.4 g of block copolymer F-127, 0.6 g of CTAB and 1.4 g of Na3PO4 were dissolved in 30 g of NaH2PO4-Na2HPO4 buffer solution with pH = 6, and reacted at 30°C for 1 h. 1.5 g of TMOS was added to the above homogeneous solution, and the reaction was continued for 12 hours. After that, the solution was hydrothermaled at 120°C for 12 hours, filtered, washed and dried. After drying, the solution was calcined at 500°C for 5 hours to obtain a pore size of 70 nm and a specific surface area of ​​174 m 2 / g, pore volume 3.1cm 3 / g porous silica nanomaterials.

[0052] Example 4: Preparation of porous silicon-based nanomaterials

[0053] 0.7g of block copolymer F-127, 0.3g of CTAB and 1.4g of Na3PO4 were dissolved in 30g of NaH2PO4-Na2HPO4 buffer solution with pH=6, and reacted at 50°C for 1h. 1.0g of TEOS was added to the above homogeneous solution, and the reaction was continued for 12 hours. Then, the solution was hydrothermaled at 120°C for 12 hours, filtered, washed and dried. After drying, the solution was calcined at 500°C for 5 hours to obtain a pore size of 10nm and a specific surface area of ​​332m 2 / g, pore volume 1.0cm 3 / g porous silica nanomaterials.

[0054] Example 5: Porous silicon-based nanomaterials for proteomics sample preparation of fetal bovine serum (FBS)

[0055] Step 1: prepare a high-concentration mother solution of porous silicon-based nanomaterials, mix an equal volume of the mother solution of porous silicon-based nanomaterials prepared in Example 1 of the present invention and an equal volume of fetal bovine serum sample, and then place the mixture in a constant temperature mixer at 37° C. and 1000 rpm for incubation for 20 min, centrifuge to remove the supernatant, and wash the precipitate three times;

[0056] Step 2: Add 10 mM sodium deoxycholate (SDC), 5 mM tris(2-carboxyethyl)phosphine hydrochloride (TCEP-HCl), and 20 mM iodoacetamide (IAA) to the sample in step 1, and place in a constant temperature mixer at 95°C for 10 min;

[0057] Step 3: After the sample returns to room temperature, add trypsin and perform enzymatic hydrolysis at 37°C, 1000 rpm for 2 h;

[0058] Step 4: After the enzymatic hydrolysis is completed, a formic acid aqueous solution with a final concentration of 1% is added to stop the enzymatic hydrolysis reaction, and the mixture is centrifuged at 13000g for 3 minutes, and the supernatant is aspirated;

[0059] Step 5: After the enzymatic hydrolysate was desalted using a C18 desalting column, the sample was concentrated, reconstituted with 0.1% formic acid aqueous solution, and protein identification was performed using LC-MS / MS.

[0060] After the protein in the sample was enriched by porous silicon-based nanomaterials and sample pretreatment, the results of LC-MS / MS detection were analyzed ( Figure 2 a) Compared with serum samples without material treatment and samples treated with commercial kits, the protein sample preparation process based on porous silicon-based nanomaterials identified more proteomes.

[0061] Example 6: Porous silicon-based nanomaterials for proteomics sample preparation of plasma samples

[0062] Step 1: prepare a high-concentration mother solution of porous silicon-based nanomaterials, mix equal volumes of the mother solution of porous silicon-based nanomaterials prepared in Example 1 of the present invention and equal volumes of plasma samples, and then place them on a constant temperature mixer at 37° C. and 1000 rpm for incubation for 20 min, centrifuge to remove the supernatant, and wash the precipitate three times;

[0063] Step 2: Add 10 mM sodium deoxycholate (SDC), 5 mM tris(2-carboxyethyl)phosphine hydrochloride (TCEP-HCl), and 20 mM iodoacetamide (IAA) to the sample in step 1, and place in a constant temperature mixer at 95°C for 5 min;

[0064] Step 3: After the sample returns to room temperature, add trypsin and perform enzymatic hydrolysis at 37°C, 1000 rpm for 2 h;

[0065] Step 4: After the enzymatic hydrolysis is completed, a formic acid aqueous solution with a final concentration of 1% is added to stop the enzymatic hydrolysis reaction, and the mixture is centrifuged at 13000g for 3 minutes, and the supernatant is aspirated;

[0066] Step 5: After the enzymatic hydrolysate was desalted using a C18 desalting column, the sample was concentrated, reconstituted with 0.1% formic acid aqueous solution, and protein identification was performed using LC-MS / MS.

[0067] After the protein in the sample was enriched by porous silicon-based nanomaterials and sample pretreatment, the results of LC-MS / MS detection were analyzed ( Figure 2 b), compared with plasma samples that have not been treated with the material and samples treated with commercial kits, the number of protein groups identified by the protein sample preparation process based on porous silicon-based nanomaterials is greater, indicating that the porous silicon-based nanomaterials in the present invention have the advantages of low cost, simplicity and high efficiency in protein sample preparation.

[0068] The protection content of the present invention is not limited to the above embodiments. Without departing from the spirit and scope of the present invention, changes and advantages that can be thought of by those skilled in the art are included in the present invention and are protected by the attached claims.

Claims

1. A porous silicon-based nanomaterial, characterized in that: The materials include silicon-based nanomaterials with mesoporous structures and / or macroporous structures and porous silicon-based nanomaterials with surface functional group modification.

2. The porous silicon-based nanomaterial according to claim 1, characterized in that: The pore size of the mesoporous structure is 2-50 nm; the pore size of the macroporous structure is >50 nm; and / or, The porous silicon-based nanomaterial has a pore size of 2-200 nm and a specific surface area of ​​100-800 m 2 / g, pore volume 0.5-5.0cm 3 / g.

3. A method for preparing a porous silicon-based nanomaterial, characterized in that: The method comprises the following steps: step (1): dissolving a surfactant and an inorganic salt in a buffer solution to obtain a uniform solution, adding a silicon source and continuing the reaction for a certain period of time to form a uniform solution; Step (2): subjecting the uniform solution obtained in step (1) to hydrothermal treatment; Step (3): Filter the solution obtained in step (2), wash, dry and then calcine to obtain a porous silica material.

4. The method according to claim 3, characterized in that In the step (1), The surfactant is selected from one or more of quaternary ammonium salt cationic surfactants, primary amine salt cationic surfactants, long-chain alkyl sulfate, phosphate, carboxylate anionic surfactants, long-chain alkylamine, polyoxyethylene ether and block copolymer nonionic surfactants; The inorganic salt is selected from one or more of sulfates, phosphates, carbonates and carboxylates; The concentration of the inorganic salt is 0.01-1.0M; The silicon source is selected from tetramethyl orthosilicate TMOS and tetraethyl orthosilicate TEOS; The mass ratio of the surfactant to the silicon source is (0.5-2):1; The reaction temperature is 25-70°C; and / or, The reaction time is 10-48 hours.

5. The method according to claim 3, characterized in that In the step (2), the temperature of the hydrothermal treatment is 50-200° C.; and / or the time of the hydrothermal treatment is 10-48 hours.

6. The method according to claim 3, characterized in that In the step (3), the drying temperature is 25-70°C; the drying time is 5-12 hours; the calcination temperature is 300-800°C; and / or the calcination time is 2-10 hours.

7. The porous silicon-based nanomaterial prepared by the method according to any one of claims 3 to 6.

8. Use of the porous silicon-based nanomaterial according to claim 1, 2 or 7 in protein enrichment and in proteomics sample preparation.

9. The use according to claim 8, characterized in that In the described application, the protein denaturation, reduction and alkylation steps are integrated into one step.

10. A method for preparing a proteomics sample in a biological sample using the porous silicon-based nanomaterial according to claim 1, 2 or 7, characterized in that: The porous silicon-based nanomaterial as claimed in claim 1, 2 or 7 is added to a protein sample, and the proteomics sample is obtained through protein enrichment, protein cleavage, reduction and alkylation, protein digestion and protein desalting.

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