Biological application of Prussian blue biological magnetic beads in protein enrichment

Abstract

The invention relates to the technical field of biomedical materials, in particular to biological application of Prussian blue biological magnetic beads in the aspect of protein enrichment. According to the Prussian blue magnetic bead and the preparation method and application thereof, different metals are doped in the Prussian blue material, the magnetism of the Prussian blue material is enhanced, and effective enrichment of low-abundance protein in plasma can be achieved.

Description

Technical Field

[0001] The invention relates to the technical field of biomedical materials, and in particular to the biological application of Prussian blue biomagnetic beads in protein enrichment. Background Art

[0002] The development of proteomics has lagged behind genomics to a large extent, mainly due to the complexity of protein molecules themselves and the lack of equivalent amplification mechanisms for low-abundance proteins. This requires complex workflows, limits scalability, and makes comprehensive investigation of the plasma proteome challenging. Despite extensive research on the plasma proteome, relatively few new candidate biomarkers have been accepted as clinically useful. Although the exact number of plasma proteomes is unknown, estimates range from >10,000 proteins to proteins that may cover all concentrations over more than 10 orders of magnitude, from 35-50 mg / mL albumin to low-abundance proteins in the pg / mL range, which make comprehensive investigation of the plasma proteome challenging.

[0003] The broad dynamic range of proteins in biological samples remains a barrier to robust identification and quantification in the context of thousands of unique proteins and even more protein variants. Comprehensive, in-depth, and unbiased proteomic analysis of plasma and other biological samples has been performed by liquid chromatography-tandem mass spectrometry (LC-MS / MS). However, these methods often involve complex sample preparation workflows. More efficient techniques, such as immunoassays and nontargeted LC-MS / MS proteomic strategies (which do not require complex fractionation methods), can increase the number of samples processed, but the breadth and depth of proteomic coverage are still insufficient.

[0004] There are several strategies for removing high-abundance proteins in plasma: ① Add chemical reagents that can cause protein aggregation and precipitation to plasma samples, remove precipitated proteins by centrifugation, and analyze the supernatant by mass spectrometry. Its main advantages are simple operation, low cost, and no species restrictions. Although the precipitation method is simple to operate and widely used, its low specificity and the easy loss of low-abundance proteins limit its wide application. ② Use monoclonal or polyclonal antibodies to specifically remove high-abundance proteins in plasma, thereby purifying plasma samples. Antibody-based immunoaffinity methods have defects such as high cost, limited sample loading capacity, and decreased column efficiency when overloaded, and immobilized antibodies may not be able to recognize certain subtypes of high-abundance proteins. ③ Nanomaterials have attracted much attention in the field of low-abundance protein enrichment. The main principle is that after specially modified nanomaterials come into contact with plasma, a large amount of protein can be adsorbed on the surface to form a nanomaterial-protein complex. According to the "Vroman effect", proteins with stronger binding affinity will competitively replace proteins adsorbed earlier, thereby enriching a variety of low-abundance proteins and effectively avoiding the loss of protein information. However, the Au and Ag nanoparticles currently used cannot be magnetically enriched. Although ferroferric oxide nanomaterials can be magnetically enriched, the synthesized materials are prone to aggregation (ACS Nano. 14(11): 15723-15737. (2020); Nat Commun. 11(1): 3662. (2020); Adv Mater. e2206008. (2022)). Therefore, it is necessary to develop a more stable magnetic enrichment material for the enrichment of low-abundance proteins in plasma. Summary of the invention

[0005] In view of this, the technical problem to be solved by the present invention is to provide biological applications of Prussian blue biomagnetic beads in protein enrichment.

[0006] The magnetic beads provided by the invention have a particle size of 5nm-50μm, contain manganese ions and Prussian blue, and the manganese ions and the cyano groups in the Prussian blue are coordinated and arranged with each other.

[0007] Prussian blue is an important complex with rich chemical and physical properties. It has a simple chemical structure and is easy to prepare. Its structure is a typical bridge coordination system, in which iron ions and cyanide ions are connected to each other through oxygen atoms. This structure gives Prussian blue strong reducing properties, anion exchange capacity and dispersion ability, as well as high strength and good transparency. It can be widely used in electrochemistry, biology, materials science and other fields.

[0008] The present invention can improve the magnetism of Prussian blue by doping and modifying Prussian blue with manganese ions, and can further improve the performance of magnetic beads by adjusting the ratio of manganese ions to cyano groups. In some embodiments, the molar ratio of manganese ions to cyano groups is 1:(1-5). For example, the molar ratio of manganese ions to cyano groups is 1:1, or 1:2, or 1:3, or 1:4, or 1:5.

[0009] The present invention does not limit the morphology of the magnetic beads. For example, the morphology of the magnetic beads includes at least one of spherical, fibrous, rod-shaped, block-shaped, layered, star-shaped, shuttle-shaped, polyhedral, nanoflower-shaped, hollow block-shaped, cubic, and needle-shaped.

[0010] The diameter of the magnetic beads of the present invention is 5 nm to 50 μm. In a specific embodiment, the particle size of the magnetic beads is 50 to 200 nm, non-limiting examples include: 5 nm, 50 nm, 80 nm, 100 nm, 200 nm, 500 nm, 1 μm, 2 μm, 4 μm, 5 μm, 10 μm or 50 μm.

[0011] Prussian blue, chemically known as ferric ferrocyanide or ferric hexacyanoferrate, is used in the preparation of magnetic beads using Prussian blue, which can overcome the problems of poor monodispersity, easy aggregation, poor particle size uniformity caused by surface modification, complex preparation process, high cost, etc. of existing ferroferric oxide magnetic beads. In order to further improve the magnetism, manganese ions are also doped in the magnetic beads, thereby improving the controllability of the Prussian blue magnetic beads. Furthermore, the present invention uses the prepared magnetic beads to achieve effective enrichment of low-abundance proteins; and the preparation method of the magnetic beads provided by the present invention is simple.

[0012] The preparation method of pullulan magnetic beads provided by the present invention comprises:

[0013] Mix Triton-100, n-hexanol (or butanol / pentanol / hexanol) and cyclohexane (or cyclohexanone, toluene-cyclohexanone) to obtain a solvent;

[0014] Dissolving a polymerization agent and potassium ferrocyanide in the solvent to obtain a solution A;

[0015] Dissolving a manganese salt and a divalent iron salt in the solvent to obtain a solution B;

[0016] The solution A and the solution B are mixed and reacted to obtain pullulan magnetic beads.

[0017] The synthesis principle of the Prussian blue magnetic beads of the present invention is as follows Figure 6The preparation method has a simple process, does not require complicated reaction equipment and operation means, has low cost, is suitable for industrial mass production, and has a stable application effect, solving the problems of large batch differences and complex preparation of existing magnetic beads.

[0018] The polymerizing agent is selected from at least one of polyacrylamide, polymethyl methacrylate, polystyrene, polymethylstyrene, polyimide, polyethylene terephthalate, polyvinyl pyrrolidone, polyethylene glycol, and polylactic acid. As a feasible case, the polymerizing agent is polyvinyl pyrrolidone.

[0019] As a feasibility case, the divalent iron salt is selected from at least one of ferrous chloride or ferrous sulfate; in some embodiments, the molar ratio of the divalent iron salt to the potassium ferrocyanide is 1:(1-5). Non-limiting examples include: 1:1, 1:2, 1:3, 1:5, etc., preferably 1:1; by limiting the molar ratio of the divalent iron salt to potassium ferrocyanide, it can be ensured that the metal ions are uniformly doped on the surface of the magnetic beads; if the content of potassium ferrocyanide is too high or too low, it will cause an imbalance between the trivalent iron ions and the divalent iron ions of the Prussian blue magnetic beads, thereby affecting the magnetic properties of the Prussian blue magnetic beads;

[0020] As a feasible case, the manganese salt is manganese chloride. Manganese ions will cause changes in the microstructure of Prussian blue magnetic beads, affecting their magnetism. In some embodiments, the amount of manganese salt added is 0.01-0.1 mol / L; non-limiting examples include: 0.01 mol / L, 0.02 mol / L, 0.05 mol / L, 0.08 mol / L, 0.1 mol / L, etc.

[0021] As a feasible example, the mass ratio of Triton-100, n-hexanol and cyclohexane in the solvent is (2-6):(1-3):(1-3). For non-limiting example, the mass ratio of Triton-100, n-hexanol and cyclohexane is 2:1:1.

[0022] As a feasible example, the mass ratio of the polymerization agent to the potassium ferrocyanide is (1.2-2):1; non-limiting examples include: 1.2:1, 1.5:1, 1.8:1, 2:1, etc.;

[0023] As a feasibility case, the reaction temperature of the solution A and the solution B is 20°C to 400°C. Preferably, the reaction temperature of the solution A and the solution B is 120°C to 160°C. Non-limiting examples include: 20°C, 80°C, 100°C, 120°C, 150°C, 200°C, 280°C, 300°C, 350°C, 400°C, etc.; the reaction time is 10min to 20h. Preferably, the reaction time is 2h to 12h. Non-limiting examples include: 10min, 30min, 1h, 2h, 5h, 8h, 10h, 12h, 15h, 20h, etc. In the embodiment of the present invention, the controllability of the size and morphology of the Prussian blue core particles can be achieved by controlling the reaction temperature and the reaction time, and the Prussian blue magnetic beads developed thereby have multiple sizes and multiple morphologies.

[0024] Specifically, the method for preparing the Prussian blue material of the embodiment of the present invention comprises the following steps:

[0025] (1) Mixing Triton-100, n-hexanol and cyclohexane to obtain a solvent;

[0026] (2) adding a polymerization agent and potassium ferrocyanide to the solvent to obtain a solution A;

[0027] (3) adding a manganese salt and a divalent iron salt to the solvent to obtain a solution B;

[0028] (4) The solution A and the solution B are mixed and reacted to obtain Prussian blue magnetic beads.

[0029] In some embodiments, step (2) further comprises the step of adding a surface modifier to the solvent. Preferably, the surface modifier comprises at least one of polyethyleneimine and aminopolyethylene glycol carboxyl. Preferably, the amount of the surface modifier is 0.01 to 0.5 mmol / L.

[0030] Furthermore, the present invention also provides a protein enrichment reagent, which includes the Prussian blue magnetic beads, dilution buffer and magnetic bead lysis buffer as described above;

[0031] The dilution buffer is PBS buffer or a mixture of Tris-HCl, EDTA, KCl and CHAPS.

[0032] The magnetic bead lysis buffer comprises water and sodium deoxycholate, 3-(2-carboxyethyl)phosphine, 2-chloroacetamide and Tris-HCl.

[0033] In the present invention, the Prussian blue magnetic beads are in the form of a suspension, and the suspension buffer is a PBS buffer. In the present invention, the content of the magnetic beads is 1 to 10 mg / ml, for example, the content is 1 mg / ml, 2 mg / ml, 3 mg / ml, 4 mg / ml, 5 mg / ml, 6 mg / ml, 7 mg / ml, 8 mg / ml, 9 mg / ml or 10 mg / ml.

[0034] In the present invention, the magnetic bead lysis buffer comprises water and 0.1wt% to 2wt% sodium deoxycholate, 5 to 15mM 3-(2-carboxyethyl)phosphine, 30 to 50mM 2-chloroacetamide and 50 to 150mM Tris-HCl.

[0035] As a feasibility case, the mass of sodium deoxycholate in the magnetic bead lysis buffer is 0.1wt%, 0.5wt%, 0.8wt%, 0.9wt%, 1.0wt%, 1.2wt%, 1.5wt% or 2.0wt%.

[0036] As a feasibility case, the concentration of 3-(2-carboxyethyl)phosphine in the magnetic bead lysis buffer is 5mM, 6mM, 7mM, 8mM, 9mM, 10mM, 11mM, 12mM, 13mM, 14mM or 15mM.

[0037] As a feasibility case, the concentration of 2-chloroacetamide in the magnetic bead lysis buffer is 30mM, 35mM, 37mM, 38mM, 39mM, 40mM, 41mM, 42mM, 43mM, 45mM or 50mM.

[0038] As a feasibility case, the concentration of Tris-HCl in the magnetic bead lysis buffer is 50mM, 60mM, 70mM, 80mM, 90mM, 100mM, 110mM, 120mM, 130mM, 140mM or 150mM.

[0039] In a specific embodiment, the magnetic bead lysis buffer includes water and 1wt% sodium deoxycholate, 10mM 3-(2-carboxyethyl)phosphine, 40mM 2-chloroacetamide and 100mM Tris-HCl;

[0040] As a feasibility case, the enrichment reagent also includes an extraction reagent, which contains trifluoroacetic acid, acetonitrile and water, wherein the volume ratio of acetonitrile to water is (75-85): (15-25), and the mass fraction of trifluoroacetic acid is 0.05%-0.15%. In a specific embodiment, the volume ratio of acetonitrile to water is 75:25, 80:20 or 85:15, and the mass fraction of trifluoroacetic acid is 0.05%, 0.1% or 0.15%.

[0041] As a feasibility case, the enrichment reagent also includes a pretreatment reagent. The pretreatment reagent includes a tissue lysis reagent. In some embodiments, the tissue lysis reagent is a mixture of RIPA solution and PMSF solution. Preferably, the sample to solution ratio is (50-100 mg): 100 μL.

[0042] Furthermore, the present invention also provides a method for enriching proteins, which comprises: enriching proteins in a sample with the enrichment reagent as described above.

[0043] Specifically, the enrichment of the present invention includes:

[0044] The Prussian blue magnetic beads prepared by the preparation method described above are washed with the dilution buffer, added to the sample, and separated from the magnetic beads after incubation;

[0045] The magnetic beads are treated with a magnetic bead lysis buffer, hydrolyzed with a protease, and then extracted to obtain enriched proteins.

[0046] In the present invention, the protease is trypsin.

[0047] In the present invention, the sample includes blood, body fluid, animal tissue and / or secretion.

[0048] In the present invention, the volume ratio of the Prussian blue magnetic bead suspension to the diluted sample is 1:(15-25). For example, the volume ratio is 1:15, 1:20 or 1:25.

[0049] In the present invention, the incubation temperature is 37°C, the incubation time is 30min to 3h, and the shaking speed of the incubation device is 300 to 1000rpm;

[0050] In the present invention, the lysis buffer has the function of reducing alkylation, and the amount of the lysis buffer added is 10-100 μL / 1-10 mg magnetic beads. The incubation temperature after adding the solution is 95° C., and the incubation time is 5 min to 30 min.

[0051] In the present invention, the amount of trypsin added is 0.5-2 μL / 1-10 mg magnetic beads, the incubation temperature after adding the trypsin is 37° C., and the incubation time is 30 min to 3 h.

[0052] In a specific embodiment, the sample is blood, and the enrichment method includes:

[0053] (a) diluting plasma with a diluent;

[0054] (b) washing the magnetic beads as described above with the diluent, and then separating and collecting the Prussian blue magnetic beads;

[0055] (c) adding the Prussian blue magnetic beads obtained in step (b) to the diluted plasma in step (a), incubating on a shaking table, then separating by magnetic treatment to discard the supernatant, reducing and alkylating, and adding trypsin for incubation;

[0056] (d) After the incubation in step (c) is completed, the extract is eluted with an extraction solution, and the supernatant is collected to obtain the enriched protein.

[0057] In this embodiment, in step (a), the solution used to dilute the plasma is a mixture of Tris-HCl, EDTA, KCl and CHAPS. Preferably, the volume ratio of the solution to the plasma is (30-40):1.

[0058] In this embodiment, in the step (c), the volume ratio of the Prussian blue magnetic beads to the diluted plasma is 1:(15-25);

[0059] In this embodiment, in the step (d), the extracting solution comprises 60% to 90% acetonitrile solution.

[0060] In a specific embodiment, the sample is saliva, and the enrichment method includes:

[0061] (a) diluting saliva with a diluent;

[0062] (b) washing the magnetic beads as described above with the diluent, and then separating and collecting the Prussian blue magnetic beads;

[0063] (c) adding the Prussian blue magnetic beads obtained in step (b) to the diluted saliva in step (a), incubating on a shaking table, then separating by magnetic treatment to discard the supernatant, reducing and alkylating, and adding trypsin for incubation;

[0064] (d) After the incubation in step (c) is completed, elution is performed using an elution buffer, and the supernatant is collected to obtain enriched salivary proteins.

[0065] In this embodiment, in the step (a), the solution used to dilute the saliva is a mixture of Tris-HCl, EDTA, KCl and CHAPS. Preferably, the volume ratio of the solution to the saliva is (30-40):1.

[0066] In this embodiment, in the step (c), the volume ratio of the Prussian blue magnetic beads to the diluted saliva is 1:(15-25);

[0067] In this embodiment, in the step (d), the extracting solution comprises 60% to 90% acetonitrile solution.

[0068] In a specific embodiment, the sample is an animal tissue, and the enrichment method includes:

[0069] (a) Sample pretreatment;

[0070] (b) washing the magnetic beads as described above with the diluent, and then separating and collecting the Prussian blue magnetic beads;

[0071] (c) adding the Prussian blue magnetic beads obtained in step (b) to the diluted sample solution in step (a), incubating on a shaking table, then separating by magnetic treatment to discard the supernatant, reducing and alkylating, and adding trypsin for incubation;

[0072] (d) After the incubation in step (c) is completed, elution is performed using an elution buffer, and the supernatant is collected to obtain enriched tissue proteins.

[0073] In this embodiment, the animal tissue is a tissue from the human body or a tissue from an animal other than humans. The animal includes but is not limited to mice, rats, guinea pigs, rabbits, dogs, cats, rhesus monkeys, cynomolgus monkeys, miniature pigs, golden hamsters, Mongolian gerbils, quail, chickens, zebrafish, African clawed frogs, fruit flies, nematodes (Caenorhabditis elegans) or amphioxus. The animal tissue includes but is not limited to synovial tissue, skin tissue, muscle tissue, bone tissue, liver tissue, heart tissue, lung tissue, kidney tissue, spleen tissue, stomach tissue, intestinal tissue, pancreatic tissue, bladder tissue, gallbladder tissue, brain tissue, spinal cord tissue, vascular tissue, adipose tissue, eyeball tissue, thyroid tissue or adrenal tissue.

[0074] In this embodiment, the sample pretreatment includes lysis and dilution of the sample tissue, the solution for lysing tissue protein is radioimmunoprecipitation buffer (RIPA) and phenylmethylsulfonyl fluoride (PMSF) solution, preferably, the sample to solution ratio is (50-100 mg): 100 μL. The solution used for diluting the tissue is a mixture of Tris-HCl, EDTA, KCl and CHAPS, preferably, the volume ratio of the solution to plasma is (30-40): 1.

[0075] In this embodiment, in the step (c), the volume ratio of the Prussian blue magnetic beads to the diluted tissue is 1:(15-25);

[0076] In this embodiment, in the step (d), the extracting solution comprises 60% to 90% acetonitrile solution.

[0077] The method for enriching proteins provided in the embodiment of the present invention can produce a Prussian blue material that can effectively enrich sample proteins. The method in the embodiment of the present invention is simple and easy to operate, and has a good protein enrichment effect.

[0078] Furthermore, the present invention also provides a method for detecting protein, which comprises enriching the protein in the sample by the enrichment method as described above and then detecting the protein.

[0079] In the present invention, the protein detection method adopts chromatography, mass spectrometry, chromatography-mass spectrometry, Kjeldahl nitrogen determination, biuret method, Coomassie brilliant blue method, ultraviolet absorption method, fluorescence method, SDS-polyacrylamide gel electrophoresis, isoelectric focusing electrophoresis, two-dimensional electrophoresis, immunoblotting, enzyme-linked immunosorbent assay or radioimmunoassay.

[0080] In the present invention, protein detection is performed by DIA detection. The DIA loading solution is a 0.05% to 0.2% formic acid solution. For example, it is a 0.05% formic acid solution, or a 0.1% formic acid solution, or a 0.15% formic acid solution, or a 0.2% formic acid solution.

[0081] In the present invention, the detection of the protein may be for diagnostic purposes or non-diagnostic purposes, which is not limited in the present invention. The non-diagnostic purposes include, but are not limited to, detection for scientific research purposes or detection of proteins that cannot be used as disease markers.

[0082] The invention provides Prussian blue magnetic beads and a preparation method and application thereof. The invention enhances the magnetic properties of the Prussian blue material by doping different metals into the Prussian blue material, thereby enabling effective enrichment of low-abundance proteins in plasma. BRIEF DESCRIPTION OF THE DRAWINGS

[0083] Figure 1 is a performance diagram of the Prussian blue material obtained in Example 1, wherein Figure 1 Figure a is a SEM image. Figure 1 b is the magnetization intensity diagram, Figure 1 In the figure c is the particle size distribution diagram. Figure 1 d in the figure is the infrared spectrum;

[0084] Figure 2 This is a diagram showing the enrichment effect of plasma and commercially available products on plasma proteins in Example 1, wherein Figure 2 The figure a in the middle is the number of identified proteins. Figure 2 Middle b is a graph showing the percentage of high / low abundance proteins in the identified proteins;

[0085] Figure 3 This is a diagram showing the enrichment effect of plasma and commercially available products on high-abundance proteins in plasma in Example 1;

[0086] Figure 4 This is a diagram showing the enrichment effect of saliva and commercially available products on salivary proteins in Example 1, wherein Figure 4 The figure a in the middle is the number of identified proteins. Figure 4b is a Venn diagram. Figure 4 Middle c is the classification diagram of the identified proteins;

[0087] Figure 5 This is a diagram showing the enrichment effect of synovial protein in patients with osteoarthritis and rheumatoid arthritis in Example 1, wherein Figure 5 In the middle, a is a volcano map. Figure 5 b is the principal component analysis diagram, Figure 5 Middle c is the differential protein heat map, Figure 5 d in the middle is the classification diagram of the identified proteins;

[0088] Figure 6 It is the technical route for the synthesis of Prussian blue magnetic beads. DETAILED DESCRIPTION

[0089] The present invention provides biological applications of Prussian blue biomagnetic beads in protein enrichment. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It should be particularly noted that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications of this article without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0090] Where values ​​are described herein as a range, it should be understood that such disclosure includes disclosure of all possible sub-ranges within that range, as well as specific values ​​falling within that range, regardless of whether a specific value or a specific sub-range is explicitly stated.

[0091] Herein, the words "comprises" and "includes" and variations thereof mean that other elements or integers which are permitted but not specifically described may be included.

[0092] In this article, the term "and / or" is merely a term used to describe the association relationship of associated objects, indicating that three relationships may exist. For example, A and / or B may represent three situations: A exists alone, A and B exist at the same time, and B exists alone.

[0093] The application of protein enrichment based on Prussian blue provided in an embodiment of the present invention comprises the following steps:

[0094] (1) Synthesize Prussian blue biomagnetic beads; (2) Incubate with samples; (3) Wash and enzymatically digest the incubated magnetic beads; (4) Protein spectrum analysis. This separation method is simple to operate and is applicable to multiple fields such as plasma, saliva, and tissues, and can effectively enrich low-abundance proteins in plasma.

[0095] In this article, the term "magnetic beads" refers to a tiny, magnetic particle material, whose core is composed of Prussian blue and metal ions, which can respond to and be effectively manipulated under the action of an external magnetic field. The surface of the magnetic beads can be modified with specific functional groups or bioactive molecules. These modifications give the magnetic beads unique functions in biochemical detection. For example, they can specifically bind to target biological molecules (such as nucleic acids, proteins, antigens, antibodies, etc.), and use magnetic separation technology to quickly separate the magnetic beads bound to target molecules from complex biological sample systems, so as to facilitate subsequent detection, analysis and identification of target molecules, thereby greatly improving the efficiency, accuracy and sensitivity of biochemical detection.

[0096] In this article, the term "reagent" refers to a functional composition used for biochemical detection. Among them, the reagent containing magnetic beads can also include other auxiliary reagents. For example, in addition to magnetic beads, the reagent also contains a buffer system to maintain appropriate pH and ionic strength to ensure the activity of biological molecules and the smooth progress of reactions; it may contain enzyme substances to catalyze specific biochemical reactions; there will also be protective agents to prevent the degradation or inactivation of biological molecules; and some auxiliary components to help improve the sensitivity, accuracy and stability of the detection, etc., and the overall synergistic effect can achieve efficient detection and analysis of target components in biological samples.

[0097] In this article, the term "enrichment" is a general term for a series of operations aimed at increasing the concentration, purity or detectability of a target in a sample. It covers operations such as extraction, separation, and purification, for example: selectively extracting or transferring a target from a complex mixed system using the magnetic beads of the present invention; and may also include steps to further achieve a higher purity or uniformity of the target.

[0098] The numerical ranges and parameters involved in the present invention have been presented in the specific embodiments as accurately as possible. However, any numerical value inherently inevitably contains standard deviations due to individual testing methods. Therefore, unless otherwise explicitly stated, it should be understood that all numerical ranges or specific data used in this disclosure may have certain reasonable deviations within a certain range, for example: within ±10%, ±5%, ±1% or ±0.5%.

[0099] In this document, the terms "one embodiment", "some embodiments", "examples", "specific examples", or "some examples" etc. mean that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, unless they are contradictory.

[0100] It should be understood that in various embodiments of the present application, the size of the sequence number of each process does not mean the order of execution, some or all of the steps can be executed in parallel or in sequence, and the execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present application. The test materials used in the present invention are all common commercial products and can be purchased on the market. The present invention is further described below in conjunction with the embodiments:

[0101] Example 1

[0102] (1) 4 g of Triton-100, 2 g of n-hexanol, and 2 g of cyclohexane were thoroughly mixed to obtain a first solution, wherein the mass ratio of Triton-100: n-hexanol: cyclohexane was 2:1:1;

[0103] (2) adding 5 g of PVP and 3.3 g (0.01 mol) of potassium ferrocyanide to the first solution, stirring and dissolving, to obtain a second solution;

[0104] (3) adding 0.6 g (0.01 mol) of manganese chloride and 2.6 g (0.01 mol) of ferrous chloride to the first solution, stirring and dissolving, to obtain a third solution, wherein the molar ratio of manganese chloride, ferrous chloride and potassium ferrocyanide is 1:1:1;

[0105] (4) mixing the second solution and the third solution and reacting them in an oil bath at 120° C. with stirring for 2 h;

[0106] (5) The product obtained in step (4) was sonicated for 15 min, suspended in 20 mL of ultrapure water, and centrifuged at 5000 rpm for 10 min. This was repeated three times to obtain Mn-doped magnetic Prussian blue nanoparticles (hereinafter referred to as NPs).

[0107] Example 2

[0108] (1) Place ~1 mL of fresh blood in a vacuum coagulation tube and centrifuge at 1300 g for 10 min at room temperature to remove blood cells and debris. Collect the supernatant;

[0109] (2) Take the supernatant obtained in step (1), centrifuge at 4000g for 15 min at room temperature to deplete platelets and collect plasma.

[0110] (3) Take 50 μL of the plasma obtained in step (2) and dilute it with PBS buffer to a 200 μL system. The NPs prepared in Example 1 were ultrasonically treated in deionized water for 10 min and then vortexed for 3-5 s. 20 μL of NP suspension (5 mg / ml, resuspended in PBS) was mixed with 200 μL of the diluted biological sample and incubated at 37°C with shaking at 300 rpm for 1 h. After incubation, the centrifuge tube was placed on a magnetic collection device for 5 min and magnetic separation was performed to remove unbound proteins in the supernatant. The magnetic beads were magnetically separated three times with 200 μL of PBS buffer.

[0111] (4) The separated magnetic beads were lysed by adding 200 μL of lysis buffer containing 1% sodium deoxycholate (DOC), 10 mM 3-(2-carboxyethyl)phosphine (TCEP), 40 mM 2-chloroacetamide (CAA) and 100 mM Tris-HCL (pH 8.5). After thorough vortexing, the mixture was heated at 95°C for 5 min. Ultrasonic scanning was performed at 30% power for 5 minutes using a handheld ultrasonic device. The lysate was then centrifuged at 16,000 g for 10 min.

[0112] (5) Transfer the supernatant obtained in step (4) to a new centrifuge tube, add 1 μg of trypsin, and digest at 37°C for 4 h.

[0113] (6) Add 200 μL of 80% acetonitrile and 0.1% trifluoroacetic acid solution for extraction, incubate at 37°C with shaking at 300 rpm for 5 min, repeat twice, heat dry, add 100 μL of 0.1% trifluoroacetic acid to completely dissolve, and then desalt.

[0114] (7) After adding 12 μL of loading solution (0.1% formic acid solution) to completely dissolve, the peptide concentration was determined. 2 μg was loaded and the effective detection time of DIA was 90 min.

[0115] Example 3

[0116] (1) Participants were asked to refrain from eating, chewing, and brushing their teeth for at least 2 hours. Each person was asked to rinse their mouth with water for about 2 minutes, then wait for 10 minutes, collect the sample, and centrifuge at 10,000 g for 10 minutes to collect saliva;

[0117] (2) The sample was suspended in lysis buffer (1% sodium deoxycholate (SDS), 8 M urea), mixed thoroughly, and then placed on ice for 30 min. During this period, the sample was vortexed once every 10 min and centrifuged at 12,000 g for 20 min at 4°. The supernatant was collected and the concentration was determined by BCA.

[0118] (3) 50 μg of protein was extracted and diluted with PBS buffer to a 200 μL system. The NPs prepared in Example 1 were ultrasonicated in deionized water for 10 min and then vortexed for 3-5 s. 20 μL of NP suspension (5 mg / ml) was mixed with 200 μL of the diluted biological sample and incubated at 37°C with shaking at 300 rpm for 1 h. After incubation, the centrifuge tube was placed on a magnetic collection device for 5 min and magnetic separation was performed to remove unbound proteins in the supernatant. The magnetic beads were magnetically separated three times with 200 μL of PBS buffer.

[0119] (4) The separated magnetic beads were lysed by adding 200 μL of lysis buffer containing 1% sodium deoxycholate (DOC), 10 mM tris(2-carboxyethyl)phosphine (TCEP), 40 mM 2-chloroacetamide (CAA) and 100 mM Tris-HCL (pH 8.5). After thorough vortexing, the mixture was heated at 95°C for 5 min. Ultrasonic scanning was performed at 30% power for 5 minutes using a handheld ultrasonic device. The lysate was then centrifuged at 16,000 g for 10 min.

[0120] (5) Transfer the supernatant to a new centrifuge tube and add 1 μg of trypsin. Digest at 37°C for 16 h.

[0121] (6) Add 200 μL of 80% acetonitrile and 0.1% trifluoroacetic acid solution for extraction, incubate at 37°C with shaking at 300 rpm for 5 min, repeat twice, heat dry, add 100 μL of 0.1% trifluoroacetic acid to completely dissolve, and then desalt.

[0122] (7) After adding 12 μL of loading solution (0.1% formic acid solution) to completely dissolve, the peptide concentration was determined. 2 μg was loaded and the effective detection time of DIA was 90 min.

[0123] Example 4

[0124] 100 mg of synovium from patients after surgery was ground with liquid nitrogen, and 200 μL of RIPA lysis buffer was added and reacted on ice for 30 minutes. During this period, the sample was vortexed once every 10 minutes and centrifuged at 12,000 g for 20 minutes at 4°. The supernatant was collected and the concentration was determined by BCA.

[0125] Extract 20 μg of protein and dilute it to 200 μL system with PBS buffer + 0.05% 3-[3-(cholamidopropyl)dimethylamino]propanesulfonic acid inner salt (CHAPS). Ultrasonicate the NPs prepared in Example 1 in deionized water for 10 min and then vortex for 3-5 s. Mix 20 μL of NP suspension (5 mg / ml) with 200 μL of diluted biological sample and incubate at 37 ° C with 300 rpm shaking for 2 h. After incubation, place the centrifuge tube on a magnetic collection device for 5 min and perform magnetic separation to remove unbound proteins in the supernatant. Magnetic separation of magnetic beads was performed three times with 200 μL PBS buffer.

[0126] The separated magnetic beads were lysed in 200 μL of lysis buffer containing 1% sodium deoxycholate (DOC), 10 mM 3-(2-carboxyethyl)phosphine (TCEP), 40 mM 2-chloroacetamide (CAA) and 100 mM Tris-HCL (pH 8.5). After sufficient vortexing, the mixture was heated at 95°C for 5 min. Ultrasonic scanning was performed at 30% power for 5 minutes using a handheld ultrasonic device. The lysate was then centrifuged at 16000 g for 10 min.

[0127] The supernatant was transferred to a new centrifuge tube, and 1 μg of trypsin was added to digest the cells at 37°C for 6 h.

[0128] 200 μL of 80% acetonitrile 0.1% trifluoroacetic acid solution was added for extraction, and incubated at 37°C with shaking at 300 rpm for 5 min, repeated twice, and heat-dried. After complete dissolution, 100 μL of 0.1% trifluoroacetic acid was added and desalted.

[0129] After adding 12 μL of loading solution (0.1% formic acid solution) to completely dissolve, the peptide concentration was determined. 2 μg was loaded and the effective detection time of DIA was 60 min.

[0130] Performance Characterization

[0131] 1. The NPs prepared in Example 1 were subjected to magnetization intensity and infrared spectroscopy analysis, and the results were as follows: Figure 1 shown.

[0132] from Figure 1 It can be seen from a that the Prussian blue material prepared in Example 1 is evenly distributed. Figure 1 As can be seen from Figure b, manganese doping can maximize the magnetic properties of Prussian blue materials, thereby improving the enrichment effect of low-abundance proteins in plasma. Figure 1 As can be seen from c, the particle size of Prussian blue biomagnetic beads is about 100nm. Figure 1 It can be seen from the d that 2081 -1 and 2160cm -1The characteristic peaks at correspond to the cyanide bonds associated with Prussian blue and CN-FeII.

[0133] 2. The NPs prepared in Example 1 and the magnetic beads of commercially available products are used to enrich low-abundance proteins in plasma, comprising the following steps:

[0134] (1) Dilute 5 μL of plasma to 200 μL of solution B using solution A containing 10 mM Tris-HCl, 1 mM EDTA, 150 mM KCl, and 0.05% CHAPS;

[0135] (2) The NP prepared in Example 1 was prepared into a 10 mg / mL solution and the commercially available product was placed in centrifuge tubes, and then ultrasonicated for 10 min and vortexed for 1-2 min; then, 200 μL of solution A was added to the centrifuge tubes, the magnetic beads were suspended and placed on a magnetic collection device for 1 min, and the supernatant was discarded. This was repeated 3 times to collect the Prussian blue magnetic beads and the commercially available product respectively;

[0136] (3) Add 10 μL of Prussian blue magnetic beads / commercially available product to 200 μL of solution B, incubate on a shaker at 37°C and 300 rpm for 1 h, place on a magnetic collection device, discard the supernatant, and add 1 μg of trypsin and incubate at 37°C for 30 min;

[0137] (4) Elute three times with 75 μL 80% acetonitrile solution and collect the supernatant;

[0138] (5) The supernatant was dried by heat and then fully dissolved with 10 μL of 0.1% formic acid solution, and the peptide concentration was measured using Nanotrap;

[0139] (6) Proteomics detection was performed with a standard mass of 1 μg peptide, and the effective detection time was 60 min.

[0140] The enrichment effects of plasma, NPs prepared in Example 1 and commercially available products on low-abundance proteins are as follows: Figure 2-Figure 3 As shown. Figure 2 It can be seen from a that the amount of enriched protein in NP prepared in Example 1 is significantly better than that in commercial products. After five repeated experiments, the total amount of protein enriched in NP prepared in Example 1 exceeds that in pure plasma and commercial products. Figure 2 As can be seen from b, the proportion of low-abundance proteins in Example 1 exceeds 80%, the low-abundance proteins in commercially available products are less than 50%, and the low-abundance proteins detected in pure plasma account for less than 40% of the total proteins; Figure 3It can be seen that among the 10 common high-abundance proteins in plasma proteins, the high-abundance proteins enriched in Example 1 account for 11% of the total proteins, the high-abundance protein ratio of commercially available products is 46%, and the high-abundance proteins detected in pure plasma account for significantly higher than 60% of the total proteins, indicating that the NP prepared in Example 1 has a better enrichment effect on low-abundance proteins and is more suitable for enriching low-abundance proteins.

[0141] 3. The NPs prepared in Example 1 and commercially available products were used for the enrichment of salivary proteins, comprising the following steps:

[0142] (1) The sample was suspended in lysis buffer (1% sodium deoxycholate (SDS), 8 M urea), mixed thoroughly, and then placed on ice for 30 min. During this period, the sample was vortexed once every 10 min and centrifuged at 12,000 g for 20 min at 4°C. The supernatant was collected and the concentration was determined by BCA.

[0143] (2) 50 μg of protein was extracted and diluted with PBS buffer to a 200 μL system. The two magnetic nanoparticles were sonicated in deionized water for 10 min and then vortexed for 3-5 s.

[0144] (3) The NP prepared in Example 1 was prepared into a 10 mg / mL solution and the commercially available product were respectively placed in centrifuge tubes, ultrasonicated for 10 min, and then vortexed for 1-2 min; then, 200 μL of solution A was added to the centrifuge tubes, the magnetic beads were suspended and placed on a magnetic collection device for 1 min, the supernatant was discarded, and the process was repeated 3 times to collect the Prussian blue magnetic beads and the commercially available product respectively;

[0145] (4) Add 10 μL of Prussian blue magnetic beads / commercially available product of Example 1 to 200 μL of solution B, incubate on a shaker at 37°C and 300 rpm for 1 h, place on a magnetic collection device, and discard the supernatant.

[0146] (5) Add 200 μL of lysis buffer containing 1% sodium deoxycholate (DOC), 10 mM 3-(2-carboxyethyl)phosphine (TCEP), 40 mM 2-chloroacetamide (CAA) and 100 mM Tris-HCL (pH 8.5) for lysis. After thorough vortexing, the mixture was heated at 95°C for 5 min. Ultrasonic scanning was performed at 30% power for 5 min using a handheld ultrasonic device. The lysate was then centrifuged at 16,000 g for 10 min.

[0147] (6) Add 1 μg of trypsin and incubate at 37°C for 30 min;

[0148] (7) Elute three times with 75 μL 80% acetonitrile solution and collect the supernatant;

[0149] (8) The supernatant was dried by heat and then fully dissolved with 10 μL of 0.1% formic acid solution, and the peptide concentration was measured using Nanotrap;

[0150] (9) Proteomics detection was performed with a standard mass of 1 μg peptide, and the effective detection time was 60 min.

[0151] The enrichment effects of saliva, NPs prepared in Example 1 and commercially available products on salivary proteins are as follows: Figure 4 As shown. Figure 4 As can be seen from a, the amount of protein enrichment in the NP prepared in Example 1 is significantly better than that in commercially available products; Figure 4 As can be seen from b, the enriched protein of Example 1 basically contains salivary proteins of commercially available products; Figure 4 As can be seen from c, Example 1 tends to enrich acute phase proteins, and commercially available products tend to enrich immunoglobulins.

[0152] 4. The NP prepared in Example 1 is used for enrichment of synoviolin, comprising the following steps:

[0153] (1) 100 mg of synovium was ground in liquid nitrogen, and 200 μL of RIPA lysis buffer was added. The mixture was reacted on ice for 30 min. During this period, the sample was vortexed once every 10 min and centrifuged at 12,000 g for 20 min at 4°C. The supernatant was collected and the concentration was determined by BCA.

[0154] (2) Extract 20 μg of protein and dilute it into 200 μL system with 10 mM Tris-HCl, 1 mM EDTA, 150 mM KCl and 0.05% CHAPS;

[0155] (3) The NP prepared in Example 1 was prepared into a 10 mg / mL solution and placed in a centrifuge tube. After ultrasonication for 10 min, the solution was vortexed for 1 to 2 min. Then, 200 μL of solution A was added to the centrifuge tube, the Prussian blue magnetic beads prepared in Example 1 were suspended and placed on a magnetic collection device for 1 min, the supernatant was discarded, and the process was repeated 3 times to obtain Prussian blue magnetic beads.

[0156] (4) Add 10 μL of Prussian blue magnetic beads to 200 μL of solution B, incubate on a shaker at 37°C and 300 rpm for 1 h, place on a magnetic collection device, and discard the supernatant;

[0157] (5) Add 200 μL of lysis buffer containing 1% sodium deoxycholate (DOC), 10 mM 3-(2-carboxyethyl)phosphine (TCEP), 40 mM 2-chloroacetamide (CAA) and 100 mM Tris-HCL (pH 8.5) for lysis. After thorough vortexing, the mixture was heated at 95°C for 5 min. Ultrasonic scanning was performed at 30% power for 5 min using a handheld ultrasonic device. The lysate was then centrifuged at 16,000 g for 10 min.

[0158] (6) Add 1 μg of trypsin and incubate at 37°C for 30 min;

[0159] (7) Elute three times with 75 μL 80% acetonitrile solution and collect the supernatant;

[0160] (8) The supernatant was dried by heat and then fully dissolved with 10 μL of 0.1% formic acid solution, and the peptide concentration was measured using Nanotrap;

[0161] (9) Proteomics detection was performed with a standard mass of 1 μg peptide, and the effective detection time was 60 min.

[0162] The enrichment effect of NPs prepared in Example 1 on synoviolin is as follows: Figure 4 As shown. Figure 5 As can be seen from a, the enriched rheumatoid arthritis and osteoarthritis proteins in Example 1 differ by more than 120 species; Figure 5 As can be seen from b, the scatter points corresponding to the two groups of samples of rheumatoid arthritis and osteoarthritis are clustered within the group, indicating that the repeatability within the group is relatively good, the sample data are very similar, and there is a good degree of distinction between the groups; Figure 5 As can be seen from c, the heat map analysis shows that the expression of proteins in rheumatoid arthritis is upregulated by more than 20 times compared with osteoarthritis; Figure 5 As can be seen from Figure d, compared with patients with osteoarthritis, the expression of acute phase proteins in patients with rheumatoid arthritis was significantly increased.

[0163] The above are only preferred embodiments of the present invention. It should be pointed out that, for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Prussian blue magnetic beads, with a diameter of 5nm to 50μm, contain manganese ions and Prussian blue, and the manganese ions and the cyanide in the Prussian blue are coordinated and arranged with each other.

2. The method for preparing the Prussian blue magnetic beads according to claim 1, comprising: Mixing Triton-100, n-hexanol and cyclohexane to obtain a solvent; Dissolving a polymerization agent and potassium ferrocyanide in the solvent to obtain a solution A; Dissolving a manganese salt and a divalent iron salt in the solvent to obtain a solution B; The solution A is mixed with the solution B to react to obtain pullulan magnetic beads; The polymerizing agent is selected from at least one of polyacrylamide, polymethyl methacrylate, polystyrene, polymethylstyrene, polyimide, polyethylene terephthalate, polyvinyl pyrrolidone, polyethylene glycol, and polylactic acid.

3. The preparation method according to claim 2, characterized in that: The polymerizing agent is polyvinyl pyrrolidone; The divalent iron salt is selected from at least one of ferrous chloride and ferrous sulfate; The mass ratio of Triton-100, n-hexanol and cyclohexane in the solvent is (2-6): (1-3): (1-3); The mass ratio of the polymerization agent to the potassium ferrocyanide is (1.2-2):1; The amount of manganese salt added is 0.01-0.1 mol / L; The molar ratio of the divalent iron salt to the potassium ferrocyanide is 1:(1-5).

4. The preparation method according to claim 2 or 3, characterized in that: The reaction temperature of the solution A and the solution B is 120° C. to 160° C., and the reaction time is 2 h to 12 h.

5. A protein enrichment reagent, comprising the Prussian blue magnetic beads according to claim 1, a dilution buffer and a magnetic bead lysis buffer; The dilution buffer is PBS buffer; The magnetic bead lysis buffer comprises water and sodium deoxycholate, 3-(2-carboxyethyl)phosphine, 2-chloroacetamide and Tris-HCl.

6. The enrichment reagent according to claim 5, characterized in that The magnetic bead lysis buffer includes water and 1wt% sodium deoxycholate, 10mM 3-(2-carboxyethyl)phosphine, 40mM 2-chloroacetamide and 100mM Tris-HCl; The enrichment reagent also includes an extraction reagent, which contains trifluoroacetic acid, acetonitrile and water, wherein the volume ratio of acetonitrile to water is (75-85): (15-25), and the mass fraction of trifluoroacetic acid is 0.05%-0.15%.

7. A method for enriching a protein, comprising: The protein in the sample is enriched using the enrichment reagent described in claim 6.

8. The enrichment method according to claim 7, characterized in that: The samples include blood, body fluids, animal tissues and / or secretions.

9. The enrichment method according to claim 7, characterized in that: The enrichment includes: The Prussian blue magnetic beads according to claim 1 are washed with the dilution buffer and then added to the sample, and the magnetic beads are separated after incubation; The magnetic beads are treated with a magnetic bead lysis buffer, hydrolyzed with a protease, and then extracted to obtain enriched proteins.

10. A method for detecting protein, comprising enriching the protein in a sample by the enrichment method according to any one of claims 7 to 9 and then detecting the protein.

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