Streptavidin dextran magnetic beads and preparation method

Abstract

The application discloses a streptavidin dextran magnetic bead and a preparation method thereof, and belongs to the technical field of biological nanomaterials. Fe3+ / Fe2+ The molar ratio is 1.8-2.2:1.The innovation of the application is that a 4-oxygen-TEMPO / NaClO / KBr ternary oxidation system (pH 6.5-7.0) is adopted, a hydroxyl radical-mediated directional oxidation mechanism is adopted, and a dextran C6 hydroxyl group is directly converted into a carboxyl group.Through regulation of the 4-oxygen-TEMPO concentration (4-6 mM) and the reaction time, the product structure stability is improved, and the sugar ring structure is not damaged in the oxidation process.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological nanomaterials, and particularly relates to a streptavidin dextran nanomagnetic particle, a preparation method and application thereof. BACKGROUND

[0002] Magnetic beads are a kind of functional material used in the field of biomedicine, which are usually made of superparamagnetic materials and can be modified with specific biomolecules (such as antibodies, ligands, etc.) on the surface for labeling and separating target cells or molecules. Magnetic beads can be magnetized under the action of a magnetic field, thereby realizing the separation and purification of target substances. It is widely used in cell sorting, protein purification, nucleic acid extraction and other fields. The sorting method of magnetic beads can be divided into two types: column type and column-free type. Column-free magnetic bead sorting is more direct and simple, which directly places the sample labeled by magnetic beads in a magnetic field to realize the separation of cells through the adsorption of the magnetic field. Column-free sorting is further divided into positive selection and negative selection. Positive selection uses antibody-coupled magnetic beads to label target cells, and negative selection uses antibody-coupled magnetic beads to label non-target cells. Magnetic bead sorting technology has the advantages of simple operation, rapidness, low cost, etc. Compared with the traditional density gradient centrifugation method, magnetic bead sorting can obtain higher cell yield and purity. In addition, the magnetic bead sorting process is mild and does not cause adverse effects such as cell activation, and a large number of cells with high activity and good function can be obtained.

[0003] Dextran is a high molecular weight polysaccharide formed by the linkage of glucose units through glycosidic bonds, which has good biocompatibility and stability. According to the type of glycosidic bond, dextran can be divided into α-dextran and β-dextran. Dextran is electrically neutral, easily soluble in water, and has low toxicity, which is suitable for biomedical applications. Dextran has a wide range of applications in the field of biomedicine. Dextran can also be used to prepare magnetic nanoparticles, which have good stability and biocompatibility and can be used for cell separation, immune recognition, drug carriers, and negative contrast agents for nuclear magnetic resonance imaging, etc.

[0004] Streptavidin dextran magnetic beads are a kind of composite material combining streptavidin and dextran. Streptavidin is a protein with high affinity to biotin, which can form a very firm bond with biotin. Dextran provides good biocompatibility and stability, while magnetic beads endow the material with magnetism, which is convenient for separation and operation in a magnetic field. Streptavidin dextran magnetic beads have the following characteristics: high hydrophilicity, superparamagnetism, fast magnetic response, excellent redispersion stability and magnetic stability. It can effectively ensure high biotin protein coupling amount and low non-specific adsorption, as well as reaction uniformity and detection consistency. Streptavidin dextran magnetic beads are widely used in the fields of biomolecular recognition, separation and detection, etc.

[0005] However, the existing sodium periodate oxidized dextran magnetic beads have the core problems of significant decrease in polymerization degree (according to the patent (molecular weight reduction of 30-50%)) [1, 2] and insufficient carboxyl density (<0.5 mmol / g).

[0006] Prior art documents

[0007] Prior non-patent documents [1] Insight on the periodate oxidation of dextran and its structural vicissitudes;

[0008] Prior patent documents [2] Selective oxidation of broadleaf wood pulp by sodium periodate. SUMMARY

[0009] To solve the above problems, the present application proposes a new controllable oxidation process, which uses a 4-oxo-TEMPO / NaClO / KBr ternary oxidation system (pH 6.5-7.0) to directly convert the C6 hydroxyl group of dextran into carboxyl group through a free radical mediated hydroxyl directed oxidation mechanism.

[0010] The purpose of the present application is achieved by the following technical solutions:

[0011] A streptavidin dextran magnetic bead, which is carboxylated and modified on the surface of a magnetic core by a 4-oxo-TEMPO / NaClO / KBr system; Fe 3+ / Fe 2+ The molar ratio is 1.8-2.2:1.

[0012] The present application also provides a preparation method of the above-mentioned streptavidin dextran magnetic bead, comprising the following steps:

[0013] S1, preparation of a dextran magnetic core, which is synthesized by a chemical co-precipitation method to obtain a dextran magnetic core with a diameter of 15 nm;

[0014] S2, carboxylated modification of the surface of the dextran magnetic core, comprising the following steps:

[0015] The magnetic core is dispersed in 40-60 mM PBS with a pH of 6.5-7.0, 0.08-0.15 g of 4-oxo-TEMPO, 1.3-1.7 g of NaClO and 0.6-0.9 g of KBr are added, and the reaction is carried out at 20-80℃ in the dark with 100-600 rpm stirring for 5-7 hours, then the carboxylated magnetic bead is obtained by magnetic separation and washing with PBS for 2-4 times;

[0016] S3, streptavidin coupling, comprising the following steps:

[0017] Carboxylated magnetic beads were reacted with EDC / NHS (EDC: 1-ethyl-(3-dimethylaminopropyl) carbodiimide, NHS: N-hydroxysuccinimide) at a molar ratio of 1:1.2-1:1.8 at room temperature for 20-60 minutes, the pH of the reaction solution was maintained at 4.6-8.0, 0.8-1.5 mg / mL SA solution was added, and the solution was incubated at room temperature for 2-16 hours. Non-specific sites were blocked with 0.8-1.5% BSA at room temperature for 0.5-2 hours. Unbound SA and BSA (bovine serum albumin) were removed to obtain streptavidin dextran magnetic beads.

[0018] Preferably, the method of chemical co-precipitation in step S1 comprises the following steps:

[0019] Weigh 2.0-6.0 g of dextran, 3.0-3.5 g of FeCl3·6H2O, and 1.3-1.7 g of FeCl2·4H2O, and dissolve them in 40-60 mL of deionized water. Stir at 100-700 rpm under nitrogen protection for 20-40 minutes. Add 8-12 mL of concentrated ammonia water with a concentration of 25-28% dropwise under nitrogen protection at a rate of 1-3 mL / min. Then, heat to 50-60°C and stir for 1.5-2.5 hours for maturation. Perform magnetic separation by applying an external magnetic field. Wash alternately with deionized water and ethanol for 2-4 times to obtain Fe3O4 magnetic cores coated with dextran.

[0020] Preferably, the reaction pH in step S2 is 6.8.

[0021] Preferably, the step S2 dextran magnetic core surface carboxylation modification comprises the following steps:

[0022] Disperse the magnetic core in 50 mM PBS at pH 6.8, add 0.08 g of 4-oxo-TEMPO, 1.3 g of NaClO, and 0.6 g of KBr. Stir at 300 rpm at 60°C in the dark for 6 hours. Perform magnetic separation and wash with PBS for 2-4 times to obtain carboxylated magnetic beads.

[0023] Preferably, the step S2 dextran magnetic core surface carboxylation modification comprises the following steps:

[0024] Disperse the magnetic core in 50 mM PBS at pH 6.8, add 0.12 g of 4-oxo-TEMPO, 1.5 g of NaClO, and 0.8 g of KBr. Stir at 300 rpm at 60°C in the dark for 6 hours. Perform magnetic separation and wash with PBS for 2-4 times.

[0025] Preferably, the step S2 dextran magnetic core surface carboxylation modification comprises the following steps:

[0026] The magnetic core is dispersed in a 50mM PBS solution with pH of 6.8, 0.15g 4-oxy-TEMPO, 1.7g NaClO and 0.9g KBr are added, and the reaction is carried out at 60°C in dark for 6 hours, the stirring speed is 300rpm, and the magnetic separation is carried out and washed with PBS for 2-4 times.

[0027] Compared with the prior art, the present application has the following beneficial effects:

[0028] 1. Magnetic core structure optimization: by controlling Fe 3+ / Fe 2+ The molar ratio is 1.8-2.2:1.

[0029] 2. Directional oxidation process: using 4-oxy-TEMPO / NaClO / KBr system with pH of 6.5-7.0, the selective oxidation of dextran hydroxyl is realized.

[0030] The innovation of the present application is to use 4-oxy-TEMPO / NaClO / KBr ternary oxidation system (pH 6.5-7.0), and through the free radical mediated hydroxyl directional oxidation mechanism, the C6 hydroxyl of dextran is directly converted into carboxyl. By adjusting the 4-oxy-TEMPO concentration (4-6mM) and the reaction time, the product structure stability is improved, and the sugar ring structure is not damaged in the oxidation process. BRIEF DESCRIPTION OF DRAWINGS

[0031] Figure 1 : SA magnetic bead synthesis process flow diagram;

[0032] Figure 2 : 4-oxy-TEMPO oxidation reaction mechanism diagram;

[0033] Figure 3 : Oxidized dextran magnetic bead hydration particle size distribution;

[0034] Figure 4 : Oxidized dextran magnetic bead scanning electron microscope image;

[0035] Figure 5 : The magnetic performance of the oxidized dextran magnetic bead is measured by VSM method at room temperature. DETAILED DESCRIPTION

[0036] With reference to the accompanying drawings, the technical solutions in the embodiments of the present application will be described clearly and completely. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments of the present application. Based on the embodiments in the present application, all the other embodiments obtained by a person of ordinary skill in the art without creative work are within the protection scope of the present application. In the present application, unless specified, the adopted equipment and raw materials can be purchased from the market or are commonly used in the art. In the following embodiments, the methods are the conventional methods in the art, unless specified.

[0037] Embodiment 1

[0038] S1, 3.0 g of dextran was weighed, 3.2435 g of FeCl3·6H2O and 1.6250 g of FeCl2·4H2O were dissolved in 50 mL of deionized water, and the reaction was carried out at room temperature for 30 min under the protection of nitrogen and 300 rpm stirring. 10 mL of concentrated ammonia water (28%) was added dropwise under the condition of continuous nitrogen flow, and the dropwise rate was controlled at 3 mL / min. Then, the temperature was raised to 60°C for 2 hours for curing, and the magnetic separation was carried out by an external magnetic field. The magnetic core coated with dextran Fe3O4 was obtained by washing with deionized water and ethanol alternately for 3 times.

[0039] S2, the magnetic core was dispersed in 50 mM PBS solution with pH of 6.8, 0.08 g of 4-oxy-TEMPO, 1.3 g of NaClO and 0.6 g of KBr were added, and the reaction was carried out at 60°C for 6 hours in the dark, with a stirring speed of 300 rpm. The magnetic separation was carried out and washed with PBS for 3 times.

[0040] S3, SA coupling: EDC / NHS activation time 6 h, SA concentration 1.0-1.4 mg / mL, carboxylated magnetic beads were reacted with EDC / NHS (molar ratio 1:1.5) for 30 min, and the activated carboxyl group generated an active ester intermediate. During the activation process, the pH of the solution was maintained at 6.1, and the activation was carried out for 30 min. Then, 0.1 mg / mL SA solution was added, and the oscillation incubation was carried out at room temperature for 4 hours. The non-specific sites were blocked with 1% BSA at room temperature for 1.5 hours. After removing the unbound SA and BSA, the streptavidin dextran magnetic beads were obtained.

[0041] Embodiment 2

[0042] S1, weigh 3.0 g of dextran, 3.2435 g of FeCl3-6H2O and 1.6250 g of FeCl2-4H2O are dissolved in 50 mL of deionized water, nitrogen protection, 300 rpm stirring, room temperature reaction for 30 min, continuous aeration, 10 mL of concentrated ammonia water (28%) is added dropwise under nitrogen protection, the dropwise rate is controlled at 3 mL / min, then the temperature is raised to 60°C and stirred for 2 hours for curing, magnetic separation is performed by an external magnetic field, and the product is washed with deionized water and ethanol alternately for 3 times to obtain dextran-coated Fe3O4 magnetic nuclei.

[0043] S2, the magnetic nuclei are dispersed in a 50 mM PBS solution with a pH of 6.8, 0.12 g of 4-oxy-TEMPO, 1.5 g of NaClO and 0.8 g of KBr are added, and the reaction is carried out at 60°C for 6 hours in the dark, the stirring speed is 300 rpm, and the product is washed with PBS for 3 times after magnetic separation.

[0044] S3, SA coupling: EDC / NHS activation time is 6 h, SA concentration is 1.0-1.4 mg / mL, carboxylated magnetic beads are reacted with EDC / NHS (molar ratio 1:1.5) for 30 min, the activated carboxyl group generates an active ester intermediate, and the pH of the solution is maintained at 6.1 during the activation process. After 30 min of activation, 0.1 mg / mL of SA solution is added, and the product is incubated at room temperature for 4 hours, then 1% BSA is added to block the non-specific sites for 1.5 hours, and after removing the unbound SA and BSA, the product is obtained.

[0045] Example 3

[0046] S1, weigh 3.0 g of dextran, 3.2435 g of FeCl3-6H2O and 1.6250 g of FeCl2-4H2O are dissolved in 50 mL of deionized water, nitrogen protection, 300 rpm stirring, room temperature reaction for 30 min, continuous aeration, 10 mL of concentrated ammonia water (28%) is added dropwise under nitrogen protection, the dropwise rate is controlled at 3 mL / min, then the temperature is raised to 60°C and stirred for 2 hours for curing, magnetic separation is performed by an external magnetic field, and the product is washed with deionized water and ethanol alternately for 3 times to obtain dextran-coated Fe3O4 magnetic nuclei.

[0047] S2, the magnetic nuclei are dispersed in a 50 mM PBS solution with a pH of 6.8, 0.12 g of 4-oxy-TEMPO, 1.5 g of NaClO and 0.8 g of KBr are added, and the reaction is carried out at 60°C for 6 hours in the dark, the stirring speed is 300 rpm, and the product is washed with PBS for 3 times after magnetic separation.

[0048] S3, SA coupling: EDC / NHS activation time 6h, SA concentration 1.0-1.4mg / mL, carboxylated magnetic beads reacted with EDC / NHS (molar ratio 1:1.5) for 30min, activated carboxyl to generate active ester intermediates, during the activation process, the solution pH was maintained at 6.1, activated for 30min, 0.1mg / mL SA solution was added, incubated at room temperature for 4 hours, non-specific sites were blocked with 1% BSA for 1.5 hours, and after removing unbound SA and BSA, streptavidin dextran magnetic beads were obtained.

[0049] Comparative Example 1 (according to patent CN 105478087 B)

[0050] S1, weigh 3g of dextran (molecular weight 40000), dissolve in 100mL of pure water, and add 0.1M NaIO4 to make the molar ratio of NaIO4 to dextran 1:2, shake at 30°C for 2 hours in the dark, obtain aldehyde dextran; dialyze 5 times to remove unreacted NaIO4. Aldehyde dextran and glutaraldehyde are grafted onto the surface of amino magnetic beads at a mass ratio of 1:5 to prepare aldehyde magnetic beads. The inner core is ferroferric oxide, the shell is polyvinyl alcohol, and the surface has modified free amino groups (primary amines). Take 1mL (10mg / mL) of amino magnetic beads, magnetically separate, wash the magnetic beads with ultrapure water 2-3 times, magnetically separate, and discard the supernatant. Weigh 20mg of aldehyde dextran, dissolve in 1900μl of 0.2MPB (PH9.0), add to the amino magnetic beads, and then add 100μL of glutaraldehyde to the amino magnetic beads. Shake at room temperature for 4 hours in the dark. After the reaction is completed, wash the magnetic beads with PBS (PH7.4) 3 times.

[0051] S2, magnetically separate the aldehyde magnetic beads, discard the supernatant, wash the magnetic beads with ultrapure water 3 times, weigh 10mg of glutamic acid, dissolve in 2mL of PBS (PH7.4), add to the centrifuge tube containing the aldehyde magnetic beads, shake at room temperature for 4 hours, then magnetically separate the magnetic beads, discard the supernatant, add 2mL of sodium borohydride (5mg / mL, dissolved in PBS, PH7.4) to the magnetic beads, shake for 2 hours, after the reaction is completed, wash the magnetic beads with PBS (PH7.4) 3 times, and suspend the magnetic beads in 1mL of PBS (PH7.4) to obtain the prepared carboxyl magnetic beads.

[0052] S3, SA coupling: EDC / NHS activation time 6h, SA concentration 1.0-1.4mg / mL, carboxylated magnetic beads reacted with EDC / NHS (molar ratio 1:1.5) for 30min, activated carboxyl to generate active ester intermediates, during the activation process, the solution pH was maintained at 6.1, activated for 30min, 0.1mg / mL SA solution was added, incubated at room temperature for 4 hours, non-specific sites were blocked with 1% BSA for 1.5 hours, after removing unbound SA and BSA, streptavidin dextran magnetic beads were obtained.

[0053] The degree of polymerization was determined using a glucose (GOPOD oxidase method) content kit, and the carboxyl concentration was determined using the conductivity method. The results are shown in Table 1.

[0054] Table 1

[0055]

[0056] Conclusion: By analyzing Table 1, the products obtained in Examples 1-3 solve the problem of excessive oxidation of dextran magnetic beads in the background, which leads to degradation of dextran polymerization degree and insufficient carboxyl density. According to the determination of the degree of polymerization using a glucose (GOPOD oxidase method) content kit and the determination of the carboxyl concentration using the conductivity method, we can obtain a scheme that maintains a relatively high degree of polymerization and high carboxyl density under the same conditions, different doses of 4-oxo-TEMPO, and comparison with sodium periodate oxidized dextran magnetic beads.

[0057] It can be understood that, for those skilled in the art, equivalent replacements or changes can be made according to the technical solutions of the present application and the concept of the present application, and all these changes or replacements shall belong to the protection scope of the appended claims of the present application. The present application can be implemented within a wider range under equivalent parameters, concentrations and conditions without departing from the purpose and scope of the present application, and without unnecessary experiments. Although the present application gives special examples, it should be understood that further improvements can be made to the present application. In summary, according to the principle of the present application, the present application includes any changes, uses or improvements of the present application, including changes made by conventional techniques known in the art, which deviate from the range disclosed in the present application. Some basic features can be applied within the scope of the following appended claims.

Claims

1. A method for preparing streptavidin-dextran magnetic beads, characterized in that, Includes the following steps: S1. Preparation of dextran magnetic cores: Dextran magnetic cores with a diameter of 15 nm were synthesized by chemical coprecipitation. The chemical coprecipitation method includes the following steps: Weigh 2.0-6.0 g of dextran, 3.0-3.5 g of FeCl3·6H2O, and 1.3-1.7 g of FeCl2·4H2O, dissolve them in 40-60 mL of deionized water, stir at 100-700 rpm for 20-40 minutes under nitrogen protection, add 8-12 mL of concentrated ammonia solution with a concentration of 25-28% dropwise at a dropping rate of 1-3 mL / min under nitrogen protection, then heat to 50-60℃ and stir for 1.5-2.5 hours for maturation, perform magnetic separation by applying an external magnetic field, and wash alternately with deionized water and ethanol 2-4 times to obtain dextran-coated Fe3O4 magnetic cores. S2. Carboxylation modification of the surface of dextran magnetic core, including the following steps: The magnetic nuclei were dispersed in 40-60 mM PBS at pH 6.8, and 0.08-0.15 g of 4-oxo-TEMPO, 1.3-1.7 g of NaClO and 0.6-0.9 g of KBr were added. The mixture was stirred at 100-600 rpm for 5-7 hours in the dark at 20-80℃. After magnetic separation, the mixture was washed 2-4 times with PBS to obtain carboxylated magnetic beads. S3, streptavidin coupling, includes the following steps: Carboxylated magnetic beads were reacted with EDC / NHS at a molar ratio of 1:1.2-1:1.8 at room temperature for 20-60 minutes, with the pH of the reaction solution maintained at 4.6-8.

0. 0.8-1.5 mg / mL SA solution was added, and the mixture was incubated with shaking at room temperature for 2-16 hours. Non-specific sites were blocked with 0.8-1.5% BSA at room temperature for 0.5-2 hours. Unbound SA and BSA were removed to obtain streptavidin-glucan magnetic beads.

2. The preparation method according to claim 1, characterized in that, Step S2, carboxylation modification of the dextran magnetic core surface, includes the following steps: The magnetic nuclei were dispersed in 50 mM PBS at pH 6.8, and 0.08 g of 4-oxy-TEMPO, 1.3 g of NaClO and 0.6 g of KBr were added. The mixture was stirred at 300 rpm for 6 hours in the dark at 60 °C. The mixture was then magnetically separated and washed 2-4 times with PBS to obtain carboxylated magnetic beads.

3. The preparation method according to claim 1, characterized in that, Step S2, carboxylation modification of the dextran magnetic core surface, includes the following steps: The magnetic nuclei were dispersed in a 50 mM PBS solution at pH 6.8, and 0.12 g of 4-oxy-TEMPO, 1.5 g of NaClO and 0.8 g of KBr were added. The mixture was reacted at 60 °C in the dark for 6 hours with a stirring speed of 300 rpm. The mixture was then magnetically separated and washed 2-4 times with PBS.

4. The preparation method according to claim 1, characterized in that, Step S2, carboxylation modification of the dextran magnetic core surface, includes the following steps: The magnetic nuclei were dispersed in a 50 mM PBS solution at pH 6.8, and 0.15 g of 4-oxy-TEMPO, 1.7 g of NaClO and 0.9 g of KBr were added. The mixture was reacted at 60 °C in the dark for 6 hours with a stirring speed of 300 rpm. The mixture was then magnetically separated and washed 2-4 times with PBS.

5. A streptavidin-dextran magnetic bead, characterized in that, Streptavidin-glucan magnetic beads obtained by the preparation method according to any one of claims 1-4.

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