CN119505124A - Superparamagnetic strong anion exchange magnetic beads for virus enrichment, preparation method and application thereof - Google Patents
Superparamagnetic strong anion exchange magnetic beads for virus enrichment, preparation method and application thereof Download PDFInfo
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- CN119505124A CN119505124A CN202411751613.9A CN202411751613A CN119505124A CN 119505124 A CN119505124 A CN 119505124A CN 202411751613 A CN202411751613 A CN 202411751613A CN 119505124 A CN119505124 A CN 119505124A
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Abstract
本发明涉及生物纳米材料技术领域，尤其涉及用于病毒富集的超顺磁强阴离子交换磁珠、其制备方法及应用。本发明公开了一种用于病毒富集的超顺磁强阴离子交换磁珠及其制备方法。所述超顺磁强阴离子交换磁珠以聚甲基丙烯酸缩水甘油酯磁性微球为原料，首先通过活性环氧基团与多氨基化合物反应引入大量的氨基基团，随后在碱性条件下，通过氨基和环氧基季铵盐反应，引入丰富的季铵盐基团。通过两次环氧开环反应，在引入足够多的季铵盐基团的同时，在磁性微球表面引入大量的亲水性羟基，从而改善了阴离子交换磁珠的亲水性，将其应用于病毒的富集，富集效率和洗脱效率高，应用前景好。The present invention relates to the technical field of bio-nanomaterials, and in particular to superparamagnetic strong anion exchange magnetic beads for virus enrichment, a preparation method and an application thereof. The present invention discloses a superparamagnetic strong anion exchange magnetic bead for virus enrichment and a preparation method thereof. The superparamagnetic strong anion exchange magnetic bead uses poly(glycidyl methacrylate) magnetic microspheres as raw materials, firstly introduces a large number of amino groups by reacting active epoxy groups with polyamino compounds, and then introduces abundant quaternary ammonium salt groups by reacting amino groups with epoxy quaternary ammonium salts under alkaline conditions. Through two epoxy ring-opening reactions, while introducing enough quaternary ammonium salt groups, a large number of hydrophilic hydroxyl groups are introduced on the surface of the magnetic microspheres, thereby improving the hydrophilicity of the anion exchange magnetic beads, and applying them to virus enrichment, the enrichment efficiency and elution efficiency are high, and the application prospect is good.
Description
Technical Field
The invention relates to the technical field of biological nano materials, in particular to a super-paramagnetic strong anion exchange magnetic bead for virus enrichment, a preparation method and application thereof.
Background
Rapid, accurate, efficient, economical and simple virus enrichment and detection are prerequisites for early warning and prevention of viral outbreaks. Biological studies of viruses typically require isolation of intact viral particles from diluted samples. The most commonly used virus separation method at present is a polyethylene glycol precipitation method, a filter membrane adsorption method or an ultrafiltration method, and is limited by a method principle, and the method is complex in operation, difficult to realize and difficult to realize automation.
The method can be used for rapidly and efficiently enriching the viruses. The method realizes the enrichment and elution of viruses by controlling the combination and separation of magnetic beads and viruses under the action of a magnetic field. CN114200127a couples the hepatitis b virus antibody to the carboxyl modified superparamagnetic nano-beads, and uses it to bind with hepatitis b virus positive serum or plasma, and then obtains enriched and concentrated hepatitis b virus. CN108486069A is used for enriching Porcine Epidemic Diarrhea Virus (PEDV) by coupling PEDV membrane protein specific single domain antibodies to the surfaces of the nano magnetic beads, and can be rapidly and effectively separated by utilizing the immunomagnetic beads. Then, immunomagnetic beads can only enrich and separate for a single virus, and expensive antibodies are needed, so that the cost is high, and the simultaneous enrichment and separation requirements of multiple viruses cannot be met.
The anion exchange magnetic beads and electronegative virus particles are combined reversibly, but the existing anion exchange magnetic beads have complex preparation process, hydrophobic irreversible combination possibly occurs between the magnetic beads and viruses, so that elution is difficult, or the problems of low adsorption efficiency, low elution efficiency and the like exist.
Disclosure of Invention
In view of the above, the technical problem to be solved by the present invention is to provide a super-paramagnetic anion exchange magnetic bead for virus enrichment, a preparation method and application thereof.
The invention provides a super-paramagnetic strong anion exchange magnetic bead which is obtained by sequentially modifying polymer magnetic microspheres with epoxy by amino groups and quaternary ammonium salts;
the polymeric magnetic microsphere with epoxy group includes:
Fe 3O4 magnetic core, and
SiO 2 coated on Fe 3O4 core, and
Gamma-methacryloxypropyl trimethoxysilane modified on SiO 2, and
Polyglycidyl methacrylate formed by in situ polymerization of gamma-methacryloxypropyl trimethoxysilane.
Specifically, the polymer magnetic microsphere with the epoxy group is prepared by the following steps of;
Step 1, preparing Fe 3O4 magnetic core through FeCl 3·6H2 O;
step 2, preparing a core-shell structure Fe 3O4@SiO2 magnetic microsphere by taking Fe 3O4 as a magnetic core and tetraethyl orthosilicate (TEOS) as a silicon source;
Step 3, using gamma-methacryloxypropyl trimethoxy silane (MPS) as a surface modification reagent to carry out surface modification on the Fe 3O4@SiO2 magnetic microsphere to prepare the Fe 3O4@SiO2 -MPS magnetic microsphere with the surface rich in double bonds;
and 4, performing in-situ polymerization of Glycidyl Methacrylate (GMA) on the surface of the Fe 3O4@SiO2 -MPS magnetic microsphere to prepare the Fe 3O4@SiO2 @PGMA magnetic microsphere with (surface-rich) epoxy groups.
The mass ratio of FeCl 3.6H2 O, TEOS, MPS, GMA is 1:0.35-0.65:0.40-0.60:0.60-1.20, and specifically 1:0.40:0.50:0.80.
More specifically, the polymer magnetic microsphere with epoxy group (the magnetic microsphere also becomes the poly glycidyl methacrylate) is synthesized by adopting the method in the patent CN112007620A, and the specific synthesis steps are as follows:
1. FeCl 3·6H2 O is used as a raw material, and a solvothermal method is used for preparing Fe 3O4 magnetic cores;
2. Fe 3O4 is used as a magnetic core, tetraethyl orthosilicate (TEOS) is used as a silicon source, and the Fe 3O4@SiO2 magnetic microsphere is prepared by a St method;
3. Performing surface modification on the Fe 3O4@SiO2 magnetic microsphere by using gamma-methacryloxypropyl trimethoxysilane (MPS) to prepare the Fe 3O4@ SiO2 -MPS magnetic microsphere;
4. on the surface of Fe 3O4@ SiO2 -MPS magnetic microsphere, azodiisobutyronitrile (AIBN) is used as an initiator, glycidyl Methacrylate (GMA) is used as a polymerization monomer, N, N-Methylenebisacrylamide (MBA) is used as a cross-linking agent, and in-situ polymerization of GMA is carried out on the surface of Fe 3O4@SiO2 magnetic microsphere to prepare Fe 3O4@ SiO2 @PGMA;
The mass ratio of FeCl 3.6H2 O, TEOS, MPS, AIBN, GMA, MBA is 1:0.35-0.65:0.40-0.60:0.015-0.040:0.60-1.20:0.15-0.35, and specifically 1:0.40:0.50:0.025:0.80:0.25.
And (3) separating carboxyl magnetic microspheres through a magnetic separator after preparing the poly (glycidyl methacrylate) magnetic microspheres, repeatedly washing the poly (glycidyl methacrylate) magnetic microspheres for at least 5 times by using purified water, and storing the poly (glycidyl methacrylate) magnetic microspheres in the purified water, wherein the quality of the microspheres is qualified, and the particle size is 400-800 nm.
Further, the particle size of the super-paramagnetic strong anion exchange magnetic beads is 400-800 nm;
in the invention, the super-paramagnetic strong anion exchange magnetic bead has the following structure:
X-R1- (CH 2)n -R2-Y (formula I), wherein,
X is a polymer magnetic microsphere with carboxyl;
y is a quaternary ammonium salt group;
r1- (CH 2) n-R2 is a spacer;
r1 is selected from primary amine groups or secondary amine groups, and specifically R1 is a primary amine group;
r2 is selected from primary amine groups or secondary amine groups, and specifically, R2 is a primary amine group;
Wherein n is an integer of 1 to 6, and further, in the embodiment of the invention, n is 3.
The invention provides a preparation method of a super-paramagnetic strong anion exchange magnetic bead, which comprises the following steps:
Step1, polymer magnetic microspheres with epoxy groups are modified by amino groups to obtain amino modified polymer microspheres;
and 2, modifying the amino modified polymeric microsphere by quaternary ammonium salt to obtain the super-paramagnetic anion exchange magnetic bead.
Further, the method comprises the steps of,
The amino modification can introduce primary amine or secondary amine groups with different contents on the surface of the polymer magnetic microsphere, and the number of the primary amine or secondary amine groups can be regulated and controlled by adjusting the type, the amount, the reaction temperature or the reaction time of an amination reagent as a spacer arm with a structure shown in a formula I.
The amination reagent is a polyamino compound;
In the invention, the amino group is modified into a polymer magnetic microsphere with carboxyl group to react with a polyamino compound;
The polyamino compound is selected from one or more of ethylenediamine, 3-diaminodipropylamine, diethylenetriamine, triethylenetetramine and/or polyethyleneimine;
In a specific embodiment of the invention, the polyamino compound is 3, 3-diaminodipropylamine;
The mass ratio of the polymer magnetic microsphere with epoxy to the 3, 3-diaminodipropylamine is 1:1-1:2, and the mass ratio of the polymer magnetic microsphere with epoxy to the 3, 3-diaminodipropylamine is 1:1;
the reaction condition is that ethanol is used as a reaction solvent, and the reaction is carried out for 4-24 hours at 40-80 ℃ in a nitrogen environment.
In a specific embodiment of the invention, the reaction conditions are that ethanol is used as a reaction solvent, and the reaction is carried out under the condition of nitrogen gas at 70 ℃ for 8 h.
The amino group is modified and then comprises the steps of magnetic separation and washing;
The washed reagent is ethanol and pure water;
the washing is sequentially performed by using ethanol and pure water, and the number of times of washing can be adjusted by a person skilled in the art according to actual conditions, which is not limited in the present invention.
The quaternary ammonium salt is modified into the amino modified polymeric microsphere to react with epoxy quaternary ammonium salt;
The epoxy quaternary ammonium salt is selected from at least one of 2, 3-epoxypropyl trimethyl ammonium chloride and/or 3, 4-epoxybutyl trimethyl ammonium chloride;
In a specific embodiment of the invention, the epoxy quaternary ammonium salt is 2, 3-epoxypropyl trimethyl ammonium chloride;
The mass ratio of the amino modified polymeric microspheres to the 2, 3-epoxypropyl trimethyl ammonium chloride is 1:0.5-1:2.0, and the mass ratio of the amino modified polymeric microspheres to the 2, 3-epoxypropyl trimethyl ammonium chloride is 1:1;
The reaction condition is that the reaction is carried out for 4-24 hours in an alkaline environment at 30-80 ℃.
Specifically, sodium hydroxide with the final concentration of 0.1M-1.0M is added to maintain an alkaline environment;
in the specific embodiment of the invention, the reaction condition is that 0.5M sodium hydroxide solution is used as a reaction solvent, and the reaction is carried out for 4 hours at 40 ℃.
The modified quaternary ammonium salt also comprises the steps of magnetic separation and washing;
The washing is to sequentially wash with ethanol and pure water until the washing liquid is neutral, and the times of washing can be adjusted by a person skilled in the art according to actual conditions, which is not limited by the invention.
In the invention, in each independent reaction, the substances participating in the reaction are fully and uniformly mixed into the conventional steps adopted by the technicians in the field, wherein the mixing method comprises ultrasonic treatment, vibration or stirring and the like, and the mixing time can be properly adjusted by the technicians in the field according to the actual situation, so that the invention is not limited.
In a specific embodiment of the invention, the preparation method of the super-paramagnetic strong anion exchange magnetic bead comprises the following steps:
S1, dispersing polymer magnetic microspheres with epoxy groups in an organic solvent or pure water, performing ultrasonic dispersion for 5-10 min, adding a certain amount of polyamino compounds, transferring into a constant-temperature water bath, heating to 40-80 ℃ in nitrogen atmosphere, and stirring for reaction for 4-24 h. After the reaction, the mixture was magnetically separated, washed with ethanol and pure water several times in order, and stored in pure water to obtain a dispersion containing amino-modified polymer magnetic microspheres.
S2, adding a certain amount of sodium hydroxide into the dispersion liquid containing the amino modified polymer magnetic microspheres, adjusting the concentration of the sodium hydroxide to be 0.1-1.0M, performing ultrasonic dispersion for 5-10 minutes, adding a certain amount of epoxy quaternary ammonium salt, transferring into a constant temperature water bath, heating to 30-80 ℃ in a nitrogen atmosphere, and stirring for reaction for 4-24 hours. After the reaction, magnetic separation is carried out, ethanol and pure water are used for washing to be neutral in sequence, and the mixture is stored in 10 mM sodium chloride solution, thus obtaining the superparamagnetic strong anion exchange magnetic beads.
The super-cis-magnetic strong anion exchange magnetic beads are prepared by using poly (styrene-glycidyl methacrylate) copolymer magnetic microspheres, poly (glycidyl methacrylate) magnetic microspheres and silicon hydroxyl magnetic microspheres and cores, and test results show that when the poly (glycidyl methacrylate) magnetic microspheres, namely the polymer magnetic microspheres with epoxy groups, are poly (glycidyl methacrylate) magnetic microspheres, the prepared super-cis-magnetic strong anion exchange magnetic beads have the best adsorption capacity and the eluting capacity after adsorption and are obviously superior to those of other two magnetic microspheres;
The invention also adjusts the raw material proportion (such as the adding proportion of the poly glycidyl methacrylate magnetic microsphere and the 3, 3-diamino dipropylamine and the 2, 3-epoxypropyl trimethyl ammonium chloride) and the parameters of the synthesis step (reaction time and the like) in the preparation process of the super-cis-magnetic strong anion exchange magnetic bead, and the experimental result shows that the super-cis-magnetic strong anion exchange magnetic bead prepared by the steps and the material proportion has the best performance, excellent virus adsorption and elution capacity, and can adsorb various viruses, and has wide application range and excellent adsorption and elution effects.
In the present invention, quaternary ammonium salt groups can be introduced to the surface of silica magnetic beads or polymer magnetic beads by various methods. However, the method is limited by the number of silicon hydroxyl groups on the surface of silicon dioxide, and the silicon hydroxyl groups are directly modified by a silane coupling agent containing quaternary ammonium salt, so that the super-clockwise magnetic strong anion exchange magnetic beads of the poly glycidyl methacrylate magnetic microsphere vegetation can be obtained by screening, the steric hindrance can be obviously reduced, the density of introduced quaternary ammonium salt groups is improved, the hydrophilicity of the polymer magnetic microsphere matrix is obviously improved, the polymer magnetic microsphere matrix is easier to disperse in an aqueous solution, and meanwhile, the epoxy groups on the surface of the polymer magnetic microsphere matrix are easy to modify, so that the difficulty of subsequent quaternary ammonium salt modification is reduced. Through the modification of the polyamino compound, more reaction sites can be provided for the subsequent introduction of quaternary ammonium salt groups. Through the modification of epoxy quaternary ammonium salt, quaternary ammonium salt groups can be controllably introduced under mild reaction conditions. In the process of amination and quaternary ammonium salt modification, the hydrophilic hydroxyl formed by ring opening of the epoxy group can further improve the hydrophilicity of the anion exchange magnetic beads, and the introduced hydrophilic hydroxyl group can further improve the hydrophilicity of the anion cross-linked magnetic beads, so that the anion cross-linked magnetic beads are more beneficial to automation operation and the elution efficiency is enhanced.
The invention provides a reagent containing magnetic beads, which comprises auxiliary materials and at least one of the super-paramagnetic and anion-exchange magnetic beads or the super-paramagnetic and anion-exchange magnetic beads prepared by the preparation method.
Further, the auxiliary materials comprise at least one of buffer solution, stabilizer, antioxidant, preservative and/or anticoagulant.
The invention provides a kit which comprises at least one of a binding solution, a cleaning solution and/or an eluent and at least one of the following I) to III):
i) The super-paramagnetic strong anion exchange magnetic bead provided by the invention;
II), the superparamagnetic strong anion exchange magnetic beads prepared by the preparation method disclosed by the invention;
III) the reagent according to the invention.
The invention provides the application of at least one of the following i) to iv) in virus enrichment:
i) The super-paramagnetic strong anion exchange magnetic bead provided by the invention;
ii) the superparamagnetic strong anion exchange magnetic beads prepared by the preparation method;
iii) The reagent of the invention;
iv) the kit according to the invention.
The virus includes lentivirus, adenovirus, new coronavirus, african swine fever virus, virus vector and the like, and the invention is not limited thereto.
The invention provides a virus enrichment method, which comprises the following steps of carrying out virus enrichment by using at least one of the following A) to D):
a) The super-paramagnetic strong anion exchange magnetic bead provided by the invention;
b) The super-paramagnetic strong anion exchange magnetic bead prepared by the preparation method disclosed by the invention;
c) The reagent of the invention;
d) The kit provided by the invention.
The invention discloses a super-paramagnetic strong anion exchange magnetic bead for virus enrichment and a preparation method thereof. The super-paramagnetic strong anion exchange magnetic beads take poly glycidyl methacrylate magnetic microspheres as raw materials, a large amount of amino groups are introduced through the reaction of active epoxy groups and polyamino compounds, and then abundant quaternary ammonium salt groups are introduced through the reaction of amino groups and epoxy quaternary ammonium salt under alkaline conditions. Through two epoxy ring-opening reactions, a large amount of hydrophilic hydroxyl groups are introduced on the surface of the magnetic microsphere while enough quaternary ammonium salt groups are introduced, so that the hydrophilicity of the anion exchange magnetic bead is improved, and the anion exchange magnetic bead is applied to virus enrichment, and has high enrichment efficiency and elution efficiency and good application prospect.
Drawings
FIG. 1 shows an SEM image of the anion exchange beads obtained in example 1;
FIG. 2 shows an SEM image of the anion exchange beads obtained in comparative example 1;
FIG. 3 shows an SEM image of the anion exchange beads obtained in comparative example 2;
FIG. 4 shows Bioclone magnetic bead SEM images.
Detailed Description
The invention provides a super-paramagnetic strong anion exchange magnetic bead for virus enrichment, a preparation method and application thereof, and a person skilled in the art can properly improve the realization of technological parameters by referring to the content of the text. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and are deemed to be included in the present invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that the invention can be practiced and practiced with modification and alteration and combination of the methods and applications herein without departing from the spirit and scope of the invention.
In the invention, the magnetic microsphere of the poly glycidyl methacrylate can also be a polymer magnetic microsphere formed by copolymerizing the poly glycidyl methacrylate and other monomers. The magnetic microsphere of the poly (glycidyl methacrylate) is produced by the biological medical engineering Co-Ltd of the beaver in Suzhou and has a multi-layer core-shell structure. The core is superparamagnetic ferroferric oxide with the diameter of about 200-400 nm, the core is synthesized by a solvothermal method, the middle layer is a silicon dioxide protective layer and is synthesized by a St-amber method and used for protecting magnetic substances, the outermost layer is a polymer layer formed by polymerizing glycidyl methacrylate or copolymerizing glycidyl methacrylate and other monomers, and the surface of the shell layer is rich in active epoxy groups. Preferably, the polymer magnetic microsphere has a three-layer core-shell structure and superparamagnetism, and the surface of the polymer magnetic microsphere has a large number of active epoxy groups and is not modified by other functional groups. The particle size of the magnetic microsphere of the polyglycidyl methacrylate used in the invention is preferably 400-800 nm.
The test materials adopted by the invention are all common commercial products and can be purchased in the market. The invention is further illustrated by the following examples:
EXAMPLE 1 preparation of Supercisive strong anion exchange magnetic beads
The polymer magnetic microspheres used in this example are poly glycidyl methacrylate magnetic microspheres, which are produced by the company limited biomedical engineering of beaver, su zhou. The polymer magnetic microsphere consists of a three-layer core-shell structure, wherein the inner core of the polymer magnetic microsphere is a superparamagnetic ferroferric oxide microsphere with the diameter of 200-400 nm, the polymer magnetic microsphere is synthesized by a solvothermal method, the middle shell is a silicon dioxide protection layer, the thickness of the shell is about 50nm, the polymer magnetic microsphere is synthesized by a St method for protecting magnetic substances, the outermost layer is a polymer shell, and the thickness of the outermost layer is about 30-nm, and the polymer magnetic microsphere is prepared by a free radical polymerization method.
The super-paramagnetic anion exchange magnetic beads are prepared according to the following steps:
S1, adding 20 g polymer magnetic microspheres and 3200 mL absolute ethyl alcohol into a 5000 mL three-neck flask, stirring uniformly by ultrasonic, placing into a constant-temperature water bath kettle, adding 35 mL of 3, 3-diaminodipropylamine, introducing nitrogen to deoxidize for 10 minutes, heating to 70 ℃, reacting for 8 hours at the temperature, washing the obtained amino polymer magnetic microspheres with the absolute ethyl alcohol for 1 time, washing with distilled water for 3 times, and storing in distilled water for later use.
S2, adding the amino polymer magnetic microsphere obtained in the step S1 and 1600 mL of 0.5M sodium hydroxide solution into a 3000 mL three-neck flask, stirring uniformly by ultrasound, placing into a constant-temperature water bath kettle, adding 20 g of 2, 3-epoxypropyl trimethyl ammonium chloride, heating to 40 ℃, and reacting for 4 hours at the temperature. The obtained quaternary ammonium salt modified polymer magnetic microsphere is named mPGMA-Q, is washed to be neutral by pure water, and is stored in 10mM NaCl solution for standby.
SEM detection is carried out on mPGMA-Q obtained in the step S2, the detection result is shown in figure 1, the obtained anion exchange magnetic beads are spherical nano-particles, the surface is smooth, no obvious agglomeration exists, the particle size distribution is uniform, the average particle size is 484 nm, and the content of quaternary ammonium salt groups is 250 mu mol/g measured by a titration method.
Comparative example 1 preparation of comparative anion exchange magnetic beads 1
The magnetic beads are poly (styrene-glycidyl methacrylate) copolymer magnetic microspheres (the synthesis process of the magnetic beads is basically consistent with that of PGMA microspheres in the embodiment, the difference is that monomer GMA is replaced by St (styrene) and GMA (GMA is added after St is added for one hour), the rest steps and the used reagents are not different, wherein the mass ratio of FeCl 3, TEOS, gamma-methacryloxypropyl trimethoxysilane (MPS), st (styrene) and Glycidyl Methacrylate (GMA) is 1:0.40:0.50:0.60:0.205), the magnetic beads are produced by the company limited by the biomedical engineering of beaver in Suzhou, the magnetic beads are copolymer magnetic microspheres consisting of a three-layer core-shell structure, the inner core of the magnetic beads is super-paramagnetic ferroferric oxide microspheres, the diameter of the magnetic beads is 200-400 nm, the magnetic beads are synthesized by a solvothermal method, the middle shell layer is silicon dioxide, the thickness is about 50 nm, the magnetic beads are synthesized by a St protective layer method, the outermost layer is used for protecting magnetic substances, and the free radical polymerization of the styrene and the glycidyl methacrylate is formed by adopting a free radical polymerization method nm.
Comparative anion exchange beads 1 were prepared as follows:
S1, adding 20 g parts of amination modified copolymer magnetic microsphere and 3200 parts of mL absolute ethyl alcohol into a 5000 and mL three-neck flask, stirring uniformly by ultrasonic, placing into a constant-temperature water bath kettle, adding 30mL parts of ethylenediamine, introducing nitrogen to deoxidize for 10 minutes, heating to 70 ℃, reacting for 8 hours at the temperature, washing the obtained amination modified copolymer magnetic microsphere with absolute ethyl alcohol for 1 time, washing with distilled water for 3 times, and preserving in distilled water for standby.
S2, adding the amination modified polymer magnetic microsphere obtained in the step S1 and 1600 mL of 0.5M sodium hydroxide solution into a 3000 mL three-neck flask, stirring and dispersing uniformly by ultrasound, placing into a constant-temperature water bath, adding 20 g of 2, 3-epoxypropyl trimethyl ammonium chloride, heating to 40 ℃, and reacting for 4 hours at the temperature. The obtained quaternary ammonium salt modified copolymer magnetic microsphere is named as mP (St-GMA) -Q, is washed to be neutral by a large amount of pure water, and is stored in a 10 mM NaCl solution for standby.
And (2) carrying out SEM (scanning electron microscope) detection on the magnetic microsphere obtained in the step (S2), wherein the detection result is shown in fig. 2, the obtained anion exchange magnetic bead is spherical nano-particles, has the advantages of rough surface, no obvious agglomeration, uniform particle size distribution and average particle size of 546 nm, and the content of quaternary ammonium salt groups is 130 mu mol/g as determined by a titration method.
Comparative example 2 preparation of comparative anion exchange magnetic beads 2
The magnetic beads are silicon hydroxyl magnetic microspheres, which are produced by the company of biomedical engineering of beaver, suzhou (silicon dioxide hydroxyl magnetic beads (Super 500 nm), cat# 70304). The silicon hydroxyl magnetic microsphere consists of a core-shell structure, wherein the core of the core-shell structure is superparamagnetic ferroferric oxide with the diameter of 300-400 nm, the core is synthesized by adopting a solvothermal method, the middle shell layer is a silicon dioxide protection layer, the thickness of the shell layer is about 50 nm, and the core-shell structure is synthesized by adopting a St method and is used for protecting magnetic substances.
Comparative anion exchange beads 2 were prepared as follows:
S1, adding 4.0 g silicon hydroxyl magnetic microspheres, 560 mL ethanol and 48 mL distilled water into a 1000 mL three-neck flask, stirring uniformly by ultrasonic, placing into a constant-temperature water bath kettle, adding 2.4 mL ammonia water and 6.0 mL gamma-methacryloxypropyl trimethoxysilane, introducing nitrogen to deoxidize for 10 minutes, heating to 70 ℃, reacting for 6 hours under the temperature condition, washing the obtained double-bond modified silicon hydroxyl magnetic beads with absolute ethyl alcohol for 3 times, and storing in the absolute ethyl alcohol for standby.
S2, preparing the magnetic microsphere of the polymethacryloyloxy propyl trimethoxy silane, namely adding 4.0g of double bond modified silicon hydroxyl magnetic beads obtained in the step S1, 560 mL of distilled water, 24 of mL of ethanol, 0.168 g of sodium dodecyl benzene sulfonate, 10.2 mL of methacryloyloxyethyl trimethyl ammonium chloride, 2.4 g of methylene bisacrylamide and 0.24 of g AIBN into a three-neck flask of 1000 mL, introducing nitrogen to deoxidize for 10 minutes, then heating to 70 ℃ and reacting for 6 hours at the temperature, wherein the obtained magnetic microsphere of the polymethacryloyloxy propyl trimethoxy silane is named mPMAC, washing 3 times by absolute ethyl alcohol, washing 3 times by distilled water, and preserving in a 10 mM NaCl solution for standby.
And (3) carrying out SEM (scanning electron microscope) detection on the magnetic microsphere obtained in the step (S2), wherein the detection result is shown in figure 3, the obtained anion exchange magnetic bead is spherical nano-particles, the surface is smooth, the particle size distribution is nonuniform, obvious agglomeration exists among the particles, the average particle size is 468 nm, and the content of quaternary ammonium salt groups is 480 mu mol/g by a titration method.
EXAMPLE 3 enrichment Effect of magnetic beads on New coronaviruses
The viruses used in this test example were novel coronavirus pseudoviruses, purchased from Xiamen Biotechnology Co., ltd, and used magnetic beads to refer to mPGMA-Q, mP (St-GMA) -Q and mPMAC prepared in example 1 and comparative example.
The test method comprises the steps of adding a certain volume of magnetic bead suspension into a centrifuge tube, wherein the concentration of the magnetic beads is 10 mg/mL, magnetically sucking off liquid, adding a certain volume of virus suspension, uniformly dispersing by vortex, and uniformly mixing on a vertical mixer for a certain time. After magnetic attraction, 300. Mu.L of the supernatant was taken for analysis, and the rest was discarded. 300. Mu.L of eluent (1 XPBS solution containing 0.75M KCl) was added into the centrifuge tube, and after vortex dispersion was uniform, the mixture was placed on a vertical mixer and mixed well for 15: 15min, and after magnetic attraction, all the supernatant was taken for analysis. The virus DNA is extracted by using an animal virus DNA/RNA extraction kit (product number: 70416 II) produced by the biological medical engineering Co., ltd. In the state of Suzhou, and the virus concentration before and after adsorption is determined by using an established quantitative PCR reaction system.
The enrichment efficiency and elution efficiency were calculated as follows:
(1) The effect of the different beads on the virus enrichment efficiency was determined according to the test method described above under conditions of 10 mg for the beads, 1X 10 5 copy/mL for the virus concentration and 10 mL for the virus suspension, and the results are shown in Table 1, as mPGMA-Q and mPMAC beads were more closely enriched for the novel coronavirus, 99.95% and 99.50%, respectively, significantly higher than mP (St-GMA) -Q (79.46%), probably due to the lower amount of mP (St-GMA) -Q quaternary ammonium groups, resulting in lower affinity for the virus. The elution data showed that mPGMA-Q was 49.16% more efficient than mPMAC (32.26%) and mP (St-GMA) -Q (6.63%) when the PBS solution containing 0.75M KCl was used as the eluent, probably because 1) mPGMA-Q had a uniform particle size and smoother morphology and a more moderate quaternary ammonium salt group content, resulting in a more moderate binding force to the virus, facilitating elution, and 2) mPGMA-Q was more hydrophilic with respect to mPMAC and mP (St-GMA) -Q, reducing the hydrophobic forces between the beads and virus, facilitating elution.
TABLE 1 different magnetic bead to virus enrichment efficiency
(2) The enrichment efficiency of mPGMA-Q magnetic beads of different masses for viruses was determined according to the test method described above at a virus concentration of 1X 10 8 copy/mL and a virus suspension volume of 300. Mu.L, and the results are shown in Table 2. With the increase of the magnetic bead dosage, the adsorption efficiency is gradually increased, when the magnetic bead dosage reaches 0.1 mg (the magnetic bead concentration is 0.33 mg/mL at the moment), the adsorption efficiency reaches 99.65%, the magnetic bead dosage is continuously increased, and the enrichment efficiency is basically maintained unchanged.
TABLE 2 enrichment efficiency of mPGMA-Q magnetic beads of different masses for viruses
(3) The enrichment efficiency of mPGMA-Q magnetic beads (0.1 mg) at various enrichment times was determined at a virus concentration of 1X 10 8 copy/mL and a virus suspension volume of 300. Mu.L according to the test method described above, and the results are shown in Table 3. It can be seen that at 5min, the enrichment efficiency of mPGMA-Q magnetic beads on viruses can reach the maximum value of 99.9%, and the enrichment efficiency is kept unchanged as time increases.
The data indicate that mPGMA-Q magnetic beads can efficiently enrich viruses in a short time.
TABLE 3 enrichment efficiency of New crowns at different enrichment times
Comparative example 3 comparison with existing magnetic beads
Bidding magnetic bead Bioclone magnetic bead (goods number: FU-101)
The enrichment and elution effects of the super-paramagnetic anion exchange magnetic beads and the competitive product magnetic beads on the real African swine fever viruses in different buffer solutions are shown in the table 3, and the enrichment and elution effects of the magnetic beads and the competitive product magnetic beads of the embodiment of the invention on the novel coronaviruses in urban sewage are shown in the table 4.
TABLE 3 enrichment and elution effects of BioBioclone magnetic beads and the super-paramagnetic anion exchange magnetic beads of the present invention on real African swine fever virus in different buffer solutions
TABLE 4 comparison of Bioclone magnetic beads with the super-paramagnetic Strong anion exchange magnetic beads of the invention
The result shows that under different conditions, the enrichment effect of the super-paramagnetic and strong-anion exchange magnetic beads on the African swine fever virus is better than that of the competitive magnetic beads (Ct difference value 1 is larger, the enrichment capacity is stronger), and particularly in a sewage sample, the super-paramagnetic and strong-anion exchange magnetic beads still have certain enrichment capacity, but the competitive magnetic beads can not be enriched almost. Meanwhile, even under almost the same enrichment conditions, the super-paramagnetic anion exchange magnetic beads of the invention are easier to elute (Ct difference 2, smaller difference, indicates better eluting capacity).
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (10)
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