CN112007620A - Preparation method of streptavidin magnetic microspheres - Google Patents
Preparation method of streptavidin magnetic microspheres Download PDFInfo
- Publication number
- CN112007620A CN112007620A CN201910466538.4A CN201910466538A CN112007620A CN 112007620 A CN112007620 A CN 112007620A CN 201910466538 A CN201910466538 A CN 201910466538A CN 112007620 A CN112007620 A CN 112007620A
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- magnetic microspheres
- magnetic
- microspheres
- streptavidin
- preparing
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- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/02—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material
- B01J20/10—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material comprising silica or silicate
- B01J20/103—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material comprising silica or silicate comprising silica
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/28—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties
- B01J20/28002—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties characterised by their physical properties
- B01J20/28009—Magnetic properties
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/28—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties
- B01J20/28014—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties characterised by their form
- B01J20/28016—Particle form
- B01J20/28021—Hollow particles, e.g. hollow spheres, microspheres or cenospheres
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/76—Chemiluminescence; Bioluminescence
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
Abstract
The invention discloses a preparation method of streptavidin magnetic microspheres, which comprises the following preparation steps: 1) preparing carboxyl magnetic microspheres; 2) preparing the streptavidin magnetic microspheres by activating and coupling the carboxyl magnetic microspheres with the cochain avidin; thus, the streptavidin magnetic microsphere which is used for chemiluminescence immunoassay and has good biological activity, high sensitivity and low nonspecific adsorption can be obtained.
Description
Technical Field
The invention relates to the technical field of immunodiagnosis, in particular to a preparation method of streptavidin magnetic microspheres.
Background
Immunodiagnosis (immunodiagnosis) is the application of immunological theories, techniques and methods to diagnose a variety of diseases and determine immune status. The immunodiagnostic reagent has the most varieties in a diagnostic kit, is widely applied to hospitals, blood stations and physical examination centers, and is mainly used for hepatitis detection, venereal disease detection, tumor detection, pregnancy detection and the like. Among them, immunodiagnosis includes radioimmunoassay, enzyme-linked immunosorbent assay, chemiluminescence, etc.
The enzyme-linked immunosorbent assay reagent is low in cost, but is long in reaction time and complicated in steps, and is not beneficial to large-scale use. The radioimmunity has high sensitivity and strong specificity, but has the problems of radiation, pollution and the like. The chemiluminescence reagent has the advantages of sensitivity, rapidness, stability, strong selectivity, good reproducibility, easy operation and flexible and various methods, and is clinically used on a large scale nowadays. Therefore, a preparation method of streptavidin magnetic microspheres which are used for chemiluminescence immunoassay, good in biological activity, high in sensitivity and low in nonspecific adsorption is urgently needed.
Disclosure of Invention
In order to solve the technical problems, the invention provides a preparation method of streptavidin magnetic microspheres for chemiluminescence immunoassay, which has good biological activity, high sensitivity and low non-specific adsorption.
In order to achieve the purpose, the technical scheme of the invention is as follows:
a preparation method of streptavidin magnetic microspheres is characterized by comprising the following preparation steps:
1) preparing carboxyl magnetic microspheres;
2) the carboxyl magnetic microspheres are activated and coupled with cochain avidin to prepare the streptavidin magnetic microspheres.
The invention provides a preparation method of streptavidin magnetic microspheres for chemiluminescence immunoassay, which has good biological activity, high sensitivity and low nonspecific adsorption.
On the basis of the technical scheme, the following improvements can be made:
as a preferred scheme, the preparation of the carboxyl magnetic microsphere comprises the following steps:
1) by FeCl3·6H2Preparation of O to obtain Fe3O4A magnetic core;
2) with Fe3O4Being a magnetic core, SiO2For the shell, Fe is prepared3O4@SiO2Magnetic microspheres;
3) MPS is added, the MPS is used as modified polystyrene, and Fe is used3O4Is magnetic core, MPS and SiO2The polymer of (A) is a shell, and Fe is prepared3O4@SiO2-MPS magnetic microspheres;
4) with Fe3O4@SiO2Preparing Fe by taking MPS as a magnetic core, taking polymer of GMA and MBA as a shell, GMA as glycidyl methacrylate and MBA as 2-mercaptobenzoic acid3O4@SiO2-MPS@P(C7H10O3-C7H6O2S)；
5) Adding sodium carbonate, sodium bicarbonate, 6-aminocaproic acid and sodium borohydride, and reacting to obtain Fe3O4@SiO2-MPS@P(C7H10O3-C7H6O2S) -COOH, namely the carboxyl magnetic microsphere.
As a preferable scheme, the step 5) of preparing the magnetic carboxyl microspheres further comprises the following preparation steps of separating the magnetic carboxyl microspheres by a magnetic separator, repeatedly washing the magnetic carboxyl microspheres with purified water for at least 5 times, storing the magnetic carboxyl microspheres in the purified water, and putting the magnetic carboxyl microspheres into the next step for preparing the streptavidin magnetic microspheres, wherein the quality of the magnetic carboxyl microspheres is qualified.
Preferably, the particle size of the carboxyl magnetic microsphere is 250-300 nm.
As a preferred embodiment, the preparation of streptavidin magnetic microspheres comprises the following steps:
1) washing the carboxyl magnetic microspheres by an activated buffer solution;
2) adding an activation buffer solution containing carbodiimide and an activation buffer solution containing N-hydroxy thiosuccinimide for activation for 13-17 min;
3) adding streptavidin solution to react for 1.5-2.5 h;
4) and (3) blocking the microspheres for 1.5 to 2.5 hours by using a bovine serum albumin solution, washing and storing the microspheres, and preparing the streptavidin magnetic microspheres.
Preferably, the concentration of the carbodiimide in the step 2) is 15-25 mg/ml; the concentration of N-hydroxy thiosuccinimide is 15-25 mg/ml.
Preferably, the concentration of the bovine serum albumin solution in the step 4) is 0.5mg/ml to 1.0 mg/ml.
Preferably, the activation buffer solution comprises: 0.025% -0.075% polysorbate-20 and 0.05-0.1M morpholine ethanesulfonic acid.
Preferably, the pH value of the activation buffer solution is 5.0-6.0.
Preferably, streptavidin has a 127AA core structure, binds 4 biotin molecules per SA molecule, and has an international specific activity of 12-15U/mg.
A preparation method of streptavidin magnetic microsphere, firstly preparing a carboxyl magnetic microsphere with short magnetic response time, long suspension time, uniform particle size and high enough ligand content, and then forming the streptavidin magnetic microsphere by activating and coupling SA protein (streptavidin).
The preparation method of the carboxyl magnetic microsphere with the particle size of 300nm comprises the following steps of firstly using FeCl3 & 6H2Preparation of Fe from O3O4Magnetic core, then coating the magnetic core with SiO2Form Fe3O4@SiO2And then MPS reacts to form Fe3O4@SiO2MPS, subsequent coating with GMA, MBA to Fe3O4@SiO2-MPS @ P (GMA-MBA), and finally sodium carbonate, sodium bicarbonate and 6-aminocaproic acid, sodium borohydride to form Fe3O4@SiO2MPS @ P (GMA-MBA) -COOH, a carboxylic magnetic microsphere used in the present invention. And (3) after the reaction is finished, removing the reaction device, separating the carboxyl magnetic microspheres by using a magnetic separator, repeatedly washing the carboxyl magnetic microspheres for at least 5 times by using purified water, finally storing the carboxyl magnetic microspheres in the purified water, and testing the mass concentration. After the concentration of the carboxyl magnetic microspheres is calibrated, taking a carboxyl magnetic micro-racket electron microscope, observing the coating effect of the polymer and the uniform degree of the particle size, and testing whether residual magnetism exists or not. And testing the magnetic attraction time and the suspension time. And after all quality tests reach the standard, putting the product into the next step for use.
The activation of the carboxyl magnetic microspheres adopts a method of EDC/Sulfo-NHS aqueous phase activation. EDC is a water-soluble carbodiimide that is used as a carboxyl activating reagent in amide synthesis, as well as for the activation of phosphate groups, the cross-linking of proteins with nucleic acids, and the preparation of immunoconjugates. The pH range of use is 4.0-6.0, and is often used in combination with N-hydroxysuccinimide (NHS) or N-hydroxythiosuccinimide (Sulfo-NHS) to improve coupling efficiency. NHS is an additive in the improved amidation and peptide coupling reactions to activate the carbonyl group upon amide bond formation. Sulfo-NHS is a derivative of NHS, has better water solubility than NHS, and has negative charge to prevent polymerization of the attached substrate (such as protein). The reaction can be selected from NHS and Sulfo-NHS, and the effect of the Sulfo-NHS is better. The preparation method comprises the steps of washing 10mg/mL carboxyl magnetic microspheres by using MEST buffer (PH5.0-6.0), adding half volume of 20mg/mL EDC in MEST and 20mg/mL Sulfo-NHS in MEST for activation for 15min, adding 1.0mg/mL streptavidin solution for reaction for two hours, then sealing by using 1% BSA (bovine serum albumin) solution for two hours, and finally washing and storing to form the streptavidin magnetic microspheres.
The streptavidin is a homotetrameric protein secreted by Streptomyces avidinii. The product has high affinity with biotin, has an optimal core structure of 127AA, is combined with 4 biotin molecules per molecule of SA, and has the specific activity of 12-15U/mg. SA is more specific than Avidin (AV), with a half-life of up to 6 hours after binding to biotin, whereas the AV half-life is only 1 hour. Since SA contains no sugar groups and the isoelectric point is close to neutral, SA has a lower non-specific background than AV in assay applications. The used streptavidin comes from escherichia coli fermentation engineering strains, the molecular weight is about 60kDa, and the product purity is more than or equal to 95 percent.
A preparation method of streptavidin magnetic microspheres comprises the following preparation steps:
1) preparation method of 300nm carboxyl magnetic microspheres
Firstly, the selection of the particle size of the magnetic microspheres is carried out, the particle size of the common immune magnetic microspheres in the market is more than or equal to 1 mu m, the magnetic microspheres with larger particle size generally have the problems of short suspension time and unfavorable subsequent reaction, and the 300nm magnetic microspheres have very good suspension performance and long suspension time through testingFor more than three hours. Secondly, coating SiO with magnetic core2The temperature is controlled, water is changed for the ultrasonic instrument every 30-40min in the reaction process, the reaction temperature is prevented from being higher than 50 ℃, magnetic core oxidation is easily caused when the reaction temperature is too high, the magnetic property of the magnetic microsphere is reduced by the magnetic core oxidation, the magnetic microsphere with weaker magnetic property is slowly lost in the magnetic attraction process, and the use standard cannot be reached. In addition, the 300nm carboxyl magnetic microsphere is coated with two layers of polymers in the preparation process, on one hand, a net structure can be formed, magnetic core leakage is reduced, on the other hand, the acid resistance of the magnetic microsphere is higher, the magnetic microsphere can be used under an acidic condition, and the structure of the magnetic microsphere is not damaged. Finally, it is very important that the polymers added in the preparation process of the magnetic microsphere are various, and by-products which are difficult to clean are easily formed, and the performance of the magnetic microsphere is directly influenced, so that the adding amount, concentration and proportion of GMA (glycidyl methacrylate), MBA (2-mercaptobenzoic acid) and AIBN (azobisisobutyronitrile), such as 3g Fe, are strictly controlled when coating the polymer layer3O4@SiO2The MPS magnetic microsphere reaction system is 9mL GMA, 9.0g MBA and 360mg AIBN, and when the preparation amount of the magnetic microspheres is increased, the amplification is carried out according to the proportion. The temperature of the step is also very critical and needs to be strictly controlled. After the time of deoxidization and stirring is finished, an oil bath pot switch and a cooling circulating water switch are opened, the temperature is raised to 75 ℃, the consistency between the temperature displayed by the instrument and the actual temperature needs to be concerned, and a thermometer needs to be used for self-detection and verification if necessary. The magnetic microspheres with uniform particle size and no by-products can be prepared only by ensuring that the whole system is uniformly heated.
2) Selection of carboxyl magnetic microsphere activation mode
NHS is an additive in the improved amidation and peptide coupling reactions to activate the carbonyl group upon amide bond formation. Sulfo-NHS is a sulfonated product of NHS, is more water soluble than NHS, and has a negative charge that is less likely to cause polymerization of the attached substrate (e.g., protein). And not only NHS but also both organic activation and aqueous activation can be selected in the manner of activation. DMF (N, N-dimethylformamide) is selected as a solvent for organic activation, water is strictly removed from the DMF (N, N-dimethylformamide) before use, and water is quickly prevented from entering the DMF as much as possible during the activation process, so that the intermediate product is hydrolyzed. Through repeated experiments and comparison of the activation mode and the activation conditions, an optimal activation coupling mode, namely a method for aqueous phase activation of EDC/Sulfo-NHS, is found. Firstly, organic solvents with pungent odor and slight toxicity such as DMF (N, N-dimethylformamide), DMAC (dimethylacetamide) and the like are avoided being used in the experimental process, so that the method is more friendly to the health of operators; secondly, the water phase activation can greatly improve the amount of the protein coated by the carboxyl magnetic microspheres, so that the carrying capacity of the immune magnetic microspheres is obviously improved; moreover, the step of water phase activation is simple and convenient, so that the preparation of an intermediate product NHS magnetic microsphere is avoided, and the immune magnetic microsphere is directly prepared; and the phenomena of magnetic microsphere adhesion and agglomeration and the like can not occur in the activation process, and the physical and chemical properties of the magnetic microsphere are well maintained.
3) Selection of streptavidin
The Biotin-Avidin System (BAS) is a novel biological reaction amplification System that can be combined with a variety of markers that have been successfully studied. The strong binding with high affinity between biotin and avidin and the multi-stage amplification effect make BAS (bovine serum albumin) immune labeling and related tracer analysis more sensitive. It has become a new technology widely used for qualitative and quantitative detection and positioning observation research of trace antigens and antibodies. Streptavidin (SA) is a protein secreted by streptomyces avidinii and has a molecular weight of 65 kD. The streptavidin molecule consists of 4 identical peptide chains, each of which can bind one biotin and is free of any sugar groups, so that, like avidin, one streptavidin molecule can also bind 4 biotin molecules, both with an affinity constant (K) of 1015 mol/L. Streptavidin has a wider application range than avidin. The types of streptavidin on the market are many, and wild types and modified types exist. The wild type streptomycin avidinii secreted by Streptomyces avidinii is selected, and the wild type streptomycin avidinii is subjected to purity test, concentration test and specific activity test before use, and is used after all the streptomycin avidinii reaches the standard.
The invention takes 300nm carboxyl magnetic microspheres as raw materials, selects proper streptavidin for coupling, and forms the streptavidin magnetic microspheres. The magnetic response time of the streptavidin magnetic microsphere is less than or equal to 20 s; the suspension time is more than or equal to 40 min; the binding capacity of the free biotin is more than or equal to 1000 pmol/mg; the binding capacity of Biotin-IgG is more than or equal to 20 ug/mg; the binding capacity of the Biotin-Probe (Biotin Probe) was 450pmol/mg or more. The standard curve has good linearity in a chemiluminescence test, R2 is more than or equal to 0.99, S1/S0 is more than 2.6, S7/S0 is more than 500, and the CV value is less than or equal to 10 percent after the same sample is detected for 10 times.
Drawings
FIG. 1 is a graph of a MYO luminescence assay of streptavidin magnetic microspheres provided by an embodiment of the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Unless otherwise specified, the reagents used in the following examples are commercially available from normal sources.
Preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings.
In order to achieve the purpose of the invention, the invention provides a preparation method of streptavidin magnetic microspheres, which is characterized by comprising the following preparation steps:
1) preparing carboxyl magnetic microspheres;
2) the carboxyl magnetic microspheres are activated and coupled with cochain avidin to prepare the streptavidin magnetic microspheres.
The invention provides a preparation method of streptavidin magnetic microspheres for chemiluminescence immunoassay, which has good biological activity, high sensitivity and low nonspecific adsorption.
On the basis of the technical scheme, the following improvements can be made:
in some embodiments, preparing the carboxyl magnetic microspheres comprises the steps of:
1) by FeCl3·6H2Preparation of O to obtain Fe3O4A magnetic core;
2) with Fe3O4Being a magnetic core, SiO2For the shell, Fe is prepared3O4@SiO2Magnetic microspheres;
3) MPS is added, the MPS is used as modified polystyrene, and Fe is used3O4Is magnetic core, MPS and SiO2The polymer of (A) is a shell, and Fe is prepared3O4@SiO2-MPS magnetic microspheres;
4) with Fe3O4@SiO2Preparing Fe by taking MPS as a magnetic core, taking polymer of GMA and MBA as a shell, GMA as glycidyl methacrylate and MBA as 2-mercaptobenzoic acid3O4@SiO2-MPS@P(C7H10O3-C7H6O2S)；
5) Adding sodium carbonate, sodium bicarbonate, 6-aminocaproic acid and sodium borohydride, and reacting to obtain Fe3O4@SiO2-MPS@P(C7H10O3-C7H6O2S) -COOH, namely the carboxyl magnetic microsphere.
In some embodiments, the step 5) of preparing the magnetic carboxyl microspheres further comprises a step of separating the magnetic carboxyl microspheres by a magnetic separator, repeatedly washing the magnetic carboxyl microspheres with purified water for at least 5 times, storing the magnetic carboxyl microspheres in the purified water, and putting the magnetic carboxyl microspheres into the next step to prepare the streptavidin magnetic microspheres, wherein the quality of the magnetic carboxyl microspheres is qualified.
In some embodiments, the particle size of the carboxyl magnetic microsphere is 250-300 nm.
In some embodiments, preparing streptavidin magnetic microspheres comprises the steps of:
1) washing the carboxyl magnetic microspheres by an activated buffer solution;
2) adding an activation buffer solution containing carbodiimide and an activation buffer solution containing N-hydroxy thiosuccinimide for activation for 13-17 min;
3) adding streptavidin solution to react for 1.5-2.5 h;
4) and (3) blocking the microspheres for 1.5 to 2.5 hours by using a bovine serum albumin solution, washing and storing the microspheres, and preparing the streptavidin magnetic microspheres.
In some embodiments, the concentration of carbodiimide in step 2) is from 15 to 25 mg/ml; the concentration of N-hydroxy thiosuccinimide is 15-25 mg/ml.
In some embodiments, the concentration of the bovine serum albumin solution in step 4) is from 0.5mg/ml to 1.0 mg/ml.
In some embodiments, the activation buffer solution is 0.05M to 0.1M morpholine ethanesulfonic acid containing 0.025% to 0.075% polysorbate-20.
In some embodiments, the pH of the activation buffer is 5.0 to 6.0.
In some embodiments, streptavidin is a 127AA core structure that binds 4 biotin molecules per molecule of SA, with an international specific activity of 12-15U/mg.
The preparation method of the 300nm carboxyl magnetic microsphere comprises the following preparation steps:
(1)Fe3O4preparation of magnetic cores
50g FeCl was weighed3·6H2Dissolving O in 1000mL of glycol;
sequentially adding 1g of sodium citrate and 55g of sodium acetate into the solution respectively, and continuously stirring until the sodium citrate and the sodium acetate are dissolved to obtain a precursor solution;
the precursor solution is respectively filled into a 250mL hydrothermal reaction kettle;
putting the installed reaction kettle into an oven, setting the reaction temperature to be 200 ℃, and reacting for 20 hours;
collecting Fe after the reaction3O4Repeatedly washing the magnetic microspheres for more than ten times by using ethanol and purified water;
the washed Fe3O4The magnetic microspheres were stored in purified water and tested for mass concentration.
(2)Fe3O4@SiO2Preparation of
2500mL of ethanol, 700mL of purified water, and 30mL of water were placed in a 5-L round-bottom flaskAmmonia and 6g Fe3O4Magnetic microspheres;
installing a mechanical stirring device on the ultrasonic cleaning instrument, turning on a switch, setting the ultrasonic time to be 6.5h, carrying out ultrasonic treatment on the reaction solution while stirring, wherein the stirring speed is 200 rpm;
diluting 9mL TEOS (tetraethyl orthosilicate) solution in 100mL ethanol, and then adding the solution into a 100mL constant-pressure dropping funnel;
after ultrasonic treatment is carried out for 30min, a constant-pressure funnel is arranged on the round-bottom flask, a TEOS (tetraethylorthosilicate)/ethanol mixed solution is dropwise added into a reaction system, and the ultrasonic stirring reaction is continuously maintained for 6 h;
in the reaction process, water is changed for the ultrasonic instrument at intervals to prevent overhigh temperature;
after the reaction is finished, the device is dismantled, and a magnetic separator is used for collecting Fe3O4@SiO2Magnetic microsphere, washing with ethanol and purified water for more than 10 times, and washing with Fe3O4@SiO2The magnetic microspheres were stored in purified water and tested for mass concentration.
(3)Fe3O4@SiO2Preparation of-MPS
1000mL of ethanol, 500mL of purified water, 10mL of ammonia water and Fe were added to a 2L round-bottom flask3O4@SiO2Magnetic microspheres;
carrying out ultrasonic treatment on the reaction solution for 30 min;
a mechanical stirring device is arranged on the experiment table, and the stirring speed is set to be 200 rpm;
taking 9mL of MPS (modified polystyrene) to dilute in 100mL of ethanol, and adding into a 100mL constant pressure dropping funnel;
a constant-pressure funnel is arranged on a round-bottom flask, MPS (modified polystyrene)/ethanol mixed solution is dripped into a reaction system, and the mixture is stirred and reacted for 20 hours;
after the reaction is finished, the reaction device is dismantled, and a magnetic separator is used for collecting Fe3O4@SiO2-MPS magnetic microspheres, washed 5 times with ethanol and finally Fe3O4@SiO2MPS magnetic microspheres were stored in ethanol and tested for mass concentration.
(4)Fe3O4@SiO2Preparation of-MPS @ P (GMA-MBA)
3g of Fe3O4@SiO2Dispersing and washing MPS magnetic microspheres in ethanol in a centrifugal tube for 3 times, and finally dispersing in 50mL of ethanol;
respectively adding 3L of purified water and Fe3O4@ SiO2-MPS magnetic microsphere dispersion and 300mg of sodium dodecyl benzene sulfonate into a 5L round-bottom flask, then carrying out ultrasonic treatment for 10min, then installing an oil bath reaction device, setting the stirring speed to be 300rpm, and stirring for 1 h;
adding 9mLGMA (glycidyl methacrylate), 9.0g MBA (2-mercaptobenzoic acid) and 360mg AIBN (azobisisobutyronitrile) into a reaction system, continuing stirring for 1h, and introducing nitrogen into a flask to remove oxygen;
after the time of deoxidization and stirring is finished, opening an oil bath pot switch and a cooling circulating water switch, heating to 75 ℃, continuing introducing nitrogen for protection and stirring, and reacting for 16 hours;
after the reaction is finished, the reaction device is dismantled, and a magnetic separator is used for separating Fe3O4@SiO2-MPS @ P (GMA-MBA) magnetic microspheres, followed by repeated washing with ethanol and purified water for more than 10 times, and finally Fe3O4@SiO2MPS @ P (GMA-MBA) magnetic microspheres were stored in purified water and tested for mass concentration.
(5)Fe3O4@SiO2Preparation of-MPS @ P (GMA-MBA) -COOH
Taking a beaker, adding 20g of sodium carbonate, 3g of sodium bicarbonate and 40g of 6-aminocaproic acid into the beaker respectively, adding 1200mL of purified water, magnetically stirring the mixture until the mixture is dissolved, then adding 1.9g of sodium borohydride into the mixture to continue dissolving the mixture, finally controlling the pH value of the solution to be about 10.6, and transferring the solution into a 2L round-bottom flask;
mixing Fe3O4@SiO2Adding MPS @ P (GMA-MBA) magnetic microspheres into the reaction system, and performing ultrasonic treatment for 20 min;
installing an oil bath reaction and mechanical stirring device, setting the stirring speed to be 200rpm, introducing nitrogen into the flask, and deoxidizing for 30 min;
after deoxygenation is finished, opening an oil bath pot switch and a cooling circulating water switch, heating to 70 ℃, continuing introducing nitrogen for protection and stirring, and reacting for 16 hours;
after the reaction is finished, the reaction device is dismantled, and a magnetic separator is used for separating Fe3O4@SiO2-MPS @ P (GMA-MBA) -COOH magnetic microspheres, followed by repeated washing with purified water for more than 5 times, and finally Fe3O4@SiO2-MPS @ P (GMA-MBA) -COOH magnetic microspheres were stored in purified water and tested for mass concentration.
Experiment for activating and coupling (di) carboxyl 300nm magnetic microspheres with streptavidin
Reagent:
washing buffer solution: MEST buffer, TBST buffer (0.05M TBS +0.15M NaCl + 0.05% Tween (polysorbate-20), pH7.5)
Activation buffer: MEST buffer (0.1M MES (morpholine ethanesulfonic acid) + 0.05% Tween (polysorbate-20), pH5.0)
Coupling buffer: MEST buffer (0.1M MES (morpholine ethanesulfonic acid) + 0.05% Tween (polysorbate-20), pH5.0)
Sealing liquid: TBST buffer containing 10mg/mL BSA (bovine serum Albumin), i.e., TBST buffer containing 1% BSA
Preservation solution: PBS (phosphate buffered saline) buffer containing 0.02% sodium azide
The method comprises the following steps:
(1) taking 15mg of 300nm carboxyl magnetic microspheres in a 2mL EP tube, and washing for 3 times by using MEST buffer (1.5 mL);
(2) preparing 1mL each of 20mg/mL EDC (carbodiimide) in MEST (activation buffer) and 20mg/mL Sulfo-NHS (N-hydroxy thiosuccinimide) in MEST (activation buffer) (now ready for use);
(3) respectively adding 750 mu L of EDC and Sulfo-NHS into a magnetic microsphere centrifuge tube, uniformly mixing, and placing in a vertical mixer for reaction and activation for 15 min;
(4) a1 mg/mL solution of streptavidin was prepared using a MEST buffer.
(5) After activation of the magnetic microspheres is finished, magnetically absorbing supernatant, adding 1.5mL of prepared antibody solution, uniformly mixing, and placing on a vertical mixer for mixing reaction for 2 h;
(6) preparing 10mg/mL BSA blocking solution: weighing 16mg BSA, adding into 1.6mL TBST buffer, and uniformly mixing for later use;
(7)Fe3O4@SiO2after the-MPS @ P (GMA-MBA) -COOH magnetic microsphere grafting SA reaction is finished, magnetically absorbing to remove a supernatant, adding 1.5mL of BSA confining liquid, uniformly mixing, placing on a vertical mixer, mixing, reacting and confining for 1 h;
(8) after the blocking reaction was completed, the supernatant was magnetically aspirated, and the magnetic microspheres were repeatedly washed with TBST buffer (10 times or more);
(9) washing with 1.5mL SA storage buffer (SA storage buffer) was repeated 3 times;
(10) the supernatant was removed by magnetic aspiration and 1.5mL of SA storage buffer was added precisely to the volume to maintain a concentration of 10 mg/mL.
And (III) a streptavidin magnetic microsphere binding biotinylation oligonucleotide capability test experiment.
Reagent:
1 × PBST (0.2% Tween (polysorbate) -20): 100mL of 10 XPBS (phosphate buffered saline) was diluted to 1L with ultrapure water, and 2mL of Tween-20 was added thereto and mixed well. The mixture was filtered through a 0.22 μm filter and stored at 4 ℃.
Biotin-Probe (Biotin-labeled Probe) and NTC-Probe (unlabeled Biotin Probe): both oligonucleotide probes were synthesized by Shanghai, and the probe dry powders were prepared as stock solutions with a concentration of 100. mu.M according to the instructions and diluted 50-fold to 2 pmol/. mu.L with 1 XPBST at the time of use.
The method comprises the following steps:
(1) dividing 4 inlet 1.5mL EP tubes into two groups A and B, wherein each group comprises two tubes (for parallel experiment), and adding 20 mu L (namely 200 mu g) of magnetic microspheres to be detected into the 4 EP tubes;
(2) add 200. mu.L of 1 XPBST to each tube of magnetic microspheres and shake with vortexes. Magnetically separating, removing supernatant, and repeatedly cleaning for 2 times;
(3) mu.L of 100. mu.M (100 pmol/. mu.L) Biotin-Probe was added 245. mu.L 1 XPBST and mixed until homogeneous, at which time the OD260 was approximately 0.8, and labeled S1. NTC-Probe stock solutions were formulated in the same way, with an OD260 of about 0.8, labelled S2;
note: "2 pmol/. mu.L" represents only the concentration labeled by the synthesis company, and should be converted according to the actual A260 value during actual measurement; the Probe stock solution needs to be adjusted to OD260 of about 0.8 by 1 xPBST;
(4) 100 mu L S1 of each group A2 tube magnetic microsphere and 100 mu L S2 of each group B2 tube magnetic microsphere are added and mixed evenly. Placing an EP tube on a vertical mixer, and carrying out rotary mixing reaction for 2 hours at room temperature (which is within the range of 22-28 ℃);
(5) after the reaction is finished, placing the 4 tubes of magnetic microspheres together with the residual S1 and S2 after sample addition in a centrifuge for 12,000g for centrifugation for 5min (4 ℃), taking the supernatant after centrifugation, and measuring the ssDNAA260 value of each tube by using OneDrap 2000(Nucleic Acid module, ssDNA mode) (repeatedly measuring and averaging for three times);
(6) recording the measurement result and calculating the amount of the single-stranded oligonucleotide bound to the magnetic microsphere by the following method:
binding capacity of 1mg magnetic microspheres to 2 Probe probes:
Biotin-Probe binding amount (pmo 1): 5S 1- (A1+ A2)/2S 4.54S 100
NTC-Probe binding amount (pmo 1): 5S 2- (B1+ B2)/2S 4.54S 100
Amount of Biotin-Probe bound per mg of magnetic microspheres (pmo 1): Biotin-Probe binding amount-NTC-Probe binding amount.
The calculation results are as follows:
TABLE C.1 calculation of the amount of bound single stranded oligonucleotides of the magnetic microspheres
The results show that: the streptavidin magnetic microsphere provided by the invention has good capability of combining with Biotin-Probe and low nonspecific adsorption.
(IV) streptavidin magnetic microsphere binding biotin IgG ability test experiment.
Reagent:
1×PBST(0.2％Tween20)：
100mL of 10 XPBS was diluted to 1L with ultrapure water, and 2mL of Tween-20 was added and mixed well. The mixture was filtered through a 0.22 μm filter and stored at 4 ℃.
5% BSA solution:
0.6g BSA was weighed and dissolved in 12mL 1 XPBST. Mix well and store at 4 ℃. The shelf life is 24 h.
TMB (tetramethylbenzidine) reaction termination solution:
2M H2SO454.5mL of a 98% sulfuric acid solution was measured and added dropwise to 350mL of ultrapure water, and the mixture was stirred while being added dropwise. After cooling to room temperature, the ultrapure water is added continuously until the volume is up to 500 mL.
Biotin (Biotin) -rabbit IgG working solution (3. mu.g/mL):
biotin (Biotin) -rabbit IgG was diluted to 3. mu.g/mL with 1 XPBST. The microspheres were stored at 4 ℃ for 24h (approximately 4mL of magnetic microspheres were prepared per batch tested).
Biotin-rabbit IgG standard stock (100 ng/mL):
the Biotin-rabbit IgG working solution (3. mu.g/mL) was diluted 30-fold, i.e., 100. mu.L of Biotin-rabbit IgG working solution was taken, and 1 XPBST was added to the solution to make the total volume 3m L. The product is stored at 4 deg.C for 24 h.
HRP (horseradish peroxidase) -goat anti-rabbit IgG:
HRP (horse radish peroxidase) -goat anti-rabbit IgG was diluted 10000 times with 5% BSA solution. The product is stored at 4 deg.C for 24 h.
The method comprises the following steps:
(1) 50uL of magnetic microspheres to be detected (10mg/mL) are measured by a pipettor and placed in a 1.5mL EP tube, 900uL of 1 xPBST is added for even mixing, the magnetic microspheres are washed, and the supernatant is removed after magnetic separation. Washing for 3 times, adding 500uL of 1 xPBST, mixing, and diluting by 10 times (namely 1 mg/mL);
(2) taking three EP tubes with 2.0mL inlets, adding 100 mu L (namely 100 mu g) of magnetic microspheres (for parallel experiments) into each tube, carrying out magnetic separation, and removing supernatant;
(3) the prepared Biotin-rabbit IgG working solution is marked as S. Adding 1.5mL Biotin-rabbit IgG working solution into each tube of magnetic microspheres, placing an EP tube on a vertical mixer, and rotationally mixing for 2 hours at room temperature (which is within the range of 22-28 ℃). The supernatants were collected into new EP tubes, labeled F1, F2, F3, respectively. Finally, S is diluted 150 times by 1 XPBST (S concentration can also be directly replaced by initial addition concentration of 3 ug/ml), F1, F2 and F3 are diluted 120 times for standby. Diluting 10 to 1.5ml or 1.2 ml;
(4) 5 Streptavidin Coated plates (Streptavidin Coated plates), 96-well enzyme-labeled Plate, each well with 200L 1 x PBST, placed in the horizontal shaker washing 3 min. And removing the washing liquid, and beating the residual liquid in the removed plate holes. Washing is repeated for 2 more times according to the steps;
(5) the Biotin-rabbit IgG working solution was diluted to 35ng/mL, 30ng/mL, 25ng/mL, 20ng/mL, 15ng/mL, 10ng/mL, 5ng/mL, and 0ng/mL with 1 XPBST to prepare a standard curve (the Biotin-rabbit IgG concentration was plotted on the abscissa, and the OD450 value measured at the corresponding concentration was plotted on the ordinate);
TABLE C.2
(6) Centrifuging the sample (more than 200 uL) diluted with S, F1 and F2 in C.3 at 17,000g for 5min (4 ℃) for later use;
(7) according to the loading layout of Table C.3, the solution prepared in two steps 5.6 was added to a Streptavidin Coated Plate, 96-well enzyme-labeled Plate, for 4-step spot tests, at 100uL per well;
TABLE C.3
(8) The ELISA plate was placed on a horizontal shaker and incubated at room temperature for 2 h. (it may also be left overnight at 4 ℃);
(9) after the reaction, 200. mu.L of 1 XPBST was added to each well, and the mixture was placed on a shaker at room temperature and washed for 3 min. Removing supernatant, and washing for 2 times;
(10) adding 200 μ L of 5% BSA into each well, placing on a shaker at room temperature, mixing and sealing for 90min, and removing supernatant;
(11) adding 100 mu L of HRP-goat anti-rabbit IgG working solution into each hole, placing on a shaker at room temperature, mixing and incubating for 2h, and removing supernatant;
(12) add 200. mu.L of 1 XPBST to each well, place on a shaker at room temperature, and wash for 3 min. Removing supernatant, and washing for 4 times;
(13) adding 100 μ L of TMB color developing solution into each well, and developing for 5min in dark; after the reaction, 50. mu.L of TMB reaction termination solution (2M concentrated sulfuric acid) is added into each hole; placing the microspheres in an enzyme labeling instrument to measure an OD450 value, and calculating the Biotin-rabbit IgG binding amount of each milligram of magnetic microspheres;
the calculation method comprises the following steps:
(14) taking the Biotin-rabbit IgG concentration (ng/mL) as the abscissa, taking the OD450 value (the average value of the No. 1 lath and the No. 2 lath and the No. 3 lath) measured under the corresponding concentration as the ordinate to make an XY scatter diagram, and calculating the linear equation of the standard curve;
(15) calculating the OD450 mean value of each diluted sample, calculating the concentration of each diluted sample according to a standard curve equation, and respectively multiplying the concentration by the sample dilution times to calculate the concentrations of the stock solution samples C (S) and C (F) (ng/mL);
remarking: c (F) represents: the average of the results calculated for OD450 of C (F1), C (F2), C (F3);
(16) the amount of Biotin-rabbit IgG bound per 100 μ g of magnetic microspheres was (ng): (c(s) -c (f) -1.5;
remarking: 1.5 represents the volume of C.3.3 added to Biotin-rabbit IgG working solution;
(17) the amount of Biotin-rabbit IgG bound per mg of magnetic microspheres was (μ g): (c(s) -c (f) -1.5 x 10/1000, i.e. (c)(s) -c (f) -0.015;
the calculation results are as follows:
TABLE C.4
| Name of magnetic microsphere | Biotin-IgG binding Capacity (ug/mg) |
| SA300nm20191004 | 23.81 |
The results show that: the 300nm streptavidin magnetic microsphere has good capacity of combining with Biotin-IgG, and can reach more than or equal to 20 ug/mg.
And (V) application of streptavidin magnetic beads in a Myoglobin (MYO) chemiluminescence detection system.
Reagent:
wash Buffer (Wash): TBST (Wash) buffer (20mM Tris (triisopropylethanesulfonyl) and 30mM NaCl and 0.1% Tween20)
Enzyme-labeled antibody diluent: PBST containing 5% BSA, ALP-MYO antibody were diluted 1: 2000, and 1ul was diluted to 2 ml.
Biotinylated antibody diluent: 1 PBS containing 0.1% Tween20, 10ug/ml, 10ul antibody diluted to 1 ml.
Antigen dilution: 1 × PBS containing 0.1% Tween20, from 1000ng dilution, adding blank total 10 holes, using the 8 hole.
Free biotin: 2 μ g/ml, diluted with 1 × PBS containing 0.1% Tween 20. (8 ul of D-Biotin at 1mg/ml was diluted to 4ml)
Substrate solution: shenzhen meikanite
Streptavidin magnetic microspheres: after coupling with biotinylated antibody, the cells were blocked with free organisms.
TABLE C.5 antigen preparation methods
Experimental procedure
1) Preparing 1ml of 10ug/ml of biotinylated antibody, and diluting 10ul of antibody to 1 ml;
2) taking 25 mu l (10mg/ml) of each 2-tube streptavidin magnetic microsphere, magnetically sucking the supernatant (1-tube control magnetic bead, 1-tube SA300nm), washing once, adding 500 mu l of 10ug/ml biotinylated antibody, and incubating for 1h at room temperature;
3) after washing for 3 times, 100 mu l of free biotin with the concentration of 2 mu g/ml is added respectively, and the mixture is incubated for 1h at room temperature; after washing for 3 times, each is diluted to 0.5mg/ml with 500. mu.l PBST;
4) taking 2 pieces of chemiluminescent plates, adding streptavidin magnetic microspheres at 50 muL/well into a 96-well plate, adding a test standard at 50 muL/well (3-10), and adding an enzyme-labeled antibody at 100 muL per well; fully shaking and resuspending streptavidin magnetic microspheres, incubating for 15min in a thermostat at 37 ℃, performing magnetic separation, sucking supernatant by using a pipettor, and taking down a 96-well plate from a magnetic separator;
5) adding 200 mu L of Washing buffer into each hole, fully oscillating the streptavidin magnetic microspheres in the resuspension tube, carrying out magnetic separation, sucking supernatant by using a pipettor, taking down a 96-hole plate from the magnetic separator, repeating the step for 2 times, and Washing for 3 times in total;
6) adding 150 mu L of substrate solution into each hole, fully oscillating the streptavidin magnetic microspheres of the resuspension tube, and incubating for 5min in a dark place;
7) the 96-well plate is put into a chemiluminescence apparatus for reading, figure 2.0 is arranged, the user name is logged in, quick measurement is carried out, oscillation is allowed (low intensity 5S, all the wells), measurement is started, Ctr1-C, Ctrl-V is carried out to a U disk.
The experimental results are as follows:
TABLE C.6 results of streptavidin magnetic beads in Myoglobin (MYO) chemiluminescence detection system
TABLE C.7 Experimental results of streptavidin magnetic beads in Myoglobin (MYO) chemiluminescence detection system
| Degree of separation | Controlling magnetic microspheres (degree of separation) | 300nm streptavidin magnetic microsphere (degree of separation) |
| S1/S0 | 8.761 | 11.030 |
| S2/S0 | 19.517 | 12.913 |
| S3/S0 | 45.167 | 27.895 |
| S4/S0 | 102.075 | 61.386 |
| S5/S0 | 235.894 | 154.772 |
| S6/S0 | 547.873 | 330.983 |
| S7/S0 | 972.698 | 632.695 |
From the experimental results and fig. 1, it is shown that: the standard curve of the streptavidin magnetic microsphere in a chemiluminescence test has good linearity, wherein R2 is more than or equal to 0.99, S1/S0 is more than 2.6, and S7/S0 is more than 500.
The above is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, many variations and modifications can be made without departing from the inventive concept of the present invention, which falls into the protection scope of the present invention.
Claims (10)
1. A preparation method of streptavidin magnetic microspheres is characterized by comprising the following preparation steps:
1) preparing carboxyl magnetic microspheres;
2) the carboxyl magnetic microspheres are activated and coupled with cochain avidin to prepare the streptavidin magnetic microspheres.
2. The method for preparing streptavidin magnetic microspheres according to claim 1, wherein the preparation of carboxyl magnetic microspheres comprises the following steps:
1) by FeCl3·6H2Preparation of O to obtain Fe3O4A magnetic core;
2) with Fe3O4Being a magnetic core, SiO2For the shell, Fe is prepared3O4@SiO2Magnetic microspheres;
3) MPS is added, the MPS is used as modified polystyrene, and Fe is used3O4Is magnetic core, MPS and SiO2The polymer of (A) is a shell, and Fe is prepared3O4@SiO2-MPS magnetic microspheres;
4) with Fe3O4@SiO2Preparing Fe by taking MPS as a magnetic core, taking polymer of GMA and MBA as a shell, GMA as glycidyl methacrylate and MBA as 2-mercaptobenzoic acid3O4@SiO2-MPS@P(C7H10O3-C7H6O2S)；
5) Adding sodium carbonate, sodium bicarbonate, 6-aminocaproic acid and sodium borohydride, and reacting to obtain Fe3O4@SiO2-MPS@P(C7H10O3-C7H6O2S) -COOH, namely the carboxyl magnetic microsphere.
3. The method for preparing streptavidin magnetic microspheres according to claim 2, wherein the step 5) of preparing the carboxyl magnetic microspheres is followed by a step of separating the carboxyl magnetic microspheres by a magnetic separator, washing the carboxyl magnetic microspheres with purified water repeatedly for at least 5 times, storing the carboxyl magnetic microspheres in the purified water, and putting the carboxyl magnetic microspheres into the next step for preparing the streptavidin magnetic microspheres after the quality inspection of the carboxyl magnetic microspheres is qualified.
4. The method for preparing streptavidin magnetic microsphere according to claim 3, wherein the particle size of the carboxyl magnetic microsphere is 250-300 nm.
5. The method for preparing streptavidin magnetic microspheres according to claim 4, wherein the preparation of streptavidin magnetic microspheres comprises the following steps:
1) washing the carboxyl magnetic microspheres by an activated buffer solution;
2) adding an activation buffer solution containing carbodiimide and an activation buffer solution containing N-hydroxy thiosuccinimide for activation for 13-17 min;
3) adding streptavidin solution to react for 1.5-2.5 h;
4) and (3) blocking the microspheres for 1.5 to 2.5 hours by using a bovine serum albumin solution, washing and storing the microspheres, and preparing the streptavidin magnetic microspheres.
6. The method for preparing streptavidin magnetic microspheres according to claim 5, wherein the carbodiimide concentration in step 2) is 15-25 mg/ml; the concentration of N-hydroxy thiosuccinimide is 15-25 mg/ml.
7. The method for preparing streptavidin magnetic microspheres according to claim 5, wherein the concentration of the bovine serum albumin solution in step 4) is 0.5mg/ml to 1.0 mg/ml.
8. The method for preparing streptavidin magnetic microspheres according to claim 5, wherein the activation buffer solution comprises: 0.025% -0.075% polysorbate-20 and 0.05-0.1M morpholine ethanesulfonic acid.
9. The method for preparing streptavidin magnetic microspheres according to claim 8, wherein the PH of the activation buffer solution is 5.0 to 6.0.
10. The method for preparing streptavidin magnetic microspheres according to any one of claims 1 to 9, wherein streptavidin has a 127AA core structure, 4 biotin molecules are bound per SA molecule, and the specific activity is 12 to 15U/mg.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114200126A (en) * | 2021-12-09 | 2022-03-18 | 牟奕 | Solid phase matrix for detecting N-type penicillin and cephalosporin antibiotic antibodies and preparation method thereof |
| CN114231596A (en) * | 2021-12-12 | 2022-03-25 | 徐州医科大学 | Gene detection method based on CRISPR/dcas9 and magnetic nano material and application |
Citations (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5840500A (en) * | 1996-07-11 | 1998-11-24 | Trega Biosciences, Inc. | Quinoline derivatives and quinoline combinatorial libraries |
| CN102159949A (en) * | 2008-06-18 | 2011-08-17 | 加州理工学院 | Multi-ligand capture agents and related compositions, methods and systems |
| US20120097194A1 (en) * | 2002-09-09 | 2012-04-26 | Reactive Surfaces, Ltd. | Polymeric Coatings Incorporating Bioactive Enzymes for Catalytic Function |
| CN102492075A (en) * | 2011-12-08 | 2012-06-13 | 天津医科大学 | Nanometer carboxylated polystyrene microsphere with spacer arm and preparation method thereof |
| US20130084652A1 (en) * | 2010-05-14 | 2013-04-04 | Beckman Coulter, Inc. | Homogeneous Chemiluminescence Assay Methods with Increased Sensitivity |
| CN103278651A (en) * | 2013-06-14 | 2013-09-04 | 博奥赛斯（天津）生物科技有限公司 | Kit for chemiluminescence immunity quantitative detection of MYO (myohaemoglobinnano) nano magnetic particle and preparation method of kit |
| CN103285838A (en) * | 2013-07-02 | 2013-09-11 | 景德镇陶瓷学院 | Preparation method of functional magnetic absorbent used for treating industrial wastewater |
| CN105556289A (en) * | 2013-10-02 | 2016-05-04 | 古河电气工业株式会社 | Method for detecting target substance |
| CN106405075A (en) * | 2016-08-31 | 2017-02-15 | 上海美吉生物医药科技有限公司 | Immunomagnetic bead and preparation method thereof |
| CN106518956A (en) * | 2016-10-24 | 2017-03-22 | 苏州海狸生物医学工程有限公司 | Method for removing nucleic acid pollution from protein purification system |
| CN109225084A (en) * | 2018-10-18 | 2019-01-18 | 郑州安图生物工程股份有限公司 | The preparation method of the core-shell type boryl magnetic microsphere of glycoprotein can be largely enriched with |
-
2019
- 2019-05-30 CN CN201910466538.4A patent/CN112007620B/en active Active
Patent Citations (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5840500A (en) * | 1996-07-11 | 1998-11-24 | Trega Biosciences, Inc. | Quinoline derivatives and quinoline combinatorial libraries |
| US20120097194A1 (en) * | 2002-09-09 | 2012-04-26 | Reactive Surfaces, Ltd. | Polymeric Coatings Incorporating Bioactive Enzymes for Catalytic Function |
| CN102159949A (en) * | 2008-06-18 | 2011-08-17 | 加州理工学院 | Multi-ligand capture agents and related compositions, methods and systems |
| US20130084652A1 (en) * | 2010-05-14 | 2013-04-04 | Beckman Coulter, Inc. | Homogeneous Chemiluminescence Assay Methods with Increased Sensitivity |
| CN102492075A (en) * | 2011-12-08 | 2012-06-13 | 天津医科大学 | Nanometer carboxylated polystyrene microsphere with spacer arm and preparation method thereof |
| CN103278651A (en) * | 2013-06-14 | 2013-09-04 | 博奥赛斯（天津）生物科技有限公司 | Kit for chemiluminescence immunity quantitative detection of MYO (myohaemoglobinnano) nano magnetic particle and preparation method of kit |
| CN103285838A (en) * | 2013-07-02 | 2013-09-11 | 景德镇陶瓷学院 | Preparation method of functional magnetic absorbent used for treating industrial wastewater |
| CN105556289A (en) * | 2013-10-02 | 2016-05-04 | 古河电气工业株式会社 | Method for detecting target substance |
| CN106405075A (en) * | 2016-08-31 | 2017-02-15 | 上海美吉生物医药科技有限公司 | Immunomagnetic bead and preparation method thereof |
| CN106518956A (en) * | 2016-10-24 | 2017-03-22 | 苏州海狸生物医学工程有限公司 | Method for removing nucleic acid pollution from protein purification system |
| CN109225084A (en) * | 2018-10-18 | 2019-01-18 | 郑州安图生物工程股份有限公司 | The preparation method of the core-shell type boryl magnetic microsphere of glycoprotein can be largely enriched with |
Non-Patent Citations (8)
| Title |
|---|
| HAO DONG ET AL.: "Novel sandwich-structured electrochemiluminescence immunosensing platform via CdTe quantum dots-embedded mesoporous silica nanospheres as enhanced signal labels and Fe3O4@SiO2@PS nanocomposites as magnetic separable carriers", 《JOURNAL OF ELECTROANALYTICAL CHEMISTRY》 * |
| JUAN WANG ET AL: "Development of multimodal membrane adsorbers for antibody purification using atom transfer radical polymerization", 《POLYMER》 * |
| ZHAO J. ET AL.: "Preparation and characterization of thermoresponsive poly(N-isopropylacrylamide-co-acrylicacid)-grafted hollow Fe3O4/SiO2 microspheres with surface holes for BSA release", 《MATERIZALS》 * |
| 温汉华: "磁性高分子微球的制备及其应用研究", 《万方数据》 * |
| 童乃虎: "亲水性Fe3O4@SiO2复合微球制备及生物应用研究", 《万方数据》 * |
| 胡林 著: "《有序介孔材料与电化学传感器》", 31 December 2013, 合肥工业大学出版社 * |
| 谢月辉 等: "利用生物信息学数据及工具合成表达链霉亲和素基因", 《暨南大学学报（自然科学与医学版）》 * |
| 邱广明 等: "Fe3O4/P(St-AL)磁性微球的制备和复合微相结构", 《电子显微学报》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114200126A (en) * | 2021-12-09 | 2022-03-18 | 牟奕 | Solid phase matrix for detecting N-type penicillin and cephalosporin antibiotic antibodies and preparation method thereof |
| CN114231596A (en) * | 2021-12-12 | 2022-03-25 | 徐州医科大学 | Gene detection method based on CRISPR/dcas9 and magnetic nano material and application |
| CN114231596B (en) * | 2021-12-12 | 2023-11-21 | 徐州医科大学 | Gene detection method based on CRISPR/dcas9 and magnetic nano material and application thereof |
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Inventor after: Ren Hui Inventor after: Wan Dongju Inventor after: Zhang Yujie Inventor before: Ren Hui Inventor before: Wan Dongju Inventor before: Zhang Yujie |