阅读说明：本技术 一种链霉亲和素修饰γ-Fe2O3磁珠的制备方法 (Preparation method of streptavidin modified gamma-Fe 2O3 magnetic beads ) 是由 李长全 谢宇 李诗琪 于 2020-04-07 设计创作，主要内容包括：本发明公开了一种链霉亲和素修饰γ-Fe2O3磁珠的制备方法,其包括以下步骤：步骤一、制备纳米γ-Fe2O3磁珠；步骤二、用AEAPS修饰纳米粒子表面；步骤三、在纳米γ-Fe2O3磁珠表面连接链霉亲和素。本发明的有益效果有：得到的成品粒径均一、形貌规整,可配套自动化设备进行高通量操作；吸附无过多现象,能确保在实验应用和检测中,结果重复性好。(The invention discloses a preparation method of streptavidin modified gamma-Fe 2O3 magnetic beads, which comprises the following steps: step one, preparing nano gamma-Fe 2O3 magnetic beads; modifying the surface of the nanoparticle by using AEAPS; and step three, connecting streptavidin to the surface of the nano gamma-Fe 2O3 magnetic bead. The invention has the following beneficial effects: the obtained finished product has uniform particle size and regular appearance, and can be matched with automatic equipment for high-throughput operation; the adsorption has no excessive phenomenon, and the result repeatability is good in experimental application and detection.)

1. A preparation method of streptavidin modified gamma-Fe 2O3 magnetic beads is characterized by comprising the following steps: which comprises the following steps:

step one, preparing nano gamma-Fe 2O3 magnetic beads;

modifying the surface of the nanoparticle by using AEAPS;

and step three, connecting streptavidin to the surface of the nano gamma-Fe 2O3 magnetic bead.

2. The method for preparing streptavidin modified gamma-Fe 2O3 magnetic beads according to claim 1, wherein the method comprises the following steps: the method for preparing the nano gamma-Fe 2O3 magnetic beads in the first step comprises the following steps: mixing Fe (NO3)3 and ethylene glycol, refluxing, removing sol, drying at 80 ℃, and putting into a muffle furnace at 400 ℃ to obtain the product.

3. The method for preparing streptavidin modified gamma-Fe 2O3 magnetic beads according to claim 2, wherein the method comprises the following steps: the method for preparing the nano gamma-Fe 2O3 magnetic beads in the first step comprises the following steps: mixing Fe (NO3)3 and ethylene glycol according to a molar ratio of 1:15, refluxing for 12h, removing sol, drying at 80 ℃ for 24h, and placing in a 400 ℃ muffle furnace for 2h to obtain the product.

4. The method for preparing streptavidin modified gamma-Fe 2O3 magnetic beads according to claim 1, wherein the method comprises the following steps: the method for modifying the surfaces of the nanoparticles by using AEAPS in the second step comprises the following steps: adding the nano gamma-Fe 2O3 magnetic beads prepared in the step one into a mixed solution of methanol and glycerol, carrying out ultrasonic treatment, adding AEAPS into the mixed solution, and carrying out ultrasonic treatment to uniformly mix the solution; reacting at 5-90 deg.C, separating out particles, washing with methanol, vacuum drying for two hr, and collecting particles.

5. The method for preparing streptavidin-modified gamma-Fe 2O3 magnetic beads according to claim 4, wherein the method comprises the following steps: the method for modifying the surfaces of the nanoparticles by using AEAPS in the second step comprises the following steps: adding 0.1g of the nano gamma-Fe 2O3 magnetic beads prepared in the step one into a mixed solution of methanol and glycerol, carrying out ultrasonic treatment for 1h, adding AEAPS into the mixed solution, and carrying out ultrasonic treatment for 1h to uniformly mix the solution; reacting at 5-90 deg.C for 2-3 hr, separating out particles, washing with methanol for 2-3 times, vacuum drying for two hr, and collecting particles.

6. The method for preparing streptavidin modified gamma-Fe 2O3 magnetic beads according to claim 1, wherein the method comprises the following steps: the method for connecting streptavidin to the surface of the nano gamma-Fe 2O3 magnetic bead in the third step is as follows: dissolving streptavidin in a PB solution, adding the amino-modified particles in the step two into the PB solution, dispersing by using ultrasound, adding a PB suspension of the amino-modified particles into the streptavidin solution, carrying out oscillation reaction at room temperature, washing and washing by using a PB buffer solution after the reaction is completed, finally dispersing the particles in the PB solution, and storing at 4 ℃ for later use.

7. The method for preparing streptavidin-modified gamma-Fe 2O3 magnetic beads according to claim 6, wherein the method comprises the following steps: the method for connecting streptavidin to the surface of the nano gamma-Fe 2O3 magnetic bead in the third step is as follows: dissolving 2mg streptavidin in 1mL PB solution, wherein the concentration of the PB solution is 0.1mol/L, the pH value is 7, adding 5mg of particles modified with amino groups in the step two into 2mL PB solution, performing ultrasonic dispersion for 10min, adding a suspension of particles modified with amino groups PB into the streptavidin solution, performing oscillation reaction for 24h at room temperature, after the reaction is completed, washing with a PB buffer solution for 2 times, finally dispersing the particles in the PB solution, and storing at 4 ℃ for later use.

Technical Field

The invention relates to a preparation method of streptavidin modified gamma-Fe 2O3 magnetic beads.

Background

In recent years, nucleic acid detection plays an important role in prevention and control of infectious diseases, early diagnosis and accurate treatment of malignant tumors, early screening of genetic diseases, prevention and control of animal and plant viruses, transgene detection and the like, and streptavidin magnetic beads have wide application in the biological field due to extremely high binding affinity. The streptavidin magnetic bead is formed by covalently bonding recombinant medium-sized streptavidin to magnetic microspheres with regular shapes and uniform particle sizes by adopting a directional immobilization technology to form a monomolecular immobilization layer, and can be used for separating and detecting biotinylated nucleic acid, antibody or other biotinylated ligands and target molecules. The demand for streptavidin magnetic beads is increasing at present.

Disclosure of Invention

The technical problem to be solved by the invention is to provide a method for preparing streptavidin modified gamma-Fe 2O3 magnetic beads,

the invention is realized by the following scheme: a preparation method of streptavidin modified gamma-Fe 2O3 magnetic beads comprises the following steps:

step one, preparing nano gamma-Fe 2O3 magnetic beads;

modifying the surface of the nanoparticle by using AEAPS;

and step three, connecting streptavidin to the surface of the nano gamma-Fe 2O3 magnetic bead.

The method for preparing the nano gamma-Fe 2O3 magnetic beads in the first step comprises the following steps: mixing Fe (NO3)3 and ethylene glycol, refluxing, removing sol, drying at 80 ℃, and putting into a muffle furnace at 400 ℃ to obtain the product.

The method for preparing the nano gamma-Fe 2O3 magnetic beads in the first step comprises the following steps: mixing Fe (NO3)3 and ethylene glycol according to a molar ratio of 1:15, refluxing for 12h, removing sol, drying at 80 ℃ for 24h, and placing in a 400 ℃ muffle furnace for 2h to obtain the product.

The method for modifying the surfaces of the nanoparticles by using AEAPS in the second step comprises the following steps: adding the nano gamma-Fe 2O3 magnetic beads prepared in the step one into a mixed solution of methanol and glycerol, carrying out ultrasonic treatment, adding AEAPS into the mixed solution, and carrying out ultrasonic treatment to uniformly mix the solution; reacting at 5-90 deg.C, separating out particles, washing with methanol, vacuum drying for two hr, and collecting particles.

The method for modifying the surfaces of the nanoparticles by using AEAPS in the second step comprises the following steps: adding 0.1g of the nano gamma-Fe 2O3 magnetic beads prepared in the step one into a mixed solution of methanol and glycerol, carrying out ultrasonic treatment for 1h, adding AEAPS into the mixed solution, and carrying out ultrasonic treatment for 1h to uniformly mix the solution; reacting at 5-90 deg.C for 2-3 hr, separating out particles, washing with methanol for 2-3 times, vacuum drying for two hr, and collecting particles.

The method for connecting streptavidin to the surface of the nano gamma-Fe 2O3 magnetic bead in the third step is as follows: dissolving streptavidin in a PB solution, adding the amino-modified particles in the step two into the PB solution, dispersing by using ultrasound, adding a PB suspension of the amino-modified particles into the streptavidin solution, carrying out oscillation reaction at room temperature, washing and washing by using a PB buffer solution after the reaction is completed, finally dispersing the particles in the PB solution, and storing at 4 ℃ for later use.

The method for connecting streptavidin to the surface of the nano gamma-Fe 2O3 magnetic bead in the third step is as follows: dissolving 2mg streptavidin in 1mL PB solution, wherein the concentration of the PB solution is 0.1mol/L, the pH value is 7, adding 5mg of particles modified with amino groups in the step two into 2mL PB solution, performing ultrasonic dispersion for 10min, adding a suspension of particles modified with amino groups PB into the streptavidin solution, performing oscillation reaction for 24h at room temperature, after the reaction is completed, washing with a PB buffer solution for 2 times, finally dispersing the particles in the PB solution, and storing at 4 ℃ for later use.

The invention has the beneficial effects that:

1. the invention relates to a preparation method of streptavidin modified gamma-Fe 2O3 magnetic beads, which comprises the steps of preparing nano gamma-Fe 2O3 magnetic beads, and modifying the surfaces of the nano particles by using AEAPS; the streptavidin is connected to the surface of the nano gamma-Fe 2O3 magnetic bead, so that the obtained finished product has uniform particle size and regular appearance, and can be matched with automatic equipment for high-throughput operation;

2. the streptavidin-modified gamma-Fe 2O3 magnetic bead has no excessive adsorption phenomenon, and can ensure good result repeatability in experimental application and detection.

Detailed Description

The present invention is further illustrated below, but the scope of the invention is not limited to the disclosure.

In the following description, for purposes of clarity, not all features of an actual implementation are described, well-known functions or constructions are not described in detail since they would obscure the invention with unnecessary detail, it being understood that in the development of any actual embodiment, numerous implementation details must be set forth in order to achieve the developer's specific goals, such as compliance with system-related and business-related constraints, changing from one implementation to another, and it being recognized that such development effort might be complex and time consuming, but would nevertheless be a routine undertaking for those of ordinary skill in the art.

A preparation method of streptavidin modified gamma-Fe 2O3 magnetic beads comprises the following steps:

step one, preparing nano gamma-Fe 2O3 magnetic beads: mixing Fe (NO3)3 and ethylene glycol according to a molar ratio of 1:15, refluxing for 12h, removing sol, drying at 80 ℃ for 24h, and placing in a 400 ℃ muffle furnace for 2h to obtain the product.

Step two, modifying the surface of the nanoparticle by using AEAPS: adding 0.1g of the nano gamma-Fe 2O3 magnetic beads prepared in the step one into a mixed solution of methanol and glycerol, carrying out ultrasonic treatment for 1h, adding AEAPS into the mixed solution, and carrying out ultrasonic treatment for 1h to uniformly mix the solution; reacting at 5-90 deg.C for 2-3 hr, separating out particles, washing with methanol for 2-3 times, vacuum drying for two hr, and collecting particles.

Step three, connecting streptavidin to the surface of the nano gamma-Fe 2O3 magnetic bead: dissolving 2mg streptavidin in 1mL PB solution, wherein the concentration of the PB solution is 0.1mol/L, the pH value is 7, adding 5mg of particles modified with amino groups in the step two into 2mL PB solution, performing ultrasonic dispersion for 10min, adding a suspension of particles modified with amino groups PB into the streptavidin solution, performing oscillation reaction for 24h at room temperature, after the reaction is completed, washing with a PB buffer solution for 2 times, finally dispersing the particles in the PB solution, and storing at 4 ℃ for later use.

Although the invention has been described and illustrated in some detail, it should be understood that various modifications may be made to the described embodiments or equivalents may be substituted, as will be apparent to those skilled in the art, without departing from the spirit of the invention.