A method for preparing a magnetic core-shell structure microsphere with pitting pattern

Abstract

The application provides a preparation method of a pimple-like magnetic core-shell structure microsphere, and belongs to the technical field of semiconductor preparation, and comprises the following steps: step S1, preparation of a pimple-like magnetic microsphere; step S2, surface modification of the pimple-like magnetic microsphere; and step S3, preparation of a pimple-like magnetic core-shell structure microsphere (ferroferric oxide@indium gallium oxide@silicon dioxide). The preparation method of the pimple-like magnetic core-shell structure microsphere is characterized in that: the indium gallium oxide particles are deposited on the surface of the microsphere to form a pimple-like magnetic microsphere, and the pimple-like magnetic microsphere is modified, coated with silicon dioxide, and modified by antibody / affinity, so that a magnetic ferroferric oxide@silicon dioxide core-indium gallium oxide@silicon dioxide magnetic microsphere and a magnetic ferroferric oxide@silicon dioxide core-indium gallium oxide@silicon dioxide@anti-sperm antibody magnetic microsphere are successfully prepared.

Description

Technical Field

[0001] This invention belongs to the field of semiconductor fabrication technology, specifically relating to a method for fabricating a speckled magnetic core-shell structured microsphere. Background Technology

[0002] Magnetic microspheres are functional microspheres composed of magnetic and non-magnetic polymer materials.

[0003] In recent years, with the rapid development of nucleic acid diagnostics and chemiluminescence immunoassay, magnetic microspheres, as an important raw material for technical platforms, have been widely used. They are a novel type of functionalized solid carrier, possessing the characteristics of both solid carriers and magnetic materials. Under the influence of an external magnetic field, they can move and concentrate directionally. When the external magnetic field is removed, they can be uniformly dispersed in a liquid with slight oscillation or suction, making solid-liquid phase separation very quick and convenient. High-purity target substances can be obtained through simple elution. The surface of magnetic microspheres is modified with various functional groups such as amino, carboxyl, epoxy, aldehyde, and thiol groups. These groups on the surface of the magnetic microspheres are covalently coupled with target biomolecules such as proteins, enzymes, and nucleic acids. After blocking unbound sites, they can be applied to protein purification, nucleic acid extraction, molecular diagnostics, and immunodiagnostic reagents.

[0004] Surface modification and functionalization of magnetic materials have always been a research hotspot to broaden their application range and achieve better results. Introducing charged ionized groups onto the surface of magnetic beads is an important research direction. Chinese patent CN104759260A discloses an amino-functionalized magnetic silica-iron oxide composite nanomaterial and its preparation method, which introduces cations by amino-functionalizing the magnetic silica-iron oxide composite nanomaterial. Chinese patent CN104014163A discloses a preparation method and application of aminoethylaminopropyl modified iron oxide, which discloses a strategy of introducing positively charged amino groups by modifying iron oxide with N-β(aminoethyl)-γ-aminopropyltrimethoxysilane. In addition, there are reports of directly using quaternary ammonium salt polymers to modify magnetic materials to introduce cations. However, all of the above methods suffer from uncontrollable modification processes and low ionization levels. Moreover, there are currently no reports on anion functionalization of magnetic materials.

[0005] Furthermore, covalent organic framework (COF) materials have become a research hotspot in recent years due to their excellent chemical and thermal stability, high specific surface area, and regular pores. For example, Chinese patent CN106117474A discloses a core-shell structured magnetic composite microsphere of covalent organic framework and its preparation method, but this method is only applicable to the introduction of uncharged covalent organic frameworks.

[0006] However, the specific surface area of ​​magnetic solid fillers obtained by existing preparation methods is not high enough, which greatly limits the further application of magnetic microspheres. Furthermore, how to further increase the specific surface area of ​​spheres, thereby effectively improving their adsorption performance and loading capacity, has become an urgent practical problem to be solved. Summary of the Invention

[0007] Based on the technical problems existing in the prior art, the present invention provides a method for fabricating speckled magnetic core-shell structured microspheres, specifically a method for fabricating speckled magnetic core-shell structured microspheres and their application in cell sorting, enrichment and purification of biomacromolecules such as DNA, and immobilization of enzymes.

[0008] According to the technical solution of the present invention, a method for fabricating speckled magnetic core-shell structured microspheres is provided, which includes the following steps:

[0009] Step S1, fabrication of speckled magnetic microspheres;

[0010] Step S2, surface modification of the speckled magnetic microspheres;

[0011] Step S3: Fabrication of speckled magnetic core-shell structured microspheres (Fe3O4@Silicon dioxide@Indium gallium oxide@Silicon dioxide).

[0012] The preparation of the pitted magnetic microspheres in step S1 includes: mixing indium antimonide or gallium antimonide / indium antimonide with a certain amount of magnetic microspheres at a mass ratio of 1:1, wherein the particle size of the magnetic microspheres is between 300 nanometers and 20 micrometers; placing the microspheres mixed with the indium antimonide or gallium antimonide / indium antimonide mixture in a reaction box; placing the reaction box in a reaction furnace; simultaneously introducing oxygen and argon; applying a predetermined gas pressure to the reaction furnace; and heating and calcining at a predetermined temperature between 300°C and 1500°C for 1 min to 15 min to obtain the pitted magnetic microspheres.

[0013] Preferably, step S1, the preparation of the speckled magnetic microspheres, includes: taking 0.2g to 0.8g of indium antimonide or gallium antimonide / indium antimonide at a mass ratio of 1:1, taking 0.1g to 3g of 500nm to 10μm magnetic microspheres, placing the microspheres mixed with the indium antimonide or gallium antimonide / indium antimonide mixture in a reaction box, placing the reaction box in a reaction furnace, with an oxygen flow rate of 1sccm to 3sccm, an argon flow rate of 50sccm to 100sccm, a reaction pressure of 5Kpa to 30Kpa, and heating and calcining at 600℃ to 900℃ for 1min to 5min, and then obtaining speckled magnetic microspheres.

[0014] More preferably, step S1, the preparation of the speckled magnetic microspheres, includes: taking 0.3g to 0.6g of indium antimonide or gallium antimonide / indium antimonide at a mass ratio of 1:1, taking 0.1g to 2g of 700nm to 8μm microspheres, placing the microspheres mixed with the indium antimonide or gallium antimonide / indium antimonide mixture in a reaction box, placing the reaction box in a reaction furnace, with an oxygen flow rate of 2 to 2.5sccm, an argon flow rate of 80 to 90sccm, a reaction pressure of 7 to 10Kpa, and heating and calcining at 700℃ to 800℃ for 2min to 3min, and then obtaining speckled magnetic microspheres.

[0015] Preferably, a certain amount of the speckled magnetic microspheres prepared in step S1 is taken, and the magnetic microspheres are enriched using a magnetic separation method. The microspheres are washed 1-5 times with deionized water and 1-6 times with anhydrous ethanol, and then dried in a vacuum oven at 25℃-80℃. Further, 0.2-3.5g of powder is added to 80-300ml of 0.5M citric acid solution, ultrasonically dispersed for 10-40 minutes, and stirred for 8-15 hours under nitrogen atmosphere at 60℃. The magnetic beads are separated using a magnetic separation method, washed 1-5 times with acetone, and then dispersed in a certain amount of deionized water to obtain an aqueous magnetic fluid with a concentration of 30 mg / mL.

[0016] Preferably, 1g to 3g of microspheres prepared in step S1 are taken, and magnetic microspheres are enriched using magnetic separation. The microspheres are washed 2 to 4 times with deionized water and 2 to 5 times with anhydrous ethanol, and then dried in a vacuum oven at 30℃ to 56℃. 0.5 to 1.5g of powder is taken and added to 100ml of 0.5M citric acid solution. The powder is ultrasonically dispersed for 15 to 30 minutes and stirred for 6 hours under nitrogen atmosphere at 60℃. The magnetic beads are separated using magnetic separation, washed 2 to 4 times with acetone, and dispersed in a certain amount of deionized water to obtain an aqueous magnetic fluid with a concentration of 30 mg / ml.

[0017] Furthermore, 2g of microspheres prepared in step S1 were taken, and magnetic microspheres were enriched using magnetic separation. The microspheres were washed three times with deionized water and twice with anhydrous ethanol, and dried in a vacuum oven at 40℃~45℃. 1g of powder was taken and added to 80ml of 0.5M citric acid solution, ultrasonically dispersed for 20 minutes, and stirred for 12 hours under nitrogen atmosphere at 60℃. The magnetic beads were separated using magnetic separation, washed three times with acetone, and dispersed in a certain amount of deionized water to obtain an aqueous magnetic fluid with a concentration of 30 mg / ml.

[0018] On the other hand, the reactor includes an inlet pipe, a vacuum chamber, a ceramic boat, a silicon oxide substrate, source materials, a vacuum pump, and a heat insulation layer. Furthermore, different gases participate in the reaction through the inlet pipe, which connects to the vacuum chamber and introduces Ar / O2 gas. The vacuum chamber is the main site for the raw material reaction and is made of high-temperature resistant glass material, with dimensions of 80mm inner diameter * 3mm wall thickness * 1000mm length.

[0019] Compared with the prior art, the method for fabricating the speckled magnetic core-shell structured microspheres of the present invention has the following advantages:

[0020] First, according to the method for fabricating speckled magnetic core-shell structured microspheres of the present invention, speckled magnetic microspheres are fabricated by depositing indium gallium oxide nanoparticles on the surface of microspheres. Through modification of the speckled magnetic microspheres, silica coating, and modification with functional groups such as antibodies / avidin, magnetic magnetite@silicon dioxide magnetic core-indium gallium oxide@silicon dioxide magnetic microspheres and magnetic magnetite@silicon dioxide magnetic core-indium gallium oxide@silicon dioxide@antisperm antibody magnetic microspheres were successfully prepared.

[0021] Secondly, the method for fabricating the speckled magnetic core-shell structured microspheres of the present invention proposes a method for modifying the surface of magnetic microspheres, and characterizes the structure, composition, magnetism, and sperm adsorption of the obtained microspheres by SEM, EDS, XRD, infrared spectroscopy, and microscopy, which effectively improves the adsorption performance and loading capacity of the magnetic microspheres. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0023] Figure 1 This is a SEM schematic diagram of the speckled magnetic microspheres of the present invention;

[0024] Figure 2 This is based on the elemental distribution in the speckled magnetic microsphere material of the present invention;

[0025] Figure 3 This is a schematic diagram of the X-ray energy dispersive spectroscopy (EDS) test results of the micro-area elemental composition of the material according to the present invention;

[0026] Figure 4 This is a magnetic force map of the speckled magnetic microspheres according to the present invention;

[0027] Figure 5This is a specific surface area diagram of the speckled magnetic microspheres according to the present invention;

[0028] Figure 6 The Fourier transform infrared spectrum is based on the present invention;

[0029] Figure 7 This is a schematic diagram of the single-temperature zone tube furnace and material synthesis process according to the present invention;

[0030] Figure 8 This is a schematic diagram of the reactor used in this invention;

[0031] Figure 9 This is a schematic diagram of the heating process of the reactor used in this invention. Detailed Implementation

[0032] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0033] It should also be noted that, for ease of description, only the parts relevant to the invention are shown in the accompanying drawings. Unless otherwise specified, the embodiments and features described herein can be combined with each other.

[0034] It should be noted that the concepts of "first" and "second" mentioned in this invention are only used to distinguish different devices, modules or units, and are not used to limit the order of functions performed by these devices, modules or units or their interdependencies.

[0035] It should be noted that the terms "a" and "a plurality of" used in this invention are illustrative rather than restrictive. Those skilled in the art should understand that, unless otherwise expressly indicated in the context, they should be understood as "one or more".

[0036] The names of the messages or information exchanged between the multiple devices in the various embodiments of the present invention are for illustrative purposes only and are not intended to limit the scope of these messages or information.

[0037] To effectively improve the specific surface area, adsorption performance, and loading capacity of magnetite magnetic microspheres, the technical problem to be solved by this invention is to grow nanowires / dots on the surface of magnetite magnetic microspheres, coat the surface of the microspheres@nanowires / dots with silica, and connect active functional groups such as antibodies and avidin to the surface of silica, and apply them to fields such as DNA enrichment and cell separation.

[0038] Through research, the applicant discovered that the magnetic component of magnetic microspheres is primarily iron(III) oxide (Fe3O4), while the non-magnetic polymer materials include synthetic polymers (such as polystyrene), natural polymers (such as proteins and agarose), and inorganic polymers (such as silica). Magnetic microspheres are widely used in cell sorting, enrichment and purification of biomolecules such as DNA, and enzyme immobilization. The magnetic strength and surface functional group binding ability of the microspheres are crucial factors determining their application effectiveness. The magnetic nanoparticles of iron(III) oxide are mostly spherical, rhombic, or amorphous, with spherical particles exhibiting the highest specific surface area.

[0039] This invention provides a method for fabricating speckled magnetic core-shell structured microspheres, and their application in nucleic acid separation and purification, and cell sorting. The method for fabricating speckled magnetic core-shell structured microspheres includes the following steps:

[0040] Step S1, Fabrication of speckled magnetic microspheres:

[0041] Indium antimonide and gallium antimonide in a mass ratio of 1:1 were mixed with a certain amount of magnetic microspheres. The particle size of the magnetic microspheres was between 300 nanometers and 20 micrometers. The microspheres mixed with the indium antimonide and gallium antimonide mixture were placed in a reaction box, and the reaction box was placed in a reaction furnace. Oxygen and argon were introduced at the same time, and a predetermined gas pressure was applied to the reaction furnace. The mixture was heated and calcined at a predetermined temperature between 300°C and 1500°C for 1 min to 15 min, and then the pitted magnetic microspheres were obtained.

[0042] Preferred Example 1-1: Step S1, the preparation of the speckled magnetic microspheres, includes: taking 0.2g to 0.8g of indium antimonide and gallium antimonide (mass ratio 1:1, i.e., 0.2g to 0.8g mixed), and 0.1g to 3g of 500nm to 10μm magnetic microspheres (Fe3O4 microspheres or Fe3O4@silicon dioxide microspheres). The microspheres mixed with the indium antimonide and gallium antimonide mixture are placed in a reaction box, which is then placed in a reaction furnace. The oxygen flow rate is 1sccm to 3sccm, the argon flow rate is 50sccm to 100sccm, the reaction pressure is 5Kpa to 30Kpa, and the mixture is heated and calcined at 600℃ to 900℃ for 1min to 5min to obtain speckled magnetic microspheres.

[0043] Preferred Examples 1-2: Step S1, preparation of the speckled magnetic microspheres, includes: taking 0.3g to 0.6g of indium antimonide and gallium antimonide (mass ratio 1:1, i.e., 0.3g to 0.6g mixed), and 0.1g to 2g of 700nm to 8μm microspheres (commercial magnetic beads, iron oxide microspheres, or iron oxide@silicon dioxide microspheres). The microspheres mixed with the indium antimonide and gallium antimonide mixture are placed in a reaction chamber, which is then placed in a reactor. The oxygen flow rate is 2 to 2.5 sccm, the argon flow rate is 80 to 90 sccm, the reaction pressure is 7 to 10 kPa, and the mixture is heated and calcined at 700℃ to 800℃ for 2 to 3 minutes to obtain speckled magnetic microspheres.

[0044] like Figure 7 The diagram shows the single-temperature zone tube furnace and the material synthesis process of the present invention. Figure 8 The diagram shows the structural schematic of the reactor used in this invention. Figure 8 The reactor apparatus shown is preferably a single-temperature zone tube furnace. The reactor apparatus includes an inlet pipe 1, a vacuum tube cavity 2, a ceramic boat 3, a silicon oxide substrate 4, a source material 5, a vacuum pump 6, and a heat insulation layer 7.

[0045] In the reactor of the present invention, different gases participate in the reaction through the inlet pipe 1, and the inlet pipe 1 is connected to the vacuum tube cavity 2; Ar / O2 gas is introduced through the inlet pipe.

[0046] Vacuum tube 2 is the main place where the raw material reaction takes place. The vacuum tube is made of high temperature resistant glass material. The vacuum tube used in this invention has an inner diameter of 80mm * wall thickness of 3mm * length of 1000mm.

[0047] The ceramic boat 3 is a boat-shaped container composed of two sets of inverted trapezoidal containers. The adjacent edges of the two sets of inverted trapezoids are connected, forming a centrally symmetrical, transparent edge. Different powder mixtures of indium antimonide and gallium antimonide are placed in each inverted trapezoidal section, meaning the ceramic boat 3 contains a total of 0.16g of indium antimonide and gallium antimonide powder mixtures. The ceramic boat 3 is made of ceramic, is extremely heat-resistant, and suitable for experiments in high-temperature environments. The ceramic boat used in this invention has dimensions of 88mm long * 9mm wide (top width) * 10mm high. The ceramic boat 3 is positioned in the center of the vacuum tube cavity 2, which is beneficial for chemical vapor deposition.

[0048] The silicon dioxide substrate 4 is used as a substrate for growing nanowires. As an insulator, it is suitable for fabricating MSM (Mesh-based) photodetectors. In this invention, the silicon dioxide substrate 4 is disposed on the upper end of the ceramic boat 3, allowing direct reception of chemical vapor deposition of a powder mixture of indium antimonide and gallium antimonide.

[0049] Source material 5 is a chemical vapor deposition material with a purity >99.99%. Preferably, this invention uses 0.16g of a uniformly mixed powder mixture of indium antimonide and gallium antimonide. Source material 5 is placed directly inside the ceramic boat 3, i.e., inside the "boat" of the ceramic boat 3.

[0050] Vacuum pump 6 continuously extracts air from vacuum chamber 2 throughout the experiment to maintain a high vacuum level within the chamber, creating a suitable atmosphere for subsequent chemical vapor deposition reactions. Vacuum pump 6 is located at the second end of vacuum chamber 2, opposite to inlet pipe 1. The vacuum pump used in this invention has a power of 1800W, a flow rate of 300L / min, and external dimensions of 315*165*320mm.

[0051] The heat insulation layer 7 is used because the temperature is high during the reaction and the temperature of the surrounding environment is also affected. Therefore, a heat insulation material needs to be wrapped around the vacuum tube cavity 2. The heat insulation material used in this invention is a high-temperature resistant aluminum silicate ceramic fiber board.

[0052] The air inlet pipe 1 is located at the front end of the vacuum tube cavity 2; the source material 5 is placed in the silicon dioxide substrate 4, and the silicon dioxide substrate 4 is placed upside down on the ceramic boat 3 and then placed in the middle position inside the vacuum tube cavity 2; the vacuum pump 6 is located at the rear end of the vacuum tube cavity 2 to extract the air in the cavity; the heat insulation layer 7 covers the entire vacuum tube cavity 2.

[0053] Furthermore, such as Figure 7 The reactor shown is a single-temperature zone tube furnace. Indium antimonide and gallium antimonide are mixed uniformly at a 1:1 mass ratio and placed in a reaction boat, which is then placed in the center of the single-temperature zone tube furnace. Ferric oxide microspheres are placed separately in another reaction boat, approximately 5 cm behind the indium antimonide and gallium antimonide boats. Heating is performed according to a set temperature curve, and reactions are carried out for 1 min, 2 min, 3 min, 4 min, and 5 min respectively to prepare speckled magnetic microspheres. This embodiment uses... Figure 9 The temperature rise curve shown indicates that the initial temperature of the reactor was 25 °C, which was increased to 500 °C at a rate of 23.75 °C / min. After equilibration for 3 min, the temperature was increased to the maximum within 15 min, the oxygen valve was opened, and reactions were carried out for different durations to prepare the material. Figure 9 The temperature rise curve shown indicates that a large number of Si-OH molecules are formed on the surface of the microspheres under high salt conditions, and nucleic acid molecules are loaded onto the surface of the magnetic beads by interaction with silanol groups.

[0054] Step S2, surface modification of the speckled magnetic microspheres:

[0055] Take a certain amount of the speckled magnetic microspheres prepared in step S1, enrich the magnetic microspheres using magnetic separation, wash with deionized water 1-5 times, wash with anhydrous ethanol 1-6 times, and then dry in a vacuum oven at 25℃-80℃ (vacuum degree preferably below 100 MPa). Take 0.2-3.5g of powder, add it to 80mL-300mL of 0.5M citric acid solution, ultrasonically disperse for 10-40 minutes, and stir and react at 60℃ under nitrogen atmosphere for 8-15 hours. Separate the magnetic beads using magnetic separation, wash with acetone 1-5 times, and then disperse in a certain amount of deionized water to obtain an aqueous magnetic fluid with a concentration of 30 mg / mL.

[0056] Preferred Example 2-1: Take 1g-3g of microspheres prepared in step S1, enrich the magnetic microspheres using magnetic separation, wash with deionized water 2-4 times, wash with anhydrous ethanol 2-5 times, and dry in a vacuum oven at 30℃-56℃. Take 0.5-1.5g of powder, add it to 100ml of 0.5M citric acid solution, ultrasonically disperse for 15-30 minutes, and stir and react for 6 hours at 60℃ under nitrogen atmosphere. Separate the magnetic beads using magnetic separation, wash with acetone 2-4 times, and disperse in a certain amount of deionized water to obtain an aqueous magnetic fluid with a concentration of 30 mg / mL.

[0057] Preferred Example 2-2: 2g of microspheres prepared in step S1 were taken, and the magnetic microspheres were enriched using magnetic separation. The microspheres were washed three times with deionized water and twice with anhydrous ethanol, and then dried in a vacuum oven at 40℃~45℃. 1g of powder was added to 80mL of 0.5M citric acid solution, ultrasonically dispersed for 20 minutes, and stirred for 12 hours under nitrogen atmosphere at 60℃. The magnetic beads were separated using magnetic separation, washed three times with acetone, and dispersed in a certain amount of deionized water to obtain an aqueous magnetic fluid with a concentration of 30 mg / mL.

[0058] Step S3: Fabrication of speckled magnetic core-shell structured microspheres (Fe3O4@Silicon dioxide@Indium gallium oxide@Silicon dioxide); specifically, 2–5 mL of aqueous magnetic fluid is added to 4–10 mL of ammonia water and ethanol-deionized water in a 4:1 ratio solution, and ultrasonically dispersed for 5–40 minutes. The mixture is then mechanically stirred at 20–60°C for 15–30 minutes. 0.5–5 mL of orthosilicate is added, and the mixture is mechanically stirred at 20–60°C for 2–12 hours. The magnetic beads are separated using magnetic separation, washed 2–8 times with anhydrous ethanol and deionized water respectively, and dispersed in a certain amount of deionized water to obtain an aqueous magnetic fluid with a concentration of 30 mg / mL. The structure, composition, and magnetic properties of the obtained microspheres are characterized using SEM, EDS, XRD, and infrared spectroscopy.

[0059] Preferred Example 3-1: Take 3-5 mL of aqueous magnetic fluid and add it to 5-8 mL of ammonia water and ethanol-deionized water solution in a 4:1 ratio. Disperse the mixture ultrasonically for 15-30 minutes, then mechanically stir for 15-30 minutes at 20°C-60°C. Add 0.5-5 mL of orthosilicate and mechanically stir for 5-10 hours at 25°C-40°C. Separate the magnetic beads using magnetic separation. Wash the beads 3-6 times with anhydrous ethanol and deionized water respectively, and disperse them in a certain amount of deionized water to obtain an aqueous magnetic fluid with a concentration of 30 mg / mL. Characterize the structure, composition, and magnetic properties of the obtained microspheres using SEM, EDS, XRD, and infrared spectroscopy.

[0060] Preferred Example 3-2: Take 4 ml or 2 ml of aqueous magnetic fluid and add it to 5 ml of ammonia water and ethanol-deionized water solution in a 4:1 ratio. Disperse the mixture ultrasonically for 15-30 minutes, then mechanically stir at 40°C for 15-30 minutes. Add 0.5 ml of orthosilicate and mechanically stir at 40°C for 12 hours. Separate the magnetic beads using magnetic separation. Wash the beads 2-3 times with anhydrous ethanol and deionized water respectively, and disperse them in a certain amount of deionized water to obtain an aqueous magnetic fluid with a concentration of 30 mg / ml.

[0061] This invention provides a method for fabricating speckled magnetic core-shell structured microspheres, offering applications for these microspheres in nucleic acid separation and purification, surface functionalization of speckled magnetic iron(III) oxide@silicon dioxide core-indium gallium oxide@silicon dioxide shell structured microspheres, and the application of speckled magnetic iron(III) oxide@silicon dioxide core-indium gallium oxide@silicon dioxide@antisperm antibody magnetic beads in sperm enrichment in sperm-epithelial cell hybrid cells. The method for fabricating speckled magnetic core-shell structured microspheres of this invention includes the following technical contents or applications.

[0062] 1. Application of speckled magnetic core-shell structured microspheres in nucleic acid separation and purification:

[0063] Add 2 μL of whole blood to a 2 mL EP tube, add 100 μL of cell lysis buffer (containing guanidine thiocyanate and SDS), and 0.1 μL of proteinase K. Incubate at 56°C for 30 minutes to 1 hour. Add 500 μL of cell adsorption buffer (containing guanidine thiocyanate) to the solution, mix well, then add 20 μL of aqueous magnetic fluid (containing prepared speckled magnetic core-shell structure microspheres). Mix for 10 to 15 minutes, adsorb the magnetic beads using a magnetic rack, discard the liquid, wash twice with 80% ethanol, wash once with anhydrous ethanol, heat at 56°C for 5 to 10 minutes to dry the anhydrous ethanol, add 20 to 100 μL of DNA preservation solution (containing TE buffer), heat at 56°C for 5 to 10 minutes to fully elute the DNA, adsorb the magnetic beads using a magnetic rack, and recover the DNA preservation solution (containing TE buffer). The concentration and purity of the DNA were detected using a UV spectrophotometer. Commercially available magnetic beads were used, and a control parallel experiment was conducted according to the kit instructions to compare the DNA concentration and purity.

[0064] Whole blood refers to all the components of blood in the human body. Whole blood is composed of plasma and blood cells. Whole blood has functions such as transport, regulation, immunity, defense, coagulation, and hemostasis. The composition, classification, and functions of plasma and blood cells are as follows: (1) Plasma consists of water, albumin, globulin, various electrolytes, small molecule organic compounds, and some gases. Small molecule organic compounds include blood glucose, blood lipids, and proteins. Proteins are further divided into albumin, globulin, and fibrinogen. Electrolytes are the main substances that constitute the osmotic pressure of plasma crystals and maintain the water balance inside and outside blood cells; proteins constitute the osmotic pressure of plasma colloids and maintain the water balance inside and outside blood vessels. (2) Blood cells are divided into three categories: red blood cells, white blood cells, and platelets. White blood cells mainly participate in various immune reactions, various allergic reactions, and parasitic infections. Red blood cells participate in the transport of oxygen; platelets mainly participate in hemostasis and coagulation.

[0065] 2. Surface functionalization of speckled magnetic magnetite@silicon dioxide core-indium gallium oxide@silicon dioxide shell structured microspheres:

[0066] The silica particles produced by the hydrolysis of orthosilicate are rich in hydroxyl groups on their surface, which can react with other organic molecules (such as antibodies, biotin, etc.) to functionalize the silica surface, that is, to achieve the surface functionalization of the speckled magnetic iron(III) oxide@silica core-indium gallium oxide@silica shell structure microspheres.

[0067] Take 2 mL of aqueous magnetic fluid and add 10 mmol / L 3-aminopropyl-3-ethoxysilane ethanol solution. React for 15–30 minutes to silanize the silica surface. Separate the magnetic beads using magnetic separation, wash 2–3 times with anhydrous ethanol, and dry in a vacuum oven at 30–56°C. Add the magnetic beads to 10 mmol / L terephthalaldehyde-acetone solution, stir mechanically for 30 minutes, separate the magnetic beads using magnetic separation, wash 2–3 times with anhydrous ethanol, dry in a vacuum oven at 30–56°C, and then suspend the magnetic beads in PBS buffer. Add antisperm antibody, mix and incubate at 37°C for 6–12 hours, separate the magnetic beads using magnetic separation, and remove free antibody to obtain magnetite@silica core-indium gallium oxide@silica@antisperm antibody magnetic beads.

[0068] 3. Application of speckled magnetic magnetite (Fe3O4@Silicon Dioxide core-InGaNO@Silicon Dioxide@Antisperm antibody magnetic beads in sperm enrichment in sperm-epithelial mixed cells.

[0069] A certain amount of sperm and epithelial cells were mixed, centrifuged at 10,000 rpm for 5 min, and the liquid was discarded to obtain a sperm and epithelial cell precipitate. The precipitate was washed three times with 1 ml of PBS buffer, and then resuspended in 500 μL of PBS buffer. 20 μL of the magnetic beads obtained in step 5) were added, and the mixture was incubated at 37°C for 6–12 hours. The magnetic beads were then separated using magnetic separation, and the liquid was discarded to obtain ferric oxide@silicon dioxide core-indium gallium oxide@silicon dioxide@antisperm antibody@sperm. 2 μL of the ferric oxide@silicon dioxide core-indium gallium oxide@silicon dioxide@antisperm antibody@sperm solution was placed on a glass slide, air-dried at room temperature (25°C), stained with hematoxylin and eosin (HE), and observed under a microscope. Add 200 μL of sperm lysis buffer, 2 μL of proteinase K, and 2 μL of DTT to iron(II) oxide@silicon dioxide core-indium gallium oxide@silicon dioxide@antisperm antibody@sperm. Incubate at 56°C for 1 hour. Separate the magnetic beads using magnetic separation. After recovering the liquid, purify the DNA to obtain a sperm DNA solution.

[0070] This invention utilizes a magnetic microsphere surface modification method to deposit indium gallium oxide (IGaO) particles onto the surface of microspheres, creating speckled magnetic microspheres. Through modification of the speckled magnetic microspheres, silica coating, and coupling with functional groups such as antibodies / avidin, magnetic magnetite@silica core-IGaO@silica magnetic microspheres and magnetic magnetite@silica core-IGaO@silica@antisperm antibody magnetic microspheres were successfully prepared. These microspheres have been applied in DNA enrichment and purification, and sperm sorting in a sperm-epithelial cell mixed system.

[0071] The performance advantages of the speckled magnetic microspheres of the present invention will be explained below with reference to the accompanying drawings.

[0072] Figure 1This is a SEM schematic diagram of the speckled magnetic microspheres of the present invention. As can be seen from the image, indium gallium oxide particles are diffusely distributed on the surface of the magnetic microspheres. The particles have a uniform morphology, which can significantly increase the specific surface area of ​​the magnetic beads, thereby enhancing their adsorption capacity. Furthermore, Figure 1 The image shown is a scanning electron microscope image of iron(II,III) oxide@silicon(II) oxide@indium gallium(II) oxide. Figure 1 As can be seen, magnetite@silicon dioxide microspheres were functionalized, resulting in a surface covered with densely packed pits, thus preparing pitted magnetic microspheres.

[0073] Figure 2 This is based on the elemental distribution in the speckled magnetic microsphere material of the present invention; Figure 2 The diagram shows the elemental distribution in the material, indicating the presence of five elements: gallium (Ga), iron (Fe), indium (In), silicon (Si), and oxygen (O). Magnetite (Fe3O4) exhibits strong magnetic properties. Indium gallium oxide (GaO) particles are dispersed on the surface of the magnetite beads, increasing their specific surface area. Silica covers the surface of the speckled magnetic microspheres; its silanol groups can adsorb DNA under specific solution conditions. The aforementioned techniques can enhance the DNA adsorption capacity of the magnetic beads. Furthermore, Figure 2 The figure shows the elemental distribution of the iron(II) oxide@silicon(II) oxide@indium gallium(II) oxide microspheres. As can be seen from the figure, the prepared material contains five elements: gallium (Ga), Fe (iron), In (indium), silicon (Si), and oxygen (O). It can be considered that the iron(II) oxide@silicon(II) oxide microspheres have been successfully functionalized and contain indium gallium(II) oxide on the surface.

[0074] Figure 3 This is a schematic diagram of the X-ray energy dispersive spectroscopy (EDS) test results of the micro-area elemental composition of the material according to the present invention; Figure 3 The elemental composition of the material in the micro-area X-ray energy dispersive spectroscopy (EDS) test results are data collected using the characteristic wavelengths of the X-rays emitted by the elements themselves, and these characteristic wavelengths depend on the energy released during energy level transitions. Figure 3 EDS results showed that the material contained five elements: gallium (Ga), iron (Fe), indium (In), silicon (Si), and oxygen (O), with a mass ratio (Si:O:Fe:In:Ga) of 36.56:31.26:14.91:13.3:0.89. The magnetite (Fe3O4) exhibits strong magnetic properties, and indium gallium oxide (IGaO) particles are dispersed on the surface of the magnetite beads, increasing their specific surface area. Silica covers the surface of the speckled magnetic microspheres, and its silanol groups can adsorb DNA under specific solution conditions. The above techniques can enhance the DNA adsorption capacity of the magnetic beads. Figure 3 This demonstrates the successful preparation of iron(II) oxide@silicon(II) oxide@indium gallium(II) oxide.

[0075] Figure 4The magnetic force diagram of the speckled magnetic microspheres according to the present invention shows that after high-temperature calcination in a tubular furnace and silica modification on the surface of the magnetic beads, the effect on the magnetic force of the magnetic beads is minimal. Even with a slight decrease in magnetic force, the speckled magnetic microspheres can improve their surface adsorption capacity. Furthermore, Figure 4 For the magnetic force map of the material, from Figure 4 As can be seen, the magnetic force of the iron(III) oxide@silicon dioxide microspheres decreased to some extent after functionalizing indium gallium oxide, but the magnetic force was significantly improved after post-modification with silicon dioxide. Even though it was slightly lower than that of the raw material, it still had a large magnetic force. The change in magnetic force did not affect the subsequent experimental operations.

[0076] Figure 5 The diagram shows the specific surface area of ​​the speckled magnetic microspheres according to the present invention. It can be seen that after high-temperature calcination in a tubular furnace and modification with silica on the surface of the magnetic beads, the specific surface area of ​​the speckled magnetic microspheres increases, and this increased specific surface area is expected to achieve high-performance DNA adsorption. Furthermore, from... Figure 5 The left, middle, and right images in the diagram show the specific surface areas of magnetite@silicon dioxide, magnetite@silicon dioxide@indium gallium oxide, and magnetite@silicon dioxide@indium gallium oxide@silicon dioxide, respectively. Their specific surface areas are 4.0007 m² / sq m. 2 / g、6.6827 m 2 / g and 16.2820 m 2 The / g figure indicates that the specific surface area of ​​the iron(III) oxide@silica microspheres gradually increases with surface modification. This increased specific surface area holds promise for achieving high-performance DNA adsorption.

[0077] Figure 6 The Fourier transform infrared spectrum is based on the present invention; as can be seen from the figure, the infrared peak of the speckled magnetic microspheres after high-salt modification is at 927.7 cm⁻¹. -1 The absorption peak at this location is that of the silicon-hydroxyl group, indicating that the high-efficiency separation is achieved through the interaction between the silicon-hydroxyl group and DNA. Furthermore, Figure 6 These are the infrared spectra of iron(II,III) oxide, iron(II,III) oxide@silicon dioxide, and iron(II,III) oxide@silicon dioxide after high-salt modification. Figure 6 This indicates that after modification with high-salt iron(III) oxide@silicon dioxide, the infrared peak is at 927.7 cm⁻¹. -1 The absorption peak at this location is that of silicon-hydroxyl groups, indicating that the high-efficiency separation of DNA is achieved through the interaction between silicon-hydroxyl groups and DNA during DNA adsorption.

[0078] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for fabricating speckled magnetic core-shell structured microspheres, characterized in that, It includes the following steps: Step S1, fabrication of speckled magnetic microspheres; Step S2, surface modification of the speckled magnetic microspheres; Step S3, fabrication of speckled magnetic core-shell structured microspheres, namely, fabrication of iron tetroxide@silicon dioxide@indium gallium oxide@silicon dioxide microspheres; Step S1 involves the preparation of the speckled magnetic microspheres as follows: Indium antimonide and gallium antimonide are mixed with a certain amount of magnetic microspheres in a mass ratio of 1:

1. The magnetic microspheres are made of iron(II,III) oxide@silicon dioxide and have a particle size between 300 nanometers and 20 micrometers. The microspheres mixed with the indium antimonide and gallium antimonide mixture are placed in a reaction box, and the reaction box is placed in a reaction furnace. Oxygen and argon are introduced at the same time, and a predetermined gas pressure is applied to the reaction furnace. The mixture is heated and calcined at a predetermined temperature between 300°C and 1000°C for 1 min to 15 min to obtain speckled magnetic microspheres. Step S2, surface modification of the pitted magnetic microspheres, includes: taking a certain amount of the pitted magnetic microspheres prepared in step S1, enriching the magnetic microspheres using a magnetic separation method, washing with deionized water 1 to 5 times, washing with anhydrous ethanol 1 to 6 times, and then drying in a vacuum oven at a temperature of 25℃ to 80℃. Take 0.2–3.5 g of powder obtained by vacuum drying oven, add it to 80 mL–300 mL of 0.5 M citric acid solution, ultrasonically disperse for 10–40 minutes, and stir and react at 60 °C under nitrogen atmosphere for 8–15 hours; separate the magnetic beads using magnetic separation method, wash with acetone 1–5 times, and then disperse in a certain amount of deionized water to obtain an aqueous magnetic fluid with a concentration of 30 mg / mL; Step S3 involves the preparation of the speckled magnetic core-shell structured microspheres: 2–5 mL of aqueous magnetic fluid is added to a solution of 4–10 mL ammonia, ethanol, and deionized water in a 4:1 ratio. The mixture is ultrasonically dispersed for 5–40 minutes, mechanically stirred at 20°C–60°C for 15–30 minutes, and then 0.5–5 mL of orthosilicate is added. The mixture is then mechanically stirred at 20°C–60°C for 2–12 hours. The magnetic beads are separated using a magnetic separation method, washed 2–8 times with anhydrous ethanol and deionized water respectively, and dispersed in a certain amount of deionized water to obtain the aqueous magnetic fluid with a concentration of 30 mg / mL.

2. The method for fabricating speckled magnetic core-shell structured microspheres according to claim 1, characterized in that, Step S1 involves preparing the speckled magnetic microspheres by taking 0.2g to 0.8g of indium antimonide and gallium antimonide in a mass ratio of 1:1, and 0.1g to 3g of magnetic microspheres with a particle size of 500 nm to 10 μm. The magnetic microspheres are made of iron(II,III) oxide@silicon dioxide. The microspheres mixed with the indium antimonide and gallium antimonide mixture are placed in a reaction chamber, which is then placed in a reactor. The oxygen flow rate is 1 sccm to 3 sccm, the argon flow rate is 50 sccm to 100 sccm, the reaction pressure is 5 kPa to 30 kPa, and the mixture is heated and calcined at 600℃ to 900℃ for 1 min to 5 min to obtain the speckled magnetic microspheres.

3. The method for fabricating speckled magnetic core-shell structured microspheres according to claim 1, characterized in that, Step S1 involves preparing the speckled magnetic microspheres as follows: 0.3g to 0.6g of indium antimonide and gallium antimonide are taken in a mass ratio of 1:1, and 0.1g to 2g of microspheres with a particle size of 700 nm to 8 μm are taken. The magnetic microspheres are made of iron(II,III) oxide@silicon dioxide. The microspheres mixed with the indium antimonide and gallium antimonide mixture are placed in a reaction chamber, which is then placed in a reactor. The oxygen flow rate is 2 to 2.5 sccm, the argon flow rate is 80 to 90 sccm, the reaction pressure is 7 to 10 kPa, and the mixture is heated and calcined at 700°C to 800°C for 2 to 3 minutes to obtain the speckled magnetic microspheres.

4. The method for fabricating speckled magnetic core-shell structured microspheres according to claim 1, characterized in that, Take 2g of microspheres prepared in step S1, enrich the magnetic microspheres using magnetic separation, wash 3 times with deionized water, wash 2 times with anhydrous ethanol, and dry in a vacuum oven at 40℃~45℃; take 1g of powder obtained from vacuum drying, add it to 80ml of 0.5M citric acid solution, ultrasonically disperse for 20 minutes, and stir and react for 12 hours at 60℃ under nitrogen atmosphere. Magnetic beads were separated using a magnetic separation method, washed three times with acetone, and dispersed in a certain amount of deionized water to obtain an aqueous magnetic fluid with a concentration of 30 mg / mL.

5. The method for fabricating speckled magnetic core-shell structured microspheres according to claim 1, characterized in that, The reactor includes an inlet pipe, a vacuum chamber, a ceramic boat, a silicon oxide substrate, source materials, a vacuum pump, and a heat insulation layer.

6. The method for fabricating speckled magnetic core-shell structured microspheres according to claim 5, characterized in that, Different gases participate in the reaction through the inlet pipe, which is connected to the vacuum tube cavity and through which Ar / O2 gas is introduced.

7. The method for fabricating speckled magnetic core-shell structured microspheres according to claim 6, characterized in that, The vacuum tube is the main site for the raw material reaction. The vacuum tube is made of high-temperature resistant glass material and has the following dimensions: 80mm inner diameter * 3mm wall thickness * 1000mm length.

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