Magnetic controllable response liquid phase floating microspheres and preparation method thereof

Abstract

The application belongs to the technical field of magnetic nanometer material, and discloses a liquid-phase floating microsphere capable of magnetic control response and a preparation method.The preparation method of the liquid-phase floating microsphere capable of magnetic control response comprises the following steps: adopting a layer-by-layer self-assembly method to coat a polymer layer on the surface of HGMs, assembling HGMs and MNPs, and assembling superparamagnetic magnetic nanoparticles to hollow glass microspheres through material modification to obtain HGMs@MNPs composite material, namely the liquid-phase floating microsphere capable of magnetic control response.The liquid-phase floating microsphere capable of magnetic control response can be simultaneously regulated by buoyancy and magnetic force, the preparation process is simple, and a large number of carboxyl groups on the surface can be used for combining various recognition molecules.The microsphere prepared by the application can be used for detection, separation, even catalysis and other fields in liquid phase (whether water or organic solvent, as long as the liquid is denser than the microsphere).

Description

Technical Field

[0001] This invention belongs to the field of magnetic nanomaterials technology, and particularly relates to a magnetically responsive liquid-phase floating microsphere and its preparation method. Background Technology

[0002] Currently, magnetic beads, whether micron-sized microspheres or nanoparticles, possess excellent magnetic responsiveness. Under the influence of a magnetic field, they can move directionally to specific locations and rapidly separate from the surrounding medium, thus showing great promise for applications including cell separation, protein separation and purification, enzyme immobilization, immunoassay, targeted drug delivery, DNA separation, and nucleic acid hybridization. However, when used in a liquid phase, they tend to settle due to gravity, often requiring external mechanical forces (such as shaking) to ensure contact with the target substance. However, due to the inherent properties of magnetic beads (mass, volume, magnetic responsiveness, etc.), external mechanical forces may cause stress damage to the target / separated substance; furthermore, some environments may not be suitable for mechanical actions such as vibration. Therefore, a strategy is needed to control the movement or sedimentation behavior of magnetic microspheres in a liquid phase to facilitate convenient and effective action on target substances while minimizing damage caused by mechanical stress.

[0003] Based on the above analysis, the problems and defects of the existing technology are as follows: the existing magnetic beads require external mechanical force to be applied in the liquid phase, which limits the application scenarios; at the same time, the magnetic beads will be damaged due to mechanical stress, resulting in poor application effect. Summary of the Invention

[0004] To address the problems existing in the prior art, this invention provides a magnetically responsive liquid-phase floating microsphere and its preparation method.

[0005] This invention is achieved as follows: a magnetically responsive liquid-phase floating microsphere, wherein the outer layer of the magnetically responsive liquid-phase floating microsphere is a hollow glass microsphere, and superparamagnetic magnetic nanoparticles are assembled inside the hollow glass microsphere; the microsphere is based on the hollow glass microsphere, and from the inside out are a polyethyleneimine (PEI) layer, a magnetite (MNPs) magnetic nanoparticle layer, a polyethyleneimine (PEI) layer, a polyacrylic acid (PAA) layer, and a recognition molecule layer (antibody, aptamer, peptide);

[0006] The magnetically responsive liquid-phase floating microspheres can be simultaneously controlled by buoyancy and magnetic force.

[0007] Another object of the present invention is to provide a method for preparing the magnetoresponsive liquid-phase floating microspheres, wherein the method for preparing the magnetoresponsive liquid-phase floating microspheres includes:

[0008] Superparamagnetic nanoparticles were assembled into hollow glass microspheres by layer-by-layer self-assembly, including coating HGMs with polymer layers, assembling HGMs with MNPs, and material modification, to obtain HGMs@MNPs composite materials.

[0009] The HGMs@MNPs composite material is a magnetically responsive liquid-phase floating microsphere.

[0010] Furthermore, the method for preparing the magnetoresponsive liquid-phase floating microspheres includes the following steps:

[0011] Step 1: By incubating clean HGMs with polyethyleneimine, positively charged HGMs with a PEI layer on the surface are obtained.

[0012] Step 2: Incubate the negatively charged magnetic nanoparticles with the modified HGMs, and assemble the MNPs onto the surface of the HGMs through electrostatic interaction.

[0013] Step 3: The assembled HGMs@MNPs are first incubated with PEI and then with polyacrylic acid to obtain an HGMs@MNPs composite material with a large number of carboxyl groups on the surface; and the HGMs@MNPs composite material with a large number of carboxyl groups on the surface is further modified to obtain a magnetically responsive liquid-phase floating microsphere.

[0014] Furthermore, the process of obtaining positively charged HGMs by incubating clean HGMs with polyethyleneimine includes:

[0015] First, weigh a certain amount of HGMs, rinse it three times with NaCl buffer solution at pH 8, and then collect the clean HGMs by taking advantage of the floating property of HGMs.

[0016] Next, a certain amount of polyethyleneimine was weighed and dissolved in the buffer solution to prepare a polyethyleneimine solution with a concentration of 1 mg / ml;

[0017] Finally, mix the prepared polyethyleneimine solution with HGMS, shake and incubate for 30 minutes; then wash repeatedly with buffer to remove excess reactants, and finally pipette the solution for later use.

[0018] Furthermore, the step of incubating negatively charged magnetic nanoparticles with modified HGMs and assembling MNPs onto the surface of HGMs via electrostatic interaction includes:

[0019] Negatively charged magnetic nanoparticles were dispersed in a buffer solution; the MNPs solution was mixed with HGMs and incubated with shaking at room temperature for 30 min; then the excess reactants were removed by repeated washing with the buffer to obtain HGMs@MNPs.

[0020] Furthermore, the process of incubating the assembled HGMs@MNPs with PEI followed by incubation with polyacrylic acid to obtain an HGMs@MNPs composite material with a large number of carboxyl groups on the surface includes:

[0021] (1) Weigh a certain amount of polyacrylic acid (PAA), dissolve it in NaCl buffer solution with pH 8, and prepare a PAA solution with a concentration of 2.4 mg / ml;

[0022] (2) The prepared HGMs@MNPs were first shaken and incubated in PEI solution at room temperature for 30 min. The excess PEI was removed by repeated washing using the magnetic responsiveness of the material. Then, the HGMs@MNPs were shaken and incubated in PAA solution at room temperature for 30 min. The washing was repeated.

[0023] Furthermore, the HGMs@MNPs composite material with a large number of carboxyl groups on its surface is further modified to obtain magneto-responsive liquid-phase floating microspheres, including:

[0024] HGMs@MNPs composite materials with a large number of carboxyl groups on the surface are modified by condensation reaction / electrostatic assembly / physical adsorption.

[0025] Another objective of this invention is to provide an application of the magnetically responsive liquid-phase floating microspheres in liquid phase detection and liquid phase separation.

[0026] Another object of the present invention is to provide an application of the magnetically responsive liquid-phase floating microspheres in liquid-phase catalysis.

[0027] Another object of the present invention is to provide an application of the magnetically responsive liquid-phase floating microspheres in in vitro biological detection.

[0028] Based on the above technical solutions and the technical problems solved, please analyze the advantages and positive effects of the technical solution to be protected by this invention from the following aspects:

[0029] First, addressing the technical problems existing in the prior art and the difficulty in solving them, this paper closely analyzes, in conjunction with the technical solution to be protected by this invention and the results and data obtained during the research and development process, how the technical solution of this invention solves the technical problems, and the inventive technical effects brought about by solving these problems. The specific description is as follows:

[0030] The magnetically responsive liquid-phase floating microspheres of the present invention can be simultaneously controlled by buoyancy and magnetic force, and the preparation process is simple. The large number of carboxyl groups on the surface can be used to bind various recognition molecules.

[0031] This invention provides a novel material and incubation method for in vitro biological detection. The preparation process is simple, allows for mass production, reduces damage to biomarkers (cells) during incubation, and facilitates downstream analysis of the biomarkers.

[0032] This invention obtains HGMs with a positively charged PEI layer on the surface by incubating clean HGMs (10 mg / ml) with polyethyleneimine (PEI, 1 mg / ml). The PEI layer provides the ability to further adsorb / bind magnetically responsive components (Fe3O4 magnetic nanoparticles, or other magnetic nanoparticles).

[0033] This invention involves incubating negatively charged magnetic nanoparticles (MNPs, with a particle size of approximately 20 nm and a concentration of 15 mg / ml) with modified HGMs; the magnetic nanoparticles are then physically bonded to the composite material via electrostatic adsorption, thereby enabling the material to acquire magnetic responsiveness.

[0034] This invention involves incubating assembled HGMs@MNPs with PEI followed by incubation with polyacrylic acid (PAA, 2.4 mg / ml) to obtain an HGMs@MNPs composite material with a large number of carboxyl groups on the surface. Further modification of the periphery further stabilizes the bound magnetic nanoparticles and introduces a large number of carboxyl groups on the material surface. This allows the material to specifically bind to or recognize target molecules (antigens, protein receptors, aptamers, etc.) through condensation reaction / electrostatic assembly / physical adsorption modification.

[0035] Second, considering the technical solution as a whole or from a product perspective, the technical effects and advantages of the technical solution to be protected by this invention are specifically described as follows:

[0036] This invention creatively proposes the concept of magnetically responsive, buoyant microspheres. The magnetically controllable, liquid-phase floating microspheres of this invention can be magnetically controlled, providing an alternative method to "manipulate" the microspheres compared to external mechanical forces. The microspheres prepared by this invention can be used in detection, separation, and even catalysis in liquid phases (whether water or organic solvents, as long as the liquid density is greater than the microsphere density).

[0037] Third, as supplementary evidence of the inventive step of the claims of this invention, it is also reflected in the following important aspects:

[0038] The technical solution of this invention fills a technological gap in the domestic and international industry: In existing technologies, magnetic beads are often used in conjunction with shakers or mechanical vibrations to ensure sufficient contact with the target material. However, during the incubation of the magnetic beads and the target material, the intense mechanical collisions can damage some of the target material. Furthermore, intense mechanical vibrations are not suitable for some scenarios. This invention proposes and successfully prepares a concept of magnetically responsive liquid-phase floating microspheres. Since the behavior of the magnetically responsive liquid-phase floating microspheres is simultaneously controlled by magnetic force and buoyancy, this invention can use an external periodic magnetic field to induce periodic movement of the material under the combined action of buoyancy, achieving sufficient contact between the magnetic beads and the target material. This provides a new method and approach for the incubation of magnetically separated materials. Attached Figure Description

[0039] Figure 1 This is a flowchart of the preparation method of magnetoresponsive liquid-phase floating microspheres provided in the embodiments of the present invention;

[0040] Figure 2 This is an optical microscope image of HGMs before assembly, provided in an embodiment of the present invention;

[0041] Figure 3 This is an optical microscope image of the assembled HGMs@MNPs provided in an embodiment of the present invention;

[0042] Figure 4 This is a statistical diagram of the particle size distribution of HGMs@MNPs provided in an embodiment of the present invention;

[0043] Figure 5 This is a SEM image of a single HGM provided in an embodiment of the present invention;

[0044] Figure 6 These are SEM images of different regions magnified according to embodiments of the present invention;

[0045] Figure 7 This is a schematic diagram of EDS elemental analysis of the surface of HGMs@MNPs material provided in an embodiment of the present invention;

[0046] Figure 8 This is a schematic diagram of unmodified HGMs@MNPs@PAA confocal analysis provided in an embodiment of the present invention;

[0047] Figure 9 This is a schematic diagram of the modified HGMs@MNPs@PAA confocal analysis provided in an embodiment of the present invention;

[0048] Figure 10 This is a schematic diagram of the movement of HGMs@MNPs and the substance to be acted upon, provided in an embodiment of the present invention.

[0049] Figure 11This is a confocal image of circulating tumor cells captured by HGMs@MNPs according to an embodiment of the present invention;

[0050] Figure 12 This is a schematic diagram illustrating the behavioral changes of the modified device with and without a magnetic field, provided in an embodiment of the present invention. Detailed Implementation

[0051] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0052] I. Explanatory and Illustrative Embodiments. To enable those skilled in the art to fully understand how the present invention is specifically implemented, this section provides an explanatory and illustrative description of the embodiments described in the claims.

[0053] The method for preparing magnetosensitive liquid-phase floating microspheres provided in this invention includes:

[0054] A layer-by-layer self-assembly method was used to assemble superparamagnetic magnetic nanoparticles (HGMs) onto hollow glass microspheres via polymer coating on the surface of HGMs, assembly of HGMs and MNPs, and material modification, resulting in HGMs@MNPs composite liquid-phase floating microspheres that can be magnetically controlled. The microspheres, with hollow glass microspheres as the substrate, consist of, from the inside out, a polyethyleneimine (PEI) layer, a magnetite (MNPs) layer, another polyethyleneimine (PEI) layer, a polyacrylic acid (PAA) layer, and a recognition molecule layer (antibody, aptamer, peptide).

[0055] like Figure 1 As shown, the method for preparing magnetically responsive liquid-phase floating microspheres provided in this embodiment of the invention includes the following steps:

[0056] S101, positively charged HGMs are obtained by incubating clean HGMs with polyethyleneimine;

[0057] S102, magnetic nanoparticles with negatively charged surfaces are incubated with modified HGMs, and MNPs are assembled onto the surface of HGMs through electrostatic interaction.

[0058] S103, the assembled HGMs@MNPs were first incubated with PEI and then with polyacrylic acid to obtain an HGMs@MNPs composite material with a large number of carboxyl groups on the surface; and the recognition molecule was modified by condensation reaction.

[0059] The present invention provides a method for obtaining positively charged HGMs by incubating clean HGMs with polyethyleneimine, comprising:

[0060] First, weigh a certain amount of HGMs, rinse it three times with NaCl buffer solution at pH 8, and then collect the clean HGMs by taking advantage of the floating property of HGMs.

[0061] Next, a certain amount of polyethyleneimine was weighed and dissolved in the buffer solution to prepare a polyethyleneimine solution with a concentration of 1 mg / ml;

[0062] Finally, mix the prepared polyethyleneimine solution with HGMS, shake and incubate for 30 minutes; then wash repeatedly with buffer to remove excess reactants, and finally pipette the solution for later use.

[0063] The present invention provides a method for incubating negatively charged magnetic nanoparticles with modified HGMs and assembling MNPs onto the surface of HGMs via electrostatic interaction, comprising:

[0064] Negatively charged magnetic nanoparticles were dispersed in a buffer solution; the MNPs solution was mixed with HGMs and incubated with shaking at room temperature for 30 min; then the excess reactants were removed by repeated washing with the buffer to obtain HGMs@MNPs.

[0065] The present invention provides that assembled HGMs@MNPs are first incubated with PEI and then with polyacrylic acid to obtain an HGMs@MNPs composite material with a large number of carboxyl groups on the surface, and the recognition molecules are modified by condensation reaction.

[0066] (1) Weigh a certain amount of polyacrylic acid (PAA), dissolve it in NaCl buffer solution with pH 8, and prepare a PAA solution with a concentration of 2.4 mg / ml;

[0067] (2) The prepared HGMs@MNPs were first incubated with PEI solution at room temperature for 30 min by shaking. The excess PEI was removed by repeatedly washing with the magnetic responsiveness of the material. Then, the HGMs@MNPs were incubated with PAA solution at room temperature for 30 min by shaking. The washing was repeated.

[0068] The technical solution of the present invention will be further described below with reference to specific embodiments.

[0069] Example 1:

[0070] This invention employs a layer-by-layer self-assembly method to assemble superparamagnetic magnetic nanoparticles (MNPs, approximately 20 nm) onto hollow glass microspheres (HGMs, approximately 30 μm), forming an HGMs@MNPs composite material. The specific steps include the following.

[0071] 1. HGMs surface coated with polymer layer: Clean HGMs are incubated with polyethyleneimine (PEI) to make the HGMs positively charged.

[0072] 2. Assembly of HGMs and MNPs: Magnetic nanoparticles with negatively charged surfaces are incubated with modified HGMs, and MNPs are assembled onto the surface of HGMs through electrostatic interaction.

[0073] 3. Further modification of the material: The assembled HGMs@MNPs were first incubated with PEI and then with poly(acrylic acid) (PAA) to give the material surface a large number of carboxyl groups, which can be modified to recognize molecules through condensation reaction.

[0074] 4. Specific experimental procedures and characterization diagrams:

[0075] (1) Polymer coating on HGMs surface:

[0076] 1) Weigh a certain amount of HGMs, rinse three times with NaCl buffer (pH=8) to remove dust and impurities, and collect the HGMs for later use by taking advantage of their floating property.

[0077] 2) Weigh a certain amount of polyethyleneimine (PEI, molecular weight 24000) and dissolve it in buffer solution to prepare a PEI solution with a concentration of 1 mg / ml.

[0078] 3) Mix the prepared PEI solution with HGMS, shake and incubate for 30 min. Then wash repeatedly with buffer to remove excess reactants, and pipette the solution for later use.

[0079] (2) Assembly of HGMs and MNPs:

[0080] 1) Negatively charged magnetic nanoparticles (approximately 20 nm in diameter) are dispersed in a buffer solution.

[0081] 2) Mix the MNPs solution with HGMs, shake and incubate at room temperature for 30 min, then wash repeatedly with buffer to remove excess reactants to obtain HGMs@MNPs.

[0082] (3) Further modification of materials:

[0083] 1) Weigh a certain amount of polyacrylic acid (PAA) (molecular weight 240,000), dissolve it in NaCl buffer (pH=8), and prepare a PAA solution with a concentration of 2.4 mg / ml.

[0084] 2) The prepared HGMs@MNPs were first incubated with PEI solution at room temperature for 30 min by shaking. The excess PEI was removed by repeated washing using the magnetic responsiveness of the material. Then, the HGMs@MNPs were incubated with PAA solution at room temperature for 30 min by shaking. Finally, the HGMs were washed repeatedly and then put into use.

[0085] II. Application Examples. To demonstrate the inventiveness and technical value of the present invention, this section provides application examples of the claimed technical solutions applied to specific products or related technologies.

[0086] Application Example 1:

[0087] The magnetically responsive liquid-phase floating microspheres provided in this application embodiment of the invention, along with a centrifuge tube containing the substance to be reacted, are periodically passed over a fixed magnetic field. During this process, the buoyancy and gravity of the HGMs@MNPs alternately dominate, causing the HGMs@MNPs to float up and down, thus achieving full interaction with the substance to be reacted. Figure 10 As shown, when HGMs@MNP is close to the top of the magnet, gravity dominates and the microspheres descend; when they are far from the top of the magnet, the magnet rises, and so on, achieving up-and-down floating.

[0088] Application Example 2:

[0089] The magnetically responsive liquid-phase floating microspheres provided in the application examples of this invention are used for the enrichment and separation of circulating tumor cells (CTCs).

[0090] Circulating tumor cells (CTCs) are tumor cells that detach from primary tumor tissue and enter the human bloodstream, and are a major cause of tumor metastasis. The biological information carried by CTCs has important implications for the prognosis and treatment of cancer patients; therefore, enriching and isolating CTCs from peripheral blood is of great significance. This invention is applied to the capture of circulating tumor cells (CTCs). Experimental verification shows that HGMs@MNPs, surface-modified with antibodies / aptamers, can successfully enrich and separate CTCs. By regulating their behavior through a combination of magnetic and buoyancy forces, the microspheres achieve sufficient contact with target cells, followed by antibody binding to the cells, thus achieving highly efficient enrichment of CTCs.

[0091] Figure 11 This is a confocal image of HGMs@MNPs after capturing circulating tumor cells; the blue areas represent cell nuclei.

[0092] III. Evidence of the Relevant Effects of the Embodiments. The embodiments of the present invention have achieved some positive effects during research and development or use, and indeed possess significant advantages compared to existing technologies. The following description, in conjunction with data, charts, and other materials from the experimental process, illustrates these advantages.

[0093] Material characterization:

[0094] First, the behavior of the material of the present invention under different magnetic field conditions was investigated. Then, optical microscopy characterization and SEM characterization were performed to observe its surface morphology. EDS analysis was used to analyze and verify its surface elemental composition. Finally, confocal microscopy was used to verify its surface functional groups and to confirm the possibility of modifying the surface of the material of the present invention with identification functional groups.

[0095] The results are as follows:

[0096] (1) Physical properties

[0097] The density of the microspheres was measured using the water displacement method, and the specific steps are as follows:

[0098] 1) Take a certain amount of microspheres, dry them, weigh them to a mass of m1, and put them into a dry and clean sample bottle.

[0099] 2) Due to the properties of microspheres, microspheres will float on the surface of the liquid phase if added directly. Therefore, the sample bottle containing microspheres should be placed above the magnet so that the microspheres are adsorbed at the bottom of the container. Then, add V1 volume of H2O. At this time, because the microspheres occupy part of the volume, the liquid level in the sample bottle will be higher than the normal liquid level of V1 volume of H2O.

[0100] 3) Measure the volume of the liquid above the normal liquid level using a micro-measuring cylinder, and denote this volume as V2. The density of the microspheres can be calculated using the formula: V2 / m1. The density of the microspheres is approximately 0.443 g / cm³. 3 .

[0101] When the microspheres are away from the magnetic field, buoyancy dominates, and the microspheres float on the surface of the liquid. When the microspheres are above the magnet, magnetic force dominates, and the microspheres sink to the bottom of the container. When the microspheres are away from the magnetic field, buoyancy returns to its dominant role, and the microspheres rise rapidly. Therefore, when the material periodically approaches and moves away from the magnetic field, it will move up and down, thus agitating the liquid.

[0102] 4) Behavioral research of microspheres

[0103] Figure 12 The behavior of the microspheres changes when there is no magnetic field. It can be seen that the microspheres will sink when placed above the magnetic field and float when away from the magnetic field.

[0104] (2) Optical microscope images

[0105] Figure 2 This represents the HGMs before assembly. Figure 3 This represents the assembled HGMs@MNPs. Figure 4 This is a statistical analysis of the particle size distribution of HGMs@MNPs. From... Figures 2 to 4It can be seen that HGMs did not undergo significant morphological changes before and after assembly, and no agglomeration occurred. The particle size distribution was between 26 μm and 30 μm, indicating that the material has good dispersibility and uniform particle size distribution.

[0106] 1) SEM surface morphology observation

[0107] Figure 5 SEM images of individual HGMs; Figure 6 Figure 5 shows SEM images of different regions after magnification. Figure 6 It can be seen that the magnetic nanoparticles are uniformly distributed on the surface of HGMs.

[0108] 2) EDS elemental analysis

[0109] Figure 7 The EDS elemental analysis of the HGMs@MNPs material surface shows that the Fe element signal is uniformly distributed on the material surface, which also indicates that the MNPs were successfully assembled onto the HGMs surface.

[0110] 3) Confocal analysis

[0111] The assembled PAA material (HGMs@MNPs@PAA) has a large number of carboxyl groups on its surface. Recognition molecules that specifically bind to CTCs are introduced through a condensation reaction with amino groups. This is verified using an aptamer, which has an amino group at one end and a Cy3 fluorescent molecule (NH2-Apt-Cy3) at the other end. The specific experimental steps are as follows:

[0112] A certain amount of HGMs@MNPs@PAA was dispersed in a buffer, and EDCl / NHS condensing agent was added to bring the final concentration to 10 nM. The mixture was then incubated at 4 °C with shaking for 1 h to activate the carboxyl groups. After activation, aptamers were added to a final concentration of 0.5 Mm, and the mixture was then incubated at room temperature (around 25 °C) with shaking for 24 h.

[0113] Figure 8 The unmodified HGMs@MNPs@PAA did not show a corresponding fluorescence signal under confocal microscopy. Figure 9 The modified material shows obvious fluorescent signals on its surface, indicating that the aptamer has been successfully modified onto the material surface.

[0114] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any modifications, equivalent substitutions, and improvements made by those skilled in the art within the scope of the technology disclosed in the present invention, and within the spirit and principles of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A magnetically responsive liquid-phase floating microsphere, characterized in that, The magnetically responsive liquid-phase floating microspheres are based on hollow glass microspheres, and superparamagnetic magnetic nanoparticles are assembled on the surface of the hollow glass microspheres. The magnetically responsive liquid-phase floating microspheres can be simultaneously controlled by buoyancy and magnetic force; The method for preparing the magnetically responsive liquid-phase floating microspheres includes: Superparamagnetic nanoparticles were assembled into hollow glass microspheres using a layer-by-layer self-assembly method through coating polymer layers on the surface of HGMs, assembling HGMs with MNPs, and material modification, to obtain HGMs@MNPs composite material; the HGMs@MNPs composite material is a magnetically responsive liquid-phase floating microsphere. The method for preparing the magnetically responsive liquid-phase floating microspheres includes the following steps: Step 1: By incubating clean HGMs with polyethyleneimine, positively charged HGMs with a PEI layer on the surface are obtained. Step 2: Incubate the negatively charged magnetic nanoparticles with the modified HGMs, and assemble the MNPs onto the surface of the HGMs through electrostatic interaction. Step 3: The assembled HGMs@MNPs are first incubated with PEI and then with polyacrylic acid to obtain an HGMs@MNPs composite material with a large number of carboxyl groups on the surface; and the HGMs@MNPs composite material with a large number of carboxyl groups on the surface is further modified to obtain a magnetically responsive liquid-phase floating microsphere.

2. The magnetically responsive liquid-phase floating microspheres as described in claim 1, characterized in that, The process of obtaining positively charged HGMs by incubating clean HGMs with polyethyleneimine includes: First, weigh a certain amount of HGMs, rinse it three times with NaCl buffer solution at pH 8, and then collect the clean HGMs by taking advantage of the floating property of HGMs. Next, a certain amount of polyethyleneimine was weighed and dissolved in the buffer solution to prepare a polyethyleneimine solution with a concentration of 1 mg / ml; Finally, mix the prepared polyethyleneimine solution with HGMS, shake and incubate for 30 minutes; then wash repeatedly with buffer to remove excess reactants, and finally pipette the solution for later use.

3. The magnetically responsive liquid-phase floating microspheres as described in claim 1, characterized in that, The step of incubating negatively charged magnetic nanoparticles with modified HGMs and assembling MNPs onto the surface of HGMs via electrostatic interaction includes: Negatively charged magnetic nanoparticles were dispersed in a buffer solution; the MNPs solution was mixed with HGMs and incubated with shaking at room temperature for 30 min; then the excess reactants were removed by repeated washing with the buffer to obtain HGMs@MNPs.

4. The magnetically responsive liquid-phase floating microspheres as described in claim 1, characterized in that, The process of incubating the assembled HGMs@MNPs with PEI and then with polyacrylic acid to obtain an HGMs@MNPs composite material with a large number of carboxyl groups on the surface includes: (1) Weigh a certain amount of polyacrylic acid (PAA), dissolve it in NaCl buffer solution with pH 8, and prepare a PAA solution with a concentration of 2.4 mg / ml; (2) The prepared HGMs@MNPs were first incubated with PEI solution at room temperature for 30 min by shaking. The excess PEI was removed by repeatedly washing with the magnetic responsiveness of the material. Then, the HGMs@MNPs were incubated with PAA solution at room temperature for 30 min by shaking. The washing was repeated.

5. The magnetically responsive liquid-phase floating microspheres as described in claim 1, characterized in that, The HGMs@MNPs composite material with a large number of carboxyl groups on its surface is further modified to obtain magnetically responsive liquid-phase floating microspheres, including: HGMs@MNPs composite materials with a large number of carboxyl groups on the surface are modified by condensation reaction / electrostatic assembly / physical adsorption.

6. An application of the magnetically responsive liquid-phase floating microspheres as described in claim 1 in liquid phase detection and liquid phase separation.

7. An application of the magnetoresponsive liquid-phase floating microspheres as described in any one of claims 1-5 in liquid-phase catalysis.

8. An application of the magnetoresponsive liquid-phase floating microspheres as described in any one of claims 1-5 in in vitro biological detection.

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