CN113304700B

CN113304700B - Method for preparing magnetic polymer microsphere and application thereof

Info

Publication number: CN113304700B
Application number: CN202110655120.5A
Authority: CN
Inventors: 刘一凡; 李璐瑶; 张蓉
Original assignee: ShanghaiTech University
Current assignee: ShanghaiTech University
Priority date: 2021-06-11
Filing date: 2021-06-11
Publication date: 2023-06-02
Anticipated expiration: 2041-06-11
Also published as: CN113304700A

Abstract

The invention provides a method for preparing magnetic polymer microspheres and application thereof. Firstly, preparing multilayer polymer micro-droplets with magnetic nano particles coated on the outer layers by using a droplet generation technology; then solidifying or semi-solidifying and demulsifying to obtain the multi-layer polymer microsphere coated with the magnetic shell layer; and dissolving at least one layer of the inner layer polymer of the multi-layer polymer microsphere to obtain the magnetic polymer microsphere containing the hollow cavity. The preparation method can be used in any biochemical reaction, the magnetic polymer microsphere serves as a reaction container, and the shell layer of the magnetic polymer microsphere serves as a passive screen to retain the encapsulated reaction substrate and isolate invasion of external pollution, and simultaneously allows a small molecule reaction reagent to diffuse through and enter the microsphere to react with the reaction substrate. The invention can realize the biochemical reaction with high efficiency and high flux under the condition of isolating external pollution; it is ensured that the sample is not cross-contaminated and exogenously contaminated between the multi-step biochemical reactions.

Description

Method for preparing magnetic polymer microsphere and application thereof

Technical Field

The invention relates to a method for preparing magnetic polymer microspheres and application thereof, belonging to the technical field of biochemistry.

Background

With iterative updating of molecular biology techniques, modern molecular biology research is increasingly relying on high-throughput analysis methods to process complex samples with single cell or single molecule resolution. In contrast to microwells, capturing individual cells, DNA, enzymes, or biomolecules in water-in-oil microdroplets or other forms of microscopic compartments allows for large-scale high-throughput parallel analysis. However, in performing many complex biochemical reactions, samples are processed in a certain order to start, modify or terminate the biochemical reactions, but these complex multi-step operations are difficult to perform in droplets. Although a multi-step process can be achieved by manipulating the droplets (droplet fusion, droplet reinjection, droplet separation and sorting), the use of such methods requires specialized microfluidic technical knowledge, and the complexity of the fluid manipulation also limits the use of such techniques. In microbiological experiments, the separation and capture of bacteria or genetic material of bacteria in micro-droplets for a corresponding series of biochemical treatments, achieving the engagement between multiple experimental operations and ensuring that there is no cross-contamination between samples and no exogenous contamination, is quite challenging. For example, in the case of genetic material amplification and analysis of microorganisms, cell lysis is performed on the microorganisms, but the reagents used in this step may affect subsequent enzymatic reactions, and thus have limitations.

Disclosure of Invention

The technical problems to be solved by the invention are as follows: how to provide a technical problem of an experimental method which can perform high-efficiency and high-flux biochemical reaction and can effectively isolate external pollution.

In order to solve the above problems, the present invention provides a method for preparing magnetic polymer microspheres, comprising the steps of:

step 1: the method comprises the steps of preparing multilayer polymer micro-droplets with magnetic nanoparticles coated on the outer layer by using a droplet generation technology and adopting raw materials comprising monomers and magnetic nanoparticles to undergo chemical crosslinking polymerization or raw materials comprising polymers and magnetic nanoparticles to undergo physical crosslinking; the multi-layer polymer micro-droplet with adjustable pore diameter can be prepared by adjusting the type or concentration of the monomer/polymer in the raw materials;

step 2: and solidifying or semi-solidifying the multi-layer polymer micro-droplets, and demulsifying to obtain the multi-layer polymer microsphere wrapped with the magnetic shell layer.

The method for preparing the multilayer polymer micro-droplet with the magnetic nano-particles coated on the outer layer in the step 1 is any one of the following methods:

the method comprises the following steps: preparing polymer micro-droplets by utilizing a droplet generation technology, solidifying or semi-solidifying and demulsifying the polymer micro-droplets to obtain polymer microspheres, and wrapping a dispersion liquid containing magnetic nano-particles (a dispersion liquid obtained by dispersing the magnetic nano-particles in a polymer) on the outer layer of the microspheres to obtain multi-layer polymer micro-droplets with the magnetic nano-particles wrapped on the outer layer;

The second method is as follows: preparing polymer or a substrate for preparing the polymer and magnetic particles into polymer micro-droplets containing magnetic nanoparticles by using a droplet generation technology, preparing the micro-droplets containing the magnetic nanoparticles into multi-layer polymer micro-droplets by using a droplet production technology after solidification or semi-solidification and demulsification, and migrating the magnetic nanoparticles in the multi-layer polymer micro-droplets to the outer layers of the multi-layer polymer micro-droplets by using a physical or chemical method to obtain the multi-layer polymer micro-droplets with the outer layers wrapped with the magnetic nanoparticles;

preferably, step 3: and (2) dissolving at least one layer of the inner layer polymer of the multi-layer polymer microsphere prepared in the step (2) to obtain the magnetic polymer microsphere containing the hollow cavity.

Preferably, the multi-layer micro-droplet prepared in the step 1 is a double-layer micro-droplet, and correspondingly, the multi-layer polymer microsphere prepared in the step 2 is a double-layer polymer microsphere.

Preferably, the specific process of step 1 includes:

step 1.1: preparing micro-droplets by adopting a high-flux microfluidic technology and adopting raw materials comprising monomers to undergo chemical crosslinking polymerization or raw materials comprising polymers to undergo physical crosslinking, and obtaining polymer microspheres after curing or semi-curing and demulsification of the micro-droplets;

Step 1.2: preparing a magnetic outer shell layer on the outer surface of the polymer microsphere obtained in the step 1.1 by adopting a high-flux microfluidic technology and adopting polymer dispersion liquid of magnetic nano particles through physical or chemical crosslinking, so as to prepare double-layer micro-droplets of which the outer layer is a polymer coated with the magnetic nano particles and the inner layer is a polymer; the outer layer polymer and the inner layer polymer have different physical and/or chemical properties.

Preferably, the polymer microsphere in the step 1.1 includes any one of polyacrylamide gel microsphere, polyethylene glycol gel microsphere, agarose gel microsphere, sodium alginate gel microsphere and gelatin gel microsphere.

Preferably, the polyacrylamide gel microsphere is prepared by forming a water-in-oil system by an oil phase containing a TEMED catalyst and a surfactant and a water phase containing an acrylamide monomer, an ammonium persulfate initiator and an N, N' -bis (acrylamide) cystamine cross-linking agent, preparing polyacrylamide gel micro-droplets through chemical cross-linking polymerization, and solidifying or semi-solidifying and demulsifying the polyacrylamide gel micro-droplets; the agarose gel microsphere is a water-in-oil system formed by an oil phase containing a surfactant and a water phase containing agarose, and is prepared by physical crosslinking to obtain agarose gel micro-droplets, and solidifying or semi-solidifying and demulsifying the agarose gel micro-droplets; the sodium alginate gel microsphere is prepared by forming a water-in-oil system from an oil phase containing a surfactant and a water phase containing sodium alginate, preparing sodium alginate gel micro-droplets through chemical crosslinking, and solidifying or semi-solidifying and demulsifying the sodium alginate gel micro-droplets; the gelatin gel microsphere is prepared by forming a water-in-oil system by an oil phase containing a surfactant and a water phase containing gelatin only or a water phase containing gelatin and one or more of the following crosslinking agents of formaldehyde, glutaric acid, EDC, genipin, methacrylic anhydride and polyethylene glycol diacrylate, and preparing gelatin micro-droplets through physical crosslinking or chemical crosslinking, and solidifying or semi-solidifying and demulsifying the gelatin micro-droplets.

Preferably, the surfactant comprises any one or more of nonionic surfactants PEG-PFPE, castor oil polyoxyl ester, polyoxyethylene 40 hydrogenated castor oil, poloxamer and ionic surfactants Krytox, sodium dodecyl sulfate and Janus nano particles; the oil phase comprises any one or more of HFE-7500 fluorinated oil phase, squarane oil phase, silicone oil phase and mineral oil phase.

Preferably, the polymer dispersion liquid of the magnetic nanoparticles in the step 1.2 includes any one of a polyacrylamide gel solution, a polyethylene glycol gel solution, an agarose gel solution, a sodium alginate gel solution and a gelatin gel solution of magnetic nanoparticles, and the magnetic nanoparticles include any one or more of single metal magnetic nanoparticles, magnetic iron oxide nanoparticles, bimetallic magnetic nanoparticles and alloy magnetic nanoparticles.

Preferably, the double-layer micro-droplet prepared in the step 1.2 comprises any one of the following double-layer micro-droplets:

bilayer micro droplet a: the outer layer is polyacrylamide gel coated with magnetic nano particles, and the inner layer is double-layer micro-droplets of the polyacrylamide gel;

Bilayer micro-droplet B: the outer layer is polyethylene glycol gel coated with magnetic nano particles, and the inner layer is double-layer micro-droplets of polyacrylamide gel;

bilayer micro droplet C: the outer layer is agarose gel coated with magnetic nano particles, and the inner layer is double-layer micro-droplets of polyacrylamide gel;

bilayer micro droplet D: the outer layer is sodium alginate gel coated with magnetic nano particles, and the inner layer is double-layer micro-droplets of polyacrylamide gel;

bilayer micro-droplet E: the outer layer is gelatin gel coated with magnetic nano particles, and the inner layer is double-layer micro-droplets of polyacrylamide gel;

bilayer micro-droplet F: the outer layer is polyacrylamide gel coated with magnetic nano particles, and the inner layer is double-layer micro-droplets of agarose gel;

bilayer micro droplet G: the outer layer is polyethylene glycol gel coated with magnetic nano particles, and the inner layer is double-layer micro-droplets of agarose gel;

bilayer microdroplet H: the outer layer is agarose gel coated with magnetic nano particles, and the inner layer is double-layer micro-droplets of the agarose gel;

bilayer micro-droplet I: the outer layer is sodium alginate gel coated with magnetic nano particles, and the inner layer is double-layer micro-droplets of agarose gel;

bilayer micro-droplet J: the outer layer is gelatin gel coated with magnetic nano particles, and the inner layer is double-layer micro-droplets of agarose gel;

Double layer microdroplet K: the outer layer is polyacrylamide gel coated with magnetic nano particles, and the inner layer is double-layer micro-droplets of sodium alginate gel;

bilayer microdroplet L: the outer layer is polyethylene glycol gel coated with magnetic nano particles, and the inner layer is double-layer micro-droplets of sodium alginate gel;

bilayer microdroplet M: the outer layer is agarose gel coated with magnetic nano particles, and the inner layer is double-layer micro-droplets of sodium alginate gel;

bilayer microdroplet N: the outer layer is sodium alginate gel coated with magnetic nano particles, and the inner layer is double-layer micro-droplets of the sodium alginate gel;

bilayer micro-droplet O: the outer layer is gelatin gel coated with magnetic nano particles, and the inner layer is double-layer micro-droplets of sodium alginate gel;

bilayer micro-droplet P: the outer layer is polyacrylamide gel coated with magnetic nano particles, and the inner layer is double-layer micro-droplets of gelatin gel;

bilayer micro-droplet Q: the outer layer is polyethylene glycol gel coated with magnetic nano particles, and the inner layer is double-layer micro-droplets of gelatin gel;

bilayer micro-droplet R: the outer layer is agarose gel coated with magnetic nano particles, and the inner layer is double-layer micro-droplets of gelatin gel;

bilayer microdroplet S: the outer layer is sodium alginate gel coated with magnetic nano particles, and the inner layer is double-layer micro-droplets of gelatin gel;

Double layer micro droplet T: the outer layer is gelatin gel coated with magnetic nano particles, and the inner layer is double-layer micro-droplets of gelatin gel.

Preferably, the double-layer micro-droplets A, F, K and P are prepared by chemical cross-linking polymerization of an oil phase containing a TEMED catalyst and a surfactant, a polyacrylamide gel solution containing an acrylamide monomer, an ammonium persulfate initiator, an N, N' -methylene bisacrylamide cross-linking agent and magnetic nano particles and a system containing polymer microspheres prepared in the step 1.1 to form water-in-oil spheres; the double-layer micro-droplets B, G, L and Q are prepared by forming a water-in-oil ball-in-oil system from an oil phase containing a TEMED catalyst and a surfactant, a polyethylene glycol gel solution containing polyethylene glycol, an ammonium persulfate initiator and magnetic nano particles and polymer microspheres prepared in the step 1.1, and performing chemical cross-linking polymerization to prepare corresponding double-layer micro-droplets; the double-layer micro-droplets C, H, M and R are prepared by forming a water-in-oil ball system from an oil phase containing a surfactant, an agarose gel solution containing agarose and magnetic nano particles and polymer microspheres prepared in the step 1.1, and performing physical crosslinking to prepare corresponding double-layer micro-droplets; the double-layer micro-droplets D, I, N and S are a system in which a water-in-oil ball is formed by a sodium alginate gel solution containing a surfactant, sodium alginate and magnetic nano particles and polymer microspheres prepared in the step 1.1, and the corresponding double-layer micro-droplets are prepared through physical crosslinking; the double-layer micro-droplets E, J, O and T are prepared by forming a water-in-oil ball system from an oil phase containing a surfactant, a gelatin gel solution containing gelatin and magnetic nano particles or a gelatin gel solution containing gelatin and magnetic nano particles and one or more of the cross-linking agents formaldehyde, glutaric acid, EDC, genipin, methacrylic anhydride and polyethylene glycol diacrylate in the following steps and polymer microspheres prepared in the step 1.1 through physical crosslinking or chemical crosslinking.

Preferably, the method of curing or semi-curing in step 1.1 and step 2 includes any one of standing, UV irradiation, heating, changing PH value and changing ion concentration; the demulsification method comprises the following steps: the procedure was followed by 1 wash with HFE-7500 containing 20% v/v 1H, 2H-perfluoctanol, 2 washes with n-hexane containing 1% v/v Span-80, 2 washes with TE buffer containing 0.1% v/v Triton X-100, and 3 washes with TE buffer containing 0.1% v/v Tween 20.

Preferably, the method used for dissolving the inner polymer in the step 3 includes any one of UV irradiation, heating, changing PH, changing ion concentration, and using dithiothreitol solution.

The invention also provides application of the method for preparing the magnetic polymer microsphere in biochemical reaction, biochemical analysis and culture of microorganisms.

Preferably, the application in biochemical reactions specifically comprises the following steps:

step 1: preparing micro-droplets encapsulated with a substrate by using a droplet generation technology;

step 2: preparing a multi-layer polymer microsphere coated with a magnetic outer shell layer;

step 3: directly adding a reactant to perform biochemical reaction, or dissolving at least one layer of the inner layer polymer of the multi-layer polymer microsphere to form a hollow cavity, and then adding the reactant to perform biochemical reaction.

Preferably, the substrate encapsulated in step 1 comprises at least one of nucleic acid, protein, cell and microorganism.

The technical principle of the invention is as follows:

the invention utilizes a droplet generation technology, in particular to a micro-fluidic chip to prepare a magnetic polymer microsphere with adjustable aperture, and a hollow cavity can be formed after an inner polymer of the magnetic polymer microsphere is dissolved. When the magnetic polymer microsphere is used for biochemical reaction, the magnetic polymer microsphere with the pore diameter suitable for the reaction substrate can be prepared by adjusting the types or the concentrations of the raw materials for preparing the magnetic polymer microsphere according to the molecular weight of the reaction substrate, so that the magnetic polymer microsphere can encapsulate the reaction substrate and allow small molecular reaction reagents to enter the microsphere, the shell layer of the microsphere is similar to a semipermeable membrane structure, and exogenous and cross-contamination can be isolated when the biochemical reaction is carried out; when complex multi-step biochemical reaction is carried out, magnetic beads can be utilized to adsorb magnetic polymer microspheres for magnetic cleaning, and after unreacted micromolecular reaction reagent in the previous step is washed off, reagents required in the next step are added for continuous reaction, so that the connection between multi-step experimental operation can be realized and the cross contamination and exogenous pollution between samples are avoided due to the biochemical reaction based on the magnetic polymer microspheres.

Compared with the prior art, the invention has the beneficial effects that:

1. the invention provides a method for preparing magnetic polymer microspheres, which can prepare polymer microspheres with adjustable volume and aperture according to requirements, and can be used in any biochemical reaction, including but not limited to single cell technology such as single bacteria and single virus culture.

2. The magnetic microsphere serves as a reaction container with semi-permeable membrane characteristics, a shell layer of the magnetic microsphere can serve as a passive screen to retain encapsulated large-molecular-weight compounds (such as nucleic acid and the like), smaller molecules (such as polymerase, restriction endonuclease and the like) are allowed to diffuse through, and substances (such as moisture, inorganic salts and other nutritional ingredients, biochemical reaction reagents and the like) can be exchanged with the outside in a cavity of the magnetic microsphere, but invasion of external pollutants (such as exogenous bacteria, cells, viruses and the like) can be isolated.

3. According to the method for preparing the magnetic polymer microsphere, when complex multi-step biochemical reaction is carried out, the magnetic polymer microsphere can be adsorbed by the magnetic beads to carry out magnetic cleaning, and after unreacted micromolecular reaction reagent in the previous step is washed off, reagents required in the next step are added to continue the reaction, so that the connection between multi-step experimental operations can be realized and no cross contamination and exogenous pollution between samples are ensured due to the biochemical reaction of the magnetic polymer microsphere, and a powerful scientific experimental method is provided for realizing high-efficiency and high-flux biochemical reaction under the condition of isolating external pollution.

Drawings

FIG. 1 is a schematic diagram of a microfluidic chip for generating soluble microdroplets;

FIG. 2 is a flow chart of the generation and experimental application of magnetic microspheres with hollow structures, which is performed according to the sequence of a-b-c-d-e;

FIG. 3 is a schematic diagram of a microfluidic chip for generating bilayer gel microdroplets;

FIG. 4 is a diagram of a close-packed double layer microdroplet;

FIG. 5 is a graph of the dynamic dissolution process of the inner microspheres, the numbers in the graph representing time in seconds;

FIG. 6 is a diagram of a magnetic microsphere of hollow structure after dissolution of the inner layer material;

FIG. 7 is a schematic cross-sectional view of a magnetic hollow structure microsphere;

FIG. 8 is a diagram of double layer gel microdroplets of closely packed different materials;

FIG. 9 is a gel microsphere view of a hollow structure of closely packed dissimilar materials;

FIG. 10 is a graph of PCR product staining prior to magnetic washing, wherein the light dots indicated by the arrows are fluorescent genomic clusters;

FIG. 11 is a graph of PCR product staining after magnetic washing, wherein the light dots indicated by the arrows are genomic clusters with fluorescence;

FIG. 12 is a graph showing agarose electrophoresis results of PCR products;

FIG. 13 is a fluorescence plot of PCR products for ten pairs of different primers;

FIG. 14 is a view of violin from fluorescence quantitative analysis of PCR products for ten pairs of different primers;

FIG. 15 is a plot of MDA product staining after magnetic washing;

FIG. 16 is a graph showing the results of magnetically washed MDA-post PCR products;

FIG. 17 is a graph showing the results of magnetically purged after MDA FISH products;

FIG. 18 is a graph showing the results of plasmid expression in a cell-free expression system;

FIG. 19 is a schematic diagram of bacterial biosensing;

FIG. 20 is a graph showing the results of bacterial culture in double-layered agarose gel microspheres;

FIG. 21 is a graph showing the results of bacterial culture in single-layer agarose gel microspheres;

FIG. 22 is a graph showing the effect of incubation time on valerolactam bioresponse behavior;

FIG. 23 is a graph of valerolactam bioresponse dose-response results;

wherein, fig. 4-9 in the above figures are all pictures taken under a microscope.

Detailed Description

In order to make the invention more comprehensible, preferred embodiments accompanied with figures are described in detail below.

The sources of materials used in each example are as follows:

HFE-7500 fluorinated oil was purchased from Minnesota mining manufacturing (Shanghai) International trade company Limited. Biocompatible surfactants (PEG-PFPE) were purchased from Zhejiang Dapu biotechnology Co. Acrylamide (AM), N, N '-methylenebisacrylamide, N, N' -bis (acryloyl) cystamine, ammonium Persulfate (APS), N, N, N ', N' -tetramethylethane-1, 2-diamine (TEMED), 1H, 2H-Perfluorooctanol, span-80, tween20, polyethylene glycol monomer (Poly (ethylene glycol) diacrylate average Mn 575), low melting point agarose, SDS (Sodium dodecyl sulfate), naCl, 300nm magnetic nanoparticles were purchased from Sigma-Aldrich. N-hexane (Hexanes) was purchased from Thermo/Fisher Chemical. Triton X-100 was purchased from Shanghai pan Biotechnology Co. The 1M dithiothreitol solution was purchased from Beijing Boaosen Biotechnology Co., ltd (Bioss). High melting agarose, formamide (Formamide) was purchased from the company of the holy biosciences of the next year (Shanghai). PCR kit (Phanta Max Super-Fidelity DNA Polymerase), phi29 DNA polymerase buffer, dNTP (10 mM) were purchased from Nanjinozan Biotech Co., ltd. Oligonucleotide primers, FISH fluorescent probes were produced intelligently by Jin Wei. EVA Green (20 x) was purchased from Biotium (USA). Random primers (EXO-RESISTANT RANDOM PRIMER), PCR fluorescent primer sequences, and non-ribozyme water (NUCLEASE-FREE WATER 10X 50 ML) were purchased from Sesamer Feishmanic technologies (China). Tris-HCl (pH 7.5) was purchased from Beijing Soy Co., ltd. L-Arabinosl- (+) -Arabinose was purchased from the further Biotech Co., ltd (BIOSYNTH). Valerolactam is supplied by the group of subjects Zhang Jingwei, university of double denier.

Example 1

A method for preparing magnetic polymer microspheres, the preparation flow is shown in fig. 2a-2c, comprising the following steps:

1) Firstly preparing polyacrylamide soluble gel microspheres by utilizing a microfluidic technology, specifically inputting HFE-7500 fluorinated oil phase containing 0.4% v/v of N, N, N ', N' -tetramethyl ethane-1, 2-diamine (TEMED) and 2%g/mL of surfactant (PEG-PFPE) into a first flow input channel of a microfluidic chip shown in figure 1; the second flow input channel was fed with a mixed aqueous solution containing 6%g/mL acrylamide monomer, 0.392% g/mL N, N' -bis (acryloyl) cystamine, and 0.6% g/mL ammonium persulfate; and collecting the soluble gel micro-droplets by a third output channel of the microfluidic chip. The collected droplets were allowed to stand at room temperature for 7 hours to gel, and after gel formation, the microdroplets were demulsified and washed sequentially with HFE-7500 fluorinated oil containing 20% v/v 1H, 2H-perfluoctanol 1 time, n-hexane containing 1% v/v Span-80 2 times, TE buffer containing 0.1% v/v Triton X-100 2 times, and TE buffer containing 0.1% v/v Tween20 3 times to obtain closely packed polyacrylamide hydrogel microdroplets (diameter: 40 μm). After the polyacrylamide hydrogel microdroplet is solidified, the polyacrylamide hydrogel microdroplet can be dissolved under specific conditions.

2) Preparing double-layer gel microspheres coated with a magnetic layer by utilizing a microfluidic technology (wherein the outer layer is polyacrylamide gel coated with magnetic nano particles, the inner layer is polyacrylamide gel), specifically, inputting a polyacrylamide gel solution containing 0.4% v/v of N, N, N ', N ' -tetramethyl ethane-1, 2-diamine (TEMED) and 2%g/mL of surfactant (PEG-PFPE) into a first flow input channel of a microfluidic chip shown in FIG. 3, inputting a soluble gel microsphere prepared in the first step into a second flow input channel, wherein the second flow input channel is used for inputting a polyacrylamide gel solution containing 4%g/mL of acrylamide monomer, 0.4% g/mL of N, N ' -methylenebisacrylamide, 0.3% g/mL of ammonium persulfate and ferroferric oxide magnetic nano particles; the fourth output channel collects the double-layer micro-droplets wrapped with the magnetic layer, and the obtained double-layer micro-droplets wrapped with the magnetic layer are in a tightly arranged structure as shown in fig. 4. The collected gel microdroplets were left at room temperature for 7 hours to gel, after gel formation, the microdroplets were de-emulsified, and sequentially washed 1 time with HFE-7500 containing 20% v/v 1H, 2H-perfluoacoctanol, 2 times with n-hexane containing 1% v/v Span-80, 2 times with TE buffer containing 0.1% v/v Triton X-100, and 3 times with TE buffer containing 0.1% v/v Tween20 to obtain bilayer gel microspheres having an inner layer of polyacrylamide gel and an outer layer of polyacrylamide gel encapsulating magnetic nanoparticles, as shown in FIG. 8.

3) The double-layer gel microsphere is soaked in a Thermopol buffer containing 50mM dithiothreitol, and is kept stand for 5 minutes, the polyacrylamide hydrogel core is dissolved, the dissolving process of the inner-layer polyacrylamide gel is shot under a microscope, the inner-layer gel is almost completely dissolved after every 10 seconds, as shown in fig. 5, and the hollow polyacrylamide gel microsphere is obtained after the dissolving, as shown in fig. 6, and the cross section view of the hollow polyacrylamide gel microsphere is shown in fig. 7.

Example 2

1) Firstly, preparing polyacrylamide soluble gel microspheres by using a microfluidic technology, wherein the preparation process is the same as that of the step 1) of the embodiment 1;

2) Preparing double-layer gel microspheres coated with a magnetic layer by utilizing a microfluidic technology (wherein the outer layer is polyethylene glycol gel coated with magnetic nano particles, the inner layer is polyacrylamide gel), specifically, inputting an HFE-7500 fluorinated oil phase containing 0.4% v/v of N, N, N ', N' -tetramethyl ethane-1, 2-diamine (TEMED) and 2%g/mL of surfactant (PEG-PFPE) into a first flow input channel of a microfluidic chip shown in FIG. 3, and inputting a polyethylene glycol gel solution containing 5% g/mL of polyethylene glycol, 0.6% g/mL of ammonium persulfate and ferroferric oxide magnetic nano particles into a second flow input channel; the third flow input channel inputs the soluble gel microspheres prepared in the first step; and collecting double-layer micro-droplets wrapped with a magnetic layer through a fourth output channel, standing the collected gel micro-droplets at room temperature for 7 hours to form gel, and after the gel forming, performing demulsification on the micro-droplets (the demulsification method is the same as that of the embodiment 1), so as to obtain the double-layer gel microsphere with the inner layer of polyacrylamide gel and the outer layer of polyethylene glycol gel wrapped with magnetic nano particles, as shown in fig. 8.

Step 3) the specific operation procedure is the same as in step 3) of example 1, and finally hollow polyethylene glycol gel microspheres are obtained, as shown in fig. 9.

Example 3

1) Firstly, preparing soluble polyacrylamide gel microspheres by using a microfluidic technology, wherein the preparation process is the same as that of the step 1) of the embodiment 1;

2) Preparing double-layer gel microspheres coated with a magnetic layer by utilizing a microfluidic technology (wherein the outer layer is agarose gel coated with magnetic nano particles, and the inner layer is polyacrylamide gel), specifically, inputting HFE-7500 fluorinated oil phase containing 2%g/mL surfactant (PEG-PFPE) into a first flow input channel of a microfluidic chip shown in FIG. 3; the second flow input channel was fed with agarose solution containing 2%g/mL agarose and containing ferroferric oxide magnetic nanoparticles (agarose was completely dissolved at 90 ℃ for 30 min); the third flow input channel inputs the soluble gel microspheres prepared in the first step; and collecting double-layer micro-droplets wrapped with the magnetic layer through a fourth output channel to obtain double-layer micro-droplets wrapped with the magnetic layer. The collected gel droplets were allowed to stand at 4℃for 1 hour to gel, and after the gel formation, the droplets were demulsified (the method of demulsification was the same as that in example 1). The double-layer gel microsphere with the inner layer of polyacrylamide gel and the outer layer of agarose gel coated with magnetic nano particles is obtained, as shown in figure 8.

Step 3) the specific procedure was the same as in step 3) of example 1, and finally hollow agarose gel microspheres were obtained as shown in fig. 9.

Example 4

1) Firstly, preparing soluble agarose gel microspheres by utilizing a microfluidic technology, specifically, inputting an HFE-7500 fluorinated oil phase containing 2%g/mL surfactant (PEG-PFPE) into a first flow input channel of a microfluidic chip shown in FIG. 1; the second flow input channel was fed with an aqueous agarose solution containing 1%g/mL agarose (agarose was completely dissolved in water at 90 ℃ for 30 minutes); and collecting the soluble gel micro-droplets by a third output channel of the microfluidic chip. The collected droplets were allowed to stand at 4℃for 1 hour to gel, and after gel formation, the microdroplets were demulsified and washed sequentially with HFE-7500 fluorinated oil containing 20% v/v 1H, 2H-perfluoctanol 1 times, n-hexane containing 1% v/v Span-80 times, TE buffer containing 0.1% v/v Triton X-100 times, and TE buffer containing 0.1% v/v Tween20 times to obtain closely packed agarose hydrogel microspheres (diameter: 40 μm). After the agarose water gel micro-droplets are solidified, the agarose water gel micro-droplets can be dissolved under specific conditions;

2) The preparation method comprises the steps of preparing double-layer gel microspheres coated with a magnetic layer by using a microfluidic technology (the outer layer is polyacrylamide gel coated with magnetic nano particles, the inner layer is agarose gel), and the specific operation process is the same as that of step 2 of the embodiment 1. The double-layer gel microsphere with the inner layer of agarose gel and the outer layer of agarose gel coated with magnetic nano particles is obtained, and is shown in figure 8.

3) And 2) soaking the double-layer gel microsphere prepared in the step 2) in TE buffer solution, standing at 90 ℃ for 30 minutes, and obtaining the hollow polyacrylamide gel microsphere finally by using an agarose hydrogel core solution, wherein the hollow polyacrylamide gel microsphere is shown in figure 9.

Example 5

A method for preparing a magnetic microsphere with a hollow structure, the preparation flow is shown in fig. 2a-2c, and the method comprises the following steps:

1) Firstly, preparing soluble agarose gel microspheres by utilizing a microfluidic technology, wherein the specific operation process is the same as that in the step 1) of the embodiment 4;

2) The preparation method comprises the steps of preparing double-layer gel microspheres coated with a magnetic layer by using a microfluidic technology (the outer layer is polyethylene glycol gel coated with magnetic nanoparticles, the inner layer is agarose gel), and the specific operation process is the same as that of step 2 of example 2. The double-layer gel microsphere with the inner layer of agarose gel and the outer layer of polyethylene glycol gel coated with magnetic nano particles is obtained, and is shown in figure 8.

3) The specific operation procedure is the same as in step 3) of example 4, and finally hollow polyethylene glycol gel microspheres are obtained, as shown in fig. 9.

Example 6

2) The preparation method of the double-layer gel microsphere coated with the magnetic layer by using the microfluidic technology (the outer layer is agarose gel coated with magnetic nano particles, the inner layer is agarose gel), and the specific operation process is the same as that of the step 2 of the embodiment 3, and the difference from the embodiment 3 is that agarose is selected from agarose with low melting point. The double-layer gel microsphere with the inner layer of agarose gel and the outer layer of agarose gel coated with magnetic nano particles is obtained, and is shown in figure 8.

3) The specific procedure was the same as in step 3) of example 4, except that the reaction was carried out at 60℃for 30 minutes, and finally hollow agarose gel microspheres were obtained, as shown in FIG. 9.

In this example, the outer layer was selected from a high melting point agarose gel solution, the inner layer was selected from a low melting point agarose gel solution, and then the inner layer agarose gel was dissolved at a lower temperature.

Application example 1

The application of the method for preparing the magnetic polymer microsphere in PCR amplification of escherichia coli plasmid genome comprises the following steps:

1) The extracted E.coli plasmid genome was first packaged in a single micro-droplet (containing 6%g/mL acrylamide monomer, 0.392% g/mL N, N ' -bis (acryl) cystamine and 0.6% g/mL ammonium persulfate) using microfluidic technology, specifically, HFE-7500 fluorinated oil phase containing 0.4% v/v N, N, N ', N ' -tetramethyl ethane-1, 2-diamine (TEMED) and 2%g/mL surfactant (PEG-PFPE) was input to the first flow input channel of the microfluidic chip as shown in FIG. 1; the second flow input channel inputs E.coli plasmid genome and mixed aqueous solution containing 6%g/mL acrylamide monomer, 0.392% g/mL N, N' -bis (acryloyl) cystamine and 0.6% g/mL ammonium persulfate; the gel micro-droplets capturing the E.coli plasmid genome were collected in the third output channel of the microfluidic chip, the collected droplets were left at room temperature for 7 hours to gel, and after gel formation, the micro-droplets were demulsified, and were washed sequentially with HFE-7500 containing 20% v/v 1H, 2H-perfluotococtanol 1 times, n-hexane containing 1% v/v Span-80 times, TE buffer containing 0.1% v/v Triton X-100 times, and TE buffer containing 0.1% v/v Tween20 times to obtain closely arranged soluble polyacrylamide hydrogel microspheres (diameter: 40 μm) encapsulating E.coli plasmid genome.

2) Re-encapsulating the dissolvable polyacrylamide hydrogel microspheres using microfluidic technology, specifically, inputting 0.4% v/v of N, N '-tetramethyl ethane-1, 2-diamine (TEMED) and 2%g/mL of surfactant (PEG-PFPE) into a first flow input channel of the microfluidic chip shown in fig. 3, inputting 4%g/mL of acrylamide monomer, 0.4% g/mL of N, N' -methylenebisacrylamide and 0.3% g/mL of ammonium persulfate and polyacrylamide gel solution containing ferroferric oxide into a second flow input channel, and inputting the dissolvable polyacrylamide hydrogel microspheres prepared in the first step; and the fourth output channel collects gel micro-droplets wrapped with the magnetic layer. The collected gel microdroplets were allowed to stand at room temperature for 7 hours to gel, after gel formation, the microdroplets were de-emulsified, and washed sequentially 1 time with HFE-7500 containing 20% v/v1H, 2H-perfluoctanol, 2 times with n-hexane containing 1% v/v Span-80, 2 times with TE buffer containing 0.1% v/v Triton X-100, and 3 times with TE buffer containing 0.1% v/v Tween20 to obtain bilayer gel microspheres encapsulating E.coli plasmid genomes.

3) The double-layer gel microsphere is soaked in a Thermopol buffer containing 50mM dithiothreitol, and is kept stand for 5 minutes, so that the polyacrylamide hydrogel inner core is dissolved, and the hollow polyacrylamide magnetic microsphere packed with the escherichia coli plasmid genome is obtained. The dissolved hollow polyacrylamide magnetic microspheres are washed twice with water without ribozyme.

4) Specific fragment regions of the plasmid genome of E.coli were amplified using the PCR amplification system shown in Table 1. The PCR reaction was 20. Mu.L containing 6.6. Mu.L of hollow polyacrylamide gel microspheres containing E.coli plasmid genome, 10. Mu.L of DNA polymerase buffer, 0.4. Mu.L of DNA polymerase, 0.8. Mu.L of 10. Mu.M forward and reverse primer, 0.4. Mu.L of 10mM dNTP and 1. Mu.L of EVA Green (20X). The system first predenatures double-stranded DNA at 95℃for 30 seconds, secondly denatures double-stranded DNA at 95℃for 15 seconds, anneals at 58℃for 15 seconds, extends at 72℃for 3 minutes, wherein the denaturation, annealing and extension operations are carried out for 35 cycles, and finally extends thoroughly at 72℃for 5 minutes. The primer sequences are respectively as follows: primer 1-F (SEQ ID NO: 1): 5'-TGT TTA CAT CAC CGC CGA TA-3'; primer 1-R (SEQ ID NO: 2): 5'-TGA TTG TCT GGC AGC AGA AC-3'; primer 2-F (SEQ ID NO: 3): 5'-CTG GCT GAT CAC TAC CAG CA-3'; primer 2-R (SEQ ID NO: 4): 5'-GGT ACC GTC GAC TGC AGA AT-3'; primer 3-F (SEQ ID NO: 5): 5'-GGG CGA AGA AGT TGT CCA TA-3'; primer 3-R (SEQ ID NO: 6): 5'-TCC GGC CTT TAT TCA CAT TC-3'; primer 4-F (SEQ ID NO: 7): 5'-GGTAAACTGCCGGTACCTTG-3'; primer 4-R (SEQ ID NO: 8): 5'-AGC AGA ACA GGA CCA TCA CC-3'; primer 5-F (SEQ ID NO: 9): 5'-CGC ATG GTA TGG ATG AAC TG-3'; primer 5-R (SEQ ID NO: 10): 5'-TTT GAG CGT CAG ATT TCG TG-3'; primer 6-F (SEQ ID NO: 11): 5'-CTC GAG GCT TGG ATT CTC AC-3'; primer 6-R (SEQ ID NO: 12): 5'-TCC GGC CTT TAT TCA CAT TC-3'; primer 7-F (SEQ ID NO: 13): 5'-CTC GAG GCT TGG ATT CTC AC-3'; primer 7-R (SEQ ID NO: 14): 5'-ATC CCA ATG GCA TCG TAA AG-3'; primer 8-F (SEQ ID NO: 15): 5'-TCT GAC GCT CAA ATC AGT GG-3'; primer 8-R (SEQ ID NO: 16): 5'-TGT CGG CAG AAT GCT TAA TG-3'; primer 9-F (SEQ ID NO: 17): 5'-TGG GCC ATA AGC TGG AAT AC-3'; primer 9-R (SEQ ID NO: 18): 5'-AGG CGT GGA ATG AGA CAA AC-3'; primer 10-F (SEQ ID NO: 19): 5'-CTG TTG TTT GTC GGT GAA CG-3'; primer 10-R (SEQ ID NO: 20): 5'-GAT ATC GAG CTC GCT TGG AC-3'. The amplified product fragments obtained by PCR amplification using the above ten pairs of primers were 150bp, 225bp, 338bp, 434bp, 547bp, 620bp, 749bp, 866bp, 968bp and 1187bp, respectively.

Taking a fluorescent microscope photograph of the hollow polyacrylamide gel microsphere subjected to PCR amplification of primer 9, and observing a genome cluster with fluorescence, as shown in FIG. 10; after the amplification product was magnetically washed with water without ribozyme (washing by sucking gel microspheres with a magnetic column or other magnetic object), fluorescent genomic clusters without background color were seen as shown in FIG. 11 (both figures are PCR amplification result of primer 9). The results of agarose gel electrophoresis of the hollow gel microspheres containing the amplified products of the above ten pairs of primers, respectively, were shown in FIG. 12, which shows that the fragment sizes of the PCR amplified products were consistent with the expectations. Fluorescence photographing was performed on the hollow gel microspheres each containing the amplification products of the above ten pairs of primers, as shown in FIG. 13. And then carrying out fluorescence quantitative analysis on the hollow microspheres successfully amplified in the picture by using image J software, and drawing a violin chart as shown in fig. 14. The results show that when the DNA molecular fragment is larger than 547bp, the hollow gel microsphere can realize complete physical sealing of the DNA small molecule.

TABLE 1 PCR amplification System

Application example 2

The application of the method for preparing the magnetic polymer microsphere in single-cell whole genome amplification (MDA, multiple displacement amplification) of single escherichia coli cells comprises the following steps:

1) Firstly, packaging single escherichia coli cells into single soluble micro-droplets (containing 6%g/mL acrylamide monomer, 0.392% g/mL N, N' -bis (acrylamide) cystamine and 0.6% g/mL ammonium persulfate) by utilizing a microfluidic technology, and performing a demulsification operation after the soluble micro-droplets are solidified to obtain the soluble polyacrylamide gel microsphere with the size of 40 mu m, wherein the specific operation steps are shown in the example 1.

2) Re-encapsulating the soluble polyacrylamide hydrogel microspheres using a microfluidic technique, specifically, inputting 0.4% v/v of an HFE-7500 fluorinated oil phase containing N, N '-tetramethyl ethane-1, 2-diamine (TEMED) and 2%g/mL of a surfactant (PEG-PFPE) to a first flow input channel of a microfluidic chip as shown in fig. 3, inputting 4%g/mL of acrylamide monomer, 0.4% g/mL of N, N' -methylenebisacrylamide and 0.3% g/mL of ammonium persulfate and a polyacrylamide gel solution containing ferroferric oxide to a second flow input channel, and inputting the soluble polyacrylamide hydrogel microspheres prepared in the first step; and the fourth output channel collects gel micro-droplets wrapped with the magnetic layer. The collected gel microdroplets were allowed to stand at room temperature for 7 hours to gel, after gel formation, the microdroplets were de-emulsified, and washed sequentially 1 time with HFE-7500 containing 20% v/v 1H, 2H-perfluoctanol, 2 times with n-hexane containing 1% v/v Span-80, 2 times with TE buffer containing 0.1% v/v Triton X-100, and 3 times with TE buffer containing 0.1% v/v Tween20 to obtain double-layered gel microspheres encapsulating individual E.coli cells.

3) The double-layer gel microsphere is soaked in a Thermopol buffer containing 50mM dithiothreitol, and the double-layer gel microsphere is stood for 5 minutes, so that the polyacrylamide hydrogel inner core is dissolved, and the hollow polyacrylamide magnetic microsphere with the single escherichia coli cells packaged is obtained. The hollow polyacrylamide magnetic microspheres were washed twice with non-ribozyme water.

4) Individual e.coli cells were subjected to cell lysis to fully expose their genome, specifically, 30 μl of hollow polyacrylamide magnetic microspheres was taken, 30 μl of alkaline lysis solution (400 mM KOH, 10mM EDTA and 100mM DTT) was added, and reacted at 65 ℃ for 15 minutes. The cleaved hollow polyacrylamide magnetic microspheres were washed twice with water without ribozyme. To obtain the hollow polyacrylamide magnetic microsphere containing single escherichia coli genome.

5) The genome-wide amplification of individual E.coli cell genomes was performed using the MDA amplification system as shown in Table 2. The MDA reaction system was 50. Mu.L, which contained 2.5. Mu.L of Phi29 DNA polymerase, 5. Mu.L of Phi29 DNA polymerase buffer, 12.5. Mu.L of random primers (10. Mu.M), 5. Mu.L of dNTPs (10 mM) and 25. Mu.L of hollow polyacrylamide gel microspheres containing a single E.coli cell genome. The MDA procedure was: the reaction was carried out at 30℃for 8 hours. The amplified product is magnetically washed by using non-ribozyme water (washing by using a magnetic column or other magnetic object to adsorb gel microspheres). After magnetic washing, 9.5. Mu.L of the hollow polyacrylamide magnetic microspheres after whole genome amplification were added with 0.5. Mu.L of EVA Green (20 x), and after mixing uniformly, fluorescent microscopy was performed to photograph, and a genome cluster with Green fluorescence without background color was seen, as shown in FIG. 15.

TABLE 2 MDA amplification System

Component of MDA amplification System	Dosage of
		Phi29DNA polymerase	2.5μL
Phi29DNA polymerase buffer	5μL
		Random primer (10 mu M)	12.5μL
dNTP(10mM)	5μL
		Hollow polyacrylamide microsphere containing single E.coli cell genome	25μL
Totals to	50μL

Application example 3

The invention relates to an application of a method for preparing magnetic polymer microspheres in single-cell whole genome amplification (MDA, multiple displacement amplification) and combined PCR fluorescent specificity screening, which comprises the following steps:

1) Packaging single E.coli cells into a magnetic polyacrylamide gel microsphere containing a hollow structure, performing alkaline lysis on the single E.coli cells and performing MDA whole genome amplification to finally obtain the hollow polyacrylamide magnetic microsphere (hollow polyacrylamide magnetic microsphere containing an E.coli whole genome amplification product) after MDA amplification, wherein the specific operation is as shown in application example 2.

2) Fluorescent PCR-specific screening was performed on the hollow magnetic gel microspheres after MDA amplification using a PCR amplification system as shown in Table 3. The PCR reaction was 20. Mu.L containing 7.6. Mu.L of hollow polyacrylamide microspheres containing the whole genome of E.coli, 10. Mu.L of DNA polymerase buffer, 0.4. Mu.L of DNA polymerase, 0.8. Mu.L of 10. Mu.M forward and reverse primers and 0.4. Mu.L of 10mM dNTPs. The system first predenatures double-stranded DNA at 95℃for 30 seconds, secondly denatures double-stranded DNA at 95℃for 15 seconds, anneals at 55℃for 30 seconds, extends at 72℃for 3 minutes, wherein the denaturation, annealing and extension operations are performed for 40 cycles, and finally extends thoroughly at 72℃for 5 minutes. The primer sequences are as follows: primer-F (SEQ ID NO: 21): 5'-Alexa Fluor 532-GCG AGC TCG ATA TCA AAT TAC GCC CCG CCC TGC CAC TCA-3'; primer-R (SEQ ID NO: 22): 5'-GAG CTA AGG AAG CTA AAA TGG AGA AAA AAA TCA CTG GAT-3'. The amplified product is magnetically washed by using non-ribozyme water (washing by using a magnetic column or other magnetic object to adsorb gel microspheres). After magnetic cleaning, 9.5 mu L of hollow polyacrylamide gel microspheres amplified by PCR are added with 0.5 mu L of EVA Green fluorescent dye (20 x), uniformly mixed and photographed by a fluorescence microscope, so that genome clusters with Green fluorescence without background color can be seen, and meanwhile, part of hollow magnetic gel microspheres can specifically show red fluorescence, as shown in figure 16.

TABLE 3 PCR amplification System

Components of PCR amplification system	Dosage of
		DNA polymerase	0.4μL
DNA polymerase buffer (2×)	10μL
		Upstream primer (10. Mu.M)	0.8μL
Downstream primer (10. Mu.M)	0.8μL
		dNTP(10mM)	0.4μL
Hollow polyacrylamide microsphere containing escherichia coli whole genome	7.6μL
		Totals to	20μL

Application example 4

The invention relates to an application of a method for preparing magnetic polymer microspheres in single-cell whole genome amplification (MDA, multiple displacement amplification) of single escherichia coli cells and fluorescence specificity screening combined with FISH (fluorescence in situ hybridization), which comprises the following steps:

1) Packaging single E.coli cells into a magnetic polyacrylamide gel microsphere containing a hollow structure, performing alkaline lysis on the single E.coli cells and performing MDA whole genome amplification to finally obtain the hollow polyacrylamide magnetic microsphere containing MDA amplification products (the hollow polyacrylamide magnetic microsphere containing the whole genome amplification products of the E.coli), wherein the specific operation is as shown in application example 2.

2) And carrying out FISH specific screening on the hollow magnetic gel microspheres after MDA amplification. The FISH reaction system was 50. Mu.L, which contained 20. Mu.L of hollow polyacrylamide microspheres containing the whole genome of E.coli, 5. Mu.L of 10. Mu.M CY 3-labeled fluorescent probe, and 25. Mu.L of hybridization buffer (0.9M NaCl, 20mM Tris-HCl (pH 7.5), 0.02% SDS, and 20% formamide). The FISH reaction procedure was: 95℃for 5 min; (ii) 16h at 37 ℃. The CY 3-labeled fluorescent probe has the sequence (SEQ ID NO: 23): 5'-CY3-AGG GCT TCA AGC GCA GCA CG-3'. And magnetically cleaning the product after FISH by using non-ribozyme water (cleaning by using a magnetic column or other magnetic objects to adsorb gel microspheres). After magnetic cleaning, 9.5 mu L of hollow magnetic polyacrylamide gel microspheres after FISH are added with 0.5 mu L of EVA Green (20 x), uniformly mixed and photographed by a fluorescence microscope, and the genome clusters with Green fluorescence without background color can be seen, and meanwhile, part of hollow magnetic gel microspheres can specifically show red fluorescence, as shown in figure 17.

Application example 5

The application of the method for preparing the magnetic polymer microsphere in CFPS (cell-free expression system) comprises the following steps:

1) Firstly, a plasmid and a cell extract capable of expressing Green Fluorescent Protein (GFP) are packaged in a soluble micro-droplet (containing 6%g/mL acrylamide monomer, 0.392% g/mL N, N' -bis (acrylamide) cystamine and 0.6% g/mL ammonium persulfate) by utilizing a microfluidic technology, and after the soluble micro-droplet is solidified, the soluble micro-droplet is subjected to a demulsification operation, so that a soluble polyacrylamide gel microsphere with the size of 40 mu m is obtained, wherein the specific operation steps are shown in an example 1.

2) Re-encapsulating the soluble polyacrylamide gel microspheres using a microfluidic technique, specifically, inputting 0.4% v/v of an HFE-7500 fluorinated oil phase containing N, N, N ', N ' -tetramethyl ethane-1, 2-diamine (TEMED) and 2%g/mL of a surfactant (PEG-PFPE) to a first flow input channel of a microfluidic chip as shown in FIG. 3, inputting 4%g/mL of an acrylamide monomer, 0.4% g/mL of N, N ' -methylenebisacrylamide and 0.3% g/mL of ammonium persulfate and a polyacrylamide gel solution containing ferroferric oxide to a second flow input channel, and inputting the soluble polyacrylamide gel microspheres prepared in the first step; and the fourth output channel collects gel micro-droplets wrapped with the magnetic layer. The collected gel microdroplets were allowed to stand at room temperature for 3 hours to gel, after which the microdroplets were de-emulsified and washed sequentially 1 time with HFE-7500 containing 20% v/v 1H, 2H-perfluoacoctanol, 2 times with n-hexane containing 1% v/v Span-80, 2 times with TE buffer containing 0.1% v/v Triton X-100, and 3 times with TE buffer containing 0.1% v/v Tween20 to obtain magnetic bilayer gel microspheres encapsulating plasmids and cell extracts capable of expressing green fluorescent proteins.

3) And (3) soaking the magnetic double-layer gel microsphere in a Thermopol buffer containing 50mM dithiothreitol, standing for 5 minutes, and dissolving a polyacrylamide gel core to obtain the hollow magnetic polyacrylamide gel microsphere. The dissolved hollow polyacrylamide microspheres were washed twice with water without ribozyme.

4) 40. Mu.L of hollow magnetic gel microspheres containing plasmids and cell extracts capable of expressing green fluorescent proteins are added into 45. Mu.L of a cell-free expression system (no plasmids), the expression is carried out for 6 hours at 30 ℃, and the fluorescence intensity of the expressed green fluorescent proteins is detected by an enzyme-labeled instrument. The expressed hollow magnetic gel microsphere is washed three times by using the non-ribozyme water, a new 45 mu L non-cell expression system (no plasmid) is added, the expression is continued for 6 hours at 30 ℃, and then the fluorescence intensity of the expressed green fluorescent protein is detected by using an enzyme-labeled instrument. As a result, as shown in FIG. 18, the plasmid was recovered and re-expressed several times by using the hollow magnetic gel microspheres.

Application example 6

The application of the method for preparing the magnetic polymer microsphere in constructing the artificial biosensing cells with lactam specific response comprises the following steps:

1) As shown in FIG. 19, the modified strain expressed the protein of ChunR, which was able to specifically bind to the lactam (lactam) small molecule, activates the Pb promoter, and thus expresses mCherry fluorescent protein, so that the lactam can be specifically detected. The engineered E.coli strain was mixed with 2%g/mL agarose solution at a 1:1 volume to finally obtain 1%g/mL agarose solution. Inputting HFE-7500 fluorinated oil phase containing 2%g/mL surfactant (PEG-PFPE) into a first flow input channel of a microfluidic chip as shown in FIG. 1; the second flow input channel inputs agarose bacteria liquid containing 1%g/mL agarose; and collecting the soluble gel micro-droplets by a third output channel of the microfluidic chip. The collected droplets were allowed to stand at 4℃for 30 minutes to gel, and after gel formation, the microdroplets were de-emulsified, and washed sequentially 1 time with HFE-7500 fluorinated oil containing 20% v/v 1H, 2H-perfluooroctanol, 2 times with TE buffer containing 0.1% v/v Triton X-100, and 3 times with TE buffer containing 0.1% v/v Tween20 to obtain closely packed agarose gel microspheres (diameter: 40 μm).

2) And preparing the double-layer gel microsphere coated with the magnetic layer by utilizing a microfluidic technology. Inputting HFE-7500 fluorinated oil phase containing 2%g/mL surfactant (PEG-PFPE) into a first flow input channel of a microfluidic chip as shown in FIG. 3; the second flow input channel inputs agarose solution containing 2%g/mL agarose and ferroferric oxide magnetic nano particles; the agarose gel microsphere containing the engineering strain prepared in the first step is input into a third flow input channel; and the fourth output channel is used for collecting double-layer micro-droplets wrapped with the magnetic layer to obtain the double-layer micro-droplets wrapped with the magnetic layer, and the double-layer micro-droplets are of a compact arrangement structure. The collected gel micro-droplets are placed at 4 ℃ for 30 minutes to form gel, and the micro-droplets are de-emulsified after the gel is formed. The demulsification method was performed by washing with HFE-7500 fluorinated oil containing 20% v/v 1H, 2H-perfluoctanol 1 time, n-hexane containing 1% v/v Span-80 2 times, TE buffer containing 0.1% v/v Triton X-100 2 times, and TE buffer containing 0.1% v/v Tween20 3 times in this order. Finally, the magnetic double-layer agarose gel microsphere coated with the agarose gel microsphere is obtained.

3) The magnetic bilayer agarose gel microspheres (50. Mu.L) containing the engineered strain obtained in step 2 were added to 1mL LB medium (containing 25. Mu.g/mL chloramphenicol resistance, 10. Mu.L 0.2% arabinose, and 10. Mu.L 10M valerolactam) and incubated at 30℃for 16 hours. Then, a small amount of bacteria escaping from the double-layer gel microsphere into the environment are washed off by a magnetic washing method, and finally the double-layer agarose gel microsphere with the inner layer full of coliform bacteria is obtained, as shown in figure 20; as a control, the single-layer agarose gel microspheres (50. Mu.L) containing the engineering strain obtained in the step 1) are added with the same culture solution, and the single-layer agarose gel microspheres full of escherichia coli flora are finally obtained through the same culture step and the cleaning step, and as shown in figure 21, part of escherichia coli can diffuse outwards and escape to the outside. During the double-layer microsphere bacteria culture, the fluorescence intensity in the hollow magnetic gel microsphere was detected every 4 hours, and the effect of the culture time on the valerolactam biological response behavior was analyzed, as shown in fig. 22. From the trend line, the fluorescence signal of valerolactam increased exponentially before entry into the plateau, and over time there was no longer a significant change in fluorescence. In addition, the magnetic bilayer agarose gel microspheres (50. Mu.L) containing the engineered strain obtained in step 2 were added to 1mL of LB medium (additionally containing 25. Mu.g/mL chloramphenicol resistance and 10. Mu.L of 0.2% arabinose) containing different concentrations of valerolactam (0 mM, 5mM, 10mM, 20mM, 50mM and 100 mM), respectively, and incubated at 30℃for 16 hours. And then respectively detecting the fluorescence intensity of the strain in the hollow magnetic gel microsphere under 5 valerolactam concentrations under a fluorescence microscope, and finally analyzing to obtain a valerolactam biological response dose-response graph, wherein the trend line basically shows the linear relation of the valerolactam biological response dose-response as shown in fig. 23.

The above embodiments are only preferred embodiments of the present invention and are not limiting in any way and in essence, it should be noted that modifications and additions may be made to the person skilled in the art without departing from the scope of the invention, which is also considered as being within the scope of the invention.

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Claims

1. The application of the magnetic polymer microsphere in biochemical reaction is characterized in that the preparation method of the magnetic polymer microsphere comprises the following steps:

step 1: the method comprises the steps of preparing multilayer polymer micro-droplets with magnetic nanoparticles coated on the outer layer by using a droplet generation technology and adopting raw materials comprising monomers and magnetic nanoparticles to undergo chemical crosslinking polymerization or raw materials comprising polymers and magnetic nanoparticles to undergo physical crosslinking;

step 2: solidifying or semi-solidifying the multi-layer polymer micro-droplets, and demulsifying to obtain multi-layer polymer microspheres wrapped with a magnetic shell layer;

the method of application comprises the following steps:

step S1: preparing micro-droplets encapsulated with a substrate by using a droplet generation technology;

step S2: preparing a multi-layer polymer microsphere coated with a magnetic outer shell layer by adopting the preparation method of the magnetic polymer microsphere;

Step S3: directly adding a reactant to perform biochemical reaction, or dissolving at least one layer of the inner layer polymers of the multi-layer polymer microsphere to form a hollow cavity, and then adding the reactant to perform biochemical reaction;

when the magnetic polymer microsphere is used for biochemical reaction, the magnetic polymer microsphere with the pore diameter being suitable for the reaction substrate is prepared by adjusting the types or the concentrations of the raw materials for preparing the magnetic polymer microsphere according to the molecular weight of the reaction substrate, so that the magnetic polymer microsphere can encapsulate the reaction substrate and allow small molecule reaction reagents to enter the microsphere, and the shell layer of the microsphere can isolate exogenous and cross contamination; when complex multi-step biochemical reaction is carried out, magnetic polymer microspheres are adsorbed by magnetic beads to carry out magnetic cleaning, and after unreacted small molecule reaction reagents in the previous step are washed off, reagents required in the next step are added to continue the reaction.

2. The use of claim 1, wherein the multi-layer microdroplet produced in step 1 is a bi-layer microdroplet, and correspondingly, the multi-layer polymeric microsphere produced in step 2 is a bi-layer polymeric microsphere.

3. The application according to claim 2, wherein the specific process of step 1 comprises:

4. The use according to claim 3, wherein the polymer microspheres of step 1.1 comprise any one of polyacrylamide gel microspheres, polyethylene glycol gel microspheres, agarose gel microspheres, sodium alginate gel microspheres, and gelatin gel microspheres.

5. The use according to claim 4, wherein the polyacrylamide gel microspheres are prepared by forming a water-in-oil system from an oil phase containing a TEMED catalyst and a surfactant and an aqueous phase containing an acrylamide monomer, an ammonium persulfate initiator and an N, N' -bis (acryl) cystamine cross-linking agent, and performing chemical cross-linking polymerization to obtain polyacrylamide gel microdroplets, and curing or semi-curing and demulsification the polyacrylamide gel microdroplets; the agarose gel microsphere is a water-in-oil system formed by an oil phase containing a surfactant and a water phase containing agarose, and is prepared by physical crosslinking to obtain agarose gel micro-droplets, and solidifying or semi-solidifying and demulsifying the agarose gel micro-droplets; the sodium alginate gel microsphere is prepared by forming a water-in-oil system from an oil phase containing a surfactant and a water phase containing sodium alginate, preparing sodium alginate gel micro-droplets through chemical crosslinking, and solidifying or semi-solidifying and demulsifying the sodium alginate gel micro-droplets; the gelatin gel microsphere is a water-in-oil system formed by an oil phase containing a surfactant and a water phase containing gelatin only or a water phase containing gelatin and one or more of crosslinking agents of formaldehyde, glutaric acid, EDC, genipin, methacrylic anhydride and polyethylene glycol diacrylate, and is prepared into gelatin micro-droplets through physical crosslinking or chemical crosslinking, and the gelatin micro-droplets are prepared after solidification or semi-solidification and demulsification.

6. The use according to claim 5, wherein the surfactant comprises any one or more of PEG-PFPE, castor oil polyoxyl ester, polyoxyethylene 40 hydrogenated castor oil, poloxamer, krytox, sodium dodecyl sulfate, and Janus nanoparticles; the oil phase comprises any one or more of HFE-7500 fluorinated oil phase, squarane oil phase, silicone oil phase and mineral oil phase.

7. The use according to claim 6, wherein the polymer dispersion of magnetic nanoparticles in step 1.2 comprises any one of a polyacrylamide gel solution, a polyethylene glycol gel solution, an agarose gel solution, a sodium alginate gel solution, and a gelatin gel solution of magnetic nanoparticles comprising any one or more of single metal magnetic nanoparticles, magnetic iron oxide nanoparticles, bimetallic magnetic nanoparticles, and alloy magnetic nanoparticles.

8. The use of claim 7, wherein the bilayer microdroplet produced in step 1.2 comprises any one of the following bilayer microdroplets:

9. The use according to claim 8, wherein the bilayer microdroplets A, F, K and P are prepared by chemical cross-linking polymerization of an oil phase comprising a TEMED catalyst and a surfactant with a polyacrylamide gel solution comprising an acrylamide monomer, an ammonium persulfate initiator, an N, N' -methylenebisacrylamide cross-linker and magnetic nanoparticles, and a system comprising polymer microspheres prepared in step 1.1 to form water-in-oil spheres; the double-layer micro-droplets B, G, L and Q are prepared by forming a water-in-oil ball-in-oil system from an oil phase containing a TEMED catalyst and a surfactant, a polyethylene glycol gel solution containing polyethylene glycol, an ammonium persulfate initiator and magnetic nano particles and polymer microspheres prepared in the step 1.1, and performing chemical cross-linking polymerization to prepare corresponding double-layer micro-droplets; the double-layer micro-droplets C, H, M and R are prepared by forming a water-in-oil ball system from an oil phase containing a surfactant, an agarose gel solution containing agarose and magnetic nano particles and polymer microspheres prepared in the step 1.1, and performing physical crosslinking to prepare corresponding double-layer micro-droplets; the double-layer micro-droplets D, I, N and S are a system in which a water-in-oil ball is formed by a sodium alginate gel solution containing a surfactant, sodium alginate and magnetic nano particles and polymer microspheres prepared in the step 1.1, and the corresponding double-layer micro-droplets are prepared through physical crosslinking; the double-layer micro-droplets E, J, O and T are a system in which oil phase containing surfactant and gelatin gel solution containing gelatin and magnetic nano particles only or gelatin gel solution containing gelatin and magnetic nano particles and one or more of cross-linking agents of formaldehyde, glutaric acid, EDC, genipin, methacrylic anhydride and polyethylene glycol diacrylate form water-in-oil ball by polymer microspheres prepared in the step 1.1, and the corresponding double-layer micro-droplets are prepared by physical crosslinking or chemical crosslinking.

10. The use according to claim 9, wherein the method of curing or semi-curing in steps 1.1 and 2 comprises any one of standing, UV irradiation, heating, changing PH and changing ion concentration; the demulsification method comprises the following steps: the procedure was followed by 1 wash with HFE-7500 containing 20% v/v1H, 2H-perfluoctanol, 2 washes with n-hexane containing 1% v/v Span-80, 2 washes with TE buffer containing 0.1% v/v Triton X-100, and 3 washes with TE buffer containing 0.1% v/v Tween 20.

11. The use according to claim 10, wherein the method used for dissolving the inner polymer in step 3 comprises any one of UV irradiation, heating, PH change, ion concentration change and dithiothreitol solution.

12. The use of claim 1, wherein the substrate encapsulated in step S1 comprises at least one of a nucleic acid, a protein, a cell, and a microorganism.