CN113304700A - Method for preparing magnetic polymer microspheres and application thereof - Google Patents
Method for preparing magnetic polymer microspheres and application thereof Download PDFInfo
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- CN113304700A CN113304700A CN202110655120.5A CN202110655120A CN113304700A CN 113304700 A CN113304700 A CN 113304700A CN 202110655120 A CN202110655120 A CN 202110655120A CN 113304700 A CN113304700 A CN 113304700A
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J13/00—Colloid chemistry, e.g. the production of colloidal materials or their solutions, not otherwise provided for; Making microcapsules or microballoons
- B01J13/02—Making microcapsules or microballoons
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J13/00—Colloid chemistry, e.g. the production of colloidal materials or their solutions, not otherwise provided for; Making microcapsules or microballoons
- B01J13/02—Making microcapsules or microballoons
- B01J13/06—Making microcapsules or microballoons by phase separation
- B01J13/14—Polymerisation; cross-linking
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
- C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
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- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
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Abstract
The invention provides a method for preparing magnetic polymer microspheres and application thereof. Firstly, preparing multilayer polymer micro-droplets with magnetic nano-particles wrapped on the outer layer by using a droplet generation technology; then obtaining the multilayer polymer microspheres wrapped with the magnetic shell layer after solidification or semi-solidification and emulsion breaking; at least one layer of inner layer polymers of the multilayer polymer microspheres is dissolved to obtain the magnetic polymer microspheres containing the hollow cavities. The preparation method can be used in any biochemical reaction, the magnetic polymer microsphere serves as a reaction container, the outer shell layer of the magnetic polymer microsphere serves as a passive sieve to retain the encapsulated reaction substrate and isolate the invasion of external pollution, and meanwhile, the small molecule reaction reagent is allowed to diffuse and pass through and enter the microsphere to react with the reaction substrate. The invention can realize the high-efficiency and high-flux biochemical reaction under the condition of isolating external pollution; can ensure that the sample is not subjected to cross contamination and exogenous pollution among multiple biochemical reactions.
Description
Technical Field
The invention relates to a method for preparing magnetic polymer microspheres and application thereof, belonging to the technical field of biochemistry.
Background
As molecular biology techniques are iteratively updated, modern molecular biology research increasingly relies on high throughput analytical methods to process complex samples with single cell or single molecule resolution. In contrast to microplates, large-scale, high-throughput parallel analysis can be achieved by trapping individual cells, DNA, enzymes or biomolecules in water-in-oil microdroplets or other forms of microscopic compartments. However, in performing many complex biochemical reactions, the sample is processed in a sequence to initiate, modify or terminate the biochemical reaction, but these complex multi-step operations are difficult to perform in droplets. Although a multi-step process can be achieved by manipulating droplets (droplet fusion, droplet re-injection, droplet separation and sorting), the use of such methods requires expert knowledge of microfluidic technology, and the complexity of fluid manipulation limits the use of such techniques. In microbiological experiments, when bacteria or genetic materials of bacteria are separated and captured in micro-droplets to perform a corresponding series of biochemical treatments, the connection between multi-step experimental operations is realized, and the samples are ensured to have no cross contamination and exogenous contamination, so that the microbiological experiments have considerable challenges. For example, in the case of genetic material amplification and analysis of microorganisms, the microorganisms are subjected to a cell lysis procedure, but the reagents used in this procedure may affect the subsequent enzymatic reaction, and thus have limitations.
Disclosure of Invention
The technical problem to be solved by the invention is as follows: how to provide an experimental method which can carry out 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: by utilizing a droplet generation technology, carrying out chemical crosslinking polymerization on raw materials comprising monomers and magnetic nanoparticles or carrying out physical crosslinking on the raw materials comprising polymers and magnetic nanoparticles to prepare a multi-layer polymer micro-droplet with the magnetic nanoparticles wrapped on the outer layer; multilayer polymer micro-droplets with adjustable pore diameters can be prepared by adjusting the types or the concentrations of monomers/polymers in the raw materials;
step 2: and the multilayer polymer micro-droplets are solidified or semi-solidified and demulsified to obtain the multilayer polymer microspheres wrapped with the magnetic shell layer.
The method for preparing the multilayer polymer micro-droplets coated with the magnetic nanoparticles in the step 1 is any one of the following methods:
the method comprises the following steps: preparing polymer micro-droplets by using a droplet generation technology, solidifying or semi-solidifying the polymer micro-droplets, demulsifying 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 a multi-layer polymer micro-droplet wrapped with the magnetic nano-particles on the outer layer;
the second method comprises the following steps: preparing a polymer or a substrate for preparing the polymer and magnetic particles into polymer micro-droplets containing magnetic nano-particles by using a droplet generation technology, solidifying or semi-solidifying the micro-droplets containing the magnetic nano-particles, demulsifying, and then preparing into multi-layer polymer micro-droplets by using a droplet production technology, transferring the magnetic nano-particles in the multi-layer polymer micro-droplets to the outer layer of the multi-layer polymer micro-droplets by using a physical or chemical method, so as to obtain the multi-layer polymer micro-droplets with the outer layer wrapped by the magnetic nano-particles;
preferably, via step 3: and (3) dissolving at least one layer of inner layer polymers of the multilayer polymer microspheres prepared in the step (2) to obtain the magnetic polymer microspheres containing the hollow cavities.
Preferably, the multilayer micro-droplets prepared in step 1 are double-layer micro-droplets, and correspondingly, the multilayer polymeric microspheres prepared in step 2 are double-layer polymeric microspheres.
Preferably, the specific process of step 1 includes:
step 1.1: by utilizing a high-flux microfluidic technology, carrying out chemical crosslinking polymerization on a raw material containing a monomer or physical crosslinking on a raw material containing a polymer to prepare micro-droplets, and solidifying or semi-solidifying and demulsifying the micro-droplets to obtain polymer microspheres;
step 1.2: secondly, preparing a magnetic shell layer on the outer surface of the polymer microsphere obtained in the step 1.1 by physically or chemically crosslinking polymer dispersion liquid of magnetic nano particles by utilizing a high-flux microfluidic technology to prepare a double-layer micro-droplet with the outer layer being a polymer wrapped with the magnetic nano particles and the inner layer being a polymer; the outer layer polymer and the inner layer polymer have different physical and/or chemical properties.
Preferably, the polymer microspheres of step 1.1 include any one of polyacrylamide gel microspheres, polyethylene glycol gel microspheres, agarose gel microspheres, sodium alginate gel microspheres, and gelatin gel microspheres.
Preferably, the polyacrylamide gel microspheres are 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 (acryloyl) cysteamine cross-linking agent, performing chemical cross-linking polymerization to prepare polyacrylamide gel microdroplets, and then performing curing or semi-curing and emulsion breaking on the polyacrylamide gel microdroplets; the agarose gel microspheres are prepared by forming a water-in-oil system by an oil phase containing a surfactant and a water phase containing agarose, preparing agarose gel micro-droplets through physical crosslinking, and solidifying or semi-solidifying and demulsifying the agarose gel micro-droplets; the sodium alginate gel microspheres are prepared by forming a water-in-oil system by 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 microspheres are prepared by forming a water-in-oil system by an oil phase containing a surfactant and a water phase only containing gelatin or a water phase containing gelatin and one or more of the following crosslinking agents formaldehyde, glutaric acid, EDC, genipin, methacrylic anhydride and polyethylene glycol diacrylate, carrying out physical crosslinking or chemical crosslinking to prepare gelatin micro-droplets, and carrying out solidification or semi-solidification and emulsion breaking on the gelatin micro-droplets.
Preferably, the surfactant comprises one or more of a nonionic surfactant PEG-PFPE, a castor oil polyoxyl ester, polyoxyethylene 40 hydrogenated castor oil, a poloxamer and an ionic surfactant Krytox, sodium lauryl sulfate and Janus nanoparticles; the oil phase comprises one or more of HFE-7500 fluorinated oil phase, Squalane oil phase, silicone oil phase and mineral oil phase.
Preferably, the polymer dispersion liquid of the magnetic nanoparticles in the step 1.2 comprises any one of polyacrylamide gel solution, polyethylene glycol gel solution, agarose gel solution, sodium alginate gel solution and gelatin gel solution of the magnetic nanoparticles, and the magnetic nanoparticles comprise any one or more of monometallic magnetic nanoparticles, magnetic iron oxide nanoparticles, bimetallic magnetic nanoparticles and alloy magnetic nanoparticles.
Preferably, the double-layer micro-droplet prepared in step 1.2 comprises any one of the following double-layer micro-droplets:
double-layer micro-droplet A: the outer layer is polyacrylamide gel wrapped with magnetic nano particles, and the inner layer is double-layer micro-droplets of the polyacrylamide gel;
double-layer micro-droplet B: the outer layer is polyethylene glycol gel wrapped with magnetic nano particles, and the inner layer is double-layer micro-droplets of polyacrylamide gel;
double-layer micro-droplet C: the outer layer is agarose gel wrapped with magnetic nano particles, and the inner layer is double-layer micro-droplets of polyacrylamide gel;
double-layer micro-droplet D: the outer layer is sodium alginate gel wrapped with magnetic nano particles, and the inner layer is double-layer micro-droplets of polyacrylamide gel;
double-layer micro-droplets E: the outer layer is gelatin gel wrapped with magnetic nano particles, and the inner layer is double-layer micro-droplets of polyacrylamide gel;
double-layer micro-droplets F: the outer layer is polyacrylamide gel wrapped with magnetic nano particles, and the inner layer is double-layer micro-droplets of agarose gel;
double-layer micro-droplet G: the outer layer is polyethylene glycol gel wrapped with magnetic nano particles, and the inner layer is double-layer micro-droplets of agarose gel;
double-layer micro-droplets H: the outer layer is agarose gel wrapped with magnetic nano particles, and the inner layer is double-layer micro-droplets of the agarose gel;
double-layer micro-droplet I: the outer layer is sodium alginate gel wrapped with magnetic nano particles, and the inner layer is double-layer micro-droplets of agarose gel;
double-layer micro-droplet J: the outer layer is gelatin gel wrapped with magnetic nano particles, and the inner layer is double-layer micro-droplets of agarose gel;
double-layer micro-droplet K: the outer layer is a double-layer micro-droplet which is wrapped with magnetic nano-particles and is made of sodium alginate gel;
double-layer micro-droplets L: the outer layer is a polyethylene glycol gel wrapped with magnetic nano particles, and the inner layer is a double-layer micro-droplet of sodium alginate gel;
double-layer micro-droplet M: the outer layer is agarose gel wrapped with magnetic nano particles, and the inner layer is double-layer micro-droplets of sodium alginate gel;
double-layer micro-droplet N: the outer layer is sodium alginate gel wrapped with magnetic nano particles, and the inner layer is double-layer micro-droplets of the sodium alginate gel;
double-layer micro-droplet O: the outer layer is gelatin gel wrapped with magnetic nano particles, and the inner layer is double-layer micro-droplets of sodium alginate gel;
double-layer micro-droplet P: the outer layer is a double-layer micro-droplet which is wrapped with magnetic nano-particles and the inner layer is gelatin gel;
double-layer micro-droplet Q: the outer layer is polyethylene glycol gel wrapped with magnetic nano particles, and the inner layer is double-layer micro-droplets of gelatin gel;
double-layer micro-droplet R: the outer layer is agarose gel wrapped with magnetic nano particles, and the inner layer is double-layer micro-droplets of gelatin gel;
double-layer micro-droplet S: the outer layer is sodium alginate gel wrapped 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 wrapped with magnetic nanoparticles, and the inner layer is double-layer micro-droplets of gelatin gel.
Preferably, the bilayer microdroplets A, F, K and P are prepared by forming a water-in-oil encapsulated sphere system by using 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' -methylenebisacrylamide cross-linking agent and magnetic nanoparticles, and polymer microspheres prepared in step 1.1, and performing chemical cross-linking polymerization to prepare corresponding bilayer microdroplets; the double-layer micro-droplets B, G, L and Q are water-in-oil ball-wrapped systems formed by oil phase containing TEMED catalyst and surfactant, polyethylene glycol gel solution containing polyethylene glycol, ammonium persulfate initiator and magnetic nanoparticles and polymer microspheres prepared in the step 1.1, and are subjected to chemical cross-linking polymerization to prepare corresponding double-layer micro-droplets; the double-layer micro-droplets C, H, M and R are water-in-oil ball-coated systems formed by oil phase containing surfactant, agarose gel solution containing agarose and magnetic nanoparticles and polymer microspheres prepared in the step 1.1, and are physically cross-linked to prepare corresponding double-layer micro-droplets; the double-layer micro-droplets D, I, N and S are water-in-oil ball-wrapped systems 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 by physical crosslinking; the bilayer microdroplets E, J, O and T are prepared by forming a water-in-oil ball-in-oil system by using an oil phase containing a surfactant, a gelatin gel solution containing gelatin and magnetic nanoparticles only or a gelatin gel solution containing gelatin and magnetic nanoparticles and one or more of formaldehyde, glutaric acid, EDC, genipin, methacrylic anhydride and polyethylene glycol diacrylate as crosslinking agents and polymer microspheres prepared in the step 1.1, and carrying out physical crosslinking or chemical crosslinking to prepare corresponding bilayer microdroplets.
Preferably, the curing or semi-curing method in step 1.1 and step 2 comprises any one of standing, UV irradiation, heating, PH change and ion concentration change; the demulsification method comprises the following steps: washing sequentially with HFE-7500 containing 20% v/v 1H,1H,2H,2H-Perfluorooctanol 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.
Preferably, the method for dissolving the inner layer polymer in the step 3 includes any one of UV irradiation, heating, PH change, ion concentration change and using a dithiothreitol solution.
The invention also provides the application of the method for preparing the magnetic polymer microspheres in biochemical reaction, biochemical analysis and microbial culture.
Preferably, said application in biochemical reactions comprises in particular the following steps:
step 1: preparing micro-droplets encapsulating substrates by using a droplet generation technology;
step 2: preparing multilayer polymer microspheres wrapped with a magnetic shell layer;
and step 3: directly adding a reaction reagent to carry out biochemical reaction, or dissolving at least one layer of inner layer polymers of the multilayer polymer microspheres to form a hollow cavity, and then adding the reaction reagent to carry out biochemical reaction.
Preferably, the substrate encapsulated in step 1 comprises at least one of nucleic acids, proteins, cells and microorganisms.
The technical principle of the invention is as follows:
the invention utilizes a droplet generation technology, in particular to a magnetic polymer microsphere with adjustable pore diameter prepared by a microfluidic chip, and an inner layer polymer of the magnetic polymer microsphere can form a hollow cavity after being dissolved. When the magnetic polymer microsphere is used for biochemical reaction, the magnetic polymer microsphere with the aperture matched with a reaction substrate is prepared by adjusting the type or concentration of 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 a small-molecule reaction reagent to enter the microsphere, the outer 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 reactions are carried out, magnetic beads can be used for adsorbing magnetic polymer microspheres to carry out magnetic cleaning, after unreacted small molecule reaction reagents in the previous step are washed away, reagents required by the next step of reaction are added to continue the reaction, and therefore the biochemical reactions based on the magnetic polymer microspheres can realize the connection among multi-step experimental operations and ensure that no cross contamination or exogenous pollution exists among samples.
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 the polymer microspheres with adjustable volume and pore size according to requirements, and can be used in any biochemical reaction, including but not limited to single cell technology, such as culture of single bacteria and single viruses.
2. The magnetic microsphere is used as a reaction container with the characteristic of a semipermeable membrane, a shell layer of the magnetic microsphere can be used as a passive sieve 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 and pass, and substances (such as moisture, inorganic salt and other nutrient components and biochemical reaction reagents and the like) can be exchanged between the inner part of a cavity and the outer part of the cavity, but the invasion of external pollution (such as exogenous bacteria, cells, viruses and the like) can be isolated.
3. According to the method for preparing the magnetic polymer microspheres, disclosed by the invention, when complex multi-step biochemical reactions are carried out, the magnetic beads can be used for adsorbing the magnetic polymer microspheres to carry out magnetic cleaning, after unreacted micromolecule reaction reagents in the previous step are washed away, reagents required by the next step of reaction are added to continue the reaction, so that the biochemical reactions based on the magnetic polymer microspheres can realize the linkage of multi-step experimental operations and ensure that no cross contamination and exogenous pollution exist among samples, and a powerful scientific experimental method is provided for realizing high-efficiency and high-flux biochemical reactions under the condition of isolating external pollution.
Drawings
FIG. 1 is a schematic diagram of a microfluidic chip for generating soluble micro-droplets;
FIG. 2 is a flow chart of the generation and experimental application of hollow magnetic microspheres, which is performed in the order of a-b-c-d-e;
FIG. 3 is a schematic diagram of a microfluidic chip for generating double-layered gel microdroplets;
FIG. 4 is a diagram of closely packed double-layer microdroplets;
FIG. 5 is a graph showing the dynamic dissolution process of inner layer microspheres, wherein the numbers represent time in seconds;
FIG. 6 is a diagram of a magnetic microsphere with a hollow structure after an inner layer substance is dissolved;
FIG. 7 is a schematic cross-sectional view of a magnetic hollow microsphere;
FIG. 8 is a diagram of closely packed double-layered gel microdroplets of different materials;
FIG. 9 is a diagram of closely packed hollow structured gel microspheres of different materials;
FIG. 10 is a graph of the staining of PCR products before magnetic washing, wherein the bright dots indicated by the arrows indicate the genomic clusters with fluorescence;
FIG. 11 is a staining pattern of the PCR product after magnetic washing, wherein the bright dots indicated by arrows indicate the genomic clusters with fluorescence;
FIG. 12 is a drawing showing the result of agarose electrophoresis of the PCR product;
FIG. 13 is a fluorescent plot of PCR products for ten different pairs of primers;
FIG. 14 is a violin diagram of the quantitative fluorescent analysis of PCR products for ten pairs of different primers;
FIG. 15 is a graph of staining of MDA product after magnetic washing;
FIG. 16 is a graph of the results of PCR products after magnetic washing of MDA;
FIG. 17 is a graph of FISH product results after magnetic washing of MDA;
FIG. 18 is a diagram showing the results of expression of plasmids in a cell-free expression system;
FIG. 19 is a schematic bacterial biosensing diagram;
FIG. 20 is a diagram showing the results of bacterial culture in double-layered agarose gel microspheres;
FIG. 21 is a diagram showing the results of bacterial culture in single-layer agarose gel microspheres;
FIG. 22 is a graph showing the results of the effect of incubation time on valerolactam bioresponse behavior;
FIG. 23 is a graph of valerolactam bioresponse dose-response results;
wherein, figures 4 to 9 in the above figures are all pictures taken under a microscope.
Detailed Description
In order to make the invention more comprehensible, preferred embodiments are described in detail below with reference to the accompanying drawings.
The sources of materials used in the examples are as follows:
HFE-7500 fluorinated oil was purchased from Minnesota mining, Inc., International trade, Shanghai. Biocompatible surfactants (PEG-PFPE) were purchased from Daphne Biotech, Inc., Zhejiang. Acrylamide (AM), N, N '-methylenebisacrylamide, N, N' -bis (acryloyl) cystamine, Ammonium Persulfate (APS), N, N, N ', N' -tetramethylethane-1, 2-diamine (TEMED), 1H,2H,2H-Perfluorooctanol, Span-80, Tween20, polyethylene glycol monomer (poly (ethylene glycol) diacid average Mn 575), low melting agarose, SDS (sodium dodecyl sulfate), NaCl, 300nm magnetic nanoparticles were purchased from Sigma-Aldrich. Hexan-hexane (Hexanes) was purchased from Thermo/Fisher Chemical. Triton X-100 was purchased from Shanghai Pan Biotechnology Ltd. 1M dithiothreitol solution was purchased from Beijing Boolsen Biotechnology, Inc. (Bioss). High melting point agarose, Formamide (Formamide) was purchased from next saint biotechnology (shanghai) ltd. PCR kit (Phanta Max Super-Fidelity DNA Polymerase), Phi29DNA Polymerase, Phi29DNA Polymerase buffer, dNTP (10mM) was purchased from Nanjing Novozam Biotech GmbH. Oligonucleotide primers, FISH fluorescent probes were produced by Jinzhi. EVA Green (20X) was purchased from Biotium (USA). Random primers (EXO-RESISTANT RANDOM PRIMER), PCR fluorescent primer sequences, and ribozyme-free water (NUCLEASE-FREE WATER 10X 50ML) were purchased from Saimer Feishell science and technology (China) Ltd. Tris-HCl (pH 7.5) was purchased from Beijing Sorboard technologies, Inc. L-Arabidopsis L- (+) -Arabinose was purchased from Biotechnology, Inc. (BIOSYNTH) at end level. Valerolactam was provided by the Zhang Longitude project group at the university of Compound Dan.
Example 1
A method for preparing magnetic polymer microspheres, the preparation process is shown in fig. 2a-2c, and the method comprises the following steps:
1) firstly, preparing polyacrylamide soluble gel microspheres by using a microfluidic technology, specifically, inputting an HFE-7500 fluorinated oil phase containing 0.4% v/v of N, N, N ', N' -tetramethylethane-1, 2-diamine (TEMED) and 2% g/mL of a surfactant (PEG-PFPE) into a first flow input channel of a microfluidic chip shown in figure 1; inputting a mixed aqueous solution containing 6% g/mL of acrylamide monomer, 0.392% g/mL of N, N' -bis (acryloyl) cystamine and 0.6% g/mL of ammonium persulfate into a second flow input channel; 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, after which the microdroplets were demulsified, washed sequentially 1 time with HFE-7500 fluorinated oil containing 20% v/v 1H,1H,2H,2H-Perfluorooctanol, 2 times with 1% v/v Span-80 in n-hexane, 2 times with 0.1% v/v Triton X-100 in TE buffer, and 3 times with 0.1% v/v Tween20 in TE buffer to obtain tightly packed polyacrylamide hydrogel microdroplets (40 μm diameter). The polyacrylamide hydrogel microdroplets can be dissolved under specific conditions after being solidified.
2) Preparing double-layer gel microspheres wrapped with magnetic layers by using a microfluidic technology (the outer layer is polyacrylamide gel wrapped with magnetic nanoparticles, and the inner layer is polyacrylamide gel), specifically, inputting a HFE-7500 fluorinated oil phase containing 0.4% v/v of N, N, N ', N ' -tetramethylethane-1, 2-diamine (TEMED) and 2% g/mL of a surfactant (PEG-PFPE) into a first flow input channel of a microfluidic chip shown in figure 3, inputting a second flow input channel containing 4% g/mL of an acrylamide monomer and 0.4% g/mL of N, N ' -methylenebisacrylamide, 0.3% g/mL ammonium persulfate and polyacrylamide gel solution containing ferroferric oxide magnetic nanoparticles, and inputting the soluble gel microspheres prepared in the first step into a third flow input channel; 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 compact arrangement structure as shown in fig. 4. Placing the collected gel microdroplets at room temperature for 7 hours to gelatinize, after the gelatinization, demulsifying the microdroplets, sequentially washing with HFE-7500 containing 20% v/v 1H,1H,2H,2H-Perfluorooctanol for 1 time, n-hexane containing 1% v/v Span-80 for 2 times, TE buffer containing 0.1% v/v Triton X-100 for 2 times and TE buffer containing 0.1% v/v Tween20 for 3 times to obtain the double-layer gel microspheres with the inner layer of polyacrylamide gel and the outer layer of polyacrylamide gel wrapped with magnetic nanoparticles, as shown in FIG. 8.
3) Soaking the double-layer gel microspheres in Thermopol buffer containing 50mM dithiothreitol, standing for 5 minutes to dissolve the inner core of the polyacrylamide hydrogel, shooting the dissolution process of the inner layer polyacrylamide gel under a microscope, and shooting the dissolution process once every 10 seconds, wherein after about 90 seconds, the inner layer gel is almost completely dissolved, as shown in figure 5, and obtaining hollow polyacrylamide gel microspheres after dissolution, as shown in figure 6, and the cross section of the hollow polyacrylamide gel microspheres is shown in figure 7.
Example 2
A method for preparing magnetic polymer microspheres, the preparation process is shown in fig. 2a-2c, and the method comprises the following steps:
1) firstly, preparing polyacrylamide soluble gel microspheres by using a microfluidic technology, wherein the preparation process is the same as the step 1) of the embodiment 1);
2) preparing a double-layer gel microsphere wrapped with a magnetic layer by using a microfluidic technology (the outer layer is polyethylene glycol gel wrapped with magnetic nano particles, and the inner layer is polyacrylamide gel), specifically, inputting a HFE-7500 fluorinated oil phase containing 0.4% v/v of N, N, N ', N' -tetramethylethane-1, 2-diamine (TEMED) and 2% g/mL of a surfactant (PEG-PFPE) into a first flow input channel of a microfluidic chip shown in figure 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; inputting the soluble gel microspheres prepared in the first step into a third flow input channel; the fourth output channel collects the double-layer micro-droplets wrapped with the magnetic layer, the collected gel micro-droplets are placed at room temperature for 7 hours to be gelatinized, and after the gelatinization, the micro-droplets are demulsified (the demulsification method is the same as that of example 1), so that the double-layer gel microspheres with the inner layers of polyacrylamide gel and the outer layers of polyethylene glycol gel wrapped with magnetic nano-particles are obtained, as shown in fig. 8.
The specific operation process of step 3) is the same as that of step 3) of example 1, and finally the hollow polyethylene glycol gel microspheres are obtained, as shown in fig. 9.
Example 3
A method for preparing magnetic polymer microspheres, the preparation process is shown in fig. 2a-2c, and the method comprises the following steps:
1) firstly, preparing soluble polyacrylamide gel microspheres by using a microfluidic technology, wherein the preparation process is the same as the step 1) of the embodiment 1);
2) preparing a double-layer gel microsphere wrapped with a magnetic layer by using a microfluidic technology (the outer layer is agarose gel wrapped with magnetic nanoparticles, and the inner layer is polyacrylamide gel), and specifically, inputting an HFE-7500 fluorinated oil phase containing 2% g/mL of surfactant (PEG-PFPE) into a first flow input channel of a microfluidic chip shown in figure 3; the second flow input channel inputs agarose solution containing 2% g/mL agarose and ferroferric oxide magnetic nanoparticles (the agarose is completely dissolved at 90 ℃ for 30 minutes); inputting the soluble gel microspheres prepared in the first step into a third flow input channel; and collecting the double-layer micro-droplets wrapped with the magnetic layer by the fourth output channel to obtain the double-layer micro-droplets wrapped with the magnetic layer. The collected gel microdroplets were allowed to stand at 4 ℃ for 1 hour to gel, and after gelling, the microdroplets were demulsified (the method of demulsification was the same as that in example 1). The obtained double-layer gel microspheres with the inner layer of polyacrylamide gel and the outer layer of agarose gel wrapped with magnetic nanoparticles are shown in figure 8.
Step 3) the specific procedure is the same as in step 3) of example 1, and finally the hollow sepharose microspheres are obtained, as shown in FIG. 9.
Example 4
A method for preparing magnetic polymer microspheres, the preparation process is shown in fig. 2a-2c, and the method comprises the following steps:
1) firstly, preparing a soluble agarose gel microsphere by utilizing a microfluidic technology, specifically, inputting an HFE-7500 fluorinated oil phase containing 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 input containing 1% g/mL agarose in agarose aqueous solution (agarose at 90 degrees C30 minutes to make it completely dissolved in water); 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, after which the micro-droplets were demulsified, washed 1 time with HFE-7500 fluorinated oil containing 20% v/v 1H,1H,2H,2H-Perfluorooctanol, 2 times with 1% v/v Span-80 in n-hexane, 2 times with 0.1% v/v Triton X-100 in TE buffer and 3 times with 0.1% v/v Tween20 in TE buffer in this order to obtain tightly packed agarose hydrogel microspheres (diameter 40 μm). The agarose hydrogel microdroplets can be dissolved under specific conditions after being solidified;
2) the micro-fluidic technology is utilized to prepare the double-layer gel microspheres wrapped with the magnetic layer (the outer layer is polyacrylamide gel wrapped with magnetic nano-particles, and the inner layer is agarose gel), and the specific operation process is the same as the step 2 of the embodiment 1. Obtaining the double-layer gel microsphere with the inner layer of the agarose gel and the outer layer of the agarose gel wrapped with the magnetic nanoparticles, as shown in figure 8.
3) Soaking the double-layer gel microspheres prepared in the step 2) in TE buffer solution, standing at 90 ℃ for 30 minutes, dissolving agarose hydrogel cores, and finally obtaining hollow polyacrylamide gel microspheres, as shown in figure 9.
Example 5
A method for preparing magnetic microspheres containing hollow structures, the preparation process is shown in figures 2a-2c, and the method comprises the following steps:
1) firstly, preparing the soluble agarose gel microspheres by using a microfluidic technology, wherein the specific operation process is the same as the step 1) of the embodiment 4;
2) the microfluidic technology is used for preparing the double-layer gel microspheres wrapped with the magnetic layer (the outer layer is polyethylene glycol gel wrapped with the magnetic nanoparticles, and the inner layer is agarose gel), and the specific operation process is the same as the step 2 of the embodiment 2. The obtained bilayer gel microspheres with agarose gel as the inner layer and polyethylene glycol gel wrapped with magnetic nanoparticles as the outer layer are shown in figure 8.
3) The specific operation process is the same as that of step 3) of example 4, and finally the hollow polyethylene glycol gel microspheres are obtained, as shown in fig. 9.
Example 6
A method for preparing magnetic polymer microspheres, the preparation process is shown in fig. 2a-2c, and the method comprises the following steps:
1) firstly, preparing the soluble agarose gel microspheres by using a microfluidic technology, wherein the specific operation process is the same as the step 1) of the embodiment 4;
2) the method is characterized in that a microfluidic technology is utilized to prepare double-layer gel microspheres wrapped with magnetic layers (the outer layer is agarose gel wrapped with magnetic nano-particles, and the inner layer is agarose gel), the specific operation process is the same as the step 2 of the embodiment 3, and the difference of the embodiment 3 is that agarose with low melting point is selected as the agarose. The obtained double-layer gel microspheres with the inner layer of agarose gel and the outer layer of agarose gel wrapped with magnetic nanoparticles are shown in figure 8.
3) The specific procedure was the same as in step 3) of example 4, except that the mixture was left at 60 ℃ for 30min to obtain hollow Sepharose beads as shown in FIG. 9.
In this example, the outer layer was a high melting point agarose gel solution and the inner layer was a low melting point agarose gel solution, and the inner layer agarose gel was dissolved at a lower temperature.
Application example 1
The application of the method for preparing the magnetic polymer microspheres in the PCR amplification of escherichia coli plasmid genome comprises the following steps:
1) firstly, encapsulating an extracted escherichia coli plasmid genome in a single micro-droplet (containing 6% g/mL of acrylamide monomer, 0.392% g/mL of N, N ' -bis (acryloyl) cystamine and 0.6% g/mL of ammonium persulfate) by using a microfluidic technology, and specifically, inputting an HFE-7500 fluorinated oil phase containing 0.4% v/v of N, N, N ', N ' -tetramethylethane-1, 2-diamine (TEMED) and 2% g/mL of a surfactant (PEG-PFPE) into a first flow input channel of a microfluidic chip shown in figure 1; the second flow input channel inputs escherichia coli plasmid genome and mixed aqueous solution containing 6% g/mL of acrylamide monomer, 0.392% g/mL of N, N' -bis (acryloyl) cystamine and 0.6% g/mL of ammonium persulfate; collecting gel micro-droplets capturing escherichia coli plasmid genomes through a third output channel of the microfluidic chip, placing the collected droplets for 7 hours at room temperature to gelatinize, de-emulsifying the micro-droplets, sequentially washing the micro-droplets for 1 time with HFE-7500 containing 20% v/v 1H,1H,2H,2H-Perfluorooctanol, washing the micro-droplets for 2 times with n-hexane containing 1% v/v Span-80, washing the micro-droplets for 2 times with TE buffer containing 0.1% v/v Triton X-100 and washing the micro-droplets for 3 times with TE buffer containing 0.1% v/v Tween20 to obtain the closely-arranged soluble polyacrylamide hydrogel microspheres (the diameter of which is 40 mu m) wrapping the escherichia coli plasmid genomes.
2) The method comprises the following steps of (1) encapsulating soluble polyacrylamide hydrogel microspheres again by using a microfluidic technology, specifically, inputting a HFE-7500 fluorinated oil phase containing 0.4% v/v of N, N, N ', N ' -tetramethylethane-1, 2-diamine (TEMED) and 2% g/mL of a surfactant (PEG-PFPE) into a first flow input channel of a microfluidic chip shown in figure 3, inputting a polyacrylamide gel solution containing 4% g/mL of an acrylamide monomer, 0.4% g/mL of N, N ' -methylenebisacrylamide, 0.3% g/mL of ammonium persulfate and ferroferric oxide into a second flow input channel, and inputting the soluble polyacrylamide hydrogel microspheres prepared in the first step into a third flow input channel; the fourth output channel collects the gel micro-droplets wrapped with the magnetic layer. Placing the collected gel microdroplets at room temperature for 7 hours to gelatinize, after the gelatinization, demulsifying the microdroplets, sequentially washing 1 time with HFE-7500 containing 20% v/v 1H,1H,2H,2H-Perfluorooctanol, 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 the double-layer gel microspheres wrapped with the Escherichia coli plasmid genome.
3) And (3) soaking the double-layer gel microspheres in Thermopol buffer containing 50mM dithiothreitol, standing for 5 minutes, and dissolving the polyacrylamide hydrogel core to obtain the hollow polyacrylamide magnetic microspheres encapsulated with the escherichia coli plasmid genome. And (3) cleaning the dissolved hollow polyacrylamide magnetic microspheres twice by using ribozyme-free water.
4) Specific regions of the plasmid genome of E.coli were amplified using the PCR amplification system shown in Table 1. The PCR reaction system 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 primers, 0.4. mu.L of 10mM dNTP and 1. mu.L of EVA Green (20X). The system first pre-denatures double-stranded DNA at 95 ℃ for 30 seconds, secondly denatures double-stranded DNA at 95 ℃ for 15 seconds, anneals at 58 ℃ for 15 seconds, and extends at 72 ℃ for 3 minutes, wherein the operations of denaturation, annealing, and extension are performed for 35 cycles, and finally complete extension is performed 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 sizes of the amplified product fragments obtained by PCR amplification by using the ten pairs of primers are respectively 150bp, 225bp, 338bp, 434bp, 547bp, 620bp, 749bp, 866bp, 968bp and 1187 bp.
Taking a photograph of the hollow polyacrylamide gel microsphere after PCR amplification of primer 9 by using a fluorescence microscope, wherein a genome cluster with fluorescence can be seen, as shown in FIG. 10; after the amplification product is magnetically washed with ribozyme-free water (the gel microspheres are absorbed by a magnetic column or other magnetic objects for washing), background color-free fluorescent genome clusters can be seen, as shown in FIG. 11 (both graphs are the PCR amplification result of primer 9). The hollow gel microspheres containing the amplification products of the above ten pairs of primers were washed and subjected to agarose gel electrophoresis, respectively, and the results are shown in fig. 12, indicating that the sizes of the PCR amplification product fragments are consistent with those expected. The hollow gel microspheres containing the amplification products of the above ten pairs of primers were subjected to fluorescence photography, as shown in FIG. 13. Then, image J software is used for carrying out fluorescence quantitative analysis on the successfully amplified hollow microspheres in the picture, and a violin graph shown in figure 14 is drawn. The results show that when the DNA molecular fragment is larger than 547bp, the hollow gel microspheres can realize complete physical sealing of DNA small molecules.
TABLE 1 PCR amplification System
Application example 2
The application of the method for preparing the magnetic polymer microspheres in single cell whole genome amplification (MDA, multiple displacement amplification) of single escherichia coli cells comprises the following steps:
1) firstly, single escherichia coli cells are encapsulated in single soluble micro-droplets (containing 6% g/mL of acrylamide monomer, 0.392% g/mL of N, N' -bis (acryloyl) cystamine and 0.6% g/mL of ammonium persulfate) by using a microfluidic technology, and after the soluble micro-droplets are solidified, demulsification operation is carried out to obtain soluble polyacrylamide gel microspheres with the size of 40 micrometers, wherein the specific operation steps are shown in example 1.
2) The method comprises the following steps of (1) utilizing a microfluidic technology to encapsulate soluble polyacrylamide hydrogel microspheres again, specifically, inputting a HFE-7500 fluorinated oil phase containing 0.4% v/v of N, N, N ', N ' -tetramethylethane-1, 2-diamine (TEMED) and 2% g/mL of a surfactant (PEG-PFPE) into a first flow input channel of a microfluidic chip shown in figure 3, inputting a polyacrylamide gel solution containing 4% g/mL of an acrylamide monomer, 0.4% g/mL of N, N ' -methylenebisacrylamide, 0.3% g/mL of ammonium persulfate and ferroferric oxide into a second flow input channel, and inputting the soluble polyacrylamide hydrogel microspheres prepared in the first step into a third flow input channel; the fourth output channel collects the gel micro-droplets wrapped with the magnetic layer. Placing the collected gel microdroplets at room temperature for 7 hours to gelatinize, after the gelatinization, demulsifying the microdroplets, sequentially washing 1 time with HFE-7500 containing 20% v/v 1H,1H,2H,2H-Perfluorooctanol, 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 the double-layer gel microspheres coated with single escherichia coli cells.
3) And (3) soaking the double-layer gel microspheres in Thermopol buffer containing 50mM dithiothreitol, standing for 5 minutes, and dissolving the polyacrylamide hydrogel core to obtain the hollow polyacrylamide magnetic microspheres encapsulated with single escherichia coli cells. The hollow polyacrylamide magnetic microspheres are washed twice by ribozyme-free water.
4) Individual E.coli cells were subjected to cell lysis to sufficiently expose their genomes, and specifically, 30. mu.L of hollow polyacrylamide magnetic microspheres were taken, and 30. mu.L of an alkaline lysis solution (400mM KOH, 10mM EDTA, and 100mM DTT) was added to react at 65 ℃ for 15 minutes. And (3) cleaning the cracked hollow polyacrylamide magnetic microspheres twice by using ribozyme-free water. Obtaining the hollow polyacrylamide magnetic microspheres containing single escherichia coli genome.
5) Whole genome amplification of a single E.coli cell genome was performed using the MDA amplification system as shown in Table 2. The MDA reaction system was 50. mu.L containing 2.5. mu.L Phi29DNA polymerase, 5. mu.L Phi29DNA polymerase buffer, 12.5. mu.L random primers (10. mu.M), 5. mu.L dNTPs (10mM) and 25. mu.L hollow polyacrylamide gel microspheres containing a single E.coli cell genome. The MDA program is: reacting at 30 ℃ for 8 h. The amplification product is washed magnetically with ribozyme-free water (washing with magnetic column or other magnetic substance adsorbing gel microspheres). After magnetic cleaning, 9.5 μ L of the hollow polyacrylamide magnetic microspheres after whole genome amplification are added with 0.5 μ L of EVA Green (20 ×), and after uniform mixing, fluorescent microscope photographing is performed, and genome clusters without background color and with Green fluorescence can be seen, as shown in fig. 15.
TABLE 2 MDA amplification System
| Components of MDA amplification systems | 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 escherichia coli cell genome | 25μL |
| Total of | 50μL |
Application example 3
The method for preparing the magnetic polymer microspheres is applied to single cell whole genome amplification (MDA, multiple displacement amplification) and PCR fluorescence specificity screening, and comprises the following steps:
1) encapsulating a single escherichia coli cell into a magnetic polyacrylamide gel microsphere with a hollow structure, performing alkaline lysis on the single escherichia coli, performing MDA whole genome amplification, and finally obtaining a hollow polyacrylamide magnetic microsphere (a hollow polyacrylamide magnetic microsphere with an escherichia coli whole genome amplification product) after the MDA amplification, wherein the specific operation is as shown in application example 2.
2) The PCR amplification system shown in Table 3 was used to perform fluorescence PCR specificity screening on the hollow magnetic gel microspheres after MDA amplification. The PCR reaction system was 20. mu.L, which contained 7.6. mu.L of hollow polyacrylamide microspheres containing the entire 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 dNTP. The system first pre-denatures double-stranded DNA at 95 ℃ for 30 seconds, secondly denatures double-stranded DNA at 95 ℃ for 15 seconds, anneals at 55 ℃ for 30 seconds, and extends at 72 ℃ for 3 minutes, wherein the operations of denaturation, annealing, and extension are performed for 40 cycles, and finally complete extension is performed at 72 ℃ for 5 minutes. The primer sequence is 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' are provided. The amplification product is washed magnetically with ribozyme-free water (washing with magnetic column or other magnetic substance adsorbing gel microspheres). After magnetic cleaning, 9.5 μ L of the hollow polyacrylamide gel microspheres subjected to PCR amplification is added with 0.5 μ L of EVA Green fluorescent dye (20 ×), and after uniform mixing, fluorescent microscope photographing is performed, so that genome clusters without background color and with Green fluorescence can be seen, and meanwhile, part of the hollow magnetic gel microspheres can specifically show red fluorescence, as shown in FIG. 16.
TABLE 3 PCR amplification System
| Components of PCR amplification System | Dosage of |
| DNA polymerase | 0.4μL |
| DNA polymerase buffer (2X) | 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 |
| Total of | 20μL |
Application example 4
The invention discloses 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) encapsulating a single escherichia coli cell into a magnetic polyacrylamide gel microsphere with a hollow structure, performing alkaline lysis on the single escherichia coli, performing MDA whole genome amplification, and finally obtaining a hollow polyacrylamide magnetic microsphere with an MDA amplification product (the hollow polyacrylamide magnetic microsphere with the escherichia coli whole genome amplification product), wherein the specific operation is as shown in application example 2.
2) And (4) 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 entire 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 program is: (i) 95 ℃ for 5 minutes; (ii) 16h at 37 ℃. The sequence of the CY 3-labeled fluorescent probe was (SEQ ID NO: 23): 5 '-CY 3-AGG GCT TCA AGC GCA GCA CG-3'. And (4) carrying out magnetic cleaning on the product after the FISH by using ribozyme-free water (cleaning by using a magnetic column or other magnetic objects to adsorb gel microspheres). After magnetic cleaning, 9.5 μ L of FISH-added hollow magnetic polyacrylamide gel microspheres are added with 0.5 μ L of EVA Green (20 ×), mixed uniformly and photographed by a fluorescence microscope, so that background color-free genome clusters with Green fluorescence can be seen, and meanwhile, part of the hollow magnetic gel microspheres can specifically show red fluorescence, as shown in fig. 17.
Application example 5
The invention discloses an application of a method for preparing magnetic polymer microspheres in CFPS (cell-free expression system), which comprises the following steps:
1) firstly, a plasmid capable of expressing Green Fluorescent Protein (GFP) and a cell extract are encapsulated in a soluble micro-droplet (containing 6% g/mL of acrylamide monomer, 0.392% g/mL of N, N' -bis (acryloyl) cystamine and 0.6% g/mL of ammonium persulfate) by utilizing a microfluidic technology, and after the soluble micro-droplet is solidified, a demulsification operation is carried out to obtain a soluble polyacrylamide gel microsphere with the size of 40 mu m, wherein the specific operation steps are shown in example 1.
2) Re-encapsulating the soluble polyacrylamide gel microspheres by using a microfluidic technology, specifically, inputting a HFE-7500 fluorinated oil phase containing 0.4% v/v of N, N, N ', N ' -tetramethylethane-1, 2-diamine (TEMED) and 2% g/mL of a surfactant (PEG-PFPE) into a first flow input channel of a microfluidic chip shown in FIG. 3, inputting a polyacrylamide gel solution containing 4% g/mL of an acrylamide monomer, 0.4% g/mL of N, N ' -methylenebisacrylamide, 0.3% g/mL of ammonium persulfate and ferroferric oxide into a second flow input channel, and inputting the soluble polyacrylamide gel microspheres prepared in the first step into a third flow input channel; the fourth output channel collects the gel micro-droplets wrapped with the magnetic layer. And (2) placing the collected gel micro-droplets at room temperature for 3 hours to gelatinize, after the gel is gelatinized, demulsifying the micro-droplets, sequentially washing 1 time with HFE-7500 containing 20% v/v 1H,1H,2H,2H-Perfluorooctanol, washing 2 times with n-hexane containing 1% v/v Span-80, washing 2 times with TE buffer containing 0.1% v/v Triton X-100 and washing 3 times with TE buffer containing 0.1% v/v Tween20 to obtain the magnetic double-layer gel microspheres coated with plasmids and cell extracts capable of expressing green fluorescent protein.
3) And (3) soaking the magnetic double-layer gel microspheres in Thermopol buffer containing 50mM dithiothreitol, standing for 5 minutes, and dissolving polyacrylamide gel cores to obtain hollow magnetic polyacrylamide gel microspheres. And (3) washing the dissolved hollow polyacrylamide microspheres twice by using ribozyme-free water.
4) Adding 40 mu L of hollow magnetic gel microspheres containing plasmids capable of expressing green fluorescent protein and cell extracts into 45 mu L of cell-free expression system (without plasmids), expressing for 6 hours at 30 ℃, and detecting the fluorescence intensity of the expressed green fluorescent protein by using a microplate reader. And (3) washing the expressed hollow magnetic gel microspheres for three times by using ribozyme-free water, adding a new 45-microliter cell-free expression system (without plasmids), continuing to express for 6 hours at 30 ℃, and then detecting the fluorescence intensity of the expressed green fluorescent protein by using an enzyme-labeling instrument. As a result, as shown in FIG. 18, the hollow magnetic gel microspheres were used to recover and re-express the plasmid many times.
Application example 6
The invention discloses an application of a method for preparing magnetic polymer microspheres in construction of lactam specific response artificial biosensing cells, which comprises the following steps:
1) as shown in fig. 19, the protein of ChnR expressed by the engineered strain can specifically bind to a small lactam (lactam) molecule, activate the Pb promoter, and thereby express mCherry fluorescent protein, so that the lactam can be specifically detected. The engineered Escherichia coli strain and 2% g/mL agarose solution are mixed together in a volume of 1:1 to finally obtain 1% g/mL agarose bacterial solution. Inputting a HFE-7500 fluorinated oil phase containing 2% g/mL of a surfactant (PEG-PFPE) into a first flow input channel of the microfluidic chip shown in FIG. 1; the second flow input channel inputs agarose bacterial 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 left at 4 ℃ for 30 minutes to gel, after which the micro-droplets were demulsified, washed 1 time with HFE-7500 fluorinated oil containing 20% v/v 1H,1H,2H,2H-Perfluorooctanol, 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, in this order, to obtain densely arranged Sepharose microspheres (diameter 40 μm).
2) The double-layer gel microspheres wrapped with the magnetic layer are prepared by a microfluidic technology. Inputting a HFE-7500 fluorinated oil phase containing 2% g/mL of a surfactant (PEG-PFPE) into a first flow input channel of the microfluidic chip shown in FIG. 3; the second flow input channel inputs agarose solution containing 2% g/mL of agarose and ferroferric oxide magnetic nanoparticles; inputting the agarose gel microspheres containing the engineered strain prepared in the first step into a third flow input channel; the fourth output channel collects the 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. And standing the collected gel micro-droplets at 4 ℃ for 30 minutes to form gel, and after the gel is formed, demulsifying the micro-droplets. The demulsification method comprises washing sequentially with HFE-7500 fluorinated oil containing 20% v/v 1H,1H,2H,2H-Perfluorooctanol 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. Finally obtaining the magnetic double-layer agarose gel microspheres wrapped with the agarose gel microspheres.
3) The magnetic double-layer agarose gel microspheres (50. mu.L) containing the engineered strain obtained in step 2 were added to 1mL of LB medium (containing 25. mu.g/mL chloramphenicol resistance, 10. mu.L of 0.2% arabinose and 10. mu.L of 10M valerolactam) and cultured at 30 ℃ for 16 hours. Then washing off a small amount of bacteria escaping from the double-layer gel microspheres to the environment by using a magnetic cleaning method, and finally obtaining the double-layer agarose gel microspheres with the inner layers full of escherichia coli flora, as shown in figure 20; as a control, the monolayer agarose gel microspheres (50 μ L) containing the engineered strain obtained in step 1) are added into the same culture solution and subjected to the same culture step and washing step to finally obtain the monolayer agarose gel microspheres full of Escherichia coli flora, and as shown in FIG. 21, part of Escherichia coli can diffuse outwards and escape to the outside. In the process of double-layer microsphere bacterial culture, the fluorescence intensity in the hollow magnetic gel microspheres is detected every 4 hours, and the influence of the culture time on the response behavior of the valerolactam is analyzed, as shown in fig. 22. As can be seen from the trend lines, the fluorescence signal of valerolactam increases exponentially before entering the plateau phase, and no longer changes significantly with time. In addition, the magnetic double-layered 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 of chloramphenicol resistance and 10. mu.L of 0.2% arabinose) containing varying concentrations of valerolactam (0mM, 5mM, 10mM, 20mM, 50mM, and 100mM), respectively, and incubated at 30 ℃ for 16 hours. Then, the fluorescence intensity of the bacterial strains in the hollow magnetic gel microspheres under the concentration of 5 kinds of valerolactams is respectively detected under a fluorescence microscope, and finally, a valerolactam biological response dose-response graph is obtained through analysis, as shown in fig. 23, a trend line basically shows the linear relationship of valerolactam biological response dose-response.
The above embodiments are only preferred embodiments of the present invention, and are not intended to limit the present invention in any way and substantially, it should be noted that those skilled in the art may make several modifications and additions without departing from the scope of the present invention, and these modifications and additions should also be regarded as the protection scope of the present invention.
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Claims (15)
1. A method for preparing magnetic polymeric microspheres, comprising the steps of:
step 1: by utilizing a droplet generation technology, carrying out chemical crosslinking polymerization on raw materials comprising monomers and magnetic nanoparticles or carrying out physical crosslinking on the raw materials comprising polymers and magnetic nanoparticles to prepare a multi-layer polymer micro-droplet with the magnetic nanoparticles wrapped on the outer layer;
step 2: and the multilayer polymer micro-droplets are solidified or semi-solidified and demulsified to obtain the multilayer polymer microspheres wrapped with the magnetic shell layer.
2. The method for preparing magnetic polymeric microspheres according to claim 1, wherein the magnetic polymeric microspheres are subjected to step 3: and (3) dissolving at least one layer of inner layer polymers of the multilayer polymer microspheres prepared in the step (2) to obtain the magnetic polymer microspheres containing the hollow cavities.
3. The method of claim 2, wherein the multi-layered micro-droplets produced in step 1 are bi-layered micro-droplets, and correspondingly, the multi-layered micro-droplets produced in step 2 are bi-layered micro-spheres.
4. The method for preparing magnetic polymer microspheres according to claim 3, wherein the specific process of the step 1 comprises:
step 1.1: by utilizing a high-flux microfluidic technology, carrying out chemical crosslinking polymerization on a raw material containing a monomer or physical crosslinking on a raw material containing a polymer to prepare micro-droplets, and solidifying or semi-solidifying and demulsifying the micro-droplets to obtain polymer microspheres;
step 1.2: secondly, preparing a magnetic shell layer on the outer surface of the polymer microsphere obtained in the step 1.1 by physically or chemically crosslinking polymer dispersion liquid of magnetic nano particles by utilizing a high-flux microfluidic technology to prepare a double-layer micro-droplet with the outer layer being a polymer wrapped with the magnetic nano particles and the inner layer being a polymer; the outer layer polymer and the inner layer polymer have different physical and/or chemical properties.
5. The method for preparing magnetic polymer microspheres according to claim 4, 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.
6. The method for preparing magnetic polymer microspheres according to claim 5, wherein the polyacrylamide gel microspheres are prepared by forming a water-in-oil system by 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 (acryloyl) cysteamine cross-linking agent, performing chemical cross-linking polymerization to prepare polyacrylamide gel micro-droplets, and performing curing or semi-curing and emulsion breaking on the polyacrylamide gel micro-droplets; the agarose gel microspheres are prepared by forming a water-in-oil system by an oil phase containing a surfactant and a water phase containing agarose, preparing agarose gel micro-droplets through physical crosslinking, and solidifying or semi-solidifying and demulsifying the agarose gel micro-droplets; the sodium alginate gel microspheres are prepared by forming a water-in-oil system by 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 microspheres are prepared by forming a water-in-oil system by an oil phase containing a surfactant and a water phase only containing gelatin or a water phase containing gelatin and one or more of the following crosslinking agents formaldehyde, glutaric acid, EDC, genipin, methacrylic anhydride and polyethylene glycol diacrylate, carrying out physical crosslinking or chemical crosslinking to prepare gelatin micro-droplets, and carrying out solidification or semi-solidification and emulsion breaking on the gelatin micro-droplets.
7. The method of claim 6, wherein the surfactant comprises one or more of PEG-PFPE, castor oil polyoxyl ester, polyoxyethylene 40 hydrogenated castor oil, poloxamer, Krytox, sodium lauryl sulfate, and Janus nanoparticles; the oil phase comprises one or more of HFE-7500 fluorinated oil phase, Squalane oil phase, silicone oil phase and mineral oil phase.
8. The method for preparing magnetic polymer microspheres according to claim 7, wherein the polymer dispersion of magnetic nanoparticles in step 1.2 comprises any one of polyacrylamide gel solution, polyethylene glycol gel solution, agarose gel solution, sodium alginate gel solution and gelatin gel solution of magnetic nanoparticles comprising any one or more of monometallic magnetic nanoparticles, magnetic iron oxide nanoparticles, bimetallic magnetic nanoparticles and alloy magnetic nanoparticles.
9. The method for preparing magnetic polymeric microspheres of claim 8, wherein the bilayer microdroplets prepared in step 1.2 comprise any one of the following bilayer microdroplets:
double-layer micro-droplet A: the outer layer is polyacrylamide gel wrapped with magnetic nano particles, and the inner layer is double-layer micro-droplets of the polyacrylamide gel;
double-layer micro-droplet B: the outer layer is polyethylene glycol gel wrapped with magnetic nano particles, and the inner layer is double-layer micro-droplets of polyacrylamide gel;
double-layer micro-droplet C: the outer layer is agarose gel wrapped with magnetic nano particles, and the inner layer is double-layer micro-droplets of polyacrylamide gel;
double-layer micro-droplet D: the outer layer is sodium alginate gel wrapped with magnetic nano particles, and the inner layer is double-layer micro-droplets of polyacrylamide gel;
double-layer micro-droplets E: the outer layer is gelatin gel wrapped with magnetic nano particles, and the inner layer is double-layer micro-droplets of polyacrylamide gel;
double-layer micro-droplets F: the outer layer is polyacrylamide gel wrapped with magnetic nano particles, and the inner layer is double-layer micro-droplets of agarose gel;
double-layer micro-droplet G: the outer layer is polyethylene glycol gel wrapped with magnetic nano particles, and the inner layer is double-layer micro-droplets of agarose gel;
double-layer micro-droplets H: the outer layer is agarose gel wrapped with magnetic nano particles, and the inner layer is double-layer micro-droplets of the agarose gel;
double-layer micro-droplet I: the outer layer is sodium alginate gel wrapped with magnetic nano particles, and the inner layer is double-layer micro-droplets of agarose gel;
double-layer micro-droplet J: the outer layer is gelatin gel wrapped with magnetic nano particles, and the inner layer is double-layer micro-droplets of agarose gel;
double-layer micro-droplet K: the outer layer is a double-layer micro-droplet which is wrapped with magnetic nano-particles and is made of sodium alginate gel;
double-layer micro-droplets L: the outer layer is a polyethylene glycol gel wrapped with magnetic nano particles, and the inner layer is a double-layer micro-droplet of sodium alginate gel;
double-layer micro-droplet M: the outer layer is agarose gel wrapped with magnetic nano particles, and the inner layer is double-layer micro-droplets of sodium alginate gel;
double-layer micro-droplet N: the outer layer is sodium alginate gel wrapped with magnetic nano particles, and the inner layer is double-layer micro-droplets of the sodium alginate gel;
double-layer micro-droplet O: the outer layer is gelatin gel wrapped with magnetic nano particles, and the inner layer is double-layer micro-droplets of sodium alginate gel;
double-layer micro-droplet P: the outer layer is a double-layer micro-droplet which is wrapped with magnetic nano-particles and the inner layer is gelatin gel;
double-layer micro-droplet Q: the outer layer is polyethylene glycol gel wrapped with magnetic nano particles, and the inner layer is double-layer micro-droplets of gelatin gel;
double-layer micro-droplet R: the outer layer is agarose gel wrapped with magnetic nano particles, and the inner layer is double-layer micro-droplets of gelatin gel;
double-layer micro-droplet S: the outer layer is sodium alginate gel wrapped 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 wrapped with magnetic nanoparticles, and the inner layer is double-layer micro-droplets of gelatin gel.
10. The method of claim 9, wherein the bilayer microdroplets A, F, K and P are formed by chemical cross-linking polymerization of an oil phase containing TEMED catalyst and surfactant, a polyacrylamide gel solution containing acrylamide monomer, ammonium persulfate initiator, N' -methylenebisacrylamide crosslinker and magnetic nanoparticles, and a water-in-oil encapsulated system containing the polymer microspheres obtained in step 1.1 to obtain corresponding bilayer microdroplets; the double-layer micro-droplets B, G, L and Q are water-in-oil ball-wrapped systems formed by oil phase containing TEMED catalyst and surfactant, polyethylene glycol gel solution containing polyethylene glycol, ammonium persulfate initiator and magnetic nanoparticles and polymer microspheres prepared in the step 1.1, and are subjected to chemical cross-linking polymerization to prepare corresponding double-layer micro-droplets; the double-layer micro-droplets C, H, M and R are water-in-oil ball-coated systems formed by oil phase containing surfactant, agarose gel solution containing agarose and magnetic nanoparticles and polymer microspheres prepared in the step 1.1, and are physically cross-linked to prepare corresponding double-layer micro-droplets; the double-layer micro-droplets D, I, N and S are water-in-oil ball-wrapped systems 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 by physical crosslinking; the bilayer microdroplets E, J, O and T are prepared by forming a water-in-oil ball-in-oil system by using an oil phase containing a surfactant, a gelatin gel solution containing gelatin and magnetic nanoparticles only or a gelatin gel solution containing gelatin and magnetic nanoparticles and one or more of formaldehyde, glutaric acid, EDC, genipin, methacrylic anhydride and polyethylene glycol diacrylate as crosslinking agents and polymer microspheres prepared in the step 1.1, and carrying out physical crosslinking or chemical crosslinking to prepare corresponding bilayer microdroplets. .
11. The method for preparing magnetic polymer microspheres according to claim 10, wherein the curing or semi-curing method in step 1.1 and step 2 comprises any one of standing, UV irradiation, heating, PH change and ion concentration change; the demulsification method comprises the following steps: washing sequentially with HFE-7500 containing 20% v/v 1H,1H,2H,2H-Perfluorooctanol 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.
12. The method for preparing magnetic polymer microspheres according to claim 11, wherein the dissolving of the inner layer polymer in step 3 comprises any one of UV irradiation, heating, PH change, ion concentration change and dithiothreitol solution.
13. Use of the method of any one of claims 1 to 12 for the preparation of magnetic polymeric microspheres for biochemical reactions, biochemical analysis and culture of microorganisms.
14. Use according to claim 13, characterized in that it comprises in particular the following steps:
step 1: preparing micro-droplets encapsulating substrates by using a droplet generation technology;
step 2: preparing multilayer polymer microspheres wrapped with a magnetic shell layer;
and step 3: directly adding a reaction reagent to carry out biochemical reaction, or dissolving at least one layer of inner layer polymers of the multilayer polymer microspheres to form a hollow cavity, and then adding the reaction reagent to carry out biochemical reaction.
15. The use of claim 14, wherein the substrate encapsulated in step 1 comprises at least one of a nucleic acid, a protein, a cell, and a microorganism.
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