WO2023274252A1 - 一种聚合物修饰的磁性纳米材料、其制备方法及应用 - Google Patents

一种聚合物修饰的磁性纳米材料、其制备方法及应用 Download PDF

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WO2023274252A1
WO2023274252A1 PCT/CN2022/102012 CN2022102012W WO2023274252A1 WO 2023274252 A1 WO2023274252 A1 WO 2023274252A1 CN 2022102012 W CN2022102012 W CN 2022102012W WO 2023274252 A1 WO2023274252 A1 WO 2023274252A1
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magnetic
polymer
modified
nanomaterial
solvent
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French (fr)
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陈炳地
乐文俊
崔征
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Tongji University
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Tongji University
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Definitions

  • the invention relates to a polymer-modified magnetic nanometer material, its preparation method and application.
  • Circulating tumor cells are widely considered to be a general term for various tumor cells that come off from the tumor site of patients with solid tumors and enter the blood circulation system of patients, and are also generally considered to be the main factor leading to cancer metastasis. Metastasis is the most fundamental cause of patient death and an important factor for postoperative recurrence. Many studies have proved that surgery, chemotherapy and other treatment methods are also important factors that cause tumor cells to detach from the lesion into the blood and form circulating tumor cells. Circulating tumor cells are considered to be the most potential multifunctional biomarker. Circulating tumor cells have been found in many different types of cancers such as breast cancer, lung cancer, colorectal cancer and even prostate cancer. The detection of circulating tumor cells, Counting and correlation analysis are of great significance for the judgment of patients' conditions, and are expected to be applied to early detection of tumors, adjuvant therapy, efficacy evaluation, and prognosis judgment.
  • the reported methods for CTC enrichment mainly include physical methods and biological methods.
  • the cell filter enriches CTC, allowing small blood cells to pass through to intercept large tumor cells; based on antibody recognition of cell surface proteins, such as using epithelial cell adhesion molecule (EpCAM) to capture cancerous epithelial cancer cells.
  • EpCAM epithelial cell adhesion molecule
  • these methods are not based on the unique properties of tumor cells to capture, there are false positives or false negatives in the detection, and they are not broad-spectrum, so they cannot be widely used.
  • Our previous research found that due to the large amount of lactic acid produced by the glycolysis of tumor cells, the surface of tumor cells has a large amount of negative charge, while normal cells are electrically neutral or slightly positively charged. Therefore, based on the unique charge difference between tumor cells and normal cells, efficient and selective enrichment of CTCs can be achieved with a broad spectrum. Constructing high-performance positively charged nanomaterials is the key to trap negatively charged CTCs.
  • iron ferric oxide (Fe 3 O 4 ) magnetic nanoparticles have received extensive attention and research in the fields of biotechnology and medicine because of their special structure and excellent performance.
  • Such materials are usually based on magnetic nanoparticles prepared by fairly mature methods, coated with inorganic materials such as silica or other organic polymer materials on the surface, and then further reacted or surface modified to prepare ferromagnetic polysaccharides.
  • Functional composite nanoparticles This kind of nanomaterials is usually easy to control in terms of material particle size, magnetic strength, etc., has good biocompatibility and stability, and is easy to modify, which greatly expands its application range.
  • biological magnetic separation, magnetic hyperthermia, magnetic resonance imaging and many other fields have research applications.
  • the development of simple, mass-preparable, biocompatible, and positively charged surface modification methods can provide high-quality nanoprobes for CTC enrichment, detection, and treatment guidance. Needle.
  • the nanomaterials reported in the prior art have deficiencies such as insufficient polymer content, poor stability, and long response time.
  • the technical problem to be solved by the present invention is to overcome defects such as insufficient polymer content, poor stability, and long response time of magnetic materials (positively charged magnetic nanoparticles (PCMNs)) in the prior art, and provide a Polymer-modified magnetic nanomaterials, their preparation methods and applications.
  • the polymer-modified magnetic nanomaterial of the present invention has good stability and fast response time, and can achieve high selectivity and high repeatability for glycosylated proteins, polypeptides, nucleic acids, circulating tumor cells, and exosomes from complex samples High-throughput and high-throughput enrichment; it can be applied to the preparation of in vivo fluorescence and magnetic resonance dual-modal imaging contrast agents or photothermal therapeutic agents for cancer treatment.
  • the present invention solves the above-mentioned technical problems through the following technical solutions.
  • the invention provides a polymer-modified magnetic nanomaterial, which includes the following structure:
  • the polymer is a cationic polymer; the polymer is coated on the surface of the magnetic nanoparticle (that is, the outer layer of the shell, referred to as the coating), forming a positively charged polymer-modified magnetic nanomaterials;
  • the magnetic nanomaterial is a core-shell structure, the core is a magnetic nanoparticle (core), and the shell is a modified layer; the modified layer is attached or coated on the magnetic nanoparticle surface, forming a modified layer of composite magnetic nanoparticles;
  • the mass ratio of the polymer to the magnetic nanomaterial is 1:10-20:1.
  • the mass ratio of the polymer to the magnetic nanomaterial may be 1:5 to 3:1; for example, 1:3.
  • the potential of the polymer-modified magnetic nanomaterial may be +5 to +60mV, such as +10 to +50mV, preferably +20 to +40mV (also such as +35mV).
  • the magnetic nanomaterial is a negatively charged magnetic nanomaterial, for example, its potential can be -10 to -60mV; for example -20 to -40mV.
  • the particle size of the polymer-modified magnetic nanomaterial may be 10nm to 600nm; for example, the particle size is 300nm to 500nm, and for example, 350nm to 400nm.
  • the particle size of the magnetic nanomaterial may be 5nm to 500nm; for example, 300nm to 350nm.
  • the thickness of the shell may be 1 nm to 100 nm, such as 40 nm to 60 nm.
  • the particle size of the magnetic nanoparticles may be 5nm to 500nm; for example, 250nm to 300nm.
  • said polymer is a (dendritic) dendritic polymer.
  • the polymer may have a weight average molecular weight MW between 2,000 and 300,000.
  • the polymer is a conventional cationic polymer in the art; for example, polyethyleneimine (PEI, Polyethyleneimine), chitosan ( ⁇ -chitosan) and polypyrrole one or more of .
  • PEI polyethyleneimine
  • chitosan ⁇ -chitosan
  • polypyrrole one or more of .
  • the magnetic nanoparticles can be conventional magnetic nanoparticles in the art, such as oxide magnetic nanoparticles (also such as Fe 3 O 4 , ⁇ -Fe 2 O 3 ), magnetic metal nanoparticles, magnetic One or more of sulfide nanoparticles and magnetic composite particles; another example is magnetic Fe 3 O 4 nanoparticles (hereinafter referred to as Fe 3 O 4 ).
  • the magnetic nanoparticles can be prepared by conventional methods in the art, such as solvothermal method, co-precipitation method and the like.
  • the magnetic nano-particles enable the polymer-modified magnetic nano-material to have magnetism, and then move under the action of a magnet, which can be used as a probe.
  • the magnetic nanomaterials can be conventional magnetic nanomaterials in the art, wherein the modified layer is wrapped on the surface of the magnetic nanoparticles to form a composite magnetic nanomaterial with a core-shell structure ;
  • the shell (layer) formed by the modified layer can prevent its agglomeration, prevent it from being destroyed, and can also carry out surface functionalization on it.
  • the material of the modified layer can be conventional organic and/or inorganic modified layer materials in the field; for example, silicon dioxide or labeled fluorescent and/or surfactant modified silica; such as silica or fluorescently labeled silica.
  • the magnetic nanomaterials may be silicon dioxide (SiO 2 ) composite magnetic nanoparticles, or labeled fluorescent and/or surfactant-modified silicon dioxide composite magnetic nanoparticles.
  • the magnetic nanoparticles composited with a silica modified layer are silica modified Layer composite magnetic Fe 3 O 4 nanoparticles (hereinafter referred to as Fe 3 O 4 @SiO 2 , ferric oxide/silicon dioxide composite microspheres).
  • the surface of the modified layer (such as the silica modified layer) contains modified amino groups, which in turn makes it capable of reacting with further modified (modified) substances.
  • the modification can use conventional surface modifiers in the art; for example, by modifying the amino group on the surface of the silica, so that it has the basis of being able to perform amide reaction with the fluorescent dye having a carboxyl group.
  • the fluorescent dye is bonded to the silica-modified layer, for example, through amide reaction.
  • the silica layer is modified by modifying the surface of the silica layer with an amino group by a surface chemical modifier.
  • the surface chemical modifier can be a conventional surface modifier capable of amino-modifying the surface of the silica-composite magnetic nanoparticles in the art; for example, ammonia water and/or APTES (3-aminopropyltriethoxysilane); Another example is ammonia water.
  • the mass ratio of the modified layer to the magnetic nanoparticles may be 50:1 ⁇ 1:10; for example, 10:1.
  • the polymer-modified magnetic nanomaterial is stable for 2 years.
  • the response time of the polymer-modified magnetic nanomaterial is 3S to 2min.
  • the magnetic nanomaterial is a silica-composite magnetic nanoparticle labeled with fluorescence
  • the fluorescent dye (or fluorescent label) in the silica-composite magnetic nanoparticle labeled with fluorescence substance) can be a conventional fluorescent dye in this type of material in the art, for example, a fluorescent dye with a carboxyl group or capable of amide reaction with an amino group, such as fluorescein isothiocyanate (fluorescenceisothiocyanate, FITC) and/or rhodamine dyes, and / or its modified substance;
  • the fluorescent dye can be fluorescein isothiocyanate, rhodamine B, rhodamine B isothiocyanate (Rhodamine B 5-isothiocyanate, RBITC) and tetramethyl rhodamine isothiocyanate One or more of tetramethylrhodamineisothiocyanate (TRITC).
  • the modification can be APS-modified fluorescein isothiocyanate and/or APS-modified rhodamine dyes.
  • the APS may be 3-aminopropyltriethoxysilane (APTES) and/or 3-aminopropyltrimethylsilane (APTMS)).
  • APTES 3-aminopropyltriethoxysilane
  • APITMS 3-aminopropyltrimethylsilane
  • the FITC modified substance can be APS-FITC (or called FITC-APS/APS modified FITC), and for example, the fluorescent dye is APS-FITC.
  • the fluorescent dyes (or fluorescent markers) in the fluorescent-labeled silica-composite magnetic nanoparticles are modified on the surface of the silica-composite nanoparticles in a conventional manner in this field to form fluorescent-labeled two Silica-composite magnetic nanoparticles; for example, attached to the surface of the silica-composite magnetic nanoparticles via linkages such as amide bonds as described above.
  • the fluorescent-labeled silicon dioxide composite magnetic nanoparticle is APS-FITC-labeled Fe 3 O 4 @SiO 2 .
  • the magnetic nanoparticles compounded by the silica modified layer are magnetic Fe 3 O 4 nanoparticles compounded by the silica modified layer (hereinafter referred to as Fe 3 O 4 @SiO 2 , ferroferric oxide/silicon dioxide composite Microspheres)
  • the magnetic nanomaterial may be a surfactant-modified silica composite magnetic nanoparticle.
  • Described surfactant can comprise sodium acetate, trisodium citrate, chitosan, polyvinylpyrrolidone, polyethylene terephthalate, stearic acid, gum arabic, hydroxypropyl methylcellulose, seaweed Sodium lauryl sulfate, sodium dodecyl sulfate, sodium dodecylbenzenesulfonate, polyvinyl alcohol, long-chain fatty acid, starch and dodecyl mercaptan, or a combination of two or more.
  • the surfactant modification for example, the agglomeration of the formed nanoparticles can be avoided, so as to achieve the control of the particle size of the polymer-modified magnetic nanomaterial.
  • the magnetic nanomaterial is a composite magnetic nanoparticle (such as APS-FITC-labeled Fe 3 O 4 @SiO 2 ) labeled with a fluorescent silica modified layer
  • the The mass ratio of the silica-composite magnetic nanoparticles to the fluorescent dye (such as APS-FITC) can be 20.
  • PEI polyethyleneimine
  • APS-FITC fluorescently labeled Fe 3 O 4 @SiO 2 magnetic nanomaterial
  • the mass ratio of described ⁇ -chitosan and described magnetic nano material can be 1: 3;
  • the particle diameter of the magnetic nano material of described polymer modification can be 20nm ⁇ 500nm; its potential can be +10 ⁇ +60mV.
  • the polymer-modified magnetic nanomaterial is APS-FITC fluorescently labeled Fe 3 O 4 @SiO 2 magnetic nanomaterial modified by polypyrrole; wherein, the weight of the polypyrrole The average molecular weight can be 5000; the mass ratio of the polypyrrole to the magnetic nanomaterial can be 1:3; the particle diameter of the polymer-modified magnetic nanomaterial can be 20nm ⁇ 500nm; its potential can be +10 ⁇ +60mV.
  • the present invention also provides a method for preparing a polymer-modified magnetic nanomaterial, which comprises the steps of:
  • the definitions of the polymer and the magnetic nanomaterial are as shown in any scheme of the above-mentioned polymer-modified magnetic nanomaterial.
  • the solvent in the mixture of the polymer and the solvent, can be a conventional solvent in this field, such as an alcohol solvent, and the alcohol solvent can be methanol.
  • the mass-volume ratio of the polymer in the mixture with the solvent can be a conventional mass-volume ratio in the art, such as 5 mg/mL.
  • the atomized form of the mixture of the polymer and the solvent can be obtained by conventional methods in the art, for example, by heating the mixture of the polymer and the solvent, preferably, the mixture of the polymer and the solvent
  • the atomized form is obtained by heating by plasma method.
  • volume flow rate of the atomizing gas can be 3-5 sccm.
  • sccm volume flow unit, also known as mass flow unit (Mass flow), which means standard milliliter/minute: mL/min).
  • the modification temperature may be from 100 to 300°C; for example, 200°C.
  • the modification is carried out in the presence of an inert atmosphere.
  • the inert atmosphere can be nitrogen and/or argon.
  • the modification is preferably, for example, using a plasma method for modification.
  • the conditions and operations of the plasma method can be the conditions and operations of the conventional plasma method in the art.
  • the following steps are preferred, in the presence of an inert atmosphere, in the presence of plasma glow, the mixture of the polymer and the solvent is heated to obtain an atomized form, and the magnetic nanomaterial is modified and modified; the obtained The polymer-modified magnetic nanomaterials mentioned above can be used.
  • the plasma glow can be obtained by the following steps. Under the inert atmosphere, the radio frequency power is adjusted to generate plasma glow in the plasma reaction chamber; the inert atmosphere The pressure can be between 300-400Pa; the power of the radio frequency can be 10W ⁇ 5W; preferably, under vacuum, the radio frequency power supply is preheated, and then the inert atmosphere is passed to the plasma reaction chamber; the The vacuum may be below 200Pa, such as 150-200Pa.
  • reaction time may be 1-2 hours.
  • the magnetic nanomaterials can be prepared by conventional preparation methods in the art.
  • the present invention preferably as follows:
  • the magnetic nanomaterial is silicon dioxide (SiO 2 ) composite magnetic nanoparticles or fluorescently labeled silicon dioxide composite magnetic nanoparticles
  • the silicon dioxide composite when the magnetic nanoparticles are Fe 3 O 4 @SiO 2 , it is preferably prepared by the following steps in the present invention:
  • Step (a) in the presence of an alkaline reagent, adding a silicon reagent to a system of Fe 3 O 4 magnetic nanoparticles and a solvent for a modification reaction to obtain the Fe 3 O 4 @SiO 2 ; and/ or,
  • Step (b) in a solvent and an alkaline reagent, perform a fluorescent labeling reaction on the Fe 3 O 4 @SiO 2 obtained in step (a) with a fluorescent dye to obtain the magnetic compound of the fluorescently labeled silica Nanoparticles do.
  • the solvent may be water, or water and an alcoholic solvent, and the alcoholic solvent may be ethanol.
  • the alkaline reagent can be ammonia water.
  • the silicon reagent can be ethyl orthosilicate (TEOS) or methyl orthosilicate; for example TEOS.
  • TEOS ethyl orthosilicate
  • methyl orthosilicate for example TEOS
  • the mass-to-volume ratio of the Fe 3 O 4 magnetic nanoparticles to the silica reagent may be 1500 g/L.
  • the silica reagent can be used in a mixture with the solvent; for example, 100 ⁇ l TEOS is dissolved in 2 mL ethanol.
  • the amount of the alkaline agent may be such that the pH of the system of the Fe 3 O 4 magnetic nanoparticles and the solvent is 9.5 ⁇ 0.5.
  • the modification reaction is preferably carried out under ultrasonic and/or mechanical stirring conditions.
  • step (a) it may also include a post-treatment step, the post-treatment may be the following step, after the reaction is completed, the Fe 3 O 4 @SiO 2 obtained through magnetic separation-assisted conditions is washed, That’s it; the washing can be done with ethanol and deionized water, for example, three times; preferably, after washing, the obtained Fe 3 O 4 @SiO 2 is dispersed in deionized water to prepare the required concentration
  • the solution is ready for use, for example, a solution with a concentration of 100 mg/mL.
  • the solvent may be a mixture of alcohol solvent and water.
  • the water can be deionized water.
  • the alcoholic solvent can be ethanol.
  • the volume ratio of the alcohol solvent to water may be 9:1 ⁇ 10:1 (eg 9.7:1).
  • the mass-to-volume ratio of the silica-composite magnetic nanoparticles (such as Fe 3 O 4 @SiO 2 ) to the solvent may be 0.56 to 0.6 g/L.
  • the alkaline reagent can be ammonia water.
  • the mass-to-volume ratio of the silica composite magnetic nanoparticles to the ammonia water may be 42 to 45 g/L.
  • the fluorescent dye can be used in the form of a mixture (such as a solution) with the solvent, and the solvent in the solution can be an alcoholic solvent, such as ethanol.
  • the volume mass of the solvent and the fluorescent dye may be 1.7mL/mg.
  • the fluorescent dye is APS-FITC
  • the APS-FITC can be in the form of a solution, such as an ethanol solution of APS-FITC, and for example, 1.5 mg FITC in 2.5 mL ethanol.
  • the fluorescent labeling reaction can be carried out under ultrasonic and mechanical stirring conditions.
  • the fluorescent labeling reaction can be carried out under the condition of avoiding light.
  • the above-mentioned silicon reagent in the fluorescent labeling reaction, can also be added (that is, the coating reaction is carried out at the same time as the fluorescent labeling reaction), that is, the silicon dioxide is further coated at the same time. quilt.
  • the mass-to-volume ratio of the silica composite magnetic nanoparticles to the silicon reagent may be 1000 g/L.
  • the silicon reagent can be in the form of a mixture with the solvent; for example, an ethanol solution of ethyl orthosilicate, and for example, 30 ⁇ l of ethyl orthosilicate is contained in 1 mL of ethanol.
  • the fluorescently labeled silica composite magnetic nanoparticles contain APS-FITC fluorescent markers
  • the silica composite magnetic nanoparticles such as Fe 3 O 4 @SiO 2
  • the following steps are used to prepare them. Add TEOS and APS-FITC to the mixed system of Fe 3 O 4 @SiO 2 , solvent and ammonia water in sequence The reaction is carried out to obtain the above-mentioned fluorescently labeled silica-composite magnetic nanoparticles.
  • the preparation method of the silica-composited magnetic nanoparticles labeled with fluorescence it may also include a post-treatment step, and the operation and conditions of the post-treatment may be routine in the art operation and conditions, the post-treatment described in the present invention can be the following steps, after the end of the reaction, wash the magnetic nanoparticles obtained through magnetic separation auxiliary conditions; the washing can be respectively Wash with ethanol and deionized water, for example three times.
  • the APS-FITC in the polymer-modified magnetic nanomaterial, when the nanoparticles are labeled with a fluorescent dye and the fluorescent dye is APS-FITC, the APS-FITC It can be prepared by the following steps: adding APS to FITC ethanol solution for reaction to obtain the APS-FITC. Wherein, the reaction is preferably carried out under the condition of avoiding light; the reaction is sufficient to obtain a clear solution, such as mixing overnight, such as 8-24 hours.
  • the mass volume ratio of FITC to APS may be 300g/L.
  • the system of the Fe 3 O 4 magnetic nanoparticles and the solvent is prepared by the following steps. Under the condition of ultrasonic and mechanical stirring, in the solvent, the Fe 3 O 4 nano magnetic beads are sequentially Wash with hydrochloric acid and deionized water until the pH of the supernatant is neutral.
  • the hydrochloric acid may be 3.6%-36% hydrochloric acid.
  • the magnetic nanoparticles in the magnetic nanomaterials are Fe 3 O 4
  • it is preferably prepared by the following steps in the present invention, FeCl 3 6H 2 O and ethyl
  • the diol solution is reacted to obtain the Fe 3 O 4 nanoparticles.
  • the alkali metal salt can be selected from trisodium citrate and/or NaAc.
  • the molar ratio of FeCl 3 .6H 2 O to NaAc may be 1:10.
  • the volume molar ratio of the solvent to FeCl 3 ⁇ 6H 2 O may be 10L/mol.
  • the temperature of the reaction may be 200°C.
  • the reaction time can be 8 hours.
  • a post-treatment step may also be included, and the post-treatment may be as follows. After the reaction is finished, the described magnetic separation assisted condition is washed. Fe 3 O 4 magnetic nanoparticles, that is enough; the washing can be washed with ethanol and deionized water, for example, three times; preferably, after washing, the obtained Fe 3 O 4 magnetic nanoparticles are dispersed in a deionized In deionized water, a solution with a required concentration can be prepared for use, for example, a solution with a concentration of 100 mg/mL.
  • the present invention also provides a polymer-modified magnetic nanomaterial, which is prepared by any scheme in the above-mentioned preparation method;
  • the polymer-modified magnetic nanomaterial is shown in any scheme of the above-mentioned polymer-modified magnetic nanomaterial.
  • the present invention also provides an application of a plasma method in the preparation of polymer-modified magnetic nanomaterials; the application may be as follows: in the presence of plasma glow, the mixture of polymer and solvent is mixed with the nanomaterial Carry out modification modification reaction.
  • the operations and conditions can be shown as the conditions and operations described in any scheme of the above-mentioned polymer-modified magnetic nanomaterials.
  • the definition of the polymer-modified magnetic nanomaterial and the polymer and the magnetic nanomaterial can be described in any scheme of the above-mentioned polymer-modified magnetic nanomaterial.
  • the present invention also provides an application of the polymer-modified magnetic nanomaterial as described above in the enrichment and separation of glycosylated proteins, polypeptides, nucleic acids, circulating tumor cells, and exosomes.
  • the application can be that the polymer-modified magnetic nanomaterials are used in the preparation of fluorescence and magnetic resonance MRI dual-modal imaging contrast agents, electrochemical cell sensors, and for capturing circulating tumor cells.
  • pharmaceuticals and/or medical products for example, for cell tracking, tumor tracking imaging, magnetic hyperthermia imaging or vascular imaging.
  • the (circulating) tumor cells may be folate receptor-positive tumor cells; preferably, the tumor cells are selected from one or more of the following: ovarian cancer tumor cells, cervical cancer tumor cells, non-small cell lung cancer tumors Cells, colon cancer cells, lung cancer cells, rectal cancer cells, gastric cancer cells, breast cancer cells (triple negative breast cancer tumor cells), esophageal cancer cells, liver cancer cells, leukemia; e.g. ovarian cancer tumor cells, cervical cancer tumor cells, triple negative Breast cancer tumor cells, colon cancer tumor cells, non-small cell lung cancer tumor cells, leukemia.
  • the tumor cells are selected from one or more of the following: ovarian cancer tumor cells, cervical cancer tumor cells, non-small cell lung cancer tumors Cells, colon cancer cells, lung cancer cells, rectal cancer cells, gastric cancer cells, breast cancer cells (triple negative breast cancer tumor cells), esophageal cancer cells, liver cancer cells, leukemia; e.g. ovarian cancer tumor cells, cervical cancer tumor cells, triple negative Breast cancer tumor cells, colon
  • the application may be the application of the polymer-modified magnetic nanomaterial in the preparation of drugs or reagents for capturing circulating tumor cells.
  • the detection object of the drug or reagent is a peripheral blood/body fluid sample;
  • the body fluid can be urine, pleural fluid, ascites, cerebrospinal fluid, etc.
  • circulating tumor cells include ovarian cancer tumor cells, cervical cancer tumor cells, non-small cell lung cancer tumor cells, colon cancer cells, lung cancer cells, rectal cancer cells, gastric cancer cells, breast cancer cells (triple negative breast cancer tumor cells), Esophageal cancer cells, liver cancer cells, leukemia.
  • the method for trapping circulating tumor cells in peripheral blood samples by drugs or reagents specifically includes the following steps:
  • the peripheral blood sample is diluted 3-4 times with PBS before density gradient centrifugation in S1.
  • the adsorption reaction in S3, the magnetic field separation and enrichment reaction in S4 are all carried out at 4°C.
  • the magnetic nanomaterial modified by the polymer comprises: a magnetic nanoparticle core, a shell of the modified layer, and a coating of a cationic polymer; the polymer is attached or coated on the surface of the magnetic nanomaterial to form The positively charged magnetic nanomaterial modified by the polymer; the magnetic nanomaterial has a core-shell structure, the core is a magnetic nanoparticle, and the shell is a modified layer; the modified layer Adhere to or cover the surface of the magnetic nano-particles to form modified layer-composite magnetic nano-particles.
  • the mass ratio of the polymer to the magnetic nanomaterial is 1:10 to 20:1.
  • Deionized water means pure water from which impurities in the form of ions have been removed.
  • deionization stipulated by the International Organization for Standardization ISO/TC 147 is: "Deionized water completely or incompletely removes ionized substances.
  • the reagents and raw materials used in the present invention are all commercially available.
  • the positive and progressive effects of the present invention are: (1) traditional surface modification methods (such as surface coating, surface oxidation, high-energy ray treatment, surface graft modification, etc.) exist such as surface structure damage, shape and thickness cannot be controlled, and materials Surface original performance disappears, post-treatment is complicated and other defects, and surface modification by gas-phase free radical polymerization can avoid these shortcomings, but there are also disadvantages such as thin polymer concentration, vacuum conditions are required, and the polymerization time is too long.
  • traditional surface modification methods such as surface coating, surface oxidation, high-energy ray treatment, surface graft modification, etc.
  • the present invention proposes the concept of mist polymerization modification, that is, after the polymer is dissolved in an organic solvent, the polymer solution is atomized to form a mist-like polymer droplet that condenses on the plasma-treated surface and reacts to realize Surface modification of polymer materials.
  • the polymer material with a special morphology surface is prepared mainly by plasma treatment of the surface to induce mist polymerization modification.
  • Polyethyleneimine (PEI), chitosan, and polypyrrole are respectively used as substrates, and after plasma treatment, the polymerization reaction of atomized polymers on the surface of the substrates is triggered to improve the surface properties of the materials.
  • the polymer-modified magnetic nanomaterials prepared in the present invention can be used in the detection of circulating tumor cells, specifically for the preparation of drugs or reagents for capturing circulating tumor cells in peripheral blood, and the detection object is peripheral blood samples.
  • the present invention has one or more of the following advantages: (1) The new application of the polymer-modified magnetic nanomaterial of the present invention has the advantages of high sensitivity, high detection rate and good specificity , and the captured CTCs are active and can be used for follow-up research; (2) Compared with the existing CTC detection methods, the new application uses less blood, rapid detection, and easy operation; (3) The new application only The cost of each detection is low, and only a microscope and a magnetic separator are needed, thereby reducing the medical burden; (4) The new application is applicable to various scenarios such as the curative effect evaluation, recurrence warning and prognosis value of tumor patients, providing doctors with A reference to medication and treatment.
  • Figure 1 is the potential characterization and fluorescence spectrum diagrams of various nanomaterials in Examples 1-7; where, A is the potential characterization; followed by Fe 3 O 4 @SiO 2 , PEI positive electromagnetic beads, plasma polymerization PEI positive Electromagnetic beads, chitosan positive electromagnetic beads, plasma polymerization chitosan positive electromagnetic beads, polypyrrole positive electromagnetic beads, plasma polymerization polypyrrole positive electromagnetic beads; B is the fluorescence spectrum.
  • Figure 2 is the relationship between the potential of the plasma polymerization method PEI positive electromagnetic beads and pH in Example 3.
  • Fig. 3 is the picture of plasma polymerization positive electromagnetic beads before and after magnetic separation in embodiment 3, (A) before magnetic separation; (B) after magnetic separation.
  • FIG. 4 is a TEM image of magnetic particles of PEI modified by plasma polymerization in Example 3.
  • FIG. 4 is a TEM image of magnetic particles of PEI modified by plasma polymerization in Example 3.
  • Fig. 5 is the stability comparison of the materials of Examples 2 and 3, (A) potential comparison, (B) particle size comparison.
  • Fig. 6 is the response performance of the materials of Examples 2 and 3 - the comparison of the recovery rate of CTC captured at different times.
  • Fig. 7 is a comparison of the graft modification percentages of the polymers of the materials of Examples 2 and 3.
  • Fig. 9 is a comparison chart of detection rates between normal people and malignant tumor patients in application example 2.
  • Figure 10 is an optical microscope image of tumor cells in Application Example 1;
  • FIG. 11 is a graph showing the results of culturing CTCs captured in Example 1 for 10 days, 20 days, and 30 days.
  • the flow unit sccm (Standard Cubic Centimeter per Minute) means standard milliliters per minute.
  • Ferric chloride FeCl 3 ⁇ 6H 2 O
  • ammonia water NH 3 ⁇ H 2 O
  • concentrated hydrochloric acid HCl, 37%)
  • absolute ethanol etc.
  • TEOS orthoethyl silicate
  • NaAc Sodium acetate
  • EG ethylene glycol
  • PEI 3-aminopolyethyleneimine
  • PEI ethylene glycol
  • PEI 3-aminopolyethyleneimine
  • APTES 3- Aminopropyl triethoxysilane
  • APTES fluorescein isothiocyanate
  • fluorescein isothiocyanate Fluorescein isothiocyanate
  • FITC fluorescein isothiocyanate
  • Solvothermal preparation of iron ferric oxide nanoparticles Accurately weigh 0.81g of FeCl 3 6H 2 O (ferric chloride hexahydrate, 0.003mol) and 2.56g of NaAc (anhydrous sodium acetate, 0.03mol) and magnetically stir for 30min Dissolve it completely in 30mL PEG (ethylene glycol) to obtain a brown-yellow mixed solution.
  • FeCl 3 6H 2 O ferric chloride hexahydrate, 0.003mol
  • NaAc anhydrous sodium acetate, 0.03mol
  • Adopt HCl to process the prepared iron ferric oxide nanoparticles add 1 mL of 36% concentrated hydrochloric acid to 9 mL of the above-mentioned ferric oxide solution dispersed in deionized water, and ultrasonically (temperature 30-40 ° C, power 80- 120W) stirred for 10-15min, removed the aqueous solution by magnetic separation, washed 6-7 times with deionized water, and stopped until the pH of the supernatant was neutral; weighed 83.8g of ethanol and 25.7g of deionized water into a three-necked flask, added hydrochloric acid to wash and used Wash 150 mg of Fe3O4 nano-magnetic beads with deionized water, mechanically stir for about 15 minutes under ultrasonic-assisted conditions (temperature 30-40 °C, power 80-120 W), add ammonia water to adjust the pH to about 9.5, and then dissolve 100 ⁇ l with 2 mL of ethanol TEOS was added and used in the above reaction,
  • fluorescent negative electromagnetic beads have been obtained from the reaction, which are marked as "fluorescent negative electromagnetic beads-production date", the concentration is calculated and marked, and prepared into a 10mg/mL dispersion, stored in categories, and stored in the refrigerator at 4°C in the dark.
  • Embodiment 3 The preparation of plasma polymerization method PEI positive electromagnetic beads
  • fluorescent negative electromagnetic beads have been obtained from the reaction, which are marked as "fluorescent negative electromagnetic beads-production date", the concentration is calculated and marked, and prepared into a 10mg/mL dispersion, stored in categories, and stored in the refrigerator at 4°C in the dark.
  • the specific experimental operation steps are as follows: Put 18mg of fluorescent negative electromagnetic bead powder into the plasma reaction chamber, and check the overall air tightness—turn on the mechanical pump to evacuate to below 200Pa, and turn on the radio frequency power supply to preheat for 15-20 minutes—turn on the nitrogen
  • the valve is to pass nitrogen gas while the mechanical pump is running, so that the nitrogen pressure is stable between 300-400Pa——turn on the radio frequency equipment, adjust the radio frequency current and voltage, so that plasma glow is generated in the reaction chamber, and at the same time adjust the radio frequency power to stabilize at 10W Left and right; heat the reaction to volatilize PEI (10mg dissolved in 2mL methanol and pass it into the plasma reaction chamber), and adjust the monomer flow rate to 3-5sccm through a flow meter—keep the reaction conditions stable and react for 1-2 hours.
  • fluorescent negative electromagnetic beads have been obtained from the reaction, which are marked as "fluorescent negative electromagnetic beads-production date", the concentration is calculated and marked, and prepared into a 10mg/mL dispersion, stored in categories, and stored in the refrigerator at 4°C in the dark.
  • Sample treatment Label the aqueous dispersion with "Fluorescent Positive Electromagnetic Beads-Production Date", measure the concentration, and prepare a 10 mg/mL dispersion; store in categories, and store in the refrigerator at 4 degrees Celsius in the dark.
  • fluorescent negative electromagnetic beads have been obtained from the reaction, which are marked as "fluorescent negative electromagnetic beads-production date", the concentration is calculated and marked, and prepared into a 10mg/mL dispersion, stored in categories, and stored in the refrigerator at 4°C in the dark.
  • the specific experimental operation steps are as follows: Put 18mg of fluorescent negative electromagnetic bead powder into the plasma reaction chamber, and check the overall air tightness—turn on the mechanical pump to evacuate to below 200Pa, and turn on the radio frequency power supply to preheat for 15-20 minutes—turn on the nitrogen
  • the valve is to pass nitrogen gas while the mechanical pump is running, so that the nitrogen pressure is stable between 300-400Pa——turn on the radio frequency equipment, adjust the radio frequency current and voltage, so that plasma glow is generated in the reaction chamber, and at the same time adjust the radio frequency power to stabilize at 10W Left and right—heating reaction to volatilize ⁇ -chitosan (10mg is dissolved in 2mL methanol and passed into the plasma reaction chamber), and adjust the monomer flow rate to 3-5sccm through a flow meter——keep the reaction conditions stable, and react 1-2 Hour.
  • the aqueous dispersion is labeled "Plasma Polymerized Chitosan-Positive Electromagnetic Beads-Production Date", the concentration is calculated, and a 10mg/mL dispersion is prepared; classified storage, kept in the dark at 4 degrees in the refrigerator.
  • fluorescent negative electromagnetic beads have been obtained from the reaction, which are marked as "fluorescent negative electromagnetic beads-production date", the concentration is calculated and marked, and prepared into a 10mg/mL dispersion, stored in categories, and stored in the refrigerator at 4°C in the dark.
  • fluorescent negative electromagnetic beads have been obtained from the reaction, which are marked as "fluorescent negative electromagnetic beads-production date", the concentration is calculated and marked, and prepared into a 10mg/mL dispersion, stored in categories, and stored in the refrigerator at 4°C in the dark.
  • the specific experimental operation steps are as follows: Put 18mg of fluorescent negative electromagnetic bead powder into the plasma reaction chamber, and check the overall air tightness—turn on the mechanical pump to evacuate to below 200Pa, and turn on the radio frequency power supply to preheat for 15-20 minutes—turn on the nitrogen
  • the valve is to pass nitrogen gas while the mechanical pump is running, so that the nitrogen pressure is stable between 300-400Pa——turn on the radio frequency equipment, adjust the radio frequency current and voltage, so that plasma glow is generated in the reaction chamber, and at the same time adjust the radio frequency power to stabilize at 10W Left and right—heating reaction to volatilize polypyrrole (10mg dissolved in 2mL methanol, and pass it into the plasma reaction chamber), and adjust the monomer flow rate to 3-5 sccm through a flow meter—keep the reaction conditions stable and react for 1-2 hours.
  • the TEM image of the magnetic particles modified by the plasma polymerization method in Example 3 is shown in FIG. 4 .
  • Example 2 The stability of the materials in Example 2 and Example 3 is compared, as shown in Figure 5, (A) The potential comparison shows that the potential in Example 2 declines significantly with time, from 40 to about 15 in 200 days. The potential of the material obtained in Example 3 can remain unchanged for 2 years, which is significantly better than that of Example 2. (B) In particle size comparison, the hydrated particle size of the material obtained in Example 3 can be kept without significant change for 2 years, which is significantly better than that of Example 2.
  • the response time of the material in Example 3 produced by the plasma polymerization of the present invention can reach 3 seconds, which is significantly faster than that obtained by the conventional non-plasma polymerization method.
  • Example 7 The comparison of the grafting percentages of the polymers of the materials in Example 2 and Example 3 is shown in FIG. 7 .
  • the ratio of the mass of the polymer to the feeding amount can reach more than 60%.
  • the ratio of the mass of the polymer to the feeding amount is only about 15%.
  • Embodiment 5 and 7 are compared with embodiment 4 and 6, can obtain similar effect.
  • the cells are large in size; the ratio of nuclei to cytoplasm is high; the nuclei have different shapes, such as meganuclei, binuclei, or multinucleate; the nuclei are deeply stained and stained unevenly; fat particles are common in the cytoplasm; the surface of the cell membrane is wrinkled or has clear borders.
  • the above are the morphological characteristics of tumor cells, and those meeting the above 4 or more characteristics are considered as tumor cells.
  • this example only requires 4 mL of peripheral blood, and the detection time is completed within 2 hours; while the Johnson & Johnson CellSearch technology requires 7.5 mL of peripheral blood, and the detection takes at least 6 hours.
  • the selection criteria for volunteers in this example are as follows:

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Abstract

本发明公开了一种聚合物修饰的磁性纳米材料、其制备方法及应用。本发明提供了一种聚合物修饰的磁性纳米材料的制备方法，其包括如下步骤：在惰性气氛存在下，在等离子体辉光存在下，将所述的聚合物与溶剂的混合物加热得到雾化形态，与所述的磁性纳米材料进行改性修饰；得到所述的聚合物修饰的磁性纳米材料即可。本发明提供的聚合物修饰的磁性纳米材料的聚合物修饰量高、稳定性好，可在糖基化蛋白、多肽类物质、核酸、循环肿瘤细胞、外泌体等的富集分离中应用，响应时间快；如可用作制备捕获外周血/尿液等体液中循环肿瘤细胞的药物或试剂。

Description

一种聚合物修饰的磁性纳米材料、其制备方法及应用
本申请要求申请日为2021/6/30的中国专利申请2021107388501的优先权。本申请引用上述中国专利申请的全文。
技术领域
本发明涉及一种聚合物修饰的磁性纳米材料、其制备方法及应用。
背景技术
随着人口数量的增长和老龄化以及环境问题等原因，我国癌症的发病率和死亡率一直在不断提高，癌症在中国乃至世界范围内都是致死的主要疾病之一，长期以来难以取得有效的治疗效果，因此对癌症的研究长期以来都是全球各地科学技术研究人员所热衷的领域。早期诊断和治疗对于挽救患者生命具有极其重要的意义，众多研究致力于癌症的早期诊断，通过早期诊断和治疗可以延长患者生存期、增加生存率和挽救患者的生命。
循环肿瘤细胞(CTC)是被广泛认为是从实体瘤患者肿瘤部位处脱落并进入患者血液循环系统中的各类肿瘤细胞的统称，也被普遍认为是导致癌症发生转移的主要因素，而癌症的转移是引发病患死亡的最根本原因，也是术后复发的重要因素，很多研究证明手术、化疗等治疗手段也是导致肿瘤细胞从病灶脱落入血从而形成循环肿瘤细胞的重要因素。循环肿瘤细胞被认为是最有潜力的多功能生物标志物，在乳腺癌、肺癌、结直肠癌乃至前列腺癌等众多不同类型的癌症中都有循环肿瘤细胞被发现，循环肿瘤细胞的检出、计数及相关分析对于患者病情判断有很重要的意义，有望应用于肿瘤早期检测、辅助治疗、疗效评估以及预后判断。
目前，已报道的CTC富集的方法主要有物理法和生物法。如细胞过滤器富集CTC，让小的血细胞通过截留大的肿瘤细胞；基于抗体识别细胞表面蛋白，如利用上皮细胞黏附分子(EpCAM)将癌变的上皮癌细胞捕获。但这些方法都不是基于肿瘤细胞独有的性质进行捕获的，检测存在假阳性或假阴性，且不具有广谱性，无法广泛应用。我们前期研究发现，由于肿瘤细胞的糖酵解产生大量乳酸，使其表面带大量负电荷，而正常细胞呈现电中性或带少量正电荷。因此，基于肿瘤细胞与正常细胞之间独特的电荷差异，可实现CTC的高效高选择性富集，且具有广谱性。构建高性能的正电荷纳米材料是捕获负电荷CTC的关键。
其中，四氧化三铁(Fe 3O 4)磁性纳米颗粒由于具有特殊的结构和优异的性能，在生 物技术与医学相关领域受到广泛关注和研究。这类材料通常以已经相当成熟的方法制备得到的磁性纳米粒子为基础，表面包覆二氧化硅等无机材料或其它有机高分子材料，然后进一步进行反应或表面修饰，制备得到具有铁磁性的多功能复合纳米微粒。这种纳米材料通常在材料粒径大小、磁强度等特征易于控制，生物相容性以及稳定性都很好，且易于修饰，极大的拓展了其应用范围，在药物靶向运输、基因载体、生物磁分离、磁热疗、磁共振成像等诸多领域均有研究应用。基于Fe 3O 4纳米颗粒优良的物理化学性质，开发简单、可大量制备、生物相容性好、表面带正电荷的修饰方法，可为CTC富集、检测以及指导治疗提供高质量的纳米探针。
但是，目前现有技术报道的纳米材料存在聚合物量不足、稳定性差，响应时间长等不足。
发明内容
本发明所要解决的技术问题在于克服现有技术中磁性材料(带正电荷的磁性纳米粒子(positively charged magnetic nanoparticles，PCMNs))聚合物量不足、稳定性差，响应时间长等缺陷，而提供了一种聚合物修饰的磁性纳米材料、其制备方法及应用。本发明的聚合物修饰的磁性纳米材料稳定性好，响应时间快，能够实现从复杂样品中对糖基化蛋白、多肽类物质、核酸、循环肿瘤细胞、外泌体的高选择性、高重复性和高通量地富集；可应用于制备活体荧光和磁共振双模态成像显影剂或用于治疗癌症的光热治疗剂。
本发明是通过下述技术方案来解决上述技术问题的。
本发明提供了一种聚合物修饰的磁性纳米材料，其包括如下结构：
所述的聚合物为阳离子型聚合物；所述的聚合物包覆于磁性纳米粒子表面(即，位于所述壳的外层，简称涂层)，形成带正电荷的所述聚合物修饰的磁性纳米材料；
所述的磁性纳米材料为核-壳结构，所述的核为磁性纳米粒子(核)，所述的壳为改性层；所述的改性层附着或包覆于所述磁性纳米粒子的表面，形成改性层复合的磁性纳米粒子；
其中，所述的聚合物与所述的磁性纳米材料的质量比为1∶10～20∶1。
在本发明的某一方案中，所述的聚合物与所述的磁性纳米材料的质量比可为1∶5至3∶1；例如1∶3。
在本发明的某一方案中，所述的聚合物修饰的磁性纳米材料的电位可为+5至+60mV，例如+10至+50mV，优选+20至+40mV(又例如+35mV)。
在本发明的某一方案中，所述的磁性纳米材料为带负电荷的磁性纳米材料，例如其 电位可为-10至-60mV；例如-20至-40mV。
在本发明的某一方案中，所述的聚合物修饰的磁性纳米材料的粒径可为10nm至600nm；例如粒径为300nm至500nm，又例如350nm至400nm。
在本发明的某一方案中，所述的磁性纳米材料的粒径可为5nm至500nm；例如，300nm至350nm。
在本发明的某一方案中，所述的壳的厚度可为1nm至100nm，例如40nm至60nm。
在本发明的某一方案中，所述的磁性纳米粒子的粒径可为5nm至500nm；例如250nm至300nm。
在本发明的某一方案中，所述的聚合物为(树)支状聚合物。
在本发明的某一方案中，所述的聚合物可为重均分子量MW在2000至300000之间。
在本发明的某一方案中，所述的聚合物为本领域常规的阳离子型聚合物；例如，聚乙烯亚胺(PEI，Polyethylenimine)、壳聚糖(β-壳聚糖)和聚吡咯中的一种或多种。
在本发明的某一方案中，所述的聚合物可为：重均分子量可为2000-100000的聚乙烯亚胺，例如为MW＝10000，99％纯度；重均分子量可为50000-300000的β-壳聚糖，例如MW＝50000；重均分子量可为5000的聚吡咯。
在本发明中，所述的磁性纳米粒子可为本领域中常规的磁性纳米粒子，例如氧化物磁性纳米粒子(又例如Fe 3O 4、γ-Fe 2O 3)、磁性金属纳米粒子、磁性硫化物纳米粒子、磁性复合粒子中的一种或几种；再例如磁性Fe 3O 4纳米粒子(以下简称Fe 3O 4)。所述磁性纳米粒子可采用本领域常规的方法制备得到，例如溶剂热法、共沉淀法等。
所述磁性纳米粒子使得所述聚合物修饰的磁性纳米材料能够具有磁性，进而能够在磁铁的作用下进行移动，可作为探针。
在本发明中，所述的磁性纳米材料可为本领域中常规的磁性纳米材料，其中，所述的改性层包裹于所述的磁性纳米粒子的表面形成核-壳结构复合的磁性纳米材料；所述的改性层形成的壳(层)既可以阻止其团聚，又能防止其被破坏，还可以对其进行表面功能化。
在本发明的某一方案中，所述的改性层的材料可为本领域常规的为有机物和/或无机物的改性层材料；例如二氧化硅或标记荧光和/或表面活性剂修饰的二氧化硅；例如二氧化硅或标记荧光的二氧化硅。即，所述的磁性纳米材料可为二氧化硅(SiO 2)复合的磁性纳米粒子、或、标记荧光和/或表面活性剂修饰的二氧化硅复合的磁性纳米粒子。
在本发明的某一方案中，当所述的磁性纳米材料为二氧化硅改性层复合的磁性纳米粒子时，所述的二氧化硅改性层复合的磁性纳米粒子为二氧化硅改性层复合的磁性Fe 3O 4 纳米粒子(以下简称Fe 3O 4@SiO 2，四氧化三铁/二氧化硅复合微球)。
在本发明的某一方案中，所述的改性层(例如所述二氧化硅改性层)的表面含有修饰得到的氨基，进而使得其具有能够与进一步修饰(改性)的物质进行反应的基础。所述的修饰可采用本领域常规的表面修饰剂；例如，通过在二氧化硅的表面修饰氨基，进而使得其具有能够与具有羧基的荧光染料进行酰胺反应的基础。所述的标记荧光的二氧化硅复合的磁性纳米粒子中，荧光染料例如通过酰胺反应与二氧化硅改性层键合。在本发明的某一方案中，所述的二氧化硅复合的磁性纳米粒子中，所述二氧化硅层进行修饰是通过表面化学修饰剂在二氧化硅层的表面修饰上氨基。所述表面化学修饰剂可为本领域常规的能够对二氧化硅复合的磁性纳米粒子表面进行氨基修饰的表面修饰剂；例如氨水和/或APTES(3-氨丙基三乙氧基硅烷)；又例如氨水。
在本发明的某一方案中，所述的改性层与所述的磁性纳米粒子的质量比可为50∶1～1∶10；例如10∶1。
在本发明的某一方案中，所述聚合物修饰的磁性纳米材料的稳定时长为2年。
在本发明的某一方案中，所述聚合物修饰的磁性纳米材料的响应时间为3S至2min。
在本发明的某一方案中，所述的磁性纳米材料为标记荧光的二氧化硅复合的磁性纳米粒子，所述的标记荧光的二氧化硅复合的磁性纳米粒子中的荧光染料(或荧光标记物)可为本领域该类材料中常规的荧光染料，例如具有羧基或能够与氨基发生酰胺反应的荧光染料，例如异硫氰酸荧光素(fluorescenceisothiocyanate，FITC)和/或罗丹明类染料，和/或其被修饰物；所述荧光染料可为异硫氰酸荧光素、罗丹明B、罗丹明B异硫氰酸酯(Rhodamine B 5-isothiocyanate，RBITC)和四甲基异硫氰酸罗丹明(tetramethylrhodamineisothiocyanate，TRITC)中的一种或多种。所述的修饰物可为APS修饰的异硫氰酸荧光素和/或APS修饰的罗丹明类染料。所述APS可为3-氨基丙基三乙氧基硅烷(APTES)和/或3-氨基丙基三甲基硅烷(APTMS))。例如，所述的FITC被修饰物可为APS-FITC(或称FITC-APS/APS修饰FITC)，又例如，所述的荧光染料为APS-FITC。
所述的标记荧光的二氧化硅复合的磁性纳米粒子中的荧光染料(或荧光标记物)采用该领域常规的方式修饰于所述的二氧化硅复合的纳米粒子的表面，形成标记荧光的二氧化硅复合的磁性纳米粒子；例如，通过连接键(例如如上所述的酰胺键)连接于二氧化硅复合的磁性纳米粒子的表面。例如，所述的标记荧光的二氧化硅复合的磁性纳米粒子为APS-FITC标记的Fe 3O 4@SiO 2。所述的二氧化硅改性层复合的磁性纳米粒子为二氧化硅改性层复合的磁性Fe 3O 4纳米粒子(以下简称Fe 3O 4@SiO 2，四氧化三铁/二氧化硅复合 微球)
在本发明的某一方案中，所述的磁性纳米材料可为表面活性剂修饰的二氧化硅复合的磁性纳米粒子。所述的表面活性剂可包括乙酸钠、柠檬酸三钠、壳聚糖、聚乙烯吡咯烷酮、聚对苯二甲酸乙二醇酯、硬脂酸、阿拉伯树胶、羟丙基甲基纤维素、海藻酸钠、十二烷基硫酸钠、十二烷基苯磺酸钠、聚乙烯醇、长链脂肪酸、淀粉和十二硫醇中的一种或两种以上的组合。通过上述表面活性剂修饰，例如能够避免形成的纳米粒子进行团聚，从而达到对聚合物修饰的磁性纳米材料的粒径的控制。
在本发明的某一方案中，当所述的磁性纳米材料为标记荧光的二氧化硅改性层复合的磁性纳米粒子(例如APS-FITC标记的Fe 3O 4@SiO 2)时，所述的二氧化硅复合的磁性纳米粒子与所述的荧光染料(例如APS-FITC)的质量比值可为20。
在本发明的某一方案中，所述的聚合物修饰的磁性纳米材料为聚乙烯亚胺(PEI，Polyethylenimine)修饰的APS-FITC荧光标记的Fe 3O 4@SiO 2磁性纳米材料，其中，所述的PEI重均分子量MW＝10000，99％(纯度)；所述聚乙烯亚胺与所述磁性纳米材料的质量比可为1∶3；所述的聚合物修饰的磁性纳米材料的粒径可为20nm～500nm；其电位可为+10mV～+60mV。
在本发明的某一方案中，所述的聚合物修饰的磁性纳米材料为β-壳聚糖修饰的APS-FITC荧光标记的Fe 3O 4@SiO 2磁性纳米材料；其中，所述的β-壳聚糖的重均分子量MW＝50000；所述β-壳聚糖与所述磁性纳米材料的质量比可为1∶3；所述的聚合物修饰的磁性纳米材料的粒径可为20nm～500nm；其电位可为+10～+60mV。
在本发明的某一方案中，所述的聚合物修饰的磁性纳米材料为聚吡咯修饰的APS-FITC荧光标记的Fe 3O 4@SiO 2磁性纳米材料；其中，所述的聚吡咯的重均分子量可为5000；所述聚吡咯与所述磁性纳米材料的质量比可为1∶3；所述的聚合物修饰的磁性纳米材料的粒径可为20nm～500nm；其电位可为+10～+60mV。
本发明还提供了一种聚合物修饰的磁性纳米材料的制备方法，其包括如下步骤：
将聚合物与溶剂的混合物与磁性纳米材料进行改性修饰，得到所述的聚合物修饰的磁性纳米材料即可；其中，所述的聚合物与溶剂的混合物为雾化形态；
其中，所述的聚合物、所述的磁性纳米材料的定义如上所述的聚合物修饰的磁性纳米材料中任一方案所示。
其中，
在本发明的某一方案中，所述的聚合物与溶剂的混合物中，所述的溶剂可为该领域常规的溶剂，例如醇类溶剂，所述的醇类溶剂可为甲醇。所述的聚合物在所述的与溶剂 的混合物中的质量体积比可为本领域常规的质量体积比，例如5mg/mL。
所述的聚合物与溶剂的混合物的雾化形态可为本领域常规的方法得到，例如通过加热所述的聚合物与溶剂的混合物得到，较佳地，将所述的聚合物与溶剂的混合物通过等离子体法加热得到所述的雾化形态。
所述的聚合物与溶剂的混合物的加入可为控制通入所述的雾化气体的体积流量在3-5sccm。(sccm为体积流量单位，又称质流单位(Mass flow)，表示标准毫升/分钟：mL/min)。
所述的改性修饰的温度可为100至300℃；例如200℃。
较佳地，所述的改性修饰在惰性气氛存在下进行。所述的惰性气氛可为氮气和/或氩气。
本发明中，所述的改性修饰，较佳地，例如采用等离子体法进行改性修饰，所述的等离子体法的条件和操作可为本领域常规的等离子体法的条件和操作，本发明中优选如下步骤，在惰性气氛存在下，在等离子体辉光存在下，将所述的聚合物与溶剂的混合物加热得到雾化形态，与所述的磁性纳米材料进行改性修饰；得到所述的聚合物修饰的磁性纳米材料即可。
在本发明的某一方案中，所述的等离子体辉光可为如下步骤得到，在所述的惰性气氛下，调节射频功率，使等离子反应腔内产生等离子体辉光；所述惰性气氛的压力可为在300-400Pa之间；所述射频的功率可为10W±5W；较佳地，在真空下，射频电源预热，再向等离子体反应腔通所述的惰性气氛；所述的真空可为200Pa以下，例如150-200Pa。
在本发明的某一方案中，所述的反应的时间可为1-2小时。
所述的磁性纳米材料可为本领域常规的制备方法制备得到。本发明中优选如下：
在本发明的某一方案中，当所述的磁性纳米材料为二氧化硅(SiO 2)复合的磁性纳米粒子或标记荧光的二氧化硅复合的磁性纳米粒子、所述的二氧化硅复合的磁性纳米粒子为Fe 3O 4@SiO 2时，本发明中优选采用如下步骤制备得到：
步骤(a)、在碱性试剂存在下，将硅试剂加入到Fe 3O 4磁性纳米微粒与溶剂的体系中进行改性反应，得到所述的Fe 3O 4@SiO 2即可；和/或，
步骤(b)、在溶剂和碱性试剂中，将步骤(a)得到所述的Fe 3O 4@SiO 2与荧光染料进行荧光标记反应，得到所述的标记荧光的二氧化硅复合的磁性纳米粒子即可。
步骤(a)中，所述的溶剂可为水，或水和醇类溶剂，所述的醇类溶剂可为乙醇。
所述的碱性试剂可为氨水。
所述的硅试剂可为正硅酸乙酯(TEOS)或正硅酸甲酯；例如TEOS。
所述的Fe 3O 4磁性纳米微粒与所述的二氧化硅试剂质量体积比可为1500g/L。
所述的二氧化硅试剂可以与所述的溶剂的混合物形式使用；例如2mL乙醇溶解100μl TEOS。
所述的碱性试剂的用量可为使所述的Fe 3O 4磁性纳米微粒与溶剂的体系的pH为9.5±0.5即可。
所述的改性反应，较佳地，在超声和/或机械搅拌条件下进行。
步骤(a)中，其还可包括后处理步骤，所述的后处理可为如下步骤，所述的反应结束后，洗涤经磁分离辅助条件获得的所述的Fe 3O 4@SiO 2，即可；所述的洗涤可为分别使用乙醇和去离子水洗涤，例如洗涤三次；较佳地，洗涤后，将得到的Fe 3O 4@SiO 2分散在去离子水中，配制成所需浓度的溶液待用即可，例如浓度为100mg/mL的溶液。
步骤(b)中，所述的溶剂可为醇类溶剂和水的混合物。所述的水可为去离子水。所述的醇类溶剂可为乙醇。所述的醇类溶剂和水的体积比可为9∶1～10∶1(例如9.7∶1)。
所述的二氧化硅复合的磁性纳米粒子(例如Fe 3O 4@SiO 2)与所述的溶剂的质量体积比可为0.56至0.6g/L。
所述的碱性试剂可为氨水。所述的二氧化硅复合的磁性纳米粒子与所述的氨水的质量体积比可为42至45g/L。
所述的荧光染料可为与所述的溶剂的混合物形式(例如溶液)使用，所述的溶液中的溶剂可为醇类溶剂，例如乙醇。所述的荧光染料与溶剂的混合物中，所述的溶剂与所述的荧光染料的体积质量可为1.7mL/mg。例如，当所述的荧光染料为APS-FITC时，所述的APS-FITC可为溶液形式，例如APS-FITC的乙醇溶液形式，又例如2.5mL乙醇中含1.5mg FITC。
所述的荧光标记反应可在超声和机械搅拌条件下进行。
所述的荧光标记反应可在避光条件下进行。
在本发明的某一方案中，所述的荧光标记反应中，还可加入如上所述的硅试剂(即，在荧光标记反应的同时，进行包被反应)，即同时进行二氧化硅进一步包被。其中，所述的二氧化硅复合的磁性纳米粒子与所述的硅试剂的质量体积比可为1000g/L。所述的硅试剂可为与所述的溶剂的混合物形式；例如，正硅酸乙酯的乙醇溶液，又例如1mL乙醇中含30μl正硅酸乙酯。
在本发明的某一方案中，当所述的磁性纳米材料为标记荧光的二氧化硅复合的磁性纳米粒子，所述的标记荧光的二氧化硅复合的磁性纳米粒子为含APS-FITC荧光标记的二氧化硅复合的磁性纳米粒子(例如Fe 3O 4@SiO 2)时，采用如下步骤制备得到，将TEOS 和APS-FITC依次加入到Fe 3O 4@SiO 2与溶剂和氨水的混合体系中进行反应，得到所述的标记荧光的二氧化硅复合的磁性纳米粒子即可。较佳地，在避光条件下，在超声和机械搅拌条件下，在TEOS缓慢地逐滴加入Fe 3O 4@SiO 2与乙醇和氨的混合体系中后，快速加入APS-FITC的溶液，进行所述的荧光标记反应。
在本发明的某一方案中，所述的标记荧光的二氧化硅复合的磁性纳米粒子的制备方法中，其还可包括后处理步骤，所述的后处理的操作和条件可为本领域常规的操作和条件，本发明中所述的后处理可为如下步骤，所述的反应结束后，洗涤经磁分离辅助条件获得的所述的磁性纳米粒子，即可；所述的洗涤可为分别使用乙醇和去离子水洗涤，例如洗涤三次。
在本发明的某一方案中，所述的聚合物修饰的磁性纳米材料中，当所述的纳米粒子为被荧光染料标记、所述的荧光染料为APS-FITC时，所述的APS-FITC可为如下步骤制备得到：将APS加入到FITC的乙醇溶液中反应，得到所述的APS-FITC即可。其中，所述的反应较佳地在避光条件下进行；所述的反应以得到澄清的溶液即可，例如混合过夜，例如8-24小时。FITC与APS的质量体积比可为300g/L。
在本发明的某一方案中，所述的Fe 3O 4磁性纳米微粒与溶剂的体系采用如下步骤制备得到，在超声和机械搅拌条件下，在溶剂中，将Fe 3O 4纳米磁珠依次用盐酸、去离子水洗涤至上清液pH中性，即可。所述的盐酸可为3.6％～36％盐酸。
在本发明的某一方案中，当所述的磁性纳米材料中的磁性纳米粒子为Fe 3O 4时，本发明中优选如下步骤制备得到，将FeCl 3·6H 2O和碱金属盐的乙二醇溶液进行反应，得到所述的Fe 3O 4纳米微粒即可。
其中，所述的碱金属盐可选自柠檬酸三钠和/或NaAc。
所述的FeCl 3·6H 2O与NaAc的摩尔比可为1∶10。
所述的溶剂与FeCl 3·6H 2O的体积摩尔比可为10L/mol。
所述的反应的温度可为200℃。
所述的反应的时间可为8小时。
所述的Fe 3O 4磁性纳米微粒的制备方法中，还可包括后处理步骤，所述的后处理可为如下步骤，所述的反应结束后，洗涤经磁分离辅助条件获得的所述的Fe 3O 4磁性纳米微粒，即可；所述的洗涤可为分别使用乙醇和去离子水洗涤，例如洗涤三次；较佳地，洗涤后，将得到的Fe 3O 4磁性纳米微粒分散在去离子水中，配制成所需浓度的溶液待用即可，例如浓度为100mg/mL的溶液。
本发明还提供了一种聚合物修饰的磁性纳米材料，其采用如上所述的制备方法中任 一方案制备得到；
较佳的，所述的聚合物修饰的磁性纳米材料为如上所述的聚合物修饰的磁性纳米材料中任一方案所示。
本发明还提供了一种等离子体法在制备聚合物修饰的磁性纳米材料中的应用；所述的应用可为如下步骤：在等离子体辉光存在下，将聚合物与溶剂的混合物与纳米材料进行改性修饰反应。
其中，所述的操作和条件可如上所述的聚合物修饰的磁性纳米材料中任一方案所述的条件和操作所示。所述的聚合物修饰的磁性纳米材料以及所述的聚合物和所述的磁性纳米材料的定义可为如上所述的聚合物修饰的磁性纳米材料中任一方案所述。
本发明还提供了一种如上所述的聚合物修饰的磁性纳米材料在糖基化蛋白、多肽类物质、核酸、循环肿瘤细胞、外泌体的富集分离中的应用。
在本发明的某一方案中，所述的应用可为所述的聚合物修饰的磁性纳米材料在制备荧光和磁共振MRI双模态成像显影剂、电化学细胞传感器、用于循环肿瘤细胞捕获的药物和/或医疗产品(试剂)、或用于治疗癌症的光热治疗剂中的应用。例如，用于细胞示踪、肿瘤示踪成像、磁热疗成像或血管成像。
所述(循环)肿瘤细胞例如可为叶酸受体阳性的肿瘤细胞；优选地，所述肿瘤细胞选自以下的一种或多种：卵巢癌肿瘤细胞、宫颈癌肿瘤细胞、非小细胞肺癌肿瘤细胞、结肠癌细胞、肺癌细胞、直肠癌细胞、胃癌细胞、乳腺癌细胞(三阴性乳腺癌肿瘤细胞)、食管癌细胞、肝癌细胞、白血病；例如卵巢癌肿瘤细胞、宫颈癌肿瘤细胞、三阴性乳腺癌肿瘤细胞、结肠癌肿瘤细胞、非小细胞肺癌肿瘤细胞、白血病。
在本发明的某一方案中，所述的应用可为所述的聚合物修饰的磁性纳米材料在制备捕获循环肿瘤细胞的药物或试剂中的应用。
优选的，所述药物或试剂的检测对象为外周血/体液样本；所述的体液可为尿液、胸腔液、腹水、脑脊液等。
如上所述循环肿瘤细胞包括卵巢癌肿瘤细胞、宫颈癌肿瘤细胞、非小细胞肺癌肿瘤细胞、结肠癌细胞、肺癌细胞、直肠癌细胞、胃癌细胞、乳腺癌细胞(三阴性乳腺癌肿瘤细胞)、食管癌细胞、肝癌细胞、白血病。
优选的，所述药物或试剂捕获外周血样本中循环肿瘤细胞的方法具体包括以下步骤：
S1、取外周血样本，用密度梯度液进行密度梯度离心，取中间段的白细胞层，去掉血浆和红细胞；
S2、将白细胞层的细胞稀释离心，重悬细胞获得细胞悬浮液，去掉蛋白及杂质；
S3、超声活化所述药物或试剂，并将活化后的药物或试剂与S2获得的细胞悬浮液以体积比3∶100混合，进行吸附反应；
S4、磁场分离S3中吸附细胞悬浮液后的药物或试剂，富集循环肿瘤细胞，然后将细胞重悬、甩片、迪夫快速染色；
S5、显微镜下阅片，并根据肿瘤形态学进行鉴定和计数。
优选地，S1中密度梯度离心前外周血样本采用PBS稀释3-4倍。
优选地，S3中的吸附反应、S4中的磁场分离及富集反应均是在4℃条件下进行。
优选的，所述聚合物修饰的磁性纳米材料包括：磁性纳米粒子核，改性层的壳，和阳离子型聚合物的涂层；所述的聚合物附着或包覆于磁性纳米材料表面，形成带正电荷的所述聚合物修饰的磁性纳米材料；所述的磁性纳米材料为核-壳结构，所述的核为磁性纳米粒子，所述的壳为改性层；所述的改性层附着或包覆于所述磁性纳米粒子的表面，形成改性层复合的磁性纳米粒子。其中，所述的聚合物修饰的磁性纳米材料中，所述的聚合物与所述的磁性纳米材料的质量比为1∶10至20∶1。
术语
“去离子水”是指是指除去了呈离子形式杂质后的纯水。国际标准化组织ISO/TC 147规定的“去离子”定义为：“去离子水完全或不完全地去除离子物质。
在不违背本领域常识的基础上，上述各优选条件，可任意组合，即得本发明各较佳实例。
本发明所用试剂和原料均市售可得。
本发明的积极进步效果在于：(1)传统的表面改性方法(如表面涂覆、表面氧化、高能射线处理、表面接枝改性等)存在如表面结构破坏，形态和厚度无法控制，材料表面原性能消失，后处理繁杂等缺陷，而气相自由基聚合法进行表面改性可规避这些不足，但也有聚合物浓度稀薄，需要真空条件，聚合时间过长等缺点。作为气相聚合的改良方法，本发明提出了雾聚合修饰的概念，即将聚合物溶于有机溶剂后，雾化该聚合物溶液，形成雾状聚合物液滴凝结于等离子体处理表面发生反应，实现对高分子材料的表面改性。主要通过等离子体处理表面引发雾聚合修饰，制备具有特殊形态表面的高分子材料。分别以聚乙烯亚胺(PEI)、壳聚糖、聚吡咯为基质，使用等离子体处理后，引发雾化聚合物在基质表面的聚合反应，改善材料的表面性能。
(2)本发明制得的聚合物修饰的磁性纳米材料可在循环肿瘤细胞检测中的应用，具体是用作制备捕获外周血中循环肿瘤细胞的药物或试剂，且检测对象为外周血样本。与现有技术相比，本发明具有以下一种或多种优点：(1)本发明所述聚合物修饰的磁性纳 米材料的新用途具有敏感度高、检出率高、特异性好的优势，且捕获的CTC具有活性，可用于后续研究；(2)所述新用途与现有的CTC检测方法相比，所用的样本血少、检测快速、操作简便；(3)所述新用途单次检测成本低，仅需配备显微镜和磁分离器即可，从而降低医疗负担；(4)所述新用途适用于肿瘤病人的疗效评估、复发预警及预后值达到等多种场景，为医生提供用药和治疗的参考。
附图说明
图1为实施例1-7中各种纳米材料的电位表征和荧光光谱图；其中，A为电位表征；依次分别为Fe 3O 4@SiO 2，PEI正电磁珠，等离子体聚合法PEI正电磁珠，壳聚糖正电磁珠，等离子体聚合法壳聚糖正电磁珠，聚吡咯正电磁珠，等离子体聚合法聚吡咯正电磁珠；B为荧光光谱图。
图2为实施例3中等离子体聚合法PEI正电磁珠电位与pH的关系。
图3为实施例3中磁分离前后等离子体聚合法正电磁珠的图片，(A)磁分离前；(B)磁分离后。
图4为实施例3中等离子体聚合法修饰PEI的磁性颗粒TEM图。
图5为实施例2和3材料的稳定性比较，(A)电位比较、(B)粒径比较。
图6为实施例2和3材料的响应性能-不同时间捕获CTC的回收率比较。
图7为实施例2和3材料的聚合物的接枝修饰百分量比较。
图8为应用实施例1中循环肿瘤细胞检测流程示意图；
图9为应用实施例2中正常人和恶性肿瘤患者的检出率比较图；
图10应用实施例1中肿瘤细胞光学显微镜图；
图11是应用实施例1中捕获的CTC培养10天、20天、30天的结果图。
具体实施方式
下面通过实施例的方式进一步说明本发明，但并不因此将本发明限制在所述的实施例范围之中。下列实施例中未注明具体条件的实验方法，按照常规方法和条件，或按照商品说明书选择。
流量单位sccm(Standard Cubic Centimeter per Minute)是表示每分钟标准毫升。
实验试剂
氯化高铁(FeCl 3·6H 2O)、氨水(NH 3·H 2O)、浓盐酸(HCl，37％)、无水乙醇等购自于国药集团；正硅酸乙酯(TEOS)、乙酸钠(NaAc)、乙二醇(EG)、3-氨聚乙烯亚胺(PEI) (MW＝10000)、β-壳聚糖(MW＝50000)、聚吡咯(MW＝5000)、3-氨丙基三乙氧基硅烷((3-Aminopropyl)triethoxysilane，APTES)和异硫氰酸荧光素(Fluorescein isothiocyanate，FITC)等购自Sigma公司；实验过程中的去离子水(Deionized water，DIW，18.2MΩ·cm)由实验室Thermo Easypure II UF纯水制备系统自制。
实验主要仪器设备
表1 实验仪器设备
Figure PCTCN2022102012-appb-000001
实施例1 多功能磁性纳米材料的制备
①超顺磁性四氧化三铁纳米颗粒的制备
四氧化三铁纳米粒子的溶剂热法制备：准确称取FeCl 3·6H 2O 0.81g(六水合三氯化铁，0.003mol)和NaAc 2.56g(无水乙酸钠，0.03mol)磁力搅拌30min使之完全溶解于30mL PEG(乙二醇)中，得到棕黄色混合溶液，将上述溶液转移入耐高温高压的不锈钢反应釜中，放入高温烘箱，温度调整至200℃，恒温反应8h；反应结束后，取出反应釜，使用流动水快速冷却至室温；通过磁铁吸附将产物从反应液中分离出来，去掉反应液，然后在磁铁辅助分离条件下，分别使用乙醇和去离子水各洗涤三次，最后得到黑色产物， 将洗涤后的产物重新在去离水中稀释分散，根据粗估配制成100mg/mL的粗浓度，采用固含量测定方法测定相对准确浓度，标记，统一存放。
②四氧化三铁/二氧化硅复合微球的制备
采用HCl处理制备好的四氧化三铁纳米微粒：将1mL 36％浓盐酸加入9mL上述分散于去离子水的四氧化三铁溶液，于圆底烧瓶中超声(温度30-40℃，功率80-120W)搅拌处理10-15min，磁分离去掉水溶液，去离子水洗6-7次，至上清液pH中性停止；称取乙醇83.8g、去离子水25.7g于三口烧瓶中，加入经盐酸洗涤并用去离子水清洗的四氧化三铁纳米磁珠150mg，超声辅助条件(温度30-40℃，功率80-120W)下机械搅拌15分钟左右，加入氨水调节pH至9.5左右，然后用2mL乙醇溶解100μl TEOS并加入用于上述反应中，继续机械搅拌12小时，然后通过磁分离获得产物并在磁分离辅助下使用无水乙醇和去离子水各洗三次，将洗涤后的产物分散在去离子水中，根据粗估配制成100mg/mL的粗浓度，采用固含量测定方法测定相对准确浓度，标记室温保存。
实施例2 PEI正电磁珠的制备
(1)APS-FITC的制备
称量1.5mg的FITC染料在1.5mL离心管中，用0.5mL无水乙醇溶解FITC，然后转入小玻璃反应瓶中，使用2mL无水乙醇稀释，磁力搅拌1分钟使其混合均匀。然后加入5μL APS，体系颜色立刻变成橘黄色，继续避光磁力搅拌过夜，直至产物状态变为为澄清溶液，得到APS-FITC溶液，实验过程中注意避光。
(2)荧光标记和TEOS包被制备负电荧光磁珠
量取无水乙醇45mL，去离子水5mL，加入氨水0.7mL，机械搅拌混匀，加入30mg实施例1制备所得Fe 3O 4@SiO 2继续超声搅拌30min左右至分散均匀；将30μl TEOS溶于1mL无水乙醇中，在超声和机械搅拌条件下缓慢地逐滴加入上述溶液，继续超声搅拌15min后将APS-FITC溶液快速加入上述反应体系，在避光条件下继续超声和机械搅拌4小时，然后停止超声，机械搅拌18小时，磁分离辅助条件获得产物并分别使用乙醇和去离子水洗涤三次。至此反应得到了荧光负电磁珠，标记为“荧光负电磁珠-生产日期”，测算和标记浓度，配制成10mg/mL分散液，分类存放，冰箱4度下避光保存。
(3)PEI修饰制备正电荧光磁珠
称取甲醇25mL，加入上一步反应得到的负电荧光磁珠18mg，避光条件下边超声边机械搅拌10min至混合均匀，然后称量PEI(MW＝10000，99％，购买于阿拉丁)10mg用2mL甲醇溶解，加入上述反应溶液，继续超声搅拌2小时，磁分离获得产物并用甲醇 洗涤一次水洗三册。样品处理：水分散液标记“荧光正电磁珠-生产日期”，测算浓度，配制成10mg/mL分散液；分类存放，冰箱4度下避光保存。
实施例3 等离子体聚合法PEI正电磁珠的制备
(1)APS-FITC的制备
称量1.5mg的FITC染料在1.5mL离心管中，用0.5mL无水乙醇溶解FITC，然后转入小玻璃反应瓶中，使用2mL无水乙醇稀释，磁力搅拌1分钟使其混合均匀。然后加入5μL APS，体系颜色立刻变成橘黄色，继续避光磁力搅拌过夜，直至产物状态变为为澄清溶液，得到APS-FITC溶液，实验过程中注意避光。
(2)荧光标记和TEOS包被制备荧光负电磁珠
量取无水乙醇45mL，去离子水5mL，加入氨水0.7mL，机械搅拌混匀，加入30mg实施例1制备所得Fe 3O 4@SiO 2继续超声搅拌30min左右至分散均匀；将30μl TEOS溶于1mL无水乙醇中，在超声和机械搅拌条件下缓慢地逐滴加入上述溶液，继续超声搅拌15min后将APS-FITC溶液快速加入上述反应体系，在避光条件下继续超声和机械搅拌4小时，然后停止超声，机械搅拌18小时，磁分离辅助条件获得产物并分别使用乙醇和去离子束洗涤三次。至此反应得到了荧光负电磁珠，标记为“荧光负电磁珠-生产日期”，测算和标记浓度，配制成10mg/mL分散液，分类存放，冰箱4度下避光保存。
(3)PEI修饰制备正电荧光磁珠
具体实验操作步骤如下：将荧光负电磁珠粉末18mg放入等离子体反应腔，检测整体气密性——开启机械泵抽真空至200Pa以下，同时打开射频电源预热15-20分钟——打开氮气阀门，在机械泵运转的同时通氮气，使氮气压力稳定在300-400Pa之间——打开射频设备，调节射频电流和电压，使反应腔体内产生等离子体辉光，同时调节射频功率稳定在10W左右；加热反应使PEI(10mg溶于2mL甲醇，通入等离子反应腔中)挥发，通过流量计调节单体流量至3-5sccm——保持各反应条件稳定，反应1-2小时。
实施例4 壳聚糖正电磁珠的制备
(1)APS-FITC的制备
称量1.5mg的FITC染料在1.5mL离心管中，用0.5mL无水乙醇溶解FITC，然后转入小玻璃反应瓶中，使用2mL无水乙醇稀释，磁力搅拌1分钟使其混合均匀。然后加入5μL APS，体系颜色立刻变成橘黄色，继续避光磁力搅拌过夜，直至产物状态变为澄清溶液，得到APS-FITC溶液，实验过程中注意避光。
(2)荧光标记和TEOS包被制备负电荧光磁珠
量取无水乙醇45mL，去离子水5mL，加入氨水0.7mL，机械搅拌混匀，加入30mg实施例1制备所得Fe 3O 4@SiO 2继续超声搅拌30min左右至分散均匀；将30μl TEOS溶于1mL无水乙醇中，在超声和机械搅拌条件下缓慢地逐滴加入上述溶液，继续超声搅拌15min后将APS-FITC溶液快速加入上述反应体系，在避光条件下继续超声和机械搅拌4小时，然后停止超声，机械搅拌18小时，磁分离辅助条件获得产物并分别使用乙醇和去离子束洗涤三次。至此反应得到了荧光负电磁珠，标记为“荧光负电磁珠-生产日期”，测算和标记浓度，配制成10mg/mL分散液，分类存放，冰箱4度下避光保存。
(3)壳聚糖修饰制备正电荧光磁珠
称取甲醇25mL，加入上一步反应得到的负点荧光磁珠18mg，避光条件下边超声边机械搅拌10min至混合均匀，然后称量β-壳聚糖10mg用2mL甲醇溶解，加入上述反应溶液，继续超声搅拌2小时，磁分离获得产物并用甲醇洗涤一次水洗三次。样品处理：水分散液标记“荧光正电磁珠-生产日期”，测算浓度，配制成10mg/mL分散液；分类存放，冰箱4度下避光保存。
实施例5 等离子体聚合法壳聚糖正电磁珠的制备
(1)APS-FITC的制备
称量1.5mg的FITC染料在1.5mL离心管中，用0.5mL无水乙醇溶解FITC，然后转入小玻璃反应瓶中，使用2mL无水乙醇稀释，磁力搅拌1分钟使其混合均匀。然后加入5μL APS，体系颜色立刻变成橘黄色，继续避光磁力搅拌过夜，直至产物状态变为为澄清溶液，得到APS-FITC溶液，实验过程中注意避光。
(2)荧光标记和TEOS包被制备负电荧光磁珠
量取无水乙醇45mL，去离子水5mL，加入氨水0.7mL，机械搅拌混匀，加入30mg实施例1制备所得Fe 3O 4@SiO 2继续超声搅拌30min左右至分散均匀；将30μl TEOS溶于1mL无水乙醇中，在超声和机械搅拌条件下缓慢地逐滴加入上述溶液，继续超声搅拌15min后将APS-FITC溶液快速加入上述反应体系，在避光条件下继续超声和机械搅拌4小时，然后停止超声，机械搅拌18小时，磁分离辅助条件获得产物并分别使用乙醇和去离子水洗涤三次。至此反应得到了荧光负电磁珠，标记为“荧光负电磁珠-生产日期”，测算和标记浓度，配制成10mg/mL分散液，分类存放，冰箱4度下避光保存。
(3)壳聚糖修饰制备正电荧光磁珠
具体实验操作步骤如下：将荧光负电磁珠粉末18mg放入等离子体反应腔，检测整 体气密性——开启机械泵抽真空至200Pa以下，同时打开射频电源预热15-20分钟——打开氮气阀门，在机械泵运转的同时通氮气，使氮气压力稳定在300-400Pa之间——打开射频设备，调节射频电流和电压，使反应腔体内产生等离子体辉光，同时调节射频功率稳定在10W左右——加热反应使β-壳聚糖(10mg用2mL甲醇溶解，通入等离子反应腔中)挥发，通过流量计调节单体流量至3-5sccm——保持各反应条件稳定，反应1-2小时。
最后得到的样品处理：水分散液标记“等离子体聚合法壳聚糖-正电磁珠-生产日期”，测算浓度，配制成10mg/mL分散液；分类存放，冰箱4度下避光保存。
实施例6 聚吡咯正电磁珠的制备
(1)APS-FITC的制备
称量1.5mg的FITC染料在1.5mL离心管中，用0.5mL无水乙醇溶解FITC，然后转入小玻璃反应瓶中，使用2mL无水乙醇稀释，磁力搅拌1分钟使其混合均匀。然后加入5μL APS，体系颜色立刻变成橘黄色，继续避光磁力搅拌过夜，直至产物状态变为为澄清溶液，得到APS-FITC溶液，实验过程中注意避光。
(2)荧光标记和TEOS包被制备负电荧光磁珠
量取无水乙醇45mL，去离子水5mL，加入氨水0.7mL，机械搅拌混匀，加入30mg实施例1制备所得Fe 3O 4@SiO 2继续超声搅拌30min左右至分散均匀；将30μl TEOS溶于1mL无水乙醇中，在超声和机械搅拌条件下缓慢地逐滴加入上述溶液，继续超声搅拌15min后将APS-FITC溶液快速加入上述反应体系，在避光条件下继续超声和机械搅拌4小时，然后停止超声，机械搅拌18小时，磁分离辅助条件获得产物并分别使用乙醇和去离子水洗涤三次。至此反应得到了荧光负电磁珠，标记为“荧光负电磁珠-生产日期”，测算和标记浓度，配制成10mg/mL分散液，分类存放，冰箱4度下避光保存。
(3)聚吡咯修饰制备正电荧光磁珠
称取甲醇25mL，加入上一步反应得到的负点荧光磁珠18mg，避光条件下边超声边机械搅拌10min至混合均匀，然后称量聚吡咯10mg，用2mL甲醇溶解，加入上述反应溶液，继续超声搅拌2小时，磁分离获得产物并用甲醇洗涤一次水洗三次。样品处理：水分散液标记“荧光正电磁珠-生产日期”，测算浓度，配制成10mg/mL分散液；分类存放，冰箱4度下避光保存。
实施例7 等离子体聚合法聚吡咯正电磁珠的制备
(1)APS-FITC的制备
称量1.5mg的FITC染料在1.5mL离心管中，用0.5mL无水乙醇溶解FITC，然后转入小玻璃反应瓶中，使用2mL无水乙醇稀释，磁力搅拌1分钟使其混合均匀。然后加入5μL APS，体系颜色立刻变成橘黄色，继续避光磁力搅拌过夜，直至产物状态变为为澄清溶液，得到APS-FITC溶液，实验过程中注意避光。
(2)荧光标记和TEOS包被制备负点荧光磁珠
量取无水乙醇45mL，去离子水5mL，加入氨水0.7mL，机械搅拌混匀，加入30mg实施例1制备所得Fe 3O 4@SiO 2继续超声搅拌30min左右至分散均匀；将30μl TEOS溶于1mL无水乙醇中，在超声和机械搅拌条件下缓慢地逐滴加入上述溶液，继续超声搅拌15min后将APS-FITC溶液快速加入上述反应体系，在避光条件下继续超声和机械搅拌4小时，然后停止超声，机械搅拌18小时，磁分离辅助条件获得产物并分别使用乙醇和去离子束洗涤三次。至此反应得到了荧光负电磁珠，标记为“荧光负电磁珠-生产日期”，测算和标记浓度，配制成10mg/mL分散液，分类存放，冰箱4度下避光保存。
(3)聚吡咯修饰制备正电荧光磁珠
具体实验操作步骤如下：将荧光负电磁珠粉末18mg放入等离子体反应腔，检测整体气密性——开启机械泵抽真空至200Pa以下，同时打开射频电源预热15-20分钟——打开氮气阀门，在机械泵运转的同时通氮气，使氮气压力稳定在300-400Pa之间——打开射频设备，调节射频电流和电压，使反应腔体内产生等离子体辉光，同时调节射频功率稳定在10W左右——加热反应使聚吡咯(10mg溶于2mL甲醇，通入等离子反应腔中)其挥发，通过流量计调节单体流量至3-5sccm——保持各反应条件稳定，反应1-2小时。
效果实施例1 多功能磁性纳米材料的性能
通过将实施例1-7制备所得多功能磁性纳米材料分散在水溶液中观察材料的分散性，将磁铁放置与装有制备的磁性纳米材料的样品瓶一侧，通过磁场对材料的吸引大致确定材料的磁力学性能；材料水合粒径大小和表面电位的测定采用激光粒度仪Zetasizer Nano-ZS(Malvern，UK)，使用动态光散射法测定纳米颗粒粒径大小分布情况及纳米颗粒表面电位情况；通过透射电子显微镜观察分析纳米颗粒的形貌；用荧光分光光度仪测制备所得纳米材料的荧光发射情况。
(1)多功能磁性纳米材料Zeta电位及荧光分析
实施例1-7中各种Fe 3O 4纳米材料的电位表征和荧光光谱图，如图1所示。
(2)溶液pH对纳米材料Zeta电位的影响
实施例3中等离子体聚合法PEI正电磁珠电位与pH的关系，如图2所示。
(3)磁性纳米材料在溶液中的分散性和磁性能观察
实施例3中磁分离前后等离子体聚合法正电磁珠，如图3所示，(A)磁分离前；(B)磁分离后
(4)纳米材料的电镜
实施例3中等离子体聚合法修饰PEI的磁性颗粒TEM图，如图4所示。
效果实施例2 非等离子体聚合法与等离子体聚合的比较
(1)材料稳定性：电位(A)、粒径(B)比较
实施例2与实施例3材料的稳定性比较，如图5所示，(A)电位比较显示，实施例2中电位随着时间显著衰退，在200天时，从40衰退至15左右。实施例3中得到的材料的电位可保持在2年没有明显变化，显著优于实施例2。(B)粒径比较中，实施例3中得到的材料的水合粒径可保持在2年没有明显变化，显著优于实施例2。
(2)响应性能：不同时间捕获CTC的回收率比较
实施例2与实施例3材料的不同时间捕获CTC的回收率比较，如图6所示。
采用本发明的等离子体聚合发的实施例3中的材料的响应时间可达3S，显著快于采用常规的非等离子体聚合法得到的材料。
(3)聚合物的接枝百分量比较
实施例2与实施例3材料的聚合物的接枝百分量比较，如图7所示。
实施例3所得聚合物修饰的磁性纳米材料中聚合物的质量与投料量的比例可达60％以上。
实施例2所得聚合物修饰的磁性纳米材料中聚合物的质量与投料量的比例仅在15％左右。
实施例5和7与实施例4和6相比，可得类似效果。
应用实施例1
聚合物修饰的磁性纳米材料在制备捕获循环肿瘤细胞的药物或试剂中的应用。如图8所示，具体应用步骤如下：
(1)取外周血样本4mL；
(2)将密度梯度分离液(Percoll细胞分离液)按顺序分层缓慢加入15mL离心管中；
(3)将外周血样本混匀，取1mL外周血样本用PBS稀释3-4倍；
(4)将稀释后的外周血样本缓慢加入上述装有梯度分离液的离心管中，离心1600r ×30min；
(5)离心后，取白细胞层于15mL离心管中，加4-6mL PBS重悬细胞，离心1600r×7min；
(6)离心后，去上清，用1mL PBS重悬细胞，并同步转移至1.5mL离心管中；
(7)超声活化药物或试剂(实施例3，PEI涂层的磁性纳米颗粒)，加30μL药物或试剂至上述1.5mL离心管中，并置于迷你旋转培养器上，4℃条件下4rpm/min孵育10分钟；
(8)孵育后，将离心管插入多功能磁分离器上，4℃磁吸附10分钟；
(9)去上清，加入1mL PBS混匀，再将离心管插入多功能磁分离器上，4℃磁吸附10分钟；
(10)去上清，加入200μL PBS，重悬混匀，甩片1-2张；
(11)用迪夫快速染色液(Diff-Quik Stain)进行染色；
(12)光学显微镜下判定计数。
如图10所示：细胞体积大；核质比高；核形态不一，可出现巨核、双核或多核现象；核深染且染色不均；细胞质常见脂肪粒；细胞膜表面褶皱或边界清楚。以上为肿瘤细胞的形态特征，满足上述4个及4个以上特征即判定为肿瘤细胞。
本实施例与市售强生CellSearch产品相比，本实施例仅需4mL外周血，且检测时间在2小时内完成；而强生CellSearch技术需要7.5mL外周血，检测至少需要6小时。
如图11所示，通过对捕获的CTC进行培养，可以看到培养10天、20天、30天后有明显的增殖，表明捕获的CTC是活细胞。
应用实施例2
采用应用实施例1的应用方法分别检测158例健康志愿者和853例恶性肿瘤志愿者，收集2020年1月至2021年12月于泉州第一医院确诊的215例结肠癌患者，年龄34-86周岁，男性149例，女性66例；188例肺癌患者，年龄32-85周岁，男性113例，女性75例；145例直肠癌患者，年龄42-78岁，男性104例，女性41例；94例胃癌患者，年龄30-87岁，男性61例，女性33例；86例乳腺癌患者，年龄33-74岁，男性1例，女性85例；74例食管癌患者，年龄51-82岁，男性55例，女性19例；51例肝癌患者，年龄43-76岁，男性40例，女性11例；并选择同时期该院的158例健康体检者为健康对照，年龄在22-75周岁，男性101例，女性57例。
本实施例中志愿者的选择标准如下：
1.纳入标准：
1.1恶性肿瘤志愿者
a、年龄在18岁以上(包括18岁)，性别不限；
b、经影像学及病理学确认的结肠癌、肺癌、直肠癌、胃癌、乳腺癌、食管癌、肝癌的单一恶性肿瘤患者；
c、仍处于放化疗、免疫治疗或靶向治疗中。
1.2健康志愿者
a、年龄在18岁以上(包括18岁)，性别不限；
b、血常规或尿常规结果正常。
2.排除标准
2.1恶性肿瘤志愿者
a、有其他并发症的恶性肿瘤患者；
b、结肠癌/肺癌/直肠癌/胃癌/乳腺癌/食管癌/肝癌外，既往有其他恶性肿瘤史。
2.2健康志愿者
a、长期服用药物；
b、有慢性病或肿瘤家族史；
c、体检发现结节或疑似肿瘤。
结果如图9所示，其中健康对照组的158例中仅有1例检出，假阳性率极低，仅为0.6％；215例结肠癌组中检出205例，检出率达95.3％；188例肺癌组检出183例，检出率达97.3％；145例直肠癌组检出139例，检出率达95.9％；94例胃癌组检出92例，检出率达97.9％；86例乳腺癌组检出76，检出率达88.4％；74食管癌组检出68，检出率达91.9％；51例肝癌组检出50例，检出率高达98％。

Claims (12)

  1. 一种聚合物修饰的磁性纳米材料，其特征在于，其包括如下结构：
    所述的聚合物为阳离子型聚合物；所述的聚合物附着或包覆于磁性纳米材料表面，形成带正电荷的所述聚合物修饰的磁性纳米材料；
    所述的磁性纳米材料为核-壳结构，所述的核为磁性纳米粒子，所述的壳为改性层；所述的改性层附着或包覆于所述磁性纳米粒子的表面，形成改性层复合的磁性纳米粒子；
    其中，所述的聚合物修饰的磁性纳米材料中，所述的聚合物与所述的磁性纳米材料的质量比为1∶10至20∶1。
  2. 如权利要求1所述的聚合物修饰的磁性纳米材料，其特征在于，所述聚合物修饰的磁性纳米材料满足下述条件中的至少一个：
    (1)所述的聚合物与所述的磁性纳米材料的质量比为1∶5至3∶1；例如1∶3；
    (2)所述的聚合物修饰的磁性纳米材料的电位为+5～+60mV，例如+10至+50mV，优选+20至+40mV；
    (3)所述的磁性纳米材料为带负电荷的磁性纳米材料，例如其电位可为-10～-60mV；例如-20～-40mV；
    (4)所述的聚合物修饰的磁性纳米材料的粒径为10nm至600nm；例如300nm至500nm，又例如350nm至400nm；
    (5)所述的磁性纳米材料的粒径可为5nm至500nm；例如，300nm至350nm；
    (6)所述的壳的厚度为1nm至100nm，例如40nm至60nm；
    (7)所述的磁性纳米粒子的粒径为5nm至500nm；例如250nm至300nm；
    (8)所述的聚合物为聚乙烯亚胺、壳聚糖和聚吡咯中的一种或多种；
    (9)所述的聚合物为支状聚合物；
    (10)所述的聚合物为重均分子量MW在2000至300000之间；
    (11)所述的磁性纳米粒子为氧化物磁性纳米粒子、磁性金属纳米粒子、磁性硫化物纳米粒子、磁性复合粒子中的一种或几种；所述氧化物磁性纳米粒子可为Fe 3O 4或γ-Fe 2O 3；例如磁性Fe 3O 4纳米粒子；
    (12)所述的改性层的材料为二氧化硅或标记荧光和/或表面活性剂修饰的二氧化硅；例如二氧化硅或标记荧光的二氧化硅；
    (13)所述的改性层的表面含有修饰得到的氨基；
    (14)所述的改性层与所述的磁性纳米粒子的质量比为50∶1～1∶10；例如1∶2至10∶1；
    (15)所述聚合物修饰的磁性纳米材料的稳定时长为2年；
    (16)所述聚合物修饰的磁性纳米材料的响应时间为3S至2min。
  3. 如权利要求2所述的聚合物修饰的磁性纳米材料，其特征在于，所述聚合物修饰的磁性纳米材料满足下述条件中的至少一个：
    (1)当所述的聚合物为聚乙烯亚胺时，所述的聚乙烯亚胺的重均分子量为2000-100000；例如MW＝10000，99％纯度；
    (2)当所述的聚合物为β-壳聚糖时，所述的β-壳聚糖重均分子量为50000-300000，例如MW＝50000；
    (3)当所述的聚合物为聚吡咯时，所述的聚吡咯的重均分子量为5000；
    (4)当所述的磁性纳米材料为标记荧光的二氧化硅复合的磁性纳米粒子时，所述的标记荧光的二氧化硅复合的磁性纳米粒子中的荧光染料为异硫氰酸荧光素和/或罗丹明类染料，和/或其被修饰物；例如异硫氰酸荧光素、罗丹明B、罗丹明B异硫氰酸酯和四甲基异硫氰酸罗丹明中的一种或多种；所述的修饰物可为APS修饰的异硫氰酸荧光素和/或APS修饰的罗丹明类染料；所述APS可为3-氨基丙基三乙氧基硅烷和/或3-氨基丙基三甲基硅烷；又例如所述荧光染料为APS-FITC；
    (5)所述的磁性纳米材料为表面活性剂修饰的二氧化硅复合的磁性纳米粒子；所述的表面活性剂可包括乙酸钠、柠檬酸三钠、壳聚糖、聚乙烯吡咯烷酮、聚对苯二甲酸乙二醇酯、硬脂酸、阿拉伯树胶、羟丙基甲基纤维素、海藻酸钠、十二烷基硫酸钠、十二烷基苯磺酸钠、聚乙烯醇、长链脂肪酸、淀粉和十二硫醇中的一种或两种以上的组合；
    (6)当所述的磁性纳米材料为标记荧光的二氧化硅复合的磁性纳米粒子时，所述的二氧化硅复合的磁性纳米粒子与所述的荧光染料的质量比值为20；
    (7)当所述的磁性纳米材料为标记荧光的二氧化硅复合的磁性纳米粒子时，所述的聚合物修饰的磁性纳米材料的荧光强度为40-1200；
    (8)当所述的磁性纳米材料为二氧化硅改性层复合的磁性纳米粒子时，所述的二氧化硅改性层复合的磁性纳米粒子为Fe 3O 4@SiO 2；
    (9)当所述的磁性纳米材料为标记荧光的二氧化硅改性层复合的磁性纳米粒子时，所述的标记荧光的二氧化硅改性层复合的磁性纳米粒子为APS-FITC标记的Fe 3O 4@SiO 2。
  4. 如权利要求1所述的聚合物修饰的磁性纳米材料，其特征在于，
    所述的聚合物修饰的磁性纳米材料选自如下任一方案：
    方案1、
    所述的聚合物修饰的磁性纳米材料为聚乙烯亚胺修饰的APS-FITC荧光标记的 Fe 3O 4@SiO 2；其中，所述的聚乙烯亚胺重均分子量MW＝10000，所述聚乙烯亚胺与所述磁性纳米材料的质量比可为1∶3；所述的聚合物修饰的磁性纳米材料的粒径可为20nm～500nm；其电位可为+10mV～+60mV；
    方案2、
    所述的聚合物修饰的磁性纳米材料为β-壳聚糖修饰的APS-FITC荧光标记的Fe 3O 4@SiO 2；其中，所述的β-壳聚糖的重均分子量MW＝50000；所述β-壳聚糖与所述磁性纳米材料的质量比可为1∶3；所述的聚合物修饰的磁性纳米材料的粒径可为20nm～500nm；其电位可为+10～+60mV；
    方案3、
    所述的聚合物修饰的磁性纳米材料为聚吡咯修饰的APS-FITC荧光标记的Fe 3O 4@SiO 2；其中，所述的聚吡咯的重均分子量为5000；所述聚吡咯与所述磁性纳米材料的质量比可为1∶3；所述的聚合物修饰的磁性纳米材料的粒径可为20nm～500nm；其电位可为+10～+60mV。
  5. 一种聚合物修饰的磁性纳米材料的制备方法，其特征在于，其包括如下步骤：
    将聚合物与溶剂的混合物与磁性纳米材料进行改性修饰，得到聚合物修饰的磁性纳米材料即可；其中，所述的聚合物与溶剂的混合物为雾化形态；
    所述聚合物、所述磁性纳米材料的定义如权利要求1-4中任一项所述。
  6. 如权利要求5所述的制备方法，其特征在于，所述制备方法满足下述条件中的至少一个：
    (1)所述的混合物与所述的磁性纳米材料采用等离子体法进行改性修饰；
    (2)所述的溶剂为醇类溶剂，所述的醇类溶剂可为甲醇；
    (3)所述的聚合物在所述的混合物中的质量体积比为5mg/mL；
    (4)所述的雾化形态通过加热所述的聚合物与溶剂的混合物得到；例如，将所述的聚合物与溶剂的混合物通过等离子体法加热得到所述的雾化形态；
    (5)所述的聚合物与溶剂的混合物的加入为控制所述的混合物体积流量在3-5sccm；
    (6)所述的改性修饰的温度为100至300℃；例如200℃；
    (7)所述的改性修饰在惰性气氛存在下进行；所述的惰性气氛可为氮气和/或氩气；
    (8)所述的改性修饰的反应的时间为1-2小时；
    (9)当所述的磁性纳米材料为二氧化硅复合的磁性纳米粒子或标记荧光的二氧化硅复合的磁性纳米粒子、所述的二氧化硅复合的磁性纳米粒子为Fe 3O 4@SiO 2时，所述的磁性纳米材料采用如下步骤制备得到：
    步骤(a)在碱性试剂存在下，将硅试剂加入到Fe 3O 4磁性纳米微粒与溶剂的体系中进行改性反应，得到所述的Fe 3O 4@SiO 2即可；和/或，
    步骤(b)将所述的Fe 3O 4@SiO 2与荧光染料进行荧光标记反应，得到所述的标记荧光的二氧化硅复合的磁性纳米粒子即可。
  7. 如权利要求6所述的制备方法，其特征在于，所述的磁性纳米材料的制备中，满足下述条件中的至少一个：
    (1)所述的聚合物修饰的磁性纳米材料的制备方法，其包括如下步骤：在惰性气氛存在下，在等离子体辉光存在下，将所述的聚合物与溶剂的混合物加热得到雾化形态，与所述的磁性纳米材料进行改性修饰；得到所述的聚合物修饰的磁性纳米材料即可；
    其中，所述的等离子体辉光可为如下步骤得到，在所述的惰性气氛下，调节射频功率，使等离子反应腔内产生等离子体辉光；所述惰性气氛的压力可为在300-400Pa之间；所述射频的功率可为10W±5W；较佳地，在真空下，射频电源预热，再向等离子体反应腔通所述的惰性气氛；所述的真空可为200Pa以下，例如150-200Pa；
    (2)步骤(a)中，所述的溶剂为水，或水和醇类溶剂，所述的醇类溶剂可为乙醇；
    (3)所述的碱性试剂为氨水；
    (4)所述的硅试剂为正硅酸乙酯或正硅酸甲酯；例如正硅酸乙酯；
    (5)所述的Fe3O4磁性纳米微粒与所述的二氧化硅试剂质量体积比为1500g/L；
    (6)所述的二氧化硅试剂以与所述的溶剂的混合物形式使用；例如2mL乙醇溶解100μl正硅酸乙酯；
    (7)所述的碱性试剂的用量为使所述的Fe 3O 4磁性纳米微粒与溶剂的体系的pH为9.5±0.5即可；
    (8)所述的改性反应在超声和/或机械搅拌条件下进行；
    (9)步骤(a)中，其还包括后处理步骤，所述的后处理为如下步骤，所述的反应结束后，洗涤经磁分离辅助条件获得的所述的Fe 3O 4@SiO 2，即可；所述的洗涤可为分别使用乙醇和去离子水洗涤，例如洗涤三次；较佳地，洗涤后，将得到的Fe 3O 4@SiO 2分散在去离子水中，配制成所需浓度的溶液待用即可，例如浓度为100mg/mL的溶液；
    (10)步骤(b)中，所述的溶剂为醇类溶剂和水的混合物；所述的水可为去离子水；所述的醇类溶剂可为乙醇；所述的醇类溶剂和水的体积比可为9∶1～10∶1；例如9.7∶1；
    (11)步骤(b)中，所述的二氧化硅复合的磁性纳米粒子与所述的溶剂的质量体积比为0.56至0.6g/L；
    (12)步骤(b)中，所述的碱性试剂为氨水；所述的二氧化硅复合的磁性纳米粒子与所 述的氨水的质量体积比可为42至45g/L；
    (13)步骤(b)中，所述的荧光染料为与所述的溶剂的混合物形式使用；所述的溶剂与所述的荧光染料的体积质量可为1.7mL/mg；
    (14)所述的荧光标记反应在超声和机械搅拌条件下进行；
    (15)所述的荧光标记反应在避光条件下进行；
    (16)步骤(b)中，所述的荧光标记反应中，还加入如步骤(a)所述的二氧化硅试剂，即同时进行二氧化硅进一步包被；所述的二氧化硅复合的磁性纳米粒子与所述的二氧化硅试剂的质量体积比可为1000g/L；所述的硅试剂可为与所述的溶剂的混合物形式；例如1mL乙醇中含30μl正硅酸乙酯；
    (17)步骤(b)中，其还包括后处理步骤，所述的后处理为如下步骤，所述的反应结束后，洗涤经磁分离辅助条件获得的所述的磁性纳米粒子，即可；所述的洗涤可为分别使用乙醇和去离子水洗涤，例如洗涤三次；
    (18)当所述的荧光染料为APS-FITC时，为如下步骤制备得到：将APS加入到FITC的乙醇溶液中反应，得到所述的APS-FITC即可；其中，所述的反应较佳地在避光条件下进行；所述的反应以得到澄清的溶液即可，例如混合过夜；FITC与APS的质量体积比可为300g/L；
    (19)步骤(a)中，所述的Fe 3O 4磁性纳米微粒与溶剂的体系采用如下步骤制备得到：在超声和机械搅拌条件下，在溶剂中，将Fe 3O 4纳米磁珠依次用盐酸、去离子水洗涤至上清液pH中性，即可；所述的盐酸可为3.6％～36％盐酸；
    (20)步骤(a)中，当所述的磁性纳米材料中的磁性纳米粒子为Fe 3O 4时，其由如下步骤制备得到：将FeCl 3·6H 2O和碱金属盐的乙二醇溶液进行反应，得到所述的Fe 3O 4纳米微粒即可；其中，所述的碱金属盐可选自柠檬酸三钠和/或NaAc；所述的FeCl 3·6H 2O与NaAc的摩尔比可为1∶10；所述的溶剂与FeCl 3·6H 2O的体积摩尔比可为10L/mol；所述的反应的温度可为200℃；其还可包括后处理步骤，所述的后处理可为如下步骤，所述的反应结束后，洗涤经磁分离辅助条件获得的所述的Fe 3O 4磁性纳米微粒，即可；所述的洗涤可为分别使用乙醇和去离子水洗涤，例如洗涤三次；较佳地，洗涤后，将得到的Fe 3O 4磁性纳米微粒分散在去离子水中，配制成所需浓度的溶液待用即可，例如浓度为100mg/mL的溶液。
  8. 一种聚合物修饰的磁性纳米材料，其特征在于，其采用如权利要求6或7中任一方案制备得到；
    较佳的，所述的聚合物修饰的磁性纳米材料为如权利要求1-4中任一项所述的聚合 物修饰的磁性纳米材料中任一方案所示。
  9. 一种等离子体法在制备聚合物修饰的磁性纳米材料中的应用；较佳地，所述的应用中，在等离子体辉光存在下，将聚合物与溶剂的混合物与纳米材料进行改性修饰反应；
    所述的混合物、所述的磁性纳米材料及所述的制备方法的操作和条件可如权利要求6或7所述的聚合物修饰的磁性纳米材料的制备方法中任一方案所述的混合物、所述的磁性纳米材料及所述的条件和操作所示；
    和/或，相应的聚合物修饰的磁性纳米材料的定义可如权利要求1-4或8中任一项所述的聚合物修饰的磁性纳米材料中任一方案所示。
  10. 一种聚合物修饰的磁性纳米材料在糖基化蛋白、多肽类物质、核酸、循环肿瘤细胞、外泌体的富集分离中的应用方法，其特征在于，所述的聚合物修饰的磁性纳米材料的定义如权利要求1-4或8中任一项所述的聚合物修饰的磁性纳米材料中任一方案所示；
    所述的应用可为所述的聚合物修饰的磁性纳米材料在制备活体荧光和磁共振双模态成像显影剂、电化学细胞传感器、用于循环肿瘤细胞捕获的药物和/或医疗产品、或用于治疗癌症的光热治疗剂中的应用；例如，用于细胞示踪、肿瘤示踪成像、磁热疗成像或血管成像；
    所述肿瘤细胞可选自以下一种或多种：卵巢癌肿瘤细胞、宫颈癌肿瘤细胞、非小细胞肺癌肿瘤细胞、结肠癌细胞、肺癌细胞、直肠癌细胞、胃癌细胞、乳腺癌细胞、食管癌细胞、肝癌细胞、白血病。
  11. 如权利要求10所述的应用，其特征在于，所述的应用可为所述的聚合物修饰的磁性纳米材料在制备捕获循环肿瘤细胞的药物或试剂中的应用。
  12. 如权利要求10所述的应用，其特征在于，
    所述药物或试剂的检测对象为外周血/体液样本；
    和/或，所述循环肿瘤细胞包括卵巢癌肿瘤细胞、宫颈癌肿瘤细胞、非小细胞肺癌肿瘤细胞、结肠癌细胞、肺癌细胞、直肠癌细胞、胃癌细胞、乳腺癌细胞、食管癌细胞、肝癌细胞、白血病；
    和/或，所述药物或试剂捕获外周血样本中循环肿瘤细胞的方法具体包括以下步骤：
    S1、取外周血样本，用密度梯度液进行密度梯度离心，取中间段的白细胞层，去掉血浆和红细胞；
    S2、将白细胞层的细胞稀释离心，重悬细胞获得细胞悬浮液，去掉蛋白及杂质；
    S3、超声活化所述药物或试剂，并将活化后的药物或试剂与S2获得的细胞悬浮液以体积比3∶100混合，进行吸附反应；
    S4、磁场分离S3中吸附细胞悬浮液后的药物或试剂，富集循环肿瘤细胞，然后将细胞重悬、甩片、迪夫快速染色；
    S5、显微镜下阅片，并根据肿瘤形态学进行鉴定和计数；
    优选地，S1中密度梯度离心前外周血样本采用PBS稀释3-4倍；
    优选的，S3中的吸附反应、S4中的磁场分离及富集反应均是在4℃条件下进行。
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