CN105126714A - Functional nano particle composite microsphere, preparation and applications thereof - Google Patents

Abstract

The invention discloses a functional nanoparticle composite microsphere, the functional nanoparticle composite microsphere is a functional nanoparticle composite PEG microsphere, and the functional nanoparticle composite PEG microsphere contains functional nanoparticles and The polymer of the PEG chain segment, the average particle diameter of the functional nanoparticle composite PEG microsphere is 0.1-1000 μm, and the variation coefficient of particle size distribution is less than 10%. The present invention prepares composite microspheres with a uniform particle size by preparing functional nanoparticles and polymers grafted with PEG segments, and its PEG segment components can effectively act as non-specific adsorption inhibitors, and can be used in biological detection and biological The field of medicine has broad application prospects.

Description

A kind of functional nanoparticle composite microsphere and its preparation and application

technical field

The invention relates to the field of preparation and application of micro-nano materials, in particular to functional nanoparticle composite microspheres and applications thereof.

Background technique

Functional nanoparticle composite microsphere refers to a functional composite microsphere obtained by combining functional nanoparticles and microspheres by a certain method. At present, nanoparticles prepared by various methods have various special optical, electrical, magnetic and biological characteristics, so these characteristics are often endowed to the microsphere itself after being combined with the microsphere. Microspheres also provide support and effective protection for these nanoparticles. At the same time, the chemical and physical properties of the microspheres themselves, such as light sensitivity, pH responsiveness, temperature sensitivity, adsorption properties, and surface active functional groups, also provide possibilities for the application of nanoparticles in various complex fields. Nanoparticle composite microspheres with different special functions have great application potential in the fields of biomedicine, industrial catalysis, chemical synthesis, electronic information, and building materials.

At this stage, functional nanoparticle composite polymer microspheres have been applied in various fields: Nie's research team embedded semiconductor nanoparticle quantum dots into porous microspheres by using the swelling infiltration method, and used the carboxyl functional groups on the surface of the microspheres to bind DNA segments Linked to the surface of the microsphere as a probe, DNA-specific detection was carried out. JunLin's group prepared up-conversion particle hydrogel core-shell structure microspheres NaYF4:Yb ³⁺ /Er ³⁺ Hydrogel, and used the fluorescence properties of fluorescent up-conversion nanoparticles to realize near-infrared excitation cell imaging, while encapsulating drugs in the shell and The pH responsiveness of the hydrogel is used to achieve the purpose of controlled drug release. ChunhuiDeng's group synthesized magnetic polymer composite microspheres Fe ₃ O ₄ SiO ₂ PMMA, and successfully used it for the magnetic separation and enrichment of biomolecular nucleic acids and proteins. This technology has important application value in the fields of mass spectrometry and other fields.

Among them, in application fields such as targeted imaging, targeted drug release, immunoassay, and immunomagnetic separation, microspheres are required to have the effect of inhibiting non-specific adsorption so as to improve the application effect. Various natural polymers such as chitosan, albumin have been shown to act as potent non-specific inhibitors. However, natural polymers have obvious disadvantages, that is, they may contain unpredictable viruses, and in many cases, their non-specific adsorption inhibition effect is not good. Therefore, synthetic polymer brushes (polymer brushes) acting as non-specific adsorption inhibitors have received increasing attention.

So far, there have been no reports on non-specific adsorption on the surface of nanoparticle composite microspheres.

Contents of the invention

In view of the defects of the prior art, the present invention provides a functional nanoparticle composite microsphere, and the adopted technical scheme is as follows:

A functional nanoparticle composite microsphere, the functional nanoparticle composite microsphere is a functional nanoparticle composite PEG microsphere, and the functional nanoparticle composite PEG microsphere includes functional nanoparticles and PEG chain segments For the polymer, the average particle size of the functional nanoparticle composite PEG microsphere is 0.1-1000 μm, and the variation coefficient of particle size distribution is less than 10%. PEG is polyethylene glycol; the coefficient of variation of particle size distribution is a statistic to measure the degree of variation in the particle size of each particle.

Further, the functional nanoparticles are one or more of the following: quantum dots, magnetic nanoparticles, fluorescent nanoparticles, metal nanoparticles, metal oxide nanoparticles and semiconductor nanoparticles; the quantum dots are the following One or more of: CdS, HgS, CdSe, CdTe, ZnSe, HgSe, ZnTe, ZnO, PbSe, HgTe, CaAs, InP, InCaAs, CdSe/CdS, CdSe/ZnS, CdSe/ZnSe, CdS/ZnS, Cd/Ag ₂ S, CdS/Cd(OH) ₂ , CdTe/ZnS, CdTe/CdS, CdSe/ZnSe, CdS/HgS, CdS/HgS/CdS, ZnS/CdS, ZnS/CdS/ZnS, ZnS/HgS/ ZnS/CdS, CdSe/CuSe, CdSeTe, CdSeTe/CdS/ZnS, CdSe/CdS/ZnS, CuInS ₂ , CuInSe ₂ , CuInS ₂ /ZnS, CuInSe ₂ /ZnS, and doped quantum dots CdS:Mn, CdS:Mn , CdS:Cu, ZnS:Cu, CdS:Tb, ZnS:Tb.

Further, the PEG segment is grafted to one or more of the following polymers: polystyrene, polyacrylic acid, polymethacrylic acid, polymethylmethacrylate, polyethylmethacrylate, polyamide , polyacrylonitrile, polycarbonate, polycaprolactone, polyurethane, polylactic acid, chitosan, albumin, collagen, polystyrene maleic anhydride copolymer, polyethyl acetate, polystyrene acrylic acid copolymer, poly Styrene methacrylic acid copolymer or polystyrene-methyl methacrylate copolymer, poly(styrene-divinylbenzene-methacrylic acid) and maleic anhydride-1-octadecene copolymer. The polymer containing the PEG segment can be the following two possibilities: one is to graft the PEG segment on the polymer before forming into a ball, and then compound it with nanoparticles to form a ball; the other is to first combine the polymer and the nano The particles are composited into spheres, and then PEG segments are grafted on the surface of the microspheres.

Further, the PEG segment includes PEG or PEG derivatives with a molecular weight in the range of 500 to 10,000 Da, and the PEG derivatives include monofunctional PEG, homogeneous bifunctional PEG, hetero(base) bifunctional PEG, and multi-arm PEG. , Y-shaped structure PEG, dendritic structure PEG or a mixture thereof.

Further, the functional nanoparticle composite PEG microspheres are surface-modified with functional groups; the surface modification is one or more of the following: hydrolysis, chemical grafting or sulfonation; the functional groups are the following One or more of: carboxyl, amino, sulfonate, nitro, hydroxyl, mercapto, chlorine or ester.

Further, one or more of the following linkers are connected to the functional group: N-hydroxysuccinimide, biotin, avidin and streptavidin.

Further, the preparation method includes swelling method, microfluidic method and membrane emulsification-emulsion solvent evaporation method. Swelling method: that is, cross-linked microspheres with a pore structure are synthesized in advance, and then the microspheres are swollen in a good solvent, and the surface micropores are expanded. At this time, functional nanoparticles are added, and the functional nanoparticles can Penetrate into the microspheres through the pore structure and adsorb on the inner wall of the microspheres. When the external solvent is removed, the microspheres shrink, thereby embedding functional nanoparticles into the microspheres. Microfluidic method: If the nanoparticles and polymers used in the preparation of functional nanoparticle composite microspheres are hydrophobic, the polymers and nanoparticles are dissolved in a non-polar organic solvent in advance, and the polymer fluid is In the microfluidic channel, the water phase at a certain flow rate is broken and emulsified to form droplets, and the formed droplets are solidified into balls after the solvent volatilizes. Membrane emulsification-emulsion solvent evaporation method: If the nanoparticles and polymers used in the preparation of functional nanoparticle composite microspheres are hydrophobic, the polymers and nanoparticles are dissolved in non-polar organic solvents in advance as the dispersed phase , and the aqueous solution containing surfactant is used as the continuous phase, the dispersed phase and the continuous phase are placed on both sides of the rigid porous membrane, and the dispersed phase is pressed into the continuous phase through the uniform microporous structure on the porous membrane under a certain nitrogen pressure. At this time, the stabilizer in the continuous phase will be adsorbed on the surface of the droplet to reduce the surface tension and stabilize the droplet, and then the formed droplet will be solidified into a ball after the solvent volatilizes.

Application of the functional nanoparticle composite microsphere in detecting one or more target objects in a sample.

A biological detection probe based on functional nanoparticle composite microspheres, said biological detection probe comprising functional nanoparticle composite PEG microspheres according to any one of claims 1-7 and coupling in said functional Probe molecules on the surface of the nanoparticle composite PEG microsphere; the probe molecules are selected from one or more of the following: proteins, protein fragments and nucleic acids.

The application of the biological detection probe in the detection of one or more target objects in a sample.

The present invention will be further described below in conjunction with the accompanying drawings, in order to fully illustrate the purpose, technical features and technical effects of the present invention.

Description of drawings

Fig. 1 is the composite PEG microsphere scanning electron micrograph of embodiment 4;

Fig. 2 is the composite PEG microsphere laser confocal micrograph of embodiment 8;

Fig. 3 is the result that the biodetection probe of embodiment 9 detects tumor marker AFP, and the comparison with the detection result based on non-PEG microsphere bioprobe;

FIG. 4 is a partially enlarged view of FIG. 3 .

Detailed ways

The specific embodiments provided by the present invention will be described in detail below in conjunction with the accompanying drawings.

1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide (EDC) activation of carboxyl groups on the surface of composite microspheres, attachment of probe molecules, phycoerythrin labeling of antigens such as alpha-fetoprotein (AFP) Such operations are the basic operations of clinical immunological detection and diagnosis. For details, please refer to "Guidelines for Clinical Immunological Testing and Experiments", author: Lu Shijing, publisher: China Medical Science Press.

Embodiment 1: the preparation of PEG graft polymer (one)

Dissolve 1 g of polystyrene maleic anhydride copolymer (PSMA) and 2 g of polyethylene glycol monomethyl ether (mPEG1000) with a molecular weight of 1000 in 20 ml of dimethylformamide (DMF), and add 100 mg of catalyst p-toluenesulfonic acid , dissolved at room temperature for 2 hours, mixed evenly, and heated to 100 degrees Celsius under nitrogen protection for 12 hours. After the reaction was completed, anhydrous ether was added to precipitate the obtained polymer PSMA-PEG, and the polymer was freeze-dried after repeated centrifugation and washing for three times.

Embodiment 2: the preparation of PEG graft polymer (two)

Dissolve 1g of polystyrene acrylic acid copolymer and 5g of carboxy-terminated polyethylene glycol monomethyl ether (mPEG-COOH) with a molecular weight of 5000 in 30ml of chloroform, add a few drops of concentrated sulfuric acid, heat and condense under _N2 protection conditions and reflux for 12h, react After completion, anhydrous ether was added to precipitate the polymer, and after repeated centrifugation and washing three times, the polymer was freeze-dried for use.

Embodiment 3: the preparation of PEG graft polymer (three)

Dissolve maleic anhydride/1-octadecene alternating copolymer (PMAO) and 10 g of one-end amino-terminated PEG (mPEG-NH ₂ ) with a molecular weight of 2000 in 30 ml of chloroform, stir for 24 hours, then add absolute ethanol to precipitate the polymer out, and remove the unreacted PEG molecules, centrifuge and wash three times, and freeze-dry the polymer for use.

Embodiment 4: the preparation of composite PEG microsphere powder (one)

The membrane emulsification device used in this example is a pressure type membrane emulsification device, which is purchased from Japan SPGtechnology company; the specific process is to dissolve the PSMA-PEG in Example 1 and the CdS/ZnS quantum dots with an emission wavelength of 518nm in toluene, polymerize The concentration of quantum dots is 1.5g/mL, and the concentration of quantum dots is 2nM/L, which is used as the dispersed phase. The SPG porous membrane with a pore diameter of 5 μm is adopted, and the dispersed phase is extruded through the membrane by nitrogen gas with a pressure of 15KPa, and enters a water continuous phase containing an emulsifier SDS concentration of 1wt.%. The flow velocity of the continuous phase is 0.35m/s, and the liquid is obtained. Oil-in-water emulsion with uniform particle size. Stir and volatilize at 25° C. and 350 rpm under magnetic stirring. After the toluene in the solution is completely volatilized, the obtained suspension of quantum dot-labeled fluorescent microspheres is collected by centrifugation. After that, it was centrifuged and washed three times with deionized water and three times with absolute ethanol, and freeze-dried to obtain the solid powder of quantum dot composite microspheres.

Observation by scanning electron microscope shows that the prepared quantum dot composite fluorescent microspheres are spherical particles with smooth surface, the average particle size is 7.1 μm, the coefficient of variation of particle size distribution CV is about 9.7%, and the monodispersity is good, see Figure 1.

Embodiment 5: the preparation of composite PEG microsphere powder (two)

What used in this example is a self-made microfluidic device with a T-shaped joint. The specific process is that the PEG _- grafted polystyrene acrylic acid copolymer and Fe in Example ₂ are dissolved in chlorine, and the concentration of the polymer is 1g/mL, Fe ₃ O ₄ concentration is 1nM/L, with it as dispersed phase; Containing the aqueous solution that concentration is 0.5wt.% emulsifier PVA and concentration is 0.5wt.% SDS as continuous phase, and dispersed phase from The injections from different channels converge and emulsify at the T-shaped interface. The flow rate of the dispersed phase was 30 mL/h, and the flow rate of the continuous phase was 1 L/h, to obtain an oil-in-water emulsion with uniform droplet size. Stir and volatilize at 25° C. and 350 rpm under magnetic stirring. After the chloroform in the solution is completely volatilized, collect the obtained magnetic bead suspension by magnetic separation. After that, it was washed 3 times, washed 3 times with absolute ethanol, and freeze-dried to obtain solid powder of magnetic beads.

Observed by scanning electron microscope, the prepared quantum dot composite fluorescent microspheres are spherical particles with smooth surface, the average particle size is 300 μm, the variation coefficient CV of particle size distribution is about 8.7%, and the monodispersity is good.

The preparation of embodiment 6 composite PEG microsphere powder (three)

The PMAO-PEG among the example ₃ and the NaYF4 rare earth nanoparticle that excitation wavelength is 980nm are dissolved in toluene, PMAO-PEG copolymer concentration is 2g/mL, NaYF4 rare earth nanoparticle concentration is 1nM/L, with it as dispersed phase . The SPG porous membrane with a pore diameter of 3 μm is adopted, and the dispersed phase is extruded through the membrane by nitrogen gas with a pressure of 20KPa, and enters a water continuous phase containing an emulsifier SDS concentration of 1wt.%. The flow velocity of the continuous phase is 0.40m/s, and the liquid is obtained. Oil-in-water emulsion with uniform particle size. Stir and volatilize at 25° C. and 350 rpm magnetic stirring. After the toluene in the solution is completely volatilized, the obtained rare earth nanoparticle composite fluorescent microsphere suspension is collected by centrifugation. Afterwards, it was centrifuged and washed three times with deionized water and three times with absolute ethanol, and freeze-dried to obtain solid powder of rare earth nanoparticle composite fluorescent microspheres.

Observed by scanning electron microscope, the prepared quantum dot composite fluorescent microspheres are spherical particles with smooth surface, the average particle size is 4.1 μm, the CV value of the particle size distribution variation coefficient is about 6.2%, and the monodispersity is good.

The preparation of embodiment 7 composite PEG microsphere powder (four)

Prepare porous polymer microspheres first, and the first step is to prepare seed polystyrene (PS) microspheres, which is to dissolve 2g polyvinylpyrrolidone (PVP) in 100g continuous phase (mixed solvent of absolute ethanol and ethylene glycol methyl ether) ); Then add the styrene monomer containing the oil-soluble initiator AIBN, under the magnetic stirring at a speed of 120rpm, the temperature is raised to 70° C. after passing nitrogen for 15 minutes, and the reaction is carried out under nitrogen protection for 12 hours. Finally, the obtained seed polystyrene (PS) microsphere suspension was centrifuged at 4000 rpm, washed with deionized water and ethanol three times each, freeze-dried and stored for later use.

Add 0.2g of PS seed microspheres and 0.1mL of chlorododecane (CD, as an activator) to 25mL of 0.25wt.% sodium dodecyl sulfate (SDS) aqueous solution, and ultrasonically disperse them for 5 minutes. Mix the two, and then sonicate for 10 minutes and then swell for 12 hours under magnetic stirring at 30°C. Then the mixed monomer (comprising styrene monomer, functional monomer methacrylic acid MAA, crosslinking agent divinylbenzene DVB) that is dissolved with 0.06gBPO is added in the aqueous solution that is dissolved with 0.25wt.%SDS, ultrasonic Disperse for 15 minutes, then mix with the activated seed microsphere solution and swell for 12 hours. Then add PVA, the concentration is 1wt.% of the whole solution, and then heat up to 70° C. after 15 minutes of blowing nitrogen, and react under nitrogen protection for 12 hours. The microsphere suspension obtained after the reaction was centrifuged at 4000 rpm, washed three times with deionized water and ethanol, and then freeze-dried to obtain surface carboxylated polystyrene microspheres. Finally, in the Soxhlet extractor, the microspheres were extracted with dichloromethane for 48 hours. After the linear PS molecules in the microspheres were extracted, the porous monodisperse poly(styrene-divinyl benzene-methacrylic acid) (PSDM) microspheres.

Add 0.5 mL of 10.0 nM TiO ₂ in chloroform to 1 mL of chloroform containing 10 ⁷ PSDM porous microspheres, seal the bottle, and swell the microspheres for 2 hours under magnetic stirring at 400 rpm. Then remove the seal, and continue to stir the solution at room temperature. With the volatilization of chloroform in the solution, the quantum dots inside the solution are gradually concentrated, and _TiO2 continuously penetrates into the microsphere under the effect of the concentration difference between the inside and outside of the microsphere. In the pore structure, stop stirring after the chloroform in the solution is completely volatilized. The microspheres were centrifuged and washed at 4000rpm with xylene, ethanol and water respectively to remove the quantum dots remaining on the outside of the microspheres. Finally, the microspheres collected by centrifugation were vacuum freeze-dried and stored in a solid powder state for later use.

Under the activation of EDC, the carboxyl groups on the surface of the obtained TiO ₂ /PSDM microspheres were connected to N-hydroxysuccinimide, and then connected to PEG with a molecular weight of 1000 and PEG with a molecular weight of 2000. The molar ratio of PEG molecules is 3:1.

Observed by scanning electron microscope, the prepared quantum dot composite fluorescent microspheres are spherical particles with smooth surface, the average particle size is 6 μm, the variation coefficient CV of particle size distribution is about 5.7%, and the monodispersity is good.

The preparation of embodiment 8 composite PEG microsphere powder (five)

The PSMA-PEG in Example 1 and CuInS ₂ /ZnS quantum dots and Fe ₃ O ₄ magnetic nanoparticles with an emission wavelength of 680nm were dissolved in toluene, the concentration of PSMA-PEG was 2g/mL, and the concentration of quantum dots was 1nM/L , the concentration of Fe ₃ O ₄ magnetic nanoparticles is 1nM/L, which is used as the dispersed phase. The SPG porous membrane with a pore size of 5 μm is adopted, and the dispersed phase is extruded through the membrane by nitrogen gas with a pressure of 20KPa, and enters a water continuous phase containing an emulsifier SDS concentration of 1wt.%. The flow velocity of the continuous phase is 0.40m/s, and the liquid is obtained. Oil-in-water emulsion with uniform particle size. Stir and volatilize at 25° C. and 350 rpm magnetic stirring. After the toluene in the solution is completely volatilized, the obtained suspension of quantum dot-labeled fluorescent microspheres is separated and collected by magnetic force. Then centrifuge washing with deionized water for 3 times and absolute ethanol for 3 times, and freeze-dry to obtain the solid powder of quantum dots/Fe ₃ O ₄ composite fluorescent magnetic microspheres with carboxyl groups on the surface. Observation by scanning electron microscope, the prepared quantum dots/Fe ₃ O ₄ composite fluorescent magnetic microspheres are spherical particles with smooth surface, the average particle size is 6.8 μm, the variation coefficient CV of particle size distribution is about 9%, and the monodispersity is good. .

Through laser confocal observation, the distribution of quantum dots inside the microspheres is uniform, as shown in Figure 2.

Example 9 Biodetection probe based on functional nanoparticles composite PEG microspheres (1)

The surface carboxyl groups of the quantum dots/Fe ₃ O ₄ composite PEG microspheres prepared in Example 8 were connected to N-hydroxysuccinimide under EDC, and then connected to the alpha-fetoprotein antibody AFP-Ab as a probe molecule. The quantum dot composite fluorescent microsphere forms a biological detection probe for specific detection of alpha-fetoprotein AFP after the surface is connected with AFP-Ab. After the biological detection probe is put into the sample containing alpha-fetoprotein AFP for reaction, the target alpha-fetoprotein AFP connected to the probe is labeled with fluorescein isothiocyanate. The biodetection probe after the final reaction emits a fluorescent signal under laser excitation, and the alpha-fetoprotein AFP in the sample is qualitatively compared with the fluorescent signal of the quantum dot inside the microsphere and the labeled fluorescent signal intensity of the target object connected to the probe molecule. quantitative analysis. At the same time, a control group experiment was carried out. The control group experiment used carboxylated polystyrene-maleic anhydride microspheres (non-PEG microspheres), and its internal nanoparticle concentration and biological probe experiments were consistent. The test results are shown in Figure 3. It can be seen from the figure that the two types of microspheres can perform highly sensitive quantitative detection of alpha-fetoprotein, while the detection sensitivity of the PEG microsphere probe is about an order of magnitude higher. The fluorescent signal intensity was significantly lower, indicating its effect in suppressing non-specific adsorption.

Example 10 Biodetection probe based on functional nanoparticles composite PEG microspheres (2)

The rare earth nanoparticle composite PEG microsphere prepared in Example 6 was activated by EDC, and a DNA segment with the sequence 5'-TCAAGGCTCAGTTCGAATGCACCATA-3' was connected as a probe molecule to form a biological detection probe for specific detection of DNA. Put this biological detection probe into the sample containing the DNA segment of 5'-TATGGTGCATTCGAACTGAGCCTTGA-3' for reaction, and then the target connected to the probe, that is, the DNA with the sequence of 5'-TATGGTGCATTCGAACTGAGCCTTGA-3' Segments are labeled with Cascade blue. Finally, the treated rare earth nanoparticle composite microsphere emits a fluorescent signal under the excitation of infrared laser. The fluorescent signal of the rare earth nanoparticle inside the microsphere and the labeled fluorescent signal of the target connected to the probe molecule are paired with 5'-TATGGTGCATTCGAACTGAGCCTTGA-3 'Qualitative and quantitative analysis of DNA segments.

Example 11 Biodetection probe based on functional nanoparticles composite PEG microspheres (3)

The surface carboxyl groups of the quantum dot composite PEG microspheres prepared in Example 3 were linked with N-hydroxysuccinimide under the activation of EDC. Then, the 518nm quantum dot composite microsphere is further connected with the anti-alpha-fetoprotein antibody AFP-Ab as the detection probe molecule. The 518nm quantum dot composite microsphere forms a biological detection probe for specific detection of alpha-fetoprotein AFP after the surface is connected with alpha-fetoprotein antibody AFP-Ab. The 680nm quantum dot composite microsphere is connected with the anti-carcinoembryonic antigen antibody CEA-Ab as the detection probe molecule. The 680nm quantum dot composite microsphere forms a biological detection probe for specific detection of carcinoembryonic antigen CEA after the surface is connected with antibody CEA-Ab. After the two biological detection probes are put into a sample containing both alpha-fetoprotein AFP and carcinoembryonic antigen CEA for reaction, the target connected to the probe is then labeled with phycoerythrin. The reacted biological detection probe emits a fluorescent signal under laser excitation. The quantum dot fluorescence inside the microsphere at 518nm is the detection result of AFP, and the quantum dot fluorescence inside the microsphere at 680nm is the detection result of CEA. The labeled fluorescence intensity of the attached target was quantified for each of AFP and CEA within the sample.

For the above-mentioned embodiments 1-8, it should be noted that the above-mentioned embodiments are not exhaustive, and those skilled in the art should know that the functional nanoparticles can also be semiconductor nanoparticles or metal nanoparticles; Functional nanoparticles can also be the following quantum dots: HgS, CdSe, CdTe, ZnSe, HgSe, ZnTe, ZnO, PbSe, HgTe, CaAs, InP, InCaAs, CdSe/CdS, CdSe/ZnS, CdSe/ZnSe, CdS/ZnS , Cd/Ag ₂ S, CdS/Cd(OH) ₂ , CdTe/ZnS, CdTe/CdS, CdSe/ZnSe, CdS/HgS, CdS/HgS/CdS, ZnS/CdS, ZnS/CdS/ZnS, ZnS/HgS /ZnS/CdS, CdSe/CuSe, CdSeTe, CdSeTe/CdS/ZnS, CdSe/CdS/ZnS, CuInS ₂ , CuInSe ₂ , CuInSe ₂ /ZnS, and doped quantum dots CdS:Mn, CdS:Mn, CdS:Cu , ZnS:Cu, CdS:Tb, ZnS:Tb, etc.; the polymer used can also be polystyrene, polyacrylic acid, polymethyl methacrylate, polyethyl methacrylate, polyamide, polyacrylonitrile, poly Carbonate, polycaprolactone, polyurethane, polylactic acid, chitosan, albumin, collagen, polyethyl acetate, polystyrene-acrylic acid copolymer, polystyrene-methacrylic acid copolymer, or polystyrene-methacrylic acid Methyl ester copolymers, etc.; the PEG derivatives used can also include multi-arm PEG, Y-shaped structure, branched structure, PEG mixed system and other derivatives; the surface modification method used can also be sulfonation, etc., after Surface modification can also be connected with one or more of the following functional groups: amino group, sulfonate group, nitro group, hydroxyl group, mercapto group, chlorine group or ester group, etc., and the following linkers can be connected through functional groups: biotin, hydrophilic And prime or streptavidin, etc.

The biological detection probe based on functional nanoparticle composite PEG microspheres of the present invention can be used to detect one or more targets in a sample, such as detecting cytokines, allergens and autoimmune reactions, HLA typing in disease diagnosis , SNP detection, tumor-specific antigen quantitative detection, multiple microbial transport detection, etc.; or used in basic research such as genotyping, protein expression typing, enzyme-substrate analysis, nucleic acid research, etc.; can also be applied to food safety, Fields such as multiple quantitative detection of pesticide and veterinary drug residues and forensic identification.

Specifically, the method for using the biological detection probe based on composite PEG microspheres of the present invention to detect one or more target objects in a sample is:

(1) Put one or more combinations of the biological detection probes of the present invention into a sample containing a target, and the probe molecule is specifically bonded to the target;

(2) further fluorescently labeling the target object connected to the biological detection probe with a fluorescent substance;

(3) Analyzing the detection results of the biological probes with the instrument.

Targets described in step (1) include proteins, protein fragments or nucleic acids; fluorescent substances described in step (2) include: fluorescein isothiocyanate (FITC), phycoerythrin (PE), propidium iodide Pyridine (PI), cyanidin (CY5), chlorophyll protein (preCP), phycoerythrin-Texas Red, Cascade blue, and surface-modified quantum dots; in step (3), use the instrument to detect the biological probe The analysis of the results refers to the use of instruments to qualitatively analyze the target in the test sample by measuring the properties of the nanoparticles inside the microsphere, and at the same time to measure the detection by marking the fluorescence intensity of the target connected to the biological detection probe. Quantitative analysis of the target in the sample; commonly used instruments for detection include: flow cytometer, Luminex suspension array detection system (Luminex, USA), fluorescence spectrophotometer, fiber optic spectrometer, laser confocal microscope, fluorescence microscope, Shake the sample magnetometer.

Analyzing the detection results of the biological probe further includes: using the determination of the properties of the nanoparticles inside the composite PEG microsphere of the present invention to qualitatively analyze the target in the detection sample; The intensity of the target substance in the detection sample is quantitatively analyzed, wherein the intensity of the fluorescent label of the target substance attached to the biological detection probe is directly proportional to the concentration of the target substance in the detection sample.

The preferred specific embodiments of the present invention have been described in detail above. It should be understood that those skilled in the art can make many modifications and changes according to the concept of the present invention without creative efforts. Therefore, all technical solutions that can be obtained by those skilled in the art based on the concept of the present invention through logical analysis, reasoning or limited experiments on the basis of the prior art shall be within the scope of protection defined by the claims.

Claims (10)

1. A functional nanoparticle composite microsphere, characterized in that, the functional nanoparticle composite microsphere is a functional nanoparticle composite PEG microsphere, and the functional nanoparticle composite PEG microsphere comprises a functional nanoparticle As for the polymer containing PEG chain segments, the average particle diameter of the functional nanoparticle composite PEG microsphere is 0.1-1000 μm, and the variation coefficient of particle diameter distribution is less than 10%. 2. The functional nanoparticle composite microsphere according to claim 1, wherein the functional nanoparticle is one or more of the following: quantum dots, magnetic nanoparticles, fluorescent nanoparticles, metal nanoparticles Particles, metal oxide nanoparticles and semiconductor nanoparticles; wherein the quantum dots are one or more of the following: CdS, HgS, CdSe, CdTe, ZnSe, HgSe, ZnTe, ZnO, PbSe, HgTe, CaAs, InP , InCaAs, CdSe/CdS, CdSe/ZnS, CdSe/ZnSe, CdS/ZnS, Cd/Ag ₂ S, CdS/Cd(OH) ₂ , CdTe/ZnS, CdTe/CdS, CdSe/ZnSe, CdS/HgS, CdS /HgS/CdS, ZnS/CdS, ZnS/CdS/ZnS, ZnS/HgS/ZnS/CdS, CdSe/CuSe, CdSeTe, CdSeTe/CdS/ZnS, CdSe/CdS/ZnS, CuInS ₂ , CuInSe ₂ , CuInS ₂ / ZnS, CuInSe ₂ /ZnS, and doped quantum dots CdS:Mn, CdS:Mn, CdS:Cu, ZnS:Cu, CdS:Tb, ZnS:Tb. 3. The functional nanoparticle composite microsphere according to claim 1, wherein the PEG segment is grafted to one or more of the following polymers: polystyrene, polyacrylic acid, polymethyl Acrylic, polymethyl methacrylate, polyethyl methacrylate, polyamide, polyacrylonitrile, polycarbonate, polycaprolactone, polyurethane, polylactic acid, chitosan, albumin, collagen, polystyrene horse Anhydride copolymer, polyethyl acetate, polystyrene acrylic acid copolymer, polystyrene methacrylic acid copolymer or polystyrene-methyl methacrylate copolymer, poly(styrene-divinylbenzene-methyl acrylic acid) and maleic anhydride-1-octadecene copolymer. 4. The functional nanoparticle composite microsphere according to claim 1, wherein the PEG segment includes PEG or PEG derivatives with a molecular weight in the range of 500 to 10,000 Da, and the PEG derivatives include monofunctional PEG , homogeneous bifunctional PEG, hetero(base) bifunctional PEG, multi-arm PEG, Y-shaped structure PEG, branched structure PEG or a mixture thereof. 5. functional nanoparticle composite microsphere according to claim 1, is characterized in that, described functional nanoparticle composite PEG microsphere is connected with functional group through surface modification; Described surface modification is a kind of in following or several: hydrolysis, chemical grafting or sulfonation; the functional group is one or more of the following: carboxyl, amino, sulfonate, nitro, hydroxyl, mercapto, chlorine or ester. 6. The functional nanoparticle composite microsphere according to claim 5, characterized in that, the functional group is also connected with one or more of the following linkers: N-hydroxysuccinimide, biotin, affinity and streptavidin. 7. The preparation method of functional nanoparticle composite microsphere according to any one of claims 1-6, characterized in that the preparation method comprises swelling method, microfluidic method and membrane emulsification-emulsion solvent evaporation method. 8. Use of the functional nanoparticle composite microsphere according to any one of claims 1-6 in detecting one or more target objects in a sample. 9. A biological detection probe based on functional nanoparticle composite microspheres, characterized in that, said biological detection probe comprises functional nanoparticle composite PEG microspheres according to any one of claims 1-6 and A probe molecule coupled to the surface of the functional nanoparticle composite PEG microsphere; the probe molecule is selected from one or more of the following: protein, protein fragment and nucleic acid. 10. Use of the biological detection probe according to claim 9 in detecting one or more target substances in a sample.