CN105126714B - Functional nano-particle composite microsphere and preparation and application thereof - Google Patents
Functional nano-particle composite microsphere and preparation and application thereof Download PDFInfo
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Abstract
The invention discloses a functional nanoparticle composite microsphere, which is a functional nanoparticle composite PEG microsphere, and comprises functional nanoparticles and a polymer containing a PEG chain segment, wherein the average particle size of the functional nanoparticle composite PEG microsphere is 0.1-1000 mu m, and the coefficient of variation of particle size distribution is less than 10%. The invention prepares the functional nano-particles and the polymer grafted with the PEG chain segment into the composite microspheres with uniform particle size, and the PEG chain segment component can effectively play the role of nonspecific adsorption inhibitor, thereby having wide application prospect in the fields of biological detection and biological medicine.
Description
Technical Field
The invention relates to the field of preparation and application of micro-nano materials, in particular to a functional nano-particle composite microsphere and application thereof.
Background
The functional nano-particle composite microsphere is a functional composite microsphere obtained by combining functional nano-particles and microspheres by a certain method. Nanoparticles prepared by various routes at present have various special optical, electric, magnetic and biological properties, so that the properties are often endowed to the microspheres after the nanoparticles are combined with the microspheres. The microspheres also provide a support carrier and effective protection for these nanoparticles. Meanwhile, the chemical and physical properties of the microsphere, such as photosensitivity, pH responsiveness, temperature sensitivity, adsorption characteristics and surface active functional groups, also provide possibility for the application of the nanoparticles in various complex fields. The nano-particle composite microspheres with different special functions have great application potential in the fields of biological medicine, industrial catalysis, chemical synthesis, electronic information, building materials and the like.
At present, functional nanoparticle composite polymer microspheres have been used in various fields: nie research group embedded semiconductor nano-particle quantum dots into porous microspheres by swelling penetration method, and connected DNA chain segments on the microsphere surface as probes by using carboxyl functional groups on the microsphere surfaceAnd (4) detecting the specificity. Jun Lin group preparation of upconversion particles @ hydrogel core-shell structure microspheres NaYF4 Yb3+/Er3+@ Hydrogel, the fluorescence property of the fluorescence upconversion nanoparticles is utilized to realize near-infrared excited cell imaging, and meanwhile, the drug is encapsulated in the shell layer, and the purpose of controlled release of the drug is achieved by utilizing the pH responsiveness of the Hydrogel. Chunhui Deng group synthesis of magnetic polymer composite microsphere Fe3O4@SiO2@ PMMA, and the method is successfully applied to magnetic separation and enrichment of biomolecule nucleic acid and protein, and the technology has important application value in the fields of mass spectrometry and the like.
Among them, microspheres are required to have an effect of suppressing nonspecific adsorption to improve applications in application fields such as targeted imaging, drug targeted controlled release, immunodetection, immunomagnetic separation, and the like. Various natural polymers such as chitosan, albumin, have been shown to act as potent nonspecific inhibitors. However, natural polymers have significant drawbacks in that they may contain unpredictable viruses and their nonspecific adsorption-inhibiting effect is poor in many cases. Synthetic polymer brushes (polymerbrushes) are therefore receiving increasing attention as nonspecific adsorption inhibitors.
At present, no report related to the nonspecific adsorption of the surface of the nanoparticle composite microsphere is found.
Disclosure of Invention
In view of the defects of the prior art, the invention provides a functional nanoparticle composite microsphere, which adopts the following technical scheme:
the functional nanoparticle composite microsphere is a functional nanoparticle composite PEG microsphere, and comprises functional nanoparticles and a polymer containing a PEG chain segment, wherein the average particle size of the functional nanoparticle composite PEG microsphere is 0.1-1000 mu m, and the coefficient of variation of particle size distribution is less than 10%. PEG, i.e., polyethylene glycol; the variation coefficient of the particle size distribution is a statistic for measuring the variation degree of the particle size of each particle.
Further, the functional nanoparticles are one or more of the following: quantum dots, magnetic nanoparticles, fluorescenceLight nanoparticles, metal oxide nanoparticles, and semiconductor nanoparticles; 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/Ag2S、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、CuInS2、CuInSe2、CuInS2/ZnS、CuInSe2The quantum dots are doped with Mn, CdS, Cu, Tb and 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, polystyrene methacrylic acid copolymer or polystyrene-methylmethacrylate copolymer, poly (styrene-divinylbenzene-methacrylic acid) and maleic anhydride-1-octadecene copolymer. The polymer containing PEG chain segment can be divided into the following two possibilities: firstly, grafting a PEG chain segment on a polymer before balling, and then compounding the PEG chain segment with nano particles to form balls; secondly, firstly, the polymer and the nano particles are compounded into a sphere, and then a PEG chain segment is grafted on the surface of the microsphere.
Further, the PEG chain segment comprises PEG or PEG derivatives with the molecular weight of 500-10000 Da, and the PEG derivatives comprise monofunctional PEG, homogeneous bifunctional PEG, hetero (radical) bifunctional PEG, multi-arm PEG, Y-shaped structure PEG, dendritic structure PEG or a mixture thereof.
Further, the functional nanoparticle composite PEG microspheres are connected with functional groups through surface modification; the surface modification is one or more of the following: hydrolysis, chemical grafting or sulfonation; the functional group is one or more of the following: carboxyl, amino, sulfonate, nitro, hydroxyl, mercapto, chlorine or ester.
Further, one or more of the following linkers are connected to the functional group: n-hydroxysuccinimidyl, biotin, avidin and streptavidin.
Further, the preparation method includes a swelling method, a microfluidic method, and a membrane emulsification-emulsion solvent evaporation method. Swelling method: the method comprises the steps of synthesizing a cross-linked microsphere with a pore structure in advance, swelling the microsphere in a good solvent, expanding surface micropores, adding functional nanoparticles, permeating the functional nanoparticles into the microsphere through the pore structure under the action of concentration difference or hydrophobicity, adsorbing the functional nanoparticles on the inner wall of the microsphere, and shrinking the microsphere after an external solvent is removed, so that the functional nanoparticles are embedded into the microsphere. A micro-fluidic method: if the nano particles and the polymer used by the prepared functional nano particle composite microsphere are both hydrophobic, the polymer and the nano particles are dissolved in a nonpolar organic solvent in advance, the polymer fluid is broken up by a water phase with a certain flow velocity in a microfluidic channel to form droplets through emulsification, and the formed droplets are volatilized by the solvent to be solidified into spheres. Membrane emulsification-emulsion solvent evaporation method: if the nano-particles and the polymer used by the prepared functional nano-particle composite microsphere are both hydrophobic, the polymer and the nano-particles are dissolved in a non-polar organic solvent in advance to be used as a dispersion phase, a water solution containing a surfactant is used as a continuous phase, the dispersion phase and the continuous phase are placed on two sides of a rigid porous membrane, the dispersion phase is pressed into the continuous phase through a uniform microporous structure on the porous membrane under the action of certain nitrogen pressure to form uniform liquid drops, at the moment, a stabilizing agent in the continuous phase is adsorbed on the surface of the liquid drops to play roles in reducing surface tension and stabilizing the liquid drops, and the formed liquid drops are solidified into spheres through volatilization of the solvent.
The functional nano-particle composite microsphere is applied to detecting one or more targets in a sample.
A biological detection probe based on a functional nanoparticle composite microsphere, the biological detection probe comprising the functional nanoparticle composite PEG microsphere according to any one of claims 1 to 7 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: proteins, protein fragments, and nucleic acids.
Use of the biological detection probe for detecting one or more targets in a sample.
The present invention will be further described with reference to the accompanying drawings to fully illustrate the objects, technical features and technical effects of the present invention.
Drawings
FIG. 1 is a scanning electron micrograph of the composite PEG microspheres of example 4;
FIG. 2 is a confocal laser micrograph of the composite PEG microspheres of example 8;
FIG. 3 is the results of the biological detection probe of example 9 for the detection of the tumor marker AFP, and comparison with the detection results of the non-PEG microsphere-based biological probe;
fig. 4 is a partially enlarged view of fig. 3.
Detailed Description
The following detailed description of the present invention will be made with reference to the accompanying drawings.
The 1- (3-dimethylaminopropyl) -3-Ethylcarbodiimide (EDC) activation of carboxyl on the surface of the composite microsphere, the connection of probe molecules, phycoerythrin marking of antigens such as Alpha Fetoprotein (AFP) and the like are all basic operations of clinical immunodetection and diagnosis, and the following operations can be specifically referred to the clinical immunology test experiment guidance, and the authors: lushi jing, press: the Chinese medicine science publishing house.
Example 1: preparation of PEG graft Polymer
1g of polystyrene maleic anhydride copolymer (PSMA) and 2g of polyethylene glycol monomethyl ether (mPEG1000) with the molecular weight of 1000 are dissolved in 20ml of Dimethylformamide (DMF), 100mg of catalyst p-toluenesulfonic acid is added, dissolved at normal temperature for 2h and uniformly mixed, and then heated to 100 ℃ under the protection of nitrogen to react for 12 h. After the reaction is finished, anhydrous ether is added to precipitate the obtained polymer PSMA-PEG, and the polymer is frozen and dried for later use after repeated centrifugal washing for three times.
Example 2: preparation of PEG graft Polymer (II)
1g of polystyrene acrylic copolymer and 5g of carboxyl terminated polyethylene glycol monomethyl ether (mPEG-COOH) with the molecular weight of 5000 are dissolved in 30ml of chloroform, and a plurality of drops of concentrated sulfuric acid are added in N2Heating, condensing and refluxing for 12h under the protection condition, adding anhydrous ether to precipitate the polymer after the reaction is finished, repeatedly centrifuging and washing for three times, and freeze-drying the polymer for later use.
Example 3: preparation of PEG graft Polymer (III)
Maleic anhydride/1-octadecene alternating copolymer (PMAO) and 10g PEG (mPEG-NH) with molecular weight of 2000 and end capped with one amino group2) Dissolving in 30ml chloroform, stirring for 24h, adding anhydrous ethanol to precipitate out polymer, removing unreacted PEG molecule, repeatedly centrifuging and washing for three times, and freeze drying the polymer for later use.
Example 4: preparation of composite PEG microsphere powder
The membrane emulsification apparatus used in this example was a pressure type membrane emulsification apparatus available from SPG technology corporation of japan; the specific process is that PSMA-PEG and CdS/ZnS quantum dots with the emission wavelength of 518nM in the embodiment 1 are dissolved in toluene, the concentration of the polymer is 1.5g/mL, the concentration of the quantum dots is 2nM/L, and the quantum dots are used as the dispersion phase. An SPG porous membrane with the pore diameter of 5 mu m is adopted, the dispersed phase is extruded through the membrane by using nitrogen with the pressure of 15KPa, and enters an aqueous continuous phase with the concentration of 1 wt.% of emulsifier SDS, and the flow rate of the continuous phase is 0.35m/s, so that the oil-in-water emulsion with uniform droplet size is obtained. Stirring and volatilizing at 25 ℃ and under the magnetic stirring of 350rpm, and after toluene in the solution is completely volatilized, carrying out centrifugal collection on the obtained quantum dot labeled fluorescent microsphere suspension. And then centrifugally washing the microspheres for 3 times by using deionized water, centrifugally washing the microspheres for 3 times by using absolute ethyl alcohol, and freeze-drying the microspheres to obtain solid powder of the quantum dot composite microspheres.
The quantum dot composite fluorescent microspheres prepared by observation of a scanning electron microscope are spherical particles with smooth surfaces, the average particle size is 7.1 mu m, the coefficient of variation CV of particle size distribution is about 9.7 percent, and the monodispersity is better, as shown in figure 1.
Example 5: preparation of composite PEG microsphere powder
The use in this case being made by self-made with T-jointsThe specific process of the microfluidic device is that the PEG grafted polystyrene acrylic acid copolymer in the example 2 and Fe3O4Dissolved in chlorine, the polymer concentration was 1g/mL, Fe3O4The concentration is 1nM/L, which is used as the dispersion phase; and taking an aqueous solution containing 0.5 wt.% emulsifier PVA and 0.5 wt.% SDS as a continuous phase, and injecting dispersed phases from different channels to converge and emulsify at the T-shaped interface. The flow rate of the dispersed phase is 30mL/h and the flow rate of the continuous phase is 1L/h, and an oil-in-water emulsion with uniform droplet size is obtained. Stirring and volatilizing at 25 ℃ under the magnetic stirring of 350rpm, and carrying out magnetic separation and collection on the obtained magnetic bead suspension after chloroform in the solution is completely volatilized. And then washing for 3 times, washing for 3 times by using absolute ethyl alcohol, and freeze-drying to obtain the solid powder of the magnetic beads.
The scanning electron microscope observation shows that the prepared quantum dot composite fluorescent microsphere is spherical particles with smooth surfaces, the average particle size is 300 mu m, the coefficient of variation CV of particle size distribution is about 8.7 percent, and the monodispersity is good.
EXAMPLE 6 preparation of composite PEG microsphere powder (III)
PMAO-PEG of example 3 and NaYF with excitation wavelength of 980nm4The rare earth nanoparticles are dissolved in toluene, the concentration of the PMAO-PEG copolymer is 2g/mL, the concentration of the NaYF4 rare earth nanoparticles is 1nM/L, and the rare earth nanoparticles are used as a dispersion phase. An SPG porous membrane with the pore diameter of 3 mu m is adopted, the dispersed phase is extruded through the membrane by using nitrogen with the pressure of 20KPa, and enters an aqueous continuous phase with the concentration of 1 wt.% of emulsifier SDS, and the flow rate of the continuous phase is 0.40m/s, so that the oil-in-water emulsion with uniform droplet size is obtained. The volatilization was carried out with stirring at 25 ℃ and magnetic stirring at 350 rpm. And after the toluene in the solution is completely volatilized, centrifugally collecting the obtained rare earth nanoparticle composite fluorescent microsphere suspension. And then centrifugally washing the mixture for 3 times by using deionized water, centrifugally washing the mixture for 3 times by using absolute ethyl alcohol, and freeze-drying the mixture to obtain the solid powder of the rare earth nanoparticle composite fluorescent microspheres.
The scanning electron microscope observation shows that the prepared quantum dot composite fluorescent microsphere is spherical particles with smooth surfaces, the average particle size is 4.1 mu m, the CV value of the particle size distribution variation coefficient is about 6.2 percent, and the monodispersity is good.
EXAMPLE 7 preparation of composite PEG microsphere powder (IV)
Firstly, preparing porous polymer microspheres, namely preparing seed Polystyrene (PS) microspheres, namely dissolving 2g of polyvinylpyrrolidone (PVP) in 100g of continuous phase (a mixed solvent of absolute ethyl alcohol and ethylene glycol monomethyl ether); then adding a styrene monomer containing an oil-soluble initiator AIBN, introducing nitrogen for 15 minutes under the magnetic stirring at the speed of 120rpm, then heating to 70 ℃, and reacting for 12 hours under the protection of the nitrogen. And finally, centrifugally separating the obtained seed Polystyrene (PS) microsphere suspension at 4000rpm, washing the seed Polystyrene (PS) microsphere suspension three times by using deionized water and ethanol, and storing the seed Polystyrene (PS) microsphere suspension for later use after freeze drying.
0.2g of PS seed microspheres and 0.1mL of chlorododecane (CD, as an activator) were added to 25mL of an aqueous solution of 0.25 wt.% Sodium Dodecyl Sulfate (SDS) dissolved therein, respectively, and after ultrasonic dispersion for 5 minutes, the two were mixed, followed by ultrasonic dispersion for 10 minutes and swelling for 12 hours under magnetic stirring at 30 ℃. Then, 0.06g of BPO in a mixed monomer (including styrene monomer, functional monomer methacrylic acid MAA, crosslinking agent divinylbenzene DVB) was added to 50mL of an aqueous solution in which 0.25 wt.% SDS was dissolved, and ultrasonic dispersion was performed for 15 minutes, followed by mixing with the activated seed microsphere solution and swelling for 12 hours. Then adding PVA with the concentration of 1 wt% of the whole solution, introducing nitrogen for 15 minutes, heating to 70 ℃, and reacting for 12 hours under the protection of nitrogen. The microsphere suspension obtained after the reaction was centrifuged at 4000rpm, washed three times with deionized water and ethanol, and then freeze-dried to obtain surface-carboxylated polystyrene microspheres. And finally, extracting the microspheres with dichloromethane in a soxhlet extractor for 48 hours to obtain porous monodisperse poly (styrene-divinylbenzene-methacrylic acid) (PSDM) microspheres after linear PS molecules in the microspheres are extracted.
0.5mL of 10.0nM TiO2The chloroform solution of (2) was added to 1mL of a solution containing 107After the reaction solution is added into the chloroform solution of the PSDM porous microspheres, the bottle mouth is sealed, and the microspheres are swelled for 2 hours under the magnetic stirring of 400 rpm. Then removing the seal, continuously stirring the solution at room temperature, gradually concentrating the quantum dots in the solution along with the volatilization of the trichloromethane in the solution, and TiO2In a microContinuously permeating into the pore structure of the microsphere under the action of the concentration difference between the inside and the outside of the microsphere, and stopping stirring after the trichloromethane in the solution is completely volatilized. And carrying out 4000rpm centrifugal washing on the microspheres by utilizing dimethylbenzene, ethanol and water respectively to remove quantum dots remained outside the microspheres, and finally, carrying out vacuum freeze-drying on the microspheres collected by centrifugation and storing the microspheres in a solid powder state for later use.
The obtained TiO is mixed2Connecting N-hydroxyl succinimido to carboxyl on the surface of PSDM microsphere under EDC activation, and then connecting PEG with molecular weight of 1000 and end amino terminated and PEG with molecular weight of 2000 and end amino terminated, wherein the molar ratio of the two PEG molecules is 3: 1.
the scanning electron microscope observation shows that the prepared quantum dot composite fluorescent microsphere is spherical particles with smooth surfaces, the average particle size is 6 mu m, the coefficient of variation CV of particle size distribution is about 5.7 percent, and the monodispersity is good.
EXAMPLE 8 preparation of composite PEG microsphere powder (V)
PSMA-PEG of example 1 and CuInS with an emission wavelength of 680nm2/ZnS quantum dots and Fe3O4The magnetic nano-particles are dissolved in toluene, the concentration of PSMA-PEG is 2g/mL, the concentration of quantum dots is 1nM/L, and Fe3O4The concentration of the magnetic nanoparticles was 1nM/L, which was used as the dispersed phase. An SPG porous membrane with the pore diameter of 5 mu m is adopted, the dispersed phase is extruded through the membrane by using nitrogen with the pressure of 20KPa, and enters an aqueous continuous phase with the concentration of 1 wt.% of emulsifier SDS, and the flow rate of the continuous phase is 0.40m/s, so that the oil-in-water emulsion with uniform droplet size is obtained. The volatilization was carried out with stirring at 25 ℃ and magnetic stirring at 350 rpm. And after the toluene in the solution is completely volatilized, separating and collecting the obtained quantum dot marked fluorescent microsphere suspension by magnetic force. And centrifugally washing with deionized water for 3 times, centrifugally washing with anhydrous ethanol for 3 times, and freeze-drying to obtain quantum dots/Fe with carboxyl on the surface3O4Solid powder of composite fluorescent magnetic microspheres. The quantum dots/Fe are obtained by scanning observation of an electron microscope3O4The composite fluorescent magnetic microsphere is spherical particle with smooth surface, the average particle size is 6.8 μm, the coefficient of variation CV of particle size distribution is about 9 percent, and the monodispersity is better.
The quantum dots inside the microspheres are uniformly distributed by laser confocal observation, which is shown in figure 2.
Example 9 biological detection probes based on functional nanoparticles composite PEG microspheres
The quantum dots/Fe prepared in example 83O4The surface carboxyl of the composite PEG microsphere is connected with N-hydroxysuccinimide under EDCylation, and then is connected with an alpha fetoprotein antibody AFP-Ab as a probe molecule. After the surface of the quantum dot composite fluorescent microsphere is connected with AFP-Ab, a biological detection probe for specifically detecting Alpha Fetoprotein (AFP) is formed. After the biological detection probe is put into a sample containing alpha fetoprotein AFP for reaction, a target object alpha fetoprotein AFP connected on the probe is marked by fluorescein isothiocyanate. And finally, the reacted biological detection probe emits a fluorescent signal under the excitation of laser, and qualitative and quantitative analysis is carried out on Alpha Fetoprotein (AFP) in the sample through the fluorescent signal of the quantum dots in the microspheres and the intensity of the labeled fluorescent signal of a target object connected on the probe molecules. Meanwhile, a control group experiment is carried out, the control group experiment adopts carboxylated polystyrene-maleic anhydride microspheres (non-PEG microspheres), and the concentration of nanoparticles in the control group experiment and the bioprobe experiment are consistent. The detection result is shown in figure 3, and it can be seen from the figure that both microspheres can carry out high-sensitivity quantitative detection on alpha fetoprotein, the detection sensitivity of the PEG microsphere probe is about one order of magnitude higher, the labeled fluorescence signal intensity is obviously lower under low concentration, and the effect of inhibiting nonspecific adsorption is shown.
Example 10 biological detection probes based on functional nanoparticles composite PEG microspheres
The rare earth nanoparticle composite PEG microspheres prepared in the example 6 are connected with a DNA chain segment with the sequence of 5'-TCA AGG CTC AGT TCG AAT GCA CCA TA-3' under the activation of EDC to form a biological detection probe for specifically detecting DNA. After the bioassay probe was put into a sample containing 5'-TAT GGT GCA TTC GAA CTG AGC CTTGA-3' DNA fragments for reaction, the target substance connected to the probe, that is, 5'-TAT GGT GCATTC GAA CTG AGC CTT GA-3' DNA fragment was labeled with Cascade blue. And finally, the treated rare earth nanoparticle composite microspheres emit fluorescent signals under the excitation of infrared laser, and the DNA chain segments of 5'-TAT GGT GCA TTC GAA CTG AGC CTT GA-3' are qualitatively and quantitatively analyzed through the fluorescent signals of the rare earth nanoparticles in the microspheres and the labeled fluorescent signals of the target objects connected on probe molecules.
Example 11 biological detection probes based on functional nanoparticles composite PEG microspheres (III)
And connecting the surface carboxyl of the quantum dot composite PEG microsphere prepared in the example 3 with N-hydroxysuccinimide under the activation of EDC. Then, the 518nm quantum dot composite microspheres are further connected with an anti-alpha fetoprotein antibody AFP-Ab serving as a detection probe molecule. The 518nm quantum dot composite microspheres are connected with an alpha fetoprotein antibody AFP-Ab on the surface to form a biological detection probe for specifically detecting the alpha fetoprotein AFP. The 680nm quantum dot composite microspheres are connected with an anti-carcinoembryonic antigen antibody CEA-Ab to serve as detection probe molecules. The 680nm quantum dot composite microsphere is connected with an antibody CEA-Ab on the surface to form a biological detection probe for specifically detecting carcinoembryonic antigen CEA. After the two biological detection probes are put into a sample containing alpha fetoprotein AFP and carcinoembryonic antigen CEA for reaction, the target object connected with the probes is marked by phycoerythrin. The reacted biological detection probe emits a fluorescence signal under the excitation of laser, the fluorescence of the quantum dots in the microspheres at 518nm is an AFP detection result, the fluorescence of the quantum dots in the microspheres at 680nm is a CEA detection result, and then the AFP and CEA in the sample are respectively quantitatively analyzed through the labeled fluorescence intensity of a target object connected to the probe molecules.
With respect to the above embodiments 1-8, it should be noted that the above embodiments are not exhaustive, and those skilled in the art should understand that the functional nanoparticles may also be semiconductor nanoparticles, metal nanoparticles; the functional nano-particles 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/Ag2S、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、CuInS2、CuInSe2、CuInSe2The material is characterized by comprising/ZnS, doped quantum dots CdS: Mn, CdS: Cu, ZnS: Cu, CdS: Tb, ZnS: Tb and the like; the polymer used may also be polystyrene, polyacrylic acid, polymethyl methacrylate, polyethyl methacrylate, polyamide, polyacrylonitrile, polycarbonate, polycaprolactone, polyurethane, polylactic acid, chitosan, albumin, collagen, polyvinyl acetate, polystyrene acrylic acid copolymer, polystyrene methacrylic acid copolymer, polystyrene-methyl methacrylate copolymer, or the like; the used PEG derivative can also be multi-arm PEG, Y-shaped structure, dendritic structure, PEG mixed system and other derivatives; the surface modification method can also be sulfonation and the like, and one or more of the following functional groups can be connected through surface modification: amino, sulfonate, nitro, hydroxyl, mercapto, chloro, or ester groups, and the like, and the following linkers may be attached through the functional group: biotin, avidin or streptavidin, and the like.
The biological detection probe based on the functional nano-particle composite PEG microsphere can be used for detecting one or more target objects in a sample, such as cytokine, allergen and autoimmune reaction detection in disease diagnosis, HLA typing, SNP detection, tumor specific antigen quantitative detection, multiple microorganism carrying capacity detection and the like; or in basic research such as genotyping, protein expression typing, enzyme-substrate analysis, nucleic acid research, etc.; can also be applied to the fields of food safety, multiple quantitative detection of pesticide and veterinary drug residues, judicial identification and the like.
Specifically, the method for detecting one or more target objects in a sample by using the biological detection probe based on the composite PEG microsphere comprises the following steps:
(1) one or more combinations of the biological detection probes of the invention are put into a sample containing a target, and the probe molecules are specifically bonded with the target;
(2) the target object connected with the biological detection probe is further fluorescently labeled by fluorescent substances;
(3) and analyzing the detection result of the biological probe by using an instrument.
The target substance in the step (1) comprises protein, protein fragments or nucleic acid; the fluorescent substance described in the step (2) includes: fluorescein Isothiocyanate (FITC), Phycoerythrin (PE), Propidium Iodide (PI), cyanidin (CY5), chlorophyll protein (precP), phycoerythrin-Texas red, Cascade blue and surface modified quantum dots; in the step (3), the step of analyzing the detection result of the biological probe by using an instrument refers to that the instrument is used for qualitatively analyzing the target object in the detection sample by measuring the performance of the nanoparticles in the microspheres, and quantitatively analyzing the target object in the detection sample by marking the fluorescence intensity of the target object connected to the biological detection probe; common instruments for detection include: flow cytometry, a Luminex suspension array detection system (Luminex corporation, USA), a fluorescence spectrophotometer, a fiber optic spectrometer, a laser confocal microscope, a fluorescence microscope and a vibration sample magnetometer.
Analyzing the results of the biological probe further comprises: the qualitative analysis of the target object in the detection sample is carried out by utilizing the measurement of the performance of the nanoparticles in the composite PEG microsphere; and quantitatively analyzing the target in the detection sample through the intensity of the fluorescence of the target marker connected to the biological detection probe, wherein the intensity of the fluorescence of the target marker connected to the biological detection probe is in a direct relation with the concentration of the target in the detection sample.
The foregoing detailed description of the preferred embodiments of the invention has been presented. It should be understood that numerous modifications and variations could be devised by those skilled in the art in light of the present teachings without departing from the inventive concepts. Therefore, the technical solutions available to those skilled in the art through logic analysis, reasoning and limited experiments based on the prior art according to the concept of the present invention should be within the scope of protection defined by the claims.
Claims (7)
1. The functional nanoparticle composite microsphere is characterized by being a functional nanoparticle composite PEG microsphere, wherein the functional nanoparticle composite PEG microsphere comprises functional nanoparticles and a polymer containing a PEG chain segment, the average particle size of the functional nanoparticle composite PEG microsphere is 0.1-1000 mu m, and the coefficient of variation of particle size distribution is less than 10%;
the functional nano-particles are one or more of the following: quantum dots, magnetic nanoparticles, fluorescent nanoparticles, 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/Ag2S、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、CuInS2、CuInSe2、CuInS2/ZnS、CuInSe2The material is/ZnS, and doped quantum dots CdS: Mn, CdS: Cu, ZnS: Cu, CdS: Tb and ZnS: Tb;
the PEG chain segment is grafted to a polymer, namely a polystyrene maleic anhydride copolymer; the functional nano-particle composite microsphere is prepared by grafting a PEG chain segment on a polymer before balling and then compounding the PEG chain segment and the nano-particles into balls; the preparation method comprises a membrane emulsification-emulsion solvent volatilization method;
the PEG chain segment is polyethylene glycol monomethyl ether.
2. The functional nanoparticle composite microspheres according to claim 1, wherein the functional nanoparticle composite PEG microspheres are surface modified with functional groups attached; the surface modification is one or more of the following: hydrolysis, chemical grafting or sulfonation; the functional group is one or more of the following: carboxyl, amino, sulfonate, nitro, hydroxyl, mercapto, chlorine or ester.
3. The functional nanoparticle composite microsphere according to claim 2, wherein one or more of the following linkers are further attached to the functional group: n-hydroxysuccinimidyl, biotin, avidin and streptavidin.
4. A preparation method of a functional nanoparticle composite microsphere is provided, wherein the functional nanoparticle composite microsphere is a functional nanoparticle composite PEG microsphere, the functional nanoparticle composite PEG microsphere comprises a functional nanoparticle and a polymer containing a PEG chain segment, the average particle size of the functional nanoparticle composite PEG microsphere is 0.1-1000 mu m, the coefficient of variation of particle size distribution is less than 10%, and the functional nanoparticle is one or more of the following: quantum dots, magnetic nanoparticles, fluorescent nanoparticles, 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/Ag2S、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、CuInS2、CuInSe2、CuInS2/ZnS、CuInSe2The material is/ZnS, and doped quantum dots CdS: Mn, CdS: Cu, ZnS: Cu, CdS: Tb and ZnS: Tb;
the PEG chain segment is grafted to a polymer, namely a polystyrene maleic anhydride copolymer; the preparation method is characterized by comprising a membrane emulsification-emulsion solvent volatilization method; grafting a PEG chain segment on the polymer before balling, and then compounding the PEG chain segment with the nano particles to form balls;
the PEG chain segment is polyethylene glycol monomethyl ether.
5. Use of the functional nanoparticle composite microspheres of any one of claims 1-3 for the detection of one or more targets in a sample.
6. A biological detection probe based on functional nanoparticle composite microspheres, which is characterized in that the biological detection probe comprises the functional nanoparticle composite PEG microspheres according to any one of claims 1 to 3 and probe molecules coupled on the surfaces of the functional nanoparticle composite PEG microspheres; the probe molecule is selected from one or more of the following: proteins, protein fragments, and nucleic acids.
7. Use of the biological detection probe of claim 6 for detecting one or more targets in a sample.
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