WO2014040353A1 - 功能性纳米颗粒复合微球粉末及其制备方法和应用 - Google Patents
功能性纳米颗粒复合微球粉末及其制备方法和应用 Download PDFInfo
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- the invention relates to the field of preparation and application of micro/nano materials, in particular to functional nanoparticle composite microspheres and preparation methods and applications thereof.
- Functional nanoparticle composite microspheres are functional composite microspheres obtained by combining functional nanoparticles with microspheres by some method.
- nanoparticles prepared by various routes have various special optical, electrical, magnetic and biological properties, and therefore these characteristics are often imparted to the microspheres themselves after being combined with the microspheres.
- Microspheres also provide support carriers and effective protection for these nanoparticles.
- the chemical and physical properties of the microspheres such as photosensitivity, pH responsiveness, temperature sensitivity, adsorption characteristics, and surface active functional groups also provide possibilities for the application of nanoparticles in a variety of complex fields.
- Nanoparticle composite microspheres with different special functions have great application potential in biomedicine, industrial chemistry, chemical synthesis, electronic information, building materials and other fields.
- In-situ polymerization The carrier microspheres are synthesized in advance, and then the microspheres are used as microreactors to synthesize various functional particles in situ or on the surface. However, due to the influence of the microspheres themselves, the type and performance of the functional particles prepared by the method are limited.
- Swelling and permeation method A crosslinked microsphere having a pore structure is synthesized in advance, and then the microsphere is swollen in a good solvent, and the surface micropores are expanded. At this time, functional particles are added, and the microspheres penetrate into the microspheres through the pore structure under the difference of concentration or hydrophobicity and adsorb on the inner wall of the microspheres. When the external solvent is removed, the microspheres shrink, thereby embedding the functional particles into the microspheres.
- This method is simple and straightforward, but requires that the surface of the microspheres have micropores that are functionally infiltrated. Due to the existence of such a microporous structure, the embedded functional particles are easily leaked out again, and the functional particles embedded therein cannot provide sufficient protection to reduce external influences. The performance stability of the nanoparticles within the sphere is poor.
- the assembly template method the functional particles or the microspheres are used as the core template, and the functional particles and the materials constituting the microspheres are sequentially bonded to the core by electrostatic action, hydrophobic action, complexation and hydrogen bonding.
- This method can accurately control the particle size of the composite microspheres and the number of nanoparticles, but the preparation steps are complicated. And the reaction environment during the preparation process has a certain influence on the performance of some functional particles.
- Polymerization method that is, the use of emulsion polymerization, suspension polymerization, dispersion polymerization or bulk polymerization to introduce functional particles while preparing microspheres.
- the functional particles are dispersed in the polymerizable monomer, and as the polymerization progresses, the functional particles are embedded in the gradually formed microsphere structure.
- the advantages of this method are simple preparation and high yield. However, this method still faces the following problems: When preparing micron-sized composite microspheres, the particle size distribution is wide, and the functional particle properties are susceptible to the polymerization reaction ring. The effect of the environment, the functional particles and the formed microsphere structure may be phase separated.
- Ugelstad's research team has prepared magnetic nanoparticles in styrene microspheres by in-situ polymerization, which has a high magnetic content and has been widely used in cancer detection and treatment.
- the Me research team carried out DNA-specific detection by embedding semiconductor nanoparticle quantum dots into porous microspheres by swelling and permeation method, and using the carboxyl functional groups on the surface of the microspheres to attach DNA segments to the surface of the microspheres as probes.
- the Ahjeong Son group used a template self-assembly method to coat a quantum dot shell on the surface of a magnetic microsphere by an amide bond to form a composite microsphere with magneto-optical properties, and was applied to nucleic acid detection.
- Yong Zhang's research group used microemulsion polymerization to prepare microspheres with up-converted luminescent rare earth nanoparticles.
- the microspheres have composite microspheres with special fluorescence properties under infrared excitation, which has broad application prospects in medical imaging.
- 200810019950.3 entitled “Nano-micron Composite Microsphere Preparation Method” proposes to chemically bond nanoparticles to the surface of microspheres to prepare nanoparticle composite
- the method of microspheres Chinese Patent Application No.: 201010593883.3, entitled “Preparation of Preparation Method of Organic/Inorganic Composite Microspheres”
- the inorganic nanoparticles are prepared by first swelling the polymer microspheres and then heating and volatilizing the swelling solvent.
- Composite polymeric microspheres are prepared by first swelling the polymer microspheres and then heating and volatilizing the swelling solvent.
- the principle of the method for preparing the composite microspheres is still based on the various conventional methods for preparing the inorganic nanoparticle composite microspheres described above, and the inherent system defects exist, and the particle size is micron-sized, uniformly controllable, and dispersed. Good functional nanoparticle composite microspheres.
- Membrane emulsification technology is the simplest and most effective method for preparing monodisperse emulsions with uniform particle size. It is through the inorganic membrane micropores to press the dispersed phase under the action of external pressure, and press into the continuous phase to form an emulsion, by controlling the dispersion pressure and membrane.
- the pore size enables monodispersity of the emulsion droplets. 0 / W, W / 0 type single emulsion can be prepared by membrane emulsification method, or multiple emulsions can be formed by secondary emulsification on the basis of single emulsion, and then liquid droplets can be converted into solid phase by some physicochemical reaction.
- the water-insoluble droplets can be converted into polymer microspheres by solvent evaporation.
- Monodisperse polymer microspheres prepared by membrane emulsification technology have many unique characteristics: small size and volume, uniform particle size and controllable particle size distribution, large specific surface area, excellent porosity, distribution of functional groups It has an ideal surface density, stable dispersion, and the like.
- Gasparini et al. prepared a biodegradable monodisperse drug carrier microsphere-polylactic acid I glycolic acid copolymer (PLGA) microsphere by membrane emulsification technique.
- PLGA polylactic acid I glycolic acid copolymer
- An object of the present invention is to provide a functional nanoparticle composite non-crosslinked microsphere powder in view of the deficiencies in the prior art.
- Another object of the present invention is to provide a use of a functional nanoparticle composite non-crosslinked microsphere powder.
- a fourth object of the present invention is to provide a biodetection probe based on functional nanoparticle composite non-crosslinked microsphere powder.
- a fifth object of the present invention is to provide a biodetection probe based on a functional nanoparticle composite non-crosslinked microsphere powder.
- a sixth object of the present invention is to provide a functional nanoparticle composite crosslinked microsphere powder.
- a seventh object of the present invention is to provide a method for preparing a functional nanoparticle composite crosslinked microsphere powder.
- An eighth object of the present invention is to provide a use of a functional nanoparticle composite crosslinked microsphere powder.
- a ninth object of the present invention is to provide a biodetection probe based on functional nanoparticle composite crosslinked microsphere powder.
- a tenth object of the present invention is to provide a biodetection probe based on a functional nanoparticle composite crosslinked microsphere powder.
- a functional nanoparticle composite non-crosslinked microsphere powder comprising functional nanoparticle composite non-crosslinked microspheres, the functional nanoparticle composite non-crosslinked microspheres comprising functional nanoparticles and a polymer, average
- the particle size is 0.1-20 ⁇ , and the coefficient of variation of the particle size distribution is 9.1%.
- the functional nanoparticles are one or more of the following: quantum dots, magnetic nanoparticles, fluorescent nanometers Particles, metal nanoparticles, metal oxide nanoparticles or 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/Ag 2S, 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, and
- the polymer is one or more of the following: polystyrene, polyacrylic acid, polymethacrylic acid, polymethyl methacrylate, polyethyl methacrylate, polyamide, polyacrylonitrile, polycarbonate Ester, polycaprolactone, polyurethane, polylactic acid, chitosan, albumin, collagen, polystyrene-maleic anhydride copolymer, polyethyl acetate, polystyrene-acrylic acid copolymer, polystyrene-methyl Acrylic copolymer or polystyrene-methyl methacrylate copolymer.
- the functional nanoparticle composite non-crosslinked microspheres are functionally bonded by 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: a carboxyl group, an amino group, a sulfonate group, a nitro group, a hydroxyl group, a chlorine group or an ester group.
- linkages are also attached to the functional group: N-hydroxysuccinimide, biotin, avidin or streptavidin.
- a continuous phase comprising deionized water and a water-soluble stabilizer and/or emulsifier; c) using a membrane emulsification apparatus to squeeze the dispersed phase under gas pressure through the porous membrane in the form of droplets Entering into the continuous phase, obtaining a monodisperse emulsion having uniform droplet size under the action of continuous phase shearing force;
- the polymer solution in the step a) is a solution in which a polymer is dissolved in an organic solvent.
- the organic solvent is a hydrophobic organic solvent.
- the hydrophobic organic solvent is selected from one or more of the following: toluene, xylene, p-chlorotoluene, methylene chloride, chloroform, tetrachloromethane, petroleum ether, n-hexane or cyclohexane.
- the concentration of the polymer solution is 0.5-2 g/mL.
- the concentration of the functional nanoparticles in the step a) is 0.5-1 nM/L.
- the stabilizer and/or emulsifier in step b) is selected from one or more of the following: sodium lauryl sulfate, Polyvinyl alcohol or Tween 20.
- the porous membrane in the step c) is an SPG porous membrane, a ceramic porous membrane or an MPG porous membrane.
- the porous membrane has a pore diameter of 0.5 to 5 ⁇ m.
- the gas pressure in the step c) is 15-30 KPa.
- the target is a biological macromolecule or a compound such as a protein and a fragment thereof, or a nucleic acid.
- a biodetection probe based on a functional nanoparticle composite non-crosslinked microsphere powder comprising the functional nanoparticle composite non-crosslinked microsphere powder as described above, the function The surface of the nanoparticle-composite non-crosslinked microspheres is coupled with a probe molecule.
- the probe molecule is selected from one or more of the following: a protein, a protein fragment or a nucleic acid.
- the biodetection probe as described above is used in detecting one or more targets in a sample.
- the target is a biological macromolecule or a compound such as a protein and a fragment thereof, or a nucleic acid.
- a functional nanoparticle composite crosslinked microsphere powder comprising functional nanoparticle composite crosslinked microspheres, wherein the functional nanoparticle composite crosslinked microsphere comprises functional nanoparticles and a monomer, a crosslinking agent and
- the initiator has an average particle diameter of 0.1-20 ⁇ m and a particle size distribution coefficient of variation of 9.6%.
- the functional nanoparticles are one or more of the following: quantum dots, magnetic nanoparticles, fluorescent nanoparticles, metal nanoparticles, metal oxide nanoparticles or 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/ZnS, CdSe/ZnSe, CdS /ZnS, Cd/Ag 2S, CdS/Cd(OH) 2 , CdTe/ZnS, CdTeSe/CdS, CdTe/CdS, CdSe/ZnSe, CdS/HgS, CdS/HgS/CdS ZnS/CdS, ZnS/CdS/ZnS ZnS/HgS/ZnS/CdS CdSe/CuSe, CdSeTe, CdSeTe/CdS/ZnS, and doped quantum dots CdS: Mn,
- the monomer is one or more of the following: styrene, methacrylic acid, acrylic acid, methyl acrylate, methyl methacrylate, ethylene, propylene, butene, butadiene, maleic anhydride or propylene. Amide.
- the crosslinking agent is one or more of the following: divinylbenzene, propylenediamine, glutaraldehyde, methylol acrylamide, ethylenediamine or genipin.
- the initiator is azobisisobutyronitrile, benzoyl peroxide or potassium persulfate.
- the functional nanoparticle composite crosslinked microspheres are functionally bonded by surface modification.
- the surface modification is selected from one or more of the following: hydrolysis, chemical grafting or sulfonation.
- the functional group is one or more of the following: a carboxyl group, an amino group, a sulfonate group, a nitro group, a hydroxyl group, a chlorine group or an ester group.
- linkages are also attached to the functional group: N-hydroxysuccinimide, biotin, avidin or streptavidin.
- a continuous phase comprising deionized water and a water-soluble stabilizer and/or emulsifier; c) using a membrane emulsification device to squeeze the dispersed phase through the porous membrane to droplets under gas pressure The form enters into the continuous phase, and a monodisperse emulsion having uniform droplet size is obtained under continuous phase shearing force;
- the monomer solution in the step a) is a solution in which a monomer, a crosslinking agent and an initiator are dissolved in an organic solvent.
- the organic solvent is a hydrophobic organic solvent.
- the hydrophobic organic solvent is one or more of the following: toluene, xylene, p-chlorotoluene, methylene chloride, chloroform, tetrachloromethane, petroleum ether, n-hexane or cyclohexane.
- the stabilizer and/or emulsifier in step b) is selected from one or more of the following: sodium lauryl sulfate, polyvinyl alcohol or Tween 20.
- the concentration of the cross-linking agent is 0.2-0.75 g/mL.
- the concentration of the initiator is from 0.001 to 0.02 g/mL.
- the concentration of the functional nanoparticles in the step a) is 0.2-1 nM/L.
- the porous membrane in the step c) is an SPG porous membrane, a ceramic porous membrane or an MPG porous membrane.
- the porous membrane has a pore diameter of 0.5 to 5 ⁇ m.
- the gas pressure in the step c) is 18-28 KPa.
- the target is a biological macromolecule or a compound such as a protein and a fragment thereof, or a nucleic acid.
- a biodetection probe based on a functional nanoparticle composite crosslinked microsphere powder comprising the functional nanoparticle composite crosslinked microsphere powder as described above, the functional nanometer
- the surface of the particle composite crosslinked microsphere is coupled with a probe molecule.
- the probe molecule is selected from one or more of the following: a protein, a protein fragment or a nucleic acid.
- the biodetection probe as described above is used in detecting one or more targets in a sample.
- the target is a biological macromolecule or a compound such as a protein and a fragment thereof, or a nucleic acid.
- Figure 1 is a scanning electron micrograph of a composite non-crosslinked microsphere of Example 2.
- Figure 2 is a photomicrograph of a composite non-crosslinked microsphere of Example 7.
- Figure 3 is a graph showing the results of detecting the HBsAg of hepatitis B virus surface antigen by the biodetection probe of Example 7.
- Figure 4 is a scanning electron micrograph of the composite crosslinked microsphere of Example 23.
- the membrane emulsification apparatus used in the following examples was a pressure membrane emulsification apparatus, which was purchased from SPG Technology, Japan; the MPG membrane was purchased from Ise Chemical Co., Japan, and was a hydrophilic membrane suitable for a pressure membrane emulsification apparatus; PG membrane was purchased from Japan.
- SPG technology a hydrophilic membrane suitable for pressure membrane emulsification devices; ceramic membranes purchased from Membraflow, Germany, is a hydrophilic membrane suitable for pressure membrane emulsification devices;
- the average particle diameter was calculated by randomly averaging the particle diameters of 200 microspheres and the average particle diameter as D av .
- the calculation method of the particle size distribution coefficient of variation CV is: 1 ⁇ - ⁇ ID m wherein A is the particle diameter of the i-th microsphere, and D av is the average particle diameter of the microsphere.
- the polystyrene-acrylic acid copolymer and the gold nanoparticles were dissolved in toluene, the polystyrene-acrylic acid copolymer concentration was 1 g/mL, and the gold nanoparticle concentration was 1 nM/L, which was used as a dispersed phase.
- a pore size of 0.5 ⁇ m and a porosity of 0.4 The MPG porous membrane was extruded through a membrane with a pressure of 30 KPa of nitrogen gas into a continuous phase of water containing an emulsifier SDS concentration of 1 wt.%, and the flow rate of the continuous phase was 0.35 m/s to obtain droplets.
- the polystyrene-maleic anhydride copolymer and CdSe/CdS quantum dots with an emission wavelength of 528 nm were dissolved in toluene at a polymer concentration of 1 g/mL and a quantum dot concentration of 1 nM/L, which was used as a dispersed phase. .
- the dispersed phase was extruded through a membrane with a pressure of 15 KPa of nitrogen into a continuous phase of water containing an emulsifier SDS concentration of 1 wt.%.
- quantum dot composite fluorescent microspheres were dispersed in 15 mL of 0.01 mol/L hydrochloric acid to prepare a suspension, and magnetically stirred for 8 h. After the anhydride functional group on the polymer was hydrolyzed to carboxyl group, a part of the suspension was extracted and the volume ratio was used. 1/1 deionized water washing (until the pH of the suspension of the hydrolyzed microspheres is stabilized at about 6.2), followed by freeze-drying to obtain surface-carboxy functionalized quantum dot composite fluorescent microspheres.
- the polystyrene and Fe 3 0 4 magnetic nanoparticles were dissolved in chloroform at a polystyrene concentration of 0.5 g/mL, and the Fe 3 0 4 magnetic nanoparticles were at a concentration of 1 nM/L, which was used as a dispersed phase.
- the dispersed phase was extruded through a membrane with a pressure of 21 KPa of nitrogen into a continuous flow of water containing 0.5 wt.% emulsifier SDS and 0.5 wt.% stabilizer PVA.
- the flow rate of the continuous phase was 0.40 m/s, and an oil-in-water emulsion having a uniform droplet size was obtained.
- the polymethacrylic acid and silver nanoparticles were dissolved in dichloromethane to have a polymethacrylic acid concentration of 2 g/mL and a silver nanoparticle concentration of 0.5 nM/L as a dispersed phase.
- a SPG porous membrane with a pore size of 1 ⁇ and a porosity of 0.5 the dispersed phase was extruded through a membrane with a pressure of 32 KPa of nitrogen into the SDS containing 0.1 wt.% emulsifier and 0.1 wt.% stabilizer Tween-20.
- the continuous phase of water, the flow rate of the continuous phase is 0.35 m/s, and an oil-in-water emulsion having a uniform droplet size is obtained.
- the dichloromethane was completely volatilized in the solution, the obtained silver nanoparticle composite microsphere suspension was collected by centrifugation. Thereafter, the mixture was washed three times with deionized water, washed three times with water-free ethanol, and freeze-dried to obtain a solid powder of silver nanoparticle composite microspheres. Scanning electron microscopy showed that the prepared silver nanoparticle composite microspheres were spherical particles with smooth surface, the average particle size was 1.1 ⁇ , and the particle size distribution coefficient of variation CV was about 7.2%, and the monodispersity was good.
- the polymethyl methacrylate and Ti0 2 nanoparticles was dissolved in chloroform, polymethyl methacrylate at a concentration of 0.5 g / mL, the concentration of particles of nano Ti02 0.5 nM / L, as in the dispersed phase.
- the dispersed phase was extruded through a membrane with a pressure of 21 KPa of nitrogen into the water containing 0.1 wt.% emulsifier SDS and 1 wt.% stabilizer PVA.
- the flow rate of the continuous phase was 0.38 m/s, and an oil-in-water emulsion having a uniform droplet size was obtained.
- the obtained Ti0 2 nanoparticle composite microsphere suspension was collected by centrifugation. Thereafter, the mixture was washed three times with deionized water, washed three times with absolute ethanol, and freeze-dried to obtain a solid powder of Ti0 2 nanoparticle composite microspheres.
- the polystyrene-methacrylic acid copolymer and the CdSe/CdS/ZnS quantum dots and gold nanoparticles having an emission wavelength of 550 nm were dissolved in xylene, and the polystyrene-methacrylic acid copolymer concentration was 1.5 g/mL.
- the quantum dot concentration was 0.5 nM/L
- the gold nanoparticle concentration was 0.2 nM/L, which was used as the dispersed phase.
- the dispersed phase was extruded through a membrane with a pressure of 18 KPa of nitrogen into the water continuous phase of 0.5 wt.% emulsifier SDS and 0.5 wt.% stabilizer PVA.
- the flow rate of the continuous phase was 0.35 m/s, and an oil-in-water emulsion having a uniform droplet size was obtained.
- the polystyrene-acrylic acid copolymer and CdSeTe quantum dots and Fe 3 0 4 magnetic nanoparticles with emission wavelength of 680 nm were dissolved in toluene, the concentration of polystyrene-acrylic acid copolymer was 2 g/mL, and the concentration of quantum dots was l.
- the nM/L, Fe 3 0 4 magnetic nanoparticles have a concentration of 1 nM/L, which is used as a dispersed phase.
- the dispersed phase was extruded through a membrane with a pressure of 20 KPa of nitrogen into a continuous phase of water containing an emulsifier SDS concentration of 1 wt.%.
- the flow rate of the continuous phase was At 0.40 m/s, an oil-in-water emulsion having a uniform droplet size was obtained.
- the mixture was washed three times with deionized water, washed three times with absolute ethanol, and freeze-dried to obtain a solid powder of quantum dots/Fe 3 0 4 composite fluorescent magnetic microspheres having carboxyl groups on the surface.
- the quantum dot/Fe 3 0 4 composite fluorescent magnetic microspheres prepared by fluorescence microscopy and electron microscopy were spherical particles with smooth surface, the average particle size was 6.3 ⁇ , and the coefficient of variation of particle size distribution CV was about 9%. Good sex, see Figure 2.
- Example 8 Preparation of Composite Non-Crosslinked Microsphere Powder (8)
- the polystyrene-maleic anhydride copolymer and NaYF 4 rare earth nanoparticles with excitation wavelength of 580 nm were dissolved in toluene, the concentration of polystyrene-maleic anhydride copolymer was 2 g/mL, and the concentration of NaYF 4 rare earth nanoparticles was I nM/L, which is used as a dispersed phase.
- the dispersed phase was extruded through a membrane with a pressure of 20 KPa of nitrogen into a continuous phase of water containing an emulsifier SDS concentration of 1 wt.%.
- the flow rate of the continuous phase was 0.40 m/s, and an oil-in-water emulsion having a uniform droplet size was obtained.
- the obtained rare earth nanoparticle composite fluorescent microsphere suspension is collected by centrifugation. Thereafter, the mixture was washed three times with deionized water, washed three times with absolute ethanol, and freeze-dried to obtain a solid powder of rare earth nanoparticle composite fluorescent microspheres.
- rare earth nanoparticles composite fluorescent microspheres were dispersed in 15 mL of 0.01 mol/L hydrochloric acid to prepare a suspension, and magnetically stirred for 8 h. After the anhydride functional group on the polymer was hydrolyzed to carboxyl group, a part of the suspension was extracted and used. It is washed with 1/1 deionized water (until the pH of the suspension of the hydrolyzed microspheres is stabilized at about 6.2), and then freeze-dried to obtain a surface-carboxy functionalized rare earth nanoparticle composite fluorescent microsphere.
- the first one was prepared by dissolving polystyrene-maleic anhydride copolymer and CdSe/CdS quantum dots with excitation wavelength of 528 nm in toluene, and the concentration of polystyrene-maleic anhydride was 1 g/mL. , the quantum dot concentration is 1 nM / L; the second polystyrene-maleic anhydride copolymer and CdSeTe quantum dots with excitation wavelength of 680 nm are dissolved in toluene, polystyrene-maleic anhydride copolymerization concentration is 1 g /mL, the quantum dot concentration is 1 nM/L.
- the two dispersed phases were respectively extruded through a membrane with a pressure of 15 KPa of nitrogen, each entering a continuous phase of water containing an emulsifier SDS concentration of 1 wt.%, continuous phase.
- the flow rate was 0.37 m/s, resulting in an oil-in-water emulsion with uniform droplet size.
- the two emulsions are each at 25! Stir and volatilize with magnetic stirring at 350 rpm. After the toluene in the solution is completely volatilized, the obtained quantum dot-labeled fluorescent microsphere suspension is collected by centrifugation.
- quantum dot composite fluorescent microspheres with different emission wavelengths were dispersed in 15 mL of 0.01 mol/L hydrochloric acid to prepare a suspension, and magnetically stirred for 8 h. After the anhydride functional group on the polymer was hydrolyzed to carboxyl group, the fraction was extracted. The suspension was washed with a volume ratio of 1/1 deionized water (until the pH of the suspension of the hydrolyzed microspheres was stabilized at about 6.2), and freeze-dried to obtain a surface-carboxy functionalized quantum dot composite microsphere.
- the two kinds of quantum dot composite microspheres prepared by scanning electron microscopy were smooth spherical particles with an average particle size of 6.5-6.6 ⁇ .
- the coefficient of variation of particle size distribution CV was 8.7%-8.8%, and the monodispersity was good.
- the polystyrene-maleic anhydride copolymer and CdSe/CdS quantum dots with an emission wavelength of 528 nm were dissolved in toluene at a polymer concentration of 1 g/mL and a quantum dot concentration of 1 nM/L, which was used as a dispersed phase. .
- the dispersed phase was extruded through a membrane with a pressure of 8 KPa of nitrogen into a continuous phase of water containing an emulsifier SDS concentration of 1 wt.%.
- quantum dot composite fluorescent microspheres were dispersed in 15 mL of 0.01 mol/L hydrochloric acid to prepare a suspension, and magnetically stirred for 8 h. After the anhydride functional group on the polymer was hydrolyzed to carboxyl group, a part of the suspension was extracted and the volume ratio was used. 1/1 deionized water washing (until the pH of the suspension of the hydrolyzed microspheres is stabilized at about 6.2), followed by freeze-drying to obtain surface-carboxy functionalized quantum dot composite fluorescent microspheres.
- the polymethacrylic acid and silver nanoparticles were dissolved in dichloromethane to have a polymethacrylic acid concentration of 2 g/mL and a silver nanoparticle concentration of 0.5 nM/L as a dispersed phase.
- a SPG porous membrane with a pore size of 0.1 ⁇ and a porosity of 0.4 the dispersed phase was extruded through a membrane with a pressure of 45 KPa of nitrogen into the SDS containing 0.1 wt.% emulsifier and 0.1 wt.% stabilizer Tween-20.
- the continuous phase of water, the flow rate of the continuous phase is 0.35 m/s, and the water with uniform droplet size is obtained.
- the surface carboxyl group of the quantum dot/Fe 3 4 composite microsphere prepared in Example 7 was activated by EDC, N-hydroxysuccinimide was attached thereto, and then a hepatitis B virus surface antibody (HBsAb) was ligated as a probe molecule.
- Quantum dot composite fluorescent microspheres form a biodetection probe for specific detection of hepatitis B virus surface antigen (HBsAg) after surface-bound HBsAb.
- the biodetection probe After the biodetection probe is put into a sample containing hepatitis B virus surface antigen (HBsAg), the target which is attached to the probe, that is, hepatitis B virus surface antigen (HBsAg), is labeled with fluorescein isothiocyanate.
- the final bioassay probe emits a fluorescent signal under laser excitation, and the HBV surface antigen (HBsAg) in the sample is detected by the fluorescence signal intensity of the quantum dot inside the microsphere and the labeled fluorescent signal intensity of the target attached to the probe molecule.
- the concentration of hepatitis B virus surface antigen (HBsAg) detected is 0-500 ng/ml
- the intensity of fluorescent label ie, the intensity of fluorescein isothiocyanate labeling
- the concentration of antigen (HBsAg) increases, it has a proportional relationship.
- the rare earth nanoparticle composite microsphere prepared in Example 8 was activated by EDC, and the DNA fragment of the sequence 5'-TCA AGG CTC AGT TCG AAT GCA CCA TA-3 ' was used as a probe molecule to form a specific detection.
- Biodetection probe for DNA After the biodetection probe is put into a sample containing a DNA segment of 5'-TAT GGT GCA TTC GAA CTG AGC CTT GA-3 ', the target attached to the probe, that is, the sequence is 5 The DNA segment of '-TAT GGT GCA TTC GAA CTG AGC CTT GA-3 ' was labeled with Cascade Blue.
- the treated rare earth nanoparticle composite microspheres emit a fluorescent signal under the excitation of infrared laser, and the fluorescent signal of the rare earth nanoparticle inside the microsphere and the labeled fluorescent signal of the target attached to the probe molecule are paired with 5'-TAT GGT GCA.
- 5'-TAT GGT GCA Qualitative and quantitative analysis of the DNA segments of TTC GAA CTG AGC CTT GA-3 '.
- the surface carboxyl group of the quantum dot composite microsphere prepared in Example 9 was attached to N-hydroxysuccinimide under EDC activation. Then, the 528 nm quantum dot composite microspheres are further linked to hepatitis B virus surface antigen (HBsAg) as a detection probe molecule.
- the 528 nm quantum dot composite microspheres form a biodetection probe for the specific detection of hepatitis B virus surface antibody (HBsAb) after surface-attached antigen (HbsAg).
- the 690 nm quantum dot composite microspheres are linked to hepatitis B virus e antigen (HBeAg) as a detection probe molecule.
- HbeAg hepatitis B virus e antibody
- HbsAb hepatitis B virus surface antibody
- HBeAb hepatitis B virus e antibody
- the quantum dot fluorescence of the microsphere is detected by HBsAb at 528 nm, and the quantum dot fluorescence of the microsphere is detected by HBeAb at 680 nm, and then passed through the probe molecule.
- the labeled fluorescence intensity of the target attached to the sample was quantified for each of the HBsAb and HBeAb in the sample.
- Styrene, divinylbenzene, methacrylic acid, benzoyl peroxide and CdSe/CdS quantum dots with an emission wavelength of 528 nm were dissolved in toluene with a styrene concentration of lg/mL and a divinylbenzene concentration of 0.75.
- methacrylic acid concentration was 0.5 g/mL
- benzoyl peroxide concentration was 0.001 g/mL
- quantum dot concentration was 1 nM/L, which was used as the dispersed phase.
- the dispersed phase was extruded through a membrane with a pressure of 18 KPa of nitrogen into a continuous phase of water containing an emulsifier SDS concentration of 1 wt.%, the flow rate of the continuous phase. It is 0.30 m/s, and an oil-in-water emulsion having a uniform droplet size is obtained.
- the emulsion was heated to 70 ° C and reacted under nitrogen for 12 h.
- the methyl acrylate, divinylbenzene, azobisisobutyronitrile and Fe 3 0 4 magnetic nanoparticles were dissolved in chloroform, the methyl acrylate concentration was 1 g/mL, and the divinylbenzene concentration was 0.5 g/mL.
- the concentration of azobisisobutyronitrile was 0.01 g/mL, and the concentration of Fe 3 0 4 magnetic nanoparticles was 0.5 nM/L, which was used as a dispersed phase.
- the dispersed phase was extruded through a membrane with a pressure of 21 KPa of nitrogen into a continuous phase of water containing an emulsifier SDS concentration of 0.5 wt.%. It is 0.40 m/s, and an oil-in-water emulsion having a uniform droplet size is obtained.
- the emulsion was heated to 70 ° C and reacted under nitrogen for 12 h.
- the microsphere suspension obtained was then centrifuged three times with deionized water, washed three times with absolute ethanol, and freeze-dried to obtain a solid powder of Fe 3 0 4 magnetic nanoparticle composite microspheres.
- the prepared Fe 3 0 4 magnetic nanoparticle composite microspheres were spherical particles with smooth surface, the average particle size was 5.8 ⁇ , the coefficient of variation of particle size distribution CV was about 8.7%, and the monodispersity was better.
- the styrene, divinylbenzene, methacrylic acid and NaYF 4 rare earth nanoparticles are dissolved in xylene with a styrene concentration of 1 g/mL, a divinylbenzene concentration of 0.5 g/mL, and a methacrylic acid concentration of 0.5. g/mL.
- the NaYF 4 rare earth nanoparticles have a concentration of 1 nM/L as a dispersed phase.
- the dispersed phase was extruded through a membrane using a pressure of 25 KPa of nitrogen to enter a concentration of 0.001 g/mL of initiator.
- the emulsion was heated to 65 ° C and reacted under nitrogen for 12 h.
- the microsphere suspension obtained was then centrifuged three times with deionized water, washed three times with absolute ethanol, and lyophilized to obtain a solid powder of NaYF 4 rare earth nanoparticle composite microspheres.
- the prepared NaYF 4 rare earth nanoparticles composite microspheres were spherical particles with smooth surface, the average particle size was 3.4 ⁇ , the coefficient of variation of particle size distribution CV was about 8.9%, and the monodispersity was better.
- Styrene, divinylbenzene, maleic anhydride, benzoyl peroxide and gold nanoparticles are dissolved in toluene with a styrene concentration of 1 g/mL and a divinylbenzene concentration of 0.5 g/mL.
- Maleic anhydride The concentration was 0.5 g/mL, the concentration of azobisisobutyronitrile was 0.01 g/mL, and the concentration of gold nanoparticles was 1 nM/L, which was used as the dispersed phase.
- the dispersed phase was extruded through a membrane with a pressure of 25 KPa of nitrogen into the membrane containing a concentration of 0.1 wt.% emulsifier SDS and 1 wt.% stabilizer PVA.
- the flow rate of the continuous phase was 0.35 m/s, and an oil-in-water emulsion having a uniform droplet size was obtained.
- the emulsion was heated to 85 ° C and reacted under nitrogen for 12 h.
- the obtained microsphere suspension was washed by centrifugal washing three times with deionized water, centrifuged three times with absolute ethanol, and freeze-dried to obtain a solid powder of gold nanoparticle composite microspheres.
- the gold nanoparticle composite microspheres prepared by electron microscopy were spherical particles with smooth surface, the average particle size was 5.5 ⁇ , the coefficient of variation of particle size distribution CV was about 8.5%, and the monodispersity was better.
- Styrene, methylol acrylamide, benzoyl peroxide, CdTeSe/CdS quantum dots with a wavelength of 680 nm and Fe 3 0 4 magnetic nanoparticles were dissolved in toluene at a styrene concentration of 1.5 g/mL.
- concentration of acrylamide was 0.5 g/mL
- concentration of benzoyl peroxide was 0.005 g/mL
- the concentration of quantum dots was 1 nM/L.
- the concentration of Fe 3 0 4 magnetic nanoparticles was 0.5 nM/L, which was used as a dispersed phase.
- the dispersed phase was extruded through a membrane with a pressure of 22 KPa into a continuous phase of water containing a concentration of 1 wt.% emulsifier SDS. It is 0.35 m/s, and an oil-in-water emulsion having a uniform droplet size is obtained.
- the emulsion was heated to 70 ° C and reacted under nitrogen for 12 h.
- the obtained microsphere suspension was centrifuged three times with deionized water, washed three times with absolute ethanol, and freeze-dried to obtain a solid powder of quantum dot/Fe 3 4 nanoparticle composite fluorescent magnetic microspheres.
- the prepared quantum dot/Fe 3 0 4 nanoparticle composite fluorescent magnetic microspheres were spherical particles with smooth surface, the average particle size was 5.8 ⁇ , and the coefficient of variation of particle size distribution CV was about 7.8%.
- the monodispersity was compared by electron microscopy. it is good.
- Styrene, acrylic acid, azobisisobutyronitrile, glutaraldehyde, Fe 3 0 4 magnetic nanoparticles and gold nanoparticles were dissolved in chloroform with a styrene concentration of 1 g/mL and an acrylic acid concentration of 0.5 g/mL.
- Glutaraldehyde concentration is 0.2 g/mL
- azo The concentration of diisobutyronitrile was 0.01 g/mL
- the concentration of Fe 3 0 4 magnetic nanoparticles was 0.5 nM/L
- the concentration of gold nanoparticles was 0.2 nM/L, which was used as a dispersed phase.
- the dispersed phase was extruded through a membrane with a pressure of 23 KPa of nitrogen into a continuous phase of water containing a concentration of 1 wt.% emulsifier SDS. It is 0.35 m/s, and an oil-in-water emulsion having a uniform droplet size is obtained.
- the emulsion was heated to 70 ° C and reacted under nitrogen for 12 h.
- the microsphere suspension obtained was then centrifuged three times with deionized water, washed three times with absolute ethanol, and lyophilized to obtain a solid powder of Fe 3 0 4 magnetic nanoparticles/gold nanoparticle composite microspheres.
- the prepared Fe 3 0 4 magnetic nanoparticles/gold nanoparticle composite microspheres were spherical particles with smooth surface, the average particle size was 3.7 ⁇ , and the coefficient of variation of particle size distribution CV was about 7.9%.
- the monodispersity was compared by electron microscopy. it is good.
- Styrene, methyl methacrylate, potassium persulfate, divinylbenzene, Ti 2 nanoparticles and gold nanoparticles are dissolved in toluene with a styrene concentration of 1.5 g/mL and a methyl methacrylate concentration of 0.5 g.
- concentration of /mL divinylbenzene was 0.5 g/mL
- concentration of Ti0 2 nanoparticles was 1 nM/L, which was used as the dispersed phase.
- the dispersed phase was extruded through a membrane with a pressure of 23 KPa of nitrogen into a concentration of 0.001 g/mL of initiator potassium persulfate at a concentration of 0.75 wt.%.
- the aqueous continuous phase of the emulsifier SDS, the flow rate of the continuous phase was 0.38 m/s, and an oil-in-water emulsion having a uniform droplet size was obtained.
- the emulsion was heated to 70 ° C and reacted under nitrogen for 12 h.
- the prepared TiO 2 nanoparticles/gold nanoparticle composite microspheres were spherical particles with smooth surface, the average particle size was 6.1 ⁇ , and the coefficient of variation of particle size distribution CV was about 8.2%. The monodispersity was better.
- Styrene, divinylbenzene, azobisisobutyronitrile, silver nanoparticles were dissolved in chloroform, styrene concentration was 1.5 g/ml, divinylbenzene concentration was 0.5 g/ml, azobisisobutyronitrile The concentration was 0.02 g/ml, and the concentration of silver nanoparticles was 0.5 nM/L, which was used as the dispersed phase.
- the dispersed phase was extruded through a membrane with a pressure of 28 KPa of nitrogen into the membrane containing a concentration of 0.1 wt.% emulsifier SDS and 1 wt.% stabilizer PVA.
- the flow rate of the continuous phase was 0.35 m/s, and an oil-in-water emulsion having a uniform droplet size was obtained.
- the emulsion was heated to 70 ° C and reacted under nitrogen for 12 h.
- microsphere suspension was washed by centrifugal washing with deionized water for 3 times, centrifuged 3 times with absolute ethanol, and freeze-dried to obtain a solid powder of silver nanoparticle composite microspheres.
- the silver nanoparticle composite microspheres prepared by electron microscopy showed smooth spherical particles with an average particle size of 3.5 ⁇ .
- the coefficient of variation of particle size distribution CV was about 8.6%, and the monodispersity was good.
- the quantum dots and the quantum dots of CdTeSe/CdS with a wavelength of 755 nm are dissolved in toluene, the concentration of styrene is 1 g/ml, the concentration of divinylbenzene is 0.75 g/ml, and the concentration of methacrylic acid is 0.5 g/ml.
- the concentration of benzoyl peroxide is 0.002 g/ml
- the concentration of CdSe/CdS/ZnS quantum dots at a wavelength of 515 nm is 0.5 nM/L
- the concentration of CdTeSe/CdS quantum dots at a wavelength of 755 nm is 0.5 nM/L, which is used as a dispersion. phase.
- the dispersed phase was extruded through a membrane with a pressure of 20 KPa of nitrogen into a solution containing a concentration of 1 wt.% emulsifier SDS and 0.1 wt.% stabilizer Tween.
- the water continuous phase of -20, the flow rate of the continuous phase was 0.35 m/s, and an oil-in-water emulsion having a uniform droplet size was obtained.
- the emulsion was heated to 70 ° C and reacted under nitrogen for 12 h.
- the microsphere suspension obtained was then centrifuged three times with deionized water, washed three times with absolute ethanol, and lyophilized to obtain a solid powder of double-emission wavelength quantum dot composite microspheres.
- the prepared quantum dot composite microspheres are spherical particles with smooth surface. As shown in Fig. 4, the average particle size is 6.5 ⁇ , the coefficient of variation of particle size distribution CV is about 7.5%, and the monodispersity is good.
- Styrene, divinylbenzene, methacrylic acid, benzoyl peroxide and CdSe/CdS quantum dots with an emission wavelength of 528 nm were dissolved in toluene with a styrene concentration of lg/mL and a divinylbenzene concentration of 0.75.
- methacrylic acid concentration was 0.5 g/mL
- benzoyl peroxide concentration was 0.001 g/mL
- quantum dot concentration was 1 nM/L, which was used as the dispersed phase.
- the dispersed phase was extruded through a membrane with a pressure of 45 KPa of nitrogen into a continuous phase of water containing an emulsifier SDS concentration of 1 wt.%. It is 0.30 m/s, and an oil-in-water emulsion having a uniform droplet size is obtained.
- the emulsion was heated to 70 ° C and reacted under nitrogen for 12 h.
- Styrene, divinylbenzene, methacrylic acid, benzoyl peroxide, CdSe/CdS/ZnS quantum dots with a wavelength of 515 nm and CdTeSe/CdS quantum dots with a wavelength of 755 nm are dissolved in toluene, styrene Concentration 1 g/ml, divinylbenzene concentration 0.75 g/ml, methacrylic acid concentration 0.5 g/ml, benzoyl peroxide concentration 0.002 g/ml, CdSe/CdS/ZnS quantum with a wavelength of 515 nm
- concentration of CdTeSe/CdS quantum dots with a wavelength of 0.5 nM/L and a wavelength of 755 nm was 0.5 nM/L, which was used as a dispersed phase.
- the dispersed phase was extruded through a membrane using a nitrogen pressure of 12 KPa into a Tween containing a concentration of 1 wt.% emulsifier SDS and 0.1 wt.% stabilizer.
- the water continuous phase of -20, the flow rate of the continuous phase was 0.35 m/s, and an oil-in-water emulsion having a uniform droplet size was obtained.
- the emulsion was heated to 70 ° C and reacted under nitrogen for 12 h.
- the microsphere suspension obtained was then centrifuged three times with deionized water, washed three times with absolute ethanol, and lyophilized to obtain a solid powder of double-emission wavelength quantum dot composite microspheres.
- the prepared quantum dot composite microspheres are spherical particles with smooth surface and average particles. The diameter is 20 ⁇ , and the coefficient of variation of the particle size distribution CV is about 6.8%, and the monodispersity is good.
- the quantum dot composite crosslinked microsphere powder prepared in Example 18 was prepared, and 0.1 g of quantum dot composite fluorescent microspheres were dispersed in 15 mL of 0.01 mol/L hydrochloric acid to prepare a suspension, magnetically stirred for 8 h, and the anhydride on the polymer was prepared. After the functional group is hydrolyzed to a carboxyl group, a part of the suspension is extracted and washed with a volume ratio of 1/1 deionized water until the pH of the suspension of the hydrolyzed microsphere is stabilized at about 6.2, and then freeze-dried to obtain a surface-carboxy functionalized quantum dot compound cross. Union microspheres.
- HBsAb hepatitis B virus surface antibody
- Quantum dot-labeled fluorescent microspheres form a quantum dot-labeled bioassay probe that specifically detects hepatitis B virus surface antigen (HBsAg) after surface-linked antibodies (HBsAb).
- the quantum dot-labeled biodetection probe After the quantum dot-labeled biodetection probe is put into a sample containing hepatitis B virus surface antigen (HBsAg), the target attached to the probe, namely hepatitis B virus surface antigen (HBsAg), is phosphorylated with isothiocyanate. Prime label. Finally, the processed quantum dot-labeled biodetection probe emits a fluorescent signal under laser excitation, and passes through the fluorescent signal inside the microsphere and the labeled fluorescent signal of the target attached to the probe molecule (ie, fluorescein isothiocyanate). Marking) Qualitative and quantitative analysis of hepatitis B virus surface antigen (HBsAg) in the sample.
- HBsAg hepatitis B virus surface antigen
- any surface carboxylated functional nanoparticle composite crosslinked microsphere powder prepared in Examples 15, 17, 20, 23 and 24 the surface carboxyl group of the prepared composite crosslinked microsphere was activated under EDC and connected.
- the DNA sequence of the upper sequence of 5'-TCA AGG CTC AGT TCG AAT GCA CCA TA-3 ' acts as a probe molecule to form a biodetection probe for specific detection of DNA.
- the target attached to the probe that is, the sequence is 5
- the DNA segment of '-TAT GGT GCA TTC GAA CTG AGC CTT GA-3 ' was labeled with Cascade Blue.
- the treated functional nanoparticle composite crosslinked microspheres emit a fluorescent signal under the excitation of infrared laser, and the fluorescent signal of the rare earth nanoparticle inside the microsphere and the labeled fluorescent signal of the target attached to the probe molecule are 5'- Qualitative and quantitative analysis of the DNA segments of TAT GGT GCA TTC GAA CTG AGC CTT GA-3'.
- the functional nanoparticles may also be semiconductor nanoparticles or the like;
- the particles may also be the following quantum dots: CdS, HgS, CdSe, CdTe, ZnSe, HgSe, ZnTe, ZnO, PbSe, HgTe, CaAs, InP, InCaAs, CdSe/ZnS, CdSe/ZnSe, CdS/ZnS, Cd/Ag 2S , 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
- the bio-detection probe based on the functional nanoparticle composite microsphere of the invention can be used for detecting one or more targets in a sample, such as detecting cytokines, allergens and autoimmune reactions, HLA typing, in disease diagnosis, SP detection, tumor specific antigen quantitative detection, multiplex microbial quantitative 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, agricultural and veterinary drugs Residual multiple quantitative testing and forensic identification.
- the method for detecting one or more targets in a sample using the bio-detection probe based on the functional nanoparticle composite microsphere of the present invention is:
- the target substance described in the step (1) includes a protein, a protein fragment or a nucleic acid
- the fluorescent substance described in the step (2) includes: fluorescein isothiocyanate (FITC), phycoerythrin (PE), and propidium iodide Pyridine (PI), phthalocyanin (CY5), chlorophyll protein (preCP), phycoerythrin-Texas Red, Cascade Blue, and surface-modified quantum dots
- instrumentation for bioprobe detection refers to the qualitative analysis of the target in the test sample by measuring the performance of the nanoparticles inside the microsphere by using the instrument, and simultaneously detecting the fluorescence intensity of the target attached to the biodetection probe.
- Quantitative analysis of the target in the sample common instruments used for detection include: flow cytometry, Luminex suspension array detection system (Luminex, USA), fluorescence spectrophotometer, laser confocal microscope, fluorescence microscope, vibration sample magnetic Strong measure.
- the analysis of the bioprobe detection result further comprises: utilizing the inner nanoparticle of the composite microsphere of the invention Qualitative analysis to qualitatively analyze the target in the test sample; quantitatively analyze the target in the test sample by the intensity of the labeled fluorescent light of the target attached to the biological detection probe, wherein the biological detection probe is connected The intensity of the target label fluorescence is proportional to the concentration of the target in the test sample.
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Abstract
公开了功能性纳米颗粒复合非交联微球粉末、交联微球粉末及其制备方法和应用。该功能性纳米颗粒复合非交联微球粉末包含功能性纳米颗粒复合非交联微球,该复合非交联微球包含功能性纳米颗粒和聚合物,平均粒径为0.1-20μm,粒径分布变异系数≤9.1%;该功能性纳米颗粒复合交联微球粉末包含有功能性纳米颗粒复合交联微球,该复合交联微球包含功能性纳米颗粒和单体、交联剂及引发剂,平均粒径为0.1-20μm,粒径分布变异系数≤9.6%。上述微球粉末是利用膜乳化技术结合溶剂挥发法或乳液聚合法制备得到。
Description
功能性纳米颗粒复合微球粉末及其制备方法和应用
技术领域
本发明涉及微纳米材料的制备与应用领域, 具体地说, 是功能性纳米颗粒复合微球 及其制备方法和应用。
背景技术
功能性纳米颗粒复合微球是指通过某种方法将功能性纳米颗粒与微球结合在一起所 得到的一种功能性复合微球。 现阶段通过各种途径制备的纳米颗粒具备各种特殊的光、 电、 磁及生物学等特性, 因此在与微球结合后往往会将这些特性赋予到微球本身。 而微 球也为这些纳米颗粒提供了支承载体和有效保护。 同时, 微球自身的化学和物理性能如 光敏感性、 pH响应性、 温度敏感性、 吸附特性以及表面活性官能团也为纳米颗粒在各种 复杂领域的应用提供可能。 具备不同特殊功能的纳米颗粒复合微球在生物医药、 工业催 化、 化工合成、 电子信息、 建筑材料等领域具有巨大的应用潜力。
现有技术中制备功能性纳米颗粒复合微球的制备方法主要有以下几种:
原位聚合法: 即事先合成载体微球, 之后以微球作为微型反应器, 在其内部或表面 原位合成各种功能性颗粒。 但此方法由于受到微球自身条件影响, 其所制备出的功能性 颗粒种类及性能会受到一定限制。
溶胀渗透法: 即事先合成具备孔结构的交联微球, 之后微球在良溶剂中被溶胀, 表 面微孔得到扩张。 此时加入功能性颗粒, 在浓度差或疏水作用下微球通过孔结构渗透进 微球并吸附在微球内壁。 当外部溶剂去除后, 微球收缩, 从而将功能性颗粒包埋进入微 球。 此种方法简单易行, 但要求微球的表面具有可供功能性渗透进入的微孔。 而由于这 种微孔结构的存在, 会令包埋进的功能性颗粒容易重新泄漏出来, 且不能为包埋在内的 功能性颗粒提供足够保护以减少外界影响, 因而此方法制备的复合微球内的纳米颗粒的 性能稳定性欠佳。
组装模板法: 即以功能性颗粒或微球为核心模板, 通过静电作用、 疏水作用、 络合 作用和氢键作用, 将功能性颗粒和构成微球的材料依次结合在核心上。 这种方法可以精 确控制复合微球的粒径和纳米颗粒数量, 但制备步骤较为复杂。 且制备过程中反应环境 对部分功能性颗粒的性能有一定影响。
聚合法: 即在利用乳液聚合、 悬浮聚合、 分散聚合或本体聚合在制备微球的同时引 入功能性颗粒。 功能性颗粒分散在可聚合单体中, 随着聚合的进行, 功能性颗粒被包埋 在逐渐形成的微球结构之内。 此种方法优点是制备简单、 产率较高。 但是此种方法仍然 面临以下问题: 制备微米级复合微球时粒度分布较宽, 功能性颗粒性能易受聚合反应环
境的影响, 功能性颗粒与形成的微球结构可能存在相分离。
现阶段,通过各种方法制备的功能性纳米颗粒复合聚合物微球已被应用于各个领域:
Ugelstad研究小组通过原位聚合法在苯乙烯微球中制备了磁性纳米粒子, 其具备较高的 磁含量已被广泛应用于癌症检测及治疗领域。 Me研究小组通过采用溶胀渗透法将半导体 纳米颗粒量子点包埋进多孔微球,利用微球表面羧基官能团将 DNA链段连接在微球表面 作为探针, 进行了 DNA特异性检测。 Ahjeong Son小组采用模板自组装法, 在磁性微球 表面通过酰胺键包覆量子点壳层从而形成具备光磁双重特性复合微球, 并应用于核酸检 测。 Yong Zhang研究小组, 利用微乳液聚合法制备了复合有上转换发光稀土纳米颗粒的 微球。 这种微球在红外激发下具备特殊荧光性能的复合微球, 其在医学成像领域具有广 阔的应用前景。
经对现有技术的文献搜索发现, 中国专利申请号: 200410073449.7, 名称为 "一种高 分子 /无机物复合微球的制备方法" 的发明, 提出首先将活性预聚物吸附在无机颗粒表 面, 之后引发聚合形成无机颗粒复合微球的方法, 中国专利申请号 200810019950.3, 名 称为 "纳米微米复合微球制备方法" 的发明, 提出通过化学方法将纳米颗粒键合到微球 表面制得纳米颗粒复合微球的方法, 中国专利申请号: 201010593883.3, 名称为 "制备有 机 /无机复合微球的制备方法" 的发明, 采用先溶胀聚合物微球, 后将溶胀溶剂加热挥发 的方法制备了无机纳米颗粒复合聚合物微球。 但以上制备复合微球所采用方法的原理仍 基于上面所述的各种传统制备无机纳米颗粒复合微球的方法, 存在其固有系统缺陷, 无 法制备得到粒径为微米级、 均一可控、 分散性好的功能性纳米颗粒复合微球。
膜乳化技术是制备均一粒径的单分散乳液最简单有效的方法, 它是通过无机膜微孔 将分散相在外加压力的作用下, 压入连续相中形成乳状液, 通过控制分散压力和膜孔径, 实现乳状液滴的单分散性。 通过膜乳化法可以制备 0 / W、 W / 0型单重乳状液, 或在 单重乳状液的基础上经过二次乳化形成多重乳状液, 再经过某些物化反应将液滴转换为 固相, 例如非水溶性液滴通过溶剂蒸发可以转化为高分子微球。 通过膜乳化技术制备的 单分散高分子微球有许多独特的特点: 小的尺寸和体积, 均一的粒径且粒径分布可控, 大的比表面积, 优良的孔隙率, 各功能团体的分布具有理想表面密度, 稳定的分散性等。
包德才等使用 PS (聚苯乙烯)二氯甲烷溶液作为分散相,含有十二烷基硫酸钠( SDS ) 的水溶液为连续相, 在外加压力作用下, 将分散相通过 SPG膜微孔分散到连续相中, 制 备出呈单分散的 0 / W型乳状液。 然后利用液中干燥法, 将膜乳化法制备的 0 / W型乳 状液迅速转移至 40°C的恒温槽中, 低速搅拌 4-6 h。 随着分散相溶剂二氯甲烷不断向连续 相扩散并逐渐挥发除去, PS逐渐析出, 最终固化成 Ps微球。 采用膜乳化-液中干燥法所
制备的 PS微球经扫描电子显微镜观测发现其表面光滑, 球形度好, 粒径呈很好的单分散 性。
Gasparini等采用膜乳化技术制备了一种可生物降解的单分散药物载体微球一聚乳酸 I乙醇酸共聚物 (PLGA) 微球。
中国期刊 《功能高分子学报》 2011年 3月 Vol.24 No. l刊出的文章 "膜乳化-溶剂挥 发法制备表面羧基功能化苯乙烯-马来酸酐共聚物微球" , 以苯乙烯-马来酸酐共聚物 (PSMA) 为原料, 利用膜乳化-溶剂挥发法, 成功制备了表面光滑、 尺寸均一的表面羧 基功能化聚合物微球。
但是目前还未见利用膜乳化技术成功制备功能性纳米颗粒复合微球的相关报道。 发明内容
本发明的目的是针对现有技术中的不足, 提供一种功能性纳米颗粒复合非交联微球 粉末。
本发明的再一的目的是, 提供一种功能性纳米颗粒复合非交联微球粉末的制备方 法。
本发明的另一的目的是 , 提供一种功能性纳米颗粒复合非交联微球粉末的用途。 本发明的第四个目的是, 提供一种基于功能性纳米颗粒复合非交联微球粉末的生物 检测探针。
本发明的第五个目的是 , 提供一种基于功能性纳米颗粒复合非交联微球粉末的生物 检测探针的用途。
本发明的第六个目的是 , 提供一种功能性纳米颗粒复合交联微球粉末。
本发明的第七个目的是 , 提供一种功能性纳米颗粒复合交联微球粉末的制备方法。 本发明的第八个目的是 , 提供一种功能性纳米颗粒复合交联微球粉末的用途。 本发明的第九个目的是 , 提供一种基于功能性纳米颗粒复合交联微球粉末的生物检 测探针。
本发明的第十个目的是 , 提供一种基于功能性纳米颗粒复合交联微球粉末的生物检 测探针的用途。
为实现上述目的, 本发明采取的技术方案是:
一种功能性纳米颗粒复合非交联微球粉末,它包含功能性纳米颗粒复合非交联微球, 所述的功能性纳米颗粒复合非交联微球包含功能性纳米颗粒和聚合物, 平均粒径为 0.1-20μηι, 粒径分布变异系数 9.1%。
所述的功能性纳米颗粒是下列中的一种或几种: 量子点、 磁性纳米颗粒、 荧光纳米
颗粒、 金属纳米颗粒、 金属氧化物纳米颗粒或半导体纳米颗粒。
所述的量子点是下列中的一种或几种: CdS、 HgS、 CdSe、 CdTe、 ZnSe、 HgSe、 ZnTe、 ZnO、 PbSe、 HgTe、 CaAs、 InP、 InCaAs、 CdSe/CdS、 CdSe/ZnS、 CdSe/ZnSe、 CdS/ZnS、 Cd/Ag 2S、 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, 以及掺杂量子点 CdS:Mn、 CdS:Mn、 CdS:Cu、 ZnS:Cu、 CdS:Tb、 ZnS:Tb。
所述的聚合物是下列中的一种或几种: 聚苯乙烯、 聚丙烯酸、 聚甲基丙烯酸、 聚甲 基丙烯酸甲酯、 聚甲基丙烯酸乙酯、 聚酰胺、 聚丙烯腈、 聚碳酸酯、 聚己内酯、 聚氨酯、 聚乳酸、 壳聚糖、 白蛋白、 胶原、 聚苯乙烯 -马来酸酐共聚物、 聚乙酸乙酯、 聚苯乙烯- 丙烯酸共聚物、 聚苯乙烯-甲基丙烯酸共聚物或聚苯乙烯 -甲基丙烯酸甲酯共聚物。
所述的功能性纳米颗粒复合非交联微球经表面改性连有官能团。
所述的表面改性是下列中的一种或几种: 水解、 化学接枝或磺化。
所述的官能团是下列中的一种或几种: 羧基、 氨基、 磺酸根基、 硝基、 羟、 氯基或 酯基。
所述的官能团上还连接有以下连接物中的一种或几种: N-羟基琥珀酰亚胺、生物素、 亲和素或抗生蛋白链菌素。
为实现上述第二个目的, 本发明采取的技术方案是:
一种如上任一所述的功能性纳米颗粒复合非交联微球粉末的制备方法, 所述的制备 方法包括以下步骤:
a) 制备分散相, 所述的分散相包括功能性纳米颗粒和聚合物溶液;
b) 制备连续相, 所述的连续相包括去离子水及溶于水的稳定剂和 /或乳化剂; c)利用膜乳化装置在气体压力作用下挤压分散相通过多孔膜以液滴形式进入到连续 相中, 在连续相剪切力作用下得到液滴粒径均一的单分散乳液;
d) 通过溶剂蒸发法获得功能性纳米颗粒复合非交联微球粉末。
所述的步骤 a) 中聚合物溶液为有机溶剂中溶解有聚合物的溶液。
所述的有机溶剂是疏水性有机溶剂。
所述的疏水性有机溶剂选自下列中的一种或几种: 甲苯、 二甲苯、 对氯甲苯、 二氯 甲烷、 三氯甲烷、 四氯甲烷、 石油醚、 正己烷或环己烷。
所述的聚合物溶液的浓度是 0.5-2 g/mL。
所述的步骤 a) 中功能性纳米颗粒的浓度是 0.5-1 nM/L。
所述的步骤 b) 中稳定剂和 /或乳化剂选自下列中的一种或几种: 十二烷基硫酸钠、
聚乙烯醇或吐温 20。
所述的步骤 c) 中多孔膜是 SPG多孔膜、 陶瓷多孔膜或 MPG多孔膜。
所述的多孔膜的孔径是 0.5-5 μιη。
所述的步骤 c) 中气体压力的大小是 15-30 KPa。
为实现上述第三个目的, 本发明采取的技术方案是:
如上任一所述的功能性纳米颗粒复合非交联微球粉末在检测样品中一种或多种目 标物中的应用。
所述目标物是蛋白质及其片段、 核酸等生物大分子或化合物。
为实现上述第四个目的, 本发明采取的技术方案是:
一种基于功能性纳米颗粒复合非交联微球粉末的生物检测探针, 所述的生物检测探 针包含如上任一所述的功能性纳米颗粒复合非交联微球粉末, 所述的功能性纳米颗粒复 合非交联微球的表面偶联有探针分子。
所述的探针分子选自下列中的一种或几种: 蛋白质、 蛋白质片段或核酸。
为实现上述第五个目的, 本发明采取的技术方案是:
如上所述的生物检测探针在检测样品中一种或多种目标物中的应用。
所述目标物是蛋白质及其片段、 核酸等生物大分子或化合物。
为实现上述第六个目的, 本发明采取的技术方案是:
一种功能性纳米颗粒复合交联微球粉末, 它包含功能性纳米颗粒复合交联微球, 所 述的功能性纳米颗粒复合交联微球包含功能性纳米颗粒和单体、 交联剂及引发剂, 平均 粒径为 0.1-20μιη, 粒径分布变异系数 9.6%。
所述的功能性纳米颗粒是下列中的一种或几种: 量子点、 磁性纳米颗粒、 荧光纳米 颗粒、 金属纳米颗粒、 金属氧化物纳米颗粒或半导体纳米颗粒。
所述的量子点是下列中的一种或几种: CdS、 HgS、 CdSe、 CdTe、 ZnSe、 HgSe、 ZnTe、 ZnO、 PbSe、 HgTe、 CaAs、 InP、 InCaAs、 CdSe/ZnS、 CdSe/ZnSe、 CdS/ZnS、 Cd/Ag 2S、 CdS/Cd(OH)2、 CdTe/ZnS、 CdTeSe/CdS、 CdTe/CdS、 CdSe/ZnSe、 CdS/HgS、 CdS/HgS/CdS ZnS/CdS、 ZnS/CdS/ZnS ZnS/HgS/ZnS/CdS CdSe/CuSe、 CdSeTe、 CdSeTe/CdS/ZnS , 以及掺杂量子点 CdS:Mn、 CdS:Mn、 CdS:Cu、 ZnS:Cu、 CdS:Tb、 ZnS:Tb。
所述的单体是下列中的一种或几种: 苯乙烯、 甲基丙烯酸、 丙烯酸、 丙烯酸甲酯、 甲基丙烯酸甲酯、 乙烯、 丙烯、 丁烯、 丁二烯、 马来酸酐或丙烯酰胺。
所述的交联剂是下列中的一种或几种: 二乙烯基苯、 丙二胺、 戊二醛、 羟甲基丙烯 酰胺、 乙二胺或京尼平。
所述的引发剂是偶氮二异丁腈、 过氧化苯甲酰或过硫酸钾。
所述的功能性纳米颗粒复合交联微球经表面改性连有官能团。
所述的表面改性选自下列中的一种或几种: 水解、 化学接枝或磺化。
所述的官能团是下列中的一种或几种: 羧基、 氨基、 磺酸根基、 硝基、 羟、 氯基或 酯基。
所述的官能团上还连接有以下连接物中的一种或几种: N-羟基琥珀酰亚胺、生物素、 亲和素或抗生蛋白链菌素。
为实现上述第七个目的, 本发明采取的技术方案是:
一种如上任一所述的功能性纳米颗粒复合交联微球粉末的制备方法, 所述的制备方 法包括以下步骤:
a) 制备分散相, 所述的分散相包括功能性纳米颗粒和单体溶液;
b) 制备连续相, 所述的连续相包括去离子水及溶于水的稳定剂和 /或乳化剂; c)利用膜乳化装置, 在气体压力作用下挤压分散相通过多孔膜以液滴形式进入到连 续相中, 在连续相剪切力作用下得到液滴粒径均一的单分散乳液;
d)对得到的单分散乳液加热升温, 进行乳液聚合, 形成功能性纳米颗粒复合交联微 球粉末。
所述的步骤 a) 中单体溶液是有机溶剂中溶解有单体、 交联剂和引发剂的溶液。 所述的有机溶剂为疏水性有机溶剂。
所述的疏水性有机溶剂是下列中的一种或几种: 甲苯、 二甲苯、 对氯甲苯、 二氯甲 烷、 三氯甲烷、 四氯甲烷、 石油醚、 正己烷或环己烷。
所述的步骤 b) 中稳定剂和 /或乳化剂选自下列中的一种或几种: 十二烷基硫酸钠、 聚乙烯醇或吐温 20。
所述的交联剂的浓度为 0.2-0.75 g/mL。
所述的引发剂的浓度为 0.001-0.02 g/mL。
所述的步骤 a) 中功能性纳米颗粒的浓度是 0.2-1 nM/L。
所述的步骤 c) 中多孔膜是 SPG多孔膜、 陶瓷多孔膜或 MPG多孔膜。
所述的多孔膜的孔径是 0.5-5 μιη。
所述的步骤 c) 中气体压力的大小是 18-28 KPa。
为实现上述第八个目的, 本发明采取的技术方案是:
如上任一所述的功能性纳米颗粒复合交联微球粉末在检测样品中一种或多种目标物 中的应用。
所述目标物是蛋白质及其片段、 核酸等生物大分子或化合物。
为实现上述第九个目的, 本发明采取的技术方案是:
一种基于功能性纳米颗粒复合交联微球粉末的生物检测探针, 所述的生物监测探针 包含如上任一所述的功能性纳米颗粒复合交联微球粉末, 所述的功能性纳米颗粒复合交 联微球的表面偶联有探针分子。
所述的探针分子选自下列中的一种或几种: 蛋白质、 蛋白质片段或核酸。
为实现上述第十个目的, 本发明采取的技术方案是:
如上所述的生物检测探针在检测样品中一种或多种目标物中的应用。
所述目标物是蛋白质及其片段、 核酸等生物大分子或化合物。
附图说明
附图 1是实施例 2的复合非交联微球扫描电镜照片。
附图 2是实施例 7的复合非交联微球荧光显微镜照片。
附图 3是实施例 7的生物检测探针检测乙肝病毒表面抗原 HBsAg结果。
附图 4是实施例 23的复合交联微球扫描电镜照片。
具体实施方式
下面结合附图对本发明提供的具体实施方式作详细说明。
以下实施例中使用的膜乳化装置为压力式膜乳化装置, 购自日本 SPG technology 公司; MPG膜购自日本 Ise Chemical公司, 为适用于压力式膜乳化装置的亲水膜; PG膜 购自日本 SPG technology公司, 为适用于压力式膜乳化装置的亲水膜; 陶瓷膜购自德国 Membraflow公司, 为适用于压力式膜乳化装置的亲水膜;
下述实施例中, 平均粒径的计算方法是: 随机将 200个微球的粒径取平均值, 平均 粒径记为 Dav。 粒径分布变异系数 CV的计算方法是:
1 Ν-ΐγ I Dm 其中 A是第 i个微球的粒径, Dav是微球的平 均粒径。
复合微球表面羧基的 EDC ( 1-(3-二甲氨基丙基 )-3-乙基碳二亚胺)活化、探针分子的 连接、抗原 (HBsAg)的异硫氰酸荧光素标记等操作均为临床免疫检测诊断的基本操作, 具 体可参照 《临床免疫学检验实验指导》, 作者: 吕世静, 出版社: 中国医药科学出版社。 实施例 1 复合非交联微球粉末的制备 (一)
将聚苯乙烯-丙烯酸共聚物和金纳米颗粒溶于甲苯中,聚苯乙烯-丙烯酸共聚物浓度为 1 g/mL, 金纳米颗粒浓度为 1 nM/L, 以之作为分散相。 采用孔径为 0.5μιη、 孔隙率为 0.4
的 MPG多孔膜, 利用压力为 30 KPa的氮气将分散相挤压过膜, 进入含有乳化剂 SDS浓 度为 1 wt.%的水连续相, 连续相的流速是 0.35 m/s, 得到液滴粒径均一的水包油乳液。 于 25 t^P 350 rpm磁力搅拌下进行搅拌挥发。待溶液中甲苯完全挥发后, 对得到的金纳 米颗粒复合微球悬浮液进行离心收集。 之后经去离子水离心洗涤 3次, 无水乙醇离心洗 涤 3次, 冷冻干燥得到表面带有羧基的金纳米颗粒复合微球的固体粉末。 经扫描电镜观 察, 所制备的金纳米颗粒复合微球为表面光滑的球形颗粒, 平均粒径为 2.0 μιη, 粒径分 布变异系数 CV为 9.1%左右。
实施例 2 复合非交联微球粉末的制备 (二)
将聚苯乙烯-马来酸酐共聚物和发射波长为 528 nm的 CdSe/CdS量子点溶于甲苯中, 聚合物浓度为 1 g/mL, 量子点浓度为 1 nM/L, 以之作为分散相。 采用孔径为 5 μιη、 孔 隙率为 0.5的 SPG多孔膜, 利用压力为 15 KPa的氮气将分散相挤压过膜, 进入含有乳化 剂 SDS浓度为 1 wt.%的水连续相, 连续相的流速是 0.35 m/s, 得到液滴粒径均一的水包 油乳液。 于 25 t^P 350 rpm磁力搅拌下进行搅拌挥发。待溶液中甲苯完全挥发后, 对得 到的量子点标记荧光微球悬浮液进行离心收集。 之后经去离子水离心洗涤 3次, 无水乙 醇离心洗涤 3次, 冷冻干燥得到量子点复合微球的固体粉末。
将 0.1 g量子点复合荧光微球分散在 15mL 0.01 mol/L的盐酸中制成悬浮液, 磁力搅 拌 8 h, 待聚合物上的酸酐官能团水解为羧基后, 提取出部分悬浮液, 用体积比 1/1去离 子水洗涤 (直至水解微球的悬浮液 pH稳定在 6.2左右), 之后冷冻干燥, 得到表面羧基 功能化的量子点复合荧光微球。
经扫描电镜观察, 所制备的量子点复合荧光微球为表面光滑的球形颗粒, 平均粒径 为 6.5 μιη, 粒径分布变异系数 CV为 8.7%左右, 单分散性较好, 见附图 1。
实施例 3 复合非交联微球粉末的制备 (三)
将聚苯乙烯和 Fe304磁性纳米颗粒溶于氯仿中, 聚苯乙烯浓度为 0.5 g/mL, Fe304磁 性纳米颗粒浓度为 1 nM/L, 以之作为分散相。采用孔径为 4 μιη、孔隙率为 0.5的 SPG多 孔膜, 利用压力为 21 KPa的氮气将分散相挤压过膜, 进入含有 0.5 wt.% 乳化剂 SDS 和 0.5 wt.% 稳定剂 PVA的水连续相, 连续相的流速是 0.40 m/s, 得到液滴粒径均一的水包 油乳液。 于 25 t^P 350 rpm磁力搅拌下进行搅拌挥发。待溶液中氯仿完全挥发后, 对得 到的微球悬浮液用磁力进行分离收集。并用去离子水离心洗涤 3次,无水乙醇离心洗涤 3 次, 冷冻干燥得到 Fe304纳米颗粒复合磁性微球的固体粉末。 经扫描电镜观察, 制备的 Fe304纳米颗粒复合磁性微球为表面光滑的球形颗粒, 平均粒径为 4.2 μιη, 粒径分布变异 系数 CV为 7.9%左右, 单分散性较好。
实施例 4 复合非交联微球粉末的制备 (四)
将聚甲基丙烯酸和银纳米颗粒溶于二氯甲烷中, 聚甲基丙烯酸浓度为 2g/mL, 银纳 米颗粒浓度为 0.5 nM/L, 以之作为分散相。 采用孔径为 1 μιη、 孔隙率为 0.5的 SPG多孔 膜,利用压力为 32 KPa的氮气将分散相挤压过膜,进入含有 0.9 wt.% 乳化剂 SDS 和 0.1 wt.% 稳定剂吐温 -20的水连续相, 连续相的流速是 0.35 m/s, 得到液滴粒径均一的水包 油乳液。 于 25 t^P 350 rpm磁力搅拌下进行搅拌挥发。 待溶液中二氯甲烷完全挥发后, 对得到的银纳米颗粒复合微球悬浮液进行离心收集。 之后经去离子水离心洗涤 3次, 无 水乙醇离心洗涤 3次, 冷冻干燥得到银纳米颗粒复合微球的固体粉末。 经扫描电镜观察, 制备的银纳米颗粒复合微球为表面光滑的球形颗粒, 平均粒径为 1.1 μιη, 粒径分布变异 系数 CV为 7.2%左右, 单分散性较好。
实施例 5 复合非交联微球粉末的制备 (五)
将聚甲基丙烯酸甲酯和 Ti02纳米颗粒溶于氯仿中, 聚甲基丙烯酸甲酯浓度为 0.5 g/mL, Ti02纳米颗粒浓度为 0.5 nM/L, 以之作为分散相。 采用孔径为 4 μ m、 孔隙率为 0.5的 SPG多孔膜,利用压力为 21 KPa的氮气将分散相挤压过膜,进入含有 0.1 wt.% 乳 化剂 SDS 和 1 wt.% 稳定剂 PVA的水连续相, 连续相的流速是 0.38 m/s, 得到液滴粒径 均一的水包油乳液。于 25 t^P 350 rpm磁力搅拌下进行搅拌挥发。待溶液中氯仿完全挥 发后, 对得到的 Ti02纳米颗粒复合微球悬浮液进行离心收集。 之后经去离子水离心洗涤 3次, 无水乙醇离心洗涤 3次, 冷冻干燥得到 Ti02纳米颗粒复合微球的固体粉末。 经扫 描电镜观察,制备的 Ti02纳米颗粒复合微球为表面光滑的球形颗粒,平均粒径为 5.2 μιη, 粒径分布变异系数 CV为 7.7%左右, 单分散性较好。
实施例 6 复合非交联微球粉末的制备 (六)
将聚苯乙烯 -甲基丙烯酸共聚物和发射波长为 550 nm的 CdSe/CdS/ZnS量子点和金纳 米颗粒溶于二甲苯中, 聚苯乙烯 -甲基丙烯酸共聚物浓度为 1.5 g/mL, 量子点浓度为 0.5 nM/L, 金纳米颗粒浓度为 0.2 nM/L, 以之作为分散相。 采用孔径为 5 μιη、 孔隙率为 0.6 的陶瓷多孔膜,利用压力为 18 KPa的氮气将分散相挤压过膜,进入 0.5 wt.% 乳化剂 SDS 和 0.5 wt.% 稳定剂 PVA的水连续相, 连续相的流速是 0.35 m/s, 得到液滴粒径均一的水 包油乳液。 于 25 t^P 350 rpm磁力搅拌下进行搅拌挥发。 待溶液中二甲苯完全挥发后, 对得到的量子点 /金纳米颗粒复合微球悬浮液进行离心分离收集。 并用去离子水离心洗涤 3次, 无水乙醇离心洗涤 3次, 冷冻干燥得到表面带有羧基的量子点 /金纳米颗粒复合微 球的固体粉末。 经扫描电镜观察, 制备的的量子点 /金纳米颗粒复合微球为表面光滑的球 形颗粒, 平均粒径为 6.2 μιη, 粒径分布变异系数 CV为 9.1%左右, 单分散性较好。
实施例 7 复合非交联微球粉末的制备 (七)
将聚苯乙烯-丙烯酸共聚物和发射波长为 680 nm的 CdSeTe量子点和 Fe304磁性纳米 颗粒溶于甲苯中, 聚苯乙烯-丙烯酸共聚物浓度为 2 g/mL, 量子点浓度为 l nM/L, Fe304 磁性纳米颗粒浓度为 1 nM/L, 以之作为分散相。 采用孔径为 5μιη、 孔隙率为 0.5的 SPG 多孔膜, 利用压力为 20 KPa的氮气将分散相挤压过膜, 进入含有乳化剂 SDS浓度为 1 wt.%的水连续相, 连续相的流速是 0.40 m/s, 得到液滴粒径均一的水包油乳液。 于 25 V 和 350 rpm磁力搅拌下进行搅拌挥发。 待溶液中甲苯完全挥发后, 对得到的量子点标记 荧光微球悬浮液用磁力进行分离收集。并用去离子水离心洗涤 3次,无水乙醇离心洗涤 3 次, 冷冻干燥得到表面带有羧基的量子点 /Fe304复合荧光磁性微球的固体粉末。 经荧光 显微镜观察和电镜扫描观察, 制备的量子点 /Fe304复合荧光磁性微球为表面光滑的球形 颗粒, 平均粒径为 6.3μιη, 粒径分布变异系数 CV为 9%左右, 单分散性较好, 见附图 2。 实施例 8 复合非交联微球粉末的制备 (八)
将聚苯乙烯-马来酸酐共聚物和激发波长为 580 nm的 NaYF4稀土纳米颗粒溶于甲苯 中, 聚苯乙烯-马来酸酐共聚物浓度为 2 g/mL, NaYF4稀土纳米颗粒浓度为 I nM/L, 以之 作为分散相。 采用孔径为 3 μ ιη, 孔隙率为 0.5的 SPG多孔膜, 禾 I」用压力为 20 KPa的氮 气将分散相挤压过膜, 进入含有乳化剂 SDS浓度为 1 wt.%的水连续相, 连续相的流速是 0.40 m/s, 得到液滴粒径均一的水包油乳液。 于 25 和 350rpm磁力搅拌下进行搅拌挥 发。 待溶液中甲苯完全挥发后, 对得到的稀土纳米颗粒复合荧光微球悬浮液进行离心收 集。 之后经去离子水离心洗涤 3次, 无水乙醇离心洗涤 3次, 冷冻干燥得到稀土纳米颗 粒复合荧光微球的固体粉末。
将 0.1 g稀土纳米颗粒复合荧光微球分散在 15mL 0.01 mol/L的盐酸中制成悬浮液, 磁力搅拌 8 h,待聚合物上的酸酐官能团水解为羧基后,提取出部分悬浮液,用体积比 1/1 去离子水洗涤 (直至水解微球的悬浮液 pH稳定在 6.2左右), 之后冷冻干燥, 得到表面 羧基功能化的稀土纳米颗粒复合荧光微球。
经扫描电镜观察, 制备的稀土纳米颗粒复合荧光微球为表面光滑的球形颗粒, 平均 粒径为 5.8 μιη, 粒径分布变异系数 CV为 8.9%左右, 单分散性较好。
实施例 9 复合非交联微球粉末的制备 (九)
配制两种分散相: 第一种将聚苯乙烯-马来酸酐共聚物和激发波长为 528 nm 的 CdSe/CdS量子点溶于甲苯中, 聚苯乙烯-马来酸酐共聚浓度为 1 g/mL, 量子点浓度为 1 nM/L; 第二种将聚苯乙烯-马来酸酐共聚物和激发波长为 680 nm的 CdSeTe量子点溶于 甲苯中, 聚苯乙烯-马来酸酐共聚浓度为 1 g/mL, 量子点浓度为 1 nM/L。 之后采用孔径
为 5 μιη、 孔隙率为 0.5的 SPG多孔膜, 利用压力为 15 KPa的氮气分别将两种分散相挤 压过膜, 各自进入含有乳化剂 SDS浓度为 1 wt.%的水连续相, 连续相的流速是 0.37 m/s, 从而得到两种液滴粒径均一的水包油乳液。两种乳液各自于 25 !和 350 rpm磁力搅拌下 进行搅拌挥发。 待溶液中甲苯完全挥发后, 对得到的量子点标记荧光微球悬浮液进行离 心收集。 之后经去离子水离心洗涤 3次, 无水乙醇离心洗涤 3次, 冷冻干燥得到发射波 长分别在 528 nm和 680 nm的两种量子点标记复合微球的固体粉末。
之后将不同发射波长的 0.1 g量子点复合荧光微球分别分散在 15mL 0.01 mol/L的盐 酸中制成悬浮液, 磁力搅拌 8 h, 待聚合物上的酸酐官能团水解为羧基后, 提取出部分悬 浮液, 用体积比 1/1去离子水洗涤(直至水解微球的悬浮液 pH稳定在 6.2左右), 冷冻干 燥, 得到表面羧基功能化的量子点复合微球。
经扫描电镜观察, 制备的两种量子点复合微球为表面光滑的球形颗粒, 平均粒径为 6.5-6.6 μιη, 粒径分布变异系数 CV为 8.7%-8.8%左右, 单分散性较好。
实施例 10 复合非交联微球粉末的制备 (十)
将聚苯乙烯-马来酸酐共聚物和发射波长为 528 nm的 CdSe/CdS量子点溶于甲苯中, 聚合物浓度为 1 g/mL, 量子点浓度为 1 nM/L, 以之作为分散相。 采用孔径为 16 μιη、 孔 隙率为 0.4的 SPG多孔膜, 利用压力为 8 KPa的氮气将分散相挤压过膜, 进入含有乳化 剂 SDS浓度为 1 wt.%的水连续相, 连续相的流速是 0.35 m/s, 得到液滴粒径均一的水包 油乳液。 于 25 !和 350 rpm磁力搅拌下进行搅拌挥发。待溶液中甲苯完全挥发后, 对得 到的量子点标记荧光微球悬浮液进行离心收集。 之后经去离子水离心洗涤 3次, 无水乙 醇离心洗涤 3次, 冷冻干燥得到量子点复合微球的固体粉末。
将 0.1 g量子点复合荧光微球分散在 15mL 0.01 mol/L的盐酸中制成悬浮液, 磁力搅 拌 8 h, 待聚合物上的酸酐官能团水解为羧基后, 提取出部分悬浮液, 用体积比 1/1去离 子水洗涤 (直至水解微球的悬浮液 pH稳定在 6.2左右), 之后冷冻干燥, 得到表面羧基 功能化的量子点复合荧光微球。
经扫描电镜观察, 所制备的量子点复合荧光微球为表面光滑的球形颗粒, 平均粒径 为 20 μιη, 粒径分布变异系数 CV为 8.2%左右, 单分散性较好。
实施例 11 复合非交联微球粉末的制备 (十一)
将聚甲基丙烯酸和银纳米颗粒溶于二氯甲烷中, 聚甲基丙烯酸浓度为 2g/mL, 银纳 米颗粒浓度为 0.5 nM/L, 以之作为分散相。 采用孔径为 0.1 μιη、 孔隙率为 0.4的 SPG多 孔膜, 利用压力为 45 KPa的氮气将分散相挤压过膜, 进入含有 0.9 wt.% 乳化剂 SDS 和 0.1 wt.% 稳定剂吐温 -20的水连续相, 连续相的流速是 0.35 m/s, 得到液滴粒径均一的水
包油乳液。于 25 !和 350 rpm磁力搅拌下进行搅拌挥发。待溶液中二氯甲烷完全挥发后, 对得到的银纳米颗粒复合微球悬浮液进行离心收集。 之后经去离子水离心洗涤 3次, 无 水乙醇离心洗涤 3次, 冷冻干燥得到银纳米颗粒复合微球的固体粉末。 经扫描电镜观察, 制备的银纳米颗粒复合微球为表面光滑的球形颗粒, 平均粒径为 0.1 μιη, 粒径分布变异 系数 CV为 7.4%左右, 单分散性较好。
实施例 12 基于功能性纳米颗粒复合非交联微球粉末的生物检测探针 (一)
将实施例 7所制备的量子点 /Fe304复合微球的表面羧基在 EDC活化下, 连接上 N- 羟基琥珀酰亚胺,然后再连接乙肝病毒表面抗体 (HBsAb)作为探针分子。量子点复合荧光 微球在表面连接 HBsAb后形成特异性检测乙肝病毒表面抗原 (HBsAg)的生物检测探针。 将这种生物检测探针投入到含有乙肝病毒表面抗原 (HBsAg)中的样品中反应后,再将探针 上连接的目标物即乙肝病毒表面抗原 (HBsAg)用异硫氰酸荧光素标记。最后反应后的生物 检测探针在激光激发下发出荧光信号, 通过微球内部量子点荧光信号和探针分子上所连 接的目标物的标记荧光信号强度对样品内的乙肝病毒表面抗原 (HBsAg)进行定性与定量 分析。 检测结果见附图 3, 从图中可以看出, 当检测的乙肝病毒表面抗原 (HBsAg)的浓度 为 0-500 ng/ml时, 其荧光标记强度 (即异硫氰酸荧光素标记的强度) 随着抗原 (HBsAg) 浓度的增加而增加, 呈现正比关系。
实施例 13 基于功能性纳米颗粒复合非交联微球粉末的生物检测探针 (二)
将实施例 8所制备的稀土纳米颗粒复合微球在 EDC活化下, 连接上序列为 5'-TCA AGG CTC AGT TCG AAT GCA CCA TA-3 '的 DNA链段作为探针分子, 形成特异性检测 DNA的生物检测探针。将这种生物检测探针投入到含有 5'-TAT GGT GCA TTC GAA CTG AGC CTT GA-3 '的 DNA链段的样品中反应后, 再将探针上所连接的目标物, 即序列为 5'-TAT GGT GCA TTC GAA CTG AGC CTT GA-3 '的 DNA链段用 Cascade蓝标记。 最后 经处理过的稀土纳米颗粒复合微球在红外激光激发下发出荧光信号, 通过微球内部稀土 纳米颗粒荧光信号和探针分子上所连接的目标物的标记荧光信号对 5'-TAT GGT GCA TTC GAA CTG AGC CTT GA-3 '的 DNA链段进行定性与定量分析。
实施例 14 基于功能性纳米颗粒复合非交联微球粉末的生物检测探针 (三)
将实施例 9所制备的量子点复合微球的表面羧基在 EDC活化下, 连接上 N-羟基琥 珀酰亚胺。 然后, 528 nm量子点复合微球进一步连接乙肝病毒表面抗原 (HBsAg)作为检 测探针分子。 528 nm量子点复合微球在表面连接抗原 (HbsAg)后形成特异性检测乙肝病 毒表面抗体 (HBsAb)的生物检测探针。 690 nm 量子点复合微球连接乙肝病毒 e 抗原 (HBeAg)作为检测探针分子。 690 nm量子点复合微球在表面连接抗原 (HbeAg)后形成特异
性检测乙肝病毒 e抗体 (HBeAb)的生物检测探针。将这两种生物检测探针一起投入到同时 含有乙肝病毒表面抗体 (HbsAb)和乙肝病毒 e抗体 (HBeAb)中的样品中反应后, 将探针上 连接的目标物再用藻红蛋白标记。 反应后的生物检测探针在激光激发下发出荧光信号, 微球内部量子点荧光在 528 nm的为 HBsAb检测结果,微球内部量子点荧光在 680 nm的 为 HBeAb检测结果,再通过探针分子上所连接的目标物的标记荧光强度对样品内 HBsAb 和 HBeAb各自进行定量分析。
实施例 15 复合交联微球粉末的制备 (一)
将苯乙烯、二乙烯基苯、甲基丙烯酸、过氧化苯甲酰和发射波长为 528 nm的 CdSe/CdS 量子点溶于甲苯中, 苯乙烯浓度为 l g/mL, 二乙烯基苯浓度为 0.75g/mL, 甲基丙烯酸浓 度为 0.5 g/mL, 过氧化苯甲酰浓度 0.001 g/mL, 量子点浓度为 1 nM/L, 以之作为分散相。 采用孔径为 0.5 μιη、 孔隙率为 0.6的陶瓷多孔膜, 利用压力为 18 KPa的氮气将分散相挤 压过膜, 进入含有乳化剂 SDS浓度为 1 wt.%的水连续相, 连续相的流速是 0.30 m/s, 得 到液滴粒径均一的水包油乳液。将乳液加热升温至 70 °C, 在氮气保护下反应 12 h。之后 得到的复合微球悬浮液经去离子水离心洗涤 3次, 无水乙醇离心洗涤 3次, 冷冻干燥得 到量子点复合微球的固体粉末。 经扫描电镜观察, 制备的量子点复合微球为表面光滑的 球形颗粒, 平均粒径为 1.5 μιη, 粒径分布变异系数 CV为 9.6%左右, 单分散性较好。 实施例 16 复合交联微球粉末的制备 (二)
将丙烯酸甲酯、 二乙烯基苯、 偶氮二异丁腈和 Fe304磁性纳米颗粒溶于氯仿中, 丙 烯酸甲酯浓度为 1 g/mL,二乙烯基苯浓度为 0.5 g/mL,偶氮二异丁腈浓度 0.01 g/mL, Fe304 磁性纳米颗粒浓度为 0.5 nM/L, 以之作为分散相。采用孔径为 5 μιη、孔隙率为 0.5的 SPG 多孔膜, 利用压力为 21 KPa的氮气将分散相挤压过膜, 进入含有乳化剂 SDS浓度为 0.5 wt.%的水连续相, 连续相的流速是 0.40 m/s, 得到液滴粒径均一的水包油乳液。 将乳液 加热升温至 70 °C, 在氮气保护下反应 12 h。之后得到的微球悬浮液经去离子水离心洗涤 3次, 无水乙醇离心洗涤 3次, 冷冻干燥得到 Fe304磁性纳米颗粒复合微球的固体粉末。 经扫描电镜观察, 制备的 Fe304磁性纳米颗粒复合微球为表面光滑的球形颗粒, 平均粒 径为 5.8 μιη, 粒径分布变异系数 CV为 8.7%左右, 单分散性较好。
实施例 17 复合交联微球粉末的制备 (三)
将苯乙烯、 二乙烯基苯、 甲基丙烯酸和 NaYF4稀土纳米颗粒溶于二甲苯中, 苯乙烯 浓度为 1 g/mL, 二乙烯基苯浓度为 0.5 g/mL, 甲基丙烯酸浓度为 0.5 g/mL。 NaYF4稀土 纳米颗粒浓度为 1 nM/L, 以之作为分散相。采用孔径为 3 μιη、孔隙率为 0.5的 SPG多孔 膜, 利用压力为 25 KPa的氮气将分散相挤压过膜, 进入含有浓度为 0.001g/mL引发剂过
硫酸钾, 浓度为 0.5 wt.% 乳化剂 SDS 和 0.5 wt.% 稳定剂 PVA的水连续相, 连续相的流 速是 0.35 m/s, 得到液滴粒径均一的水包油乳液。 将乳液加热升温至 65 °C, 在氮气保护 下反应 12 h。之后得到的微球悬浮液经去离子水离心洗涤 3次, 无水乙醇离心洗涤 3次, 冷冻干燥得到 NaYF4稀土纳米颗粒复合微球的固体粉末。经电镜扫描观察,制备的 NaYF4 稀土纳米颗粒复合微球为表面光滑的球形颗粒, 平均粒径为 3.4 μιη, 粒径分布变异系数 CV为 8.9%左右, 单分散性较好。
实施例 18 复合交联微球粉末的制备 (四)
将苯乙烯、 二乙烯基苯、 马来酸酐、 过氧化苯甲酰和金纳米颗粒溶于甲苯中, 苯乙 烯浓度为 1 g/mL,二乙烯基苯浓度为 0.5 g/mL,马来酸酐浓度为 0.5 g/mL, 偶氮二异丁腈 浓度 0.01 g/mL, 金纳米颗粒浓度为 1 nM/L, 以之作为分散相。 采用孔径为 4 μιη、 孔隙 率为 0.5的 MPG多孔膜, 利用压力为 25 KPa的氮气将分散相挤压过膜, 进入含有浓度 为 0.1 wt.% 乳化剂 SDS 和 1 wt.% 稳定剂 PVA的水连续相, 连续相的流速是 0.35 m/s, 得到液滴粒径均一的水包油乳液。 将乳液加热升温至 85°C, 在氮气保护下反应 12 h。 之 后得到的微球悬浮液经去离子水离心洗涤 3次, 无水乙醇离心洗涤 3次, 冷冻干燥得到 金纳米颗粒复合微球的固体粉末。 经电镜扫描观察, 制备的金纳米颗粒复合微球为表面 光滑的球形颗粒, 平均粒径为 5.5μιη, 粒径分布变异系数 CV为 8.5%左右, 单分散性较 好。
实施例 19 复合交联微球粉末的制备 (五)
将苯乙烯、 羟甲基丙烯酰胺、 过氧化苯甲酰、 波长为 680 nm的 CdTeSe/CdS量子点 和 Fe304磁性纳米颗粒溶于甲苯中, 苯乙烯浓度为 1.5 g/mL, 羟甲基丙烯酰胺浓度为 0.5 g/mL, 过氧化苯甲酰浓度 0.005g/mL, 量子点浓度为 1 nM/L Fe304磁性纳米颗粒浓度为 0.5 nM/L, 以之作为分散相。 采用孔径为 5 μιη、 孔隙率为 0.5的 SPG多孔膜, 利用压力 为 22 KPa的氮气将分散相挤压过膜, 进入含有浓度为 1 wt.% 乳化剂 SDS的水连续相, 连续相的流速是 0.35 m/s, 得到液滴粒径均一的水包油乳液。 将乳液加热升温至 70 °C, 在氮气保护下反应 12 h。 之后得到的微球悬浮液经去离子水离心洗涤 3次, 无水乙醇离 心洗涤 3 次, 冷冻干燥得到量子点 /Fe304纳米颗粒复合荧光磁性微球的固体粉末。 经电 镜扫描观察, 制备的量子点 /Fe304纳米颗粒复合荧光磁性微球为表面光滑的球形颗粒, 平均粒径为 5.8 μιη, 粒径分布变异系数 CV为 7.8%左右, 单分散性较好。
实施例 20 复合交联微球粉末的制备 (六)
将苯乙烯、 丙烯酸、 偶氮二异丁腈、 戊二醛、 Fe304磁性纳米颗粒和金纳米颗粒溶于 氯仿中, 苯乙烯浓度为 1 g/mL, 丙烯酸浓度为 0.5 g/mL, 戊二醛浓度为 0.2 g/mL, 偶氮
二异丁腈浓度 0.01 g/mL, Fe304磁性纳米颗粒浓度为 0.5 nM/L, 金纳米颗粒浓度为 0.2 nM/L, 以之作为分散相。 采用孔径为 3 μιη、 孔隙率为 0.5的 SPG多孔膜, 利用压力为 23 KPa的氮气将分散相挤压过膜, 进入含有浓度为 1 wt.% 乳化剂 SDS的水连续相, 连 续相的流速是 0.35 m/s, 得到液滴粒径均一的水包油乳液。 将乳液加热升温至 70 °C, 在 氮气保护下反应 12 h。 之后得到的微球悬浮液经去离子水离心洗涤 3次, 无水乙醇离心 洗涤 3次, 冷冻干燥得到 Fe304磁性纳米颗粒 /金纳米颗粒复合微球的固体粉末。 经电镜 扫描观察, 制备的 Fe304磁性纳米颗粒 /金纳米颗粒复合微球为表面光滑的球形颗粒, 平 均粒径为 3.7 μιη, 粒径分布变异系数 CV为 7.9%左右, 单分散性较好。
实施例 21 复合交联微球粉末的制备 (七)
将苯乙烯、 甲基丙烯酸甲酯、 过硫酸钾、 二乙烯基苯、 Ti02纳米颗粒和金纳米颗粒 溶于甲苯中, 苯乙烯浓度为 1.5 g/mL, 甲基丙烯酸甲酯浓度为 0.5 g/mL二乙烯基苯浓度 为 0.5 g/mL, Ti02纳米颗粒浓度为 1 nM/L, 以之作为分散相。 采用孔径为 5 μιη、 孔隙率 为 0.5的陶瓷多孔膜,利用压力为 23 KPa的氮气将分散相挤压过膜,进入含有浓度为 0.001 g/mL引发剂过硫酸钾, 浓度为 0.75 wt.% 乳化剂 SDS的水连续相, 连续相的流速是 0.38 m/s,得到液滴粒径均一的水包油乳液。将乳液加热升温至 70 °C,在氮气保护下反应 12 h。 之后得到的微球悬浮液经去离子水离心洗涤 3次, 无水乙醇离心洗涤 3次, 冷冻干燥得 到 Ti02纳米颗粒 /金纳米颗粒复合微球的固体粉末。 经电镜扫描观察, 制备的 Ti02纳米 颗粒 /金纳米颗粒复合微球为表面光滑的球形颗粒, 平均粒径为 6.1 μιη, 粒径分布变异系 数 CV为 8.2%左右, 单分散性较好。
实施例 22 复合交联微球粉末的制备 (八)
将苯乙烯、 二乙烯基苯、 偶氮二异丁腈、 银纳米颗粒溶于氯仿中, 苯乙烯浓度为 1.5 g/ml,二乙烯基苯浓度为 0.5 g/ml,偶氮二异丁腈浓度为 0.02 g/ml, 银纳米颗粒浓度为 0.5 nM/L, 以之作为分散相。 采用孔径为 5 μιη、 孔隙率为 0.4的 MPG多孔膜, 利用压力为 28 KPa的氮气将分散相挤压过膜,进入含有浓度为 0.1 wt.% 乳化剂 SDS 和 1 wt.% 稳定 剂 PVA的水连续相, 连续相的流速是 0.35 m/s, 得到液滴粒径均一的水包油乳液。 将乳 液加热升温至 70 °C, 在氮气保护下反应 12 h。之后得到的微球悬浮液经去离子水离心洗 涤 3次, 无水乙醇离心洗涤 3次, 冷冻干燥得到银纳米颗粒复合微球的固体粉末。 经电 镜扫描观察, 制备的银纳米颗粒复合微球为表面光滑的球形颗粒, 平均粒径为 3.5 μιη, 粒径分布变异系数 CV为 8.6%左右, 单分散性较好。
实施例 23 复合交联微球粉末的制备 (九)
将苯乙烯、二乙烯基苯、甲基丙烯酸、过氧化苯甲酰,波长为 515 nm的 CdSe/CdS/ZnS
量子点和波长为 755 nm的 CdTeSe/CdS的量子点溶于甲苯中, 苯乙烯浓度为 1 g/ml, 二 乙烯基苯浓度为 0.75 g/ml,甲基丙烯酸浓度为 0.5 g/ml, 过氧化苯甲酰浓度 0.002 g/ml, 波 长为 515 nm的 CdSe/CdS/ZnS量子点浓度为 0.5nM/L, 波长为 755 nm的 CdTeSe/CdS量 子点浓度为 0.5 nM/L, 以之作为分散相。采用孔径为 5 μιη、 孔隙率为 0.6的陶瓷多孔膜, 利用压力为 20 KPa的氮气将分散相挤压过膜, 进入含有浓度为 1 wt.%乳化剂 SDS和 0.1 wt.%稳定剂吐温 -20的水连续相, 连续相的流速是 0.35 m/s, 得到液滴粒径均一的水包油 乳液。 将乳液加热升温至 70 °C, 在氮气保护下反应 12 h。 之后得到的微球悬浮液经去离 子水离心洗涤 3次, 无水乙醇离心洗涤三次, 冷冻干燥得到双发射波长量子点复合微球 的固体粉末。经电镜扫描观察, 制备的量子点复合微球为表面光滑的球形颗粒, 如附图 4 所示, 平均粒径为 6.5 μιη, 粒径分布变异系数 CV为 7.5%左右, 单分散性较好。
实施例 24 复合交联微球粉末的制备 (十)
将苯乙烯、二乙烯基苯、甲基丙烯酸、过氧化苯甲酰和发射波长为 528 nm的 CdSe/CdS 量子点溶于甲苯中, 苯乙烯浓度为 l g/mL, 二乙烯基苯浓度为 0.75g/mL, 甲基丙烯酸浓 度为 0.5 g/mL, 过氧化苯甲酰浓度 0.001 g/mL, 量子点浓度为 1 nM/L, 以之作为分散相。 采用孔径为 0.1 μιη、 孔隙率为 0.6的陶瓷多孔膜, 利用压力为 45 KPa的氮气将分散相挤 压过膜, 进入含有乳化剂 SDS浓度为 1 wt.%的水连续相, 连续相的流速是 0.30 m/s, 得 到液滴粒径均一的水包油乳液。将乳液加热升温至 70 °C, 在氮气保护下反应 12 h。之后 得到的复合微球悬浮液经去离子水离心洗涤 3次, 无水乙醇离心洗涤 3次, 冷冻干燥得 到量子点复合微球的固体粉末。 经扫描电镜观察, 制备的量子点复合微球为表面光滑的 球形颗粒, 平均粒径为 Ο. ΐ μιη, 粒径分布变异系数 CV为 8.3%左右, 单分散性较好。 实施例 25 复合交联微球粉末的制备 (十一)
将苯乙烯、二乙烯基苯、甲基丙烯酸、过氧化苯甲酰,波长为 515 nm的 CdSe/CdS/ZnS 量子点和波长为 755 nm的 CdTeSe/CdS的量子点溶于甲苯中, 苯乙烯浓度为 1 g/ml, 二 乙烯基苯浓度为 0.75 g/ml,甲基丙烯酸浓度为 0.5 g/ml, 过氧化苯甲酰浓度 0.002 g/ml, 波 长为 515 nm的 CdSe/CdS/ZnS量子点浓度为 0.5nM/L, 波长为 755 nm的 CdTeSe/CdS量 子点浓度为 0.5 nM/L, 以之作为分散相。采用孔径为 18 μιη、孔隙率为 0.6的陶瓷多孔膜, 利用压力为 12 KPa的氮气将分散相挤压过膜, 进入含有浓度为 1 wt.%乳化剂 SDS和 0.1 wt.%稳定剂吐温 -20的水连续相, 连续相的流速是 0.35 m/s, 得到液滴粒径均一的水包油 乳液。 将乳液加热升温至 70 °C, 在氮气保护下反应 12 h。 之后得到的微球悬浮液经去离 子水离心洗涤 3次, 无水乙醇离心洗涤三次, 冷冻干燥得到双发射波长量子点复合微球 的固体粉末。 经电镜扫描观察, 制备的量子点复合微球为表面光滑的球形颗粒, 平均粒
径为 20 μιη, 粒径分布变异系数 CV为 6.8%左右, 单分散性较好。
实施例 26 功能性纳米颗粒复合交联微球表面的羧基化
取实施例 18制备的量子点复合交联微球粉末, 将 0.1 g量子点复合荧光微球分散在 15mL 0.01 mol/L的盐酸中制成悬浮液, 磁力搅拌 8 h, 待聚合物上的酸酐官能团水解为 羧基后, 提取出部分悬浮液, 用体积比 1/1去离子水洗涤, 直至水解微球的悬浮液 pH稳 定在 6.2左右, 之后冷冻干燥, 得到表面羧基功能化的量子点复合交联微球。
实施例 27 基于功能性纳米颗粒复合交联微球粉末的生物检测探针 (一)
取实施例 15、 17、 20、 23和 24所制备的任一表面羧基化的功能性纳米颗粒复合交 联微球粉末, 将所制备的复合交联微球的表面羧基在 EDC活化下, 连接上 N-羟基琥珀 酰亚胺,然后再连接乙肝病毒表面抗体 (HBsAb)作为探针分子。量子点标记荧光微球在表 面连接抗体 (HBsAb)后形成特异性检测乙肝病毒表面抗原 (HBsAg)的量子点标记生物检 测探针。将这种量子点标记生物检测探针投入到含有乙肝病毒表面抗原 (HBsAg)中的样品 中反应后, 再将探针上连接的目标物即乙肝病毒表面抗原 (HBsAg)用异硫氰酸荧光素标 记。 最后经处理过的量子点标记生物检测探针在激光激发下发出荧光信号, 通过微球内 部量子点荧光信号和探针分子上所连接的目标物的标记荧光信号 (即异硫氰酸荧光素标 记) 对样品内的乙肝病毒表面抗原 (HBsAg)进行定性与定量分析。
实施例 28 基于功能性纳米颗粒复合交联微球粉末的生物检测探针 (二)
取实施例 15、 17、 20、 23和 24所制备的任一表面羧基化的功能性纳米颗粒复合交 联微球粉末,将所制备的复合交联微球的表面羧基在 EDC活化下,连接上序列为 5'-TCA AGG CTC AGT TCG AAT GCA CCA TA-3 '的 DNA链段作为探针分子, 形成特异性检测 DNA的生物检测探针。将这种生物检测探针投入到含有 5'-TAT GGT GCA TTC GAA CTG AGC CTT GA-3 '的 DNA链段的样品中反应后, 再将探针上所连接的目标物, 即序列为 5'-TAT GGT GCA TTC GAA CTG AGC CTT GA-3 '的 DNA链段用 Cascade蓝标记。 最后 经处理过的功能性纳米颗粒复合交联微球在红外激光激发下发出荧光信号, 通过微球内 部稀土纳米颗粒荧光信号和探针分子上所连接的目标物的标记荧光信号对 5'-TAT GGT GCA TTC GAA CTG AGC CTT GA-3'的 DNA链段进行定性与定量分析。
针对上述实施例 1-28, 需要说明的是, 上述实施例并非穷举, 所属技术领域的技术 人员应当知道, 所述的功能性纳米颗粒还可以是半导体纳米颗粒等; 所述的功能性纳米 颗粒还可以是以下量子点: CdS、 HgS、 CdSe、 CdTe、 ZnSe、 HgSe、 ZnTe、 ZnO、 PbSe、 HgTe、 CaAs、 InP、 InCaAs、 CdSe/ZnS、 CdSe/ZnSe、 CdS/ZnS、 Cd/Ag 2S、 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/CdS/ZnS, 以及掺杂量子点 CdS:Mn、 CdS:Mn、 CdS:Cu、 ZnS:Cu、 CdS:Tb、 ZnS:Tb; 制备功能性纳米颗粒复合交联微球所使用的单体还 可以是下列中的一种或几种: 甲基丙烯酸甲酯、 乙烯、 丙烯、 丁烯、 丁二烯或丙烯酰胺; 制备功能性纳米颗粒复合交联微球所使用的交联剂还可以是丙二胺、 乙二胺或京尼平; 制备功能性纳米颗粒复合交联微球所使用的有机溶剂还可以是: 对氯甲苯、 四氯甲烷、 石油醚、 正己烷或环己烷; 制备功能性纳米颗粒复合非交联微球所使用的聚合物还可选 自: 聚丙烯酸、 聚甲基丙烯酸乙酯、 聚酰胺、 聚丙烯腈、 聚碳酸酯、 聚己内酯、 聚氨酯、 聚乳酸、 壳聚糖、 白蛋白、 胶原、 聚乙酸乙酯或聚苯乙烯 -甲基丙烯酸甲酯共聚物; 制得 的复合微球表面改性的方法还可以是化学接枝或磺化; 经表面改性还可连接有以下官能 团中的一种或几种: 氨基、 磺酸根基、 硝基、 羟、 氯基或酯基, 并可通过官能团连接以 下连接物: 生物素、 亲和素或抗生蛋白链菌素。
本发明的基于功能性纳米颗粒复合微球的生物检测探针可用于检测样品中一种或多 种目标物, 如用于疾病诊断中检测细胞因子、 过敏原和自身免疫反应、 HLA分型、 S P 检测、 肿瘤特异抗原定量检测、 多重微生物定量检测等; 或用于基础研究中如基因分型、 蛋白表达分型、 酶 -底物分析、 核酸研究等; 还可运用到食品安全、 农兽药残留多重定量 检测和司法鉴定等领域。
具体地, 采用本发明的基于功能性纳米颗粒复合微球的生物检测探针检测样品中一 种或多种目标物的方法是:
( 1 )将本发明的生物检测探针中的一种或多种组合投入到含有目标物的样品中, 探 针分子与目标物特异性成键;
(2) 对连接在生物检测探针上的目标物进一步用荧光物质进行荧光标记;
(3 ) 利用仪器对生物探针检测结果进行分析。
步骤 (1 ) 中所述的目标物包括蛋白质、 蛋白质片段或核酸; 步骤 (2) 中所述的荧 光物质包括: 异硫氰酸荧光素(FITC)、藻红蛋白(PE)、碘化丙啶(PI)、化青素(CY5 )、 叶绿素蛋白 (preCP)、 藻红蛋白-德克萨斯红、 Cascade蓝及表面改性量子点; 步骤 (3 ) 中, 利用仪器对生物探针检测结果进行分析指的是利用仪器, 通过对微球内部纳米颗粒 性能的测定来对检测样品中的目标物进行定性分析, 同时通过对生物检测探针上所连接 的目标物标记荧光强度来对检测样品中的目标物进行定量分析; 用于检测的常用仪器包 括: 流式细胞仪、 Luminex悬浮式阵列检测系统(美国 Luminex公司)、荧光分光光度计、 激光共聚焦显微镜、 荧光显微镜、 震动样品磁强计。
对生物探针检测结果进行分析进一步包括: 利用本发明的复合微球内部纳米颗粒性
能的测定来对检测样品中的目标物进行定性分析; 通过生物检测探针上所连接的目标物 标记荧光的强度来对检测样品中的目标物进行定量分析, 其中生物检测探针上所连接的 目标物标记荧光的强度与检测样品中的目标物浓度成正比关系。
Claims
1. 一种功能性纳米颗粒复合非交联微球粉末,它包含功能性纳米颗粒复合非交联微 球, 其特征在于, 所述的功能性纳米颗粒复合非交联微球包含功能性纳米颗粒和聚合物, 平均粒径为 0.1-20μιη, 粒径分布变异系数 9.1%。
2. 根据权利要求 1所述的功能性纳米颗粒复合非交联微球粉末, 其特征在于, 所述 的功能性纳米颗粒是下列中的一种或几种: 量子点、 磁性纳米颗粒、 荧光纳米颗粒、 金 属纳米颗粒、 金属氧化物纳米颗粒或半导体纳米颗粒。
3. 根据权利要求 2所述的功能性纳米颗粒复合非交联微球粉末, 其特征在于, 所述 的量子点是下列中的一种或几种: CdS、 HgS、 CdSe、 CdTe、 ZnSe、 HgSe、 ZnTe、 ZnO、 PbSe、 HgTe、 CaAs、 InP、 InCaAs、 CdSe/CdS、 CdSe/ZnS、 CdSe/ZnSe、 CdS/ZnS、 Cd/Ag 2S、 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, 以及掺杂量子点 CdS:Mn、 CdS:Mn、 CdS:Cu、 ZnS:Cu、 CdS:Tb、 ZnS:Tb。
4. 根据权利要求 1所述的功能性纳米颗粒复合非交联微球粉末, 其特征在于, 所述 的聚合物是下列中的一种或几种: 聚苯乙烯、 聚丙烯酸、 聚甲基丙烯酸、 聚甲基丙烯酸 甲酯、 聚甲基丙烯酸乙酯、 聚酰胺、 聚丙烯腈、 聚碳酸酯、 聚己内酯、 聚氨酯、 聚乳酸、 壳聚糖、 白蛋白、 胶原、 聚苯乙烯 -马来酸酐共聚物、 聚乙酸乙酯、 聚苯乙烯 -丙烯酸共聚 物、 聚苯乙烯-甲基丙烯酸共聚物或聚苯乙烯 -甲基丙烯酸甲酯共聚物。
5. 根据权利要求 1所述的功能性纳米颗粒复合非交联微球粉末, 其特征在于, 所述 的功能性纳米颗粒复合非交联微球经表面改性连有官能团。
6. 根据权利要求 5所述的功能性纳米颗粒复合非交联微球粉末, 其特征在于, 所述 的表面改性是下列中的一种或几种: 水解、 化学接枝或磺化。
7. 根据权利要求 5所述的功能性纳米颗粒复合非交联微球粉末, 其特征在于, 所述 的官能团是下列中的一种或几种: 羧基、 氨基、 磺酸根基、 硝基、 羟、 氯基或酯基。
8. 根据权利要求 5所述的功能性纳米颗粒复合非交联微球粉末, 其特征在于, 所述 的官能团上还连接有以下连接物中的一种或几种: N-羟基琥珀酰亚胺、 生物素、 亲和素 或抗生蛋白链菌素。
9. 一种权利要求 1-8任一所述的功能性纳米颗粒复合非交联微球粉末的制备方法, 其特征在于, 所述的制备方法包括以下步骤:
a) 制备分散相, 所述的分散相包括功能性纳米颗粒和聚合物溶液;
b) 制备连续相, 所述的连续相包括去离子水及溶于水的稳定剂和 /或乳化剂;
C)利用膜乳化装置在气体压力作用下挤压分散相通过多孔膜以液滴形式进入到连续 相中, 在连续相剪切力作用下得到液滴粒径均一的单分散乳液;
d) 通过溶剂蒸发法获得功能性纳米颗粒复合非交联微球粉末。
10.根据权利要求 9所述的制备方法, 其特征在于, 所述的步骤 a) 中聚合物溶液为 有机溶剂中溶解有聚合物的溶液。
11.根据权利要求 10所述的制备方法, 其特征在于, 所述的有机溶剂是疏水性有机 溶剂。
12.根据权利要求 11所述的制备方法, 其特征在于, 所述的疏水性有机溶剂选自下 列中的一种或几种: 甲苯、 二甲苯、 对氯甲苯、 二氯甲烷、 三氯甲烷、 四氯甲烷、 石油 醚、 正己烷或环己烷。
13.根据权利要求 9-12任一所述的制备方法, 其特征在于, 所述的聚合物溶液的浓 度是 0.5-2 g/mL。
14.根据权利要求 9所述的制备方法, 其特征在于, 所述的步骤 a) 中功能性纳米颗 粒的浓度是 0.5-1 nM/L。
15.根据权利要求 9所述的制备方法, 其特征在于, 所述的步骤 b) 中稳定剂和 /或乳 化剂选自下列中的一种或几种: 十二烷基硫酸钠、 聚乙烯醇或吐温 20。
16.根据权利要求 9所述的制备方法, 其特征在于, 所述的步骤 c) 中多孔膜是 SPG 多孔膜、 陶瓷多孔膜或 MPG多孔膜。
17.根据权利要求 16所述的制备方法,其特征在于,所述的多孔膜的孔径是 0.5-5 μιη。
18.根据权利要求 9所述的制备方法, 其特征在于, 所述的步骤 c) 中气体压力的大 小是 15-30 KPa。
19.权利要求 1-8任一所述的功能性纳米颗粒复合非交联微球粉末在检测样品中一种 或多种目标物中的应用。
20.一种基于功能性纳米颗粒复合非交联微球粉末的生物检测探针, 其特征在于, 所 述的生物检测探针包含权利要求 1-8任一所述的功能性纳米颗粒复合非交联微球粉末, 所述的功能性纳米颗粒复合非交联微球的表面偶联有探针分子。
21.根据权利要求 20所述的生物检测探针, 其特征在于, 所述的探针分子选自下列 中的一种或几种: 蛋白质、 蛋白质片段或核酸。
22.权利要求 20或 21 所述的生物检测探针在检测样品中一种或多种目标物中的应 用。
23.一种功能性纳米颗粒复合交联微球粉末, 它包含功能性纳米颗粒复合交联微球,
其特征在于, 所述的功能性纳米颗粒复合交联微球包含功能性纳米颗粒和单体、 交联剂 及引发剂, 平均粒径为 0.1-20μιη, 粒径分布变异系数 9.6%。
24.根据权利要求 23所述的功能性纳米颗粒复合交联微球粉末, 其特征在于, 所述 的功能性纳米颗粒是下列中的一种或几种: 量子点、 磁性纳米颗粒、 荧光纳米颗粒、 金 属纳米颗粒、 金属氧化物纳米颗粒或半导体纳米颗粒。
25.根据权利要求 24所述的功能性纳米颗粒复合交联微球粉末, 其特征在于, 所述 的量子点是下列中的一种或几种: CdS、 HgS、 CdSe、 CdTe、 ZnSe、 HgSe、 ZnTe、 ZnO、 PbSe、HgTe、 CaAs、InP、InCaAs、 CdSe/ZnS、 CdSe/ZnSe、 CdS/ZnS Cd/Ag 2S、 CdS/Cd(OH)2、 CdTe/ZnS、 CdTeSe/CdS、 CdTe/CdS、 CdSe/ZnSe、 CdS/HgS、 CdS/HgS/CdS ZnS/CdS、 ZnS/CdS/ZnS ZnS/HgS/ZnS/CdS CdSe/CuSe、 CdSeTe、 CdSeTe/CdS/ZnS, 以及掺杂量 子点 CdS:Mn、 CdS:Mn、 CdS:Cu、 ZnS:Cu、 CdS:Tb、 ZnS:Tb。
26.根据权利要求 23所述的功能性纳米颗粒复合交联微球粉末, 其特征在于, 所述 的单体是下列中的一种或几种: 苯乙烯、 甲基丙烯酸、 丙烯酸、 丙烯酸甲酯、 甲基丙烯 酸甲酯、 乙烯、 丙烯、 丁烯、 丁二烯、 马来酸酐或丙烯酰胺。
27.根据权利要求 23所述的功能性纳米颗粒复合交联微球粉末, 其特征在于, 所述 的交联剂是下列中的一种或几种: 二乙烯基苯、 丙二胺、 戊二醛、 羟甲基丙烯酰胺、 乙 二胺或京尼平。
28.根据权利要求 23所述的功能性纳米颗粒复合交联微球粉末, 其特征在于, 所述 的引发剂是偶氮二异丁腈、 过氧化苯甲酰或过硫酸钾。
29.根据权利要求 23所述的功能性纳米颗粒复合交联微球粉末, 其特征在于, 所述 的功能性纳米颗粒复合交联微球经表面改性连有官能团。
30.根据权利要求 29所述的功能性纳米颗粒复合交联微球粉末, 其特征在于, 所述 的表面改性选自下列中的一种或几种: 水解、 化学接枝或磺化。
31.根据权利要求 29所述的功能性纳米颗粒复合交联微球粉末, 其特征在于, 所述 的官能团是下列中的一种或几种: 羧基、 氨基、 磺酸根基、 硝基、 羟、 氯基或酯基。
32.根据权利要求 29所述的功能性纳米颗粒复合交联微球粉末, 其特征在于, 所述 的官能团上还连接有以下连接物中的一种或几种: N-羟基琥珀酰亚胺、 生物素、 亲和素 或抗生蛋白链菌素。
33.一种权利要求 23-32任一所述的功能性纳米颗粒复合交联微球粉末的制备方法, 其特征在于, 所述的制备方法包括以下步骤:
a) 制备分散相, 所述的分散相包括功能性纳米颗粒和单体溶液;
b) 制备连续相, 所述的连续相包括去离子水及溶于水的稳定剂和 /或乳化剂; c) 利用膜乳化装置, 在气体压力作用下挤压分散相通过多孔膜以液滴形式进入到 连续相中, 在连续相剪切力作用下得到液滴粒径均一的单分散乳液;
d) 对得到的单分散乳液加热升温, 进行乳液聚合, 形成功能性纳米颗粒复合交联 微球粉末。
34.根据权利要求 33所述的制备方法, 其特征在于, 所述的步骤 a)中单体溶液是有 机溶剂中溶解有单体、 交联剂和引发剂的溶液。
35.根据权利要求 34所述的制备方法, 其特征在于, 所述的有机溶剂为疏水性有机 溶剂。
36.根据权利要求 35所述的制备方法, 其特征在于, 所述的疏水性有机溶剂是下列 中的一种或几种: 甲苯、 二甲苯、 对氯甲苯、 二氯甲烷、 三氯甲烷、 四氯甲烷、 石油醚、 正己烷或环己烷。
37.根据权利要求 33所述的制备方法, 其特征在于, 所述的步骤 b) 中稳定剂和 /或 乳化剂选自下列中的一种或几种: 十二烷基硫酸钠、 聚乙烯醇或吐温 20。
38.根据权利要求 34所述的制备方法, 其特征在于, 所述的交联剂的浓度为 0.2-0.75 g mL o
39.根据权利要求 34所述的制备方法,其特征在于,所述的引发剂的浓度为 0.001-0.02 g mL o
40.根据权利要求 33所述的制备方法, 其特征在于, 所述的步骤 a)中功能性纳米颗 粒的浓度是 0.2-1 nM/L。
41.根据权利要求 33所述的制备方法, 其特征在于, 所述的步骤 c)中多孔膜是 SPG 多孔膜、 陶瓷多孔膜或 MPG多孔膜。
42.根据权利要求 41所述的制备方法,其特征在于,所述的多孔膜的孔径是 0.5-5 μιη。
43.根据权利要求 33所述的制备方法, 其特征在于, 所述的步骤 c)中气体压力的大 小是 18-28 KPa。
44.权利要求 23-32任一所述的功能性纳米颗粒复合交联微球粉末在检测样品中一种 或多种目标物中的应用。
45.一种基于功能性纳米颗粒复合交联微球粉末的生物检测探针, 其特征在于, 所述 的生物检测探针包含权利要求 23-32任一所述的功能性纳米颗粒复合交联微球粉末, 所 述的功能性纳米颗粒复合交联微球的表面偶联有探针分子。
46.根据权利要求 45所述的生物检测探针, 其特征在于, 所述的探针分子选自下列
种或多种目标物中的应
Applications Claiming Priority (4)
Application Number | Priority Date | Filing Date | Title |
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CN201210330685.7A CN102908960B (zh) | 2012-09-10 | 2012-09-10 | 功能性纳米颗粒复合交联微球粉末及其制备方法和应用 |
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EP3875562A1 (en) * | 2020-03-02 | 2021-09-08 | Hangzhou ShineDo Biotech Co., Ltd. | Method for preparing fluorescent-encoded microspheres coated with metal nanoshells |
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