A fluorescent microsphere synthesized in reverse phase, a tracer and its preparation method

Abstract

This application provides a reverse-phase synthesized fluorescent microsphere, a tracer, and a method for preparing the same. The method allows for the one-step synthesis of micron-sized silica fluorescent microspheres, which exhibit good shape, high stability, and controllable particle size. The preparation method includes the following steps: S1, providing an aqueous solution comprising a silica hydrolysate and a hydrophilic fluorescent material; providing an oil phase solution comprising an oil solvent and a water-in-oil surfactant; the water-in-oil surfactant is a nonionic type with an HLB value of 1.5-5.8; S2, mixing the aqueous solution and the oil phase solution, and emulsifying the mixture; S3, subjecting the emulsified mixture to a condensation reaction to form a silica shell coating the hydrophilic fluorescent material, thereby obtaining fluorescent microspheres.

Description

Technical Field

[0001] This application belongs to the technical field of fluorescent microspheres, specifically, it relates to a reverse-phase synthesized fluorescent microsphere, a tracer, and a method for preparing the same. Background Technology

[0002] Fluorescent microspheres are microspheres in which fluorescent materials are encapsulated inside or attached to the surface of the microsphere. Fluorescent materials mainly include organic fluorescent dyes, quantum dots, and metal oxides. These fluorescent materials are easily affected by the environment; for example, they are prone to degradation, photobleaching, and are susceptible to high temperatures / strong light exposure, as well as water and oxygen. Therefore, current technologies mostly use inorganic or organic polymer shells to encapsulate fluorescent materials. Fluorescent materials emit fluorescence when excited by light or electricity. By utilizing different types and concentrations of fluorescent components, multiple different emission bands (colors) can be formed, providing encoding capabilities. Therefore, they are widely used in fields such as biomarking, disease diagnosis, tracers, solid-phase chips, liquid-phase chips, immunochromatography, and Raman scattering.

[0003] For example, fluorescent microspheres are used as tracers in PIV (Particle Image Velocimetry) technology. This technology disperses tracer particles with good tracking or reflectivity in a flow field, illuminates a cross-sectional area of ​​the flow field with laser light, and captures two or more consecutively exposed particle images using an imaging recording system. The image cross-correlation method is then used to analyze the captured PIV images to obtain the average displacement of the particle image in each small region, thereby determining the two-dimensional fluid velocity distribution across the entire cross-section of the flow field. This technology has traditionally used PS-QD (polystyrene quantum dot) microspheres. However, when testing high-density or high-viscosity fluids, polystyrene, due to its low density, tends to float on top of the fluid, making it impossible to simulate the motion of the fluid below. Inorganic materials (such as silica) have a higher density than organic materials like polystyrene, and silica also has the characteristics of being non-toxic and biocompatible. Existing technologies for coating fluorescent materials with silica include various methods, such as the Stober method. The synthesized silica fluorescent microspheres are mostly nanoscale. To prepare micron-sized silica fluorescent microspheres using this method, multiple layer-by-layer coatings are required, resulting in a lengthy experimental design and complex process. For example, the emulsion method, using commonly available surfactants, produces fluorescent microspheres with a particle size of 200 micrometers or larger. Such large particle sizes lead to instability. To reduce the particle size of micron-sized fluorescent microspheres, existing technologies employ microfluidic technology, which is costly and has low synthesis efficiency.

[0004] In view of this, this application provides a reverse-phase synthesized fluorescent microsphere, a tracer, and a method for preparing the same, which synthesizes micron-sized silica fluorescent microspheres in one step. The fluorescent microspheres have good shape, high stability, and controllable particle size. Summary of the Invention

[0005] The purpose of this application is to provide a reverse-phase synthesized fluorescent microsphere, tracer and its preparation method, which synthesizes micron-sized silica fluorescent microspheres in one step. The fluorescent microspheres have good shape, high stability and controllable particle size.

[0006] A first aspect of this application provides a method for preparing fluorescent microspheres synthesized in reverse phase, comprising the steps of:

[0007] S1, providing an aqueous phase solution comprising: silica hydrolysate and a hydrophilic fluorescent material; providing an oil phase solution comprising: an oil phase solvent and a water-in-oil surfactant; wherein the water-in-oil surfactant is a nonionic type and has an HLB value of 1.5-5.8;

[0008] S2, the aqueous solution and the oil solution are mixed, and the mixture is emulsified;

[0009] S3, the emulsified mixture undergoes a condensation reaction to form a silica shell coating the hydrophilic fluorescent material, thereby obtaining fluorescent microspheres.

[0010] In some embodiments, in step S1, the silicon source is mixed with an acid solution, and the silicon source is hydrolyzed to form the silica hydrolysate sol.

[0011] Furthermore, the silicon source is mixed with the acid solution and stirred at room temperature for 1-72 hours to hydrolyze the silicon source and form a silica hydrolysate sol.

[0012] Furthermore, the silicon source includes one of the following: silicate ester, oxysilane, aminosilane, and mercaptosilane. Preferably, the silicon source is mercaptosilane.

[0013] Furthermore, the acid solution includes one of the following: hydrochloric acid aqueous solution, sulfuric acid aqueous solution, phosphoric acid aqueous solution, oxalic acid aqueous solution, or formic acid aqueous solution.

[0014] In some embodiments, the pH value of the silica hydrolysate is 5.5-7.5.

[0015] In some embodiments, the fluorescent material is modified with a hydrophilic ligand to form the hydrophilic fluorescent material.

[0016] Furthermore, the hydrophilic ligand comprises a hydrophilic group, which includes at least one of the following: carboxyl, phosphate, amino, imino, quaternary ammonium, amide, hydroxyl, and aldehyde groups.

[0017] Preferably, the hydrophilic ligand includes: mercapto-Tween, mercapto-PEG, polyoxyethylene fatty acid ester, polyoxyethylene fatty acid alcohol ether, higher fatty alcohol sulfate ester, aliphatic sulfonate, and alkyl aryl sulfonate.

[0018] In some embodiments, the aqueous solution further includes a viscosity modifier that adjusts the viscosity of the aqueous solution.

[0019] Furthermore, the viscosity of the aqueous solution is 1,000–100,000 mPa·s.

[0020] In some embodiments, in step S1, the oil phase solvent is an alkane solvent. The oil phase solvent is stirred with a water-in-oil surfactant until the water-in-oil surfactant is completely dissolved to obtain an oil phase solution.

[0021] In some embodiments, the water-in-oil surfactant comprises a lipophilic group and a hydrophilic group. The hydrophilic group includes one of polyethylene glycol, polyglycerol, and polyoxyethylene, and the lipophilic group includes one of polyhydroxystearate, polyricinoleic acid condensate, polyoleate, polyfatty acid ester, and polyoxybutylene. The fluorescent microspheres have a particle size of 20-160 μm.

[0022] Preferably, the water-in-oil surfactant comprises lipophilic and hydrophilic groups. The hydrophilic group comprises one of polyethylene glycol, polyglycerol, and polyoxyethylene, and the lipophilic group comprises one of polyhydroxystearate, condensed ricinoleic acid, and polyoxybutylene. The fluorescent microspheres have a particle size of 20-110 μm.

[0023] Preferably, when the lipophilic group of the water-in-oil surfactant is dihydroxystearate, the hydrophilic group is polyethylene glycol or polyglycerol. The water-in-oil surfactant includes: polyethylene glycol (30) dihydroxystearate (P135) and polyglycerol-2 dihydroxystearate (PGPH).

[0024] Preferably, when the lipophilic group of the water-in-oil surfactant is polyricinoleic acid condensation, the hydrophilic group is polyglycerol. The water-in-oil surfactant includes: polyglycerol-3 polyricinoleate, polyglycerol-4 polyricinoleate, polyglycerol-6 polyricinoleate, polyglycerol-10 polyricinoleate, and polyethylene glycol-2 polyricinoleate.

[0025] Preferably, when the lipophilic group of the water-in-oil surfactant is a polyoleate or a polyfatty acid ester, the hydrophilic group is polyethylene glycol or polyglycerol. The water-in-oil surfactant includes: dipolyglycerol dioleate, hexaglycerol pentastearate, and decaglycerol decaoleate.

[0026] Preferably, when the lipophilic group of the water-in-oil surfactant is polyoxybutylene, the hydrophilic group is polyoxyethylene. The water-in-oil surfactant includes: polyoxyethylene-polyoxybutylene copolymer.

[0027] Furthermore, in the oil phase solution, the water-in-oil surfactant accounts for 2-15% by mass.

[0028] In some embodiments, in step S2, emulsification of the mixture includes stirring the mixture at a speed of 300-1000 r / min for 0.3-2 h.

[0029] In some embodiments, the mass ratio of the aqueous phase solution to the oil phase solution is 1:(10-30).

[0030] In some embodiments, the aqueous phase solution is added to the oil phase solution at a rate of 1-3 ml / hour.

[0031] In some embodiments, in step S3, the emulsified mixture is subjected to catalytic treatment, and a condensation reaction is performed to form a silica shell coating the hydrophilic fluorescent material.

[0032] In some embodiments, the fluorescent microspheres have a particle size of 20-160 μm, and the particle size of the fluorescent microspheres is controllable. Preferably, the particle size of the fluorescent microspheres is 20-110 μm.

[0033] Furthermore, the catalytic treatment includes at least one of the following: adding a catalyst, heating, or vacuum distillation. The catalyst includes: ammonia, triethylamine, or trioctylamine.

[0034] In some embodiments, the method for preparing the reverse-phase synthesized fluorescent microspheres further includes step S4, which involves connecting the fluorescent microspheres with functional ligands to form functional ligand-modified fluorescent microspheres.

[0035] Furthermore, the surface of the fluorescent microspheres contains silicon-oxygen bonds, and the functional ligands contain silicon-oxygen bonds and functional groups.

[0036] Furthermore, the functional groups include at least one of water-soluble functional groups, oil-soluble functional groups, and coupling functional groups. Water-soluble functional groups impart good water solubility to the quantum dot microspheres, oil-soluble functional groups impart good oil solubility, and coupling functional groups enable the quantum dot microspheres to connect to biomolecules.

[0037] A second aspect of this application provides a reverse-synthesized fluorescent microsphere, which is obtained using the aforementioned preparation method.

[0038] In some embodiments, the fluorescent microspheres have a particle size of 20–160 μm, and the particle size of the fluorescent microspheres is controllable. Preferably, the particle size of the fluorescent microspheres is 20–110 μm.

[0039] A third aspect of this application provides a tracer comprising the aforementioned fluorescent microspheres and a functional ligand connected to the surface of the fluorescent microspheres, the functional ligand comprising a silicon-oxygen bond and a water-soluble functional group and / or an oil-soluble functional group.

[0040] In some embodiments, the tracer is a groundwater tracer, and the functional ligand includes a silicon-oxygen bond and a water-soluble functional group.

[0041] In some embodiments, the tracer is a petroleum tracer, and the functional ligand includes a silicon-oxygen bond and an oil-soluble functional group.

[0042] A fourth aspect of this application provides a method for preparing a tracer, wherein the aforementioned fluorescent microspheres are linked to a functional ligand, the functional ligand comprising a silicon-oxygen bond and a water-soluble or oil-soluble functional group.

[0043] In a fifth aspect of this application, the reverse-synthesized fluorescent microspheres are used in biomedicine or as tracers, wherein the biomedicine includes: drug loading, biological probes, biological markers, disease diagnosis, solid-phase chips, liquid-phase chips, and Raman scattering; and the tracers include: gas-phase tracers and liquid-phase tracers.

[0044] The reverse-synthesized fluorescent microspheres and their preparation method of this application have at least the following advantages compared with the prior art:

[0045] (1) The technical solution of this application is to synthesize micron-sized silica fluorescent microspheres in one step. First, the silicon source is hydrolyzed to form a silica hydrolysate sol, which is then mixed with a hydrophilic fluorescent material to form an aqueous solution. Then, it is mixed with an oil solution to form water-in-oil microdroplets. Under catalysis, the hydroxylation products in the silica hydrolysate sol undergo a condensation reaction in the inner phase solution (i.e., the aqueous solution) of the microdroplets. This separates the "hydrolysis reaction" from the "condensation reaction", thus synthesizing silica microspheres coated with fluorescent material in one step.

[0046] (2) The fluorescent microspheres of this application have a particle size of 20-160 micrometers, which is relatively small and stable. Through extensive experimentation, the applicant has determined that, in addition to meeting the requirements of an HLB value of 1.5-5.8 for the water-in-oil surfactant, indicating it is a nonionic water-in-oil surfactant, and that the hydrophilic groups of the water-in-oil surfactant include one of polyethylene glycol, polyglycerol, or polyoxyethylene, and the lipophilic groups include one of polyhydroxystearate, condensed castor oil acid, polyoleate, polyfatty acid ester, or polyoxybutylene, the applicant can achieve both strong emulsifying ability and high group compatibility with the silicon source and oil phase solvent of this application, as well as high component compatibility, thus enabling the preparation of fluorescent microspheres of 20-160 micrometers with high stability.

[0047] (3) The hydrophilic ligands of the hydrophilic fluorescent microspheres of this application can bind to hydrolyzed silica, be stably dispersed in the silica hydrolysate sol, and will not escape to the edge of the fluorescent microspheres in the subsequent condensation reaction.

[0048] (4) Based on the selection of silicon source type and / or the addition of viscosity modifier in aqueous solution, this application makes it less likely for quantum dots to agglomerate and improves dispersibility; and it is beneficial to maintain good fluorescent microsphere formation and uniform sphericity during subsequent condensation reaction.

[0049] (5) The fluorescent microspheres of this application have controllable particle size. This application controls the particle size of the synthesized fluorescent microspheres by controlling the type and concentration of the water-in-oil surfactant, the mass ratio of the aqueous solution to the oil solution, and the stirring speed, so that the particle size of the fluorescent microspheres is controllable in the range of 20-160 micrometers.

[0050] (6) The method for preparing fluorescent microspheres in this application does not require the use of microfluidic technology to synthesize fluorescent microspheres of 20-160 micrometers, resulting in low production cost and high production efficiency.

[0051] (7) The fluorescent microspheres synthesized in this application have good biocompatibility and high density, making them suitable for tracing fluids with high density or high viscosity. Since the density of fluorescent microspheres is also related to their particle size, while the particle size of the fluorescent microspheres in this application is controllable, they are suitable for tracing various fluids with high density or high viscosity. Attached Figure Description

[0052] Combine with the following appendix Figure 1 The above and other features of this application will be more fully described when the drawings are read. It is understood that these drawings only depict a few embodiments of the application and should not be considered as limiting the scope of the application. The application will be explained more clearly and in more detail through the use of the drawings.

[0053] Figure 1 This is a fluorescence microscope image of the fluorescent microspheres of Example 1 of this application.

[0054] Figure 2 This is a fluorescence microscope image of the fluorescent microspheres of Example 2 of this application.

[0055] Figure 3 This is a fluorescence microscope image of the fluorescent microspheres of Example 3 of this application.

[0056] Figure 4 This is a fluorescence microscope image of the fluorescent microspheres of Example 4 of this application.

[0057] Figure 5 This is a fluorescence microscope image of the fluorescent microspheres of Example 5 of this application.

[0058] Figure 6 This is a fluorescence microscope image of the fluorescent microspheres of Example 6 of this application.

[0059] Figure 7 This is a fluorescence microscope image of the fluorescent microspheres of Example 7 of this application.

[0060] Figure 8 This is a fluorescence microscope image of the fluorescent microspheres of Example 8 of this application.

[0061] Figure 9 This is a fluorescence microscope image of the fluorescent microspheres of Example 9 of this application.

[0062] Figure 10 The image shown is a fluorescence microscope image of the fluorescent microspheres of Comparative Example 1 of this application.

[0063] Figure 11 The image shown is a fluorescence microscope image of the fluorescent microspheres of Comparative Example 2 of this application.

[0064] Figure 12 This is a fluorescence microscope image of the fluorescent microspheres of Comparative Example 3 of this application.

[0065] Figure 13 This is a fluorescence microscope image of the fluorescent microspheres of Comparative Example 4 of this application. Detailed Implementation

[0066] The following embodiments are described to aid in understanding this application. These embodiments are not, and should not be, construed in any way as limiting the scope of protection of this application.

[0067] As used herein, expressions such as "at least one" modify the entire list of elements without modifying any individual elements of the list when placed before or after it. Unless otherwise defined, all terms in this specification (including technical and scientific terms) are to be defined as commonly understood by one of ordinary skill in the art. Terms defined in common dictionaries should be interpreted as consistent with their meaning in the context of the relevant art and in this disclosure, and should not be interpreted ideally or overly broadly unless clearly defined. Furthermore, unless expressly stated to the contrary, the terms "comprising" and "including," when used in this specification, indicate the presence of the stated features, regions, wholes, steps, operations, elements, and / or components, but do not exclude the presence or addition of one or more other features, regions, wholes, steps, operations, elements, components, and / or sets thereof. Therefore, the above terms should be understood to mean that the stated elements are included, but not that any other elements are excluded.

[0068] As described in the background section, silica microspheres coated with fluorescent materials are typically nanoscale, while those synthesized via the Stobe method are mostly nanoscale. However, preparing micron-sized microspheres requires multiple layer-by-layer coating processes, which involve lengthy experimental design and complex procedures. Emulsion synthesis, using common surfactants, produces fluorescent microspheres with diameters of 200 micrometers or larger; however, excessively large microspheres become unstable. To reduce the particle size of micron-sized fluorescent microspheres, microfluidic technology is employed to prepare microspheres in the 1-160 micrometer range, but this method is costly and has low synthesis efficiency.

[0069] A first aspect of this application provides a method for preparing fluorescent microspheres synthesized in reverse phase, comprising the steps of:

[0070] S1, providing an aqueous phase solution comprising: silica hydrolysate and a hydrophilic fluorescent material; providing an oil phase solution comprising: an oil phase solvent and a water-in-oil surfactant; wherein the water-in-oil surfactant is a nonionic type and has an HLB value of 1.5-5.8;

[0071] S2, the aqueous solution and the oil solution are mixed, and the mixture is emulsified;

[0072] S3, the emulsified mixture undergoes a condensation reaction to form a silica shell coating the hydrophilic fluorescent material, thereby obtaining fluorescent microspheres.

[0073] In some embodiments, in step S1, the silicon source is mixed with an acid solution, and the silicon source is hydrolyzed to form the silica hydrolysate sol.

[0074] Furthermore, the silicon source is mixed with the acid solution and stirred at room temperature for 1-72 hours to hydrolyze the silicon source, forming a silica hydrolysate sol. Preferably, the mixture is stirred at room temperature for 11-24 hours.

[0075] The silicon source is hydrolyzed to generate Si(OH)4 hydroxylated products, which is the main component of the silica hydrolysate sol.

[0076] Furthermore, the silicon source includes one of the following: silicate ester, oxygen-containing silane, aminosilane, and mercaptosilane. Preferably, the silicon source is mercaptosilane. The silicon source includes: tetraethyl orthosilicate, methyl orthosilicate, butyl orthosilicate, methyltrimethoxysilane, methyltriethoxysilane, tetraethoxysilane, 3-aminopropyltriethoxysilane, mercaptopropyltrimethylsilane, and 3-mercaptopropylmethyldimethoxysilane.

[0077] The thiol groups of mercaptosilane can chelate with the metals on the surface of quantum dots, forming an interaction force that makes the quantum dots uniformly dispersed. This makes the quantum dots less prone to aggregation and highly dispersed. Furthermore, due to the uniform dispersion of the quantum dots, the fluorescent microspheres formed in the subsequent condensation reaction have good morphology and uniform spherical shape.

[0078] Furthermore, the acid solution includes one of the following: hydrochloric acid aqueous solution, sulfuric acid aqueous solution, phosphoric acid aqueous solution, oxalic acid aqueous solution, or formic acid aqueous solution. The concentration of the acid solution is 0.05-0.3 mol / L.

[0079] Furthermore, in the aqueous solution, the mass ratio of the acid solution to the silicon source is 1:(30-70).

[0080] In some embodiments, the pH value of the silica hydrolysate is 5.5-7.5.

[0081] Furthermore, an alkaline pH adjuster is added to the silica hydrolysate to make the pH value of the silica hydrolysate sol 5.5-7.5.

[0082] The alkaline pH adjuster includes at least one of sodium hydroxide, potassium hydroxide, sodium carbonate, potassium carbonate, phosphate buffer, and borate buffer.

[0083] Freshly hydrolyzed silica sol is highly acidic and cannot be directly added to quantum dots, as the quantum dots will be quenched by the strong acid. Therefore, the pH of the silica sol needs to be adjusted to 5.5-7.5 before adding quantum dots.

[0084] In some embodiments, the fluorescent material is modified with a hydrophilic ligand to form the hydrophilic fluorescent material.

[0085] Furthermore, the fluorescent material includes at least one of fluorescent nanoparticles, fluorescent polymers, and organic fluorescent dyes, wherein the fluorescent nanoparticles include at least one of quantum dots, metal oxide nanoparticles, nanorods, or nanosheets.

[0086] Quantum dots include at least one of the following groups: IIB-VIA, IIIA-VA, IVA-VIA, IVA, IB-IIIA-VIA, VIII-VIA, perovskite materials, and carbon quantum dots. For example, Group II-VI compounds may include: CdSe, CdTe, ZnS, ZnSe, ZnTe, ZnO, HgS, HgSe, HgTe, MgSe, MgS, CdSeS, CdSeTe, CdSTe, ZnSeS, ZnSeTe, ZnSTe, HgSeS, HgSeTe, HgSTe, CdZnS, CdZnSe, CdZnT e. CdHgS, CdHgSe, CdHgTe, HgZnS, HgZnSe, HgZnTe, MgZnSe, MgZnS, HgZnTeS, CdZnSeS, CdZnSeTe, CdZnSTe, CdHgSeS, CdHgSeTe, CdHgSTe, HgZnSeS, HgZnSeTe, HgZnSTe or combinations thereof. Group III-V compounds may include: GaN, GaP, GaAs, GaSb, AlN, AlP, AlAs, AlSb, InN, InP, InAs, InSb, GaNP, GaNAs, GaNSb, GaPAs, GaPSb, AlNP, AlNAs, AlNSb, AlPAs, AlPSb, InNP, InNAs, InNSb, InPAs, InPSb, InZnP, GaAlNP, GaAlNAs, GaAlNSb, GaAlPAs, GaAlPSb, GaInNP, GaInNAs, GaInNSb, GaInPAs, GaInPSb, InAlNP, InAlNAs, InAlNSb, InAlPAs, InAlPSb, or combinations thereof. Quantum dots include nanocrystals having a homogeneous or substantially homogeneous composition, such as a core, and heterogeneous nanocrystals, such as core / shell quantum dots comprising a core and one or more shells surrounding the core. A shell is defined as the material surrounding the core and may include one or more shell layers. Metal oxides include: Zn, Cr, Co, Dy, Er, Eu, Fe, Gd, Pr, Nd, Ni, In, Sm, Tb, Tm, and combinations thereof. Fluorescent polymers contain fluorescent functional groups (such as fluorescein) and monomers capable of polymerization. These monomers polymerize with each other or with other non-fluorescent monomers to prepare fluorescent polymers. Organic fluorescent dyes include: fluorescein (stilbene derivatives, coumarins, fluoranes, benzoxazoles, naphthalenedicarboxylamides, thiophene dicarboxylic amides, fused-ring aromatics, perylenetetracarboxylamides, etc.), aromatic fused-ring compounds, intramolecular charge-transfer compounds, metal complex fluorescent materials, enzymes, and rare earth metal chelates.

[0087] "Ligand" refers to any molecule or ion that can interact weakly or strongly with a quantum dot (e.g., through covalent interactions, ionic interactions, van der Waals interactions, or through interactions with any other molecule on the outer surface of the quantum dot).

[0088] Furthermore, the hydrophilic ligand comprises a hydrophilic group, which includes at least one of the following: carboxyl, phosphate, amino, imino, quaternary ammonium, amide, hydroxyl, and aldehyde groups.

[0089] Preferably, the hydrophilic ligand includes: mercapto-Tween, mercapto-PEG, polyoxyethylene fatty acid ester, polyoxyethylene fatty acid alcohol ether, higher fatty alcohol sulfate ester, aliphatic sulfonate, and alkyl aryl sulfonate.

[0090] Preferably, the content of the hydrophilic ligand in the hydrophilic fluorescent material is 50–300 μmol / g.

[0091] The hydrophilic ligands described above are preferred because not all hydrophilic ligands are suitable for this application. The hydrophilic ligand needs to be stably dispersed in the silica hydrolysate sol and able to bind to the hydrolyzed silica (containing alcoholic or silanol groups), and it must not escape to the edge of the fluorescent microspheres during the subsequent condensation reaction. For example, hexadecyltrimethylammonium bromide (CTAB) will escape to the edge of the fluorescent microspheres during the subsequent condensation reaction.

[0092] In some embodiments, the aqueous solution further includes a viscosity modifier that adjusts the viscosity of the aqueous solution.

[0093] The viscosity modifiers include: PEG20000 (polyethylene glycol 20000), Tween 80, PVA (polyvinyl alcohol), PVP (polyvinylpyrrolidone), cellulose, alginate, agarose, starch, and xanthan gum.

[0094] Furthermore, the viscosity of the aqueous solution is 1,000–100,000 mPa·s.

[0095] The viscosity modifier is a water-soluble polymer that can increase the viscosity of the aqueous solution. The higher viscosity of the aqueous solution makes it less likely for quantum dots to agglomerate and improves dispersibility. Furthermore, it helps to maintain the good morphology and uniform spherical shape of the formed fluorescent microspheres during subsequent condensation reactions.

[0096] In some embodiments, in step S1, the oil phase solvent is an alkane solvent.

[0097] Furthermore, the oil phase solvent includes: liquid paraffin, hexadecane, dodecane, tetradecane, cyclohexane, n-octanol, and n-decanol.

[0098] In some embodiments, the oil phase solvent and the water-in-oil surfactant are stirred until the water-in-oil surfactant is completely dissolved to obtain an oil phase solution.

[0099] In some embodiments, the water-in-oil surfactant is a nonionic water-in-oil surfactant with an HLB value of 1.5-5.8.

[0100] Furthermore, the water-in-oil surfactant includes lipophilic and hydrophilic groups. The hydrophilic group includes one of polyethylene glycol, polyglycerol, and polyoxyethylene, and the lipophilic group includes one of polyhydroxystearate, polyricinoleic acid condensate, polyoleate, polyfatty acid ester, and polyoxybutylene.

[0101] Preferably, the water-in-oil surfactant includes lipophilic and hydrophilic groups. The hydrophilic group includes one of polyethylene glycol, polyglycerol, and polyoxyethylene, and the lipophilic group includes one of polyhydroxystearate, condensed ricinoleic acid, and polyoxybutylene.

[0102] Preferably, when the lipophilic group of the water-in-oil surfactant is dihydroxystearate, the hydrophilic group is polyethylene glycol or polyglycerol. The water-in-oil surfactant includes: polyethylene glycol (30) dihydroxystearate (P135) and polyglycerol-2 dihydroxystearate (PGPH). The prepared fluorescent microspheres have a particle size of 20-70 μm.

[0103] Preferably, when the lipophilic group of the water-in-oil surfactant is condensed ricinoleic acid, the hydrophilic group is polyglycerol. The water-in-oil surfactant includes at least one of polyglycerol-3 polyricinoleate, polyglycerol-4 polyricinoleate, polyglycerol-6 polyricinoleate, polyglycerol-10 polyricinoleate, and polyethylene glycol-2 polyricinoleate. The prepared fluorescent microspheres have a particle size of 60-110 μm.

[0104] Preferably, when the lipophilic group of the water-in-oil surfactant is a polyoleate or a polyfatty acid ester, the hydrophilic group is polyethylene glycol or polyglycerol. The water-in-oil surfactant includes at least one of diglycerol dioleate, hexaglycerol pentastearate, and decaglycerol decaoleate. The prepared fluorescent microspheres have a particle size of 70-160 μm.

[0105] Preferably, when the lipophilic group of the water-in-oil surfactant is polyoxybutylene, the hydrophilic group is polyoxyethylene. The water-in-oil surfactant includes: polyoxyethylene-polyoxybutylene copolymer.

[0106] Typically, water-in-oil surfactants are chosen to form water-in-oil droplets, and the lower the HLB value (hydrophilic-lipophilic balance value) of the surfactant, the stronger its lipophilicity. However, in this application, it is not simply a matter of choosing a water-in-oil surfactant with a low HLB value. For example, Span 80 has an HLB value of 4.3, which falls within the HLB value range of 1.5-5.8 in this application. It is also a nonionic water-in-oil surfactant, but using the preparation method of this application, the fluorescent microspheres prepared have a particle size of approximately 300 μm and above, and the structure of the fluorescent microspheres is unstable. Through extensive experimentation, the applicant has determined that, in addition to meeting the following requirements for water-in-oil surfactants: an HLB value of 1.5-5.8, indicating a nonionic water-in-oil surfactant, and the presence of hydrophilic groups including polyethylene glycol, polyglycerol, and polyoxyethylene, and lipophilic groups including polyhydroxystearate, condensed castor oil, polyoleate, polyfatty acid ester, and polyoxybutylene, the applicant can achieve both strong emulsifying ability and high group compatibility and component matching with the silicon source and oil phase solvent of this application. This enables the preparation of fluorescent microspheres with a size of 20-160 micrometers and high stability.

[0107] Furthermore, in the oil phase solution, the water-in-oil surfactant accounts for 2-15% by mass.

[0108] By controlling the mass ratio of water-in-oil surfactants added, the particle size of the synthesized fluorescent microspheres can be finely adjusted.

[0109] In some embodiments, in step S2, emulsification of the mixture includes stirring the mixture at a speed of 300-1000 r / min for 0.3-2 h.

[0110] By controlling the stirring speed and time during emulsification, the particle size of the synthesized fluorescent microspheres can be finely adjusted.

[0111] In some embodiments, the aqueous phase solution is added to the oil phase solution at a rate of 1-3 ml / hour.

[0112] Add the aqueous solution to the oil solution, rather than the other way around, and control the rate at which the aqueous solution is added to ensure that the mixture is easily and evenly mixed.

[0113] In some embodiments, the mass ratio of the aqueous phase solution to the oil phase solution is 1:(10-30).

[0114] By controlling the mass ratio of the aqueous solution to the oil solution, the particle size of the synthesized fluorescent microspheres can be finely adjusted.

[0115] In some embodiments, in step S3, the emulsified mixture is subjected to catalytic treatment, and a condensation reaction is performed to form a silica shell coating the hydrophilic fluorescent material.

[0116] Furthermore, the catalytic treatment includes at least one of the following: adding a catalyst, heating, or vacuum distillation. The catalyst includes: ammonia, triethylamine, or trioctylamine.

[0117] In the silica hydrolysate sol, the hydroxylated products Si-OH and Si-OR, or Si-OH groups, undergo a condensation reaction under catalysis in the inner phase solution (i.e., aqueous solution) of the microdroplets to form silica micronuclei. These silica micronuclei collide, condense, and gradually aggregate to form silica microspheres coated with fluorescent materials.

[0118] In some embodiments, the fluorescent microspheres have a particle size of 20-160 μm, and the particle size of the fluorescent microspheres is controllable.

[0119] In some embodiments, the method for preparing the reverse-phase synthesized fluorescent microspheres further includes step S4, which involves connecting the fluorescent microspheres with functional ligands to form functional ligand-modified fluorescent microspheres.

[0120] Furthermore, the surface of the fluorescent microspheres contains silicon-oxygen bonds, and the functional ligands contain silicon-oxygen bonds and functional groups.

[0121] Furthermore, the functional groups include at least one of water-soluble functional groups, oil-soluble functional groups, and coupling functional groups. Water-soluble functional groups impart good water solubility to the quantum dot microspheres, oil-soluble functional groups impart good oil solubility, and coupling functional groups enable the quantum dot microspheres to connect to biomolecules.

[0122] The water-soluble functional groups include at least one of the following: carboxyl, phosphate, amino, imino, quaternary ammonium, amide, ether, hydroxyl, and aldehyde. The oil-soluble functional groups include: hydrocarbon, ester, aromatic, polyoxybutylene, long-chain perfluoroalkyl, and polysiloxane. The coupling functional groups include: amino, carboxyl, epoxy, azide, and aldehyde.

[0123] A second aspect of this application provides a reverse-synthesized fluorescent microsphere, which is obtained using the aforementioned preparation method.

[0124] In some embodiments, the fluorescent microspheres have a particle size of 20–160 μm, and the particle size of the fluorescent microspheres is controllable.

[0125] A third aspect of this application provides a tracer comprising the aforementioned fluorescent microspheres and a functional ligand attached to the surface of the fluorescent microspheres.

[0126] In some embodiments, the tracer is a groundwater tracer, and the functional ligand includes a silicon-oxygen bond and a water-soluble functional group.

[0127] In some embodiments, the tracer is a petroleum tracer, and the functional ligand includes a silicon-oxygen bond and an oil-soluble functional group.

[0128] A fourth aspect of this application provides a method for preparing a tracer, wherein the aforementioned fluorescent microspheres are linked to a functional ligand, the functional ligand comprising a silicon-oxygen bond and a water-soluble functional group and / or an oil-soluble functional group.

[0129] The fluorescent microspheres of this application, as tracers, have high density and are suitable for tracing fluids with high density or high viscosity. Since the density of fluorescent microspheres is also related to their particle size, and the particle size of the fluorescent microspheres of this application is controllable, they are suitable for tracing various fluids with high density or high viscosity.

[0130] In a fifth aspect of this application, the reverse-synthesized fluorescent microspheres are used in biomedicine or as tracers, wherein the biomedicine includes: drug loading, biological probes, biological markers, disease diagnosis, solid-phase chips, liquid-phase chips, and Raman scattering; and the tracers include: gas-phase tracers and liquid-phase tracers.

[0131] In some embodiments, applied in the biomedical field, the functional ligands modified on the surface of the fluorescent microspheres include silicon-oxygen bonds, water-soluble functional groups, and coupling functional groups.

[0132] The fluorescent microspheres of this application are intended for use in the biomedical field. The silica shell is non-toxic and biocompatible. Water-soluble functional groups and coupling functional groups can also be modified onto the surface of the silica shell. Furthermore, a polymer shell can be coated onto the outer surface of the silica shell.

[0133] The present invention will be further described in detail below with reference to specific embodiments and comparative examples. However, the present invention is not limited to the following embodiments. The implementation conditions used in the embodiments can be further adjusted according to different requirements of specific use. The conditions not specified are conventional conditions in the industry.

[0134] Example 1:

[0135] Step S1: Prepare aqueous and oil phase solutions

[0136] In a capped glass bottle equipped with a magnetic stirrer, 2.5 ml of tetraethoxysilane was added. While stirring magnetically, 0.5 ml of hydrochloric acid aqueous solution (0.1 mol / L concentration) was added dropwise. The bottle was capped and the mixture was magnetically stirred at room temperature for 12 hours (100 rpm) to hydrolyze the silicon source, forming a silica hydrolysate. At room temperature, sodium hydroxide aqueous solution was added to adjust the pH, and the pH of the silica hydrolysate was measured to be 6. 0.1 ml of a 50 mg / ml aqueous solution of cadmium selenide red quantum dots (surface-modified mercapto Tween 80 ligand) was added, and the mixture was magnetically stirred until no quantum dot particles were visible. Then, 0.5 g of PEG20000 (polyethylene glycol 20000) was added, and the mixture was magnetically stirred until homogeneous, yielding an aqueous solution.

[0137] In a five-necked flask equipped with a thermometer, mechanical stirrer, reflux condenser, nitrogen inlet and feed port, add 38.7g of liquid paraffin and 2g of polyethylene glycol (30) dihydroxystearate (P135). Stir at room temperature until P135 is completely dissolved to obtain an oil phase solution.

[0138] Step S2: Emulsification

[0139] The aqueous phase solution was slowly added to the oil phase solution (in the five-necked flask mentioned above) at a rate of 2 ml / hour. The mixture was stirred at room temperature for 30 minutes at a speed of 300 rpm to emulsify and obtain water-in-oil microdroplets.

[0140] Step S3: Condensation reaction

[0141] Add a mixture of 0.2g triethylamine and 4.3g liquid paraffin to the five-necked flask in step S2 at once to start the reaction. Stir at room temperature for 3 hours at a stirring speed of 300 rpm to obtain fluorescent microspheres.

[0142] The stock solution containing fluorescent microspheres prepared in Example 1 was dropped onto a glass slide. Using a fluorescence microscope, the average particle size of the fluorescent microspheres was measured to be 50 μm, and images of the microspheres under the fluorescence microscope were taken. Figure 1 As shown.

[0143] Example 2:

[0144] Example 2 is largely the same as Example 1, except that the 2.5 ml of tetraethoxysilane in step S1 of Example 1 is replaced with 2.5 ml of mercaptopropyltrimethylsilane.

[0145] The stock solution containing fluorescent microspheres prepared in Example 2 was dropped onto a glass slide. Using a fluorescence microscope, the average particle size of the fluorescent microspheres was measured to be 60 μm, and images of the microspheres under the fluorescence microscope were taken. Figure 2 As shown.

[0146] Example 3:

[0147] Example 3 is largely the same as Example 1, except that the 2g of polyethylene glycol (30) dihydroxystearate in step S1 of Example 1 is replaced with 5g of polyethylene glycol (30) dihydroxystearate.

[0148] The stock solution containing fluorescent microspheres prepared in Example 3 was dropped onto a glass slide. Using a fluorescence microscope, the average particle size of the fluorescent microspheres was measured to be 20 μm, and images of the microspheres under the fluorescence microscope were taken. Figure 3 As shown.

[0149] Example 4:

[0150] Example 4 is largely the same as Example 1, except that: the 2g of polyethylene glycol (30) dihydroxy stearate in step S1 of Example 1 is replaced with 3g of polyethylene glycol (30) dihydroxy stearate; and the stirring speed of 300rpm in step S2 of Example 1 is replaced with a stirring speed of 400rpm.

[0151] The stock solution containing fluorescent microspheres prepared in Example 4 was dropped onto a glass slide. Using a fluorescence microscope, the average particle size of the fluorescent microspheres was measured to be 45 μm, and images of the microspheres under the fluorescence microscope were taken. Figure 4 As shown.

[0152] Example 5:

[0153] Example 5 is largely the same as Example 1, except that the 2g of polyethylene glycol (30) dihydroxystearate in step S1 of Example 1 is replaced with 1.5g of diglycerol dioleate.

[0154] The stock solution containing fluorescent microspheres prepared in Example 5 was dropped onto a glass slide. Using a fluorescence microscope, the average particle size of the fluorescent microspheres was measured to be 150 μm, and images of the microspheres under the fluorescence microscope were taken. Figure 5 As shown.

[0155] Example 6:

[0156] Step S1: Prepare aqueous and oil phase solutions

[0157] In a capped glass bottle equipped with a magnetic stirrer, 2.5 ml of tetraethoxysilane was added. While stirring magnetically, 0.5 ml of hydrochloric acid aqueous solution (0.1 mol / L concentration) was added dropwise. The bottle was capped and the mixture was magnetically stirred at room temperature for 12 hours (100 rpm) to hydrolyze the silicon source, forming a silica hydrolysate. At room temperature, sodium bicarbonate aqueous solution was added to adjust the pH, and the pH of the silica hydrolysate was measured to be 6. 0.1 ml of a 50 mg / ml aqueous solution of cadmium selenide red quantum dots (surface-modified with mercapto Tween 80 ligand) was added, and the mixture was magnetically stirred until the quantum dot particles were no longer visible. Then, 0.5 g of Tween 80 was added, and the mixture was magnetically stirred until homogeneous, yielding an aqueous solution.

[0158] In a five-necked flask equipped with a thermometer, mechanical stirrer, reflux condenser, nitrogen inlet, and feed port, 38.7 g of liquid paraffin and 1.5 g of polyglycerol-4 polyricinoleate (PGPR) were added. The mixture was stirred at room temperature until the PGPR was completely dissolved, resulting in an oil phase solution.

[0159] Step S2: Emulsification

[0160] The aqueous phase solution was slowly added to the oil phase solution (in the five-necked flask mentioned above) at a rate of 2 ml / hour. The mixture was stirred at room temperature for 10 minutes at a stirring speed of 600 rpm to emulsify and obtain a water-in-oil microemulsion.

[0161] Step S3: Condensation reaction

[0162] Add a mixture of 0.2g triethylamine and 4.3g liquid paraffin to the five-necked flask in step S2 at once to start the reaction. Stir at room temperature for 2 hours at a stirring speed of 600 rpm to obtain fluorescent microspheres.

[0163] The stock solution containing fluorescent microspheres prepared in Example 6 was dropped onto a glass slide. Using a fluorescence microscope, the average particle size of the fluorescent microspheres was measured to be 100 μm, and images of the microspheres under the fluorescence microscope were taken. Figure 6 As shown.

[0164] Example 7:

[0165] Example 7 is largely the same as Example 6, except that the 1.5g of polyglycerol-4 polyricinoleate in step S1 of Example 6 is replaced with 5g of polyglycerol-4 polyricinoleate.

[0166] The stock solution containing fluorescent microspheres prepared in Example 7 was dropped onto a glass slide. Using a fluorescence microscope, the average particle size of the fluorescent microspheres was measured to be 80 μm, and images of the microspheres under the fluorescence microscope were taken. Figure 7 As shown.

[0167] Example 8:

[0168] Example 8 is largely the same as Example 1, except that the 0.1 ml of 50 mg / ml aqueous solution of cadmium selenide red quantum dots (surface-modified mercapto Tween 80 ligand) in step S1 of Example 1 is replaced with 0.01 g of sodium fluorescein (with its own carboxylic acid group).

[0169] The stock solution containing fluorescent microspheres prepared in Example 8 was dropped onto a glass slide. Using a fluorescence microscope, the average particle size of the fluorescent microspheres was measured to be 45 μm, and images of the microspheres under the fluorescence microscope were taken. Figure 8 As shown.

[0170] Example 9:

[0171] Example 9 is largely the same as Example 1, except that the 0.1 ml of 50 mg / ml aqueous solution of cadmium selenide red quantum dots (surface-modified mercapto Tween 80 ligand) in step S1 of Example 1 is replaced with 0.01 g of Rhodamine B (with its own amine and hydroxyl groups).

[0172] The stock solution containing fluorescent microspheres prepared in Example 9 was dropped onto a glass slide. Using a fluorescence microscope, the average particle size of the fluorescent microspheres was measured to be 46 μm, and images of the microspheres under the fluorescence microscope were taken. Figure 9 As shown.

[0173] Comparative Example 1:

[0174] Comparative Example 1 is largely the same as Example 1, except that the 0.1 ml of 50 mg / ml aqueous solution of cadmium selenide red quantum dots (surface-modified mercapto Tween 80 ligand) in step S1 of Example 1 is replaced with 0.1 ml of 50 mg / ml aqueous solution of cadmium selenide red quantum dots (surface-modified hexadecyltrimethylammonium bromide ligand CTAB).

[0175] The stock solution containing fluorescent microspheres prepared in Comparative Example 1 was dropped onto a glass slide, and an image was taken under a fluorescence microscope, as shown below. Figure 10 As shown. From Figure 10 It can be seen that in the prepared fluorescent microspheres, most quantum dots are distributed at the edges of the microspheres. In contrast, in the fluorescent microspheres prepared in Examples 1-9, the quantum dots are more uniformly distributed within the microspheres. Comparative Example 2:

[0176] Comparative Example 2 is largely the same as Example 1, except that 0.5g of PEG20000 (polyethylene glycol 20000) is not added in step S1 of Example 1.

[0177] The stock solution containing fluorescent microspheres prepared in Comparative Example 2 was dropped onto a glass slide, and an image was taken under a fluorescence microscope, as shown below. Figure 11As shown. From Figure 11 It can be seen that large clusters of fluorescent light are formed together, and spherical fluorescent microspheres cannot be observed.

[0178] Comparative Example 3:

[0179] Comparative Example 3 is largely the same as Example 1, except that the 2g of polyethylene glycol (30) dihydroxystearate in step S1 of Example 1 is replaced with 5g of sorbitan monooleate (span80).

[0180] The stock solution containing fluorescent microspheres prepared in Comparative Example 3 was dropped onto a glass slide, and an image was taken under a fluorescence microscope, as shown below. Figure 12 As shown. From Figure 12 It can be seen that there are fluorescent aggregates with large particle size and irregular shape, indicating that the fluorescent microspheres are too large in size and have poor morphology.

[0181] Comparative Example 4:

[0182] Comparative Example 4 is largely the same as Example 1, except that the 2g of polyethylene glycol (30) dihydroxy stearate in step S1 of Example 1 is replaced with 5g of triglyceride monostearate.

[0183] The stock solution containing fluorescent microspheres prepared in Comparative Example 4 was dropped onto a glass slide, and its image was taken under a fluorescence microscope, as shown in the image. Figure 13 As shown. From Figure 13 It can be seen that there are fluorescent aggregates with large particle size and irregular shape, indicating that the fluorescent microspheres are too large in size and have poor morphology.

[0184] Although this application discloses several aspects and embodiments, other aspects and embodiments will be obvious to those skilled in the art. Various modifications and improvements can be made without departing from the concept of this application, and these all fall within the scope of protection of this application. The various aspects and embodiments disclosed in this application are for illustrative purposes only and are not intended to limit this application. The actual scope of protection of this application is determined by the claims.

Claims

1. A method for preparing fluorescent microspheres synthesized in reverse phase, characterized in that, Including the following steps: S1, providing an aqueous phase solution comprising: a silica hydrolysate and a hydrophilic fluorescent material; the silica hydrolysate is formed by hydrolysis of a silicon source, the silicon source comprising: a silicate ester, an oxysilane, an aminosilane, or a mercaptosilane; the viscosity of the aqueous phase solution is 1,000,000 to 100,000 mPa·s; providing an oil phase solution comprising: an oil phase solvent and a water-in-oil surfactant; the water-in-oil surfactant is a nonionic type with an HLB value of 1.5-5.8; the water-in-oil surfactant comprises lipophilic and hydrophilic groups, the hydrophilic group comprising: polyethylene glycol, polyglycerol, or polyoxyethylene; the lipophilic group comprising: polyhydroxystearate, polyricinoleic acid condensate, polyoleate, polyfatty acid ester, or polyoxybutylene; S2, the aqueous solution and the oil solution are mixed to obtain a mixture, and the mixture is emulsified; S3, the emulsified mixture undergoes a condensation reaction to form a silica shell coating the hydrophilic fluorescent material, thereby obtaining fluorescent microspheres.

2. The method for preparing fluorescent microspheres synthesized by reverse phase synthesis as described in claim 1, characterized in that, In step S1, one or more features selected from the group consisting of: (1) The silicon source is mixed with an acid solution, and the silicon source is hydrolyzed to form the silica hydrolysate; the pH value of the silica hydrolysate is 5.5-7.5; (2) Modify the fluorescent material with a hydrophilic ligand to form the hydrophilic fluorescent material; (3) The aqueous solution further includes a viscosity modifier, which adjusts the viscosity of the aqueous solution.

3. The method for preparing fluorescent microspheres synthesized by reverse phase synthesis as described in claim 1, characterized in that, Includes one or more features selected from the following group: (1) When the lipophilic group of the water-in-oil surfactant is a dihydroxy stearate, the hydrophilic group is polyethylene glycol or polyglycerol; (2) When the lipophilic group of the water-in-oil surfactant is condensed ricinoleic acid, the hydrophilic group is polyglycerol; (3) When the lipophilic group of the water-in-oil surfactant is a polyoleate or a polyfatty acid ester, the hydrophilic group is polyethylene glycol or polyglycerol; (4) When the lipophilic group of the water-in-oil surfactant is polyoxybutylene, the hydrophilic group is polyoxyethylene; (5) In the oil phase solution, the mass percentage of the water-in-oil surfactant is 2-15%.

4. The method for preparing fluorescent microspheres synthesized by reverse phase synthesis as described in claim 1, characterized in that, In step S2, the mass ratio of the aqueous phase solution to the oil phase solution is 1:(10-30); the mixture is emulsified, including stirring the mixture at a speed of 300-1000 r / min for 0.3-2 h.

5. The method for preparing fluorescent microspheres synthesized by reverse phase synthesis as described in claim 4, characterized in that, Add the aqueous phase solution to the oil phase solution at a rate of 1-3 ml / hour.

6. The method for preparing fluorescent microspheres synthesized by reverse phase synthesis as described in claim 1, characterized in that, In step S3, the emulsified mixture is subjected to catalytic treatment, and a condensation reaction is performed to form a silica shell coating the hydrophilic fluorescent material.

7. The method for preparing fluorescent microspheres synthesized by reverse phase synthesis as described in claim 1, characterized in that, The method for preparing fluorescent microspheres by reverse-phase synthesis further includes step S4, which involves connecting the fluorescent microspheres with functional ligands to form functional ligand-modified fluorescent microspheres.

8. A reverse-synthesized fluorescent microsphere, wherein the fluorescent microsphere is obtained by the preparation method according to any one of claims 1-7.

9. A tracer comprising fluorescent microspheres as described in claim 8 and a functional ligand attached to the surface of the fluorescent microspheres, the functional ligand comprising a water-soluble functional group and / or an oil-soluble functional group.

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