CN116143964A

CN116143964A - Monodisperse superparamagnetism fluorescent coding microsphere, preparation method and application

Info

Publication number: CN116143964A
Application number: CN202310162551.7A
Authority: CN
Inventors: 王红蕾; 陈婕妤
Original assignee: N Research Center Private Investment Co ltd
Current assignee: N Research Center Private Investment Co ltd
Priority date: 2023-02-23
Filing date: 2023-02-23
Publication date: 2023-05-23

Abstract

The invention relates to the technical field of coded microspheres, in particular to a monodisperse superparamagnetic fluorescent coded microsphere, a preparation method and application thereof; the application discloses monodisperse superparamagnetic fluorescent coding microspheres, which comprise a hollow spherical supporting layer formed by a polystyrene polymer matrix material; superparamagnetic Fe is arranged on the inner surface and the outer surface of the supporting layer ₃ O ₄ A nanoparticle; a coating layer is arranged on the outer surface of the supporting layer; the coating layer comprises a first coating layer and a second coating layer; the first coating layer is coated on the super-compliant layer arranged on the outer surface of the supporting layerMagnetic Fe ₃ O ₄ The nanoparticle is coated on the first coating layer by the second coating layer; the second coating layer is coated with a functional layer which can capture the luminescent substance and provide a functional group capable of further coupling with the biomarker. The monodisperse superparamagnetic fluorescent coding microsphere prepared by the method can be used for IVD (IVD) determination.

Description

Monodisperse superparamagnetism fluorescent coding microsphere, preparation method and application

Technical Field

The invention relates to the technical field of coded microspheres, in particular to a monodisperse superparamagnetic fluorescent coded microsphere, a preparation method and application thereof.

Background

Microspheres are widely used in various fields of in vitro diagnostics. The microsphere has large specific surface area, and can capture the biomarker efficiently. With the development of a large number of in vitro detection reagents, microspheres are widely used for rapid and accurate multiplex detection of nucleic acids, proteins, circulating Tumor Cells (CTCs), bacteria, and the like. The finely designed microspheres may be provided with different codes, particularly in the form of morphological (size, shape) or optical (colour, fluorescence, raman signal) differences. The luminescent color or luminescent lifetime of the fluorescently encoded microspheres is used as an identification code for the surface chemistry of the microspheres, and then the capture targets of subsequent microspheres are quantified by means of additional spectrally distinguishable fluorescent labels. The microspheres with different codes are adopted in-vitro detection, so that multiplexing high-flux detection can be realized. The high-throughput analysis technique is capable of simultaneously detecting a plurality of targets in a short time, and is capable of effectively saving sample cost and reducing detection cost compared with conventional batch analysis.

The monodisperse superparamagnetic microbeads are materials with superparamagnetism based on the microspheres as carriers and are also common materials in diagnostic kits. By applying an external magnetic field, the magnetic beads can be used to quickly and easily perform antibody purification and separation of mammalian cells, bacteria, viruses, subcellular organelles, and individual proteins. Compared with the coded microspheres, the coded magnetic beads can purify the biomarker in the sample, and the detection sensitivity is remarkably improved. Clinically, a series of coded magnetic beads are often required to extract a plurality of biomarkers of a disease, and then monitor and quantify the biomarkers to reliably diagnose the disease. If there is no high-throughput detection based on fluorescent-encoded magnetic beads, the overall detection efficiency is limited. In addition, because the magnetic beads which are coded based on fluorescence gradient can be directly used on mature flow cytometry equipment, the development and popularization of the in-vitro diagnostic reagent are greatly accelerated.

While one common method of polymer fluorescent coding microspheres is to expand preformed Polystyrene (PS) or polymethyl methacrylate (PMMA) microspheres by adding a nonpolar organic solvent containing the luminophores, which allows the luminophores to penetrate and attach into the microsphere polymer network. The magnetic fluorescent coding microsphere prepared by CN110187115A is also manufactured based on the method, and fluorescent signal molecules such as Fluorescein Isothiocyanate (FITC), rhodamine isothiocyanate (RBITC), polymethylalgae chlorophyll protein (PerCP) or CdSe/ZnS quantum dots are soaked into the magnetic microsphere in a solvent swelling mode, so that the fluorescent dye can permeate and adhere to a microsphere polymer network, the scheme cannot control the distribution of the fluorescent dye molecules in the magnetic microsphere, the magnetic particles can block fluorescence to a certain extent, and the prepared magnetic microsphere has low magnetic saturation, which is only 20% of that of commercial magnetic beads, so that the moving speed in a magnetic field is slow and is not in line with the requirements of most applications; in addition, the codes of the patent use magnetic beads with different particle sizes to select different fluorescent dyes, and the method does not achieve that one fluorescent dye generates different gradients. Furthermore, the polymethylchlorophyll protein (PerCP) used in this patent is a protein that can only be dissolved in a buffer system, and has a great limitation in use in an organic phase.

Another approach is to add organic dyes or quantum dots during the polymerization reaction. Organic dye or quantum dot luminescent compound is dissolved or dispersed in monomer solution or added into reaction mixture to form microsphere and encapsulate luminescent chemical. This process requires a sufficiently stable luminescent compound that no chemical structural changes occur under the polymerization conditions and that there is suitable solubility or dispersibility in the system. From the results, this method has a limitation in that the steps are too simple, and all problems need to be solved in one-step reaction, so that the reaction system is too complex and very uncontrollable. Moreover, this method is limited to emulsion polymerization, and thus cannot produce microspheres of a micrometer scale, let alone magnetic beads. CN100442052C also describes a similar method, but in magnetic particle polymerization a two-step emulsion polymerization is used. The first step is to produce microsphere seeds containing magnetic particles, the second step is to add organic solvent into the seed solution to make the microsphere seeds swell, so as to obtain a swelling particle mixture containing fluorescent dye, and then to add initiator to raise temperature to make copolymerization so as to obtain the magnetic fluorescent microsphere.

The two schemes can not control the distribution of fluorescent dye molecules in the magnetic microsphere, and the obtained fluorescent microsphere has the greatest characteristic that the fluorescent dye is distributed in the whole magnetic microsphere framework. However, in practical use, the fluorescent light is only emitted from the surface of the microsphere, but not the whole microsphere, so that the expensive fluorescent dye is wasted and the high fluorescent intensity cannot be achieved by using the scheme. In addition, certain magnetic nanoparticles present in the microsphere may also block the excitation light source, resulting in non-excitation of the fluorescent dye distributed inside the sphere. Secondly, for the magnetic microspheres, the doping polymerization has strong randomness, so that the magnetic particle ratio is relatively low, and the magnetic particle ratio in each microsphere is different. In addition, the two schemes have no advantage in preparing a series of magnetic fluorescent microspheres with different fluorescence gradients. Since each fluorescent gradient magnetic fluorescent microsphere must correspond to one polymerization reaction, if 5 fluorescent gradient microspheres are required to be produced, 5 polymerization reactions are required, and each fluorescent dye molecule added into the reaction solution is not completely utilized, which is a great waste for expensive fluorescent dyes. In addition, every time a gradient is generated, a swelling polymerization reaction is needed, so that the particle size of the microspheres or the magnetic beads with 5 fluorescence gradients has a certain deviation, and precise regulation and repetition cannot be performed. The free radicals used in the final reaction may also affect the fluorescent molecules, which may cause chemical structural changes under the polymerization conditions (i.e., initiator and high temperature conditions), affecting the excitation wavelength and signal of the final product.

Another fluorescent coding microsphere is based on a core-shell structure design. There are a number of ways in which the core shell can be designed. And wrapping the microspheres layer by layer. This layer-by-layer encapsulation method utilizes oppositely charged polyelectrolyte layers containing colloidal semiconductor Quantum Dots (QDs) or organic dyes that can be deposited stepwise on the microsphere surface. This approach can achieve the deposition of fluorescent dyes or quantum dots on the microsphere surface, but the challenge to follow is the need to modify the microsphere. For example, as a carrier for in vitro detection, the microsphere surface needs to carry a certain density of amino or carboxyl groups to further modify the antigen or antibody.

CN102120168B invented another core-shell structure, the core of this structure is ferroferric oxide nano-particle, its surface and modified fluorescent dye are combined by means of silica, and the grain size of the finally formed magnetic microsphere is less than 320nm. The magnetic microsphere volume produced by this method is determined by the core of the ferroferric oxide. Such magnetic microspheres have major limitations such as solution hydrolysis of the silica-based shell in a pH-high buffer, and in addition, particle sizes too small to match conventional flow cytometry, requiring separate development equipment if applied to in vitro assays.

CN101650998B describes a fluorescent magnetic microsphere, coupling magnetic nanoparticles to the microsphere surface, the main feature being that 50% or less of the magnetic microsphere surface is covered with magnetic material. Such magnetic materials include particles or aggregates of particles having a particle size of 10-1000 nanometers; and a polymer coating the magnetic material and the core microsphere, and adding fluorescent dye into the polymer. The design of the magnetic microsphere employs large clusters of magnetic nanoparticles, the main starting point being to prevent the coverage of the entire microsphere surface by small magnetic nanoparticles, which absorb light resulting in little light emission from the core. In one specific example, a microsphere surface of 7 microns is completely coated with 5nm diameter magnetic nanoparticles, and photon transport into and out of the microsphere is severely inhibited by the magnetic particles.

Considering the above cases comprehensively, the coding strategies of fluorescent dyes with various excitation wavelengths are not lacked. This strategy appears to be simple but is inconvenient for practical use. Firstly, the use of multiple fluorescent dyes requires that the detection device have multiple independent excitation light channels and channels for collecting luminescence signals; secondly, the spectra of the fluorescent dyes are easy to interfere with each other, for example, the fluorescence excitation wavelength of FITC can excite other adjacent fluorescent dyes, but the excitation efficiency is different, but the mixing inevitably causes the increase of background signals; third, magnetic beads or microspheres containing different fluorescent dyes, when in close proximity, can produce fluorescence resonance energy transfer. Based on the above limitations, the available fluorochromes are very limited, and the strategy of encoding with a plurality of different fluorochromes is difficult to put into practice in combination with the extremely high requirements on the application end.

The fluorescent dyes mentioned in the above cases are both organic dyes and quantum dots; the essential difference between the two is that the quantum dots are nanocrystals, while the fluorescent organic dye is a small molecule organic compound. Quantum Dots (QDs) are luminescent nanocrystals with dimensions <10nm, typically having a core/shell particle structure, consisting of an inorganic core, an inorganic surface passivating shell, and a stable organic ligand shell, ensuring dispersibility and colloidal stability. Its advantages are wide absorption spectrum, narrow emission band and high light stability. But since the surface chemistry of QDs is not only very important for their colloidal stability, but also to a large extent they control their photoluminescent properties, in particular their photoluminescent quantum yields. Its addition to the polymer microsphere faces agglomeration of QDs and loss of light reflection efficiency. Meanwhile, during the encoding process, the ligands on the QD surface are likely to be modified during incorporation into the microsphere or bead, thereby introducing additional trap states through ligand removal, or ligand exchange first is required to ensure QD surface chemistry with monomer// polymer phase, possibly leading to luminescence quenching, and it is found that while the photoluminescence quantum yield of these microspheres is reduced to 35% compared to QDs that emit very strongly initially, their photoluminescence quantum yield is still not high.

The preparation of fluorescent microspheres based on organic dyes has the advantages of high quantum yield, multiple types and low cost, and the organic dyes are widely applied to flow cytometry, and the laser and the channel of the organic dyes can be matched with the standard flow cytometry, so that the fluorescent microspheres based on the organic dyes are most widely applied, and an effective strategy can be to effectively combine the fluorescent organic dyes and the magnetic microspheres together to form the coded magnetic beads.

Ideally, an efficient and reliable manufacturing strategy must be easy to implement, have high batch-to-batch reproducibility, uniform particle size, easy to surface modify, and have excellent magnetic response. In addition, the fluorescent surface microbeads thus produced should be readily functionalized, coupled or captured for biomarkers. Finally, the strategy should be able to comprehensively prepare fluorescent magnetic beads with different luminous gradients and different particle diameters in a rapid and simple manner, so as to realize multi-dimensional and multi-detection.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention provides the monodisperse superparamagnetic fluorescent coding microsphere, a preparation method and application thereof, and the prepared monodisperse superparamagnetic fluorescent coding microsphere has a polystyrene core and can provide functional groups for IVD measurement.

The technical scheme adopted for solving the technical problems is as follows:

it is a first object of the present application to provide monodisperse superparamagnetic fluorescent encoding microspheres comprising:

a support layer of hollow structure formed from a polystyrene polymer matrix material;

superparamagnetic Fe is arranged on the inner surface and the outer surface of the supporting layer ₃ O ₄ A nanoparticle; a coating layer is arranged on the outer surface of the supporting layer;

the coating layer comprises a first coating layer and a second coating layer; the first coating layer coats superparamagnetism Fe arranged on the outer surface of the supporting layer ₃ O ₄ The second coating layer is coated on the first coating layer on the nano particles;

the polystyrene polymer matrix material comprises styrene monomer and crosslinking monomer;

the second coating layer is coated with a functional layer which can capture the luminescent substance and provide a functional group capable of further coupling with the biomarker.

In the technical scheme, the magnetic fluorescent coding microsphere is provided with a supporting layer with a hollow structure. The hollow structure avoids a large amount of luminescent substances from adhering to the core part of the inner polymer. Due to superparamagnetism Fe ₃ O ₄ The nano particles are randomly gathered on the surface of the core part in a high density, and superparamagnetic Fe ₃ O ₄ Absorption of light by the nanoparticle results in little luminescence from the core portion, resulting in the luminescence of the fluorescent-encoded magnetic beadsThe light efficiency becomes low.

Further, the raw materials of the coating layer comprise styrene monomers and/or hydrophilic functional group monomers.

Further, the raw materials of the functional layer comprise styrene monomers and/or hydrophilic functional group monomers.

Further, the crosslinking monomer is one or more of divinylbenzene, ethylene glycol dimethacrylate, bisphenol a dimethacrylate, butanediol dimethacrylate, tricyclodecane dimethanol diacrylate, pentaerythritol triacrylate, tripropylene glycol diacrylate, propoxylated neopentyl diacrylate, ditrimethylolpropane tetraacrylate, tripropylene diacrylate, trimethylolpropane ethoxylated triacrylate, trimethylolpropane propoxylated triacrylate, ditrimethylolpropane tetraacrylate, glycerol propoxylated triacrylate, pentaerythritol propoxylated triacrylate, poly (ethylene glycol) diacrylate, poly (propylene glycol) diacrylate, tri (propylene glycol) diacrylate, N' -methylenebis (acrylamide), bisphenol a-bis (2-hydroxypropyl) acrylate, bisphenol a ethoxy diacrylate;

Further, the styrene monomer comprises styrene and/or a styrene derivative; the styrene derivative is one or more of 4-methyl styrene, 3-methyl styrene and 4-tertiary butyl styrene.

Still further, the hydrophilic functional group monomer is one or more of acrylic acid, methacrylic acid, 2-carboxyethyl acrylate, acrylamide, methacrylamide, allylamine, (hydroxyethyl) methacrylate, hydroxypropyl methacrylate, 4-hydroxybutyl acrylate, glycidyl methacrylate, allyl glycidyl ether, 1, 2-epoxy-5-hexene, maleic anhydride, 2-hydroxyethyl methacrylate, bisphenol a-bis (2-hydroxypropyl) acrylate, 2-carboxyethyl acrylate oligomer.

Further, superparamagnetic Fe is generated in situ on the inner surface of the supporting layer and the outer surface of the supporting layer ₃ O ₄ And (3) nanoparticles.

Further, dye molecules for fluorescent coding including, but not limited to, one or several of the following are absorbed on the functional layer: rhodamine WT, fluorescein, BDY 650-X, SE, coumarin, nile red, coumarin 6, coumarin 4, rhodamine B, nile blue, oxazine 725, oxazine 750.

A second object of the present application is to provide a method for preparing monodisperse superparamagnetic fluorescent encoding microspheres, the method comprising the steps of:

step 1) synthesizing to obtain the microsphere with the highly monodisperse hollow structure through a single-kettle multistage polymerization process;

step 2) swelling the microspheres with hollow structures by using an organic solvent to generate superparamagnetism Fe in situ ₃ O ₄ The nano particles are uploaded to the inner surface and the outer surface of the supporting layer to obtain superparamagnetic microspheres;

step 3) preparing a coating layer which can form a network structure with the supporting layer on the superparamagnetic microsphere;

step 4) using a monomer which can form a network structure with the coating layer to functionalize the superparamagnetic microsphere base material;

and 5) slightly swelling the magnetic microspheres by using an organic solvent to enable the magnetic microspheres to absorb organic fluorescent dyes with different concentration gradients, so as to obtain the monodisperse superparamagnetic fluorescent coding microspheres.

Further, in step 1), the highly monodisperse hollow-structured microspheres (support layer) are synthesized by a single-pot multistage copolymerization process using three monomers (styrene, crosslinking monomer and hydrophilic functional group monomer), specifically:

(a) The first stage is dispersion polymerization of styrene in a solvent. Firstly, forming a mixture of an initiator, a polymer stabilizer, a surfactant and alcohol, generating macromolecular free radicals, and then adding styrene for dispersion polymerization to generate a polystyrene core. The surfactant and polymer stabilizer control the size and hollow structure of the polystyrene core. The nuclei that form large-sized hollow microspheres are responsible for controlling the nucleation process. The first stage styrene can be adjusted by the viscosity of the solution and the surfactant, thereby forming hollow microspheres of uniform size. The solvent comprises one or more of methanol, ethanol, isopropanol and isobutanol, and further comprises 1 or more alcohol hydroxyl group surfactant, which can be Triton series, such as one or more of Triton X-100, triton X-165, triton X-305, tween 20 and Tween 80. The weight ratio of the additive in the solvent may be any of 0.01 to 40%, for example 0.01%, 0.2%, 20%, 40%. The total polymerization time of the first stage is controlled to be 2 to 24 hours.

(b) The second stage is to add a crosslinking monomer for crosslinking. The weight percentage of crosslinking monomer relative to styrene may be 1-40%. In this stage, the polymerization time was controlled to 1 to 8 hours.

(c) In the third stage, a functional monomer (hydrophilic functional group) is added to copolymerize with the remaining amount of the crosslinking monomer and styrene, thereby introducing the hydrophilic functional group to the microsphere surface. In addition, more than one functional group monomer (such as two or three functional monomers) may be added together at this stage, introducing a plurality of functional groups at a time. The weight percentage of functional monomer relative to styrene may be from 1 to 30%, such as from 5 to 20%. The polymerization time may be 8 to 24 hours at this stage.

The polymerization process of the single-pot multistage copolymerization process can be carried out at any suitable temperature, and is preferably 40-80 ℃. By adjusting the formulation, such as the ratio of each component, the particle size of the monodisperse microsphere with the particle size range (outer diameter) of 0.9-10 μm, such as 6.5 μm and CV <10%, can be prepared;

specifically, an initiator may be used in the above-described process, if necessary. The initiator may be selected from azo initiators such as AIBN, AMBN, 4' -azobis (4-cyanovaleric acid). The initiator may also be selected from peroxide initiators and persulfates, the peroxide initiators including t-amyl hydroperoxide, potassium persulfate, sodium persulfate, and ammonia persulfate.

The body of the support layer comprises styrene and/or a styrene derivative; wherein the styrene derivative is selected from one or more of 4-methyl styrene, 3-methyl styrene and 4-tertiary butyl styrene;

the cross-linking monomer of the supporting layer is one or more of divinylbenzene, ethylene glycol dimethacrylate, bisphenol A-bis (2-hydroxypropyl) acrylate, bisphenol A ethoxydiacrylate and N, N' -methylenebis (acrylamide);

the functional groups on the support layer functional monomer may be one or more from the group consisting of amino, carboxyl, epoxy, and hydroxyl: the functional monomer of the support layer may be one or more of acrylic acid, methacrylic acid, 2-carboxyethyl acrylate, acrylamide, methacrylamide, allylamine, (hydroxyethyl) methacrylate, hydroxypropyl methacrylate, 4-hydroxybutyl acrylate, glycidyl methacrylate, allyl glycidyl ether, 1, 2-epoxy-5-hexene, maleic anhydride, 2-hydroxyethyl methacrylate, 2-carboxyethyl acrylate oligomer.

In the process directly above, the weight ratio of styrene monomer to crosslinking monomer may be 20:1 to 1:2, such as 10:1 to 1:1. For example, the weight ratio of styrene monomer to first functional monomer is from 20:1 to 1:2, preferably from 10:1 to 1:1.

The solvent used in the polymerization of the support layer contains: the solvent may be methanol, ethanol, isopropanol, isobutanol or a mixture thereof, etc., and the solvent may further contain additives of 1 or more alcoholic hydroxyl groups, such as Triton series, such as Triton X-100, triton X-165, triton X-305, tween 20, tween 80, etc., and the solvent may further contain additive polymer stabilizers, specifically one or more of polyvinylpyrrolidone (PVP), polyethyleneimine (PEI), polyacrylic acid (PAA), polyvinyl alcohol (PVA), hydroxypropyl methylcellulose (HPC), and chitosan.

Further, in step 2), the washed hollow-structured microspheres (support layer) are mixed in a proper molar ratio (e.g., fe ³⁺ :Fe ²⁺ In a molar ratio of 2.5:1, 2:1 or 1.8:1) in a dispersion containing Fe ³⁺ And Fe (Fe) ²⁺ For example, an aqueous solution, such as a mixture of water and a polar solvent (e.g., ethanol); the temperature of the solution may then be raised (e.g., to 50-80 ℃) for several hours with agitationSo that both ions interact with functional groups on the surface of the support layer. Part of Fe ³⁺ And Fe (Fe) ²⁺ Will penetrate into the polymer matrix of the microspheres with a portion of Fe ³⁺ And Fe (Fe) ²⁺ By interaction with functional groups on the surface of the formed microsphere (i.e., the first shell surface), will be adsorbed onto the surface of the microsphere. Next, ammonia is added to charge Fe ³⁺ And Fe (Fe) ² ⁺ In situ conversion to Fe ₃ O ₄ And (3) nanoparticles. The superparamagnetic polymer microspheres obtained by centrifugation are then washed with water to remove residual ammonia and any loose Fe ₃ O ₄ And (3) nanoparticles.

Specifically, in the magnetizing process of the supporting layer in the step 2), the microspheres with the hollow structures are placed into a solution containing Fe (III) salt and Fe (II) salt, and then alkali is added to form the naked monodisperse superparamagnetic microspheres. Fe on paramagnetic microspheres ₃ O ₄ The nanometer particles have particle size smaller than 50nm and are distributed in single layer.

Directly in the above process:

(a) The Fe (III) salt may be selected from FeCl ₃ And/or Fe ₂ (SO ₄ ) ₃ The method comprises the steps of carrying out a first treatment on the surface of the And/or

(b) The Fe (II) salt may be selected from one or more of the following raw materials: feCl ₂ ，FeSO ₄ ，Fe(OAC) ₂ The method comprises the steps of carrying out a first treatment on the surface of the And/or

(c) The base may be selected from one or more of the following raw materials: ammonium hydroxide, naOH, KOH and amines; and/or

(d) The solution may further comprise CoCl ₂ And/or MnCl ₂ 。

Further, in step 3), by adding Fe ₃ O ₄ A polymer network is formed around the nanoparticles, and a coating is prepared on the superparamagnetic polymer microspheres. In order to achieve intimate bonding with the support layer, preventing delamination from layer to layer, it is desirable to polymerize at least one or more non-functional monomers that may be used with the support layer. The monomer forms the intertwining of the outer layer network and the supporting layer network through the penetration of the original supporting layer. Unlike the prior art However, in the present application, since the support layer has crosslinks of the crosslinking monomer, the microspheres of the hollow structure do not swell significantly due to permeation of the monomer, but rather form an interlaced network structure. Meanwhile, in the polymerization process, other monomers with functional groups can be continuously added, and the monomers are polymerized under the free radical polymerization condition. Such monomers may provide anchor points for the functional layer. Without adding functional groups, the addition of the functional layer needs to be achieved by means of sufficient interleaving of the polymer network.

In step 3), the polymerization time may be 3 to 30 hours, such as 5 to 16 hours. The free radical polymerization may be carried out at a temperature of 40-80 ℃ (such as 50-70 ℃). The weight percentage of crosslinking monomer relative to the bulk monomer may be from 5 to 70%, preferably from 10 to 50%.

The purpose of step 3) is to achieve coating of the magnetic nanoparticles, and free radical activity gradually decreases with time during the process of full growth of the coating layer, so that the functional groups formed in this step can only be used as anchor points for the functionalization of the magnetic microspheres in combination with the next layer, because this step cannot effectively adjust the functional group density.

Specifically, the non-functional monomer of the coating layer in the step 3) may be selected from monomers having the same or similar structure as the monomer of the support layer: non-functional monomers such as the coating layer include styrene and/or styrene derivatives; the styrene derivative is selected from one or more of the following raw materials:

4-methylstyrene, 3-methylstyrene, 4-t-butylstyrene;

the coating layer crosslinking monomer may be selected from one or more of the following raw materials:

divinylbenzene, ethylene glycol dimethacrylate, bisphenol a dimethacrylate, N' -methylenebis (acrylamide);

the functional groups on the coating layer functional monomer may be independently selected from one or more of amino, epoxy and hydroxyl groups: the functional monomer of the coating layer is for example selected from one or more of acrylamide, methacrylamide, allylamine, (hydroxyethyl) methacrylate, hydroxypropyl methacrylate, 4-hydroxybutyl acrylate, glycidyl methacrylate, allyl glycidyl ether, 1, 2-epoxy-5-hexene, maleic anhydride, 2-hydroxyethyl methacrylate, 2-carboxyethyl acrylate oligomer.

The emulsifier of the coating may be selected from one or more of glycolic acid ethoxylate, 4-t-butylphenyl ether (having an aromatic hydrophobic tail), glycolic acid ethoxylated lauryl phenyl ether (having an alkylphenyl hydrophobic tail), glycolic acid ethoxylated oleyl ether (having only an alkyl tail), dioctyl sodium sulfosuccinate, sodium dodecyl sulfate, sodium dodecyl tetraoxyethylene sulfate, sodium dodecyl ether sulfate, sodium dodecyl benzene sulfonate, sodium methyl cocoyl taurate, sodium lauryl sulfate.

The solvent of the coating layer is deionized water

Further, in step 4), a polymer network is formed around the coating layer, so as to achieve the mutual winding of the functional layer and the network of the coating layer, further balance the elasticity and rigidity of the superparamagnetic microsphere, increase the thickness of the polymer on the outer layer of the superparamagnetic nanoparticle, and adsorb more organic fluorescent dye. The preparation of this layer requires the use of at least one non-functional monomer used in the coating layer or one monomer capable of reacting with the anchor point of the coating layer, a crosslinking monomer being polymerized by free radicals and a monomer having a functional group being added at the end of the reaction. The monomer is added in the later stage of the reaction, so that all functional groups can be better gathered on the surface, but not in the polymer network, and the functional groups are favorably coupled with the functional protein in the later stage. The addition of functional groups to the surface of the superparamagnetic microspheres and the adjustment of the density of functional groups are also formed at this stage.

In step 4), the first polymerization time may be 3 to 8 hours and the second polymerization time may be 3 to 24 hours, such as 5 to 17 hours. The free radical polymerization may be carried out at a temperature of 40-80 ℃ (such as 50-70 ℃). The weight percentage of crosslinking monomer relative to the bulk monomer may be from 5 to 70%, preferably from 10 to 50%. The weight percentage of functional group monomer relative to the bulk monomer may be from 1 to 30%, such as from 5 to 20%.

Specifically, in step 4), the non-functional monomer of the functional group layer may be selected from the same monomers as the coating layer or monomers having similar structures: the non-functional monomers of the functional group layer include styrene derivatives; the styrene derivative is selected from one or more of the following raw materials: 4-methylstyrene, 3-methylstyrene and 4-tert-butylstyrene.

The functional layer crosslinking monomer may be selected from one or more of the following raw materials:

divinylbenzene, ethylene glycol dimethacrylate, bisphenol a dimethacrylate and N, N' -methylenebis (acrylamide), bisphenol a-bis (2-hydroxypropyl) acrylate, bisphenol a ethoxy diacrylate;

the emulsifier of the functional layer may be comprised of one or more anionic surfactants including glycolic acid ethoxylate, 4-t-butylphenyl ether (having an aromatic hydrophobic tail), glycolic acid ethoxylated lauryl phenyl ether (having an alkylphenyl hydrophobic tail), glycolic acid ethoxylated oleyl ether (having only an alkyl tail), dioctyl sodium sulfosuccinate, sodium dodecyl sulfate, sodium dodecyl tetraoxol sulfate, sodium dodecyl ether sulfate, sodium dodecyl benzenesulfonate, sodium methyl cocoyl taurate, sodium lauryl sulfate.

The functional group of the functional group monomer of the functional layer can be independently selected from one or more of amino, carboxyl, epoxy and hydroxyl, and can be from the following raw materials: acrylic acid, methacrylic acid, 2-carboxyethyl acrylate, acrylamide, methacrylamide, allylamine, (hydroxyethyl) methacrylate, hydroxypropyl methacrylate, 4-hydroxybutyl acrylate, glycidyl methacrylate, allyl glycidyl ether, 1, 2-epoxy-5-hexene, maleic anhydride, 2-hydroxyethyl methacrylate, 2-carboxyethyl acrylate oligomers.

The solvent of the functional layer is deionized water

Further, step 5) dye molecules for fluorescent encoding include, but are not limited to, one or more of the following: rhodamine WT, fluorescein, BDY 650-X, SE, coumarin, nile red, coumarin 6, coumarin 4, rhodamine B, nile blue, oxazine 725, oxazine750.

It is a third object of the present application to provide the use of monodisperse superparamagnetic fluorescent-encoded microspheres for in vitro diagnostic assays.

The beneficial effects of the invention are as follows:

the monodisperse superparamagnetic microsphere of the present invention has a polystyrene core covered by a crosslinked layer of styrene-crosslinked monomer-functional monomer, and further covered by superparamagnetic Fe ₃ O ₄ The nanoparticle layer covers and only a small part of the nanoparticle layer is dispersed in Fe ₃ O ₄ Functional coatings are prepared on the surface of the pass-through microspheres in nanoparticle polymer matrices to bind and cover superparamagnetic Fe ₃ O ₄ Nanoparticles, and provide functional groups for IVD assays, can be used in vitro diagnostic assays.

Drawings

The invention will be further described with reference to the drawings and examples.

FIG. 1 is a schematic diagram of the design of a magnetically fluorescent encoded microsphere according to the present invention;

FIG. 2 is a schematic diagram of the preparation process of the magnetic fluorescent coding microsphere according to the present invention;

FIG. 3 is an SEM image (scale 10 μm) of the support layer A-2 of the washed microspheres according to example 1 of the invention;

FIG. 4 is an SEM image (scale 1 μm) of a magnetized microsphere support layer B according to example 2 of the present invention;

FIG. 5 is an SEM image (scale 1 μm) of superparamagnetic microspheres of example 3 of the present invention after forming a coating layer;

FIG. 6-1 is a graph showing the fluorescence intensity of E1-E5 samples according to example 6 of the present invention;

FIG. 6-2 is a graph showing fluorescence intensity of samples EC1 through EC5 according to example 6 of the present invention;

FIG. 7 is an SEM image (scale 1 μm) of the washed microsphere support layer E-2 according to example 8 of the invention;

FIG. 8 is an SEM image (scale 1 μm) of superparamagnetic microspheres of example 8 of the present invention after forming a coating layer;

FIG. 9 is an SEM image of the interior of a magnetic bead fragment according to example 8 of the present invention (scale 1 μm);

FIG. 10 is an SEM image (scale 1 micron) of a 1 micron control solid microsphere template according to comparative example 2 of the present invention;

FIG. 11 is an SEM image of the interior of a fragment of a magnetic bead according to comparative example 2 of the present invention (1 micron control magnetic bead, scale 1 micron);

FIG. 12 is a graph showing the fluorescence intensity (1 micron, single gradient) of J-1 superparamagnetic fluorescent microspheres according to comparative example 2 of the present invention;

FIG. 13 is an SEM image of 6.5 μm polystyrene microspheres (scale bar 10 μm) according to example 11 of the present invention;

FIG. 14 is an SEM image (scale 1 micron) of a 6.5 micron superparamagnetic microsphere substrate according to example 11 of the present invention;

FIG. 15 is a graph showing the fluorescence intensity of M-1 to M-7 superparamagnetic fluorescent encoding microspheres according to example 11 of the present invention.

Detailed Description

The invention will be further illustrated by the following examples, which are not intended to limit the scope of the invention, in order to facilitate the understanding of those skilled in the art.

These materials were purchased from the sources provided below, as well as the instruments and test methods described below:

styrene: tokyo chemical industries, inc (TCI), stabilized with TBC (4-t-butylcatechol) >99.0% (GC);

Azobisisobutyronitrile (AIBN): sigma-Aldrich (12% in acetone);

sodium persulfate (Na) ₂ S ₂ O ₈ SPS): alfa Aesar, crystalline, 98%;

polyvinylpyrrolidone (PVP, K30, mw=40,000): TCI, total nitrogen 12.0% -12.8% (on anhydrous basis); maximum water content 7.0%, K value 26.0-34.0;

divinylbenzene (DVB, meta-and para-mixtures): TCI,50.0% (GC) (containing ethylvinylbenzene, diethylbenzene) (stabilized with 4-t-butylcatechol);

acrylic acid: TCI, >99.0% (GC) (stabilized with monomethyl ether hydroquinone);

glycidyl methacrylate: sigma-Aldrich, > 97.0% (GC);

2-hydroxyethyl methacrylate: sigma-Aldrich, > 99.0%;

2-carboxyethyl acrylate oligomer: sigma-Aldrich,2000ppm MEHQ as inhibitor;

ethanol: 99 percent;

citric acid: sigma-Aldrich,98%;

trimethylolpropane triacrylate: sigma-Aldrich;

methyl methacrylate: TCI;

2- (2-aminoethoxy) ethanol: sigma-Aldrich,98%;

Triton X-100:Sigma-Aldrich；

sodium dodecyl sulfonate: sigma-Aldrich, >99%;

deionized (DI) water: purchased from ELGA Ultrapure Water Treatment Systems (PURELAB option);

scanning Electron Microscope (SEM) imaging using JEOL JSM 6700F;

Transmission Electron Microscope (TEM) imaging using JEOL 2100F;

mechanical stirrer: wiggens, WB2000-M overhead stirrer;

flow cytometer Cytoflex flow cytometer, beckman Coulter

Automatic potentiometric titrator T5-Mettler Toledo

SEPMAG biomagnetic separator biological magnetic separator

Calculating a Coefficient of Variation (CV)% of the polystyrene microspheres according to the SEM image (x 10,000) measurement result; 100 microspheres were measured using IMAGE J and then the average diameter and CV were calculated.

The monodisperse superparamagnetic fluorescent coding microsphere comprises a support layer 100, wherein the support layer 100 comprises a support layer body 101, an inner surface 102 and an outer surface 103. The inner surface 102 and the outer surface 103 are provided with compact superparamagnetic Fe ₃ O ₄ Nanoparticles 104. The support layer body 101 is made of polystyrene and crosslinked singleThe polymer matrix material is formed, and the network structure of the polymer matrix material contains part of superparamagnetic Fe ₃ O ₄ And (3) nanoparticles. The inner surface 102 and the outer surface 103 of the support layer are modified with a coating layer of a functional polymer. The coating layer can be made of Fe ₃ O ₄ The nanoparticles coordinate.

The coating layer 110 contains a first coating layer 111 or a first coating layer 111 and a second coating layer 112. The first coating layer is formed by crosslinking a non-functional monomer with a styrene monomer. The second coating layer is formed by a styrene monomer crosslinking functional group monomer. The first coating layer 111 and the supporting layer form a network cross-linked polymer, which can encapsulate Fe ₃ O ₄ And (3) nanoparticles. The second coating layer 112 forms a crosslinked polymer with the first coating layer 111, completely covers the first coating layer 111, and provides functional groups that can react with the functional layer.

Functional layer 120 the functional layer is formed from a non-functional monomer used in the coating and/or a functional monomer that reacts with the anchor point of the coating. The functional layer may capture luminescent substances and provide functional groups that may be further coupled to a biomarker.

The light emitting substances may be distributed in the coating layer 110 and the functional layer 120 in different ratios.

The preparation process of the encoded microsphere is described in examples 1-11 and comparative examples 1-2.

Example 1

Synthesis of support layer

Described below is a "single pot multistage" polymerization process for the synthesis of monodisperse microspheres with-COOH and-OH groups.

To a 250mL three neck round bottom glass reactor equipped with a mechanical stirrer was added AIBN solution (2 g, 12% in acetone); PVP (0.8 g), isopropanol (100 mL) and Triton X-100 (0.2 g) were then added, and the mixture was stirred at room temperature for 5 minutes to obtain a clear solution; the solution was then heated to 60 ℃ with an oil bath, followed by the addition of styrene (7.5 mL), resulting in a white colloidal solution after half an hour; after 5 hours of reaction, DVB solution (2 mL DVB in 7mL ethanol) was slowly added using a constant pressure dropping funnel, and after the DVB solution addition was completed, the reaction (crosslinking polymerization reaction) was continued for 3 hours; then, a syringe was used to add a monomer solution of 2-hydroxyethyl methacrylate (0.75 g in 5mL ethanol) and 2-carboxyethyl acrylate oligomer (0.75 g in 5mL ethanol, neutralized with ammonia solution, 25% W/W); the reaction (polymerization) was continued for 6 hours to obtain a milky colloidal solution containing microspheres. SEM images showed that the synthesized microspheres were spherical and in a monodisperse state, with a diameter of about 4.5 μm (FIG. 3). The obtained microsphere is called as "A-1", and the unreacted impurities and solvent are removed by centrifugal separation and washing, so that the obtained microsphere is called as "A-2".

Example 2

Magnetization of support layer

To a 250mL three neck round bottom glass reactor equipped with a mechanical stirrer, feCl was added ₃ (8.0 g) and 100mL of water, stirred to form a brown solution, followed by addition of FeCl ₂ (3.1 g) and EtOH (20 mL). The mixture was stirred for 30 minutes to form a solution, to the resulting solution was added an aqueous solution (50 mL, about 8% by mass) of microsphere A-2, followed by stirring at 70℃for 3 hours. After the mixture was cooled to room temperature, aqueous ammonia (25%, 30 mL) was added with vigorous stirring to immediately obtain a black slurry, and then the black slurry was heated to 70 ℃ for 1 hour to complete the reaction. The superparamagnetic microspheres were purified by several centrifugation to remove unbound superparamagnetic nanoparticles, which were finally re-dispersed in deionized water (100 mL, about 40 mg/mL). The resulting blackish brown superparamagnetic microsphere was designated "B-1". The obtained magnetic microsphere has Fe ₃ O ₄ The nanoparticles are uniformly coated on the outside of the support layer (fig. 4).

Example 3

Preparation of the coating layer

Emulsion polymerization coating step: the magnetic microsphere solution (10 mL) was added to a 50mL centrifuge tube containing 30mL deionized water. The mixture was then added to a 250mL three neck round bottom glass reactor equipped with a mechanical stirrer. The reactor was purged with nitrogen to remove oxygen and then SPS (30 mg, dissolved in 2mL of water) was added. The mixture dispersion was heated to 60 ℃ and stirred at 60 ℃ for 10 minutes. Subsequently, a mixture of monomers containing styrene (400 mg), trimethylolpropane triacrylate (200 mg), deionized water (10 ml) and sodium dodecyl sulfate (0.5%) was slowly added over a period of 1 hour. After the addition of the monomer mixture was completed, the reaction (coating polymerization) was continued at 60℃for 6 hours. After cooling to room temperature, the coated superparamagnetic microspheres were separated with a magnetic separator, then washed with ethanol (50 ml×2) and deionized water (50 ml×2), the last wash and 30mL deionized water were added, and the beads were resuspended. The superparamagnetic microsphere substrate with the coating is referred to as C-1.

The SEM image in fig. 5 clearly shows that the surface of the microspheres containing superparamagnetic nanoparticles becomes smooth after coating. The edges of which are completely covered with a layer of polymer, which prevents leaching of the magnetic particles.

Example 4

Preparation of functional layer

Emulsion polymerization functionalization step: the magnetic microsphere solution C-1 of example 3 was added to a 250mL three-necked round bottom glass reactor equipped with a mechanical stirrer. The reactor was purged with nitrogen to remove oxygen and then SPS (30 mg, dissolved in 2mL of water) was added. The mixture dispersion was heated to 60 ℃ and stirred at 60 ℃ for 10 minutes. Subsequently, a mixture of a monomer containing styrene (400 mg) and sodium dodecylsulfate (0.5%) was slowly added over a period of 1 hour, and after two hours of reaction, an aqueous solution of a monomer containing acrylic acid (50 mg) was slowly added over a period of 1 hour thereafter. After the addition of the monomer mixture was completed, the reaction was continued at 60℃for 2 hours. After cooling to room temperature, the coated superparamagnetic microspheres were separated with a magnetic separator, then washed with ethanol (50 ml×2) and deionized water (50 ml×2), the last wash and 120mL deionized water added, and the beads resuspended. The superparamagnetic microsphere substrate with the coating is called D-1.

Example 5

Fluorescent coding in functional layers

5 parts of the solution containing the same dry weight of magnetic beads, e.g., 1mg, are placed in a 2ml centrifuge tube. The mixture was washed repeatedly three times with 1ml of acetonitrile on a magnetic separator and finally resuspended in 0.5ml of acetonitrile solution. 1mg/mL Alexa 647 acetonitrile mother liquor was prepared, the mother liquor was further diluted with acetonitrile stepwise 5-fold to form 5 concentration gradients of fluorochromes, 0.5mL of fluorochrome solution 0.1mg/mL, 0.05mg/mL, 0.01mg/mL, 0.005/mL, 0.0001mg/mL and 0.5mL of magnetic bead solution 1: 1. After shaking the tube on a shaker for 2 hours, it was then washed with ethanol (50 mL. Times.2) and deionized water (50 mL. Times.2), the last wash and 1mL deionized water was added. The superparamagnetic fluorescent encoded microsphere is called E-1to E-5.

Comparative example 1

Fluorescent coding of coating

Superparamagnetic microsphere base material C-1 (without a functional layer) with a coating layer is directly subjected to fluorescent coding according to example 5 to obtain a superparamagnetic fluorescent coding microsphere called EC-1to EC-5.

Example 6

Flow cytometer Cytoflex flow cytometer, beckman Coulter performed fluorescence intensity measurements on two sets of samples E1-E5 and EC1-EC5 5 samples with fluorescence gradients, and R660-APC was selected to collect the signal. The fluorescence intensity of E1 to E5 decreases from right to left, and the fluorescence can reach a span of 4 orders of magnitude (see FIG. 6-1, fluorescence intensity of E1 to E5 decreases from right to left). Whereas the fluorescence of EC1-EC5 can only reach spans of 3 orders of magnitude. In particular, the peak value of the contrast fluorescence, E1, is increased by more than 10 times over EC1, which indicates that the functional layer thickness reaches a saturated fluorescence loading of at least 10 times that of the coating layer. The larger the fluorescence order span, the more encoded gradients are possible in that span, and the encoded bands are also more easily separated by far enough margins to effectively avoid signal crossings. Comparing the bandwidths of the respective gradient bands of the two graphs, FIG. 6-1 is also significantly better than FIG. 6-2 (FIG. 6-2, fluorescence intensities of EC1 through EC5 decrease from right to left);

Example 7

Magnetic response test

The magnetic response of the superparamagnetic beads was measured using SEPMAG biomagnetic separator. An aqueous dispersion of magnetic beads having about 5mg/ml was placed in SEPMAG biomagnetic separator, the transmittance of the solution was monitored for 200s, and commercial magnetic beads were used as a control, and tested in parallel. Since the beads in the buffer are usually opaque when in suspension, the gradual change is only transparent when the beads are enriched in the tube wall by the magnet to achieve solid-liquid separation, and thus can be used in optical monitoring processes. After the test, the beads were re-shaken and suspended in water and repeated twice. The speed of the magnetic response is expressed in terms of the time required to separate half of the beads (t 50). The test results (see Table 1) show that three tests with CV lower than 5% can determine that no beads adhere to the wall under the repeated action of the magnetic field or irreversible bead agglomerates are formed in the solution due to hysteresis. In addition, the magnet recovered the beads faster than the commercial beads, and the commercial beads used as control samples had a t50 of only 14s.

TABLE 1D1 magnetic bead substrate magnetic response curve detection results

Sample of	t50(s)
		D1-test1	9.08
D1-test2	8.89
		D1-test3	8.93
AVG	8.97
		CV(％)	0.91

Example 8

Synthesis of support layer

Described below is a polymerization process for a 2.8 micron microsphere support layer for the synthesis of monodisperse microspheres with-COOH and-OH groups.

To a 250mL three neck round bottom glass reactor equipped with a mechanical stirrer was added AIBN solution (2 g, 12% in acetone). PVP (0.4 g), isopropanol (100 mL), triton X-100 (0.05 g) were then added and the mixture was stirred at room temperature for 5 minutes to give a clear solution; the solution was then heated to 60 ℃ with an oil bath, followed by the addition of styrene (5 mL). After half an hour a white colloidal solution was produced. After 5 hours of reaction, DVB solution (1 mL DVB in 7mL ethanol) was slowly added using a constant pressure dropping funnel. After the DVB solution addition was completed, the reaction (crosslinking polymerization) was continued for 3 hours. Then, a syringe was used to add a monomer solution of 2-hydroxyethyl methacrylate (0.25 g in 5mL ethanol) and 2-carboxyethyl acrylate oligomer (0.25 g in 5mL ethanol, neutralized with ammonia solution, 25% W/W). The reaction (polymerization) was continued for 6 hours to obtain a milky colloidal solution. SEM images showed that the synthesized microspheres were spherical and in a monodisperse state, with a diameter of about 2.8 μm (FIG. 7). The obtained microsphere was designated as "F-1". Centrifugal separation and cleaning are adopted to remove unreacted impurities and solvent, and the obtained microsphere is F-2;

the support layer was magnetized, the coating layer and the functional layer were carried out as described in examples 2, 3 and 4, and the final superparamagnetic microsphere substrate with coating layer was designated as G-1. The SEM image in fig. 8 clearly shows that the 2.8 micron superparamagnetic nanoparticle microspheres become smooth and dense on the surface after coating. After the magnetic beads were intentionally crushed by external force, the interior thereof was observed by SEM, see fig. 9. Fig. 9 shows that the superparamagnetic microsphere is hollow, the supporting layer body 101 is a layer of uniform and compact polymer, and the densities of the magnetic nanoparticles on the inner surface 102 and the outer surface 103 are different. In particular, after coating, the inner magnetic nano particles are very fluffy due to the fact that the coating layer is not arranged, the outer magnetic nano particles are filled in gaps by the coating layer, and the whole layer of the composite material is very compact.

Example 9

Carboxyl groups on the surface of the polystyrene particles were measured by acid titration (Journal of Colloid and Interlace Science, 1974, 49, 3, 425-432). In a 50mL container, accurately weighed magnetic microsphere sample G-1 (containing about 120mg dry weight of magnetic microsphere dispersion) was diluted with deionized water to a volume of 25 mL. The conductivity was adjusted by adding dilute NaOH solution and the sample was placed on an automatic potentiometric titrator T5, then the conductivity probe was placed in the diluted dispersion. The samples were titrated with standard 0.1N aqueous HCl in 0.005mL increments with mechanical agitation and the dispersion was checked for conductivity. The temperature was maintained at about 25 ℃ throughout the titration.

The titration endpoint was determined according to literature (Journal of Colloid and Interlace Science, 1974, 49, 3, 425-432). The concentration of acid is expressed in milliequivalents of charge per gram of polymer solids (MEQ/g). The concentration of the "surface bound" acid can be calculated by the following formula:

wherein: vsb is the volume of HCl titrant (in cc) required to neutralize the "surface bound" acid, N is the equivalent concentration of HCl titrant, W is the total weight of the dispersion sample, and S is the mass fraction of solids in the sample.

The experiment shows that the surface binding acid density is 296 mu mol/g, which indicates that the method can be used for realizing rich surface carboxylation.

Comparative example 2

Synthesis of support layer

Described below is a polymerization process for a 1 micron microsphere support layer for the synthesis of monodisperse microspheres with-COOH and-OH groups.

To a 250mL three neck round bottom glass reactor equipped with a mechanical stirrer was added AIBN solution (0.7 g, 12% in acetone). PVP (0.4 g), triton X-100 (0.33 g), ethanol (100 mL) were then added and the mixture was stirred at room temperature for 5 min to give a clear solution; the solution was then heated to 60 ℃ with an oil bath, followed by the addition of styrene (10 mL). After half an hour a white colloidal solution was produced. After 8 hours of reaction, the DVB solution (0.2 mL DVB in 7mL ethanol) was slowly added using a constant pressure dropping funnel. After the DVB solution addition was completed, the reaction (crosslinking polymerization) was continued for 3 hours. Then, a syringe was used to add a monomer solution of 2-hydroxyethyl methacrylate (0.05 g in 5mL ethanol) and 2-carboxyethyl acrylate oligomer (0.05 g in 5mL ethanol, neutralized with ammonia solution, 25% W/W). The reaction (polymerization) was continued for 6 hours to obtain a milky colloidal solution. SEM images showed that the synthesized microspheres were spherical and in a monodisperse state, with a diameter of about 6.5 μm (FIG. 10). The microspheres obtained are referred to as "H-1". Centrifugal separation and cleaning are adopted to remove unreacted impurities and solvent, and the obtained microsphere is H-2;

The support layer was magnetized, the coating layer and the functional layer were carried out as described in examples 2, 3 and 4, and the final superparamagnetic microsphere substrate with coating layer was designated as I-1. The SEM image in fig. 11 clearly shows that after coating, the superparamagnetic nanoparticle microspheres of 1 micron, which are solid and coated with a thick whole shell, appear very thick as a composite material after deliberately crushing the beads with external force, are observed inside by SEM.

1mg of the coated magnetic beads were placed in a 2ml centrifuge tube. The mixture was washed repeatedly three times with 1ml of acetonitrile on a magnetic separator and finally resuspended in 0.5ml of acetonitrile solution. 0.1mg/mLAlexa 647 acetonitrile mother liquor was prepared, and 0.5ml of a fluorescent dye solution and 0.5ml of a magnetic bead solution 1 were taken: 1. After shaking the tube on a shaker for 2 hours, it was then washed with ethanol (50 mL. Times.2) and deionized water (50 mL. Times.2), the last wash and 1mL deionized water was added. The superparamagnetic fluorescent microsphere is called J-1.

The samples were tested by flow cytometer Cytoflex flow cytometer, beckman Coulter, with only a single channel R660-APC acquisition signal selected, see FIG. 12. The maximum intensity of the fluorescent signal of this sample is much lower than that of sample E1. In addition, the fluorescence signal spans 1.5 orders of magnitude, and the bandwidth is very wide, so that multi-gradient coding is almost impossible. This is primarily the case because the fluorescent molecules are distributed within the solid core, which results in the fluorescent light not being able to penetrate, and in addition, the fluorescent molecules slowly enter the microsphere core, which results in fewer fluorescent molecules remaining on the surface than the hollow spheres, so that the fluorescent signal is significantly lower and the fluorescent signal distribution is very uneven.

Example 11

Synthesis of support layer

Described below is the polymerization process of a 6.5 micron microsphere support layer for the synthesis of monodisperse microspheres with-COOH and-OH groups.

To a 250mL three neck round bottom glass reactor equipped with a mechanical stirrer was added AIBN solution (0.7 g, 12% in acetone). PVP (0.4 g) and ethanol (100 mL) were then added, and the mixture was stirred at room temperature for 5 minutes to obtain a clear solution; the solution was then heated to 60 ℃ with an oil bath, followed by the addition of styrene (1 mL). After half an hour a white colloidal solution was produced. After 8 hours of reaction, the DVB solution (0.2 mL DVB in 7mL ethanol) was slowly added using a constant pressure dropping funnel. After the DVB solution addition was completed, the reaction (crosslinking polymerization) was continued for 3 hours. Then, a syringe was used to add a monomer solution of 2-hydroxyethyl methacrylate (0.05 g in 5mL ethanol) and 2-carboxyethyl acrylate oligomer (0.05 g in 5mL ethanol, neutralized with ammonia solution, 25% W/W). The reaction (polymerization) was continued for 6 hours to obtain a milky colloidal solution. SEM images showed that the synthesized microspheres were spherical and in a monodisperse state, with a diameter of about 6.5 μm (FIG. 13). The obtained microsphere was referred to as "K-1". Centrifugal separation and cleaning are adopted to remove unreacted impurities and solvent, and the obtained microsphere is K-2;

The support layer was magnetized, the coating layer and the functional layer were carried out as described in examples 2, 3 and 4, and the final superparamagnetic microsphere substrate with coating layer was designated as L-1 (FIG. 14).

7 parts of the solution containing the same dry weight of magnetic beads, e.g., 1mg, are placed in a 2ml centrifuge tube. The mixture was washed repeatedly three times with 1ml of acetonitrile on a magnetic separator and finally resuspended in 0.5ml of acetonitrile solution. 1mg/mL of Alexa 647 mother liquor was prepared from acetonitrile and dioxane (volume ratio 2:3), the mother liquor was further diluted with acetonitrile stepwise 7-fold to form 7 concentration gradients of fluorescent dye, 0.5mL of fluorescent dye solution 0.1mg/mL, 0.05mg/mL, 0.01mg/mL, 0.005/mL, 0.0001mg/mL, 0.0005mg/mL and 0.00001mg/mL of 0.5mL of magnetic bead solution 1: 1. After shaking the tube on a shaker for 2 hours, it was then washed with ethanol (50 mL. Times.2) and deionized water (50 mL. Times.2), the last wash and 1mL deionized water was added. The superparamagnetic fluorescent encoded microspheres are designated M-1 to M-7.

The samples were tested by flow cytometer Cytoflex flow cytometer, beckman Coulter, with only a single channel R660-APC acquisition signal selected, see fig. 15: the bandwidth of the fluorescence encoding signal for this set of samples is very narrow, and the fluorescence signal achieves 7 fluorescence gradients (M-1 to M-7) within 4 orders of magnitude, with the fluorescence intensities of M-1 to M-7 decreasing from right to left.

Design considerations for the above examples and comparative examples:

all polymerization processes used mechanical stirring (200 rpm), and before use, deionized water and ethanol were bubbled with nitrogen for 20 minutes to remove oxygen; all chemicals (including AIBN, SPS, PVP, acrylic acid, allylamine, DVB, styrene, glycidol, glycidyl methacrylate, bisphenol a diglycidyl ether, and 2-hydroxyethyl methacrylate) were used as received without purification; all polymerizations and reactions were performed under nitrogen protection using standard Schlenk line techniques.

The foregoing embodiments are illustrative of the preferred embodiments of the present invention, and the invention may be embodied in other forms without departing from the spirit or essential characteristics thereof, and any apparent structure, process or formulation substitutions are intended to fall within the scope of the invention.

Claims

1. Monodisperse superparamagnetic fluorescent encoding microspheres, characterized by comprising:

forming a hollow spherical support layer from a polystyrene polymer matrix material;

the coating layer comprises a first coating layer and a second coating layer; the first coating layer coats superparamagnetism Fe arranged on the outer surface of the supporting layer ₃ O ₄ NanoparticlesThe first coating layer is arranged on the first substrate;

the raw materials of the polystyrene polymer matrix material comprise styrene monomer, crosslinking monomer and hydrophilic functional group monomer;

2. The monodisperse superparamagnetic fluorescent encoded microsphere according to claim 1, wherein the raw material of the coating layer comprises a styrene monomer and/or a hydrophilic functional group monomer.

3. The monodisperse superparamagnetic fluorescent encoded microsphere according to claim 1, wherein the raw materials of the functional layer comprise styrene monomers and/or hydrophilic functional group monomers.

4. The monodisperse superparamagnetic fluorescent-encoded microsphere according to claim 1, wherein the crosslinking monomer is one or more of divinylbenzene, ethylene glycol dimethacrylate, bisphenol a dimethacrylate, butanediol dimethacrylate, tricyclodecane dimethanol diacrylate, pentaerythritol triacrylate, tripropylene glycol diacrylate, propoxylated neopentyl diacrylate, ditrimethylolpropane tetraacrylate, tripropylene diacrylate, trimethylolpropane ethoxylated triacrylate, trimethylolpropane propoxylated triacrylate, ditrimethylolpropane tetraacrylate, glycerol propoxylated triacrylate, pentaerythritol propoxylated triacrylate, poly (ethylene glycol) diacrylate, poly (propylene glycol) diacrylate, tri (propylene glycol) diacrylate, N' -methylenebis (acrylamide), bisphenol a-bis (2-hydroxypropyl) acrylate, bisphenol a ethoxylated diacrylate.

5. A monodisperse superparamagnetic fluorescent encoded microsphere according to any of claims 1-3, wherein said styrene monomer comprises styrene and/or a styrene derivative; the styrene derivative is one or more of 4-methyl styrene, 3-methyl styrene and 4-tertiary butyl styrene.

6. A monodisperse superparamagnetic fluorescent encoded microsphere according to any of claims 1-3, wherein said hydrophilic functional group monomer is one or more of acrylic acid, methacrylic acid, 2-carboxyethyl acrylate, acrylamide, methacrylamide, allylamine, (hydroxyethyl) methacrylate, hydroxypropyl methacrylate, 4-hydroxybutyl acrylate, glycidyl methacrylate, allyl glycidyl ether, bisphenol a-bis (2-hydroxypropyl) acrylate, 1, 2-epoxy-5-hexene, maleic anhydride, 2-hydroxyethyl methacrylate, 2-carboxyethyl acrylate oligomer.

7. The monodisperse superparamagnetic fluorescent encoded microsphere according to claim 1, wherein superparamagnetic Fe is generated in situ on an inner surface of the support layer and an outer surface of the support layer ₃ O ₄ And (3) nanoparticles.

8. The monodisperse superparamagnetic fluorescent-encoded microsphere according to claim 1, wherein dye molecules for fluorescent encoding are absorbed on the functional layer, the dye molecules for fluorescent encoding including, but not limited to, one or more of the following: rhodamine WT, fluorescein, BDY 650-X, SE, coumarin, nile red, coumarin 6, coumarin 4, rhodamine B, nile blue, oxazine 725, oxazine 750.

9. A method for preparing the monodisperse superparamagnetic fluorescent-encoded microsphere according to claim 1, comprising the steps of:

step 2) swelling the microspheres with the hollow structures to generate superparamagnetism Fe in situ ₃ O ₄ Nanoparticles and upload to the supportThe inner surface and the outer surface of the support layer are provided with superparamagnetism microspheres;

step 3) preparing a coating layer capable of forming a network structure with the supporting layer on the superparamagnetic microspheres to obtain a superparamagnetic microsphere base material;

step 4) using a monomer forming a network structure with the coating layer to functionalize the superparamagnetic microsphere base material to obtain a coated microsphere base material;

and 5) swelling the coated microsphere base material to enable the coated microsphere base material to absorb organic fluorescent dyes with different concentration gradients, so as to obtain the monodisperse superparamagnetic fluorescent coding microsphere.

10. Use of monodisperse superparamagnetic fluorescence-encoding microspheres, wherein the monodisperse superparamagnetic fluorescence-encoding microspheres are derived from the monodisperse superparamagnetic fluorescence-encoding microspheres according to any one of claims 1 to 8 or from the monodisperse superparamagnetic fluorescence-encoding microspheres prepared by the preparation method according to claim 9, and the monodisperse superparamagnetic fluorescence-encoding microspheres are used in-vitro diagnostic assays.