CN112111268B

CN112111268B - Fluorescent coding microsphere and array and preparation method thereof

Info

Publication number: CN112111268B
Application number: CN202011030055.9A
Authority: CN
Inventors: 王耀; 徐宏; 古宏晨
Original assignee: Shanghai Mag Gene Nanotech Co ltd; Shanghai Jiao Tong University
Current assignee: Shanghai Mag Gene Nanotech Co ltd
Priority date: 2020-09-27
Filing date: 2020-09-27
Publication date: 2021-11-23
Anticipated expiration: 2040-09-27
Also published as: WO2022063337A1; CN112111268A

Abstract

The present invention provides a fluorescent coded microsphere and an array and a preparation method, wherein the coded microsphere comprises a microsphere carrier, the microsphere carrier is a mesoporous microsphere, and at least one fluorescent light is arranged inside the microsphere carrier A marker, the fluorescent marker is formed by connecting a fluorescent dye and an intermediary substance, and the central emission wavelengths of the fluorescent dyes corresponding to each of the fluorescent markers are different, so that each fluorescent dye defines one of the encoded microspheres Encoding information for the dimension.

Description

Fluorescent coding microsphere and array and preparation method thereof

Technical Field

The invention relates to the field of micro-nano biomaterials and multi-index detection, in particular to a fluorescent coding microsphere and array and a preparation method thereof.

Background

The suspension array technology based on the coding microspheres is one of the multi-index detection technologies which are widely concerned in recent years, can realize simultaneous identification and quantitative analysis of various target molecules (including nucleic acid, protein, small molecular compounds and the like) to be detected in a single reaction, and has important application value in the fields of disease diagnosis, drug development, environmental pollution monitoring and the like. Compared with the traditional solid phase chip, the suspension array technology replaces a flat plate carrier with micro-nano-scale coding microspheres, each type of microspheres carries coding information which can be identified by an instrument so as to identify the type of a probe fixed on the surface of the microspheres, and after the microspheres specifically react with corresponding target molecules, an obtained compound has a detection signal which can be read by the instrument, so that the detection result analysis can be carried out. The coded microspheres are used as a core carrier of a suspension array technology, have a three-dimensional structure and a large specific surface area, and have a reaction with target molecules similar to a liquid phase reaction kinetic process and high detection sensitivity; the probes on the surfaces of the coded microspheres can be customized according to the requirements of detection items, and the coded microspheres with the surfaces connected with different probes are mixed to realize single-reaction multi-index detection, so that the method has higher combination flexibility.

The microsphere carrier is endowed with a coding function, and the method is the basis for realizing single-reaction multi-index detection. Researchers have proposed different encoding methods to encode microsphere carriers, and compared with pattern encoding, physical encoding, magnetic encoding, and the like, the optical encoding method based on fluorescent elements is most widely used because encoding is flexible and convenient, and decoding method is mature and fast. Among them, fluorescent dyes are still the most popular fluorescent coding elements at present due to the advantages of easy preparation, low cost, good solvent tolerance, low biological toxicity, controllable coding process, etc. The xMap system developed by Luminex corporation in the united states is a successfully commercialized multi-index detection platform based on fluorescence-encoded microspheres, and organic fluorescent dye is loaded in polymer microspheres by using a swelling method. The preparation method is disclosed in U.S. Pat. No. 5, 6514295, 1, and comprises soaking polymer microspheres in organic solvent for swelling, allowing fluorescent dye molecules dissolved in the organic solvent to diffuse into the microspheres through the gaps of the polymer chains of the microspheres, removing the solvent, shrinking the microspheres, and retaining the dye molecules in the microspheres, and adjusting the type and concentration of the dye to obtain a series of different fluorescent coding microspheres. However, the swelling method must select a solvent which can simultaneously swell the polymer microsphere and dissolve the fluorescent dye, and has the problem of system compatibility; meanwhile, the method is only suitable for organic microspheres, cannot be expanded to the preparation of fluorescent coding microspheres by using inorganic microsphere carriers, and has limitation. Chinese patent publication No. CN106634945A discloses a method for preparing fluorescent coding microspheres, which comprises blending monomers, fluorescent dyes, initiators, dispersants, etc., and wrapping dye molecules in polymer microspheres by polymerization to obtain the fluorescent coding microspheres. However, the doping of the fluorescent dye increases the difficulty of controlling the particle size of the microsphere in the polymerization process, and the reduction of the uniformity of the particle size can affect the encoding accuracy. Chinese patent No. CN101846676B discloses a fluorescence encoding method for aminated microspheres, which utilizes covalent bonding of fluorescent dye and the amino groups on the surface of the microspheres to realize fluorescence encoding. Although the method has the advantages of simple operation, stable fluorescence and the like, the method also limits the functionalized groups on the surface of the microsphere, and only utilizes the outer surface of the microsphere to label fluorescent molecules, so that the coding capacity is greatly limited.

Therefore, the technical personnel in the field are dedicated to develop a fluorescent dye doped coding microsphere with simple and controllable preparation process, wide application range, accurate coding and high coding capacity and a preparation method thereof.

Disclosure of Invention

In view of the problems in the prior art, the present invention provides an encoded microsphere, which comprises a microsphere carrier, wherein the microsphere carrier is a mesoporous microsphere, and the matrix component and the pore diameter of the microsphere carrier are used for defining the first dimension encoding information of the encoded microsphere.

Further, the matrix component adopts inorganic substances or polymers.

Further, the matrix component employs silica or titania.

Further, the matrix component adopts polystyrene, polyacrylic acid, polymethyl acrylate, polymethacrylic acid, polymethyl methacrylate, polydivinylbenzene and/or copolymer formed by two or more monomers involved in the above polymers.

Further, the diameter of the microsphere carrier is selected within the range of 0.2-20 μm, and the preferable range is 3-6 μm.

Furthermore, the pore diameter of the microsphere carrier is selected within the range of 2-100 nm, and the preferable range is 10-60 nm.

Further, the first dimension information is adjusted according to the matrix composition and/or pore size of the microsphere carrier, and different first dimension information is distinguished by the signal distribution of FSC-SSC (forward scattered light-side scattered light) two-dimensional scatter diagram obtained by the detection of the encoded microspheres by a flow cytometer.

Furthermore, an intermediate substance is arranged in the microsphere carrier.

Further, the encoded microspheres further comprise at least one fluorescent material, each of the fluorescent materials having a central emission wavelength that is different from each other, such that each of the fluorescent materials defines encoded information for one dimension of the encoded microspheres.

Further, the difference of the central emission wavelength between the fluorescent materials is more than 30 nm.

Further, the fluorescent material is disposed inside and/or outside the microsphere carrier.

Further, the encoded microspheres also include a first fluorescent material that defines second dimension encoded information of the encoded microspheres.

Further, the first fluorescent material is disposed inside the microsphere carrier.

Further, the second dimension coding information is adjusted according to the content of the first fluorescent material.

Further, the first fluorescent material is a fluorescent dye and/or a rare earth complex, preferably a green fluorescent dye Fluorescein Isothiocyanate (FITC) with an emission wavelength of 517 nm.

Further, the first fluorescent material is connected with an intermediate substance to form a fluorescent marker, and the first fluorescent material is arranged inside the microsphere carrier in the form of the fluorescent marker.

Further, the intermediary material is a polymer.

Furthermore, the intermediate substance is arranged in the microsphere carrier, and the content of the first fluorescent material is adjusted according to the ratio of the intermediate substance to the fluorescent marker. In one embodiment of the invention, the intermediary material is an amino polymer.

Further, the encoded microsphere further comprises a second fluorescent material defining third dimension encoded information of the encoded microsphere.

Further, the second fluorescent material is disposed outside the microsphere carrier.

Further, the third dimension coding information is adjusted according to the content of the second fluorescent material.

Further, the second fluorescent material is a quantum dot, a conjugated polymer fluorescent nanoparticle, an aggregation-induced emission nanoparticle or an up-conversion fluorescent nanoparticle, and is preferably a CdSe/ZnS red quantum dot with an emission wavelength of 600 nm.

Further, the encoded microspheres also include magnetic nanoparticles.

Further, the magnetic nanoparticles are disposed outside the microsphere carrier.

Further, the magnetic nano-particles adopt Fe₃O₄Nanoparticles or gamma-Fe₂O₃Nanoparticles, preferably Fe₃O₄And (3) nanoparticles.

Further, the outer surface of the coding microsphere is a silicon oxide coating layer.

Furthermore, the outer surface of the coding microsphere is a silicon oxide coating layer and a functional molecule modifying layer, the functional molecule modifying layer is arranged on the outermost layer, and the preferable functional molecules are gamma-aminopropyl triethoxysilane (APTES) and polyacrylic acid (PAA).

The invention also provides a coded microsphere array, which comprises at least two kinds of coded microspheres, wherein different coded information exists among the coded microspheres; the coded microsphere comprises a microsphere carrier, wherein the microsphere carrier is a mesoporous microsphere, and the matrix component and the pore diameter of the microsphere carrier are used for limiting the first dimension coding information of the coded microsphere.

Further, the microsphere carriers of each of the encoded microspheres have substantially similar diameter dimensions. In some embodiments, the variation in diameter between different types of the microsphere carriers is defined as ≦ 15%.

Further, the array of encoded microspheres includes at least two different matrix compositions of the encoded microspheres.

Furthermore, different pore sizes are respectively selected for a plurality of the coded microspheres with the same matrix component.

Further, the matrix component adopts inorganic substances or polymers.

Further, the matrix component employs silica or titania.

Further, each encoding microsphere is detected by a flow cytometer, and the first dimension of each encoding microsphere is distinguished according to the signal distribution of the FSC-SSC two-dimensional scatter diagram obtained by detection.

Furthermore, an intermediate substance is arranged in the microsphere carrier.

Further, the intermediary material is a polymer.

Further, the encoded microspheres also include a second fluorescent material, the first fluorescent material defining third dimension encoded information of the encoded microspheres.

Further, the encoded microspheres also include magnetic nanoparticles.

Further, the magnetic nano-particles adopt Fe₃O₄Nanoparticles or gamma-Fe₂O₃And (3) nanoparticles.

The invention also provides a preparation method of the coding microsphere, which comprises the following steps:

selecting a microsphere carrier, wherein the microsphere carrier is a mesoporous microsphere, and determining the matrix component, the diameter and the pore diameter of the microsphere carrier so as to limit the first dimension coding information of the coding microsphere.

Further, the matrix component adopts inorganic substances or polymers.

Further, the matrix component employs silica or titania.

Further, the preparation method further comprises the following steps:

step two, arranging a fluorescent dye in the microsphere carrier to limit second-dimension coding information of the coding microsphere; or disposing an intermediary substance inside the microsphere carrier.

Further, in the second step, the fluorescent dye is disposed inside the microsphere carrier, and specifically includes:

connecting a fluorescent dye and an intermediate substance to form a fluorescent marker, and arranging the fluorescent marker or the mixture of the fluorescent marker and the intermediate substance in the inner space of the microsphere carrier through physical/chemical action.

Further, the intermediary material is a polymer. If the negatively charged microsphere carrier is used in the second step in some embodiments, the surface modification may be performed on the negatively charged microsphere carrier to obtain a microsphere carrier with negatively charged modified molecules in the inner space and the outer surface, and the positively charged amino polymer is used in the second step to form the fluorescence labeling polymer and is mixed with the fluorescence labeling polymer, so that the fluorescence labeling polymer can be adsorbed in the inner space of the microsphere carrier.

Further, the fluorescent dye forming the fluorescent marker is linked to the intermediate substance through a covalent bond.

Further, the second dimension encoding information is adjusted according to the ratio of the fluorescent marker to the intermediary substance in the mixture.

Further, the preparation method further comprises the following steps:

and thirdly, arranging the magnetic nano-particles on the outer surface of the microsphere carrier.

Further, the third step in the preparation method specifically comprises:

and coating the magnetic nano-particles on the outer surface of the microsphere carrier through physical/chemical action. If the positively charged amino polymer is used in the second step, the positively charged amino polymer is also adsorbed on the outer surface of the microsphere carrier after the second step is completed, and the negatively charged magnetic nanoparticles can be used in the third step, so that the magnetic nanoparticles are coated on the outer surface of the current microsphere carrier.

Further, the third step further includes:

and coating the magnetic nanoparticles on the outer surface of the microsphere carrier, and then coating the polymer on the outer surface of the microsphere carrier.

Further, the preparation method further comprises the following steps:

and step four, arranging the fluorescent nano particles on the outer surface of the microsphere carrier to limit the third-dimensional coding information of the coding microsphere.

Further, the fluorescent nanoparticles are quantum dots, conjugated polymer fluorescent nanoparticles, aggregation-induced emission nanoparticles or up-conversion fluorescent nanoparticles, preferably CdSe/ZnS red quantum dots with an emission wavelength of 600 nm.

Further, the fourth step in the preparation method specifically comprises:

and coating the fluorescent nano-particles on the outer surface of the microsphere carrier through physical/chemical action.

Further, the fourth step further includes:

and coating the polymer on the outer surface of the microsphere carrier after coating the fluorescent nano-particles on the outer surface of the microsphere carrier.

Further, the physical/chemical action includes electrostatic action, hydrophilic-hydrophobic action, hydrogen bonding action, coordination action, and/or covalent bonding action.

Further, after all the steps are finished, the outer surface of the microsphere carrier is coated with silicon oxide.

Further, after the outer surface of the microsphere carrier is coated with silicon oxide, the outer surface of the microsphere carrier is modified with functional molecules, so as to obtain the microsphere carrier with functionalized surface, wherein the preferred functional molecules are gamma-aminopropyl triethoxysilane (APTES) and polyacrylic acid (PAA).

The invention also provides a fluorescent dye-doped coding microsphere, which comprises a microsphere carrier, wherein the microsphere carrier is a mesoporous microsphere, at least one fluorescent marker is arranged in the microsphere carrier, the fluorescent marker is formed by connecting fluorescent dyes and an intermediate substance, the central emission wavelengths of the fluorescent dyes corresponding to the fluorescent markers are different, and therefore, each fluorescent dye limits the coding information of one dimension of the coding microsphere.

Further, the matrix component of the microsphere carrier adopts inorganic substances or polymers.

Further, the matrix component of the microsphere carrier adopts silicon dioxide or titanium dioxide.

Further, the matrix component of the microsphere carrier adopts polystyrene, polyacrylic acid, polymethyl acrylate, polymethacrylic acid, polymethyl methacrylate, polydivinylbenzene and/or copolymer formed by two or more monomers involved in the above polymers.

Furthermore, the diameter of the microsphere carrier is selected within the range of 0.1-100 μm.

Furthermore, the pore diameter selection range of the microsphere carrier is 2-100 nm.

Further, the difference of the central emission wavelengths of the fluorescent dyes corresponding to the fluorescent markers is more than 30 nm.

Further, the intermediary material is a polymer.

Further, the encoding information of the defined dimension is adjusted according to the content of the fluorescent marker.

Furthermore, the intermediate substance is also arranged in the microsphere carrier, and the content of each fluorescent marker is adjusted according to the ratio of the intermediate substance to each fluorescent marker.

Further, the encoded microspheres also include magnetic nanoparticles.

Furthermore, the outer surface of the coding microsphere is a silicon oxide coating layer and a functional molecule modification layer, and the functional molecule modification layer is positioned on the outermost layer.

The invention also provides a preparation method of the fluorescent dye-doped coding microsphere, which comprises the following steps:

selecting a microsphere carrier, wherein the microsphere carrier is a mesoporous microsphere;

step two, respectively connecting at least one fluorescent dye with an intermediate substance to form at least one fluorescent marker, wherein each fluorescent dye limits the coded information of one dimension of the coded microsphere;

and step three, arranging the fluorescent marker in the microsphere carrier.

Further, the microsphere carrier is one or more of porous silica microspheres, carboxylated porous polystyrene microspheres, epoxy group-modified porous silica microspheres, epoxy group-modified porous polystyrene microspheres, aminated porous silica microspheres and aminated porous polystyrene microspheres.

Further, the intermediary material is a polymer.

Furthermore, the functional group contained in the molecular structure of the fluorescent dye is an amino group, and the functional group contained in the molecular structure of the intermediate substance is one or more of a carboxyl group and an epoxy group.

Further, the functional group contained in the molecular structure of the fluorescent dye is one or more of isothiocyanate, carboxyl, N-hydroxysuccinimide ester and epoxy group, and the functional group contained in the molecular structure of the intermediate substance is amino.

Further, the fluorescent dye adopts one or more of Fluorescein Isothiocyanate (FITC), rhodamine isothiocyanate B (RITC), Cy 5-N-hydroxysuccinimide ester (Cy5-NHS) and 5-aminofluorescein (5-AF).

Further, the intermediate material is Polyethyleneimine (PEI) and/or polyacrylic acid (PAA).

Further, the third step in the preparation method specifically comprises:

disposing the fluorescently labeled polymer or the mixture of fluorescently labeled polymer and polymer in the interior space of the microsphere carrier by physical/chemical action.

Further, the coding information of each dimension is adjusted according to each fluorescent labeling polymer in the mixture and the proportion of the polymers.

Further, the preparation method further comprises the following steps:

and step four, arranging the magnetic nano particles on the outer surface of the microsphere carrier.

Further, the fourth step in the preparation method specifically comprises:

and coating the magnetic nano-particles on the outer surface of the microsphere carrier through physical/chemical action.

Further, the fourth step further includes:

Further, after the outer surface of the microsphere carrier is coated with silicon oxide, the outer surface of the microsphere carrier is modified with functional molecules, so that the microsphere carrier with the functionalized surface is obtained.

The invention also provides an encoding microsphere array, which comprises at least two encoding microspheres doped with the fluorescent dye, or at least two encoding microspheres prepared by the preparation method of the encoding microspheres doped with the fluorescent dye, wherein different encoding information exists among the encoding microspheres.

The coding microspheres and the array and the preparation method have the beneficial effects that:

1. aiming at the problem of insufficient coding capacity of the existing coding microsphere array, the invention firstly develops a new coding mode, namely, different internal structures of microsphere carriers are utilized for coding. In the field of construction of encoded microsphere arrays, the internal structure has not been utilized as an encoding element as an inherent property of microsphere carriers. The invention only changes the internal structure (comprising different matrix components and/or pore sizes) of microsphere carriers with similar diameters, simultaneously reads forward scattered light (FSC) and side scattered light (SSC) signals of the microsphere carriers on a flow cytometer, obtains different signal groups of different types of microsphere carriers in a two-dimensional scatter diagram of FSC-SSC, and realizes the structure coding. Once the internal structure of the microsphere carrier is determined, the FSC-SSC optical signal generated by the microsphere carrier is also determined, and the FSC-SSC optical signal is stable encoded information and hardly influenced by the external environment, and the structural encoding is a brand new encoding mode.

2. In the invention, different types of microspheres with similar diameters are used as carriers, the diameter deviation is less than or equal to 15 percent, only the internal structures of the different types of microspheres are used for coding, and the similar diameters ensure that the microsphere carriers corresponding to different detection indexes have similar reaction kinetics, so that the method is a 'fair' reaction environment for a multi-index detection process; in addition, the diameter of the microsphere carrier is limited to be 0.2-20 microns, and in the actual detection application process, the microsphere carrier with a smaller size has better suspension characteristics in a liquid phase and is not easy to settle, so that the microsphere carrier has faster reaction kinetics and is beneficial to improving the detection sensitivity.

3. Aiming at the problem of insufficient coding capacity of the existing coding microsphere array and other technical defects, the invention further provides a novel structure-fluorescence joint coding strategy on the basis of developing new coding element structure coding (namely, expanding coding dimension), and realizes the preparation of the coding microsphere array with ultrahigh coding capacity; the invention combines originally independent different coding elements by utilizing the internal structure difference of the microsphere carrier, the fluorescence intensity level of the internal space of the microsphere and the fluorescence intensity level of the external surface of the microsphere, thereby obviously improving the coding capacity of the microsphere carrier.

4. The microsphere carrier in the coding microsphere array provided by the invention can simultaneously have a plurality of functional characteristics such as structural coding, fluorescence coding of an internal space, fluorescence coding of an external surface, magnetic responsiveness and the like. The invention fully utilizes the porous structure characteristics of the carrier microsphere and three areas thereof, namely a carrier material framework, an internal pore passage and the outer surface of the carrier, and carries out reasonable regional functional design, and specifically comprises the following steps: (1) specific structure coding information is obtained by changing the internal constitution of the microsphere carrier, namely the matrix component and/or the pore size; (2) fluorescent dye molecules can be loaded by utilizing the porous structure (namely the internal space) of the microsphere carrier, the microsphere is modified by functional groups while the fluorescent coding function is realized, and polymer molecules are mainly combined on the wall of the internal hole through physical/chemical actions, so that the influence on the structural coding performance of the microsphere carrier is favorably reduced; (3) the magnetic nano-particles and the fluorescent nano-particles represented by quantum dots can be assembled by utilizing the outer surface of the microsphere carrier, so that the magnetic response performance and the fluorescent coding performance of the microsphere carrier are endowed, and the internal structure coding performance of the microsphere carrier is protected as far as possible. The functional structure design of the subareas realizes that each independent area of the microsphere carrier plays the respective function and reduces the mutual influence, thereby ensuring the controllability and the repeatability of the coding process.

5. The preparation method adopted by the invention is a layer-by-layer self-assembly method, firstly, the amino polymer marked with the fluorescent dye is assembled with the microsphere carrier through physical/chemical action, so that the fluorescent dye is loaded in the inner space, and meanwhile, the amination modification of the microsphere surface is realized; secondly, assembling the magnetic nanoparticles on the outer surface of the microsphere carrier through the physical/chemical action of the magnetic nanoparticles and the amino polymer; furthermore, fluorescent nanoparticles represented by quantum dots and the like are self-assembled on the outer surface of the microsphere carrier through coordination or electrostatic interaction of the fluorescent nanoparticles and amino polymers; and finally, coating a silicon oxide protective layer on the outermost layer and modifying functional molecules. The loading process of the fluorescent dye, the magnetic nanoparticles and the fluorescent nanoparticles adopts a layer-by-layer assembly method, and the self-assembly solvent environment is changed according to different molecular action modes, so that the preparation method is simple and convenient, and the repeatability is good.

6. In the application process of the coding microsphere array provided by the invention, appropriate fluorescent coding elements (such as green fluorescent dye FITC loaded in the internal space of the preferable microsphere carrier and CdSe/ZnS red quantum dots assembled on the outer surface of the microsphere carrier) can be selected, and the relation between the carrier microsphere structure coding and FSC-SSC scattered light signals is combined, so that the simultaneous excitation of monochromatic laser on all coding signals (namely, the structure signals, the green fluorescent signals and the red fluorescent signals can be excited by a 488nm laser source) can be realized. The decoding cost can be greatly reduced, and the decoding process becomes simpler and more convenient.

The coding microsphere and array doped with fluorescent dye and the preparation method thereof have the beneficial effects that:

1. the fluorescent dye doping method mediated by the intermediate substance has universality on different types of microsphere carriers (including inorganic microspheres and polymer microspheres). In commercial products, a swelling method is generally adopted to diffuse fluorescent molecules into polymer microspheres to prepare coding microspheres, but the method is not suitable for inorganic microsphere carriers and has no universality. The invention uses the intermediate substance marked by fluorescent molecule covalent bond as the doping raw material, and combines the doping raw material into the porous microsphere through physical/chemical action, thereby realizing the loading of the microsphere carrier on the fluorescent dye; the physical/chemical action can be selected according to the physicochemical properties of the microsphere and the polymer molecule, including electrostatic action, hydrophilic and hydrophobic action, hydrogen bond action, coordination action and covalent bond action, and has applicability to both inorganic microspheres and polymer microspheres.

2. According to the invention, the porous microspheres with high specific surface area and large pore volume are taken as carriers, the internal space of the microspheres is fully utilized to load fluorescent dye, so that higher fluorescent molecule loading capacity can be realized, and a wider fluorescent intensity adjusting range can be realized; and by combining the common doping of different fluorescent dyes, higher fluorescent coding capacity can be finally realized, and the requirement of actual multi-index detection is met.

3. The coding method provided by the invention is simple and the coding control is accurate. The covalent bond labeling of the intermediate substance is carried out only by using different fluorescent dyes, and then the intermediate substance is mixed with polymer molecules which are not fluorescently labeled according to different proportions, so that the fluorescence encoding intensity of the microsphere can be conveniently and accurately regulated; in addition, the polymer molecules marked by the fluorescent dye can be directly loaded in the microspheres through physical/chemical action with the porous microspheres without further purification, free fluorescent molecules can be removed in the subsequent washing process, and the preparation method and the encoding process are very simple, convenient and controllable.

4. On the basis of fluorescence encoding, the magnetic nanoparticles can be assembled on the outer surface of the microsphere through physical/chemical action, so that the encoded microsphere is endowed with superparamagnetism, and separation operation under the action of a subsequent magnetic field is facilitated.

5. The surface of the fluorescent coding microsphere prepared by the method is a silicon dioxide protective shell layer, so that the leakage of fluorescent dye in the microsphere can be prevented; meanwhile, the silicon hydroxyl on the surface of the shell layer is beneficial to further modifying various functional groups, and provides reaction sites for the coded microsphere coupling probe.

The conception, the specific structure and the technical effects of the present invention will be further described with reference to the accompanying drawings to fully understand the objects, the features and the effects of the present invention.

Drawings

FIG. 1 is a diagram of the stream decoding result corresponding to a 5-recoded microsphere array obtained by using five types of microspheres including SiO2-17, SiO2-48, PS-14, PS-37 and PS-51 and encoding the microspheres by using the structures thereof in one embodiment of the present invention;

FIG. 2 is a hysteresis loop of five types of encoded microspheres prepared by using five types of microspheres including SiO2-17, SiO2-48, PS-14, PS-37 and PS-51, performing combined encoding by using the internal structure + internal space fluorescence + external surface fluorescence, and endowing the microspheres with magnetic response performance through self-assembly of magnetic nanoparticles in one embodiment of the invention;

FIG. 3 is a flow decoding result diagram corresponding to the 300 recoded microsphere array prepared by adopting five types of microspheres including SiO2-17, SiO2-48, PS-14, PS-37 and PS-51, performing combined coding by using the internal structure + internal space fluorescence + external surface fluorescence, and endowing the microspheres with magnetic response performance through self-assembly of magnetic nanoparticles in one embodiment of the invention;

FIG. 4 is a flow decoding result diagram of 7-fold fluorescence-encoded microspheres prepared by using porous silica with a diameter of 1.7 μm as a microsphere carrier, using FITC as a doping dye and endowing the microspheres with magnetic response performance through self-assembly of magnetic nanoparticles in one embodiment of the present invention;

FIG. 5 is a diagram of the flow decoding result of 6-fold fluorescence-encoded microspheres prepared according to an embodiment of the present invention, in which porous silica having a diameter of 5.5 μm is used as a microsphere carrier, RITC is used as a doping dye, and magnetic response performance is imparted to the microspheres through self-assembly of magnetic nanoparticles;

FIG. 6 is a diagram showing the flow decoding result of 32-fold fluorescence-encoded microspheres prepared according to an embodiment of the present invention, in which porous silica with a diameter of 3.3 μm is used as a microsphere carrier, FITC and RITC are used as doping dyes, and magnetic response performance is imparted to the microspheres through self-assembly of magnetic nanoparticles;

FIG. 7 is a magnetic field separation photograph of a fluorescence-encoded microsphere prepared according to an embodiment of the present invention using porous silica having a diameter of 3.3 μm as a microsphere carrier, FITC and RITC as doping dyes, and magnetic response property imparted to the microsphere by self-assembly of magnetic nanoparticles;

FIG. 8 is a graph of the flow decoding results of 51-fold fluorescence-encoded microspheres prepared according to an embodiment of the present invention using porous silica with a diameter of 5.5 μm as a microsphere carrier, FITC and RITC as doping dyes, and magnetic response properties imparted to the microspheres by self-assembly of magnetic nanoparticles;

FIG. 9 is a two-dimensional array layout under a confocal laser fluorescence microscope of 51-fold fluorescence encoded microspheres prepared according to an embodiment of the present invention by using porous silica with a diameter of 5.5 μm as a microsphere carrier, using FITC and RITC as doping dyes, and by endowing the microspheres with magnetic response performance through self-assembly of magnetic nanoparticles.

Detailed Description

Example one

In the encoded microspheres of the embodiment, a group of microsphere carriers have similar diameters and different internal structures, and the change of the internal structures of the microsphere carriers is used as first-dimension encoded information; the microsphere carrier is a mesoporous microsphere with a porous structure, and different internal structures can be different in matrix components, different in pore size, or different in matrix components and pore size.

The diameter of the microsphere carrier is selected within the range of 0.2-20 μm, and the preferable range is 3-6 μm. The pore diameter of the microsphere carrier is selected within the range of 2-100 nm, and the preferable range is 10-60 nm. The matrix component of the microsphere carrier comprises inorganic substances and polymers. The inorganic substance includes silica and/or titania. The polymer comprises polystyrene, polyacrylic acid, polymethyl acrylate, polymethacrylic acid, polymethyl methacrylate, polydivinylbenzene and/or copolymers of the above polymers.

In order to effectively distinguish the structure coding information, different types of microsphere carriers should have significantly different signal groups in the two-dimensional scattergram of the flow FSC-SSC, and for better effect, the following five types of microsphere carriers with the diameter of 5 μm are preferred in the embodiment:

(1) mesoporous silica microspheres with a diameter of 5.2 μm and a pore diameter of 17nm, hereinafter denoted as SiO₂-17；

(2) Mesoporous silica microspheres with a diameter of 5.3 μm and a pore diameter of 48nm, hereinafter denoted as SiO₂-48；

(3) The mesoporous polystyrene microsphere with the diameter of 4.8 mu m and the aperture of 14nm is marked as PS-14 below;

(4) the mesoporous polystyrene microsphere with the diameter of 5.5 mu m and the aperture of 37nm is marked as PS-37 below;

(5) the mesoporous polystyrene microsphere with the diameter of 5.0 mu m and the pore diameter of 51nm is hereinafter referred to as PS-51.

Fluorescent materials can be loaded in the inner space and the outer surface of the microsphere carrier in this embodiment, the difference between the central emission wavelength of the fluorescent material in the inner space and the central emission wavelength of the fluorescent material in the outer surface is more than 30nm, in order to achieve a better effect, green fluorescent dye FITC with an emission wavelength of 517nm is preferably selected as a coding element of a second dimension for the fluorescent element contained in the inner space of the microsphere carrier in this embodiment, and CdSe/ZnS red quantum dots with an emission wavelength of 600nm is preferably selected as a coding element of a third dimension for the fluorescent element contained in the outer surface of the microsphere carrier. It should be noted that the present invention can be embodied in many different forms and is not limited to the preferred parameters and embodiments described herein. These preferred parameters and embodiments are provided so that this disclosure will be thorough and complete.

The following details a method for preparing the encoded microspheres in this example.

Step 1, selecting a microsphere carrier.

The first dimension encoding information of the encoded microspheres may be defined according to the matrix composition, diameter and pore size of the microsphere support. As in this example, five types of microsphere carriers were selected as described above with a diameter of 5 μm.

And 2, performing carboxyl functional modification on the selected mesoporous polystyrene microspheres, and skipping the step if the mesoporous silica microspheres are selected in the step 1.

Take 8X 10⁹Mesoporous polystyrene microsphere andadding into 5mL chloroform, ultrasonic dispersing for 30min, adding chloroform solution containing 500mg PSMA, and ultrasonic mixing for 40 min. 35mL of NaOH solution (0.1M) was added, emulsified with stirring, and sonicated for an additional 40 min. The microsphere sample was centrifuged to remove the supernatant and washed with ethanol and water sequentially three times. Finally, dispersing the obtained microspheres in 10mL of aqueous solution to obtain the carboxyl-modified mesoporous polystyrene microspheres.

And 3, loading fluorescent dye in the inner space of the microsphere carrier.

First, 150mg of PEI (molecular weight 750K) was dissolved in 15mL of NaCl solution (0.5M) and the pH was adjusted to 8.0 and recorded as a blank PEI solution. To the above blank PEI solution was added 4.4mg of FITC and reacted overnight at 30 ℃ with shaking in the dark to obtain a PEI solution labeled with FITC (designated as FITC-PEI). Next, the FITC-PEI solution and the blank PEI solution were mixed at different ratios to prepare a mixed solution having a total volume of 1.5mL, and 2X 10 was added⁷And (3) carrying out ultrasonic mixing on the mesoporous silica microspheres or the mesoporous polystyrene microspheres modified with carboxyl obtained in the step (2), and then carrying out dark rotation reaction for 20 min. And centrifuging to remove supernatant after reaction, washing with water for three times, and dispersing the obtained microspheres in 0.4mL of aqueous solution to obtain the microsphere carrier with the fluorescent dye loaded in the inner space.

And 4, assembling the magnetic nano-particles on the outer surface of the microsphere carrier.

Adding 0.4mL of microsphere dispersion obtained in step 3 dropwise into 1.1mL of microsphere dispersion containing Fe under ultrasonic condition₃O₄And (3) carrying out a light-shielding rotation reaction for 30min in an aqueous solution of magnetic nanoparticles (with the particle size of 8nm and carboxyl on the surface). After the reaction, the supernatant was magnetically separated and washed three times with water. Then adding the obtained microspheres into 1.5mL of blank PEI solution mentioned in step 2, carrying out a light-shielding rotary reaction for 20min, carrying out magnetic separation after the reaction to remove supernatant, and washing with water for three times to obtain the product with Fe assembled on the outer surface₃O₄The magnetic nano-particles are modified with PEI microsphere carriers on the outermost layer.

And 5, assembling quantum dots on the outer surface of the microsphere carrier.

Preparing a chloroform/n-butanol mixed solution of the quantum dots: taking a certain amount of quantum dots (with the diameter ofCdSe/ZnS quantum dots of 6nm, emission wavelength 600nm as an example) were added to a chloroform/n-butanol (v/v 1:20) mixed solution of 1mL in total volume, prepared at different quantum dot concentrations and mixed uniformly. Take 2X 10⁷And (4) centrifuging or magnetically separating the PEI-modified microspheres obtained in the step (3) or (4) to remove the supernatant, washing the microspheres with absolute ethyl alcohol for three times, adding the microspheres into the chloroform/n-butanol mixed solvent containing the quantum dots, carrying out a light-shielding rotary reaction for 1h, removing the supernatant after the reaction, and sequentially washing the microspheres with the chloroform/n-butanol mixed solvent, ethanol and water. And adding the microspheres into 1mL of a NaCl solution (0.1M, pH 8.0) containing 9mg/mL of PEI (molecular weight 25K), carrying out a rotation reaction for 1h in a dark place, removing a supernatant after the reaction, and washing with water for three times to obtain the microsphere carrier with the quantum dots assembled on the outer surface and the PEI modified on the outermost layer.

And 6, silicon oxide coating and surface functional modification of the microsphere carrier.

Take 2X 10⁷And (3) centrifuging or magnetically separating the PEI-modified microspheres obtained in the step (3), step (4) or step (5) to obtain supernatant, washing the microspheres twice by using absolute ethyl alcohol, adding the microspheres into a mixed system containing 3mL of ethyl alcohol, 0.3mL of water and 40 mu L of TEOS, and carrying out rotation reaction for 30min in a dark place. Then adding 22 mu L of concentrated ammonia water, continuing to carry out dark rotary reaction for 22h at the temperature of 30 ℃, removing supernatant after reaction, and sequentially washing with absolute ethyl alcohol and water for three times to obtain the microsphere carrier coated with silicon oxide on the surface. Dispersing the obtained microspheres in 0.25mL of ethanol, adding 11.5 mu L of concentrated ammonia water and 50 mu L of ethanol solution containing APTES (4.2 mu L of APTES is diluted in 1.5mL of ethanol), carrying out rotary reaction for 4h at 40 ℃, removing supernatant after reaction, and washing with absolute ethanol for three times to obtain the microsphere carrier with the modified amino on the surface. Washing the obtained microspheres with MEST (10mM, pH 5.0) solution for three times, dispersing in 1mL of MEST (10mM, pH 5.0), adding 2.5mg of PAA (molecular weight 5K) and 0.5mg of EDC, carrying out rotary reaction for 2h, removing supernatant after reaction, and washing with water for three times to obtain the microsphere carrier with the modified carboxyl on the surface.

According to the preparation method, the coding microsphere which adopts at most three coding modes can be obtained, wherein the coding modes comprise a coding mode based on the internal structure of the microsphere carrier, a coding mode based on the fluorescent material in the microsphere carrier and a coding mode based on the fluorescent material outside the microsphere carrier. Either or both of these encoding modes can be selected as desired, and magnetic nanoparticles can also be optionally added.

For the coding dimension related to the coding microspheres, besides the coding information of one dimension can be defined according to the internal structure of the microsphere carrier, the coding information of multiple dimensions can be defined inside and outside the microsphere carrier according to the selection of fluorescent materials, for example, two fluorescent dyes with different central emission wavelengths are adopted inside the microsphere carrier, namely the coding information of two dimensions can be defined; two different central emission wavelength quantum dots can be loaded by repeating the method of step 5 to define two dimensions of encoded information.

When microsphere supports of different diameters (e.g., -1 μm, -3 μm, -7 μm, etc.) are used, the preparation of the encoded microspheres is essentially the same as described above, with only minor adjustments to the amounts of reactants involved based on changes in the surface area of the microspheres.

Combinations of sets of encoded microsphere arrays are listed below to facilitate a further understanding of the multidimensional encoded information of the encoded microspheres, as well as the differences in encoded information between the various encoded microspheres in the encoded microsphere array.

Coded microsphere array 1 (SiO)₂-17、SiO₂Structural codes of five types of microspheres, namely 48, PS-14, PS-37 and PS-51):

1. by means of SiO₂17 number of codes I of internal space of original microsphere directly as microsphere support, divided according to level of fluorescence intensity₁Number of codes O on the outer surface of microspheres divided by the level of fluorescence intensity ═ 1₁1, the coding capacity X of such microspheres₁＝I₁×O₁＝1。

2. By means of SiO₂48 encoded number I of microspheres internal space divided according to fluorescence intensity level, with original microspheres directly as microsphere support₂Number of codes O on the outer surface of microspheres divided by the level of fluorescence intensity ═ 1₂1, the coding capacity X of such microspheres₂＝I₂×O₂＝1。

3. Adopting the carboxylated PS-14 microspheres obtained in the step 2 as microsphere carriers, and dividing the coding number I of the internal space of the microspheres according to the fluorescence intensity level₃Number of codes O on the outer surface of microspheres divided by the level of fluorescence intensity ═ 1₃1, the coding capacity X of such microspheres₃＝I₃×O₃＝1。

4. Adopting the carboxylated PS-37 microspheres obtained in the step 2 as microsphere carriers, and dividing the coding number I of the internal space of the microspheres according to the fluorescence intensity level₄Number of codes O on the outer surface of microspheres divided by the level of fluorescence intensity ═ 1₄1, the coding capacity X of such microspheres₄＝I₄×O₄＝1。

5. Adopting the carboxylated PS-51 microspheres obtained in the step 2 as microsphere carriers, and dividing the coding number I of the internal space of the microspheres according to the fluorescence intensity level₅Number of codes O on the outer surface of microspheres divided by the level of fluorescence intensity ═ 1₅1, the coding capacity X of such microspheres₅＝I₅×O₅＝1。

6. Combining the five types of microspheres, namely introducing structural coding information, decoding the structural information through two scattered light channels of FSC and SSC of a flow cytometer, and finally constructing a coding microsphere array with 5 times, namely Y ═ (I ═₁×O₁)+(I₂×O₂)+(I₃×O₃)+(I₄×O₄)+(I₅×O₅)＝5。

As shown in fig. 1, five types of microsphere carriers with diameters of about-5 μm are used, and a flow cytometer is used as a decoding platform to read characteristic scattered optical signals (FSC and SSC) of the microspheres by changing the internal structures (including different matrix components and/or pore sizes) of the microspheres, so as to obtain five groups with significant signal intensity differences in a FSC-SSC two-dimensional decoding diagram, thereby revealing that the internal structures of the microsphere carriers can be effectively converted into coded information. Although the five types of microspheres have similar diameters, each should have a close FSC value, as is common in the art; however, after analysis, the difference of the internal structure of the microsphere carrier influences the SSC signal value, and the FSC signal value of partial carrier also shows a significant difference, which is not found in the prior art. That is to say, the internal structure of the microsphere carrier can be effectively converted into optical coding information as the inherent attribute, and the novel coding dimension element can be reasonably combined with other coding elements, so that the coding capacity is greatly improved.

Coded microsphere array 2 (SiO)₂-17、SiO₂Structural codes of five types of magnetic microspheres, namely-48, PS-14, PS-37 and PS-51):

1. by means of SiO₂-17 using the original microsphere as a carrier matrix, assembling magnetic nanoparticles on the outer surface of the microsphere carrier according to the

steps

3, 4 and 6 in the preparation method, coating and modifying the surface, and preparing by using blank PEI in the step 3, wherein the coding number I of the microsphere internal space is divided according to the fluorescence intensity level₁Number of codes O on the outer surface of microspheres divided by the level of fluorescence intensity ═ 1₁1, the coding capacity X of the magnetic microsphere₁＝I₁×O₁＝1。

2. By means of SiO₂-48 original microspheres as carrier matrix, assembling magnetic nanoparticles on the outer surface of the microsphere carrier according to the

steps

3, 4 and 6 of the preparation method, coating and modifying the surface, and preparing by using blank PEI in the step 3, wherein the coding number I of the microsphere inner space divided according to the fluorescence intensity level₂Number of codes O on the outer surface of microspheres divided by the level of fluorescence intensity ═ 1₂1, the coding capacity X of the magnetic microsphere₂＝I₂×O₂＝1。

3. Adopting the carboxylated PS-14 microspheres obtained in the step 2 in the preparation method as a carrier matrix, assembling magnetic nano particles on the outer surface of the microsphere carrier according to the step 3, the step 4 and the step 6, coating and modifying the surface, adopting blank PEI (polyetherimide) in the step 3 for preparation, and dividing the coding quantity I of the inner space of the microspheres according to the fluorescence intensity level₃Number of codes O on the outer surface of microspheres divided by the level of fluorescence intensity ═ 1₃1, the coding capacity X of the magnetic microsphere₃＝I₃×O₃＝1。

4. Adopting the carboxylated PS-37 microspheres obtained in the step 2 in the preparation method as a carrier matrix, assembling magnetic nano particles on the outer surface of the microsphere carrier according to the step 3, the step 4 and the step 6, coating and modifying the surface, adopting blank PEI (polyetherimide) in the step 3 for preparation, and dividing the coding quantity I of the inner space of the microspheres according to the fluorescence intensity level₄Number of codes O on the outer surface of microspheres divided by the level of fluorescence intensity ═ 1₄1, the coding capacity X of the magnetic microsphere₄＝I₄×O₄＝1。

5. Adopting the carboxylated PS-51 microspheres obtained in the step 2 in the preparation method as a carrier matrix, assembling magnetic nano particles on the outer surface of the microsphere carrier according to the step 3, the step 4 and the step 6, coating and modifying the surface, adopting blank PEI (polyetherimide) in the step 3 for preparation, and dividing the coding quantity I of the inner space of the microspheres according to the fluorescence intensity level₅Number of codes O on the outer surface of microspheres divided by the level of fluorescence intensity ═ 1₅1, the coding capacity X of the magnetic microsphere₅＝I₅×O₅＝1。

6. Combining the five types of magnetic microspheres, namely introducing structure coding information, decoding the structure information through two scattered light channels of FSC and SSC of a flow cytometer, and finally constructing a magnetic coding microsphere array with 5 weight, namely Y ═ (I ═₁×O₁)+(I₂×O₂)+(I₃×O₃)+(I₄×O₄)+(I₅×O₅)＝5。

Coded microsphere array 3 (SiO)₂-17、SiO₂-structure of four classes of microspheres-48, PS-14 and PS-51 + joint coding of internal spatial fluorescence):

1. by means of SiO₂-17 original microspheres are used as carrier matrixes, fluorescent dye FITC is loaded in the internal space of the microsphere carriers according to the steps 3 and 6 in the preparation method, surface coating and modification are carried out, and the mixing of FITC-PEI solution and blank PEI solution is adjusted in the step 3Mixing proportion to obtain 11 recoded microspheres coded by the microsphere internal space divided according to the FITC fluorescence intensity level, namely I₁Number of codes O on the outer surface of microspheres divided by the level of fluorescence intensity ═ 11₁1, the coding capacity X of such microspheres₁＝I₁×O₁＝11。

2. By means of SiO₂-48 original microspheres are used as a carrier matrix, a fluorochrome FITC is loaded in the internal space of the microsphere carrier according to the steps 3 and 6 in the preparation method, surface coating and modification are carried out, 10 recoded microspheres coded by the internal space of the microspheres divided according to the FITC fluorescence intensity level, namely I recoded microspheres, are obtained by adjusting the mixing ratio of the FITC-PEI solution and the blank PEI solution in the step 3₂Number of codes O on the outer surface of microspheres divided by the level of fluorescence intensity ═ 10₂1, the coding capacity X of such microspheres₂＝I₂×O₂＝10。

3. Adopting the carboxylated PS-14 microspheres obtained in the step 2 in the preparation method as a carrier matrix, loading a fluorochrome FITC in the internal space of the microsphere carrier according to the step 3 and the step 6, carrying out surface coating and modification, and obtaining 10 recoded microspheres coded by the internal space of the microspheres divided according to the FITC fluorescence intensity level, namely I, by adjusting the mixing ratio of the FITC-PEI solution and the blank PEI solution in the step 3₃Number of codes O on the outer surface of microspheres divided by the level of fluorescence intensity ═ 10₃1, the coding capacity X of such microspheres₃＝I₃×O₃＝10。

4. Adopting the carboxylated PS-51 microspheres obtained in the step 2 in the preparation method as a carrier matrix, loading a fluorescent dye FITC in the internal space of the microsphere carrier according to the step 3 and the step 6, and carrying out surface coating and modification, wherein the 9-recoded microspheres coded in the internal space of the microspheres divided according to the FITC fluorescence intensity level, namely I, are obtained by adjusting the mixing ratio of the FITC-PEI solution and the blank PEI solution in the step 3₄Number of codes O on the outer surface of microspheres divided by the level of fluorescence intensity ═ 9₄1, the coding capacity X of such microspheres₄＝I₄×O₄＝9。

5. Combining the four types of microspheres, namely introducing structure coding information, decoding the structure information through two scattering light channels of FSC and SSC of a flow cytometer, decoding the internal space fluorescence information through a fluorescence detection channel, and finally constructing a 40-fold coding microsphere array, namely Y ═ (I ═ I)₁×O₁)+(I₂×O₂)+(I₃×O₃)+(I₄×O₄)＝40。

Coded microsphere array 4 (SiO)₂-17、SiO₂-structure of three types of magnetic microspheres, 48 and PS-51 + joint encoding of internal spatial fluorescence):

1. by means of SiO₂-17 original microspheres are used as a carrier matrix, a fluorescent dye FITC is loaded in the internal space of the microsphere carrier according to the

steps

3, 4 and 6 in the preparation method, magnetic nanoparticles are assembled on the outer surface of the microsphere carrier, surface coating and modification are carried out, 11 recoded microspheres coded by the internal space of the microspheres divided according to the FITC fluorescence intensity level, namely I recoded microspheres coded by the internal space of the microspheres divided according to the FITC fluorescence intensity level are obtained by adjusting the mixing ratio of the FITC-PEI solution and the blank PEI solution in the step 3₁Number of codes O on the outer surface of microspheres divided by the level of fluorescence intensity ═ 11₁1, the coding capacity X of the magnetic microsphere₁＝I₁×O₁＝11。

2. By means of SiO₂-48 original microspheres are used as a carrier matrix, a fluorescent dye FITC is loaded in the internal space of the microsphere carrier according to the

steps

3, 4 and 6 in the preparation method, magnetic nanoparticles are assembled on the outer surface of the microsphere carrier, surface coating and modification are carried out, 10 recoded microspheres coded by the internal space of the microspheres divided according to the FITC fluorescence intensity level, namely I, are obtained by adjusting the mixing ratio of the FITC-PEI solution and the blank PEI solution in the step 3₂Number of codes O on the outer surface of microspheres divided by the level of fluorescence intensity ═ 10₂1, the coding capacity X of the magnetic microsphere₂＝I₂×O₂＝10。

3. Adopting the carboxylated PS-51 microsphere obtained in the step 2 in the preparation method as a carrier matrix, and loading the microspheres in the internal space of the microsphere carrier according to the

steps

3, 4 and 6Performing surface coating and modification on the fluorescent dye FITC, and in step 3, adjusting the mixing ratio of the FITC-PEI solution and the blank PEI solution to obtain 9 recoded microspheres coded by the microsphere internal space divided according to the FITC fluorescence intensity level, namely I₃Number of codes O on the outer surface of microspheres divided by the level of fluorescence intensity ═ 9₃1, the coding capacity X of the magnetic microsphere₃＝I₃×O₃＝9。

4. Combining the three types of magnetic microspheres, namely introducing structure coding information, decoding the structure information through two scattering light channels of FSC and SSC of a flow cytometer, decoding the internal space fluorescence information through a fluorescence detection channel, and finally constructing a 30-fold magnetic coding microsphere array, namely Y ═ (I ═₁×O₁)+(I₂×O₂)+(I₃×O₃)＝30。

Coded microsphere array 5 (SiO)₂-structure of microspheres of three types, 17, PS-14 and PS-51 + joint coding of external surface fluorescence):

1. by means of SiO₂-17, using the original microsphere as a carrier matrix, assembling quantum dots on the outer surface of the microsphere carrier according to the

steps

3, 5 and 6 in the preparation method, coating and modifying the surface, and preparing by using blank PEI in the step 3, wherein the coding quantity I of the inner space of the microsphere is divided according to the fluorescence intensity level₁In step 5, 6 recoded microspheres coded on the outer surfaces of the microspheres divided according to the fluorescence intensity level of the quantum dots, namely O, are obtained by adjusting the adding concentration of the quantum dots₁6, the coding capacity X of this type of microspheres₁＝I₁×O₁＝6。

2. Adopting the carboxylated PS-14 microspheres obtained in the step 2 in the preparation method as a carrier matrix, assembling quantum dots on the outer surface of the microsphere carrier according to the step 3, the step 5 and the step 6, coating and modifying the surface, adopting blank PEI (polyetherimide) in the step 3 for preparation, and dividing the coding number I of the inner space of the microspheres according to the fluorescence intensity level₂In step 5, the external surface code of the microsphere divided according to the fluorescence intensity level of the quantum dots is obtained by adjusting the adding concentration of the quantum dots as 16 recoded microspheres, i.e. O₂6, the coding capacity X of this type of microspheres₂＝I₂×O₂＝6。

3. Adopting the carboxylated PS-51 microspheres obtained in the step 2 in the preparation method as a carrier matrix, assembling quantum dots on the outer surface of the microsphere carrier according to the

steps

3, 5 and 6, coating and modifying the surface, adopting blank PEI (polyetherimide) in the step 3 for preparation, and dividing the coding number I of the microsphere inner space according to the fluorescence intensity level₃In step 5, 6 recoded microspheres coded on the outer surfaces of the microspheres divided according to the fluorescence intensity level of the quantum dots, namely O, are obtained by adjusting the adding concentration of the quantum dots₃6, the coding capacity X of this type of microspheres₃＝I₃×O₃＝6。

4. Combining the three types of microspheres, namely introducing structure coding information, decoding the structure information through two scattering light channels of FSC and SSC of a flow cytometer, decoding the fluorescence information on the outer surface through a fluorescence detection channel, and finally constructing an 18-fold coding microsphere array, namely Y ═ (I ═₁×O₁)+(I₂×O₂)+(I₃×O₃)＝18。

Coded microsphere array 6 (SiO)₂-17、SiO₂-structure of four types of magnetic microspheres of 48, PS-14 and PS-51 + joint encoding of external surface fluorescence):

1. by means of SiO₂-17 using the original microsphere as a carrier matrix, assembling magnetic nanoparticles and quantum dots on the outer surface of the microsphere carrier according to the

steps

3, 4, 5 and 6 in the preparation method, coating and modifying the surface, and preparing by using blank PEI in the step 3, wherein the coding number I of the microsphere internal space is divided according to the fluorescence intensity level₁In step 5, 6 recoded microspheres coded on the outer surfaces of the microspheres divided according to the fluorescence intensity level of the quantum dots, namely O, are obtained by adjusting the adding concentration of the quantum dots₁6, the coding capacity X of the magnetic microsphere₁＝I₁×O₁＝6。

2. By means of SiO₂-48 original microspheres as support matrix, according to the aboveIn the preparation method, the magnetic nano particles and the quantum dots are assembled on the outer surface of the microsphere carrier in the

steps

3, 4, 5 and 6, the surface is coated and modified, blank PEI is adopted in the step 3 for preparation, and the coding quantity I of the microsphere internal space divided according to the fluorescence intensity level₂In step 5, 6 recoded microspheres coded on the outer surfaces of the microspheres divided according to the fluorescence intensity level of the quantum dots, namely O, are obtained by adjusting the adding concentration of the quantum dots₂6, the coding capacity X of the magnetic microsphere₂＝I₂×O₂＝6。

3. Adopting the carboxylated PS-14 microspheres obtained in the step 2 in the preparation method as a carrier matrix, assembling magnetic nano particles and quantum dots on the outer surface of the microsphere carrier according to the step 3, the step 4, the step 5 and the step 6, coating and modifying the surface, adopting blank PEI (polyetherimide) in the step 3 for preparation, and dividing the coding quantity I of the inner space of the microspheres according to the fluorescence intensity level₃In step 5, 6 recoded microspheres coded on the outer surfaces of the microspheres divided according to the fluorescence intensity level of the quantum dots, namely O, are obtained by adjusting the adding concentration of the quantum dots₃6, the coding capacity X of the magnetic microsphere₃＝I₃×O₃＝6。

4. Adopting the carboxylated PS-51 microspheres obtained in the step 2 in the preparation method as a carrier matrix, assembling magnetic nano particles and quantum dots on the outer surface of the microsphere carrier according to the

steps

3, 4, 5 and 6, coating and modifying the surface, adopting blank PEI (polyetherimide) in the step 3 for preparation, and dividing the coding quantity I of the inner space of the microspheres according to the fluorescence intensity level₄In step 5, 6 recoded microspheres coded on the outer surfaces of the microspheres divided according to the fluorescence intensity level of the quantum dots, namely O, are obtained by adjusting the adding concentration of the quantum dots₄6, the coding capacity X of the magnetic microsphere₄＝I₄×O₄＝6。

5. Combining the four types of magnetic microspheres, namely introducing structure coding information, decoding the structure information through two scattering light channels of FSC and SSC of a flow cytometer, and then performing fluorescence communication on the outer surface through a fluorescence detection channelDecoding the information to finally construct a 24-fold magnetically encoded microsphere array, namely Y ═ I₁×O₁)+(I₂×O₂)+(I₃×O₃)+(I₄×O₄)＝24。

Coded microsphere array 7 (SiO)₂-17、SiO₂-structure of four types of magnetic microspheres of 48, PS-14 and PS-37 + joint encoding of external surface fluorescence):

in the combination, CdSe/ZnS quantum dots with the emission wavelength of 600nm, which are preferably assembled on the outer surface of the microsphere carrier, are adjusted to NaYF with the emission wavelength of 535nm₄Er and Yb upconversion fluorescent nanoparticles. The step 5 in the preparation method is adjusted as follows: and assembling the upconversion fluorescent nanoparticles on the outer surface of the microsphere carrier through coordination reaction. Preparing a chloroform/n-butanol mixed solution of the upconversion fluorescent nanoparticles: taking a certain amount of upconversion fluorescent nanoparticles (NaYF with diameter of 25nm and emission wavelength of 535 nm)₄Er, Yb upconversion fluorescent nanoparticles) were added to a chloroform/n-butanol (v/v ═ 1:20) mixed solution with a total volume of 1mL, and different upconversion fluorescent nanoparticle concentrations were prepared and mixed uniformly. Take 2X 10⁷And (4) magnetically separating the PEI-modified microspheres obtained in the step (4) to obtain supernatant, washing the microspheres for three times by using absolute ethyl alcohol, adding the microspheres into the chloroform/n-butanol mixed solvent containing the up-conversion fluorescent nanoparticles, carrying out a rotation reaction for 1h in a dark place, removing the supernatant after the reaction, and sequentially washing the microspheres by using the chloroform/n-butanol mixed solvent, ethanol and water. And adding the microspheres into 1mL of a NaCl solution (0.1M, pH 8.0) containing 9mg/mL of PEI (molecular weight 25K), carrying out a rotation reaction for 1h in a dark place, removing a supernatant after the reaction, and washing with water for three times to obtain the microsphere carrier with the outer surface being provided with the upconversion fluorescent nanoparticles and the outermost layer being modified with PEI.

1. By means of SiO₂17 original microspheres as carrier matrix, assembling magnetic nanoparticles and up-conversion fluorescent nanoparticles on the outer surface of the microsphere carrier according to the

steps

3, 4, 5 (step 5 adjusted according to the coded microsphere array 7) and 6 in the preparation method, coating and modifying the surface, and preparing by using blank PEI in the step 3 according to the fluorescence intensity levelCoded number of partitioned microsphere internal spaces I₁1, in the step 5 (step 5 adjusted according to the coding microsphere array 7), 5 recoded microspheres coded by the microsphere outer surface and divided according to the fluorescence intensity level of the upconversion fluorescent nanoparticles, namely O, are obtained by adjusting the adding concentration of the upconversion fluorescent nanoparticles ₁5, the coding capacity X of the magnetic microsphere₁＝I₁×O₁＝5。

2. By means of SiO₂-48 original microspheres as carrier matrix, assembling magnetic nanoparticles and up-conversion fluorescent nanoparticles on the outer surface of the microsphere carrier according to the

steps

3, 4, 5 (step 5 adjusted according to the coded microsphere array 7) and 6 in the preparation method, and performing surface coating and modification, wherein blank PEI is adopted in the step 3 for preparation, and the coding number I of the microsphere internal space divided according to the fluorescence intensity level ₂1, in the step 5 (step 5 adjusted according to the coding microsphere array 7), 5 recoded microspheres coded by the microsphere outer surface and divided according to the fluorescence intensity level of the upconversion fluorescent nanoparticles, namely O, are obtained by adjusting the adding concentration of the upconversion fluorescent nanoparticles ₂5, the coding capacity X of the magnetic microsphere₂＝I₂×O₂＝5。

3. Adopting the carboxylated PS-14 microspheres obtained in the step 2 in the preparation method as a carrier matrix, assembling magnetic nanoparticles and up-conversion fluorescent nanoparticles on the outer surface of the microsphere carrier according to the

steps

3, 4, 5 (the step 5 adjusted according to the coded microsphere array 7) and 6, coating and modifying the surface, adopting blank PEI (polyetherimide) in the step 3, and dividing the coding number I of the inner space of the microspheres according to the fluorescent intensity level ₃1, in the step 5 (step 5 adjusted according to the coding microsphere array 7), 5 recoded microspheres coded by the microsphere outer surface and divided according to the fluorescence intensity level of the upconversion fluorescent nanoparticles, namely O, are obtained by adjusting the adding concentration of the upconversion fluorescent nanoparticles ₃5, the coding capacity X of the magnetic microsphere₃＝I₃×O₃＝5。

4. The carboxyl obtained by the step 2 in the preparation method is adoptedPS-37 microspheres are used as a carrier matrix, magnetic nanoparticles and up-conversion fluorescent nanoparticles are assembled on the outer surface of the microsphere carrier according to the

steps

3, 4, 5 (the step 5 is adjusted according to the coded microsphere array 7) and 6, the surface is coated and modified, blank PEI is adopted in the step 3 for preparation, and then the coding quantity I of the microsphere internal space divided according to the fluorescence intensity level ₄1, in the step 5 (step 5 adjusted according to the coding microsphere array 7), 5 recoded microspheres coded by the microsphere outer surface and divided according to the fluorescence intensity level of the upconversion fluorescent nanoparticles, namely O, are obtained by adjusting the adding concentration of the upconversion fluorescent nanoparticles ₄5, the coding capacity X of the magnetic microsphere₄＝I₄×O₄＝5。

5. Combining the four types of magnetic microspheres, namely introducing structure coding information, decoding the structure information through two scattering light channels of FSC and SSC of a flow cytometer, decoding the fluorescence information on the outer surface through a fluorescence detection channel, and finally constructing a magnetic coding microsphere array with 20 weight, namely Y ═ (I ═ I ═₁×O₁)+(I₂×O₂)+(I₃×O₃)+(I₄×O₄)＝20。

Coded microsphere array 8 (SiO)₂-17、SiO₂-structure of microspheres of three types 48 and PS-51 + joint coding of internal space fluorescence + external surface fluorescence):

steps

3, 5 and 6 in the preparation method, quantum dots are assembled on the outer surface of the microsphere carrier, surface coating and modification are carried out, 11 recoded microspheres coded by the internal space of the microspheres divided according to the FITC fluorescence intensity level, namely I recoded microspheres coded by the internal space of the microspheres divided according to the FITC fluorescence intensity level are obtained by adjusting the mixing ratio of the FITC-PEI solution and the blank PEI solution in the step 3₁In step 5, 6 recoded microspheres coded on the outer surfaces of the microspheres divided according to the fluorescence intensity level of the quantum dots, namely O, are obtained by adjusting the adding concentration of the quantum dots₁6, the coding capacity X of this type of microspheres₁＝I₁×O₁＝66。

steps

3, 5 and 6 in the preparation method, quantum dots are assembled on the outer surface of the microsphere carrier, surface coating and modification are carried out, 10 recoded microspheres coded by the internal space of the microspheres divided according to the FITC fluorescence intensity level, namely I, are obtained by adjusting the mixing ratio of the FITC-PEI solution and the blank PEI solution in the step 3₂In step 5, 6 recoded microspheres coded on the outer surfaces of the microspheres divided according to the fluorescence intensity level of the quantum dots, namely O, are obtained by adjusting the adding concentration of the quantum dots₂6, the coding capacity X of this type of microspheres₂＝I₂×O₂＝60。

3. Adopting the carboxylated PS-51 microspheres obtained in the step 2 in the preparation method as a carrier matrix, loading fluorescent dye FITC in the internal space of the microsphere carrier according to the step 3, the step 5 and the step 6, assembling quantum dots on the external surface, coating and modifying the surface, and obtaining 9 recoded microspheres coded by the internal space of the microspheres divided according to the FITC fluorescence intensity level, namely I recoded microspheres coded according to the FITC-PEI solution and the blank PEI solution by adjusting the mixing ratio of the FITC-PEI solution and the blank PEI solution in the step 3₃In step 5, 6 recoded microspheres coded by the outer surface of the microspheres divided according to the fluorescence intensity level of the quantum dots, namely O, are obtained by adjusting the adding concentration of the quantum dots₃6, the coding capacity X of this type of microspheres₃＝I₃×O₃＝54。

4. Combining the three types of microspheres, namely introducing structure coding information, decoding the structure information through two scattering light channels of FSC and SSC of a flow cytometer, decoding the fluorescence information of an inner space and an outer surface through a fluorescence detection channel respectively, and finally constructing a 180-weight coding microsphere array, namely Y ═ (I ═₁×O₁)+(I₂×O₂)+(I₃×O₃)＝180。

Coded microsphere array 9 (SiO)₂-17、SiO₂-structure of microspheres of three types 48 and PS-37 + joint coding of internal space fluorescence + external surface fluorescence):

in the combination, CdSe/ZnS quantum dots with the emission wavelength of 600nm, which are preferably assembled on the outer surface of the microsphere carrier, are adjusted to be conjugated polymer fluorescent nanoparticles with the emission wavelength of 580 nm. The step 5 is adjusted as follows: assembling the conjugated polymer fluorescent nano-particles on the outer surface of the microsphere carrier through electrostatic reaction. And (3) dropwise adding 0.4mL of microsphere dispersion obtained in the step (3) into 1.1mL of aqueous solution containing the conjugated polymer fluorescent nanoparticles (with the particle size of 30nm and carboxyl on the surface) with different concentrations under an ultrasonic condition, and carrying out a rotation reaction for 30min in a dark place. After the reaction, the supernatant was centrifuged and washed with water three times. And then adding the obtained microspheres into 1.5mL of blank PEI solution mentioned in the step 3, carrying out rotation reaction for 20min in a dark place, centrifuging after reaction, removing supernatant, and washing with water for three times to obtain the microsphere carrier with the outer surface assembled with the conjugated polymer fluorescent nanoparticles and the outermost layer modified with PEI.

1. By means of SiO₂-17 original microspheres are used as a carrier matrix, fluorescent dye FITC is loaded in the inner space of the microsphere carrier according to the steps 3, 5 (the step 5 is adjusted according to the coded microsphere array 9) and 6 in the preparation method, conjugated polymer fluorescent nanoparticles are assembled on the outer surface of the microsphere carrier, surface coating and modification are carried out, 11 recoded microspheres coded by the microsphere inner space divided according to the FITC fluorescence intensity level, namely I recoded microspheres are obtained by adjusting the mixing ratio of the FITC-PEI solution and the blank PEI solution in the step 3₁In step 5 (step 5 adjusted according to the coded microsphere array 9), 5 recoded microspheres coded by the outer surface of the microspheres divided according to the fluorescence intensity level of the conjugated polymer fluorescent nanoparticles, namely O, are obtained by adjusting the adding concentration of the conjugated polymer fluorescent nanoparticles in step 11₁5, the coding capacity X of this type of microspheres₁＝I₁×O₁＝55。

2. By means of SiO₂-48 original microspheres are used as a carrier matrix, fluorescent dye FITC is loaded in the inner space of the microsphere carrier according to the steps 3, 5 (the step 5 is adjusted according to the coded microsphere array 9) and 6 in the preparation method, conjugated polymer fluorescent nanoparticles are assembled on the outer surface of the microsphere carrier, surface coating and modification are carried out, and the mixing ratio of the FITC-PEI solution and the blank PEI solution is adjusted in the step 3, so that the microsphere inner space divided according to the FITC fluorescence intensity level is obtainedCoded 10 recoded microspheres, i.e. I₂10, in the step 5 (step 5 adjusted according to the coding microsphere array 9), 5 recoded microspheres coded by the outer surface of the microspheres divided according to the fluorescence intensity level of the conjugated polymer fluorescent nanoparticles, namely O, are obtained by adjusting the adding concentration of the conjugated polymer fluorescent nanoparticles ₂5, the coding capacity X of this type of microspheres₂＝I₂×O₂＝50。

3. Adopting the carboxylated PS-37 microspheres obtained in the step 2 in the preparation method as a carrier matrix, loading a fluorescent dye FITC in the internal space of the microsphere carrier according to the step 3, the step 5 (the step 5 is adjusted according to the coded microsphere array 9) and the step 6, assembling conjugated polymer fluorescent nanoparticles on the outer surface, coating and modifying the surface, and obtaining 10 recoded microspheres coded by the internal space of the microspheres divided according to the FITC fluorescence intensity level, namely I recoded microspheres, by adjusting the mixing ratio of the FITC-PEI solution and the blank PEI solution in the step 3₃10, in the step 5 (step 5 adjusted according to the coding microsphere array 9), 5 recoded microspheres coded by the outer surface of the microspheres divided according to the fluorescence intensity level of the conjugated polymer fluorescent nanoparticles, namely O, are obtained by adjusting the adding concentration of the conjugated polymer fluorescent nanoparticles ₃5, the coding capacity X of this type of microspheres₃＝I₃×O₃＝50。

4. Combining the three types of microspheres, namely introducing structure coding information, decoding the structure information through two scattering light channels of FSC and SSC of a flow cytometer, decoding the fluorescence information of the inner space and the outer surface through a fluorescence detection channel respectively, and finally constructing an 155-weight coding microsphere array, namely Y ═ (I ═ I-₁×O₁)+(I₂×O₂)+(I₃×O₃)＝155。

Coded microsphere array 10 (SiO)₂-17、SiO₂-combined encoding of structure + internal space fluorescence + external surface fluorescence of five classes of magnetic microspheres of 48, PS-14, PS-37 and PS-51):

1. by means of SiO₂-17 original microspheres as support matrix according to step 3, step 4, step 5 and step 6 of the above preparation methodLoading a fluorescent dye FITC in the internal space of the microsphere carrier, assembling magnetic nanoparticles and quantum dots on the outer surface of the microsphere carrier, coating and modifying the surface of the microsphere carrier, and adjusting the mixing ratio of a FITC-PEI solution and a blank PEI solution in step 3 to obtain 11 recoded microspheres which are divided according to the FITC fluorescence intensity level and are coded in the internal space of the microsphere, namely I recoded microspheres₁In step 5, 6 recoded microspheres coded on the outer surfaces of the microspheres divided according to the fluorescence intensity level of the quantum dots, namely O, are obtained by adjusting the adding concentration of the quantum dots₁6, the coding capacity X of the magnetic microsphere₁＝I₁×O₁＝66。

steps

3, 4, 5 and 6 in the preparation method, magnetic nano particles and quantum dots are assembled on the outer surface of the microsphere carrier, the surface of the microsphere carrier is coated and modified, and 10 recoded microspheres coded by the internal space of the microspheres divided according to the FITC fluorescence intensity level, namely I, 10 recoded microspheres coded by the internal space of the microspheres divided according to the FITC fluorescence intensity level are obtained by adjusting the mixing ratio of the FITC-PEI solution and the blank PEI solution in the step 3₂In step 5, 6 recoded microspheres coded on the outer surfaces of the microspheres divided according to the fluorescence intensity level of the quantum dots, namely O, are obtained by adjusting the adding concentration of the quantum dots₂6, the coding capacity X of the magnetic microsphere₂＝I₂×O₂＝60。

3. Adopting the carboxylated PS-14 microspheres obtained in the step 2 in the preparation method as a carrier matrix, loading a fluorescent dye FITC in the internal space of the microsphere carrier, assembling magnetic nano-particles and quantum dots on the outer surface of the microsphere carrier according to the

steps

3, 4, 5 and 6, and carrying out surface coating and modification, wherein 10 recoded microspheres coded by the internal space of the microspheres divided according to the FITC fluorescence intensity level, namely I recoded microspheres coded according to the FITC fluorescence intensity level are obtained by adjusting the mixing ratio of the FITC-PEI solution and the blank PEI solution in the step 3₃In step 5, 6 recoded microspheres coded on the outer surfaces of the microspheres divided according to the fluorescence intensity level of the quantum dots, namely O, are obtained by adjusting the adding concentration of the quantum dots₃6, the coding capacity X of the magnetic microsphere₃＝I₃×O₃＝60。

4. Adopting the carboxylated PS-37 microspheres obtained in the step 2 in the preparation method as a carrier matrix, loading a fluorescent dye FITC in the internal space of the microsphere carrier, assembling magnetic nano-particles and quantum dots on the outer surface of the microsphere carrier according to the

steps

3, 4, 5 and 6, and carrying out surface coating and modification, wherein 10 recoded microspheres coded by the internal space of the microspheres divided according to the FITC fluorescence intensity level, namely I recoded microspheres coded according to the FITC fluorescence intensity level are obtained by adjusting the mixing ratio of the FITC-PEI solution and the blank PEI solution in the step 3₄In step 5, 6 recoded microspheres coded on the outer surfaces of the microspheres divided according to the fluorescence intensity level of the quantum dots, namely O, are obtained by adjusting the adding concentration of the quantum dots₄6, the coding capacity X of the magnetic microsphere₄＝I₄×O₄＝60。

5. Adopting the carboxylated PS-51 microspheres obtained in the step 2 in the preparation method as a carrier matrix, loading a fluorescent dye FITC in the internal space of the microsphere carrier, assembling magnetic nano-particles and quantum dots on the outer surface of the microsphere carrier according to the

steps

3, 4, 5 and 6, and carrying out surface coating and modification, wherein 9 recoded microspheres coded by the internal space of the microspheres divided according to the FITC fluorescence intensity level, namely I recoded microspheres coded according to the FITC fluorescence intensity level are obtained by adjusting the mixing ratio of the FITC-PEI solution and the blank PEI solution in the step 3₅In step 5, 6 recoded microspheres coded by the outer surface of the microspheres divided according to the fluorescence intensity level of the quantum dots, namely O, are obtained by adjusting the adding concentration of the quantum dots₅6, the coding capacity X of the magnetic microsphere₅＝I₅×O₅＝54。

6. Combining the five types of magnetic microspheres, namely introducing structure coding information, decoding the structure information through two scattering light channels of FSC and SSC of a flow cytometer, decoding the fluorescence information of the inner space and the outer surface through a fluorescence detection channel respectively, and finally constructing a magnetic coding microsphere array with the weight of 300, namely Y ═ (I ═ is-₁×O₁)+(I₂×O₂)+(I₃×O₃)+(I₄×O₄)+(I₅×O₅)＝300。

As shown in fig. 2, the hysteresis loops of the five types of magnetically encoded microspheres show that none of the five types of microspheres have remanence and zero coercivity, and have typical superparamagnetic characteristics, which facilitate the manipulation of the microspheres in a magnetic field. In addition, the saturation magnetization of the five types of magnetic coding microspheres is 0.81-1.71emu/g, and good magnetic response performance is embodied.

As shown in fig. 3, five types of microspheres with different internal structures are used as a carrier matrix, a fluorescent dye FITC is loaded in the internal space of the microsphere carrier, magnetic nanoparticles and quantum dots are assembled on the outer surface of the microsphere carrier in sequence, and surface coating and modification are performed, so that the five types of finally obtained magnetic coding microspheres still have significant signal intensity difference in a flow-type FSC-SSC two-dimensional scattering optical decoding diagram. Although the positions are changed compared with the original microspheres, the mutual grouping of the five types of microspheres is obvious, and the structure of the microspheres is proved to have a corresponding relation with FSC-SSC scattering signals again; meanwhile, each type of microsphere contains 54-66 weight of fluorescent coding microspheres, but the FSC-SSC coding positions of the same type of microsphere are distributed in a smaller range, so that the controllability and the repeatability of the preparation method are proved. On the basis of decoding of the five types of microsphere structures, the fluorescence information of the FITC channel and the QDs channel of each type of microsphere is continuously read for decoding, five independent two-dimensional coding arrays with the coding weight of 54-66 can be obtained, and each scattering point is obviously distinguished. And combining the two-dimensional coding arrays of the five types of magnetic coding microspheres to obtain a 300-weight ultrahigh-flux magnetic coding microsphere array. The result shows that the structural coding of the microsphere carrier is used as a new coding element developed by the invention, and the coding capacity of the coding microsphere array can be obviously improved by combining with fluorescence coding. In addition, four decoding parameters related to the coded microsphere array, namely FSC intensity, SSC intensity, FITC fluorescence intensity and QDs fluorescence intensity, can be excited by 488nm monochromatic excitation light, so that the decoding cost is greatly reduced, the decoding process is simpler and more convenient, and 300 times of decoding capacity is the highest-weight coding capacity obtained by the currently reported monochromatic laser excitation. The coding microsphere array provided by the invention has great application prospect in ultrahigh-flux multi-index detection.

Example two

As described above, according to the preparation method of example one, step 5 can be repeated to assemble multiple layers of quantum dots with different central emission wavelengths on the outer surface of the microsphere carrier, thereby further increasing the dimension of the code and amplifying the number of the codes.

Similarly, based on the preparation method of the first embodiment, two or more fluorescent dyes with different central emission wavelengths can be loaded inside the microsphere carrier. The focus of this embodiment is to further detail the preparation method of the coding microsphere with one or more fluorochromes loaded inside the microsphere carrier, and the coding microsphere in this embodiment can be applied alone, or the method of this embodiment can be applied to step 2 of the first embodiment, thereby expanding the coding dimension of the coding microsphere in the first embodiment.

The design of the preparation method of the coding microsphere mainly comprises the following steps:

step 1, carrying out covalent bond labeling on polymer molecules by using X fluorescent dyes to obtain X fluorescent labeled polymer molecule solutions, wherein X is more than or equal to 1; mixing the obtained X polymer molecule solutions with the polymer molecule solutions which are not fluorescently labeled according to different proportions to obtain a mixed solution M;

step 2, adding porous microspheres into the mixed solution M obtained in the step 1, combining polymer molecules into the microspheres through physical/chemical action, centrifuging and washing with deionized water to obtain fluorescent dye doped microspheres;

step 3, adding the magnetic nanoparticles into the fluorescent dye-doped microspheres obtained in the step 2, performing physical adsorption to assemble the magnetic nanoparticles on the outer surfaces of the microspheres, and cleaning after reaction; then carrying out physical adsorption on the product and an amino polymer to obtain a fluorescent dye doped microsphere with magnetic nano particles assembled on the outer surface and the amino polymer on the outermost layer; the addition amount of the magnetic nano-particles accounts for more than or equal to 0 percent of the mass proportion of the fluorescent dye doped microspheres;

and 4, coating a silicon oxide protective shell layer on the surface of the microsphere obtained in the

step

2 or 3, thereby obtaining the fluorescent dye doped coding microsphere. After the silicon oxide coating is finished, the outer surface of the microsphere carrier can be modified with functional molecules, so that the surface functionalized coding microsphere is obtained.

Wherein the diameter of the porous microsphere is 0.1-100 μm, the pore diameter is 2-100 nm, and the matrix component of the microsphere comprises inorganic substances and polymers. The inorganic substance includes silica and/or titania. The polymer comprises polystyrene, polyacrylic acid, polymethyl acrylate, polymethacrylic acid, polymethyl methacrylate, polydivinylbenzene and/or copolymers of the above polymers.

The physical/chemical action includes electrostatic action, hydrophilic and hydrophobic action, hydrogen bonding action, coordination action, covalent bonding action, preferably electrostatic action.

The magnetic nanoparticles are Fe₃O₄Nanoparticles or gamma-Fe₂O₃Nanoparticles, preferably Fe₃O₄And (3) nanoparticles.

The functional group contained in the molecular structure of the fluorescent dye is one or more of isothiocyanate, carboxyl, N-hydroxysuccinimide ester and epoxy group, and the functional group contained in the chain segment structure of the polymer molecule is amino; or the functional group contained in the molecular structure of the fluorescent dye is amino, and the functional group contained in the molecular structure of the polymer is one or more of carboxyl and epoxy group.

The fluorescent dye comprises Fluorescein Isothiocyanate (FITC), rhodamine isothiocyanate B (RITC), Cy 5-N-hydroxysuccinimide ester (Cy5-NHS) and 5-aminofluorescein (5-AF); the polymer molecules include Polyethyleneimine (PEI), polyacrylic acid (PAA); the porous microspheres comprise porous silica microspheres, carboxylated porous polystyrene microspheres, porous silica microspheres for modifying epoxy groups, porous polystyrene microspheres for modifying epoxy groups, aminated porous silica microspheres and aminated porous polystyrene microspheres.

The following is a further description of a specific production method.

The preparation method 1 comprises the following steps:

1. 150mg of PEI (molecular weight 750K) was dissolved in 15mL of NaCl solution (0.5M), the pH was adjusted to 8.0 and the solution was recorded as a blank PEI solution; adding 4.4mg of FITC into the blank PEI solution, and carrying out shaking overnight reaction at the temperature of 30 ℃ in the dark to obtain a PEI solution (marked as FITC-PEI) marked with FITC; mixing a FITC-PEI solution and a blank PEI solution in different proportions to prepare a mixed solution with the total volume of 1.5 mL;

2. mixing 6.4X 10⁸Adding porous silica microspheres with the diameter of 1.7 mu m into the mixed solution, carrying out ultrasonic mixing uniformly, then carrying out dark rotary reaction for 20min, and bonding FITC-PEI and PEI into the microspheres through electrostatic action; centrifuging to remove supernatant after reaction, washing with water for three times, and dispersing the obtained microspheres in 0.4mL of aqueous solution to obtain microspheres with fluorescent dye loaded in the inner space;

3. 0.4mL of the microsphere dispersion obtained above was added dropwise to 1.1mL of a dispersion containing Fe under ultrasonic conditions₃O₄And (3) carrying out a light-shielding rotation reaction for 30min in an aqueous solution of magnetic nanoparticles (with the particle size of 8nm and carboxyl on the surface). After the reaction, the supernatant was magnetically separated and washed with water three times; then adding the obtained microspheres into 1.5mL of blank PEI solution mentioned in the step 1, carrying out a light-shielding rotary reaction for 20min, carrying out magnetic separation after the reaction to remove supernatant, and washing with water for three times to obtain the product with Fe assembled on the outer surface₃O₄The magnetic nanoparticles are modified with PEI fluorescent microspheres on the outermost layer;

4. magnetically separating the microspheres obtained in the step 3 to obtain supernatant, washing the microspheres twice by using absolute ethyl alcohol, adding the microspheres into a mixed system containing 3mL of ethyl alcohol, 0.3mL of water and 40 mu L of TEOS, and carrying out a light-proof rotation reaction for 30 min; then, adding 22 mu L of concentrated ammonia water, continuously carrying out dark rotary reaction for 22h at the temperature of 30 ℃, removing supernatant after the reaction, and sequentially washing with absolute ethyl alcohol and water for three times to obtain the FITC fluorescent dye doped magnetic coding microspheres coated with silicon oxide on the surface.

In the magnetic fluorescence coding microsphere prepared by the preparation method 1, fluorescence coding information of FITC dye can be excited by 488nm excitation light of a flow cytometer, and an emission waveband received by an optical filter is 515 +/-10 nm; can also be excited by 488nm exciting light of a fluorescence microscope, and the emission waveband received by the filter is 515 +/-15 nm.

As shown in FIG. 4, porous silica with a diameter of 1.7 μm is used as a microsphere carrier, FITC is used as a doping dye, and 7 fluorescence dye doping coding microspheres which can be distinguished on a flow cytometer are prepared.

The preparation method 2 comprises the following steps:

1. 150mg of PEI (molecular weight 750K) was dissolved in 15mL of NaCl solution (0.5M), the pH was adjusted to 8.0 and the solution was recorded as a blank PEI solution; adding 7.0mg of Cy 5-N-hydroxysuccinimide ester (Cy5-NHS) into the blank PEI solution, and oscillating the mixture overnight at the temperature of 30 ℃ in the dark to react to obtain a PEI solution (marked as Cy5-PEI) marked with Cy 5; mixing Cy5-PEI solution and blank PEI solution according to different proportions to prepare a mixed solution with the total volume of 1.5 mL;

2. mix 8X 10⁷Adding the carboxylated porous polystyrene microspheres with the diameter of 3.3 mu m into the mixed solution, carrying out ultrasonic mixing uniformly, then carrying out dark rotary reaction for 20min, and combining Cy5-PEI and PEI into the microspheres through electrostatic action; centrifuging to remove supernatant after reaction, washing with water for three times, and dispersing the obtained microspheres in 0.4mL of aqueous solution to obtain microspheres with fluorescent dye loaded in the inner space;

3. centrifuging the microspheres obtained in the step 2 to remove supernatant, washing the microspheres twice by using absolute ethyl alcohol, adding the microspheres into a mixed system containing 3mL of ethyl alcohol, 0.3mL of water and 40 mu L of TEOS, and carrying out a light-proof rotation reaction for 30 min; and then adding 22 mu L of concentrated ammonia water, continuously carrying out dark rotary reaction for 22h at the temperature of 30 ℃, removing the supernatant after the reaction, and sequentially washing with absolute ethyl alcohol and water for three times to obtain the silica-coated Cy5 fluorescent dye-doped coding microsphere.

In the fluorescence-encoded microspheres prepared by the preparation method 2, the fluorescence-encoded information of the Cy5 dye can be excited by 640nm excitation light of a flow cytometer, and the emission waveband received by the optical filter is 675 +/-12.5 nm; can also be excited by 633nm exciting light of a fluorescence microscope, and the emission waveband received by the filter is 675 +/-25 nm.

The preparation method 3 comprises the following steps:

1. 150mg of PEI (molecular weight 750K) was dissolved in 15mL of NaCl solution (0.5M), the pH was adjusted to 8.0 and the solution was recorded as a blank PEI solution; adding 11.9mg of RITC into the blank PEI solution, and carrying out light-shielding oscillation overnight reaction at the temperature of 30 ℃ to obtain a PEI solution (marked with RITC (RiTC-PEI)); mixing RITC-PEI solution and blank PEI solution according to different proportions to prepare mixed solution with the total volume of 1.5 mL;

2. 2 x 10 to⁷Adding porous silica microspheres with the diameter of 5.5 microns into the mixed solution, carrying out ultrasonic mixing uniformly, then carrying out dark rotary reaction for 20min, and bonding RITC-PEI and PEI into the microspheres through electrostatic action; centrifuging to remove supernatant after reaction, washing with water for three times, and dispersing the obtained microspheres in 0.4mL of aqueous solution to obtain microspheres with fluorescent dye loaded in the inner space;

4. magnetically separating the microspheres obtained in the step 3 to obtain supernatant, washing the microspheres twice by using absolute ethyl alcohol, adding the microspheres into a mixed system containing 3mL of ethyl alcohol, 0.3mL of water and 40 mu L of TEOS, and carrying out a light-proof rotation reaction for 30 min; then, adding 22 mu L of concentrated ammonia water, continuously carrying out dark rotary reaction for 22h at the temperature of 30 ℃, removing supernatant after the reaction, and sequentially washing with absolute ethyl alcohol and water for three times to obtain the RITC fluorescent dye-doped magnetic coding microspheres coated with silicon oxide on the surfaces.

In the magnetic fluorescent coding microsphere prepared by the preparation method 3, the fluorescence coding information of the RITC dye can be excited by 488nm excitation light of a flow cytometer, and the emission waveband received by the optical filter is 565 +/-10 nm; can also be excited by 488nm exciting light of a fluorescence microscope, and the emission band received by the filter is 585 +/-15 nm.

As shown in FIG. 5, the distinguishable fluorochrome-doped coded microspheres of 6-fold on the flow cytometer were prepared using porous silica with a diameter of 5.5 μm as the microsphere carrier and RITC as the doping dye.

The preparation method 4 comprises the following steps:

2. 2 x 10 to⁷Adding porous polystyrene microspheres with modified epoxy groups and the diameter of 5.5 microns into the mixed solution, uniformly mixing by ultrasonic waves, rotating in the dark for reacting overnight, and bonding RITC-PEI and PEI into the microspheres through covalent bonds; centrifuging to remove supernatant after reaction, washing with water for three times, and dispersing the obtained microspheres in 0.4mL of aqueous solution to obtain microspheres with fluorescent dye loaded in the inner space;

3. centrifuging the microspheres obtained in the step 2 to remove supernatant, washing the microspheres twice by using absolute ethyl alcohol, adding the microspheres into a mixed system containing 3mL of ethyl alcohol, 0.3mL of water and 40 mu L of TEOS, and carrying out a light-proof rotation reaction for 30 min; then, adding 22 mu L of concentrated ammonia water, continuously carrying out dark rotary reaction for 22h at the temperature of 30 ℃, removing supernatant after the reaction, and sequentially washing with absolute ethyl alcohol and water for three times to obtain the RITC fluorescent dye doped coding microspheres coated with silicon oxide on the surfaces.

In the fluorescence-encoded microspheres prepared by the preparation method 4, the fluorescence-encoded information of the RITC dye can be excited by 488nm excitation light of a flow cytometer, and the emission waveband received by the optical filter is 565 +/-10 nm; can also be excited by 488nm exciting light of a fluorescence microscope, and the emission band received by the filter is 585 +/-15 nm.

The preparation method 5 comprises the following steps:

1. 200mg of polyacrylic acid (PAA, molecular weight 5K) was dissolved in 15mL of NaCl solution (0.5M), pH was adjusted to 6.0, and the solution was designated as a blank PAA solution; adding 3.9mg of 5-aminofluorescein (5-AF) and 40mg of carbodiimide (EDC) into the blank PAA solution, and oscillating the mixture overnight at the temperature of 30 ℃ in a dark place for reaction to obtain a PAA solution (marked as 5-AF-PAA) marked with 5-AF; mixing the 5-AF-PAA solution and the blank PAA solution in different proportions to prepare a mixed solution with the total volume of 1.5 mL;

2. 2 x 10 to⁷Adding aminated porous polystyrene microspheres with the diameter of 5.5 microns into the mixed solution, carrying out ultrasonic mixing uniformly, then carrying out rotation reaction for 20min in a dark place, and adsorbing 5-AF-PAA and PAA into the microspheres through electrostatic action; centrifuging to remove supernatant after reaction, washing with water for three times, and dispersing the obtained microspheres in 0.4mL of aqueous solution to obtain microspheres with fluorescent dye loaded in the inner space;

3. 150mg of PEI (molecular weight 750K) was dissolved in 15mL of NaCl solution (0.5M), the pH was adjusted to 8.0 and the solution was recorded as a blank PEI solution; centrifuging the microspheres obtained in the step 2 to remove supernatant, adding the microspheres into 1.5mL of blank PEI solution, carrying out a light-shielding rotary reaction for 20min, centrifuging the reaction product to remove supernatant, and washing the reaction product with water for three times to obtain the PEI-modified fluorescent microspheres;

4. centrifuging the microspheres obtained in the step 3 to remove supernatant, washing the microspheres twice by using absolute ethyl alcohol, adding the microspheres into a mixed system containing 3mL of ethyl alcohol, 0.3mL of water and 40 mu L of TEOS, and carrying out a light-proof rotation reaction for 30 min; then, adding 22 mu L of concentrated ammonia water, continuously carrying out dark rotary reaction for 22h at the temperature of 30 ℃, removing the supernatant after the reaction, and sequentially washing with absolute ethyl alcohol and water for three times to obtain the 5-AF fluorescent dye doped coding microsphere with the surface coated with silicon oxide.

In the fluorescent coding microsphere prepared by the preparation method 5, the fluorescent coding information of the 5-AF dye can be excited by 488nm exciting light of a flow cytometer, and the emission waveband received by the optical filter is 515 +/-10 nm; can also be excited by 488nm exciting light of a fluorescence microscope, and the emission waveband received by the filter is 515 +/-15 nm.

The preparation method 6 comprises the following steps:

1. 150mg of PEI (molecular weight 750K) was dissolved in 15mL of NaCl solution (0.5M), the pH was adjusted to 8.0 and the solution was recorded as a blank PEI solution; respectively adding 4.4mg of FITC and 7.0mg of Cy5-NHS into the blank PEI solution, and oscillating the mixture overnight at the temperature of 30 ℃ in the dark to react to obtain a PEI solution (marked as FITC-PEI) marked with FITC and a PEI solution (marked as Cy5-PEI) marked with Cy 5; mixing a FITC-PEI solution, a Cy5-PEI solution and a blank PEI solution in different proportions to prepare a mixed solution with the total volume of 1.5 mL;

2. mixing 6.4X 10⁸Adding carboxylated porous polystyrene microspheres with the diameter of 1.7 mu m into the mixed solution, carrying out ultrasonic mixing uniformly, then carrying out dark rotary reaction for 20min, and adsorbing FITC-PEI, Cy5-PEI and PEI into the microspheres through electrostatic action; centrifuging to remove supernatant after reaction, washing with water for three times, and dispersing the obtained microspheres in 0.4mL of aqueous solution to obtain microspheres with fluorescent dye loaded in the inner space;

3. centrifuging the microspheres obtained in the step 2 to remove supernatant, washing the microspheres twice by using absolute ethyl alcohol, adding the microspheres into a mixed system containing 3mL of ethyl alcohol, 0.3mL of water and 40 mu L of TEOS, and carrying out a light-proof rotation reaction for 30 min; and then adding 22 mu L of concentrated ammonia water, continuously carrying out dark rotary reaction for 22h at the temperature of 30 ℃, removing the supernatant after the reaction, and sequentially washing with absolute ethyl alcohol and water for three times to obtain the FITC and Cy5 two-color fluorescent dye doped coding microspheres with the surfaces coated with silicon oxide.

In the fluorescence-encoded microspheres prepared by the preparation method 6, fluorescence-encoded information of the FITC dye can be excited by 488nm excitation light of a flow cytometer, and an emission waveband received by an optical filter is 515 +/-10 nm; the fluorescence encoding information of the Cy5 dye can be excited by 640nm exciting light of a flow cytometer, and the emission waveband received by the filter is 675 +/-12.5 nm. Or the fluorescence coding information of the FITC dye can also be excited by 488nm exciting light of a fluorescence microscope, and the emission waveband received by the optical filter is 515 +/-15 nm; the fluorescence encoding information of the Cy5 dye can also be excited by 633nm exciting light of a fluorescence microscope, and the emission waveband received by the filter is 675 +/-25 nm.

The preparation method 7 comprises the following steps:

1. 150mg of PEI (molecular weight 750K) was dissolved in 15mL of NaCl solution (0.5M), the pH was adjusted to 8.0 and the solution was recorded as a blank PEI solution; respectively adding 4.4mg of FITC and 11.9mg of RITC into the blank PEI solution, and carrying out shaking overnight reaction at the temperature of 30 ℃ in the dark to obtain a PEI solution (marked as FITC-PEI) marked with FITC and a PEI solution (marked as RITC-PEI) marked with RITC; mixing a FITC-PEI solution, a RITC-PEI solution and a blank PEI solution in different proportions to prepare a mixed solution with the total volume of 1.5 mL;

2. mix 8X 10⁷Adding porous silica microspheres with the diameter of 3.3 microns into the mixed solution, carrying out ultrasonic mixing uniformly, then carrying out dark rotary reaction for 20min, and adsorbing FITC-PEI, RITC-PEI and PEI into the microspheres through electrostatic action; centrifuging to remove supernatant after reaction, washing with water for three times, and dispersing the obtained microspheres in 0.4mL of aqueous solution to obtain microspheres with fluorescent dye loaded in the inner space;

4. magnetically separating the microspheres obtained in the step 3 to obtain supernatant, washing the microspheres twice by using absolute ethyl alcohol, adding the microspheres into a mixed system containing 3mL of ethyl alcohol, 0.3mL of water and 40 mu L of TEOS, and carrying out a light-proof rotation reaction for 30 min; then, adding 22 mu L of concentrated ammonia water, continuously carrying out dark rotary reaction for 22h at the temperature of 30 ℃, removing supernatant after the reaction, and sequentially washing with absolute ethyl alcohol and water for three times to obtain the FITC and RITC two-color fluorescent dye doped magnetic coding microspheres with the surfaces coated with silicon oxide.

In the magnetic fluorescence coding microsphere prepared by the preparation method 7, fluorescence coding information of FITC dye can be excited by 488nm excitation light of a flow cytometer, and an emission waveband received by an optical filter is 515 +/-10 nm; the fluorescence encoding information of the RITC can be excited by 488nm exciting light of a flow cytometer, and the emission waveband received by the optical filter is 565 +/-10 nm. Or the fluorescence coding information of the FITC dye can also be excited by 488nm exciting light of a fluorescence microscope, and the emission waveband received by the optical filter is 515 +/-15 nm; the fluorescence coding information of the RITC dye can also be excited by 488nm exciting light of a fluorescence microscope, and the emission waveband received by the filter is 585 +/-15 nm.

As shown in FIG. 6, 32 fluorescence dye-doped encoding microspheres distinguishable on a flow cytometer were prepared by using porous silica with a diameter of 3.3 μm as a microsphere carrier and FITC and RITC as doping dyes. As shown in FIG. 7, the fluorescent dye-doped encoded microspheres obtained by the preparation method 7 can be magnetically separated rapidly within 2 minutes by a magnetic field, and exhibit good magnetic response performance.

The preparation method 8 comprises the following steps:

2. 2 x 10 to⁷Adding porous silica microspheres with the diameter of 5.5 microns into the mixed solution, carrying out ultrasonic mixing uniformly, then carrying out dark rotary reaction for 20min, and adsorbing FITC-PEI, RITC-PEI and PEI into the microspheres through electrostatic action; centrifuging to remove supernatant after reaction, washing with water for three times, and dispersing the obtained microspheres in 0.4mL of aqueous solution to obtain microspheres with fluorescent dye loaded in the inner space;

In the magnetic fluorescence coding microsphere prepared by the preparation method 8, fluorescence coding information of FITC dye can be excited by 488nm excitation light of a flow cytometer, and an emission waveband received by an optical filter is 515 +/-10 nm; the fluorescence encoding information of the RITC can be excited by 488nm exciting light of a flow cytometer, and the emission waveband received by the optical filter is 565 +/-10 nm. Or the fluorescence coding information of the FITC dye can also be excited by 488nm exciting light of a fluorescence microscope, and the emission waveband received by the optical filter is 515 +/-15 nm; the fluorescence coding information of the RITC dye can also be excited by 488nm exciting light of a fluorescence microscope, and the emission waveband received by the filter is 585 +/-15 nm.

As shown in FIG. 8, 51-fold flow cytometry distinguishable fluorochrome-doped encoded microspheres were prepared using porous silica with a diameter of 5.5 μm as the microsphere carrier and FITC and RITC as the doping dyes. As shown in fig. 9, the fluorescent dye-doped encoded microspheres prepared by the preparation method 8 can also be decoded and analyzed by using a fluorescence microscope, and a two-dimensional array arrangement of 51 re-encoded microspheres can be obtained according to the fluorescence intensity.

For the encoded microspheres with silica-coated surfaces prepared by the preparation methods 1 to 8 in this embodiment, functional molecules can be modified on the microsphere surfaces by adopting the specific manner (regarding the preparation process of surface functionalized modification) in step 6 of the preparation method in the first embodiment, so as to obtain the encoded microspheres with carboxyl groups modified on the surfaces.

The foregoing detailed description of the preferred embodiments of the invention has been presented. It should be understood that numerous modifications and variations could be devised by those skilled in the art in light of the present teachings without departing from the inventive concepts. Therefore, the technical solutions available to those skilled in the art through logic analysis, reasoning and limited experiments based on the prior art according to the concept of the present invention should be within the scope of protection defined by the claims.

Claims

1. The coded microsphere is characterized by comprising a microsphere carrier, wherein the microsphere carrier is a mesoporous microsphere, at least one fluorescent marker is arranged in the microsphere carrier, the fluorescent marker is formed by connecting fluorescent dyes and polymers through covalent bonds, the central emission wavelengths of the fluorescent dyes corresponding to the fluorescent markers are different, and therefore each fluorescent dye limits the coded information of one dimension of the coded microsphere.

2. The encoded microsphere of claim 1, wherein the matrix component of the microsphere support is inorganic or polymeric.

3. The encoded microsphere of claim 1, wherein the matrix component of the microsphere support comprises silica or titania.

4. The encoded microsphere of claim 1, wherein the matrix component of the microsphere carrier comprises polystyrene, polyacrylic acid, polymethyl acrylate, polymethacrylic acid, polymethyl methacrylate, polydivinylbenzene, and/or copolymers of two or more monomers selected from the group consisting of the above polymers.

5. The encoded microsphere of claim 1, wherein the microsphere carrier has a diameter selected from the range of 0.1 to 100 μm.

6. The encoded microsphere of claim 1, wherein the pore size of the microsphere carrier is selected from the range of 2 to 100 nm.

7. The encoded microsphere of claim 1, wherein the fluorescent dyes corresponding to each of the fluorescent labels differ in central emission wavelength by greater than 30 nm.

8. The encoded microsphere of claim 1, wherein the encoded information of the defined dimension is adjusted based on the amount of the fluorescent label.

9. The encoded microsphere of claim 1, wherein the polymer is further disposed inside the microsphere carrier, and wherein the amount of each fluorescent marker is adjusted according to the ratio of the polymer to each fluorescent marker.

10. The encoded microsphere of claim 1, further comprising magnetic nanoparticles.

11. The encoded microsphere of claim 10, wherein the magnetic nanoparticle is disposed outside of the microsphere carrier.

12. The encoded microsphere of claim 10, wherein the magnetic nanoparticles are Fe3O4 nanoparticles or γ -Fe2O3 nanoparticles.

13. The encoded microsphere of claim 1, wherein the outer surface of the encoded microsphere is a silica-coated layer.

14. The coded microsphere of claim 1, wherein the outer surface of the coded microsphere is a silica coating layer and a functional molecule modification layer, and the functional molecule modification layer is arranged on the outermost layer.

15. A method for preparing coded microspheres, comprising:

step two, respectively connecting at least one fluorescent dye with a polymer through a covalent bond to form at least one fluorescent marker, wherein each fluorescent dye limits the coding information of one dimension of the coding microsphere;

and step three, arranging the fluorescent marker in the microsphere carrier.

16. The method of claim 15, wherein the matrix component of the microsphere carrier is inorganic or polymeric.

17. The method of claim 15, wherein the matrix component of the microsphere carrier is silica or titania.

18. The method of claim 15, wherein the matrix component of the microsphere carrier is polystyrene, polyacrylic acid, polymethyl acrylate, polymethacrylic acid, polymethyl methacrylate, polydivinylbenzene, and/or a copolymer of two or more monomers selected from the group consisting of the above polymers.

19. The method of claim 15, wherein the microsphere carrier has a diameter selected from the range of 0.1 to 100 μm.

20. The method of claim 15, wherein the pore size of the microsphere carrier is selected from the range of 2 to 100 nm.

21. The method according to claim 15, wherein the microsphere carrier is one or more of porous silica microspheres, carboxylated porous polystyrene microspheres, epoxy group-modified porous silica microspheres, epoxy group-modified porous polystyrene microspheres, aminated porous silica microspheres, and aminated porous polystyrene microspheres.

22. The method of claim 15, wherein the fluorescent dyes of each of the fluorescent labels have central emission wavelengths that differ by more than 30 nm.

23. The method of claim 15, wherein the encoded information of the defined dimension is adjusted according to the amount of the fluorescent marker.

24. The method according to claim 15, wherein the fluorescent dye has one or more functional groups selected from the group consisting of isothiocyanate, carboxyl, N-hydroxysuccinimide ester and epoxy group in its molecular structure, and wherein the polymer has an amino group in its molecular structure.

25. The method according to claim 15, wherein the functional group contained in the molecular structure of the fluorescent dye is an amino group, and the functional group contained in the molecular structure of the polymer is one or more of a carboxyl group and an epoxy group.

26. The method of claim 15, wherein the fluorescent dye is one or more of fluorescein isothiocyanate, rhodamine B, Cy 5-N-hydroxysuccinimide ester isothiocyanate, and 5-aminofluorescein isothiocyanate.

27. The method of claim 15, wherein the polymer is polyethyleneimine and/or polyacrylic acid.

28. The method of claim 15, wherein step three specifically comprises:

disposing the fluorescent marker or the mixture of the fluorescent marker and the polymer in the inner space of the microsphere carrier by physical/chemical action.

29. The method of claim 28, wherein the encoded information for each dimension is adjusted based on the ratio of each fluorescent label and the polymer in the mixture.

30. The method of claim 15, further comprising:

31. The method of claim 30, wherein the magnetic nanoparticles are Fe3O4 nanoparticles or γ -Fe2O3 nanoparticles.

32. The method of claim 30, wherein the fourth step specifically comprises:

33. The method of claim 28 or 32, wherein the physical/chemical interaction comprises electrostatic interaction, hydropathic/hydrophobic interaction, hydrogen bonding interaction, complexation, and/or covalent bonding interaction.

34. The method of claim 30, wherein the fourth step further comprises:

35. The method of claim 15 or 34, wherein after all steps are completed, the outer surface of the microsphere carrier is coated with silica.

36. The method of claim 35, wherein the surface functionalized microsphere carrier is obtained by coating silica on the outer surface of the microsphere carrier and then modifying functional molecules on the outer surface of the microsphere carrier.

37. An encoded microsphere array, comprising at least two encoded microspheres according to any one of claims 1 to 14 or at least two encoded microspheres prepared by the preparation method according to any one of claims 15 to 36, wherein each encoded microsphere has different encoded information.