WO2022063336A1

WO2022063336A1 - Encoding microspheres and array and preparation method

Info

Publication number: WO2022063336A1
Application number: PCT/CN2021/128883
Authority: WO
Inventors: 王耀; 徐宏; 古宏晨; 陈藏
Original assignee: 上海交通大学; 上海迈景纳米科技有限公司
Priority date: 2020-09-27
Filing date: 2021-11-05
Publication date: 2022-03-31
Also published as: CN112179885B; CN112179885A

Abstract

Encoding microspheres and an array and a preparation method, the encoding microspheres comprising microsphere carriers, and the microsphere carriers being mesoporous microspheres. The matrix ingredients and pore diameters of the microsphere carriers are used to define a first dimension of encoding information of the encoding microspheres. The encoding microsphere array comprises at least two of the described encoding microspheres.

Description

Encoded microspheres and arrays and methods of making

technical field

The invention relates to the fields of micro-nano biological materials and multi-index detection, in particular to a coding microsphere and an array and a preparation method.

Background technique

In recent years, with the rapid development of life sciences, clinical diagnosis, drug analysis, environmental monitoring and other scientific fields, single-tube multi-index detection technology has become the focus of many researchers. This technology can realize simultaneous screening and parallel analysis of multiple substances to be tested in the same sample, so that the testing personnel can obtain high-density information in the shortest time, the smallest sample volume and the most convenient and sensitive way. Among them, the suspension lattice technology with encoded microspheres as the core carrier is one of the most advantageous and widely used multi-index detection technologies. In actual detection, the carrier used is a mixture of microspheres with different encoded information, and each type of microspheres carries an identifiable encoded information in order to identify the probe species immobilized on its surface. After the corresponding target substance, the obtained complex can be read by the decoding system and the detection result can be further analyzed.

Based on the suspension lattice technology of encoded microspheres, the number of multi-index detections directly depends on the encoding capacity of the carrier microspheres. Among many encoding methods, the fluorescence encoding method based on fluorescent signal is still the most mainstream encoding method due to its flexible design, convenient preparation, and good match with mature decoding and detection systems such as flow cytometer and fluorescence microscope. . Fluorescence coding usually loads different fluorescent elements (such as fluorescent dyes or new luminescent nanoparticles such as quantum dots) on the surface or inside of the microsphere, and realizes coding by adjusting the loading amount of fluorescent elements (that is, changing the fluorescence intensity) and changing the fluorescence emission wavelength. . At present, commercial encoding microspheres mainly use fluorescent dyes for encoding, but when the excitation wavelengths of different fluorescent dyes are different, more lasers need to be added, which greatly increases the decoding cost; and the fluorescence emission spectrum of dye molecules is generally wide, and at the same time The use of multiple fluorescent dyes can easily lead to spectral overlap and interference, which limits the improvement of encoding capacity. Although the encoded microspheres prepared by using fluorescent nanoparticles represented by quantum dots have high theoretical coding capacity, in fact, it is difficult to simultaneously load many kinds of quantum dots in the limited space of the microsphere carrier, and the types of quantum dots The increase of α also affects the adjustment range of the fluorescence intensity of the microspheres, resulting in the decrease of the encoding capacity. After literature search, Chan's research group (Angew.Chem.Int.Ed., Vol. 47, pp. 5577-5581, 2008) prepared 105 recoded microspheres with five different color quantum dots and three intensities, which is the current quantum One of the largest known reports of point coding capacity.

With the development of multidisciplinary technology and the improvement of throughput requirements for detection indicators, a single fluorescent coding strategy is slightly insufficient in ultra-high-throughput multi-indicator detection. In response to this problem, researchers have tried to combine fluorescence with other different encoding methods in order to obtain encoded microsphere arrays with higher encoding capacity. The Chinese patent with the announcement number CN101912757B discloses a preparation method of fluorescent-magnetic double-coded microspheres. However, the complex decoding process of this system limits its popularization and application. The Chinese patent with the announcement number CN100565185C discloses a photonic crystal composite coding microsphere and a preparation method. The photonic crystal coding microsphere is an orderly assembly of nano-scale colloidal particles to form a microsphere with a larger particle size, and the detection is a microsphere. The characteristic reflection peak wavelength of the patent is to use the reflection peak position of the photonic crystal and the fluorescence intensity of the quantum dot to expand the encoding capacity. However, the size of photonic crystal-based encoded microspheres is usually larger than 100 μm, which is not conducive to maintaining a suspended state in a liquid-phase detection environment, and thus the reaction kinetics are significantly lower than that of micrometer-scale encoded microspheres. Lu et al. (Chem. Mater., Vol. 29, pp. 10398-10408, 2017) publicly reported a strategy for co-encoding microsphere size and fluorescence by utilizing two different sizes of magnetic microspheres (2.9 μm and 6.2 μm) As carriers, two fluorescent substances, red quantum dots and green fluorescent dyes, were used as coding elements to construct a three-dimensional coding microsphere array with a coding capacity of 100. Although the multi-dimensional encoding strategy of microsphere size combined with fluorescence can improve the encoding capacity, the surface area of microspheres of different sizes is significantly different, and the number of probes attached to the surface is also significantly different, which will lead to the reaction kinetics between different microspheres and target substances. The huge difference in response consistency is an "unfair" response environment for the multi-index detection process.

Therefore, those skilled in the art are devoted to developing new coding elements and developing a coding strategy that can greatly increase coding capacity, so as to obtain coding microsphere arrays that can be used for ultra-high-throughput multi-index detection.

SUMMARY OF THE INVENTION

In view of the problems in the prior art, the present invention provides a coded microsphere, the coded microsphere includes a microsphere carrier, the microsphere carrier is a mesoporous microsphere, and the matrix composition and pore size of the microsphere carrier are determined by First dimension encoding information for defining the encoded microspheres.

Further, the matrix component adopts inorganic substances or polymers.

Further, the matrix component adopts silicon dioxide or titanium dioxide.

Further, the matrix component adopts polystyrene, polyacrylic acid, polymethyl acrylate, polymethacrylic acid, polymethyl methacrylate, polydivinyl benzene and/or two kinds of polymers involved in the above-mentioned polymers. or a copolymer of two or more monomers.

Further, the diameter of the microsphere carrier is selected in the range of 0.2-20 μm, preferably in the range of 3-6 μm.

Further, the pore size of the microsphere carrier is selected in the range of 2-100 nm, preferably in the range of 10-60 nm.

Further, the first dimension information is adjusted according to the matrix composition and/or pore size of the microsphere carrier, and the FSC- SSC (forward scattered light-side scattered light) two-dimensional scattergram of the signal distribution to distinguish.

Further, an intermediary substance is also arranged inside the microsphere carrier.

Further, the encoded microspheres further include at least one fluorescent material, and the central emission wavelengths of the fluorescent materials are different, so that each of the fluorescent materials defines the encoded information of one dimension of the encoded microspheres.

Further, the difference between the central emission wavelengths of the fluorescent materials is greater than 30 nm.

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

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

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

Further, the second dimension encoding 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 intermediary 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 substance is a polymer.

Further, the intermediary substance is further disposed inside the microsphere carrier, and the content of the first fluorescent material is adjusted according to the ratio of the intermediary substance to the fluorescent marker. In one embodiment of the present invention, the intermediary substance is an amino polymer.

Further, the encoded microspheres further include a second fluorescent material, and the second fluorescent material defines third-dimensional encoded information of the encoded microspheres.

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

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

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

Further, the encoded microspheres also include magnetic nanoparticles.

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

Further, the magnetic nanoparticles are Fe ₃ O ₄ nanoparticles or γ-Fe ₂ O ₃ nanoparticles, preferably Fe ₃ O ₄ nanoparticles.

Further, the outer surface of the encoded microspheres is a silicon oxide coating layer.

Further, the outer surface of the encoded microspheres is a silicon oxide coating layer and a functional molecule modified layer, the functional molecule modified layer is in the outermost layer, and the preferred functional molecule is γ-aminopropyl triethoxysilane ( APTES) and polyacrylic acid (PAA).

The present invention also provides an encoded microsphere array, the encoded microsphere array includes at least two types of encoded microspheres, and each of the encoded microspheres has different encoded information; the encoded microspheres include a microsphere carrier, The microsphere carrier is a mesoporous microsphere, and the matrix composition and pore size of the microsphere carrier are used to define the first dimension encoded information of the encoded microsphere.

Further, the microsphere carriers of the various encoded microspheres have substantially similar diameters. In some embodiments, the variation in diameter between different species of the microsphere carrier is defined as < 15%.

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

Further, different pore sizes of the encoded microspheres of the same matrix component are selected respectively.

Further, the matrix component adopts inorganic substances or polymers.

Further, the matrix component adopts silicon dioxide or titanium dioxide.

Further, flow cytometry is used to detect the encoded microspheres, and the encoded microspheres are distinguished in the first dimension according to the signal distribution of the FSC-SSC two-dimensional scatter plot obtained by the detection.

Further, the intermediary substance is a polymer.

Further, the encoded microspheres further include a second fluorescent material, and the first fluorescent material defines third-dimensional encoded information of the encoded microspheres.

Further, the encoded microspheres also include magnetic nanoparticles.

Further, the magnetic nanoparticles are Fe ₃ O ₄ nanoparticles or γ-Fe ₂ O ₃ nanoparticles.

The present invention also provides a preparation method of encoded microspheres, the preparation method comprises:

Step 1: Select a microsphere carrier, which is a mesoporous microsphere, and determine the matrix composition, diameter and pore size of the microsphere carrier, so as to define the encoding information of the first dimension of the encoded microsphere.

Further, the matrix component adopts inorganic substances or polymers.

Further, the matrix component adopts silicon dioxide or titanium dioxide.

Further, the preparation method also includes:

In step 2, the fluorescent dye is arranged inside the microsphere carrier to define the second dimension encoding information of the encoded microsphere; or an intermediary substance is arranged inside the microsphere carrier.

Further, in the second step, the fluorescent dye is arranged inside the microsphere carrier, which specifically includes:

The fluorescent dye and the intermediate substance are connected to form a fluorescent marker, and the fluorescent marker or the mixture of the fluorescent marker and the intermediate substance is arranged in the inner space of the microsphere carrier through physical/chemical action.

Further, the intermediary substance is a polymer. For example, in some embodiments, a negatively charged microsphere carrier is used in step 2, and surface modification can be performed on the non-negatively charged microsphere carrier to obtain a microsphere carrier with negatively charged modified molecules both in the inner space and on the outer surface. In the second, a positively charged amino polymer is used to form a fluorescently labeled polymer, and to form a mixture with the fluorescently labeled polymer, so that the fluorescently labeled polymer can be adsorbed on the inner space of the microsphere carrier.

Further, the fluorescent dye forming the fluorescent label is connected with the intermediary substance through a covalent bond.

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

Further, the preparation method also includes:

Step 3: Disposing the magnetic nanoparticles on the outer surface of the microsphere carrier.

Further, the step 3 in the preparation method specifically includes:

The magnetic nanoparticles are coated on the outer surface of the microsphere carrier by physical/chemical action. For example, when a positively charged amino polymer is used in the second step, the outer surface of the microsphere carrier after the second step is also adsorbed with a positively charged amino polymer, then a negatively charged magnetic polymer can be used in the third step. nanoparticles, so that the magnetic nanoparticles are coated on the outer surface of the current microsphere carrier.

Further, the step 3 also includes:

After the magnetic nanoparticles are coated on the outer surface of the microsphere carrier, the outer surface of the microsphere carrier is then coated with a polymer.

Further, the preparation method also includes:

Step 4: Disposing the fluorescent nanoparticles on the outer surface of the microsphere carrier to define the third-dimensional encoded information of the encoded microspheres.

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

Further, the step 4 in the preparation method specifically includes:

The fluorescent nanoparticles are coated on the outer surface of the microsphere carrier by physical/chemical action.

Further, the step 4 also includes:

After the fluorescent nanoparticles are coated on the outer surface of the microsphere carrier, the outer surface of the microsphere carrier is then coated with a polymer.

Further, the physical/chemical interactions include electrostatic interactions, hydrophobic interactions, hydrogen bonding interactions, coordination interactions and/or covalent bonding interactions.

Further, after all the steps are completed, 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, functional molecules are modified on the outer surface of the microsphere carrier, thereby obtaining a surface-functionalized microsphere carrier, and the preferred functional molecule is γ-ammonia. Propyltriethoxysilane (APTES) and polyacrylic acid (PAA).

The present invention also provides an encoded microsphere doped with fluorescent dyes, the encoded microsphere includes a microsphere carrier, the microsphere carrier is a mesoporous microsphere, and at least one fluorescent marker is arranged inside the microsphere carrier The fluorescent label is formed by connecting a fluorescent dye and an intermediary substance, and the central emission wavelength of the fluorescent dye corresponding to each fluorescent label is different, so that each fluorescent dye defines one dimension of the encoded microsphere encoding information.

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

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

Further, the matrix component of the microsphere carrier adopts polystyrene, polyacrylic acid, polymethyl acrylate, polymethacrylic acid, polymethyl methacrylate, polydivinyl benzene and/or those obtained from the above-mentioned polymers. A copolymer of two or more monomers involved.

Further, the diameter selection range of the microsphere carrier is 0.1-100 μm.

Further, the pore size selection range of the microsphere carrier is 2-100 nm.

Further, the central emission wavelengths of the fluorescent dyes corresponding to the fluorescent labels differ by more than 30 nm.

Further, the intermediary substance is a polymer.

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

Further, the intermediary substance is also arranged inside the microsphere carrier, and the content of each fluorescent marker is adjusted according to the ratio of the intermediary substance and each of the fluorescent markers.

Further, the encoded microspheres also include magnetic nanoparticles.

Further, the outer surfaces of the encoded microspheres are a silicon oxide coating layer and a functional molecule modification layer, and the functional molecule modification layer is at the outermost layer.

The present invention also provides a method for preparing encoded microspheres doped with fluorescent dyes, the preparation method comprising:

Step 1, selecting a microsphere carrier, the microsphere carrier is a mesoporous microsphere;

Step 2, connecting at least one fluorescent dye with an intermediary substance to form at least one fluorescent marker, and each fluorescent dye defines the encoded information of one dimension of the encoded microsphere;

Step 3: Disposing the fluorescent marker inside the microsphere carrier.

Further, the pore size selection range of the microsphere carrier is 2-100 nm.

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

Further, the intermediary substance is a polymer.

Further, 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 group, N-hydroxysuccinimide ester, and epoxy group, and the molecule of the intermediary substance is The functional group contained in the structure is an amino group.

Further, the fluorescent dye adopts fluorescein isothiocyanate (FITC), rhodamine B isothiocyanate (RITC), Cy5-N-hydroxysuccinimide ester (Cy5-NHS), 5-aminofluorescein One or more of (5-AF).

Further, the intermediary substance is polyethyleneimine (PEI) and/or polyacrylic acid (PAA).

Further, the step 3 in the preparation method specifically includes:

The fluorescently labeled polymer or the mixture of the fluorescently labeled polymer and the polymer is disposed in the inner space of the microsphere carrier through physical/chemical action.

Further, the encoded information of each dimension is adjusted according to the ratio of each fluorescently labeled polymer and the polymer in the mixture.

Further, the preparation method also includes:

Step 4: Disposing the magnetic nanoparticles on the outer surface of the microsphere carrier.

Further, the step 4 in the preparation method specifically includes:

The magnetic nanoparticles are coated on the outer surface of the microsphere carrier by physical/chemical action.

Further, the step 4 also includes:

Further, after the outer surface of the microsphere carrier is coated with silicon oxide, functional molecules are modified on the outer surface of the microsphere carrier, thereby obtaining a surface-functionalized microsphere carrier.

The present invention also provides a coded microsphere array, which includes at least two of the above-mentioned coded microspheres doped with fluorescent dyes, or at least two kinds of codes prepared by the above-mentioned preparation method of coded microspheres doped with fluorescent dyes Microspheres, the encoded microspheres have different coding information.

The beneficial effects of the encoded microspheres, arrays and preparation methods of the present invention include:

1. Aiming at the problem of insufficient coding capacity of the existing coding microsphere array, the present invention first develops a new coding method, that is, coding is performed by using different internal structures of the microsphere carrier. In the field of coding microsphere array construction, the internal structure, as the inherent property of microsphere carrier, has not been utilized as coding element. The present invention only changes the internal structure of the microsphere carrier with similar diameters (including the matrix composition and/or the pore size is different), and simultaneously reads the forward scattered light (FSC) and side scatter of the microsphere carrier on the flow cytometer Light (SSC) signal, different signal groups of different kinds of microsphere carriers were obtained in the two-dimensional scatter plot of FSC-SSC, and the structure encoding was realized. Once the internal structure of the microsphere carrier is determined, the FSC-SSC optical signal generated by it will also be determined. This is a stable encoded information that is hardly affected by the external environment. Structure encoding is a brand-new encoding method.

2. In the present invention, different types of microspheres with similar diameters are used as carriers, and the diameter deviation is less than or equal to 15%. Only the internal structures of different types of microspheres are used for coding, and the similar diameters ensure that the microsphere carriers corresponding to different detection indicators have similar properties. It is a "fair" reaction environment for the multi-index detection process; in addition, the present invention limits the diameter of the microsphere carrier to 0.2-20 μm, in the actual detection application process, the smaller size The microsphere carrier has better suspension characteristics in the liquid phase and is less prone to sedimentation, so it has faster reaction kinetics, which is beneficial to improve the detection sensitivity.

3. Aiming at the problem of insufficient coding capacity of the existing coding microsphere array and other technical defects, the present invention further proposes a new structure-fluorescence combination on the basis of developing a new coding element structure coding (that is, expanding the coding dimension). The coding strategy realizes the preparation of the coding microsphere array with ultra-high coding capacity; the present invention utilizes the internal structure difference of the microsphere carrier, the fluorescence intensity level of the inner space of the microsphere and the fluorescence intensity level of the outer surface of the microsphere, and the original independent The combination of different coding elements of the microspheres significantly increases the coding capacity of the microsphere carrier.

4. The microsphere carrier in the encoded microsphere array proposed by the present invention can simultaneously have multiple functional properties such as structural coding, fluorescent coding of the inner space, fluorescent coding of the outer surface, and magnetic responsiveness. The space volume of the microsphere carrier is limited, and the present invention makes full use of the porous structure characteristics of the carrier microsphere and its three regions, namely, the skeleton of the carrier material, the internal pores and the outer surface of the carrier, and carries out a reasonable functional design of sub-regions , as follows: (1) By changing the internal composition of the microsphere carrier, that is, the matrix composition and/or pore size, specific structure-encoding information is obtained; (2) The porous structure (ie, the inner space) of the microsphere carrier can be used to load Fluorescent dye molecules, while realizing the fluorescent coding function, also make the microspheres modified with functionalized groups, and the polymer molecules are mainly bound to the inner pore wall through physical/chemical interactions, which is beneficial to reduce the coding performance of the microsphere carrier structure. (3) Magnetic nanoparticles and fluorescent nanoparticles represented by quantum dots can be assembled by using the outer surface of the microsphere carrier, which not only endows the microsphere carrier with magnetic responsiveness and fluorescence encoding performance, but also protects as much as possible. The internal structure-encoding properties of microsphere carriers were investigated. The functional structure design of the above sub-regions realizes that each independent region of the microsphere carrier exerts its own functions and reduces mutual influence, and ensures the controllability and repeatability of the coding process.

5. The preparation method used in the present invention is a layer-by-layer self-assembly method. First, the amino polymer labeled with fluorescent dye is assembled with the microsphere carrier through physical/chemical action, so that the fluorescent dye is loaded in the inner space, and the microsphere is realized at the same time. Amination modification of the spherical surface; secondly, the magnetic nanoparticles are assembled on the outer surface of the microsphere carrier through the physical/chemical interaction between the magnetic nanoparticles and the amino polymer; thirdly, the fluorescent nanoparticles represented by quantum dots are combined with The coordination or electrostatic action of the amino polymer self-assembles it on the outer surface of the microsphere carrier; finally, the outermost layer is coated with a silicon oxide protective layer and modified with functional molecules. The loading process of the above-mentioned fluorescent dyes, magnetic nanoparticles and fluorescent nanoparticles all use a layer-by-layer assembly method, and the solvent environment for self-assembly is changed according to different molecular action modes, and the preparation method is simple and repeatable.

6. In the application process of the encoded microsphere array proposed by the present invention, suitable fluorescent encoding elements (such as the green fluorescent dye FITC loaded in the inner space of the preferred microsphere carrier of the present invention, and the CdSe/CdSe/CdSe/substrate assembled on the outer surface of the microsphere carrier) can be selected. ZnS red quantum dots), combined with the relationship between the carrier microsphere structure encoding and the FSC-SSC scattered light signal, can realize the simultaneous excitation of all encoded signals by a monochromatic laser (ie, structural signal, green fluorescence signal and red fluorescence signal, all can be excited by a 488 nm laser source). It can greatly reduce the decoding cost and make the decoding process simpler and more convenient.

The beneficial effects of the encoded microspheres and arrays doped with fluorescent dyes and the preparation method of the present invention include:

1. The fluorescent dye doping method mediated by the intermediary substance proposed in the present invention is universal to different kinds of microsphere carriers (including inorganic microspheres and polymer microspheres). In commercial products, the swelling method is usually used to diffuse fluorescent molecules into polymer microspheres to prepare encoded microspheres, but this method is not suitable for inorganic microsphere carriers and is not universal. In the present invention, the intermediary substance marked by the covalent bond of fluorescent molecules is used as the doping raw material, and it is combined into the interior of the porous microsphere through physical/chemical action, so as to realize the loading of the fluorescent dye on the microsphere carrier; The physicochemical properties of spheres and polymer molecules are selected, including electrostatic interaction, hydrophobic interaction, hydrogen bonding interaction, coordination interaction, and covalent bonding interaction, and are applicable to both inorganic microspheres and polymer microspheres.

2. In the present invention, the porous microspheres with high specific surface area and large pore volume are used as carriers, and the inner space of the microspheres is fully utilized to load fluorescent dyes, which can achieve a higher fluorescent molecular load, and can achieve a wider fluorescence spectrum. The intensity adjustment range; combined with the co-doping of different fluorescent dyes, a higher fluorescence encoding capacity can be finally achieved to meet the needs of practical multi-index detection.

3. The coding method proposed by the present invention is simple, and the coding control is precise. It is only necessary to use different fluorescent dyes to covalently label the intermediary substances, and then mix them with non-fluorescently labeled polymer molecules in different proportions, so that the fluorescence encoding intensity of the microspheres can be easily and precisely regulated; The dye-labeled polymer molecules do not require further purification, and can be directly loaded into the microspheres through physical/chemical interactions with the porous microspheres, and free fluorescent molecules can be removed in the subsequent washing process. The preparation method and coding process are very simple and feasible. control.

4. On the basis of fluorescent coding, the present invention can assemble magnetic nanoparticles on the outer surface of the microsphere through physical/chemical action, and endow the coded microsphere with superparamagnetic properties, which facilitates the separation and manipulation under the action of a subsequent magnetic field.

5. The surface of the fluorescent coding microspheres prepared by the present invention is a silica protective shell layer, which can prevent the leakage of fluorescent dyes inside the microspheres; at the same time, the silanols on the surface of the shell layer are conducive to further modifying various functional groups, which are the coding microspheres. Sphere-coupled probes provide reaction sites.

The concept, specific structure and technical effects of the present invention will be further described below in conjunction with the accompanying drawings, so as to fully understand the purpose, characteristics and effects of the present invention.

Description of drawings

Fig. 1 adopts SiO2-17, SiO2-48, PS-14, PS-37 and PS-51 five types of microspheres in one embodiment of the present invention, and utilizes the flow corresponding to the 5-fold coding microsphere array obtained by its structural coding formula decoding result graph;

2 is an embodiment of the present invention using SiO2-17, SiO2-48, PS-14, PS-37 and PS-51 five types of microspheres, using its internal structure + internal space fluorescence + external surface fluorescence for joint coding , and endows the microspheres with magnetic response properties through the self-assembly of magnetic nanoparticles, and prepares the obtained hysteresis loops of the five types of encoded microspheres;

3 is an embodiment of the present invention using SiO2-17, SiO2-48, PS-14, PS-37 and PS-51 five types of microspheres, using its internal structure + internal space fluorescence + external surface fluorescence for joint coding , and endows the microspheres with magnetic response properties through the self-assembly of magnetic nanoparticles, and prepares the corresponding flow decoding result map of the obtained 300-fold encoded microsphere array;

Fig. 4 is an embodiment of the present invention using porous silica with a diameter of 1.7 μm as a microsphere carrier, using FITC as a doping dye, and giving the microspheres magnetic response properties through the self-assembly of magnetic nanoparticles, prepared. Flow-through decoding results of 7-fold fluorescently encoded microspheres;

Fig. 5 is an embodiment of the present invention using porous silica with a diameter of 5.5 μm as a microsphere carrier, using RITC as a doping dye, and giving the microspheres magnetic response properties through the self-assembly of magnetic nanoparticles, prepared. Flow decoding results of 6-fold fluorescently encoded microspheres;

Fig. 6 is an embodiment of the present invention using porous silica with a diameter of 3.3 μm as a microsphere carrier, using FITC and RITC as doping dyes, and imparting magnetic response properties to the microspheres through the self-assembly of magnetic nanoparticles. The flow decoding result diagram of the obtained 32-fold fluorescent encoded microspheres;

Fig. 7 is an embodiment of the present invention using porous silica with a diameter of 3.3 μm as a microsphere carrier, using FITC and RITC as doping dyes, and imparting magnetic response properties to the microspheres through the self-assembly of magnetic nanoparticles. Magnetic field separation photos of the obtained fluorescently encoded microspheres;

8 is an embodiment of the present invention using porous silica with a diameter of 5.5 μm as the microsphere carrier, using FITC and RITC as doping dyes, and imparting magnetic response properties to the microspheres through the self-assembly of magnetic nanoparticles. The flow decoding result diagram of the obtained 51-fold fluorescent encoded microspheres;

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

detailed description

Example 1

In the encoded microspheres of this embodiment, a group of microsphere carriers with similar diameters and different internal structures are used, and the change in the internal structure of the microsphere carriers is used as the first dimension encoding information; wherein the microsphere carriers are mesoporous microspheres , which has a porous structure, and different internal structures may have different matrix components, or may have different pore sizes, or may also have different matrix components and pore sizes.

The diameter of the microsphere carrier is selected in the range of 0.2 to 20 μm, preferably in the range of 3 to 6 μm. The pore size of the microsphere carrier is selected in the range of 2-100 nm, preferably in the range of 10-60 nm. The matrix components of the microsphere carrier include inorganic substances and polymers. Inorganic substances include silica and/or titania. Polymers include polystyrene, polyacrylic acid, polymethyl acrylate, polymethacrylic acid, polymethyl methacrylate, polydivinylbenzene and/or copolymers of the foregoing polymers.

In order to effectively distinguish the structural coding information, different types of microsphere carriers should have significantly different signal groups in the two-dimensional scattergram of the flow FSC-SSC. In order to achieve a better effect, in this embodiment, the following five types of diameters are preferably: ~5μm microsphere carrier:

(1) Mesoporous silica microspheres with a diameter of 5.2 μm and a pore diameter of 17 nm, hereinafter referred to as SiO ₂ -17;

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

(3) Mesoporous polystyrene microspheres with a diameter of 4.8 μm and a pore diameter of 14 nm, hereinafter referred to as PS-14;

(4) Mesoporous polystyrene microspheres with a diameter of 5.5 μm and a pore diameter of 37 nm, hereinafter referred to as PS-37;

(5) Mesoporous polystyrene microspheres with a diameter of 5.0 μm and a pore diameter of 51 nm, hereinafter referred to as PS-51.

The inner space and outer surface of the microsphere carrier of this embodiment can be loaded with fluorescent materials, and the central emission wavelength of the fluorescent material in the inner space and the central emission wavelength of the fluorescent material on the outer surface are different by more than 30 nm. The fluorescent element contained in the inner space of the sphere carrier preferably emits a green fluorescent dye FITC with a wavelength of 517 nm as the coding element of the second dimension, and the fluorescent element contained on the outer surface of the microsphere carrier preferably emits a CdSe/ZnS red quantum dot with a wavelength of 600 nm as the third dimension. The encoded element of the dimension. It should be noted that the present invention can be implemented in many different forms and is not limited to the preferred parameters and embodiments described herein. These preferred parameters and examples are provided for the purpose of providing a thorough and complete understanding of the present disclosure.

The preparation method of a coded microsphere in this embodiment is described in detail below.

Step 1. Select the microsphere carrier.

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

Step 2. Perform carboxyl functional modification on the selected mesoporous polystyrene microspheres. If mesoporous silica microspheres are selected in step 1, this step can be skipped.

8×10 ⁹ mesoporous polystyrene microspheres were added into 5 mL of chloroform and dispersed by ultrasonic for 30 min, then a solution of chloroform dissolved in 500 mg of PSMA was added, and the mixed solution was ultrasonicated for 40 min. An additional 35 mL of NaOH solution (0.1 M) was added, emulsified with stirring, and sonication was continued for 40 min. The microsphere samples were centrifuged to remove the supernatant and washed with ethanol and water three times in turn. Finally, the obtained microspheres were dispersed in 10 mL of aqueous solution to obtain mesoporous polystyrene microspheres modified with carboxyl groups.

Step 3. Load the fluorescent dye in the inner space of the microsphere carrier.

First, 150 mg of PEI (molecular weight: 750K) was dissolved in 15 mL of NaCl solution (0.5 M), the pH was adjusted to 8.0, and it was recorded as a blank PEI solution. 4.4 mg of FITC was added to the above blank PEI solution, and the reaction was performed overnight at 30°C in the dark to obtain a PEI solution labeled with FITC (referred to as FITC-PEI). Next, mix the FITC-PEI solution and the blank PEI solution in different ratios to prepare a mixed solution with a total volume of 1.5 mL, and add 2×10 ⁷ mesoporous silica microspheres or the mesoporous polymer modified with carboxyl groups obtained in step 2. Styrene microspheres, ultrasonically mixed uniformly, and then rotated in the dark for 20 min. After the reaction, the supernatant was removed by centrifugation, washed three times with water, and the obtained microspheres were dispersed in 0.4 mL of an aqueous solution to obtain a microsphere carrier with a fluorescent dye loaded in the inner space.

Step 4, assembling magnetic nanoparticles on the outer surface of the microsphere carrier.

0.4 mL of the microsphere dispersion obtained in step 3 was added dropwise to 1.1 mL of an aqueous solution containing Fe ₃ O ₄ magnetic nanoparticles (8 nm in particle size, with carboxyl groups on the surface) under ultrasonic conditions, and the reaction was rotated in the dark for 30 min. After the reaction, the supernatant was removed by magnetic separation and washed three times with water. Then, the obtained microspheres were added to 1.5 mL of the blank PEI solution mentioned in step 2, and the reaction was rotated in the dark for 20 min. After the reaction, the supernatant was removed by magnetic separation, and washed with water for three times to obtain magnetic nanoparticles with Fe ₃ O ₄ on the outer surface. A microsphere carrier with PEI modified in the outermost layer.

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

Prepare a chloroform/n-butanol mixed solution of quantum dots: take a certain amount of quantum dots (take CdSe/ZnS quantum dots with a diameter of 6 nm and an emission wavelength of 600 nm as an example) and add them to a total volume of 1 mL of chloroform/n-butanol ( v/v=1:20) in the mixed solution, prepare different quantum dot concentrations and mix them evenly. Take 2 × 10 ⁷ microspheres with PEI modified on the surface obtained in

steps

3 or 4, centrifuge or magnetically separate the supernatant, wash the microspheres with absolute ethanol three times, and then add them to the above-mentioned chloroform/n-butanol containing quantum dots. In the mixed solvent, the reaction was rotated in the dark for 1 h. After the reaction, the supernatant was removed, and the mixture was washed with a mixed solvent of chloroform/n-butanol, ethanol and water in turn. The obtained microspheres were then added to 1 mL of NaCl solution (0.1 M, pH=8.0) containing PEI (molecular weight: 25K) with a concentration of 9 mg/mL, and the reaction was rotated in the dark for 1 h. After the reaction, the supernatant was removed and washed with water three times, namely A microsphere carrier with quantum dots assembled on the outer surface and PEI modified on the outermost layer was obtained.

Step 6: Silica coating and surface functional modification of the microsphere carrier.

Take 2 × 10 ⁷ microspheres with PEI modified on the surface obtained in

steps

3 or 4 or 5, centrifuge or magnetically separate the supernatant, wash the microspheres twice with absolute ethanol, add them to a solution containing 3 mL of ethanol, 0.3 mL of water and In the mixed system of 40 μL TEOS, the reaction was rotated in the dark for 30 min. Then, 22 μL of concentrated ammonia water was added, and the reaction was continued for 22 h under the condition of 30 °C in the dark. After the reaction, the supernatant was removed, and washed three times with absolute ethanol and water in turn to obtain a microsphere carrier coated with silicon oxide. The obtained microspheres were dispersed in 0.25 mL of ethanol, 11.5 μL of concentrated ammonia water and 50 μL of APTES-containing ethanol solution (4.2 μL of APTES diluted in 1.5 mL of ethanol) were added, and the reaction was rotated at 40 °C for 4 h. The supernatant was washed three times with absolute ethanol to obtain a microsphere carrier whose surface was modified with amino groups. The obtained microspheres were washed three times with MEST (10 mM, pH=5.0) solution, dispersed in 1 mL of MEST (10 mM, pH=5.0), 2.5 mg of PAA (molecular weight 5K) and 0.5 mg of EDC were added, and the reaction was rotated. 2h, after the reaction, the supernatant was removed, and washed with water for three times to obtain a microsphere carrier whose surface was modified with carboxyl groups.

According to the above preparation method, encoded microspheres with at most three encoding methods can be obtained, including the encoding method based on the internal structure of the microsphere carrier, the encoding method based on the fluorescent material inside the microsphere carrier, and the encoding method based on the fluorescent material outside the microsphere carrier Way. Likewise, any one or two encoding modes can be selected according to needs, and magnetic nanoparticles can also be selectively added.

For the encoding dimensions involved in encoding microspheres, in addition to the encoding information of one dimension that can be defined according to the internal structure of the microsphere carrier, the encoding information of multiple dimensions can be defined inside and outside the microsphere carrier according to the choice of fluorescent material, such as in the microsphere carrier. Two fluorescent dyes with different central emission wavelengths are used inside the spherical carrier, that is, the encoded information in two dimensions can be defined; the quantum dots with two different central emission wavelengths can be loaded by repeating the method of step 5 to define the encoded information in two dimensions. .

When using microsphere carriers of different diameters (such as ~1 μm, ~3 μm, ~7 μm, etc.), the preparation method of the encoded microspheres is basically the same as the above, and only needs to be slightly adjusted according to the changes in the surface area of the microspheres.

The following lists the combinations of several groups of encoded microsphere arrays, so as to further understand the multi-dimensional encoded information of encoded microspheres and the difference of encoded information among various encoded microspheres in the encoded microsphere array.

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

1. Using SiO ₂ -17 original microspheres directly as the microsphere carrier, then the number of codes I ₁ =1 for the inner space of the microspheres divided according to the level of fluorescence intensity, and the number of codes O ₁ for the outer surface of the microspheres divided by the level of fluorescence intensity =1, the encoding capacity of this type of microspheres is X ₁ =I ₁ ×O ₁ =1.

2. Using SiO ₂ -48 original microspheres directly as the microsphere carrier, the number of codes in the inner space of the microspheres divided by the level of fluorescence intensity I ₂ =1, and the number of codes on the outer surface of the microspheres divided by the level of fluorescence intensity O ₂ =1, the encoding capacity of this type of microspheres is X ₂ =I ₂ ×O ₂ =1.

3. Using the carboxylated PS-14 microspheres obtained in step 2 as the microsphere carrier, the number of codes in the inner space of the microspheres divided according to the level of fluorescence intensity I ₃ =1, and the number of codes on the outer surface of the microspheres divided according to the level of fluorescence intensity The number of codes O ₃ =1, and the code capacity of this type of microspheres is X ₃ =I ₃ ×O ₃ =1.

4. Using the carboxylated PS-37 microspheres obtained in step 2 as the microsphere carrier, the number of codes in the inner space of the microspheres divided according to the fluorescence intensity level I ₄ =1, and the number of codes on the outer surface of the microspheres divided according to the fluorescence intensity levels. The number of codes O ₄ =1, and the code capacity of this type of microspheres is X ₄ =I ₄ ×O ₄ =1.

5. Using the carboxylated PS-51 microspheres obtained in step 2 as the microsphere carrier, the number of codes I ₅ =1 in the inner space of the microspheres divided by the fluorescence intensity The number of codes O ₅ =1, and the code capacity of this type of microspheres is X ₅ =I ₅ ×O ₅ =1.

6. Combining the above five types of microspheres, that is, introducing structural coding information, decoding the structural information through the FSC and SSC scattering light channels of the flow cytometer, and finally constructing a 5-fold coded microsphere array, namely Y=(I ₁ ×O ₁ )+(I ₂ ×O ₂ )+(I ₃ ×O ₃ )+(I ₄ ×O ₄ )+(I ₅ ×O ₅ )=5.

As shown in Figure 1, five types of microsphere carriers with a diameter of about 5 μm are used to read the microspheres by changing the internal structure of the microspheres (including the difference in matrix composition and/or pore size), using a flow cytometer as a decoding platform. The characteristic scattered optical signals (FSC and SSC) of the spheres yielded five clusters with significant signal intensity differences in the FSC-SSC 2D decoded map, revealing that the internal structure of the microsphere carrier can be efficiently transformed into encoded information. Although the five types of microspheres have similar diameters, according to the technical knowledge known in the art, each microsphere should have a similar FSC value; however, after analysis, it was found that the different internal structures of the microsphere carriers will not only affect the SSC signal value, but also cause some There was also a significant difference in the FSC signal values of the vectors, which was not found in the art. That is to say, the internal structure of the microsphere carrier, as its inherent property, can be effectively transformed into optically encoded information, and this new encoding dimension element can be reasonably combined with other encoding elements to greatly increase the encoding capacity.

Coded microsphere array 2 (structure coding of five types of magnetic microspheres of SiO ₂ -17, SiO ₂ -48, PS-14, PS-37 and PS-51):

1. Using SiO ₂ -17 original microspheres as the carrier matrix, assemble magnetic nanoparticles on the outer surface of the microsphere carrier according to

steps

3, 4 and 6 in the above preparation method, and carry out surface coating and modification. Blank PEI is prepared, then the number of codes I ₁ =1 for the inner space of the microspheres divided according to the level of fluorescence intensity, the number of codes for the outer surface of the microspheres divided according to the level of fluorescence intensity O ₁ =1, the coding capacity of this type of magnetic microspheres X ₁ =I ₁ ×O ₁ =1.

2. Using SiO ₂ -48 original microspheres as the carrier matrix, assemble magnetic nanoparticles on the outer surface of the microsphere carrier according to

steps

3, 4 and 6 in the above-mentioned preparation method, and carry out surface coating and modification. If blank PEI is prepared, the number of codes in the inner space of the microspheres divided according to the level of fluorescence intensity I ₂ =1, the number of codes on the outer surface of the microspheres divided by the level of fluorescence intensity O ₂ =1, the coding capacity of this type of magnetic microspheres X ₂ =I ₂ ×O ₂ =1.

3. Using the carboxylated PS-14 microspheres obtained in step 2 of the above preparation method as the carrier matrix, assembling magnetic nanoparticles on the outer surface of the microsphere carrier according to

steps

3, 4 and 6, and performing surface coating and modification , in step 3, blank PEI is used for preparation, then the number of codes in the inner space of the microspheres divided according to the level of fluorescence intensity I ₃ =1, and the number of codes on the outer surface of the microspheres divided by the level of fluorescence intensity O ₃ =1, this type of magnetic The encoding capacity of the microsphere is X ₃ =I ₃ ×O ₃ =1.

4. Using the carboxylated PS-37 microspheres obtained in step 2 of the above preparation method as the carrier matrix, assembling magnetic nanoparticles on the outer surface of the microsphere carrier according to

steps

3, 4 and 6, and carrying out surface coating and modification , in step 3, blank PEI is used for preparation, then the number of codes in the inner space of the microspheres divided according to the level of fluorescence intensity I ₄ =1, and the number of codes on the outer surface of the microspheres divided by the level of fluorescence intensity O ₄ =1, this type of magnetic The encoding capacity of the microsphere is X ₄ =I ₄ ×O ₄ =1.

5. Using the carboxylated PS-51 microspheres obtained in step 2 of the above preparation method as the carrier matrix, assembling magnetic nanoparticles on the outer surface of the microsphere carrier according to

steps

3, 4 and 6 and performing surface coating and modification , in step 3, blank PEI is used for preparation, then the number of codes in the inner space of the microspheres divided according to the level of fluorescence intensity I ₅ =1, and the number of codes on the outer surface of the microspheres divided by the level of fluorescence intensity O ₅ =1, this type of magnetic The encoding capacity of the microsphere is X ₅ =I ₅ ×O ₅ =1.

6. Combine the above five types of magnetic microspheres, that is, introduce structural coding information, decode the structural information through the two scattered light channels of FSC and SSC of the flow cytometer, and finally construct a magnetic coding microsphere array with 5 layers. , that is, Y=(I ₁ ×O ₁ )+(I ₂ ×O ₂ )+(I ₃ ×O ₃ )+(I ₄ ×O ₄ )+(I ₅ ×O ₅ )=5.

Coding microsphere array 3 (structure of four types of microspheres of SiO ₂ -17, SiO ₂ -48, PS-14 and PS-51 + joint coding of internal space fluorescence):

1. Using SiO ₂ -17 original microspheres as the carrier matrix, according to steps 3 and 6 in the above preparation method, the fluorescent dye FITC was loaded into the inner space of the microsphere carrier and surface coated and modified. In step 3, by adjusting the FITC- The mixing ratio of PEI solution and blank PEI solution can obtain 11-recoded microspheres encoded by the inner space of the microspheres according to the level of FITC fluorescence intensity, that is, I ₁ =11, and the number of codes on the outer surface of the microspheres divided by the level of fluorescence intensity O ₁ =1, the encoding capacity of this type of microspheres is X ₁ =I ₁ ×O ₁ =11.

2. Using SiO ₂ -48 original microspheres as the carrier matrix, according to steps 3 and 6 in the above preparation method, the fluorescent dye FITC was loaded into the inner space of the microsphere carrier and surface coated and modified. In step 3, by adjusting the FITC- The mixing ratio of PEI solution and blank PEI solution can obtain 10-encoded microspheres encoded by the inner space of the microspheres according to the level of FITC fluorescence intensity, that is, I ₂ =10, and the number of codes on the outer surface of the microspheres divided by the level of fluorescence intensity O ₂ =1, the encoding capacity of this type of microspheres is X ₂ =I ₂ ×O ₂ =10.

3. Using the carboxylated PS-14 microspheres obtained in step 2 of the above preparation method as the carrier matrix, loading the fluorescent dye FITC in the inner space of the microsphere carrier according to steps 3 and 6, and carrying out surface coating and modification, step 3. By adjusting the mixing ratio of the FITC-PEI solution and the blank PEI solution, the 10-encoded microspheres encoded by the inner space of the microspheres according to the level of FITC fluorescence intensity are obtained, that is, I ₃ =10, while the microspheres divided according to the level of fluorescence intensity The number of codes on the outer surface O ₃ =1, and the code capacity of this type of microspheres is X ₃ =I ₃ ×O ₃ =10.

4. Using the carboxylated PS-51 microspheres obtained in step 2 of the above preparation method as the carrier matrix, loading the fluorescent dye FITC in the inner space of the microsphere carrier according to steps 3 and 6 and carrying out surface coating and modification, step 3 By adjusting the mixing ratio of the FITC-PEI solution and the blank PEI solution, the 9-encoded microspheres encoded by the inner space of the microspheres according to the level of FITC fluorescence intensity are obtained, that is, I ₄ =9, while the microspheres divided according to the level of fluorescence intensity The number of codes on the outer surface O ₄ =1, and the code capacity of this type of microspheres is X ₄ =I ₄ ×O ₄ =9.

5. Combining the above four types of microspheres, that is, introducing structural coding information, decoding the structural information through the FSC and SSC scattered light channels of the flow cytometer, and then decoding the internal space fluorescence information through the fluorescence detection channel, Finally, an encoded microsphere array with 40 layers was constructed, ie, Y=(I ₁ ×O ₁ )+(I ₂ ×O ₂ )+(I ₃ ×O ₃ )+(I ₄ ×O ₄ )=40.

Coded microsphere array 4 (structure of three types of magnetic microspheres of SiO ₂ -17, SiO ₂ -48 and PS-51 + combined coding of internal space fluorescence):

1. Using SiO ₂ -17 original microspheres as the carrier matrix, according to

steps

3, 4 and 6 in the above preparation method, the inner space of the microsphere carrier is loaded with the fluorescent dye FITC, and the outer surface is assembled with magnetic nanoparticles and surface-coated and modification, in step 3, by adjusting the mixing ratio of the FITC-PEI solution and the blank PEI solution, the 11-recoded microspheres encoded by the internal space of the microspheres divided according to the level of FITC fluorescence intensity are obtained, that is, I ₁ =11, and according to the fluorescence intensity The number of codes on the outer surface of the horizontally divided microspheres is O ₁ =1, and the coding capacity of this type of magnetic microspheres is X ₁ =I ₁ ×O ₁ =11.

2. Using SiO ₂ -48 original microspheres as the carrier matrix, according to

steps

3, 4 and 6 in the above preparation method, the inner space of the microsphere carrier is loaded with the fluorescent dye FITC, and the outer surface is assembled with magnetic nanoparticles and surface-coated and modification, in step 3, by adjusting the mixing ratio of the FITC-PEI solution and the blank PEI solution, the 10-recoded microspheres encoded by the internal space of the microspheres divided according to the level of FITC fluorescence intensity are obtained, that is, I ₂ =10, and according to the fluorescence intensity The number of codes on the outer surface of the horizontally divided microspheres is O ₂ =1, and the coding capacity of this type of magnetic microspheres is X ₂ =I ₂ ×O ₂ =10.

3. Using the carboxylated PS-51 microspheres obtained in step 2 of the above preparation method as the carrier matrix, load the fluorescent dye FITC in the inner space of the microsphere carrier according to

steps

3, 4 and 6, and carry out surface coating and modification , in step 3, by adjusting the mixing ratio of the FITC-PEI solution and the blank PEI solution, the 9-encoded microspheres encoded by the internal space of the microspheres divided according to the level of FITC fluorescence intensity are obtained, that is, I ₃ =9, and the level of fluorescence intensity is divided according to The number of codes on the outer surface of the microspheres is O ₃ =1, and the code capacity of this type of magnetic microspheres is X ₃ =I ₃ ×O ₃ =9.

4. Combine the above three types of magnetic microspheres, that is, introduce structural coding information, decode the structural information through the FSC and SSC scattered light channels of the flow cytometer, and then decode the internal spatial fluorescence information through the fluorescence detection channel. , and finally a magnetically encoded microsphere array with 30 layers was constructed, that is, Y=(I ₁ ×O ₁ )+(I ₂ ×O ₂ )+(I ₃ ×O ₃ )=30.

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

1. Using SiO ₂ -17 original microspheres as the carrier matrix, according to

steps

3, 5 and 6 in the above preparation method, assemble quantum dots on the outer surface of the microsphere carrier and carry out surface coating and modification, in step 3, use blank PEI is prepared, then the number of codes I ₁ = 1 in the inner space of the microspheres divided according to the level of fluorescence intensity. In step 5, by adjusting the concentration of quantum dots added, the 6 codes of the outer surface of the microspheres divided according to the level of fluorescence intensity of quantum dots are obtained. Recoded microspheres, ie O ₁ =6, the encoding capacity of this type of microspheres is X ₁ =I ₁ ×O ₁ =6.

2. Using the carboxylated PS-14 microspheres obtained in step 2 of the above preparation method as a carrier matrix, assembling quantum dots on the outer surface of the microsphere carrier according to

steps

3, 5 and 6, and carrying out surface coating and modification, In step 3, blank PEI is used for preparation, then the number of codes in the inner space of the microspheres divided according to the level of fluorescence intensity I ₂ =1, and in step 5 by adjusting the concentration of quantum dots added, the microspheres divided according to the level of fluorescence intensity of quantum dots are obtained For the 6-encoded microspheres encoded on the outer surface, that is, O ₂ =6, the encoding capacity of this type of microspheres is X ₂ =I ₂ ×O ₂ =6.

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

steps

3, 5 and 6, and performing surface coating and modification, In step 3, blank PEI is used for preparation, then the number of codes in the inner space of the microspheres divided according to the level of fluorescence intensity I ₃ =1, and in step 5 by adjusting the concentration of quantum dots added, the microspheres divided according to the level of fluorescence intensity of quantum dots are obtained For the 6-encoded microspheres encoded on the outer surface, that is, O ₃ =6, the encoding capacity of this type of microspheres is X ₃ =I ₃ ×O ₃ =6.

4. Combining the above three types of microspheres, that is, introducing structural coding information, decoding the structural information through the FSC and SSC scattered light channels of the flow cytometer, and then decoding the external surface fluorescence information through the fluorescence detection channel, and finally. An encoded microsphere array with 18-fold was constructed, ie Y=(I ₁ ×O ₁ )+(I ₂ ×O ₂ )+(I ₃ ×O ₃ )=18.

Coding microsphere array 6 (structure of four types of magnetic microspheres of SiO ₂ -17, SiO ₂ -48, PS-14 and PS-51 + combined coding of external surface fluorescence):

1. Using SiO ₂ -17 original microspheres as the carrier matrix, assemble magnetic nanoparticles and quantum dots on the outer surface of the microsphere carrier according to

steps

3, 4, 5 and 6 in the above preparation method, and carry out surface coating and Modification, in step 3, blank PEI is used for preparation, then the number of codes I ₁ = 1 in the inner space of the microspheres divided according to the level of fluorescence intensity, and in step 5 by adjusting the concentration of quantum dots added, the number of codes divided according to the level of fluorescence intensity of quantum dots is obtained. For the 6-encoded microspheres encoded on the outer surface of the microspheres, that is, O ₁ =6, the encoding capacity of this type of magnetic microspheres is X ₁ =I ₁ ×O ₁ =6.

2. Using SiO ₂ -48 original microspheres as the carrier matrix, assemble magnetic nanoparticles and quantum dots on the outer surface of the microsphere carrier according to

steps

3, 4, 5 and 6 in the above preparation method, and perform surface coating and Modification, in step 3, blank PEI is used for preparation, then the number of codes in the inner space of the microspheres divided according to the level of fluorescence intensity I ₂ =1, and in step 5 by adjusting the concentration of quantum dots added, the number of codes divided according to the level of fluorescence intensity of quantum dots is obtained. For the 6-encoded microspheres coded on the outer surface of the microspheres, that is, O ₂ =6, the coding capacity of this type of magnetic microspheres is X ₂ =I ₂ ×O ₂ =6.

3. Using the carboxylated PS-14 microspheres obtained in step 2 of the above preparation method as the carrier matrix, assembling magnetic nanoparticles and quantum dots on the outer surface of the microsphere carrier according to

steps

3, 4, 5 and 6 and then assembling magnetic nanoparticles and quantum dots. Surface coating and modification are carried out. In step 3, blank PEI is used for preparation, and the number of codes in the inner space of the microsphere divided according to the level of fluorescence intensity I ₃ =1. The 6-encoded microspheres coded on the outer surface of the microspheres divided by the level of fluorescence intensity, namely O ₃ =6, the coding capacity of this type of magnetic microspheres is X ₃ =I ₃ ×O ₃ =6.

4. Use the carboxylated PS-51 microspheres obtained in step 2 of the above preparation method as the carrier matrix, and assemble magnetic nanoparticles and quantum dots on the outer surface of the microsphere carrier according to

steps

3, 4, 5 and 6. Surface coating and modification are carried out. In step 3, blank PEI is used for preparation, and the number of codes in the inner space of the microsphere divided according to the level of fluorescence intensity I ₄ =1. The 6-encoded microspheres coded on the outer surface of the microspheres divided by the level of fluorescence intensity, namely O ₄ =6, the coding capacity of this type of magnetic microspheres is X ₄ =I ₄ ×O ₄ =6.

5. Combine the above four types of magnetic microspheres, that is, introduce structural coding information, decode the structural information through the FSC and SSC scattered light channels of the flow cytometer, and then decode the outer surface fluorescence information through the fluorescence detection channel, Finally, a magnetically encoded microsphere array with 24 layers was constructed, namely Y=(I ₁ ×O ₁ )+(I ₂ ×O ₂ )+(I ₃ ×O ₃ )+(I ₄ ×O ₄ )=24.

Coding microsphere array 7 (structure of four types of magnetic microspheres of SiO ₂ -17, SiO ₂ -48, PS-14 and PS-37 + combined coding of external surface fluorescence):

In this combination, the CdSe/ZnS quantum dots preferably assembled on the outer surface of the microsphere carrier with an emission wavelength of 600 nm are adjusted to NaYF ₄ :Er,Yb up-conversion fluorescent nanoparticles with an emission wavelength of 535 nm. Step 5 in the above preparation method is adjusted to: assemble upconversion fluorescent nanoparticles on the outer surface of the microsphere carrier through a coordination reaction. Preparation of chloroform/n-butanol mixed solution of up-converting fluorescent nanoparticles: take a certain amount of up-converting fluorescent nanoparticles (NaYF ₄ :Er, Yb up-converting fluorescent nanoparticles with a diameter of 25 nm and an emission wavelength of 535 nm) and add it to the total volume In 1 mL of chloroform/n-butanol (v/v=1:20) mixed solution, different concentrations of up-converting fluorescent nanoparticles were prepared and mixed uniformly. Take 2 × 10 ⁷ microspheres with PEI modified on the surface obtained in step 4, magnetically separate to remove the supernatant, wash the microspheres with absolute ethanol three times, and then add to the above-mentioned chloroform/n-butanol mixture containing up-converting fluorescent nanoparticles. In the solvent, the reaction was rotated in the dark for 1 h. After the reaction, the supernatant was removed, and the mixture was washed with a mixed solvent of chloroform/n-butanol, ethanol and water in turn. The obtained microspheres were then added to 1 mL of NaCl solution (0.1 M, pH=8.0) containing PEI (molecular weight: 25K) with a concentration of 9 mg/mL, and the reaction was rotated in the dark for 1 h. After the reaction, the supernatant was removed and washed with water three times, namely The microsphere carrier whose outer surface is equipped with up-conversion fluorescent nanoparticles and whose outermost layer is modified with PEI is obtained.

1. Using SiO ₂ -17 original microspheres as the carrier matrix, according to the

steps

3, 4, 5 (step 5 adjusted according to the encoded microsphere array 7) and step 6 in the above-mentioned preparation method on the outer surface of the microsphere carrier Assemble magnetic nanoparticles and up-converting fluorescent nanoparticles and carry out surface coating and modification. In step 3, blank PEI is used to prepare, then the number of codes I ₁ = 1 in the inner space of the microsphere divided according to the fluorescence intensity level, step 5 (according to In step 5) of the adjustment of the encoded microsphere array 7, by adjusting the added concentration of the up-conversion fluorescent nanoparticles, the 5-fold encoded microspheres encoded on the outer surface of the microspheres divided according to the fluorescence intensity levels of the up-conversion fluorescent nanoparticles are obtained, that is, O ₁ =5, the encoding capacity of the magnetic microspheres is X ₁ =I ₁ ×O ₁ =5.

2. Using SiO ₂ -48 original microspheres as the carrier matrix, according to

steps

3, 4, 5 (step 5 adjusted according to the coding microsphere array 7) and step 6 in the above-mentioned preparation method on the outer surface of the microsphere carrier Assemble magnetic nanoparticles and up-converting fluorescent nanoparticles and carry out surface coating and modification. In step 3, blank PEI is used for preparation, and the number of codes in the inner space of the microspheres divided according to the level of fluorescence intensity I ₂ =1, step 5 (according to In step 5) adjusted by the encoded microsphere array 7, by adjusting the added concentration of the up-conversion fluorescent nanoparticles, the 5-fold encoded microspheres encoded on the outer surface of the microspheres divided according to the fluorescence intensity levels of the up-conversion fluorescent nanoparticles are obtained, namely O ₂ =5, the encoding capacity of the magnetic microspheres is X ₂ =I ₂ ×O ₂ =5.

3. Use the carboxylated PS-14 microspheres obtained in step 2 of the above preparation method as the carrier matrix, and follow

steps

3, 4, 5 (step 5 adjusted according to the encoded microsphere array 7) and step 6 in the microspheres. The outer surface of the spherical carrier is assembled with magnetic nanoparticles and up-converted fluorescent nanoparticles, and the surface is coated and modified. In step 3, blank PEI is used for preparation, and the number of codes in the inner space of the microsphere divided according to the level of fluorescence intensity I ₃ =1 In step 5 (step 5 adjusted according to the encoding microsphere array 7), by adjusting the added concentration of the up-converted fluorescent nanoparticles, the 5-fold encoded microspheres encoded on the outer surface of the microspheres divided according to the fluorescence intensity levels of the up-converted fluorescent nanoparticles are obtained. ball, namely O ₃ =5, the encoding capacity of this type of magnetic microsphere is X ₃ =I ₃ ×O ₃ =5.

4. Use the carboxylated PS-37 microspheres obtained in step 2 of the above preparation method as the carrier matrix, and follow

steps

3, 4, 5 (step 5 adjusted according to the encoded microsphere array 7) and step 6 in the microspheres. Magnetic nanoparticles and up-conversion fluorescent nanoparticles are assembled on the outer surface of the sphere carrier, and the surface is coated and modified. In step 3, blank PEI is used for preparation, and the number of codes in the inner space of the microsphere divided according to the level of fluorescence intensity I ₄ =1 In step 5 (step 5 adjusted according to the encoding microsphere array 7), by adjusting the added concentration of the up-converted fluorescent nanoparticles, the 5-fold encoded microspheres encoded on the outer surface of the microspheres divided according to the fluorescence intensity levels of the up-converted fluorescent nanoparticles are obtained. ball, namely O ₄ =5, the encoding capacity of this type of magnetic microsphere is X ₄ =I ₄ ×O ₄ =5.

5. Combine the above four types of magnetic microspheres, that is, introduce structural coding information, decode the structural information through the FSC and SSC scattered light channels of the flow cytometer, and then decode the outer surface fluorescence information through the fluorescence detection channel, Finally, a magnetically encoded microsphere array with 20 layers was constructed, ie, Y=(I ₁ ×O ₁ )+(I ₂ ×O ₂ )+(I ₃ ×O ₃ )+(I ₄ ×O ₄ )=20.

Coded microsphere array 8 (structure of three types of microspheres of SiO ₂ -17, SiO ₂ -48 and PS-51 + combined coding of inner space fluorescence + outer surface fluorescence):

1. Using SiO ₂ -17 original microspheres as the carrier matrix, according to

steps

3, 5 and 6 in the above preparation method, the inner space of the microsphere carrier is loaded with the fluorescent dye FITC, the outer surface is assembled with quantum dots, and surface coating and Modification, in step 3, by adjusting the mixing ratio of FITC-PEI solution and blank PEI solution, the 11-recoded microspheres encoded by the internal space of the microspheres divided according to the level of FITC fluorescence intensity are obtained, that is, I ₁ =11, and in step 5, by adjusting The added concentration of quantum dots can obtain 6-encoded microspheres encoded on the outer surface of the microspheres according to the level of fluorescence intensity of the quantum dots, that is, O ₁ =6, and the encoding capacity of this type of microspheres is X ₁ =I ₁ ×O ₁ =66 .

2. Using SiO ₂ -48 original microspheres as the carrier matrix, according to

steps

3, 5 and 6 in the above preparation method, the inner space of the microsphere carrier is loaded with the fluorescent dye FITC, the outer surface is assembled with quantum dots, and surface coating and Modification, in step 3, by adjusting the mixing ratio of FITC-PEI solution and blank PEI solution, 10-encoded microspheres with internal space encoding of microspheres divided according to the level of FITC fluorescence intensity are obtained, that is, I ₂ =10, and in step 5, by adjusting The added concentration of quantum dots can obtain 6-encoded microspheres encoded on the outer surface of the microspheres according to the level of fluorescence intensity of the quantum dots, that is, O ₂ =6, and the encoding capacity of this type of microspheres is X ₂ =I ₂ ×O ₂ =60 .

3. Using the carboxylated PS-51 microspheres obtained in step 2 of the above preparation method as the carrier matrix, according to

steps

3, 5 and 6, the inner space of the microsphere carrier is loaded with the fluorescent dye FITC, and the outer surface is assembled with quantum dots. Surface coating and modification are carried out. In step 3, by adjusting the mixing ratio of the FITC-PEI solution and the blank PEI solution, 9-encoded microspheres with internal space encoding of the microspheres divided according to the level of FITC fluorescence intensity are obtained, that is, I ₃ =9, In step 5, by adjusting the concentration of quantum dots added, 6-encoded microspheres encoded on the outer surface of the microspheres divided according to the level of fluorescence intensity of the quantum dots are obtained, that is, O ₃ =6, and the encoding capacity of this type of microspheres is X ₃ =I ₃ ×O ₃ =54.

4. Combine the above three types of microspheres, that is, introduce structural coding information, decode the structural information through the FSC and SSC scattered light channels of the flow cytometer, and then use the fluorescence detection channel to detect the inner space and outer surface respectively. The fluorescence information was decoded, and finally an encoded microsphere array with 180 layers was constructed, ie, Y=(I ₁ ×O ₁ )+(I ₂ ×O ₂ )+(I ₃ ×O ₃ )=180.

Coded microsphere array 9 (structure of three types of microspheres of SiO ₂ -17, SiO ₂ -48 and PS-37 + combined coding of inner space fluorescence + outer surface fluorescence):

In this combination, the CdSe/ZnS quantum dots preferably assembled on the outer surface of the microsphere carrier with an emission wavelength of 600 nm are adjusted to conjugated polymer fluorescent nanoparticles with an emission wavelength of 580 nm. Step 5 is adjusted to: assemble the conjugated polymer fluorescent nanoparticles on the outer surface of the microsphere carrier by electrostatic reaction. 0.4 mL of microsphere dispersion obtained in step 3 was added dropwise to 1.1 mL of aqueous solution containing different concentrations of conjugated polymer fluorescent nanoparticles (30 nm in particle size, with carboxyl groups on the surface) under ultrasonic conditions, and the reaction was rotated in the dark. 30min. After the reaction, the supernatant was removed by centrifugation and washed three times with water. Then, the obtained microspheres were added to 1.5 mL of the blank PEI solution mentioned in step 3, and rotated for 20 minutes in the dark. After the reaction, the supernatant was removed by centrifugation, and washed with water three times to obtain fluorescent nanoparticles equipped with conjugated polymers on the outer surface. And the outermost layer is modified with PEI microsphere carrier.

1. Using SiO ₂ -17 original microspheres as the carrier matrix, load fluorescent dyes in the inner space of the microsphere carrier according to steps 3, 5 (step 5 adjusted according to the coding microsphere array 9) and step 6 in the above-mentioned preparation method FITC and the outer surface are assembled with conjugated polymer fluorescent nanoparticles, and the surface is coated and modified. In step 3, by adjusting the mixing ratio of the FITC-PEI solution and the blank PEI solution, the inner space code of the microspheres divided according to the level of FITC fluorescence intensity is obtained. 11 re-encoded microspheres, that is, I ₁ =11, in step 5 (step 5 adjusted according to the encoded microsphere array 9), by adjusting the added concentration of the conjugated polymer fluorescent nanoparticles, the conjugated polymer fluorescent nanoparticles are obtained according to The 5-fold coded microspheres encoded on the outer surface of the microspheres divided by the level of particle fluorescence intensity, namely O ₁ =5, the coding capacity of this type of microspheres is X ₁ =I ₁ ×O ₁ =55.

2. Using SiO ₂ -48 original microspheres as the carrier matrix, load fluorescent dyes in the inner space of the microsphere carrier according to steps 3, 5 (step 5 adjusted according to the coding microsphere array 9) and step 6 in the above-mentioned preparation method FITC and the outer surface are assembled with conjugated polymer fluorescent nanoparticles, and the surface is coated and modified. In step 3, by adjusting the mixing ratio of the FITC-PEI solution and the blank PEI solution, the inner space code of the microspheres divided according to the level of FITC fluorescence intensity is obtained. The 10-fold encoded microspheres, that is, I ₂ =10, in step 5 (step 5 adjusted according to the encoded microsphere array 9), by adjusting the added concentration of the conjugated polymer fluorescent nanoparticles, the conjugated polymer fluorescent nanoparticles are obtained according to The 5-fold coded microspheres encoded on the outer surface of the microspheres divided by the level of particle fluorescence intensity, that is, O ₂ =5, the coding capacity of this type of microspheres is X ₂ =I ₂ ×O ₂ =50.

3. Use the carboxylated PS-37 microspheres obtained in step 2 in the above preparation method as the carrier matrix, and follow steps 3, 5 (step 5 adjusted according to coding microsphere array 9) and step 6 in the microsphere carrier. The inner space is loaded with the fluorescent dye FITC, and the outer surface is equipped with conjugated polymer fluorescent nanoparticles, and the surface is coated and modified. The 10-fold encoded microspheres encoded by the internal space of the microspheres, that is, I ₃ =10, in step 5 (step 5 adjusted according to the encoded microsphere array 9 ), by adjusting the added concentration of the conjugated polymer fluorescent nanoparticles, according to the The 5-fold coded microspheres encoded on the outer surface of the microspheres with the fluorescence intensity level division of the conjugated polymer fluorescent nanoparticles, namely O ₃ =5, the coding capacity of this type of microspheres is X ₃ =I ₃ ×O ₃ =50.

4. Combine the above three types of microspheres, that is, introduce structural coding information, decode the structural information through the FSC and SSC scattered light channels of the flow cytometer, and then use the fluorescence detection channel to detect the inner space and outer surface respectively. The fluorescence information was decoded, and finally an encoded microsphere array with 155 layers was constructed, ie, Y=(I ₁ ×O ₁ )+(I ₂ ×O ₂ )+(I ₃ ×O ₃ )=155.

Coded microsphere array 10 (structure of five types of magnetic microspheres of SiO ₂ -17, SiO ₂ -48, PS-14, PS-37 and PS-51 + combined coding of inner space fluorescence + outer surface fluorescence):

1. Using SiO ₂ -17 original microspheres as the carrier matrix, according to

steps

3, 4, 5 and 6 in the above preparation method, the inner space of the microsphere carrier is loaded with the fluorescent dye FITC, and the outer surface is assembled with magnetic nanoparticles and quantum particles. Point and carry out surface coating and modification. In step 3, by adjusting the mixing ratio of FITC-PEI solution and blank PEI solution, the 11-recoded microspheres encoded by the internal space of the microspheres divided according to the level of FITC fluorescence intensity are obtained, that is, I ₁ = 11. In step 5, by adjusting the concentration of the quantum dots added, the 6-fold encoded microspheres encoded on the outer surface of the microspheres divided according to the level of the fluorescence intensity of the quantum dots are obtained, that is, O ₁ =6, and the encoding capacity of this type of magnetic microspheres is X ₁ =I ₁ ×O ₁ =66.

2. Using SiO ₂ -48 original microspheres as the carrier matrix, according to

steps

3, 4, 5 and 6 in the above preparation method, the inner space of the microsphere carrier is loaded with the fluorescent dye FITC, and the outer surface is assembled with magnetic nanoparticles and quantum particles. Point and carry out surface coating and modification. In step 3, by adjusting the mixing ratio of FITC-PEI solution and blank PEI solution, 10-encoded microspheres with internal space encoding of the microspheres divided according to the level of FITC fluorescence intensity are obtained, that is, I ₂ = 10. In step 5, by adjusting the concentration of quantum dots added, 6-fold encoded microspheres encoded on the outer surface of the microspheres divided according to the level of fluorescence intensity of the quantum dots are obtained, that is, O ₂ =6, and the encoding capacity of this type of magnetic microspheres is X ₂ =I ₂ ×O ₂ =60.

3. The carboxylated PS-14 microspheres obtained in step 2 of the above preparation method are used as the carrier matrix, and the fluorescent dye FITC is loaded in the inner space of the microsphere carrier according to

steps

3, 4, 5 and 6, and the outer surface is assembled The magnetic nanoparticles and quantum dots are coated and modified on the surface. In step 3, by adjusting the mixing ratio of the FITC-PEI solution and the blank PEI solution, 10-encoded microspheres with internal space encoding of the microspheres divided according to the level of FITC fluorescence intensity are obtained. , that is, I ₃ =10. In step 5, by adjusting the concentration of quantum dots added, the 6-encoded microspheres coded on the outer surface of the microspheres divided according to the level of the fluorescence intensity of the quantum dots are obtained, that is, O ₃ =6. This kind of magnetic microspheres The encoding capacity of X ₃ =I ₃ ×O ₃ =60.

4. The carboxylated PS-37 microspheres obtained in step 2 of the above preparation method are used as the carrier matrix, and the fluorescent dye FITC is loaded in the inner space of the microsphere carrier according to

steps

3, 4, 5 and 6, and the outer surface is assembled The magnetic nanoparticles and quantum dots are coated and modified on the surface. In step 3, by adjusting the mixing ratio of the FITC-PEI solution and the blank PEI solution, 10-encoded microspheres with internal space encoding of the microspheres divided according to the level of FITC fluorescence intensity are obtained. , that is, I ₄ =10. In step 5, by adjusting the concentration of quantum dots added, 6-encoded microspheres coded on the outer surface of the microspheres divided according to the level of the fluorescence intensity of the quantum dots are obtained, that is, O ₄ =6. This kind of magnetic microspheres The encoding capacity of X ₄ =I ₄ ×O ₄ =60.

5. Using the carboxylated PS-51 microspheres obtained in step 2 of the above preparation method as the carrier matrix, according to

steps

3, 4, 5 and 6, the inner space of the microsphere carrier is loaded with fluorescent dye FITC, and the outer surface is assembled Magnetic nanoparticles and quantum dots are coated and modified on the surface. In step 3, by adjusting the mixing ratio of FITC-PEI solution and blank PEI solution, 9-encoded microspheres with internal space encoding of the microspheres divided according to the level of FITC fluorescence intensity are obtained. , that is, I ₅ =9. In step 5, by adjusting the concentration of quantum dots added, the 6-encoded microspheres coded on the outer surface of the microspheres divided according to the level of the fluorescence intensity of the quantum dots are obtained, that is, O ₅ =6. This kind of magnetic microspheres The encoding capacity of X ₅ =I ₅ ×O ₅ =54.

6. The above five types of magnetic microspheres are combined, that is, the structural coding information is introduced, the structural information is decoded through the FSC and SSC scattered light channels of the flow cytometer, and the inner space and the outer surface are respectively analyzed through the fluorescence detection channel. Decode the fluorescence information of , and finally construct a 300-fold magnetically encoded microsphere array, that is, Y=(I ₁ ×O ₁ )+(I ₂ ×O ₂ )+(I ₃ ×O ₃ )+(I ₄ × O ₄ )+(I ₅ ×O ₅ )=300.

As shown in Figure 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 are convenient for the manipulation of the microspheres under a magnetic field. . In addition, the saturation magnetization of the five types of magnetically encoded microspheres is 0.81-1.71 emu/g, reflecting good magnetic response performance.

As shown in Fig. 3, five types of microspheres with different internal structures are used as the carrier matrix, the inner space of the microsphere carrier is loaded with the fluorescent dye FITC, the outer surface is assembled with magnetic nanoparticles and quantum dots, and the surface is coated and modified. , the final obtained five types of magnetically encoded microspheres still have significant signal intensity differences in the flow-through FSC-SSC two-dimensional scattered light decoding map. Although the positions of the microspheres have changed compared with the original microspheres, the five types of microspheres are clearly grouped with each other, which once again proves that the structure of the microspheres has a corresponding relationship with the FSC-SSC scattering signal; at the same time, each type of microspheres contains 54-66 The fluorescent coding microspheres are heavy, but the FSC-SSC coding positions of the same type of microspheres are distributed in a small range, which proves the controllability and repeatability of the preparation method in the present invention. On the basis of decoding the structure of five types of microspheres, continue to read the fluorescence information of the FITC channel and QDs channel of each type of microspheres for decoding, and five independent two-dimensional coding arrays with coding multiples of 54 to 66 can be obtained. And the distinction between each scatter point is obvious. Combining the two-dimensional encoded arrays of five types of magnetically encoded microspheres, a 300-fold ultra-high-throughput magnetically encoded microsphere array was obtained. This result shows that the structural encoding of the microsphere carrier is a new coding element developed by the present invention, and the combination of fluorescent coding can significantly improve the coding capacity of the coding microsphere array. In addition, the four decoding parameters involved in the encoded microsphere array, namely FSC intensity, SSC intensity, FITC fluorescence intensity and QDs fluorescence intensity, can all be excited by 488nm monochromatic excitation light, which greatly reduces the decoding cost and enables decoding The process becomes simpler and more convenient, and 300 multiples is also the highest coding capacity of the single-color laser excitation that has been reported so far. The encoded microsphere array proposed by the present invention has a great application prospect in the ultra-high-throughput multi-index detection.

Embodiment 2

As mentioned above, according to the preparation method of Embodiment 1, 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, so as to further increase the dimension of the encoding and amplify the encoding quantity.

Similarly, on the basis of the preparation method in Example 1, two or more fluorescent dyes with different central emission wavelengths can also be loaded inside the microsphere carrier. The key point of this embodiment is to further describe the preparation method of encoded microspheres loaded with one or more fluorescent dyes in the microsphere carrier. The encoded microspheres in this embodiment can be used alone, or this The method of the embodiment is applied in step 2 of the first embodiment, thereby expanding the encoding dimension of the encoded microspheres in the first embodiment.

The design of the preparation method of the encoded microspheres of the present embodiment mainly includes the following steps:

Step 1. Covalently label the polymer molecules with X kinds of fluorescent dyes to obtain X kinds of fluorescently labeled polymer molecule solutions, X≥1; and then combine the obtained X kinds of polymer molecule solutions with unfluorescently labeled polymer molecules. The molecular solutions are mixed in different proportions to obtain a mixed solution M;

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

Step 3, adding the magnetic nanoparticles into the fluorescent dye-doped microspheres obtained in step 2 to carry out physical adsorption to assemble them on the outer surface of the microspheres, and cleaning after the reaction; Fluorescent dye-doped microspheres with magnetic nanoparticles assembled on the outer surface and amino polymers on the outermost layer are obtained; the added amount of the magnetic nanoparticles accounts for ≥0% of the mass proportion of the fluorescent dye-doped microspheres;

Step 4. In

step

2 or 3, the surface of the microsphere obtained in

step

2 or 3 is covered with a silicon oxide protective shell layer, thereby obtaining a fluorescent dye-doped encoded microsphere. After the silicon oxide coating is completed, the outer surface of the microsphere carrier can be modified with functional molecules, thereby obtaining surface-functionalized encoded microspheres.

The diameter of the porous microspheres is 0.1-100 μm, the pore diameter is 2-100 nm, and the matrix components of the microspheres include inorganic substances and polymers. Inorganic substances include silica and/or titania. Polymers include polystyrene, polyacrylic acid, polymethyl acrylate, polymethacrylic acid, polymethyl methacrylate, polydivinylbenzene and/or copolymers of the foregoing polymers.

Physical/chemical interactions include electrostatic interactions, hydrophilic-hydrophobic interactions, hydrogen bonding interactions, coordination interactions, covalent bonding interactions, preferably electrostatic interactions.

The magnetic nanoparticles are Fe ₃ O ₄ nanoparticles or γ-Fe ₂ O ₃ nanoparticles, preferably Fe ₃ O ₄ nanoparticles.

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

Fluorescent dyes include fluorescein isothiocyanate (FITC), rhodamine B isothiocyanate (RITC), Cy5-N-hydroxysuccinimide ester (Cy5-NHS), 5-aminofluorescein (5-AF) ; polymer molecules include polyethyleneimine (PEI), polyacrylic acid (PAA); porous microspheres include porous silica microspheres, carboxylated porous polystyrene microspheres, and porous silica modified with epoxy groups Microspheres, Porous Polystyrene Microspheres Modified with Epoxy Groups, Aminated Porous Silica Microspheres, Aminated Porous Polystyrene Microspheres.

The specific preparation method is further described below.

Preparation method 1 includes the following steps:

1. Dissolve 150mg of PEI (molecular weight: 750K) in 15mL of NaCl solution (0.5M), adjust the pH to 8.0, and record it as a blank PEI solution; add 4.4mg of FITC to the above blank PEI solution, at 30°C Under the condition of avoiding light and shaking overnight, the PEI solution labeled with FITC (referred to as FITC-PEI) was obtained; the FITC-PEI solution and the blank PEI solution were mixed in different ratios to prepare a mixed solution with a total volume of 1.5 mL;

2. Add 6.4×10 ⁸ porous silica microspheres with a diameter of 1.7 μm to the above mixed solution, mix uniformly by ultrasonic, and rotate for 20 minutes in the dark, and bind FITC-PEI and PEI to the interior of the microspheres by electrostatic action. After the reaction, the supernatant was removed by centrifugation, washed three times with water, and the obtained microspheres were dispersed in 0.4 mL of aqueous solution to obtain microspheres with fluorescent dyes loaded in the inner space;

3. Add 0.4 mL of the above-obtained microsphere dispersion dropwise to 1.1 mL of the aqueous solution containing Fe ₃ O ₄ magnetic nanoparticles (with a particle size of 8 nm and a carboxyl group on the surface) under ultrasonic conditions, and rotate for 30 min in the dark. . After the reaction, the supernatant was removed by magnetic separation, and washed three times with water; then the obtained microspheres were added to 1.5 mL of the blank PEI solution mentioned in step 1, and rotated in the dark for 20 min. After the reaction, the supernatant was removed by magnetic separation, and washed with water three times. That is, fluorescent microspheres with Fe ₃ O ₄ magnetic nanoparticles assembled on the outer surface and PEI modified on the outermost layer are obtained;

4. Magnetically separate the microspheres obtained in the above step 3 to remove the supernatant, wash the microspheres twice with absolute ethanol, and add them to a mixed system containing 3 mL of ethanol, 0.3 mL of water and 40 μL of TEOS, and rotate in the dark for 30 min; then , add 22 μL of concentrated ammonia water, continue to rotate in the dark at 30 °C for 22 h, remove the supernatant after the reaction, and wash three times with absolute ethanol and water in turn to obtain a FITC fluorescent dye doped with silica coated on the surface. Magnetically encoded microspheres.

In the magnetic fluorescent coding microspheres prepared by preparation method 1, the fluorescent coding information of the FITC dye can be excited by the excitation light of 488nm of the flow cytometer, and the emission band received by the filter is 515±10nm; The excitation light is excited, and the emission band received by the filter is 515±15nm.

As shown in Figure 4, using porous silica with a diameter of 1.7 μm as the microsphere carrier and using FITC as the doping dye, seven fluorescent dye-doped coded microspheres distinguishable on the flow cytometer were prepared.

Preparation method 2 includes the following steps:

1. Dissolve 150mg of PEI (molecular weight 750K) in 15mL of NaCl solution (0.5M), adjust the pH to 8.0, and record it as a blank PEI solution; add 7.0mg of Cy5-N-hydroxyl to the above blank PEI solution Succinimidyl ester (Cy5-NHS) was reacted overnight at 30°C in the dark to obtain a PEI solution labeled with Cy5 (denoted as Cy5-PEI); Cy5-PEI solution and blank PEI solution were mixed in different ratios , to prepare a mixed solution with a total volume of 1.5 mL;

2. Add 8 × 10 ⁷ carboxylated porous polystyrene microspheres with a diameter of 3.3 μm to the above mixed solution, mix them uniformly by ultrasonic, and rotate for 20 min in the dark, and bind Cy5-PEI and PEI to the microspheres by electrostatic action. Inside the sphere; after the reaction, the supernatant was removed by centrifugation, washed with water three times, and the obtained microspheres were dispersed in 0.4 mL of aqueous solution, that is, the microspheres with fluorescent dyes loaded in the inner space were obtained;

3. The microspheres obtained in the above step 2 were centrifuged to remove the supernatant, the microspheres were washed twice with absolute ethanol, added to a mixed system containing 3 mL of ethanol, 0.3 mL of water and 40 μL of TEOS, and rotated in the dark for 30 min; then, 22 μL of concentrated ammonia water was added, and the reaction was continued at 30 °C for 22 h in the dark. After the reaction, the supernatant was removed, and washed three times with absolute ethanol and water in turn to obtain the Cy5 fluorescent dye-doped code coated with silicon oxide on the surface. Microspheres.

In the fluorescence-encoded microspheres prepared by preparation method 2, the fluorescence-encoded information of Cy5 dye can be excited by the excitation light of 640 nm of the flow cytometer, and the emission band received by the filter is 675±12.5 nm; it can also be excited by the 633 nm of the fluorescence microscope. The excitation light is excited, and the emission band received by the filter is 675±25nm.

Preparation method 3 includes the following steps:

1. Dissolve 150mg of PEI (molecular weight: 750K) in 15mL of NaCl solution (0.5M), adjust the pH to 8.0, and record it as blank PEI solution; add 11.9mg of RITC to the above blank PEI solution, at 30°C Under the conditions, the reaction was performed overnight in the dark, to obtain a PEI solution marked with RITC (referred to as RITC-PEI); the RITC-PEI solution and the blank PEI solution were mixed in different ratios to prepare a mixed solution with a total volume of 1.5 mL;

2. Add 2 × 10 ⁷ porous silica microspheres with a diameter of 5.5 μm to the above mixed solution, mix them uniformly by ultrasonic, and rotate for 20 minutes in the dark, and bind RITC-PEI and PEI to the interior of the microspheres by electrostatic action. After the reaction, the supernatant was removed by centrifugation, washed three times with water, and the obtained microspheres were dispersed in 0.4 mL of aqueous solution to obtain microspheres with fluorescent dyes loaded in the inner space;

4. Magnetically separate the microspheres obtained in the above step 3 to remove the supernatant, wash the microspheres twice with absolute ethanol, and add them to a mixed system containing 3 mL of ethanol, 0.3 mL of water and 40 μL of TEOS, and rotate in the dark for 30 min; then , 22 μL of concentrated ammonia water was added, and the reaction was continued to rotate in the dark for 22 h at 30 °C. After the reaction, the supernatant was removed, and washed with absolute ethanol and water for three times in turn to obtain a RITC fluorescent dye doped with silicon oxide on the surface. Magnetically encoded microspheres.

In the magnetic fluorescent coding microspheres prepared by preparation method 3, the fluorescent coding information of the RITC dye can be excited by the 488nm excitation light of the flow cytometer, and the emission band received by the filter is 565±10nm; The excitation light is excited, and the emission band received by the filter is 585±15nm.

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

Preparation method 4 includes the following steps:

2. Add 2 × 10 ⁷ porous polystyrene microspheres with a diameter of 5.5 μm modified epoxy groups into the above mixed solution, mix uniformly by ultrasonic, and rotate overnight in the dark to react. PEI and PEI are bound to the interior of the microspheres; after the reaction, the supernatant is removed by centrifugation, washed with water three times, and the obtained microspheres are dispersed in 0.4 mL of aqueous solution, that is, the microspheres with fluorescent dyes loaded in the inner space are obtained;

3. The microspheres obtained in the above step 2 were centrifuged to remove the supernatant, the microspheres were washed twice with absolute ethanol, added to a mixed system containing 3 mL of ethanol, 0.3 mL of water and 40 μL of TEOS, and rotated in the dark for 30 min; then, Add 22 μL of concentrated ammonia water, and continue to rotate at 30 °C for 22 h in the dark. After the reaction, remove the supernatant and wash with absolute ethanol and water three times in turn to obtain the RITC fluorescent dye doped code coated with silicon oxide on the surface. Microspheres.

For the fluorescence-encoded microspheres prepared by preparation method 4, the fluorescence-encoded information of the RITC dye can be excited by the 488nm excitation light of the flow cytometer, and the emission band received by the filter is 565±10nm; it can also be excited by the 488nm excitation light of the fluorescence microscope Light excitation, the emission band received by the filter is 585±15nm.

Preparation method 5 includes the following steps:

1. Dissolve 200mg of polyacrylic acid (PAA, molecular weight of 5K) in 15mL of NaCl solution (0.5M), adjust the pH to 6.0, and record as blank PAA solution; add 3.9mg of 5- Aminofluorescein (5-AF) and 40 mg of carbodiimide (EDC) were reacted overnight at 30°C in the dark to obtain a PAA solution labeled with 5-AF (denoted as 5-AF-PAA); Mix 5-AF-PAA solution and blank PAA solution in different proportions to prepare a mixed solution with a total volume of 1.5 mL;

2. Add 2 × 10 ⁷ aminated porous polystyrene microspheres with a diameter of 5.5 μm to the above mixed solution, mix uniformly by ultrasonic, and rotate for 20 min in the dark, and adsorb 5-AF-PAA and PAA by electrostatic action. to the interior of the microspheres; after the reaction, the supernatant was removed by centrifugation, washed with water for three times, and the obtained microspheres were dispersed in 0.4 mL of aqueous solution, that is, the microspheres with fluorescent dyes loaded in the inner space were obtained;

3. Dissolve 150 mg of PEI (molecular weight 750K) in 15 mL of NaCl solution (0.5M), adjust the pH to 8.0, and record it as a blank PEI solution; centrifuge the microspheres obtained in the above step 2 to remove the supernatant, add to In 1.5 mL of blank PEI solution, the reaction was rotated in the dark for 20 min, centrifuged to remove the supernatant after the reaction, and washed with water three times to obtain the outermost fluorescent microspheres modified with PEI;

4. The microspheres obtained in the above step 3 were centrifuged to remove the supernatant, the microspheres were washed twice with absolute ethanol, added to a mixed system containing 3 mL of ethanol, 0.3 mL of water and 40 μL of TEOS, and rotated for 30 min in the dark; then, 22 μL of concentrated ammonia water was added, and the reaction was continued at 30 °C for 22 h in the dark. After the reaction, the supernatant was removed, and washed with absolute ethanol and water three times in turn to obtain a 5-AF fluorescent dye doped with silicon oxide on the surface. coded microspheres.

In the fluorescence-encoded microspheres prepared by preparation method 5, the fluorescence-encoded information of the 5-AF dye can be excited by the 488nm excitation light of the flow cytometer, and the emission band received by the filter is 515±10nm; 488nm excitation light is excited, and the emission band received by the filter is 515±15nm.

Preparation method 6 includes the following steps:

1. Dissolve 150 mg of PEI (molecular weight: 750K) in 15 mL of NaCl solution (0.5M), adjust the pH to 8.0, and record as blank PEI solution; add 4.4 mg of FITC and 7.0 mg of FITC to the above blank PEI solution, respectively The Cy5-NHS was reacted overnight at 30°C in the dark, to obtain PEI solution labeled with FITC (referred to as FITC-PEI) and PEI solution labeled with Cy5 (referred to as Cy5-PEI); in different ratios Mix FITC-PEI solution, Cy5-PEI solution and blank PEI solution to prepare a mixed solution with a total volume of 1.5 mL;

2. Add 6.4×10 ⁸ carboxylated porous polystyrene microspheres with a diameter of 1.7 μm to the above mixed solution, mix them uniformly by ultrasonic, and rotate for 20 min in the dark. The FITC-PEI, Cy5-PEI and FITC-PEI, Cy5-PEI and PEI is adsorbed to the inside of the microspheres; after the reaction, the supernatant is removed by centrifugation, washed with water three times, and the obtained microspheres are dispersed in 0.4 mL of aqueous solution, that is, the microspheres with fluorescent dyes loaded in the inner space are obtained;

3. The microspheres obtained in the above step 2 were centrifuged to remove the supernatant, the microspheres were washed twice with absolute ethanol, added to a mixed system containing 3 mL of ethanol, 0.3 mL of water and 40 μL of TEOS, and rotated in the dark for 30 min; then, 22 μL of concentrated ammonia water was added, and the reaction was continued in the dark at 30°C for 22 hours. After the reaction, the supernatant was removed, and washed three times with absolute ethanol and water in turn to obtain the FITC and Cy5 dual-color fluorescent dyes coated with silica on the surface. Miscellaneous coded microspheres.

For the fluorescence-encoded microspheres prepared by preparation method 6, the fluorescence-encoded information of the FITC dye can be excited by the 488nm excitation light of the flow cytometer, and the emission band received by the filter is 515±10nm; the fluorescence-encoded information of the Cy5 dye can be The 640nm excitation light of the flow cytometer was excited, and the emission band received by the filter was 675±12.5nm. Alternatively, the fluorescence encoded information of the FITC dye can also be excited by the 488nm excitation light of the fluorescence microscope, and the emission band received by the filter is 515±15nm; the fluorescence encoded information of the Cy5 dye can also be excited by the 633nm excitation light of the fluorescence microscope. The received emission band is 675±25nm.

Preparation method 7 includes the following steps:

1. Dissolve 150mg of PEI (molecular weight 750K) in 15mL of NaCl solution (0.5M), adjust the pH to 8.0, and record it as blank PEI solution; add 4.4mg of FITC and 11.9mg of FITC to the above blank PEI solution, respectively The RITC was reacted overnight in the dark at 30°C to obtain a PEI solution labeled with FITC (referred to as FITC-PEI) and a PEI solution labeled with RITC (referred to as RITC-PEI); FITC was mixed in different ratios. -PEI solution, RITC-PEI solution and blank PEI solution to prepare a mixed solution with a total volume of 1.5 mL;

2. Add 8 × 10 ⁷ porous silica microspheres with a diameter of 3.3 μm to the above mixed solution, mix uniformly by ultrasonic, and rotate for 20 min in the dark, and adsorb FITC-PEI, RITC-PEI and PEI by electrostatic action to the interior of the microspheres; after the reaction, the supernatant was removed by centrifugation, washed with water for three times, and the obtained microspheres were dispersed in 0.4 mL of aqueous solution, that is, the microspheres with fluorescent dyes loaded in the inner space were obtained;

4. Magnetically separate the microspheres obtained in the above step 3 to remove the supernatant, wash the microspheres twice with absolute ethanol, and add them to a mixed system containing 3 mL of ethanol, 0.3 mL of water and 40 μL of TEOS, and rotate in the dark for 30 min; then , add 22 μL of concentrated ammonia water, continue to rotate at 30 °C for 22 h in the dark, remove the supernatant after the reaction, and wash three times with absolute ethanol and water in turn to obtain FITC and RITC dual-color fluorescent dyes coated with silica on the surface Doped magnetically encoded microspheres.

In the magnetic fluorescent coding microspheres prepared by preparation method 7, the fluorescent coding information of the FITC dye can be excited by the excitation light of 488 nm of the flow cytometer, and the emission band received by the filter is 515 ± 10 nm; the fluorescent coding information of the RITC dye can be Excited by the 488nm excitation light of the flow cytometer, the emission band received by the filter is 565±10nm. Or the fluorescence encoded information of FITC dye can also be excited by the 488nm excitation light of the fluorescence microscope, and the emission band received by the filter is 515±15nm; the fluorescence encoded information of the RITC dye can also be excited by the 488nm excitation light of the fluorescence microscope, the filter The received emission band is 585±15nm.

As shown in Fig. 6, using porous silica with a diameter of 3.3 μm as the microsphere carrier, and using FITC and RITC as doping dyes, a 32-fold fluorescent dye-doped coding microarray distinguishable on the flow cytometer was prepared. ball. As shown in Figure 7, the fluorescent dye-doped coded microspheres obtained by the preparation method 7 can be rapidly magnetically separated by a magnetic field within 2 minutes, reflecting good magnetic response performance.

Preparation method 8 includes the following steps:

2. Add 2 × 10 ⁷ porous silica microspheres with a diameter of 5.5 μm to the above mixed solution, mix them uniformly by ultrasonic, and rotate for 20 min in the dark, and adsorb FITC-PEI, RITC-PEI and PEI by electrostatic action to the interior of the microspheres; after the reaction, the supernatant was removed by centrifugation, washed with water for three times, and the obtained microspheres were dispersed in 0.4 mL of aqueous solution, that is, the microspheres with fluorescent dyes loaded in the inner space were obtained;

In the magnetic fluorescent coding microspheres prepared by preparation method 8, the fluorescent coding information of the FITC dye can be excited by the excitation light of 488 nm of the flow cytometer, and the emission band received by the filter is 515 ± 10 nm; the fluorescent coding information of the RITC dye can be Excited by the 488nm excitation light of the flow cytometer, the emission band received by the filter is 565±10nm. Or the fluorescence encoded information of FITC dye can also be excited by the 488nm excitation light of the fluorescence microscope, and the emission band received by the filter is 515±15nm; the fluorescence encoded information of the RITC dye can also be excited by the 488nm excitation light of the fluorescence microscope, the filter The received emission band is 585±15nm.

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

For the coded microspheres whose surfaces are coated with silicon oxide prepared according to preparation methods 1 to 8 in this example, the specific method in step 6 of the preparation method in Example 1 (about the preparation process of surface functionalization modification) can be used. ), and functional molecules are modified on the surface of the microspheres to obtain encoded microspheres with carboxyl groups modified on the surface.

The preferred embodiments of the present invention have been described in detail above. It should be understood that those skilled in the art can make many modifications and changes according to the concept of the present invention without creative efforts. Therefore, any technical solutions that can be obtained by those skilled in the art through logical analysis, reasoning or limited experiments on the basis of the prior art according to the concept of the present invention shall fall within the protection scope determined by the claims.

Claims

An encoded microsphere, characterized in that it comprises a microsphere carrier, the microsphere carrier is a mesoporous microsphere, and the matrix composition and aperture of the microsphere carrier are used to define the first dimension code of the encoded microsphere information.
The coded microsphere according to claim 1, wherein the matrix component is an inorganic substance or a polymer.
The encoded microsphere of claim 1, wherein the matrix component is silicon dioxide or titanium dioxide.
The encoded microsphere according to claim 1, wherein the matrix component is made of polystyrene, polyacrylic acid, polymethyl acrylate, polymethacrylic acid, polymethyl methacrylate, polydivinylbenzene and /or a copolymer formed by two or more monomers involved in the above-mentioned polymers.
The encoded microsphere according to claim 1, wherein the diameter of the microsphere carrier is selected in the range of 0.2-20 μm.
The encoded microsphere according to claim 1, wherein the pore diameter of the microsphere carrier is selected in the range of 2-100 nm.
The encoded microsphere according to claim 1, wherein the first dimension information is adjusted according to the matrix composition and/or pore size of the microsphere carrier, and the different first dimension information is analyzed by a flow cytometer. The detection of the encoded microspheres can be distinguished by the signal distribution of the FSC-SSC two-dimensional scatter plot obtained.
The encoded microsphere according to claim 1, wherein an intermediate substance is further provided inside the microsphere carrier.
The encoded microspheres of claim 1, further comprising at least one fluorescent material, each of which has a different central emission wavelength, so that each of the fluorescent materials defines the encoded microspheres One dimension of encoded information.
The encoded microsphere according to claim 9, wherein the central emission wavelengths of the fluorescent materials differ by more than 30 nm.
The encoded microsphere of claim 9, wherein the fluorescent material is disposed inside and/or outside the microsphere carrier.
The encoded microsphere of claim 1, further comprising a first fluorescent material, the first fluorescent material defining second-dimensional encoded information of the encoded microsphere.
The encoded microsphere of claim 12, wherein the first fluorescent material is disposed inside the microsphere carrier.
The encoded microsphere of claim 12, wherein the second dimension encoding information is adjusted according to the content of the first fluorescent material.
The encoded microsphere of claim 12, wherein the first fluorescent material is a fluorescent dye and/or a rare earth complex.
The encoded microsphere according to claim 13, wherein the first fluorescent material is connected with an intermediary substance to form a fluorescent marker, and the first fluorescent material is disposed on the microsphere in the form of the fluorescent marker. The interior of the ball carrier.
The encoded microsphere of claim 8 or 16, wherein the intermediary substance is a polymer.
The encoded microsphere according to claim 14, wherein the intermediary substance is further provided inside the microsphere carrier, and the first fluorescent material is adjusted according to the ratio of the intermediary substance to the fluorescent marker content.
The encoded microsphere of claim 12, further comprising a second fluorescent material, the second fluorescent material defining third-dimensional encoded information of the encoded microsphere.
The encoded microsphere of claim 19, wherein the second fluorescent material is disposed outside the microsphere carrier.
The encoded microsphere of claim 19, wherein the third dimension encoded information is adjusted according to the content of the second fluorescent material.
The encoded microsphere of claim 19, wherein the second fluorescent material is quantum dots, conjugated polymer fluorescent nanoparticles, aggregation-induced luminescence nanoparticles, or upconversion fluorescent nanoparticles.
The encoded microsphere of claim 1, further comprising magnetic nanoparticles.
The encoded microsphere of claim 23, wherein the magnetic nanoparticles are disposed outside the microsphere carrier.
The encoded microsphere of claim 23, wherein the magnetic nanoparticles are Fe 3 O 4 nanoparticles or γ-Fe 2 O 3 nanoparticles.
The encoded microsphere of claim 1, wherein the outer surface of the encoded microsphere is a silicon oxide coating layer.
The coded microsphere according to claim 1, wherein the outer surface of the coded microsphere is a silicon oxide coating layer and a functional molecule modification layer, and the functional molecule modification layer is at the outermost layer.
A coded microsphere array, characterized in that it includes at least two kinds of coded microspheres, and each of the coded microspheres has different coding information; the coded microspheres include a microsphere carrier, and the microsphere carrier is an intermediary Pore microspheres, the matrix composition and pore size of the microsphere carrier are used to define the first dimension encoded information of the encoded microspheres.
The coded microsphere array of claim 28, wherein the microsphere carriers of the various coded microspheres have substantially similar diameters.
The encoded microsphere array of claim 28, wherein the encoded microspheres comprise at least two different matrix components.
The coded microsphere array according to claim 28, wherein the plurality of coded microspheres of the same matrix component are selected with different aperture sizes respectively.
The encoded microsphere array according to claim 28, wherein the matrix component is an inorganic substance or a polymer.
The encoded microsphere array of claim 28, wherein the matrix component is silicon dioxide or titanium dioxide.
The encoded microsphere array according to claim 28, wherein the matrix component is polystyrene, polyacrylic acid, polymethyl acrylate, polymethacrylic acid, polymethyl methacrylate, polydivinyl benzene and/or copolymers formed by two or more monomers involved in the above polymers.
The encoded microsphere array according to claim 28, wherein the diameter of the microsphere carrier is selected in the range of 0.2-20 μm.
The coded microsphere array according to claim 28, wherein the pore size selection range of the microsphere carrier is 2-100 nm.
The encoded microsphere array according to claim 28, wherein each encoded microsphere is detected by a flow cytometer, and each encoded microsphere is detected according to the signal distribution of the FSC-SSC two-dimensional scatter plot obtained by detection. The microspheres make the distinction of the first dimension.
The encoded microsphere array according to claim 28, wherein an intermediate substance is further provided inside the microsphere carrier.
The coded microsphere array according to claim 28, wherein the coded microspheres further comprise at least one fluorescent material, and the central emission wavelengths of the fluorescent materials are different, so that each fluorescent material has different central emission wavelengths. Encoding information that defines one dimension of the encoded microspheres.
The encoded microsphere array according to claim 39, wherein the central emission wavelengths of the fluorescent materials differ by more than 30 nm.
The encoded microsphere array of claim 39, wherein the fluorescent material is disposed inside and/or outside the microsphere carrier.
30. The encoded microsphere array of claim 28, wherein the encoded microspheres further comprise a first fluorescent material, the first fluorescent material defining second dimension encoded information of the encoded microspheres.
The encoded microsphere array of claim 42, wherein the first fluorescent material is disposed inside the microsphere carrier.
The encoded microsphere array of claim 42, wherein the second dimension encoded information is adjusted according to the content of the first fluorescent material.
The encoded microsphere array of claim 42, wherein the first fluorescent material is a fluorescent dye and/or a rare earth complex.
The encoded microsphere array according to claim 43, wherein the first fluorescent material is connected with an intermediary substance to form a fluorescent marker, and the first fluorescent material is arranged on the fluorescent marker in the form of the fluorescent marker. The interior of the microsphere carrier.
The encoded microsphere array of claim 38 or 46, wherein the intermediary substance is a polymer.
The encoded microsphere array according to claim 47, wherein the intermediary substance is further provided inside the microsphere carrier, and the first fluorescence is adjusted according to the ratio of the intermediary substance to the fluorescent marker material content.
The encoded microsphere array of claim 42, wherein the encoded microspheres further comprise a second fluorescent material, the second fluorescent material defining third-dimensional encoded information of the encoded microspheres.
The encoded microsphere array of claim 49, wherein the second fluorescent material is disposed outside the microsphere carrier.
The encoded microsphere array of claim 49, wherein the third dimension encoded information is adjusted according to the content of the second fluorescent material.
The encoded microsphere array of claim 49, wherein the second fluorescent material is quantum dots, conjugated polymer fluorescent nanoparticles, aggregation-induced luminescence nanoparticles, or upconverting fluorescent nanoparticles.
The encoded microsphere array of claim 28, wherein the encoded microspheres further comprise magnetic nanoparticles.
The encoded microsphere array of claim 53, wherein the magnetic nanoparticles are disposed outside the microsphere carrier.
The encoded microsphere array of claim 53, wherein the magnetic nanoparticles are Fe 3 O 4 nanoparticles or γ-Fe 2 O 3 nanoparticles.
The coded microsphere array according to claim 28, wherein the outer surface of the coded microspheres is a silicon oxide coating layer.
The coded microsphere array according to claim 28, wherein the outer surface of the coded microspheres is a silicon oxide coating layer and a functional molecule modification layer, and the functional molecule modification layer is at the outermost layer.
A method for preparing encoded microspheres, comprising:

Step 1: Select a microsphere carrier, which is a mesoporous microsphere, and determine the matrix composition and pore size of the microsphere carrier, so as to define the first dimension encoding information of the encoded microsphere.
The preparation method according to claim 58, wherein the matrix component is inorganic or polymer.
The preparation method of claim 58, wherein the matrix component is silicon dioxide or titanium dioxide.
The preparation method according to claim 58, wherein the matrix components are polystyrene, polyacrylic acid, polymethyl acrylate, polymethacrylic acid, polymethyl methacrylate, polydivinylbenzene and/or Or a copolymer formed by two or more monomers involved in the above-mentioned polymers.
The preparation method of claim 58, wherein the diameter of the microsphere carrier is selected in the range of 0.2 to 20 µm.
The preparation method according to claim 58, wherein the pore size of the microsphere carrier is selected in the range of 2-100 nm.
The preparation method of claim 58, further comprising:

In step 2, the fluorescent dye is arranged inside the microsphere carrier to define the second dimension encoding information of the encoded microsphere; or an intermediary substance is arranged inside the microsphere carrier.
The preparation method according to claim 64, wherein in the second step, the fluorescent dye is arranged inside the microsphere carrier, which specifically includes:

The fluorescent dye and the intermediary substance are connected to form a fluorescent label, and the fluorescent label or the mixture of the fluorescent label and the intermediary substance is arranged in the inner space of the microsphere carrier through physical/chemical action.
The preparation method of claim 65, wherein the intermediary substance is a polymer.
The preparation method according to claim 65, wherein the fluorescent dye forming the fluorescent label is connected with the intermediary substance through a covalent bond.
The preparation method of claim 65, wherein the second dimension encoded information is adjusted according to the ratio of the fluorescent marker and the intermediary substance in the mixture.
The preparation method of claim 58, further comprising:

Step 3: Disposing the magnetic nanoparticles on the outer surface of the microsphere carrier.
The preparation method of claim 69, wherein the magnetic nanoparticles are Fe 3 O 4 nanoparticles or γ-Fe 2 O 3 nanoparticles.
The preparation method of claim 69, wherein the step 3 specifically comprises:

The magnetic nanoparticles are coated on the outer surface of the microsphere carrier by physical/chemical action.
The preparation method of claim 71, wherein the step 3 further comprises:

After the magnetic nanoparticles are coated on the outer surface of the microsphere carrier, the outer surface of the microsphere carrier is then coated with a polymer.
The preparation method of claim 58, further comprising:

Step 4: Disposing the fluorescent nanoparticles on the outer surface of the microsphere carrier to define the third-dimensional encoded information of the encoded microspheres.
The encoded microsphere of claim 73, wherein the fluorescent nanoparticles are quantum dots, conjugated polymer fluorescent nanoparticles, aggregation-induced luminescence nanoparticles, or upconverting fluorescent nanoparticles.
The preparation method of claim 73, wherein the step 4 specifically comprises:

The fluorescent nanoparticles are coated on the outer surface of the microsphere carrier by physical/chemical action.
The preparation method of claim 75, wherein the step 4 further comprises:

After the fluorescent nanoparticles are coated on the outer surface of the microsphere carrier, the outer surface of the microsphere carrier is then coated with a polymer.
The preparation method according to claim 65 or 71 or 75, wherein the physical/chemical interactions include electrostatic interactions, hydrophilic and hydrophobic interactions, hydrogen bonding interactions, coordination interactions and/or covalent bonding interactions.
The preparation method according to claim 65, 72 or 76, characterized in that, after all steps are completed, the outer surface of the microsphere carrier is coated with silicon oxide.
The preparation method of claim 78, wherein after the outer surface of the microsphere carrier is coated with silicon oxide, functional molecules are modified on the outer surface of the microsphere carrier to obtain a surface-functionalized microsphere carrier. Ball vector.