Fluorescent encoding microsphere, preparation method thereof and application of fluorescent encoding microsphere in immunoassay analyzer

Abstract

The invention discloses a fluorescence coding microsphere, a preparation method thereof and application of the fluorescence coding microsphere in an immunoassay analyzer, the fluorescence coding microsphere is prepared by adding 40-150 [mu] g of a coding compound and 0.05-0.15 mL of a swelling agent into every 0.5-1.5 mg of carrier microsphere, and the coding compound comprises an energy supply compound, a medium compound, a luminescent compound A and a luminescent compound B; the total mass of the energy supply compound and the medium compound is 10-30 [mu] g, and the mass ratio of the energy supply compound to the medium compound is 1: (0.5-1.5); the total mass of the luminescent compound A and the luminescent compound B is 30-120 [mu] g, and the mass ratio of the luminescent compound A to the luminescent compound B is (1-10): (10-1); the preparation method comprises the following steps: taking 0.5-1.5 mg of carrier microspheres for separation, adding 40-150 [mu] g of a coding compound into 0.05-0.15 mL of a swelling agent, mixing, adding the mixture into the carrier microspheres, oscillating, and cleaning; the application in an immunoassay analyzer is as follows: a magnetic sphere compound for immunoassay comprises the fluorescent encoding microsphere disclosed by the invention. The two light-emitting compounds in the fluorescent coded microspheres do not generate crosstalk in the same laser light source, so that available coding points on the fluorescent coded microspheres are increased.

Description

Technical Field

[0001] The present invention relates to the technical field of biocomposites, and particularly relates to a fluorescently encoded microsphere and a preparation method thereof. Background Art

[0002] Encoded microspheres refer to using microspheres as carriers and accurately encoding the microspheres through a certain method, so that the microspheres correspond to the analyte to be measured, achieving the effect of high-throughput detection. Many encoding markers have been developed, such as: magnetic field encoding, chemical encoding, spatial encoding, and optical encoding. Currently, the most widely used application scenario is optical encoding. By using different types A of dyes and different concentrations N of dyes, N A types can be achieved. For single-fluorescent encoded microspheres, the selectivity of dyes is strong, but for dual-fluorescent encoded microspheres, the selection of dyes is particularly important. It is necessary to ensure that no crosstalk occurs between the two dyes under the same laser light source. Therefore, the Stokes shift difference between the two dyes is relatively large, and they have similar quantum yields, so as to achieve N A types. Two dyes that meet these conditions are very rare, so the entire laser scatter pattern will be wedge-shaped and the number of encodable points will decrease. To solve the crosstalk problem, using different laser light sources for the two dyes can effectively avoid this problem, but adding a laser increases the instrument cost and the instrument volume, resulting in a decrease in market competitiveness. To reduce the crosstalk problem, one of the fluorescent dyes can also be replaced with upconversion nanomaterials, so that under the same excitation light source, the two dyes have Stokes shifts to both sides of the excitation light source. However, the quantum yield of upconversion nanomaterials is low and the microsphere accommodation capacity is small, resulting in a small number of encoding points and restricting its development. Summary of the Invention

[0003] The technical problem to be solved by the present invention is to provide a fluorescently encoded microsphere and a preparation method thereof.

[0004] A fluorescently encoded microsphere is prepared by adding 40 - 150 μg of an encoding compound and 0.05 - 0.15 mL of a swelling agent to every 0.5 - 1.5 mg of carrier microspheres. The encoding compound includes an energy-providing compound, a mediator compound, a luminescent compound A, and a luminescent compound B; the total mass of the energy-providing compound and the mediator compound is 10 - 30 μg, and the mass ratio between them is 1:(0.5 - 1.5); the total mass of the luminescent compound A and the luminescent compound B is 30 - 120 μg, and the mass ratio between them is (1 - 10):(10 - 1);

[0005] The energy-providing compound is methylene blue, rose bengal, porphyrin, or phthalocyanine, and the mediator compound is dimethylthiophene, 9-alkylidene-N-9,10-dihydroacridine, dioxacyclic compound, or oxalate compound.

[0006] Preferably, the dioxocyclic compound is dioxocyclohexene, and the oxalate compound is bis(2,4,6-trichlorophenyl) oxalate.

[0007] Preferably, the luminescent compound A is an organic fluorescent dye A or a quantum dot. The organic fluorescent dye A is silicon-based rhodamine, styrene-boron dipyrrole or Cy5 dye. The quantum dot is a carbon quantum dot, a CdTe quantum dot, a CdS quantum dot, a PbS quantum dot or a MoS 2 quantum dot.

[0008] Preferably, the luminescent compound B is an organic fluorescent dye B, a quantum dot or a rare earth chelate. The organic fluorescent dye B is 2,5,8,11-tetra-tert-butylperylene, AMCA dye or DiO dye. The quantum dot is a carbon quantum dot, a CdTe quantum dot, a CdS quantum dot, a PbS quantum dot or a MoS 2 quantum dot, and the rare earth chelate is a terbium chelate.

[0009] Preferably, the swelling agent is N,N-dimethylformamide, methyl chloride, dichloromethane, chloroform, carbon tetrachloride, toluene or acetone.

[0010] Preferably, the particle size of the carrier microspheres is 1-100 microns, and the groups of the carrier microspheres are carboxyl, amino, epoxy or tosyl groups.

[0011] A method for preparing the above-mentioned fluorescently encoded microspheres, comprising the following steps:

[0012] S1. Take 0.5-1.5 mg of carrier microspheres for separation, and remove the supernatant;

[0013] S2. Add 40-150 μg of the encoding compound to 0.05-0.15 mL of the swelling agent to obtain a mixture; the encoding compound includes an energy-providing compound, a mediator compound, a luminescent compound A and a luminescent compound B; the total mass of the energy-providing compound and the mediator compound is 10-30 μg, and the mass ratio between the two is 1:(0.5-1.5); the total mass of the luminescent compound A and the luminescent compound B is 30-120 μg, and the mass ratio between the two is (1-10):(10-1);

[0014] S3. Add the mixture to the separated carrier microspheres, mix and shake, and then wash to obtain the fluorescently encoded microspheres.

[0015] Preferably, in step S3, the shaking speed is 30-70 rpm and the shaking time is 10-60 min.

[0016] Preferably, in step S3, the washing is carried out 2-4 times with an aqueous ethanol solution with a mass fraction of 50-100%.

[0017] An application of the above-mentioned fluorescently encoded microspheres in an immunoassay analyzer, the immunoassay analyzer comprising:

[0018] A sheath flow module for causing M*N kinds of magnetic bead complexes to pass through the detection area of the sheath flow module one by one, wherein the magnetic bead complexes comprise the substance to be measured and fluorescently encoded microspheres bound to the substance to be measured, and the fluorescently encoded microspheres comprise a luminescent compound A having M excitation intensity levels and a luminescent compound B having N laser intensity levels;

[0019] A first laser for emitting a first laser to the detection area to excite the magnetic bead complexes to generate fluorescence for quantification;

[0020] A scattered light receiving module for receiving, at a first side of the sheath flow module, the fluorescence for quantification generated after passing through the detection area and converting it into a counting parameter;

[0021] A second laser for emitting a second laser to the detection area to excite the M intensity levels of luminescent compound A to correspondingly generate M intensity levels of classification fluorescence A and the N intensity levels of luminescent compound B to correspondingly generate N intensity levels of classification fluorescence B;

[0022] A classification fluorescence A receiving module for receiving, at a second side of the sheath flow module, the M intensity levels of classification fluorescence A after passing through the detection area and converting it into M classification fluorescence A intensity classification parameters;

[0023] A classification fluorescence B receiving module for receiving, at a second side of the sheath flow module, the N intensity levels of classification fluorescence B after passing through the detection area and converting it into N classification fluorescence B intensity classification parameters;

[0024] A signal processing module electrically connected to the scattered light receiving module, the classification fluorescence A receiving module, and the classification fluorescence B receiving module, for receiving the counting parameter, the M classification fluorescence A intensity classification parameters, and the N classification fluorescence B intensity classification parameters, and converting them into the counting results corresponding to each of the M*N kinds of magnetic bead complexes.

[0025] Advantages of the present invention:

[0026] The fluorescently encoded microspheres of the present invention use an energy-providing compound, a mediator compound, and two luminescent compounds as encoding compounds. By using the energy-providing compound and the mediator compound to transfer light energy to one of the luminescent compounds, the two luminescent compounds can be free from crosstalk in the same laser light source, thereby increasing the available encoding points on the fluorescently encoded microspheres. In addition, only one laser light source is required, which can save the instrument cost.

[0027] The preparation method of the fluorescently encoded microspheres of the present invention is simple and feasible, with low production cost. The prepared fluorescently encoded microspheres can achieve the purpose of no crosstalk between the two luminescent compounds by using only one laser light source.

[0028] The fluorescently encoded microspheres of the present invention can be applied to an immunoassay analyzer. By configuring the fluorescently encoded microspheres of the magnetic bead complex with M excitation intensity levels of luminescent compound A and N laser intensity levels of luminescent compound B, and only one classification laser can be used to count each of the M*N magnetic bead complexes, which reduces costs while improving the classification performance and shortening the detection time. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] The present invention will be further described below in conjunction with the drawings and embodiments. In the drawings:

[0030] Figure 1 is a scatter plot of the fluorescently encoded microspheres of Example 1 of the present invention under red light excitation. DETAILED DESCRIPTION OF THE INVENTION

[0031] For a clearer understanding of the technical features, objectives, and effects of the present invention, the present invention will be further described in detail below in conjunction with embodiments. These embodiments are only used to explain the present invention and do not constitute a limitation on the protection scope of the present invention.

[0032] The present invention provides a fluorescently encoded microsphere, which is prepared by adding 40-150 μg of a coding compound and 0.05-0.15 mL of a swelling agent to every 0.5-1.5 mg of carrier microspheres. Among them, the mass of the carrier microspheres can be, but is not limited to, 0.5 mg, 0.7 mg, 0.9 mg, 1 mg, 1.3 mg, 1.5 mg; the mass of the coding compound can be, but is not limited to, 40 μg, 60 μg, 80 μg, 100 μg, 120 μg, 150 μg; and the volume of the swelling agent can be, but is not limited to, 0.05 mL, 0.08 mL, 0.1 mL, 0.13 mL, 0.15 mL.

[0033] The above-mentioned encoded compounds include an energy-providing compound, a mediator compound, a luminescent compound A, and a luminescent compound B. The total mass of the energy-providing compound and the mediator compound is 10 - 30 μg, and this total mass can be, but is not limited to, 10 μg, 15 μg, 20 μg, 25 μg, 30 μg. The mass ratio between the energy-providing compound and the mediator compound is 1:(0.5 - 1.5). Understandably, the mass ratio between the energy-providing compound and the mediator compound can be set to, but is not limited to, 1:0.5, 1:0.7, 1:0.9, 1:1, 1:1.3, and 1:1.5. The total mass of the luminescent compound A and the luminescent compound B is 30 - 120 μg, and this total mass can be, but is not limited to, 30 μg, 60 μg, 80 μg, 100 μg, 120 μg. The mass ratio between the luminescent compound A and the luminescent compound B is (1 - 10):(10 - 1). Understandably, the mass ratio between the luminescent compound A and the luminescent compound B can be set to, but is not limited to, 1:10, 2:9, 3:8, 4:7, 5:6, 5:5, 6:5, 7:4, 8:3, 9:2, and 10:1.

[0034] Specifically, the energy-providing compound is methylene blue, rose bengal, porphyrin, or phthalocyanine, and the mediator compound is dimethylthiophene, 9-alkylene-N-9,10-dihydroacridine, dioxacyclic compound, or oxalate compound. Among them, the dioxacyclic compound is preferably dioxacyclohexene, and the oxalate compound is preferably bis(2,4,6-trichlorophenyl) oxalate.

[0035] The luminescent compound A is an organic fluorescent dye A or a quantum dot. Among them, the organic fluorescent dye A is silicon-based rhodamine, styrene-boron dipyrrole, or Cy5 dye. The organic fluorescent dye has a higher fluorescence quantum yield than the upconversion nanomaterial. The quantum dot is a carbon quantum dot or a metal quantum dot. The metal quantum dot is preferably a CdTe quantum dot, a CdS quantum dot, a PbS quantum dot, or MoS 2 quantum dot.

[0036] The emission wavelength of the luminescent compound B is less than the emission wavelength of the excitation light source, and the luminescent compound B has transferability with the mediator compound, that is, the mediator compound can transfer energy to the luminescent compound B. The luminescent compound B is an organic fluorescent dye B, a quantum dot, or a rare earth chelate. The organic fluorescent dye B is 2,5,8,11-tetra-tert-butylperylene, AMCA dye, or DiO dye. The quantum dot is a carbon quantum dot, a CdTe quantum dot, a CdS quantum dot, a PbS quantum dot, or MoS 2 quantum dot, and the rare earth chelate is preferably a terbium chelate. In some embodiments, the rare earth chelate can also be a chelate of other rare earth elements except terbium.

[0037] The swelling agent is N,N-dimethylformamide, methyl chloride, dichloromethane, chloroform, carbon tetrachloride, toluene, or acetone.

[0038] The carrier microspheres have a particle size of 1 - 100 microns, and the groups of the carrier microspheres are carboxyl, amino, epoxy or tosyl groups. In some embodiments, the carrier microspheres may have magnetism, such as magnetic microspheres.

[0039] For the fluorescently encoded microspheres proposed by the present invention, an energy-providing compound and a mediator compound are added to two luminescent compounds. Under the same laser light source, luminescent compound A receives the light source, and the Stokes shift shifts to the right, emitting the light source; the energy-providing compound also receives the light source, and the generated high-energy oxygen is transferred to the mediator compound through the Brownian motion of molecules. The mediator compound is excited by the high-energy oxygen, and the generated energy is transferred to luminescent compound B, and luminescent compound B emits the light source, and the wavelength of the emitted light source is less than the wavelength of the excitation light source. Therefore, luminescent compound A and luminescent compound B do not interfere with each other, and only one excitation light source is required to meet the requirement. The fluorescently encoded microspheres of the present invention can use red or green laser.

[0040] The present invention also provides a preparation method of the above fluorescently encoded microspheres, comprising the following steps:

[0041] S1. Take 0.5 - 1.5 mg of carrier microspheres for separation, and remove the supernatant. Specifically, the separation method is magnetic separation, centrifugal separation or ultrafiltration separation. In some embodiments, the carrier microspheres are magnetic microspheres, and magnetic separation is performed on the magnetic microspheres, such as separation by a magnetic separation rack. It can be understood that the separation method of the carrier microspheres is a prior art and can be selected and set according to actual needs, and will not be elaborated here.

[0042] S2. Add 40 - 150 μg of encoding compounds to 0.05 - 0.15 mL of swelling agent to obtain a mixture. The encoding compounds include an energy-providing compound, a mediator compound, luminescent compound A and luminescent compound B; the total mass of the energy-providing compound and the mediator compound is 10 - 30 μg, and the mass ratio between the two is 1:(0.5 - 1.5); the total mass of luminescent compound A and luminescent compound B is 30 - 120 μg, and the mass ratio between the two is (1 - 10):(10 - 1).

[0043] S3. Add the mixture to the separated carrier microspheres, mix and shake, and then wash to obtain the fluorescence-encoded microspheres. Specifically, during the shaking operation, the shaking speed is 30 - 70 rpm, and the shaking speed can be, but is not limited to, 30 rpm, 40 rpm, 50 rpm, 60 rpm, 70 rpm; the shaking time is 10 - 60 min, and the shaking time can be, but is not limited to, 10 min, 20 min, 30 min, 40 min, 50 min, 60 min. During the washing operation, wash with an ethanol aqueous solution with a mass fraction of 50 - 100% for 2 - 4 times. The mass fraction of the ethanol aqueous solution can be, but is not limited to, 50%, 60%, 70%, 80%, 90%, 100%, and the number of washing times can be 2 times, 3 times, 4 times.

[0044] The present invention also provides an application of the above fluorescence-encoded microspheres in an immunoassay analyzer. The magnetic bead complex for immunoassay includes the fluorescence-encoded microspheres of the present invention. The immunoassay analyzer includes a sheath flow module, a first laser, a scattered light receiving module, a second laser, a classification fluorescence A receiving module, a classification fluorescence B receiving module, and a signal processing module. Among them, the signal processing module is electrically connected to the scattered light receiving module, the classification fluorescence A receiving module, and the classification fluorescence B receiving module.

[0045] The sheath flow module is used to make M×N kinds of magnetic bead complexes pass through the detection area of the sheath flow module one by one. Among them, the magnetic bead complex includes the substance to be detected and the fluorescence-encoded microspheres bound to the substance to be detected. The fluorescence-encoded microspheres include a luminescent compound A with M kinds of excitation intensity levels and a luminescent compound B with N kinds of laser intensity levels. The substance to be detected can be an antigen, an antibody, a protein drug, a small molecule compound, etc., or other substances that need to be classified and counted.

[0046] The first laser is used to emit a first laser to the detection area to excite the magnetic bead complex to generate fluorescence for quantification. The second laser is used to emit a second laser to the detection area to excite the M kinds of intensity-level luminescent compound A to generate M kinds of intensity-level classification fluorescence A and the N kinds of intensity-level luminescent compound B to generate N kinds of intensity-level classification fluorescence B.

[0047] The scattered light receiving module is used to receive the fluorescence for quantification generated after passing through the detection area on the first side of the sheath flow module and convert it into a counting parameter. The scattered light receiving module includes conventional optical elements such as a diaphragm, a lens, a filter, a signal detector, etc. It can be understood that the scattered light receiving module can adopt the existing technology, and the present invention will not elaborate here.

[0048] The classification fluorescence A receiving module is used to receive M classification fluorescences A with different intensity levels after passing through the detection area on the second side of the sheath flow module and convert them into M classification fluorescence A intensity classification parameters. The classification fluorescence B receiving module is used to receive N classification fluorescences B with different intensity levels after passing through the detection area on the second side of the sheath flow module and convert them into N classification fluorescence B intensity classification parameters. Both the classification fluorescence A receiving module and the classification fluorescence B receiving module may include optical elements such as a collection lens group, a collimating lens group, a focusing lens group, a band-pass filter, and a fluorescence detector. The collimating lens group can be an aspherical lens or multiple lenses, which is used to collimate the fluorescence into parallel light or approximately parallel light. The focusing lens group can be an aspherical lens or multiple lenses, which is used to focus the fluorescence on the fluorescence detector. It can be understood that the structures of the classification fluorescence A receiving module and the classification fluorescence B receiving module can both adopt the existing technologies, and the present invention will not elaborate here.

[0049] The signal processing module is used to receive the above counting parameters, M classification fluorescence A intensity classification parameters, and N classification fluorescence B intensity classification parameters, and convert them into the counting results corresponding to each of the M×N magnetic bead complexes.

[0050] The following is illustrated by specific examples:

[0051] As shown in Table 1, the compositions of the fluorescence-coded microspheres in Examples 1-3 and Comparative Examples 1-5 of the present invention. Among them, the coding compound in Comparative Example 1 only contains the luminescent compound A, the coding compound in Comparative Example 2 only contains the energy-providing compound, the medium compound, and the luminescent compound B, the coding compound in Comparative Example 3 is two common fluorescent dyes, APC-Cy7 dye and APC dye, the coding compound in Comparative Example 4 is APC dye, and the coding compound in Comparative Example 5 is APC-Cy7 dye.

[0052] Table 1 Compositions of the fluorescence-coded microspheres in Examples 1-3 and Comparative Examples 1-5

[0053]

[0054]

[0055] Note: " / " indicates not added.

[0056] The preparation methods of the fluorescence-coded microspheres in the above Examples 1-3 and Comparative Examples 1-5 are as follows:

[0057] Example 1

[0058] The preparation method of the fluorescence-coded microspheres in this example includes the following steps:

[0059] S1. Take 1 mg of carrier microspheres (carboxyl) with a particle size of 5 μm and perform centrifugal separation to remove the supernatant.

[0060] S2. Add 60 μg of the coding compound (including 10 μg of methylene blue, 10 μg of dimethylthiophene, 20 μg of silicon rhodamine, and 20 μg of terbium chelate) to 0.1 mL of chloroform to obtain a mixture.

[0061] S3. Add the mixture to the separated carrier microspheres, shake and react at a speed of 50 rpm for 10 min, and then wash 3 times with an aqueous ethanol solution with a mass fraction of 80% to obtain fluorescently encoded microspheres.

[0062] The fluorescently encoded microspheres of this example can be applied to an immunoassay analyzer, and the fluorescently encoded microspheres combine with the substance to be measured to form a magnetic sphere complex for immunoassay detection.

[0063] In this example, an energy-providing compound, a mediator compound, and two luminescent compounds are used to optically encode the carrier microspheres to prepare fluorescently encoded microspheres. Under the same excitation light source, one luminescent compound directly receives the light source, and the other luminescent compound receives the light source through the energy transfer of the energy-providing compound and the mediator compound, so that crosstalk between the two luminescent compounds can be avoided under the same excitation light source, and thus the available coding points on the fluorescently encoded microspheres are increased.

[0064] Comparative Example 1

[0065] The preparation method of the fluorescently encoded microspheres of this example includes the following steps:

[0066] S1. Take 1 mg of carrier microspheres (amino group) with a particle size of 5 μm for centrifugal separation to remove the supernatant.

[0067] S2. Add 20 μg of the coding compound (20 μg of silicon rhodamine) to 0.1 mL of dichloromethane to obtain a mixture.

[0068] S3. Add the mixture to the separated carrier microspheres, shake and react at a speed of 50 rpm for 60 min, and then wash 3 times with an aqueous ethanol solution with a mass fraction of 100% to obtain fluorescently encoded microspheres.

[0069] Comparative Example 2

[0070] The preparation method of the fluorescently encoded microspheres of this example includes the following steps:

[0071] S1. Take 1 mg of carrier microspheres (amino group) with a particle size of 5 μm for centrifugal separation to remove the supernatant.

[0072] S2. Add 40 μg of the coding compound (including 10 μg of methylene blue, 10 μg of dimethylthiophene, and 20 μg of terbium chelate) to 0.1 mL of dichloromethane to obtain a mixture.

[0073] S3. Add the mixture to the separated carrier microspheres, shake the reaction at a speed of 50 rpm for 60 min, and then wash three times with an ethanol aqueous solution with a mass fraction of 100%, to obtain fluorescently encoded microspheres.

[0074] Comparative Example 3

[0075] The preparation method of the fluorescently encoded microspheres in this example includes the following steps:

[0076] S1. Take 1 mg of carrier microspheres (tosyl group) with a particle size of 5 μm for centrifugal separation, and remove the supernatant.

[0077] S2. Add 60 μg of coding compounds (including 30 μg of APC-Cy7 dye and 30 μg of APC dye) to 0.1 mL of toluene to obtain a mixture.

[0078] S3. Add the mixture to the separated carrier microspheres, shake the reaction at a speed of 50 rpm for 30 min, and then wash three times with an ethanol aqueous solution with a mass fraction of 50%, to obtain fluorescently encoded microspheres.

[0079] Comparative Example 4

[0080] The preparation method of the fluorescently encoded microspheres in this example includes the following steps:

[0081] S1. Take 1 mg of carrier microspheres (tosyl group) with a particle size of 5 μm for centrifugal separation, and remove the supernatant.

[0082] S2. Add 30 μg of coding compounds (30 μg of APC dye) to 0.1 mL of toluene to obtain a mixture.

[0083] S3. Add the mixture to the separated carrier microspheres, shake the reaction at a speed of 50 rpm for 30 min, and then wash three times with an ethanol aqueous solution with a mass fraction of 50%, to obtain fluorescently encoded microspheres.

[0084] Comparative Example 5

[0085] The preparation method of the fluorescently encoded microspheres in this example includes the following steps:

[0086] S1. Take 1 mg of carrier microspheres (tosyl group) with a particle size of 5 μm for centrifugal separation, and remove the supernatant.

[0087] S2. Add 30 μg of coding compounds (30 μg of APC-Cy7 dye) to 0.1 mL of toluene to obtain a mixture.

[0088] S3. Add the mixture to the separated carrier microspheres, shake and react at a speed of 50 rpm for 30 min, and then wash three times with an ethanol aqueous solution with a mass fraction of 50%, to obtain fluorescently encoded microspheres.

[0089] Example 2

[0090] The preparation method of the fluorescently encoded microspheres in this example includes the following steps:

[0091] S1. Take 1.5 mg of microspheres (amino group) with a particle size of 1 μm for ultrafiltration separation, and remove the supernatant.

[0092] S2. Add 150 μg of the encoding compound (including 30 μg in total, with a mass ratio of 1:0.5 of rose bengal and dioxane, and 120 μg in total, with a mass ratio of 1:10 of carbon quantum dots and AMCA dye) to 0.15 mL of carbon tetrachloride to obtain a mixture.

[0093] S3. Add the mixture to the separated carrier microspheres, shake and react at a speed of 70 rpm for 10 min, and then wash twice with an ethanol aqueous solution with a mass fraction of 100%, to obtain fluorescently encoded microspheres.

[0094] The fluorescently encoded microspheres in this example can be applied to an immunoanalyzer, and the fluorescently encoded microspheres combine with the substance to be detected to form a magnetic bead complex for immunoassay detection.

[0095] Example 3

[0096] The preparation method of the fluorescently encoded microspheres in this example includes the following steps:

[0097] S1. Take 0.5 mg of magnetic microspheres (epoxy group) with a particle size of 100 μm for magnetic separation, and remove the supernatant. The magnetic separation is carried out on a magnetic separation rack for 1 min.

[0098] S2. Add 40 μg of the encoding compound (including 10 μg in total, with a mass ratio of 1:1.5 of porphyrin and bis(2,4,6-trichlorophenyl) oxalate, and 30 μg in total, with a mass ratio of 10:1 of CdTe quantum dots and PbS quantum dots) to 0.05 mL of N,N-dimethylformamide to obtain a mixture.

[0099] S3. Add the mixture to the separated carrier microspheres, shake and react at a speed of 30 rpm for 60 min, and then wash four times with an ethanol aqueous solution with a mass fraction of 50%, to obtain fluorescently encoded microspheres.

[0100] The fluorescently encoded microspheres in this example can be applied to an immunoanalyzer, and the fluorescently encoded microspheres combine with the substance to be detected to form a magnetic bead complex for immunoassay detection.

[0101] Crosstalk test:

[0102] 1. A crosstalk test was conducted on the fluorescently encoded microspheres of Example 1 and Comparative Examples 1-2, and the test results are shown in Table 2.

[0103] Test instrument and parameter selection: Flow cytometer (Dinamica F1200), the laser light source was selected as 630 nm red light, the fluorescence receiving channel 1 was selected as the APC channel, and the filter was selected as 660 / 10BP; the fluorescence receiving channel 2 was selected as the FITC channel, and the filter was selected as 525 / 40BP.

[0104] 2. A crosstalk test was conducted on the fluorescently encoded microspheres of Comparative Examples 3-5, and the test results are shown in Table 3.

[0105] Test instrument and parameter selection: Flow cytometer (Dinamica F1200), the laser light source was selected as 630 nm red light, the fluorescence receiving channel 1 was selected as the APC channel, and the filter was selected as 660 / 10BP; the fluorescence receiving channel 2 was selected as the APC-Cy7 channel, and the filter was selected as 780 / 60BP.

[0106] Table 2 Crosstalk test results of Example 1 and Comparative Examples 1-2

[0107] Fluorescence reception channel 1 Fluorescence reception channel 2 Example 1 (fluorescence intensity) 423214 456321 Comparative example 1 (fluorescence intensity) 548632 <![CDATA[40 [2] > Comparative example 2 (fluorescence intensity) <![CDATA[34 [1] > 623158

[0108] Note: [1] is the background value of fluorescence receiving channel 1, and [2] is the background value of fluorescence receiving channel 2.

[0109] Table 3 Crosstalk test results of Comparative Examples 3-5

[0110] Fluorescence reception channel 1 Fluorescence reception channel 2 Comparative example 3 (fluorescence intensity) 442568 325469 Comparative example 4 (fluorescence intensity) 456232 32321 Comparative example 5 (fluorescence intensity) 52356 502314

[0111] As can be seen from Table 2, the dual-fluorescently encoded microspheres of Example 1 have a certain fluorescence intensity in both fluorescence receiving channels. The single-fluorescently encoded microspheres of Comparative Example 1 and Comparative Example 2 only have fluorescence intensity in one fluorescence receiving channel corresponding to the luminescent compound, while there is no fluorescence intensity in the other fluorescence receiving channel, indicating that there is no crosstalk between the two luminescent compounds.

[0112] As can be seen from Table 3, the dual-fluorescently encoded microspheres of Comparative Example 3 have a certain fluorescence intensity in both fluorescence receiving channels. The single-fluorescently encoded microspheres of Comparative Example 4 and Comparative Example 5 not only have fluorescence intensity in one fluorescence receiving channel corresponding to the luminescent compound, but also have a certain fluorescence intensity in the other fluorescence receiving channel, indicating that there is crosstalk between the two luminescent compounds.

[0113] By adjusting the total mass and mass ratio of luminescent compound A and luminescent compound B in the fluorescently encoded microspheres of the present invention, the scatter plots of the results of the dual-fluorescence receiving channels of the obtained fluorescently encoded microspheres under red light excitation are asFigure 1 as shown, where the abscissa is the fluorescence intensity of fluorescence receiving channel 1 (APC channel) and the ordinate is the fluorescence intensity of fluorescence receiving channel 2 (FITC channel). From Figure 1 it can be seen that the scatter plot in the array arrangement is generally square, which indicates that there is no crosstalk between luminescent compound A and luminescent compound B. Otherwise, the scatter plot would generally be wedge-shaped or diamond-shaped.

[0114] It can be understood that the above embodiments only represent the preferred embodiments of the present invention, and the description is relatively specific and detailed. However, it should not be construed as a limitation on the scope of the patent for the present invention. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, the above technical features can be freely combined, and several deformations and improvements can also be made, which all fall within the protection scope of the present invention. Therefore, all equivalent transformations and modifications made to the scope of the claims of the present invention shall fall within the scope covered by the claims of the present invention.

Claims

1. A fluorescently encoded microsphere, characterized in that, it is prepared by adding 40 - 150 μg of a coding compound and 0.05 - 0.15 mL of a swelling agent to every 0.5 - 1.5 mg of carrier microspheres, and the coding compound includes an energy - supplying compound, a mediator compound, a luminescent compound A and a luminescent compound B; the total mass of the energy - supplying compound and the mediator compound is 10 - 30 μg, and the mass ratio between them is 1:(0.5 - 1.5); the total mass of the luminescent compound A and the luminescent compound B is 30 - 120 μg, and the mass ratio between them is (1 - 10):(10 - 1); the energy - supplying compound is methylene blue, rose bengal, porphyrin or phthalocyanine, and the mediator compound is dimethylthiophene, 9 - alkylene - N - 9,10 - dihydroacridine, dioxocyclic compound or oxalate compound.

2. The fluorescently encoded microsphere according to claim 1, characterized in that, the dioxocyclic compound is dioxocyclohexene, and the oxalate compound is bis(2,4,6 - trichlorophenyl) oxalate.

3. The fluorescently encoded microsphere according to claim 1, characterized in that, The luminescent compound A is an organic fluorescent dye A or a quantum dot. The organic fluorescent dye A is a silicon-based rhodamine, styryl-boron dipyrrole, or Cy5 dye. The quantum dot is a carbon quantum dot, a CdTe quantum dot, a CdS quantum dot, a PbS quantum dot, or a MoS 2 quantum dot.

4. The fluorescently encoded microsphere according to claim 1, characterized in that, The luminescent compound B is an organic fluorescent dye B, a quantum dot or a rare earth chelate. The organic fluorescent dye B is 2,5,8,11-tetra-tert-butylperylene, an AMCA dye or a DiO dye. The quantum dot is a carbon quantum dot, a CdTe quantum dot, a CdS quantum dot, a PbS quantum dot or a MoS 2 quantum dot. The rare earth chelate is a terbium chelate.

5. The fluorescently encoded microsphere according to claim 1, characterized in that, the swelling agent is N,N - dimethylformamide, methyl chloride, methylene chloride, chloroform, carbon tetrachloride, toluene or acetone.

6. The fluorescently encoded microsphere according to claim 1, characterized in that, the particle size of the carrier microspheres is 1 - 100 microns, and the groups of the carrier microspheres are carboxyl, amino, epoxy or tosyl.

7. A method for preparing the fluorescently encoded microsphere according to any one of claims 1 to 6, characterized in that, it comprises the following steps: S1. Take 0.5 - 1.5 mg of carrier microspheres for separation and remove the supernatant; S2. Add 40 - 150 μg of a coding compound to 0.05 - 0.15 mL of a swelling agent to obtain a mixture; the coding compound includes an energy - supplying compound, a mediator compound, a luminescent compound A and a luminescent compound B; the total mass of the energy - supplying compound and the mediator compound is 10 - 30 μg, and the mass ratio between them is 1:(0.5 - 1.5); the total mass of the luminescent compound A and the luminescent compound B is 30 - 120 μg, and the mass ratio between them is (1 - 10):(10 - 1); S3. Add the mixture to the separated carrier microspheres, mix and shake, and then wash to obtain the fluorescently encoded microspheres.

8. The method for preparing the fluorescently encoded microsphere according to claim 7, characterized in that, in step S3, the shaking speed is 30 - 70 rpm and the shaking time is 10 - 60 min.

9. The method for preparing the fluorescently encoded microsphere according to claim 7, characterized in that, in step S3, the washing is carried out by washing 2 - 4 times with an ethanol aqueous solution with a mass fraction of 50 - 100%.

10. The application of the fluorescently encoded microsphere according to any one of claims 1 to 6 in an immuno - analyzer, characterized in that, The immunoassay analyzer includes: A sheath flow module for enabling M×N types of magnetic bead complexes to pass through the detection area of the sheath flow module one by one, wherein the magnetic bead complex includes a substance to be measured and a fluorescence-encoded microsphere bound to the substance to be measured, and the fluorescence-encoded microsphere includes a luminescent compound A with M excitation intensity levels and a luminescent compound B with N laser intensity levels; A first laser for emitting a first laser to the detection area to excite the magnetic bead complex to generate fluorescence for quantification; A scattered light receiving module for receiving the fluorescence for quantification generated after passing through the detection area on the first side of the sheath flow module and converting it into a counting parameter; A second laser for emitting a second laser to the detection area to excite the M intensity levels of the luminescent compound A to correspondingly generate M intensity levels of classification fluorescence A and the N intensity levels of the luminescent compound B to correspondingly generate N intensity levels of classification fluorescence B; A classification fluorescence A receiving module for receiving the M intensity levels of the classification fluorescence A after passing through the detection area on the second side of the sheath flow module and converting it into M classification fluorescence A intensity classification parameters; A classification fluorescence B receiving module for receiving the N intensity levels of the classification fluorescence B after passing through the detection area on the second side of the sheath flow module and converting it into N classification fluorescence B intensity classification parameters; A signal processing module electrically connected to the scattered light receiving module, the classification fluorescence A receiving module, and the classification fluorescence B receiving module, for receiving the counting parameter, the M classification fluorescence A intensity classification parameters, and the N classification fluorescence B intensity classification parameters, and converting them into the counting result corresponding to each of the M×N types of magnetic bead complexes.

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