Fluorescence coding microsphere as well as preparation method and application thereof

Abstract

The invention discloses a fluorescent encoding microsphere and a preparation method and application thereof, the preparation method comprises the following steps: mixing magnetic polystyrene microspheres, a surfactant, a composite swelling agent and a fluorescent dye to prepare a mixed solution; wherein the composite swelling agent comprises methanol and chlorododecane; and heating the mixed solution to swell the magnetic polystyrene microspheres and enable the fluorescent dye to enter the swollen magnetic polystyrene microspheres, and cooling to prepare the fluorescent encoding microspheres. According to the preparation method, the combination of methanol and chlorododecane in different proportions is regulated and controlled, so that the microspheres cannot be infinitely swelled and cannot be broken or deformed, the original form of the microspheres can be kept to the maximum extent, and the swelling uniformity can be improved.

Description

Technical Field

[0001] The present application relates to the field of biomaterials, and in particular to a fluorescent coded microsphere and a preparation method and application thereof. Background Art

[0002] Fluorescently coded microspheres are micron-sized particles with specific fluorescent markers, which can be used in research fields such as multiple analysis and high-throughput screening. Its materials include organic and inorganic substances. Organic substances include polystyrene, dextran, polymethyl methacrylate, etc., and inorganic substances include silica, glass, etc. The synthesis methods of fluorescently coded microspheres mainly include sol-gel method, microemulsion polymerization method, inverse emulsion polymerization method, water / oil coating method, etc. When synthesized by sol-gel method, microspheres will aggregate, resulting in uneven particle dispersion. The sol-gel method is to add fluorescent dyes and other functional molecules to stable polymer microspheres and mark them through reactions; this method is simple and easy to operate, but during the reaction process, microspheres will aggregate, resulting in uneven particle dispersion. The microemulsion polymerization method reacts fluorescent dyes in microemulsions and forms microspheres during the reaction; the microspheres prepared by this method are uniform in size and have good dispersibility, but the preparation process of microemulsions is relatively complicated. The inverse emulsion polymerization method adds water-dispersed fluorescent dyes to the oil phase to form an inverse emulsion, and forms fluorescent coded microspheres during the reaction process; the surface properties of the microspheres prepared by this method are adjustable, but the immersion time and reaction time of the emulsifier are long. The water / oil coating method forms fluorescent coded microspheres by encapsulating fluorescent dyes in water droplets and then coating them with an oil phase; the microspheres prepared by this method are small in size and smooth in surface, but the fluorescence signal intensity is weak.

[0003] In summary, various traditional synthesis methods of fluorescent coded microspheres have some problems. Therefore, it is urgent to develop a new synthesis method of fluorescent coded microspheres. Summary of the invention

[0004] Based on this, it is necessary to provide a fluorescent coding microsphere and a preparation method and application thereof. The fluorescent coding microsphere has better uniformity, more uniform encapsulated fluorescent dye, and more convenient fluorescent coding.

[0005] The first aspect of the present application provides a method for preparing fluorescent coded microspheres, comprising the following steps:

[0006] The magnetic polystyrene microspheres, a surfactant, a composite swelling agent and a fluorescent dye are mixed to prepare a mixed solution; wherein the composite swelling agent comprises methanol and chlorododecane in a volume ratio of (1-4):1;

[0007] The mixed solution is heated to make the magnetic polystyrene microspheres swell and the fluorescent dye enter the swollen magnetic polystyrene microspheres, and then cooled to prepare fluorescent coded microspheres; wherein the fluorescent coded microspheres include the magnetic polystyrene microspheres and the fluorescent dye encapsulated in the magnetic polystyrene microspheres.

[0008] In some embodiments, the composite swelling agent includes methanol and chlorododecane in a volume ratio of (1-2):1.

[0009] In some embodiments, in the mixed solution, the mass volume fraction of the magnetic polystyrene microspheres in the composite swelling agent is 0.1% to 0.2%; optionally, the mass volume fraction of the magnetic polystyrene microspheres in the composite swelling agent is 0.14% to 0.2%.

[0010] In some embodiments, in the mixed solution, the mass ratio of the magnetic polystyrene microspheres to the fluorescent dye is 100:(1-80).

[0011] In some embodiments, in the mixed solution, the mass volume fraction of the surfactant is 0.2% to 0.3%.

[0012] In some embodiments, the surfactant is selected from sodium dodecyl sulfate.

[0013] In some embodiments, the average particle size of the magnetic polystyrene microspheres is 5000nm~8000nm.

[0014] In some embodiments, the surface of the magnetic polystyrene microspheres carries functional groups, and optionally, the functional groups are selected from one or more of carboxyl, amino and p-toluene cycloacyl.

[0015] In some embodiments, the fluorescent dye is an oil-based fluorescent dye.

[0016] In some embodiments, the oily fluorescent dye is selected from at least one of cyanine dyes, fluorescein isothiocyanate and allophycocyanin dyes.

[0017] In some embodiments, the step of heating the mixed solution comprises heating and stirring the mixed solution; optionally, heating and stirring the mixed solution satisfies one or more of the following conditions:

[0018] The heating temperature is 70℃~90℃;

[0019] The stirring speed is 300rpm~500rpm;

[0020] The stirring time is 5h~7h.

[0021] In some embodiments, the cooling further includes separation and cleaning steps; optionally, the separation and cleaning meet one or more of the following conditions:

[0022] The separation speed is 3000rpm~4000rpm; the separation time is 3min~5min; and anhydrous ethanol and ultrapure water are used for cleaning.

[0023] The second aspect of the present application provides a fluorescent coded microsphere, which is prepared by the preparation method of the fluorescent coded microsphere described in the first aspect of the present application.

[0024] The third aspect of the present application provides the use of the fluorescent coded microspheres prepared by the preparation method of the fluorescent coded microspheres described in the first aspect of the present application or the fluorescent coded microspheres described in the second aspect of the present application in the preparation of nucleic acid or protein detection kits.

[0025] By adjusting the combination of poor solvent (methanol) and good solvent (chlorododecane) in different proportions, the microspheres can be prevented from swelling infinitely, the microspheres will not break or deform, the original shape of the microspheres can be maintained to the greatest extent, and the uniformity of swelling can be improved.

[0026] The fluorescent coding microspheres prepared by the aforementioned preparation method have better uniformity, more uniform encapsulated fluorescent dyes, and more convenient fluorescent coding. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the technical solutions in the implementation methods and examples of the present application and to more completely understand the present application and its beneficial effects, the following is a brief introduction to the drawings required for use in the description of the implementation methods or examples. Obviously, the drawings described below are only some embodiments of the present application, and those skilled in the art can obtain other drawings based on these drawings without creative work.

[0028] Figure 1 The fluorescence distribution histogram of the fluorescent coded microspheres prepared in Example 1;

[0029] Figure 2 The fluorescence distribution histogram of the fluorescent coded microspheres prepared in Example 2;

[0030] Figure 3 The fluorescence distribution histogram of the fluorescent encoded microspheres prepared in Example 3;

[0031] Figure 4 The fluorescence distribution histogram of the fluorescent encoded microspheres prepared in Example 4;

[0032] Figure 5 The fluorescence distribution histogram of the fluorescent encoded microspheres prepared in Example 5;

[0033] Figure 6 The fluorescence distribution histogram of the fluorescent encoded microspheres prepared in Example 6;

[0034] Figure 7 The fluorescence distribution histogram of the fluorescent encoded microspheres prepared in Comparative Example 1;

[0035] Figure 8 The fluorescence distribution histogram of the fluorescence encoded microspheres prepared in Comparative Example 2;

[0036] Fig. 9 This is the fluorescence distribution histogram of the fluorescence encoded microspheres prepared in Comparative Example 3. DETAILED DESCRIPTION

[0037] In order to facilitate the understanding of the present application, the present application will be described more fully below with reference to the relevant drawings. The preferred embodiments of the present application are given in the drawings. However, the present application can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present application more thorough and comprehensive.

[0038] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this application belongs. The terms used herein in the specification of this application are only for the purpose of describing specific embodiments and are not intended to limit this application. The term "and / or" used herein includes any and all combinations of one or more of the related listed items.

[0039] In this application, "optionally", "optional", and "optional" mean optional or dispensable, that is, any one of the two parallel schemes of "yes" or "no". If multiple "options" appear in a technical solution, unless otherwise specified and there is no contradiction or mutual restriction, each "optional" is independent.

[0040] The terms "having", "containing", "including" and "comprising" used in this application are synonymous, which are inclusive or open-ended and do not exclude additional, uncited members or features. Members or features include materials or components, structures, elements, instruments, etc.; non-limiting examples of members or features include actions, conditions for the occurrence of actions, timing, states, etc.

[0041] In this application, the technical features or technical solutions described in an open language include closed technical features or technical solutions composed of the listed contents, and also include open technical features or technical solutions containing the listed contents.

[0042] In this application, if the unit of the data range is followed only by the right endpoint, it means that the units of the left endpoint and the right endpoint are the same. For example, 5~7h means that the units of the left endpoint "5" and the right endpoint "7" are both h, which have the same meaning as 5h~7h. In addition, similar descriptions involving other parameters also apply to the above understanding.

[0043] In this application, if there are multiple steps involved in the method flow, unless there is a clear different description in this document, there is no strict order restriction for the execution of these steps, and they can be executed in other orders than described. Moreover, any step can include multiple sub-steps or multiple stages, and these sub-steps or stages are not necessarily executed at the same time, but can be executed at different times, and their execution order is not necessarily sequential, but can be executed in turn, alternating or simultaneously with other steps or parts of sub-steps or stages of other steps.

[0044] In the present application, exemplary descriptions such as "in some implementation modes (or examples)" and "in one implementation mode (or example)" may include but are not limited to the following meanings: these solutions may be combined with other solutions in a suitable manner to form new technical solutions.

[0045] In the present application, the terms "first", "second", "third", etc. in "the first aspect", "the second aspect", "the third aspect", etc. are used only for descriptive purposes and cannot be understood as indicating or implying relative importance or quantity, nor can they be understood as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first", "second", "third", etc. only serve the purpose of non-exhaustive enumeration and description, and it should be understood that they do not constitute a closed limitation on quantity.

[0046] In the present application, when it comes to a numerical interval (i.e., a numerical range), unless otherwise specified, the distribution of the optional numerical values ​​in the numerical interval is considered to be continuous, and includes the two numerical endpoints (i.e., the minimum and maximum values) of the numerical interval, and each numerical value between the two numerical endpoints. Unless otherwise specified, when the numerical interval only refers to an integer in the numerical interval, it includes the two endpoint integers of the numerical range, and each integer between the two endpoints, which is equivalent to directly listing each integer. When multiple numerical ranges are provided to describe features or characteristics, these numerical ranges can be merged. In other words, unless otherwise specified, the numerical range disclosed herein should be understood to include any and all sub-ranges included therein. The "numerical value" in the numerical interval can be any quantitative value, such as a number, a percentage, a ratio, etc. "Numerical interval" allows broadly including numerical interval types such as percentage intervals, ratio intervals, and ratio intervals.

[0047] There are many shortcomings in the current preparation methods of fluorescent coded microspheres, such as the synthesized fluorescent coded microspheres have a small number of coding weights, uneven swelling of fluorescent dyes, and many process steps, which result in greater impact on the synthesized microspheres.

[0048] Based on this, the embodiments of the present application at least provide a fluorescent coded microsphere and a preparation method and application thereof.

[0049] In some embodiments, a method for preparing fluorescently coded microspheres is provided, comprising the following steps: mixing magnetic polystyrene microspheres, a composite swelling agent, and a fluorescent dye to prepare a mixed solution; wherein the composite swelling agent comprises methanol and chlorododecane; heating the mixed solution to swell the magnetic polystyrene microspheres and allow the fluorescent dye to enter the swollen magnetic polystyrene microspheres, and cooling to prepare fluorescently coded microspheres.

[0050] In some embodiments, a method for preparing fluorescently coded microspheres is provided, comprising the following steps: mixing magnetic polystyrene microspheres, a surfactant, a composite swelling agent, and a fluorescent dye to prepare a mixed solution; wherein the composite swelling agent comprises methanol and chlorododecane; heating the mixed solution to swell the magnetic polystyrene microspheres and allow the fluorescent dye to enter the swollen magnetic polystyrene microspheres, and cooling to prepare fluorescently coded microspheres.

[0051] In some embodiments, in the composite swelling agent, the volume ratio of methanol to chlorododecane may be (1-4):1.

[0052] In some embodiments, in the composite swelling agent, the volume ratio of methanol to chlorododecane may be (1-2):1.

[0053] In some embodiments, a method for preparing fluorescently coded microspheres is provided, comprising the following steps: mixing magnetic polystyrene microspheres, a surfactant, a composite swelling agent, and a fluorescent dye to prepare a mixed solution; wherein the composite swelling agent comprises methanol and chlorododecane in a volume ratio of (1-4):1; heating the mixed solution to swell the magnetic polystyrene microspheres and allow the fluorescent dye to enter the swollen magnetic polystyrene microspheres, and cooling to prepare fluorescently coded microspheres.

[0054] In some embodiments, a swelling method is used, by adding a certain proportion of good solvent and poor solvent as swelling agents, and adding oily fluorescent dyes under heating and stirring conditions, the absorption of fluorescent dyes can be achieved through swelling, and finally the microspheres are coded for fluorescent dyes by removing the swelling agent. This method can use commercial magnetic microspheres as base balls to achieve the synthesis of fluorescent coded microspheres, and can also be used for fluorescent coding of self-synthesized magnetic microspheres.

[0055] In some embodiments, commercially available polystyrene microspheres with functional groups modified on the magnetic surface are used as base balls, oily fluorescent dyes are added, a combination of poor solvents and good solvents are added as swelling agents, and a small amount of surfactant is added. Under heating and stirring conditions, the microspheres will swell to a certain extent and have holes, allowing the fluorescent dye to enter the interior. After removing the swelling agent, the swollen microspheres will return to their original form, causing the fluorescent dye that has entered the interior to be retained inside to achieve the purpose of fluorescent coding. Compared with a simple good solvent, the use of a combination of more than one poor solvent and a good solvent can prevent the microspheres from swelling infinitely, prevent the microspheres from breaking and deforming, and can maximize the original form of the microspheres, which can improve the uniformity of swelling.

[0056] In the present application, unless otherwise specified, the surface of the magnetic polystyrene microspheres carries functional groups for binding with nucleic acids or proteins in subsequent applications. In a non-limiting manner, the functional groups on the surface of the magnetic polystyrene microspheres are selected from at least one of carboxyl, amino and p-toluene cycloacyl, and further, the surface of the magnetic polystyrene microspheres is modified with carboxyl groups.

[0057] In the present application, unless otherwise specified, "swelling" refers to the phenomenon that a polymer material expands in volume in a solvent.

[0058] In a first aspect of the present application, a method for preparing fluorescent coded microspheres is provided.

[0059] In some embodiments, the method for preparing fluorescent coded microspheres may include the following steps:

[0060] S100: mixing magnetic polystyrene microspheres, a surfactant, a composite swelling agent and a fluorescent dye to prepare a mixed solution; wherein the composite swelling agent includes a poor solvent and a good solvent, and may further include a poor solvent and a good solvent in a volume ratio of (1-4):1;

[0061] S200: heating the mixed solution to make the magnetic polystyrene microspheres swell and allow the fluorescent dye to enter the swollen magnetic polystyrene microspheres, and cooling the mixed solution to prepare fluorescent coded microspheres; wherein the fluorescent coded microspheres include magnetic polystyrene microspheres and fluorescent dyes encapsulated in the magnetic polystyrene microspheres.

[0062] In some embodiments, the poor solvent may be selected from one or more of ethanol, methanol, isopropanol, ethanol, ethylene glycol, propionate alcohol, n-octanol, undecanol, isobutanol, and n-propanol.

[0063] In some embodiments, the good solvent may be selected from one or more of dichloromethane, n-hexane, chloroform, petroleum ether and chlorododecane.

[0064] In some embodiments, the poor solvent may be methanol, and the good solvent may be chlorododecane.

[0065] In some embodiments, the method for preparing fluorescent coded microspheres may include the following steps:

[0066] S100': mixing magnetic polystyrene microspheres, a surfactant, a composite swelling agent and a fluorescent dye to prepare a mixed solution; wherein the composite swelling agent includes methanol and chlorododecane, and may further include methanol and chlorododecane in a volume ratio of (1-4):1;

[0067] S200': heating the mixed solution to make the magnetic polystyrene microspheres swell and allow the fluorescent dye to enter the swollen magnetic polystyrene microspheres, and cooling to prepare fluorescent coded microspheres; wherein the fluorescent coded microspheres include magnetic polystyrene microspheres and fluorescent dyes encapsulated in the magnetic polystyrene microspheres.

[0068] In some embodiments, the volume ratio of methanol to chlorododecane may be, but is not limited to, 1:1, 1.33:1, 2:1, 3:1, 4:1, or a ratio or range between any two of the above ratios.

[0069] In this application, unless otherwise specified, "methanol" is used as a poor solvent and "chlorododecane" is used as a good solvent. By adjusting the ratio of methanol to chlorododecane in the swelling system, the swelling degree of the microspheres can be controlled, and the number of molecules of the fluorescent dye entering the microspheres can be adjusted, so that the swelling of the microspheres will not be infinitely swollen, the microspheres will not be broken or deformed, the original shape of the microspheres can be maintained to the maximum extent, and the uniformity of the swelling can be improved.

[0070] In some embodiments, in the mixed solution, the mass volume fraction of the magnetic polystyrene microspheres in the composite swelling agent can be 0.1% to 0.2%. Non-limitingly, the mass volume fraction of the magnetic polystyrene microspheres in the composite swelling agent can be, but not limited to, 0.1%, 0.12%, 0.14%, 0.16%, 0.18%, 0.2%, or a value or range between any two of the above values.

[0071] In some embodiments, in the mixed solution, the mass ratio of the magnetic polystyrene microspheres to the fluorescent dye can be 100:(1~80). Without limitation, the mass ratio of the magnetic polystyrene microspheres to the fluorescent dye can be but not limited to 100:1, 100:2, 100:4, 100:8, 100:16, 100:20, 100:30, 100:32, 100:64, 100:80 or a ratio or range between any two of the above ratios.

[0072] In some embodiments, in the mixed solution, the mass volume fraction of the surfactant is 0.2% to 0.3%. In a non-limiting manner, the mass volume fraction of the surfactant can be, but is not limited to, 0.2%, 0.25%, 0.3% or a value or range between any two of the above values.

[0073] In some embodiments, the surfactant may be selected from sodium dodecyl sulfate.

[0074] In the present application, unless otherwise specified, the surfactant can reduce the tension at the interface of the magnetic polystyrene microsphere emulsion droplets and maintain the stability of the emulsion. In addition, the surfactant can also help the fluorescent dye to be evenly encapsulated inside the magnetic polystyrene microspheres, which helps to improve the stability and uniformity of the fluorescently encoded microspheres.

[0075] In some embodiments, the average particle size of the magnetic polystyrene microspheres may be 5000 nm to 8000 nm. Non-limitingly, the average particle size of the magnetic polystyrene microspheres may be, but not limited to, 5000 nm, 6000 nm, 7000 nm, 8000 nm, or a value or range between any two of the above values.

[0076] In some embodiments, the fluorescent dye is an oily fluorescent dye, and further, the oily fluorescent dye is selected from at least one of cyanine dyes, fluorescein isothiocyanate and allophycocyanin dyes.

[0077] In some embodiments, in step S200', the step of heating the mixed solution includes heating and stirring the mixed solution.

[0078] In some embodiments, in the step of heating and stirring the mixed solution, the heating temperature is 70° C. to 90° C. In a non-limiting manner, the heating temperature may be, but is not limited to, 70° C., 80° C., 90° C., or a temperature or range between any two of the above temperatures.

[0079] In some embodiments, in the step of heating and stirring the mixed solution, the stirring speed is 300 rpm to 500 rpm. In a non-limiting manner, the stirring speed can be but is not limited to 300 rpm, 400 rpm, 500 rpm or a value or range between any two of the above values.

[0080] In some embodiments, in the step of heating and stirring the mixed solution, the stirring time is 5 h to 7 h. In a non-limiting manner, the stirring time can be but is not limited to 5 h, 6 h, 7 h, or a time or range between any two of the above time periods.

[0081] In some embodiments, in step S200', separation and cleaning steps are further included after cooling.

[0082] In some embodiments, the separation rotation speed is 3000 rpm to 4000 rpm. Non-limitingly, the separation rotation speed can be but not limited to 3000 rpm, 3500 rpm, 4000 rpm or a value or range between any two of the above values.

[0083] In some embodiments, the separation time is 3 min to 5 min. Without limitation, the separation time can be 3 min, 4 min, 5 min, or a time period between any two of the above time periods.

[0084] In some embodiments, the cleaning step comprises cleaning with anhydrous ethanol and ultrapure water.

[0085] In a second aspect of the present application, a fluorescent coded microsphere is provided, which is prepared by the method for preparing fluorescent coded microspheres according to the first aspect of the present application.

[0086] The fluorescent coding microspheres prepared by the above method have better uniformity, more uniform encapsulated fluorescent dyes, and more convenient fluorescent coding.

[0087] In the third aspect of the present application, provided is the use of the fluorescently coded microspheres prepared by the method for preparing the fluorescently coded microspheres of the first aspect of the present application or the fluorescently coded microspheres of the second aspect of the present application in preparing a nucleic acid or protein detection kit.

[0088] Some examples are provided below.

[0089] The embodiments of the present application will be described in detail below in conjunction with examples. It should be understood that these examples are only used to illustrate the present application and are not intended to limit the scope of the present application. The experimental methods for which the conditions are not specified in the following examples are preferably referred to the guidance given in the present application, and can also be based on the experimental manual or normal conditions in the art, or can also be based on the conditions recommended by the manufacturer, or with reference to experimental methods known in the art.

[0090] In the following examples, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument testing accuracy or operating accuracy are allowed.

[0091] In the following examples, SDS water refers to an aqueous solution containing sodium dodecyl sulfate (SDS).

[0092] Example 1

[0093] 1. Synthesis of fluorescent coded microspheres by swelling method

[0094] (1) Add 10 mL of 0.25% SDS aqueous solution into a 100 mL flask, and then add 0.1 mg of commercial magnetic carboxyl polystyrene microspheres (purchased from MBL).

[0095] (2) Add 60 µL of a mixed solvent of methanol and chlorododecane (volume ratio = 2:1).

[0096] (3) Add 1, 2, 4, 8, 16, 32, 64, and 80 μg of Cy5.5 fluorescent dye respectively.

[0097] (4) Heat in an oil bath at 80°C and stir at 400 rpm for 6 hours.

[0098] (5) Rapidly cool to room temperature, transfer to a 50 mL centrifuge tube and centrifuge at 3500 rpm for 5 min for cleaning. Wash twice with anhydrous ethanol and twice with ultrapure water, and finally store in ultrapure water.

[0099] Example 2

[0100] (1) Add 10 mL of 0.25% SDS aqueous solution to a 100 mL flask and then add 0.1 mg of commercial magnetic carboxyl polystyrene microspheres.

[0101] (2) Add 60 µL of a mixed solvent of methanol and chlorododecane (volume ratio = 2:1).

[0102] (3) Add 8 μg of Cy5 fluorescent dye.

[0103] (4) Heat in an oil bath at 80°C and stir at 400 rpm for 6 hours.

[0104] (5) Rapidly cool to room temperature, transfer to a 50 mL centrifuge tube and centrifuge at 3500 rpm for 5 min for cleaning. Wash twice with anhydrous ethanol and twice with ultrapure water, and finally store in ultrapure water.

[0105] Example 3

[0106] (1) Add 10 mL of 0.25% SDS aqueous solution to a 100 mL flask and then add 0.1 mg of commercial magnetic carboxyl polystyrene microspheres.

[0107] (2) Add 60 µL of a mixed solvent of methanol and chlorododecane (volume ratio = 1:1).

[0108] (3) Add 4 μg of Cy5 fluorescent dye.

[0109] (4) Heat in an oil bath at 80°C and stir at 400 rpm for 6 hours.

[0110] (5) Rapidly cool to room temperature, transfer to a 50 mL centrifuge tube and centrifuge at 3500 rpm for 5 min for cleaning. Wash twice with anhydrous ethanol and twice with ultrapure water, and finally store in ultrapure water.

[0111] Example 4

[0112] (1) Add 10 mL of 0.25% SDS aqueous solution to a 100 mL flask and then add 0.1 mg of commercial magnetic carboxyl polystyrene microspheres.

[0113] (2) Add 60 µL of a mixed solvent of methanol and chlorododecane (volume ratio = 2:1).

[0114] (3) Add 4 μg of Cy5 fluorescent dye.

[0115] (4) Heat in an oil bath at 80°C and stir at 400 rpm for 6 hours.

[0116] (5) Rapidly cool to room temperature, transfer to a 50 mL centrifuge tube and centrifuge at 3500 rpm for 5 min for cleaning. Wash twice with anhydrous ethanol and twice with ultrapure water, and finally store in ultrapure water.

[0117] Example 5

[0118] (1) Add 10 mL of 0.25% SDS aqueous solution to a 100 mL flask and then add 0.1 mg of commercial magnetic carboxyl polystyrene microspheres.

[0119] (2) Add 70 µL of a mixed solvent of methanol and chlorododecane (volume ratio = 4:3).

[0120] (3) Add 4 μg of Cy5 fluorescent dye.

[0121] (4) Heat in an oil bath at 80°C and stir at 400 rpm for 6 hours.

[0122] (5) Rapidly cool to room temperature, transfer to a 50 mL centrifuge tube and centrifuge at 3500 rpm for 5 min for cleaning. Wash twice with anhydrous ethanol and twice with ultrapure water, and finally store in ultrapure water.

[0123] Example 6

[0124] (1) Add 10 mL of 0.25% SDS aqueous solution to a 100 mL flask and then add 0.1 mg of commercial magnetic carboxyl polystyrene microspheres.

[0125] (2) Add 50 µL of a mixed solvent of methanol and chlorododecane (volume ratio = 4:1).

[0126] (3) Add 2 μg of Cy5 fluorescent dye.

[0127] (4) Heat in an oil bath at 80°C and stir at 400 rpm for 6 hours.

[0128] (5) Rapidly cool to room temperature, transfer to a 50 mL centrifuge tube and centrifuge at 3500 rpm for 5 min for cleaning. Wash twice with anhydrous ethanol and twice with ultrapure water, and finally store in ultrapure water.

[0129] Comparative Example 1. Only good solvent chlorododecane was used as the swelling agent.

[0130] (1) Add 30 mL of 0.25% SDS aqueous solution to a 100 mL flask, and then add 0.1 mg of commercial magnetic carboxyl polystyrene microspheres.

[0131] (2) Add 60 µL of chlorododecane.

[0132] (3) Add 1, 2, 4, 8, 16, 32, 64, and 80 μg of Cy5.5 fluorescent dye.

[0133] (4) Heat in an oil bath at 80°C and stir at 400 rpm for 6 hours.

[0134] (5) Rapidly cool to room temperature, transfer to a 50 mL centrifuge tube and centrifuge for cleaning at 3500 rpm for 5 min. Wash twice with anhydrous ethanol and twice with ultrapure water, and finally store in ultrapure water.

[0135] Comparative Example 2. Changing the composition of the composite swelling agent.

[0136] (1) Add 10 mL of 0.25% SDS aqueous solution to a 100 mL flask and then add 0.1 mg of commercial magnetic carboxyl polystyrene microspheres.

[0137] (2) Add 60 µL of a mixed solvent of n-hexane and chlorododecane (volume ratio = 2:1).

[0138] (3) Add 8 μg of Cy5 fluorescent dye.

[0139] (4) Heating in an oil bath at 80°C and stirring at 400 rpm for 6 hours;

[0140] (5) Rapidly cool to room temperature, transfer to a 50 mL centrifuge tube and centrifuge at 3500 rpm for 5 min for cleaning. Wash twice with anhydrous ethanol and twice with ultrapure water, and finally store in ultrapure water.

[0141] Comparative Example 3. Changing the type of good solvent.

[0142] (1) Add 10 mL of 0.25% SDS aqueous solution to a 100 mL flask and then add 0.1 mg of commercial magnetic carboxyl polystyrene microspheres.

[0143] (2) Add 60 µL of a mixed solvent of n-hexane and methanol (volume ratio = 2:1).

[0144] (3) Add 8 μg of Cy5 fluorescent dye.

[0145] (4) Heating in an oil bath at 80°C and stirring at 400 rpm for 6 hours;

[0146] (5) Rapidly cool to room temperature, transfer to a 50 mL centrifuge tube and centrifuge at 3500 rpm for 5 min for cleaning. Wash twice with anhydrous ethanol and twice with ultrapure water, and finally store in ultrapure water.

[0147] Performance test

[0148] 1. Test and analysis methods

[0149] Carboxylated magnetic microspheres were purchased from MBL, SDS, methanol, ethanol, and chlorododecane were purchased from Sinopharm Reagent. The instruments were Attune NxT flow cytometer (Thermo Fisher), A5000 cantilever stirrer, and G3000 magnetic stirrer (Ohaus). The encoded microspheres in Examples 1 to 6 and Comparative Examples 1 to 3 were washed with ethanol and water and stored in an aqueous solution. The fluorescence intensity was detected using a flow cytometer, and the detection results were displayed using a flow fluorescence distribution histogram. Figure 1 and Figure 7 The results of the fluorescent coded microspheres prepared in Example 1 and Comparative Example 1 in the RL2 channel of the flow cytometer, wherein the horizontal axis represents the fluorescence intensity, and the vertical axis "percent of max" represents the maximum percentage of the number of fluorescent coded microspheres obtained using different masses of Cy5.5 fluorescent dyes, which is used to observe the distribution of fluorescence intensity of different fluorescent coded microspheres. In Example 1 and Comparative Example 1, the fluorescent coded microspheres obtained using 8 groups of different masses of Cy5.5 fluorescent dyes were labeled as 1μg Cy5.5, 2μg Cy5.5, 4μg Cy5.5, 8μg Cy5.5, 16μg Cy5.5, 32μg Cy5.5, 64μg Cy5.5 and 80μg Cy5.5, respectively.

[0150] Figure 2~Figure 6 The results of the fluorescent coded microspheres obtained in Examples 2 to 6 in the RL1 channel of the flow cytometer are as follows: Figure 8 and Fig. 9 The results of the fluorescence-encoded microspheres obtained in Comparative Example 2 and Comparative Example 3 in the RL1 channel of the flow cytometer are shown in FIG. 1 , wherein the horizontal axis represents the fluorescence intensity, and the vertical axis "count" represents the number of the fluorescence-encoded microspheres.

[0151] 2. Test results analysis

[0152] From the results of Example 1 and Comparative Example 1, it can be seen that the fluorescence intensity distribution of some coded microspheres obtained in Comparative Example 1 is uneven and wide, and several coded microsphere signals overlap and cannot be completely distinguished. The distribution uniformity of the fluorescence intensity obtained by the method of Example 1 is significantly improved, the distribution is narrow, and the codes can be basically completely separated.

[0153] From the results of Comparative Examples 2 and 3, it can be seen that the fluorescence intensity distribution of the encoded microspheres obtained by using other combinations of poor solvents and good solvents is relatively wide.

[0154] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0155] The above-mentioned embodiments only express several implementation methods of the present application, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present application, and these all belong to the protection scope of the present application. Therefore, the protection scope of the patent of the present application shall be subject to the attached claims, and the description and drawings may be used to interpret the scope of the claims.

Claims

1. A method for preparing fluorescent coded microspheres, characterized in that: The following steps are involved: The magnetic polystyrene microspheres, a surfactant, a composite swelling agent and a fluorescent dye are mixed to prepare a mixed solution; wherein the composite swelling agent comprises methanol and chlorododecane in a volume ratio of (1-4):1; The mixed solution is heated to make the magnetic polystyrene microspheres swell and the fluorescent dye enter the swollen magnetic polystyrene microspheres, and then cooled to prepare fluorescent coded microspheres; wherein the fluorescent coded microspheres include the magnetic polystyrene microspheres and the fluorescent dye encapsulated in the magnetic polystyrene microspheres.

2. The method for preparing fluorescent coded microspheres according to claim 1, characterized in that: The composite swelling agent comprises methanol and chlorododecane in a volume ratio of (1-2):

1.

3. The method for preparing fluorescent coded microspheres according to claim 2, characterized in that: The preparation method meets one or more of the following conditions: In the mixed solution, the mass volume fraction of the magnetic polystyrene microspheres in the composite swelling agent is 0.1% to 0.2%; optionally, the mass volume fraction of the magnetic polystyrene microspheres in the composite swelling agent is 0.14% to 0.2%; In the mixed solution, the mass ratio of the magnetic polystyrene microspheres to the fluorescent dye is 100:(1-80); In the mixed solution, the mass volume fraction of the surfactant is 0.2% to 0.3%; Optionally, the surfactant is selected from sodium lauryl sulfate.

4. The method for preparing fluorescent coded microspheres according to claim 3, characterized in that: The magnetic polystyrene microspheres meet one or more of the following conditions: The average particle size of the magnetic polystyrene microspheres is 5000nm~8000nm; The surface of the magnetic polystyrene microspheres carries functional groups, and optionally, the functional groups are selected from one or more of carboxyl, amino and p-toluene cycloacyl.

5. The method for preparing fluorescent coded microspheres according to claim 4, characterized in that: The fluorescent dye is an oily fluorescent dye; Optionally, the oily fluorescent dye is selected from at least one of cyanine dyes, fluorescein isothiocyanate and allophycocyanin dyes.

6. The method for preparing fluorescent coded microspheres according to any one of claims 1 to 5, characterized in that: The step of heating the mixed solution includes heating and stirring the mixed solution.

7. The method for preparing fluorescent coded microspheres according to claim 6, characterized in that: The mixed solution is heated and stirred to meet one or more of the following conditions: The heating temperature is 70℃~90℃; The stirring speed is 300rpm~500rpm; The stirring time is 5h~7h.

8. The method for preparing fluorescent coded microspheres according to claim 7, characterized in that: The cooling step also includes separation and cleaning steps; Optionally, the separation and cleaning satisfy one or more of the following conditions: The separation speed is 3000rpm~4000rpm; the separation time is 3min~5min; Use anhydrous ethanol and ultrapure water for cleaning.

9. A fluorescent coded microsphere, characterized in that: The fluorescent coded microspheres are prepared by the preparation method of any one of claims 1 to 8.

10. Use of the fluorescent coded microspheres prepared by the method for preparing the fluorescent coded microspheres according to any one of claims 1 to 9 or the fluorescent coded microspheres according to claim 10 in preparing a nucleic acid or protein detection kit.

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