CN112048096B - Preparation method of polystyrene hydrophilic fluorescent microspheres, prepared microspheres and application - Google Patents
Preparation method of polystyrene hydrophilic fluorescent microspheres, prepared microspheres and application Download PDFInfo
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Abstract
The preparation method comprises the steps of carrying out GMA surface modification on the polystyrene microsphere, then carrying out fluorescent dyeing, and then adding methacrylic acid for polymerization reaction to form the polystyrene hydrophilic fluorescent microsphere with the surface provided with the long-chain structure carboxyl polymerization coating. The preparation method of the polystyrene hydrophilic fluorescent microsphere is simple in process steps, simple and easily available in raw materials, low in cost and easy to widely popularize and use. The cTnI content detection card prepared from the polystyrene hydrophilic fluorescent microspheres has higher detection sensitivity and stability and stronger practicability.
Description
Technical Field
The application relates to the technical field of medical materials, in particular to a preparation method of a polystyrene hydrophilic fluorescent microsphere, the prepared microsphere and application.
Background
As a novel functionalized solid carrier, the polystyrene fluorescent microsphere has the characteristics of a solid carrier, a fluorescent material and the like, and is widely applied to detection devices such as flow cells, biosensors, immunodiagnostic reagents and the like at present. The polystyrene fluorescent microspheres can emit fluorescence with different wavelengths under the action of external illumination, so that a target can be analyzed and detected through a fluorescent signal. The surface of the polystyrene fluorescent microsphere is modified with various functional groups, and the polystyrene fluorescent microsphere is coupled with target biomolecules (such as protein, enzyme, nucleic acid and the like) through covalent bonding of the functional groups.
Carboxyl is a common functional group for modifying fluorescent microspheres, and can be coupled with biological ligands under certain conditions to obtain the immunofluorescent microspheres. The principle of the carboxyl modified polystyrene fluorescent microsphere is that carboxyl on the surface of the fluorescent microsphere is used for activation treatment, EDC (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride) and NHS (N-hydroxysuccinimide) are used for group activation, and the generated active group and amino-containing biomolecules are subjected to covalent bonding reaction.
However, when amino biomolecule detection is carried out, the existing carboxyl modified polystyrene fluorescent microspheres have a large amount of non-specific adsorption and low protein loading capacity, so that a reaction signal is weak in the detection process, and the detection sensitivity is further influenced.
Disclosure of Invention
The application provides a preparation method of a polystyrene hydrophilic fluorescent microsphere, the prepared microsphere and application thereof, and aims to solve the problems that when the existing carboxyl fluorescent microsphere is used for amino biomolecule detection, a large amount of nonspecific adsorption exists, the protein loading capacity is low, a reaction signal is weak in the detection process, and the detection sensitivity is further influenced.
The application provides a preparation method of a polystyrene hydrophilic fluorescent microsphere, which comprises the following steps:
preparing GMA modified polystyrene microspheres;
carrying out fluorescent dyeing on the GMA modified polystyrene microspheres to prepare polystyrene fluorescent microspheres;
modifying a carboxyl polymerization coating on the surface of the polystyrene fluorescent microsphere through polymerization reaction to prepare the polystyrene hydrophilic fluorescent microsphere, wherein the prepared polystyrene hydrophilic fluorescent microsphere has a simple structure formula as follows:
Optionally, the step of preparing GMA modified polystyrene microspheres specifically comprises the following steps,
adding polystyrene microspheres into a mixed reagent of concentrated sulfuric acid and nitric acid, stirring and reacting for 15-25h at 20-40 ℃ to enable the surfaces of the polystyrene microspheres to have nitro groups, and centrifugally cleaning to prepare nitro microspheres;
pouring the product microspheres of the nitro microspheres into a three-neck flask, diluting the nitro microspheres to 5-15mg/ml with purified water, adding NaOH powder while stirring, adding Na2S2O4, continuously stirring, reacting for 2-4h at 70-90 ℃, centrifuging, and washing with purified water to obtain amino microspheres;
adding the amino microspheres into GMA and an organic solvent reagent, stirring and reacting for 15-25h at the temperature of 60-80 ℃, and then centrifugally cleaning the suspension to prepare the GMA modified polystyrene microspheres.
Optionally, the mass ratio of the polystyrene microspheres to the concentrated sulfuric acid to the nitric acid is 1: (1-1.5): (2-3);
the mass ratio of the nitro microspheres to the Na2S2O4 to the NaOH is 1: (1.5-3): (2-5).
Optionally, the amino microspheres, the GMA and the organic reagent are added in a mass ratio of 1: (0.1-0.5): (5-50).
Optionally, the step of preparing the polystyrene fluorescent microspheres by fluorescent dyeing of the GMA-modified polystyrene microspheres specifically includes the following steps:
dissolving oil-soluble dye in dichloromethane solvent,
suspending GMA modified polystyrene microspheres in SDS aqueous solution,
mixing and emulsifying the prepared two solutions, stirring the emulsion at room temperature for reaction for 15-25h, and centrifugally cleaning to obtain the polystyrene fluorescent microspheres.
Optionally, the oil-soluble fluorescent dye comprises one or a combination of any two or more of NBD, Bodipy, rhodamine, and coumarin.
Optionally, the step of modifying the carboxyl polymeric coating on the surface of the polystyrene fluorescent microsphere through a polymerization reaction to obtain the polystyrene hydrophilic fluorescent microsphere specifically includes the following steps:
suspending the polystyrene fluorescent microspheres in an ethanol solvent, adding methacrylic acid and azobisisobutyronitrile, stirring and reacting for 16-20h at 70-80 ℃, and then centrifugally cleaning to obtain the polystyrene hydrophilic fluorescent microspheres.
Optionally, the mass ratio of the fluorescent microspheres to ethanol to methacrylic acid to azobisisobutyronitrile is 1: (30-100): (0.5-3): (0.1-0.3).
The application provides a polystyrene hydrophilic fluorescent microsphere prepared by the method.
The application provides an application of the polystyrene hydrophilic fluorescent microsphere prepared by the method in a cTnI content detection card.
The preparation method comprises the steps of carrying out GMA surface modification on polystyrene microspheres, then carrying out fluorescent dyeing, and then adding methacrylic acid to carry out polymerization reaction to form the polystyrene hydrophilic fluorescent microspheres with the long-chain structure carboxyl polymerization coating on the surfaces. The polystyrene hydrophilic fluorescent microsphere contains a carboxyl polymerization coating of a carboxyl coating with a long-chain structure, wherein the carboxyl polymerization coating on the surface is formed by polymerizing a carboxyl monomer on the surface of the microsphere, the length of a spacer arm is longer than that of a space arm of the microsphere modified by a common functional group, the long-chain carboxyl spacer ratio is long, coupling and combination of biomolecules can be performed to the maximum extent, and the protein coupling amount is high. In addition, the carboxyl polymerization coating on the surface of the polystyrene hydrophilic fluorescent microsphere contains high-density carboxyl, so that the hydrophilic performance of the polystyrene hydrophilic fluorescent microsphere is better, and the polystyrene hydrophilic fluorescent microsphere has lower nonspecific adsorption in the application process, so that a large amount of nonspecific adsorption can be eliminated, a series of problems caused by the nonspecific adsorption are reduced, and the stability is better.
The preparation method of the polystyrene hydrophilic fluorescent microsphere is simple in process steps, simple and easily available in raw materials, low in cost and easy to widely popularize and use. The cTnI content detection card prepared from the polystyrene hydrophilic fluorescent microspheres has higher detection sensitivity and stability and stronger practicability.
The cTnI content detection card prepared from the polystyrene hydrophilic fluorescent microspheres has higher detection sensitivity and stability and stronger practicability.
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In order to more clearly explain the technical solution of the present application, the drawings needed to be used in the embodiments will be briefly described below, and it is obvious to those skilled in the art that other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 is a flow chart of an example of a method for preparing polystyrene hydrophilic fluorescent microspheres according to the present application.
Detailed Description
Fig. 1 is a flowchart of an example of a preparation method of a polystyrene hydrophilic fluorescent microsphere according to the present application, and as shown in fig. 1, the preparation method of the polystyrene hydrophilic fluorescent microsphere includes:
step S100, preparing glycidyl methacrylate (hereinafter abbreviated as GMA) modified polystyrene microspheres.
In the present application, step S100, preparing GMA-modified polystyrene microspheres specifically includes,
step S110, adding the polystyrene microspheres into a mixed reagent of concentrated sulfuric acid and nitric acid, stirring and reacting for 15-25h at the temperature of 20-40 ℃ to enable the surfaces of the polystyrene microspheres to be provided with nitro groups, and centrifugally cleaning to obtain the nitro microspheres. In this example, the mass ratio of the polystyrene microspheres to the concentrated sulfuric acid to the nitric acid is 1: (1-1.5): (2-3). It should be noted that the stirring temperature and stirring time in this step can be adjusted by those skilled in the art according to actual needs, for example, stirring reaction at 30 ℃ for 20h, which falls within the protection scope of the present application.
Step S120, pouring the nitro microsphere product microspheres into a three-neck flask, diluting the nitro microspheres to 5-15mg/ml by purified water, adding NaOH powder while stirring, and adding Na 2 S 2 O 4 And continuously stirring, reacting for 2-4h under the condition of 70-90 ℃, centrifuging, and washing by purified water to obtain the amino microsphere. In this example, nitro-microsphere, Na 2 S 2 O 4 And the mass ratio of NaOH is 1: (1.5-3): (2-5). It should be noted that the volume of the diluted nitro-microsphere in this step can be adjusted by those skilled in the art according to actual needs, for example, the nitro-microsphere is diluted to 10mg/ml by purified water, which is within the protection scope of the present application. The stirring temperature and stirring time in this step can be adjusted by those skilled in the art according to actual needs, for example, stirring reaction at 80 ℃ for 3h, which is within the scope of the present application. The number of times of the purified water washing in this step can be adjusted by those skilled in the art according to actual needs, for example, 3 times of the purified water washing, which all fall within the scope of the present application.
Step S130, adding the amino microspheres into GMA and an organic solvent reagent, stirring and reacting for 15-25h at the temperature of 60-80 ℃, and then centrifugally cleaning the suspension to prepare GMA modified polystyrene microspheres. In the example, the mass ratio of the amino microspheres, GMA and the organic reagent is 1: (0.1-0.5): (5-50). In this example, N-dimethylformamide is used as the organic solvent reagent, and other reagents can be selected by those skilled in the art according to actual needs, and all of them are within the scope of the present application. It should be noted that the stirring temperature and stirring time in this step can be adjusted by those skilled in the art according to actual needs, for example, stirring reaction at 70 ℃ for 20h, which falls within the protection scope of the present application.
And step S200, carrying out fluorescent dyeing on the GMA modified polystyrene microsphere to obtain the polystyrene fluorescent microsphere.
In this application, step S200 is to prepare the polystyrene fluorescent microsphere through fluorescent dyeing the GMA-modified polystyrene microsphere, and specifically includes:
step S210, dissolving oil-soluble dye in dichloromethane solvent;
step S220, suspending the GMA modified polystyrene microsphere in Sodium Dodecyl Sulfate (SDS) aqueous solution, wherein the oil-soluble fluorescent dye comprises one or the combination of any two or more of NBD, Bodipy, rhodamine and coumarin;
and step S230, mixing and emulsifying the prepared two solutions, stirring the emulsion at room temperature for reaction for 15-25h, and centrifugally cleaning to obtain the polystyrene fluorescent microspheres. It should be noted that the stirring time in this step can be adjusted by those skilled in the art according to actual needs, for example, the stirring time is 20h, which all fall into the protection scope of the present application.
Step S300, modifying a carboxyl polymerization coating on the surface of the polystyrene fluorescent microsphere through polymerization reaction to prepare the polystyrene hydrophilic fluorescent microsphere, wherein the prepared polystyrene hydrophilic fluorescent microsphere has a structural formula as follows:
In the application, step S300, modifying a carboxyl polymeric coating on the surface of the polystyrene fluorescent microsphere through a polymerization reaction to obtain the polystyrene hydrophilic fluorescent microsphere, specifically including suspending the polystyrene fluorescent microsphere in an ethanol solvent, adding methacrylic acid and azobisisobutyronitrile into the ethanol solvent, stirring and reacting for 16-20 hours at 70-80 ℃, and then performing centrifugal cleaning to obtain the polystyrene hydrophilic fluorescent microsphere. It should be noted that the stirring time in this step can be adjusted by those skilled in the art according to actual needs, for example, stirring the reaction for 18h at 75 ℃, which is within the scope of the present application.
In step S300, the mass ratio of the fluorescent microsphere, ethanol, methacrylic acid, and azobisisobutyronitrile is 1: (30-100): (0.5-3): (0.1-0.3). In addition, in step S300, the fluorescent microspheres are repeatedly washed by centrifugation using a centrifuge to remove all unreacted and bonded polymers and inorganic salts. And (4) analyzing and determining by using a conductivity meter in the cleaning process, and indicating that the cleaning is complete if the cleaning standard is met. The skilled person can set the washing standard according to the actual need, for example, the washing standard is that the conductivity meter measurement result is less than 10us/cm, and the washing is considered to be complete.
In order to facilitate the technical solution better understood by those skilled in the art, the following will further describe the preparation method of the polystyrene hydrophilic fluorescent microsphere with an example.
(1) Preparing a mechanical stirrer and a water bath, adding 15ml of concentrated sulfuric acid and 10ml of nitric acid into an installed three-neck flask, adding 2g of polystyrene microspheres under the condition of continuous stirring, stirring for 18 hours at 50 ℃, slowly pouring the polystyrene microspheres into 200ml of purified water after the reaction is finished, centrifuging, and washing with the purified water until the ph of a washing solution is neutral.
Pouring 1g of product microspheres into a three-neck flask, diluting the microspheres to 10mg/ml with purified water, adding 2.5g of NaOH powder while stirring, and adding 2g of Na 2 S 2 O 4 After the reaction at 80 ℃ for 3 hours, the reaction was completed, and the reaction mixture was centrifuged and washed with purified water 3 times.
Pouring 1g of amino microspheres into a three-neck flask, adding 2ml of GMA and 20ml of N, N-dimethylformamide, stirring at 70 ℃ for reaction for 20h, and after the reaction is finished, centrifugally cleaning the suspension for 3 times to obtain GMA modified polystyrene microspheres.
(2) 10mg of rhodamine dye is dissolved in 1ml of dichloromethane solvent, 1g of GMA microsphere is suspended in 100ml of 0.25% SDS aqueous solution, the rhodamine dye and the GMA microsphere are mixed and ultrasonically emulsified, the emulsion is stirred and reacts for 20 hours at room temperature, the SDS and the dichloromethane are centrifugally cleaned to remove, and the fluorescent microsphere is obtained after 5 times of washing.
(3) Suspending 1g of fluorescent microspheres in 50ml of ethanol solvent, adding 2ml of methacrylic acid and 150mg of azodiisobutyronitrile into a fluorescent microsphere system, stirring and reacting for 18h at 75 ℃, and centrifugally cleaning and suspending until the conductivity of the solution measured by a conductivity meter is less than 10us/cm to prepare the hydrophilic fluorescent microspheres.
The application also provides a polystyrene hydrophilic fluorescent microsphere prepared by the method, which contains high-density carboxyl, wherein a carboxyl polymerization coating on the surface of the polystyrene hydrophilic fluorescent microsphere is formed by polymerizing a carboxyl monomer on the surface of the microsphere, the length of a spacer arm is longer than that of a space arm of a microsphere modified by a common functional group, the spacing ratio of the long-chain carboxyl is longer, so that biomolecules can be coupled and combined to the maximum extent, and the protein coupling amount is high. In addition, the high-density carboxyl groups on the surface of the polystyrene hydrophilic fluorescent microsphere prepared by the method form a hydrophilic polymer structure with good hydrophilicity, the existence of the structure ensures that the surface of the fluorescent microsphere has better hydrophilicity, and the polystyrene hydrophilic fluorescent microsphere has lower nonspecific adsorption in the application process and can eliminate a large amount of nonspecific adsorption, thereby reducing a series of problems caused by nonspecific adsorption and having better stability.
The application also provides a polystyrene hydrophilic fluorescent microsphere prepared by the method, which is applied to a fluorescence immunochromatography reagent product, namely a cTnI content detection card, and specifically, the polystyrene hydrophilic fluorescent microsphere is used for quantitatively detecting the content of cardiac troponin I (cTnI) in human blood. It should be noted that the process of preparing the detection card using the fluorescent microspheres is prior art, and the process is not improved here, and thus will not be described in detail.
When the fluorescent label is actually used, after a sample is added into a cTnI content detection card, the polystyrene hydrophilic fluorescent microspheres and the cTnI monoclonal antibody I which is fluorescently labeled on the detection card are specifically combined to form a fluorescently-labeled antibody-antigen complex. The compound moves forward along a nitrocellulose membrane on a test card along with chromatography, and is captured by a cTnI monoclonal antibody coated on a detection area on the nitrocellulose membrane and a rabbit anti-mouse immunoglobulin G antibody in a quality control area respectively to form a fluorescence-labeled double-antibody sandwich compound, and the content of cTnI in human blood is quantitatively detected by applying highly specific antigen-antibody reaction and a fluorescence immunochromatography technology.
To illustrate that the cTnI content detection card prepared from the polystyrene hydrophilic fluorescent microsphere of the present application has superior performance to the cTnI content detection card prepared from the conventional polystyrene fluorescent microsphere, the following description will be made in detail by using test data.
In the test process, the cTnI content detection card prepared from the polystyrene hydrophilic fluorescent microsphere and the traditional polystyrene fluorescent microsphere are respectively used, and signal value detection is carried out on a fluorescence quantitative immunoassay instrument.
Table 1 shows the signal value detection of samples with different concentrations, and as can be seen from the data in table 1, the signal values of the cTnI content detection cards prepared from the polystyrene hydrophilic fluorescent microspheres of the present application (referred to as the detection cards of the present application in the table for short) are all greater than those of the cTnI content detection cards prepared from the conventional polystyrene fluorescent microspheres (referred to as the conventional detection cards in the table for short), and the lowest detectable sample concentration of the cTnI content detection cards prepared from the polystyrene hydrophilic fluorescent microspheres of the present application is 0.01ng/ml, while the lowest detectable sample concentration of the cTnI content detection cards prepared from the conventional polystyrene fluorescent microspheres is 0.03 ng/ml. Therefore, the cTnI content detection card prepared from the polystyrene hydrophilic fluorescent microspheres has higher sensitivity.
TABLE 1 detection of signal values for samples of different concentrations
Table 2 shows the signal value detection of samples with different concentrations in different acceleration times, and from the data in table 2, that is, when the signal values of three samples with different concentrations, namely 0.3ng/ml, 5ng/ml and 45ng/ml, are detected within 0-7 days of the acceleration time, from the overall data, the signal values of the cTnI content detection card prepared from the polystyrene hydrophilic fluorescent microsphere of the present application (referred to as the detection card of the present application in the table) are all greater than the signal values of the cTnI content detection card prepared from the conventional polystyrene fluorescent microsphere (referred to as the conventional detection card in the table in the short), so that it can be shown that the cTnI content detection card prepared from the polystyrene hydrophilic fluorescent microsphere of the present application has better performance in product stability than the conventional polystyrene fluorescent microsphere.
Specifically, the signal values of the cTnI content detection card prepared by the polystyrene hydrophilic fluorescent microspheres after being stored for 7 days are compared with the signal values of the cTnI content detection card after being stored for 7 days at 37 ℃, and compared with the signal values of the cTnI content detection card after being stored for 0 day, the test reduction amplitude of a 0.3ng/ml sample is 14.69%, the test reduction amplitude of a 5ng/ml sample is 9.81%, and the test reduction amplitude of a 45ng/ml sample is 7.37%. Compared with the signal value of the day 0 after the cTnI content detection card prepared by the traditional polystyrene fluorescent microspheres is stored for 7 days at 37 ℃, the test reduced amplitude of a 0.3ng/ml sample is 22.88 percent, the test reduced amplitude of a 5ng/ml sample is 17.42 percent, and the test reduced amplitude of a 45ng/ml sample is 13.12 percent. Compared with the prior art, the detection signal value of the cTnI content detection card prepared from the polystyrene hydrophilic fluorescent microsphere is reduced by a smaller amount than that of the cTnI content detection card prepared from the traditional polystyrene hydrophilic fluorescent microsphere, so that the polystyrene hydrophilic fluorescent microsphere has better stability.
TABLE 2 detection of signal values of samples of different concentrations at different acceleration times
The preparation method comprises the steps of carrying out GMA surface modification on polystyrene microspheres, then carrying out fluorescent dyeing, and then adding methacrylic acid to carry out polymerization reaction to form the polystyrene hydrophilic fluorescent microspheres with the long-chain carboxyl polymerization coating on the surfaces. The preparation method of the polystyrene hydrophilic fluorescent microsphere is simple in process steps, simple and easily available in raw materials, low in cost and easy to widely popularize and use.
The polystyrene hydrophilic fluorescent microsphere prepared by the method contains high-density carboxyl, a carboxyl polymerization coating on the surface of the polystyrene hydrophilic fluorescent microsphere is formed by polymerizing a carboxyl monomer on the surface of the microsphere, the length of a spacer arm is longer than that of a space arm of a microsphere modified by a common functional group, the carboxyl spacer ratio of a long chain is long, the polystyrene hydrophilic fluorescent microsphere can be coupled and combined with biomolecules to the maximum extent, and the protein coupling amount is high. In addition, the high-density carboxyl groups on the surface of the polystyrene hydrophilic fluorescent microsphere prepared by the method form a hydrophilic polymer structure with good hydrophilicity, the existence of the structure enables the surface of the fluorescent microsphere to have better hydrophilicity, and the polystyrene hydrophilic fluorescent microsphere has lower nonspecific adsorption in the application process and can eliminate a large amount of nonspecific adsorption, thereby reducing a series of problems caused by nonspecific adsorption and having better stability.
The cTnI content detection card prepared from the polystyrene hydrophilic fluorescent microspheres has higher detection sensitivity and stability and stronger practicability.
The above-described embodiments of the present application do not limit the scope of the present application.
Claims (10)
1. A preparation method of a polystyrene hydrophilic fluorescent microsphere is characterized by comprising the following steps:
preparing GMA modified polystyrene microspheres;
carrying out fluorescent dyeing on the GMA modified polystyrene microspheres to prepare polystyrene fluorescent microspheres;
modifying a carboxyl polymerization coating on the surface of the polystyrene fluorescent microsphere through polymerization reaction to prepare the polystyrene hydrophilic fluorescent microsphere, wherein the prepared polystyrene hydrophilic fluorescent microsphere has a simple structure formula:
2. The method for preparing polystyrene hydrophilic fluorescent microspheres according to claim 1, wherein the step of preparing GMA modified polystyrene microspheres specifically comprises the following steps,
adding polystyrene microspheres into a mixed reagent of concentrated sulfuric acid and nitric acid, stirring and reacting for 15-25h at the temperature of 20-40 ℃ to ensure that the surfaces of the polystyrene microspheres are provided with nitro groups, and centrifugally cleaning to prepare the nitro microspheres;
pouring the nitro microsphere product microspheres into a three-neck flask, diluting the nitro microspheres to 5-15mg/ml by purified water, adding NaOH powder while stirring, and adding Na 2 S 2 O 4 Continuously stirring, reacting for 2-4h at 70-90 ℃, centrifuging, washing with purified water, and obtaining amino microspheres;
adding the amino microspheres into GMA and an organic solvent reagent, stirring and reacting for 15-25h at the temperature of 60-80 ℃, and then centrifugally cleaning the suspension to prepare the GMA modified polystyrene microspheres.
3. The preparation method of the polystyrene hydrophilic fluorescent microsphere according to claim 2, wherein the mass ratio of the polystyrene microsphere to the concentrated sulfuric acid to the nitric acid is 1: (1-1.5): (2-3);
the nitro microspheres and Na 2 S 2 O 4 And the mass ratio of NaOH is 1: (1.5-3): (2-5).
4. The preparation method of the polystyrene hydrophilic fluorescent microsphere as claimed in claim 2, wherein the mass ratio of the amino microsphere, GMA and organic reagent is 1: (0.1-0.5): (5-50).
5. The method for preparing polystyrene hydrophilic fluorescent microspheres according to claim 1, wherein the step of preparing the polystyrene fluorescent microspheres from GMA modified polystyrene microspheres by fluorescent dyeing comprises the following steps:
dissolving oil-soluble dye in dichloromethane solvent,
suspending GMA modified polystyrene microspheres in SDS aqueous solution,
mixing and emulsifying the prepared two solutions, stirring the emulsion at room temperature for reaction for 15-25h, and centrifugally cleaning to obtain the polystyrene fluorescent microspheres.
6. The method for preparing polystyrene hydrophilic fluorescent microspheres according to claim 5, wherein the oil-soluble fluorescent dye comprises one or a combination of any two or more of NBD, Bodipy, rhodamine and coumarin.
7. The method for preparing the polystyrene hydrophilic fluorescent microsphere according to claim 1, wherein the step of preparing the polystyrene hydrophilic fluorescent microsphere by modifying the surface of the polystyrene fluorescent microsphere with a carboxyl polymeric coating through a polymerization reaction specifically comprises the following steps:
suspending the polystyrene fluorescent microspheres in an ethanol solvent, adding methacrylic acid and azobisisobutyronitrile, stirring and reacting for 16-20h at 70-80 ℃, and centrifuging and cleaning to obtain the polystyrene hydrophilic fluorescent microspheres.
8. The preparation method of the polystyrene hydrophilic fluorescent microsphere as claimed in claim 7, wherein the mass ratio of the fluorescent microsphere to the ethanol to the methacrylic acid to the azobisisobutyronitrile is 1: (30-100): (0.5-3): (0.1-0.3).
9. A polystyrene hydrophilic fluorescent microsphere prepared by the method of any one of claims 1 to 8.
10. Use of the polystyrene hydrophilic fluorescent microspheres prepared by the method of any one of claims 1 to 8 in a cTnI content detection card.
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