A fluorescent composite microsphere, its preparation method and application

Abstract

The present invention relates to fluorescent composite microspheres, a preparation method and applications thereof, and belongs to the field of in vitro diagnosis. In view of the problems of nonspecific adsorption, high hydrophobicity and whitening of the fluorescent microspheres used in the prior art, the surfaces of the fluorescent composite microspheres of the present invention are modified with p-toluenesulfonyl functional groups, and zwitterionic compounds with resistance to nonspecific adsorption are introduced. A serrated modification layer is formed on the surface of the fluorescent composite microspheres, which reduces the steric hindrance effect on antigens, antibodies or ligands, improves activation efficiency and coupling efficiency, and solves the problem of difficulty in large-scale production.

Description

Technical Field

[0001] The present invention relates to a microsphere for in vitro diagnostic reagent, and in particular to a fluorescent composite microsphere and a preparation method and application thereof. Background Art

[0002] Fluorescence immunochromatography has become an indispensable quantitative testing method in clinical testing due to its high sensitivity, low cost, and rapid detection speed. Currently, a large number of clinical testing projects based on fluorescence immunochromatography have been approved by the National Medical Products Administration and have entered into clinical practice, such as those for inflammation, myocardial function, thyroid function, hormones, and drug screening.

[0003] As a key component in the analytical system, the properties of fluorescent composite microspheres play a crucial role in the results of immunoassays. Fluorescent composite microspheres rely on surface functional groups (such as carboxyl and amino groups) to covalently bind to antibodies or antigens, thereby capturing the corresponding analyte. Subsequently, upon excitation, they generate a fluorescent signal, enabling quantitative detection of the corresponding analyte.

[0004] Currently, the surface functional groups of fluorescent latex microspheres on the market are mainly carboxyl groups, and the coupling method commonly used for covalent bonding of microspheres with antigens / antibodies is the EDC / NHS method. First, water-soluble EDC reacts with the carboxyl functional groups on the microspheres to form a first active ester intermediate. Then, the first active ester intermediate reacts with N-hydroxysuccinimide (NHS) to form a second active ester intermediate. Finally, the second active ester intermediate undergoes a substitution reaction with the amino group to form an amide bond, completing the coupling. However, in the actual coupling process, there are often problems such as low activation efficiency, low coupling efficiency, microsphere agglomeration, and difficulty in large-scale production. In addition, the labeled microspheres often have problems such as high background, slow fading, and low sensitivity during subsequent chromatography.

[0005] Modifying the surface of fluorescent composite microspheres with active groups is an effective method for solving the above-mentioned coupling technology problems. CN113150352A discloses fluorescent latex microspheres with epoxy groups modified on the surface. However, the detection of the epoxy content of the microspheres is still relatively difficult. The detection method has poor specificity and is difficult to effectively calibrate. At the same time, the epoxy group has the problem of poor stability. When stored in solution, it is easy to undergo ring-opening hydrolysis. Therefore, freeze-drying is often required for storage, which increases the difficulty of preparation. In addition, no method is mentioned to improve the surface steric effect or to control the hydrophilicity and hydrophobicity of the microsphere surface.

[0006] The document "Ultrasensitive Detection of COVID-19 Virus N Protein Based on p-Toluenesulfonyl Modified Fluorescent Microspheres Immunoassay" proposes the preparation of p-toluenesulfonyl-modified immunofluorescent composite microspheres and their application in COVID-19 detection. However, since p-toluenesulfonyl chloride monomer is directly used in the preparation of microspheres, on the one hand, p-toluenesulfonyl chloride is easily hydrolyzed into p-toluenesulfonic acid in the presence of water and high temperature during the polymerization process. On the other hand, the functional groups are embedded in the microspheres during the polymerization process, resulting in a limited content of effective groups on the surface of the microspheres. In addition, the microspheres have obvious steric hindrance and poor control of hydrophilicity and hydrophobicity. Therefore, there is still much room for improvement in the detection results and efficiency. Summary of the Invention

[0007] To address these technical issues, the present invention optimizes the surface modification of microspheres using long-chain p-toluenesulfonyl compounds, reducing steric hindrance. Further modification with zwitterionic compounds improves hydrophilicity and hydrophobicity, prevents aggregation of traditional microspheres, and reduces nonspecific adsorption. When used for detection, these microspheres exhibit increased coupling efficiency with analytes, low background interference, and extremely high precision, resulting in more accurate and reliable test results.

[0008] One aspect of the present invention is to provide a method for preparing fluorescent composite microspheres, first providing hydroxylated fluorescent latex microspheres, functionalizing the hydroxylated fluorescent latex microspheres, and then modifying the hydroxylated fluorescent latex microspheres with Tosyl-ester and zwitterionic compounds to obtain fluorescent composite microspheres. One embodiment is as follows Figure 1 shown.

[0009] Specifically, the method for preparing fluorescent composite microspheres comprises the following steps:

[0010] S01 provides hydroxylated fluorescent latex microspheres, which are dispersed in a solution;

[0011] The hydroxylated fluorescent latex microspheres are fluorescent latex microspheres with hydroxyl groups on the surface. One way to obtain them is to purchase commercially available products. Another way is to prepare them by the following method:

[0012] The emulsifier is dissolved in water, styrene and / or its derivative monomers and functional monomers are added to the water, and after nitrogen replacement, an initiator is added to react. The reaction is filtered, and the filtrate is collected and centrifuged to obtain a microsphere core with a surface modified with a hydroxyl group; preferably, the reaction temperature is 60°C-90°C, and the reaction time is 12-24 hours.

[0013] The mass ratio of the styrene and / or its derivative monomer, the functional monomer, the initiator and the emulsifier is 100:(1-100):(0.1-10):(0.1-10); and the functional monomer is an acrylic acid ester compound with a hydroxyl group.

[0014] The microsphere core modified with hydroxyl groups and the fluorescent dye are dispersed in a swelling agent for reaction. After the reaction is completed, the reaction is filtered and the filtrate is centrifuged to obtain a solid phase, which is then washed to obtain hydroxylated fluorescent latex microspheres. Preferably, the reaction temperature is 25°C-50°C and the reaction time is 12-48 hours.

[0015] The mass ratio of the microsphere core with a surface modified with hydroxyl groups, the fluorescent dye and the swelling agent is 100: (1-100): (1-100).

[0016] S02. Modifying the hydroxylated latex microspheres with a siloxane coupling agent having an alkenyl group to obtain bifunctionally modified fluorescent latex microspheres;

[0017] Specifically, a solution of a siloxane coupling agent with an alkenyl group is added to a hydroxylated latex microsphere solution, and then the two solutions are mixed to form a reaction system. Ammonia water is then added to the reaction system, and the reaction is followed by filtration. The filtrate is centrifuged, and the solid phase is collected and washed to obtain bifunctionally modified fluorescent latex microspheres.

[0018] Preferably, the alkenyl siloxane coupling agent is added to ethanol to form an alkenyl siloxane coupling agent solution, and the hydroxylated fluorescent latex microspheres are added to a mixed solvent of anhydrous ethanol and PVP to form a solution; preferably, the two solutions are continuously stirred for 5-30 minutes; preferably, the stirring reaction is continued for 2-6 hours after the addition of ammonia water.

[0019] The mass ratio of the alkenyl-containing siloxane coupling agent to the hydroxylated fluorescent latex microspheres is (1-100):100, and the volume ratio of ammonia water to the reaction system is (1-80):100.

[0020] S03. Adding the bifunctionally modified fluorescent latex microspheres to a solvent, stirring and dispersing them uniformly, then adding an initiator, replacing the atmosphere with nitrogen while continuing to stir, then adding a tosyl ester or a tosyl ester and a zwitterionic compound, filtering after the reaction, centrifuging the filtrate, collecting the solid phase, and washing the solid phase to obtain fluorescent composite microspheres;

[0021] Preferably, the solvent is a mixed solvent formed by pure water, an emulsifier and / or PVP; preferably, the reaction temperature after adding Tosyl-ester is 60°C-90°C, and the reaction time is 12-48 hours;

[0022] The mass ratio of the Tosyl-ester to the bifunctionally modified fluorescent latex microspheres is (0.1-20):1, or the mass ratio of the Tosyl-ester, the zwitterionic compound, and the bifunctionally modified fluorescent latex microspheres is (0.1-20):(0.1-20):1.

[0023] The "Tosyl-ester" of the present invention is a toluenesulfonyl compound having the following structure: ; The zwitterionic compound is selected from , , and One or more of;

[0024] Wherein, R1 is hydrogen or one of C1-C4 alkyl or alkoxy groups, n is an integer ranging from 0 to 200, M represents an oxygen atom, R2 is one of C1-C4 alkyl or alkoxy groups, and R3 and R4 are independently C2-C6 alkyl groups.

[0025] Furthermore, in the method for preparing the microsphere core, the reflux reaction temperature is preferably 70°C-85°C, such as 72°C, 75°C, 78°C, 81°C or 83°C; preferably, the reaction time is 14 hours, 16 hours or 20 hours; preferably, the mass ratio of the styrene and / or its derivative monomer, functional monomer, initiator and emulsifier is 100:(5~50):(0.5~8):(0.5~8), or 100:10:2:4, or 100:35:6:6.

[0026] Furthermore, the initiator is one or more of dibenzoyl peroxide, dicumyl peroxide, dodecyl peroxide, di-tert-butyl peroxide, azobisisoheptonitrile, dimethyl azobisisobutyrate, azobisisobutyronitrile, ammonium persulfate and potassium persulfate, preferably potassium persulfate; the emulsifier is an anionic surfactant, such as one or more of sodium dodecyl sulfate and sodium dodecylbenzenesulfonate.

[0027] Furthermore, during the swelling reaction of the fluorescent dye, the reaction temperature is preferably 30°C-40°C, such as 32°C, 34°C, 36°C or 38°C; preferably, the reaction time is 16-32 hours, such as 18 hours, 20 hours, 24 hours, 26 hours or 30 hours; preferably, the mass ratio of the microsphere core modified with hydroxyl groups, the fluorescent dye and the swelling agent is 100:(5~60):(5~60), such as 100:10:10, 100:30:30 or 100:50:20; the reaction temperature is preferably 30°C-40°C, such as 32°C, 34°C, 36°C or 38°C; the reaction time is preferably 20-32 hours, such as 22 hours, 24 hours, 26 hours, 28 hours or 30 hours.

[0028] Furthermore, the fluorescent dye is selected from lanthanide fluorescent dyes such as tris(dinaphthylmethylene)monophenanthroline europium (III), tris(1,3-diphenyl-1,3-propanedione) (1,10-o-phenanthroline) europium (III), (1,10-phenanthroline)tris[4,4,4-trifluoro-1-(2-thienyl)-1,3-butanedione] europium (III), tris(dibenzoylmethane)mono(5-amino-1,10-phenanthroline) europium (III), or one or more organic small molecule fluorescent dyes such as fluorescein, rhodamine, and BODIPY; preferably, lanthanide fluorescent dyes such as (1,10-phenanthroline)tris[4,4,4-trifluoro-1-(2-thienyl)-1,3-butanedione] europium (III); the fluorescent dye enters the microsphere core with active sites through swelling to obtain hydroxylated fluorescent latex microspheres.

[0029] Furthermore, the swelling agent is selected from one or more of dichloromethane, tetrahydrofuran, chloroform, acetone, toluene, N,N-dimethylformamide, and dimethyl sulfoxide.

[0030] Furthermore, the mass ratio of the siloxane coupling agent with an alkenyl group to the fluorescent latex microspheres is (5-80):100, such as 10:100, 30:100, 50:100 or 70:100; preferably, the volume ratio of the ammonia water to the reaction system is (4-60):100, such as 10:100, 20:100, 30:100, 40:100 or 50:100.

[0031] Further, in S03, preferably, the reaction temperature after adding Tosyl-ester is 65°C, 70°C, 73°C, 75°C, 78°C, 80°C or 85°C; preferably, the reaction time is 16 hours, 20 hours, 24 hours, 28 hours, 32 hours, 36 hours or 42 hours.

[0032] Further, R1 is methyl or ethyl; preferably, R2 is methyl or ethyl; preferably, n is an integer of 10-100; preferably, R1 is methyl; R2 is methyl; n is an integer of 20-50.

[0033] Preferably, the styrene derivative monomer is one or more materials selected from 4-methylstyrene, 3-methylstyrene and 4-tert-butylstyrene; the functional monomer is selected from one or more of hydroxyethyl acrylate, hydroxypropyl acrylate, hydroxybutyl acrylate, hydroxyethyl methacrylate, hydroxypropyl methacrylate and hydroxybutyl methacrylate.

[0034] Furthermore, the mass ratio of the Tosyl ester and the bifunctionally modified fluorescent latex microspheres is (0.3-10):1, or the mass ratio of the Tosyl ester, the zwitterionic compound and the bifunctionally modified fluorescent latex microspheres is (0.3-10):(0.3-10):1.

[0035] Preferably, the siloxane coupling agent with an alkenyl group is selected from one or more of acryloxypropyltrimethoxysilane, 3-(methacryloyloxy)propyltrimethoxysilane, 3-(methacryloyloxy)propyltriethoxysilane, (3-methacrylamidopropyl)triethoxysilane, 3-butenetriethoxysilane, 5-hexenyltriethoxysilane, styrylethyltrimethoxysilane and 2-(divinylmethylsilyl)ethyltriethoxysilane.

[0036] It is understood that the purpose of filtration is to eliminate agglomerated products or products of unexpected particle size during the reaction process. Those skilled in the art will select the filtration mesh size within the range disclosed in the present invention in each preparation step. One embodiment is to maintain consistency in the filtration mesh size in each preparation step. A specific embodiment is to use 80-400 mesh filter cloth for filtration.

[0037] Another aspect of the present invention is to provide a fluorescent composite microsphere comprising: 1) a polymer core, and 2) a structure coated with a Tosyl-ester modified structure on the surface of the core, wherein the Tosyl-ester has the following structure: ; wherein R1 is hydrogen or one of a C1-C4 alkyl or alkoxy group, and n is an integer ranging from 0 to 200.

[0038] Furthermore, the surface of the fluorescent composite microsphere is also coated with a structure modified by a zwitterionic compound, and the zwitterionic compound is selected from , , and wherein M represents an oxygen atom, R2 is one of a C1-C4 alkyl group or an alkoxy group, and R3 and R4 are independently a C2-C6 alkyl group.

[0039] Preferably, R1 is a methyl group or an ethyl group, and / or R2 is a methyl group or an ethyl group, and / or n is an integer of 10-100; more preferably, R1 is a methyl group, and / or R2 is a methyl group, and / or n is an integer of 20-50.

[0040] It can be understood that the Tosyl-ester and the zwitterionic compound react with the alkenyl groups on the surface of the bifunctionally modified fluorescent latex microspheres through the alkenyl groups to obtain the corresponding fluorescent composite microspheres modified with the Tosyl-ester and the zwitterionic compound.

[0041] In one embodiment, the zwitterionic compound is ZWC1, ZWC2 and / or ZWC3, and the structural formula of ZWC1 is 、ZWC2 structural formula is 、ZWC3 structural formula is .

[0042] Furthermore, the polymer core is prepared by polymerization reaction of styrene and / or its derivative monomers with acrylates having hydroxyl groups.

[0043] Furthermore, the particle size of the fluorescent composite microspheres is 20 nm-5 μm; preferably, the particle size is preferably 50 nm-2 μm.

[0044] Furthermore, the fluorescent composite microspheres are prepared by the aforementioned method for preparing fluorescent composite microspheres.

[0045] Another aspect of the present invention provides the use of the fluorescent composite microspheres in preparing in vitro diagnostic reagents.

[0046] In one embodiment, the fluorescent composite microspheres of the present invention are coupled with antibodies for use in the detection of antigens to be detected.

[0047] In another embodiment, the fluorescent composite microspheres of the present invention are coupled with biotin, avidin or streptavidin for use in in vitro diagnostic testing.

[0048] The surface of the fluorescent composite microspheres described in the present invention is modified with a p-toluenesulfonyl functional group having a steric de-hindrance effect, and a zwitterionic compound having resistance to nonspecific adsorption is introduced. A serrated modification layer is formed on the surface of the fluorescent composite microspheres, which reduces the steric hindrance effect on the antigen, antibody, or ligand, improves the activation efficiency and coupling efficiency, and solves the problem of difficulty in scale-up production. In addition, due to the introduction of the zwitterionic polymer layer, the nonspecific adsorption of the microspheres on the analyte during the coupling process is reduced, avoiding the problem of microsphere agglomeration that occurs in traditional activation coupling processes. The hydrophilicity and hydrophobicity of the microsphere surface are also improved, avoiding the problem of microsphere agglomeration that occurs in traditional activation coupling processes, and reducing the nonspecific adsorption of the microspheres on the antigen, antibody, or ligand during the chromatography process after the microspheres are coupled to the biomacromolecules. Advantages such as low background and fast fading in chromatographic detection ensure the accuracy of the detection results and the consistency of batch detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following is a brief introduction to the drawings required for the embodiments or the description of the prior art. In all drawings, similar elements or parts are generally identified by similar reference numerals. In the drawings, the various elements or parts are not necessarily drawn according to the actual scale. Obviously, the drawings described below are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can also be obtained based on these drawings without inventive work.

[0050] Figure 1 The figure shows the preparation process of the fluorescent composite microspheres of the present invention;

[0051] Figure 2 This is a scanning electron microscope image of the fluorescent composite microspheres after labeling according to the present invention;

[0052] Figure 3 The reaction kinetic curves of different labeled fluorescent composite microsphere detection reagents;

[0053] Figure 4 The fluorescence signal curves of different labeled fluorescent composite microspheres are shown. DETAILED DESCRIPTION

[0054] To make the purpose, technical solutions, and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0055] As used herein, "and / or" includes any and all combinations of one or more of the associated listed items.

[0056] Herein, "plurality" means two or more than two, ie, it includes two, three, four, five, etc.

[0057] The term "anti-nonspecific adsorption" as used herein refers to the ability of the composite magnetic microspheres prepared herein to significantly reduce nonspecific adsorption to antibodies, antigens, or protein conjugates, thereby reducing background. Those skilled in the art will understand that "background" refers to the nonspecific fluorescent signal present on the entire nitrocellulose membrane (NC membrane) during the assay, excluding the test line (T line) and the control line (C line). "Bleaching" refers to the disappearance of background signal.

[0058] Example 1 Preparation of fluorescent composite microspheres

[0059] (1) Preparation of Tosyl-ester

[0060] To a three-necked flask, add 35g of hydroxyethyl methacrylate, 38g of pyridine, and 300mL of dichloromethane. Under nitrogen, cool to 5°C. Weigh 63g of p-toluenesulfonyl chloride into a beaker and dissolve in 100mL of dichloromethane. Once completely dissolved, slowly add it to the reaction flask. Stir overnight at room temperature. Pour the reaction solution into 600mL of dichloromethane and extract with 1M dilute hydrochloric acid. Separate the layers and collect the organic phase. Extract with saturated sodium bicarbonate and then pure water, dry over anhydrous sodium sulfate, filter, concentrate, and purify by chromatography to obtain a short-chain tosyl-ester, referred to as Tosyl-ester 1.

[0061] To a three-necked flask, add 50g of polyethylene glycol methacrylate (average molecular weight approximately 900), 50g of pyridine, and 1000mL of N,N-dimethylformamide. Under nitrogen, cool to 5°C. Weigh 80g of p-toluenesulfonyl chloride into a beaker and add 100mL of N,N-dimethylformamide. Once completely dissolved, slowly add it to the three-necked flask. Stir at room temperature for 2 days. Pour the reaction solution into 2000mL of pure water and extract multiple times with ethyl acetate. The organic phases are combined and extracted sequentially with dilute hydrochloric acid, saturated sodium bicarbonate, and pure water. The mixture is dried over anhydrous sodium sulfate, filtered, concentrated, and purified by chromatography to obtain a long-chain tosyl ester, referred to as Tosyl ester 2.

[0062] Referring to the preparation process of Tosyl-ester 2, polyethylene glycol methacrylate with an average molecular weight of about 2000 and about 2500 was selected to prepare Tosyl-ester 3 and Tosyl-ester 4 with different chain lengths, respectively.

[0063] (2) Hydroxylated fluorescent latex microspheres

[0064] To a three-necked flask, add 2000g of pure water and 10g of sodium lauryl sulfate and stir to dissolve. Then, add 300g of styrene and 60g of hydroxyethyl methacrylate, replace the atmosphere with nitrogen three times, and stir for 30 minutes. Then, add 2.7g of potassium persulfate and replace the atmosphere with nitrogen three more times. Then, heat the mixture to 80°C and reflux, stirring for 15 hours. After the reaction is complete, cool to room temperature and filter through a 200-mesh filter cloth. The filtrate is collected and centrifuged to obtain the surface-hydroxyl-modified microsphere core.

[0065] Add 100g of the hydroxyl-modified microsphere cores prepared in the above steps to a three-necked flask and start stirring. Then, weigh 10g of (1,10-phenanthroline)tris[4,4,4-trifluoro-1-(2-thienyl)-1,3-butanedione]europium(III), 15g of tetrahydrofuran, and 20g of dichloromethane in a beaker. After ultrasonic dissolution, add all of the mixture to the flask. Heat to 35°C, seal, and stir overnight in the dark. After completion of the reaction, filter through a 200-mesh filter cloth, collect the filtrate, and centrifuge it to collect the solids. Wash them with ethanol and purified water to obtain hydroxylated fluorescent latex microspheres.

[0066] (3) Dual-functional modified fluorescent latex microspheres

[0067] Add 150g of the hydroxylated fluorescent latex microspheres prepared in step (2) to the three-necked flask, add 300g of anhydrous ethanol and 30g of PVP K30 to the three-necked flask, and stir for 15 minutes to form a hydroxylated fluorescent latex microsphere solution; weigh 30g of 3-(methacryloyloxy)propyltrimethoxysilane and 30g of anhydrous ethanol in a beaker and stir and dissolve to form a siloxane coupling agent solution with alkenyl groups. Pour the siloxane coupling agent solution with alkenyl groups into the three-necked flask containing the hydroxylated fluorescent latex microsphere solution and continue stirring for 15 minutes. Take 60mL of concentrated ammonia water and dilute it with 60mL of pure water, then add it dropwise to the three-necked flask. After the addition is complete, continue stirring for 3 hours. After stirring, filter with a 200-mesh filter cloth, collect the filtrate, and centrifuge the filtrate. Collect the solid and wash it by centrifugation with ethanol and purified water to obtain bifunctional modified fluorescent latex microspheres.

[0068] (4) Fluorescent composite microspheres

[0069] Take 5g (dry weight) of the bifunctional modified fluorescent latex microspheres prepared in step (3), add 100mL of pure water, 0.5g of sodium dodecyl sulfate and 1g of PVP, stir for 30 minutes to dissolve, then add 0.1g of azobisisobutyronitrile, replace with nitrogen, continue stirring for 15 minutes, after stirring, add 7.5g of Tosyl-ester 1, and ultrasonically stir for 15 minutes. After stirring, heat to 80℃ and stir overnight. After the reaction is completed, filter with 200 mesh filter cloth, collect the filtrate, centrifuge the filtrate, collect the solid, and centrifuge and wash with ethanol and purified water to obtain fluorescent composite microspheres 1.

[0070] Referring to the above preparation method, when the molar amount of sulfonyl group is basically the same, Tosyl-ester 1 is replaced by Tosyl-ester 2, Tosyl-ester-3 and Tosyl-ester 4, respectively, to prepare fluorescent composite microspheres 2, fluorescent composite microspheres 3 and fluorescent composite microspheres 4, respectively.

[0071] Take another 5g (dry weight) of the bifunctional modified fluorescent latex microspheres prepared in step (3), add 100mL of pure water, 0.5g of sodium dodecyl sulfate and 1g of PVP, stir for 30 minutes to dissolve, then add 0.15g of azobisisobutyronitrile, replace with nitrogen, continue stirring for 15 minutes, after stirring, add 7.5g of Tosyl-ester 3 and 7.5g of ZWC3, and stir ultrasonically for 15 minutes. After stirring, heat to 80℃ and stir overnight. After the reaction is complete, filter with 200 mesh filter cloth, collect the filtrate, centrifuge the filtrate, collect the solid, and centrifuge and wash with ethanol and purified water to obtain fluorescent composite microspheres 5.

[0072] Example 2

[0073] (1) Antibody-labeled microspheres

[0074] 100 μg of NT-proBNP monoclonal antibody (Chongqing Aishengsi Bioengineering Co., Ltd.) and 500 μg of the fluorescent composite microspheres described in Example 1 were respectively reacted at 37°C with stirring for 24 hours. After a stable covalent bond was formed between the fluorescent composite microspheres and the antibody, 1% BSA was added, and the mixture was blocked and stirred at 37°C for 24 hours. The mixture was then centrifuged at 14,000 rpm for 20 minutes, the supernatant was discarded, and the pellet was restored to volume in fluorescent antibody storage solution (100 mM Tris, 1% Tween 20, 5 mg / mL bovine serum albumin, 2% sucrose) to prepare the corresponding labeled microspheres. These were numbered as labeled microspheres 1-5 according to the fluorescent composite microsphere number and stored at 2-8°C for future use.

[0075] According to the above method, commercially available carboxyl fluorescent composite microspheres (Weidu Bio, FT0300CC) were used to replace the fluorescent composite microspheres described in the present invention, and labeled according to the general method, that is, NT-proBNP antibody was added to the commercially available carboxyl fluorescent composite microspheres at an amount of 100 μg NT-proBNP monoclonal antibody / 500 μg fluorescent composite microspheres, 10 μg EDC was added, and the reaction was stirred at room temperature for 2 hours. Then 5% BSA was added, and the mixture was blocked and stirred at room temperature for 1 hour. Then, it was centrifuged at 14000 rpm for 20 minutes, the supernatant was discarded, and the precipitate was restored to volume with fluorescent antibody storage solution (100 mM Tris, 1% Tween 20, 5 mg / mL bovine serum albumin, 2% sucrose) to prepare labeled microspheres 0.

[0076] (2) Biotin-labeled antibodies

[0077] Weigh 0.001 g of biotin and add it to 175 μL of DMSO to dissolve it into a biotin working solution. Take 0.5 mg of NT-proBNP antibody and add 7 μL of biotin working solution. Mix immediately. Rotate and label at room temperature in the dark. Then dialyze or pass through a column to remove free biotin to obtain the antibody-biotin complex. Store at 2-8°C until use.

[0078] (3) Detection reagents

[0079] Dilute the antibody-labeled microspheres to 0.34 mg / mL in detection reagent storage solution (20 mM Tris, 1% Tween 20, 5 mg / mL sodium caseinate, 5% trehalose) and dilute the antibody-biotin complex to 0.032 mg / mL. Mix equal volumes of the two solutions to prepare the detection reagent and store at 2-8°C until use.

[0080] (4) Detection reagent card

[0081] Dilute streptavidin to 0.2 mg / mL in PBS buffer. Draw two capture detection lines 5-15 mm from the left end of the nitrocellulose membrane. Assemble a universal chromatography support by tightly overlapping the sample pad, nitrocellulose membrane, and absorbent pad on a substrate. Cut the assembled universal chromatography support into the desired width using a chopper and load it into the cartridge.

[0082] Effect verification example

[0083] First, determine the performance of the fluorescent composite microspheres, and then characterize the effects of different fluorescent composite microspheres on the detection indicators through specific test items.

[0084] The test process involves mixing the test reagent and the serum sample to be tested in a preset ratio and incubating at room temperature for 3 minutes. Through specific antigen-antibody recognition, the test reagent and the analyte form a reaction complex consisting of "fluorescent composite microspheres-NT-proBNP antibody-NT-proBNP analyte-NT-proBNP antibody-biotin." 40 μL of the reaction solution is transferred to the sample loading area of the test reagent card. Capillary action moves the reaction solution to the detection area. Streptavidin on the test line specifically binds to biotin, capturing the reaction complex onto the test line. The detection device collects the tracer signal on the test line, processes the data, and outputs the analyte content result. The details are as follows.

[0085] Effect Example 1

[0086] The polydispersity coefficient is often used to characterize the dispersibility of nanomaterials. A smaller polydispersity coefficient indicates better dispersibility, and a polydispersity coefficient < 0.1 is generally considered a monodisperse system. The polydispersity coefficients of the fluorescent composite microspheres described herein and commercially available microspheres were analyzed and compared before and after labeling. The commercially available microspheres were carboxyl fluorescent composite microspheres (Weidu Biotechnology, FT0300CC), denoted as fluorescent composite microspheres 0.

[0087] Table 1 Polydispersity analysis of fluorescent composite microspheres before and after labeling

[0088]

[0089] The results in Table 1 show that the polydispersity coefficient of the commercially available microspheres increased after antibody labeling and was greater than 0.1, indicating that some microspheres aggregated during the labeling process, which is consistent with theoretical analysis. The polydispersity coefficient of the fluorescent composite microspheres of the present invention remained almost unchanged before and after antibody labeling, especially the microspheres further modified with zwitterionic compounds (fluorescent composite microspheres 5), with a polydispersity coefficient of 0.02, indicating that they have good dispersibility and are not affected by the labeling process. Scanning electron microscopy images show that their particle size is regular and there is no agglomeration ( Figure 2 ).

[0090] Effect Example 2

[0091] Serum samples with low, medium, and high NT-proBNP values within the linear range of 10-35,000 pg / mL were selected. Each sample was tested three times, and the average value was calculated. Using labeled microsphere 0 as the control, the deviations of the test results of different labeled microspheres relative to the control were calculated. A positive relative deviation indicates that the sensitivity is higher than that of the control labeled microsphere, and a larger positive deviation indicates a higher sensitivity. The test results are shown in Table 2 below:

[0092] Table 2 Sensitivity of different labeled microspheres

[0093]

[0094] *Determined by the Roche Elecsys® NT-proBNP assay

[0095] According to the test results, the sensitivity of short-chain Tosyl-ester-modified labeled microspheres 1 (Tosyl-ester polymerization degree 0) at all three concentrations was lower than that of commercially available products. However, the sensitivity of the labeled microspheres modified with long-chain Tosyl-esters was significantly improved due to the improved steric effect. Using the same long-chain Tosyl-ester, the sensitivity of labeled microspheres 5, formed by further surface modification of the fluorescent composite microspheres with zwitterionic compounds, was significantly better than that of the unmodified labeled microspheres 3. The inventors found similar results with other zwitterionic-modified fluorescent composite microspheres, indicating that zwitterionic modification of the microspheres improves nonspecific adsorption and thus enhances sensitivity.

[0096] Effect Example 3

[0097] To further illustrate the effect of the fluorescent composite microspheres of the present invention, labeled microspheres 1, 2, 5, and 0 were used as detection reagents for exemplary illustration. Linear gradient samples were prepared according to the dilution ratios of high and low NT-proBNP samples. Each sample was tested three times, and the average value was calculated. The linear correlation coefficient r was calculated based on the theoretical concentration-T / C signal average, and the reaction kinetics curve was plotted. The sample dilution ratios and concentrations are shown in Table 3, and the reaction kinetics curve is shown in Table 3. Figure 3 shown.

[0098] Table 3 Sample dilution ratio and concentration

[0099]

[0100] Combined with Table 3 and Figure 3 It can be seen that in the detection practice, the modified fluorescent composite microspheres have a good linear relationship, and the linear correlation coefficient r meets the acceptance standard (r>0.99), which is not much different from the commercial microspheres on the market.

[0101] Effect Example 4

[0102] Using labeled microspheres 1, 2, 5, and 0 as detection reagents, serum samples within the linear range of NT-proBNP (10-35,000 pg / mL) were selected. Ten replicates were performed for each sample, and the precision (CV) was calculated. Smaller concentration CV values indicate higher precision. Sample information and test results are shown in Table 4.

[0103] Table 4 Precision test sample information and results

[0104]

[0105] *Determined by the Roche Elecsys® NT-proBNP assay

[0106] Table 4 shows that in terms of precision, labeled microsphere 5, dually modified with tosyl ester and zwitterionic compounds, meets CV < 5% across the entire linear range. Labeled microsphere 1 meets CV < 5% only at the high end, while labeled microsphere 2 and commercially available microspheres primarily fail to meet CV < 5% at the low end. These results further confirm that appropriately increasing the tosyl ester chain length can improve test accuracy, and further combining it with zwitterionic modification can significantly enhance test reliability.

[0107] Effect Example 5

[0108] Using labeled microspheres 1, 2, 5, and 0 as detection reagents, a zero-value buffer matrix was selected instead of the serum sample, and the test was repeated three times. The average value of the original fluorescence value signal at each scanning point within the reagent card window was calculated, and the range was calculated. Among them, the smaller the range value, the better the effect of the reagent in background whitening. Some scanning point signals are shown in Table 5 and Figure 4 shown.

[0109] Table 5 Original fluorescence value signal at each scanning point

[0110]

[0111] Through Table 5 and Figure 4 It can be seen that in terms of background fading, the labeled microspheres 5 double-modified with Tosyl-ester and zwitterionic compounds have the best effect, with low background fluorescence and a relatively flat fluorescence curve.

Claims

1. A method for preparing fluorescent composite microspheres, characterized in that: The method comprises the following steps: S01. Dispersing hydroxylated fluorescent latex microspheres in a solution; S02. Modifying the hydroxylated fluorescent latex microspheres with a siloxane coupling agent having an alkenyl group to obtain bifunctionally modified fluorescent latex microspheres; S03. The bifunctionally modified fluorescent latex microspheres are added to the solvent, stirred and dispersed uniformly, and then an initiator is added. After nitrogen replacement, stirring is continued, and then Tosyl-ester or Tosyl-ester and zwitterionic compound are added to prepare fluorescent composite microspheres; The Tosyl-ester has the following structure: , the zwitterionic compound is selected from 、 、 and wherein one or more of: R1 is hydrogen or one of C1-C4 alkyl or alkoxy; n is an integer from 0 to 200; M is an oxygen atom; R2 is one of C1-C4 alkyl or alkoxy; and R3 and R4 are independently C2-C6 alkyl.

2. The preparation method according to claim 1, wherein The R1 is a methyl group or an ethyl group, and / or the R2 is a methyl group or an ethyl group, and / or the n is an integer of 10-100.

3. The preparation method according to claim 2, wherein The R1 is a methyl group, and / or the R2 is a methyl group, and / or the n is an integer of 20-50.

4. The preparation method according to claim 1, wherein The hydroxylated fluorescent latex microspheres are added to a mixed solvent of anhydrous ethanol and PVP to form a solution; and / or a siloxane coupling agent with alkenyl groups is added to ethanol to form an alkenyl siloxane coupling agent solution, which is then reacted with the hydroxylated fluorescent latex microspheres.

5. The preparation method according to claim 1 or 4, characterized in that The mass ratio of the alkenyl-containing siloxane coupling agent to the hydroxylated fluorescent latex microspheres is (1-100):100, and / or the mass ratio of the Tosyl-ester to the bifunctionally modified fluorescent latex microspheres is (0.1-20):1, or the mass ratio of the Tosyl-ester, the zwitterionic compound, and the bifunctionally modified fluorescent latex microspheres is (0.1-20):(0.1-20):

1.

6. The preparation method according to claim 5, wherein The mass ratio of the alkenyl-containing siloxane coupling agent to the hydroxylated fluorescent latex microspheres is (5-50):100, and / or the mass ratio of the Tosyl-ester to the bifunctionally modified fluorescent latex microspheres is (0.3-10):1, or the mass ratio of the Tosyl-ester, the zwitterionic compound, and the bifunctionally modified fluorescent latex microspheres is (0.3-10):(0.3-10):

1.

7. The preparation method according to claim 5, wherein The siloxane coupling agent with an alkenyl group is selected from one or more of acryloxypropyltrimethoxysilane, 3-(methacryloyloxy)propyltrimethoxysilane, 3-(methacryloyloxy)propyltriethoxysilane, (3-methacrylamidopropyl)triethoxysilane, 3-butenetriethoxysilane, 5-hexenyltriethoxysilane, styrylethyltrimethoxysilane and 2-(divinylmethylsilyl)ethyltriethoxysilane.

8. A fluorescent composite microsphere, characterized in that: The fluorescent composite microspheres contain: 1) a polymer core, and 2) a structure coated with a Tosyl-ester modified structure on the surface of the core, wherein the Tosyl-ester has the following structure: ; Wherein, R1 is hydrogen or a C1-C4 alkyl or alkoxy group, and n is an integer ranging from 0 to 200; The Tosyl-ester is grafted onto the polymer core via the alkenyl group.

9. The fluorescent composite microsphere according to claim 8, wherein The invention is prepared by the method according to any one of claims 1 to 7.

10. Use of the fluorescent composite microspheres according to claim 8 or 9 in the preparation of in vitro diagnostic reagents.

Images