Preparation method of photosensitive microspheres, homogeneous chemiluminescence detection kit and detection method thereof

Abstract

The invention discloses a preparation method of photosensitive microspheres, a homogeneous chemiluminiscence detection kit and a detection method thereof, and belongs to the technical field of homogeneous chemiluminiscence detection.The preparation method of the photosensitive microspheres comprises the steps that copper phthalocyanine is pretreated, then an antibody-acrylate conjugate solution is prepared, and the antibody-acrylate conjugate solution is prepared; finally, styrene, alkyl substituted methyl acrylate, a copper phthalocyanine photosensitive monomer solution and the antibody-acrylate conjugate are subjected to photopolymerization under the protection of a nitrogen atmosphere. The kit comprises a photosensitive microsphere reagent, a luminous microsphere reagent and a buffer solution. During detection, a homogeneous chemiluminescence detection method is adopted, an antigen-antibody-microsphere bridge type compound can be forcibly formed through molecular design, the HOOK effect is fundamentally avoided, and meanwhile the high sensitivity characteristic of low-concentration detection is kept.

Description

Technical Field

[0001] The present invention relates to the technical field of homogeneous chemiluminescence detection, in particular to a preparation method of photosensitive microspheres, a homogeneous chemiluminescence detection kit and a detection method thereof. Background Art

[0002] Homogeneous chemiluminescence detection technology is widely used in clinical diagnosis (such as infectious disease markers and tumor marker detection) due to its advantages of no wash-off and high throughput. However, traditional detection systems are prone to the HOOK effect in the presence of high target concentrations: when the antigen concentration exceeds the number of antibody binding sites, the excess antigen will occupy the binding sites of both the donor microspheres and the acceptor microspheres, forming an "antigen-antibody-antigen" linear complex rather than a bridge complex. This results in the ineffective transfer of singlet oxygen, ultimately manifesting as a false-negative result with an abnormally low signal value.

[0003] In the prior art, improvements to the HOOK effect are mainly concentrated in the following two categories: (1) Antibody affinity optimization method: For example, CN113030476A attempts to expand the linear detection range by screening paired antibody combinations with different affinities. However, this method is limited by the antibody library resources and still has a blind spot for ultra-high concentration samples (such as serum from patients in the acute infection period).

[0004] (2) Microsphere surface modification and regulation method: A typical example is US20210063514A1, which uses polystyrene microspheres copolymerized with acrylic acid monomers to adjust the antibody loading by controlling the carboxyl density. However, this technology only delays the threshold of the HOOK effect by reducing the number of antibodies per microsphere, but it leads to a significant decrease in the sensitivity of low-concentration samples and cannot achieve the compatibility of wide dynamic range and high sensitivity.

[0005] More importantly, the existing microsphere preparation process generally ignores the decisive role of steric hindrance effect on the surface topology of the microspheres during the polymerization process. For example, when conventional emulsion polymerization is used, the copolymerization of hydrophobic monomers (such as styrene) and hydrophilic functional monomers (such as acrylates) is prone to form surface heterogeneous regions due to phase separation, resulting in uneven distribution of antibody coupling sites. At high antigen concentrations, this disordered arrangement will increase the probability of forming "linear complexes". In addition, if the photosensitive components (such as phthalocyanine photosensitizers) are not molecularly dispersed through steric hindrance design, aggregation quenching is prone to occur, further weakening the efficiency of singlet oxygen generation.

[0006] This patent can regulate the steric hindrance polymerization process through monomer design, achieve structural optimization in the microsphere formation stage, and fundamentally avoid the HOOK effect while maintaining the high sensitivity characteristics of low-concentration detection. Summary of the Invention

[0007] To address the above technical issues, the present invention has developed a method for preparing photosensitive microspheres, a homogeneous chemiluminescence detection kit, and a detection method thereof. The technical solutions are as follows:

[0008] A method for preparing photosensitive microspheres, comprising the following steps:

[0009] (1) Pretreatment of copper phthalocyanine: Dissolve polyethylene glycol PEG-2000 in deionized water, heat to 50-60°C and stir until completely dissolved, and maintain for 45 minutes to obtain a PEG-2000 aqueous dispersion. Slowly add copper phthalocyanine CuPC powder to the PEG-2000 solution while stirring and pre-mixing to wet it, thereby obtaining a copper phthalocyanine photosensitive monomer solution;

[0010] (2) Preparation of antibody-acrylate conjugate solution: dissolve the antibody in degassed PBS buffer, add a reducing agent, and incubate at 37°C for a certain period of time. Remove the excess reducing agent, then add Tris-HCl buffer (pH 8.0-8.5), add a monomer containing acrylate, and add the molar ratio of antibody:monomer = 1:10-1:20. Add the catalyst triethylamine, and incubate at 4-25°C under nitrogen protection. Stir and react for 12-48 hours in the dark. Remove the unreacted monomer and small molecule by-products. After concentration and purification, obtain the antibody-acrylate conjugate, which is stored at 4-25°C for future use.

[0011] (3) Microsphere polymerization: Styrene, alkyl-substituted methyl acrylate, copper phthalocyanine photosensitive monomer solution, and antibody-acrylate conjugate solution were added dropwise to the aqueous dispersion of polyvinyl alcohol and stirred for photopolymerization under nitrogen atmosphere. The light source was a visible light source with a wavelength of 600-700 nm and an illumination intensity of 130 mW / cm 2 , temperature 25-30℃, time 30 minutes to 2 hours. After the reaction is completed, remove the light source and cool to room temperature to terminate the polymerization, remove unreacted monomers and small molecular impurities, and the nanoparticles obtained after polymerization are 200-300nm.

[0012] Preferably, the acrylate-containing monomer is selected from glycidyl methacrylate GMA, hydroxyethyl methacrylate HEMA, succinimidyl methacrylate NHS-MA, maleimide-methacrylate bonded compound, and NHS-acrylate.

[0013] Preferably, the alkyl-substituted methyl acrylate, wherein the alkyl group is selected from n-butyl, isopropyl, tert-butyl, and cyclohexyl.

[0014] Preferably, the ratios of styrene, alkyl-substituted methyl acrylate, copper phthalocyanine photosensitive monomer solution, and antibody-acrylate conjugate solution are as follows: styrene accounts for 70-90% of the total monomer mass, alkyl-substituted methyl acrylate accounts for 13-28.5% of the total monomer mass, copper phthalocyanine accounts for 1-5% of the total monomer mass, and antibody-acrylate conjugate accounts for 0.5-2% of the total monomer mass.

[0015] Preferably, the mass concentration of the aqueous dispersion of polyvinyl alcohol is 2-5%.

[0016] The present invention also provides a homogeneous chemiluminescence detection kit, which includes a photosensitive microsphere reagent, a luminescent microsphere reagent and a buffer solution. The photosensitive microsphere is prepared according to the method of claim 1, 2 or 4. The preparation method of the luminescent microsphere is as follows: adding a dimethylthiophene derivative and a europium (III) complex MTTA-EU3+, ethanol, magnetic stirring, and heating the water bath to 70°C to obtain a complex solution; styrene is used as a matrix monomer and alkyl-substituted methyl acrylate is used as a functional monomer, and the monomers are added dropwise to a polyvinyl alcohol-dispersed aqueous dispersion for emulsion polymerization, wherein styrene is 50-70wt%; alkyl-substituted methyl acrylate is 30-50wt%; and the total monomer amount accounts for 1% of the emulsion. The invention relates to a method for preparing a luminescent microsphere comprising: a pre-emulsion comprising 20-40% of the total mass of the monomers; a PVA aqueous dispersion concentration of 2-5% by weight; an initiator (APS / KPS): 0.5-1.5% by weight, based on the total mass of the monomers; heating the pre-emulsion to 70°C under nitrogen protection, adding a portion of the initiator to initiate prepolymerization; slowly dripping the remaining monomers and initiator through a constant pressure dropping funnel for 2-4 hours, controlling the dripping speed to maintain a stable polymerization; after the dripping is completed, heating the funnel to 80-85°C and maintaining the temperature for 1-2 hours to allow the monomers to fully react; after the reaction is completed, cooling the funnel to room temperature, removing the residual monomers, PVA and initiator, and obtaining luminescent microspheres having a size of 200-300 nm; and coupling the luminescent microspheres with another antibody to complete the antibody coating.

[0017] Preferably, the alkyl group is selected from n-butyl, isopropyl, tert-butyl and cyclohexyl.

[0018] Preferably, the buffer solution comprises polyvinyl alcohol with a molecular weight of 4000-8000.

[0019] The present invention also provides a detection method of a homogeneous chemiluminescence detection kit, using the aforementioned homogeneous chemiluminescence detection kit, the detection method comprises the following steps:

[0020] (1) Reagent and sample preparation: Take out the photosensitive microsphere reagent and luminescent microsphere reagent from the kit and equilibrate them to room temperature; dilute the sample to be tested with buffer solution to an appropriate concentration;

[0021] (2) Reaction system construction: Add the sample, luminescent microsphere reagent, and photosensitive microsphere reagent to the reaction system in sequence to ensure that the microspheres are evenly dispersed;

[0022] (3) Incubation: Incubate at 37°C in the dark for 5-30 min.

[0023] (4) Excitation and signal detection: Laser excitation: Use 680nm laser to irradiate the reaction system to excite the photosensitizer in the photosensitive microspheres to produce singlet oxygen 1 O2, singlet oxygen diffusion triggers the luminescent substance inside the luminescent microspheres within the steric hindrance distance range to produce chemiluminescence.

[0024] (5) Signal reading: Detect the luminescence signal at a wavelength of 615 nm, establish a signal-concentration curve using a standard of known concentration, and calculate the concentration of the target in the sample by fitting.

[0025] Compared with the prior art, the technical solution of the present invention has the following beneficial effects:

[0026] (1) Improving the density of antibodies: By polymerizing antibodies on microspheres through a "one-step" polymerization reaction under mild reaction conditions, the antibody density can be designed and improved. Compared with the prior art of reassembling antibodies on the surface of carboxylated microspheres, the antibody density is higher and the designability and controllability are better.

[0027] (2) Antibody spacing regulation: Long-chain alkyl-substituted methyl acrylate is introduced as a steric monomer. Its side chain stretches during the polymerization process to form a "molecular fence", forcing the antibody-acrylate conjugate to be arranged at intervals (about 5-10nm), effectively inhibiting the linear binding tendency of high-concentration antigens.

[0028] (3) During the copolymerization of copper phthalocyanine photosensitive monomer and styrene / acrylate, the spatial repulsion of the steric monomer can prevent the aggregation of phthalocyanine molecules, ensuring that they are evenly distributed in the hydrophobic core of the microspheres in the form of monomers, thereby improving the singlet oxygen quantum yield.

[0029] (4) Visible light-induced polymerization (600-700 nm) and nitrogen protection reduce the free radical reaction rate, allowing each monomer to autonomously assemble into a core-shell structure based on hydrophilic and hydrophobic interactions, ultimately obtaining homogeneous microspheres with a surface functional group distribution standard deviation of less than 15%. This design ensures that even at high antigen concentrations, the spatial isolation of antibodies on the microsphere surface can still force the formation of an "antigen-antibody-microsphere" bridge complex, fundamentally avoiding the HOOK effect while maintaining the high sensitivity of low-concentration detection. DETAILED DESCRIPTION

[0030] In order to further illustrate the content of the technical solution, the technical solution of the present invention is further explained below.

[0031] Example 1 Preparation of Photosensitive Microspheres

[0032] A method for preparing photosensitive microspheres, comprising the following steps:

[0033] (1) Pretreatment of copper phthalocyanine: Dissolve polyethylene glycol PEG-2000 in deionized water, heat to 50°C and stir until completely dissolved, and maintain for 45 minutes to obtain a PEG-2000 aqueous dispersion. Slowly add copper phthalocyanine CuPC powder to the PEG-2000 solution and pre-mix it with a high-speed stirrer at a speed of 1000 rpm to wet it, thereby obtaining a copper phthalocyanine photosensitive monomer solution.

[0034] (2) Preparation of antibody-acrylate conjugate solution: The antibody was dissolved in degassed PBS buffer, a reducing agent was added, and the solution was incubated at 37°C for a certain period of time. The excess reducing agent was removed, and then Tris-HCl buffer (pH 8.0-8.5) was added. Glycidyl methacrylate (GMA) was added at a molar ratio of antibody to monomer of 1:15, and triethylamine was added as a catalyst. The reaction was stirred at 4°C in the dark under nitrogen protection for 48 hours. Glycine was added to quench the unreacted acrylic acid monomer, and the unreacted acrylic acid monomer and small molecule byproducts were removed. The antibody-acrylate conjugate was obtained after concentration and purification, and stored at 4°C for future use.

[0035] Microsphere polymerization: Styrene, alkyl-substituted methyl acrylate, copper phthalocyanine photosensitive monomer solution, and antibody-acrylate conjugate solution were added dropwise to the aqueous dispersion of polyvinyl alcohol. The mixture was stirred and photopolymerized under nitrogen atmosphere. The light source was a visible light source with a wavelength of 600 nm and an illumination intensity of 130 mW / cm 2 , temperature 25 ° C, time 2 hours. After the reaction is completed, the light source is removed and the reaction is cooled to room temperature to terminate the polymerization and remove unreacted monomers and small molecular impurities. The nanoparticles obtained after polymerization are 240 nm.

[0036] Example 2 Preparation of Photosensitive Microspheres

[0037] A method for preparing photosensitive microspheres, comprising the following steps:

[0038] (1) Pretreatment of copper phthalocyanine: Dissolve polyethylene glycol PEG-2000 in deionized water, heat to 60°C and stir until completely dissolved, and maintain for 45 minutes to obtain a PEG-2000 aqueous dispersion. Slowly add copper phthalocyanine CuPC powder to the PEG-2000 solution and pre-mix it with a high-speed stirrer at a speed of 1000 rpm to wet it, thereby obtaining a copper phthalocyanine photosensitive monomer solution.

[0039] (2) Preparation of antibody-acrylate conjugate solution: The antibody was dissolved in degassed PBS buffer, a reducing agent was added, and the mixture was incubated at 37°C for a certain period of time. The excess reducing agent was removed, and Tris-HCl buffer (pH 8.0-8.5) was added. Hydroxyethyl methacrylate (HEMA) was added at a molar ratio of antibody to monomer of 1:20, and triethylamine was added as a catalyst. The reaction was stirred at 25°C under nitrogen protection for 12 hours in the dark. Glycine was added to quench the unreacted acrylic acid monomer, and the unreacted monomer and small molecule by-products were removed. The antibody-acrylate conjugate was obtained after concentration and purification, and stored at 25°C for future use.

[0040] Microsphere polymerization: Styrene, alkyl-substituted methyl acrylate, copper phthalocyanine photosensitive monomer solution, and antibody-acrylate conjugate solution were added dropwise to the aqueous dispersion of polyvinyl alcohol. The mixture was photopolymerized under nitrogen atmosphere with stirring. The light source was a visible light source with a wavelength of 700 nm and an illumination intensity of 130 mW / cm 2 , temperature 30℃, time 30 minutes. After the reaction is completed, the light source is removed and the mixture is cooled to room temperature to terminate the polymerization. Unreacted monomers and small molecular impurities are removed. The nanoparticles obtained after polymerization are 200nm.

[0041] Example 3 Preparation of Photosensitive Microspheres

[0042] A method for preparing photosensitive microspheres, comprising the following steps:

[0043] (1) Pretreatment of copper phthalocyanine: Dissolve polyethylene glycol PEG-2000 in deionized water, heat to 55°C and stir until completely dissolved, and maintain for 45 minutes to obtain a PEG-2000 aqueous dispersion. Slowly add copper phthalocyanine CuPC powder to the PEG-2000 solution and pre-mix it with a high-speed stirrer at a speed of 1000 rpm to wet it, thereby obtaining a copper phthalocyanine photosensitive monomer solution.

[0044] (2) Preparation of antibody-acrylate conjugate solution: The antibody was dissolved in degassed PBS buffer, a reducing agent was added, and the solution was incubated at 37°C for a certain period of time. The excess reducing agent was removed, and Tris-HCl buffer (pH 8.0-8.5) was added. Maleimide-methacrylate bond was added at a molar ratio of antibody: monomer = 1:25, and a catalyst triethylamine was added. The reaction was stirred at 10°C under nitrogen protection for 16 hours in the dark. Glycine was added to quench the unreacted acrylic acid monomer, and the unreacted monomer and small molecule by-products were removed. The antibody-acrylate conjugate was obtained after concentration and purification, and stored at 10°C for future use.

[0045] Microsphere polymerization: Styrene, alkyl-substituted methyl acrylate, copper phthalocyanine photosensitive monomer solution, and antibody-acrylate conjugate solution were added dropwise to the aqueous dispersion of polyvinyl alcohol. The mixture was stirred and photopolymerized under nitrogen atmosphere. The light source was a visible light source with a wavelength of 640 nm and an illumination intensity of 130 mW / cm 2 , temperature 25 ° C, time 60 minutes. After the reaction is completed, the light source is removed and the mixture is cooled to room temperature to terminate the polymerization and remove unreacted monomers and small molecular impurities. The nanoparticles obtained after polymerization are 300 nm.

[0046] Example 4 Preparation of Photosensitive Microspheres

[0047] A method for preparing photosensitive microspheres, comprising the following steps:

[0048] (1) Pretreatment of copper phthalocyanine: Dissolve polyethylene glycol PEG-2000 in deionized water, heat to 50°C and stir until completely dissolved, and maintain for 45 minutes to obtain a PEG-2000 aqueous dispersion. Slowly add copper phthalocyanine CuPC powder to the PEG-2000 solution and pre-mix it with a high-speed stirrer at a speed of 1000 rpm to wet it, thereby obtaining a copper phthalocyanine photosensitive monomer solution.

[0049] (2) Preparation of antibody-acrylate conjugate solution: The antibody was dissolved in degassed PBS buffer, a reducing agent was added, and the solution was incubated at 37°C for a certain period of time. The excess reducing agent was removed, and Tris-HCl buffer (pH 8.0-8.5) was added. NHS-acrylate was added at a molar ratio of antibody: monomer = 1:20, and triethylamine was added as a catalyst. The reaction was stirred at 4°C under nitrogen protection for 12 hours in the dark. Glycine was added to quench the unreacted acrylic acid monomer, and the unreacted monomer and small molecule by-products were removed. The antibody-acrylate conjugate was obtained after concentration and purification, and stored at 4°C for future use.

[0050] Microsphere polymerization: Styrene, alkyl-substituted methyl acrylate, copper phthalocyanine photosensitive monomer solution, and antibody-acrylate conjugate solution were added dropwise to the aqueous dispersion of polyvinyl alcohol. The mixture was stirred and photopolymerized under nitrogen atmosphere. The light source was a visible light source with a wavelength of 600 nm and an illumination intensity of 130 mW / cm 2 , temperature 25 ° C, time 30 minutes, after the reaction is completed, remove the light source, cool to room temperature to terminate the polymerization, remove unreacted monomers and small molecular impurities, the nanoparticles obtained after polymerization are 296nm.

[0051] Example 5 Preparation of Photosensitive Microspheres

[0052] A method for preparing photosensitive microspheres, comprising the following steps:

[0053] (1) Pretreatment of copper phthalocyanine: Dissolve polyethylene glycol PEG-2000 in deionized water, heat to 50°C and stir until completely dissolved, and maintain for 45 minutes to obtain a PEG-2000 aqueous dispersion. Slowly add copper phthalocyanine CuPC powder to the PEG-2000 solution and pre-mix it with a high-speed stirrer at a speed of 1000 rpm to wet it, thereby obtaining a copper phthalocyanine photosensitive monomer solution.

[0054] (2) Preparation of antibody-acrylate conjugate solution: The antibody was dissolved in degassed PBS buffer, a reducing agent was added, and the solution was incubated at 37°C for a certain period of time. The excess reducing agent was removed, and then Tris-HCl buffer (pH 8.0-8.5) was added. Succinimidyl methacrylate NHS-MA, maleimide-methacrylate bond, and NHS-acrylate were added at a molar ratio of antibody: monomer = 1:22. The catalyst triethylamine was added. The reaction was stirred at 4°C under nitrogen protection for 48 hours in the dark. Glycine was added to quench the unreacted acrylic acid monomer, and the unreacted monomer and small molecule by-products were removed. The antibody-acrylate conjugate was obtained after concentration and purification, and stored at 4°C for future use.

[0055] Microsphere polymerization: Styrene, alkyl-substituted methyl acrylate, copper phthalocyanine photosensitive monomer solution, and antibody-acrylate conjugate solution were added dropwise to the aqueous dispersion of polyvinyl alcohol. The mixture was stirred and photopolymerized under nitrogen atmosphere. The light source was a visible light source with a wavelength of 600 nm and an illumination intensity of 130 mW / cm 2 , temperature 25 ° C, time 2 hours. After the reaction is completed, the light source is removed and the reaction is cooled to room temperature to terminate the polymerization and remove unreacted monomers and small molecular impurities. The nanoparticles obtained after polymerization are 256 nm.

[0056] Example 6 Discussion on Alkyl Substitution of Photosensitive Microspheres

[0057] The difference from Examples 1-5 is that the alkyl group in each example is selected from n-butyl, isopropyl, tert-butyl, and cyclohexyl, forming corresponding orthogonal examples.

[0058] After testing, under the same polymerization conditions, when the substituent is tert-butyl, the polymerized microspheres are applied to the photosensitive kit, and their test precision and linear range and accuracy are higher. The linear range and accuracy of the n-butyl product are also slightly better than those of commercially available products, while the linear range and accuracy of the cyclohexyl-substituted microspheres applied to the photosensitive kit are slightly reduced. It can be shown that the introduction of long-chain alkyl groups on the photosensitive microspheres causes the side chains to stretch during the polymerization process to form "molecular fences", forcing the antibody-acrylate conjugates to be arranged at intervals (about 5-10nm), effectively inhibiting the linear binding tendency of high-concentration antigens, but excessively large long-chain groups will affect the binding efficiency between antibodies, or excessive distances will affect singlet oxygen 1The conduction of O2 energy causes the linear range of detection to decrease. The specific detection data will be discussed in other embodiments.

[0059] Example 7 Discussion on polymerization conditions

[0060] In the above facts, the ratios of styrene, alkyl-substituted methyl acrylate, copper phthalocyanine photosensitive monomer solution, and antibody-acrylate conjugate solution are adjusted as follows:

[0061] (1) Styrene accounts for 70% of the total monomer mass, alkyl-substituted methyl acrylate accounts for 28.5% of the total monomer mass, copper phthalocyanine accounts for 1% of the total monomer mass, and antibody-acrylate conjugate accounts for 0.5% of the total monomer mass.

[0062] (2) Styrene accounts for 90% of the total monomer mass, alkyl-substituted methyl acrylate accounts for 6.5% of the total monomer mass, copper phthalocyanine accounts for 2.5% of the total monomer mass, and antibody-acrylate conjugate accounts for 1% of the total monomer mass.

[0063] (3) Styrene accounts for 80% of the total monomer mass, alkyl-substituted methyl acrylate accounts for 13% of the total monomer mass, copper phthalocyanine accounts for 5% of the total monomer mass, and antibody-acrylate conjugate accounts for 2% of the total monomer mass.

[0064] The mass concentration of the polyvinyl alcohol aqueous dispersion in the reaction system is 2-5%.

[0065] The reaction temperature should be kept as low as possible. The reaction temperature of the antibody-acrylate conjugate is 4°C and the reaction time is 48 hours, which is the preferred solution to maintain the activity of the antibody and ensure the complete reaction. The polymerization wavelength of the photosensitive microspheres is 600nm and the light intensity is 130mW / cm 2 , temperature 25 ° C, time 2 hours is the preferred option, which can ensure the polymerization efficiency and reduce the free radical reaction rate, so that each monomer can autonomously assemble into a core-shell structure according to the hydrophilic and hydrophobic effects, and finally obtain homogeneous microspheres with a surface functional group distribution standard deviation of <15%. The diameter of the microspheres is 200-300nm, which is also relatively reasonable, and more preferably 200-220nm, which has better uniformity and practical application effects.

[0066] Example 8 Preparation of Luminescent Microspheres

[0067] The preparation method of the luminescent microspheres is as follows: adding dimethylthiophene derivatives and europium (III) complex MTTA-EU3+, ethanol, magnetic stirring, and heating the water bath to 70°C to obtain a complex solution; styrene as a matrix monomer and alkyl-substituted methyl acrylate as a functional monomer are added dropwise to a polyvinyl alcohol-dispersed aqueous dispersion for emulsion polymerization, wherein styrene: 50-70wt%; alkyl-substituted methyl acrylate: 30-50wt%; the total monomer amount accounts for 20-40% of the total mass of the emulsion; the concentration of the PVA aqueous dispersion is 2-5wt%; the initiator (APS / KPS): 0.5-1.5wt% based on the total weight of the monomers; the pre-emulsion is heated to 70°C under nitrogen protection, and a portion of the initiator is added to initiate prepolymerization; the remaining monomers and initiator are slowly added dropwise through a constant pressure dropping funnel for 2-4 hours, and the addition rate is controlled to maintain smooth polymerization; after the addition is complete, the temperature is raised to 80-85°C and maintained for 1-2 hours to allow the monomers to fully react. After the reaction is complete, the temperature is cooled to room temperature, and the residual monomers, PVA, and initiator are removed; the buffer solution includes polyvinyl alcohol with a molecular weight of 4000-8000, and the resulting luminescent microspheres are 200-300nm. After the preparation of the luminescent microspheres, they are coupled with another antibody to complete the antibody coating.

[0068] After comparative experiments, the diameter of the luminescent microspheres is 200-220nm, with better uniformity and practical application effects.

[0069] The method for preparing luminescent microspheres in this example partially utilizes existing techniques, employing a two-step synthesis process: first, carboxyl-grouped microspheres are synthesized, followed by coating with another antibody using existing methods. However, unlike existing methods, the methyl acrylate monomer used in the microsphere preparation is alkyl-substituted, resulting in long-chain alkyl groups on the polymer surface in addition to carboxyl groups.

[0070] The alkyl group is selected from n-butyl, isopropyl, tert-butyl, and cyclohexyl, forming corresponding orthogonal embodiments.

[0071] After testing, under the same polymerization conditions, the same as the photosensitive microspheres, when the substituent is tert-butyl, the polymerized luminescent microspheres are used in conjunction with the photosensitive microspheres in the photosensitive kit, and the test accuracy and linear range and accuracy are relatively high. The linear range and accuracy of the product substituted with n-butyl are also slightly better than those of commercially available products, while the linear range and accuracy of the luminescent microspheres substituted with cyclohexyl when used in the photosensitive kit are slightly reduced. Long-chain alkyl groups are introduced to the luminescent microspheres, and their side chains stretch during the polymerization process to form "molecular fences", forcing the antibody-acrylate conjugates to be arranged at intervals (about 5-10nm), effectively inhibiting the linear binding tendency of high-concentration antigens, but excessively large long-chain groups will affect the binding efficiency between antibodies, or excessive distances will affect singlet oxygen 1The conduction of O2 energy causes the linear range of detection to decrease. The specific detection data will be discussed in other embodiments.

[0072] Example 9 Kit and Test Method

[0073] A homogeneous chemiluminescence detection kit includes a photosensitive microsphere reagent, a luminescent microsphere reagent, and a buffer solution. The buffer solution is an aqueous dispersion of polyvinyl alcohol with a molecular weight of 4000-8000 and a solid content of 0.5%. The buffer solution uses a standard reagent in the prior art and is mainly used for dilution and dispersion.

[0074] Specific antibody screening is carried out according to the different antigens to be detected. The two antibodies are generally different. The antibody on the photosensitive microsphere and the other antibody on the luminescent microsphere form a double antibody sandwich structure with the antigen to be tested, and the luminescence is stimulated to complete the test.

[0075] For example, in kit A, antibodies were purchased from Biosin Biotechnology, where antibody 1 was Cat: V2705 and antibody 2 was Cat: V2707. The kit was prepared as described in Examples 4 and 8 above, wherein the alkyl groups were both n-butyl, and the particle size of the luminescent or photosensitive microspheres was 200-210 nm. Antibody 1 was labeled on the photosensitive microspheres, and antibody 2 was labeled on the luminescent microspheres. Antibody diluent was prepared by weighing 8 g NaCl, 0.2 g KCl, 1.44 g Na2HPO4, and 0.24 g KH2PO4, dissolving them in 800 ml distilled water, adjusting the pH of the solution to 7.4 with HCl, and then adding distilled water to make up to 1 L.

[0076] The photosensitive microspheres were diluted into the above antibody diluent to make the concentration of the photosensitive microspheres 1 μg / mL, which was called R1; the luminescent microspheres were diluted into the above antibody diluent to make the concentration of the luminescent microspheres 2 μg / mL, which was called R2.

[0077] Purchase the HCG national standard from the China National Institute for Food and Drug Control and Inspection, and prepare 9 calibrators according to the instructions, with calibrator 0 being the antibody diluent.

[0078] Human Chorionic Gonadotropin (HCG), a national standard for human chorionic gonadotropin immunoassay, is prepared from high-purity HCG extracted from the urine of pregnant women. It is used for HCG immunoassay and is suitable for accuracy evaluation of enzyme-linked immunosorbent assay, chemiluminescence assay, and time-resolved immunofluorescence assay.

[0079] Calibrators Theoretical concentration (mIU / mL) Calibrator 9 10000 Calibrator 8 5000 Calibrator 7 1000 Calibrator 6 500 Calibrator 5 100 Calibrator 4 50 Calibrator 3 10 Calibrator 2 5 Calibrator 1 1 Calibrator 0 0

[0080] Pipette 20 μL of calibrator of varying concentrations, add 100 μL of R1 reagent, and then 100 μL of R2 reagent. Incubate at 37°C for 10 min. Read the RLU values ​​on a homogeneous chemiluminescence immunoassay to generate a HCG calibration curve.

[0081] Forty clinical HCG samples were collected, with the median value measured by Roche. Aspirate 20 μL of sample, add 100 μL of R1 reagent, and then add 100 μL of R2 reagent. Incubate at 37°C for 10 minutes. Read the RLU value on a homogeneous chemiluminescence immunoassay to determine the HCG concentration of the test sample.

[0082] During the detection, the aforementioned homogeneous chemiluminescence detection kit is used, and the detection method comprises the following steps:

[0083] (1) Reagent and sample preparation: Take out the photosensitive microsphere reagent and luminescent microsphere reagent from the kit and equilibrate them to room temperature; dilute the sample to be tested to an appropriate concentration using a buffer solution;

[0084] (2) Reaction system construction: Add the sample, luminescent microsphere reagent, and photosensitive microsphere reagent to the reaction system in sequence to ensure that the microspheres are evenly dispersed;

[0085] (3) Incubation: Incubate at 37°C in the dark for 5-30 min.

[0086] (4) Excitation and signal detection: Laser excitation: Use 680nm laser to irradiate the reaction system to excite the photosensitizer in the photosensitive microspheres to produce singlet oxygen 1 O2, singlet oxygen diffusion triggers the luminescent substance inside the luminescent microspheres within the steric hindrance distance range to produce chemiluminescence.

[0087] (5) Signal reading: Detect the luminescence signal at a wavelength of 615 nm, establish a signal-concentration curve using a standard of known concentration, and calculate the concentration of the target in the sample by fitting.

[0088] After testing 40 clinical samples, the error between the HCG detection value of this embodiment and the Roche test value was -2.66%, indicating that the detection method of this embodiment has a high linear range and accuracy.

[0089]

[0090]

[0091] Example 10 Kit and Test Method

[0092] The difference between Example 10 and Example 9 is that the antibodies were purchased from Bio-antigen, wherein Antibody 1 was Cat: AB0069-3 and Antibody 2 was Cat: AB0069-1.

[0093] According to the description of the above-mentioned Example 4 and Example 8, the alkyl groups are all tert-butyl, the particle size of the luminescent or photosensitive microspheres is 200-210 nm, antibody 1 is labeled on the photosensitive microspheres, and antibody 2 is labeled on the luminescent microspheres.

[0094] Prepare antibody diluent: weigh 8g NaCl, 0.2g KCl, 1.44g Na2HPO4 and 0.24g KH2PO4, dissolve in 800ml distilled water, adjust the pH of the solution to 7.4 with HCl, and then add distilled water to 1L.

[0095] Antibody 1 was diluted into the above antibody diluent so that the concentration of the photosensitive microspheres was 0.5 μg / mL, which was called R1. Antibody 2 was diluted into the above antibody diluent so that the concentration of the luminescent microspheres was 0.8 μg / mL, which was called R2.

[0096] Purchase the AFP national standard from the China National Institute for Food and Drug Control and Inspection and prepare nine calibrators according to the instructions. Calibrator 0 is the antibody diluent. The national standard for alpha-fetoprotein immunoassays, Alpha Fetoprotein (AFP), is prepared from alpha-fetoprotein extracted from umbilical cord blood. It is used for AFP immunoassays and is suitable for methods such as chemiluminescence and time-resolved immunofluorescence. Prepare nine calibrators according to the instructions, with calibrator 0 being the antibody diluent.

[0097] Pipette 25 μL of calibrator of varying concentrations, add 100 μL of reagent R1, and then 100 μL of reagent R2. Incubate at 37°C for 15 min. Read the RLU values ​​on a homogeneous chemiluminescence immunoassay to generate an AFP calibration curve.

[0098] Calibrators Theoretical concentration (mIU / mL) Calibrator 9 1000 Calibrator 8 500 Calibrator 7 100 Calibrator 6 50 Calibrator 5 10 Calibrator 4 5 Calibrator 3 1 Calibrator 2 0.5 Calibrator 1 0.1 Calibrator 0 0

[0099] Forty clinical AFP samples were collected, with the median values ​​measured by Roche. Aspirate 25 μL of sample, add 100 μL of R1 reagent, and then add 100 μL of R2 reagent. Incubate at 37°C for 15 minutes. Read the RLU values ​​on a homogeneous chemiluminescence immunoassay to determine the AFP concentration of the test sample.

[0100] After testing 40 clinical samples, the error between the AFP detection value of this embodiment and the Roche test value was -2.24%, indicating that the detection method of this embodiment has a high linear range and accuracy.

[0101]

[0102]

[0103] The above embodiments are detailed explanations of the technical solutions of the present invention. The protection scope of the present invention shall be subject to the description of the claims.

Claims

1. A method for preparing photosensitive microspheres, characterized in that: The method comprises the following steps: (1) Pretreatment of copper phthalocyanine: Dissolve polyethylene glycol PEG-2000 in deionized water, heat to 50-60°C and stir until completely dissolved, and maintain for 45 minutes to obtain a PEG-2000 aqueous dispersion. Slowly add copper phthalocyanine CuPC powder to the PEG-2000 solution while stirring and pre-mixing to wet it, to obtain a copper phthalocyanine photosensitive monomer solution; (2) Preparation of antibody-acrylate conjugate solution: dissolve the antibody in degassed PBS buffer, add a reducing agent, and incubate at 37°C for a certain period of time. Remove the excess reducing agent, then add Tris-HCl buffer (pH 8.0-8.5), add a monomer containing acrylate, and add the molar ratio of antibody:monomer = 1:15-1:

25. Add the catalyst triethylamine, and incubate at a temperature of 4-25°C under nitrogen protection. Stir and react for 12-48 hours in the dark. Remove the unreacted monomer and small molecule by-products. After concentration and purification, obtain the antibody-acrylate conjugate, which is stored at 4-25°C for future use. (3) Microsphere polymerization: Styrene, alkyl-substituted methyl acrylate, copper phthalocyanine photosensitive monomer solution, and antibody-acrylate conjugate solution were added dropwise to the aqueous dispersion of polyvinyl alcohol and stirred for photopolymerization under nitrogen atmosphere. The light source was a visible light source with a wavelength of 600-700 nm and an illumination intensity of 130 mW / cm 2 , temperature 25-30℃, 30 minutes to 2 hours. After the reaction is completed, remove the light source and cool to room temperature to terminate the polymerization, remove unreacted monomers and small molecular impurities, and the particle size of the nanoparticles obtained after polymerization is 200-300nm.

2. The method for preparing the photosensitive microspheres according to claim 1, wherein: The monomer containing acrylate is selected from glycidyl methacrylate GMA, hydroxyethyl methacrylate HEMA, succinimidyl methacrylate NHS-MA, maleimide-methacrylate bonded compound, and NHS-acrylate.

3. The method for preparing the photosensitive microspheres according to claim 1, wherein: The alkyl-substituted methyl acrylate, wherein the alkyl group is selected from n-butyl, isopropyl, tert-butyl, and cyclohexyl.

4. The method for preparing photosensitive microspheres according to claim 1, characterized in that: The proportions of styrene, alkyl-substituted methyl acrylate, copper phthalocyanine photosensitive monomer solution, and antibody-acrylate conjugate solution are as follows: styrene accounts for 70-90% of the total monomer mass, alkyl-substituted methyl acrylate accounts for 10-30% of the total monomer mass, copper phthalocyanine accounts for 1-5% of the total monomer mass, and the antibody-acrylate conjugate accounts for 0.5-2% of the total monomer mass.

5. The method for preparing photosensitive microspheres according to claim 1, characterized in that: The mass concentration of the aqueous dispersion of polyvinyl alcohol is 2-5%.

6. A homogeneous chemiluminescence detection kit, characterized in that: The kit comprises a photosensitive microsphere reagent, a luminescent microsphere reagent and a buffer solution, wherein the photosensitive microsphere is prepared according to the method of any one of claims 1 to 5, and the luminescent microsphere is prepared as follows: a dimethylthiophene derivative and a europium (III) complex MTTA-EU3+ and ethanol are added, magnetic stirring is performed, and the water bath is heated to 70° C. to obtain a complex solution; Styrene is used as a matrix monomer and alkyl-substituted methyl acrylate is used as a functional monomer, which are added dropwise into a polyvinyl alcohol-dispersed aqueous dispersion for emulsion polymerization. The monomer content is 50-70 wt%; the alkyl-substituted methyl acrylate is 30-50 wt%; the total monomer content accounts for 20-40% of the total mass of the emulsion; the concentration of the PVA aqueous dispersion is 2-5 wt%; and the initiator (APS / KPS) is 0.5-1.5 wt%, based on the total mass of the monomers. The pre-emulsion is heated to 70 DEG C under nitrogen protection, and a portion of the initiator is added to initiate pre-polymerization. The remaining monomers and the initiator are slowly added dropwise through a constant pressure dropping funnel for 2-4 hours, and the dropping speed is controlled to maintain stable polymerization. After the dropwise addition is completed, the temperature is raised to 80-85 DEG C and kept for 1-2 hours to allow the monomers to fully react. After the reaction is completed, the mixture is cooled to room temperature, and the residual monomers, PVA and the initiator are removed. The obtained luminescent microspheres have a particle size of 200-300 nm. After the preparation of the luminescent microspheres, they are coupled with another antibody to complete antibody coating.

7. The homogeneous chemiluminescence detection kit according to claim 6, characterized in that The alkyl group is selected from n-butyl, isopropyl, tert-butyl and cyclohexyl.

8. The homogeneous chemiluminescence detection kit according to claim 6, characterized in that The buffer solution includes polyvinyl alcohol with a molecular weight of 4000-8000.

9. A detection method for a homogeneous chemiluminescence detection kit, characterized in that: Using the homogeneous chemiluminescence detection kit according to any one of claims 6 to 8, the detection method comprises the following steps: (1) Reagent and sample preparation: Take out the photosensitive microsphere reagent and luminescent microsphere reagent from the kit and equilibrate them to room temperature; dilute the sample to be tested with buffer solution to an appropriate concentration; (2) Reaction system construction: Add the sample, luminescent microsphere reagent, and photosensitive microsphere reagent to the reaction system in sequence to ensure that the microspheres are evenly dispersed; (3) Incubation: Incubate at 37°C in the dark for 5-30 min. (4) Excitation and signal detection: Laser excitation: Use 680nm laser to irradiate the reaction system to excite the photosensitizer in the photosensitive microspheres to produce singlet oxygen 1 O2, singlet oxygen diffusion triggers the luminescent substance inside the luminescent microspheres within the steric hindrance distance range to produce chemiluminescence. (5) Signal reading: Detect the luminescence signal at a wavelength of 615 nm, establish a signal-concentration curve using a standard of known concentration, and calculate the concentration of the target in the sample by fitting.

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