Preparation method of microsphere composition for light-activated chemiluminescence immunoassay technology

Abstract

The invention relates to a microsphere composition for a light-activated chemiluminescence immunoassay technology and a preparation method thereof, the microsphere composition is composed of donor microspheres and acceptor microspheres coated with biological functional substances, the donor microspheres mainly comprise a polymer matrix and a photosensitizer, and the acceptor microspheres mainly comprise a polymer matrix and a luminescent compound. And one of the donor microspheres and the acceptor microspheres also contains magnetic nanoparticles. According to the microsphere composition, the detection limit is reduced, and the sensitivity of detecting complex samples is improved.

Description

Technical Field

[0001] The invention belongs to the technical field of chemiluminescent immunoassay, and in particular relates to a method for preparing microspheres used in light-induced chemiluminescent immunoassay technology. Background Art

[0002] Photoluminescence detection technology, also known as homogeneous chemiluminescence technology, was first developed based on the LOCI (Luminescent Oxygen Channeling Assay) technology developed by Ullman et al. in 1994. Its principle is based on two types of microspheres, donor and acceptor. The donor microspheres contain photosensitizers, and the acceptor microspheres contain a variety of compounds. The photosensitive microspheres release singlet oxygen from the photosensitizer under 680nm light excitation. The propagation distance of singlet oxygen in water is about 200nm. Therefore, only when the photosensitive microspheres and the luminescent microspheres are connected through the analyte (such as antigen), can the singlet oxygen diffuse to the luminescent microspheres. At this time, a series of reactions occur between the singlet oxygen and the compounds in the luminescent microspheres to generate light signals. Qualitative or quantitative detection can be achieved by detecting the intensity of the light signal.

[0003] Currently, this technology has been commercialized by PerkinElmer. PerkinElmer's products are mainly divided into two series: AlphaScreen and AlphaLISA. Both methods use the same donor microspheres, but different acceptor microspheres. Three compounds are embedded in the AlphaScreen acceptor microspheres: dimethylthiophene, anthracene and rubrene. The final luminescent fluorescent dye rubrene will emit detectable light in the band of 520-620nm. In the AlphaLISA acceptor microspheres, anthracene and rubrene are replaced by europium complexes. Singlet oxygen reacts with dimethylthiophene to produce ultraviolet light, which excites the europium (Eu) complex to form a narrow-band high-intensity light signal around 615nm. Therefore, AlphaLISA is less susceptible to interference and more sensitive than AlphaScreen.

[0004] U.S. Patent No. 5,709,994 discloses that photosensitizers include methylene blue, chlorophyll, phthalocyanine, etc.; chemiluminescent compounds include enamines, enol ethers, etc.; rare earth complexes include Eu(fod)3, Eu(TTA)3, etc. Patent CN 200510025360.8 discloses a combination of photoinduced chemiluminescent microspheres. The particle size of the photosensitive microspheres in this patent is smaller than that of the luminescent microspheres. The combination of the photosensitive microspheres and the luminescent microspheres is used to improve the detection sensitivity. The photosensitizers in the donor microspheres in this patent include methylene blue, porphyrin, chlorophyll, etc., and the acceptor microspheres contain europium complexes and compounds containing unsaturated olefinic bonds.

[0005] Although photochemiluminescence technology is relatively mature and has been widely used in clinical testing and scientific research, when detecting complex samples, it is sometimes affected by other components of the sample and the detection limit is not low enough. Summary of the invention

[0006] As is known to all, in the ELISA method, after the steps of coating antibodies, adding samples to be tested, adding enzyme-labeled secondary antibodies, etc., multiple plate washing is required. In the chemiluminescence method, it is also necessary to use a magnetic field to perform multiple washings after the magnetic beads capture the test sample and after adding the enzyme-labeled secondary antibody. The inventors have found through research that after the ordinary photochemiluminescent microspheres are endowed with magnetic functions, the detected components are captured and separated by magnetic separation technology, and the detection is performed after only one washing, which can reduce the detection limit without losing the convenient and fast characteristics of the photochemiluminescent method, and has special advantages in detecting low-concentration analytes in certain complex samples. Therefore, the present invention provides a microsphere composition used in a photochemiluminescent detection technology in view of the deficiencies in the prior art. The main components of the donor microspheres include a polymer matrix and a photosensitizer, and the main components of the acceptor microspheres include a polymer matrix and a luminescent compound, and one of the donor microspheres and the acceptor microspheres also contains magnetic nanoparticles.

[0007] In an embodiment of the present invention, the photosensitizer contained in the body microspheres may be porphyrin, chlorophyll or phthalocyanine. The luminescent compound contained in the receptor microspheres is a mixture of dimethylthiophene, dioxine and rare earth chelate.

[0008] In an embodiment of the present invention, the polymer matrix used in the donor microspheres or the acceptor microspheres is a synthetic polymer including but not limited to polypropylene, polystyrene, and polymethyl methacrylate.

[0009] In an embodiment of the present invention, the surface of the donor microsphere or receptor is modified by different methods to obtain functional groups such as carboxyl, amino, thiol, aldehyde, etc. for coupling with biologically active molecules.

[0010] In the embodiment of the present invention, the magnetic nanoparticles may include but are not limited to Fe3O4 or CoFe2O4, and the mass of the magnetic nanoparticles accounts for 10%-50% of the total mass of the microspheres, and the preferred percentage is 20%-30%. The particle size of the nanoparticles does not exceed 200nm, and the preferred particle size range is 20-100nm.

[0011] In an embodiment of the present invention, the concentration of the donor microspheres and the acceptor microspheres is 1-20000 μg / mL, and the preferred concentration of the donor microspheres and the acceptor microspheres is 10-10000 μg / mL.

[0012] The present invention also claims a method for synthesizing the microsphere composition, comprising the following steps:

[0013] Step 1: Synthesis of magnetic nanoparticles

[0014] Using the precipitation method, ferric iron salt and ferrous iron salt are dissolved in water at a substance amount ratio of 2:1, and then an alkaline substance is added. The mixture is reacted for 20-120 minutes under heating conditions, and oleic acid is added to react for 10-60 minutes. After the reaction is completed, the mixture is washed with water.

[0015] Step 2: Synthesis of magnetic donor microspheres

[0016] The magnetic nanoparticles are ultrasonically dispersed in water with styrene, tert-butyl methacrylate, oil-soluble initiator, crosslinker, photosensitive substance, emulsifier and stabilizer, and the magnetic nanoparticles are added for ultrasonic dispersion, and reacted under heating conditions for 2-6 hours. After the reaction is completed, the product is separated by a magnetic field, and the microspheres are stirred in a sodium hydroxide solution for 1-6 hours and washed for use.

[0017] Step 3: Synthesis of receptor microspheres

[0018] Stir styrene, methacrylic acid, water-soluble initiator, crosslinking agent and surfactant in water for 10 minutes and react at 60-80°C for 1-6 hours. After the reaction, wash the microspheres with ethanol, swell the microspheres in an organic solvent containing a luminescent substance for 2-12 hours, and wash them for later use.

[0019] Step 4: Magnetic donor microspheres coated with biological functional substances

[0020] The magnetic donor microspheres were activated with NHS and EDC, and then 1%-10% of the mass of the microspheres was added with streptavidin. After reacting at room temperature for 2-3 hours, the microspheres were blocked with BSA for 0.5-4 hours, and then washed for later use.

[0021] Step 5: Receptor microspheres coated with biological functional substances

[0022] After the receptor microspheres were activated with NHS and EDC, 1%-10% of the mass of the microspheres were added with antibodies, reacted at room temperature for 2-3 hours, blocked with BSA for 0.5-4 hours, and washed for later use.

[0023] or

[0024] Step 1: Synthesis of magnetic nanoparticles

[0025] Using the precipitation method, ferric iron salt and ferrous iron salt are dissolved in water at a substance amount ratio of 2:1, and then an alkaline substance is added. The mixture is reacted for 20-120 minutes under heating conditions, and oleic acid is added to react for 10-60 minutes. After the reaction is completed, the mixture is washed with water.

[0026] Step 2: Synthesis of donor microspheres

[0027] Stir styrene, methacrylic acid, water-soluble initiator, crosslinking agent and surfactant in water for 10 minutes and react at 60-80°C for 1-6 hours. After the reaction, wash the microspheres with ethanol, swell the microspheres in an organic solvent containing photosensitive substances for 2-12 hours, and wash them for later use.

[0028] Step 3: Synthesis of magnetic receptor microspheres

[0029] The magnetic nanoparticles are ultrasonically dispersed in water with styrene, methacrylate, oil-soluble initiator, crosslinker, luminescent substance, emulsifier and stabilizer, and the magnetic nanoparticles are added for ultrasonic dispersion, and reacted under heating conditions for 2-6 hours. After the reaction is completed, the product is separated by a magnetic field, and the microspheres are stirred in a sodium hydroxide solution for 1-6 hours and washed for use.

[0030] Step 4: Coating of donor microspheres with biofunctional substances

[0031] After the donor microspheres are activated with NHS and EDC, 1%-10% of the mass of the microspheres of streptavidin are added, reacted at room temperature for 2-3 hours, blocked with BSA for 0.5-4 hours, and washed for later use.

[0032] Step 5: Coating the Magnetic Receptor Microspheres with Biofunctional Substances The magnetic receptor microspheres are activated with NHS and EDC, and then 1%-10% of the mass of the microspheres are added with antibodies. After reacting at room temperature for 2-3 hours, the microspheres are blocked with BSA for 0.5-4 hours, and then washed for later use.

[0033] The beneficial effect of the present invention is that the detection limit of the photochemiluminescence method for detecting complex biological samples is further reduced while maintaining the convenience and speed of the photochemiluminescence method. DETAILED DESCRIPTION

[0034] The present invention is further described below with reference to some embodiments.

[0035] Example 1: Synthesis of magnetic nanoparticles

[0036] Dissolve 0.1 mol FeCl3 and 0.05 mol FeCl2 in 200 mL water, heat to 70°C, add 20 mL ammonia water and react for 30 min, then add 2 mL oleic acid and continue to react for 30 min. After the reaction, separate using a magnetic field to obtain Fe3O4 magnetic nanoparticles, wash three times with deionized water for later use.

[0037] Example 2: Modification of magnetic nanoparticles

[0038] Take 200 mg of the magnetic nanoparticles synthesized in Example 1, disperse them in 30 mL of water, add 2 mL of TEOS and 0.2 g of TritonX100, and ultrasonically disperse them for 5 min. Pour the dispersion into a reaction bottle, add 10 mL of ethanol solution containing 4.5 mL of ammonia water under stirring, and react at room temperature for 5 h. Continue to add 2 mL of 3-(isobutylene acyloxy)propyltrimethoxysilane and react for 5 h.

[0039] Example 3: Synthesis of magnetic donor microspheres

[0040] 2 mL of styrene, 0.4 mL of methyl methacrylate, 10 μL of divinylbenzene, 20 mg of AIBN, 20 mg of zinc phthalocyanine, 5 mg of sodium dodecyl sulfate, and 0.5 mL of hexadecane were ultrasonically dispersed in 40 mL of water for 5 min, and then 200 mg of the magnetic nanoparticles synthesized in Example 1 were added and ultrasonically dispersed for 5 min. The dispersion was reacted at 80° C. for 3 hours. After the reaction was completed, the microspheres were separated by a magnetic field, and stirred in a 1 M sodium hydroxide solution for 4 hours, and then washed with deionized water for use.

[0041] Example 4: Synthesis of magnetic donor microspheres

[0042] 2 mL of styrene, 0.6 mL of methacrylic acid, 10 μL of divinylbenzene, 20 mg of AIBN, and 20 mg of zinc phthalocyanine were dissolved in DMF, and 200 mg of the modified magnetic nanoparticles of Example 2 were added and ultrasonically dispersed for 5 min. The dispersion was reacted at 80° C. for 4 hours. After the reaction was completed, the microspheres were separated by a magnetic field, and stirred in a 1 M sodium hydroxide solution for 4 hours, and then washed with deionized water for use.

[0043] Example 5: Synthesis of magnetic receptor microspheres

[0044] 2 mL of styrene, 0.4 mL of methyl methacrylate, 10 μL of divinylbenzene, 20 mg of AIBN, 20 mg of europium chelate, 20 mg of dimethylthiophene derivative, 5 mg of sodium dodecyl sulfate, and 0.5 mL of hexadecane were ultrasonically dispersed in 40 mL of water for 5 min, and then 200 mg of the magnetic nanoparticles synthesized in Example 1 were added and ultrasonically dispersed for 5 min. The dispersion was reacted at 80 ° C for 3 hours. After the reaction was completed, the microspheres were separated by a magnetic field, and the microspheres were stirred in a 1M sodium hydroxide solution for 4 hours, and then washed with deionized water for use.

[0045] Example 6: Magnetic donor microspheres coated with streptavidin

[0046] The first step is to prepare the following solutions: ① 20mM-MES buffer solution, PH7.0; ② 100mM-glycine + 0.3% BSA + 0.02% SDS + 0.1% Tween + 0.1% preservative, PH8.0; ③ 20mM-HEPES + 0.1% BSA + 0.1% preservative + 0.1% Tween, PH8.0.

[0047] Step 2: Take 200ul of the magnetic donor microspheres synthesized in Example 3 and 200ul of pure water and mix them evenly. Use a magnetic rack to separate the magnetic donor microspheres for 1 minute and remove the supernatant.

[0048] Step 3: Add 485ul of solution ① to the centrifuge tube, mix well, and sonicate for one minute; use solution ① to prepare 10mg / mL NHS and EDC; add 10ul of NHS solution and 5ul of EDC solution in sequence, mix well; after activation for 15 minutes, separate the magnetic donor microspheres with a magnetic stand for 1 minute, and remove the supernatant.

[0049] Step 4: Add 500ul of solution ① to the centrifuge tube and mix well. Ultrasonicate for one minute, add 15ul of streptavidin, place in a dark place, and mix well with a mixer for 2 hours.

[0050] Step 5: Separate the magnetic donor microspheres with a magnetic rack for 1 minute and discard the supernatant; add 200ul of solution ② and mix well, and sonicate for 1 minute; place in a dark place and mix well with a blood mixer for 1 hour.

[0051] Step 6: Separate the magnetic donor microspheres with a magnetic rack for 1 minute and remove the supernatant; add 200ul of solution ①, mix well, and sonicate for 1 minute; repeat the above operation twice; add 400ul of solution ③, mix well, and sonicate for 1 minute to obtain the donor microspheres coated with streptavidin.

[0052] Example 7: Magnetic receptor microspheres coated with antibody A

[0053] The first step is to prepare the following solutions: ④50mM-MES, PH7.0; ⑤100mM-glycine + 0.3% BSA + 0.02% SDS + 0.1% Tween + 0.1% preservative, PH8.0; ⑥10mM-TRIS + 0.1% BSA + 0.1% preservative + 0.1% Tween, PH8.0.

[0054] Step 2: Take 200ul of the receptor microspheres synthesized in Example 5 and 200ul of pure water, mix them evenly, separate the magnetic receptor microspheres using a magnetic rack for 1 minute, and remove the supernatant.

[0055] Step 3: Add 485ul solution ④ to the centrifuge tube, mix well, and sonicate for one minute; use solution ④ to prepare 10mg / mL NHS and EDC; add 10ul NHS solution and 5ul EDC solution in sequence, mix well; after activation for 15 minutes, separate the magnetic receptor microspheres with a magnetic stand for 1 minute and remove the supernatant.

[0056] Step 4: Add 500ul of solution ④ to the centrifuge tube and mix well. Ultrasonicate for one minute, add 10uLA antibody, place in a dark place, and mix well with a blood mixer for 2 hours.

[0057] Step 5: Separate the magnetic receptor microspheres with a magnetic rack for 1 minute and remove the supernatant; add 200ul solution⑤ and mix well, and sonicate for 1 minute; place in a dark place and mix well with a mixer for 1 hour.

[0058] Step 6: Separate the magnetic receptor microspheres with a magnetic stand for 1 minute, remove the supernatant; add 200ul solution ④, mix well, and sonicate for 1 minute; repeat the above operation twice; add 400ul solution ⑥, mix well, and sonicate for 1 minute to obtain the magnetic receptor microspheres coated with antibody A.

[0059] Example 8

[0060] Donor microspheres and acceptor microspheres were prepared according to the method of Examples 4 and 5 without adding magnetic nanoparticles, and streptavidin and antibody coating was performed according to the method of Examples 6 and 7 at the same microsphere / protein mass ratio.

[0061] Example 9

[0062] Donor microspheres and acceptor microspheres were prepared according to the method of patent CN201910743273.8, and streptavidin and antibodies were coated respectively using the methods of Examples 6 and 7.

[0063] Example 10: Selecting different microsphere combinations to test a certain antigen A in a blood sample

[0064] The magnetic donor microspheres in Example 3 and the acceptor microspheres in Example 8 were diluted to a concentration of 50 μg / mL. 10 μL of blood samples containing different concentrations of antigen A and 50 μL of magnetic donor microspheres were mixed and separated by a magnetic field for 1 minute. After removing the supernatant, 50 μL of solution ③ was added to resuspend and transferred to a microplate, and 50 μL of biotinylated antibody and 50 μL of acceptor microspheres were added in sequence. After incubation at 37°C for 10 minutes, the samples were tested using a homogeneous chemiluminescent immunoassay and the average value of ten experiments was taken.

[0065] For non-magnetic donor or acceptor microspheres, magnetic field separation was not performed, and other operations were similar to those in Example 10. The test results of various microsphere combinations are listed in Table 1.

[0066] Table 1 Signal values ​​of serum samples detected by different microsphere combinations

[0067]

[0068] B / A in Table 1 reflects the detection limit. The higher the B / A, the lower the detection limit (the more sensitive). It can be seen that when using the magnetic donor microsphere-acceptor microsphere combination or the donor microsphere-magnetic acceptor microsphere combination, only one separation and cleaning process is required. Under the premise that the operation time is not significantly increased, B / A is significantly higher than that of the ordinary donor microsphere-acceptor microsphere combination, which is helpful for the photochemiluminescence method to detect certain low-concentration analytes in complex samples.

Claims

1. A microsphere composition for use in a photochemiluminescent immunoassay technique with a low detection limit, characterized in that: The microsphere composition consists of donor microspheres coated with biological functional substances and acceptor microspheres, wherein the donor microspheres mainly include polymer matrix and photosensitizer, the acceptor microspheres mainly include polymer matrix and luminescent compounds, and one of the donor microspheres and the acceptor microspheres contains magnetic nanoparticles.

2. The microsphere composition according to claim 1, characterized in that: The photosensitizer contained in the donor microspheres is one of porphyrin, chlorophyll or phthalocyanine compounds or a combination thereof.

3. The microsphere composition according to claim 1, characterized in that: The luminescent compounds contained in the acceptor microspheres are different combinations of dimethylthiophene, dioxine, and rare earth chelates.

4. The microsphere composition according to claim 1, characterized in that: The polymer matrix includes the synthetic polymer, including one or a combination of polypropylene, polystyrene, and polymethyl methacrylate.

5. The microsphere composition according to claim 1, characterized in that: The magnetic nanoparticles are Fe3O4 or CoFe2O4.

6. The microsphere composition according to claim 1, characterized in that: The mass of the magnetic nanoparticles accounts for 10%-50% of the total mass of the microspheres. The particle size of the magnetic nanoparticles does not exceed 200nm, and the particle size distribution is monodisperse or polydisperse.

7. The microsphere composition according to claim 1, characterized in that: The surface of the donor microsphere or the acceptor microsphere is modified to obtain carboxyl, amino, thiol, and aldehyde functional groups for coupling bioactive molecules.

8. The microsphere composition according to claim 1, characterized in that: The donor microspheres and acceptor microspheres were used at a concentration of 1-20000 μg / mL.

9. A method for synthesizing the microsphere composition according to any one of claims 1 to 9, comprising the following steps: Step 1: Synthesis of magnetic nanoparticles Using the precipitation method, ferric iron salt and ferrous iron salt are dissolved in water at a mass ratio of 2:1, and then an alkaline substance is added. The mixture is reacted for 20-120 minutes under heating conditions, and oleic acid is added to react for 10-60 minutes. After the reaction is completed, the mixture is washed with water. Step 2: Synthesis of magnetic donor microspheres Ultrasonic dispersion of styrene, methacrylate, oil-soluble initiator, crosslinking agent, photosensitive substance, emulsifier and stabilizer in water, adding the magnetic nanoparticles prepared in step 1 and ultrasonic dispersion, reacting for 2-6 hours under heating conditions, separating the products by magnetic field after the reaction, stirring the microspheres in sodium hydroxide solution for 1-6 hours, and washing for later use; Step 3: Synthesis of receptor microspheres Stir styrene, methacrylic acid or methacrylic acid ester, water-soluble initiator, crosslinking agent and surfactant in water for 10 minutes, react at 60-80°C for 1-6 hours, wash the microspheres with ethanol after the reaction, swell the microspheres in an organic solvent containing a luminescent substance for 2-12 hours, and wash for later use; Step 4: Magnetic donor microspheres coated with biological functional substances After the magnetic donor microspheres are activated with NHS and EDC, 1%-10% of the mass of the microspheres of streptavidin are added, reacted at room temperature for 2-3 hours, blocked with BSA for 0.5-4 hours, and washed for later use; Step 5: Receptor microspheres coated with biological functional substances After the receptor microspheres are activated with NHS and EDC, 1%-10% of the mass of the microspheres are added with antibodies, reacted at room temperature for 2-3 hours, blocked with BSA for 0.5-4 hours, and washed for later use; or Step 1: Synthesis of magnetic nanoparticles Using the precipitation method, ferric iron salt and ferrous iron salt are dissolved in water at a mass ratio of 2:1, and then an alkaline substance is added. The mixture is reacted for 20-120 minutes under heating conditions, and oleic acid is added to react for 10-60 minutes. After the reaction is completed, the mixture is washed with water. Step 2: Synthesis of donor microspheres Stir styrene, methacrylic acid or methacrylic acid ester, water-soluble initiator, crosslinking agent and surfactant in water for 10 minutes, react at 60-80°C for 1-6 hours, wash the microspheres with ethanol after the reaction, swell the donor microspheres in an organic solvent containing a photosensitive substance for 2-12 hours, wash and set aside; Step 3: Synthesis of magnetic receptor microspheres Styrene, methacrylate, oil-soluble initiator, crosslinking agent, luminescent substance, emulsifier and stabilizer are ultrasonically dispersed in water, magnetic nanoparticles are added and ultrasonically dispersed, and the reaction is carried out under heating conditions for 2-6 hours. After the reaction is completed, the product is separated by a magnetic field, and the microspheres are continuously stirred in a sodium hydroxide solution for 1-6 hours, and then washed for use; Step 4: Coating of donor microspheres with biofunctional substances After the donor microspheres are activated with NHS and EDC, 1%-10% of the mass of the microspheres of streptavidin are added, reacted at room temperature for 2-3 hours, blocked with BSA for 0.5-4 hours, and washed for later use; Step 5: Magnetic receptor microspheres coated with biological functional substances The magnetic receptor microspheres were activated with NHS and EDC, and then 1%-10% of the mass of the microspheres were added with antibodies. After reacting at room temperature for 2-3 hours, they were blocked with BSA for 0.5-4 hours and washed for later use.

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