A method for preparing a microsphere composition used in photochemiluminescence immunoassay technology

Abstract

The present invention provides a microsphere composition for use in photochemiluminescent immunoassay technology and its preparation method. The microsphere composition comprises donor microspheres and acceptor microspheres coated with a biofunctional substance. The donor microspheres primarily comprise a polymer matrix and a photosensitizer, while the acceptor microspheres primarily comprise a polymer matrix and a luminescent compound. One of the donor and acceptor microspheres also contains magnetic nanoparticles. The microsphere composition of the present invention reduces the detection limit and improves the sensitivity of detecting complex samples.

Description

Technical Field

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

[0002] Photoluminescence detection technology, also known as homogeneous chemiluminescence, 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 a photosensitizer, while the acceptor microspheres contain various compounds. When excited by 680nm light, the photosensitizer releases singlet oxygen from the photosensitive microspheres. Singlet oxygen travels approximately 200nm in water, so it can only diffuse to the luminescent microspheres when the photosensitive and luminescent microspheres are linked by an analyte (such as an antigen). At this point, the singlet oxygen undergoes a series of reactions with the compounds in the luminescent microspheres, generating a light signal. Qualitative or quantitative detection can be achieved by measuring the intensity of this light signal.

[0003] 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 emits detectable light in the 520-620nm band. 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 at around 615nm. Therefore, AlphaLISA is less susceptible to interference and more sensitive than AlphaScreen.

[0004] U.S. Patent 5,709,994 discloses photosensitizers including methylene blue, chlorophyll, and phthalocyanine; chemiluminescent compounds including enamines and enol ethers; and rare earth complexes including Eu(fod)3 and Eu(TTA)3. 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. This combination of photosensitive and luminescent microspheres is intended to improve detection sensitivity. The photosensitizers in the donor microspheres in this patent include methylene blue, porphyrin, and chlorophyll, while the acceptor microspheres contain a europium complex and a compound containing an unsaturated olefinic bond.

[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 well known, in the ELISA method, multiple plate washing is required after steps such as coating antibodies, adding samples to be tested, and adding enzyme-labeled secondary antibodies. In the chemiluminescence method, multiple washings are also required after magnetic beads capture the test sample and after adding enzyme-labeled secondary antibodies. The inventors have found through research that after ordinary photochemiluminescent microspheres are given magnetic functions, the detected components are captured and separated using magnetic separation technology, and detection is performed after only one washing, which can reduce the detection limit without losing the convenient and fast characteristics of the photochemiluminescent method. It has special advantages in detecting low-concentration analytes in certain complex samples. Therefore, the present invention is directed to the deficiencies in the prior art and provides a microsphere composition used in a photochemiluminescent detection technology. 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, and the luminescent compound contained in the receptor microspheres may be 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 microspheres or receptors is modified by different methods to obtain functional groups such as carboxyl, amino, thiol, and aldehyde groups for coupling with bioactive molecules.

[0010] In embodiments of the present invention, the magnetic nanoparticles may include, but are not limited to, Fe₃O₄ or CoFe₂O₄. The magnetic nanoparticles may comprise 10% to 50% of the total mass of the microspheres, preferably 20% to 30%. The nanoparticles may have a particle size of no more than 200 nm, preferably 20 to 100 nm.

[0011] In an embodiment of the present invention, the concentration of the donor microspheres and the acceptor microspheres is 1-20,000 μg / mL, and the preferred concentration of the donor microspheres and the acceptor microspheres is 10-10,000 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 salt and ferrous salt are dissolved in water at a substance ratio of 2:1, and then an alkaline substance is added. The mixture is reacted under heating conditions for 20-120 minutes, 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] Magnetic nanoparticles are ultrasonically dispersed in water with styrene, tert-butyl methacrylate, an oil-soluble initiator, a crosslinker, a photosensitizer, an emulsifier, and a stabilizer. The magnetic nanoparticles are then added and ultrasonically dispersed. The mixture is then heated and allowed to react for 2-6 hours. After the reaction, the product is separated using a magnetic field. The microspheres are then stirred in a sodium hydroxide solution for another 1-6 hours and then washed for later use.

[0017] Step 3: Synthesize receptor microspheres

[0018] Styrene, methacrylic acid, a water-soluble initiator, a crosslinking agent, and a surfactant are stirred in water for 10 minutes and reacted at 60-80°C for 1-6 hours. After the reaction, the microspheres are washed with ethanol, swelled in an organic solvent containing a luminescent substance for 2-12 hours, and then washed for later use.

[0019] Step 4: Magnetic donor microspheres coated with biofunctional 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 washed for later use.

[0021] Step 5: Receptor microspheres coated with biofunctional substances

[0022] After the acceptor 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 salt and ferrous salt are dissolved in water at a substance ratio of 2:1, and then an alkaline substance is added. The mixture is reacted under heating conditions for 20-120 minutes, 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, a water-soluble initiator, a crosslinking agent, and a 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 photosensitive substance for 2-12 hours, and then wash and set aside.

[0028] Step 3: Synthesis of magnetic receptor microspheres

[0029] Magnetic nanoparticles are ultrasonically dispersed in water with styrene, methacrylate, an oil-soluble initiator, a crosslinker, a luminescent material, an emulsifier, and a stabilizer. The magnetic nanoparticles are then added and allowed to react under heating for 2-6 hours. After the reaction, the product is separated using a magnetic field. The microspheres are then stirred in a sodium hydroxide solution for another 1-6 hours and then washed for later use.

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

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

[0032] Step 5: Coating the magnetic receptor microspheres with biological functional substances. Activate the magnetic receptor microspheres with NHS and EDC, then add 1%-10% of the mass of the microspheres with antibodies. After reacting at room temperature for 2-3 hours, block with BSA for 0.5-4 hours, and wash 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 of water, heat to 70°C, add 20 mL of aqueous ammonia dropwise, and allow to react for 30 minutes. Add 2 mL of oleic acid dropwise and continue the reaction for another 30 minutes. After the reaction, separate the particles using a magnetic field to obtain Fe₃O₄ magnetic nanoparticles. Rinse three times with deionized water for later use.

[0037] Example 2: Modification of magnetic nanoparticles

[0038] 200 mg of the magnetic nanoparticles synthesized in Example 1 were dispersed in 30 mL of water. 2 mL of TEOS and 0.2 g of Triton X100 were added, and ultrasonic dispersion was performed for 5 minutes. The dispersion was poured into a reaction flask, and 10 mL of an ethanol solution containing 4.5 mL of ammonia was added dropwise with stirring. The mixture was allowed to react at room temperature for 5 hours. 2 mL of 3-(isomethylacryloyloxy)propyltrimethoxysilane was then added and allowed to react for 5 hours.

[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 minutes. 200 mg of the magnetic nanoparticles synthesized in Example 1 were then added and ultrasonically dispersed for another 5 minutes. The dispersion was reacted at 80°C for 3 hours. After the reaction, the microspheres were separated using a magnetic field. After stirring in a 1 M sodium hydroxide solution for 4 hours, the microspheres were rinsed with deionized water and set aside.

[0041] Example 4: Synthesis of magnetic donor microspheres

[0042] Dissolve 2 mL of styrene, 0.6 mL of methacrylic acid, 10 μL of divinylbenzene, 20 mg of AIBN, and 20 mg of zinc phthalocyanine in DMF. Add 200 mg of the modified magnetic nanoparticles from Example 2 and ultrasonically disperse for 5 minutes. The dispersion is reacted at 80°C for 4 hours. After the reaction, the microspheres are separated using a magnetic field. After stirring in a 1 M sodium hydroxide solution for 4 hours, the microspheres are rinsed with deionized water and set aside.

[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 a dimethylthiophene derivative, 5 mg of sodium dodecyl sulfate, and 0.5 mL of hexadecane were ultrasonically dispersed in 40 mL of water for 5 minutes. Then, 200 mg of the magnetic nanoparticles synthesized in Example 1 were added and ultrasonic dispersion continued for another 5 minutes. The dispersion was reacted at 80°C for 3 hours. After the reaction, the microspheres were separated using a magnetic field. After stirring in a 1 M sodium hydroxide solution for 4 hours, the microspheres were rinsed with deionized water and set aside.

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

[0046] The first step is to prepare the following solutions: ① 20mM-MES buffer solution, pH 7.0; ② 100mM-glycine + 0.3% BSA + 0.02% SDS + 0.1% Tween + 0.1% preservative, pH 8.0; ③ 20mM-HEPES + 0.1% BSA + 0.1% preservative + 0.1% Tween, pH 8.0.

[0047] Step 2: Take 200ul of the magnetic donor microspheres synthesized in Example 3 and 200ul of pure water, mix them evenly, separate the magnetic donor microspheres using a magnetic stand 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 and mix well; after activation for 15 minutes, separate the magnetic donor microspheres using 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 using a magnetic stand for 1 minute and discard the supernatant; add 200ul of solution ② and mix thoroughly, then sonicate for 1 minute; place in a dark place and mix thoroughly using a blood mixer for 1 hour.

[0051] Step 6: Separate the magnetic donor microspheres using a magnetic rack for 1 minute and discard the supernatant; add 200ul of solution ①, mix well, and sonicate for 1 minute; repeat the above steps 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: ④ 50mM-MES, pH 7.0; ⑤ 100mM-glycine + 0.3% BSA + 0.02% SDS + 0.1% Tween + 0.1% preservative, pH 8.0; ⑥ 10mM-TRIS + 0.1% BSA + 0.1% preservative + 0.1% Tween, pH 8.0.

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

[0055] Step 3: Add 485 μl of solution ④ to the centrifuge tube, mix well, and sonicate for one minute; use solution ④ to prepare 10 mg / mL NHS and EDC; add 10 μl of NHS solution and 5 μl of EDC solution in sequence and mix well; after activation for 15 minutes, separate the magnetic receptor microspheres using 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 10ul of antibody, place in a dark place, and mix well with a blood mixer for 2 hours.

[0057] Step 5: Separate the magnetic receptor microspheres using a magnetic stand for 1 minute and discard the supernatant; add 200ul of solution ⑤ and mix well, then sonicate for 1 minute; place in a dark place and mix well using a mixer for 1 hour.

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

[0059] Example 8

[0060] Donor microspheres and acceptor microspheres were prepared according to the methods of Examples 4 and 5 without adding magnetic nanoparticles, and streptavidin and antibody coating was performed using the methods 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 from Example 3 and the acceptor microspheres from Example 8 were diluted to a concentration of 50 μg / mL. 10 μL of blood samples containing varying concentrations of antigen A were mixed with 50 μL of the magnetic donor microspheres and separated using a magnetic field for 1 minute. After removing the supernatant, 50 μL of solution ③ was added for resuspending and transferred to a microplate. 50 μL of biotinylated antibody and 50 μL of acceptor microspheres were then added. After incubation at 37°C for 10 minutes, the samples were analyzed using a homogeneous chemiluminescence immunoassay, and the average of ten experiments was calculated.

[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] The 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. Without significantly increasing the operation time, the B / A is significantly higher than that of the ordinary donor microsphere-acceptor microsphere combination, which is conducive to the detection of certain low-concentration analytes in complex samples by photochemiluminescence.

Claims

1. A method for synthesizing a microsphere composition for use in a photochemiluminescent immunoassay technique with a low detection limit, comprising the following steps: Step 1: Synthesis of magnetic nanoparticles Using the precipitation method, dissolve ferric salt and ferrous salt in water at a mass ratio of 2:1, add alkaline substance, react under heating conditions for 20-120 minutes, continue to add oleic acid and react for 10-60 minutes, and wash with water after the reaction is completed; Step 2: Synthesis of magnetic donor microspheres Ultrasonic dispersion of styrene, methacrylate, oil-soluble initiator, crosslinker, photosensitive substance, emulsifier, and stabilizer in water, adding the magnetic nanoparticles prepared in step 1 and ultrasonic dispersion, reacting under heating conditions for 2-6 hours, separating the products using a magnetic field after the reaction, and continuing to stir the microspheres in a sodium hydroxide solution for 1-6 hours, washing, and setting aside; Step 3: Synthesize receptor microspheres Stir styrene, methacrylic acid or methacrylate, a water-soluble initiator, a crosslinking agent, and a 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 then wash and set aside; Step 4: Magnetic donor microspheres coated with biofunctional substances Activate the magnetic donor microspheres with NHS and EDC, then add 1%-10% of the mass of the microspheres in streptavidin, react at room temperature for 2-3 hours, block with BSA for 0.5-4 hours, and wash for later use; Step 5: Receptor microspheres coated with biofunctional substances After activating the acceptor microspheres with NHS and EDC, add 1%-10% of the mass of the microspheres with antibodies, react at room temperature for 2-3 hours, block with BSA for 0.5-4 hours, and wash for later use.

2. The method for synthesizing the microsphere composition according to claim 1, wherein: The photosensitizing substance in step 2 is one of porphyrin, chlorophyll or phthalocyanine compounds or a combination thereof.

3. The method for synthesizing the microsphere composition according to claim 1, wherein: The luminescent material in step three is different combinations of dimethylthiophene, dioxine, and rare earth chelate.

4. The method for synthesizing the microsphere composition according to claim 1, wherein: The magnetic nanoparticles are Fe3O4.

5. The method for synthesizing the microsphere composition according to claim 1, wherein: 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 200 nm, and the particle size distribution is monodisperse or polydisperse.

6. The method for synthesizing the microsphere composition according to claim 1, wherein: The donor and acceptor microspheres were used at a concentration of 1-20,000 μg / mL.

7. A microsphere composition prepared by the method for synthesizing a microsphere composition according to any one of claims 1 to 6.

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