A method for preparing donor microspheres for homogeneous chemiluminescence analysis

Abstract

The present invention discloses a preparation method of donor microspheres for homogeneous chemiluminescence analysis, comprising the following steps: (1) using an emulsion polymerization method, under the protection of an inert gas, first polymerizing a cyclohexyl ester monomer, a double-bond hydroxyl monomer, and a carboxyl monomer to obtain a nano polymer microsphere; (2) grafting a modified phthalocyanine photosensitizer onto the surface of the nano polymer microsphere by covalent bonding to obtain a donor microsphere. The donor microspheres prepared by the present invention dope the photosensitizer onto the surface of the polymer microspheres by a chemical grafting method, and the photosensitizer is not easy to leak, thereby effectively improving the stability of the signal value; the hydrophilic functional groups and macromolecular chains on the surface of the donor microspheres prepared by the method are conducive to reducing the nonspecific adsorption of the microspheres and improving the detection signal-to-noise ratio.

Description

Technical Field

[0001] The invention relates to a method for preparing donor microspheres for homogeneous chemiluminescence analysis. Background Art

[0002] Homogeneous photochemiluminescence immunoassay (AlphaLISA) is a homogeneous immunological detection technology that uses receptor microspheres and donor microspheres (with a particle size of less than 200 nm) coated with an affinity coating as carriers. Photosensitizers and luminescent agents are coated on the surface of microspheres with affinity coatings, and these microspheres are used as carriers for immunoadsorption for immunological analysis, thus giving birth to AlphaLISA technology. This technology originated from the photochemiluminescence technology in 1994 and was later improved by Perkin Elmer. AlphaLISA technology is not easily affected by the matrix in the test sample and has strong specificity. AlphaLISA technology has become quite mature today and is also widely used in various fields of biological research. Homogeneous photochemiluminescence immunoassay technology has the characteristics of high sensitivity, high specificity, and low sample requirement, and has become a new type of rapid detection technology.

[0003] With the advancement of detection technology, the detection industry has an increasing demand for ultrasensitive reagents and higher requirements for detection sensitivity. At present, there are two main methods to improve the detection sensitivity of immunoadsorption carrier microspheres: one is to increase the amount of photosensitizer incorporated, and the method of incorporating photosensitizers is mainly based on the swelling method, but the photosensitizer incorporated by the swelling method is easy to leak, affecting the stability of the microsphere signal value; the second is to reduce the large nonspecific adsorption between the microspheres and antigen antibodies. Nonspecific adsorption will cause the detection sensitivity to decrease and even cause errors in the detection results. The main causes of nonspecific adsorption are hydrophobic and electrostatic effects. At present, nonspecific adsorption is mainly changed from two aspects. One is to use a blocking agent to block nonspecific sites on the surface of the microspheres to hinder nonspecific adsorption; the other is to perform multi-step grafting modification on the surface of the microspheres (to improve the hydrophilicity of the microsphere surface). This method is not only complicated to operate, but also affects the quality of the microspheres after multi-step grafting modification. The modified microspheres are very easy to agglomerate, thus affecting the subsequent detection performance. Summary of the invention

[0004] Purpose of the invention: The purpose of the present invention is to provide a method for preparing donor microspheres for homogeneous chemiluminescence analysis. The donor microspheres prepared by this method can effectively improve the stability of the signal value of the photosensitive microspheres on the one hand, and on the other hand, can effectively reduce the nonspecific adsorption of the microspheres to antigens and antibodies, thereby improving the detection sensitivity of the microspheres.

[0005] Technical solution: The method for preparing donor microspheres for homogeneous chemiluminescence analysis of the present invention comprises the following steps:

[0006] (1) using an emulsion polymerization method, under the protection of an inert gas, firstly polymerizing a cyclohexyl ester monomer, a double-bond hydroxyl monomer, and a carboxyl monomer to obtain nano-polymer microspheres;

[0007] (2) The modified phthalocyanine photosensitizer is grafted onto the surface of the nano-polymer microspheres by covalent bonding to obtain donor microspheres.

[0008] Wherein, in step (1), the mass ratio of the cyclohexyl ester monomer, the double bond-containing hydroxyl monomer and the carboxyl monomer is 7-9:0.5-2.5:0.5-1.

[0009] The cyclohexyl ester monomer is 2-cyclohexyl acrylate, cyclohexyl methacrylate, 1-ethylcyclohexyl methacrylate or 3,3,5-trimethylcyclohexyl methacrylate. The present invention uses cyclohexyl ester monomers as main monomers, which can effectively reduce the non-specific adsorption of microspheres.

[0010] Wherein, the hydroxyl monomer containing double bonds is a polyethylene glycol compound with a methacrylic acid structure, such as polyethylene glycol methacrylate (PEGMA) or polyethylene glycol methyl ether methacrylate (a class of substances having both polymerizable double bonds and polyethylene glycol structure). The long carbon chain of polyethylene glycol can provide greater steric hindrance, which is conducive to blocking the substances that produce nonspecific adsorption at a position far away from the surface of the matrix (microsphere), and polyethylene glycol is electrically neutral, and the electrically neutral structure with a large molecular weight can block the electrostatic field interaction between the matrix (microsphere) and the protein. In addition, it is hydrophilic, thereby effectively reducing the nonspecific adsorption of the microspheres.

[0011] The carboxyl monomer is acrylic acid, methacrylic acid, succinic acid mono-[2-[(2-methyl-acryloyl)oxy]ethyl] ester or maleic acid mono-2-(methacryloyloxy)ethyl ester. The carboxyl monomer can make the surface of the obtained nano polymer microspheres have carboxyl groups, which is convenient for coupling with antibodies or antigens for application in biomedical detection.

[0012] Among them, in step (1), the specific process of the emulsion polymerization method is: first add water to the reaction device, introduce inert gas to replace the air in the reaction device, then add a surfactant, stir, add cyclohexyl ester monomers, double bond-containing hydroxyl monomers and carboxyl monomers, heat and stir, add an initiator after rising to the reaction temperature, cool to room temperature after the reaction is completed, and obtain nano polymer microspheres; the polymerization temperature is 75-80°C, and the polymerization time is 12-18h

[0013] Wherein, the surfactant is sodium dodecyl sulfonate or sodium dodecylbenzene sulfonate, and the dosage is 0.1-0.4% of the total mass of the reaction materials.

[0014] The initiator is potassium persulfate or ammonium persulfate, and the amount of the initiator added is 1-2% of the mass of the cyclohexyl ester monomer.

[0015] Wherein, in step (2), the modified photosensitizer is prepared by the following method: using a phthalocyanine photosensitizer (compound 1) as a matrix, first reacting it with 3-chloropropyldimethylsilyl chloride (compound 2) to obtain an intermediate product (dimethylsilyl chloride tetra-tert-butylphthalocyanine silicon hydrate-compound 3), and then reacting the intermediate product with 5-(2-aminoethyl)amino)naphthalene-1-sulfonyl azide (compound 4) to obtain a modified photosensitizer (compound 5), and the modified photosensitizer is covalently connected to the carbon-hydrogen bond on the polymer microspheres through its azide group.

[0016] Wherein, in step (2), the molar ratio of polymer microspheres to modified photosensitizer is 0.5-1.0:10; and the reaction temperature is 60-65°C.

[0017] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: (1) The donor microspheres prepared by the present invention are doped with photosensitizers onto the surface of polymer microspheres by a chemical grafting method, and the photosensitizers are not easy to leak, thereby effectively improving the stability of the signal value; (2) The hydrophilic functional groups and macromolecular chains on the surface of the donor microspheres prepared by the present method are conducive to reducing the nonspecific adsorption of the microspheres and improving the detection signal-to-noise ratio; (3) The carboxyl functional groups on the surface of the donor microspheres prepared by the present method can also specifically bind to antigens and antibodies, and there are no redundant active groups, which is not easy to cause cross-linking of microspheres and proteins, and is convenient for coupling with antibodies or antigens for application in biomedical detection; (4) The method of the present invention is simple to operate, and through a one-step polymerization method, there is no need for multi-step glycosylation modification, thereby effectively ensuring the quality of the microspheres. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The scanning electron microscope images of the polymer microspheres obtained in Examples 1, 2 and 3 of the present invention;

[0019] Figure 2 The scanning electron microscope images of the donor microspheres obtained in Examples 9 and 10 of the present invention;

[0020] Figure 3 The UV-visible absorption spectra of the modified photosensitizer prepared in Example 8 and the donor microspheres prepared in Example 10 of the present invention;

[0021] Figure 4 This is a comparison chart of the test results of using carboxylated polystyrene donor microspheres and donor microspheres prepared in Example 10 of the present invention in AlphaLISA;

[0022] Figure 5 is the mass spectrum of the modified photosensitizer obtained in Example 8 of the present invention;

[0023] Figure 6 Signal stability test of donor microspheres prepared in Example 9 and Example 10 of the present invention;

[0024] Figure 7 The standard curve and cut-off value for detecting ricin by using common carboxylated polystyrene donor microspheres of the present invention are shown.

[0025] Figure 8 The standard curve and cut-off value of the donor microspheres prepared in Example 9 of the present invention for detecting ricin.

[0026] Fig. 9 The standard curve and cut-off value of the donor microspheres prepared in Example 10 of the present invention for detecting ricin. DETAILED DESCRIPTION

[0027] Example 1

[0028] The preparation method of nano polymer microspheres of the present invention specifically comprises the following steps: adding 0.02 g of SDS (sodium dodecyl sulfate) into 50 g of deionized water, mechanically stirring for 10 minutes, then adding 9 g of cyclohexyl methacrylate, 0.5 g of PEGMA and 0.5 g of succinic acid mono[2-[(2-methyl-acryloyl)oxy]ethyl] ester thereto, driving the air in a reaction device with high-purity nitrogen, heating and stirring to 80° C., adding 0.1 g of potassium persulfate, carrying out copolymerization reaction, reacting for 12 hours, cooling to room temperature, taking out a microsphere emulsion, and centrifugally washing it with anhydrous ethanol and deionized water for 3 times to obtain nano polymer microspheres with good spherical shape and uniform particle size.

[0029] Example 2

[0030] The preparation method of nano polymer microspheres of the present invention comprises the following steps: adding 0.02 g of SDS (sodium dodecyl sulfate) into 50 g of deionized water, mechanically stirring for 10 minutes, then adding 7 g of cyclohexyl methacrylate, 2.5 g of PEGMA and 0.5 g of succinic acid mono[2-[(2-methyl-acryloyl)oxy]ethyl] ester, driving the air in a reaction device with high-purity nitrogen, heating and stirring to 80° C., adding 0.1 g of potassium persulfate, carrying out copolymerization reaction, reacting for 12 hours, cooling to room temperature, taking out a microsphere emulsion, and centrifugally washing it with anhydrous ethanol and deionized water for 3 times to obtain nano polymer microspheres with good spherical shape and uniform particle size.

[0031] Example 3

[0032] The preparation method of nano polymer microspheres of the present invention specifically comprises the following steps: adding 0.02 g of SDS (sodium dodecyl sulfate) into 50 g of deionized water, mechanically stirring for 10 minutes, then adding 7 g of cyclohexyl methacrylate, 2 g of PEGMA and 1 g of mono[2-[(2-methyl-acryloyl)oxy]ethyl] succinate therein, driving the air in a reaction device with high-purity nitrogen, heating and stirring to 80° C., adding 0.1 g of potassium persulfate, carrying out copolymerization reaction, reacting for 12 hours, cooling to room temperature, taking out a microsphere emulsion, and centrifugally washing it with anhydrous ethanol and deionized water for 3 times to obtain nano polymer microspheres with good spherical shape and uniform particle size.

[0033] Example 4

[0034] The preparation method of nano polymer microspheres of the present invention specifically comprises the following steps: adding 0.02 g of SDBS (sodium dodecylbenzene sulfonate) into 50 g of deionized water, mechanically stirring for 10 minutes, then adding 7 g of cyclohexyl methacrylate, 2.5 g of PEGMA and 0.5 g of mono[2-[(2-methyl-acryloyl)oxy]ethyl] succinate therein, driving the air in a reaction device with high-purity nitrogen, heating and stirring to 80° C., adding 0.1 g of potassium persulfate, carrying out copolymerization reaction, reacting for 12 hours, cooling to room temperature, taking out a microsphere emulsion, and centrifugally washing it with anhydrous ethanol and deionized water for 3 times to obtain nano polymer microspheres with good spherical shape and uniform particle size.

[0035] Example 5

[0036] The preparation method of nano polymer microspheres of the present invention specifically comprises the following steps: adding 0.02 g of SDS (sodium dodecyl sulfate) into 50 g of deionized water, mechanically stirring for 10 minutes, then adding 7 g of cyclohexyl methacrylate, 2.5 g of PEGMA and 0.5 g of mono[2-[(2-methyl-acryloyl)oxy]ethyl] succinate therein, driving the air in a reaction device with high-purity nitrogen, heating and stirring to 80° C., adding 0.1 g of ammonium persulfate, carrying out copolymerization reaction, reacting for 12 hours, cooling to room temperature, taking out a microsphere emulsion, and centrifugally washing it with anhydrous ethanol and deionized water for 3 times to obtain nano polymer microspheres with good spherical shape and uniform particle size.

[0037] Example 6

[0038] The preparation method of nano polymer microspheres of the present invention comprises the following steps: adding 0.02 g of SDS (sodium dodecyl sulfate) into 50 g of deionized water, mechanically stirring for 10 minutes, then adding 7 g of cyclohexyl methacrylate, 2.5 g of PEGMA and 0.5 g of mono[2-[(2-methyl-acryloyl)oxy]ethyl] succinate, driving the air in a reaction device with high-purity nitrogen, heating and stirring to 75° C., adding 0.1 g of potassium persulfate, carrying out copolymerization reaction, reacting for 12 hours, cooling to room temperature, taking out a microsphere emulsion, and centrifugally washing it with anhydrous ethanol and deionized water for 3 times to obtain nano polymer microspheres with good spherical shape and uniform particle size.

[0039] Example 7

[0040] The preparation method of nano polymer microspheres of the present invention comprises the following steps: adding 0.02 g of SDS (sodium dodecyl sulfate) into 50 g of deionized water, mechanically stirring for 10 minutes, then adding 7 g of cyclohexyl methacrylate, 2.5 g of PEGMA and 0.5 g of mono[2-[(2-methyl-acryloyl)oxy]ethyl] succinate, driving the air in the reaction device with high-purity nitrogen, heating and stirring to 75° C., adding 0.1 g of potassium persulfate, carrying out copolymerization reaction, reacting for 18 hours, cooling to room temperature, taking out the microsphere emulsion, and centrifugally washing it with anhydrous ethanol and deionized water for 3 times to obtain nano polymer microspheres with good spherical shape and uniform particle size.

[0041] Example 8

[0042] The preparation method of the modified photosensitizer of the present invention is specifically as follows: 0.625mmol of compound 1 and 2.5mmol of imidazole are added to a flask, 20mL of DMF (N,N-dimethylformamide) is added, and the mixture is stirred for 10min, and then 1.375mmol of compound 2 is added, and the mixture is reacted at room temperature in the dark for 36h; water is added to quench the reaction, and the product (compound 3) is extracted with EA resin, dried with anhydrous Na2SO4, and filtered to obtain a blue solid, and rapid column chromatography PE:EA=50:1 is performed to obtain a blue solid (compound 3) with a yield of 30%; 1mmol of the blue solid is reacted with 6mmol of compound 4 at 60°C for 6h to obtain a modified photosensitizer (compound 5).

[0043] The chemical reaction formula of the photosensitizer modification process is:

[0044]

[0045]

[0046] Example 9

[0047] A method for preparing donor microspheres, specifically comprising: using a swelling method to incorporate a photosensitizer into polymer microspheres, taking 100 mg of the polymer microspheres of Example 2 and dispersing them in 5 mL of a 0.25% SDS aqueous solution, adding 1 mL of dichloromethane dissolved with 10 mg of the photosensitizer (the mass concentration of the photosensitizer is 10 mg / mL), magnetically stirring at 30° C. for 4 hours, cooling to room temperature after the reaction is completed, centrifuging and discarding the supernatant, adding water to resuspend, and repeatedly washing 3 to 5 times to obtain donor microspheres.

[0048] Example 10

[0049] The preparation method of the donor microspheres of the present invention is specifically as follows: a photosensitizer is covalently linked to the surface of a polymer microsphere by a chemical grafting method: 100 mg of the dried polymer microspheres of Example 2 are taken, 10 mg of the photosensitizer is added, and the mixture is stirred at 60° C. overnight. After the reaction is completed, the mixture is cooled to room temperature, centrifuged with methanol to discard the supernatant, resuspended with methanol, and repeatedly washed 3 to 5 times to obtain donor microspheres.

[0050] Depend on Figure 1 and Figure 2 It can be seen that the particle size of the microspheres prepared in Example 2 is 200 nm, the surface of the donor microspheres prepared in Example 9 is not smooth and has a certain degree of adhesion, while the surface of the donor microspheres prepared in Example 10 is smooth, evenly distributed and has no agglomeration, so it can be well applied in the subsequent AlphaLISA technology.

[0051] Depend on Figure 3 It can be seen that the maximum ultraviolet absorption wavelength of the donor microspheres prepared in Example 10 is 680 nm, which is consistent with the maximum ultraviolet absorption wavelength of the photosensitizer prepared in Example 8, indicating that the photosensitizer has been successfully doped into the surface of the polymer microspheres.

[0052] Ordinary carboxylated polystyrene donor microspheres and the donor microspheres of Example 10 were applied to the AlphaLISA technique. Figure 4 It can be seen that the signal value of the donor microspheres prepared in Example 10 is slightly higher than that of ordinary donor microspheres, but the background signal value of the donor microspheres prepared in Example 10 is significantly reduced compared with that of ordinary polymer donor microspheres, indicating that polymer donor microspheres prepared with cyclohexyl esters and double-bond polyethylene glycol are beneficial to reducing the nonspecific adsorption of microspheres, thereby preparing polymer donor microspheres with a high signal-to-noise ratio.

[0053] The donor microspheres of Example 9 and Example 10 were placed at room temperature for 30 days. Figure 6It can be seen that the signal intensity of the donor microspheres prepared in Example 9 decreased significantly, indicating that the photosensitizer had some leakage. However, the signal value of the donor microspheres prepared in Example 10 did not decrease significantly and was relatively stable, indicating that the photosensitizer was grafted onto the surface of the polymer microspheres by chemical grafting, the photosensitizer of the prepared donor microspheres did not fall off, the signal intensity was stable, almost unchanged, and the microspheres had good stability.

[0054] pass Figures 7 to 9 It can be seen that the donor microspheres prepared in Example 9 of the present invention are used for the detection of ricin, with a cut-off value of 5521.76 and a detection limit of 0.2ng / mL. The cut-off value of ordinary carboxylated polystyrene donor microspheres is 20811.01, and the detection limit is 0.78ng / mL. The donor microspheres prepared in Example 10 are used for the detection of ricin, with a cut-off value of 5218.74 and a detection limit of 0.1ng / mL. The donor microspheres of Example 9 are prepared by the swelling method, and the prepared microspheres have a certain degree of agglomeration, which will affect the actual detection signal value and signal-to-noise ratio; ordinary carboxylated microspheres have high nonspecific adsorption of proteins, so the background value is high during actual detection, resulting in reduced detection sensitivity.

Claims

1. A method for preparing donor microspheres for homogeneous chemiluminescence analysis, characterized in that: The steps include: (1) Using emulsion polymerization, under the protection of inert gas, cyclohexyl ester monomers, double bond-containing hydroxyl monomers and carboxyl monomers are first polymerized to obtain nano-polymer microspheres; Wherein, the cyclohexyl ester monomer is 2-cyclohexyl acrylate, cyclohexyl methacrylate, 1-ethylcyclohexyl methacrylate or 3,3,5-trimethylcyclohexyl methacrylate; the double-bond-containing hydroxyl monomer is polyethylene glycol methacrylate or polyethylene glycol methyl ether methacrylate; the carboxyl monomer is acrylic acid, methacrylic acid, succinic acid mono-[2-[(2-methyl-acryloyl)oxy]ethyl] ester or maleic acid mono-2-(methacryloyloxy)ethyl ester; the mass ratio of the cyclohexyl ester monomer, the double-bond-containing hydroxyl monomer and the carboxyl monomer is 7-9:0.5-2.5:0.5-1; (2) grafting the modified phthalocyanine photosensitizer onto the surface of the nano-polymer microspheres by covalent bonding to obtain donor microspheres; The modified photosensitizer is prepared by the following method: a phthalocyanine photosensitizer is used as a matrix, first reacted with 3-chloropropyldimethylsilyl chloride to obtain an intermediate product, and then the intermediate product is reacted with 5-(2-aminoethyl)amino)naphthalene-1-sulfonyl azide to obtain a modified photosensitizer, and the modified photosensitizer is covalently connected to the carbon-hydrogen bond on the polymer microsphere through its azide group; The chemical structure of phthalocyanine photosensitizer is: 。 2. The method for preparing donor microspheres for homogeneous chemiluminescence analysis according to claim 1, characterized in that: In step (1), the specific process of the emulsion polymerization method is: first add water to the reaction device, introduce inert gas to replace the air in the reaction device, then add a surfactant, stir, add cyclohexyl ester monomers, double bond-containing hydroxyl monomers and carboxyl monomers, heat and stir, add an initiator after the reaction temperature is raised to the reaction temperature, cool to room temperature after the reaction is completed, and obtain nano-polymer microspheres; the polymerization temperature is 75-80°C, and the polymerization time is 12-18h.

3. The method for preparing donor microspheres for homogeneous chemiluminescence analysis according to claim 2, characterized in that: The surfactant is sodium dodecyl sulfonate or sodium dodecylbenzene sulfonate, and the amount used is 0.1-0.4% of the total mass of the reaction materials.

4. The method for preparing donor microspheres for homogeneous chemiluminescence analysis according to claim 2, characterized in that: The initiator is potassium persulfate or ammonium persulfate, and the amount of the initiator added is 1-2% of the mass of the cyclohexyl ester monomer.

5. The method for preparing donor microspheres for homogeneous chemiluminescence analysis according to claim 1, characterized in that: In step (2), the molar ratio of the polymer microspheres to the modified photosensitizer is 0.5-1.0:10; and the reaction temperature is 60-65°C.

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