A carboxyl-modified and loaded polystyrene chemiluminescent nanosphere and a preparation method thereof

Abstract

The present invention belongs to the field of material technology, and specifically relates to a carboxyl-modified and loaded polystyrene chemiluminescent nanosphere and a preparation method thereof, including the preparation of carboxyl-modified polystyrene nanospheres (hereinafter referred to as PS-COOH) and the preparation of a photoactivated chemiluminescent immunoassay receptor sphere (a chemiluminescent nanosphere loaded with Eu(DBM)3Phen and PCU, hereinafter referred to as Eu&PCU@PS-COOH). The present invention provides a preparation method of a chemiluminescent nanosphere with a perfect spherical morphology, good dispersibility, high monodispersity, and controllable morphology and particle size. The chemiluminescent nanosphere can be used as a photoactivated chemiluminescent immunoassay receptor sphere and can also be used for the detection and imaging of reactive oxygen species.

Description

Technical Field

[0001] The present invention belongs to the technical field of materials, and particularly relates to a carboxyl-modified and loaded polystyrene chemiluminescent nanosphere and a preparation method thereof. Background Art

[0002] Compared with traditional methods such as the photo-induced chemiluminescence immunoassay method and the enzyme-linked immunosorbent assay (ELISA), the fluorescence immunoassay method, etc., the photo-induced chemiluminescence immunoassay method has obvious advantages. One is the homogeneous reaction, which eliminates the washing step; the other is that the emission wavelength is shorter than the excitation wavelength, avoiding the interference of background fluorescence.

[0003] The nanoprobe used in the photo-induced chemiluminescence immunoassay method includes a donor sphere and a receptor sphere. The donor sphere is a nanosphere with a particle size of about 200 nm, loaded with a photosensitizer, and singlet oxygen is generated under the excitation of a laser with a wavelength of 680 nm. The receptor sphere is a nanosphere with a particle size of about 200 nm, loaded with a chemiluminescent agent and a fluorescent agent. When the receptor sphere contacts singlet oxygen, the chemiluminescent agent on the receptor sphere is oxidized by singlet oxygen, and the generated excited-state product excites the fluorescent agent through the chemiluminescence energy resonance transfer (CRET) process. When the fluorescent agent transitions back to the ground state, red light with a wavelength of 615 nm is emitted. Two different monoclonal antibodies of the antigen to be detected are respectively modified on the receptor sphere and the donor sphere. When the sample containing the antigen to be detected is incubated with the donor sphere and the receptor sphere in the sample pool, a "donor sphere - antibody 1 - antigen - antibody 2 - receptor sphere" sandwich complex is formed. The sample pool is irradiated with a laser with a wavelength of 680 nm. In the sandwich complex, the singlet oxygen generated by the donor sphere diffuses to the receptor sphere and reacts with the receptor sphere to emit light. Because the diffusion distance of singlet oxygen in water is only 200 nm, the nanospheres that do not form the sandwich complex do not participate in luminescence. Therefore, the luminescence intensity of the sample pool is proportional to the content of the sandwich complex, and the content of the antigen to be detected can be measured in this way, and the washing step is eliminated.

[0004] At present, there are many problems with commercially available photo-induced chemiluminescence immunoassay donor spheres and receptor spheres:

[0005] 1. Poor morphology, irregular shape.

[0006] 2. Poor monodispersity, and the particle size distribution range of the nanoparticles is very wide.

[0007] 3. Serious aggregation and adhesion.

[0008] 4. It is difficult to control the morphology and particle size of the product.

[0009] 5. There is still room for improvement in the signal intensity. Summary of the Invention

[0010] In view of the deficiencies of the prior art, a method for preparing chemiluminescent nanospheres with a perfect spherical morphology, good dispersibility, high monodispersity, and controllable morphology and particle size is provided. The chemiluminescent nanospheres can be used as acceptor spheres for photochemiluminescence immunoassay and can also be used for the detection and imaging of reactive oxygen species.

[0011] To achieve the above-mentioned invention object, the technical solution provided by the present invention is as Figure 1 shown, including the preparation of carboxyl-modified polystyrene nanospheres (hereinafter referred to as PS-COOH) and the preparation of photochemiluminescent immunoassay acceptor spheres (chemiluminescent nanospheres loaded with Eu(DBM) 3 Phen and PCU, hereinafter referred to as Eu&PCU@PS-COOH).

[0012] Specifically, the present invention is realized through the following technical solutions:

[0013] A method for preparing polystyrene chemiluminescent nanospheres, including:

[0014] (1) Preparation of carboxyl-modified polystyrene nanospheres (hereinafter referred to as PS-COOH);

[0015] (2) Loading Eu(DBM) 3 Phen and PCU on the carboxyl-modified polystyrene nanospheres obtained in step (1).

[0016] As a preferred technical solution of the present invention: step (1) includes:

[0017] (1.1) Under N 2 protection, in deionized water, add polyvinylpyrrolidone and styrene, and stir and reflux;

[0018] (1.2) Add an initiator and stir and reflux, preferably 2,2'-azobis(2-methylpropionamidine) dihydrochloride (V-50);

[0019] (1.3) Add acrylic acid and stir and reflux. After the reaction, add anhydrous ethanol and centrifuge to remove the supernatant, and then add deionized water and ultrasonically disperse.

[0020] As a preferred technical solution of the present invention: the dosage of polyvinylpyrrolidone corresponding to every 5.5 mL of styrene is 76 - 380 mg.

[0021] As a preferred technical solution of the present invention: the dosage of 2,2'-azobis(2-methylpropionamidine) dihydrochloride corresponding to every 5.5 mL of styrene is 75 mg.

[0022] As a preferred technical solution of the present invention: the dosage of acrylic acid corresponding to every 5.5 mL of styrene is 0.2 mL.

[0023] As a preferred technical solution of the present invention: acrylic acid is added 6 hours after adding the V-50 initiator, and the reaction continues for 18 hours. In this step, the stirring speed is 800 r / min and the reflux temperature is 70 °C.

[0024] Through a large number of experimental studies, by selecting this preferred technical solution, nanospheres with a size of about 200 nm can be prepared, which meet the requirements of the receptor sphere particle size for the photochemiluminescence immunoassay method. They have good dispersibility and monodispersity and can be used as the substrate for the photochemiluminescence immunoassay receptor sphere.

[0025] As a preferred technical solution of the present invention: step (2) includes:

[0026] (2.1) Prepare the swelling agent: CH 2 Cl 2 Dissolve Eu(DBM) 3 Phen and PCU to prepare the swelling agent.

[0027] Preferably, for every 1 mL of CH 2 Cl 2 Dissolve 0.5 g of Eu(DBM) 3 Phen and 0.1 g of PCU to prepare the swelling agent. After the swelling agent is prepared, it should not be left standing for a long time and should be used immediately after preparation.

[0028] (2.2) Disperse PS-COOH in an aqueous solution of sodium dodecyl sulfate (SDS): The concentrations of polystyrene (PS) and SDS are 20 mg / mL and 5 mg / mL, respectively.

[0029] (2.3) In a brown vial, take the dispersion obtained in step (2), and while stirring at 800 r / min, add the swelling agent dropwise. Cover the bottle cap and maintain the stirring speed at 800 r / min for 24 hours. Then open the cap and continue to maintain the stirring speed at 800 r / min for 24 hours to volatilize CH 2 Cl 2 ;

[0030] (2.4) After CH 2 Cl 2 has volatilized, centrifuge the dispersion obtained in step (3), discard the supernatant, and disperse the precipitate in water to obtain the Eu&PCU@PS-COOH aqueous dispersion.

[0031] Through a large number of experimental studies, by selecting this preferred technical solution, the obtained product has no visible deformation, good dispersibility and monodispersity, and strong chemiluminescence.

[0032] In this process, there is no need to wash the nanospheres with organic solvents to remove the PCU and Eu(DBM) 3 Phen that have not swollen into the nanospheres. Because CRET requires PCU molecules and Eu(DBM)3 The distance between Phen molecules is within 10 nm, so the PCU crystals and Eu(DBM) that failed to swell into the nanospheres and precipitated with the volatilization of the swelling agent 3 Phen crystals do not participate in luminescence. If the nanospheres are washed with organic solvents, PCU molecules and Eu(DBM) 3 Phen molecules are extracted from the nanospheres.

[0033] The beneficial effects of the present invention compared with the prior art include:

[0034] By a simple method, the present invention prepares carboxyl-modified polystyrene nanospheres and chemiluminescent nanospheres with good dispersibility, high monodispersity and a particle size of about 200 nm. The chemiluminescent nanospheres provided by the present invention can interact with ROS including singlet oxygen to emit red light at 615 nm, and are suitable as receptor spheres for photochemiluminescence immunoassay. The chemiluminescent nanospheres provided by the present invention overcome the problems of poor morphology, poor monodispersity, serious aggregation, and difficulty in controlling morphology and particle size of commercial photochemiluminescence immunoassay receptor spheres. In addition, the chemiluminescent nanospheres provided by the present invention can also be applied to chemiluminescent imaging of in vivo tumors and inflammation sites. ROS is overexpressed in tumor and inflammation sites, and ROS interacts with the chemiluminescent nanospheres provided by the present invention to emit red light at 615 nm for imaging. This imaging has obvious advantages. One is that no external excitation light is required, so the autofluorescence interference of tissues can be avoided, and the damage to the penetrated tissues caused by the excitation light can also be avoided; the second is that the red light at 615 nm has strong tissue penetration and good imaging effect; the third is PCU and Eu(DBM) 3 Phen has very low water solubility and is not easily leaked from the nanospheres, and has low toxicity to the human body. Description of the Drawings

[0035] Figure 1 Schematic diagram of the preparation process of PS-COOH and Eu&PCU@PS-COOH;

[0036] Figure 2 Particle size distribution of PS-COOH;

[0037] Figure 3 SEM image of PS-COOH;

[0038] Figure 4 Particle size distribution of Eu&PCU@PS-COOH;

[0039] Figure 5 SEM image of Eu&PCU@PS-COOH. Detailed Embodiments

[0040] The present invention will be further described below in conjunction with embodiments and the accompanying drawings, but the present invention is not limited thereto:

[0041] Example 1

[0042] The preparation process is as Figure 1 shown

[0043] (1) Preparation of PS-COOH

[0044] (1.1) Under N 2 protection, add 49 mL of deionized water, 380 mg of polyvinylpyrrolidone (PVP) with an average molecular weight of 58,000, and 5.5 mL of styrene into a 100 mL three-necked flask, and stir and reflux to raise the temperature to 70 °C. (Stirring speed: 800 r / min)

[0045] (1.2) After passing N 2 for 30 min, dropwise add 1 mL of an aqueous solution of 2,2'-azobis(2-methylpropionamidine) dihydrochloride (V-50 initiator) at 75 mg / mL, and continue to pass N 2 , and maintain stirring and reflux. (Stirring speed: 800 r / min, reflux temperature: 70 °C)

[0046] (1.3) After adding the V-50 initiator for 6 h, dropwise add 0.2 mL of acrylic acid, and continue to pass N 2 , and maintain stirring and reflux. (Stirring speed: 800 r / min, reflux temperature: 70 °C)

[0047] (1.4) After adding acrylic acid for 18 h, stop the reaction, roughly divide the obtained product dispersion into 4 portions, add them to 4 50 mL centrifuge tubes, add about 20 mL of absolute ethanol to each tube and balance with absolute ethanol, shake and mix well, centrifuge at 9000 r / min for 30 min, discard the supernatant, add 10 mL of absolute ethanol to each tube, stir with a glass rod while ultrasonically cleaning with an ultrasonic cleaner until the precipitate is completely dispersed. Combine the dispersions in 4 tubes into 2 tubes, make the volume of the combined 2 tubes up to about 35 mL with absolute ethanol and balance with absolute ethanol, centrifuge at 9000 r / min for 5 min, discard the supernatant, add 35 mL of absolute ethanol to each tube, stir with a glass rod while ultrasonically cleaning with an ultrasonic cleaner until the precipitate is completely dispersed, balance with absolute ethanol, centrifuge at 9000 r / min for 5 min, discard the supernatant, add 35 mL of deionized water to each tube, stir with a glass rod while ultrasonically cleaning with an ultrasonic cleaner until the precipitate is completely dispersed, thus obtaining the PS-COOH aqueous dispersion.

[0048] (1.5) Calibrate the concentration: Before calibration, to ensure that the PS-COOH dispersion obtained in step 4 has been fully mixed, first ultrasonically clean it with an ultrasonic cell disruptor at a power of 300 W for 5 min. Take a 1.5 mL centrifuge tube, weigh the mass of the empty tube (denoted as m0 ) Place it on the centrifuge tube rack, add 1 mL of PS-COOH dispersion with a pipette, then open the tube cap, dry the water completely in an oven at 60 °C for 24 h, cover the tube cap, weigh the total mass of the whole tube (denoted as m_total), and then the concentration of PS-COOH in the dispersion obtained in step 4 can be calculated as c = (m 总 - m 0 ) / 1 mL, which is approximately 60 mg / mL.

[0049] (1.6) Dynamic light scattering (DLS) measurement of PS-COOH aqueous dispersion: The measured number-average hydrated particle size is 257.4 nm, the polydispersity index (PDI) is 0.116, and the particle size distribution is as Figure 2 shown, and the average zeta potential is -24.7 mV.

[0050] (1.7) As Figure 3 shown, the scanning electron microscope (SEM) image of PS-COOH shows that its morphology is a perfect sphere, with a particle size of about 200 nm and high monodispersity.

[0051] (2). Preparation of Eu&PCU@PS-COOH

[0052] (2.1) Prepare the swelling agent: Dissolve 0.5 g of Eu(DBM) 2 Cl 2 Phen and 0.1 g of PCU in 1 mL of CH 3 to prepare the swelling agent. The prepared swelling agent should not be stored for a long time and should be used immediately after preparation.

[0053] (2.2) Disperse PS-COOH in an aqueous solution of sodium dodecyl sulfate (SDS): Use an ultrasonic cell disruptor to ultrasonically treat the PS-COOH aqueous dispersion at a power of 300 W for 5 min. Add deionized water and SDS aqueous solution according to the measured concentration of PS-COOH, so that the final concentrations of polystyrene (PS) and SDS are 20 mg / mL and 5 mg / mL, respectively.

[0054] (2.3) Use an ultrasonic cell disruptor to ultrasonically treat the dispersion obtained in step (2) at a power of 300 W for 5 min. In a 10 mL brown vial, take 4 mL of the dispersion obtained in step (2) just after ultrasonication, and while stirring at 800 r / min, add 0.1 mL of the swelling agent dropwise. Cover the bottle cap and maintain the stirring speed at 800 r / min for 24 h, then open the cap and continue to maintain the stirring speed at 800 r / min for 24 h to volatilize CH 2 Cl 2 .

[0055] (2.4) CH 2 Cl 2After evaporation, the dispersion obtained in step (3) was transferred to a 50 mL centrifuge tube, 20 mL of deionized water was added, and centrifuged at 11,000 r / min for 30 min. The supernatant was discarded, and the precipitate was dispersed in 20 mL of water to obtain the Eu&PCU@PS-COOH aqueous dispersion.

[0056] There is no need to wash the nanospheres with organic solvents to remove the PCU and Eu(DBM) that have not swollen into the nanospheres. 3 Phen. Because CRET requires the distance between PCU molecules and Eu(DBM) 3 and Phen molecules to be within 10 nm, the PCU crystals and Eu(DBM) 3 Phen crystals that failed to swell into the nanospheres and precipitated with the evaporation of the swelling agent do not participate in luminescence. If the nanospheres are washed with organic solvents, the PCU molecules and Eu(DBM) 3 Phen molecules will be extracted from the nanospheres.

[0057] (2.5) DLS measurement of Eu&PCU@PS-COOH aqueous dispersion: The measured number-average hydrated particle size was 244.3 nm, the PDI was 0.036, and the particle size distribution was as Figure 4 shown, and the average zeta potential was -13.3 mV.

[0058] (2.6) As Figure 5 shown, the SEM image of Eu&PCU@PS-COOH shows that its morphology is a perfect sphere, the particle size is about 200 nm, the monodispersity is high, and there is no obvious difference in morphology from PS-COOH.

[0059] Example 2

[0060] As shown in Table 1, a homogeneous chemiluminescence immunoassay analyzer from Aixing Biotech was used to test the chemiluminescence of PS-COOH and Eu&PCU@PS-COOH obtained in Example 1 under the action of singlet oxygen generated in an aqueous solution of methylene blue (MB) (test conditions: when using PS spheres, the PS sphere concentration was always 0.1 mg / mL; when using free dyes, the dye concentration was always 1 μg / mL; the total volume was always 0.2 mL; the excitation wavelength was 680 nm, and the emission wavelength was 615 nm), and strong chemiluminescence of Eu&PCU@PS-COOH was measured. It was shown that when Eu&PCU@PS-COOH comes into contact with reactive oxygen species (ROS) such as singlet oxygen, H 2 O 2 etc., the PCU loaded on the nanospheres is oxidized and cleaved, and the cleavage products are in an excited state, and Eu(DBM) is excited through chemiluminescence resonance energy transfer (CRET). 3Phen emits red light with a wavelength of 615 nm. Eu&PCU@PS-COOH can be used as a receptor sphere for photochemiluminescence immunoassay and can be used for the detection and imaging of reactive oxygen species.

[0061] Table 1: Photochemiluminescence of PS-COOH and Eu&PCU@PS-COOH

[0062] Donor / Acceptor Eu&PCU@PS-COOH PS-COOH Deionized water MB aqueous solution 116637 132 133 Deionized water 187 193 172

[0063] Comparative Example 1

[0064] Referring to the technical solution of Example 1, when the dosage of polyvinylpyrrolidone corresponding to every 5.5 mL of styrene is 76 mg, the average particle size of the product is about 500 nm. When the dosage of polyvinylpyrrolidone corresponding to every 5.5 mL of styrene is 152 mg, the average particle size of the product is about 410 nm.

[0065] When the dosage of polyvinylpyrrolidone corresponding to every 5.5 mL of styrene is 380 mg, the average particle size of the product is about 200 nm. The dosage of 2,2'-azobis(2-methylpropionamidine) dihydrochloride corresponding to every 5.5 mL of styrene is 75 mg. When the dosage of acrylic acid corresponding to every 5.5 mL of styrene is 0.2 mL, the dispersibility and monodispersity of the product are the best.

[0066] The above content is a further detailed description of the present invention in combination with specific preferred embodiments. It cannot be determined that the specific implementation of the present invention is only limited to these descriptions. For those of ordinary skill in the technical field to which the present invention belongs, without departing from the concept of the present invention, several simple deductions or substitutions can be made, and all should be regarded as belonging to the protection scope of the present invention.

Claims

1. A preparation method of polystyrene chemiluminescent nanospheres, characterized in that, it comprises: (1) Preparation of carboxyl-modified polystyrene nanospheres PS-COOH; The step (1) comprises: (1.1)N 2 Under protection, in deionized water, polyvinylpyrrolidone and styrene were added and stirred under reflux. The dosage range of polyvinylpyrrolidone corresponding to every 5.5 mL of styrene was 76 - 380 mg; (1.2) Adding an initiator and stirring under reflux. The initiator is azodiisobutyramidine hydrochloride (V-50), and the dosage of azodiisobutyramidine hydrochloride corresponding to every 5.5 mL of styrene is 75 mg; (1.3) Adding acrylic acid and stirring under reflux: After adding the V-50 initiator for 6 h, add acrylic acid. The dosage of acrylic acid corresponding to every 5.5 mL of styrene is 0.1 - 0.4 mL, and continue the reaction for 18 h; After the reaction, add absolute ethanol and centrifuge to remove the supernatant, and then add deionized water and disperse by ultrasonic wave; In the steps (1.2) and (1.3), the stirring speed is 800 r / min and the reflux temperature is 70 °C; (2) Load Eu(DBM) 3 Phen and 2-(4-(N,N-dimethylamino)phenyl)-3-phenyl-1,4-thioxane-2-cyclohexene PCU; The step (2) comprises: (2.1) Prepare the swelling agent: CH 2 Cl 2 Dissolve Eu(DBM) 3 Phen and PCU to prepare the swelling agent. For every 1 mL of CH 2 Cl 2 Dissolve 0.5 g of Eu(DBM) 3 Phen and 0.1 g of PCU to prepare the swelling agent; (2.2) Disperse PS-COOH in an aqueous solution of sodium dodecyl sulfate (SDS). The concentrations of polystyrene (PS) and SDS are 20 mg / mL and 5 mg / mL respectively; (2.3) In a brown vial, take the dispersion obtained in step (2.2), and while stirring at 800 r / min, add the swelling agent dropwise. Cover the bottle cap and maintain stirring at 800 r / min for 24 h. Then, open the cap and continue to stir at 800 r / min for 24 h to volatilize CH 2 Cl 2 ; (2.4) Centrifuge the dispersion obtained in the step (2.3), discard the supernatant, and disperse the precipitate in water to obtain the Eu&PCU@PS-COOH aqueous dispersion.

Images