CN113956490B - Streptavidin-modified aggregation-induced emission polymer microsphere and preparation method and application thereof - Google Patents
Streptavidin-modified aggregation-induced emission polymer microsphere and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a streptavidin modified aggregation-induced emission polymer microsphere and a preparation method and application thereof. The method comprises the following steps: dissolving AIE molecules and an auxiliary stabilizer in a mixed solution of a carboxyl functional monomer and a hydrophobic monomer to obtain an oil phase solution; adding an emulsifier aqueous solution into the oil phase solution, adding a water-soluble initiator, and reacting to obtain the surface carboxyl modified AIE fluorescent microspheres; adding the AIE fluorescent microspheres into an acidic buffer solution, adding a carbodiimide condensation reagent after ultrasonic dispersion, and putting the mixture into a mixer to activate carboxyl; and adding an alkaline buffer solution and streptavidin into the activated fluorescent microspheres, putting the fluorescent microspheres into a mixer for incubation, centrifuging to take supernatant, adding a confining liquid and a preservation liquid, and performing ultrasonic dispersion to obtain the polymer microspheres. The invention meets the application of various scenes of biological medicine based on the aggregation-induced emission phenomenon of AIE molecules; the preparation method is simple and convenient, and large-scale industrial production can be realized by adjusting the proportion of the microspheres, the carbodiimide and the streptavidin.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a streptavidin-modified aggregation-induced emission polymer microsphere, and a preparation method and application thereof.
Background
The combination of streptavidin and biotin has high specificity and stability, the affinity constant of the two is 10-100 ten thousand times higher than that of antigen-antibody immune reaction, and the non-covalent action with the highest strength is known at present, which makes the combination a very good tool in protein biochemistry. The streptavidin modified fluorescent microsphere is combined with biotinylated antibody, polypeptide, aptamer and targeting protein, can be used as a fluorescent immune reagent, and can be widely applied to the fields of in vitro diagnosis, fluorescent tracing and cell imaging.
At present, in the preparation process of fluorescent microspheres, the traditional aggregation-induced quenching (ACQ) fluorescent molecules can generate the phenomenon of fluorescence weakening and even fluorescence quenching in the aggregation process, and the ACQ phenomenon can reduce the linearity and sensitivity of fluorescent microsphere products and limit the application of the fluorescent microspheres to a certain extent. In 2001, the Tang Benzhou subject group found that aggregation-induced emission (AIE) materials, AIE fluorescent microspheres emitted intense fluorescence due to a mechanism of restricted intramolecular rotation, and the discovery of AIE materials provided a new solution for fluorescent immunoreagents.
At present, the preparation method of the fluorescent microsphere comprises a swelling adsorption method, a coating method and a chemical bonding method. The most common method is a swelling adsorption method, wherein the swelling adsorption method swells microspheres in an organic solvent, and the fluorescent material (organic micromolecular dye or quantum dots) is swollen in the microspheres by using the strong hydrophobic effect between the surfaces of the microspheres to form the fluorescent microspheres. The coating method is to uniformly disperse fluorescent molecules in a medium, and the fluorescent microspheres are prepared by using a polymerization reaction, a microencapsulation method or a self-assembly method, so that the fluorescent microspheres have high fluorescent molecule loading rate and are suitable for industrial large-scale production. Wherein, the miniemulsion polymerization is a coating method for efficiently preparing the nano fluorescent microspheres, and the miniemulsion polymerization is particularly suitable for preparing the polymer microspheres by introducing a second component due to a droplet nucleation mechanism of the miniemulsion polymerization.
Recently, Cao Shi Hai task group first prepares AIE fluorescent microspheres through miniemulsion copolymerization of 1-allyl-1-methyl-2, 3,4, 5-tetraphenylsilane (AMTPS) and styrene, the loading of AIE is up to 20 wt%, but the AIE molecules need to modify polymerizable double bonds, increasing the use cost of AIE fluorescent microspheres [ APolymer Chemistry2016, 7(35): 5571-. The subsequent preparation of polypeptide-modified AIE fluorescent microspheres by miniemulsion polymerization technology can specifically enter cells, and shows potential application value in the field of cell imagingPolymer Chemistry, 2019, 10(30): 4220-. Recently, the wanwanwan task group of plum uses polystyrene copolymer to prepare AIE fluorescent microsphere liquid drops [ cn108587609a ], and then solvent volatilization is performed to prepare monodisperse AIE fluorescent microspheres, although the SPG membrane emulsification-emulsion solvent volatilization method can effectively prepare monodisperse polymer microspheres, organic solvent cannot be avoided, and environmental cost is increased. At present, documents or patents for preparing streptavidin modified AIE fluorescent microspheres by using a green and environment-friendly miniemulsion polymerization technology are newly reported, and the application of the streptavidin modified AIE fluorescent microspheres in the field of in vitro diagnosis is limited. Therefore, it is necessary to develop AIE fluorescent microspheres that have high fluorescence intensity, good water dispersibility, good homogeneity, high streptavidin modification density, and are industrially producible.
Disclosure of Invention
In order to overcome the defects in the prior art, the invention aims to provide a novel method for preparing streptavidin modified AIE fluorescent microspheres based on miniemulsion polymerization technology.
The technical scheme adopted by the invention is as follows:
a preparation method of streptavidin modified aggregation-induced emission polymer microspheres comprises the following steps:
(1) dissolving an emulsifier in water to serve as an aqueous emulsifier solution for the continuous phase;
(2) dissolving AIE molecules and an auxiliary stabilizer in a mixed solution of a carboxyl functional monomer and a hydrophobic monomer to obtain an oil phase solution;
(3) adding the emulsifier aqueous solution in the step (1) into the oil phase solution in the step (2), performing ultrasonic treatment to obtain a monomer miniemulsion, raising the temperature, adding a water-soluble initiator, and reacting to obtain the AIE fluorescent microspheres with surface carboxyl modified;
(4) adding the AIE fluorescent microspheres prepared in the step (3) into an acid buffer solution, adding a carbodiimide condensation reagent after ultrasonic dispersion, putting the mixture into a mixer to activate carboxyl, and centrifuging to remove supernatant so as to obtain activated fluorescent microspheres;
(5) and (3) adding the activated fluorescent microspheres obtained in the step (4) into an alkaline buffer solution and streptavidin, placing the mixture into a mixer for incubation, centrifuging the mixture to obtain a supernatant, adding a confining liquid and a preservation liquid, and performing ultrasonic dispersion to obtain the streptavidin-modified aggregation-induced luminescent polymer microspheres.
The test paper strip comprises a bottom plate, a sample pad, a nitrocellulose membrane and a water absorption pad, wherein a detection line parallel to the end face of the bottom plate is coated on the exposed nitrocellulose membrane between the end part of the sample pad and the end part of the water absorption pad, an anti-streptavidin antibody is coated on the detection line, the self-made streptavidin modified AIE fluorescent microspheres are dripped into the sample pad, and a dry type fluorescence immunoassay instrument is used for detecting a fluorescent signal value.
Preferably, in the step (1), the mass usage of the emulsifier is 0.01-20% of the mass usage of water;
further preferably, the mass usage of the water-soluble emulsifier is 0.1-5% of the mass usage of water.
Preferably, in the step (1), the water is ultrapure water;
preferably, the emulsifier is selected from at least one of the following: sodium dodecyl sulfate, polyvinyl alcohol, tween 20 and sodium dodecyl benzene sulfonate.
Preferably, in the step (2), the mass usage amount of the AIE molecules is 0.05-20% of the total mass of the monomers; the mass consumption of the co-stabilizer is 0.01-8% of the total mass of the monomers; the mass usage of the carboxyl functional monomer is 5-50% of the total mass usage of the monomer; the mass usage of the hydrophobic monomer is 50-95% of the total mass usage of the monomers;
the total mass of the monomers refers to the total mass of the carboxyl functional monomer and the hydrophobic monomer.
Preferably, the AIE molecule of step (2) is selected from at least one of formula (I), formula (II), and formula (III):
preferably, the co-stabilizer is selected from at least one of the following: aliphatic hydrocarbon of C14~ C22, aliphatic alcohol of C14~ C22;
preferably, the carboxyl functional monomer is at least one of acrylic acid and methacrylic acid shown in a formula (IV);
in the formula (IV), R1Is H or CH3；
Preferably, the hydrophobic monomer is selected from at least one of the following: styrene of formula (V), an acrylate or methacrylate monomer compound of formula (VI);
in the formula (VI), R7Is H or methyl; r8Is a C1-C5 alkyl group.
Further preferably, in the step (2), in consideration of the application of the prepared AIE fluorescent microspheres in cell imaging, red AIE molecules in the formula (III) are preferably used as fluorescent components, and the mass usage amount of the AIE molecules is preferably 0.1% -4% of the total mass usage amount of the monomers;
further preferably, in the step (2), in consideration of the curing phenomenon of the colloidal particles, n-hexadecane is preferably selected as the co-stabilizer, and the mass usage of the co-stabilizer is preferably 1-6% of the mass usage of the monomer;
through intensive research of the inventor, the monomer oil-water ratio, the type of the surfactant and the dosage of the surfactant play an important role in the particle size and the stability of the AIE fluorescent microspheres, the surfactant and the monomer oil-water ratio can accurately regulate and control the particle size of the AIE fluorescent microspheres, the particle size of the microspheres can be reduced by increasing the dosage of the surfactant or reducing the monomer oil-water ratio in the polymerization reaction process, and the regulation and control interval range is 50 nm-500 nm. Therefore, the surfactant and the monomer oil-water ratio need to be selected according to different particle size requirements.
Further preferably, in the step (2), in consideration of providing enough reaction sites for subsequent biotinylation macromolecule modification and ensuring excellent narrow distribution of the fluorescent microspheres, the carboxyl functional monomer is preferably methacrylic acid, and the mass usage amount of the carboxyl functional monomer is preferably 10-30% of the mass usage amount of the monomer;
further preferably, in the step (2), considering that the AIE fluorescent microspheres are mainly used in a fluorescent immunodiagnostic reagent, the hydrophobic monomer is preferably styrene, and the mass usage amount of the hydrophobic monomer is preferably 70-90% of the total mass usage amount of the monomers.
Preferably, in the step (3), the power of the ultrasonic treatment is 100W-500W, and the time of the ultrasonic treatment is 0.5 min-60 min; the reaction temperature is 50-90 ℃, and the reaction time is 2-48 h;
preferably, in the step (3), the mass usage of the water-soluble initiator is 0.05-10% of the total mass of the monomers;
the total mass of the monomers is the total mass of the carboxyl functional monomer and the hydrophobic monomer;
preferably, in step (3), the water-soluble initiator is selected from at least one of the following: persulfates; water-soluble azo compounds; a redox system consisting of an oxidizing agent and a reducing agent. The water-soluble initiator is preferably potassium persulfate in view of stability of the polymerization reaction system;
preferably, in step (4), the carbodiimide condensing agent is selected fromN, N' -dicyclohexylcarbodiimide,N, N’-one of diisopropylcarbodiimide and 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride;
preferably, in the step (4), the acidic buffer is glycine-hydrochloric acid buffer, phthalic acid-hydrochloric acid buffer, disodium hydrogen phosphate-citric acid buffer, or 2- (N-morpholine) ethanesulfonic acid buffer; the pH value of the acidic buffer solution is 6.0-6.5; the mass usage of the carbodiimide condensation reagent is 100-400% of the usage of the carboxyl functional monomer, and the activation time is 5 min-3 h; in order to maximize the efficiency of the condensation reaction, the acidic buffer solution is preferably 2- (N-morpholine) ethanesulfonic acid buffer solution with the pH value ranging from 6.0 to 6.5, and the carbodiimide condensing agent is preferably 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride.
Preferably, the centrifugation time is 10-60min, the rotation speed is 8000-. Further preferably, the centrifugation time is 30min, and the rotating speed is 12000rpm
Preferably, in the step (5), the mass usage of the streptavidin is 10% -200% of the usage of the carboxyl functional monomer;
preferably, in the step (5), the alkaline buffer is tris-hydrochloric acid buffer, boric acid buffer, Phosphate Buffered Saline (PBS); the pH value of the alkaline buffer solution is 7.0-8.0; in order to improve the condensation reaction efficiency to the maximum extent, the pH value of the alkaline buffer solution is preferably 7.0-8.0.
Preferably, in the step (5), blocking of the activated ester is required in consideration that the activated ester is not completely reacted after activation, and the blocking solution is preferably Bovine Serum Albumin (BSA) and glycine, and more preferably BSA.
The streptavidin modified aggregation-induced emission polymer microsphere prepared by the preparation method.
The streptavidin modified aggregation-induced emission polymer microsphere is applied to the field of in vitro diagnosis.
The inventors of the present invention have made intensive studies, and as a result, carbodiimide can be used as a flocculant as well as a condensing agent, and in order to ensure colloidal stability, carbodiimide should be used in an amount suitable for the streptavidin modification density. Meanwhile, in order to prevent the protonation of carboxyl and amino groups on the surface of the condensation reaction, MES buffer with pH =6 is selected for carboxyl activation, and boric acid buffer with pH =8 is selected for streptavidin coupling.
Compared with the prior art, the invention has the following beneficial effects:
the method provides a novel method for efficiently preparing the streptavidin-modified AIE polymer fluorescent microspheres by combining a miniemulsion polymerization technology and a carbodiimide reaction, detects the effect of the microspheres in an immunochromatography mode, and verifies the potential application value of the streptavidin-modified AIE fluorescent microspheres in-vitro diagnosis. The AIE molecules are embedded in a polymer matrix, and the AIE fluorescent microspheres with surface carboxyl modified are prepared through copolymerization of carboxyl monomers and hydrophobic monomers. And (3) efficiently preparing the streptavidin-modified AIE fluorescent microspheres by using carbodiimide reaction, and finally adding confining liquid and preserving liquid to obtain the streptavidin-modified AIE fluorescent microspheres.
The method has the advantages that:
(1) the carboxyl modified polystyrene AIE fluorescent microspheres are prepared by a one-step method, and the modification density of streptavidin of the AIE fluorescent microspheres can be regulated and controlled by adjusting the ratio of the carbodiimide condensing agent to the streptavidin, so that the streptavidin modified AIE fluorescent microspheres are efficiently prepared.
(2) Compared with the traditional ACQ molecule, the AIE molecule can emit strong fluorescence in the microsphere due to the aggregation-induced emission phenomenon of the AIE molecule, and the emission wavelength of the AIE fluorescent microsphere can cover a plurality of wave bands such as red, blue and green, so that the application of various scenes of biological medicine is met.
(3) The preparation method is simple and convenient, and large-scale industrial production can be realized by adjusting the proportion of the microspheres, the carbodiimide and the streptavidin.
Drawings
FIG. 1 is a scanning electron microscope image of streptavidin-modified AIE fluorescent microspheres prepared in example 1.
FIG. 2 is a fluorescence emission spectrum of the streptavidin-modified AIE fluorescent microsphere emulsion prepared in example 1.
FIG. 3 is a diagram showing the test strip effect of streptavidin-modified AIE fluorescent microspheres prepared in example 1, comparative example 1 and comparative example 2.
Detailed Description
The invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto.
The invention uses an immunochromatographic test strip to test the effect of streptavidin modified AIE fluorescent microspheres, the test strip comprises a bottom plate, a sample pad, a nitrocellulose membrane and a water absorption pad, a detection line parallel to the end face of the bottom plate is coated on the exposed nitrocellulose membrane between the end part of the sample pad and the end part of the water absorption pad, an anti-streptavidin antibody is coated on the detection line, self-made streptavidin modified AIE fluorescent microspheres are dripped into the sample pad, and a dry type fluorescence immunoassay instrument is used for detecting a fluorescence signal value.
Example 1
0.4g of sodium lauryl sulfate was weighed out and completely dissolved in 12.5 g of ultrapure water, and used as an aqueous phase solution of a continuous phase. 0.05g of the AIE molecule of formula (1) and 0.06g of n-hexadecane were weighed out and dissolved in 0.1g of methacrylic acid and 1g of styrene to obtain an oil phase solution.
Adding the aqueous phase solution into the oil phase solution, stirring and mixing, performing ultrasonic treatment for 8min with the power of 300W to obtain a monomer miniemulsion, raising the temperature to 70 ℃, adding 0.02g of potassium persulfate, and reacting to obtain the AIE fluorescent microsphere emulsion with the carboxyl modified surface. And centrifuging the AIE fluorescent microspheres at the rotating speed of 12000rpm, then suspending the AIE fluorescent microspheres in water, and circulating the AIE fluorescent microspheres for three times to obtain pure carboxyl modified AIE fluorescent microspheres.
The AIE fluorescent microspheres are added into 200g of MES buffer (pH = 6), 0.19g of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride is added after ultrasonic dispersion, and the mixture is placed into a mixer to activate carboxyl, the rotating speed is 300 rpm, and the time is 5 min. Centrifuging at 12000rpm to remove supernatant to obtain activated AIE fluorescent microsphere, adding 60g boric acid buffer solution (pH = 8) and 0.05g streptavidin, placing into a mixer, incubating at 300 rpm for 5min, centrifuging at 12000rpm for 30min to obtain supernatant, adding blocking solution bovine serum albumin and preservation solution, ultrasonically dispersing, and preserving at 2-8 deg.C.
The spectrum of the streptavidin-modified AIE fluorescent microspheres is tested by using a microplate reader, the excitation wavelength of the streptavidin-modified AIE fluorescent microspheres is 400nm, the emission wavelength of the streptavidin-modified AIE fluorescent microspheres is 525nm, and green fluorescence is shown under the excitation of a 365nm ultraviolet lamp (shown in figure 2). The morphology of the streptavidin-modified AIE fluorescent microspheres is observed by using a field emission scanning electron microscope, the microspheres are spherical structures, and the statistical particle size is 200nm (shown in figure 1). The anti-streptavidin antibody test strip is used as a detection model, self-made streptavidin modified AIE fluorescent microspheres are dropped into the test strip, the detection line shows bright green fluorescence (as shown in figure 3A), and a fluorescence signal value is detected by using a dry-type fluorescence immunoassay instrument, and the result is 12901.
Comparative example 1
The carboxyl modified AIE polymer fluorescent microsphere is prepared by adopting the same formula and preparation conditions as example 1.
The AIE fluorescent microspheres are added into 200g of MES buffer (pH = 6), 0.19g of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride is added after ultrasonic dispersion, and the mixture is placed into a mixer to activate carboxyl, the rotating speed is 300 rpm, and the time is 5 min. Centrifuging at 12000rpm to remove supernatant to obtain activated AIE fluorescent microsphere, adding 60g boric acid buffer (pH = 8) and 0.05g bovine serum albumin, placing in a mixer, incubating at 300 rpm for 5min, centrifuging at 12000rpm for 30min to obtain supernatant, adding preservation solution, ultrasonically dispersing, and preserving at 2-8 deg.C.
The spectrum of the AIE fluorescent microspheres modified by the bovine serum albumin is tested by using a microplate reader, the excitation wavelength of the AIE fluorescent microspheres is 400nm, the emission wavelength of the AIE fluorescent microspheres is 525nm, and green fluorescence is presented under the excitation of a 365nm ultraviolet lamp. The morphology of the AIE fluorescent microspheres modified by the bovine serum albumin is observed by using a field emission scanning electron microscope, the microspheres are spherical structures, and the statistical particle size is 200 nm. The anti-streptavidin antibody test strip is used as a detection model, self-made bovine serum albumin modified AIE fluorescent microspheres are dripped in, due to the fact that bovine serum albumin and anti-streptavidin antibody are not specifically combined, the detection line shows weak green fluorescence (as shown in figure 3B), a dry-type fluorescence immunoassay instrument is used for detecting a fluorescence signal value, and the result is 2519.
Comparative example 2
The carboxyl modified AIE polymer fluorescent microsphere is prepared by adopting the same formula and preparation conditions as example 1.
The AIE fluorescent microspheres are added into 200g of MES buffer (pH = 6), 0.19g of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride is added after ultrasonic dispersion, and the mixture is placed into a mixer to activate carboxyl, the rotating speed is 300 rpm, and the time is 5 min. Centrifuging at 12000rpm to remove supernatant to obtain activated AIE fluorescent microsphere, adding 60g boric acid buffer solution (pH = 4) and 0.05g streptavidin, placing into a mixer, incubating at 300 rpm for 5min, centrifuging at 12000rpm for 30min to obtain supernatant, adding blocking solution bovine serum albumin and preservation solution, ultrasonically dispersing, and preserving at 2-8 deg.C.
And (3) testing the spectrum of the streptavidin modified AIE fluorescent microsphere by using a microplate reader, wherein the excitation wavelength is 400nm, the emission wavelength is 525nm, and green fluorescence is presented under the excitation of a 365nm ultraviolet lamp. And observing the morphology of the streptavidin modified AIE fluorescent microspheres by using a field emission scanning electron microscope, wherein the morphology of the microspheres is of a spherical structure, and the statistical particle size is 200 nm. The anti-streptavidin antibody test strip is used as a detection model, self-made streptavidin-modified AIE fluorescent microspheres are dropped into the test strip, the streptavidin-modified AIE fluorescent microspheres fail due to the fact that the pH value of a coupling reaction system is too low, the detection line shows weak green fluorescence (as shown in figure 3C), a dry-type fluorescence immunoassay instrument is used for detecting a fluorescence signal value, and the result is 3519.
Example 2
0.15g of Tween 20 was weighed out and completely dissolved in 60g of ultrapure water, and used as an aqueous phase solution of a continuous phase. 0.1g of the AIE molecule of formula (II) and 0.2g of n-hexadecane were weighed out and dissolved in 0.5g of methacrylic acid and 2.5g of methyl methacrylate to obtain an oil phase solution.
Adding the aqueous phase solution into the oil phase solution, stirring and mixing, performing ultrasonic treatment for 10min by using 500W power to obtain a monomer miniemulsion, raising the temperature to 70 ℃, adding 0.05g of azodiisobutyl amidine hydrochloride, and reacting to obtain the AIE fluorescent microsphere emulsion with the carboxyl modified on the surface. And centrifuging the AIE fluorescent microspheres at the rotating speed of 12000rpm, then suspending the AIE fluorescent microspheres in water, and circulating the AIE fluorescent microspheres for three times to obtain pure carboxyl modified AIE fluorescent microspheres.
The AIE fluorescent microspheres were added to 250g mes buffer (pH = 6), ultrasonically dispersed, followed by addition of 0.55g1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, and placed in a mixer to activate the carboxyl groups. Activating the AIE fluorescent microspheres by using a rotation speed of 12000rpm in a centrifugal mode, adding 100g of boric acid buffer solution (pH = 8) and 0.06g of streptavidin, placing the mixture into a mixer for incubation, using a rotation speed of 12000rpm for centrifugation for 30min, taking supernatant, adding blocking solution bovine serum albumin and preservation solution, performing ultrasonic dispersion, and preserving at the temperature of 2-8 ℃.
And testing the spectrum of the streptavidin modified AIE fluorescent microsphere by using a microplate reader, wherein the excitation wavelength is 365nm, the emission wavelength is 415nm, and the dark blue fluorescence is shown under the excitation of a 365nm ultraviolet lamp. And observing the morphology of the streptavidin modified AIE fluorescent microspheres by using a field emission scanning electron microscope, wherein the morphology of the microspheres is of a spherical structure, and the statistical particle size is 300 nm. The anti-streptavidin antibody test strip is used as a detection model, self-made streptavidin modified AIE fluorescent microspheres are dripped into the anti-streptavidin antibody test strip, bright dark blue fluorescence is displayed in the detection line, a dry fluorescence immunoassay instrument is used for detecting a fluorescence signal value, and the result is 13407.
Example 3
2.5g of sodium dodecylbenzenesulfonate was weighed out and completely dissolved in 125g of ultrapure water, and used as an aqueous phase solution of the continuous phase. 0.3g of the AIE molecule of the formula (III) and 0.6g of co-stabilizer were weighed out and dissolved in 2g of itaconic acid and 12g of methyl methacrylate to obtain an oil phase solution.
Adding the aqueous phase solution into the oil phase solution, stirring and mixing, performing ultrasonic treatment for 20min with 600W power to obtain a monomer miniemulsion, raising the temperature to 72 ℃, adding 0.1g of potassium persulfate, and reacting to obtain the AIE fluorescent microsphere emulsion with the carboxyl modified surface. And centrifuging the AIE fluorescent microspheres at the rotating speed of 12000rpm, then suspending the AIE fluorescent microspheres in water, and circulating the AIE fluorescent microspheres for three times to obtain pure carboxyl modified AIE fluorescent microspheres.
The AIE fluorescent microspheres are added into 300 g of glycine-hydrochloric acid buffer (pH = 6), ultrasonically dispersed, added with 2.4g of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, and placed into a mixer to activate carboxyl groups. The AIE fluorescent microspheres are centrifugally activated at the rotating speed of 12000rpm, 120 g of boric acid-borax buffer solution (pH = 8) and 0.08g of streptavidin are added, the mixture is placed into a mixer for incubation, the mixture is centrifuged at the rotating speed of 12000rpm for 30min, supernatant is taken, blocking solution bovine serum albumin and preservation solution are added, ultrasonic dispersion is carried out, and the mixture is preserved at the temperature of 2-8 ℃.
And testing the spectrum of the streptavidin modified AIE fluorescent microspheres by using a microplate reader, wherein the excitation wavelength is 510nm, the emission wavelength is 610nm, and red fluorescence is presented under the excitation of a 365nm ultraviolet lamp. And observing the morphology of the streptavidin modified AIE fluorescent microspheres by using a field emission scanning electron microscope, wherein the morphology of the microspheres is of a spherical structure, and the statistical particle size is 400 nm. The anti-streptavidin antibody test strip is used as a detection model, self-made streptavidin modified AIE fluorescent microspheres are dripped into the anti-streptavidin antibody test strip, bright red fluorescence is displayed in the detection line, and a dry fluorescence immunoassay instrument is used for detecting a fluorescence signal value, so that the result is 12709.
Example 4
5g of sodium lauryl sulfate was weighed out and completely dissolved in 250g of ultrapure water, and used as an aqueous phase solution of a continuous phase. 0.4g of the AIE molecule of formula (I) and 1.5g of n-hexadecane were weighed out and dissolved in 5g of methacrylic acid and 30g of methyl methacrylate to obtain an oil phase solution.
Adding the aqueous phase solution into the oil phase solution, stirring and mixing, performing ultrasonic treatment for 9min with 300W power to obtain a monomer miniemulsion, raising the temperature to 70 ℃, adding 0.15g of potassium persulfate, and reacting to obtain the AIE fluorescent microsphere emulsion with the carboxyl modified surface. And centrifuging the AIE fluorescent microspheres at the rotating speed of 12000rpm, then suspending the AIE fluorescent microspheres in water, and circulating the AIE fluorescent microspheres for three times to obtain pure carboxyl modified AIE fluorescent microspheres.
The AIE fluorescent microspheres are added into 400 g phthalic acid-hydrochloric acid buffer solution (pH = 6), after ultrasonic dispersion, 3.5g1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride is added, and the mixture is placed into a mixer to activate carboxyl groups. The AIE fluorescent microspheres are centrifugally activated at 12000rpm, 135g of boric acid buffer solution (pH = 8) and 0.09g of streptavidin are added, the mixture is placed into a mixer for incubation, centrifugation is carried out at 12000rpm for 30min, supernatant is taken, blocking solution bovine serum albumin and preservation solution are added, ultrasonic dispersion is carried out, and the mixture is preserved at 2-8 ℃.
And testing the spectrum of the streptavidin modified AIE fluorescent microsphere by using a microplate reader, wherein the excitation wavelength is 365nm, the emission wavelength is 415nm, and the dark blue fluorescence is shown under the excitation of a 365nm ultraviolet lamp. And observing the morphology of the streptavidin modified AIE fluorescent microspheres by using a field emission scanning electron microscope, wherein the morphology of the microspheres is of a spherical structure, and the statistical particle size is 500 nm. The anti-streptavidin antibody test strip is used as a detection model, self-made streptavidin modified AIE fluorescent microspheres are dripped into the anti-streptavidin antibody test strip, bright deep blue fluorescence is displayed in the detection line, a dry-type fluorescence immunoassay instrument is used for detecting a fluorescence signal value, and the result is 11007.
Example 5
7.5g of polyvinyl alcohol was weighed out and completely dissolved in 125g of ultrapure water, and used as an aqueous phase solution of the continuous phase. 0.2g of the AIE molecule of formula (II) and 0.3g of n-hexadecane were weighed out and dissolved in 10g of methacrylic acid and 125g of methyl methacrylate to obtain an oil phase solution.
And adding the water phase solution into the oil phase solution, stirring and mixing, carrying out ultrasonic treatment for 15min by using 400W power to obtain a monomer miniemulsion, raising the temperature to 70 ℃, adding 0.25g of azodiisobutyl amidine hydrochloride, and reacting to obtain the AIE fluorescent microsphere emulsion with the carboxyl modified on the surface. And centrifuging the AIE fluorescent microspheres at the rotating speed of 12000rpm, then suspending the AIE fluorescent microspheres in water, and circulating the AIE fluorescent microspheres for three times to obtain pure carboxyl modified AIE fluorescent microspheres.
The AIE fluorescent microspheres are added into 100g of disodium hydrogen phosphate-citric acid buffer (pH = 6), ultrasonic dispersion is carried out, 4.5g of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride is added, and the mixture is placed into a mixer to activate carboxyl. Activating the AIE fluorescent microspheres by using a rotation speed of 12000rpm in a centrifugal mode, adding 100g of boric acid buffer solution (pH = 8) and 0.12g of streptavidin, placing the mixture into a mixer for incubation, using a rotation speed of 12000rpm for centrifugation for 30min, taking supernatant, adding blocking solution bovine serum albumin and preservation solution, performing ultrasonic dispersion, and preserving at the temperature of 2-8 ℃.
And (3) testing the spectrum of the streptavidin modified AIE fluorescent microsphere by using a microplate reader, wherein the excitation wavelength is 400nm, the emission wavelength is 525nm, and green fluorescence is presented under the excitation of a 365nm ultraviolet lamp. And observing the morphology of the streptavidin modified AIE fluorescent microspheres by using a field emission scanning electron microscope, wherein the morphology of the microspheres is of a spherical structure, and the statistical particle size is 300 nm. An experimental structure of an isothermal calorimetry titration method shows that streptavidin is successfully modified on the surface of the AIE fluorescent microsphere. The anti-streptavidin antibody test strip is used as a detection model, self-made streptavidin modified AIE fluorescent microspheres are dripped in, the detection line shows bright green fluorescence, a dry-type fluorescence immunoassay instrument is used for detecting a fluorescence signal value, and the result is 13098.
Example 6
4.8g of sodium lauryl sulfate was weighed out and completely dissolved in 125g of ultrapure water, and used as an aqueous phase solution of a continuous phase. 0.15g of the AIE molecule of formula (III) and 0.5g of n-hexadecane were weighed out and dissolved in 3.5g of methacrylic acid and 15g of methyl methacrylate to obtain an oil phase solution.
And adding the water phase solution into the oil phase solution, stirring and mixing, performing ultrasonic treatment for 30min by using 400W power to obtain a monomer miniemulsion, raising the temperature to 65 ℃, adding 0.1g of azodiisobutyl amidine hydrochloride, and reacting to obtain the AIE fluorescent microsphere emulsion with the carboxyl modified on the surface. And centrifuging the AIE fluorescent microspheres at the rotating speed of 12000rpm, then suspending the AIE fluorescent microspheres in water, and circulating the AIE fluorescent microspheres for three times to obtain pure carboxyl modified AIE fluorescent microspheres.
The AIE fluorescent microspheres were added to 150g MES buffer (pH = 6), ultrasonically dispersed, and 5.5g1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride was added thereto, followed by activating the carboxyl group in a mixer. Activating the AIE fluorescent microspheres by using a rotation speed of 12000rpm in a centrifugal mode, adding 100g of boric acid buffer solution (pH = 8) and 0.14g of streptavidin, placing the mixture into a mixer for incubation, using a rotation speed of 12000rpm for centrifugation for 30min, taking supernatant, adding blocking solution bovine serum albumin and preservation solution, performing ultrasonic dispersion, and preserving at the temperature of 2-8 ℃.
And testing the spectrum of the streptavidin modified AIE fluorescent microspheres by using a microplate reader, wherein the excitation wavelength is 510nm, the emission wavelength is 610nm, and red fluorescence is presented under the excitation of a 365nm ultraviolet lamp. And observing the morphology of the streptavidin modified AIE fluorescent microspheres by using a field emission scanning electron microscope, wherein the morphology of the microspheres is of a spherical structure, and the statistical particle size is 100 nm. An experimental structure of an isothermal calorimetry titration method shows that streptavidin is successfully modified on the surface of the AIE fluorescent microsphere. The anti-streptavidin antibody test strip is used as a detection model, self-made streptavidin modified AIE fluorescent microspheres are dripped into the anti-streptavidin antibody test strip, bright green fluorescence is displayed in the detection line, a dry-type fluorescence immunoassay instrument is used for detecting a fluorescence signal value, and the result is 14097.
Example 7
4g of sodium dodecylbenzenesulfonate was weighed out and completely dissolved in 125g of ultrapure water, and used as an aqueous phase solution of the continuous phase. 0.2g of the AIE molecule of formula (II) and 0.4g of n-hexadecane were weighed out and dissolved in 4g of methacrylic acid and 40g of styrene ester to obtain an oil phase solution.
Adding the aqueous phase solution into the oil phase solution, stirring and mixing, performing ultrasonic treatment for 10min with 400W power to obtain a monomer miniemulsion, raising the temperature to 70 ℃, adding 0.1g of potassium persulfate, and reacting to obtain the AIE fluorescent microsphere emulsion with the carboxyl modified surface. And centrifuging the AIE fluorescent microspheres at the rotating speed of 12000rpm, then suspending the AIE fluorescent microspheres in water, and circulating the AIE fluorescent microspheres for three times to obtain pure carboxyl modified AIE fluorescent microspheres.
The AIE fluorescent microspheres were added to 150g MES buffer (pH = 6), ultrasonically dispersed, and after adding 4.5g1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, the carboxyl groups were activated by placing in a mixer. Activating the AIE fluorescent microspheres by using a rotation speed of 12000rpm in a centrifugal mode, adding 100g of boric acid buffer solution (pH = 8) and 0.35g of streptavidin, placing the mixture into a mixer for incubation, using a rotation speed of 12000rpm for centrifugation for 30min, taking supernatant, adding blocking solution bovine serum albumin and preservation solution, performing ultrasonic dispersion, and preserving at the temperature of 2-8 ℃.
And (3) testing the spectrum of the streptavidin modified AIE fluorescent microsphere by using a microplate reader, wherein the excitation wavelength is 400nm, the emission wavelength is 525nm, and green fluorescence is presented under the excitation of a 365nm ultraviolet lamp. And observing the morphology of the streptavidin modified AIE fluorescent microspheres by using a field emission scanning electron microscope, wherein the morphology of the microspheres is of a spherical structure, and the statistical particle size is 50 nm. An experimental structure of an isothermal calorimetry titration method shows that streptavidin is successfully modified on the surface of the AIE fluorescent microsphere. The anti-streptavidin antibody test strip is used as a detection model, self-made streptavidin modified AIE fluorescent microspheres are dripped into the anti-streptavidin antibody test strip, bright green fluorescence is displayed in the detection line, a dry-type fluorescence immunoassay instrument is used for detecting a fluorescence signal value, and the result is 12309.
The above-described embodiments of the invention are intended to be illustrative of the invention and are not to be construed as limiting the invention, and any variations that fall within the meaning and scope of the invention equivalent to the claims are intended to be embraced therein.
Claims (8)
1. A preparation method of streptavidin modified aggregation-induced emission polymer microspheres is characterized by comprising the following steps:
(1) dissolving an emulsifier in water to serve as an aqueous emulsifier solution for the continuous phase;
(2) dissolving AIE molecules and an auxiliary stabilizer in a mixed solution of a carboxyl functional monomer and a hydrophobic monomer to obtain an oil phase solution;
(3) adding the emulsifier aqueous solution in the step (1) into the oil phase solution in the step (2), performing ultrasonic treatment to obtain a monomer miniemulsion, raising the temperature, adding a water-soluble initiator, and reacting to obtain the AIE fluorescent microspheres with surface carboxyl modified;
(4) adding the AIE fluorescent microspheres prepared in the step (3) into an acid buffer solution, adding a carbodiimide condensation reagent after ultrasonic dispersion, putting the mixture into a mixer to activate carboxyl, and centrifuging to remove supernatant so as to obtain activated fluorescent microspheres;
(5) adding an alkaline buffer solution and streptavidin into the fluorescent microspheres activated in the step (4), placing the fluorescent microspheres into a mixer for incubation, centrifuging to obtain a supernatant, adding a confining liquid and a preservation liquid, and performing ultrasonic dispersion to obtain streptavidin-modified aggregation-induced luminescent polymer microspheres;
in the step (2), the mass usage amount of the AIE molecules is 0.05-20% of the total mass of the monomers; the mass consumption of the co-stabilizer is 0.01-8% of the total mass of the monomers; the mass usage of the carboxyl functional monomer is 5-50% of the total mass usage of the monomer; the mass usage of the hydrophobic monomer is 50-95% of the total mass usage of the monomers; the total mass of the monomers refers to the total mass of the carboxyl functional monomers and the hydrophobic monomers;
the AIE molecule of step (2) is selected from at least one of formula (I), formula (II) and formula (III):
the co-stabilizer is selected from at least one of the following: aliphatic hydrocarbon of C14~ C22, aliphatic alcohol of C14~ C22;
the carboxyl functional monomer is at least one of acrylic acid and methacrylic acid shown in a formula (IV);
in the formula (IV), R1Is H or CH3；
The hydrophobic monomer is selected from at least one of the following: styrene of formula (V), an acrylate or methacrylate monomer compound of formula (VI);
in the formula (VI), R7Is H or methyl; r8Is C1-C5 alkyl;
in the step (4), the acidic buffer solution is glycine-hydrochloric acid buffer solution, phthalic acid-hydrochloric acid buffer solution, disodium hydrogen phosphate-citric acid buffer solution, and 2- (N-morpholine) ethanesulfonic acid buffer solution; the pH value of the acidic buffer solution is 6.0-6.5; the mass usage of the carbodiimide condensation reagent is 100-400% of the usage of the carboxyl functional monomer, and the activation time is 5 min-3 h;
in the step (5), the mass usage of the streptavidin is 10% -200% of the usage of the carboxyl functional monomer;
in the step (5), the alkaline buffer solution is a tris-hydroxymethyl aminomethane-hydrochloric acid buffer solution, a boric acid buffer solution, or a phosphate buffer salt solution; the pH value of the alkaline buffer solution is 7.0-8.0.
2. The preparation method according to claim 1, wherein in the step (1), the mass amount of the emulsifier is 0.01-20% of the mass amount of water; the emulsifier is selected from at least one of the following: sodium dodecyl sulfate, polyvinyl alcohol, tween 20 and sodium dodecyl benzene sulfonate.
3. The production method according to claim 1,
the mass usage of the AIE molecules is 0.1-4% of the total mass usage of the monomers;
the mass usage of the co-stabilizer is 1-6% of the total mass usage of the monomers;
the mass usage of the carboxyl functional monomer is 10-30% of the total mass usage of the monomer;
the mass usage of the hydrophobic monomer is 70-90% of the total mass usage of the monomers.
4. The preparation method according to claim 1, wherein in the step (3), the power of the ultrasonic treatment is 100W-500W, and the time of the ultrasonic treatment is 0.5 min-60 min; the reaction temperature is 50-90 ℃, and the reaction time is 2-48 h;
in the step (3), the mass usage amount of the water-soluble initiator is 0.05-10% of the total mass of the monomers;
the total mass of the monomers is the total mass of the carboxyl functional monomer and the hydrophobic monomer;
in the step (3), the water-soluble initiator is selected from at least one of the following: persulfate, water-soluble azo, oxidant and reductant constitute redox system.
5. The production method according to claim 1, wherein in the step (4), the carbodiimide condensing agent is selected from the group consisting ofN, N' -dicyclohexylcarbodiimide,N, N’-one of diisopropylcarbodiimide and 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride;
the centrifugation time is 10-60min, the rotating speed is 8000-16000rpm, and the centrifugation frequency is more than three times.
6. The method according to claim 1, wherein in the step (5), the blocking solution is bovine serum albumin or glycine.
7. The streptavidin-modified aggregation-induced emission polymer microsphere prepared by the preparation method according to any one of claims 1 to 6.
8. Use of the streptavidin-modified aggregation-induced-luminescent polymer microsphere of claim 7 in the preparation of an in vitro diagnostic reagent.
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