CN114778819A - Multi-mode aggregation-induced fluorescence immunochromatographic test paper and preparation method thereof - Google Patents
Multi-mode aggregation-induced fluorescence immunochromatographic test paper and preparation method thereof Download PDFInfo
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Abstract
The invention discloses aggregation-induced fluorescence particles, a compound, multi-mode immunochromatographic test paper and a preparation method thereof. The aggregation-induced fluorescent particle is of a core-shell structure under a transmission electron microscope, has a particle size of 50-500nm, and mainly comprises an aggregation-induced fluorescent material and a polymer, wherein the aggregation-induced fluorescent material and the polymer have both color and fluorescence characteristics. The invention adopts a miniemulsion-solvent volatilization method to prepare the aggregation-induced fluorescent particles, the equipment and the process are simple, the prepared composite particles have uniform particle size, controllable conditions and good repeatability, the concentration of the aggregation-induced fluorescent material in the particles is easy to control, and most importantly, the obtained particles have two characteristics of color and fluorescence. The immunochromatographic test strip prepared by taking the particles as a signal carrier can be used for qualitative detection by naked eyes and quantitative detection by fluorescence.
Description
Technical Field
The invention belongs to the technical field of rapid immunodetection, and particularly relates to aggregation-induced fluorescent particles, a composition, multi-mode chromatography test paper and a preparation method thereof
Background
The immunochromatographic test paper has the advantages of simplicity, convenience, rapidness, low cost and the like, is a main technical means for rapid detection and analysis on site at present, and is widely applied to the technical fields of clinical diseases, food safety, environmental monitoring and the like. The basic principle is that a nitrocellulose membrane is used as a chromatography reaction membrane, an analyte to be detected in a sample and an antigen or an antibody fixed in a detection area on the membrane generate a specific immunoreaction, and the signal intensity of an immunological marker in the detection area is analyzed to realize the detection of the analyte to be detected. In the traditional immunochromatography, colloidal gold is used as a marker, and the detection result can be read by naked eyes without complex equipment and medical professionals. Although this detection method easily achieves qualitative or semi-quantitative results, immunochromatographic detection based on a colorimetric method is difficult to express in shade of color for a slight change in target concentration, and thus accurate quantification and high sensitivity cannot be achieved.
The quantitative performance of immunochromatographic assays can be improved by using fluorescent materials, including organic fluorophores or quantum dots, as labels to achieve higher signals, better contrast and lower background interference. However, the light absorption capacity of fluorescent materials is low, and thus fluorescent test strips generally lack visual detection ability. And the use of fluorescence immunochromatography detection is limited by relying on an additional excitation light source and a sensing device for signal reading. The shortcomings of colorimetric and fluorescent methods have prompted the emergence of a new universal detection method with independent strong visible/fluorescent dual signals, which is suitable for a range of application scenarios. Chongwen Wang et al reported a Polyethyleneimine (PEI) -mediated assembly method (Analytical Chemistry,2020,92(23): 15542-15549) as SiO2As a core, a shell layer formed by a layer of colloidal gold generates a strong colorimetric signal, a shell layer formed by a layer of carboxylated quantum dots provides a fluorescent signal and protein coupling surface sites, nanoparticles with good stability, high luminescence and good biocompatibility are prepared, and the immunochromatography test strip prepared by using the nanoparticles as a marker can realize detection in two modes of colorimetric-fluorescence. The defects are that the preparation process is complex, and the quantum dots have an aggregated fluorescence quenching phenomenon.
The traditional fluorescent material has an aggregate fluorescence quenching phenomenon (ACQ): the fluorescent material shows stronger fluorescence in dilute solution, but the luminous performance of the fluorescent material is often weakened or even completely disappeared when the fluorescent material is in high-concentration solution or is prepared into a solid state. While aggregation-induced emission (AIE) is a phenomenon that was discovered by the team of down loyal courtyards occasionally in 2001: the luminescence of the material is enhanced in an aggregation state, and a new idea is provided for solving the problem of fluorescence quenching of the traditional ACQ material; the advantages of the AIE materials for biomolecular labeling are mainly reflected in: low background, high signal-to-noise ratio, good sensitivity and the like. Weihua Lai et al (Biosensors and Bioelectronics,2019,135: 173-. The Li Kuh-Kuh university of Bekindskin in 2021 reports (ACS nano,2021,15(5): 8996-9004), and the method can reduce the background fluorescence interference of the sample by adopting the near infrared II-region AIE material to prepare the fluorescent microspheres for immunochromatography detection. In recent years, it has been reported that aggregation-inducing fluorescent materials also have a color visible to the naked eye, and are used for the development of organic photoelectric materials. However, the existing AIE material for preparing the fluorescent microsphere mainly utilizes the fluorescence characteristic of the AIE material to prepare the fluorescence immunochromatographic test paper, thereby limiting the application of the fluorescence immunochromatographic test paper in field naked eye detection.
Disclosure of Invention
The invention aims to solve the defects of the prior art and provides a multimode aggregation-induced fluorescent particle with good stability and high sensitivity, an aggregation-induced fluorescent particle-antibody compound, an immunochromatographic test strip coated with the aggregation-induced fluorescent particle-antibody compound and a preparation method thereof. The preparation method is simple, high in sensitivity, good in repeatability and environment-friendly.
In order to achieve the purpose, the invention adopts the following technical scheme:
an aggregation-induced fluorescent particle is of a core-shell structure under a transmission electron microscope, has a particle size of 50-500nm, and mainly comprises an aggregation-induced fluorescent material and a polymer, wherein the aggregation-induced fluorescent material and the polymer have both color and fluorescence characteristics. Preferably, the surface of the aggregation-inducing fluorescent particle further contains a carboxyl functional group.
Further, the aggregation-induced fluorescent material with both color and fluorescence characteristics is selected from one or more of a tetrastyrene AIE derivative, a silole AIE derivative, a 1, 4-stilbene AIE derivative, a five-membered heterocyclic AIE derivative and a furan AIE derivative.
Further, the polymer is selected from at least one of polystyrene-acrylic acid copolymer, polystyrene-maleic anhydride copolymer and polymethyl methacrylate-methacrylic acid copolymer.
Further, the molecular weight of the polymer is 1500-40000. Preferably, the molecular weight of the polymer is 1900-37000.
Further, the aggregation-inducing fluorescent particle has a carboxyl group on the surface thereof, the carboxyl group being derived from a polymer. The present invention also provides an aggregation-inducing fluorescent particle-antibody complex comprising the aggregation-inducing fluorescent particle and an antibody as described above.
The invention also provides an immunochromatographic test paper, wherein the coupling pad of the immunochromatographic test paper is coated with the aggregation-induced fluorescent particle-antibody complex.
The invention also provides a method for preparing the aggregation-induced fluorescence particle, which comprises the following specific steps: the oil phase comprises an aggregation-induced fluorescent material and a polymer which are dissolved in chloroform, wherein the concentration of the aggregation-induced fluorescent material is 1-20 mg/mL, and the concentration of the polymer is 5-100 mg/mL; the water phase is a surfactant aqueous solution with the mass concentration of 0.1-1%; mixing the oil phase and the water phase at a volume ratio of 1: 3-6 (namely the oil phase: the water phase: 1: 3-6), magnetically stirring for 1-10 minutes, and emulsifying for 1-10 minutes by using an ultrasonic probe to obtain a microemulsion; and removing the chloroform solvent solidified microemulsion liquid drop to obtain the aggregation-induced fluorescent particles.
Further, after the microemulsion liquid drops are solidified, the aggregation-induced fluorescent particles are centrifuged, and the precipitate is taken, washed by phosphate buffer solution and then dispersed in the phosphate buffer solution for later use.
Further, the ratio of the aggregation-inducing fluorescent material to the polymer in the oil phase is 1: 1-8.
Further, the concentration of the aggregation-induced fluorescent material in the oil phase is 2.5-5 mg/mL; the concentration of the polymer was 20 mg/mL.
Further, the surfactant is selected from at least one of sodium dodecyl sulfate, tween 20, tween 80 and triton X-100.
In another aspect, the present invention also provides a method of preparing the aggregation-induced fluorescent particle-antibody complex: dispersing the aggregation-induced fluorescent particles into a phosphate buffer solution with the pH value of 4-6, adding an activating agent, carrying out an activation reaction, centrifuging to remove a supernatant, dispersing the activated particles into the phosphate buffer solution, adding an antibody, carrying out an incubation reaction at room temperature, centrifuging to remove the supernatant, namely removing the free antibody, dispersing the precipitate particle-antibody conjugate into a confining liquid, and incubating to obtain the aggregation-induced fluorescent particle-antibody complex.
Further, the activating agent is 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC); the working concentration of EDC is 0.1-1 mg/mL.
Specifically, dispersing aggregation-induced fluorescent particles in a phosphate buffer solution with the pH value of 4-6, adding an activating agent 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride with the concentration of 0.1-1 mg/mL, performing an activation reaction for 20-40 minutes (preferably 30 minutes), centrifuging to remove a supernatant, dispersing the activated particles in the phosphate buffer solution, adding an antibody, performing incubation reaction for 1 hour at room temperature, centrifuging to remove free antibody, dispersing a precipitate particle-antibody conjugate in a confining liquid, and performing incubation for more than 2 hours (including 2 hours) at room temperature to obtain an aggregation-induced fluorescent particle-antibody complex.
Further, the aggregation-induced fluorescent particle-antibody complex obtained after blocking is added with Bovine Serum Albumin (BSA) with a mass concentration of 5% and stored for later use.
Further, the blocking solution is a casein solution. Preferably, the blocking solution is a casein solution with a concentration of 5 mg/mL.
The invention also provides an immunochromatographic test paper: comprises a sample pad, a coupling pad, a nitrocellulose membrane and absorbent paper which are sequentially overlapped on a bottom plate; wherein the coupling pad is coated with the aggregation-inducing fluorescent particle-antibody complex as described above.
Compared with the prior art, the invention has the following advantages:
1. compared with the existing immunochromatography detection method using colloidal gold as a marker, the method has the characteristics of higher sensitivity of aggregation-induced fluorescence chromatography detection, small batch-to-batch difference, more stable marker and the like.
2. Compared with the existing immunochromatography detection method using fluorescent materials including organic fluorophores or quantum dots as markers, the immunochromatography detection method has strong light absorption capacity and can be observed by naked eyes; the fluorescence intensity is high and is not easy to quench; high signal-to-noise ratio, good sensitivity and the like.
3. The invention adopts miniemulsion-solvent volatilization method to prepare the aggregation-induced fluorescent particles, compared with the particles prepared by electrostatic self-assembly method, the equipment and the process are simple, the prepared composite particles have uniform particle size, controllable conditions and good repeatability, and the material concentration in the particles is easy to control.
The conception, specific structure and technical effects of the present invention will be further described in conjunction with the accompanying drawings to fully understand the purpose, characteristics and effects of the present invention.
Drawings
FIG. 1 is a transmission electron micrograph of aggregation-induced fluorescent particles at different magnifications, 500nm on the large scale and 50 nm on the lower right small scale.
FIG. 2 shows the results of immunochromatography detection of C-reactive protein using aggregation-induced fluorescent particles having both color and fluorescence as a label, where a is the result under ultraviolet light excitation and b is the result under natural light.
FIG. 3 shows the results of detecting interleukin 6 by immunochromatography using time-resolved fluorescent microspheres as labels, where a is the result under natural light and b is the result under ultraviolet light excitation.
Detailed Description
The present invention will be further explained with reference to the drawings in order to make the technical means, inventive features, objectives and effects of the invention easy to understand. However, the present invention is not limited to the following embodiments.
It should be understood that the structures, ratios, sizes, and the like shown in the drawings and described in the specification are only used for matching the disclosure and are not used for limiting the practical limit of the present invention, so that the present invention is not limited to the essential technical meaning, and any modifications of the structures, changes of the ratio relationships, or adjustments of the sizes, should still fall within the scope of the technical disclosure of the present invention without affecting the efficacy and the achievable purpose of the present invention.
Example 1: the method for preparing aggregation-induced fluorescent particles and marking the C-reactive protein antibody comprises the following steps:
A. preparing an oil phase component: dispersing a tetrabenzoic AIE derivative and a polystyrene-maleic anhydride copolymer (molecular weight 1900, purchased from Sigma-Aldrich) in a chloroform solution, wherein the concentration of the tetrabenzoic AIE derivative is 10mg/mL, and the concentration of the polymer is 10 mg/mL;
B. preparing a water phase component: preparing aqueous solution from sodium dodecyl sulfate and Triton X-100, wherein the mass concentration of the sodium dodecyl sulfate is 0.1 percent, and the mass concentration of the Triton X-100 is 0.5 percent;
C. adding the oil phase into the water phase solution, magnetically stirring until the mixture is uniform, and then carrying out ultrasonic treatment on the mixed solution for 2 minutes by using an ultrasonic crusher to obtain uniform and stable microemulsion;
D. placing the microemulsion in an open container at room temperature, gradually volatilizing the solvent under magnetic stirring for 16 hours, centrifugally washing, and ultrasonically dispersing precipitates to obtain material-polymer composite particles; the electron micrograph is shown in figure 1, and the emulsion micrograph is shown in figure 2;
E. activating carboxyl groups on the surface of the particles: the particles were dispersed in phosphate-HCl phosphate buffer pH4.0 at a particle concentration of 20mg/mL, followed by addition of 1-ethyl- (3-dimethylaminopropyl) carbodiimide (EDC) at a concentration of 1.0mmol/L and incubation at room temperature for 30 min;
F. centrifuging to remove supernatant after incubation is finished, adding a precipitate into a phosphate buffer solution for dispersion, then adding a C-reactive protein antibody (purchased from New Albizzia technologies Co., Ltd.) into the phosphate buffer solution, wherein the concentration of the C-reactive protein antibody is 30 mu g/ml, then adding an aggregation-induced fluorescent particle solution into the C-reactive protein antibody solution, and incubating for 30 minutes at room temperature;
G. centrifuging after incubation to remove redundant antibodies, adding casein for ultrasonic dispersion, wherein the concentration is 5mg/mL, and sealing (incubating) at room temperature for more than 2 hours;
H. and adding BSA with the mass concentration of 5% after blocking to obtain the aggregation-induced fluorescent particles coupled with the C-reactive protein antibody.
Example 2: preparation of aggregation-induced fluorescent particles and labeling of goat anti-rabbit immunoglobulin (IgG) antibodies:
A. preparing oil phase components: dispersing a tetrabenzonitrile-AIE derivative and a polystyrene-acrylic acid copolymer (molecular weight 37000, purchased from Sigma-Aldrich) in a chloroform solution, wherein the concentration of the tetrabenzonitrile-AIE derivative is 10mg/mL, and the concentration of the polymer is 10 mg/mL;
B. preparing a water phase component, namely preparing sodium dodecyl sulfate into an aqueous solution, wherein the mass concentration of the sodium dodecyl sulfate is 0.5%;
C. adding the oil phase into the aqueous phase solution, magnetically stirring until the mixture is uniform, and then carrying out ultrasonic treatment on the mixed solution for 5 minutes by using an ultrasonic crusher to obtain uniform and stable microemulsion;
D. placing the microemulsion in an open container at room temperature, gradually volatilizing the solvent under magnetic stirring for 24 hours, centrifugally washing, and ultrasonically dispersing the precipitate with water to obtain material-polymer composite particles; the electron micrograph is shown in FIG. 1, and the emulsion micrograph is shown in FIG. 2;
E. activating carboxyl groups on the surface of the particles: the particles were dispersed in phosphate buffer pH4.0 at a particle concentration of 20mg/mL, followed by addition of 1-ethyl- (3-dimethylaminopropyl) carbodiimide (EDC) at a concentration of 1.0mmol/L and incubation at room temperature for 30 minutes;
F. centrifuging to remove supernatant after incubation is finished, adding phosphate buffer solution into the precipitate for dispersion, then adding a goat anti-rabbit IgG polyclonal antibody (purchased from Hangzhou Longji BioLtd.) into the phosphate buffer solution, wherein the concentration of the goat anti-rabbit IgG antibody is 50ug/ml, then adding the aggregation-induced fluorescent particle solution into the goat anti-rabbit antibody solution, and incubating for 30 minutes at room temperature;
G. centrifuging after incubation to remove redundant antibodies, adding casein for ultrasonic dispersion, wherein the concentration is 5mg/mL, and sealing at room temperature for more than 24 hours;
H. and adding BSA with the mass concentration of 5% for storage after blocking, thus obtaining the aggregation-induced particles coupled with the goat anti-rabbit IgG antibody.
Example 3: c-reactive protein sample detection
The aggregation-induced fluorescent particles labeled with the C-reactive protein antibody in example 1 (i.e., aggregation-induced fluorescent particle-C-reactive protein antibody complex) and the aggregation-induced particles labeled with the goat anti-rabbit IgG antibody in example 2 (i.e., aggregation-induced fluorescent particle-goat anti-rabbit IgG antibody complex) were used to prepare a highly sensitive C-reactive protein aggregation-induced fluorescent particle multi-mode immunochromatographic test strip, which was prepared by the following steps:
coating the aggregation-induced fluorescent particle-C reactive protein antibody compound and the aggregation-induced fluorescent particle-goat anti-rabbit IgG antibody compound on a coupling pad for later use; respectively regulating the concentration of another antibody of the C-reactive protein strain and the concentration of the anti-rabbit antibody to be 1.0mg/mL by Phosphate Buffered Saline (PBS);
spraying another antibody of the C-reactive protein on the lower part of a nitrocellulose membrane as a detection line (T line), spraying rabbit IgG on the upper part of the nitrocellulose membrane as a quality control line (C line), scribing at a membrane speed of 1 mu L/cm, and drying at 37 ℃ for later use;
and finally, overlapping the sample pad, the coupling pad, the nitrocellulose membrane and the absorbent paper on the bottom plate, and cutting into a proper width by using a knife to obtain the C-reactive protein aggregation-induced fluorescent particle multi-mode immunochromatography test paper.
The detection principle and the detection process are as follows:
the detection principle is as follows: when a positive sample is detected by a sandwich method, the marker conjugate antibody and antibodies marked by a T line are two antibodies aiming at different sites of the same antigen, the antigen to be detected is firstly combined with the marker conjugated with the corresponding antibody, and thus, when an antigen-antibody complex passes through the T line, the antigen-antibody complex is combined with the other antibody on the T line to form an antibody-antigen-antibody sandwich structure, so that the color development of the T line of the positive sample is detected. In contrast, in the negative sample, no antigen to be detected is bound to the antibody on the marker, so that the marker cannot be bound to the antibody on the T line when passing through the T line, thereby causing no color development of the T line.
And (3) detection process: 5 mul of serum/plasma sample (or 8.5 mul of whole blood sample) is aspirated, the sample is added into phosphate buffer, and the mixture is fully mixed for 30 s-1 min. Sucking 75 μ L of the solution with a pipette, adding into a sample pad of immunochromatographic test paper, waiting for sample addition reaction for 2-8 min, observing the test paper result with naked eyes (FIG. 2a) or scanning the test paper fluorescence intensity with a laser fluorescence reader to calculate the quantitative result (fluorescence excitation: FIG. 2b)
In the result, the upper line of the test paper strip is a quality control line (C line), and the lower line of the test paper strip is a detection line (T line). Because the C reactive protein is a macromolecular antigen and a sandwich method is adopted for detection, the color of the T line is darker and darker along with the increase of the detection concentration. And the rabbit IgG fixed by the independent C line and the aggregation-induced fluorescent particles marked with the goat anti-rabbit IgG antibody perform an immune binding reaction, so that the color of the C line is kept unchanged.
Comparative example 1: method for labeling interleukin 6 antibody by time-resolved fluorescent microspheres
A. Dispersing the purchased time-resolved fluorescent microsphere finished product in phosphate buffer solution with the pH value of 4.0, wherein the particle concentration is 20mg/mL, then adding 1-ethyl- (3-dimethylaminopropyl) carbodiimide (EDC) with the concentration of 1.0mmol/L, and incubating for 30 minutes at room temperature;
B. centrifuging to remove supernatant after incubation is finished, adding a phosphate buffer solution into the precipitate for dispersion, then adding the interleukin 6 antibody into the phosphate buffer solution, wherein the concentration of the antibody is 50ug/ml, then adding the time-resolved fluorescent microspheres into the interleukin 6 antibody solution, and incubating for 30 minutes at room temperature;
C. centrifuging after incubation to remove redundant antibodies, adding casein for ultrasonic dispersion, wherein the concentration is 5mg/mL, and sealing at room temperature for more than 24 hours;
D. BSA (purchased from national chemical group, Inc.) with the mass concentration of 5 percent of the preservation solution is added after blocking, and the time-resolved fluorescent microsphere coupled with the interleukin 6 antibody is obtained.
Comparative example 2: detection of Interleukin 6 samples
The interleukin 6 immunochromatographic test paper with high sensitivity is prepared by using the interleukin 6 antibody-labeled time-resolved fluorescent microspheres (i.e. time-resolved fluorescent microsphere-interleukin 6 antibody compound) obtained in the comparative example 1, and the test paper preparation steps are as follows:
coating the time-resolved microsphere-interleukin 6 antibody compound on a coupling pad for later use;
adjusting the concentration of the other antibody strain of interleukin 6 to 1.0mg/mL by using PBS solution;
spraying another antibody of interleukin 6 on the lower part of the nitrocellulose membrane as a detection line, and drying at 37 ℃ for later use, wherein the film scratching speed is 0.5 mu L/cm;
and finally, overlapping the sample pad, the coupling pad, the nitrocellulose membrane and the absorbent paper on the bottom plate, and cutting into a proper width by using a knife to obtain the interleukin 6 immunochromatographic test paper.
The detection principle is the same as that of example 3.
And (3) detection process: and adding 100 mu L of interleukin 6 quality control product onto a sample pad of the test strip, waiting for sample adding reaction for 2-8 minutes, and observing the test strip result with naked eyes (figure 3a) or scanning the fluorescence intensity of the test strip by using a laser fluorescence reader to calculate a quantitative result (fluorescence excitation result: figure 3 b).
As a result, the lower line of the test strip was a test line (T line), and a quality control line (C line) was not provided since it was a comparison of example 3. Because interleukin 6 is a macromolecular antigen and a sandwich method is adopted for detection, the color of the T line is darker and darker along with the increase of the detection concentration. Unlike the detection results observed in both modes in example 3, in comparative example 2, the detection results were observed only under excitation of excitation light, and the detection results were not observed with the naked eye.
The foregoing detailed description of the preferred embodiments of the invention has been presented. It should be understood that numerous modifications and variations could be devised by those skilled in the art in light of the present teachings without departing from the inventive concept. Therefore, the technical solutions available to those skilled in the art through logic analysis, reasoning and limited experiments based on the prior art according to the concept of the present invention should be within the scope of protection defined by the claims.
Claims (9)
1. An aggregation-induced fluorescent particle comprising an aggregation-induced fluorescent material having both color and fluorescent properties and a polymer; the aggregation-induced fluorescent material with both color and fluorescence characteristics is one or more of a tetrastyrene AIE derivative, a silole AIE derivative, a 1, 4-stilbene AIE derivative, a five-membered heterocyclic AIE derivative and a furan AIE derivative; the polymer is one or more of polystyrene-acrylic acid copolymer, polystyrene-maleic anhydride copolymer and polymethyl methacrylate-methacrylic acid copolymer.
2. The aggregation-induced fluorescent particle of claim 1, wherein the aggregation-induced fluorescent particle comprises carboxyl functional groups on the surface.
3. An aggregation-inducing fluorescent particle-antibody complex comprising the aggregation-inducing fluorescent particle according to claim 1 or 2.
4. An immunochromatographic test strip, wherein a coupling pad of the immunochromatographic test strip is coated with the aggregation-inducing fluorescent particle-antibody complex of claim 3.
5. A method for preparing the aggregation-induced fluorescent particle of claim 1 or 2, comprising the specific steps of: the oil phase comprises the aggregation-induced fluorescent material and the polymer dissolved in chloroform, wherein the concentration of the aggregation-induced fluorescent material is 1-20 mg/mL, and the concentration of the polymer is 5-100 mg/mL; the water phase is a surfactant aqueous solution with the mass concentration of 0.1-1%; the oil phase/water phase mixing volume ratio is 1: 3-6, and the microemulsion is obtained by emulsifying the mixture through an ultrasonic probe after magnetic stirring; and removing the chloroform solvent solidified microemulsion liquid drop to obtain the aggregation-induced fluorescent particles.
6. The method of claim 5, wherein the ratio of aggregation-inducing fluorescent material to polymer in the oil phase is 1: 1 to 8.
7. The method of claim 6, wherein the concentration of the aggregation-inducing fluorescent material in the oil phase is 2.5 to 5 mg/mL; the concentration of the polymer was 20 mg/mL.
8. A method of preparing the aggregation-inducing fluorescent particle-antibody complex of claim 3, comprising the steps of: dispersing the aggregation-induced fluorescent particles in a phosphate buffer solution with the pH value of 4-6, adding an activating agent, carrying out an activation reaction, centrifuging to remove a supernatant, dispersing the activated particles in the phosphate buffer solution, adding an antibody, carrying out incubation reaction at room temperature, centrifuging to remove a free antibody, dispersing the precipitated particle-antibody conjugate in a confining liquid, and incubating at room temperature to obtain an aggregation-induced fluorescent particle-antibody complex.
9. The method of claim 8, wherein the activating agent is 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride and the blocking solution is a casein solution.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115389760A (en) * | 2022-10-27 | 2022-11-25 | 艾康生物技术(杭州)有限公司 | Detection reagent for immunoassay test strip |
| CN115932248A (en) * | 2022-12-10 | 2023-04-07 | 苏州宇测生物科技有限公司 | Single-molecule immunoassay method based on aggregation-induced emission material |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115389760A (en) * | 2022-10-27 | 2022-11-25 | 艾康生物技术(杭州)有限公司 | Detection reagent for immunoassay test strip |
| CN115932248A (en) * | 2022-12-10 | 2023-04-07 | 苏州宇测生物科技有限公司 | Single-molecule immunoassay method based on aggregation-induced emission material |
| CN115932248B (en) * | 2022-12-10 | 2023-09-12 | 苏州宇测生物科技有限公司 | Single-molecule immunity detection method based on aggregation-induced emission material |
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