Aggregation-induced emission microspheres with colorimetric-fluorescence double signals as well as preparation method and application of aggregation-induced emission microspheres

Abstract

The invention discloses aggregation-induced emission microspheres with colorimetric-fluorescence double signals as well as a preparation method and application of the aggregation-induced emission microspheres. The aggregation-induced emission microsphere comprises a carboxyl polystyrene microsphere and an aggregation-induced emission dye embedded in the carboxyl polystyrene microsphere, the aggregation-induced emission microsphere has colorimetric-fluorescence double signals, is red under natural light, has strong absorption at 600nm, shows high luminous efficiency at 725nm, not only effectively avoids the interference of biological autofluorescence and reduces background interference, but also has good light stability and photobleaching resistance, and can be used for preparing the aggregation-induced emission microsphere. And due to the high molar extinction coefficient, the colorimetric and fluorescent double-signal output capability is realized at the same time. In addition, the aggregation-induced emission microspheres are high in reproducibility in the preparation process, have the potential of large-scale synthesis and meet the requirements of industrial batch production.

Description

Technical Field

[0001] The present invention relates to the field of medical testing technology, and in particular to an aggregation-induced luminescence microsphere with colorimetric-fluorescence dual signals and a preparation method and application thereof. Background Art

[0002] Lateral Flow Immunoassays (LFIA) have the characteristics of fast detection speed, simple operation and low price. They are suitable for the detection of various biomarkers and are currently widely used in many fields such as lung disease, myocardial infarction, inflammation, pregnancy, etc.

[0003] Patent CN110940814A discloses a test strip for time-resolved fluorescence quantitative detection of KL-6 and its preparation method. The antibody is labeled with lanthanide elements, and the fluorescence is measured by time-resolved technology according to the luminescence characteristics of lanthanide chelates, which can effectively eliminate the interference of non-specific fluorescence and improve the sensitivity of analysis. However, the photobleaching of time-resolved fluorescent materials leads to poor reproducibility of the test results. Aggregation-induced luminescence materials are characterized by enhanced luminescence in the solid or aggregated state, and have the advantages of photobleaching resistance and strong luminescence, and have great potential to replace traditional fluorescent dyes. Using aggregation-induced luminescence materials as fluorescent labeling probes can not only increase the loading capacity of fluorescent microspheres, but also overcome the aggregation-induced quenching effect of traditional dyes, greatly improving the signal output capacity of fluorescent microspheres and the sensitivity of detection.

[0004] However, the fluorescent signal probe requires an additional ultraviolet excitation light source, which limits the large-scale application of immunochromatographic test strips. In recent years, a large number of researchers have developed immunochromatographic test strips with colorimetric-fluorescent dual signal output, which has the ability of naked eye qualitative and fluorescent quantitative. The dual signal output mode greatly improves the flexibility of detection and is suitable for a variety of application scenarios. The colorimetric-fluorescent signals currently used are mostly self-assembled into composite materials by precious metals such as gold nanoparticles and fluorescent dyes such as quantum dots. However, the signals of the composite materials will interfere with each other, and it is difficult to achieve a perfect combination of the two signals. Patent CN114660029A discloses a colorimetric-fluorescent dual signal aggregation-induced fluorescent microsphere and its application, using a single aggregation-induced luminescent material, which not only solves the signal interference generated between multiple materials, but also provides a colorimetric-fluorescent dual signal nano-microsphere with strong absorption performance, high fluorescence intensity and low biological toxicity. However, the aggregation-induced luminescent dye absorbs at 530nm and emits at 625nm, and is still interfered by autofluorescence (within the 400-700nm band), resulting in reduced sensitivity.

[0005] Therefore, the prior art still needs to be improved and developed. Summary of the invention

[0006] In view of the above-mentioned deficiencies in the prior art, the object of the present invention is to provide an aggregation-induced luminescence microsphere with colorimetric-fluorescence dual signals and a preparation method and application thereof, aiming to solve the problem that the existing aggregation-induced luminescence microsphere with colorimetric-fluorescence dual signals is interfered by autofluorescence when used for biomarker detection.

[0007] The technical solution of the present invention is as follows:

[0008] In a first aspect of the present invention, there is provided an aggregation-induced luminescence microsphere having a colorimetric-fluorescence dual signal, the aggregation-induced luminescence microsphere comprising: a carboxyl polystyrene microsphere and an aggregation-induced luminescence dye embedded in the carboxyl polystyrene microsphere;

[0009] The structural formula of the aggregation-induced emission dye is:

[0010]

[0011] According to a preferred technical solution, in the aggregation-induced luminescence microspheres, the mass ratio of the aggregation-induced luminescence dye to the carboxyl polystyrene microspheres is 1:(0.5-10).

[0012] According to a preferred technical solution, the particle size of the aggregation-induced luminescence microspheres is 250 nm to 350 nm.

[0013] The second aspect of the present invention provides a method for preparing the aggregation-induced luminescence microspheres as described in the first aspect, comprising the steps of:

[0014] S1, dissolving the carboxyl polystyrene microspheres in an aqueous solution containing a surfactant, and after uniform dispersion, adding an organic solvent for swelling to obtain a first solution;

[0015] S2, dissolving the aggregation-induced emission dye in a good solvent and dispersing it evenly to obtain a second solution;

[0016] S3, adding the first solution to the second solution, after uniform dispersion, removing the organic phase by vacuum rotary evaporation to obtain an inorganic phase;

[0017] S4, centrifuging the inorganic phase, discarding the supernatant, adding deionized water, and centrifuging again until the supernatant has no fluorescence, thereby obtaining the aggregation-induced luminescence microspheres.

[0018] A preferred technical solution is a method for preparing the aggregation-induced emission dye, comprising:

[0019] The aggregation-induced emission dye is obtained by coupling 4,4'-dimethoxy-4"-boronic acid triphenylamine and 3,6-bis(5-bromo-2-thienyl)-2,5-bis(2-ethylhexyl)-2,5-dihydropyrrolo[3,4-C]pyrrole-1,4-dione under an inert atmosphere.

[0020] In a preferred technical solution, the surfactant is selected from one or more of dodecylsulfonamide, sodium dodecyl sulfate, and dodecylbenzenesulfonic acid; and / or the organic solvent is selected from one or more of acetone, toluene, tetrahydrofuran, and chloroform; and / or the good solvent is selected from one or more of acetone, toluene, tetrahydrofuran, and chloroform.

[0021] According to a preferred technical solution, in step S1, the swelling time is 10 to 30 min; and / or, in step S3, the reduced pressure rotary evaporation time is 15 to 30 min; and / or, in step S4, the centrifugal speed is 8000 to 15000 rpm and the centrifugal time is 10 to 20 min.

[0022] The third aspect of the present invention provides an immunofluorescent probe, which is the antibody labeled with the aggregation-induced luminescent microspheres described in the first aspect.

[0023] According to a preferred technical solution, in the immunofluorescent probe, the mass ratio of the antibody to the aggregation-induced luminescence microspheres is (0.08-0.12):1.

[0024] The fourth aspect of the present invention provides an immunochromatographic test strip, wherein the immunochromatographic test strip comprises the immunofluorescent probe as described in the third aspect, and the immunofluorescent probe specifically binds to a substance to be tested.

[0025] Beneficial effects: The present invention provides an aggregation-induced luminescent microsphere with colorimetric-fluorescent dual signals, and the aggregation-induced luminescent microsphere comprises: carboxyl polystyrene microspheres and aggregation-induced luminescent dyes embedded in the carboxyl polystyrene microspheres. Compared with the prior art, the aggregation-induced luminescent microspheres of the present invention have colorimetric-fluorescent dual signals, are red under natural light, have strong absorption at 600nm, and show high luminescence efficiency at 725nm. They not only effectively avoid the interference of biological autofluorescence and reduce background interference, but also have good photostability and photobleaching resistance. The high molar extinction coefficient enables it to have both colorimetric and fluorescent dual signal output capabilities. In addition, the preparation process of the aggregation-induced luminescent microspheres is highly reproducible and has the potential for large-scale synthesis, meeting the needs of industrial mass production.

[0026] Furthermore, the immunofluorescence probe prepared by the aggregation-induced luminescence microspheres of the present invention can simultaneously realize naked eye qualitative and fluorescent quantitative detection. In addition, the immunochromatographic test strip prepared by the immunofluorescence probe has high sensitivity (qualitative detection limit of 4.11 ng / mL, quantitative detection limit of 53.4 pg / mL), wide detection range (linear range of 15 to 33333 pg / mL), high reliability (correlation R with commercial ELISA kits) and high sensitivity (qualitative detection limit of 4.11 ng / mL, quantitative detection limit of 53.4 pg / mL). 2The detection is not limited by instruments and regions, and provides a convenient detection tool for the differential diagnosis, prognosis determination and dynamic monitoring of biomarkers. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is the hydrogen spectrum of the aggregation-induced emission dye prepared in Example 1.

[0028] Figure 2 This is the carbon spectrum of the aggregation-induced emission dye prepared in Example 1.

[0029] Figure 3 This is a high-resolution mass spectrum of the aggregation-induced emission dye prepared in Example 1.

[0030] Figure 4 is a scanning electron microscope image of the aggregation-induced fluorescent microspheres prepared in Example 1.

[0031] Figure 5 This is the absorption and emission spectrum of the aggregation-induced fluorescent microspheres prepared in Example 1.

[0032] Figure 6 1 is a diagram showing the detection principle of the immunochromatographic test strip prepared in Example 3 for detecting the sialylated sugar chain antigen KL-6.

[0033] Figure 7 This is a graph showing the test results of the immunochromatographic test strip prepared in Example 3 for detecting the sialylated sugar chain antigen KL-6. DETAILED DESCRIPTION

[0034] The present invention provides an aggregation-induced luminescence microsphere with colorimetric-fluorescence dual signals and a preparation method and application thereof. To make the purpose, technical scheme and effect of the present invention clearer and more specific, the present invention is further described in detail below.

[0035] The embodiment of the present invention provides an aggregation-induced luminescence microsphere with colorimetric-fluorescence dual signals, wherein the aggregation-induced luminescence microsphere comprises: a carboxyl polystyrene microsphere and an aggregation-induced luminescence dye embedded in the carboxyl polystyrene microsphere;

[0036] The structural formula of the aggregation-induced emission dye is:

[0037]

[0038] Specifically, the aggregation-induced luminescence dye of the present invention uses triphenylamine as a donor, 2,5-dihydropyrrolo[3,4-c]pyrrole-1,4-dione as an acceptor, and thiophene as an intermediate π bridge connecting the donor and the acceptor. The methoxy groups at both ends of the molecule promote charge transfer to a certain extent, forming a near-infrared aggregation-induced luminescence molecule with a D-π-A-π-D structure. The aggregation-induced luminescence dye is red under natural light, has strong absorption at 600nm, and exhibits high luminescence efficiency at 725nm. It not only effectively avoids the interference of biological autofluorescence and reduces background interference, but also has good photostability and resistance to photobleaching. The high molar extinction coefficient enables it to have both colorimetric and fluorescent dual signal output capabilities.

[0039] At the same time, the present invention uses carboxyl polystyrene microspheres as template balls and uses a swelling method to prepare aggregation-induced luminescence microspheres. The synthesized aggregation-induced luminescence microspheres have the advantages of both colorimetric signals and fluorescence signals, high molar extinction coefficient, and high quantum efficiency, overcoming the shortcomings of signal interference and complex preparation of traditional fluorescent microspheres. The preparation process is highly reproducible and has the potential for large-scale synthesis, meeting the needs of industrial mass production.

[0040] In one embodiment, in the aggregation-induced emission microspheres, the mass ratio of the aggregation-induced emission dye to the carboxyl polystyrene microspheres is 1:(0.5-10).

[0041] In one embodiment, the particle size of the aggregation-induced emission microspheres is 250 nm to 350 nm.

[0042] The embodiment of the present invention provides a method for preparing the aggregation-induced luminescence microspheres as described above, comprising the steps of:

[0043] S1, dissolving the carboxyl polystyrene microspheres in an aqueous solution containing a surfactant, and after uniform dispersion, adding an organic solvent for swelling to obtain a first solution;

[0044] S2, dissolving the aggregation-induced emission dye in a good solvent and dispersing it evenly to obtain a second solution;

[0045] S3, adding the first solution to the second solution, after uniform dispersion, removing the organic phase by vacuum rotary evaporation to obtain an inorganic phase;

[0046] S4, centrifuging the inorganic phase, discarding the supernatant, adding deionized water, and centrifuging again until the supernatant has no fluorescence, thereby obtaining the aggregation-induced luminescence microspheres.

[0047] In one embodiment, the method for preparing the aggregation-induced emission dye comprises:

[0048] The aggregation-induced emission dye is obtained by coupling 4,4'-dimethoxy-4"-boronic acid triphenylamine and 3,6-bis(5-bromo-2-thienyl)-2,5-bis(2-ethylhexyl)-2,5-dihydropyrrolo[3,4-C]pyrrole-1,4-dione under an inert atmosphere.

[0049] Specifically, the preparation method of the aggregation-induced emission dye and the synthesis route are as follows:

[0050]

[0051] The synthesis operation is as follows:

[0052] 4,4'-dimethoxy-4"-boronic acid triphenylamine, 3,6-bis(5-bromo-2-thienyl)-2,5-bis(2-ethylhexyl)-2,5-dihydropyrrolo[3,4-c]pyrrole-1,4-dione, a catalyst and a weak base are added to a reaction container, vacuumized and introduced with an inert gas, a reaction solvent is added, and the reaction is carried out at 60-100° C. for 12-36 hours to obtain 3,6-bis(5-(4-(bis(4-methoxyphenyl)amino)phenyl)thiophen-2-yl)-2,5-bis(2-ethylhexyl)-2,5-dihydropyrrolo[3,4-c]pyrrole-1,4-dione, i.e., the aggregation-induced emission dye.

[0053] Preferably, the catalyst is tetrakis(triphenylphosphine)palladium (Pd(PPh3)4).

[0054] Preferably, the weak base is selected from one or more of potassium carbonate, sodium carbonate and cesium carbonate.

[0055] Preferably, the reaction solvent is selected from one or more of acetone, toluene and tetrahydrofuran.

[0056] In one embodiment, the surfactant is selected from one or more of dodecylamine sulfonate, sodium dodecyl sulfate, and dodecylbenzene sulfonic acid.

[0057] In one embodiment, the organic solvent is selected from one or more of acetone, toluene, tetrahydrofuran, and chloroform.

[0058] In one embodiment, the good solvent is selected from one or more of acetone, toluene, tetrahydrofuran, and chloroform.

[0059] In one embodiment, in step S1, the swelling time is 10 to 30 minutes.

[0060] In one embodiment, in step S3, the time of reduced pressure rotary evaporation is 15 to 30 minutes.

[0061] In one embodiment, in step S4, the centrifugal speed is 8000-15000 rpm, and the centrifugal time is 10-20 min.

[0062] In one embodiment, in the steps S1 to S3, the uniform dispersion is performed by ultrasound, the power of the ultrasound is 60w to 90w, and the time of the ultrasound is 1 to 5min.

[0063] An embodiment of the present invention provides an immunofluorescent probe, which is an antibody labeled with the aggregation-induced luminescent microspheres as described above.

[0064] In one embodiment, the method for preparing the immunofluorescent probe comprises the steps of:

[0065] S1, taking the above-mentioned aggregation-induced luminescence microspheres and mixing them with a labeling buffer, and uniformly dispersing them by ultrasonication to obtain a first mixed solution;

[0066] S2, adding antibodies to the first mixed solution, and subjecting the mixture to electrostatic adsorption for 30 to 60 minutes to obtain a second mixed solution;

[0067] S3, adding a cross-linking agent to the second mixed solution, mixing thoroughly, and reacting for 30 to 60 minutes to obtain a third mixed solution;

[0068] S4. Add a blocking agent to the third mixed solution, react at 10-40° C. for 30-60 min, centrifuge to remove the supernatant, add a probe preservation solution to resuspend, and obtain an immunofluorescence probe.

[0069] Preferably, the cross-linking agent is N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC).

[0070] Preferably, the labeling buffer is 0.005-0.05M phosphate (PB) buffer (pH 5.5-7.4) or 0.01-0.05M 2-(N-morpholino)ethanesulfonic acid (MES) solution;

[0071] Preferably, the blocking agent is any one of bovine serum albumin (BSA), ovalbumin (OVA), casein or skim milk.

[0072] Preferably, the probe preservation solution is a 0.01 M phosphate (PB) buffer (pH 8.0) containing 5-25% sucrose (M:V), 10-20% trehalose (M:V), and 0.01-1% sodium azide (M:V).

[0073] In one embodiment, in the immunofluorescent probe, the mass ratio of the antibody to the aggregation-induced luminescence microspheres is (0.08-0.12):1.

[0074] According to a fourth aspect of the present invention, there is provided an immunochromatographic test strip, wherein the immunochromatographic test strip comprises the immunofluorescent probe as described above, and the immunofluorescent probe specifically binds to a substance to be tested.

[0075] Specifically, taking an immunochromatographic test strip for detecting sialylated sugar chain antigen KL-6 as an example, the immunochromatographic test strip comprises: a polyvinyl chloride base plate and a sample pad, a conjugation pad, a nitrocellulose membrane and absorbent paper fixed on the polyvinyl chloride base plate in sequence along the chromatography direction; the conjugation pad is coated with an immunofluorescent probe, and the immunofluorescent probe comprises the first antibody of sialylated sugar chain antigen KL-6 labeled with the aggregation-induced luminescent microspheres as described above; a detection line and a quality control line are sequentially arranged on the nitrocellulose membrane along the chromatography direction; the detection line is coated with a second antibody of sialylated sugar chain antigen KL-6, and the quality control line is coated with a sheep anti-mouse IgG antibody; the first antibody of sialylated sugar chain antigen KL-6 and the second antibody of sialylated sugar chain antigen KL-6 recognize different epitopes of sialylated sugar chain antigen KL-6.

[0076] The method for preparing the immunochromatographic test strip for detecting the sialolyzed sugar chain antigen KL-6 as described above comprises the steps of:

[0077] S1. Preparation of sample pad: Spread the sample pad flat on a 30×30×3 cm tray, take 50-100 mL of sample pad pretreatment solution to soak the sample pad, leave it for 1-2 min, then take it out, place the treated sample pad in an oven, and bake it at 37-50°C for 24-72 h;

[0078] Preferably, the sample pad pretreatment solution is a 0.01 M phosphate-buffered saline (PBS) buffer containing 0.1-0.5 mg / mL heterophilic antibody blocking agent, 0.2-0.5 wt % Tween-20, 0.5-2 wt % bovine serum albumin (BSA) and 0.5-1.5 wt % sucrose.

[0079] S2. Preparation of the conjugate pad: prepare a solution of the first antibody of the sialylated sugar chain antigen KL-6 labeled with aggregation-induced luminescent microspheres to a concentration of 0.05-0.5 mg / mL, spray the solution of the first antibody of the sialylated sugar chain antigen KL-6 labeled with aggregation-induced luminescent microspheres evenly on the conjugate pad using a colloidal gold spraying instrument, and vacuum dry at 37-50°C for 3-8 hours to obtain the conjugate pad.

[0080] S3. Preparation of nitrocellulose membrane: Use coating buffer to prepare a solution of 1.5 mg / mL of sialylated sugar chain antigen KL-6 secondary antibody and a solution of 1 mg / mL of goat anti-mouse IgG antibody, respectively. Strikes on the nitrocellulose membrane in parallel along the chromatography direction with a spray volume of 1 μL / cm, as the detection line and quality control line respectively, with an interval of 7 mm between the two lines. Place in a 37°C oven and dry for 12 h.

[0081] S4. Assembly of test strips: On the polyvinyl chloride base plate, stick the sample pad, binding pad, nitrocellulose membrane and absorbent paper in order along the chromatography direction to obtain a large test strip plate. Use a strip cutter to cut the large test strip plate into test strips. Fix the test strips in the plastic base card, cover and press the card surface, and store in a 25°C drying oven for later use.

[0082] The above-mentioned immunochromatographic test strip detection method for detecting sialolyzed sugar chain antigen KL-6 comprises:

[0083] The sample to be tested was added to the sample pad of the immunochromatographic test strip for detecting the sialolyzed sugar chain antigen KL-6, and after standing for 15 minutes, the fluorescence intensity of the test line was recorded by a fluorescence immunoassay analyzer.

[0084] The present invention will be further described below by means of specific examples.

[0085] Example 1

[0086] This embodiment provides an aggregation-induced luminescence microsphere with colorimetric-fluorescence dual signals, comprising: carboxyl polystyrene microspheres and an aggregation-induced luminescence dye embedded in the carboxyl polystyrene microspheres. The preparation process is as follows:

[0087] 1. Preparation of aggregation-induced luminescent dyes with colorimetric and fluorescent dual signals

[0088] The synthetic route is as follows:

[0089]

[0090] The synthesis steps are as follows:

[0091] In a 50 mL three-necked flask, 4,4'-dimethoxy-4"-boric acid triphenylamine (200 mg, 0.57 mmol), 3,6-bis(5-bromo-2-thienyl)-2,5-bis(2-ethylhexyl)-2,5-dihydropyrrolo[3,4-C]pyrrole-1,4-dione (191 mg, 0.28 mmol), Pd(PPh3)4 (35 mg, 0.03 mmol) and K2CO3 (124 mg, 0.9 mmol) were added; the mixture was evacuated and nitrogen was introduced three times, and 10 mL of tetrahydrofuran and 2.5 mL of water were added. mL, stirred at 85°C for 24 hours under nitrogen protection to obtain a reaction solution. After the reaction solution was cooled to room temperature, the solvent was removed by evaporation under reduced pressure, water was added, and the mixture was extracted three times with dichloromethane. The organic phases were combined, dried over anhydrous Na2SO4 and filtered, and the solvent was removed by evaporation under reduced pressure to obtain a crude product. The crude product was purified by column chromatography (eluent: petroleum ether: ethyl acetate = 5:1, v / v) to obtain a red solid (167 mg, yield 52.7%), which is an aggregation-induced luminescence dye with colorimetric-fluorescence dual signals. The hydrogen spectrum, carbon spectrum and high-resolution mass spectrum of the aggregation-induced luminescence dye are as follows Figures 1 to 3 shown.

[0092] 2. Preparation of aggregation-induced luminescence microspheres with colorimetric and fluorescence dual signals

[0093] 1 mg of carboxyl polystyrene microspheres (purchased from Suzhou Nanovita Technology Co., Ltd., item number: LBWNC-025) was dissolved in an aqueous solution containing 0.4% (M:V) SDS, and after ultrasonic dispersion, 2 mL of acetone was added to fully swell to obtain a mixed solution of carboxyl polystyrene microspheres; the aggregation-induced luminescence dye was dissolved in tetrahydrofuran and ultrasonically dispersed evenly to obtain a solution of the aggregation-induced luminescence dye; the mixed solution of carboxyl polystyrene microspheres was added to the solution of the aggregation-induced luminescence dye, and after ultrasonication for 3 minutes, the organic phase was removed by vacuum rotary evaporation for 20 minutes to obtain an inorganic phase; the inorganic phase was centrifuged at 12000 rpm at 25°C for 15 minutes, the supernatant was discarded, deionized water was added, and centrifuged again until the supernatant had no fluorescence, and aggregation-induced fluorescent microspheres with colorimetric-fluorescence dual signals were obtained. The scanning electron microscope of the aggregation-induced fluorescent microspheres is shown in FIG. Figure 4 The absorption and emission spectra are shown in Figure 5 shown.

[0094] Example 2

[0095] This embodiment provides an immunofluorescent probe for detecting sialylated sugar chain antigen KL-6, which is the first antibody of sialylated sugar chain antigen KL-6 labeled with aggregation-induced luminescence microspheres in Example 1. The specific preparation process is as follows:

[0096] (1) 500 μg of the aggregation-induced fluorescent microspheres prepared in Example 1 were mixed with a labeling buffer (0.01 M phosphate buffer, pH 6.0), and ultrasonically dispersed to obtain a mixed solution of aggregation-induced fluorescent microspheres; (2) 60 μg of the first antibody of the sialylated sugar chain antigen KL-6 was added to the mixed solution of aggregation-induced fluorescent microspheres, and the solution was superfused 30 times to make the antibody and microspheres evenly dispersed, followed by electrostatic adsorption for 30 minutes to obtain a pre-reaction solution; (3) 10 mg / mL of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC) solution was prepared, and 10 μL of The EDC solution was added to the pre-reaction solution, mixed thoroughly, and reacted for 45 minutes; (4) step (3) was repeated three times, followed by addition of bovine serum albumin, reaction at 25°C for 60 minutes, centrifugation at 12000 rpm for 15 minutes at 4°C, the supernatant was discarded, and 1 mL of probe preservation solution (containing 5% sucrose (M:V), 15% trehalose (M:V), 0.05% sodium azide (M:V) in 0.01 M phosphate solution (pH 8.0)) was added for resuspending to obtain the first antibody of the sialylated sugar chain antigen KL-6 labeled with aggregation-induced luminescence microspheres, i.e., the immunofluorescence probe for detecting the sialylated sugar chain antigen KL-6, which was stored at 4°C for future use.

[0097] Example 3

[0098] This embodiment provides an immunochromatographic test strip for detecting sialolytic sugar chain antigen KL-6 and a preparation method and application thereof, as follows:

[0099] 1. Preparation of immunochromatographic test strips for detecting sialolyzed sugar chain antigen KL-6

[0100] (1) Preparation of sample pad:

[0101] Spread the sample pad (glass fiber membrane) flat in a 30×30×3 cm tray, take 80 mL of sample pad pretreatment solution (0.3 mg / mL heterophilic antibody blocker, 0.3 wt% Tween-20, 1 wt% BSA and 1 wt% sucrose in 0.01 M PBS buffer) to soak the sample pad, stay for 1 min and then take it out. The treated sample pad is placed in an oven and baked at 45°C for 24 h to obtain the treated sample pad.

[0102] (2) Preparation of conjugate pad:

[0103] The first antibody labeled with sialylated sugar chain antigen KL-6 by aggregation-induced luminescence microspheres prepared in Example 2 was diluted to a final concentration of 1 mg / mL using a probe preservation solution, and the first antibody labeled with sialylated sugar chain antigen KL-6 by aggregation-induced luminescence microspheres was evenly sprayed on the conjugate pad at a spray volume of 1.04 μL / cm using a colloidal gold spraying instrument, and then vacuum-dried at 45° C. for 5 h to obtain a treated conjugate pad.

[0104] (3) Preparation of nitrocellulose membrane

[0105] The sialylated sugar chain antigen KL-6 secondary antibody and goat anti-mouse IgG antibody were diluted to 1.5 mg / mL and 1 mg / mL respectively with coating buffer (0.02 M PBS buffer (pH 7.4) containing 5% sucrose), and were streaked on the nitrocellulose membrane in parallel with a spray volume of 1 μL / cm according to the chromatography direction, as the detection line and quality control line, respectively, with an interval of 7 mm between the two lines. The membranes were placed in a 37°C oven and dried for 12 h.

[0106] (4) Assembly of immunochromatographic test strips

[0107] A sample pad, a binding pad, a nitrocellulose membrane and a blotting paper were sequentially pasted on a polyvinyl chloride bottom plate along the chromatography direction to obtain a large test paper plate. The large test paper plate was cut into 3.9 mm wide test paper strips using a strip cutter. The test paper strips were fixed in a plastic bottom card, covered and pressed on the card surface to obtain an immunochromatographic test strip for detecting the sialolyzed sugar chain antigen KL-6, and stored in a 25°C drying oven for future use.

[0108] 2. Evaluation of the sensitivity, detection range, repeatability, specificity and stability of the immunochromatographic test strip for detecting the sialolyzed sugar chain antigen KL-6

[0109] The above-mentioned immunochromatographic test strips for detecting sialoglycosylated sugar chain antigen KL-6 were used to measure different concentrations of sialoglycosylated sugar chain antigen KL-6 standards (0.002, 0.006, 0.017, 0.05, 0.15, 0.46, 1.37, 4.11, 12.34, 37.03, 111.11, 333.33, 1000 and 2000 units ng / mL). The detection principle is as follows: Figure 6 As shown, each concentration was tested three times, with the concentration of the standard as the horizontal axis and the signal ratio of the corresponding detection line T line to the quality control line C line (PL T / PL C ) as the ordinate and plotted into a standard curve. The result is as follows Figure 7 As shown, the lowest detection limit of the immunochromatographic test strip of the present invention for naked eye qualitative determination is 4.11 ng / mL, the lowest detection limit for fluorescence quantitative determination is 0.09 ng / mL, and the detection range is 0.15 to 333.33 ng / mL.

[0110] It should be understood that the application of the present invention is not limited to the above examples. For ordinary technicians in this field, improvements or changes can be made based on the above description. All these improvements and changes should fall within the scope of protection of the claims attached to the present invention.

Claims

1. An aggregation-induced luminescence microsphere with colorimetric and fluorescence dual signals, characterized in that: The aggregation-induced luminescence microspheres include: carboxyl polystyrene microspheres and aggregation-induced luminescence dyes embedded in the carboxyl polystyrene microspheres; The structural formula of the aggregation-induced emission dye is:

2. The aggregation-induced luminescence microsphere according to claim 1, characterized in that: In the aggregation-induced luminescence microspheres, the mass ratio of the aggregation-induced luminescence dye to the carboxyl polystyrene microspheres is 1:(0.5-10).

3. The aggregation-induced luminescence microsphere according to claim 1, characterized in that: The particle size of the aggregation-induced luminescence microspheres is 250nm to 350nm.

4. A method for preparing the aggregation-induced luminescence microspheres according to any one of claims 1 to 3, characterized in that: Includes steps: S1, dissolving the carboxyl polystyrene microspheres in an aqueous solution containing a surfactant, and after uniform dispersion, adding an organic solvent for swelling to obtain a first solution; S2, dissolving the aggregation-induced emission dye in a good solvent and dispersing it evenly to obtain a second solution; S3, adding the first solution to the second solution, after uniform dispersion, removing the organic phase by vacuum rotary evaporation to obtain an inorganic phase; S4, centrifuging the inorganic phase, discarding the supernatant, adding deionized water, and centrifuging again until the supernatant has no fluorescence, thereby obtaining the aggregation-induced luminescence microspheres.

5. The preparation method according to claim 4, characterized in that: The preparation method of the aggregation-induced luminescence dye comprises: The aggregation-induced emission dye is obtained by coupling 4,4'-dimethoxy-4"-boronic acid triphenylamine and 3,6-bis(5-bromo-2-thienyl)-2,5-bis(2-ethylhexyl)-2,5-dihydropyrrolo[3,4-C]pyrrole-1,4-dione under an inert atmosphere.

6. The preparation method according to claim 4, characterized in that: The surfactant is selected from one or more of dodecyl sulfonamide, sodium dodecyl sulfate, and dodecylbenzene sulfonic acid; and / or the organic solvent is selected from one or more of acetone, toluene, tetrahydrofuran, and chloroform; and / or the good solvent is selected from one or more of acetone, toluene, tetrahydrofuran, and chloroform.

7. The preparation method according to claim 4, characterized in that: In the step S1, the swelling time is 10 to 30 minutes; and / or, in the step S3, the reduced pressure rotary evaporation time is 15 to 30 minutes; and / or, in the step S4, the centrifugal speed is 8000 to 15000 rpm, and the centrifugal time is 10 to 20 minutes.

8. An immunofluorescence probe, characterized in that The immunofluorescence probe is an antibody labeled with the aggregation-induced luminescence microspheres according to any one of claims 1 to 3.

9. The immunofluorescence probe according to claim 8, characterized in that In the immunofluorescent probe, the mass ratio of the antibody to the aggregation-induced luminescent microspheres is (0.08-0.12):

1.

10. An immunochromatographic test strip, characterized in that: The immunochromatographic test strip comprises the immunofluorescent probe according to any one of claims 8 to 9, and the immunofluorescent probe specifically binds to the substance to be tested.

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