Aggregation-induced emission microspheres with colorimetric-fluorescent dual signals and preparation method and application thereof

Abstract

The application discloses an aggregation-induced emission microsphere with colorimetric-fluorescent double signals and a preparation method and application thereof. The aggregation-induced emission microsphere comprises carboxyl polystyrene microspheres and aggregation-induced emission dyes embedded in the carboxyl polystyrene microspheres; and the structural formula of the aggregation-induced emission dyes is as follows: The aggregation-induced emission microsphere has colorimetric-fluorescent double signals, is red under natural light, has strong absorption at 600 nm, and shows high light-emitting efficiency at 725 nm, effectively avoids the interference of biological autofluorescence, reduces background interference, has good light stability and light bleaching resistance, and has high molar extinction coefficient, so that the aggregation-induced emission microsphere has colorimetric and fluorescent double signal output capabilities. In addition, the preparation process of the aggregation-induced emission microsphere has high reproducibility, has the potential for large-scale synthesis, and meets the needs of industrial batch production.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of medical testing, in particular to a colorimetric-fluorescent double signal aggregation-induced emission microsphere and a preparation method and application thereof. BACKGROUND

[0002] The lateral flow immunoassay (LFIA) has the characteristics of fast detection speed, simple operation and low price, is suitable for detection of various biomarkers, and is currently widely used in many fields such as lung disease, myocardial infarction, inflammation, pregnancy and the like.

[0003] Patent CN110940814A discloses a time-resolved fluorescent quantitative detection KL-6 test strip and a preparation method thereof. The antibody is labeled with a lanthanide element. According to the luminescence characteristics of the lanthanide element chelate, the fluorescence is measured by using the time-resolved technique, which can effectively eliminate the interference of non-specific fluorescence and improve the analysis sensitivity. However, the light bleaching of the time-resolved fluorescent material leads to poor reproducibility of the detection results. The aggregation-induced emission material has the characteristics of luminescence enhancement in solid or aggregated state, and has the advantages of light bleaching resistance and strong luminescence, and has great potential to replace traditional fluorescent dyes. Using the aggregation-induced emission material as a fluorescent labeling probe can not only improve the loading capacity of the fluorescent microsphere, but also overcome the aggregation-induced quenching effect of traditional dyes, greatly improving the signal output capacity of the fluorescent microsphere and the sensitivity of the detection.

[0004] However, the fluorescent signal probe needs an additional ultraviolet excitation light source, which limits the large-scale application of the lateral flow immunoassay. In recent years, a large number of researchers have developed colorimetric-fluorescent double signal output lateral flow immunoassay test strips, which have the ability of naked eye qualitative and fluorescent quantitative. The mode of double signal output greatly improves the flexibility of detection and is suitable for various application scenarios. Most of the colorimetric-fluorescent signals used at present are composite materials self-assembled from noble metals such as gold nanoparticles and fluorescent dyes such as quantum dots. The signals of the composite material interfere with each other, and it is difficult to achieve perfect combination of the two signals. Patent CN114660029A discloses a colorimetric-fluorescent double signal aggregation-induced emission microsphere and application thereof. A single aggregation-induced emission material is used, which not only solves the signal interference between multiple materials, but also provides a colorimetric-fluorescent double signal nanomicrosphere with strong absorption performance, high fluorescence intensity and low biological toxicity. However, the aggregation-induced emission dye absorbs at 530 nm and emits at 625 nm, which is still interfered by autofluorescence (400-700 nm waveband), resulting in reduced sensitivity.

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

[0006] In view of the deficiencies of the prior art, the present application aims to provide an aggregation-induced emission microsphere with colorimetric-fluorescent dual signals, a preparation method and application thereof, and aims to solve the problem that the existing aggregation-induced emission microsphere with colorimetric-fluorescent dual signals is interfered by autofluorescence when applied to biomarker detection.

[0007] The technical scheme of the present application is as follows:

[0008] In a first aspect, the present application provides an aggregation-induced emission microsphere with colorimetric-fluorescent dual signals, comprising: carboxyl polystyrene microspheres and an aggregation-induced emission dye embedded in the carboxyl polystyrene microspheres.

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

[0010]

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

[0012] In a preferred technical scheme, the particle size of the aggregation-induced emission microsphere is 250-350 nm.

[0013] In a second aspect, the present application provides a preparation method of the aggregation-induced emission microsphere according to the first aspect, comprising the following steps:

[0014] S1. Dissolve the carboxyl polystyrene microspheres in an aqueous solution containing a surfactant, uniformly disperse, then add an organic solvent for swelling to obtain a first solution;

[0015] S2. Dissolve the aggregation-induced emission dye in a good solvent, uniformly disperse to obtain a second solution;

[0016] S3. Add the first solution to the second solution, uniformly disperse, then remove the organic phase by rotary evaporation under reduced pressure to obtain an inorganic phase;

[0017] S4. Centrifuge the inorganic phase, discard the supernatant, add deionized water, centrifuge again until the supernatant has no fluorescence, to obtain the aggregation-induced emission microsphere.

[0018] In a preferred technical scheme, the preparation method of the aggregation-induced emission dye comprises:

[0019] 4,4'-Dimethoxy-4"-boronic acid triphenylamine and 3,6-bis(5-bromo-2-thienyl)-2,5-bis(2-ethylhexyl)-2,5-dihydro-pyrrolo[3,4-C]pyrrole-1,4-dione are subjected to a coupling reaction under an inert atmosphere to obtain the aggregation-induced emission dye.

[0020] Preferably, the surface active agent is selected from one or more of dodecyl sulfonic acid amine, sodium dodecyl sulfate, and dodecyl benzene 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.

[0021] Preferably, in the step S1, the swelling time is 10-30 min; and / or, in the step S3, the time for rotary evaporation under reduced pressure is 15-30 min; and / or, in the step S4, the centrifugal speed is 8000-15000 rpm, and the centrifugal time is 10-20 min.

[0022] In a third aspect, the present application provides an immunofluorescent probe, which is an antibody labeled with the aggregation-induced emission microsphere according to the first aspect.

[0023] Preferably, in the immunofluorescent probe, the mass ratio of the antibody to the aggregation-induced emission microsphere is (0.08-0.12) ︰ 1.

[0024] In a fourth aspect, the present application provides an immunochromatography test strip, which comprises the immunofluorescent probe according to the third aspect, and the immunofluorescent probe specifically binds to a substance to be detected.

[0025] The present application provides an aggregation-induced emission microsphere with colorimetric-fluorescent dual signals, which comprises carboxyl polystyrene microspheres and aggregation-induced emission dyes embedded in the carboxyl polystyrene microspheres. Compared with the prior art, the aggregation-induced emission microsphere of the present application has colorimetric-fluorescent dual signals, is red under natural light, has strong absorption at 600 nm, and exhibits high light-emitting efficiency at 725 nm. The aggregation-induced emission microsphere not only effectively avoids the interference of biological autofluorescence and reduces background interference, but also has good light stability and light bleaching resistance. The high molar extinction coefficient enables the aggregation-induced emission microsphere to have colorimetric and fluorescent dual signal output capabilities. In addition, the preparation process of the aggregation-induced emission microsphere has high reproducibility and has the potential for large-scale synthesis, meeting the needs of industrial batch production.

[0026] Further, the immunofluorescent probe prepared by using the aggregation-induced emission microsphere of the present application can simultaneously realize naked-eye qualitative and fluorescent quantitative detection. In addition, the immunochromatography test strip prepared by using the immunofluorescent probe has high sensitivity (qualitative detection limit of 4.11 ng / mL and quantitative detection limit of 53.4 pg / mL), wide detection range (linear range of 15-33333 pg / mL), and high reliability (correlation R 2The detection is not limited by instruments and regions, and provides a convenient detection tool for differential diagnosis, prognosis determination and dynamic monitoring of biomarkers. BRIEF DESCRIPTION OF DRAWINGS

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

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

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

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

[0031] Fig. 5 is an absorption emission spectrum of the aggregation-induced fluorescence microspheres prepared in Example 1.

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

[0033] Fig. 7 is a detection result diagram of the immunochromatography test strip for detecting sialylated sugar chain antigen KL-6 prepared in Example 3. DETAILED DESCRIPTION

[0034] The present application provides an aggregation-induced emission microsphere with colorimetric-fluorescent dual signals and a preparation method and application thereof.

[0035] The present application provides an aggregation-induced emission microsphere with colorimetric-fluorescent dual signals, which comprises: carboxyl polystyrene microspheres and aggregation-induced emission dyes embedded in the carboxyl polystyrene microspheres.

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

[0037]

[0038] Specifically, the aggregation-induced emission dye of the present application adopts triphenylamine as a donor, 2,5-dihydro-pyrrolo[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 emission molecule with a D-π-A-π-D structure. The aggregation-induced emission dye is red under natural light, has strong absorption at 600 nm, and exhibits high luminescent efficiency at 725 nm. Not only does it effectively avoid the interference of biological autofluorescence and reduce background interference, but it also has good light stability and light bleaching resistance. The high molar extinction coefficient enables it to have both colorimetric and fluorescent signal output capabilities.

[0039] Meanwhile, the present application uses carboxyl polystyrene microspheres as template spheres to prepare aggregation-induced emission microspheres by a swelling method. The synthesized aggregation-induced emission microspheres have the advantages of colorimetric signal and fluorescent signal, high molar extinction coefficient, and high quantum efficiency, overcoming the disadvantages of signal interference and complex preparation of traditional fluorescent microspheres. Moreover, the preparation process has high reproducibility and has the potential for large-scale synthesis, meeting the needs of industrial batch production.

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

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

[0042] The present application provides a preparation method of the aggregation-induced emission microspheres as described above, comprising the following steps:

[0043] S1, dissolving carboxyl polystyrene microspheres in an aqueous solution containing a surfactant, uniformly dispersing, then adding an organic solvent for swelling to obtain a first solution;

[0044] S2, dissolving an aggregation-induced emission dye into a good solvent, uniformly dispersing to obtain a second solution;

[0045] S3, adding the first solution to the second solution, uniformly dispersing, then removing the organic phase by rotary evaporation under reduced pressure 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 to obtain the aggregation-induced emission microspheres.

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

[0048] The 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 are coupled under inert atmosphere to obtain the aggregation-induced emission dye.

[0049] Specifically, the preparation method of the aggregation-induced emission dye has the following synthesis route:

[0050]

[0051] The synthesis operation is as follows:

[0052] In the reaction container, 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, vacuumized and inert gas is introduced, a reaction solvent is added, and the reaction is carried out at 60-100℃ for 12-36h 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 an embodiment, the surfactant is selected from one or more of dodecylamine sulfonate, sodium dodecyl sulfate and dodecylbenzenesulfonic acid.

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

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

[0059] In an embodiment, in the step S1, the swelling time is 10-30min.

[0060] In an embodiment, in the step S3, the time for rotary evaporation under reduced pressure is 15-30min.

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

[0062] In an embodiment, the dispersion in steps S1-S3 is performed by ultrasonic, the power of the ultrasonic is 60-90 w, and the ultrasonic time is 1-5 min.

[0063] The embodiment of the present application provides an immunofluorescent probe, which is an antibody labeled by the aggregation-induced emission microsphere as described above.

[0064] In an embodiment, the preparation method of the immunofluorescent probe comprises the following steps:

[0065] S1, mixing the aggregation-induced emission microsphere as described above with a labeling buffer, uniformly dispersing by ultrasonic to obtain a first mixture;

[0066] S2, adding an antibody into the first mixture, and electrostatically adsorbing for 30-60 min to obtain a second mixture;

[0067] S3, adding a cross-linking agent into the second mixture, uniformly mixing, and then reacting for 30-60 min to obtain a third mixture;

[0068] S4, adding a blocking agent into the third mixture, reacting for 30-60 min at 10-40 ℃, centrifuging to remove supernatant, and then adding a probe storage solution to resuspend to obtain the immunofluorescent probe.

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

[0070] Preferably, the labeling buffer is a 0.005-0.05M phosphate buffer (PB) (pH 5.5-7.4) or a 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 skimmed milk.

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

[0073] In an embodiment, the mass ratio of the antibody to the aggregation-induced emission microsphere in the immunofluorescent probe is (0.08-0.12)︰1.

[0074] In a fourth aspect, the present application provides an immunochromatographic test strip comprising the immunofluorescent probe as described above, which specifically binds to the substance to be detected.

[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, sequentially fixed on the polyvinyl chloride base plate along a chromatographic direction, a sample pad, a conjugate pad, a nitrocellulose membrane and an absorbent paper; the conjugate pad is coated with an immunofluorescent probe, which comprises the aggregation-induced emission microsphere-labeled sialylated sugar chain antigen KL-6 first antibody as described above; the nitrocellulose membrane is provided with, sequentially along the chromatographic direction, a detection line and a quality control line; the detection line is coated with a sialylated sugar chain antigen KL-6 second antibody, and the quality control line is coated with a goat anti-mouse lgG antibody; the sialylated sugar chain antigen KL-6 first antibody and the sialylated sugar chain antigen KL-6 second antibody recognize different epitopes of the sialylated sugar chain antigen KL-6.

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

[0077] S1, preparation of the sample pad: spread the sample pad in a 30×30×3cm tray, immerse the sample pad in 50-100mL of sample pad pretreatment liquid, take it out after 1-2min, and place the treated sample pad in an oven for 24-72h of baking at 37-50℃;

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

[0079] S2, preparation of the conjugate pad: prepare a solution of the aggregation-induced emission microsphere-labeled sialylated sugar chain antigen KL-6 first antibody, so that the concentration of the solution is 0.05-0.5mg / mL, uniformly spray the solution of the aggregation-induced emission microsphere-labeled sialylated sugar chain antigen KL-6 first antibody on the conjugate pad by using a colloidal gold spraying instrument, and vacuum dry the conjugate pad at 37-50℃ for 3-8h.

[0080] S3, Preparation of nitrocellulose membrane: a solution of salivary mucin carbohydrate antigen KL-6 secondary antibody at a concentration of 1.5 mg / mL and a solution of goat anti-mouse IgG antibody at a concentration of 1 mg / mL were prepared with coating buffer, respectively, and were drawn in parallel on the nitrocellulose membrane in the chromatographic direction at a spraying amount of 1 μL / cm as the detection line and the quality control line, respectively, with an interval of 7 mm between the two lines, and were placed in a 37℃ oven for drying for 12 h.

[0081] S4, Assembly of test strip: a sample pad, a conjugate pad, a nitrocellulose membrane and an absorbent paper were pasted in sequence on a polyvinyl chloride bottom plate in the chromatographic direction to obtain a test strip large plate, the test strip large plate was cut into test strips by a strip cutting machine, the test strips were fixed in a plastic bottom card, the card cover was covered and pressed tightly, and the card was placed in a 25℃ drying box for storage for standby.

[0082] The detection method of the immunochromatographic test strip for detecting salivary mucin carbohydrate antigen KL-6 as described above comprises:

[0083] The sample to be detected is added to the sample pad of the immunochromatographic test strip for detecting salivary mucin carbohydrate antigen KL-6, and after standing for 15 min, the fluorescence intensity of the detection line is recorded by a fluorescence immunoassay instrument.

[0084] The application will be further described below through specific examples.

[0085] Example 1

[0086] The embodiment provides an aggregation-induced emission microsphere with colorimetric-fluorescent dual signals, comprising: a carboxyl polystyrene microsphere and an aggregation-induced emission dye embedded in the carboxyl polystyrene microsphere. The preparation process is specifically as follows:

[0087] 1. Preparation of aggregation-induced emission dye with colorimetric-fluorescent dual signals

[0088] The synthesis route is as follows:

[0089]

[0090] The synthesis steps are as follows:

[0091] In a 50 mL three-necked flask, 4,4'-dimethoxy-4"-boronic acid triphenylamine (200 mg, 0.57 mmol), 3,6-bis(5-bromo-2-thienyl)-2,5-bis(2-ethylhexyl)-2,5-dihydro-pyrrolo[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; vacuumed and purged with nitrogen three times, then tetrahydrofuran 10 mL and water 2.5 mL were added, and stirred at 85 °C for 24 h under nitrogen atmosphere to obtain the post-reaction solution. After the post-reaction solution was cooled to room temperature, the solvent was removed by evaporation under reduced pressure, water was added, and the mixture was extracted with dichloromethane three times. The organic phase was combined, dried over anhydrous Na2SO4, 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 was an aggregation-induced emission dye with colorimetric-fluorescent dual signals. The hydrogen spectrum, carbon spectrum and high resolution mass spectrum of the aggregation-induced emission dye are shown in Figs. 1-3 .

[0092] 2. Preparation of aggregation-induced emission microspheres with colorimetric-fluorescent dual signals

[0093] 1 mg of carboxyl polystyrene microspheres (purchased from Suzhou Nanometer Technology Co., Ltd., product number: LBWNC-025) was dissolved in an aqueous solution containing 0.4% (M:V) SDS, and then ultrasonically dispersed. After that, 2 mL of acetone was added to fully swell the carboxyl polystyrene microspheres to obtain a mixture of carboxyl polystyrene microspheres. The aggregation-induced emission dye was dissolved in tetrahydrofuran and ultrasonically dispersed to obtain a solution of the aggregation-induced emission dye. The mixture of carboxyl polystyrene microspheres was added to the solution of the aggregation-induced emission dye, and then ultrasonically dispersed for 3 min. After that, the organic phase was removed by rotary evaporation under reduced pressure for 20 min to obtain an inorganic phase. The inorganic phase was centrifuged at 12000 rpm for 15 min at 25 °C, and then the supernatant was discarded. Deionized water was added, and the mixture was centrifuged until the supernatant was no longer fluorescent to obtain aggregation-induced emission microspheres with colorimetric-fluorescent dual signals. The scanning electron microscope of the aggregation-induced emission microspheres is shown in Fig. 4 , and the absorption and emission spectra are shown in Fig. 5 .

[0094] Example 2

[0095] This example provides an immunofluorescence probe for detecting sialylated sugar chain antigen KL-6, which is a sialylated sugar chain antigen KL-6 primary antibody labeled by the aggregation-induced emission microspheres of Example 1. The preparation process is as follows:

[0096] (1) 500 μg of the aggregation-induced fluorescence 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 mixture of the aggregation-induced fluorescence microspheres; (2) 60 μg of the saliva liquefied sugar chain antigen KL-6 first antibody was added to the mixture of the aggregation-induced fluorescence microspheres and ultrasonically dispersed for 30 times to uniformly disperse the antibody and the microspheres, followed by electrostatic adsorption for 30 min to obtain a pre-reaction solution; (3) a 10 mg / mL N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC) solution was prepared, 10 μL of the EDC solution was added to the pre-reaction solution, and after thoroughly mixed, the reaction was carried out for 45 min; (4) Step (3) was repeated three times, followed by the addition of bovine serum albumin, and after reaction at 25°C for 60 min, centrifugation was carried out at 12,000 rpm for 15 min at 4°C, the supernatant was discarded, 1 mL of a probe storage solution (0.01 M phosphate buffer (pH 8.0) containing 5% sucrose (M:V), 15% trehalose (M:V) and 0.05% sodium azide (M:V)) was added for resuspension to obtain the aggregation-induced fluorescence microspheres labeled saliva liquefied sugar chain antigen KL-6 first antibody, i.e., the immunofluorescence probe for detecting the saliva liquefied sugar chain antigen KL-6, which was stored at 4°C for standby use.

[0097] Example 3

[0098] The present example provides an immunochromatography test strip for detecting the saliva liquefied sugar chain antigen KL-6, a preparation method and application thereof, which are specifically as follows.

[0099] 1. Preparation of the immunochromatography test strip for detecting the saliva liquefied sugar chain antigen KL-6

[0100] (1) Preparation of the sample pad:

[0101] The sample pad (glass fiber membrane) was laid in a 30×30×3 cm tray, 80 mL of a sample pad pretreatment solution (0.3 mg / mL of a heterophilic antibody blocking agent, 0.3 wt% Tween-20, 1 wt% BSA and 1 wt% sucrose in 0.01 M PBS buffer) was used to soak the sample pad, which was taken out after staying for 1 min, and the treated sample pad was placed in an oven for drying at 45°C for 24 h to obtain the treated sample pad.

[0102] (2) Preparation of the conjugate pad:

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

[0104] (3) Preparation of nitrocellulose membrane

[0105] The saliva glycochain antigen KL-6 secondary antibody and the goat anti-mouse lgG antibody were diluted to 1.5 mg / mL and 1 mg / mL respectively with a coating buffer (0.02 M PBS buffer containing 5% sucrose, pH 7.4), and were drawn in parallel on the nitrocellulose membrane in a chromatographic direction at a spraying amount of 1 μL / cm as a detection line and a quality control line respectively, with a 7 mm interval between the two lines, and were placed in a 37°C oven for drying for 12 h.

[0106] (4) Assembly of immunochromatographic test strip

[0107] A sample pad, a conjugate pad, a nitrocellulose membrane and an absorbent paper were pasted on a polyvinyl chloride base plate in sequence in a chromatographic direction to obtain a test strip large plate, the test strip large plate was cut into 3.9 mm wide test strips by a cutting machine, the test strips were fixed in a plastic bottom card, the card surface was covered and compressed, an immunochromatographic test strip for detecting saliva glycochain antigen KL-6 was obtained, and was placed in a 25°C drying box for storage for standby use.

[0108] 2. Sensitivity, detection range, repeatability, specificity and stability evaluation of the immunochromatographic test strip for detecting saliva glycochain antigen KL-6

[0109] The immunochromatographic test strip for detecting saliva glycochain antigen KL-6 described above was used to determine saliva glycochain antigen KL-6 standard samples with different concentrations (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), and the detection principle is shown in Fig. 6 Each concentration was tested three times repeatedly, the standard sample concentration was taken as the abscissa, the signal ratio (PL T / PL C ) of the fluorescence signal of the detection line T and the fluorescence signal of the quality control line C was taken as the ordinate, and a standard curve was drawn, and the results are shown in Fig. 7 It was found that the naked-eye qualitative minimum detection limit of the immunochromatographic test strip of the application was 4.11 ng / mL, the fluorescence quantitative minimum detection limit was 0.09 ng / mL, and the detection range was 0.15-333.33 ng / mL.

[0110] It should be understood that the application of the application is not limited to the above examples, and those skilled in the art can make improvements or changes according to the above description, and all these improvements and changes shall belong to the protection scope of the appended claims of the application.

Claims

1. An immunochromatographic test strip for detecting sialylated sugar chain antigen KL-6, characterized by comprising: The immunochromatography test strip comprises a polyvinyl chloride base plate and a sample pad, a conjugate pad, a nitrocellulose membrane and an absorbent paper fixed on the polyvinyl chloride base plate in sequence along a chromatographic direction; The conjugate pad is coated with an immunofluorescent probe comprising saliva liquefied sugar chain antigen KL-6 first antibody marked with aggregation-induced emission microspheres; The nitrocellulose membrane is provided with a detection line and a quality control line in sequence along the chromatographic direction; The saliva liquefied sugar chain antigen KL-6 second antibody is coated on the detection line, and the goat anti-mouse lgG antibody is coated on the quality control line; The saliva liquefied sugar chain antigen KL-6 first antibody and the saliva liquefied sugar chain antigen KL-6 second antibody recognize different epitopes of the saliva liquefied sugar chain antigen KL-6; The aggregation-induced emission microspheres comprise carboxyl polystyrene microspheres and aggregation-induced emission dyes embedded in the carboxyl polystyrene microspheres; The aggregation-induced emission dyes have the following structural formula: 。 2. The immunochromatographic test strip according to claim 1, characterized in that, In the aggregation-induced emission microspheres, the mass ratio of the aggregation-induced emission dyes to the carboxyl polystyrene microspheres is 1: (0.5-10).

3. The immunochromatographic test strip according to claim 1, characterized in that, The particle size of the aggregation-induced emission microspheres is 250-350 nm.

4. The immunochromatographic test strip according to claim 1, characterized by, The preparation method of the aggregation-induced emission microspheres comprises the following steps: S1, dissolving carboxyl polystyrene microspheres in an aqueous solution containing a surfactant, uniformly dispersing, adding an organic solvent for swelling, and obtaining a first solution; S2, dissolving aggregation-induced emission dyes into a good solvent, uniformly dispersing, and obtaining a second solution; S3, adding the first solution into the second solution, uniformly dispersing, removing the organic phase by rotary evaporation under reduced pressure, and obtaining an inorganic phase; S4, centrifuging the inorganic phase, discarding the supernatant, adding deionized water, and centrifuging until the supernatant has no fluorescence, and obtaining the aggregation-induced emission microspheres.

5. The immunochromatographic test strip according to claim 4, characterized in that, The preparation method of the aggregation-induced emission dyes comprises: 4,4'-dimethoxy-4''-boronic acid triphenylamine and 3,6-bis (5-bromo-2-thienyl)-2,5-bis (2-ethylhexyl)-2,5-dihydro-pyrrolo [3,4-C] pyrrole-1,4-dione are coupled under an inert atmosphere to obtain the aggregation-induced emission dyes.

6. The immunochromatographic test strip according to claim 4, characterized in that, The surfactant is selected from one or more of dodecyl sulfonic acid amine, sodium dodecyl sulfate and dodecyl benzene 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 immunochromatographic test strip according to claim 4, characterized in that, In step S1, the swelling time is 10-30 min; and / or, in step S3, the rotary evaporation time under reduced pressure is 15-30 min; and / or, in step S4, the centrifugation speed is 8000-15000 rpm, and the centrifugation time is 10-20 min.

8. The immunochromatographic test strip according to claim 1, characterized in that, In the immunofluorescent probe, the mass ratio of the saliva liquefied sugar chain antigen KL-6 first antibody to the aggregation-induced emission microspheres is (0.08-0.12):1.

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