Preparation method and application of HPV16 / 18 type E6 / E7 protein multi-target detection test strip

Abstract

The invention discloses a preparation method and application of an HPV16 / 18 type E6 / E7 protein multi-target detection test strip, and belongs to the technical field of biological immunodetection analysis. The detection test strip comprises a back plate, a sample pad, a combination pad, a nitrocellulose membrane and an absorption pad, AIE fluorescent microsphere labeled probes are distributed on the combination pad, and a capture antibody and an AIE solution are distributed on the nitrocellulose membrane. The preparation method comprises the following three steps: preparation of the nitrocellulose membrane, preparation of the combination pad and assembly of the test strip. The detection test strip can synchronously and quantitatively detect PV16 E6, HPV16 E7, HPV18 E6 and HPV18 E7, each target corresponds to four signal points, multi-target synchronous rapid detection is achieved, the linear range is wide, the detection limit is low, operation is easy and convenient, cost is low, and the detection test strip is expected to become a novel efficient and reliable on-site screening tool for cervical cancer and precancerous lesions.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological immune detection analysis, in particular to a preparation method and application of an HPV16 / 18 type E6 / E7 protein multi-target detection test strip. BACKGROUND

[0002] Persistent infection of high-risk human papillomavirus (HPV) is the primary cause of cervical cancer and precancerous lesions, among which HPV16 and HPV18 infections contribute to more than 80% of cervical cancer cases.

[0003] Currently, HPV nucleic acid detection is the mainstream method for cervical cancer screening, which realizes typing diagnosis by detecting viral DNA sequences and has the advantage of high sensitivity. However, this method has significant limitations: on the one hand, the HPV infection rate in the population is high, but most of them are transient infections, and nucleic acid detection cannot distinguish between persistent and transient infections, resulting in a large number of false positive results and unnecessary referral and over-treatment; on the other hand, detection requires laboratory conditions and professional operation, which is costly and time-consuming, and is difficult to promote in primary medical settings. These drawbacks highlight the urgency of developing new markers that are convenient, rapid, and meet the needs of primary screening.

[0004] In-depth research shows that E6 and E7 proteins encoded by HPV play an irreplaceable core role in the carcinogenic process. E6 protein degrades P53 protein through the ubiquitination pathway, removes the cell cycle regulation restriction, and activates telomerase to maintain cell immortalization; E7 protein targets retinoblastoma protein (Rb) and promotes abnormal cell proliferation. The persistent expression of both is a key marker for the progression of HPV infection to malignant lesions. Therefore, direct detection of E6 / E7 protein in cervical cells can more accurately reflect the risk of lesions, and compared with nucleic acid detection, it can better reflect the biological effect, providing a more clinically valuable biomarker for cervical cancer screening.

[0005] Although E6 / E7 detection has obvious advantages, the existing detection methods still have significant defects. Molecular biology detection methods, including HPV16 / 18 DNA detection and E6 / E7 mRNA detection, have high sensitivity and specificity, but the former cannot distinguish the infection state and easily leads to over-diagnosis, and the latter has strict requirements for sample quality and experimental conditions, high cost, and complex operation, and is not suitable for large-scale screening; immunological analysis methods such as Western Blot and ELISA are suitable for laboratory detection, but the process is cumbersome and time-consuming; immunochromatographic test strips (such as OncoE6) are convenient and suitable for primary screening, but have limited sensitivity and are difficult to quantify; traditional fluorescent markers (such as quantum dots and lanthanide microspheres) have aggregation-induced quenching (ACQ) phenomenon, and when the markers aggregate, the fluorescence signal will significantly decay, resulting in decreased detection stability; and the existing methods cannot detect multiple targets at the same time, and require multiple experiments or complex instruments, which are time-consuming and costly; in addition, the existing detection methods generally lack internal reference correction mechanism, and due to the difference in the sampling amount of cervical exfoliated cells, the detection results are easily affected by the fluctuation of sample amount, leading to false results. These methods cannot simultaneously meet the requirements of accuracy, convenience and cost-effectiveness for primary screening, and therefore, it is urgent to develop a new type of technology with high sensitivity, quantitative ability and on-site detection adaptability. SUMMARY

[0006] The purpose of the present application is to provide a preparation method and use of a HPV16 / 18 type E6 / E7 protein multi-target detection test strip to solve the problems existing in the prior art.

[0007] To achieve the above-mentioned purpose, the present application provides the following solutions:

[0008] In a first aspect, the present application provides a HPV16 / 18 type E6 / E7 protein multi-target detection test strip, comprising a back plate, a sample pad, a binding pad, a nitrocellulose membrane and an absorption pad, the nitrocellulose membrane is located above the back plate, the sample pad and the binding pad are sequentially arranged on the left side of the back plate above the nitrocellulose membrane, the absorption pad is arranged on the right side of the back plate, the binding pad is distributed with an aggregation-induced emission (AIE) fluorescent microsphere labeled probe, and the nitrocellulose membrane is distributed with a capture antibody and an AIE solution.

[0009] Preferably, the AIE fluorescent microsphere labeled probe comprises an HPV16 E6 AIE fluorescent microsphere labeled probe, an HPV16 E7 AIE fluorescent microsphere labeled probe, an HPV18 E6 AIE fluorescent microsphere labeled probe, an HPV18 E7 AIE fluorescent microsphere labeled probe and an internal reference protein AIE fluorescent microsphere labeled probe.

[0010] Preferably, the preparation method of the AIE fluorescent microsphere labeled probe comprises the following steps:

[0011] Take four-(4-bromobenzene) ethylene, 4-methoxycarbonyl phenyl boronic acid, tetraphenylphosphonium palladium, cesium fluoride into ethylene glycol dimethyl ether solvent dissolving, heating for 24-72 h, cooling; adding water / dichloromethane solution with a volume ratio of 2:1-5:1, recovering the organic phase, drying, reducing pressure, purifying, and reducing pressure again to obtain the AIE material; dissolving the AIE material in tetrahydrofuran to obtain an AIE solution; taking polystyrene microspheres, washing and resuspending in sodium dodecyl sulfate, adding the AIE solution, ultrasonic, oscillation, centrifugation to remove the supernatant, washing, and storing in water to obtain an AIE fluorescent microsphere stock solution;

[0012] Centrifuging the AIE fluorescent microsphere stock solution, taking the supernatant, adding 4-morpholine ethanesulfonic acid and ultrasonic treatment, centrifuging and removing the supernatant; adding 4-morpholine ethanesulfonic acid and ultrasonic resuspension, adding microsphere activation liquid A and microsphere activation liquid B, room temperature oscillation, centrifugation, and removing the supernatant; adding microsphere coupling washing liquid and ultrasonic resuspension, centrifuging and removing the supernatant, adding buffer and ultrasonic resuspension; adding the corresponding labeled antibody and diluting with buffer, oscillation, centrifugation, and removing the supernatant; adding microsphere coupling blocking liquid and ultrasonic resuspension, oscillation overnight, centrifugation, and removing the supernatant; adding microsphere coupling washing liquid and washing, adding labeled microsphere storage liquid to obtain the AIE fluorescent microsphere labeled probe.

[0013] Preferably, the capture antibody is an HPV16 E6 capture antibody, an HPV16 E7 capture antibody, an HPV18 E6 capture antibody, an HPV18 E7 capture antibody, and a β-Actin internal reference protein capture antibody;

[0014] The HPV16 E6 capture antibody is used to capture HPV16 E6 protein, the HPV16 E7 capture antibody is used to capture HPV16 E7 protein, the HPV18 E6 capture antibody is used to capture HPV18 E6 protein, the HPV18 E7 capture antibody is used to capture HPV18 E7 protein, and the β-Actin internal reference protein capture antibody is used to capture the internal reference protein.

[0015] Preferably, the preparation method of the AIE solution comprises the following steps:

[0016] Take four-(4-bromobenzene) ethylene, 4-methoxycarbonyl phenyl boronic acid, tetraphenylphosphonium palladium, cesium fluoride into ethylene glycol dimethyl ether solvent dissolving, heating for 24-72 h, cooling; adding water / dichloromethane solution with a volume ratio of 2:1-5:1, recovering the organic phase, drying, reducing pressure, purifying, and reducing pressure again to obtain the AIE material; dissolving the AIE material in tetrahydrofuran to obtain an AIE solution.

[0017] In a second aspect, the present application also provides a preparation method of the HPV16 / 18 type E6 / E7 protein multi-target detection test strip, comprising the following steps:

[0018] Preparation of the nitrocellulose membrane;

[0019] Preparation of the conjugate pad;

[0020] Assembly of the test strip.

[0021] Preferably, the preparation of the nitrocellulose membrane comprises the following steps:

[0022] 10-30 nL of HPV16 E6 capture antibody, HPV16 E7 capture antibody, HPV18 E6 capture antibody, HPV18 E7 capture antibody and β-Actin internal reference protein capture antibody with a concentration of 0.1-0.9 mg / mL are spotted on the nitrocellulose membrane in an array form; 0.1-0.2 mg / mL of AIE solution is spotted on the four corners of the nitrocellulose membrane, and dried to obtain the nitrocellulose membrane.

[0023] Preferably, the preparation of the conjugate pad comprises the following steps:

[0024] Synthesis of AIE fluorescent microsphere stock solution: four-(4-bromophenyl) ethylene, 4-methoxycarbonyl phenyl boronic acid, tetrakis triphenylphosphine palladium, cesium fluoride are added to ethylene glycol dimethyl ether solvent for dissolution, heated for 24-72 h, and cooled; add water / dichloromethane solution with a volume ratio of 2:1-5:1, recover the organic phase, dry, reduce pressure, purify, and reduce pressure again to obtain AIE material; take the AIE material and dissolve it in tetrahydrofuran to obtain AIE solution; take polystyrene microspheres, wash and resuspend in sodium dodecyl sulfate, add AIE solution, ultrasonic, oscillation, centrifugal supernatant, wash, and store in water to obtain AIE fluorescent microsphere stock solution;

[0025] Preparation of AIE fluorescent microsphere labeled probe: centrifuge the AIE fluorescent microsphere stock solution, take the supernatant, add 4-morpholine ethanesulfonic acid and ultrasonic treatment, centrifuge and remove the supernatant; resuspend by ultrasonic treatment with 4-morpholine ethanesulfonic acid again, add microsphere activation liquid A and microsphere activation liquid B, oscillate at room temperature, centrifuge and remove the supernatant; resuspend by ultrasonic treatment with microsphere coupling washing liquid, centrifuge and remove the supernatant, resuspend by ultrasonic treatment with buffer; at the same time, set five groups, add HPV16 E6 antibody, HPV16 E7 antibody, HPV18 E6 antibody, HPV18 E7 antibody and β-Actin internal reference protein antibody respectively, and use buffer to constant volume, oscillate, centrifuge, and remove the supernatant; resuspend by ultrasonic treatment with microsphere coupling blocking liquid, oscillate overnight, centrifuge and remove the supernatant; wash with microsphere coupling washing liquid, and add labeled microsphere preservation solution to obtain five kinds of AIE fluorescent microsphere labeled probes;

[0026] Spray five kinds of AIE fluorescent microsphere labeled probes on the conjugate pad with a spraying amount of 1-3 μL / cm, and dry to obtain the conjugate pad.

[0027] Preferably, the preparation of the nitrocellulose membrane comprises the following steps:

[0028] 20 nL of HPV16 E6 capture antibody, HPV16 E7 capture antibody, HPV18 E6 capture antibody, HPV18 E7 capture antibody and β-Actin internal reference protein capture antibody with a concentration of 0.5 mg / mL were spotted on a nitrocellulose membrane in the form of an array with a diameter of 800 μm and a center-to-center spacing of 1000 μm, and 4 repeated spots were set for each spot; 0.15 mg / mL of AIE solution was spotted on the four corners of the nitrocellulose membrane with a diameter of 300 μm, and the nitrocellulose membrane was dried at 37°C for 2 h to obtain the nitrocellulose membrane.

[0029] Preferably, the preparation of the binding pad comprises the following steps:

[0030] Synthesis of AIE fluorescent microsphere stock solution: 100-300 mg of tetra-(4-bromophenyl) ethylene, 278-300 mg of 4-methoxycarbonyl phenylboronic acid, 35.6-50 mg of tetrakis triphenylphosphine palladium, and 609-700 mg of cesium fluoride were dissolved in 10-20 mL of ethylene glycol dimethyl ether solvent, heated for 24-72 h, and cooled; 2:1-5:1 volume ratio of water / dichloromethane solution was added, the organic phase was recovered, dried, reduced pressure, purified, and reduced pressure to obtain AIE material; the AIE material was dissolved in tetrahydrofuran to obtain an AIE solution with a concentration of 1-7 mg / mL; 0.5-1.5 mL of polystyrene microspheres with a concentration of 50-150 mg / mL was taken, washed and resuspended in 5-15 mL of sodium dodecyl sulfate, 0.5-1.5 mL of AIE solution was added, ultrasonic, oscillation, centrifugation to remove supernatant, washing, and stored in water to obtain AIE fluorescent microsphere stock solution;

[0031] Preparation of AIE fluorescent microsphere labeled probe: 50-150 muL AIE fluorescent microsphere stock solution was centrifuged, and the supernatant was taken, 0.5-1.5 mL 4-morpholine ethanesulfonic acid with a concentration of 20-30 mM was added and ultrasonically treated, centrifuged, and the supernatant was removed; 410-450 mL of 4-morpholine ethanesulfonic acid was added and ultrasonically resuspended, 5-15 muL of microsphere activation liquid A and 132-150 muL of microsphere activation liquid B were added, and the mixture was oscillated at room temperature, centrifuged, and the supernatant was removed; 0.5-1.5 mL of microsphere coupling washing liquid was added and ultrasonically resuspended, centrifuged, and the supernatant was removed, and a buffer with a pH of 7.0 was added and ultrasonically resuspended; at the same time, five groups were set, 5-15 muL of HPV16 E6 antibody, HPV16 E7 antibody, HPV18 E6 antibody, HPV18 E7 antibody and beta-Actin internal reference protein antibody with a concentration of 0.5-1.5 mg / mL were added, the buffer was added to constant volume, oscillated, centrifuged, and the supernatant was removed; 0.5-1.5 mL of microsphere coupling blocking liquid was added and ultrasonically resuspended, oscillated overnight, centrifuged, and the supernatant was removed; 0.5-1.5 mL of microsphere coupling washing liquid was added and washed, and 0.5-1.5 mL of labeled microsphere storage solution was added, to obtain five kinds of AIE fluorescent microsphere labeled probes;

[0032] The five kinds of AIE fluorescent microsphere labeled probes were sprayed on the binding pad at a spraying amount of 1-3 muL / cm, and the binding pad was dried.

[0033] The present application discloses the following technical effects:

[0034] 1. Solve the detection accuracy problem: In view of the defects of immunohistochemical method, such as dependence on pathological section, large subjective judgment error and difficulty in quantification, the present application takes beta-Actin protein as an internal reference protein. Since beta-Actin is stably expressed in cells, the simultaneous detection of E6 / E7 tumor protein and beta-Actin by double antibody sandwich method can effectively correct sample quantity difference and operation error, realize objective and accurate quantification analysis of target protein, and improve the reliability and repeatability of detection results;

[0035] 2. Solve the problem of missed detection and sample heterogeneity: In view of the problem that the design of traditional test strip detection line is easy to cause missed detection or error due to uneven distribution of target protein in sample, the present application realizes multi-point signal integration of HPV16 / 18 type E6 / E7 protein by four signal point detection, calculates the mean value of four-point fluorescence intensity, corrects sampling error, effectively offsets the influence of local concentration fluctuation of sample, and improves the detection reliability;

[0036] 3. Solve the problem of simultaneous detection of multiple targets: existing methods are difficult to detect multiple targets at the same time, and require multiple experiments or use complex instruments, which is time-consuming and costly. The present application has five detection regions, which can simultaneously quantitatively detect HPV16 E6, HPV16 E7, HPV18 E6, HPV18 E7 and beta-Actin internal reference. Each target corresponds to four signal points, and the four-point signal is read synchronously by a fluorescence quantitative analyzer, realizing "one-time sample addition, multiple target synchronous quantification";

[0037] 4. Solve the problem of insufficient performance of test strip detection technology on the market: In view of the problems that colloidal gold immunochromatography is only qualitative and has low sensitivity, and the traditional fluorescent material in fluorescent immunochromatography has the problem of aggregation quenching leading to signal weakening, the present application uses aggregation-induced emission (AIE) fluorescent microspheres as a marker. AIE materials still maintain high fluorescence quantum yield at low concentration, so even if the sample concentration is much lower than the detection limit of colloidal gold immunochromatography, stable output of weak signal can still be achieved. In addition, AIE materials have the unique property of aggregation-enhanced luminescence, which avoids the phenomenon of false negatives caused by fluorescence quenching of ordinary fluorescent substances in high-concentration samples due to aggregation state;

[0038] 5. Simplify the operation process: In view of the problems that existing mainstream detection technologies have long detection time and rely on professional equipment, which is difficult to carry out in the grassroots, the four-point positioning multi-target quantitative detection AIE fluorescent test strip developed by the present application adopts a portable design, and the operation process is simplified to two steps of sample addition and fluorescence reading. Combined with the four-point positioning fluorescence reading technology, the detection can be completed in a short time without complex instruments, making the detection process more convenient and fast, and meeting the needs of grassroots field screening;

[0039] 6. Realize accurate quantification and qualitative combination: Existing screening methods cannot simultaneously meet the needs of quantitative and qualitative detection. The present application accurately positions the signal reading area position through four-point positioning technology, and cooperates with the stable luminescence characteristics of AIE fluorescent microspheres, which can not only accurately quantify E6 / E7 protein through fluorescence intensity, but also qualitatively judge through visual observation of color development results, providing more comprehensive information for clinical diagnosis;

[0040] 7. Optimize the performance of test strips and reduce costs: In view of the problems of non-specific adsorption and inaccurate positioning of detection signal points in ordinary test strips, the present application optimizes and screens nitrocellulose membranes, selects a type with better adsorption performance and flow rate, reduces background interference, and improves detection specificity. At the same time, by using four-point positioning technology, the signal reading area position is accurately positioned, avoiding the reading error caused by strip deviation, reducing the production and use cost while ensuring the detection performance, and improving the accessibility of the technology.

[0041] The application develops a four-point positioning multi-target quantitative AIE fluorescent test strip based on beta-Actin protein as an internal reference. The method integrates multiple innovative designs: AIE molecules are used as fluorescent markers, and their unique aggregation-enhanced luminescence characteristics can avoid the aggregation quenching problem of traditional fluorescent materials, improving the detection stability; beta-Actin, which is constantly expressed in cells, is used as an internal reference protein, and the double antibody sandwich method is used to simultaneously detect target proteins and internal reference proteins, correct sampling errors, and ensure the accuracy of the results; five detection areas simultaneously detect HPV16 E6, HPV16 E7, HPV18 E6, HPV18 E7 and beta-Actin internal reference, and each target corresponds to four signal points, and the four-point signal is read synchronously by a fluorescence quantitative analyzer, realizing "one-time sampling, multi-target synchronous quantitative"; four-point positioning fluorescence reading technology is used, combined with the optimization screening of nitrocellulose membrane and the optimization of microsphere labeling process, to realize accurate positioning of signal reading area and signal amplification. The test strip has both qualitative visual interpretation and quantitative fluorescence analysis capability, and can simultaneously and rapidly detect multiple targets with wide linear range and low detection limit, and is simple to operate and low in cost, and is expected to become a new tool for efficient and reliable on-site screening of cervical cancer and precancerous lesions. BRIEF DESCRIPTION OF DRAWINGS

[0042] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed in the embodiments will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor on the basis of these drawings.

[0043] Figure 1 The structure schematic diagram of the test strip prepared by the present application is shown in the figure.

[0044] Figure 2 The AIE fluorescent microsphere characterization result graph is shown in the figure.

[0045] Figure 3 The phosphate buffer pH value optimization result graph is shown in the figure.

[0046] Figure 4 The fluorescence reading time optimization result graph is shown in the figure.

[0047] Figure 5 The stability evaluation result graph is shown in the figure.

[0048] Figure 6 The HPV16 E6 protein detection standard curve graph is shown in the figure. DETAILED DESCRIPTION

[0049] Various exemplary embodiments of the present application will now be described in detail, with reference to the drawings, which are not to be construed as limiting the application, but rather as illustrating certain aspects, features and embodiments of the application.

[0050] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. Additionally, the use of the term "about" in relation to a value or a range of values is intended to include each and every value falling within the range and each and every value falling within the range as well as the range itself. The upper and lower limits of each range are included in the range. The upper and lower limits of each range are also included in the range.

[0051] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, preferred methods and materials are described. All publications mentioned in this specification are herein incorporated by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The citation of any reference in this specification is not an admission that it is prior art.

[0052] Many modifications and variations of this application can be made without departing from its scope or spirit. Other embodiments of the application will be apparent to those skilled in the art from consideration of the specification and practice of the application disclosed herein. The specification and examples given are exemplary only.

[0053] As used herein, the terms "comprises", "comprising", "includes", "including", "has", "having", "contains", "containing", or variations thereof, are intended to be open-ended terms that mean inclusion, but not limited to, the listed material or step.

[0054] Experimental materials and reagents: Tetrahydrofuran (item number: T821373), 0.9% sodium chloride (item number: S805275), 25 mM Tris-hydroxymethyl aminomethane (item number: T819511), 5% bovine serum albumin (item number: B824162), 1% trehalose (item number: D807342), 1% sucrose (item number: S818046) are all purchased from Shanghai McLean Biotechnology Co., Ltd.; 300 nm polystyrene microspheres (item number: 83000720100290), 1% 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide (item number: 22980), cell preservation solution (item number: 14190144), cell lysis solution (item number: 87787) are all purchased from Thermo Fisher Scientific, USA; 0.25% sodium dodecyl sulfate (item number: S108350) is purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; 4-morpholine ethanesulfonic acid (MES) solution (item number: M3671), 1% N-hydroxysuccinimide (item number: 56485), 0.05% liquid biological preservative (ProClin 300, item number: 48912-U), 0.05% Tween-20 (item number: P7949) are all purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd.; HPV16 E6 antibody (item number: RVV09005), HPV16 E7 antibody (item number: RVV08910), HPV18 E6 antibody (item number: RVV09006), HPV18 E7 antibody (item number: RVV08908), HPV16 E6 capture antibody (item number: RVV09004), HPV16 E7 capture antibody (item number: RVV08909), HPV18 E6 capture antibody (item number: RVV09003), HPV18 E7 capture antibody (item number: RVV08906) are all purchased from AntibodySystem company; β-Actin internal reference protein antibody (item number: 8457), β-Actin internal reference protein capture antibody (item number: 3700) are all purchased from Cell Signaling Technology company; HPV16 L1 protein, HPV18 L1 protein, squamous cell carcinoma antigen, human epididymal protein 4 are all purchased from Pu Jian Biotechnology (Wuhan) Co., Ltd.; CN140 nitrocellulose membrane is purchased from Germany Sartorius company; Pall vivid 90 nitrocellulose membrane is purchased from USA Pall Corporation company; YNFS nitrocellulose membrane is purchased from Shantou Inobio Membrane Co., Ltd.

[0055] The main solvent formula used in the embodiment of the application is as follows:

[0056] (1) Microsphere activation solution A: 25 mM morpholineethanesulfonic acid, 1% 1-(3- dimethylaminopropyl)-3-ethylcarbodiimide, pH 6.1.

[0057] (2) Microsphere activation solution B: 25 mM morpholineethanesulfonic acid, 1% N- hydroxysuccinimide, pH 6.1.

[0058] (3) Microsphere coupling wash solution: 0.9% sodium chloride, 25 mM tris-hydroxymethyl- aminomethane, 0.05% ProClin 300, 0.05% Tween-20, pH 7.8.

[0059] (4) Microsphere coupling blocking solution: 25 mM phosphate buffer, 5% bovine serum albumin, pH 7.0.

[0060] (5) Labeled microsphere storage solution: 25 mM tris-hydroxymethyl-aminomethane, 5% bovine serum albumin, 1% trehalose, 1% sucrose, 0.9% NaCl, 0.05% Tween-20, 0.05% ProClin 300, pH 7.2.

[0061] (6) Phosphate buffer solution A (0.2 M): Weigh 31.2 g of NaH2PO4·2H2O and dissolve in ultrapure water, and make up to 1 L.

[0062] (7) Phosphate buffer solution B (0.2 M): Weigh 71.63 g of Na2HPO4·12H2O and dissolve in ultrapure water, and make up to 1 L.

[0063] (8) Phosphate buffer (0.025 M, pH 5.0): Mix 124.25 mL of phosphate buffer solution A and 0.75 mL of phosphate buffer solution B, and dilute with ultrapure water to 0.98 L. Adjust the pH of the buffer to 5.0 with 10 M HCl, and make up to 1 L with ultrapure water.

[0064] (9) Phosphate buffer (0.025 M, pH 6.0): Mix 117.50 mL of phosphate buffer solution A and 7.50 mL of phosphate buffer solution B, and dilute with ultrapure water to make up to 1 L.

[0065] (10) Phosphate buffer (0.025 M, pH 7.0): Mix 76.25 mL of phosphate buffer solution A and 48.75 mL of phosphate buffer solution B, and make up to 1 L with ultrapure water.

[0066] (11) Phosphate buffer (0.025 M, pH 8.0): 17.10 mL of phosphate buffer A and 107.90 mL of phosphate buffer B were mixed and diluted with ultrapure water to 1 L.

[0067] (12) Phosphate buffer (0.025 M, pH 9.0): 1.95 mL of phosphate buffer A and 123.05 mL of phosphate buffer B were mixed and diluted with ultrapure water to 0.98 L, the pH of the buffer was adjusted to 9.0 by 10 M NaOH, and diluted to 1 L with ultrapure water.

[0068] Example 1 Preparation of AIE fluorescent microsphere stock solution

[0069] 1. Synthesis of AIE material

[0070] Under argon protection, 200 mg of tetra-(4-bromophenyl)ethylene, 278 mg of 4-methoxycarbonyl phenylboronic acid, 35.6 mg of tetrakis triphenylphosphine palladium, and 609 mg of cesium fluoride were dissolved in 15 mL of ethylene glycol dimethyl ether solvent, heated to 92°C and kept for 48 h, and then cooled to room temperature. Then, water / dichloromethane (2 / 1, v / v) was added to the reaction mixture. The organic phase was immediately recovered and dried with anhydrous sodium sulfate, and the solvent was removed by rotary evaporation under reduced pressure. The resulting product was purified by column chromatography, and finally the solvent was removed by rotary evaporation under reduced pressure to obtain a green AIE material.

[0071] 2. Preparation of AIE fluorescent microsphere stock solution

[0072] 40 mg of AIE material was dissolved in 10 mL of tetrahydrofuran (THF) to obtain an AIE solution of 4 mg / mL. 1 mL of polystyrene microspheres (100 mg / mL) with a particle size of 300 nm was taken, washed three times, and then precipitated and resuspended in 10 mL of 0.25% sodium dodecyl sulfate. 1 mL of AIE solution was added to the resuspended microspheres, and after ultrasonic treatment for 30 min using a probe-type ultrasonic instrument, it was placed in a constant-temperature shaker at 60°C for 4 h to remove the organic solvent. After centrifugation at 18,000 rpm for 30 min, the supernatant was removed, washed three times, and stored in 1 mL of ultrapure water. The final concentration of the AIE fluorescent microsphere stock solution was 10 mg / mL, and it was stored at 4°C.

[0073] 3. Characterization of AIE fluorescent microspheres

[0074] Take 5 μL of AIE fluorescent microsphere stock solution and dilute it to 1 mL with ultrapure water. Add an appropriate amount to the surface of a carbon film copper mesh with a specification of 400 mesh. Dry it in a drying oven overnight and then test it with a transmission electron microscope (TEM). The characterization result is shown in the following figure Figure 2 It can be seen from Figure 2 that the AIE fluorescent microspheres are successfully prepared.

[0075] Example 2 Preparation of AIE fluorescent microsphere labeled probe

[0076] 1. Preparation of HPV16 E6 AIE fluorescent microsphere labeled probe

[0077] First, centrifuge 100 μL of AIE fluorescent microsphere stock solution (10 mg / mL) prepared in Example 1 at 15,000 rpm for 15 min, remove the supernatant, add 1 mL of 25 mM 4-morpholine ethanesulfonic acid (MES) solution, treat it under ultrasonic frequency of 40 Khz for 0.5 min, centrifuge it at 15,000 rpm for 15 min, and discard the supernatant. Second, add 430 μL of 25 mM 4-morpholine ethanesulfonic acid (MES) solution and resuspend it under ultrasonic frequency, then add 10 μL of microsphere activation solution A and 132 μL of microsphere activation solution B, and oscillate it in a circular shaker at room temperature for 30 min. Centrifuge it at 18,000 rpm for 30 min, discard the supernatant, add 1 mL of microsphere coupling washing solution and resuspend it under ultrasonic frequency, then centrifuge it at 18,000 rpm for 30 min, discard the supernatant, and repeat the process twice. Add 200 μL of phosphate buffer (0.025 M, pH 7.0) and resuspend it under ultrasonic frequency. Add 10 μL of 1 mg / mL HPV16 E6 antibody, and then add phosphate buffer (0.025 M, pH 7.0) to a total volume of 500 μL, oscillate it in a circular shaker at room temperature for 2 h, centrifuge it at 18,000 rpm for 30 min, and discard the supernatant. Add 1 mL of microsphere coupling blocking solution and resuspend it under ultrasonic frequency, oscillate it in a circular shaker at room temperature overnight, centrifuge it at 18,000 rpm for 30 min, and discard the supernatant. Add 1 mL of microsphere coupling washing solution twice, then add 1 mL of labeled microsphere storage solution, and store it at 4°C for later use.

[0078] 2. Preparation of HPV16 E7 AIE fluorescent microsphere labeled probe

[0079] The only difference between the preparation of HPV16 E6 AIE fluorescent microsphere labeled probe in Example 2 and the preparation of HPV16 E7 AIE fluorescent microsphere labeled probe is that the HPV16 E6 antibody is replaced by the HPV16 E7 antibody.

[0080] 3. Preparation of HPV18 E6 AIE fluorescent microsphere-labeled probe

[0081] The only difference between this example and "1. Preparation of HPV16 E6 AIE fluorescent microsphere-labeled probe" in Example 2 is that the HPV16 E6 antibody is replaced by the HPV18 E6 antibody.

[0082] 4. Preparation of HPV18 E7 AIE fluorescent microsphere-labeled probe

[0083] The only difference between this example and "1. Preparation of HPV16 E6 AIE fluorescent microsphere-labeled probe" in Example 2 is that the HPV16 E6 antibody is replaced by the HPV18 E7 antibody.

[0084] 5. Preparation of internal reference protein AIE fluorescent microsphere-labeled probe

[0085] The only difference between this example and "1. Preparation of HPV16 E6 AIE fluorescent microsphere-labeled probe" in Example 2 is that the HPV16 E6 antibody is replaced by the β-Actin internal reference protein antibody.

[0086] Example 3 Preparation and application of lateral flow immunoassay (LFA) test strips

[0087] 1. Preparation of lateral flow immunoassay (LFA) test strips

[0088] Using a non-contact microarray spotter, 20 nL of HPV16 E6 capture antibody (for capturing HPV16 E6 protein), HPV16 E7 capture antibody (for capturing HPV16 E7 protein), HPV18 E6 capture antibody (for capturing HPV18 E6 protein), HPV18 E7 capture antibody (for capturing HPV18 E7 protein), and β-Actin internal reference protein capture antibody (for capturing β-Actin protein) with a concentration of 0.5 mg / mL were spotted on a CN140 nitrocellulose membrane in an array format with a diameter of 800 μm and a center-to-center spacing of 1000 μm, and 4 repeated spots were set for each spot. Using a non-contact microarray spotter, 0.15 mg / mL of AIE solution was spotted on the four corners of the nitrocellulose membrane with a diameter of 300 μm. The spotted nitrocellulose membrane was placed in a blast drying oven and dried at 37°C for 2 h, and then stored in a dry cabinet for standby.

[0089] Five kinds of 1 mg / mL AIE fluorescent microsphere-labeled probe solutions were sprayed on the conjugate pad at a spraying amount of 2 μL / cm, and then they were placed in a vacuum drying oven at 37°C for 4 h, and then stored in a dry cabinet for standby.

[0090] The sample pad, the binding pad, the nitrocellulose membrane and the absorption pad are pasted on the back plate to prepare the test strip, wherein the nitrocellulose membrane is located above the back plate, the sample pad and the binding pad are sequentially arranged on the left side of the back plate, and the absorption pad is arranged on the right side of the back plate, then the test strip is cut into a width of 5.0 mm by a paper cutter. Finally, each test strip is inserted into a two-piece plastic box and placed in a self-sealing bag containing a desiccant, and the LFA test strip is obtained after sealing and storing. The schematic diagram of the test strip structure can be seen Figure 1 .

[0091] 2. The use method of the lateral flow immunoassay (LFA) test strip

[0092] After the cervical swab sampling, it is placed in a centrifuge tube containing 2 mL of cell preservation solution, oscillated for 1 min, and then centrifuged at 13000 r / min for 1 min to discard the supernatant. Then 1 mL of cell lysis solution is added, oscillated for 10 min, and then centrifuged at 8000 r / min for 5 min. The supernatant is taken as a sample for detection. The finished test strip is placed in a 37°C constant temperature incubator, 100 μL of the sample to be tested is added to the sample addition hole of the finished test strip, and after incubation for 15 min, the fluorescence signals of the test strip T (detection line) and C (quality control line) are read by the test strip fluorescence reader.

[0093] Result interpretation:

[0094] 1) Positive: color development of detection line and quality control line;

[0095] 2) Negative: only the quality control line develops color;

[0096] 3) Invalid: no color development of the quality control line, invalid detection, and it is recommended to take another detection card for re-detection.

[0097] 3. Preparation process optimization of LFA test strip

[0098] 3.1 Taking HPV16 E6 AIE fluorescent microsphere labeled probe as an example, optimization of pH value of phosphate buffer

[0099] Selecting phosphate buffer with pH values of 5, 6, 7, 8 and 9, prepare the AIE fluorescent microsphere labeled probe according to the preparation process in Example 2 above, and compare the average value of the fluorescence value to obtain the best pH value.

[0100] The experimental result graph can be seen Figure 3 . From Figure 3 The results show that the HPV16 E6 AIE fluorescent microsphere labeled probe prepared by selecting the phosphate buffer with pH 7 is used for subsequent research, and the fluorescence intensity is the best, therefore, pH 7 is selected as the best pH of the phosphate buffer.

[0101] Meanwhile, the optimal pH of phosphate buffer corresponding to different fluorescent microsphere-labeled probes was verified, and the detection results are shown in Table 1.

[0102] Table 1 Optimal pH of phosphate buffer corresponding to different fluorescent microsphere-labeled probes

[0103]

[0104] 3.2 Taking HPV16 E6 AIE fluorescent microsphere-labeled probe as an example, optimization of types of nitrocellulose membrane

[0105] Three types of nitrocellulose membranes, CN140, Pall vivid 90 and YNFS, were selected to prepare test strips according to the above preparation process. According to the non-specific adsorption in the negative sample and the average fluorescence value of the sample containing 200 ng / mL of HPV16 E6 protein, the optimal nitrocellulose membrane was screened. The results are shown in Table 2.

[0106] Table 2 Experimental results of different nitrocellulose membranes

[0107]

[0108] According to Table 2, CN140 and Pall vivid 90 nitrocellulose membranes did not produce non-specific adsorption in the negative sample, and there was no noise signal. The fluorescence intensity of CN140 nitrocellulose membrane was higher in the sample containing HPV16 E6 protein. Therefore, CN140 nitrocellulose membrane was the optimal nitrocellulose membrane.

[0109] 3.3 Taking HPV16 E6 AIE fluorescent microsphere-labeled probe as an example, optimization of probe antibody and capture antibody input

[0110] HPV16 E6 antibody was selected as 1 μL, 5 μL, 10 μL, 15 μL and 20 μL. HPV16 E6 capture antibody was diluted to 0.25-1 mg / mL with phosphate buffer. The sample containing 200 ng / mL of HPV16 E6 protein was prepared according to the above test strip preparation process and detected according to the above detection process. The average value of the signal point fluorescence value obtained by the test strip reader was compared to obtain the optimal monoclonal antibody input and capture antibody input. The detection results corresponding to the input optimization system are shown in Table 3.

[0111] Table 3 Experimental results of input optimization

[0112]

[0113] According to the results in Table 3, it can be seen that when the HPV16 E6 antibody is 10 μL and the HPV16 E6 capture antibody is 0.5 mg / mL, the fluorescence intensity is the highest, and therefore, they are the optimal monoclonal antibody input amount and the optimal capture antibody input amount, respectively.

[0114] On this basis, the optimal input amount of different fluorescent microsphere-labeled probes was verified, and the results are shown in Table 4.

[0115] Table 4 Optimal input amount of different fluorescent microsphere-labeled probes

[0116]

[0117] 3.4 Taking the HPV16 E6 AIE fluorescent microsphere-labeled probe as an example, the optimization of the fluorescence reading time

[0118] According to the detection process of the immunochromatographic test strip, the fluorescence intensity of the T line within 30 min after sample addition of the test strip was monitored. The results are shown in Figure 4 .

[0119] Figure 4 It is shown that taking the HPV16 E6 AIE fluorescent microsphere-labeled probe as an example, when the test strip is added with sample for 15 min, the fluorescence intensity of the T line reaches the highest, and after that, the fluorescence intensity remains stable, and it can be seen that the optimal reading time is 15 min.

[0120] On this basis, the optimal fluorescence reading time corresponding to different fluorescent microsphere-labeled probes was verified, and the results are shown in Table 5.

[0121] Table 5 Optimal fluorescence reading time

[0122]

[0123] It can be seen that the optimal fluorescence reading time of the test strip prepared in the application corresponding to different fluorescent microsphere-labeled probes is 15 min.

[0124] Example 4 Performance evaluation of the LFA detection test strip

[0125] 1. Precision evaluation

[0126] The samples to be tested with the HPV16 E6 protein concentration of 5 ng / mL, 50 ng / mL, 100 ng / mL, 200 ng / mL and 300 ng / mL were detected, and three batches of test strips were used for repeated detection for 5 times, respectively, and the batch and inter-batch variation coefficients were calculated, respectively.

[0127] The test samples with the concentration of HPV16 E7 protein of 5 ng / mL, 50 ng / mL, 100 ng / mL, 200 ng / mL and 300 ng / mL were detected, and three batches of test strips were used to repeat the detection for 5 times, and the intra-batch and inter-batch coefficients of variation were calculated respectively;

[0128] The test samples with the concentration of HPV18 E6 protein of 5 ng / mL, 50 ng / mL, 100 ng / mL, 200 ng / mL and 300 ng / mL were detected, and three batches of test strips were used to repeat the detection for 5 times, and the intra-batch and inter-batch coefficients of variation were calculated respectively;

[0129] The test samples with the concentration of HPV18 E7 protein of 5 ng / mL, 50 ng / mL, 100 ng / mL, 200 ng / mL and 300 ng / mL were detected, and three batches of test strips were used to repeat the detection for 5 times, and the intra-batch and inter-batch coefficients of variation were calculated respectively;

[0130] The test samples with the concentration of 0.2 µg / mL, 2 µg / mL, 3 µg / mL and 4 µg / mL of the internal reference protein were detected, and three batches of test strips were used to repeat the detection for 5 times, and the intra-batch and inter-batch coefficients of variation were calculated respectively.

[0131] The results are shown in Tables 6 and 7.

[0132] Table 6 Coefficients of variation for detecting different concentrations of target proteins

[0133]

[0134] Table 7 Coefficients of variation for detecting different concentrations of internal reference proteins

[0135]

[0136] According to the results in Tables 6 and 7, it can be seen that the precision of the test strip is good, and all the CVs are less than or about equal to 10%.

[0137] 2. Accuracy evaluation

[0138] The test samples with the concentration of HPV16 E6 protein of 5 ng / mL, 50 ng / mL, 100 ng / mL, 200 ng / mL and 300 ng / mL were detected, and the recovery rate was calculated to evaluate the accuracy of the test strip;

[0139] The test samples with the concentration of HPV16 E7 protein of 5 ng / mL, 50 ng / mL, 100 ng / mL, 200 ng / mL and 300 ng / mL were detected, and the recovery rate was calculated to evaluate the accuracy of the test strip;

[0140] The test sample with the concentration of HPV18 E6 protein of 5 ng / mL, 50 ng / mL, 100 ng / mL, 200 ng / mL, and 300 ng / mL was detected, and the recovery rate was calculated to evaluate the accuracy of the test strip.

[0141] The test sample with the concentration of HPV18 E7 protein of 5 ng / mL, 50 ng / mL, 100 ng / mL, 200 ng / mL, and 300 ng / mL was detected, and the recovery rate was calculated to evaluate the accuracy of the test strip.

[0142] The test sample with the concentration of 0.2 µg / mL, 2 µg / mL, 3 µg / mL, and 4 µg / mL of the internal reference protein was detected, and the recovery rate was calculated to evaluate the accuracy of the test strip.

[0143] The results are shown in Tables 8 and 9.

[0144] Table 8 Recovery rate of detection of different concentrations of target proteins

[0145]

[0146] Table 9 Recovery rate of detection of different concentrations of internal reference proteins

[0147]

[0148] According to the results in Tables 8 and 9, it can be seen that the accuracy of the test strip is good, and the recovery rate is between 90% and 110%.

[0149] 3. Specificity evaluation

[0150] The mixed sample 1, mixed sample 2, mixed sample 3, mixed sample 4, mixed sample 5, and mixed sample 6 were detected according to the test strip detection process described in Example 3 to evaluate the specificity of the test strip.

[0151] Mixed sample 1: HPV16 L1 protein (200 ng / mL), HPV18 L1 protein (200 ng / mL), squamous cell carcinoma antigen (200 ng / mL), and human epididymal protein 4 (200 ng / mL);

[0152] Mixed sample 2: HPV16 E6 protein (200 ng / mL), HPV16 L1 protein (200 ng / mL), HPV18 L1 protein (200 ng / mL), squamous cell carcinoma antigen (200 ng / mL), and human epididymal protein 4 (200 ng / mL);

[0153] Mixed sample 3: HPV16 E7 protein (200 ng / mL), HPV16 L1 protein (200 ng / mL), HPV18 L1 protein (200 ng / mL), squamous cell carcinoma antigen (200 ng / mL), human epididymis protein 4 (200 ng / mL);

[0154] Mixed sample 4: HPV18 E6 protein (200 ng / mL), HPV16 L1 protein (200 ng / mL), HPV18 L1 protein (200 ng / mL), squamous cell carcinoma antigen (200 ng / mL), human epididymis protein 4 (200 ng / mL);

[0155] Mixed sample 5: HPV18 E7 protein (200 ng / mL), HPV16 L1 protein (200 ng / mL), HPV18 L1 protein (200 ng / mL), squamous cell carcinoma antigen (200 ng / mL), human epididymis protein 4 (200 ng / mL);

[0156] Mixed sample 6: internal reference protein (2 µg / mL), HPV16 L1 protein (200 ng / mL), HPV18 L1 protein (200 ng / mL), squamous cell carcinoma antigen (200 ng / mL), human epididymis protein 4 (200 ng / mL).

[0157] The results are shown in Table 10.

[0158] Table 10. Specificity evaluation results of different mixed samples

[0159]

[0160] According to the results in Table 10, it can be seen that the test strip has good specificity and no obvious cross reaction with common detection proteins of cervical cancer in clinic.

[0161] 4. Stability evaluation

[0162] The assembled LFA test strip was placed in a 55℃ oven for 20 days, and during this process, the test strip was taken out regularly, the mixed sample of HPV16 E6 / E7 protein and HPV18 E6 / E7 protein at 200 ng / mL and internal reference protein at 4 µg / mL was added to the sample hole, the fluorescence value was recorded, and the aging storage results of the test strip were plotted.

[0163] The results are shown in Figure 5 . According to Figure 5 , it can be seen that the fluorescence intensity of the test target protein and the internal reference protein of the test strip does not change under the above conditions for 20 days, that is, the LFA test strip prepared in the application can be stored for at least 20 days under the above conditions.

[0164] 5. Feasibility of internal reference correction of sampling amount

[0165] Five cervical cancer samples positive for HPV16 and HPV18 were taken and numbered 1-10, and were added with collagenase for treatment, cell suspensions were prepared and cell counting was performed; 10 samples were taken from each patient's cervical cancer cell suspension, and the number of cells was 4000, 6000, 8000, 10000, 12000, 14000, 16000, 18000, 20000 and 22000, respectively; after the samples were treated, they were added for detection, and after the detection results of each group were corrected by β-Actin protein, the corrected results were analyzed to verify the feasibility of β-Actin protein as an internal reference for correcting sampling errors in the present application. The results are shown in Table 11.

[0166] Table 11. Feasibility analysis results of internal reference correction of sampling amount

[0167]

[0168] According to the results in Table 11, it can be seen that the use of β-Actin protein as an internal reference for correcting the error of the sampling amount of the test strip has feasibility, and the CV is less than 10%, which can ensure the accuracy of the detection results.

[0169] 6. Limit of detection evaluation

[0170] Taking the HPV16 E6 AIE fluorescent microsphere labeled probe as an example, samples with HPV16 E6 protein concentrations of 5, 10, 20, 40, 80, 160 and 240 ng / mL were set, and each sample was detected repeatedly for 3 times, and the average value of the detection fluorescence intensity value was calculated. Taking the HPV16 E6 protein concentration as the abscissa and the T fluorescence intensity value as the ordinate, a standard curve equation (y=145.01x+150.55) was fitted by linear regression analysis, and the standard curve graph is shown in Figure 6 .

[0171] The negative sample was repeatedly measured for 20 times, the average value and the standard deviation of the fluorescence intensity value of the 20 detection results were calculated, the average value+3 times the standard deviation was substituted into the above linear equation, and the corresponding concentration value was obtained, which was the minimum detection limit of the test strip for the substance. The minimum detection limit results of different target proteins measured by this method are shown in Table 12.

[0172] Table 12. Minimum detection limit results of different target proteins

[0173]

[0174] According to the results in Table 12, the minimum detection limit of the test strip for HPV16 E6 protein is 0.65 ng / mL, the minimum detection limit of the test strip for HPV16 E7 protein is 0.91 ng / mL, the minimum detection limit of the test strip for HPV18 E6 protein is 0.77 ng / mL, and the minimum detection limit of the test strip for HPV18 E7 protein is 1.13 ng / mL.

[0175] 7. Clinical experiment

[0176] Seven samples of patients diagnosed by clinical pathology as cervical cancer (six samples of squamous cell carcinoma (SCC) and one sample of cervical adenocarcinoma (AC)), ten samples of patients with high-grade squamous intraepithelial lesion (HSIL), twelve samples of patients with low-grade squamous intraepithelial lesion (LSIL), and twenty samples of patients with negative HPV DNA were detected by the test strip prepared in the application. The experimental results are shown in Table 13.

[0177] Table 13. Results of the clinical experiment

[0178]

[0179] As shown in Table 13, the sensitivity of the test strip prepared in the application for screening cervical cancer is 100%, the sensitivity of the test strip for screening cervical cancer and HSIL is 88.2%, the sensitivity of the test strip for screening cervical cancer, HSIL and LSIL is 79.3%, and the specificity of the test strip is 90.0%.

[0180] The above-described embodiments are only used to describe the preferred modes of the application, and do not limit the scope of the application. Without departing from the design spirit of the application, various modifications and improvements to the technical solutions of the application made by those skilled in the art shall fall within the protection scope of the claims of the application.

Claims

1. A HPV16 / 18 type E6 / E7 protein multi-target detection test strip, comprising a back plate, a sample pad, a binding pad, a nitrocellulose membrane and an absorption pad, the nitrocellulose membrane is above the back plate, the sample pad and the binding pad are sequentially arranged on the left side of the back plate above the nitrocellulose membrane, and the absorption pad is arranged on the right side of the back plate, characterized in that, The AIE fluorescent microsphere labeled probe is distributed on the binding pad, and the capture antibody and the AIE solution are distributed on the nitrocellulose membrane; The AIE fluorescent microsphere labeled probe includes an HPV16 E6 AIE fluorescent microsphere labeled probe, an HPV16 E7 AIE fluorescent microsphere labeled probe, an HPV18 E6 AIE fluorescent microsphere labeled probe, an HPV18 E7 AIE fluorescent microsphere labeled probe and an internal reference protein AIE fluorescent microsphere labeled probe; The preparation method of the AIE fluorescent microsphere labeled probe includes the following steps: Take four-(4-bromophenyl) ethylene, 4-methoxycarbonyl phenyl boronic acid, tetraphenylphosphonium palladium, cesium fluoride, dissolve in ethylene glycol dimethyl ether solvent, heat for 24-72 h, cool; add water / dichloromethane solution with a volume ratio of 2:1-5:1, recover the organic phase, dry, reduce pressure, purify, and reduce pressure again to obtain AIE material; take the AIE material and dissolve it in tetrahydrofuran to obtain an AIE solution; take polystyrene microspheres, wash and resuspend them in sodium dodecyl sulfate, add the AIE solution, ultrasonic, oscillation, centrifugal to remove supernatant, wash, and store in water to obtain an AIE fluorescent microsphere stock solution; Take the AIE fluorescent microsphere stock solution, centrifugal, take the supernatant, add 4-morpholine ethanesulfonic acid and ultrasonic treatment, centrifugal, remove the supernatant; add 4-morpholine ethanesulfonic acid and ultrasonic resuspension, add microsphere activation liquid A and microsphere activation liquid B, room temperature oscillation, centrifugal, remove the supernatant; add microsphere coupling washing liquid and ultrasonic resuspension, centrifugal, remove the supernatant, add buffer and ultrasonic resuspension; add the corresponding labeled antibody and dilute with buffer, oscillation, centrifugal, remove the supernatant; add microsphere coupling blocking liquid and ultrasonic resuspension, oscillate overnight, centrifugal, remove the supernatant; add microsphere coupling washing liquid and wash, and then add labeled microsphere storage solution to obtain the AIE fluorescent microsphere labeled probe.

2. The HPV 16 / 18 type E6 / E7 protein multi-target test strip according to claim 1, characterized in that, The capture antibody is an HPV16 E6 capture antibody, an HPV16 E7 capture antibody, an HPV18 E6 capture antibody, an HPV18 E7 capture antibody and a beta-Actin internal reference protein capture antibody; The HPV16 E6 capture antibody is used for capturing HPV16 E6 protein, the HPV16 E7 capture antibody is used for capturing HPV16 E7 protein, the HPV18 E6 capture antibody is used for capturing HPV18 E6 protein, the HPV18 E7 capture antibody is used for capturing HPV18 E7 protein, and the beta-Actin internal reference protein capture antibody is used for capturing internal reference protein. 3.The HPV 16 / 18 type E6 / E7 protein multi-target test strip according to claim 1, characterized in that, The preparation method of the AIE solution includes the following steps: Take four-(4-bromophenyl) ethylene, 4-methoxycarbonyl phenyl boronic acid, tetraphenylphosphonium palladium, cesium fluoride, dissolve in ethylene glycol dimethyl ether solvent, heat for 24-72 h, cool; add water / dichloromethane solution with a volume ratio of 2:1-5:1, recover the organic phase, dry, reduce pressure, purify, and reduce pressure again to obtain AIE material; take the AIE material and dissolve it in tetrahydrofuran to obtain an AIE solution.

4. A method for preparing the HPV 16 / 18 type E6 / E7 protein multi-target test strip according to any one of claims 1-3, characterized in that, The preparation method of the AIE solution includes the following steps: The preparation of the nitrocellulose membrane; The preparation of the binding pad; The assembly of the test strip.

5. The preparation method according to claim 4, characterized in that, The preparation of the nitrocellulose membrane includes the following steps: The preparation of the nitrocellulose membrane includes the following steps: Spot 10-30 nL of HPV16 E6 capture antibody, HPV16 E7 capture antibody, HPV18 E6 capture antibody, HPV18 E7 capture antibody and beta-Actin internal protein capture antibody with concentration of 0.1-0.9 mg / mL on nitrocellulose membrane in array form; spot 0.1-0.2 mg / mL of AIE solution on four corners of the nitrocellulose membrane, dry to obtain.

6. The preparation method according to claim 4, characterized in that, The preparation of the binding pad comprises the following steps: Synthesis of AIE fluorescent microsphere stock solution: take tetrakis-(4-bromophenyl) ethylene, 4-methoxycarbonyl phenyl boronic acid, tetrakis triphenyl phosphine palladium, cesium fluoride, and dissolve them in ethylene glycol dimethyl ether solvent, heat for 24-72 h, cool; add water / dichloromethane solution with a volume ratio of 2:1-5:1, recover the organic phase, dry, reduce pressure, purify, and reduce pressure again to obtain AIE material; dissolve the AIE material in tetrahydrofuran to obtain AIE solution; take polystyrene microspheres, wash and resuspend them in sodium dodecyl sulfate, add AIE solution, ultrasonic, oscillation, centrifugal to remove supernatant, wash, and store in water to obtain AIE fluorescent microsphere stock solution; Preparation of AIE fluorescent microsphere labeled probe: centrifuge the AIE fluorescent microsphere stock solution, take the supernatant, ultrasonic treatment with 4-morpholine ethanesulfonic acid, centrifugal to remove the supernatant; resuspend with 4-morpholine ethanesulfonic acid again, add microsphere activation liquid A and microsphere activation liquid B, room temperature oscillation, centrifugal to remove the supernatant; resuspend with microsphere coupling washing liquid, centrifugal to remove the supernatant, resuspend with buffer; at the same time, set five groups, add HPV16 E6 antibody, HPV16 E7 antibody, HPV18 E6 antibody, HPV18 E7 antibody and beta-Actin internal protein antibody respectively, and use buffer to constant volume, oscillation, centrifugal to remove the supernatant; resuspend with microsphere coupling blocking liquid, oscillate overnight, centrifugal to remove the supernatant; wash with microsphere coupling washing liquid, and add labeled microsphere storage solution to obtain five kinds of AIE fluorescent microsphere labeled probes; Spray five kinds of AIE fluorescent microsphere labeled probes on the binding pad with a spraying amount of 1-3 μL / cm, and dry to obtain the binding pad.

7. The preparation method according to claim 5, characterized in that, The preparation of the nitrocellulose membrane comprises the following steps: Spot 20 nL of HPV16 E6 capture antibody, HPV16 E7 capture antibody, HPV18 E6 capture antibody, HPV18 E7 capture antibody and beta-Actin internal protein capture antibody with concentration of 0.5 mg / mL on nitrocellulose membrane in array form with a diameter of 800 μm and a center distance of 1000 μm, and set 4 repeated points for each point; spot 0.15 mg / mL of AIE solution on four corners of the nitrocellulose membrane with a diameter of 300 μm, dry the nitrocellulose membrane at 37℃ for 2 h after spotting to obtain the nitrocellulose membrane.

8. The preparation method according to claim 6, characterized in that, The preparation of the binding pad comprises the following steps: Synthesis of AIE fluorescent microsphere stock solution: 100-300 mg of tetra-(4-bromophenyl) ethylene, 278-300 mg of 4-methoxycarbonyl phenyl boronic acid, 35.6-50 mg of tetrakis triphenylphosphine palladium, 609-700 mg of cesium fluoride were dissolved in 10-20 mL of ethylene glycol dimethyl ether solvent, heated for 24-72 h, and cooled; a water / dichloromethane solution with a volume ratio of 2:1-5:1 was added, the organic phase was recovered, dried, reduced pressure, purified, and reduced pressure to obtain the AIE material; the AIE material was dissolved in tetrahydrofuran to obtain an AIE solution with a concentration of 1-7 mg / mL; 0.5-1.5 mL of polystyrene microspheres with a concentration of 50-150 mg / mL were taken, washed, and resuspended in 5-15 mL of sodium dodecyl sulfate, 0.5-1.5 mL of AIE solution was added, ultrasonic, oscillation, centrifugation to remove supernatant, washing, and storage in water to obtain AIE fluorescent microsphere stock solution; Preparation of AIE fluorescent microsphere labeled probe: 50-150 μL of AIE fluorescent microsphere stock solution was centrifuged, the supernatant was taken, 0.5-1.5 mL of 4-morpholine ethanesulfonic acid with a concentration of 20-30 mM was added and ultrasonically treated, centrifuged, and the supernatant was removed; 410-450 mL of 4-morpholine ethanesulfonic acid was added and resuspended by ultrasonication, 5-15 μL of microsphere activation liquid A and 132-150 μL of microsphere activation liquid B were added, and the mixture was oscillated at room temperature, centrifuged, and the supernatant was removed; 0.5-1.5 mL of microsphere coupling washing liquid was added and resuspended by ultrasonication, centrifuged, and the supernatant was removed, and 4-morpholine ethanesulfonic acid was added and resuspended by ultrasonication; at the same time, five groups were set, 5-15 μL of HPV16 E6 antibody, HPV16 E7 antibody, HPV18 E6 antibody, HPV18 E7 antibody, and β-Actin internal reference protein antibody with a concentration of 0.5-1.5 mg / mL were added respectively, the buffer was added to constant volume, oscillated, centrifuged, and the supernatant was removed; 0.5-1.5 mL of microsphere coupling blocking liquid was added and resuspended by ultrasonication, oscillated overnight, centrifuged, and the supernatant was removed; 0.5-1.5 mL of microsphere coupling washing liquid was added and washed, and 0.5-1.5 mL of labeled microsphere storage solution was added to obtain five kinds of AIE fluorescent microsphere labeled probes; The five kinds of AIE fluorescent microsphere labeled probes were sprayed on the binding pad at a spraying amount of 1-3 μL / cm, and dried to obtain the binding pad.

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