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

Abstract

The application 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 immune detection and analysis. The 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 capture antibodies and AIE solution are distributed on the nitrocellulose membrane. The preparation method comprises three steps of preparation of the nitrocellulose membrane, preparation of the combination pad and assembly of the test strip. The test strip can quantitatively detect PV16 E6, HPV16 E7, HPV18 E6 and HPV18 E7 simultaneously, four signal points correspond to each target, multi-target synchronous rapid detection is realized, the linear range is wide, the detection limit is low, the operation is simple and convenient, the cost is low, and the test strip is expected to become a new tool for efficient and reliable on-site screening of 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, tetrakis triphenyl phosphine 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 AIE material; taking AIE material dissolving in tetrahydrofuran to obtain AIE solution; taking polystyrene microspheres, washing and resuspending in sodium dodecyl sulfate, adding AIE solution, ultrasonic, oscillation, centrifugation to remove supernatant, washing, and storing in water to obtain AIE fluorescent microsphere stock solution;

[0012] Take AIE fluorescent microsphere stock solution, centrifugation, take supernatant, add 4-morpholine ethanesulfonic acid, ultrasonic treatment, centrifugation, and remove supernatant; add 4-morpholine ethanesulfonic acid again, ultrasonic resuspension, add microsphere activation solution A and microsphere activation solution B, room temperature oscillation, centrifugation, and remove supernatant; add microsphere coupling washing solution, ultrasonic resuspension, centrifugation, remove supernatant, add buffer, ultrasonic resuspension; add corresponding labeled antibody, and add buffer to constant volume, oscillation, centrifugation, and remove supernatant; add microsphere coupling blocking solution, ultrasonic resuspension, oscillation overnight, centrifugation, and remove supernatant; add microsphere coupling washing solution, wash again, and add labeled microsphere storage solution to obtain AIE fluorescent microsphere labeled probe.

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

[0014] 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 β-Actin internal reference protein capture antibody is used for capturing 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, tetrakis triphenyl phosphine 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 AIE material; taking AIE material dissolving in tetrahydrofuran to obtain 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 probes: 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 the present disclosure is intended to mean that quantities, dimensions, and other specified values need not be exact, but can be approximations. 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, the preferred methods and materials are described. All publications mentioned herein are incorporated by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. In case of conflict, the content of the present specification will control.

[0051] Unless otherwise indicated, 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, the preferred methods and materials are described. All publications mentioned herein are incorporated by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. In case of conflict, the present specification will control.

[0052] Various modifications and changes can be made to the specific embodiments of the present application described herein without departing from the scope or spirit of the application. Other embodiments of the application will be apparent to those of ordinary skill in the art from the description and examples presented herein. The description and examples are illustrative of the application and are not intended to limit the scope of the application.

[0053] As used herein, the terms "comprise", "comprising", "include", "including", "have", "having" and the like are open-ended and do not exclude additional elements or steps.

[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 4-morpholine ethanesulfonic acid, 1% 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, pH 6.1.

[0057] (2) Microsphere activation solution B: 25mM 4-morpholine ethanesulfonic acid, 1% N-hydroxysuccinimide, pH 6.1.

[0058] (3) Microsphere coupling washing 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 preservation 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 NaH2PO4・2H2O and dissolve it in ultrapure water, then make up to 1 L.

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

[0063] (8) Phosphate buffer (0.025 M, pH 5.0): Take 124.25 mL of phosphate buffer A and 0.75 mL of phosphate buffer B, mix them, and dilute with ultrapure water to 0.98 L. Adjust the pH of the buffer to 5.0 with 10 M HCl, and bring the volume to 1 L with ultrapure water.

[0064] (9) Phosphate buffer (0.025 M, pH 6.0): Take 117.50 mL of phosphate buffer A and 7.50 mL of phosphate buffer B, mix them, dilute with ultrapure water, and bring the volume to 1 L.

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

[0066] (11) Phosphate buffer (0.025 M, pH 8.0): Take 17.10 mL of phosphate buffer A and 107.90 mL of phosphate buffer B, mix them, dilute with ultrapure water, and bring the volume to 1 L.

[0067] (12) Phosphate buffer (0.025 M, pH 9.0): Take 1.95 mL of phosphate buffer A and 123.05 mL of phosphate buffer B, mix them, and dilute with ultrapure water to 0.98 L. Adjust the pH of the buffer to 9.0 with 10 M NaOH, and bring the volume to 1 L with ultrapure water.

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

[0069] 1. Synthesis of AIE materials

[0070] Under argon protection, 200 mg of tetra-(4-bromophenyl)ethylene, 278 mg of 4-methoxycarbonylphenylboronic acid, 35.6 mg of tetrakis(triphenylphosphine)palladium, and 609 mg of cesium fluoride were dissolved in 15 mL of ethylene glycol dimethyl ether. The mixture was heated to 92 °C and maintained for 48 h, then cooled to room temperature. Water / dichloromethane (2 / 1, v / v) was then added to the reaction mixture. The organic phase was immediately recovered and dried over anhydrous sodium sulfate. The solvent was removed under reduced pressure using a rotary evaporator. The resulting product was purified by column chromatography, and the solvent was finally removed under reduced pressure using a rotary evaporator to obtain the 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) solution to obtain an AIE solution with a concentration of 4 mg / mL. 1 mL of 300 nm polystyrene microspheres (100 mg / mL) was taken, washed three times, and the precipitate was resuspended in 10 mL of 0.25% sodium dodecyl sulfate. 1 mL of AIE solution was added to the resuspended microspheres, and the mixture was sonicated for 30 min using a probe sonicator. The mixture was then 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, and the mixture was washed three times. 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] 5 μL of AIE fluorescent microsphere stock solution was diluted to 1 mL with ultrapure water. An appropriate amount was then added dropwise to the surface of a 400-mesh carbon film copper mesh. After drying overnight in a forced-air drying oven, the solution was analyzed using a transmission electron microscope (TEM). The characterization results are shown in the figure below. Figure 2 .Depend on Figure 2 It can be concluded that AIE fluorescent microspheres have been successfully prepared.

[0075] Example 2: Preparation of AIE fluorescent microsphere labeled probes

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

[0077] First, take 100 μL of the AIE fluorescent microsphere stock solution (10 mg / mL) prepared in Example 1, centrifuge at 15,000 rpm for 15 min, remove the supernatant, add 1 mL of 25 mM 4-morpholinoethanesulfonic acid (MES) solution, treat at an ultrasonic frequency of 40 kHz for 0.5 min, centrifuge at 15,000 rpm for 15 min, and discard the supernatant. Next, add 430 μL of 25 mM 4-morpholinoethanesulfonic acid (MES) solution and resuspend by sonication. Then add 10 μL of microsphere activation solution A and 132 μL of microsphere activation solution B, and shake in a circular vibrator at room temperature for 30 min. Centrifuge at 18,000 rpm for 30 min and discard the supernatant. Add 1 mL of microsphere coupling washing buffer and resuspend by sonication. Centrifuge at 18,000 rpm for 30 min and discard the supernatant. Repeat twice. Add 200 μL of phosphate buffer (0.025 M, pH 7.0) and resuspend by sonication. Add 10 μL of 1 mg / mL HPV16 E6 antibody, and then adjust the volume to 500 μL with phosphate buffer (0.025 M, pH 7.0). Shake in a circular vibrator at room temperature for 2 h, and centrifuge at 18,000 rpm for 30 min. Discard the supernatant. Add 1 mL of microsphere coupling blocking buffer and sonicate to resuspend the microspheres. Shake overnight at room temperature using a circumferential shaker. Centrifuge at 18,000 rpm for 30 min and discard the supernatant. Wash twice with 1 mL of microsphere coupling washing buffer, then add 1 mL of labeled microsphere preservation buffer at a concentration of 1 mg / mL. Store at 4°C until use.

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

[0079] Same as "1. Preparation of HPV16 E6 AIE fluorescent microsphere labeled probe" in Example 2, the only difference being that the HPV16 E6 antibody is replaced with the HPV16 E7 antibody.

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

[0081] The preparation of HPV16 E6 AIE fluorescent microsphere labeled probe is the same as in Example 2, except that the HPV16 E6 antibody is replaced with the HPV18 E6 antibody.

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

[0083] The preparation of HPV16 E6 AIE fluorescent microsphere labeled probe is the same as in Example 2, except that the HPV16 E6 antibody is replaced with the HPV18 E7 antibody.

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

[0085] The preparation of HPV16 E6 AIE fluorescent microsphere labeled probe is the same as in Example 2, except that the HPV16 E6 antibody is replaced with β-Actin internal reference protein antibody.

[0086] Example 3: Preparation and application of lateral immunochromatographic (LFA) test strips

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

[0088] Using a non-contact microarray spotting instrument, 20 nL of 0.5 mg / mL 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) were spotted onto a CN140 nitrocellulose membrane in an array with a diameter of 800 μm and a center-to-center spacing of 1000 μm, with four replicates for each spot. Using the same instrument, 0.15 mg / mL AIE solution was spotted at the four corners of the nitrocellulose membrane with a diameter of 300 μm. The spotted nitrocellulose membranes were then placed in a forced-air drying oven and dried at 37°C for 2 h, and then stored in a desiccator for later use.

[0089] Five AIE fluorescent microsphere labeled probe solutions of 1 mg / mL were sprayed onto the conjugate pad at a spraying rate of 2 μL / cm, and then dried in a vacuum drying oven at 37°C for 4 h, and stored in a desiccant cabinet for later use.

[0090] The sample pad, conjugate pad, nitrocellulose membrane, and absorbent pad are adhered to a backing plate to prepare the test strip. The nitrocellulose membrane is located on top of the backing plate. The sample pad and conjugate pad are sequentially placed on the left side of the backing plate, and the absorbent pad is placed on the right side. The strips are then cut into 5.0 mm wide strips using a paper cutter. Finally, each strip is inserted into a two-piece plastic box, placed in a self-sealing bag containing desiccant, and sealed for storage to obtain the LFA test strip. A schematic diagram of the test strip structure can be seen below. Figure 1 .

[0091] 2. Instructions for using lateral immunochromatographic (LFA) test strips

[0092] After cervical swab sampling, the sample was placed in a centrifuge tube containing 2 mL of cell preservation solution. After shaking for 1 min, the sample was centrifuged at 13000 r / min for 1 min, and the supernatant was discarded. Then, 1 mL of cell lysis buffer was added, and the sample was shaken for 10 min, followed by centrifugation at 8000 r / min for 5 min. The supernatant was used as the sample for detection. The test strip was placed in a 37℃ incubator. 100 μL of the sample to be tested was added to the well of the test strip. After incubation for 15 min, the fluorescence signals of the T (detection line) and C (control line) lines were read using a test strip fluorescence reader.

[0093] Interpretation of results:

[0094] 1) Positive: The test line and control line show color development;

[0095] 2) Negative: Only the control line shows color;

[0096] 3) Invalid: The control line shows no color development, the test is invalid, and it is recommended to take another test card and retest.

[0097] 3. Optimization of the preparation process of LFA test strips

[0098] 3.1 Optimization of phosphate buffer pH, taking HPV16 E6 AIE fluorescent microsphere labeled probe as an example.

[0099] Phosphate buffer solutions with pH values ​​of 5, 6, 7, 8, and 9 were prepared according to the AIE fluorescent microsphere labeled probe preparation process described in Example 2 above. The optimal pH value was obtained by comparing the average fluorescence value.

[0100] The experimental results are shown in the figure. Figure 3 .Depend on Figure 3 The results showed that the HPV16 E6 AIE fluorescent microsphere labeled probe prepared with a phosphate buffer at pH 7 exhibited the best fluorescence intensity for subsequent studies. Therefore, pH 7 was selected as the optimal pH for the phosphate buffer.

[0101] Furthermore, the optimal pH of the phosphate buffer for different fluorescent microsphere labeled probes was verified, and the results are shown in Table 1.

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

[0103]

[0104] 3.2 Optimization of Nitrocellulose Membrane Types: Taking HPV16 E6 AIE Fluorescent Microsphere Labeled Probes as an Example

[0105] Three different types of nitrocellulose membranes—CN140, Pall Vivid 90, and YNFS—were selected and prepared according to the test strip preparation process described above. The optimal nitrocellulose membrane was selected based on its non-specific adsorption in negative samples and the average fluorescence value in test samples with an HPV16 E6 protein content of 200 ng / mL. The results are shown in Table 2.

[0106] Table 2 Experimental results of different nitrocellulose membranes

[0107]

[0108] Table 2 shows that CN140 and Pall Vivid 90 nitrocellulose membranes did not produce nonspecific adsorption in negative samples and showed no extraneous signals. In test samples containing HPV16 E6 protein, the CN140 nitrocellulose membrane exhibited higher fluorescence intensity. Therefore, the CN140 nitrocellulose membrane is the optimal nitrocellulose membrane.

[0109] 3.3 Taking HPV16 E6 AIE fluorescent microsphere-labeled probes as an example, the optimal dosage of probe antibody and capture antibody was optimized.

[0110] HPV16 E6 antibody was selected at doses of 1 μL, 5 μL, 10 μL, 15 μL, and 20 μL; HPV16 E6 capture antibody was selected and diluted with phosphate buffer to 0.25–1 mg / mL; samples with HPV16 E6 protein concentration of 200 ng / mL were prepared according to the above test strip preparation procedure and detected according to the above detection procedure. The average fluorescence values ​​of the signal points obtained by the test strip reader were compared to obtain the optimal monoclonal antibody dosage and capture antibody dosage. The detection results corresponding to the optimized dosage system are shown in Table 3.

[0111] Table 3 Results of the input optimization experiment

[0112]

[0113] According to the results in Table 3, the fluorescence intensity was highest when the HPV16 E6 antibody concentration was 10 μL and the HPV16 E6 capture antibody concentration was 0.5 mg / mL. Therefore, these are the optimal amounts of monoclonal antibody and capture antibody, respectively.

[0114] Based on this, the optimal dosage of different fluorescent microsphere labeled probes was verified, and the results are shown in Table 4.

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

[0116]

[0117] 3.4 Optimization of fluorescence readout time using HPV16 E6 AIE fluorescent microsphere-labeled probe as an example

[0118] Following the immunochromatographic test strip detection procedure, the change in T-line fluorescence intensity was monitored within 30 minutes after sample addition. Results are shown below. Figure 4 .

[0119] Figure 4 The results show that, taking the HPV16 E6 AIE fluorescent microsphere labeled probe as an example, the fluorescence intensity of the T line reaches its highest point 15 minutes after the sample is added to the test strip, and the fluorescence intensity remains stable thereafter. Therefore, the optimal reading time is 15 minutes.

[0120] Based on this, the optimal fluorescence reading time for different fluorescent microsphere labeled probes was verified, and the results are shown in Table 5.

[0121] Table 5 Optimal Fluorescence Reading Time

[0122]

[0123] Therefore, it can be seen that the optimal fluorescence reading time for the test strips prepared in this invention is 15 min for different fluorescent microsphere labeled probes.

[0124] Example 4: Performance Evaluation of LFA Test Strips

[0125] 1. Precision evaluation

[0126] Samples with HPV16 E6 protein concentrations of 5 ng / mL, 50 ng / mL, 100 ng / mL, 200 ng / mL, and 300 ng / mL were tested using three batches of test strips, and the intra-batch and inter-batch coefficients of variation were calculated.

[0127] Samples with HPV16 E7 protein concentrations of 5 ng / mL, 50 ng / mL, 100 ng / mL, 200 ng / mL, and 300 ng / mL were tested using three batches of test strips, and the intra-batch and inter-batch coefficients of variation were calculated.

[0128] Samples with HPV18 E6 protein concentrations of 5 ng / mL, 50 ng / mL, 100 ng / mL, 200 ng / mL, and 300 ng / mL were tested using three batches of test strips, and the intra-batch and inter-batch coefficients of variation were calculated.

[0129] Samples with HPV18 E7 protein concentrations of 5 ng / mL, 50 ng / mL, 100 ng / mL, 200 ng / mL, and 300 ng / mL were tested using three batches of test strips, and the intra-batch and inter-batch coefficients of variation were calculated.

[0130] Test samples with internal control protein concentrations of 0.2 µg / mL, 2 µg / mL, 3 µg / mL, and 4 µg / mL were tested five times using three batches of test strips, and the intra-batch and inter-batch coefficients of variation were calculated.

[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 protein

[0135]

[0136] Based on the results in Tables 6 and 7, it can be seen that the test strip has good precision, with all CVs being less than or approximately equal to 10%.

[0137] 2. Accuracy Evaluation

[0138] The recovery rate of the test strips was calculated for HPV16 E6 protein concentrations of 5 ng / mL, 50 ng / mL, 100 ng / mL, 200 ng / mL, and 300 ng / mL to evaluate the accuracy of the test strips.

[0139] The recovery rate of the test strips was calculated to evaluate the accuracy of the test strips when HPV16 E7 protein concentrations of 5 ng / mL, 50 ng / mL, 100 ng / mL, 200 ng / mL, and 300 ng / mL were detected in the test samples.

[0140] The HPV18 E6 protein concentrations of 5 ng / mL, 50 ng / mL, 100 ng / mL, 200 ng / mL, and 300 ng / mL were tested, and their recovery rates were calculated to evaluate the accuracy of the test strip.

[0141] The recovery rate of the test strips was calculated to evaluate the accuracy of the test strips when HPV18 E7 protein concentrations of 5 ng / mL, 50 ng / mL, 100 ng / mL, 200 ng / mL, and 300 ng / mL were detected in the test samples.

[0142] The recovery rates of test samples with internal reference protein concentrations of 0.2 µg / mL, 2 µg / mL, 3 µg / mL, and 4 µg / mL were calculated to evaluate the accuracy of the test strips.

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

[0144] Table 8 Recovery rates of target proteins at different concentrations

[0145]

[0146] Table 9 Recovery rates of internal reference proteins at different concentrations

[0147]

[0148] According to the results in Tables 8 and 9, the test strip has good accuracy, with recovery rates ranging from 90% to 110%.

[0149] 3. Specificity evaluation

[0150] The test strip detection process described in Example 3 was used to test mixed samples 1, 2, 3, 4, 5, and 6 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), 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), 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 epididymal 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 epididymal 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 epididymal 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), and human epididymal 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, the test strip has good specificity and no significant cross-reactivity with common proteins detected in cervical cancer clinically.

[0161] 4. Stability Evaluation

[0162] The assembled LFA test strips were stored in a 55℃ oven for 20 days. During this period, the test strips were periodically removed and mixed samples of 200 ng / mL each of HPV16 E6 / E7 protein and HPV18 E6 / E7 protein and 4 µg / mL of internal reference protein were added to the sample wells. The fluorescence values ​​were recorded and the aging and storage results of the test strips were plotted.

[0163] See results Figure 5 .in accordance with Figure 5 It can be seen that the fluorescence intensity of the target protein and internal reference protein of the test strip did not change after being stored under the above conditions for 20 days, that is, the LFA test strip prepared by the present invention can be stored under the above conditions for at least 20 days.

[0164] 5. Feasibility of sampling size for internal reference calibration

[0165] Five cancer samples from HPV16 and HPV18 positive cervical cancer patients were collected and numbered 1-10. Collagenase was added to each sample to prepare cell suspensions, and cell counts were performed. Ten samples were taken from each patient's cervical cancer cell suspension to obtain cell counts of 4000, 6000, 8000, 10000, 12000, 14000, 16000, 18000, 20000, and 22000 cells per cell, respectively. After processing, the samples were added for testing. The results were corrected using β-Actin protein, and the corrected results were analyzed to verify the feasibility of using β-Actin protein as an internal reference to correct sampling errors in this invention. The results are shown in Table 11.

[0166] Table 11 Feasibility Analysis Results of Internal Reference Calibration Sampling Quantity

[0167]

[0168] According to the results in Table 11, it is feasible to use β-Actin protein as an internal reference to correct the sampling error of the test strip. The CV is <10%, which can ensure the accuracy of the test results.

[0169] 6. Evaluation of detection limits

[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 tested. Each sample was tested three times, and the average fluorescence intensity was calculated. A standard curve equation (y = 145.01x + 150.55) was fitted using linear regression analysis with HPV16E6 protein concentration as the x-axis and T fluorescence intensity as the y-axis. The standard curve is shown in [image missing]. Figure 6 .

[0171] The negative sample was tested 20 times repeatedly. The average and standard deviation of the fluorescence intensity values ​​of the 20 tests were calculated. The average value plus three times the standard deviation was substituted into the linear equation above to obtain the corresponding concentration value, which is the limit of detection (LOD) of the test strip for the substance. The LOD results of different target proteins determined by this method are shown in Table 12.

[0172] Table 12 Results of the Limit of Detection for Different Target Proteins

[0173]

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

[0175] 7. Clinical trials

[0176] Seven samples from patients with clinically and pathologically confirmed cervical cancer (six samples of squamous cell carcinoma (SCC) and one sample of cervical adenocarcinoma (AC), ten samples from patients with high-grade squamous intraepithelial lesion (HSIL), twelve samples from patients with low-grade squamous intraepithelial lesion (LSIL), and 20 HPV DNA-negative samples were tested using the test strips prepared in this invention. The experimental results are shown in Table 13.

[0177] Table 13 Clinical Trial Test Results

[0178]

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

[0180] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A multi-target test strip for HPV16 / 18 E6 / E7 protein, comprising a backing plate, a sample pad, a conjugate pad, a nitrocellulose membrane, and an absorbent pad, wherein the nitrocellulose membrane is located above the backing plate, the sample pad and the conjugate pad are sequentially arranged on the left side of the nitrocellulose membrane backing plate, and the absorbent pad is arranged on the right side of the backing plate, characterized in that, The binding pad is covered with AIE fluorescent microsphere-labeled probes, and the nitrocellulose membrane is covered with capture antibodies and AIE solution. The AIE fluorescent microsphere labeling probes include HPV16 E6 AIE fluorescent microsphere labeling probe, HPV16 E7 AIE fluorescent microsphere labeling probe, HPV18 E6 AIE fluorescent microsphere labeling probe, HPV18 E7 AIE fluorescent microsphere labeling probe, and internal reference protein AIE fluorescent microsphere labeling probe. The preparation method of the AIE fluorescent microsphere labeled probe includes the following steps: Tetra-(4-bromophenyl)ethylene, 4-methoxycarbonylphenylboronic acid, tetra-triphenylphosphine palladium and cesium fluoride were dissolved in ethylene glycol dimethyl ether solvent, heated and maintained for 24-72 h, and then cooled. A solution of water and dichloromethane with a volume ratio of 2:1-5:1 was added, the organic phase was recovered, dried, purified under reduced pressure, and then purified again under reduced pressure to obtain AIE material. The AIE material was dissolved in tetrahydrofuran to obtain AIE solution. Polystyrene microspheres were taken, washed, and resuspended in sodium dodecyl sulfate. AIE solution was added, sonicated, oscillated, centrifuged to remove the supernatant, washed, and stored in water to obtain AIE fluorescent microsphere stock solution. After centrifuging the AIE fluorescent microsphere stock solution, collect the supernatant, add 4-morpholine ethanesulfonic acid, sonicate, centrifuge, and discard the supernatant; resuspend in 4-morpholine ethanesulfonic acid, add microsphere activation solution A and microsphere activation solution B, shake at room temperature, centrifuge, and discard the supernatant; add microsphere conjugation washing buffer, sonicate, centrifuge, and discard the supernatant; add buffer, sonicate, and resuspend; add the corresponding labeled antibody, adjust the volume with buffer, shake, centrifuge, and discard the supernatant; add microsphere conjugation blocking solution, sonicate, shake overnight, centrifuge, and discard the supernatant; wash with microsphere conjugation washing buffer, and add labeled microsphere preservation solution to obtain the AIE fluorescent microsphere labeled probe.

2. The HPV16 / 18 E6 / E7 protein multi-target test strip according to claim 1, characterized in that, The capture antibodies are HPV16 E6 capture antibody, HPV16 E7 capture antibody, HPV18 E6 capture antibody, HPV18 E7 capture antibody and β-Actin internal reference protein capture antibody; 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 internal reference protein.

3. The HPV16 / 18 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: Tetra-(4-bromophenyl)ethylene, 4-methoxycarbonylphenylboronic acid, tetra-triphenylphosphine palladium and cesium fluoride were dissolved in ethylene glycol dimethyl ether solvent, heated and maintained for 24-72 h, and then cooled. A solution of water and dichloromethane with a volume ratio of 2:1-5:1 was added, the organic phase was recovered, dried, purified under reduced pressure, and then purified again under reduced pressure to obtain AIE material. The AIE material was dissolved in tetrahydrofuran to obtain AIE solution.

4. A method for preparing a multi-target test strip for HPV16 / 18 E6 / E7 protein as described in any one of claims 1-3, characterized in that, Includes the following steps: Preparation of nitrocellulose membranes; Preparation of the binding pad; Assembly of test strips.

5. The preparation method according to claim 4, characterized in that, The preparation of the nitrocellulose membrane includes the following steps: 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 at a concentration of 0.1-0.9 mg / mL were spotted in an array onto a nitrocellulose membrane; 0.1-0.2 mg / mL of AIE solution was spotted at the four corners of the nitrocellulose membrane, and the membrane was dried.

6. The preparation method according to claim 4, characterized in that, The preparation of the conjugate pad includes the following steps: Synthesis of AIE fluorescent microsphere stock solution: Tetra-(4-bromophenyl)ethylene, 4-methoxycarbonylphenylboronic acid, tetra-triphenylphosphine palladium and cesium fluoride were dissolved in ethylene glycol dimethyl ether solvent, heated and maintained for 24-72 h, and then cooled; water and dichloromethane solution with a volume ratio of 2:1-5:1 were added, the organic phase was recovered, dried, purified under reduced pressure, and then purified under reduced pressure again to obtain AIE material; AIE material was dissolved in tetrahydrofuran to obtain AIE solution; polystyrene microspheres were taken, washed and resuspended in sodium dodecyl sulfate, AIE solution was added, sonicated, oscillated and centrifuged to remove supernatant, washed, and stored in water to obtain AIE fluorescent microsphere stock solution; Preparation of AIE fluorescent microsphere labeled probes: After centrifuging the AIE fluorescent microsphere stock solution, the supernatant was collected, and 4-morpholine ethanesulfonic acid was added for sonication. After centrifugation, the supernatant was discarded. 4-morpholine ethanesulfonic acid was added again for sonication and resuspending. Microsphere activation solution A and microsphere activation solution B were added, and the mixture was shaken at room temperature. After centrifugation, the supernatant was discarded. Microsphere coupling washing buffer was added for sonication and resuspending. After centrifugation, the supernatant was discarded. Buffer was added for sonication and resuspending. Five groups were set up simultaneously, with HPV16 E6 antibody, HPV16 E7 antibody, HPV18 E6 antibody, HPV18 E7 antibody, and β-Actin internal reference protein antibody added respectively. The mixture was brought to a final volume with buffer, shaken, and centrifuged, and the supernatant was discarded. Microsphere coupling blocking buffer was added for sonication and resuspending. The mixture was shaken overnight and centrifuged, and the supernatant was discarded. Microsphere coupling washing buffer was added for washing, and then labeled microsphere preservation solution was added to obtain five types of AIE fluorescent microsphere labeled probes. Five AIE fluorescent microsphere labeled probes were sprayed onto the conjugate pad at a spraying rate of 1-3 μL / cm and dried to obtain the conjugate pad.

7. The preparation method according to claim 5, characterized in that, The preparation of the nitrocellulose membrane includes the following steps: 20 nL of 0.5 mg / mL HPV16 E6 capture antibody, HPV16 E7 capture antibody, HPV18 E6 capture antibody, HPV18 E7 capture antibody, and β-Actin internal reference protein capture antibody were spotted onto a nitrocellulose membrane in an array with a diameter of 800 μm and a center-to-center spacing of 1000 μm, with four replicates for each spot; 0.15 mg / mL AIE solution was spotted at the four corners of the nitrocellulose membrane with a diameter of 300 μm. After spotting, the nitrocellulose membrane was dried at 37 °C for 2 h to obtain the final product.

8. The preparation method according to claim 6, characterized in that, The preparation of the conjugate pad includes the following steps: Synthesis of AIE fluorescent microsphere stock solution: Dissolve 100-300 mg tetra-(4-bromophenyl)ethylene, 278-300 mg 4-methoxycarbonylphenylboronic acid, 35.6-50 mg tetra-triphenylphosphine palladium, and 609-700 mg cesium fluoride in 10-20 mL of ethylene glycol dimethyl ether solvent, heat for 24-72 h, and cool. Add water and dichloromethane solution with a volume ratio of 2:1-5:1, recover the organic phase, dry, reduce pressure, purify, and then reduce pressure again to obtain AIE material. Dissolve the AIE material in tetrahydrofuran to obtain an AIE solution with a concentration of 1-7 mg / mL. Take 0.5-1.5 mL of polystyrene microspheres with a concentration of 50-150 mg / mL, wash, resuspend in 5-15 mL of sodium dodecyl sulfate, add 0.5-1.5 mL of AIE solution, sonicate, shake, centrifuge to remove supernatant, wash, and store in water to obtain the AIE fluorescent microsphere stock solution. Preparation of AIE fluorescent microsphere labeled probes: Centrifuge 50-150 μL of AIE fluorescent microsphere stock solution and collect the supernatant. Add 0.5-1.5 mL of 20-30 mM 4-morpholine ethanesulfonic acid and sonicate. Centrifuge again and discard the supernatant. Add 410-450 mL of 4-morpholine ethanesulfonic acid and sonicate to resuspend the microspheres. Add 5-15 μL of microsphere activation solution A and 132-150 μL of microsphere activation solution B. Shake at room temperature and centrifuge. Discard the supernatant. Add 0.5-1.5 mL of microsphere coupling washing buffer and sonicate to resuspend the microspheres. Centrifuge again and discard the supernatant. Add pH 7.0 buffer and sonicate to resuspend the microspheres. Simultaneously, set up five groups, adding 5-15 μL of 0.5-1.5 mg / mL HPV16 E6 antibody, HPV16 E7 antibody, HPV18 E6 antibody, and HPV18 antibody, respectively. E7 antibody and β-Actin internal reference protein antibody were diluted to volume with buffer, vortexed, centrifuged, and the supernatant was discarded. 0.5-1.5 mL of microsphere conjugation blocking buffer was added, and the mixture was sonicated and resuspended overnight. After centrifugation, the supernatant was discarded. 0.5-1.5 mL of microsphere conjugation washing buffer was added, followed by 0.5-1.5 mL of labeled microsphere preservation buffer to obtain five AIE fluorescent microsphere labeled probes. Five AIE fluorescent microsphere labeled probes were sprayed onto the conjugate pad at a spraying rate of 1-3 μL / cm and dried to obtain the conjugate pad.

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