A high-risk HPV detection kit and a preparation method thereof
By preparing silicon-core selenium-shell nanoparticles with a particle size of 150-200 nm as markers, the problems of high cost and low sensitivity in existing HPV detection technologies have been solved, enabling rapid and convenient detection of high-risk HPV and improving the sensitivity and specificity of the detection.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- WEIFANG GUARANTEE BIOLOGICAL TECH CO LTD
- Filing Date
- 2024-01-05
- Publication Date
- 2026-07-14
AI Technical Summary
Existing HPV testing technologies cannot achieve rapid and convenient detection of high-risk HPV, and traditional colloidal gold markers are costly and have low sensitivity, while colloidal selenium particles have poor dispersibility, making them difficult to widely apply to lateral immunochromatographic platforms.
Silicon core selenium shell nanoparticles with a particle size of 150-200 nm were prepared by seed growth method using silicon core selenium shell nanoparticles as markers. Combined with PEG modification and surface modification, they were used to prepare the labeling pad and solid-phase reaction membrane of high-risk HPV detection kits to improve detection sensitivity.
It enables low-cost, high-sensitivity testing of HPV types 16 and 18, applicable to cervical or urine samples, with high positive concordance rate and specificity, suitable for rapid and convenient screening of high-risk populations.
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Abstract
Description
Technical Field
[0001] This invention relates to the field of in vitro diagnostic technology, specifically to a high-risk HPV detection kit and its preparation method. Background Technology
[0002] Human papillomavirus (HPV) is a non-enveloped, double-stranded circular DNA virus that specifically infects and parasitizes the epithelial cells of human reproductive organs and other tissues, causing proliferative lesions of the skin and mucous membranes. Currently, more than 200 subtypes of HPV have been discovered and identified, with approximately 54 types capable of infecting the mucous membranes of the reproductive tract. Based on the different risks associated with cervical cancer, HPV types are divided into two main categories: high-risk and low-risk.
[0003] High-risk HPV types mainly include HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68. Almost all cervical cancers are caused by persistent infection with high-risk HPV types, with HPV16 and 18 being the most common. In my country, over 84.5% of cervical squamous cell carcinoma cases are related to HPV 16 and 18 infection. The "Guidelines for the Diagnosis and Treatment of Cervical Cancer (2022 Edition)" states that cervical / vaginal cytology smears and HPV testing are currently the primary screening methods for detecting early cervical cancer and precancerous lesions (CIN). HPV testing can effectively supplement cervical liquid-based thin-layer cytology (TCT), and combined testing of both is beneficial for improving screening efficiency.
[0004] The HPV genome is divided into three parts: the early gene region (E), the late gene region (L), and the long regulatory region (LCR). The early region encodes E6, E7, E1, E2, E4, and E5 proteins, which are mainly involved in viral DNA replication and transcription. Among them, E6 / E7 are oncogenes. The late region encodes L1 and L2 proteins, which are the major and minor capsid proteins of the virus, respectively. During the development of cancer from low-grade lesions, the HPV genome integrates. During this integration process, the E6 / E7 genes persist, while the L1 gene may be lost. Using the L1 gene as a detection target may result in false negatives in HPV testing. Conversely, using the E6 / E7 genes as a detection target eliminates the possibility of false negatives regardless of whether genome integration occurs.
[0005] Currently, HPV cannot be cultured in vitro, and the detection of high-risk HPV is based on molecular diagnostic techniques using HPV DNA. HPV DNA detection is equipment-dependent, requires sophisticated operation, is prone to contamination, and has a long testing cycle, making rapid or over-the-counter testing impossible. Therefore, establishing a rapid and convenient method for detecting high-risk HPV in cervical or urine samples could effectively promote proactive screening of high-risk populations during the asymptomatic stage, reducing the risk of asymptomatic infection.
[0006] With the widespread use of HPV vaccines, HPV antibody testing alone is no longer sufficient for accurate disease diagnosis. Currently, domestic patents involve lateral immunochromatography for HPV antigen detection, using colloidal gold as a marker. Colloidal gold possesses plasma properties, but its small particle size, high-temperature boiling requirements during preparation, and expensive raw materials often result in low sensitivity and high cost when used as a marker. Compared to colloidal gold, colloidal selenium, as a non-metallic colloidal particle, offers advantages such as insensitivity to electrolytes, room-temperature preparation, simple methods, and low raw material costs, and has been gradually adopted as a reagent in lateral immunochromatography. However, due to its small particle size and poor dispersibility, the development of colloidal selenium has been slow in recent years, and its application is not widespread. Therefore, there is an urgent need to prepare nanoparticles with large particle size, low cost, and good dispersibility for use in lateral immunochromatography platforms to improve detection sensitivity. Summary of the Invention
[0007] The purpose of this invention is to provide a high-risk HPV detection kit and its preparation method, which detects HPV types 16 and 18, uses cervical samples or urine samples as detection media, and employs silicon core selenium shell nanoparticles as markers. The kit has low preparation cost and high detection sensitivity.
[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A method for preparing a high-risk HPV test kit, including a method for preparing a labeled pad, wherein the method for preparing the labeled pad is as follows: HPV16 E6 / E7-2 antibody-silicon core selenium shell label and HPV18 E6 / E7-2 antibody-silicon core selenium shell label are mixed evenly at a volume ratio of 1:1 and then sprayed onto glass fiber MA0800 to obtain the labeled pad. Both HPV16 E6 / E7-2 antibody-silicon core selenium shell label and HPV18 E6 / E7-2 antibody-silicon core selenium shell label are prepared by labeling with silicon core selenium shell nanoparticle solution as a tracer.
[0009] The preparation method of the HPV16 E6 / E7-2 antibody-silicon core selenium shell label is as follows: Take 5 mL of silicon core selenium shell nanoparticle solution and adjust the pH to 8.0 with 0.1 M potassium carbonate; quickly add 0.5 mg / mL HPV16 E6 / E7-2 antibody, react fully for 10 min, add 1% BSA to the final concentration, react fully for 10 min, centrifuge at 10000 rpm for 15 min, and resuspend in 5 mL of resuspension to obtain HPV16E6 / E7-2 antibody-silicon core selenium shell label;
[0010] The preparation method of the HPV18 E6 / E7-2 antibody-silicon core selenium shell label is as follows: Take 5 mL of silicon core selenium shell nanoparticle solution and adjust the pH to 8.0 with 0.1 M potassium carbonate; quickly add 0.5 mg / mL HPV18 E6 / E7-2 antibody, react fully for 10 min, add 1% BSA to the final concentration, react fully for 10 min, centrifuge at 10000 rpm for 15 min, and resuspend in 5 mL of resuspension to obtain HPV18E6 / E7-2 antibody-silicon core selenium shell label;
[0011] The silicon core selenium shell nanoparticle solution utilizes a seed growth method. First, the surface of silica microspheres is grafted and modified. Then, PEG is applied to the surface of the modified SNPs. The PEG-modified SNPs serve as silicon core seeds, and selenium dioxide is used as the growth solution. A selenium shell layer is then grown on the surface of the silicon core seeds to obtain core-shell structured silicon core selenium shell nanoparticles.
[0012] The method for preparing the silicon core selenium shell nanoparticle solution includes the following steps:
[0013] S1. Preparation of silica microspheres: Silica microspheres were prepared using the Stober method. During the preparation process, silica microspheres with a particle size of 110 nm were obtained by adjusting the concentration of ammonia water. After acidification with HNO3 and centrifugation and washing with water, silica microspheres with a positive charge on the surface and a concentration of 1 mg / mL were finally obtained.
[0014] Preparation of S2 and PEG-modified silica microsphere solution: The silica microspheres obtained in S1 were surface-grafted and modified using succinic anhydride as a modifier to induce carboxyl functional groups on the surface, resulting in carboxylated silica microspheres. 10 mL of carboxylated silica microspheres were added to 10 mL of PEG solution, stirred at 500 rpm for 30 min at room temperature, ultrasonically dispersed for 10 min, centrifuged, washed three times with water, and resuspended in 10 mL of ultrapure water to obtain the PEG-modified silica microsphere solution.
[0015] S3. Preparation of silicon core selenium shell nanoparticle solution: Take 50 mL of ultrapure water and place it in a 100 mL beaker. Add 0.2% gum arabic and 1% PVP as stabilizers and stir to dissolve. Add 10 mL of PEG-modified silica microsphere solution prepared in S2 as silicon core seeds, add 10 mL of 0.3 M selenium dioxide as growth solution, and add 10 mL of ascorbic acid as reducing agent. Stir at 100 rpm for 30 min at room temperature to allow colloidal selenium to aggregate on the surface of the microspheres to form a selenium shell. Centrifuge and wash 3 times with water, and resuspend in 10 mL of ultrapure water to obtain silicon core selenium shell nanoparticle solution. Store in a refrigerator at 4℃ for later use.
[0016] The particle size of the silicon core selenium shell nanoparticles is 150-200nm.
[0017] As an optimized approach, the concentration of the PEG solution is 0.5%-1.5%;
[0018] As an optimized approach, the concentration of ascorbic acid is 0.6-1.5M;
[0019] As an optimized solution, a method for preparing a high-risk HPV test kit also includes a method for preparing a solid-phase reaction membrane. The method for preparing the solid-phase reaction membrane is as follows: HPV16 E6 / E7-1 antibody, HPV18 E6 / E7-1 antibody and goat anti-mouse antibody are respectively coated onto an NC membrane as the detection T1 line, detection T2 line and quality control C line.
[0020] As an optimized solution, a method for preparing a high-risk HPV test kit includes a test strip, which comprises a PVC plate. From left to right, the PVC plate is provided with absorbent paper, a solid-phase reaction membrane, a labeling pad, and a sample pad.
[0021] As an optimized approach, a method for preparing a high-risk HPV detection kit includes a lysis buffer when the detection medium is a cervical sample. The lysis buffer comprises 100-150 mM PBS solution, 0.05% NP40 by mass, and 0.01% X-100 by mass.
[0022] The present invention, employing the above technical solution, has the following advantages: The labeling agent of the present invention is a silicon-core selenium-shell nanoparticle with a particle size of 150-200 nm. Its preparation does not require expensive gold chloride raw materials or cumbersome high-temperature boiling preparation steps, resulting in lower preparation costs compared to traditional colloidal gold particles. The silica microspheres are coated with a selenium shell, increasing the particle size and surface area of the nanoparticles. This allows for more binding sites on the nanoparticle surface to the labeled protein, improving the labeling and solidification rate of the labeled protein, thereby enhancing the binding force with the coated antibody. Compared to traditional colloidal selenium labeling, the detection sensitivity is higher.
[0023] The preparation and application of silicon-core selenium-shell nanoparticles in this invention are one of the key technologies for improving the sensitivity of this reagent kit. The prepared silica microspheres are first acidified, and the numerous positive charges forming a charge layer ensure stable and uniform distribution of the microspheres in the solvent. The silica microspheres are then carboxylated, resulting in a stronger and denser bond between PEG and the silica microspheres, forming a PEG non-immune dense layer on the surface of the silica microspheres. The introduction of this PEG non-immune dense layer enhances the stability of the silica microspheres while also controlling the growth rate of colloidal selenium, thus improving the binding efficiency of colloidal selenium. By controlling the ratio of selenium growth solution to ascorbic acid, the growth rate and coverage of selenium on the surface of the silica microspheres are adjusted, thereby regulating the shape, particle size, and dispersibility of the silicon-core selenium-shell particles. Detailed Implementation
[0024] The embodiments of the present invention will be described in detail below with reference to the examples. Specific conditions not specified in the examples are based on conventional conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased on the market.
[0025] The described embodiments are merely some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention. Example 1
[0026] A method for preparing a high-risk HPV test kit includes a method for preparing a labeled pad. The method for preparing the labeled pad involves mixing HPV16 E6 / E7-2 antibody-silicon core selenium shell label and HPV18 E6 / E7-2 antibody-silicon core selenium shell label at a 1:1 volume ratio and then spraying the mixture onto a glass fiber MA0800 to obtain the labeled pad. Both the HPV16 E7-2 antibody-silicon core selenium shell label and the HPV18 E6-2 antibody-silicon core selenium shell label are prepared by labeling with a silicon core selenium shell nanoparticle solution as a tracer.
[0027] Preparation method of HPV16 E6 / E7-2 antibody-silicon core selenium shell label: Take 5 mL of silicon core selenium shell nanoparticle solution and adjust the pH to 8.0 with 0.1 M potassium carbonate; quickly add 0.5 mg / mL HPV16 E6 / E7-2 antibody, react fully for 10 min, add 1% BSA to the final concentration, react fully for 10 min, centrifuge at 10000 rpm for 15 min, and resuspend in 5 mL of resuspension to obtain HPV16 E6 / E7-2 antibody-silicon core selenium shell label;
[0028] Preparation method of HPV18 E6 / E7-2 antibody-silicon core selenium shell label: Take 5 mL of silicon core selenium shell nanoparticle solution and adjust the pH to 8.0 with 0.1 M potassium carbonate; quickly add 0.5 mg / mL HPV18 E6 / E7-2 antibody, react fully for 10 min, add 1% BSA to the final concentration, react fully for 10 min, centrifuge at 10000 rpm for 15 min, and resuspend in 5 mL of resuspension to obtain HPV18 E6 / E7-2 antibody-silicon core selenium shell label;
[0029] The silicon core selenium shell nanoparticle solution utilizes a seed growth method. First, the surface of silica microspheres (SNPs) is grafted and modified. Then, PEG is applied to the surface of the modified SNPs. The PEG-modified SNPs serve as silicon core seeds, and selenium dioxide is used as the growth solution. A selenium shell layer is then grown on the surface of the silicon core seeds to obtain core-shell structured silicon core selenium shell nanoparticles.
[0030] The preparation method of silicon core selenium shell nanoparticle solution includes the following steps:
[0031] S1. Preparation of silica microspheres: Silica microspheres were prepared using the Stober method. During the preparation process, silica microspheres with a particle size of 110 nm were obtained by adjusting the concentration of ammonia water. After acidification with HNO3 and centrifugation and washing with water, silica microspheres with a positive charge on the surface and a concentration of 1 mg / mL were finally obtained.
[0032] Preparation of S2 and PEG-modified silica microsphere solution: The silica microspheres obtained in S1 were surface-grafted and modified using succinic anhydride as a modifier to functionalize the surface carboxyl groups, resulting in carboxylated silica microspheres. 10 mL of carboxylated silica microspheres were added to 10 mL of 0.5% PEG solution, stirred at 500 rpm for 30 min at room temperature, ultrasonically dispersed for 10 min, centrifuged, washed three times with water, and resuspended in 10 mL of ultrapure water to obtain the PEG-modified silica microsphere solution.
[0033] S3. Preparation of silicon core selenium shell nanoparticle solution: Take 50 mL of ultrapure water and place it in a 100 mL beaker. Add 0.2% gum arabic and 1% PVP as stabilizers and stir to dissolve. Add 10 mL of PEG-modified silica microsphere solution prepared in S2 as silicon core seeds, add 10 mL of 0.3 M selenium dioxide as growth solution, and add 10 mL of 0.6 M ascorbic acid as reducing agent. Stir at 100 rpm for 30 min at room temperature to allow colloidal selenium to aggregate on the surface of the microspheres to form a selenium shell of a certain thickness. Centrifuge and wash with water 3 times. Resuspend in 10 mL of ultrapure water to obtain silicon core selenium shell nanoparticle solution. Store in a refrigerator at 4℃ for later use.
[0034] The silicon core selenium shell nanoparticle solution prepared by the above steps was detected by transmission electron microscopy (TEM) and showed to be spherical with a particle size of 200 nm and good dispersibility.
[0035] A method for preparing a high-risk HPV test kit, further comprising a method for preparing a solid-phase reaction membrane, wherein the solid-phase reaction membrane is prepared by coating an NC membrane with HPV16 E6 / E7-1 antibody, HPV18 E6 / E7-1 antibody and goat anti-mouse antibody as detection T1 line, detection T2 line and quality control C line, respectively.
[0036] A method for preparing a high-risk HPV test kit. The test kit includes a test strip, which includes a PVC plate. From left to right, the PVC plate is provided with absorbent paper, a solid-phase reaction membrane, a labeling pad, and a sample pad. In this embodiment, the test sample is urine. Example 2
[0037] A method for preparing a high-risk HPV test kit includes a method for preparing a labeled pad. The method for preparing the labeled pad is as follows: HPV16 E6 / E7-2 antibody-silicon core selenium shell label and HPV18 E6 / E7-2 antibody-silicon core selenium shell label are mixed evenly at a volume ratio of 1:1 and then sprayed onto glass fiber MA0800 to obtain the labeled pad.
[0038] Preparation method of HPV16 E6 / E7-2 antibody-silicon core selenium shell label: Take 5 mL of silicon core selenium shell nanoparticle solution and adjust the pH to 8.0 with 0.1 M potassium carbonate; quickly add 0.5 mg / mL HPV16 E6 / E7-2 antibody, react fully for 10 min, add 1% BSA to the final concentration, react fully for 10 min, centrifuge at 10000 rpm for 15 min, and resuspend in 5 mL of resuspension to obtain HPV16 E6 / E7-2 antibody-silicon core selenium shell label;
[0039] Preparation method of HPV18 E6 / E7-2 antibody-silicon core selenium shell label: Take 5 mL of silicon core selenium shell nanoparticle solution and adjust the pH to 8.0 with 0.1 M potassium carbonate; quickly add 0.5 mg / mL HPV18 E6 / E7-2 antibody, react fully for 10 min, add 1% BSA to the final concentration, react fully for 10 min, centrifuge at 10000 rpm for 15 min, and resuspend in 5 mL of resuspension to obtain HPV18 E6 / E7-2 antibody-silicon core selenium shell label;
[0040] The silicon core selenium shell nanoparticle solution utilizes a seed growth method. First, the surface of silica microspheres (SNPs) is grafted and modified. Then, PEG is applied to the surface of the modified SNPs. The PEG-modified SNPs serve as silicon core seeds, and selenium dioxide is used as the growth solution. A selenium shell layer is then grown on the surface of the silicon core seeds to obtain core-shell structured silicon core selenium shell nanoparticles.
[0041] The preparation method of silicon core selenium shell nanoparticle solution includes the following steps:
[0042] S1. Preparation of silica microspheres: Silica microspheres were prepared using the Stober method. During the preparation process, silica microspheres with a particle size of 110 nm were obtained by adjusting the concentration of ammonia water. After acidification with HNO3 and centrifugation and washing with water, silica microspheres with a positive charge on the surface and a concentration of 1 mg / mL were finally obtained.
[0043] Preparation of S2 and PEG-modified silica microsphere solution: The silica microspheres obtained in S1 were surface-grafted and modified using succinic anhydride as a modifier to functionalize the surface carboxyl groups, resulting in carboxylated silica microspheres. 10 mL of carboxylated silica microspheres were added to 10 mL of 0.5% PEG solution, stirred at 500 rpm for 30 min at room temperature, ultrasonically dispersed for 10 min, centrifuged, washed three times with water, and resuspended in 10 mL of ultrapure water to obtain the PEG-modified silica microsphere solution.
[0044] S3. Preparation of silicon core selenium shell nanoparticle solution: Take 50 mL of ultrapure water and place it in a 100 mL beaker. Add 0.2% gum arabic and 1% PVP as stabilizers and stir to dissolve. Add 10 mL of PEG-modified silica microsphere solution prepared in S2 as silicon core seeds, add 10 mL of 0.3 M selenium dioxide as growth solution, and add 10 mL of 0.6 M ascorbic acid as reducing agent. Stir at 100 rpm for 30 min at room temperature to allow colloidal selenium to aggregate on the surface of the microspheres to form a selenium shell of a certain thickness. Centrifuge and wash with water 3 times. Resuspend in 10 mL of ultrapure water to obtain silicon core selenium shell nanoparticle solution. Store in a refrigerator at 4℃ for later use.
[0045] The silicon core selenium shell nanoparticle solution prepared by the above steps was detected by transmission electron microscopy (TEM) and showed to be spherical with a particle size of 200 nm and good dispersibility.
[0046] A method for preparing a high-risk HPV test kit, further comprising a method for preparing a solid-phase reaction membrane, wherein the solid-phase reaction membrane is prepared by coating an NC membrane with HPV16 E6 / E7-1 antibody, HPV18 E6 / E7-1 antibody and goat anti-mouse antibody as detection T1 line, detection T2 line and quality control C line, respectively.
[0047] A method for preparing a high-risk HPV test kit. The kit includes a test strip, which comprises a PVC plate. From left to right, the PVC plate is provided with absorbent paper, a solid-phase reaction membrane, a labeling pad, and a sample pad.
[0048] A method for preparing a high-risk HPV detection kit. When the detection medium is a cervical sample, the kit also includes a lysis buffer, which comprises 100-150 mM PBS solution, 0.05% NP40 by mass, and 0.01% X-100 by mass. Example 3
[0049] A method for preparing a high-risk HPV test kit includes a method for preparing a labeled pad. The method for preparing the labeled pad is as follows: HPV16 E6 / E7-2 antibody-silicon core selenium shell label and HPV18 E6 / E7-2 antibody-silicon core selenium shell label are mixed evenly at a volume ratio of 1:1 and then sprayed onto glass fiber MA0800 to obtain the labeled pad.
[0050] Preparation method of HPV16 E6 / E7-2 antibody-silicon core selenium shell label: Take 5 mL of silicon core selenium shell nanoparticle solution and adjust the pH to 8.0 with 0.1 M potassium carbonate; quickly add 0.5 mg / mL HPV16 E6 / E7-2 antibody, react fully for 10 min, add 1% BSA to the final concentration, react fully for 10 min, centrifuge at 10000 rpm for 15 min, and resuspend in 5 mL of resuspension to obtain HPV16 E6 / E7-2 antibody-silicon core selenium shell label;
[0051] Preparation method of HPV18 E6 / E7-2 antibody-silicon core selenium shell label: Take 5 mL of silicon core selenium shell nanoparticle solution and adjust the pH to 8.0 with 0.1 M potassium carbonate; quickly add 0.5 mg / mL HPV18 E6 / E7-2 antibody, react fully for 10 min, add 1% BSA to the final concentration, react fully for 10 min, centrifuge at 10000 rpm for 15 min, and resuspend in 5 mL of resuspension to obtain HPV18 E6 / E7-2 antibody-silicon core selenium shell label;
[0052] The silicon core selenium shell nanoparticle solution utilizes a seed growth method. First, the surface of silica microspheres (SNPs) is grafted and modified. Then, PEG is applied to the surface of the modified SNPs. The PEG-modified SNPs serve as silicon core seeds, and selenium dioxide is used as the growth solution. A selenium shell layer is then grown on the surface of the silicon core seeds to obtain core-shell structured silicon core selenium shell nanoparticles.
[0053] The preparation method of silicon core selenium shell nanoparticle solution includes the following steps:
[0054] S1. Preparation of silica microspheres: Silica microspheres were prepared using the Stober method. During the preparation process, silica microspheres with a particle size of 110 nm were obtained by adjusting the concentration of ammonia water. After acidification with HNO3 and centrifugation and washing with water, silica microspheres with a positive charge on the surface and a concentration of 1 mg / mL were finally obtained.
[0055] Preparation of S2 and PEG-modified silica microsphere solution: The silica microspheres obtained in S1 were surface-grafted and modified using succinic anhydride as a modifier to induce carboxyl functional groups on the surface, resulting in carboxylated silica microspheres. 10 mL of carboxylated silica microspheres were added to 10 mL of 1.0% PEG solution, and the mixture was stirred at 500 rpm for 30 min at room temperature, ultrasonically dispersed for 10 min, centrifuged, washed three times with water, and resuspended in 10 mL of ultrapure water to obtain the PEG-modified silica microsphere solution.
[0056] S3. Preparation of silicon core selenium shell nanoparticle solution: Take 50 mL of ultrapure water and place it in a 100 mL beaker. Add 0.2% gum arabic and 1% PVP as stabilizers and stir to dissolve. Add 10 mL of PEG-modified silica microsphere solution prepared in S2 as silicon core seeds, add 10 mL of 0.3 M selenium dioxide as growth solution, and add 10 mL of 0.9 M ascorbic acid as reducing agent. Stir at 100 rpm for 30 min at room temperature to allow colloidal selenium to aggregate on the surface of the microspheres to form a selenium shell of a certain thickness. Centrifuge and wash with water 3 times. Resuspend in 10 mL of ultrapure water to obtain silicon core selenium shell nanoparticle solution and store in a refrigerator at 4℃ for later use.
[0057] The silicon core selenium shell nanoparticles prepared by the above steps were examined by transmission electron microscopy (TEM) and found to be spherical with a particle size of 180 nm and good dispersibility.
[0058] A method for preparing a high-risk HPV test kit, further comprising a method for preparing a solid-phase reaction membrane, wherein the solid-phase reaction membrane is prepared by coating an NC membrane with HPV16 E6 / E7-1 antibody, HPV18 E6 / E7-1 antibody and goat anti-mouse antibody as detection T1 line, detection T2 line and quality control C line, respectively.
[0059] A method for preparing a high-risk HPV test kit. The kit includes a test strip, which comprises a PVC plate. From left to right, the PVC plate is provided with absorbent paper, a solid-phase reaction membrane, a labeling pad, and a sample pad.
[0060] A method for preparing a high-risk HPV detection kit. When the detection medium is a cervical sample, the kit also includes a lysis buffer, which comprises 100-150 mM PBS solution, 0.05% NP40 by mass, and 0.01% X-100 by mass. Example 4
[0061] A method for preparing a high-risk HPV test kit includes a method for preparing a labeled pad. The method for preparing the labeled pad is as follows: HPV16 E6 / E7-2 antibody-silicon core selenium shell label and HPV18 E6 / E7-2 antibody-silicon core selenium shell label are mixed evenly at a volume ratio of 1:1 and then sprayed onto glass fiber MA0800 to obtain the labeled pad.
[0062] Preparation method of HPV16 E6 / E7-2 antibody-silicon core selenium shell label: Take 5 mL of silicon core selenium shell nanoparticle solution and adjust the pH to 8.0 with 0.1 M potassium carbonate; quickly add 0.5 mg / mL HPV16 E6 / E7-2 antibody, react fully for 10 min, add 1% BSA to the final concentration, react fully for 10 min, centrifuge at 10000 rpm for 15 min, and resuspend in 5 mL of resuspension to obtain HPV16 E6 / E7-2 antibody-silicon core selenium shell label;
[0063] Preparation method of HPV18 E6 / E7-2 antibody-silicon core selenium shell label: Take 5 mL of silicon core selenium shell nanoparticle solution and adjust the pH to 8.0 with 0.1 M potassium carbonate; quickly add 0.5 mg / mL HPV18 E6 / E7-2 antibody, react fully for 10 min, add 1% BSA to the final concentration, react fully for 10 min, centrifuge at 10000 rpm for 15 min, and resuspend in 5 mL of resuspension to obtain HPV18 E6 / E7-2 antibody-silicon core selenium shell label;
[0064] The silicon core selenium shell nanoparticle solution utilizes a seed growth method. First, the surface of silica microspheres (SNPs) is grafted and modified. Then, PEG is applied to the surface of the modified SNPs. The PEG-modified SNPs serve as silicon core seeds, and selenium dioxide is used as the growth solution. A selenium shell layer is then grown on the surface of the silicon core seeds to obtain core-shell structured silicon core selenium shell nanoparticles.
[0065] The preparation method of silicon core selenium shell nanoparticle solution includes the following steps:
[0066] S1. Preparation of silica microspheres: Silica microspheres were prepared using the Stober method. During the preparation process, silica microspheres with a particle size of 110 nm were obtained by adjusting the concentration of ammonia water. After acidification with HNO3 and centrifugation and washing with water, silica microspheres with a positive charge on the surface and a concentration of 1 mg / mL were finally obtained.
[0067] Preparation of S2 and PEG-modified silica microsphere solution: The silica microspheres obtained in S1 were surface-grafted and modified using succinic anhydride as a modifier to functionalize the surface carboxyl groups, resulting in carboxylated silica microspheres. 10 mL of carboxylated silica microspheres were added to 10 mL of 1.5% PEG solution, stirred at 500 rpm for 30 min at room temperature, ultrasonically dispersed for 10 min, centrifuged, washed three times with water, and resuspended in 10 mL of ultrapure water to obtain the PEG-modified silica microsphere solution.
[0068] S3. Preparation of silicon core selenium shell nanoparticle solution: Take 50 mL of ultrapure water and place it in a 100 mL beaker. Add 0.2% gum arabic and 1% PVP as stabilizers and stir to dissolve. Add 10 mL of PEG-modified silica microsphere solution prepared in S2 as silicon core seeds, add 10 mL of 0.3 M selenium dioxide as growth solution, and add 10 mL of 1.5 M ascorbic acid as reducing agent. Stir at 100 rpm for 30 min at room temperature to allow colloidal selenium to aggregate on the surface of the microspheres to form a selenium shell of a certain thickness. Centrifuge and wash with water 3 times. Resuspend in 10 mL of ultrapure water to obtain silicon core selenium shell nanoparticle solution and store in a refrigerator at 4℃ for later use.
[0069] The silicon core selenium shell nanoparticles prepared by the above steps were examined by transmission electron microscopy (TEM) and found to be spherical with a particle size of 150 nm and good dispersibility.
[0070] A method for preparing a high-risk HPV test kit, further comprising a method for preparing a solid-phase reaction membrane, wherein the solid-phase reaction membrane is prepared by coating an NC membrane with HPV16 E6 / E7-1 antibody, HPV18 E6 / E7-1 antibody and goat anti-mouse antibody as detection T1 line, detection T2 line and quality control C line, respectively.
[0071] A method for preparing a high-risk HPV test kit. The kit includes a test strip, which comprises a PVC plate. From left to right, the PVC plate is provided with absorbent paper, a solid-phase reaction membrane, a labeling pad, and a sample pad.
[0072] A method for preparing a high-risk HPV detection kit. When the detection medium is a cervical sample, the kit also includes a lysis buffer, which comprises 100-150 mM PBS solution, 0.05% NP40 by mass, and 0.01% X-100 by mass.
[0073] Cervical samples and homologous urine samples of HPV types 16 and 18, confirmed by the Roche HPV test kit (PCR fluorescence method), were tested using the kits prepared in Examples 2-4, and homologous urine samples were tested using the kit prepared in Example 1. The specific test results are shown in Table 1.
[0074] Table 1. Results of HPV clinical sample validation
[0075]
[0076] As shown in Table 1:
[0077] Example 1 showed that the positive concordance rate of homologous urine samples from patients diagnosed with HPV16 by Roche fluorescence PCR was 90%, and the positive concordance rate of homologous urine samples from patients diagnosed with HPV18 was 90%, with a specificity of 100% in both cases.
[0078] In Examples 2-4, the positive concordance rate of cervical samples from patients diagnosed with HPV16 by Roche fluorescence PCR was 96%, and the positive concordance rate of cervical samples from patients diagnosed with HPV18 was 94%, with a specificity of 100% for both.
[0079] To better demonstrate that the kit of the present invention has good sensitivity and specificity, with Example 3 as a reference, five comparative examples were used to simultaneously detect positive and negative cervical samples of types 16 and 18 diagnosed by the Roche HPV test kit (PCR fluorescence method) using the kit prepared in Example 3 of the present invention and the kits prepared in Comparative Examples 1-5. The specific detection results are shown in Table 2.
[0080] Comparative Example 1
[0081] A method for preparing a high-risk HPV test kit differs from Example 3 in that the labeling pad is prepared by mixing colloidal gold particle-labeled HPV16 E6 / E7-2 antibody and HPV18 E6 / E7-2 antibody at a 1:1 volume ratio and then spraying the mixture onto glass fiber MA0800 to obtain the labeling pad.
[0082] Comparative Example 2
[0083] A method for preparing a high-risk HPV test kit differs from Example 3 in that the labeling pad is prepared by mixing HPV16 E6 / E7-2 antibody and HPV18 E6 / E7-2 antibody labeled with colloidal selenium particles at a volume ratio of 1:1 and then spraying the mixture onto glass fiber MA0800 to obtain the labeling pad.
[0084] Comparative Example 3
[0085] A method for preparing a high-risk HPV testing kit, which differs from Example 3 in that the method for preparing silicon core selenium shell nanoparticles includes the following steps:
[0086] S1. Preparation of silica microspheres: Silica microspheres were prepared using the Stober method. During the preparation process, silica microspheres with a particle size of 110 nm were obtained by adjusting the concentration of ammonia water. After acidification with HNO3 and centrifugation and washing with water, silica microspheres with a positive charge on the surface and a concentration of 1 mg / mL were finally obtained.
[0087] S2. Preparation of silicon core selenium shell nanoparticle solution: Take 50 mL of ultrapure water and place it in a 100 mL beaker. Add 0.2% gum arabic and 1% PVP as stabilizers and stir to dissolve. Add 10 mL of the silica microsphere solution prepared in S1 as silicon core seeds, add 10 mL of 0.3 M selenium dioxide as growth solution, and add 10 mL of 0.9 M ascorbic acid as reducing agent. Stir at 100 rpm for 30 min at room temperature to allow colloidal selenium to aggregate on the surface of the microspheres to form a selenium shell of a certain thickness. Centrifuge and wash with water 3 times. Resuspend in 10 mL of ultrapure water to obtain silicon core selenium shell nanoparticle solution and store in a refrigerator at 4℃ for later use.
[0088] Comparative Example 4
[0089] A method for preparing a high-risk HPV testing kit, which differs from Example 3 in that the method for preparing silicon core selenium shell nanoparticles includes the following steps:
[0090] S1. Preparation of silica microspheres: Silica microspheres were prepared using the Stober method. During the preparation process, silica microspheres with a particle size of 110 nm were obtained by adjusting the concentration of ammonia water. After acidification with HNO3 and centrifugation and washing with water, silica microspheres with a positive charge on the surface and a concentration of 1 mg / mL were finally obtained.
[0091] Preparation of S2 and PEG-modified silica microsphere solution: The silica microspheres obtained in S1 were surface-grafted and modified using succinic anhydride as a modifier to induce carboxyl functional groups on the surface, resulting in carboxylated silica microspheres. 10 mL of carboxylated silica microspheres were added to 10 mL of 1.0% PEG solution, and the mixture was stirred at 500 rpm for 30 min at room temperature, ultrasonically dispersed for 10 min, centrifuged, washed three times with water, and resuspended in 10 mL of ultrapure water to obtain the PEG-modified silica microsphere solution.
[0092] S3. Preparation of silicon core selenium shell nanoparticle solution: Take 50 mL of ultrapure water and place it in a 100 mL beaker. Add 0.2% gum arabic and 1% PVP as stabilizers and stir to dissolve. Add 10 mL of PEG-modified silica microsphere solution prepared in S2 as silicon core seeds, add 10 mL of 0.3 M selenium dioxide as growth solution, and add 10 mL of 0.3 M ascorbic acid as reducing agent. Stir at 100 rpm for 30 min at room temperature to allow colloidal selenium to aggregate on the surface of the microspheres to form a selenium shell of a certain thickness. Centrifuge and wash with water 3 times. Resuspend in 10 mL of ultrapure water to obtain silicon core selenium shell nanoparticle solution and store in a refrigerator at 4℃ for later use.
[0093] Comparative Example 5
[0094] A method for preparing a high-risk HPV testing kit, which differs from Example 3 in that the method for preparing silicon core selenium shell nanoparticles includes the following steps:
[0095] S1. Preparation of silica microspheres: Silica microspheres were prepared using the Stober method. During the preparation process, silica microspheres with a particle size of 110 nm were obtained by adjusting the concentration of ammonia water. After acidification with HNO3 and centrifugation and washing with water, silica microspheres with a positive charge on the surface and a concentration of 1 mg / mL were finally obtained.
[0096] Preparation of S2 and PEG-modified silica microsphere solution: The silica microspheres obtained in S1 were surface-grafted and modified using succinic anhydride as a modifier to induce carboxyl functional groups on the surface, resulting in carboxylated silica microspheres. 10 mL of carboxylated silica microspheres were added to 10 mL of 1.0% PEG solution, and the mixture was stirred at 500 rpm for 30 min at room temperature, ultrasonically dispersed for 10 min, centrifuged, washed three times with water, and resuspended in 10 mL of ultrapure water to obtain the PEG-modified silica microsphere solution.
[0097] S3. Preparation of silicon core selenium shell nanoparticle solution: Take 50 mL of ultrapure water and place it in a 100 mL beaker. Add 0.2% gum arabic and 1% PVP as stabilizers and stir to dissolve. Add 10 mL of PEG-modified silica microsphere solution prepared in S2 as silicon core seeds, add 10 mL of 0.3 M selenium dioxide as growth solution, and add 10 mL of 2.1 M ascorbic acid as reducing agent. Stir at 100 rpm for 30 min at room temperature to allow colloidal selenium to aggregate on the surface of the microspheres to form a selenium shell of a certain thickness. Centrifuge and wash with water 3 times. Resuspend in 10 mL of ultrapure water to obtain silicon core selenium shell nanoparticle solution and store in a refrigerator at 4℃ for later use.
[0098] Table 2. Validation results of clinical samples from the examples and comparative examples.
[0099]
[0100] As shown in Table 2:
[0101] (1) Comparative Examples 1 and 2 are prepared using traditional processes for colloidal gold and colloidal selenium, and there are obvious omissions compared to Example 3;
[0102] (2) In Comparative Example 3, the silica microspheres were not modified with PEG. The prepared silicon core selenium shell nanoparticles were turbid and poorly dispersed. TEM detection results showed that the particle size was inconsistent and the shape was irregular.
[0103] (3) The amount of ascorbic acid added in Comparative Example 4 was too low, and the prepared silicon core selenium shell nanoparticles were of different sizes and irregular in shape, and there were false positives compared with Example 3.
[0104] (4) The amount of ascorbic acid added in Comparative Example 5 was too high, the particle size was smaller than that in Example 3, the sensitivity was low, and there was a possibility of missed detection.
[0105] Compared to Comparative Examples 1-5, the kit prepared in Example 3 of this invention has higher sensitivity and stronger specificity in actual detection applications.
[0106] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Examples of implementation methods are provided, and any parts not described in detail are common knowledge to those skilled in the art. Those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.
Claims
1. A method for preparing a high-risk HPV detection kit, characterized in that: The method for preparing the labeled pad is as follows: HPV16 E6 / E7-2 antibody-silicon core selenium shell label and HPV18 E6 / E7-2 antibody-silicon core selenium shell label are mixed evenly at a volume ratio of 1:1 and then sprayed onto glass fiber MA0800 to obtain the labeled pad. Both HPV16 E6 / E7-2 antibody-silicon core selenium shell label and HPV18 E6 / E7-2 antibody-silicon core selenium shell label are prepared by labeling with silicon core selenium shell nanoparticle solution as tracer. The preparation method of the HPV16 E6 / E7-2 antibody-silicon core selenium shell label is as follows: Take 5 mL of silicon core selenium shell nanoparticle solution and adjust the pH to 8.0 with 0.1 M potassium carbonate; quickly add 0.5 mg / mL HPV16 E6 / E7-2 antibody, react fully for 10 min, add 1% BSA to the final concentration, react fully for 10 min, centrifuge at 10000 rpm for 15 min, and resuspend in 5 mL of resuspension to obtain HPV16 E6 / E7-2 antibody-silicon core selenium shell label; The preparation method of the HPV18 E6 / E7-2 antibody-silicon core selenium shell label is as follows: Take 5 mL of silicon core selenium shell nanoparticle solution and adjust the pH to 8.0 with 0.1 M potassium carbonate; quickly add 0.5 mg / mL HPV18 E6 / E7-2 antibody, react fully for 10 min, add 1% BSA to the final concentration, react fully for 10 min, centrifuge at 10000 rpm for 15 min, and resuspend in 5 mL of resuspension to obtain HPV18 E6 / E7-2 antibody-silicon core selenium shell label; The silicon core selenium shell nanoparticle solution utilizes a seed growth method. First, the surface of silica microspheres is grafted and modified. Then, PEG is applied to the surface of the modified SNPs. The PEG-modified SNPs serve as silicon core seeds, and selenium dioxide is used as the growth solution. A selenium shell layer is then grown on the surface of the silicon core seeds to obtain core-shell structured silicon core selenium shell nanoparticles. The method for preparing the silicon core selenium shell nanoparticle solution includes the following steps: S1. Preparation of silica microspheres: Silica microspheres were prepared by the Stober method. During the preparation process, silica microspheres with a particle size of 110 nm were obtained by adjusting the concentration of ammonia water. After acidification with HNO3 and centrifugation and washing with water, silica microspheres with a positive charge on the surface with a concentration of 1 mg / mL were finally obtained. Preparation of S2 and PEG-modified silica microsphere solution: The silica microspheres obtained in S1 were surface grafted and modified using succinic anhydride as a modifier to perform surface carboxyl functional grouping, resulting in carboxylated silica microspheres; 10 mL of carboxylated silica microspheres were taken, 10 mL of PEG solution was added, and the mixture was stirred at 500 rpm for 30 min at room temperature, ultrasonically dispersed for 10 min, centrifuged and washed with water 3 times, and resuspended in 10 mL of ultrapure water to obtain PEG-modified silica microsphere solution; Preparation of S3, Silicon Core Selenium Shell Nanoparticle Solution: Take 50 mL of ultrapure water and place it in a 100 mL beaker. Add 0.2% gum arabic and 1% PVP as stabilizers and stir to dissolve. Add 10 mL of the PEG-modified silica microsphere solution prepared in S2 as silicon core seeds, add 10 mL of 0.3 M selenium dioxide as growth solution, and add 10 mL of ascorbic acid as reducing agent. Stir at 100 rpm for 30 min at room temperature to allow colloidal selenium to aggregate on the surface of the microspheres to form a selenium shell. Centrifuge and wash 3 times with water, and resuspend in 10 mL of ultrapure water to obtain the silicon core selenium shell nanoparticle solution. Store in a refrigerator at 4℃ for later use. The concentration of ascorbic acid is 0.6-1.5M.
2. The method for preparing a high-risk HPV detection kit according to claim 1, characterized in that: The silicon core selenium shell nanoparticles have a particle size of 150-200 nm.
3. The method for preparing a high-risk HPV detection kit according to claim 2, characterized in that: The concentration of the PEG solution is 0.5%-1.5%.
4. A method for preparing a high-risk HPV detection kit according to any one of claims 1-3, characterized in that: It also includes a method for preparing a solid-phase reaction membrane, which involves coating an NC membrane with HPV16 E6 / E7-1 antibody, HPV18 E6 / E7-1 antibody, and goat anti-mouse antibody as the detection T1 line, detection T2 line, and quality control C line, respectively.
5. The reagent kit prepared according to the method for preparing a high-risk HPV detection kit according to claim 4, characterized in that: The test strip consists of a PVC plate, on which absorbent paper, a solid-phase reaction membrane, a label pad, and a sample pad are arranged from left to right. The lysis buffer consists of 100-150 mM PBS solution, 0.05% NP40 by mass, and 0.01% X-100 by mass.