Preparation method and application of vaginal secretion hydrogen peroxide hypersensitivity detection reagent

Abstract

The invention discloses a preparation method of a vaginal secretion hydrogen peroxide hypersensitive detection reagent. The preparation method comprises the following steps: preparing a buffer solution with the pH value of 6.8-7.2 and the concentration of 10-100 mmol / L; the buffer solution is a 3-(N-morpholine) propanesulfonic acid buffer solution, a phosphate buffer solution, a citric acid-dipotassium phosphate buffer solution or a citric acid-disodium hydrogen phosphate buffer solution; preparing a second solution for reducing the biological entropy; the second solution is a hydroxypropyl-beta-cyclodextrin solution, a beta-cyclodextrin solution, an agarose solution, a carboxylated chitosan solution, a sericin solution or a chitosan oligosaccharide solution; preparing a third solution required for combining the hydrogen donor and the color source, wherein the third solution is a POD-hydrogen donor solution; wherein the hydrogen donor is TOPS, TOOS, DHBS, ADOS, ADPS, ALPS, DAOS, HDAOS or MADB, and the hydrogen donor is a hydrogen donor. Preparing a fourth solution required for combining the hydrogen donor and the color source, wherein the fourth solution is a color source solution; and preparing H2O2 solid-phase liquid for preparing the vaginal secretion hydrogen peroxide hypersensitivity detection reagent based on the second solution, the third solution and the fourth solution. The invention also discloses a kit as well as a preparation method and application thereof.

Description

Technical Field

[0001] The present invention relates to the technical field of vaginal secretion ultrasensitive detection, and in particular to a method for preparing a vaginal secretion hydrogen peroxide ultrasensitive detection reagent and application thereof. Background Art

[0002] Hydrogen peroxide is a component secreted by lactic acid bacteria in the vagina. Hydrogen peroxide has a bactericidal effect and can maintain the self-cleaning of the vagina. It is also a good indicator for judging whether the vaginal ecology is normal or not, and can be used to understand the vitality of the dominant bacteria in the vaginal ecology. When the hydrogen peroxide concentration in the vagina is higher than 2μmol / L, it means that the content of lactic acid bacteria is high, reflecting that the vaginal microorganisms are normal, and the vagina is in a normal and healthy state at this time; when the hydrogen peroxide concentration in the vagina is lower than 2μmol / L, it means that the lactobacillus microorganisms are unbalanced, the lactobacillus in the vagina is reduced, and other miscellaneous bacteria can grow, and the vaginal self-cleaning ability is poor. At this time, dysbacteriosis will occur, and the vagina is in a pathological state. However, vaginal secretions are complex and there are many interfering factors. For example, catalase will degrade hydrogen peroxide and interfere with the reaction, making the determination of hydrogen peroxide more difficult.

[0003] At present, the common method for detecting hydrogen peroxide is the Trinder reaction, also known as the "coupled end-point colorimetric method". Its principle is that the hydrogen peroxide (H2O2) produced by the test substance through the action of the enzyme can generate a red quinone imine compound in the presence of 4-aminoantipyrine (4-AA) and peroxidase (POD). Catalase is also a type of peroxidase and can catalyze the reaction of hydrogen peroxide. However, there are the following differences between the catalytic reactions of peroxidase (POD) and catalase: when peroxidase (POD) catalyzes a chromogenic substrate or a luminescent substrate, there are two substrates: hydrogen peroxide and Trinder's reagent as a hydrogen donor. When catalase catalyzes a chromogenic substrate or a luminescent substrate, the substrate is only hydrogen peroxide, and catalase also serves as a hydrogen donor.

[0004] Traditional Trinder's reagents mainly include phenols (such as phenol, 4-chlorophenol, 2,4-dichloro-6-phenolsulfonate sodium, etc.) and anilines (N,N-dialkylaniline, N,N-dialkyl-m-toluidine, etc.). However, due to the poor substrate specificity of peroxidase (POD) that catalyzes the Trinder reaction, reducing substances such as vitamin C, uric acid, glutathione and bilirubin also compete with chromogenic substances for H2O2, so that the H2O2 produced during the oxidation process is consumed, resulting in reduced pigment formation and low measurement results. In addition, if there is a large amount of catalase mixed in the tool enzyme, it can also consume the oxidation product H2O2, making the measured value of the analyte low and reducing the accuracy of the test.

[0005] At present, the new Trinder's reagents mainly include N-ethyl-N-(3-sulfopropyl)-3-methylaniline sodium salt (TOPS), N-(2-hydroxy-3-sulfopropyl)-3'5-dimethoxyaniline sodium salt (HDAOS), N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline sodium salt (TOOS), 3,5-dichloro-2-hydroxybenzenesulfonate sodium (DHBS), N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methoxyaniline sodium salt (ADOS), N-ethyl-N-(3-sulfopropyl)-3-methoxyaniline sodium salt (ADPS), N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline sodium salt (DAOS), N,N-di(4-sulfobutyl)-3,5-dimethylaniline sodium salt (MADB), etc. The reaction principle of these new chromogens is similar to that of traditional chromogens, but they have more advantages. In the presence of hydrogen peroxide and peroxidase, the new Trinder's reagent forms a very stable purple or blue dye during the oxidative coupling reaction with 4-aminoantipyrine (4-AA) or 3-methylbenzothiazole sulfone hydrazone (MBTH). The molar absorbance of the dye coupled with MBTH is 1.5-2 times higher than that of the dye coupled with 4-AA. The substrate is oxidized by its oxidase to produce hydrogen peroxide. The concentration of this hydrogen peroxide corresponds to the concentration of the substrate, so the amount of the substrate can be determined by the color development of the oxidative coupling reaction. The new Trinder's reagent has high water solubility and is a stable aniline analog. The pH range of the color development process and oxidation reaction is very wide, but the new Trinder's reagent still has the problem of reagent instability.

[0006] Therefore, the present invention needs to prepare a new detection reagent and a corresponding kit to solve the problems of low sensitivity of the determination reagent, many interferences in the measurement process, unstable reagents and harsh storage and transportation conditions of the reagents in the current method for determining hydrogen peroxide. Summary of the invention

[0007] The purpose of the present invention is to overcome the defects of the prior art. Aiming at the problems of low reagent sensitivity, reagent instability and harsh storage and transportation conditions in the related technology, a method for preparing a vaginal secretion hydrogen peroxide ultrasensitive detection reagent and its application are provided. Hydrogen donors TOPS, TOOS, DHBS, ADOS, ADPS, ALPS, DAOS, HDAOS, MADB and the like are combined with color sources 4-AA and MBTH; hydroxypropyl-β-cyclodextrin solution, β-cyclodextrin solution, agarose solution, carboxylated chitosan solution, sericin solution or chitosan oligosaccharide solution which can reduce biological entropy are added; amino acids, sucrose, fucose, trehalose and the like which can remove active oxygen are added; enzyme stabilizers BO3 and bovine serum albumin which can stabilize the enzyme are added; surfactants S9, dextran T70 and Triton X-100 which can reduce the surface hydrophobicity of the protein and increase the internal hydrophobicity of the protein are added. X-100), propylene glycol, inositol, etc.; add 3-(N-morpholine) propanesulfonic acid (MOPS) buffer with a pH value of 6.8-7.2 and a concentration of 10-100mmol / L, phosphate buffer (PB), citric acid-dipotassium hydrogen phosphate buffer or citric acid-disodium hydrogen phosphate buffer to eliminate the influence of metal ions, and also slow down the change of pH when a certain amount of other substances are added to maintain the activity of the enzyme.

[0008] The first aspect of the present invention is to provide a method for preparing a vaginal secretion hydrogen peroxide ultrasensitive detection reagent, comprising: S1, preparing a buffer solution with a pH value of 6.8-7.2 and a concentration of 10-100 mmol / L; the buffer solution is 3-(N-morpholino)propanesulfonic acid (MOPS) buffer, phosphate buffer (PB) buffer, citric acid-dipotassium hydrogen phosphate buffer or citric acid-disodium hydrogen phosphate buffer; S2, preparing a second solution for reducing biological entropy; the second solution is a hydroxypropyl-β-cyclodextrin solution, a β-cyclodextrin solution, an agarose solution, a carboxylated chitosan solution, a sericin solution or a chitosan oligosaccharide solution; S3, preparing a third solution required for combining a hydrogen donor with a color source, wherein the third solution is a POD-hydrogen donor solution; wherein the hydrogen donor is TOPS, TOOS, DHBS, ADOS, ADPS, ALPS, DAOS, HDAOS or MADB; S4, preparing a fourth solution required for combining the hydrogen donor with the color source, wherein the fourth solution is a color source solution; S5, preparing a H2O2 solid phase liquid based on the second solution, the third solution and the fourth solution, wherein the H2O2 solid phase liquid is used to prepare a vaginal secretion hydrogen peroxide ultrasensitive detection reagent.

[0009] Preferably, the S1 comprises: S11, using a ten-thousandth electronic balance to weigh a certain amount of a buffer matrix for preparing the buffer; wherein the buffer matrix of the 3-(N-morpholine)propanesulfonic acid (MOPS) buffer is MOPS, the buffer matrix of the phosphate (PB) buffer is PB, the buffer matrix of the citric acid-dipotassium hydrogen phosphate buffer is citric acid-dipotassium hydrogen phosphate, and the buffer matrix of the citric acid-disodium hydrogen phosphate buffer is citric acid-disodium hydrogen phosphate; S12, dissolving the buffer matrix in purified water and stirring until completely dissolved to form a first solution; S13, adding a quantitative alkaline solution to the first solution to adjust the pH value to 6.9-7.1; S14, using purified water to make up the volume with a measuring cylinder and retesting the pH value to see whether it is maintained at 6.9-7.1, thereby preparing a buffer solution with a pH value maintained at 6.9-7.1.

[0010] Preferably, S2 includes: S21, weighing a certain amount of a second solution matrix using a ten-thousandth electronic balance, wherein the second solution matrix is ​​hydroxypropyl-β-cyclodextrin, β-cyclodextrin, agarose, carboxylated chitosan, sericin or chitosan oligosaccharide; S22, adding a certain amount of purified water to the second solution matrix and dissolving by ultrasonication until fully mixed, thereby forming a quasi-second solution; S23, using purified water in a measuring cylinder to adjust the volume of the quasi-second solution to form a second solution.

[0011] Preferably, S3 includes: S31, taking a certain amount of buffer solution with a pH value maintained at 6.9-7.1, adding a certain amount of enzyme stabilizer to the buffer solution and stirring until it is completely dissolved; S32, adding a certain amount of hydrogen donor to the solution prepared in S31 and stirring until it is completely dissolved; S33, adding a certain amount of enzyme stabilizer to the solution prepared in S32 again and stirring until it is completely dissolved; S34, sequentially adding a certain amount of one or more active oxygen scavengers to the solution prepared in S33 and stirring until completely dissolved; S35, adding a certain concentration of quantitative peroxidase (POD) to the solution prepared in S34 and stirring until it is completely dissolved; The enzyme stabilizer in S31 and S33 is enzyme stabilizer BO3 or bovine serum albumin that can stabilize the enzyme. The same enzyme stabilizer or different enzyme stabilizers can be selected for the two steps. The active oxygen remover in S34 is amino acids, sucrose, fucose or trehalose that can remove active oxygen.

[0012] Preferably, S4 includes: S41, measuring a certain amount of a buffer solution having a pH value maintained at 6.9-7.1, adding a certain amount of the active oxygen scavenger and stirring until completely dissolved; S42, adding a certain amount of color source to the solution prepared in S41 and stirring until completely dissolved and mixed to form the fourth solution, wherein the color source is 4-AA or MBTH.

[0013] Preferably, S5 includes: S51, measuring a certain amount of the second solution with a certain concentration, adding a certain amount of the third solution to the second solution, and stirring until the mixture is uniform; S52, adding the fourth solution to the solution prepared in S51 and stirring until the mixture is uniformly mixed to form the H2O2 solid phase liquid, wherein the H2O2 solid phase liquid is used to prepare a vaginal secretion hydrogen peroxide ultrasensitive detection reagent.

[0014] Preferably, the S52 includes: The H2O2 solid phase liquid and the second solution are solidified and printed in the dry chemical reaction device in a constant volume manner, and then quickly dried, cooled for a certain period of time and placed at room temperature.

[0015] Preferably, the S5 further includes: S53, adding a surfactant that reduces the surface hydrophobicity of the protein and increases the internal hydrophobicity of the protein to the H2O2 solid phase liquid; the surfactant is one or more of surfactant S9, dextran T70, Triton X-100, propylene glycol and inositol.

[0016] The second aspect of the present invention is to provide a kit for ultrasensitive detection of hydrogen peroxide in vaginal secretions, the kit comprising: A dry chemical reaction device, a sample diluent and at least one color developer, wherein the color developer comprises the vaginal secretion hydrogen peroxide ultrasensitive detection reagent prepared in the first aspect; The dry chemical reaction device has at least one reaction hole for detecting hydrogen peroxide, and the reaction hole is used to add the vaginal secretion sample diluted by the sample diluent, and the sample diluent is placed in a diluent tube.

[0017] The third aspect of the present invention is to provide a method for preparing the kit for ultrasensitive detection of hydrogen peroxide in vaginal secretions according to the second aspect, comprising: (1) In a solid phase room at a temperature of 20±2°C and a relative humidity of 50±5%, use a micro-dispenser to take the H2O2 solid phase liquid-solid phase, print it into the reaction wells of the dry chemical reaction device at 8 μL / well, place it in a rapid drying device, and dry it at a high wind speed for 10-30 minutes; place the dried device in a drying room with an ambient temperature of 22±2°C and a relative humidity of <40% to cool it for 20 minutes to room temperature for use; (2) Then, 0.2-0.6% of the second solution solid phase is taken with a microdispenser, and 8 μL / well is printed into the reaction wells of the dry chemical reaction device, and the device is placed in a rapid drying device and dried at a high wind speed for 10-30 minutes; the dried device is placed in a drying room with an ambient temperature of 22±2°C and a relative humidity of <40% and cooled for 20 minutes to room temperature; (3) Pack the dry chemical reaction device into aluminum foil packaging bags, add an appropriate amount of desiccant, and store in an environment of 2-30°C; (4) Prepare sample diluent and place in the diluent tube.

[0018] The fourth aspect of the present invention is to provide use of the kit for ultrasensitive detection of hydrogen peroxide in vaginal secretions according to the third aspect in the detection of vaginal secretions.

[0019] Beneficial effects of the reagent preparation method, kit and preparation method of the present invention: (1) Combine hydrogen donors TOPS, TOOS, DHBS, ADOS, ADPS, ALPS, DAOS, HDAOS, MADB, etc. with color sources 4-AA and MBTH; (2) adding hydroxypropyl-β-cyclodextrin solution, β-cyclodextrin solution, agarose solution, carboxylated chitosan solution, sericin solution or chitosan oligosaccharide solution which can reduce biological entropy; (3) Adding amino acids, sucrose, fucose, trehalose, etc. that can remove active oxygen; (4) Add enzyme stabilizers such as BO3 and bovine serum albumin to stabilize the enzyme; (5) Add surfactants such as S9, Dextran T70, Triton X-100, glycerol, and inositol to reduce the surface hydrophobicity of the protein and increase the internal hydrophobicity of the protein; (6) Add 3-(N-morpholino)propanesulfonic acid (MOPS) buffer, phosphate buffer (PB), citric acid-dipotassium hydrogen phosphate buffer or citric acid-disodium hydrogen phosphate buffer with a pH value of 6.8-7.2 and a concentration of 10-100 mmol / L to eliminate the influence of metal ions. It can also slow down the pH change when a certain amount of other substances are added to maintain the activity of the enzyme. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the related technologies, the drawings required for use in the specific embodiments or the related technical descriptions will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0021] Figure 1 This is a flow chart of a method for preparing a vaginal secretion hydrogen peroxide ultrasensitive detection reagent according to an embodiment of the present invention; Figure 2 This is a flow chart of step S1 in the method for preparing a vaginal secretion hydrogen peroxide ultrasensitive detection reagent according to an embodiment of the present invention; Figure 3 This is a flow chart of step S2 in the method for preparing a vaginal secretion hydrogen peroxide ultrasensitive detection reagent according to an embodiment of the present invention; Figure 4 This is a flow chart of step S3 in the method for preparing a vaginal secretion hydrogen peroxide ultrasensitive detection reagent according to an embodiment of the present invention; Figure 5 This is a flow chart of step S4 in the method for preparing a vaginal secretion hydrogen peroxide ultrasensitive detection reagent according to an embodiment of the present invention; Figure 6 This is a flow chart of step S5 in the method for preparing a vaginal secretion hydrogen peroxide ultrasensitive detection reagent provided according to an embodiment of the present invention. DETAILED DESCRIPTION

[0022] The technical solution of the present invention will be described clearly and completely below in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0023] In the description of the present invention, it should be noted that the terms "center", "upper", "lower", "left", "right", "vertical", "horizontal", "inner", "outer", etc., indicating the orientation or positional relationship, are based on the orientation or positional relationship shown in the drawings, and are only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore cannot be understood as limiting the present invention. In addition, the terms "first", "second", and "third" are used for descriptive purposes only, and cannot be understood as indicating or implying relative importance.

[0024] In the description of the present invention, it should be noted that, unless otherwise clearly specified and limited, the terms "installed", "connected", and "connected" should be understood in a broad sense, for example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be a direct connection, or it can be indirectly connected through an intermediate medium, or it can be the internal communication of two components. For ordinary technicians in this field, the specific meanings of the above terms in the present invention can be understood according to specific circumstances. Embodiment 1

[0025] like Figure 1 As shown, this embodiment provides a method for preparing a vaginal secretion hydrogen peroxide ultrasensitive detection reagent, comprising: S1, preparing a buffer solution with a pH value of 6.8-7.2 and a concentration of 10-100 mmol / L; the buffer solution is used to eliminate the influence of metal ions on the detection reagent, and at the same time slow down the change of pH when a certain amount of other substances is added to maintain the activity of the enzyme; As a preferred embodiment, the buffer is 3-(N-morpholino)propanesulfonic acid (MOPS) buffer, phosphate buffer (PB), citric acid-dipotassium hydrogen phosphate buffer or citric acid-disodium hydrogen phosphate buffer.

[0026] like Figure 2 As shown, as a preferred embodiment, the S1 includes: S11, using a ten-thousandth electronic balance to weigh a certain amount of a buffer matrix for preparing the buffer; in this embodiment, the buffer matrix of the 3-(N-morpholine)propanesulfonic acid (MOPS) buffer is MOPS, the buffer matrix of the phosphate (PB) buffer is PB, the buffer matrix of the citric acid-dipotassium hydrogen phosphate buffer is citric acid-dipotassium hydrogen phosphate, and the buffer matrix of the citric acid-disodium hydrogen phosphate buffer is citric acid-disodium hydrogen phosphate.

[0027] S12, dissolving the buffer matrix in purified water and stirring until completely dissolved to form a first solution; S13, adding a quantitative alkaline solution to the first solution to adjust the pH value to 6.9-7.1; S14, using purified water to make up the volume with a measuring cylinder and retesting the pH value to see whether it is maintained at 6.9-7.1, thereby preparing a buffer solution with a pH value maintained at 6.9-7.1.

[0028] S2, preparing a second solution for reducing biological entropy; the second solution is a hydroxypropyl-β-cyclodextrin solution, a β-cyclodextrin solution, an agarose solution, a carboxylated chitosan solution, a sericin solution or a chitosan oligosaccharide solution; like Figure 3 As shown, as a preferred embodiment, the S2 includes: S21, weighing a certain amount of a second solution matrix using a ten-thousandth electronic balance, wherein the second solution matrix is ​​hydroxypropyl-β-cyclodextrin, β-cyclodextrin, agarose, carboxylated chitosan, sericin or chitosan oligosaccharide; S22, adding a certain amount of purified water to the second solution matrix and dissolving by ultrasonication until fully mixed, thereby forming a quasi-second solution; S23, using purified water in a measuring cylinder to adjust the volume of the quasi-second solution to form a second solution.

[0029] S3, preparing a third solution required for combining a hydrogen donor with a color source, wherein the third solution is a POD-hydrogen donor solution; wherein the hydrogen donor is TOPS, TOOS, DHBS, ADOS, ADPS, ALPS, DAOS, HDAOS or MADB; like Figure 4 As shown, as a preferred implementation, S3 includes: S31, taking a certain amount of buffer solution with a pH value maintained at 6.9-7.1, adding a certain amount of enzyme stabilizer to the buffer solution and stirring until it is completely dissolved; S32, adding a certain amount of hydrogen donor to the solution prepared in S31 and stirring until it is completely dissolved; S33, adding a certain amount of enzyme stabilizer to the solution prepared in S32 again and stirring until it is completely dissolved; In this embodiment, the enzyme stabilizer in S31 and S33 is the enzyme stabilizer BO3 or bovine serum albumin that can stabilize the enzyme. The same enzyme stabilizer or different enzyme stabilizers can be selected for the two steps, and those skilled in the art can conceive of using other enzyme stabilizers commonly used in the art, all of which are within the scope of protection of the present invention.

[0030] S34, sequentially adding a certain amount of one or more active oxygen scavengers to the solution prepared in S33 and stirring until completely dissolved; In this embodiment, the active oxygen scavenger of S34 is amino acid, sucrose, fucose or trehalose which can remove active oxygen.

[0031] S35, adding a certain concentration of quantitative peroxidase (POD) to the solution prepared in S34 and stirring until it is completely dissolved.

[0032] S4, preparing a fourth solution required for combining the hydrogen donor with the color source, wherein the fourth solution is a color source solution; like Figure 5 As shown, as a preferred embodiment, the S4 includes: S41, measuring a certain amount of a buffer solution having a pH value maintained at 6.9-7.1, adding a certain amount of the active oxygen scavenger and stirring until completely dissolved; S42, adding a certain amount of color source to the solution prepared in S41 and stirring until completely dissolved and mixed to form the fourth solution, wherein the color source is 4-AA or MBTH.

[0033] S5, preparing a H2O2 solid phase liquid based on the second solution, the third solution and the fourth solution, wherein the H2O2 solid phase liquid is used to prepare a vaginal secretion hydrogen peroxide ultrasensitive detection reagent; like Figure 6 As shown, as a preferred implementation, the S5 includes: S51, measuring a certain amount of the second solution with a certain concentration, adding a certain amount of the third solution to the second solution, and stirring until the mixture is uniform; S52, adding the fourth solution to the solution prepared in S51 and stirring until the mixture is uniformly mixed to form the H2O2 solid phase liquid, wherein the H2O2 solid phase liquid is used to prepare a vaginal secretion hydrogen peroxide ultrasensitive detection reagent.

[0034] As a preferred implementation, the S5 further includes: S53, adding a surfactant to the H2O2 solid phase liquid to reduce the surface hydrophobicity of the protein and increase the internal hydrophobicity of the protein.

[0035] As a preferred embodiment, the surfactant is one or more of surfactant S9, Dextran T70, Triton X-100, glycerol and inositol.

[0036] As a preferred implementation, the S52 includes: The H2O2 solid phase liquid and the second solution are solidified and printed in the dry chemical reaction device in a constant volume manner, and then quickly dried, cooled for a certain period of time and placed at room temperature. Embodiment 2

[0037] This embodiment provides a kit for ultrasensitive detection of hydrogen peroxide in vaginal secretions, the kit comprising: A dry chemical reaction device, a sample diluent and at least one color developer, wherein the color developer comprises the vaginal secretion hydrogen peroxide ultrasensitive detection reagent prepared in Example 1; The dry chemical reaction device has at least one reaction hole for detecting hydrogen peroxide, and the reaction hole is used to add the vaginal secretion sample diluted by the sample diluent, and the sample diluent is placed in a diluent tube. Embodiment 3

[0038] This embodiment provides a method for preparing the kit for ultrasensitive detection of hydrogen peroxide in vaginal secretions of embodiment 2, comprising: (1) In a solid phase room at a temperature of 20±2°C and a relative humidity of 50±5%, use a micro-dispenser to take the H2O2 solid phase liquid-solid phase, print it into the reaction wells of the dry chemical reaction device at 8 μL / well, place it in a rapid drying device, and dry it at a high wind speed for 10-30 minutes; place the dried device in a drying room with an ambient temperature of 22±2°C and a relative humidity of <40% to cool it for 20 minutes to room temperature for use; (2) Then, 0.2-0.6% of the second solution solid phase is taken with a microdispenser, and 8 μL / well is printed into the reaction wells of the dry chemical reaction device, and the device is placed in a rapid drying device and dried at a high wind speed for 10-30 minutes; the dried device is placed in a drying room with an ambient temperature of 22±2°C and a relative humidity of <40% and cooled for 20 minutes to room temperature; (3) Pack the dry chemical reaction device into aluminum foil packaging bags, add an appropriate amount of desiccant, and store in an environment of 2-30°C; (4) Prepare sample diluent and place in the diluent tube. Embodiment 4

[0039] This embodiment provides the use of the kit for ultrasensitive detection of hydrogen peroxide in vaginal secretions of embodiment 3 in the detection of vaginal secretions.

[0040] Specific application examples 1. Preparation of ultrasensitive detection reagent for hydrogen peroxide in vaginal secretions (1) Prepare pH 6.9-7.115-25mmol / L MOPS buffer (taking 20mL as an example) Weigh 0.0628-0.1046g MOPS with a ten-thousandth electronic balance, dissolve in 15mL purified water, stir with a glass rod until completely dissolved, adjust the pH value to 6.9-7.1 with 5M NaOH, use a graduated cylinder with purified water to make up to 20mL, and re-measure the pH value to 6.9-7.1. The final solution is a colorless transparent liquid with no precipitation and no visible impurities.

[0041] (2) Preparation of 0.2-0.6% carboxylated chitosan (taking 200 mL as an example) Weigh 0.4000-0.1200g of carboxylated chitosan with a ten-thousandth electronic balance, mix thoroughly in 160mL of purified water (use ultrasonic dissolution), and use a graduated cylinder with purified water to make up to 200mL. The final solution is a colorless transparent liquid with no precipitation and no visible impurities.

[0042] (3) Preparation of POD-TOOS solution (taking 6 mL as an example) Take 6mL of pH 6.9-7.115-25mmol / L MOPS buffer solution, add 0.0060-0.0180g of enzyme stabilizer B03, stir with a glass rod until completely dissolved, add 0.0600-0.1800g of TOOS, stir with a glass rod until completely dissolved, add 0.1200-0.3000g of bovine serum albumin, stir with a glass rod until completely dissolved, then add 0.6000-1.8000g of sucrose, stir with a glass rod until completely dissolved, then add 0.6000-1.8000g of D-trehalose, stir with a glass rod until completely dissolved. Take 6mL of the above mixed liquid, add POD 2400-2700U (final concentration: 400-450U / mL), stir with a glass rod until completely dissolved, the final solution color is brown-yellow, no precipitation, no visible impurities.

[0043] (4) Preparation of 4-AA solution (taking 6 mL as an example) Take 6mL of pH 6.9-7.115-25mmol / L MOPS buffer solution, add 0.1000-0.3000g of D-trehalose, stir with a glass rod until completely dissolved, add 0.0360-0.0480g of 4-AA, stir with a glass rod until completely dissolved, and mix well. The final solution is light yellow in color, with no precipitation and no visible impurities.

[0044] (5) Preparation of H2O2 solid phase liquid (taking 81 mL as an example) Take 70.2 mL of 0.2-0.6% carboxylated chitosan solution, add 5.4 mL of POD-TOOS solution, stir with a glass rod until mixed evenly, then add 5.4 mL of 4-AA solution, stir with a glass rod until mixed evenly. The final solution is light yellow in color, with no precipitation and no visible impurities.

[0045] (II) Preparation of the kit In a solid phase room at a temperature of 20±2℃ and a relative humidity of 50±5%. Use a micro-dispenser to take the H2O2 solid phase liquid-solid phase, print it in the device at 8μL / well, place it in a rapid drying device, and dry it at a high wind speed for 10-30min. Place the dried device in a drying room with an ambient temperature of 22±2℃ and a relative humidity of <40%, cool it for 20min and place it at room temperature for use. Use a micro-dispenser to take the 0.2-0.6% carboxylated chitosan solution solid phase, print it in the device at 8μL / well, place it in a rapid drying device, and dry it at a high wind speed for 10-30min. Place the dried device in a drying room with an ambient temperature of 22±2℃ and a relative humidity of <40%, cool it for 20min and place it at room temperature. Dispense the device in an aluminum foil bag, add an appropriate amount of desiccant, and store it in an environment of 2-30℃. Prepare the sample diluent and place it in a diluent tube.

[0046] (III) Instrument operation 1. Take out the test kit and equilibrate it to room temperature (10℃~30℃) before use.

[0047] 2. Obtaining and processing vaginal secretion samples: Use a swab to collect samples from the posterior vaginal fornix (about 5 cm in the vagina), rotate the swab for 10-20 seconds, and collect as much secretion as possible, based on the fact that the secretion attached to the swab is clearly visible. Put the sampled swab into a soft test tube (add 600µL of diluent first) or a diluent tube, and squeeze the swab repeatedly 5-6 times to fully remove the attachments on the swab.

[0048] 3. Vaginal secretion analyzer (JY-VFS-A) Beijing Medical Device Registration No. 20172220785 4. Detection steps: Add about 35µL of diluted sample to the reaction well of the kit, put it into the detection port, operate and interpret the results according to the instrument manual: If the reaction well turns purple, it is negative; if it turns yellow, it is positive.

[0049] (IV) Comparative test of detection effect and stability of three reagents 1. The color development under different concentrations of hydrogen peroxide is shown in Table 1: Table 1

[0050] Results: The sensitivity of the novel Trinder's reagent and the ultrasensitive detection reagent of the present invention is relatively high.

[0051] 2. After being stored at 45°C for different periods of time, the novel Trinder's reagent and the ultrasensitive detection reagent of the present invention were tested with 2 μmol / L H2O2. The results are shown in Table 2: Table 2

[0052] Test results: The ultrasensitive detection reagent of the present invention has relatively good stability.

[0053] 3. Stability test of hydrogen peroxide detection device, used to test the stability of hydrogen peroxide detection device at super normal temperature (45°C).

[0054] The prepared three batches of hydrogen peroxide detection devices (10 tests in each batch, tested by 10 testers) were tested in parallel with the newly configured hydrogen peroxide detection device at 45°C in the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth and thirteenth months to verify the stability and accuracy of the reagent.

[0055] Test method: Use 2mmol / L H2O2 to test three batches of hydrogen peroxide detection devices and a newly configured hydrogen peroxide detection device, and observe the color change. The color development of the three parallel reaction devices is also consistent.

[0056] Test results: The hydrogen peroxide detection device remains stable at super-normal temperature (45°C).

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[0068] December

[0069] 13th month

[0070] Through the description of the above implementation methods, those skilled in the art can clearly understand that the above embodiments can be implemented by software, or by means of software plus necessary general hardware platforms. Based on such understanding, the technical solutions of the above embodiments can be embodied in the form of software products, which can be stored in a non-volatile storage medium (which can be a CD-ROM, a USB flash drive, a mobile hard disk, etc.), including several instructions for enabling a computer device (which can be a personal computer, a server, or a network device, etc.) to execute the methods described in the various embodiments of the present invention.

[0071] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing a vaginal secretion hydrogen peroxide ultrasensitive detection reagent, characterized in that: include: S1, preparing a buffer solution with a pH value of 6.8-7.2 and a concentration of 10-100 mmol / L; the buffer solution is 3-(N-morpholine)propanesulfonic acid buffer, phosphate buffer, citric acid-dipotassium hydrogen phosphate buffer or citric acid-disodium hydrogen phosphate buffer; S2, preparing a second solution for reducing biological entropy; the second solution is a hydroxypropyl-β-cyclodextrin solution, a β-cyclodextrin solution, an agarose solution, a carboxylated chitosan solution, a sericin solution or a chitosan oligosaccharide solution; S3, preparing a third solution required for combining a hydrogen donor with a color source, wherein the third solution is a POD-hydrogen donor solution; wherein the hydrogen donor is TOPS, TOOS, DHBS, ADOS, ADPS, ALPS, DAOS, HDAOS or MADB; S4, preparing a fourth solution required for combining the hydrogen donor with the color source, wherein the fourth solution is a color source solution; S5, preparing a H2O2 solid phase liquid based on the second solution, the third solution and the fourth solution, wherein the H2O2 solid phase liquid is used to prepare a vaginal secretion hydrogen peroxide ultrasensitive detection reagent.

2. The method for preparing a vaginal secretion hydrogen peroxide ultrasensitive detection reagent according to claim 1, characterized in that: The S1 includes: S11, using a ten-thousandth electronic balance to weigh a certain amount of a buffer matrix for preparing the buffer; wherein the buffer matrix of the 3-(N-morpholine)propanesulfonic acid buffer is MOPS, the buffer matrix of the phosphate buffer is PB, the buffer matrix of the citric acid-dipotassium hydrogen phosphate buffer is citric acid-dipotassium hydrogen phosphate, and the buffer matrix of the citric acid-disodium hydrogen phosphate buffer is citric acid-disodium hydrogen phosphate; S12, dissolving the buffer matrix in purified water and stirring until completely dissolved to form a first solution; S13, adding a quantitative alkaline solution to the first solution to adjust the pH value to 6.9-7.1; S14, using purified water to make up the volume with a measuring cylinder and retesting the pH value to see whether it is maintained at 6.9-7.1, thereby preparing a buffer solution with a pH value maintained at 6.9-7.

1.

3. The method for preparing a vaginal secretion hydrogen peroxide ultrasensitive detection reagent according to claim 2, characterized in that: The S2 includes: S21, weighing a certain amount of a second solution matrix using a ten-thousandth electronic balance, wherein the second solution matrix is ​​hydroxypropyl-β-cyclodextrin, β-cyclodextrin, agarose, carboxylated chitosan, sericin or chitosan oligosaccharide; S22, adding a certain amount of purified water to the second solution matrix and dissolving by ultrasonication until fully mixed, thereby forming a quasi-second solution; S23, using purified water in a measuring cylinder to adjust the volume of the quasi-second solution to form a second solution.

4. The method for preparing a vaginal secretion hydrogen peroxide ultrasensitive detection reagent according to claim 3, characterized in that: The S3 includes: S31, taking a certain amount of buffer solution with a pH value maintained at 6.9-7.1, adding a certain amount of enzyme stabilizer to the buffer solution and stirring until it is completely dissolved; S32, adding a certain amount of hydrogen donor to the solution prepared in S31 and stirring until it is completely dissolved; S33, adding a certain amount of enzyme stabilizer to the solution prepared in S32 again and stirring until it is completely dissolved; S34, sequentially adding a certain amount of one or more active oxygen scavengers to the solution prepared in S33 and stirring until completely dissolved; S35, adding a certain concentration of quantitative peroxidase to the solution prepared in S34 and stirring until it is completely dissolved; The enzyme stabilizer in S31 and S33 is enzyme stabilizer BO3 or bovine serum albumin that can stabilize the enzyme. The same enzyme stabilizer or different enzyme stabilizers can be selected for the two steps. The active oxygen remover in S34 is amino acids, sucrose, fucose or trehalose that can remove active oxygen.

5. The method for preparing a vaginal secretion hydrogen peroxide ultrasensitive detection reagent according to claim 4, characterized in that: The S4 includes: S41, measuring a certain amount of a buffer solution having a pH value maintained at 6.9-7.1, adding a certain amount of the active oxygen scavenger and stirring until completely dissolved; S42, adding a certain amount of color source to the solution prepared in S41 and stirring until completely dissolved and mixed to form the fourth solution, wherein the color source is 4-AA or MBTH.

6. The method for preparing a vaginal secretion hydrogen peroxide ultrasensitive detection reagent according to claim 5, characterized in that: The S5 includes: S51, measuring a certain amount of the second solution with a certain concentration, adding a certain amount of the third solution to the second solution, and stirring until the mixture is uniform; S52, adding the fourth solution to the solution prepared in S51 and stirring until the mixture is uniformly mixed to form the H2O2 solid phase liquid, wherein the H2O2 solid phase liquid is used to prepare a vaginal secretion hydrogen peroxide ultrasensitive detection reagent.

7. The method for preparing a vaginal secretion hydrogen peroxide ultrasensitive detection reagent according to claim 6, characterized in that: The S52 includes: The H2O2 solid phase liquid and the second solution are solidified and printed in the dry chemical reaction device in a constant volume manner, and then quickly dried, cooled for a certain period of time and placed at room temperature.

8. The method for preparing a vaginal secretion hydrogen peroxide ultrasensitive detection reagent according to claim 7, characterized in that: The S5 further includes: S53, adding a surfactant to the H2O2 solid phase liquid that reduces the surface hydrophobicity of the protein and increases the internal hydrophobicity of the protein; the surfactant is one or more of surfactant S9, dextran T70, Triton X-100, glycerol and inositol.

9. A kit for ultrasensitive detection of hydrogen peroxide in vaginal secretions, characterized in that: The kit comprises: A dry chemical reaction device, a sample diluent and at least one color developer, wherein the color developer comprises a vaginal secretion hydrogen peroxide ultrasensitive detection reagent prepared by the preparation method according to any one of claims 1 to 8; The dry chemical reaction device has at least one reaction hole for detecting hydrogen peroxide, and the reaction hole is used to add the vaginal secretion sample diluted by the sample diluent, and the sample diluent is placed in a diluent tube.

10. The method for preparing a kit for ultrasensitive detection of hydrogen peroxide in vaginal secretions according to claim 9, characterized in that: include: (1) In a solid phase room at a temperature of 20±2°C and a relative humidity of 50±5%, use a micro-dispenser to take the H2O2 solid phase liquid-solid phase, print it into the reaction wells of the dry chemical reaction device at 8 μL / well, place it in a rapid drying device, and dry it at a high wind speed for 10-30 minutes; place the dried device in a drying room with an ambient temperature of 22±2°C and a relative humidity of <40% to cool it for 20 minutes to room temperature for use; (2) Then, 0.2-0.6% of the second solution solid phase is taken with a microdispenser, and 8 μL / well is printed into the reaction wells of the dry chemical reaction device, and the device is placed in a rapid drying device and dried at a high wind speed for 10-30 minutes; the dried device is placed in a drying room with an ambient temperature of 22±2°C and a relative humidity of <40% and cooled for 20 minutes to room temperature; (3) Pack the dry chemical reaction device into aluminum foil packaging bags, add an appropriate amount of desiccant, and store in an environment of 2-30°C; (4) Prepare sample diluent and place in a diluent tube.

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