Dry chemical qualitative detection method and kit for detecting seminal plasma fructose

Abstract

The invention relates to the technical field of biochemical medicine, in particular to a dry chemical qualitative detection method and a kit for detecting seminal plasma fructose. The method comprises the following steps: by adopting a room-temperature competitive color development system constructed by 3-quinoline boronic acid and alizarin red S, mixing a sample with a special diluent, and dropwise adding the mixture into the kit pre-loaded with test paper; and the judgment can be completed by comparing colors with naked eyes. The operation threshold and the cost limitation of equipment such as high performance liquid chromatography are avoided; the reaction is carried out under mild conditions, and strong corrosive reagents such as concentrated hydrochloric acid are not used, so that the operation safety is guaranteed; the sample treatment only needs simple dilution, and tedious pretreatment steps such as deproteinization, heating or derivatization are not needed, so that the detection efficiency is improved; the selected 3-quinoline boric acid, alizarin red S and auxiliary materials are conventional easily available chemical reagents, and the problem of obtaining special enzyme raw materials is solved. The technical effects of simplicity, convenience, quickness, safety, low cost and easiness in popularization are realized.

Description

Technical Field

[0001] This invention relates to the field of biochemical medical technology, and in particular to a dry chemical qualitative detection method and kit for detecting fructose in seminal plasma. Background Technology

[0002] Seminal fructose testing is primarily used to assess male reproductive system function. Based on the characteristic that fructose is specifically secreted by the seminal vesicles, this test can aid in the diagnosis of anatomical abnormalities such as ejaculatory duct obstruction and seminal vesicle developmental abnormalities. At the pathological level, abnormal fructose levels are associated with secretory dysfunction caused by low testosterone levels and seminal vesicle infections. The test results, combined with analysis of other indicators, have significant reference value in the differential diagnosis of obstructive azoospermia.

[0003] Currently, there are various methods for detecting fructose in refined products, including high-performance liquid chromatography (HPLC), indole colorimetric method, resorcinol method, thiobarbituric acid method, sorbitol dehydrogenase method, hexokinase method, and fructose dehydrogenase method. However, existing technologies have many limitations: HPLC, while highly accurate and specific, requires sophisticated equipment, is complex to operate, and is expensive, making it difficult to promote in primary healthcare institutions or for rapid on-site testing; chemical colorimetric methods such as indole, resorcinol, and thiobarbituric acid typically require the use of highly corrosive reagents such as concentrated hydrochloric acid, as well as heat treatment and cumbersome sample protein pretreatment steps, making the operation dangerous and time-consuming; the sorbitol dehydrogenase method has stringent requirements for the pH value of the reaction system, and endogenous sorbitol dehydrogenase in the human body may interfere with the results; while the hexokinase method and fructose dehydrogenase method have good sensitivity and specificity, the former requires multiple enzymatic reactions and incubation, making the operation complex, while the latter is limited by the difficulty and cost of obtaining fructose dehydrogenase raw materials (which depend on specific strains for production).

[0004] In summary, the existing technology lacks a simple, rapid method for qualitative detection of fructose in refined grains that does not require complex instruments, strong acids or bases, or complex sample pretreatment, and for which raw materials are readily available. Summary of the Invention

[0005] The purpose of this invention is to provide a dry chemical qualitative detection method and kit for detecting fructose in seminal plasma, which solves the problem that there is a lack of a simple, rapid qualitative detection method for seminal plasma fructose that does not require complex instruments, strong acids or bases, or complex sample pretreatment, and for which raw materials are readily available.

[0006] To achieve the above objectives, the present invention provides a dry chemical qualitative detection method for detecting fructose in seminal plasma, the dry chemical qualitative detection method for detecting fructose in seminal plasma comprising the following steps: Preparation of dry chemical test strips: Prepare a treatment solution containing 3-quinoline boric acid, alizarin red S and auxiliary reagents. After soaking the fiber sheet in the treatment solution, the fiber sheet is subjected to a roller brushing, drying and cutting process to obtain dry chemical test strips of a predetermined size. Sample diluent preparation: Dissolve Tween 80, polyethylene glycol 2000, preservative, sodium pyrophosphate and glycine in purified water to prepare the sample diluent; Assembly of dry chemical test strips; inserting the dry chemical test strips into the test wells and reference wells of the reagent kit; Sample dilution: Mix the seminal plasma sample to be tested with the sample diluent in the specified proportion; Detection and Judgment: The diluted sample and the quality control sample with a known concentration of fructose reference value were added to the test well and the reference well, respectively. After standing at room temperature for reaction, the fructose content of the refined grains was determined by comparing the colors of the two wells and using a colorimetric card.

[0007] The specific steps for preparing the dry chemical test strips are as follows: Preparation of treatment solution: Dissolve 3-quinoline boric acid (3QBA) in an organic solvent to obtain a 3QBA-organic solvent solution with a 3-quinoline boric acid concentration of 0.5%-1%; Alizarin Red S (ARS) was dissolved in phosphate buffer (PB) with a pH of 7.0-9.0 and a concentration of 0.1M-0.5M to obtain an ARS-PB solution with an Alizarin Red S concentration of 0.05%-0.2%. The 3QBA-organic solvent solution is mixed with the ARS-PB solution, and then 1%-5% by volume of surfactant, 1%-10% by mass of polymeric auxiliaries and 0.1%-0.5% by volume of preservative are added. The mixture is stirred at room temperature for 0.5 hours to obtain the treatment solution. Immersion treatment: Immerse the fiber sheet in the treatment solution, ensuring complete coverage, and maintain at 25℃-37℃ for 0.5-3 hours; Drying treatment: Remove the soaked fiber sheet, remove excess treatment solution and reduce wrinkles by mechanical roller brush, and then dry it with a forced air at 37℃-45℃ for 7-15 hours until it is completely dry; Cutting and shaping: Cut the dried fiber sheet into dry chemical test paper sheets of predetermined size.

[0008] In the step of preparing the treatment solution, the organic solvent is dimethyl sulfoxide; The phosphate buffer solution is prepared from disodium hydrogen phosphate dodecahydrate and sodium dihydrogen phosphate dihydrate; The surfactant is Tween 80; The polymeric additive is polyethylene glycol 2000; The preservative is Proclin 300.

[0009] The specific steps of the soaking process are as follows: Pour 200ml of the treatment solution into a treatment container, slowly immerse the A4-sized fiber sheet into the treatment solution, ensuring that the treatment solution completely submerges the fiber sheet and that there are no air bubbles under the fiber sheet, and oscillate in a constant temperature shaker at 25℃-37℃ and a speed of 100-200rpm for 0.5-3 hours to complete the soaking of the fiber sheet.

[0010] The specific contents of the drying process are as follows: take out the fiber sheet after soaking and shaking, lay it flat on the drying net, and use rollers or drums to brush the front and back of the fiber sheet at least 10 times each to remove excess treatment liquid and flatten the wrinkles. The fiber sheet is then transferred to an oven, the blower function is turned on, and it is dried continuously at a temperature of 37℃-45℃ for 7-15 hours until the fiber sheet is completely dry.

[0011] The specific steps of the cutting and forming step are as follows: the dried fiber sheet is first cut into at least two rectangular test strips of 30cm×6.5cm and at least one rectangular test strip of 30cm×7cm, and then the rectangular test strips are uniformly cut into square dry chemical test strips of 4.9mm×4.9mm using an automatic cutting device.

[0012] In the sample diluent preparation step, 1%-5% by volume of Tween 80, 1%-10% by mass of polyethylene glycol 2000, 0.1%-0.5% by volume of preservative, 1%-5% by mass of sodium pyrophosphate, and 1%-10% by mass of glycine are mixed and diluted with purified water to obtain the sample diluent.

[0013] The present invention also provides a kit for detecting seminal plasma fructose, which is applied to the dry chemical qualitative detection method for seminal plasma fructose as described above. The kit for detecting seminal plasma fructose includes an upper shell and a lower shell, the upper shell and the lower shell being snap-fitted together. The upper shell is provided with a reference C-hole and a detection T-hole, and the lower shell is provided with a C-hole groove and a T-hole groove. The reference C-hole corresponds to the C-hole groove, and the detection T-hole is adapted to the T-hole groove.

[0014] This invention discloses a dry chemical qualitative detection method and kit for detecting fructose in seminal plasma. Utilizing a room-temperature competitive colorimetric system constructed from 3-quinoline boric acid and Alizarin Red S, the method requires only mixing the sample with a dedicated diluent and adding it dropwise to a pre-packaged test strip. The result is determined by visual comparison of the color. The entire process eliminates the need for complex instruments, avoiding the operational barriers and cost limitations of equipment such as high-performance liquid chromatography (HPLC). The reaction is carried out under mild conditions, without the use of highly corrosive reagents such as concentrated hydrochloric acid, ensuring operational safety. Sample processing requires only simple dilution, eliminating cumbersome pretreatment steps such as protein removal, heating, or derivatization, significantly improving detection efficiency. The selected 3-quinoline boric acid, Alizarin Red S, and excipients are all commonly available chemical reagents, overcoming the difficulty of obtaining special enzyme raw materials such as fructose dehydrogenase. Therefore, this method achieves the technical advantages of being simple, rapid, safe, low-cost, and easy to promote. Attached Figure Description

[0015] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0016] Figure 1 This is a flowchart of the steps of the dry chemical qualitative detection method for detecting fructose in seminal plasma provided by the present invention.

[0017] Figure 2 This is a flowchart illustrating the specific steps involved in preparing the dry chemical test strips provided by the present invention.

[0018] Figure 3 This is a schematic diagram of the external structure of the reagent kit for detecting fructose in seminal plasma provided by the present invention.

[0019] Figure 4 This is a schematic diagram of the disassembled structure of the reagent kit for detecting fructose in seminal plasma provided by the present invention.

[0020] 201-Upper shell, 202-Lower shell, 203-Reference C-hole, 204-Detection T-hole, 205-C-hole groove, 206-T-hole groove. Detailed Implementation

[0021] Embodiments of the present invention are described in detail below, examples of which are illustrated in the accompanying drawings, wherein the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions throughout. The embodiments described below with reference to the accompanying drawings are exemplary and intended to explain the present invention, and should not be construed as limiting the present invention.

[0022] Please see Figure 1 and Figure 2 This invention provides a dry chemical qualitative detection method for detecting fructose in seminal plasma, the method comprising the following steps: S1. Preparation of dry chemical test strips: Prepare a treatment solution containing 3-quinoline boric acid (3QBA), alizarin red S (ARS) and auxiliary reagents. After soaking the fiber sheet in the treatment solution, the fiber sheet is subjected to a roller brushing, drying and cutting process to obtain dry chemical test strips of a predetermined size. S2. Preparation of sample diluent: Dissolve Tween 80, polyethylene glycol 2000, preservative, sodium pyrophosphate and glycine in purified water to prepare sample diluent; S3. Assembly of dry chemical test strips; insert the dry chemical test strips into the test wells and reference wells of the reagent kit; S4. Sample dilution: Mix the seminal plasma sample to be tested with the sample diluent in the specified ratio; S5. Detection and Judgment: Add the diluted sample and the fructose reference value quality control sample of known concentration to the detection well and reference well, respectively. After standing at room temperature for reaction, determine whether the fructose content of the refined fruit is normal by comparing the colors of the two wells and combining with the colorimetric card.

[0023] In this embodiment, a room-temperature competitive colorimetric system constructed using 3-quinoline boric acid and Alizarin Red S is employed. The sample is simply mixed with a dedicated diluent and added dropwise to a pre-packaged test strip kit; color comparison is then performed visually to determine the result. The entire process eliminates the need for complex instruments, avoiding the operational barriers and cost limitations of equipment such as high-performance liquid chromatography (HPLC). The reaction is conducted under mild conditions, without the use of highly corrosive reagents such as concentrated hydrochloric acid, ensuring operational safety. Sample processing requires only simple dilution, eliminating the need for cumbersome pretreatment steps such as protein removal, heating, or derivatization, significantly improving detection efficiency. The selected 3-quinoline boric acid, Alizarin Red S, and excipients are all readily available chemical reagents, overcoming the difficulty of obtaining specialized enzyme raw materials such as fructose dehydrogenase. Therefore, this method achieves the technical advantages of being simple, rapid, safe, low-cost, and easy to promote.

[0024] Working Principle: This method is based on a competitive complexation-displacement reaction between 3-quinoline boric acid (3QBA) and Alizarin Red S (ARS). Therefore, the dry chemical test strip contains a pre-loaded 3QBA-ARS complex, which is orange-yellow. When the sample is added, if the sample contains fructose, the cis-vicinal diol group in its molecular structure will specifically bind to the boric acid group of 3QBA, forming a more stable colorless 3QBA-fructose complex, simultaneously displacing free Alizarin Red S. Alizarin Red S appears purple under weakly alkaline conditions. The color of the detection well is determined by the mixing ratio of the residual orange-yellow complex and the newly formed purple dye. By comparing the color development results with those of the reference well (containing a fructose control sample of known concentration), a purple tint indicates that the fructose concentration is not lower than the reference value, while an orange-yellow tint indicates that the concentration is lower than the reference value. This reaction can be completed rapidly at room temperature without the need for external acid or alkali treatment or heating. Qualitative determination is achieved through direct color comparison, simplifying and facilitating the detection process.

[0025] Furthermore, the specific details of the preparation steps for the dry chemical test strips are as follows: S101: Dissolve 3-quinolineboronic acid in an organic solvent to obtain a 3QBA-organic solvent solution with a 3-quinolineboronic acid concentration of 0.5%-1%; S102: Dissolve Alizarin Red S in phosphate buffer (PB) with a pH of 7.0-9.0 and a concentration of 0.1M-0.5M to obtain an ARS-PB solution with an Alizarin Red S concentration of 0.05%-0.2%. S103: Mix the 3QBA-organic solvent solution with the ARS-PB solution, then add 1%-5% by volume of surfactant, 1%-10% by mass of polymeric auxiliaries and 0.1%-0.5% by volume of preservative, and stir at room temperature for 0.5 h to obtain the treatment solution; S104: Immerse the fiber sheet in the treatment solution, ensuring complete coverage, and maintain at 25℃-37℃ for 0.5-3 hours; S105: Remove the soaked fiber sheet, remove excess treatment liquid and reduce wrinkles by mechanical roller brush, and then dry it in a forced-air dryer at 37℃-45℃ for 7-15 hours until it is completely dry; S106: Cut the dried fiber sheet into dry chemical test paper sheets of a predetermined size.

[0026] In this embodiment, in step S101, the organic solvent is dimethyl sulfoxide (DMSO). In step S102, the pH value of the phosphate buffer (PB) is 7.0-9.0, and the phosphate buffer is prepared by disodium hydrogen phosphate dodecahydrate (Na2HPO4-12H2O) and sodium dihydrogen phosphate dihydrate (NaH2PO4-2H2O). In step S103, the surfactant is Tween 80; The polymeric additive is polyethylene glycol 2000; The preservative is Proclin 300.

[0027] The specific content of step S104 is as follows: pour 200ml of the treatment solution into the treatment container, slowly immerse the A4 size fiber sheet into the treatment solution, ensure that the treatment solution completely submerges the fiber sheet and there are no air bubbles under the fiber sheet, and oscillate in a constant temperature shaker at 25℃-37℃ and a speed of 100-200rpm for 0.5-3 hours to complete the soaking of the fiber sheet.

[0028] The specific content of step S105 is as follows: take out the fiber sheet after soaking and shaking treatment, lay it flat on the drying net, and use rollers or drums to perform at least 10 roller brush treatments on the front and back sides of the fiber sheet to remove excess treatment liquid and flatten the wrinkles. The fiber sheet is then transferred to an oven, the blower function is turned on, and it is dried continuously at a temperature of 37℃-45℃ for 7-15 hours until the fiber sheet is completely dry.

[0029] The specific steps of step S106 are as follows: the dried fiber sheet is first cut into at least two rectangular test strips of 30cm×6.5cm and at least one rectangular test strip of 30cm×7cm, and then the rectangular test strips are uniformly cut into square dry chemical test strips of 4.9mm×4.9mm using an automatic cutting device.

[0030] Furthermore, in the sample diluent preparation step, 1%-5% by volume of Tween 80, 1%-10% by mass of polyethylene glycol 2000, 0.1%-0.5% by volume of preservative, 1%-5% by mass of sodium pyrophosphate, and 1%-10% by mass of glycine are mixed and diluted with purified water to obtain the sample diluent.

[0031] Example 1: This invention provides a dry chemical qualitative detection method for spermatogenic fructose, comprising the following steps: 3-Quinolinoboronic acid (3QBA) was dissolved in dimethyl sulfoxide (DMSO) to obtain 3QBA-dimethyl sulfoxide (3QBA-DMSO) with a 3QBA concentration of 1%. Alizarin Red S (ARS) was dissolved in 0.1M phosphate buffer (PB) at pH 8.0 to obtain an ARS-PB solution with an ARS concentration of 0.2%. The 3QBA-DMSO was mixed with the ARS-PB solution, and then 5% Tween 80 (by volume), 3% polyethylene glycol (by mass), and 0.2% Proclin 300 (by volume) were added. The mixture was stirred at room temperature for 0.5 hours to obtain the treatment solution. Pour 200ml of the treatment solution into the treatment container, and slowly immerse the A4 size fiber sheet into the treatment solution, ensuring that the treatment solution completely submerges the fiber sheet and that there are no air bubbles under the fiber sheet. Shake the fiber sheet in a constant temperature shaker at 25°C and 200rpm for 0.5 hours to complete the soaking of the fiber sheet. Take out the soaked and shaken fiber sheet, lay it flat on the drying net, and use rollers or drums to brush the front and back of the fiber sheet at least 10 times each to remove excess treatment liquid and smooth out wrinkles. Then transfer the fiber sheet to the drying oven, turn on the blower function, and dry it continuously at 45°C for 10 hours until the fiber sheet is completely dry. After drying, the fiber sheet is first cut into two 30cm×6.5cm and at least one 30cm×7cm rectangular test strips. Then, the rectangular test strips are uniformly cut into 4.9mm×4.9mm square dry chemical test strips using an automatic cutting device. The sample dilution solution was prepared by mixing 5% Tween 80 (by volume), 3% polyethylene glycol 2000 (by mass), 0.2% Proclin 300 (by volume), 1% sodium pyrophosphate (by mass), and 1% glycine (by mass), and then diluted with purified water to a final volume. Use plastic tweezers to insert two 4.9*4.9mm square dry chemical test strips into the test well and reference well of the kit, respectively; Mix the seminal plasma sample to be tested with 10 ml of the sample diluent in the specified ratio; The diluted sample and a fructose reference value quality control sample of known concentration were added to the test well and reference well, respectively. After the reaction was allowed to stand at room temperature, the fructose content of the spermatin was determined by comparing the colors of the two wells and using a colorimetric card.

[0032] Example 2: This invention provides a dry chemical qualitative detection method for spermatogenic fructose, comprising the following steps: 3-Quinolinoboronic acid (3QBA) was dissolved in dimethyl sulfoxide (DMSO) to obtain 3QBA-dimethyl sulfoxide (3QBA-DMSO) with a 3QBA concentration of 0.5%. Alizarin Red S (ARS) was dissolved in 0.3 M phosphate buffer (PB) at pH 7.5 to obtain an ARS-PB solution with a concentration of 0.2% ARS. The 3QBA-DMSO was mixed with the ARS-PB solution, and then 1% by volume of Tween 80, 1% by mass of polyethylene glycol, and 0.1% by volume of Proclin 300 were added. The mixture was stirred at room temperature for 0.5 h to obtain the treatment solution. Pour 200ml of the treatment solution into the treatment container, and slowly immerse the A4 size fiber sheet into the treatment solution, ensuring that the treatment solution completely submerges the fiber sheet and that there are no air bubbles under the fiber sheet. Shake the fiber sheet in a constant temperature shaker at 25°C and 100rpm for 1 hour to complete the soaking of the fiber sheet. Take out the soaked and shaken fiber sheet, lay it flat on the drying net, and use rollers or drums to brush the front and back of the fiber sheet at least 10 times each to remove excess treatment liquid and smooth out wrinkles. Then transfer the fiber sheet to the drying oven, turn on the blower function, and dry it continuously at 37°C for 12 hours until the fiber sheet is completely dry. After drying, the fiber sheet is first cut into two 30cm×6.5cm and at least one 30cm×7cm rectangular test strips. Then, the rectangular test strips are uniformly cut into 4.9mm×4.9mm square dry chemical test strips using an automatic cutting device. The sample dilution solution was prepared by mixing 1% Tween 80 (by volume), 1% polyethylene glycol 2000 (by mass), 0.1% Proclin 300 (by volume), 5% sodium pyrophosphate (by mass), and 10% glycine (by mass), and then diluted with purified water to a final volume. Use plastic tweezers to insert two 4.9*4.9mm square dry chemical test strips into the test well and reference well of the kit, respectively; Mix the seminal plasma sample to be tested with 10 ml of the sample diluent in the specified ratio; The diluted sample and a fructose reference value quality control sample of known concentration were added to the test well and reference well, respectively. After the reaction was allowed to stand at room temperature, the fructose content of the spermatin was determined by comparing the colors of the two wells and using a colorimetric card.

[0033] Example 3: This invention provides a dry chemical qualitative detection method for spermatogenic fructose, comprising the following steps: 3-Quinolinoboronic acid (3QBA) was dissolved in dimethyl sulfoxide (DMSO) to obtain 3QBA-dimethyl sulfoxide (3QBA-DMSO) with a 3QBA concentration of 0.8%. Alizarin Red S (ARS) was dissolved in 0.5M phosphate buffer (PB) at pH 8.5 to obtain an ARS-PB solution with an ARS concentration of 0.08%. The 3QBA-DMSO was mixed with the ARS-PB solution, and then 3% by volume of Tween 80, 3% by mass of polyethylene glycol, and 0.2% by volume of Proclin 300 were added. The mixture was stirred at room temperature for 0.5 h to obtain the treatment solution. Pour 200ml of the treatment solution into the treatment container, and slowly immerse the A4 size fiber sheet into the treatment solution, ensuring that the treatment solution completely submerges the fiber sheet and that there are no air bubbles under the fiber sheet. Shake the fiber sheet in a constant temperature shaker at 30°C and 100rpm for 2 hours to complete the soaking of the fiber sheet. Take out the soaked and shaken fiber sheet, lay it flat on the drying net, and use rollers or drums to brush the front and back of the fiber sheet at least 10 times each to remove excess treatment liquid and smooth out wrinkles. Then transfer the fiber sheet to the drying oven, turn on the blower function, and dry it continuously at 40°C for 8 hours until the fiber sheet is completely dry. After drying, the fiber sheet is first cut into two 30cm×6.5cm and at least one 30cm×7cm rectangular test strips. Then, the rectangular test strips are uniformly cut into 4.9mm×4.9mm square dry chemical test strips using an automatic cutting device. The sample dilution solution was prepared by mixing 3% Tween 80 (by volume), 3% polyethylene glycol 2000 (by mass), 0.2% Proclin 300 (by volume), 3% sodium pyrophosphate (by mass), and 3% glycine (by mass), and then diluted with purified water to a final volume. Use plastic tweezers to insert two 4.9*4.9mm square dry chemical test strips into the test well and reference well of the kit, respectively; Mix the seminal plasma sample to be tested with 10 ml of the sample diluent in the specified ratio; The diluted sample and a fructose reference value quality control sample of known concentration were added to the test well and reference well, respectively. After the reaction was allowed to stand at room temperature, the fructose content of the spermatin was determined by comparing the colors of the two wells and using a colorimetric card.

[0034] Example 4: This invention provides a dry chemical qualitative detection method for spermatogenic fructose, comprising the following steps: 3-Quinolinoboronic acid (3QBA) was dissolved in dimethyl sulfoxide (DMSO) to obtain 3QBA-dimethyl sulfoxide (3QBA-DMSO) with a 3QBA concentration of 1%. Alizarin Red S (ARS) was dissolved in 0.1M phosphate buffer (PB) at pH 9.0 to obtain an ARS-PB solution with an ARS concentration of 0.08%. The 3QBA-DMSO was mixed with the ARS-PB solution, and then 5% by volume of Tween 80, 10% by mass of polyethylene glycol, and 0.5% by volume of Proclin 300 were added. The mixture was stirred at room temperature for 0.5 h to obtain the treatment solution. Pour 200ml of the treatment solution into the treatment container, and slowly immerse the A4 size fiber sheet into the treatment solution, ensuring that the treatment solution completely submerges the fiber sheet and that there are no air bubbles under the fiber sheet. Shake the fiber sheet in a constant temperature shaker at 37°C and 150rpm for 3 hours to complete the soaking of the fiber sheet. Take out the soaked and shaken fiber sheet, lay it flat on the drying net, and use rollers or drums to brush the front and back of the fiber sheet at least 10 times each to remove excess treatment liquid and smooth out wrinkles. Then transfer the fiber sheet to the drying oven, turn on the blower function, and dry it continuously at 45°C for 15 hours until the fiber sheet is completely dry. After drying, the fiber sheet is first cut into two 30cm×6.5cm and at least one 30cm×7cm rectangular test strips. Then, the rectangular test strips are uniformly cut into 4.9mm×4.9mm square dry chemical test strips using an automatic cutting device. The sample dilution solution was prepared by mixing 5% Tween 80 (by volume), 10% polyethylene glycol 2000 (by mass), 0.5% Proclin 300 (by volume), 3% sodium pyrophosphate (by mass), and 3% glycine (by mass), and then diluted with purified water to a final volume. Use plastic tweezers to insert two 4.9*4.9mm square dry chemical test strips into the test well and reference well of the kit, respectively; Mix the seminal plasma sample to be tested with 10 ml of the sample diluent in the specified ratio; The diluted sample and a fructose reference value quality control sample of known concentration were added to the test well and reference well, respectively. After the reaction was allowed to stand at room temperature, the fructose content of the spermatin was determined by comparing the colors of the two wells and using a colorimetric card.

[0035] Please see Figure 3 and Figure 4The present invention also provides a kit for detecting seminal plasma fructose, which is applied to the dry chemical qualitative detection method for seminal plasma fructose as described above. The kit for detecting seminal plasma fructose includes an upper shell 201 and a lower shell 202, the upper shell 201 and the lower shell 202 being snap-fitted together. The upper shell 201 is provided with a reference C-hole 203 and a detection T-hole 204. The lower shell 202 is provided with a C-hole groove 205 and a T-hole groove 206. The reference C-hole 203 corresponds to the C-hole groove 205, and the detection T-hole 204 is adapted to the T-hole groove 206.

[0036] In this embodiment, the reagent kit forms a sealed and easy-to-operate detection unit through the snap-fit ​​design of the upper shell 201 and the lower shell 202. The reference C hole 203 (reference hole in the method) and the detection T hole 204 (detection hole in the method) provided in the upper shell 201 serve as sample and quality control application windows, and their positions are precisely aligned with the corresponding grooves in the lower shell 202, ensuring that the sample liquid is accurately carried on the dry chemical test strip pre-placed in the groove. This structural design not only ensures spatial isolation of the reaction area, avoiding cross-contamination, but also facilitates color observation and comparison after the reaction. In addition, the snap-fit ​​connection method achieves stable encapsulation and convenient disassembly and assembly of the test strip.

[0037] The above description discloses only one preferred embodiment of the present invention, and should not be construed as limiting the scope of the present invention. Those skilled in the art will understand that all or part of the processes of the above embodiments can be implemented, and equivalent changes made in accordance with the claims of the present invention are still within the scope of the invention.

Claims

1. A dry chemical qualitative detection method for sperm fructose, characterized in that, Includes the following steps: Preparation of dry chemical test strips: Prepare a treatment solution containing 3-quinoline boric acid, alizarin red S and auxiliary reagents. After soaking the fiber sheet in the treatment solution, the fiber sheet is subjected to a roller brushing, drying and cutting process to obtain dry chemical test strips of a predetermined size. Sample diluent preparation: Dissolve Tween 80, polyethylene glycol 2000, preservative, sodium pyrophosphate and glycine in purified water to prepare the sample diluent; Assembly of dry chemical test strips; inserting the dry chemical test strips into the test wells and reference wells of the reagent kit; Sample dilution: Mix the seminal plasma sample to be tested with the sample diluent in the specified proportion; Detection and Judgment: The diluted sample and the quality control sample with a known concentration of fructose reference value were added to the test well and the reference well, respectively. After standing at room temperature for reaction, the fructose content of the refined grains was determined by comparing the colors of the two wells and using a colorimetric card.

2. The dry chemical qualitative detection method for detecting fructose in seminal plasma as described in claim 1, characterized in that, The specific steps for preparing the dry chemical test strips are as follows: Preparation of treatment solution: Dissolve 3-quinolineboric acid in an organic solvent to obtain a 3-quinolineboric acid-organic solvent solution with a concentration of 0.5%-1%; Alizarin Red S was dissolved in phosphate buffer solution with a pH of 7.0-9.0 and a concentration of 0.1M-0.5M to obtain an Alizarin Red S-phosphate buffer solution with a concentration of 0.05%-0.2%. The 3-quinoline boric acid-organic solvent solution was mixed with the alizarin red S-phosphate buffer solution, and then 1%-5% (v / v) of surfactant, 1%-10% (w / w) of polymeric auxiliaries and 0.1%-0.5% (v / v) of preservative were added. The mixture was stirred at room temperature for 0.5 h to obtain the treatment solution. Immersion treatment: Immerse the fiber sheet in the treatment solution, ensuring complete coverage, and maintain at 25℃-37℃ for 0.5-3 hours; Drying treatment: Remove the soaked fiber sheet, remove excess treatment solution and reduce wrinkles by mechanical roller brush, and then dry it with a forced air at 37℃-45℃ for 7-15 hours until it is completely dry; Cutting and shaping: Cut the dried fiber sheet into dry chemical test paper sheets of predetermined size.

3. The dry chemical qualitative detection method for detecting fructose in seminal plasma as described in claim 2, characterized in that, In the step of preparing the treatment solution, the organic solvent is dimethyl sulfoxide; The phosphate buffer solution is prepared from disodium hydrogen phosphate dodecahydrate and sodium dihydrogen phosphate dihydrate; The surfactant is Tween 80; The polymeric additive is polyethylene glycol 2000; The preservative is Proclin 300.

4. The dry chemical qualitative detection method for detecting fructose in seminal plasma as described in claim 3, characterized in that, The specific steps of the soaking treatment are as follows: Pour 200ml of the treatment solution into the treatment container, slowly immerse the A4 size fiber sheet into the treatment solution, ensuring that the treatment solution completely submerges the fiber sheet and that there are no air bubbles under the fiber sheet, and oscillate in a constant temperature shaker at 25℃-37℃ and a speed of 100-200rpm for 0.5-3 hours to complete the soaking of the fiber sheet.

5. The dry chemical qualitative detection method for detecting fructose in seminal plasma as described in claim 4, characterized in that, The specific contents of the drying process are as follows: take out the fiber sheet after soaking and shaking, lay it flat on the drying net, and use rollers or drums to brush the front and back of the fiber sheet at least 10 times each to remove excess treatment liquid and flatten the wrinkles. The fiber sheet is then transferred to an oven, the blower function is turned on, and it is dried continuously at a temperature of 37℃-45℃ for 7-15 hours until the fiber sheet is completely dry.

6. The dry chemical qualitative detection method for detecting fructose in seminal plasma as described in claim 5, characterized in that, The specific steps of the cutting and forming step are as follows: the dried fiber sheet is first cut into at least two rectangular test strips of 30cm×6.5cm and at least one rectangular test strip of 30cm×7cm, and then the rectangular test strips are uniformly cut into square dry chemical test strips of 4.9mm×4.9mm using an automatic cutting device.

7. The dry chemical qualitative detection method for detecting fructose in seminal plasma as described in claim 6, characterized in that, In the sample diluent preparation step, 1%-5% by volume of Tween 80, 1%-10% by mass of polyethylene glycol 2000, 0.1%-0.5% by volume of preservative, 1%-5% by mass of sodium pyrophosphate, and 1%-10% by mass of glycine are mixed and diluted with purified water to obtain the sample diluent.

8. A kit for detecting seminal plasma fructose, applied to the dry chemical qualitative detection method for seminal plasma fructose as described in claim 1, characterized in that, The reagent kit for detecting seminal plasma fructose includes an upper shell and a lower shell, which are snap-fitted together. The upper shell is provided with a reference C-hole and a detection T-hole, and the lower shell is provided with a C-hole groove and a T-hole groove. The reference C-hole corresponds to the C-hole groove, and the detection T-hole is adapted to the T-hole groove.

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