Sperm quality detection device and preparation method thereof

Abstract

The application discloses a sperm quality detection device and a preparation method thereof. The sperm quality detection device comprises a base material, a sperm activity detection module, an SP10 detection module and a PSA detection module. The sperm activity detection module comprises a containing cavity and a convex lens. The containing cavity has a detection plane. The convex lens is configured to be able to slide to a sample adding hole along with a sliding assembly and to magnify the image of the detection plane. The SP10 detection module comprises a P10 colloidal gold test strip. The PSA detection module comprises a PSA colloidal gold test strip. The application provides a detection device integrating sperm acrosome enzyme SP10 protein, prostate specific antigen PSA and special counting for a semen sample. The device is simple to operate and can provide a detection result in a short time. The detection result can be judged by the person to be detected independently, and the device is suitable for primary medical units and on-site rapid detection.

Description

Technical Field

[0001] This application relates to the field of reproductive medicine technology, and in particular to a sperm quality testing device and its preparation method. Background Technology

[0002] Sperm quality determines a man's ability to conceive. Due to environmental pollution, unhealthy diets, obesity, and other factors, male sperm quality and concentration have declined significantly, contributing to infertility and its global prevalence. In China, the incidence of infertility has risen from 11.9% to 18% in recent years. Therefore, routine semen analysis is crucial for assessing sperm quality and motility, identifying any underlying diseases, and determining the cause for targeted treatment. Routine semen analysis includes various aspects such as sperm concentration, quality, motility, and morphology.

[0003] Sperm acrosomal enzyme SP10 (Sperm Protein 10) is an immunogenic polypeptide found in the acrosome of mature sperm. As an analyte for sperm concentration detection, it can be used as an adjunct in the diagnosis and monitoring of treatment efficacy in male infertility. Currently, the standard detection method is colloidal gold qualitative analysis. Sperm PSA (prostate-specific antigen) is a protein secreted by prostate epithelial cells. Approximately 30% of seminal plasma is prostatic fluid. The degree of semen liquefaction and viscosity are related to the PSA content in the seminal plasma. PSA is an important factor involved in semen liquefaction; therefore, the secretory function of the prostate gland affects sperm quality.

[0004] A triplet test strip is currently available, in which three analytes are tested separately on three different test strips. This involves a double-antibody sandwich method forming a complex of quantum dot microspheres + capture antibody A + analyte antigen + capture antibody B. While this kit can quantitatively detect SP10 and PSA, it requires specialized equipment and technicians, is time-consuming and costly, and is not suitable for rapid screening or on-site testing. Furthermore, quantum dots are prone to quenching and aggregation, leading to reagent instability. Production costs and detection accuracy need improvement, and it does not provide effective monitoring of sperm motility and morphological characteristics. Summary of the Invention

[0005] This application aims to address at least one of the technical problems existing in the prior art. To this end, this application proposes a sperm quality testing device and its preparation method. This testing device is characterized by short testing time, low cost, high accuracy, and ease of rapid screening and on-site testing.

[0006] A first aspect of this application provides a sperm quality testing device, the sperm quality testing device comprising:

[0007] A first substrate and a second substrate are positioned opposite each other, and the first substrate is provided with a sample feeding hole;

[0008] A sperm motility detection module includes a detection component and a sliding component. The detection component includes a first cavity component and a second cavity component, which are spaced apart from each other and stacked on a second substrate to form a receiving cavity. The sliding component includes a convex lens movably connected to the first substrate. The receiving cavity has a detection plane. The convex lens is configured to slide with the sliding component to the position of the sample application hole and magnify the image of the detection plane.

[0009] SP10 detection module, SP10 detection module is fixed on the second substrate, SP10 detection module includes SP10 colloidal gold test strip;

[0010] The PSA detection module is fixed on the second substrate and includes a PSA colloidal gold test strip.

[0011] In some embodiments of this application, the SP10 colloidal gold test strip is a competitive lateral chromatography test strip.

[0012] In some embodiments of this application, the SP10 colloidal gold test strip includes a first sample pad, a first conjugation pad, a first nitrocellulose membrane, and a first absorbent paper that are sequentially overlapped. The first nitrocellulose membrane is provided with a first detection line (T line) and a first control line (C line) in the same order.

[0013] In some embodiments of this application, the first binding pad is coated with a colloidal gold-labeled SP10-specific antibody, and the first detection line is coated with the SP10 antigen. The conventional method for SP10 detection is a sandwich method, where the binding pad and the T line are coated with gold-labeled specific antibody 1 and specific antibody 2 targeting different epitopes of SP10, respectively, ultimately forming a complex of gold-labeled specific antibody 1-antigen-specific antibody 2 on the T line. When the SP10 protein content in the sample is higher than a threshold, the complex aggregates on the T line, resulting in a red band, indicating a negative result. Conversely, when the SP10 protein content is lower than the threshold, no red band appears on the T line, indicating a positive result. However, considering that this does not conform to users' interpretation habits, some embodiments of this application optimize it to a competitive method. When the SP10 protein content is below the threshold, it does not bind sufficiently to the colloidal gold-labeled specific antibody. The remaining specific antibody binds to the SP10 antigen on the T line, resulting in a red band and a positive test result. When the SP10 protein content is above the threshold, the colloidal gold-labeled specific antibody binds sufficiently to the SP10 protein and can no longer bind to the SP10 antigen on the T line, resulting in no red band and a negative test result, indicating normal sperm concentration.

[0014] In some embodiments of this application, the PSA colloidal gold test strip is a competitive lateral chromatography test strip.

[0015] In some embodiments of this application, the PSA colloidal gold test strip includes a second sample pad, a second conjugation pad, a second nitrocellulose membrane, and a second absorbent paper that are sequentially overlapped. The second nitrocellulose membrane is provided with a second detection line (T line) and a second control line (C line) in the same order.

[0016] In some embodiments of this application, the second binding pad is coated with colloidal gold-labeled PSA-specific antibodies, and the second detection line is coated with PSA antigen. Routine PSA testing uses a double-antibody sandwich method to measure PSA in blood samples (such as plasma, serum, etc.). The threshold for PSA concentration in blood samples is 4 ng / mL; concentrations above 10 ng / mL indicate a risk of prostate cancer. Semen analysis has a different significance. The threshold for PSA antigen concentration in semen is 1.2 mg / mL. Concentrations below 1.2 mg / mL reduce seminal plasma liquefaction capacity, affecting sperm quality and decreasing the chances of conception. To conform to user interpretation habits, the detection method in this application is optimized to a competitive method. During the test strip chromatography process, PSA in the sample binds to the colloidal gold-labeled specific antibody. When the PSA level in the sample is below the threshold, binding with the gold-labeled specific antibody is insufficient, and the remaining gold-labeled specific antibody binds to the PSA antigen on the T line, resulting in a red band and a positive result, indicating that the PSA antigen concentration is below the threshold (e.g., 1.2 mg / mL). When the amount of PSA in the sample is greater than or equal to the threshold, it binds sufficiently to the gold-labeled specific antibody. The gold-labeled specific antibody no longer binds to the PSA antigen on the T line, and no band appears on the T line. The result is negative, indicating that the concentration of PSA antigen in the sample is normal.

[0017] In some embodiments of this application, the distance between the convex lens and the detection plane is 1 to 1.5 mm, for example, it can be 1 mm, 1.1 mm, 1.2 mm, 1.3 mm, 1.4 mm, or 1.5 mm.

[0018] In some embodiments of this application, the height of the receiving cavity is 0.05 to 0.15 mm, for example, it can be 0.05 mm, 0.06 mm, 0.07 mm, 0.08 mm, 0.09 mm, 0.1 mm, 0.11 mm, 0.12 mm, 0.13 mm, 0.14 mm, or 0.15 mm.

[0019] In some embodiments of this application, the sliding assembly includes a first sliding component located on the side of the first substrate away from the second substrate and a second sliding component located on the side of the first substrate close to the second substrate. The first sliding component and the second sliding component are fixedly connected. A convex lens is provided on the second sliding component. The sliding assembly is configured to be able to drive the second sliding component to slide by sliding the first sliding component on the first substrate, thereby covering the sample application hole. At the same time, the convex lens in the second sliding component is aligned with the detection plane.

[0020] In some embodiments of this application, a light-transmitting hole is provided on the detection plane.

[0021] In some embodiments of this application, the diameter of the light-transmitting hole is 4 to 10 mm, for example, it can be 4 mm, 5 mm, 6 mm, 7 mm, 8 mm, 9 mm, or 10 mm.

[0022] In some embodiments of this application, the diameter of the convex lens is 1 to 5 mm, for example, it can be 1 mm, 2 mm, 3 mm, 4 mm, or 5 mm.

[0023] In some embodiments of this application, the receiving cavity is a transparent receiving cavity.

[0024] In some embodiments of this application, the first cavity component and the second cavity component forming the receiving cavity are made of glass or polymer material (e.g., PC).

[0025] In some embodiments of this application, the distance between the convex lens and the receiving cavity is 0.8 to 1.5 mm, for example, 0.8 mm, 0.9 mm, 1 mm, 1.1 mm, 1.2 mm, 1.3 mm, 1.4 mm, or 1.5 mm.

[0026] In some embodiments of this application, the thickness of the receiving cavity is 0.5 to 2 mm, for example, 0.5 mm, 0.6 mm, 0.7 mm, 0.8 mm, 0.9 mm, 1 mm, 1.1 mm, 1.2 mm, 1.3 mm, 1.4 mm, 1.5 mm, 1.6 mm, 1.7 mm, 1.8 mm, 1.9 mm, or 2 mm.

[0027] In some embodiments of this application, the distance between the cavity first component and the cavity first component is 0.05 to 0.15 mm, for example, 0.05 mm, 0.06 mm, 0.07 mm, 0.08 mm, 0.09 mm, 0.1 mm, 0.11 mm, 0.12 mm, 0.13 mm, 0.14 mm, or 0.15 mm.

[0028] In some embodiments of this application, a counting grid is provided on the convex lens or the detection plane.

[0029] In some embodiments of this application, the counting grid comprises grid lines arranged in a crisscross pattern.

[0030] In some embodiments of this application, the lateral spacing of the grid lines is 0.2 to 0.25 mm.

[0031] In some embodiments of this application, the longitudinal spacing of the grid lines is 0.2 to 0.25 mm.

[0032] In some embodiments of this application, the longitudinal and transverse spacing of the grid lines are the same.

[0033] In some embodiments of this application, the binding pad is further coated with a colloidal gold-labeled quality control antibody, and the first quality control line is coated with an anti-antibody that specifically binds to the quality control antibody.

[0034] In some embodiments of this application, the quality control antibody includes an IgG antibody.

[0035] In some embodiments of this application, the quality control antibody includes a mouse IgG antibody.

[0036] In some embodiments of this application, the anti-antibody is a goat anti-mouse IgG antibody.

[0037] In some embodiments of this application, the first sample pad of the SP10 colloidal gold test strip and the second sample pad of the PSA colloidal gold test strip are respectively connected to the detection plane of the receiving cavity.

[0038] A second aspect of this application provides a method for preparing a sperm quality testing device, the method comprising preparing SP10 colloidal gold test strips and PSA colloidal gold test strips:

[0039] The preparation of SP10 colloidal gold test strips involves coating SP10 gold standard solution onto a substrate to prepare a first binding pad.

[0040] The preparation of PSA colloidal gold test strips involves coating a PSA gold standard solution onto a substrate to prepare a second binding pad.

[0041] In some embodiments of this application, the preparation of SP10 gold labeling solution includes reacting colloidal gold solution with SP10 specific antibody to generate colloidal gold-labeled SP10 specific antibody, adding blocking solution to block binding sites, centrifuging to remove supernatant, and adding SP10 reconstitution solution to obtain SP10 gold labeling solution.

[0042] In some embodiments of this application, the SP10 reconstitution solution includes borate buffer, sucrose, sodium caseinate, Triton X-100, and PC300.

[0043] In some embodiments of this application, the concentration of borate buffer in the SP10 reconstitution solution is 0.01 to 0.1 mol / L, for example, it can be 0.01 mol / L, 0.02 mol / L, 0.03 mol / L, 0.04 mol / L, 0.05 mol / L, 0.06 mol / L, 0.07 mol / L, 0.08 mol / L, 0.09 mol / L, or 0.1 mol / L.

[0044] In some embodiments of this application, the concentration of sucrose in the SP10 reconstituted solution is 5-20 wt%, for example, it can be 5 wt%, 6 wt%, 7 wt%, 8 wt%, 9 wt%, 10 wt%, 11 wt%, 12 wt%, 13 wt%, 14 wt%, 15 wt%, 16 wt%, 17 wt%, 18 wt%, 19 wt%, or 20 wt%.

[0045] In some embodiments of this application, the concentration of sodium caseinate in the SP10 reconstituted solution is 0.2–5 wt%, for example, it can be 0.2 wt%, 0.4 wt%, 0.5 wt%, 0.6 wt%, 0.8 wt%, 1 wt%, 1.2 wt%, 1.4 wt%, 1.5 wt%, 1.6 wt%, 1.8 wt%, or 2 wt%.

[0046] In some embodiments of this application, the concentration of Triton X-100 in the SP10 reconstituted solution is 0.1–10 v / v%, for example, it can be 0.1 v / v%, 0.2 v / v%, 0.4 v / v%, 0.5 v / v%, 0.6 v / v%, 0.8 v / v%, 1 v / v%, 2 v / v%, 3 v / v%, 4 v / v%, 5 v / v%, 6 v / v%, 7 v / v%, 8 v / v%, 9 v / v%, or 10 v / v.

[0047] In some embodiments of this application, the concentration of PC300 in the SP10 reconstituted solution is 0.01 to 0.1 wt%, for example, it can be 0.01 wt%, 0.02 wt%, 0.03 wt%, 0.04 wt%, 0.05 wt%, 0.06 wt%, 0.07 wt%, 0.08 wt%, 0.09 wt%, or 0.1 wt%.

[0048] In some embodiments of this application, the pH of the SP10 reconstituted solution is 7.5 to 8.5, for example, it can be 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, or 8.5.

[0049] In some embodiments of this application, the preparation of PSA gold-labeled solution includes reacting colloidal gold solution with PSA-specific antibody to generate colloidal gold-labeled PSA-specific antibody, adding blocking solution to block binding sites, centrifuging to remove supernatant, and adding PSA reconstitution solution to obtain PSA gold-labeled solution.

[0050] In some embodiments of this application, the PSA reconstitution solution includes PB buffer, sucrose, sodium caseinate, BSA, Gly, Tween-20, and PC300.

[0051] In some embodiments of this application, the final concentration of sodium caseinate in the PSA reconstituted solution is 1.5 to 2.5 wt%, for example, it can be 1.5 wt%, 1.6 wt%, 1.7 wt%, 1.8 wt%, 1.9 wt%, 2 wt%, 2.1 wt%, 2.2 wt%, 2.3 wt%, 2.4 wt%, or 2.5 wt%.

[0052] In some embodiments of this application, the concentration of PB buffer in the PSA reconstitution solution is 0.01 to 0.1 mol / L, for example, it can be 0.01 mol / L, 0.02 mol / L, 0.03 mol / L, 0.04 mol / L, 0.05 mol / L, 0.06 mol / L, 0.07 mol / L, 0.08 mol / L, 0.09 mol / L, or 0.1 mol / L.

[0053] In some embodiments of this application, the concentration of sucrose in the PSA reconstituted solution is 5-20 wt%, for example, it can be 5 wt%, 6 wt%, 7 wt%, 8 wt%, 9 wt%, 10 wt%, 11 wt%, 12 wt%, 13 wt%, 14 wt%, 15 wt%, 16 wt%, 17 wt%, 18 wt%, 19 wt%, or 20 wt%.

[0054] In some embodiments of this application, the concentration of BSA in the PSA reconstituted solution is 0.1 to 5 wt%, for example, it can be 0.1 wt%, 0.2 wt%, 0.4 wt%, 0.5 wt%, 0.6 wt%, 0.8 wt%, 1 wt%, 2 wt%, 3 wt%, 4 wt%, or 5 wt%.

[0055] In some embodiments of this application, the concentration of glycine (Gly) in the PSA reconstituted solution is 0.1 to 1 wt%, for example, it can be 0.1 wt%, 0.2 wt%, 0.3 wt%, 0.4 wt%, 0.5 wt%, 0.6 wt%, 0.7 wt%, 0.8 wt%, 0.9 wt%, or 1 wt%.

[0056] In some embodiments of this application, the concentration of Tween-20 in the PSA reconstitution solution is 0.1–5 v / v%, for example, it can be 0.1 v / v%, 0.2 v / v%, 0.4 v / v%, 0.5 v / v%, 0.6 v / v%, 0.8 v / v%, 1 v / v%, 2 v / v%, 3 v / v%, 4 v / v%, or 5 v / v.

[0057] In some embodiments of this application, the concentration of PC300 in the PSA reconstituted solution is 0.01 to 0.1 wt%, for example, it can be 0.01 wt%, 0.02 wt%, 0.03 wt%, 0.04 wt%, 0.05 wt%, 0.06 wt%, 0.07 wt%, 0.08 wt%, 0.09 wt%, or 0.1 wt%.

[0058] In some embodiments of this application, the pH of the PSA reconstitution solution is 7 to 8, for example, it can be 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8.

[0059] In some embodiments of this application, the preparation of SP10 colloidal gold test strips includes: diluting SP10 specific antibodies with a coating solution and coating them onto a first nitrocellulose membrane to form a first detection line; the preparation of PSA colloidal gold test strips includes diluting PSA specific antibodies with a coating solution and coating them onto a second nitrocellulose membrane to form a second detection line.

[0060] In some embodiments of this application, the coating solution includes PB buffer and sucrose.

[0061] In some embodiments of this application, the final concentration of sucrose in the coating solution is 1–3 wt%, for example, it can be 1 wt%, 1.1 wt%, 1.2 wt%, 1.3 wt%, 1.4 wt%, 1.5 wt%, 1.6 wt%, 1.7 wt%, 1.8 wt%, 1.9 wt%, 2 wt%, 2.1 wt%, 2.2 wt%, 2.3 wt%, 2.4 wt%, 2.5 wt%, 2.6 wt%, 2.7 wt%, 2.8 wt%, 2.9 wt%, or 3 wt%.

[0062] In some embodiments of this application, the concentration of PB buffer in the coating solution is 0.01 to 0.1 mol / L, for example, it can be 0.01 mol / L, 0.02 mol / L, 0.03 mol / L, 0.04 mol / L, 0.05 mol / L, 0.06 mol / L, 0.07 mol / L, 0.08 mol / L, 0.09 mol / L, or 0.1 mol / L.

[0063] In some embodiments of this application, the preparation method of SP10 gold standard solution includes the following steps:

[0064] Colloidal gold solution was prepared using chloroauric acid and trisodium citrate. Potassium carbonate solution was added to the colloidal gold solution to adjust the pH, followed by the addition of SP10 antibody. The mixture was vortexed and reacted at room temperature to obtain gold-labeled SP10 antibody.

[0065] After the reaction is complete, add BSA blocking solution, vortex mix, and react at room temperature;

[0066] After the reaction was completed, the mixture was centrifuged, the supernatant was discarded, SP10 reconstitution solution was added, and the mixture was resuspended to obtain SP10 gold standard solution.

[0067] In some embodiments of this application, the preparation method of PSA gold standard solution includes the following steps:

[0068] A colloidal gold solution was prepared using chloroauric acid and trisodium citrate. Potassium carbonate solution was added to the colloidal gold solution to adjust the pH, followed by the addition of PSA antibody. The mixture was vortexed and reacted at room temperature to obtain gold-labeled PSA antibody.

[0069] After the reaction is complete, add BSA blocking solution, vortex mix, and react at room temperature;

[0070] After the reaction was complete, the mixture was centrifuged, the supernatant was discarded, PSA reconstitution solution was added, and the mixture was resuspended to obtain PSA gold standard solution.

[0071] In some embodiments of this application, chloroauric acid is used in the preparation of colloidal gold solution at a concentration of 0.04 to 0.08 wt%, for example, 0.04 wt%, 0.05 wt%, 0.06 wt%, 0.07 wt%, or 0.08 wt%.

[0072] In some embodiments of this application, during the preparation of the colloidal gold solution, the trisodium citrate content is 0.04–0.14 wt%, for example, it can be 0.04 wt%, 0.05 wt%, 0.06 wt%, 0.07 wt%, 0.08 wt%, 0.09 wt%, 0.1 wt%, 0.11 wt%, 0.12 wt%, 0.13 wt%, or 0.14 wt%.

[0073] In some embodiments of this application, the potassium carbonate solution is 0.1 to 0.3 mol / L, for example, it can be 0.1 mol / L, 0.15 mol / L, 0.2 mol / L, 0.25 mol / L, or 0.3 mol / L.

[0074] In some embodiments of this application, during the preparation of the gold-labeled antibody, the final concentrations of the SP10 antibody and the PSA antibody are independently 15–20 μg / mL, for example, 15 μg / mL, 16 μg / mL, 17 μg / mL, 18 μg / mL, 19 μg / mL, and 20 μg / mL.

[0075] In some embodiments of this application, the sealing liquid comprises 10 to 20 wt% BSA, for example, 10 wt%, 12 wt%, 14 wt%, 15 wt%, 16 wt%, 18 wt%, or 20 wt%.

[0076] In some embodiments of this application, after the sealing liquid is added and vortexed, it is reacted at room temperature for 5 to 15 minutes.

[0077] In some embodiments of this application, after the colloidal gold solution is prepared, it is centrifuged at 10,000 to 12,000 rpm for 10 to 25 minutes.

[0078] In some embodiments of this application, after centrifugation of the SP10 colloidal gold solution, the supernatant is discarded, and SP10 gold standard solution is added and resuspended to 50-100% of the original volume to obtain SP10 gold standard solution.

[0079] In some embodiments of this application, after centrifugation of the PSA colloidal gold solution, the supernatant is discarded, and a PSA gold standard solution is added and resuspended to 50-100% of the original volume to obtain a PSA gold standard solution.

[0080] In some embodiments of this application, the quality control antibody is diluted with a coating solution and coated onto a first nitrocellulose membrane to form a first quality control line, and the SP10 antigen is diluted with a coating solution and coated onto a first nitrocellulose membrane to form a first detection line.

[0081] In some embodiments of this application, the final concentration of the quality control antibody after dilution with the coating solution is 0.5 to 2 mg / mL, for example, it can be 0.5 mg / mL, 0.6 mg / mL, 0.8 mg / mL, 1 mg / mL, 1.2 mg / mL, 1.4 mg / mL, 1.5 mg / mL, 1.6 mg / mL, 1.8 mg / mL, or 2 mg / mL.

[0082] In some embodiments of this application, a coating apparatus is used to coat the antibody diluted with the coating solution onto a nitrocellulose membrane.

[0083] In some embodiments of this application, the coating is dried at 30–40°C.

[0084] In some embodiments of this application, the drying time is 24 to 48 hours.

[0085] In some embodiments of this application, the dried SP10 gold pad (first bonding pad) is cut to 3-5 mm and adhered to the above-mentioned PVC base plate, and a sample pad and absorbent paper are attached, and then cut into SP10 test strips of 2.5-4 mm.

[0086] This application also relates to a method for detecting sperm quality, including using the aforementioned sperm quality detection device, including using a sperm activity detection module to detect sperm activity, using an SP10 detection module to detect SP10 protein, and using a PSA detection module to detect PSA.

[0087] In some embodiments of this application, detecting sperm motility includes placing a light source in a light-transmitting hole, aligning a camera with a sample application hole and activating the camera mode, adjusting the distance between the camera and the sample application hole, recording video, and analyzing at least one of sperm count, concentration, and sperm motility in the video.

[0088] In some embodiments of this application, detecting sperm activity also includes analyzing at least one of sperm morphology, sperm aggregation, etc.

[0089] In some embodiments of this application, detecting SP10 protein or detecting PSA includes detecting the presence of a band on the test line, provided that a band has appeared on the control line.

[0090] In some embodiments of this application, the sperm quality testing method is for non-disease diagnosis or treatment purposes.

[0091] The sperm quality testing device provided in this application has the following beneficial effects:

[0092] This application provides a test card integrating sperm acrosomal enzyme SP10 protein and prostate-specific antigen (tPSA) detection in semen samples, along with a specially designed counting mechanism. It is simple to operate, providing results within 10 minutes. The results can be independently determined by the test subject, making it suitable for primary healthcare units and rapid on-site testing. The presence of bands in the T zone of the SP10 and PSA test strips indicates abnormal SP10 levels, with sperm counts below 15 × 10⁶ / ml and PSA levels below 1.2 mg / mL, resulting in abnormal sperm quality. The sperm motility detection module is located in the sample application area of ​​the test card and consists of a transparent sample platform with squares at the bottom and a movable sample application port cover. After sample application, sliding the cover upwards exposes the observation port of the motility detection module. Using a mobile phone or other device with photo or video recording capabilities, the observation port can be used to record sperm motility and count through the magnifying lens.

[0093] Additional aspects and advantages of this application will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of this application. Attached Figure Description

[0094] Figure 1 This is a schematic diagram of the structure of the SP10 colloidal gold test strip in one embodiment of this application.

[0095] Figure 2 This is a schematic diagram of the structure of a PSA colloidal gold test strip in one embodiment of this application.

[0096] Figure 3 This is a schematic diagram of the sperm quality testing card in one embodiment of this application. A is a top view of the sperm quality testing card, and B is a bottom view of the sperm quality testing card.

[0097] Figure 4 This is a side view of the structure of the sliding component on the first substrate in one embodiment of this application.

[0098] Figure 5 This is a line drawing diagram taken in one embodiment of this application. A and B represent the results of taking pictures at different magnifications, and C is a schematic diagram of the squares selected for counting sperm counts when the sperm count is high.

[0099] Figure 6 This is a photographic result of a semen sample from one embodiment of this application. A through J represent the photographic results of sample numbers 1 through 10, respectively.

[0100] Figure 7 The results are obtained by photographing and testing different semen samples in this application using the sperm quality test cards of Example 3 and Comparative Example 4.

[0101] Reference numerals: First PVC plate 100, First sample pad 110, SP10 gold pad 120, First nitrocellulose membrane 130, First detection line 131, First quality control line 132, First absorbent paper 140, Second PVC plate 200, Second sample pad 210, PSA gold pad 220, Second nitrocellulose membrane 230, Second detection line 231, Second quality control line 232, Second absorbent paper 240, Substrate 300, First substrate 310, Sample loading hole 311, Second substrate 320, Light transmission hole 321, Sliding assembly 330, First sliding component 331, Second sliding component 332, Convex lens 333, Receiving cavity 340, First cavity component 341, Second cavity component 342, Sample inlet 343. Detailed Implementation

[0102] The following will clearly and completely describe the concept and technical effects of this application in conjunction with embodiments, so as to fully understand the purpose, features and effects of this application. Obviously, the described embodiments are only some embodiments of this application, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of this application without creative effort are all within the scope of protection of this application.

[0103] The embodiments of this application are described in detail below. The described embodiments are exemplary and are only used to explain this application, and should not be construed as limiting this application.

[0104] In the description of this application, "several" means one or more, "multiple" means two or more, "greater than," "less than," and "exceeding" are understood to exclude the stated number, while "above," "below," and "within" are understood to include the stated number, and "approximately" means within the range of ±20%, 10%, 8%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.2%, 0.1%, etc. of the stated number. The use of "first" and "second" is merely for distinguishing technical features and should not be construed as indicating or implying relative importance, or implicitly indicating the number of indicated technical features, or implicitly indicating the order of the indicated technical features.

[0105] In the description of this application, the terms "one embodiment," "some embodiments," "illustrative embodiment," "example," "specific example," or "some examples," etc., refer to specific features, structures, materials, or characteristics described in connection with that embodiment or example, which are included in at least one embodiment or example of this application. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0106] The present application will be described below with reference to specific embodiments.

[0107] Example 1

[0108] This embodiment provides an SP10 colloidal gold test strip, the preparation process of which is as follows:

[0109] (1) Add 1L of purified water to the flask, heat to boiling, add 40mL of 1wt% chloroauric acid solution, continue heating to boiling, add 30mL of 2wt% sodium citrate solution, continue heating to boiling, react for 10min, the colloidal gold solution is ready, cool to room temperature for later use.

[0110] (2) Take 50 mL of the prepared colloidal gold solution, add 0.75 mL of 0.2 mol / L potassium carbonate, mix with a vortex mixer, add 1 mg of mouse anti-SP10 monoclonal antibody, mix well, and let stand at room temperature for 15 min.

[0111] (3) Take 1 mL of 20 wt% BSA blocking solution and add it to the reaction system of step (2). After vortexing and mixing, let it stand at room temperature for 5 min.

[0112] (4) Place the liquid that has been reacted in step (3) into a centrifuge, centrifuge at 12000 rpm for 20 min, discard the supernatant, add 50 mL of SP10 gold standard solution, and resuspend as SP10 gold standard solution for later use.

[0113] (5) Take 45mL of SP10 gold standard solution and spread it evenly on a glass cellulose membrane (25.4cm×30cm). Place it at 37℃ and dry for 24 hours to make SP10 colloidal gold pad for later use.

[0114] (6) Take 50 mL of the prepared colloidal gold solution, add 0.5 mL of 0.2 mol / L potassium carbonate, mix with a vortex apparatus, add 1 mg of mouse IgG, mix well, and let stand at room temperature for 15 min.

[0115] (7) Take 1 mL of 20 wt% BSA blocking solution and add it to the reaction system of step (6). After vortexing and mixing, let it stand at room temperature for 5 min.

[0116] (8) Place the liquid from the reaction in step (7) into a centrifuge, centrifuge at 12000 rpm for 20 min, discard the supernatant, add 100 mL of mouse IgG gold reconstitution solution, and resuspend as mouse IgG gold reconstitution solution for later use.

[0117] (9) Take 45 mL of mouse IgG gold solution and spread it evenly on a glass cellulose membrane (25.4 cm × 30 cm). Place it at 37°C and dry for 24 hours to make mouse IgG colloidal gold pad for later use.

[0118] (10) Attach the nitrocellulose membrane to a PVC substrate. Take goat anti-mouse IgG antibody and dilute it with coating diluent to a final concentration of 1 mg / mL. Take recombinant SP10 antigen and dilute it to a final concentration of 1.2 mg / mL. Using a coating apparatus, coat the goat anti-mouse IgG antibody diluent and recombinant SP10 antigen diluent onto the corresponding positions on the nitrocellulose membrane at a rate of 1 μL / cm to form the C line (first control line) and T1 line (first detection line). Dry at 37°C for 24 hours for later use.

[0119] (11) Cut the dried SP10 gold pad and mouse IgG gold pad to 3mm and paste them onto the PVC base plate in (10), and attach a 19cm×30cm sample pad and 20.5cm×30cm absorbent paper, and cut them into 3mm wide SP10 colloidal gold test strips.

[0120] In step (4), the SP10 gold standard solution is as follows: 0.05 mol / L borate buffer is added with a final concentration of 10 wt% sucrose, 1 wt% sodium caseinate, 1 v / v% Triton X-100, and 0.03 wt% PC300, and the pH is adjusted to 8.0 ± 0.2.

[0121] The sample pads were prepared as follows: 0.2 wt% Tween-20 and 0.03 wt% PC300 were added to 0.02 mol / L PB buffer. The pads were evenly spread on the glass cellulose membrane at a rate of 45 mL / sheet (25.4 cm × 30 cm) and dried at 37°C for 2 hours before use.

[0122] The coating solution was prepared as follows: 2 wt% sucrose was added to 0.02 mol / L PB buffer, and the pH was adjusted to 7.4–8.0.

[0123] refer to Figure 1 The structure of the SP10 colloidal gold test strip is as follows: it includes a first PVC plate 100, on which a first sample pad 110, an SP10 gold pad 120, a mouse IgG gold pad, a first nitrocellulose membrane 130, and a first absorbent paper 140 are sequentially overlapped. The first nitrocellulose membrane 130 is provided with a first detection line 131 and a first control line 132 in the same order.

[0124] The antigens and antibodies used for coating in the above experiment are as follows:

[0125] The recombinant SP10 antigen coated onto the T1 line in step (10) is SP10 recombiant protein, and the mouse anti-SP10 monoclonal antibody labeled onto the gold pad in steps (2) to (5) is SP10 antibody (1a1).

[0126] Example 2

[0127] This embodiment provides a PSA colloidal gold test strip, the preparation process of which is referred to in Example 1, and is briefly described below:

[0128] (1) Take 50 mL of the prepared colloidal gold solution, add 0.5 mL of 0.2 mol / L potassium carbonate, mix with a vortex apparatus, add 1 mg of PSA antibody 1, mix well, and let stand at room temperature for 15 min.

[0129] (2) Add 1 mL of 20% BSA blocking solution to the reaction system of step (1), vortex mix, and let it stand at room temperature for 5 min.

[0130] (3) Place the liquid that has been reacted in step (2) into a centrifuge, centrifuge at 12000 rpm for 20 min, discard the supernatant, add 100 mL of PSA gold standard solution, and resuspend as PSA gold standard solution for later use.

[0131] (4) Take 45 mL of PSA gold standard solution and spread it evenly on a glass cellulose membrane (25.4 cm × 30 cm). Place it at 37°C and dry for 24 hours to make PSA gold pad for later use.

[0132] (5) Place the nitrocellulose membrane on a PVC substrate, take sheep anti-mouse IgG antibody, and dilute the sheep anti-mouse IgG antibody with coating diluent to a final concentration of 1 mg / mL; take PSA antibody 2, and dilute it with coating diluent to a final concentration of 0.8 mg / mL. Use a membrane scrubbing instrument to coat the sheep anti-mouse IgG antibody diluent and PSA antibody 2 diluent onto the corresponding positions on the nitrocellulose membrane at a rate of 1 μL / cm to form C lines and T2 lines. Dry at 37°C for 24 hours for later use.

[0133] (6) Cut the dried PSA gold pad to 3mm and paste it onto the PVC base plate in (5). Then, attach a 19cm×30cm sample pad and a 20.5cm×30cm absorbent paper, and cut it into 3mm PSA colloidal gold test strips. The PSA colloidal gold test strips consist of a sample pad, a PSA gold pad, a mouse IgG gold pad, a nitrocellulose membrane, and absorbent paper that are sequentially overlapped on the PVC base plate. The nitrocellulose membrane also has T2 lines and C lines in the same order.

[0134] The preparation of the PSA gold-labeled complex solution involved adding 10 wt% sucrose, 2 wt% sodium caseinate, 1 wt% BSA, 0.5 wt% Gly, 0.5 v / v% Tween-20, and 0.03 wt% PC300 to 0.02 mol / L PB buffer at the final concentration, and adjusting the pH to 7.4 ± 0.2.

[0135] refer to Figure 2 The structure of the PSA colloidal gold test strip is as follows: it includes a second PVC plate 200, on which a second sample pad 210, a PSA gold pad 220, a second nitrocellulose membrane 230, and a second absorbent paper 140 are sequentially overlapped. The second nitrocellulose membrane 230 is provided with a second detection line 231 and a second quality control line 232 in the same order.

[0136] The antigens and antibodies used for coating in the above experiment are as follows:

[0137] The PSA antigen coated onto the T2 line in step (5) is PSA-Ag; the PSA antibody 1 labeled on the PSA gold pad in steps (1) to (4) is T-PSA-mAb.

[0138] Example 3

[0139] This embodiment provides a sperm quality testing card, for reference. Figure 3 The test kit includes a substrate comprising a first substrate 310 (upper cover of the test card) and a second substrate 320 (lower cover of the test card) disposed opposite to each other. A sperm motility detection module, a PSA colloidal gold test strip, and an SP10 colloidal gold test strip are disposed between the first substrate 310 and the second substrate 320. The first substrate 310 has a sample application hole 311, while the second substrate 320 has a light transmission hole 321. The sample application hole 311 and the light transmission hole 321 are disposed opposite to each other in a direction perpendicular to the first substrate 310 and the second substrate 320, and the projection of the sample application hole 311 onto the second substrate 320 in a direction perpendicular to the first substrate 310 covers the light transmission hole 321, meaning the area of ​​the sample application hole 311 is larger than that of the light transmission hole 321. The sample to be tested is added through the sample application hole 311 between the first substrate 310 and the second substrate 320.

[0140] The sperm motility detection module includes a detection component, which comprises a first cavity component 341 (upper cavity cover) and a second cavity component 342 (lower cavity cover). The first cavity component 341 and the second cavity component 342 are spaced apart to form a receiving cavity 340 between them. The receiving cavity 340 is open at both ends along the direction of the first cavity component 341 or the second cavity component 342. One end of the receiving cavity 340 has a sample inlet 343, and the portion of the receiving cavity 340 located between the first cavity component 341 and the second cavity component 342 constitutes a detection plane. The first cavity component 341 and the second cavity component 342 are made of transparent material. The sperm motility detection module is fixed to the substrate through the connection between the second cavity component 342 and the second substrate 320. The sperm motility detection module also includes a sliding component 330, which is movably connected to the first substrate 310. The sliding component 330 moves in a reciprocating direction, either towards or away from the sample application hole 311. A convex lens 333 is provided on the sliding component 330. By sliding the sliding component 330, the operator can align the convex lens 333 with the detection plane through the sample application hole 311, allowing observation of the image on the detection plane from one side of the sample application hole 311.

[0141] For details on the structure of the sliding component 330, please refer to [reference]. Figure 3The system includes a first sliding member 331 located on the side of the first substrate 310 away from the second substrate 320 and a second sliding member 332 located on the side of the first substrate 310 closer to the second substrate 320. The first sliding member 331 and the second sliding member 332 are fixedly connected. A convex lens 333 is provided on the second sliding member 332. The first sliding member 331 and the second sliding member 332 are fixedly connected. By sliding the first sliding member 331 on the first substrate 310, the second sliding member 332 is slid at intervals to cover the sample application hole 311. At the same time, the convex lens 333 in the second sliding member 332 is aligned with the light transmission hole 321 in the receiving cavity 340.

[0142] The first sample pad 110 and the second sample pad 210 of the PSA colloidal gold test strip and the SP10 colloidal gold test strip come into contact with the injection port 343, so that the test sample dripped from the sample application hole 311 into the injection port 343 can penetrate the PSA colloidal gold test strip and the SP10 colloidal gold test strip for detection. At the same time, the sample entering the receiving cavity from the injection port 343 can be used for sperm motility detection.

[0143] The first substrate 310 is also provided with an SP10 detection result observation window and a PSA detection result observation window. The SP10 detection result observation window exposes the first detection line 131 and the first control line 132 on the first nitrocellulose membrane 130 of the SP10 colloidal gold test strip, while the PSA detection result observation window exposes the second detection line 231 and the second control line 232 on the second nitrocellulose membrane 230 of the PSA colloidal gold test strip. This allows observation of the detection results of SP10 and PSA in the sample. Furthermore, a crisscrossing grid of lines is pre-etched on the convex lens 333 or the detection plane to form a counting grid.

[0144] refer to Figure 4 In this embodiment, the convex lens 333 has a diameter d of 2 mm and a focal length of 1.5 mm. The distance L1 between the convex lens 333 and the detection plane of the receiving cavity 340 is 1.2 mm. The distance L2 between the first cavity component 341 and the second cavity component 342 of the receiving cavity 340 is 0.1 mm. The cavity thickness L3 is 0.8 mm. The diameter D of the light-transmitting aperture 321 is 4 mm. The interval between adjacent horizontal and vertical lines of the grid is 0.2 mm. Under these parameters, sperm in the sample are less likely to stack in the receiving cavity, facilitating the formation of a single-cell layer for observation and counting. The sperm morphology and the imaging of the counting chamber are clear, making statistical analysis easier.

[0145] This embodiment also provides a method for testing using the above-mentioned sperm quality testing card, the steps of which are as follows:

[0146] (1) Use artificial stimulation or a non-toxic condom designed specifically for semen collection to collect semen. The best time to collect semen is 2-7 days after the last ejaculation.

[0147] (2) Collect the semen sample into a clean container and let it stand at room temperature for 30 to 60 minutes until the semen liquefies.

[0148] (3) Use a pipette to take 0.1 mL of completely liquefied semen sample and add it to 1.9 mL of diluent. Shake left and right to mix.

[0149] (4) Add 5 drops (about 200 μL) of the mixed sample into the sample well, avoiding air bubbles in the added liquid.

[0150] (5) After the sample is added, wait 3 minutes. Use a clean cotton swab to insert into the sample hole and wipe the top of the cavity cover 5 times to clean up the excess sample on the top of the cavity cover and avoid affecting the lens image.

[0151] (6) Press and slide the sliding component. The sample loading hole is closed by the second sliding part of the sliding component, and the convex lens is exposed. At this time, the convex lens is aligned with the sample loading cavity and the light transmission hole.

[0152] (7) Observe the test results of the T1 line, T2 line, and C line after waiting for 10 minutes for the SP10 colloidal gold test strip and the PSA colloidal gold test strip. The sperm motility detection module can be observed immediately. Place the LED light source at the light hole below the test card, point the mobile phone camera at the sample application hole, turn on the camera mode, adjust the distance between the camera and the sample application hole, focus the mobile phone camera to make the image clear, and record a 30-second video. The optimal viewing distance of the mobile phone camera (i.e., the distance between the mobile phone camera and the sample application hole) is 10mm.

[0153] (8) Result Interpretation:

[0154] SP10 Test Result Interpretation: Observe the observation window of the SP10 test strip 10 minutes after sample application. The appearance of a band in the control area (C line) indicates normal testing operation, and the test result is valid; otherwise, it is invalid. When the control area (C line) appears, observe the T1 test area. A red band in the T1 test area indicates that the sperm content in the sample is below 15 × 10⁻⁶. 6 / mL indicates that the sperm content in the sample is below the lower limit of the reference value; conversely, if no red band appears in the T1 test area, it indicates that the sperm content is above the lower limit of the reference value.

[0155] PSA test result interpretation: Observe the observation window of the PSA test strip 10 minutes after sample addition. The appearance of a band at the control line (C line) indicates that the test operation is normal and the test result is valid; otherwise, it is invalid. When a band appears at the control line (C line), observe the T2 detection area. A red band in the T2 detection area indicates that the PSA antigen content in the sample is higher than 5 mg / mL, indicating an abnormal PSA antigen content in the sample. Conversely, the absence of a red band in the T2 detection area indicates that the PSA antigen content in the sample is lower than 5 mg / mL.

[0156] Analysis of the videos captured by the vitality detection module:

[0157] Sperm count and concentration: Select a 5×5 grid area near the center of the marked area, containing 25 small squares of 0.2mm×0.2mm. When the sperm count is low, count all sperm in this area; when the sperm count is high, select four diagonal squares and one central square within the area for counting (as shown in the figure). Five times the count is the sperm count for that area. Only count complete sperm (with head and tail). Example: If the sperm count in the 5×5 grid area is X, then the concentration is 20X / 100nL, which equals 0.2X×10. 6 / mL.

[0158] Sperm motility: Three distinct regions were selected within the counting grid, with 200 sperm counted in each region. The number of progressively motile (PR) and non-progressively motile (NP) sperm (sperm whose tail movement is minimal or only tail wagging is observed) was recorded in each region. The mean number of PR+NP sperm across the three regions was calculated. The lower limit of the reference range for total sperm motility (PR+NP) is 40% (5th percentile, 95% CI, 38%–42%). The lower limit of the reference range for progressively motile (PR) sperm is 32% (5th percentile, 95% CI, 31%–34%), used to assess sperm motility.

[0159] In addition to the two key sperm quality indicators, concentration and motility, sperm morphology and sperm aggregation can also be observed initially.

[0160] For the aforementioned sperm quality testing cards, the SP10 testing module and the PSA testing module were evaluated using quality control samples:

[0161] SP10 recombinant antigen (SP10) quality control products S1-S10 (where 30 ng / mL of SP10 recombinant antigen and sperm concentration are 15 × 10⁻⁶) 6 The SP10 antigen content is consistent at 30 ng / mL, therefore 30 ng / mL is used as the threshold for clinical interpretation of sperm concentration.

[0162] Prostate-specific antigen (PSA) quality control products P1-P10.

[0163] The quality control products S1-S10 were used to test the SP10 test strip, and the quality control products P1-P10 were used to test the PSA test strip. The results are shown in Table 1, and all meet the requirements for clinical interpretation.

[0164] Table 1. Test Results

[0165]

[0166]

[0167] Example 4

[0168] Ten fresh semen samples were collected clinically. Sperm motility was tested using the sperm quality test card and corresponding method described in Example 3. Sample information and test results are shown in Table 2. The clinical test results in the table were obtained by counting and statistical analysis under a microscope using a cell counting chamber.

[0169] Table 2. Clinical Samples and Test Results

[0170]

[0171] The method for detecting sperm motility and concentration by shooting a 30-second test video with a mobile phone is as follows:

[0172] Concentration: Select a 5×5 grid area in the video, closer to the center. When the sperm count is low, count all sperm in this area (e.g., samples 1-5). When the sperm count is high, select four diagonal squares and one central square within this area (e.g., samples 6-10). Five times the counted number represents the sperm count in this area. Only count complete sperm (with head and tail). If the sperm count in the selected 5×5 grid area is X, then the concentration is 20X / 100nL = 0.2X×10 6 sperm count / mL. Normal sperm concentration >15×10⁻⁶. 6 per mL.

[0173] Vigor: Select 3 different regions within the counting grid, count 200 sperm in each region, and record the number of forward motility (PR) and non-forward motility (NP) sperm in each region (tail movement is almost unable to drive head movement, or only tail wagging is observed). The lower limit of the reference value for total sperm vigor (PR+NP) is 40%.

[0174] Based on clinical reference thresholds, the interpretation of concentration and activity in the 10 samples was consistent with clinical results.

[0175] Comparative Example 1

[0176] Comparative Example 1 provides a sperm quality test card, which differs from the sperm quality test card in Example 3 in that: the SP10 gold standard compound solution is composed of: 0.05 mol / L borate buffer with 10 wt% sucrose, 1 wt% Casein-Na, 1 wt% Tween-20, and 0.03 wt% PC300 added to a final concentration, and the pH is adjusted to 8.0 ± 0.2; that is, the 1 wt% Triton X-100 is replaced with 1 wt% Tween-20.

[0177] The test results of the sperm quality test card of Comparative Example 1 on SP10 quality control products S1-S10 are shown in Table 3:

[0178] Table 3. Test Results

[0179] Quality control products Interpretation Quality control products Interpretation S1 Positive S6 Positive S2 Negative S7 Positive S3 Negative S8 Positive S4 Positive S9 Positive S5 Positive S10 Positive

[0180] The results showed that Comparative Example 1 was more prone to false positives than the Example, indicating that replacing 1 wt% Tween-20 with 1 wt% Triton X-100 could effectively improve the detection specificity.

[0181] Comparative Example 2

[0182] This comparative example provides a sperm quality testing card, which differs from the sperm quality testing card in Example 3 in that the composition of the PSA gold-labeled reconstituted solution is different: 10 wt% sucrose, 1 wt% Casein-Na, 1 wt% BSA, 0.5 wt% Gly, 0.5 wt% Tween-20, and 0.03 wt% PC300 are added to 0.02 mol / L PB buffer, and the pH is adjusted to 7.4 ± 0.2; that is, the 2 wt% Casein-Na in the PSA gold-labeled reconstituted solution is reduced to 1 wt%.

[0183] The test results of the sperm quality test card of Comparative Example 2 on PSA quality control samples P1-P10 are shown in Table 4:

[0184] Table 4. Test Results

[0185] Quality control products Interpretation Quality control products Interpretation P1 Negative P6 Negative P2 Negative P7 Positive P3 Negative P8 Positive P4 Negative P9 Positive P5 Negative P10 Positive

[0186] The results showed that the detection limit of Comparative Example 2 was 1.3 mg / mL, lower than the 1.2 mg / mL detection threshold in Example 3, thus its sensitivity decreased significantly. This demonstrates that increasing the concentration of Casein-Na from 1% to 2% effectively improves the detection sensitivity.

[0187] Comparative Example 3

[0188] This comparative example provides a sperm quality testing card, which differs from Example 3 in that the coating solution used for the SP10 test strip and the PSA test strip is composed of 0.02 mol / L PB buffer with 2 wt% trehalose added, and the pH is adjusted to 7.4-8.0; that is, 2 wt% sucrose is replaced with 2 wt% trehalose.

[0189] The stability test results of the sperm quality test card in Comparative Example 3 for quality control samples S1-S10 and P1-P10 are shown in Table 5:

[0190] Table 5. Stability Test Results

[0191]

[0192] The results in the table show that Example 3 is superior to Comparative Example 3 in terms of room temperature stability. The SP10 test strip maintained its original detection limit of 30 ng / mL after one year, while the original detection limit of Comparative Example 3 was only maintained for 90 days, decreasing to 80 ng / mL after one year. Similarly, the PSA test strip maintained its original detection limit of 1.2 mg / mL after one year, while the original detection limit of Comparative Example 3 was only maintained for 21 days, decreasing to 1.5 mg / mL after one year (below the threshold of 1.2 mg / mL). These results indicate that replacing sucrose with trehalose in the coating solution effectively improves the stability of the test strips.

[0193] Comparative Example 4

[0194] This comparative example provides a sperm quality testing card, which differs from Example 3 in that the distance between the convex lens and the transparent sample-carrying cavity is adjusted from 1.2 mm to 2.0 mm.

[0195] The results of imaging three clinical samples using the sperm quality testing card from Comparative Example 4 are as follows: Figure 7 As shown, compared to Example 3, Comparative Example 4 exhibits significantly worse imaging performance. These results indicate that setting the object distance between the convex lens and the receiving cavity to 1.2 mm is more suitable than 2.0 mm, ensuring that the object distance is 80-90% of the focal length.

[0196] Comparative Example 5

[0197] This comparative example provides a sperm quality testing card, which differs from Example 3 in that the distance between the upper and lower covers of the sample-carrying chamber (i.e., the height for calculating the sample liquid volume) is adjusted from 0.1 mm to 0.2 mm.

[0198] The observational statistics of 10 collected samples are shown in Table 6.

[0199] Table 6. Comparison of test results for comparative examples

[0200]

[0201] Statistical calculations of the imaging results of samples 6-10 show a significant difference between the results of comparative example 5 and the clinical test results. This may be because the higher height caused sperm aggregation in the liquid sample, affecting the counting. The calculation results indicate that the example is significantly better than the comparative example in terms of statistical data, proving that a spacing of 0.1 mm between the upper and lower covers of the sample-carrying chamber is more suitable than 0.2 mm.

[0202] The sperm quality testing device provided in this embodiment includes the following components:

[0203] ①SP10 and PSA use colloidal gold color development to interpret the results: The test results can usually be judged directly by observing the color development with the naked eye, reducing the reliance on professional analytical equipment. There is no need for complicated equipment to interpret the results or for professional medical personnel to explain the results. The test subject can judge the results independently by visually observing the color development of the test area.

[0204] ② The sperm motility detection module uses a built-in convex lens for magnification and a grid function to assist in counting within the sperm cavity, recording sperm motility using an imaging device. Based on the counting grid in the image, it statistically counts sperm concentration and quantity. Operators can refer to the relevant indicators provided in the instruction manual to independently interpret sperm motility and concentration.

[0205] Therefore, this sperm quality testing device has the following advantages:

[0206] I. Detection of multiple biomarkers: By simultaneously detecting sperm SP10 protein and PSA, and observing sperm morphology and motility, this test card can provide multiple information about sperm count, quality, activity, and morphology.

[0207] II. Simplified sample addition: No complicated instruments or professional operators are required. The simple sample addition operation and reagent design for diluting samples reduce the difficulty of operation, making the test more portable and user-friendly.

[0208] III. Quick and Simple Process: Colloidal gold immunochromatographic assay can be completed in a short time, with results interpreted within 10 minutes. The integrated testing system can provide multiple indicators of sperm quality in a short time, which helps in the rapid diagnosis of male infertility and is suitable for primary healthcare institutions and on-site rapid screening.

[0209] IV. High cost-effectiveness: Since it does not require expensive equipment and professional personnel to operate, the design of the colloidal gold test card makes the overall cost relatively low.

[0210] V. User-friendly design: The test card is designed with user convenience in mind, making it easy for non-professionals to operate. The simple visual interpretation method also allows users to make a preliminary interpretation of the results on their own.

[0211] The present application has been described in detail above with reference to the embodiments. However, the present application is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present application. Furthermore, unless otherwise specified, the embodiments and features in the embodiments of the present application can be combined with each other.

Claims

1. A sperm quality testing device, characterized in that, include: A first substrate and a second substrate are disposed opposite to each other, and the first substrate is provided with a sample feeding hole; A sperm motility detection module includes a detection component and a sliding component. The detection component includes a first cavity component and a second cavity component, which are spaced apart from each other and stacked on a second substrate to form a receiving cavity. The sliding component includes a convex lens movably connected to the first substrate. The receiving cavity has a detection plane. The convex lens is configured to slide with the sliding component to the position of the sample application hole and magnify the image of the detection plane. The sliding component includes a first sliding component located on the side of the first substrate away from the second substrate and a second sliding component located on the side of the first substrate closer to the second substrate. The first sliding component and the second sliding component are fixedly connected. The convex lens is provided on the second sliding component. The sliding component is configured to be able to slide the second sliding component by sliding the first sliding component on the first substrate, thereby covering the sample application hole, while the convex lens in the second sliding component is aligned with the detection plane. The SP10 detection module is fixed on the second substrate. The SP10 detection module includes an SP10 colloidal gold test strip, which is a competitive lateral chromatography test strip. The SP10 colloidal gold test strip includes a first sample pad, a first conjugate pad, a first nitrocellulose membrane, and a first absorbent paper that are sequentially overlapped. The first conjugate pad is coated with a colloidal gold-labeled SP10-specific antibody. PSA detection module, the PSA detection module is fixed on the second substrate, the PSA detection module includes PSA colloidal gold test strip; The first binding pad of the SP10 colloidal gold test strip is prepared by coating the SP10 gold standard solution onto a substrate. The SP10 gold labeling solution is prepared by reacting colloidal gold solution with SP10-specific antibody to generate colloidal gold-labeled SP10-specific antibody, adding blocking buffer to block the binding site, centrifuging to remove the supernatant, and then adding SP10 reconstitution solution to obtain the SP10 gold labeling solution. The SP10 reconstitution solution includes 0.01~0.1mol / L borate buffer, 5~20wt% sucrose, 0.2~5wt% sodium caseinate, 0.1~10v / v% Triton X-100 and 0.01~0.1wt% PC300.

2. The sperm quality testing device according to claim 1, characterized in that, The first nitrocellulose membrane is provided with a first detection line and a first control line in the same order, and the first detection line is coated with SP10 antigen.

3. The sperm quality testing device according to claim 1, characterized in that, The distance between the convex lens and the detection plane is 1~1.5 mm.

4. The sperm quality testing device according to claim 1, characterized in that, The height of the receiving cavity is 0.05~0.15 mm.

5. A method for preparing the sperm quality testing device according to any one of claims 1 to 4, characterized in that, The invention includes the preparation of SP10 colloidal gold test strips and PSA colloidal gold test strips. The preparation of SP10 colloidal gold test strips includes coating SP10 gold standard solution onto a substrate to prepare a first conjugation pad, and the preparation of PSA colloidal gold test strips includes coating PSA gold standard solution onto a substrate to prepare a second conjugation pad.

6. The preparation method according to claim 5, characterized in that, The preparation of the PSA gold labeling solution includes reacting colloidal gold solution with PSA-specific antibody to generate colloidal gold-labeled PSA-specific antibody, adding blocking solution to block the binding site, centrifuging to remove supernatant, and adding PSA reconstitution solution to obtain PSA gold labeling solution. The PSA reconstitution solution comprises PB buffer, sucrose, sodium caseinate, BSA, Gly, Tween-20, and PC300, with the final concentration of sodium caseinate being 1.5 to 2.5 wt%.

7. The preparation method according to claim 5, characterized in that, The preparation of the SP10 colloidal gold test strip includes diluting the SP10 specific antigen with a coating solution and coating it onto a first nitrocellulose membrane to form a first detection line. The preparation of the PSA colloidal gold test strip includes diluting the PSA specific antigen with a coating solution and coating it onto a second nitrocellulose membrane to form a second detection line. The coating solution includes PB buffer and sucrose with a final concentration of 1-3 wt%.

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