Chemiluminescence reaction detection reagent and detection method for content of spermine in seminal plasma

Abstract

The present invention relates to the technical field of biology. Disclosed are a chemiluminescence reaction detection reagent and detection method for the content of spermine in seminal plasma. The chemiluminescence reaction detection reagent comprises a luminol-containing sodium hydroxide solution, a copper ion solution and a sodium borate buffer solution. According to the present invention, on the basis of the chemiluminescence reaction principle of metal ions catalyzing hydrogen peroxide to oxidize luminol, it is found that the luminescence reaction intensity of copper ions and a luminol reagent would be remarkably influenced in the presence of spermine. On this basis, the present invention develops a kit for detecting the content of spermine on the basis of static chemiluminescence analysis, and establishes a new method for static chemiluminescence analysis of spermine, wherein the method involves high sensitivity, simple operation, small sample volume and low costs, and improves the working efficiency.

Description

A chemiluminescence reaction detection reagent and detection method for spermine content in seminal plasma Technical Field

[0001] The present invention relates to the field of biotechnology, in particular to a chemiluminescence reaction detection reagent and a detection method for spermine content in seminal plasma. Background Art

[0002] Spermine, a key component of seminal plasma, is an aliphatic polyamine synthesized and secreted by the male accessory gland, the prostate. It protects DNA from free radical oxidative damage and maintains the stability of the sperm genome. It also possesses anti-infective properties, protecting sperm from bacterial and viral attacks, making it a key indicator of semen quality. Traditional methods for quantitative spermine detection include gas chromatography, thin-layer chromatography, capillary electrophoresis, electrochemical biosensors, and high-performance liquid chromatography, primarily used to detect spermine in plants and aquatic organisms. While highly sensitive and accurate, these methods impose stringent sample quality requirements, are cumbersome, time-consuming, and costly, making them difficult to meet routine clinical and scientific testing requirements. Furthermore, spermine itself lacks ultraviolet fluorescence and electrochemical activity, and traditional methods generally rely on derivatization, which not only increases analysis time but also easily generates interfering peaks, making satisfactory results difficult to achieve.

[0003] Therefore, how to establish a simple, fast and low-cost method for detecting spermine is an urgent problem to be solved.

[0004] Summary of the Invention

[0005] The present invention aims to provide a chemiluminescent reaction detection reagent and detection method for spermine content in seminal plasma to solve the problems existing in the above-mentioned prior art. The chemiluminescent reaction detection method provided by the present invention can accurately and sensitively detect spermine in solution.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The invention provides a chemiluminescence reaction detection reagent for spermine content in seminal plasma, which comprises a sodium hydroxide solution of luminol, a copper ion solution and a sodium borate buffer solution.

[0008] Furthermore, in the sodium hydroxide solution of luminol, the concentration of luminol is 5×10 -4 mol / L; the concentration of copper ions in the copper ion solution is 1×10 -4 mol / L; the pH of the sodium borate buffer solution is 9.2, wherein the concentration of sodium borate is 0.05 mol / L.

[0009] The present invention also provides application of the chemiluminescence reaction detection reagent in preparing a kit for detecting the content of spermine in seminal plasma.

[0010] The present invention also provides a chemiluminescence reaction detection kit for spermine content in seminal plasma, comprising the chemiluminescence reaction detection reagent.

[0011] The present invention also provides the use of the chemiluminescence reaction detection reagent or the chemiluminescence reaction detection kit in determining the spermine content in seminal plasma.

[0012] The present invention also provides a chemiluminescence reaction detection method for spermine content in seminal plasma, comprising the step of using the chemiluminescence reaction detection reagent or the chemiluminescence reaction detection kit to detect a sample to be detected.

[0013] Furthermore, the method specifically includes the following steps:

[0014] The copper ion solution, sodium borate buffer solution and the sample to be tested are mixed to obtain a mixture, and then a sodium hydroxide solution of luminol is added to the mixture to perform a luminol luminescence reaction. The luminescence intensity is detected, and the content of spermine in the sample to be tested is calculated according to the spermine standard curve and the luminescence intensity.

[0015] Furthermore, the reaction system of the luminol luminescence reaction is: 1 mL of copper ion solution, 1 mL of sodium borate buffer solution, 1 mL of the sample to be tested and 0.2 mL of luminol sodium hydroxide solution.

[0016] The present invention discloses the following technical effects:

[0017] The present invention is based on the principle of metal ion-catalyzed hydrogen peroxide chemiluminescent reaction, and found that the presence of spermine significantly affects the luminescent reaction intensity of copper ions and luminol reagent. When spermine and a small amount of copper ions form a saturated coordination compound, it has inhibitory activity on the hydrogen peroxide chemiluminescent reaction of luminol, while when spermine and an excess of copper ions form an unsaturated coordination compound, it has catalytic activity on the hydrogen peroxide chemiluminescent reaction of luminol (Figure 1). Based on this, the present invention has developed a kit for detecting spermine content based on static chemiluminescent analysis, and established a new method for static chemiluminescent analysis of spermine. This method has high sensitivity, simple operation, small sample amount, low cost, and improved work efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0019] FIG1 is a schematic diagram of the luminol chemiluminescence determination of spermine content;

[0020] FIG2 is a chemical reaction curve of the luminol chemiluminescence reaction for detecting the spermine content in the blank control (A) and the spermine standard solution (B);

[0021] Figure 3 is the standard curve of spermine. DETAILED DESCRIPTION

[0022] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0023] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0024] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0025] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be illustrative only.

[0026] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0027] Example 1 Detection of spermine content in clinical seminal plasma samples

[0028] 1. Sample Collection: All males had normal physical and andrological examinations and abstained from sex for 4 days before semen collection. Semen samples were collected from 12 men with a history of fertility and 12 from men with infertility.

[0029] 2. Sample processing: Place the semen sample in a 37°C water bath for 30 minutes to allow the semen to liquefy. Centrifuge at 3000g for 10 minutes and transfer the supernatant to a clean test tube, which is the seminal plasma.

[0030] 3. Drawing of spermine standard curve:

[0031] Prepare a series of spermine standard solutions with different concentrations (1×10 -3 mol / L, 2×10 -3 mol / L, 5×10 -3 mol / L, 10×10 -3 mol / L and 20×10 -3 mol / L), the copper ion solution (copper ion concentration is 1×10 -4 mol / L), sodium borate buffer (pH 9.2, concentration of 0.05 mol / L) and spermine standard solution were added into the test tube in the order of Table 1 and mixed by pipetting.

[0032] Table 1 Order and situation of adding each substance

[0033] After mixing, take out 0.1 mL of the mixed solution from each test tube into a new test tube and add 0.2 mL of luminol sodium hydroxide solution (luminol concentration is 5×10 -4 mol / L), mixed and allowed to stand for 30 min, then added to BPCL weak luminescence measuring instrument for measurement, and quantified by peak height.

[0034] The experimental results are shown in Figure 2. The chemical reaction curve of the blank control group has only one peak (A in Figure 2), which is the light emitted after the reaction between copper ions and luminol sodium hydroxide solution, while the spermine standard solution (5×10 -3 The chemical reaction curve for 100 mol / L (100 mmol / L) exhibits three peaks (Figure 2B). Peak (a) represents the light emitted by the reaction of copper ions with a sodium hydroxide solution of luminol. Peak (b) represents the light emitted by the reaction of copper hydroxide with a sodium hydroxide solution of luminol, which takes 25 seconds from the onset of luminescence to reach its maximum chemiluminescence intensity. Peak (c) represents the light emitted by the reaction of copper hydroxide with a sodium hydroxide solution of luminol in the presence of spermine, which takes 20 seconds from the onset of luminescence to reach its maximum chemiluminescence intensity. This demonstrates that the luminol chemiluminescence reaction can accurately and sensitively detect spermine in solution.

[0035] The relative luminescence intensity of the spermine standard series solutions was determined based on the peak height, and the concentration of the spermine standard solution (1×10 -3 mol / L, 2×10 -3 mol / L, 5×10 -3 mol / L, 10×10-3 mol / L and 20×10 -3 mol / L) was used to draw a standard curve of the relationship between the logarithm of the relative luminescence intensity and the spermine concentration, and the linear equation was calculated: y = 0.3786x + 2.2713, where x is the spermine concentration and y is the logarithm of the relative luminescence intensity ( Figure 3 ).

[0036] 4. Detection of spermine content in seminal plasma:

[0037] Add the following reagents to the corresponding sterile test tubes in the order listed in Table 2 and mix thoroughly by pipetting.

[0038] Table 2 Order and situation of adding each substance

[0039] After mixing, 0.1 mL of the mixed solution was taken from each test tube into a new test tube, and 0.2 mL of luminol sodium hydroxide solution (5 × 10 -4 mol / L), mixed and allowed to stand for 30 min, then added to a BPCL weak luminescence meter for measurement, with peak height being used for quantification. The experimentally measured luminescence intensity was converted to a logarithmic value and inserted into the spermine standard curve equation to obtain the results shown in Table 3.

[0040] Table 3 Luminol chemiluminescence determination of spermine content results

[0041] In summary, the use of luminol chemiluminescence reaction to detect spermine content in seminal plasma has a clear experimental principle, feasible experimental operation, simple experimental steps, reliable experimental results, and significantly improved work efficiency.

[0042] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A chemiluminescent reaction detection reagent for spermine content in seminal plasma, characterized in that: Includes luminol sodium hydroxide solution, copper ion solution and sodium borate buffer solution.

2. The chemiluminescent reaction detection reagent according to claim 1, characterized in that: In the sodium hydroxide solution of luminol, the concentration of luminol is 5×10 -4 mol / L; the concentration of copper ions in the copper ion solution is 1×10 -4 mol / L; the pH of the sodium borate buffer solution is 9.2, wherein the concentration of sodium borate is 0.05 mol / L.

3. Use of the chemiluminescent reaction detection reagent as claimed in claim 1 or 2 in preparing a kit for detecting the content of spermine in seminal plasma.

4. A chemiluminescent reaction detection kit for spermine content in seminal plasma, characterized in that: The invention comprises the chemiluminescent reaction detection reagent as described in claim 1 or 2.

5. Use of the chemiluminescent reaction detection reagent according to any one of claims 1 to 2 or the chemiluminescent reaction detection kit according to claim 4 in determining the spermine content in seminal plasma.

6. A chemiluminescent reaction detection method for spermine content in seminal plasma, characterized in that: The method comprises the step of using the chemiluminescent reaction detection reagent according to any one of claims 1 to 2 or the chemiluminescent reaction detection kit according to claim 4 to detect the sample to be detected.

7. The chemiluminescent reaction detection method according to claim 6, characterized in that: The specific steps include: The copper ion solution, the sodium borate buffer solution and the sample to be tested are mixed to obtain a mixture, and then a sodium hydroxide solution of luminol is added to the mixture to carry out a luminol luminescence reaction, and the luminescence intensity is detected. The content of spermine in the sample to be tested is calculated according to the spermine standard curve and the luminescence intensity.

8. The chemiluminescent reaction detection method according to claim 7, characterized in that: The reaction system of the luminol luminescence reaction is: 1 mL of copper ion solution, 1 mL of sodium borate buffer solution, 1 mL of the sample to be tested and 0.2 mL of luminol sodium hydroxide solution.

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