A method for detecting free amino acid content in biological tissue

Abstract

The present invention belongs to the technical field of biological tissue material detection, particularly to the field of G01N21 / 33, and specifically to a method for detecting the content of free amino acids in biological tissues. The method comprises the following steps: S1: preparing an amino acid color developer; S2: preparing a gradient standard amino acid solution; using the amino acid color developer to treat the gradient standard amino acid solution, using an ultraviolet spectrophotometer to test its absorbance under a 570nm wavelength light source, and drawing an absorbance standard curve; S3: using the amino acid color developer to treat a biological tissue sample, using an ultraviolet spectrophotometer to test its absorbance under a 570nm wavelength light source; S4: using the linear relationship between amino acid concentration and absorbance to calculate the free amino acid content in the biological tissue sample. The method quantitatively analyzes the free amino acid content in biological tissue materials, and has the advantages of simple operation, rapid detection, high detection accuracy, and low requirements for equipment.

Description

Technical Field

[0001] The invention belongs to the technical field of biological tissue material detection, in particular to the field of G01N21 / 33, and specifically to a method for detecting the content of free amino acids in biological tissues. Background Art

[0002] Biological tissue materials are important raw materials in the field of medical technology and are often used in organ transplantation and tissue repair. They have the following advantages: (1) good biocompatibility and low biological toxicity; (2) good toughness and elasticity, and strong durability; (3) three-dimensional structure, porous sponge-like, which is conducive to cell adhesion, crawling growth, and gradually integrating with host tissues. (4) Rich in bioactive factors, they provide a good microenvironment for the growth of host cells. With the above excellent properties, the research and development of biomaterials has become a hot spot in the research of medical materials.

[0003] Biological tissue materials are often fixed with glutaraldehyde or other crosslinking agents in applications to reduce immunogenicity, reduce rejection reactions, increase the stability and strength of biological tissues, and enhance resistance to enzymatic reactions. Of course, the degree of reaction of biological tissues with crosslinking agents will also affect their performance. The more complete the reaction, the better the performance in all aspects. Therefore, in the process of research and development of biological tissue materials, it is usually necessary to study the degree of reaction between biological tissue materials and crosslinking agents. The degree of reaction of biological tissues with crosslinking agents can be judged by the number of free amino acids in the biological tissues after the reaction. More free amino acids indicate a low degree of reaction, and vice versa, fewer free amino acids indicate a high degree of reaction.

[0004] At present, the main free amino acid detection methods are high performance liquid chromatography and amino acid automatic analyzer. These two methods have high equipment prices and complex operations. Many medical biomaterial research and development companies do not have the corresponding resources. In addition, free amino acid detection methods are mainly used in other industries, such as beverages, vegetables, tea, and agricultural chemical industries. For example, the Chinese patent with publication number CN108195785A discloses a method for detecting free amino acids in water-soluble fertilizers. This method deviates from the biological tissue material detection method and has limited detection accuracy. Summary of the invention

[0005] In view of the above problems, the present invention provides a method for detecting the content of free amino acids in biological tissues, comprising the following steps:

[0006] S1: preparing amino acid color developing agent;

[0007] S2: preparing a gradient standard amino acid solution; treating the gradient standard amino acid solution with the amino acid color developer, testing its absorbance with an ultraviolet spectrophotometer under a 570 nm wavelength light source, and drawing an absorbance standard curve;

[0008] S3: using the amino acid color developing agent to treat the biological tissue sample, and testing its absorbance under a 570 nm wavelength light source using an ultraviolet spectrophotometer;

[0009] S4: The free amino acid content in the biological tissue sample is calculated using the linear relationship between amino acid concentration and absorbance.

[0010] In some preferred embodiments, the biological tissue sample is one or more of pig or cow pericardium, skin, tendon, heart valve or pleura.

[0011] In some preferred embodiments, the standard amino acid used in the gradient standard amino acid solution is selected as L-lysine and / or D-lysine.

[0012] In some preferred embodiments, in step 3, the biological tissue sample is pretreated before being treated with the amino acid color developer, and the pretreatment method is to freeze-dry the biological tissue sample and rewarm it in purified water at room temperature for 50 to 100 minutes.

[0013] In some preferred embodiments, in step 3, the method of using the amino acid color developer to treat the biological tissue sample is to shake the reaction at room temperature for 10 to 60 minutes, and then heat at 80 to 150° C. for 10 to 30 minutes.

[0014] In some preferred embodiments, the amino acid color developer is prepared by mixing ninhydrin, an acidic solution with a pH value of 6, and stannous chloride, wherein the weight ratio of the ninhydrin, the acidic solution with a pH value of 6, and stannous chloride is 300:10000:3.

[0015] In some preferred embodiments, the acidic solution is a sodium citrate and citric acid solution.

[0016] In some preferred embodiments, the amino acid concentration gradient of the gradient standard amino acid solution is set to 0, 1A mg / ml, 2A mg / ml, 3A mg / ml, 4A mg / ml, 5A mg / ml, wherein A takes any value between 0.01 and 0.1.

[0017] In some preferred embodiments, the amino acid concentration gradient of the gradient standard amino acid solution is set to 0, 0.08 mg / ml, 0.16 mg / ml, 0.24 mg / ml, 0.32 mg / ml, and 0.40 mg / ml.

[0018] In some preferred embodiments, in step 2, the method for treating the gradient standard amino acid solution with the amino acid color developer is to mix the amino acid color developer and the gradient standard amino acid solution and then heat them at 80 to 150° C. for 10 to 30 minutes.

[0019] Beneficial effects: The present invention provides a method for detecting the free amino acid content in biological tissues, which performs quantitative analysis on the free amino acid content in biological tissue materials, and has the advantages of simple operation, rapid detection, high detection accuracy, and low requirements for equipment.

[0020] The present invention provides a method for detecting the content of free amino acids in biological tissue materials, which belongs to the field of detection technology methods and can quantitatively detect the content of free amino acids in biological tissues. The method comprises the following steps: preparing amino acid color developer ninhydrin solution; preparing standard amino acid solutions of different gradients, testing their absorbance at a specific absorbance with an ultraviolet spectrophotometer after being treated with ninhydrin solution, and drawing an absorbance standard curve; treating the biological tissue with the same method, testing its absorbance, and calculating the amino acid content in the biological tissue sample using the linear relationship between amino acid concentration and absorbance. The detection method has the advantages of simple operation, rapid detection, high detection accuracy, good repeatability and reproducibility, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 It is the amino acid standard curve in Example 1 of the present invention. DETAILED DESCRIPTION

[0022] Main materials and instruments:

[0023] UV-visible spectrophotometer (UNICO-3802S), freeze dryer (Fevik-TL6016, Germany), ninhydrin (Aladdin Biochemical Technology Co., Ltd.), L-lysine (Aladdin Biochemical Technology Co., Ltd.), biological tissue materials (commercially available bovine pericardium, porcine pericardium, porcine skin, bovine Achilles tendon, bovine heart valve, bovine pleura)

[0024] (1) Preparation of ninhydrin solution

[0025] Take 3g of ninhydrin and dissolve it in 100ml of sodium citrate and citric acid solution with a pH value of 6.0, add 0.03g of stannous chloride, stir until completely dissolved, and then store in the dark.

[0026] (2) Preparation of amino acid standard curve

[0027] Dissolve 0.1g of standard L-lysine in 100ml of purified water to obtain a mother solution of L-lysine with a concentration of 1mg / ml. Take 5ml of ninhydrin solution to dilute the 1mg / ml L-lysine mother solution into 0, 0.08mg / ml, 0.16mg / ml, 0.24mg / ml, 0.32mg / ml, and 0.40mg / ml lysine-ninhydrin solutions, respectively. Mix well and place in a 110℃ oven to heat for 20 minutes. After cooling to room temperature, use a UV spectrophotometer to test the absorbance at 570nm. Draw a standard curve with the concentration of the lysine standard solution as the horizontal axis and the absorbance value as the vertical axis. The obtained standard curve is as shown below. Figure 1 As shown, the regression equation of the curve is: y=2.067x+0.012, and the correlation coefficient R2=0.9995.

[0028] (3) Detection of free amino acid content in biological tissue material samples

[0029] Rinse the biological tissue with deionized water, freeze dry it in a freeze dryer, weigh 5 mg of the dried tissue, and soak the tissue in deionized water for 60 minutes. Wipe the tissue sample dry, put it in a test tube, add 5 ml of ninhydrin solution, shake and react for 30 minutes, and move the test tube to a 110°C oven and heat for 20 minutes. After cooling to room temperature, take the supernatant, test the absorbance at 570nm with a UV spectrophotometer, and calculate the free amino acid content in the biological tissue material. The relative standard deviation (RSD) of the free amino acid content in different biological tissue materials obtained by repeating the experiment 5 times is shown in Table 1.

[0030]

[0031] Table 1

[0032] It can be seen from the data in Table 1 that the relative standard deviation of the free amino acid content in each biological tissue material is less than 10%, indicating that the test method of the present invention has high accuracy and good repeatability. The deviation of the free amino acid content in different biological tissue materials is due to the different sources in the biological tissues and the differences in the cross-linking agent fixation process, which bring about the change of the free amino acid content in the biological tissue materials.

[0033] Performance Test:

[0034] Rinse the bovine pericardial tissue with deionized water, freeze dry it in a freeze dryer, weigh 6 portions of dried bovine pericardial tissue (2 mg), and soak the tissue in deionized water for 60 minutes. Wipe dry tissue samples, put them in 6 test tubes, and add 5 ml of ninhydrin solution to each. Add 0.04 ml of 1 mg / ml L-lysine solution to 3 test tubes, and add 0.08 ml of L-lysine solution to the other 3 test tubes. Shake and react for 30 minutes, then move the test tubes to a 110 ° C oven and heat for 20 minutes. Cool to room temperature, then take the supernatant, and test the absorbance at 570 nm with a UV spectrophotometer. The recovery rate is calculated, and the results are shown in Table 2.

[0035] By using the method provided by the present invention, the free amino acid content of 2 mg of bovine pericardial tissue is measured to be 0.153 mg of amino acids.

[0036] Sample amount / mg Added amount / mg Measured value / mg Spiked recovery / mg Recovery rate 1 0.153 0.08mg 0.2354 0.082 1.025 2 0.153 0.08mg 0.2253 0.072 0.900 3 0.153 0.08mg 0.2328 0.080 1.000 4 0.153 0.16mg 0.3071 0.154 0.962 5 0.153 0.16mg 0.3152 0.162 1.013 6 0.153 0.16mg 0.3274 0.174 1.088

[0037] Table 2

[0038] It can be seen from Table 2 that the recovery rate of the test results is between 0.900 and 1.088, which is within the range of 1±0.1, indicating that the test method of the present invention has high accuracy and strong reliability.

Claims

1. A method for detecting the content of free amino acids in biological tissues, characterized in that: The following steps are involved: S1: preparing amino acid colorimetric reagent; S2: preparing a gradient standard amino acid solution; treating the gradient standard amino acid solution with the amino acid color developer, testing its absorbance with an ultraviolet spectrophotometer under a 570 nm wavelength light source, and drawing an absorbance standard curve; S3: using the amino acid color developing agent to treat the biological tissue sample, and testing its absorbance under a 570 nm wavelength light source using an ultraviolet spectrophotometer; S4: Calculate the free amino acid content in the biological tissue sample using the linear relationship between amino acid concentration and absorbance; In step 3, the biological tissue sample is pretreated before being treated with the amino acid color developing agent, wherein the pretreatment method is to freeze-dry the biological tissue sample and rewarm it in purified water at room temperature for 50 to 100 minutes; The standard amino acid used in the gradient standard amino acid solution is selected as L-lysine and / or D-lysine, and the amino acid color developer is prepared by mixing ninhydrin, an acidic solution with a pH value of 6, and stannous chloride, wherein the weight ratio of ninhydrin, the acidic solution with a pH value of 6, and stannous chloride is 300:10000:

3.

2. The method for detecting the free amino acid content in biological tissue according to claim 1, characterized in that: The biological tissue sample is one or more of the pericardium, skin, tendon, heart valve or pleura of a pig or a cow.

3. The method for detecting the free amino acid content in biological tissue according to claim 1, characterized in that: In step 3, the method of using the amino acid color developer to treat the biological tissue sample is to shake the reaction at room temperature for 10 to 60 minutes, and then heat it at 80 to 150° C. for 10 to 30 minutes.

4. The method for detecting the free amino acid content in biological tissue according to claim 1, characterized in that: The acidic solution is selected from sodium citrate and citric acid solution.

5. The method for detecting the free amino acid content in biological tissue according to claim 1, characterized in that: The amino acid concentration gradient of the gradient standard amino acid solution is set to 0, 1A mg / ml, 2A mg / ml, 3A mg / ml, 4A mg / ml, 5A mg / ml, wherein A takes any value between 0.01 and 0.

1.

6. The method for detecting the free amino acid content in biological tissue according to claim 1, characterized in that: The amino acid concentration gradient of the gradient standard amino acid solution is set to 0, 0.08 mg / ml, 0.16 mg / ml, 0.24 mg / ml, 0.32 mg / ml, and 0.40 mg / ml.

7. The method for detecting the free amino acid content in biological tissue according to claim 1, characterized in that: In step 2, the method for treating the gradient standard amino acid solution with the amino acid color developer is to mix the amino acid color developer and the gradient standard amino acid solution and then heat them at 80 to 150° C. for 10 to 30 minutes.

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