Mixed solution and method for extracting hard corneal contact lens protein from mixed solution
By treating rigid gas permeable contact lenses with lysozyme solution and a mixed solution (calcium chloride, ethanol, and water), the problems of poor tear protein removal and chemical reactions in existing technologies are solved, achieving safe, effective protein extraction and accurate detection.
Patent Information
- Application Number
- CN202410756336.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-06-12
- Publication Date
- 2025-12-12
AI Technical Summary
In existing technologies, acetonitrile trifluoroacetic acid is difficult to effectively remove tear proteins on rigid gas permeable contact lenses, and may react chemically with the lens, affecting oxygen permeability and test results, posing a safety hazard.
Rigid contact lenses are treated with a lysozyme solution and a mixed solution (calcium chloride, ethanol, and water) with a pH of 10-11.4. The precipitated proteins are extracted by shaking, avoiding chemical reactions and maintaining lens performance and detection accuracy.
It achieves safe and effective removal of tear proteins from rigid gas permeable contact lenses, maintaining lens performance, providing accurate test results, being environmentally friendly and non-toxic, and having a high recycling rate.
Smart Images
Figure CN121108235A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the field of extracting contact lens protein, in particular to a mixed solution and a method for extracting hard corneal contact lens protein by using the mixed solution. BACKGROUND
[0002] Corneal contact lenses have a large number of invisible fiber oxygen pores, and the human eye secretes a large amount of tear fluid at all times, which contains a large amount of tear protein that can easily penetrate into the fiber oxygen pores, thereby reducing the DK value (oxygen permeability) of the corneal contact lens, causing corneal hypoxia, edema and other symptoms, and in severe cases, causing corneal damage, bacterial infection, corneal inflammation, and even vision impairment. This problem has attracted great attention from the eye and vision industry around the world to the safety of wearing corneal contact lenses. In China, due to the frequent cases of corneal infection caused by corneal contact lenses, contact lenses were listed as the third type of medical devices in 2012, which are subject to high-risk management.
[0003] In order to effectively remove the tear protein on the surface of the corneal contact lens and protect the eye safety of consumers, a variety of methods for removing the tear protein on the surface of the hard corneal contact lens have been developed in the market.
[0004] The existing market for protein extraction reagents for corneal contact lenses is basically 0.1% acetonitrile trifluoroacetic acid. This reagent can effectively recover the protein on the soft corneal contact lens, but the effect on the hard corneal contact lens is not ideal. And acetonitrile trifluoroacetic acid can make the hard corneal contact lens soft during the extraction process, thereby changing the shape, which may be a chemical reaction between acetonitrile trifluoroacetic acid and some substances in the hard corneal contact lens. And the acetonitrile trifluoroacetic acid extraction reagent has an effect on the absorbance of the extraction solution at A280, which usually has a trend of increasing, thereby affecting the judgment of the corneal contact lens protein extraction detection result.
[0005] In view of these problems, a new technical solution is needed to solve them. SUMMARY
[0006] To at least solve one of the technical problems existing in the prior art, the present application provides a mixed solution and a method for extracting hard corneal contact lens protein by using the mixed solution, which is convenient, effective, safe and harmless.
[0007] In one aspect, the present application provides a method for extracting hard corneal contact lens protein by using a mixed solution, which specifically comprises the following steps:
[0008] S1: preparing a lysozyme solution with a pH value ranging from 10 to 11.4;
[0009] S2: a certain amount of lysozyme solution is measured and placed in a container, the hard corneal contact lens is placed in the lysozyme solution in the container, and is cultured in a constant temperature incubator for a period of time, and the hard corneal contact lens is deposited with precipitated protein;
[0010] S3: the lysozyme solution after culture in the container is removed, a mixed solution is added to the container in which the hard corneal contact lens is placed, and is shaken for a certain period of time, and the protein content in the mixed solution is detected; wherein the mixed solution is composed of calcium chloride, ethanol and water.
[0011] As a preferred scheme of the present application, in step S2, the hard corneal contact lens is placed concave upward in the lysozyme solution in the container.
[0012] As a preferred scheme of the present application, in step S1, the concentration of the lysozyme solution is in the range of 1.9-2.2 mg / ml.
[0013] As a preferred scheme of the present application, in step S3, the molar ratio of calcium chloride, ethanol and water in the mixed solution is 1:(1-6):(3-8). Further preferably, the molar ratio of the calcium chloride, ethanol and water includes 1:2:8, 1:3:6, 1:4:5, 1:5:5, 1:6:3 or others.
[0014] As a preferred scheme of the present application, in step S2, the protein content of the lysozyme solution in the container is measured.
[0015] In step S3, the protein content of the lysozyme solution after culture removed from the container is measured, and the recovery rate of the precipitated protein is detected, wherein the calculation formula of the recovery rate of the precipitated protein is:
[0016]
[0017] Further preferably, in step S2, the protein content of the lysozyme solution in the container is measured by using an ultraviolet spectrophotometer; and / or
[0018] The protein content of the lysozyme solution after culture removed from the container is measured by using an ultraviolet spectrophotometer.
[0019] As a preferred scheme of the present application, the pH value of the lysozyme solution is 11.4.
[0020] As a preferred scheme of the present application, in step S2, the corneal contact lens is placed in the lysozyme solution in the container, and is cultured in a constant temperature incubator at 34-37°C for 12-48 hours.
[0021] As a preferred scheme of the present application, in step S3, the mixed solution is added to the container in which the corneal contact lens is placed, and is shaken at 60-70°C for 1.5-3 hours.
[0022] In another aspect, the present application also provides a mixed solution for extracting hard corneal contact lens protein, the mixed solution comprising or containing calcium chloride, ethanol and water, and the molar ratio of the calcium chloride, ethanol and water is 1:(1-6):(3-8). Specifically, the molar ratio of the calcium chloride, ethanol and water comprises 1:2:8, 1:3:6, 1:4:5, 1:5:5, 1:6:3 or others.
[0023] Compared with the prior art, the technical scheme of the present application has at least one or more of the following beneficial effects:
[0024] The mixed solution of the present application only contains CaCl2, ethanol and deionized water, and does not contain chemical substances that react with hard corneal contact lens materials, so it will not damage the performance of the hard corneal contact lens itself. The mixed solution is green and environmentally friendly, and does not contain toxic substances that pollute the environment.
[0025] The mechanism of the mixed solution of the present application for extracting hard corneal contact lens precipitated protein may be that ethanol promotes the swelling of denatured protein, and then dehydrated calcium chloride penetrates into the swollen part. Two calcium complex compounds coordinate with one amino acid molecule (such as lysine), which destroys various combinations, resulting in shrinkage, dispersion and dissolution. The protein extraction method of the present application is convenient and effective, and will not cause physical damage.
[0026] For spectrophotometric measurement of protein content, the mixed solution does not contain chemical substances that interfere with detection. The present application avoids the influence of organic chemical substances (such as acetonitrile trifluoroacetic acid) other than anhydrous ethanol on the absorbance at A280. The mixed solution does not react with the material of the hard corneal contact lens, and can extract the precipitated protein on the hard corneal contact lens and the wall of the culture container without damaging the hard corneal contact lens, and has high repeatability.
[0027] In the process of recovering the precipitated denatured tear protein on the hard corneal contact lens and the wall of the culture container by the ternary solution, shaking extraction at a temperature above 60℃ can achieve better recovery effect.
[0028] Most of the lysozyme is contained in tear protein, and the present application extracts and tests lysozyme to remove tear protein on the corneal contact lens.
[0029] Additional aspects and advantages of the present application will be partially given in the following description, partially will become obvious from the following description, or will be understood by the practice of the present application. BRIEF DESCRIPTION OF DRAWINGS
[0030] To more clearly illustrate the technical solution of the present invention, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.
[0031] Figure 1 This is a graph showing the absorbance of the lysozyme solution at different concentrations according to the present invention.
[0032] Figure 2 This is a schematic diagram of the lysozyme solution recovery rate detection process of the present invention. Detailed Implementation
[0033] Embodiments of the present invention are described in detail below, examples of which are illustrated in the accompanying drawings, wherein the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions throughout. The embodiments described below with reference to the accompanying drawings are exemplary and intended to explain the present invention, and should not be construed as limiting the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.
[0034] This invention provides a method for extracting proteins from rigid corneal contact lenses using a mixed solution, specifically comprising the following steps:
[0035] S1: Prepare a lysozyme solution with a pH range of 10-11.4;
[0036] S2: A certain amount of lysozyme solution is placed in a container, and the concentration of the lysozyme solution is measured using a UV spectrophotometer. The protein content of the lysozyme solution in the container is calculated. A rigid corneal contact lens is placed in the lysozyme solution in the container and incubated in a constant temperature incubator for a period of time. Precipitated protein is deposited on the rigid corneal contact lens. Wherein, protein content = concentration × volume;
[0037] S3: Remove the cultured lysozyme solution from the container and add the mixed solution to the container holding the rigid gas permeable contact lens. Shake for a certain period of time and use a UV spectrophotometer to detect the protein content in the mixed solution. The mixed solution consists of calcium chloride, ethanol and water.
[0038] Preferably, in step S2, the concave side of the rigid gas permeable contact lens is placed upwards when it is immersed in the lysozyme solution.
[0039] Preferably, the molar ratio of calcium chloride, ethanol and water in the mixed solution is 1:(1-6):(3-8).
[0040] Preferably, in step S1, the concentration range of the lysozyme solution is 1.9-2.2 mg / ml.
[0041] In a preferred embodiment, the pH of the lysozyme solution is 11.4.
[0042] In a preferred embodiment, in step S2, the corneal contact lens is placed in the lysozyme solution in the container and incubated in a constant temperature incubator at 34℃-37℃ for 12-48 hours; or
[0043] In step S3, the mixed solution is added to the container holding the contact lens, and the mixture is shaken at 60℃-70℃ for 1.5-3 hours. Experiments have shown that shaking extraction at temperatures above 60℃ yields better results in recovering and extracting lysozyme from the mixed solution.
[0044] In a preferred embodiment, in step S2, the protein content of the lysozyme solution in the container is measured;
[0045] In step S3, the protein content of the lysozyme solution removed from the container after culture is measured to detect the recovery rate of precipitated protein. The formula for calculating the recovery rate of precipitated protein is as follows:
[0046]
[0047] More preferably, in step S2, the protein content of the lysozyme solution in the container is measured using an ultraviolet spectrophotometer; and / or in step S3, the protein content of the cultured lysozyme solution removed from the container is measured using a spectrophotometer.
[0048] Example 1
[0049] (1) Preparation of lysozyme solution
[0050] Weigh 110 mg of lysozyme powder, add 45 mL of pure water to dissolve it completely, adjust the pH to 11.4, and then make up to 50 mL with a 50 mL volumetric flask. Store the prepared lysozyme solution in a refrigerator at 4 °C. In this example, 0.1-1 mol / L sodium hydroxide is used to adjust the pH of the lysozyme solution, but this patent is not limited to this.
[0051] (2) Cultivate protein deposits in rigid corneal contact lenses
[0052] The concentration of the lysozyme solution in step (1) was determined using an A280 ultraviolet spectrophotometer. The protein content of the lysozyme solution in the container was calculated based on the volume of the lysozyme solution in the container. Then, 1 mL of the lysozyme solution prepared in step (1) was added to a 10 mL sealable centrifuge tube. The rigid corneal contact lens was placed in the lysozyme solution in the centrifuge tube with its concave side facing up. The tube was then incubated in a constant temperature incubator at 37℃ for 24 h. The protein content of the lysozyme solution = the concentration of the lysozyme solution × the volume of the lysozyme solution.
[0053] (3) Protein extraction
[0054] The lysozyme solution after culture was removed from the centrifuge tube, and the concentration of the lysozyme solution after culture at point A280 was determined using a UV spectrophotometer.
[0055] A certain volume of mixed solution was added to a centrifuge tube and the mixture was shaken at 60°C for 2 hours to extract the precipitated proteins on the inner wall of the centrifuge tube and on the corneal contact lens. After shaking, the concentration of lysozyme in the mixed solution at point A280 was measured using a UV spectrophotometer, and the protein content of the mixed solution was calculated based on the volume of the mixed solution.
[0056] To verify the feasibility of the protein extraction method of the present invention, the recovery rate of lysozyme was determined.
[0057] 1. Relevant testing procedures are as follows: Figure 2 As shown.
[0058] 2. The formula for calculating the recovery rate is as follows:
[0059]
[0060] The protein content is calculated as: protein content = lysozyme concentration × solution volume.
[0061] The experiment was conducted according to the steps in Example 1, and the experimental data are shown in the table below:
[0062]
[0063] The experimental results showed a recovery rate of over 96%, confirming that this mixed solution can effectively extract proteins from rigid gas permeable contact lenses.
[0064] In this experiment, different concentrations of the prepared lysozyme solution showed a linear relationship when detected at an A280 UV spectrophotometer, as shown in the table below. Figure 1 As shown.
[0065]
[0066] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "yet another embodiment," "another embodiment," "other embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Furthermore, those skilled in the art can combine and integrate the different embodiments or examples described in this specification.
[0067] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications and variations to the above embodiments within the scope of the present invention.
Claims
1. A method for extracting proteins from rigid corneal contact lenses using a mixed solution, characterized in that, Specifically, the steps include the following: S1: Prepare a lysozyme solution with a pH range of 10-11.4; S2: Measure a certain amount of lysozyme solution and place it in a container. Put the rigid corneal contact lens into the lysozyme solution in the container and incubate it in a constant temperature incubator for a period of time. Precipitated protein will be deposited on the rigid corneal contact lens. S3: Remove the cultured lysozyme solution from the container and add the mixed solution to the container holding the rigid gas permeable contact lens. Shake for a certain period of time and use a UV spectrophotometer to detect the protein content in the mixed solution. The mixed solution consists of calcium chloride, ethanol and water.
2. The method for extracting rigid corneal contact lens proteins from a mixed solution according to claim 1, characterized in that, In step S2, the concave side of the rigid gas permeable contact lens is placed upwards when it is immersed in the lysozyme solution; and / or In step S1, the concentration range of the lysozyme solution is 1.9-2.2 mg / ml.
3. The method for extracting rigid corneal contact lens proteins from a mixed solution according to claim 1, characterized in that, In step S3, the molar ratio of calcium chloride, ethanol and water in the mixed solution is 1:(1-6):(3-8).
4. The method for extracting rigid corneal contact lens proteins from a mixed solution according to claim 1, characterized in that, In step S2, the protein content of the lysozyme solution in the container is measured using an ultraviolet spectrophotometer; In step S3, the protein content of the lysozyme solution removed from the container after culture is measured, and the recovery rate of precipitated protein is detected. The formula for calculating the recovery rate of precipitated protein is as follows:
5. The method for extracting rigid corneal contact lens proteins from a mixed solution according to claim 1, characterized in that, The pH value of the lysozyme solution is 11.
4.
6. The method for extracting rigid corneal contact lens proteins from a mixed solution according to claim 1, characterized in that, In step S2, the corneal contact lens is placed in the lysozyme solution in the container and incubated in a constant temperature incubator at 34℃-37℃ for 12-48 hours.
7. The method for extracting rigid corneal contact lens proteins from a mixed solution according to claim 1, characterized in that, In step S3, the mixed solution is added to the container holding the corneal contact lens and shaken at 60℃-70℃ for 1.5-3 hours.
8. A method for extracting proteins from rigid corneal contact lenses using a mixed solution, characterized in that, Specifically, the steps include the following: a. Prepare a lysozyme solution with a pH range of 10-11.4; b. Measure a certain amount of lysozyme solution and place it in a container. Put the rigid corneal contact lens into the lysozyme solution in the container and incubate it in a constant temperature incubator for a period of time. Precipitated protein will be deposited on the rigid corneal contact lens. c. Remove the cultured lysozyme solution from the container and add the mixed solution to the container holding the rigid gas permeable contact lens. Shake for a certain period of time and detect the protein content in the mixed solution. The mixed solution consists of calcium chloride, ethanol and water.
9. The method for extracting rigid corneal contact lens proteins from a mixed solution according to claim 8, characterized in that, In step S2, the protein content of the lysozyme solution in the container is measured; In step S3, the protein content of the lysozyme solution removed from the container after culture is measured, and the recovery rate of precipitated protein is detected. The formula for calculating the recovery rate of precipitated protein is as follows:
10. A mixed solution for extracting proteins from rigid corneal contact lenses, characterized in that, The mixed solution comprises calcium chloride, ethanol and water, wherein the molar ratio of calcium chloride, ethanol and water is 1:(1-6):(3-8).