WO2022143442A1 - 一种有效测量软性亲水角膜接触镜泪蛋白含量的方法 - Google Patents

一种有效测量软性亲水角膜接触镜泪蛋白含量的方法 Download PDF

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WO2022143442A1
WO2022143442A1 PCT/CN2021/141070 CN2021141070W WO2022143442A1 WO 2022143442 A1 WO2022143442 A1 WO 2022143442A1 CN 2021141070 W CN2021141070 W CN 2021141070W WO 2022143442 A1 WO2022143442 A1 WO 2022143442A1
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protein
lens
soft hydrophilic
contact lens
adsorbed
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PCT/CN2021/141070
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English (en)
French (fr)
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孙碧霞
孙瑞春
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苏州三个臭皮匠生物科技有限公司
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Priority to US18/269,576 priority Critical patent/US20240060877A1/en
Publication of WO2022143442A1 publication Critical patent/WO2022143442A1/zh

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
    • G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
    • G01N21/33—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using ultraviolet light
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/145—Extraction; Separation; Purification by extraction or solubilisation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/34—Purifying; Cleaning

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  • the invention relates to the field of biological protein detection, in particular to a method for effectively measuring the tear protein content of a soft hydrophilic corneal contact lens.
  • the material structure of contact lenses has a large number of invisible fibers Itching hole, human eyes secrete a large amount of tears all the time, tears contain a lot of tear protein, tear protein can easily penetrate into the itching hole of the fiber, resulting in a decrease in the DK value of the lens (oxygen permeability), resulting in corneal hypoxia, edema and other symptoms , serious corneal damage, bacterial infection, corneal inflammation and even vision impairment and other problems.
  • the purpose of this invention is to solve the problem that exists in the prior art, a kind of method for effectively measuring the tear protein content of soft hydrophilic contact lenses is provided, and the technical scheme adopted is as follows:
  • a method for effectively measuring the tear protein content of a soft hydrophilic contact lens detects the protein concentration in a protein extraction solution of the soft hydrophilic contact lens by a micro-ultraviolet spectrophotometer, and specifically includes:
  • a protein extraction solution is prepared, and the protein extraction solution is composed of acetonitrile, pure water and trifluoroacetic acid; the soft hydrophilic contact lens with protein adsorbed on the surface is placed in the protein extraction solution, and the protein extraction solution will
  • the protein is separated from the soft hydrophilic contact lens, the soft hydrophilic contact lens is taken out from the protein extraction solution, and the micro-ultraviolet spectrophotometer is used to detect the amount of the protein in the protein extraction solution.
  • the protein concentration is obtained to obtain the lens-adsorbed protein concentration value, and the lens-adsorbed protein content value is obtained by calculating the lens-adsorbed protein concentration value and the volume of the protein extract.
  • the protein extraction solution consists of a mixture of 50 parts acetonitrile, 50 parts pure water and 0.2 parts 100% trifluoroacetic acid.
  • the method for effectively measuring the tear protein content of a soft hydrophilic contact lens according to the present invention further comprises: preparing the soft hydrophilic contact lens with protein adsorbed on the surface, the specific method of which is include:
  • the residual protein concentration in the artificial tears is detected by the micro-ultraviolet spectrophotometer to obtain the residual protein concentration value
  • the concentration of the protein adsorbed on the soft hydrophilic contact lens is calculated by the original protein concentration value of the artificial tears and the residual protein concentration value, and the theoretical concentration value of the lens adsorbed protein is obtained.
  • the prepared artificial tears have a concentration of 2.2 mg/ml, and the original protein concentration of the artificial tears is 2.2 mg/ml.
  • the constant temperature cultivation of the soft hydrophilic contact lens in the artificial tears specifically includes:
  • the residual protein concentration in the artificial tears is detected by the micro-ultraviolet spectrophotometer to obtain the residual protein concentration value, and the detected residual protein concentration value is 1.328 mg/ml.
  • calculating the protein concentration adsorbed on the soft hydrophilic contact lens through the original protein concentration value of the artificial tears and the remaining protein concentration value to obtain the theoretical concentration value of the lens adsorbed protein specifically including:
  • the theoretical concentration value of the lens adsorption protein is obtained by subtracting the obtained residual protein concentration value from the original protein concentration value of the configured artificial tears, and the theoretical concentration value of the lens adsorption protein is calculated to be 0.872 mg/ml.
  • the soft hydrophilic contact lens with protein adsorbed on the surface is placed in the protein extraction solution, and the protein extraction solution separates the protein from the soft hydrophilic contact lens, Specifically, it includes: taking 1 ml of the protein extraction solution and placing it in a centrifuge tube, placing the soft hydrophilic contact lens in the centrifuge tube for vibration cleaning, and the protein adsorbed on the soft hydrophilic contact lens is the same as the described soft hydrophilic contact lens separation.
  • the protein concentration in the protein extraction solution is detected by the micro-ultraviolet spectrophotometer to obtain a lens-adsorbed protein concentration value, and the detected lens-adsorbed protein concentration value is 0.805 mg/ml.
  • the lens-adsorbed protein content value is calculated by the obtained lens-adsorbed protein concentration value.
  • the obtained lens-adsorbed protein concentration value and the lens-adsorbed protein theoretical concentration value are numerically calculated to obtain the elution rate of the protein from the protein extraction solution, which specifically includes:
  • the protein elution rate of the protein extraction solution was calculated by the ratio of the concentration value of the lens adsorbed protein to the theoretical concentration value of the lens adsorbed protein, and the elution rate of the protein extraction solution to the protein was calculated to be 92.3%.
  • the present invention has the following beneficial effects:
  • the invention provides a method for effectively measuring the tear protein content of a soft hydrophilic contact lens, which can solve the problem that the specific content of the tear protein absorbed by the soft hydrophilic contact lens is difficult to measure compared with the prior art.
  • the protein is separated from the soft hydrophilic corneal contact lens by a protein extraction solution.
  • the protein extraction solution can be a mixed solution of acetonitrile-pure water-trifluoroacetic acid.
  • the contact lens material does not react and does not affect the measurement value of protein. Therefore, the soft hydrophilic corneal contact lens cultured with artificial tears can be used as the detection sample, and it can be placed in the protein extraction solution to extract and isolate the protein by vibration.
  • the protein concentration in the protein extraction solution is detected by a micro-ultraviolet spectrophotometer, and the protein content value of the lens adsorbed can be obtained by simple calculation of the protein concentration. Therefore, the soft hydrophilic corneal contact can be obtained by the detection method of the present invention.
  • the elution rate of the protein by the protein extraction solution can also be calculated through experimental detection and calculation. The experimental results show that the elution rate of the tear protein adsorbed on the soft hydrophilic contact lens using this method is greater than 90%. .
  • the gist of the present invention is further described below in conjunction with the embodiments.
  • the present invention proposes a method for effectively measuring the tear protein content of soft hydrophilic contact lenses.
  • the lacrimal protein adsorbed in acetonitrile-trifluoroacetic acid-water mixed solution can be 100% extracted.
  • the method detects the protein concentration in the protein extraction solution of the soft hydrophilic corneal contact lens by a micro-ultraviolet spectrophotometer, and specifically includes:
  • the artificial tears are prepared according to the human eye environment, and the soft hydrophilic contact lens is incubated in the artificial tears at a constant temperature.
  • the spectrophotometer detects the remaining protein concentration in the artificial tears to obtain the remaining protein concentration value.
  • the soft hydrophilic corneal contact lens will adsorb proteins when incubated at a constant temperature in the artificial tears. Therefore, culturing for a period of time is also to simulate wearing by human eyes.
  • Application scenarios of soft hydrophilic contact lenses The protein concentration adsorbed on the soft hydrophilic contact lens is calculated by the original protein concentration value of artificial tears and the remaining protein concentration value, and the theoretical concentration value of the adsorbed protein on the lens is obtained. This is to quantify the protein content on the lens to assist Follow-up test calculation process.
  • the known substance that can extract protein at this stage is pbs solution (phosphate buffered solution, a standard salt solution), but it also has defects, that is, it cannot fully extract soft hydrophilic contact lenses. protein. Therefore, for the time being, there is no recognized method to test the protein content actually adsorbed on the soft hydrophilic contact lenses worn by human eyes.
  • the soft hydrophilic contact lens cultured from artificial tears is used by default on the premise that there is no method to specifically measure the protein content actually adsorbed on the soft hydrophilic contact lens.
  • the actual protein content adsorbed on the lens is the theoretical content of protein adsorbed on the lens obtained by calculation.
  • the protein extraction solution includes acetonitrile, pure water and trifluoroacetic acid, the protein extraction solution does not react with the soft hydrophilic contact lens material, and the protein can be removed from the soft hydrophilic contact lens. separation.
  • a soft hydrophilic contact lens cultured with artificial tears is placed in the protein extraction solution, which separates proteins from the soft hydrophilic contact lens, and is removed from the protein extraction solution
  • protein is dissolved in the protein extraction solution, and the protein concentration in the protein extraction solution is detected by the micro-ultraviolet spectrophotometer to obtain the concentration value of the adsorbed protein on the lens.
  • the protein extraction solution may be a solution formed by mixing 50 parts of acetonitrile, 50 parts of pure water and 0.2 parts of 100% trifluoroacetic acid.
  • the concentration of the artificial tears configured according to the human eye environment is 2.2 mg/ml, then the original protein concentration value of the artificial tears is 2.2 mg/ml.
  • the constant temperature incubation of the soft hydrophilic contact lens in the artificial tears may specifically include the following steps:
  • the contact lens may be a Class IV soft hydrophilic contact lens classified by the United States Food and Drug Administration (FDA).
  • FDA United States Food and Drug Administration
  • the soft hydrophilic contact lens is cultured in the artificial tears, the soft hydrophilic contact lens is taken out from the artificial tears, and the residual protein concentration in the artificial tears is detected by the micro-ultraviolet spectrophotometer , the remaining protein concentration value was obtained, and the remaining protein concentration value was detected to be 1.328 mg/ml.
  • calculating the protein concentration adsorbed on the soft hydrophilic contact lens through the original protein concentration value of the artificial tears and the remaining protein concentration value to obtain the theoretical concentration value of the adsorbed protein on the lens which specifically includes: using the configured artificial tears
  • placing the soft hydrophilic contact lens cultured with artificial tears in the protein extraction solution, and the protein extraction solution separates the protein from the soft hydrophilic contact lens specifically including: taking 1 ml of The protein extraction solution is placed in a centrifuge tube, the soft hydrophilic contact lens is placed in the centrifuge tube, the soft hydrophilic contact lens is vibrated and washed in the centrifuge tube, and the soft The protein adsorbed on the hydrophilic contact lens is separated from the soft hydrophilic contact lens; the protein concentration in the protein extraction solution is detected by the micro-ultraviolet spectrophotometer, and the concentration value of the adsorbed protein on the lens is obtained.
  • the lens-adsorbed protein concentration value is 0.805 mg/ml.
  • the lens-adsorbed protein concentration value can be used to calculate the lens-adsorbed protein content value, that is, the lens-adsorbed protein concentration value and protein extraction.
  • the product of the solution volume is the amount of protein adsorbed to the lens.
  • the obtained lens-adsorbed protein concentration value and the lens-adsorbed protein theoretical concentration value are numerically calculated to obtain the elution rate of the protein from the protein extraction solution, which specifically includes:
  • the elution rate of the protein from the protein extraction solution was calculated, and the protein extraction was calculated.
  • the elution rate of the solution to the protein was 92.3%
  • the soft hydrophilic contact lens can absorb a certain content of tear protein on the lens by wearing on human eyes, soaking in artificial tears, etc.
  • 1ml or 4ml of protein extraction solution (50 parts acetonitrile-50 parts pure water-0.2 part 100% trifluoroacetic acid solution) can be taken to fully dissolve the protein on the soft hydrophilic contact lens that has adsorbed tear protein , and shake at room temperature for 24 hours to further dissolve, and the protein content extracted from the protein extraction solution can also be tested by spectrophotometry.
  • the invention provides a method for effectively measuring the tear protein content of a soft hydrophilic contact lens. Compared with the prior art, it can solve the problem that the specific content of the tear protein absorbed by the soft hydrophilic contact lens is difficult to measure.
  • the extraction solution separates the protein from the soft hydrophilic contact lens, and the protein extraction solution can be a mixed solution of acetonitrile-pure water-trifluoroacetic acid, and the protein extraction solution is incompatible with the soft hydrophilic contact lens material.
  • the reaction does not affect the measurement value of the protein. Therefore, the soft hydrophilic contact lens cultured with artificial tears can be used as the detection sample, and it can be placed in the protein extraction solution to extract and isolate the protein by vibration.
  • the spectrophotometer detects the protein concentration in the protein extraction solution, and the lens adsorption protein content value can be obtained by simply calculating the protein concentration. Therefore, through the detection method of the present invention, the soft hydrophilic contact lens adsorption tear protein can be obtained specific content. Further, the elution rate of the protein by the protein extraction solution can also be calculated through experimental detection and calculation. The experimental results show that the elution rate of the tear protein adsorbed on the soft hydrophilic contact lens using this method is greater than 90%. .
  • references to the terms “one embodiment,” “some embodiments,” “example,” “specific example,” or “some examples”, etc. means a specific feature described in connection with the embodiment or example, A structure, material, or feature is included in at least one embodiment or example of the present invention.
  • schematic representations of the above terms are not necessarily directed to the same embodiment or example.
  • the particular features, structures, materials or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.
  • those skilled in the art may combine and combine the different embodiments or examples described in this specification.

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Abstract

提供了一种有效测量软性亲水角膜接触镜泪蛋白含量的方法，方法通过微量紫外分光光度计检测软性亲水角膜接触镜蛋白提取溶液中的蛋白浓度，具体包括：通过乙腈、纯水和三氟乙酸配置蛋白提取溶液；将表面吸附有蛋白的软性亲水角膜接触镜放置在蛋白提取溶液中，蛋白提取溶液将蛋白自软性亲水角膜接触镜上分离，通过微量紫外分光光度计检测蛋白提取溶液中的蛋白浓度，获得镜片吸附蛋白浓度值。还可以通过镜片吸附蛋白理论浓度值与软性亲水角膜接触镜表面吸附的蛋白浓度计算蛋白提取溶液对蛋白的洗脱率，通过测量软性亲水角膜接触镜泪蛋白含量的方法可以定量定性的检测出镜片表面的泪蛋白含量，为用户提供实验检测数据，保障用户消费使用安全。

Description

一种有效测量软性亲水角膜接触镜泪蛋白含量的方法 技术领域
本发明涉及生物蛋白检测领域，尤其涉及一种有效测量软性亲水角膜接触镜泪蛋白含量的方法。
背景技术
角膜接触镜如何除蛋白的问题困扰了行业半个多世纪，引发了各国的眼视光行业对接触镜佩戴安全的高度重视。我国也因角膜接触镜上的角膜感染案例频发，从而将接触镜在2012年便列入第三类医疗器械类，作为高风险管理，其原因在于：接触镜材质结构有大量肉眼不见的纤维透痒孔，人眼时刻分泌大量的泪液，泪液里含有大量泪蛋白，泪蛋白极易渗透到纤维透痒孔中，造成镜片DK值降低(透氧率)，造成角膜缺氧、水肿等症状，严重的会造成角膜受损，细菌感染，角膜炎症甚至是视力受损等问题。
为了有效清除接触镜表面的泪蛋白，保障消费者的眼部安全，市场也推出了多种用于去除软性亲水角膜接触镜表面泪蛋白的方法，但由于肉眼不可见泪蛋白是否被完全有效的洗脱降解，逐渐的蛋白检测方法就被重视发展起来，以使得通过科学检测有效验证是否蛋白被完全降解，但是现阶段市场上的软性亲水角膜接触镜有效除蛋白的检测方法因软性亲水角膜接触镜内部纵横交错的透氧孔特殊结构具有局限性，无法找到一种可以直接有效且精确测量软性亲水角膜接触镜所吸附泪蛋白的具体量值，因此其检测结果不全面，仅具有一定的参考意义，其并不能成为定量定性检测蛋白降解程度的专业检测依据。
因此，亟需提出一种新的技术方案来解决上述问题。
发明内容
本发明的目的是解决现有技术中存在的问题，提供一种有效测量软性 亲水角膜接触镜泪蛋白含量的方法，采用的技术方案如下：
一种有效测量软性亲水角膜接触镜泪蛋白含量的方法，所述方法通过微量紫外分光光度计检测软性亲水角膜接触镜蛋白提取溶液中的蛋白浓度，具体包括：
配置蛋白提取溶液，所述蛋白提取溶液由乙腈、纯水和三氟乙酸混合组成；将表面吸附有蛋白的软性亲水角膜接触镜放置在所述蛋白提取溶液中，所述蛋白提取溶液将所述蛋白自所述软性亲水角膜接触镜上分离，自所述蛋白提取溶液中取出所述软性亲水角膜接触镜，通过所述微量紫外分光光度计检测所述蛋白提取溶液中的蛋白浓度，得到镜片吸附蛋白浓度值，通过所述镜片吸附蛋白浓度值和蛋白提取液体积计算得到镜片吸附蛋白含量值。
在一个进一步的实施例中，所述蛋白提取溶液由50份乙腈、50份纯水和0.2份100％的三氟乙酸混合组成。
在一个进一步的实施例中，本发明所述的有效测量软性亲水角膜接触镜泪蛋白含量的方法，其还包括：制备所述表面吸附有蛋白的软性亲水角膜接触镜，其具体包括：
配置人工泪液，将软性亲水角膜接触镜在所述人工泪液中恒温培养，培养完成后，将所述软性亲水角膜接触镜自人工泪液中取出；
将所述软性亲水角膜接触镜自人工泪液中取出后，通过所述微量紫外分光光度计检测人工泪液中的剩余蛋白浓度，获得剩余蛋白浓度值，
通过人工泪液的原始蛋白浓度值与所述剩余蛋白浓度值计算所述软性亲水角膜接触镜上吸附的蛋白浓度，获得镜片吸附蛋白理论浓度值。
进一步的，配置的所述人工泪液的浓度为2.2mg/ml，所述人工泪液的原始蛋白浓度值为2.2mg/ml。
进一步的，将软性亲水角膜接触镜在所述人工泪液中恒温培养具体包括：
取1ml所述人工泪液放置在离心管中，将未被使用的软性亲水角膜接触镜放置在所述人工泪液中，恒温37℃培养1天，取出，获得表面吸附有蛋白的软性亲水角膜接触镜；
通过所述微量紫外分光光度计检测人工泪液中的剩余蛋白浓度，获得剩余蛋白浓度值，检测得到所述剩余蛋白浓度值为1.328mg/ml。
进一步的，通过人工泪液的原始蛋白浓度值与所述剩余蛋白浓度值计算所述软性亲水角膜接触镜上吸附的蛋白浓度，获得镜片吸附蛋白理论浓度值，具体包括：
利用配置的人工泪液的原始蛋白浓度值减去获得的所述剩余蛋白浓度值得到所述镜片吸附蛋白理论浓度值，计算得到所述镜片吸附蛋白理论浓度值为0.872mg/ml。
在一个进一步的实施例中，将表面吸附有蛋白的软性亲水角膜接触镜放置在所述蛋白提取溶液中，所述蛋白提取溶液将蛋白自所述软性亲水角膜接触镜上分离，具体包括：取1ml所述蛋白提取溶液放置在离心管中，将所述软性亲水角膜接触镜放置在该离心管中振动清洗，所述软性亲水角膜接触镜上吸附的蛋白与所述软性亲水角膜接触镜分离。
进一步的，通过所述微量紫外分光光度计检测所述蛋白提取溶液中的蛋白浓度，获得镜片吸附蛋白浓度值，检测得到所述镜片吸附蛋白浓度值为0.805mg/ml。
进一步的，通过获得的所述镜片吸附蛋白浓度值计算所述镜片吸附蛋白含量值。
进一步的，将获得的镜片吸附蛋白浓度值与所述镜片吸附蛋白理论浓度值进行数值计算，获得蛋白提取溶液对蛋白的洗脱率，具体包括：
通过所述镜片吸附蛋白浓度值与所述镜片吸附蛋白理论浓度值的比值计算所述蛋白提取溶液对蛋白的洗脱率，计算得到所述蛋白提取溶液对蛋白的洗脱率为92.3％。
与现有技术相比，本发明具有如下有益效果：
本发明提供一种有效测量软性亲水角膜接触镜泪蛋白含量的方法，与现有技术相比其可以解决软性亲水角膜接触镜吸附泪蛋白具体含量难以测量的问题。本发明通过蛋白提取溶液将蛋白自所述软性亲水角膜接触镜上分离，所述蛋白提取溶液可以是乙腈-纯水-三氟乙酸混合溶液，所述蛋白提取溶液与软性亲水角膜接触镜材料不发生反应，也不影响蛋白质的测量 值，因而，可以将人工泪液培养的软性亲水角膜接触镜作为检测样本，将其放置在所述蛋白提取溶液中进行振动提取分离蛋白，再通过微量紫外分光光度计检测蛋白提取溶液中的蛋白浓度，通过对该蛋白浓度进行简单计算就可以得到镜片吸附蛋白含量值，因此，通过本发明的检测方法就可以得到软性亲水角膜接触镜吸附泪蛋白的具体含量。进一步的，通过实验检测计算也可以计算得到所述蛋白提取溶液对蛋白的洗脱率，实验结果表明，使用这种方法软性亲水角膜接触镜上所吸附泪蛋白的洗脱率大于90％。
具体实施方式
下面将结合本发明实施例，对本发明的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
在本发明的描述中，需要理解的是，除非另有明确的规定和限定，术语应做广义理解，对于本领域的普通技术人员而言，可以根据具体情况理解上述术语在本发明中的具体含义。
下面结合实施例进一步说明本发明要旨。
实施例：
现有技术中无法找到一种可以直接有效且精确测量角膜接触镜所吸附泪蛋白的具体量值，我们在证明角膜接触镜清洗效果的实际应用中，亟需一种可直接将角膜接触镜所吸附泪蛋白具体量化的测试方法。因此，为了解决这一问题，本发明提出了一种有效测量软性亲水角膜接触镜泪蛋白含量的方法，所述方法依附的主要原理是软性亲水角膜接触镜表面及内部透氧孔中所吸附的泪蛋白，可以根据乙腈-三氟乙酸-水的混合溶液进行100％提取。所述方法通过微量紫外分光光度计检测软性亲水角膜接触镜蛋白提取溶液中的蛋白浓度，具体包括：
根据人眼环境配置人工泪液，将软性亲水角膜接触镜在所述人工泪液中恒温培养，培养完成后，将所述软性亲水角膜接触镜自人工泪液中取出， 通过所述微量紫外分光光度计检测人工泪液中的剩余蛋白浓度，获得剩余蛋白浓度值，所述软性亲水角膜接触镜在所述人工泪液中恒温培养会吸附蛋白，因此，培养一段时间也是为了模拟人眼佩戴软性亲水角膜接触镜的应用场景。通过人工泪液的原始蛋白浓度值与所述剩余蛋白浓度值计算所述软性亲水角膜接触镜上吸附的蛋白浓度，获得镜片吸附蛋白理论浓度值，这是为了量化镜片上蛋白含量，以辅助后续试验计算过程。其中需要说明的是，现阶段公知的可以提取蛋白质的物质是pbs溶液(磷酸盐缓冲溶液，一种标准盐溶液)，但是其也存在缺陷，即其不能充分提取软性亲水角膜接触镜上的蛋白。因而，暂时还没有一种公认的方法可以检验人眼佩戴的软性亲水角膜接触镜上实际吸附的蛋白含量，但为了证明本发明所述方法的有效性，本发明通过人工泪液培养的软性亲水角膜接触镜进行实验，且在没有方法可以具体量测软性亲水角膜接触镜上实际吸附的蛋白含量的前提下，本实验默认自人工泪液中培养出来的软性亲水角膜接触镜上吸附的实际蛋白含量即为通过计算得到的镜片吸附蛋白理论含量。
配置蛋白提取溶液，所述蛋白提取溶液包括乙腈、纯水和三氟乙酸，所述蛋白提取溶液不与软性亲水角膜接触镜材料反应，且可以将蛋白自软性亲水角膜接触镜上分离。
将通过人工泪液培养的软性亲水角膜接触镜放置在所述蛋白提取溶液中，所述蛋白提取溶液将蛋白自所述软性亲水角膜接触镜上分离，自所述蛋白提取溶液中取出所述软性亲水角膜接触镜，所述蛋白提取溶液中溶解有蛋白，通过所述微量紫外分光光度计检测所述蛋白提取溶液中的蛋白浓度，获得镜片吸附蛋白浓度值。
在一种实施例中，所述蛋白提取溶液可以是50份乙腈、50份纯水和0.2份100％的三氟乙酸混合形成的溶液。
在一种实施例中，根据人眼环境配置的所述人工泪液的浓度为2.2mg/ml，则所述人工泪液的原始蛋白浓度值即为2.2mg/ml。
在一种实施例中，将软性亲水角膜接触镜在所述人工泪液中恒温培养具体可以包括如下步骤：
先取1ml所述人工泪液放置在离心管中，再将未被使用的软性亲水角 膜接触镜放置在所述人工泪液中，恒温37℃培养1天，所述未被使用的软性亲水角膜接触镜可以是由美国食品和药物管理局(FDA)归类的IV类软性亲水性接触镜。
当软性亲水角膜接触镜在所述人工泪液中培养完成后，将所述软性亲水角膜接触镜自人工泪液中取出，通过所述微量紫外分光光度计检测人工泪液中的剩余蛋白浓度，获得剩余蛋白浓度值，检测得到所述剩余蛋白浓度值为1.328mg/ml。
进一步的，通过人工泪液的原始蛋白浓度值与所述剩余蛋白浓度值计算所述软性亲水角膜接触镜上吸附的蛋白浓度，获得镜片吸附蛋白理论浓度值，具体包括：利用配置的人工泪液的原始蛋白浓度值减去获得的所述剩余蛋白浓度值得到所述镜片吸附蛋白理论浓度值，计算得到所述镜片吸附蛋白理论浓度值为0.872mg/ml，即2.2mg/ml-1.328mg/ml＝0.872mg/ml。
进一步的，将通过人工泪液培养的软性亲水角膜接触镜放置在所述蛋白提取溶液中，所述蛋白提取溶液将蛋白自所述软性亲水角膜接触镜上分离，具体包括：取1ml所述蛋白提取溶液放置在离心管中，将所述软性亲水角膜接触镜放置在该离心管中，所述软性亲水角膜接触镜在所述离心管中振动清洗，所述软性亲水角膜接触镜上吸附的蛋白与所述软性亲水角膜接触镜分离；通过所述微量紫外分光光度计检测所述蛋白提取溶液中的蛋白浓度，获得镜片吸附蛋白浓度值，检测得到所述镜片吸附蛋白浓度值为0.805mg/ml，根据质量等于浓度乘于体积的概念可以通过获得的所述镜片吸附蛋白浓度值计算所述镜片吸附蛋白含量值，即镜片吸附蛋白浓度值和蛋白提取溶液体积的乘积即为镜片吸附蛋白含量。
进一步的，将获得的镜片吸附蛋白浓度值与所述镜片吸附蛋白理论浓度值进行数值计算，获得蛋白提取溶液对蛋白的洗脱率，具体包括：
通过所述镜片吸附蛋白浓度值(0.805mg/ml)与所述镜片吸附蛋白理论浓度值(0.872mg/ml)的比值计算所述蛋白提取溶液对蛋白的洗脱率，计算得到所述蛋白提取溶液对蛋白的洗脱率为92.3％，
即：0.805mg/ml÷0.872mg/ml＝92.3％。
在一种实施例中，所述软性亲水角膜接触镜可通过人眼佩戴、使用 人工泪液浸泡等方式使镜片上吸附上一定含量的泪蛋白。
在一种实施例中，可取1ml或4ml蛋白提取溶液(50份乙腈-50份纯水-0.2份100％三氟乙酸溶液)充分溶解已经吸附泪蛋白的软性亲水角膜接触镜上的蛋白，并在常温下振动24小时以进一步溶解，也可通过分光光度法测试蛋白提取溶液中提取出来的蛋白含量。
本发明提供一种有效测量软性亲水角膜接触镜泪蛋白含量的方法，与现有技术相比其可以解决软性亲水角膜接触镜吸附泪蛋白具体含量难以测量的问题，本发明通过蛋白提取溶液将蛋白自所述软性亲水角膜接触镜上分离，所述蛋白提取溶液可以是乙腈-纯水-三氟乙酸混合溶液，所述蛋白提取溶液与软性亲水角膜接触镜材料不发生反应，也不影响蛋白质的测量值，因而，可以将人工泪液培养的软性亲水角膜接触镜作为检测样本，将其放置在所述蛋白提取溶液中进行振动提取分离蛋白，再通过微量紫外分光光度计检测蛋白提取溶液中的蛋白浓度，通过对该蛋白浓度进行简单计算就可以得到镜片吸附蛋白含量值，因此，通过本发明的检测方法就可以得到软性亲水角膜接触镜吸附泪蛋白的具体含量。进一步的，通过实验检测计算也可以计算得到所述蛋白提取溶液对蛋白的洗脱率，实验结果表明，使用这种方法软性亲水角膜接触镜上所吸附泪蛋白的洗脱率大于90％。
在本说明书的描述中，参考术语“一个实施例”、“一些实施例”、“示例”、“具体示例”或“一些示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本发明的至少一个实施例或示例中。在本说明书中，对上述术语的示意性表述不必针对的是相同的实施例或示例。而且，描述的具体特征、结构、材料或者特点可以在任何的一个或多个实施例或示例中以合适的方式结合。此外，本领域人员可以将本说明书中描述的不同实施例或示例进行接合和组合。
尽管上面已经示出和描述了本发明的实施例，可以理解的是，上述实施例是示例性的，不能理解为对本发明的限制，本领域的普通技术人员在本发明的范围内可以对上述实施例进行变化、修改和变型。

Claims (7)

  1. 一种有效测量软性亲水角膜接触镜泪蛋白含量的方法，其特征在于，
    所述方法通过微量紫外分光光度计检测软性亲水角膜接触镜蛋白提取溶液中的蛋白浓度，具体包括：
    配置蛋白提取溶液，所述蛋白提取溶液由乙腈、纯水和三氟乙酸混合组成；
    将表面吸附有蛋白的软性亲水角膜接触镜放置在所述蛋白提取溶液中，所述蛋白提取溶液将所述蛋白自所述软性亲水角膜接触镜上分离，自所述蛋白提取溶液中取出所述软性亲水角膜接触镜，通过所述微量紫外分光光度计检测所述蛋白提取溶液中的蛋白浓度，得到镜片吸附蛋白浓度值，通过所述镜片吸附蛋白浓度值和蛋白提取液体积计算得到镜片吸附蛋白含量值。
  2. 根据权利要求1所述的有效测量软性亲水角膜接触镜泪蛋白含量的方法，其特征在于，所述蛋白提取溶液由50份乙腈、50份纯水和0.2份100％的三氟乙酸混合组成。
  3. 根据权利要求1所述的有效测量软性亲水角膜接触镜泪蛋白含量的方法，其特征在于，其还包括：
    制备所述表面吸附有蛋白的软性亲水角膜接触镜，具体包括：
    配置人工泪液，将软性亲水角膜接触镜在所述人工泪液中恒温培养，培养完成后，将所述软性亲水角膜接触镜自人工泪液中取出；
    将所述软性亲水角膜接触镜自人工泪液中取出后，通过所述微量紫外分光光度计检测人工泪液中的剩余蛋白浓度，获得剩余蛋白浓度值，
    通过人工泪液的原始蛋白浓度值与所述剩余蛋白浓度值计算所述软性亲水角膜接触镜上吸附的蛋白浓度，获得镜片吸附蛋白理论浓度值。
  4. 根据权利要求3所述的有效测量软性亲水角膜接触镜泪蛋白含量的方法，其特征在于，
    配置的所述人工泪液的浓度为2.2mg/ml，所述人工泪液的原始蛋白浓 度值为2.2mg/ml，
    将软性亲水角膜接触镜在所述人工泪液中恒温培养具体包括：
    取1ml所述人工泪液放置在离心管中，将未被使用的软性亲水角膜接触镜放置在所述人工泪液中，恒温37℃培养1天，取出，获得表面吸附有蛋白的软性亲水角膜接触镜；
    通过所述微量紫外分光光度计检测人工泪液中的剩余蛋白浓度，获得剩余蛋白浓度值，检测得到所述剩余蛋白浓度值为1.328mg/ml。
  5. 根据权利要求4所述的有效测量软性亲水角膜接触镜泪蛋白含量的方法，其特征在于，
    通过人工泪液的原始蛋白浓度值与所述剩余蛋白浓度值计算所述软性亲水角膜接触镜上吸附的蛋白浓度，获得镜片吸附蛋白理论浓度值，具体包括：
    利用配置的人工泪液的原始蛋白浓度值减去获得的所述剩余蛋白浓度值得到所述镜片吸附蛋白理论浓度值，计算得到所述镜片吸附蛋白理论浓度值为0.872mg/ml。
  6. 根据权利要求5所述的有效测量软性亲水角膜接触镜泪蛋白含量的方法，其特征在于，
    将表面吸附有蛋白的软性亲水角膜接触镜放置在所述蛋白提取溶液中，所述蛋白提取溶液将蛋白自所述软性亲水角膜接触镜上分离，具体包括：取1ml所述蛋白提取溶液放置在离心管中，将所述软性亲水角膜接触镜放置在该离心管中振动清洗，所述软性亲水角膜接触镜上吸附的蛋白与所述软性亲水角膜接触镜分离；
    通过所述微量紫外分光光度计检测所述蛋白提取溶液中的蛋白浓度，获得镜片吸附蛋白浓度值，检测得到所述镜片吸附蛋白浓度值为0.805mg/ml；
    通过获得的所述镜片吸附蛋白浓度值计算所述镜片吸附蛋白含量值。
  7. 根据权利要求6所述的有效测量软性亲水角膜接触镜泪蛋白含量的方法，其特征在于，
    将获得的镜片吸附蛋白浓度值与所述镜片吸附蛋白理论浓度值进行数值计算，获得蛋白提取溶液对蛋白的洗脱率，具体包括：
    通过所述镜片吸附蛋白浓度值与所述镜片吸附蛋白理论浓度值的比值计算所述蛋白提取溶液对蛋白的洗脱率，计算得到所述蛋白提取溶液对蛋白的洗脱率为92.3％。
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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4738790A (en) * 1984-08-21 1988-04-19 Toyo Contact Lens Co., Ltd. Method for cleaning contact lenses with composition containing amidoamine surfactant, thiourea and reducing agent
US5820696A (en) * 1995-08-02 1998-10-13 Tomey Technology Corporation Method of cleaning and disinfecting contact lens
CN1387919A (zh) * 2002-06-20 2003-01-01 上海卫康光学有限公司 一种高效清除软性亲水接触镜上残留蛋白的组合物及其应用
JP2003084248A (ja) * 2001-09-14 2003-03-19 Menicon Co Ltd コンタクトレンズ用酵素剤
JP2003121805A (ja) * 2001-10-10 2003-04-23 Tomey Corp コンタクトレンズの洗浄及び消毒方法
CN109030669A (zh) * 2018-10-31 2018-12-18 苏州赛分科技有限公司 一种分离peg修饰蛋白样品中游离peg的高效液相色谱方法

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4738790A (en) * 1984-08-21 1988-04-19 Toyo Contact Lens Co., Ltd. Method for cleaning contact lenses with composition containing amidoamine surfactant, thiourea and reducing agent
US5820696A (en) * 1995-08-02 1998-10-13 Tomey Technology Corporation Method of cleaning and disinfecting contact lens
JP2003084248A (ja) * 2001-09-14 2003-03-19 Menicon Co Ltd コンタクトレンズ用酵素剤
JP2003121805A (ja) * 2001-10-10 2003-04-23 Tomey Corp コンタクトレンズの洗浄及び消毒方法
CN1387919A (zh) * 2002-06-20 2003-01-01 上海卫康光学有限公司 一种高效清除软性亲水接触镜上残留蛋白的组合物及其应用
CN109030669A (zh) * 2018-10-31 2018-12-18 苏州赛分科技有限公司 一种分离peg修饰蛋白样品中游离peg的高效液相色谱方法

Non-Patent Citations (7)

* Cited by examiner, † Cited by third party
Title
KEITH D., HONG B., CHRISTENSEN M.: "A novel procedure for the extraction of protein deposits from soft hydrophilic contact lenses for analysis", CURRENT EYE RESEARCH, vol. 16, no. 5, 1 January 1997 (1997-01-01), US , pages 503 - 510, XP009537977, ISSN: 0271-3683, DOI: 10.1076/ceyr.16.5.503.7049 *
LI CHANGBING, ET AL.: "Analysis of Protein Precipitation on Contact Lenses Made from Two Different Materials", JOURNAL OF CLINICAL MEDICINE IN PRACTICE, vol. 13, no. 4, 31 December 2009 (2009-12-31), pages 108 - 109, XP055948255, ISSN: 1672-2353 *
LI XINMING, ET AL.: "Adsorption of BSA on Hydrogel Containing Silicane", JOURNAL OF XINYANG NORMAL UNIVERSITY (NATURAL SCIENCE EDITION), vol. 20, no. 2, 30 April 2007 (2007-04-30), pages 174 - 178, XP055948254, ISSN: 1003-0972 *
POWELL DANIEL R., THANGAVELU MIRUNALNI, CHANDLER HEATHER L., NICHOLS KELLY K., NICHOLS JASON J.: "Evaluation of Extractants and Precipitants in Tear Film Proteomic Analyses", OPTOMETRY AND VISION SCIENCE., WILLIAMS AND WILKINS, BALTIMORE, MD., US, vol. 87, no. 11, 1 November 2010 (2010-11-01), US , pages 854 - 860, XP055948293, ISSN: 1040-5488, DOI: 10.1097/OPX.0b013e3181f6fb71 *
SUBBARAMAN LAKSHMAN N., GLASIER MARY ANN, SHEARDOWN HEATHER, JONES LYNDON: "Efficacy of an Extraction Solvent Used to Quantify Albumin Deposition on Hydrogel Contact Lens Materials", EYE & CONTACT LENS: SCIENCE & CLINICAL PRACTICE, vol. 35, no. 2, 1 March 2009 (2009-03-01), US , pages 76 - 80, XP009537978, ISSN: 1542-2321, DOI: 10.1097/ICL.0b013e318199aff6 *
WEN YAN, ET AL.: "A Testing Method of Assessing the Protein-Removing Effect of the Contact Lenses' Protein -Removing Care Solution", CHINESE JOURNAL OF MEDICAL INSTRUMENTATION, SHANGHAI YILIAO QIXIE YANJIUSUO, CN, vol. 30, no. 2, 31 December 2006 (2006-12-31), CN , pages 131 - 133, XP055948296, ISSN: 1671-7104 *
ZHANG JIN, ET AL.: "The Kinetics of in Vitro Lysozyme Deposition on Rigid Gas-permeable Contact Lens for a Short Period", CHINESE JOURNAL OF TISSUE ENGINEERING RESEARCH, ZHONGGUO KANGFU YIXUEHUI,, CN, vol. 14, no. 3, 15 January 2010 (2010-01-15), CN , pages 473 - 476, XP055948249, ISSN: 1673-8225, DOI: 10.3969/j.issn.1673-8225.2010.03.022 *

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