WO2017097070A1 - 二喹啉甲酸用于海藻酸盐材料中蛋白含量的定量检测方法 - Google Patents

二喹啉甲酸用于海藻酸盐材料中蛋白含量的定量检测方法 Download PDF

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WO2017097070A1
WO2017097070A1 PCT/CN2016/104538 CN2016104538W WO2017097070A1 WO 2017097070 A1 WO2017097070 A1 WO 2017097070A1 CN 2016104538 W CN2016104538 W CN 2016104538W WO 2017097070 A1 WO2017097070 A1 WO 2017097070A1
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alginate
solution
protein content
protein
acid
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谢红国
于炜婷
谢威杨
马小军
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Dalian Institute of Chemical Physics of CAS
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    • 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
    • G01N1/38—Diluting, dispersing or mixing samples
    • 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
    • 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
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
    • G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour

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  • the invention relates to a method for determining protein content, in particular to a method for quantitatively detecting protein content in alginate materials by using quinolinic acid.
  • Alginate materials have shown broad application prospects as biomaterials for tissue regeneration due to their good biocompatibility.
  • impurity proteins in alginate materials will seriously affect their biocompatibility during application.
  • the amount of the protein component in the product will be strictly limited.
  • the quantitative characterization method for proteins in national relevant standards adopts the Coomassie Brilliant Blue method.
  • the method needs to be carried out in an acidic environment during the test, and the alginate material forms a gel state in an acidic pH environment, and a uniform solution cannot be formed, so that the conventional Coomassie Brilliant Blue protein detection method cannot be applied to alginic acid. Salt material detection of heteroproteins.
  • the method for detecting the protein of the conventional Coomassie Brilliant Blue cannot be applied to the defect in the detection of the alginate material heteroprotein, and the present invention proposes that the method for determining the protein content under alkaline conditions can be used for the impurity protein content in the alginate material. Detection.
  • a protein standard solution of 100.0 ⁇ g/ml was prepared using physiological saline.
  • the standard solution prepared in the step (1) is diluted with physiological saline to 10.0 ⁇ g/ml, 20.0 ⁇ g/ml, 30.0 ⁇ g/ml, 40.0 ⁇ g/ml, 50.0 ⁇ g/ml, 60.0 ⁇ g/ml, 70.0. Gg/ml, 80.0 ⁇ g/ml, 90.0 ⁇ g/ml.
  • Reagent A Weigh 10.0 g of BCA (diquinolinecarboxylic acid) (1%), 20.0 g of Na 2 CO 3 ⁇ H 2 O (2%), and 1.6 g of sodium tartrate (Na 2 C 4 H 4 O 6 ⁇ 2H) 2 O, 0.16%), 4.0g NaOH (0.4%), 9.5g NaHCO 3 (0.95%), the volume to 1L, with 10mol / L NaOH or solid NaHCO 3 to adjust the pH to 11.25.
  • BCA diquinolinecarboxylic acid
  • Reagent B Accurately weigh 2.0 g of CuSO 4 ⁇ 5H 2 O (4%), and make up to 50 mL of distilled water.
  • the mass ratio of the two is uniformly mixed 1:5-1:50, and the reaction is carried out in a 40-70 ° C water bath for 10-60 minutes.
  • the physiological saline containing no protein standard solution was used as a control group, and the absorbance values at 562 nm of each group were measured, and a protein content-absorbance standard curve was plotted.
  • Alginate sample solution (1-20 mg/ml) was prepared in physiological saline.
  • the mass ratio of the two is uniformly mixed 1:5-1:50, and the reaction is carried out in a 40-70 ° C water bath for 10-60 minutes.
  • the physiological saline was used as a control group, and the absorbance value of the control group and the alginate sample group at 562 nm was measured, and the protein content in the alginate solution was calculated according to the protein content-absorbance standard curve obtained in the step (4), according to the alginate.
  • the solution concentration calculates the protein content in the alginate material sample.
  • the sodium alginate sample can also be directly dissolved in the diquinolinic acid working solution, and the alginate sample is prepared at a concentration of 10 ⁇ g/ml to 10 mg/ml in a 40-70 ° C water bath for 10-60 minutes.
  • the absorbance value was measured at 562 nm, and the protein content in the alginate solution was calculated based on the protein content-absorbance standard curve obtained in the step (4).
  • the quantitative detection method for determining the protein content in alginate materials proposed by the invention is suitable for determining alginate materials having a molecular weight of 10-500 kDa.
  • the quantitative detection method for determining the protein content in the alginate material proposed by the invention is suitable for determining the sodium or potassium salt of alginic acid.
  • Reagent A Weigh 10.0 g BCA (1%), 20.0 g Na 2 CO 3 ⁇ H 2 O (2%), 1.6 g sodium tartrate (Na 2 C 4 H 4 O 6 ⁇ 2H 2 O, 0.16%) , 4.0g NaOH (0.4%), 9.5g NaHCO 3 (0.95%), the volume to 1L, with 10mol / L NaOH or solid NaHCO 3 to adjust the pH to 11.25.
  • Reagent B Accurately weigh 2.0 g of CuSO 4 ⁇ 5H 2 O (4%), and make up to 50 mL of distilled water.
  • a gradient protein standard solution prepare a 100.0 ⁇ g/ml bovine serum albumin standard solution with physiological saline, and dilute it to 10.0 ⁇ g/ml, 20.0 ⁇ g/ml, 30.0 ⁇ g/ml, 40.0 ⁇ g/ml with physiological saline. 50.0 ⁇ g/ml, 60.0 ⁇ g/ml, 70.0 ⁇ g/ml, 80.0 ⁇ g/ml, and 90.0 ⁇ g/ml.
  • the working solution of diquinoline formic acid was added to the gradient protein standard solution, and the mass ratio of the two was uniformly mixed at 1:10, and the reaction was carried out in a water bath at 60 ° C for 30 minutes.
  • the physiological saline containing no protein standard solution was used as a control group, and each group was measured at 562 nm. At the absorbance value, the protein content-absorbance standard curve is plotted.
  • Reagent A Weigh 10.0 g BCA (1%), 20.0 g Na 2 CO 3 ⁇ H 2 O (2%), 1.6 g sodium tartrate (Na 2 C 4 H 4 O 6 ⁇ 2H 2 O, 0.16%) , 4.0g NaOH (0.4%), 9.5g NaHCO 3 (0.95%), the volume to 1L, with 10mol / L NaOH or solid NaHCO 3 to adjust the pH to 11.25.
  • Reagent B Accurately weigh 2.0 g of CuSO 4 ⁇ 5H 2 O (4%), and make up to 50 mL of distilled water.
  • a gradient protein standard solution prepare a 100.0 ⁇ g/ml bovine serum albumin standard solution with physiological saline, and dilute it to 10.0 ⁇ g/ml, 20.0 ⁇ g/ml, 30.0 ⁇ g/ml, 40.0 ⁇ g/ml with physiological saline. 50.0 ⁇ g/ml, 60.0 ⁇ g/ml, 70.0 ⁇ g/ml, 80.0 ⁇ g/ml, and 90.0 ⁇ g/ml.
  • the working solution of diquinoline formic acid was added to the gradient protein standard solution, and the mass ratio of the two was uniformly mixed at 1:10, and the reaction was carried out in a water bath at 60 ° C for 30 minutes.
  • the physiological saline containing no protein standard solution was used as a control group, and each group was measured at 562 nm. At the absorbance value, the protein content-absorbance standard curve is plotted.

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Abstract

一种将二喹啉甲酸用于海藻酸盐材料中蛋白含量的定量检测方法，在碱性条件下，用蛋白标准品在微量区间绘制蛋白含量-吸光度检测标准曲线，再依此标准曲线，定量检测海藻酸盐材料中的蛋白含量。

Description

二喹啉甲酸用于海藻酸盐材料中蛋白含量的定量检测方法 技术领域
本发明涉及一种蛋白含量的测定方法，具体说是一种将二喹啉甲酸用于海藻酸盐材料中蛋白含量的定量检测方法。
背景技术
海藻酸盐材料因其良好的生物相容性，作为组织再生的生物材料显示出广阔的应用前景。但海藻酸盐材料中杂质蛋白将严重影响其在应用过程中的生物相容性。作为组织工程的原材料产品，其产品中的杂蛋白成分将被严格限制。目前，国家相关标准中针对蛋白的定量表征方法均采用考马斯亮蓝法。但，该方法在测试过程中，需要在酸性环境下进行，而海藻酸盐材料在pH酸性环境下形成凝胶态，无法形成均匀溶液，使得常规考马斯亮蓝检测蛋白的方法无法应用到海藻酸盐材料杂蛋白的检测中。
发明内容
针对常规考马斯亮蓝检测蛋白的方法无法应用到海藻酸盐材料杂蛋白的检测中的缺陷，本发明提出，将可在碱性条件下测定蛋白含量的方法用于海藻酸盐材料中杂质蛋白含量的检测。
本发明提出的具体技术方案如下：
(1)用生理盐水配制成100.0μg/ml的蛋白质标准液。
(2)将步骤(1)配制的标准液用生理盐水依此稀释成10.0μg/ml、20.0μg/ml、30.0μg/ml、40.0μg/ml、50.0μg/ml、60.0μg/ml、70.0μg/ml、80.0μg/ml、90.0μg/ml。
(3)配制二喹啉甲酸试剂及其工作液：
试剂A：分别称取10.0g BCA(二喹啉甲酸)(1％)，20.0g Na2CO3·H2O(2％)，1.6g酒石酸钠(Na2C4H4O6·2H2O，0.16％)，4.0g NaOH(0.4％)，9.5g NaHCO3(0.95％)，定容至1L，用10mol/L NaOH或固体NaHCO3调节pH值至11.25。
试剂B：精确称取2.0g CuSO4·5H2O(4％)，蒸馏水定容至50mL。
取50体积份试剂A与1体积份试剂B混合均匀，即配制成绿色的工作液。
(4)向步骤(2)获得的梯度蛋白质标准液中加入二喹啉甲酸工作液，二者质量比为1:5-1:50均匀混合，40-70℃水浴中反应10-60分钟，不含蛋白质标准液的生理盐水为对照组，测定各组在562nm处的吸光度值，绘制蛋白含量-吸光度标准曲线。
(5)生理盐水配制海藻酸盐样品溶液(1-20mg/ml)。
(6)向步骤(5)配制的海藻酸盐溶液中加入二喹啉甲酸工作液，二者质量比为1:5-1:50均匀混合，40-70℃水浴中反应10-60分钟，生理盐水为对照组，测定对照组与海藻酸盐样品组在562nm处的吸光度值，根据步骤(4)获得的蛋白含量-吸光度标准曲线，计算海藻酸盐溶液中的蛋白含量，根据海藻酸盐溶液浓度计算海藻酸盐材料样品中的蛋白含量。
(7)也可将海藻酸钠样品直接溶解在二喹啉甲酸工作液中，海藻酸盐样品的配制浓度在10μg/ml-10mg/ml，40-70℃水浴中反应10-60分钟，在562nm处测定吸光度值，根据步骤(4)获得的蛋白含量-吸光度标准曲线，计算海藻酸盐溶液中的蛋白含量。
本发明提出的测定海藻酸盐材料中蛋白含量的定量检测方法，适合测定分子量在10-500kDa的海藻酸盐材料。
本发明提出的测定海藻酸盐材料中蛋白含量的定量检测方法，适合测定海藻酸的钠盐或钾盐。
具体实施方式
实施例1：
(1)配制二喹啉甲酸试剂及其工作液：
试剂A：分别称取10.0g BCA(1％)，20.0g Na2CO3·H2O(2％)，1.6g酒石酸钠(Na2C4H4O6·2H2O，0.16％)，4.0g NaOH(0.4％)，9.5g NaHCO3(0.95％)，定容至1L，用10mol/L NaOH或固体NaHCO3调节pH值至11.25。
试剂B：精确称取2.0g CuSO4·5H2O(4％)，蒸馏水定容至50mL。
取50体积份试剂A与1体积份试剂B混合均匀，即配制成绿色的工作液。
(2)绘制蛋白含量-吸光度标准曲线：
配制梯度蛋白标准液：用生理盐水配制成100.0μg/ml的牛血清白蛋白标准液，用生理盐水依此稀释成10.0μg/ml、20.0μg/ml、30.0μg/ml、40.0μg/ml、50.0μg/ml、60.0μg/ml、70.0μg/ml、80.0μg/ml、90.0μg/ml。向梯度蛋白质标准液中加入二喹啉甲酸工作液，二者质量比为1:10均匀混合，60℃水浴中反应30分钟，不含蛋白质标准液的生理盐水为对照组，测定各组在562nm处的吸光度值，绘制蛋白含量-吸光度标准曲线。
(3)测定待测海藻酸钠样品中的蛋白含量：取海藻酸钠样品，用生理盐水配制成5mg/ml的海藻酸钠溶液，向海藻酸钠溶液中加入二喹啉甲酸工作液，二者质量比为1:10均匀混合，60℃水浴中反应30分钟，生理盐水为对照组，测定两组在562nm处的吸光度值，根据蛋白含量-吸光度标准曲线，求得海藻酸钠溶液中蛋白含量为45.0μg/ml。根据海藻酸钠溶液浓度，计算得待测海藻酸钠样品中蛋白含量为0.9％(质量分数)。
实施例2：
(1)配制二喹啉甲酸试剂及其工作液：
试剂A：分别称取10.0g BCA(1％)，20.0g Na2CO3·H2O(2％)，1.6g酒石酸钠(Na2C4H4O6·2H2O，0.16％)，4.0g NaOH(0.4％)，9.5g NaHCO3(0.95％)，定容至1L，用10mol/L NaOH或固体NaHCO3调节pH值至11.25。
试剂B：精确称取2.0g CuSO4·5H2O(4％)，蒸馏水定容至50mL。
取50体积份试剂A与1体积份试剂B混合均匀，即配制成绿色的工作液。
(2)绘制蛋白含量-吸光度标准曲线：
配制梯度蛋白标准液：用生理盐水配制成100.0μg/ml的牛血清白蛋白标准液，用生理盐水依此稀释成10.0μg/ml、20.0μg/ml、30.0μg/ml、40.0μg/ml、50.0μg/ml、60.0μg/ml、70.0μg/ml、80.0μg/ml、90.0μg/ml。向梯度蛋白质标准液中加入二喹啉甲酸工作液，二者质量比为1:10均匀混合，60℃水浴中反应30分钟，不含蛋白质标准液的生理盐水为对照组，测定各组在562nm处的吸光度值，绘制蛋白含量-吸光度标准曲线。
(3)测定待测海藻酸钾样品中的蛋白含量：取海藻酸钾样品，用二喹啉甲酸工作液配制成1mg/ml的海藻酸钾溶液，60℃水浴中反应30分钟，测定在562nm处的吸光度值，根据蛋白含量-吸光度标准曲线，求得海藻酸钾溶液中蛋白含量为28.0μg/ml。根据海藻酸钾溶液浓度，计算得待测海藻酸钾样品中蛋白含量为0.28％(质量分数)。

Claims (5)

  1. 二喹啉甲酸用于海藻酸盐材料中蛋白含量的定量检测方法，其特征在于：在碱性条件下，用蛋白质标准品绘制蛋白含量-吸光度检测标准曲线，再依此标准曲线，定量检测海藻酸盐材料中的蛋白含量。
  2. 按照权利要求1所述的测定海藻酸盐材料中蛋白含量的定量检测方法，其特征在于所述方法的具体步骤如下：
    (1)用生理盐水配制成100.0μg/ml的蛋白质标准液；
    (2)将步骤(1)配制的标准液用生理盐水依此稀释成10.0μg/ml、20.0μg/ml、30.0μg/ml、40.0μg/ml、50.0μg/ml、60.0μg/ml、70.0μg/ml、80.0μg/ml、90.0μg/ml；
    (3)配制二喹啉甲酸试剂及其工作液：
    试剂A：分别称取10.0g BCA(1％)，20.0g Na2CO3·H2O(2％)，1.6g酒石酸钠(Na2C4H4O6·2H2O，0.16％)，4.0g NaOH(0.4％)，9.5g NaHCO3(0.95％)，定容至1L，用10mol/L NaOH或固体NaHCO3调节pH值至11.25；
    试剂B：精确称取2.0g CuSO4·5H2O(4％)，蒸馏水定容至50mL；
    取50体积份试剂A与1体积份试剂B混合均匀，即配制成绿色的二喹啉甲酸工作液；
    (4)向步骤(2)获得的梯度蛋白质标准液中分别加入二喹啉甲酸工作液，蛋白质标准液与二喹啉甲酸工作液二者质量比为1:5-1:50均匀混合，40-70℃水浴中反应10-60分钟，不含蛋白质标准液的生理盐水为对照组，测定各组在562nm处的吸光度值，绘制蛋白含量-吸光度标准曲线；
    (5)生理盐水配制海藻酸盐样品溶液(1-20mg/ml)；向配制的海藻酸盐溶液中加入二喹啉甲酸工作液，海藻酸盐溶液与二喹啉甲酸工作液二者质量比为1:5-1:50均匀混合；或将海藻酸钠样品直接溶解在二喹啉甲酸工作液中，海藻酸盐样品的配制浓度在10μg/ml-10mg/ml；
    40-70℃水浴中反应10-60分钟，生理盐水为对照组，测定对照组与海藻酸盐样品组在562nm处的吸光度值，根据步骤(4)获得的蛋白含量-吸光度标准曲线，计算海藻酸盐溶液中的蛋白含量，根据海藻酸盐溶液浓度计算海藻酸盐材料样品中的蛋白含量。
  3. 按照权利要求1或2所述的测定海藻酸盐材料中蛋白含量的定量检测方法，其特征在于：该方法适合测定分子量在10-500kDa的海藻酸盐材料。
  4. 按照权利要求1或2所述的测定海藻酸盐材料中蛋白含量的定量检测方法，其特征在于：该方法适合测定海藻酸的钠盐或钾盐。
  5. 按照权利要求1或2所述的测定海藻酸盐材料中蛋白含量的定量检测方法，其特征在于：所述蛋白质为白蛋白、酪蛋白、免疫球蛋白等。
PCT/CN2016/104538 2015-12-10 2016-11-04 二喹啉甲酸用于海藻酸盐材料中蛋白含量的定量检测方法 Ceased WO2017097070A1 (zh)

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