CN105115945B - The detection method of gamma Globulin - Google Patents
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- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
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Abstract
本发明公开了一种γ‑球蛋白的检测方法，所述检测方法包括：将如式(I)所示结构的聚合物PPEASO3溶于水中制得溶剂；将不同浓度的γ‑球蛋白标准溶液置于上述制得的溶剂中，并加水和缓冲溶液定容制成不同浓度的待测溶液，测定各待测溶液的最大荧光强度；以溶剂的荧光发射峰的荧光强度和待测溶液的荧光发射峰的荧光强度的比值为纵坐标，γ‑球蛋白溶液的浓度为横坐标建立荧光吸收光谱曲线的方程；检测待测浓度的γ‑球蛋白溶液的最大荧光强度，然后根据荧光吸收光谱曲线的方程计算待测浓度的γ‑球蛋白溶液中γ‑球蛋白的浓度；其中，n为正整数。实现γ‑球蛋白的浓度检测灵敏度高、稳定性好且可重复性高，操作简单，能快速测定的效果。
The invention discloses a detection method of γ-globulin, the detection method comprising: dissolving the polymer PPEASO 3 having the structure shown in formula (I) in water to prepare a solvent; The solution is placed in the solvent prepared above, and water and buffer solution are added to constant volume to make different concentrations of the solutions to be tested, and the maximum fluorescence intensity of each solution to be tested is measured; the fluorescence intensity of the fluorescence emission peak of the solvent and the solution to be tested are The ratio of the fluorescence intensity of the fluorescence emission peak is the ordinate, and the concentration of the gamma globulin solution is the equation for setting up the fluorescence absorption spectrum curve on the abscissa; detect the maximum fluorescence intensity of the gamma globulin solution of the concentration to be measured, and then according to the fluorescence absorption spectrum The equation of curve calculates the concentration of gamma globulin in the gamma globulin solution of concentration to be tested; Wherein, n is a positive integer. The concentration detection of γ-globulin has the advantages of high sensitivity, good stability and high repeatability, simple operation and rapid determination.
Description
技术领域technical field
本发明涉及生物体内蛋白质的检测方法，具体地，涉及一种γ-球蛋白的检测方法。The invention relates to a method for detecting proteins in organisms, in particular to a method for detecting gamma-globulin.
背景技术Background technique
血清球蛋白由人体单核-吞噬细胞系统合成，它经过蛋白电泳可分为α1-、α2-、β1-、β2-和γ-球蛋白。而γ-球蛋白也称为丙类球蛋白，是具有抗体活性并能与相应抗原特异地结合的一类球蛋白，也是肝功能检查中一项重要指标，γ-球蛋白的偏高几乎见于所有肝胆疾病。当患者是病毒性肝炎时，γ-球蛋白会中度增高；当患者是重型肝炎时，γ-球蛋白可明显升高；当患者是肝硬化时，γ-球蛋白普遍增高，尤其在晚期或进行性失代偿肝硬化，γ-球蛋白可极度增高。目前常见的由免疫球蛋白异常引发的疾病有原发性免疫缺陷病和变异性免疫缺陷症。因此根据体内γ-球蛋白的含量变化可以快速的得出肝功能有没障碍，尽可能的减少肝胆疾病所带来的风险。Serum globulin is synthesized by the human mononuclear-phagocyte system, which can be divided into α1-, α2-, β1-, β2- and γ-globulins through protein electrophoresis. γ-globulin, also known as gamma globulin, is a type of globulin that has antibody activity and can specifically bind to the corresponding antigen. It is also an important indicator in liver function tests. The high level of γ-globulin is almost seen in All hepatobiliary diseases. When the patient has viral hepatitis, the γ-globulin will be moderately increased; when the patient is severe hepatitis, the γ-globulin can be significantly increased; when the patient is cirrhotic, the γ-globulin will generally increase, especially in the late stage Or progressive decompensated cirrhosis, γ-globulin can be extremely increased. At present, the common diseases caused by abnormal immunoglobulin include primary immunodeficiency disease and variable immunodeficiency disease. Therefore, according to the changes in the content of γ-globulin in the body, it is possible to quickly determine whether the liver function is impaired, and reduce the risk of liver and gallbladder diseases as much as possible.
目前针对γ-球蛋白的测定方法已经被提出来，这其中包括考马斯亮蓝法和溴酚蓝法。在这些方法中，由于敏感度、重复度等不够高，因此在实际应用时具有一定的局限性和不稳定性。一般的荧光分光光度法在实际测试中由于各类蛋白质结构特性的相似性等会不可避免的出现很多干扰，而且短波发射也会导致很多不必要的信号干扰。At present, methods for the determination of γ-globulin have been proposed, including Coomassie brilliant blue method and bromophenol blue method. Among these methods, due to insufficient sensitivity and repeatability, they have certain limitations and instability in practical application. In the actual test of general fluorescence spectrophotometry, there will inevitably be a lot of interference due to the similarity of the structural properties of various proteins, and short-wave emission will also cause a lot of unnecessary signal interference.
因此，提供一种灵敏度高、稳定性好且具有较好的可重复性，并且操作简单，能快速测得γ-球蛋白的浓度的γ-球蛋白的检测方法是本发明亟需解决的问题。Therefore, it is an urgent problem to be solved in the present invention to provide a detection method for gamma-globulin with high sensitivity, good stability, good repeatability, simple operation, and the ability to quickly measure the concentration of gamma-globulin .
发明内容Contents of the invention
针对上述现有技术，本发明的目的在于克服现有技术中γ-球蛋白在测定过程中，往往会由于各类蛋白质结构特性的相似性从而不可避免的出现很多干扰，同时短波发射也会导致很多不必要的信号干扰等问题，从而提供一种灵敏度高、稳定性好且具有较好的可重复性，并且操作简单，能快速测得γ-球蛋白的浓度的γ-球蛋白的检测方法。With regard to the above-mentioned prior art, the purpose of the present invention is to overcome the unavoidable interference of many types of proteins during the determination of gamma-globulin in the prior art due to the similarity of the structural properties of various proteins, and at the same time, short-wave emission will also cause Many unnecessary signal interference and other problems, so as to provide a detection method of γ-globulin with high sensitivity, good stability and good repeatability, and simple operation, which can quickly measure the concentration of γ-globulin .
为了实现上述目的，本发明提供了一种γ-球蛋白的检测方法，其中，所述检测方法包括：In order to achieve the above object, the present invention provides a detection method for gamma-globulin, wherein the detection method comprises:
(1)将如式(I)所示结构的聚合物PPEASO3溶于水中制得溶剂；(1) polymer PPEASO of structure shown in formula (I) is dissolved in water and makes solvent;
(2)将不同浓度的γ-球蛋白标准溶液置于步骤(1)制得的溶剂中，并加水和缓冲溶液定容制成不同浓度的待测溶液，测定各待测溶液的最大荧光强度；(2) Place gamma-globulin standard solutions of different concentrations in the solvent prepared in step (1), add water and buffer solution to constant volume to make different concentrations of test solutions, and measure the maximum fluorescence intensity of each test solution ;
(3)以溶剂的荧光发射峰的荧光强度和待测溶液的荧光发射峰的荧光强度的比值为纵坐标，γ-球蛋白溶液的浓度为横坐标建立荧光吸收光谱曲线的方程；(3) take the ratio of the fluorescence intensity of the fluorescence emission peak of the solvent and the fluorescence intensity of the fluorescence emission peak of the solution to be measured as the ordinate, and the concentration of the gamma-globulin solution as the abscissa to establish the equation of the fluorescence absorption spectrum curve;
(4)检测待测浓度的γ-球蛋白溶液的最大荧光强度，然后根据荧光吸收光谱曲线的方程计算待测浓度的γ-球蛋白溶液中γ-球蛋白的浓度；(4) Detect the maximum fluorescence intensity of the γ-globulin solution of the concentration to be measured, then calculate the concentration of γ-globulin in the γ-globulin solution of the concentration to be measured according to the equation of the fluorescence absorption spectrum curve;
其中，n为正整数。Wherein, n is a positive integer.
通过上述技术方案，本发明将聚合物PPEASO3溶于水中制得溶剂，而后通过该溶剂及缓冲溶液与不同浓度的γ-球蛋白标准溶液混合，再定容制得待测溶液，并测定上述待测溶液的最大荧光强度，并以溶剂的荧光发射峰的荧光强度和待测溶液的荧光发射峰的荧光强度的比值为纵坐标，γ-球蛋白溶液的浓度为横坐标建立荧光吸收光谱曲线方程，最后测定需要检测的γ-球蛋白溶液的最大荧光强度，并根据上述荧光吸收光谱曲线方程计算得到γ-球蛋白的浓度，从而通过这种方式，将荧光吸收光谱曲线方程建立出来后，则仅需要测定一次最大荧光强度，即可得到需要检测的γ-球蛋白溶液的浓度，实现了灵敏度高、稳定性好且具有较好的可重复性，并且操作简单，能快速测定γ-球蛋白溶液的浓度的效果。Through the above-mentioned technical scheme, the present invention dissolves the polymer PPEASO 3 in water to obtain a solvent, then mixes the solvent and buffer solution with different concentrations of γ-globulin standard solutions, then fixes the volume to prepare the solution to be tested, and measures the above-mentioned The maximum fluorescence intensity of the solution to be measured, and the ratio of the fluorescence intensity of the fluorescence emission peak of the solvent to the fluorescence intensity of the fluorescence emission peak of the solution to be measured is the ordinate, and the concentration of the gamma-globulin solution is the abscissa to establish a fluorescence absorption spectrum curve Finally, measure the maximum fluorescence intensity of the gamma-globulin solution that needs to be detected, and calculate the concentration of gamma-globulin according to the above-mentioned fluorescence absorption spectrum curve equation, so that in this way, after the fluorescence absorption spectrum curve equation is established, It only needs to measure the maximum fluorescence intensity once to obtain the concentration of the γ-globulin solution to be detected, which achieves high sensitivity, good stability and good repeatability, and is easy to operate and can quickly determine γ-globulin. The effect of the concentration of the protein solution.
本发明的其他特征和优点将在随后的具体实施方式部分予以详细说明。Other features and advantages of the present invention will be described in detail in the following detailed description.
附图说明Description of drawings
附图是用来提供对本发明的进一步理解，并且构成说明书的一部分，与下面的具体实施方式一起用于解释本发明，但并不构成对本发明的限制。在附图中：The accompanying drawings are used to provide a further understanding of the present invention, and constitute a part of the description, together with the following specific embodiments, are used to explain the present invention, but do not constitute a limitation to the present invention. In the attached picture:
图1是实施例中所提供的各浓度的γ-球蛋白溶液的荧光强度图谱；Fig. 1 is the fluorescence intensity spectrum of the gamma-globulin solution of each concentration provided in the embodiment;
图2是实施例中所提供的一种荧光吸收光谱曲线的方程；Fig. 2 is the equation of a kind of fluorescence absorption spectrum curve provided in the embodiment;
图3是实施例在不同温度下制得的荧光吸收光谱曲线的方程；Fig. 3 is the equation of the fluorescence absorption spectrum curve that embodiment makes at different temperatures;
图4是实施例中PPEASO3溶液在不同浓度下的γ-球蛋白存在时的荧光衰减情况。Fig. 4 is the fluorescence decay of PPEASO 3 solution in the embodiment in the presence of γ-globulin at different concentrations.
具体实施方式detailed description
以下对本发明的具体实施方式进行详细说明。应当理解的是，此处所描述的具体实施方式仅用于说明和解释本发明，并不用于限制本发明。Specific embodiments of the present invention will be described in detail below. It should be understood that the specific embodiments described here are only used to illustrate and explain the present invention, and are not intended to limit the present invention.
本发明提供了一种γ-球蛋白的检测方法，其中，所述检测方法包括：The present invention provides a detection method for gamma-globulin, wherein the detection method comprises:
(1)将如式(I)所示结构的聚合物PPEASO3溶于水中制得溶剂；(1) polymer PPEASO of structure shown in formula (I) is dissolved in water and makes solvent;
(2)将不同浓度的γ-球蛋白标准溶液置于步骤(1)制得的溶剂中，并加水和缓冲溶液定容制成不同浓度的待测溶液，测定各待测溶液的最大荧光强度；(2) Place gamma-globulin standard solutions of different concentrations in the solvent prepared in step (1), add water and buffer solution to constant volume to make different concentrations of test solutions, and measure the maximum fluorescence intensity of each test solution ;
(3)以溶剂的荧光发射峰的荧光强度和待测溶液的荧光发射峰的荧光强度的比值为纵坐标，γ-球蛋白溶液的浓度为横坐标建立荧光吸收光谱曲线的方程；(3) take the ratio of the fluorescence intensity of the fluorescence emission peak of the solvent and the fluorescence intensity of the fluorescence emission peak of the solution to be measured as the ordinate, and the concentration of the gamma-globulin solution as the abscissa to establish the equation of the fluorescence absorption spectrum curve;
(4)检测待测浓度的γ-球蛋白溶液的最大荧光强度，然后根据荧光吸收光谱曲线的方程计算待测浓度的γ-球蛋白溶液中γ-球蛋白的浓度；(4) Detect the maximum fluorescence intensity of the γ-globulin solution of the concentration to be measured, then calculate the concentration of γ-globulin in the γ-globulin solution of the concentration to be measured according to the equation of the fluorescence absorption spectrum curve;
其中，n为正整数。Wherein, n is a positive integer.
上述设计通过将聚合物PPEASO3溶于水中制得溶剂，而后通过该溶剂及缓冲溶液与不同浓度的γ-球蛋白标准溶液混合，再定容制得待测溶液，并测定上述待测溶液的最大荧光强度，并以溶剂的荧光发射峰的荧光强度和待测溶液的荧光发射峰的荧光强度的比值为纵坐标，γ-球蛋白溶液的浓度为横坐标建立荧光吸收光谱曲线方程，最后测定需要检测的γ-球蛋白溶液的最大荧光强度，并根据上述荧光吸收光谱曲线方程计算得到γ-球蛋白的浓度，从而通过这种方式，将荧光吸收光谱曲线方程建立出来后，则仅需要测定一次最大荧光强度，即可得到需要检测的γ-球蛋白溶液的浓度，实现了灵敏度高、稳定性好且具有较好的可重复性，并且操作简单，能快速测定γ-球蛋白溶液的浓度的效果。The above design is prepared by dissolving the polymer PPEASO 3 in water to obtain a solvent, then mixing the solvent and buffer solution with different concentrations of γ-globulin standard solutions, and then constant volume to prepare the solution to be tested, and measure the concentration of the above solution to be tested. The maximum fluorescence intensity, and the ratio of the fluorescence intensity of the fluorescence emission peak of the solvent and the fluorescence intensity of the fluorescence emission peak of the solution to be measured is the ordinate, and the concentration of the gamma-globulin solution is the abscissa to establish the fluorescence absorption spectrum curve equation, and finally determine The maximum fluorescence intensity of the γ-globulin solution that needs to be detected, and the concentration of γ-globulin is calculated according to the above-mentioned fluorescence absorption spectrum curve equation, so that in this way, after the fluorescence absorption spectrum curve equation is established, it is only necessary to measure The concentration of the γ-globulin solution to be detected can be obtained once with the maximum fluorescence intensity, which achieves high sensitivity, good stability and good repeatability, and is easy to operate, and can quickly determine the concentration of the γ-globulin solution Effect.
所述聚合物PPEASO3和所述缓冲溶液之间的比例可以不作进一步限定，但是，为了使得到的检测数据更为准确，进而提高检测的精确性，同时尽可能节省成本，在本发明的一种优选的实施方式中，相对于1mg的所述聚合物PPEASO3，所述缓冲溶液的用量为0.01-0.2μmol。The ratio between the polymer PPEASO 3 and the buffer solution may not be further limited, however, in order to make the obtained detection data more accurate, thereby improving the accuracy of detection, while saving costs as much as possible, in one aspect of the present invention In a preferred embodiment, relative to 1 mg of the polymer PPEASO 3 , the amount of the buffer solution is 0.01-0.2 μmol.
所述缓冲溶液可以为本领域常规使用的缓冲溶液类型，例如，在本发明的一种优选的实施方式中，为了使缓冲溶液的缓冲效果更好，所述缓冲溶液可以限定为磷酸氢二钠-磷酸二氢钾或三(羟甲基)氨基甲烷—盐酸缓冲溶液。当然，这里选择何种缓冲溶液主要是根据实验中具体的pH值进行选择的，本领域技术人员可以据此进行直接选择，在此不一一赘述。The buffer solution can be the type of buffer solution commonly used in the art, for example, in a preferred embodiment of the present invention, in order to make the buffer solution of the buffer solution better, the buffer solution can be limited to disodium hydrogen phosphate - potassium dihydrogen phosphate or tris (hydroxymethyl) amino methane - hydrochloric acid buffer solution. Of course, the choice of the buffer solution here is mainly based on the specific pH value in the experiment, and those skilled in the art can make a direct selection based on this, so details will not be repeated here.
当然，为了整个体系中pH值更为适宜，从而最大程度地使得测得的结果准确，在本发明的一种更为优选的实施方式中，所述磷酸氢二钠-磷酸二氢钾和三(羟甲基)氨基甲烷—盐酸缓冲溶液的pH值可以进一步限定为5-9。Of course, in order to make the pH value more suitable in the whole system, thereby making the measured results accurate to the greatest extent, in a more preferred embodiment of the present invention, the disodium hydrogen phosphate-potassium dihydrogen phosphate and trisodium hydrogen phosphate The pH value of (hydroxymethyl)aminomethane-hydrochloric acid buffer solution can be further limited to 5-9.
所述聚合物PPEASO3可以为常规使用的类型，例如，可以为纯净物，当然，为了尽可能在操作中便于保存和使用，在本发明的一种优选的实施方式中，步骤(1)中所述聚合物PPEASO3可以由聚合物PPEASO3溶液提供，溶液中的溶剂可以为本领域常规使用的溶剂类型，例如，在本发明的一种更为优选的实施方式中，所述聚合物PPEASO3溶液的溶剂可以为水。Described polymer PPEASO 3 can be the type that routinely uses, for example, can be pure thing, certainly, in order to be convenient to preserve and use in operation as far as possible, in a kind of preferred embodiment of the present invention, in step (1) Described polymer PPEASO 3 can be provided by polymer PPEASO 3 solution, and the solvent in the solution can be the solvent type conventionally used in this field, for example, in a kind of more preferred embodiment of the present invention, described polymer PPEASO 3 The solvent of the solution can be water.
聚合物PPEASO3溶液中聚合物PPEASO3的浓度可以不作进一步限定，当然，为了使得其在使用时便于保存且能满足本发明的使用，在本发明的一种优选的实施方式中，所述聚合物PPEASO3溶液中聚合物PPEASO3的浓度可以为7.5-22.5μg/mL。Polymer PPEASO The concentration of the polymer PPEASO in the solution can not be further limited, certainly, in order to make it convenient to preserve and can satisfy the use of the present invention when it is used, in a preferred embodiment of the present invention, the polymer The concentration of polymer PPEASO 3 in the solution of polymer PPEASO 3 may be 7.5-22.5 μg/mL.
为了使得检测待测溶液时得到的荧光强度数据更为准确，在整个体系中，可以对待测溶液的pH作进一步限定，例如，在本发明的一种优选的实施方式中，为了让整个体系的条件能够使检测的荧光强度更为准确和清晰，所述待测溶液的pH值可以进一步限定为5-9。In order to make the fluorescence intensity data obtained when detecting the solution to be tested more accurate, in the whole system, the pH of the solution to be tested can be further limited, for example, in a preferred embodiment of the present invention, in order to make the pH of the whole system The conditions can make the detected fluorescence intensity more accurate and clear, and the pH value of the solution to be tested can be further limited to 5-9.
为了让体系内各物质得到充分反应，进一步保证检测的荧光强度的准确，在本发明的一种优选的实施方式中，所述检测方法还可以包括将待测溶液放置1-80min后测定其荧光强度。In order to allow the substances in the system to fully react and further ensure the accuracy of the detected fluorescence intensity, in a preferred embodiment of the present invention, the detection method may also include measuring the fluorescence of the solution to be tested after placing it for 1-80min. strength.
荧光强度的检测可以按照本领域常规检测方法进行操作，同时，在本发明的一种优选的实施方式中，为了得到最大荧光强度，在步骤(2)中，检测各待测溶液的最大荧光强度波长范围为590-820nm。The detection of fluorescence intensity can be operated according to the conventional detection method in this field. Meanwhile, in a preferred embodiment of the present invention, in order to obtain the maximum fluorescence intensity, in step (2), detect the maximum fluorescence intensity of each solution to be tested The wavelength range is 590-820nm.
当然，为了使得制得的荧光吸收光谱曲线的方程的线性相关尽可能趋近于1，在本发明的一种优选的实施方式中，还可以进一步限定为在温度为293-320K的条件下测定最大荧光强度。Of course, in order to make the linear correlation of the equation of the obtained fluorescence absorption spectrum curve approach to 1 as much as possible, in a preferred embodiment of the present invention, it can also be further limited to be measured at a temperature of 293-320K maximum fluorescence intensity.
以下将通过实施例对本发明进行详细描述。以下实施例中，所述聚合物PPEASO3为根据下述制备方法制得，所述γ-球蛋白为上海晶纯生化科技股份有限公司的市售品，所述磷酸氢二钠-磷酸二氢钾缓冲溶液为根据下述制备方法制得。The present invention will be described in detail below by way of examples. In the following examples, the polymer PPEASO 3 is prepared according to the following preparation method, the γ-globulin is a commercial product of Shanghai Jingchun Biochemical Technology Co., Ltd., and the disodium hydrogen phosphate-dihydrogen phosphate Potassium buffer solution was prepared according to the following preparation method.
制备例1Preparation Example 1
根据《Microchim.Acta》期刊第177期第357-364页中记载的方法制得聚合物PPEASO3。The polymer PPEASO 3 was prepared according to the method described in the journal "Microchim. Acta" No. 177, pp. 357-364.
制备例2Preparation example 2
将3.561g磷酸氢二钠溶于100mL水中，制得0.2mol/L的磷酸氢二钠溶液，将3.06g磷酸二氢钾溶于100mL水中，制得0.2mol/L的磷酸二氢钾溶液，然后将上述31.5mL的磷酸氢二钠溶液滴加到68.5mL的磷酸二氢钾溶液中，调节pH值至6.5，从而制得磷酸氢二钠-磷酸二氢钾缓冲溶液。Dissolve 3.561 g of disodium hydrogen phosphate in 100 mL of water to obtain a 0.2 mol/L disodium hydrogen phosphate solution, and dissolve 3.06 g of potassium dihydrogen phosphate in 100 mL of water to obtain a 0.2 mol/L potassium dihydrogen phosphate solution. Then, 31.5 mL of the disodium hydrogen phosphate solution was added dropwise to 68.5 mL of the potassium dihydrogen phosphate solution, and the pH value was adjusted to 6.5, thereby preparing a disodium hydrogen phosphate-potassium dihydrogen phosphate buffer solution.
实施例1Example 1
将400μL浓度为0.5mg/mL的聚合物PPEASO3溶于浓度为0.2mol/L的上述磷酸氢二钠-磷酸二氢钾缓冲溶液中制得溶剂；取10份上述溶剂，并在每份溶剂中分别加入浓度为5μg/mL的γ-球蛋白标准溶液0μl，10μl，20μl，100μl，200μl，500μl，700μl，1000μl，1500μl和2000μl，并加水定容制成不同浓度的待测溶液，放置40min后分别测定上述待测溶液的荧光强度，如图1所示，测定各待测溶液的最大荧光强度(nm)分别为534nm，508nm，488.7nm，425.8nm，396.4nm，332.2nm，301.9nm，247nm，205.3nm和182.5nm，其中，图1中γ-球蛋白标准溶液的浓度自上而下依次为0μg/L，5μg/L，10μg/L，50μg/L，100μg/L，250μg/L，350μg/L，500μg/L，750μg/L和1000μg/L；如图2所示，以溶剂的荧光发射峰的荧光强度(荧光强度值为534nm)和待测溶液的荧光发射峰的荧光强度的比值为纵坐标，γ-球蛋白溶液的浓度为横坐标建立荧光吸收光谱曲线的方程，得到温度为293K下的荧光吸收光谱曲线的方程为：y＝1.11246+0.0019x，其中线性相关为R＝0.99789。Dissolve 400 μL of polymer PPEASO 3 with a concentration of 0.5 mg/mL in the above-mentioned disodium hydrogen phosphate-potassium dihydrogen phosphate buffer solution with a concentration of 0.2 mol/L to prepare a solvent; take 10 parts of the above solvent, and in each part of solvent Add 0 μl, 10 μl, 20 μl, 100 μl, 200 μl, 500 μl, 700 μl, 1000 μl, 1500 μl and 2000 μl of γ-globulin standard solution with a concentration of 5 μg/mL into the solution, and add water to make different concentrations of the solution to be tested, and let it stand for 40 minutes After measuring the fluorescence intensity of above-mentioned solution to be measured respectively, as shown in Figure 1, measure the maximum fluorescence intensity (nm) of each solution to be measured to be respectively 534nm, 508nm, 488.7nm, 425.8nm, 396.4nm, 332.2nm, 301.9nm, 247nm, 205.3nm and 182.5nm, where the concentration of the γ-globulin standard solution in Figure 1 from top to bottom is 0μg/L, 5μg/L, 10μg/L, 50μg/L, 100μg/L, 250μg/L , 350 μg/L, 500 μg/L, 750 μg/L and 1000 μg/L; As shown in Figure 2, with the fluorescence intensity of the fluorescence emission peak of solvent (fluorescence intensity value is 534nm) and the fluorescence intensity of the fluorescence emission peak of solution to be tested The ratio is the ordinate, and the concentration of the γ-globulin solution is the abscissa to establish the equation of the fluorescence absorption spectrum curve, and the equation of the fluorescence absorption spectrum curve under 293K to obtain the temperature is: y=1.11246+0.0019x, wherein the linear correlation is R = 0.99789.
如图3所示，按照上述方法分别制得在温度为310K和320K下的荧光吸收光谱曲线图。可以看出，在温度为293K的条件下猝灭常数最高。As shown in FIG. 3 , the fluorescence absorption spectrum curves at temperatures of 310K and 320K were respectively prepared according to the above method. It can be seen that the quenching constant is the highest at the temperature of 293K.
如图4所示，取4份PPEASO3溶液，第1份PPEASO3溶液中不加入γ-球蛋白，第2-4份PPEASO3溶液中分别加入相同体积(10μL)的浓度为100ng/mL、250ng/mL、500ng/mL的γ-球蛋白，测定其荧光衰减时间，其中，未加入γ-球蛋白的荧光衰减时间为0.781ns，加入浓度为100ng/mL的γ-球蛋白的荧光衰减时间为0.787ns，加入250ng/mL的γ-球蛋白的荧光衰减时间为0.793ns、500ng/mL的γ-球蛋白的荧光衰减时间为0.794ns，由此可以很明显地看出，荧光衰减时间几乎不变。因此，进一步确定了γ-球蛋白对PPEASO3聚合物的猝灭是静态猝灭。As shown in Figure 4, take 4 parts of PPEASO 3 solution, do not add γ-globulin to the first part of PPEASO 3 solution , add the same volume (10 μL) of 100 ng/mL, Measure the fluorescence decay time of 250ng/mL and 500ng/mL γ-globulin, among them, the fluorescence decay time of no γ-globulin is 0.781ns, and the fluorescence decay time of 100ng/mL γ-globulin is added The fluorescence decay time of 250ng/mL γ-globulin is 0.793ns, and the fluorescence decay time of 500ng/mL γ-globulin is 0.794ns. It can be clearly seen that the fluorescence decay time is almost constant. Therefore, it was further confirmed that the quenching of PPEASO 3 polymer by γ-globulin was static quenching.
应用例1Application example 1
按照实施例的方法进行操作，不同的是，所述γ-球蛋白溶液的浓度为0.2μg/ml，测得的最大荧光强度为340.8nm，按照上述方程计算得出γ-球蛋白溶液的浓度为A1＝0.196μg/ml。Operate according to the method of the embodiment, the difference is that the concentration of the gamma-globulin solution is 0.2 μg/ml, the maximum fluorescence intensity measured is 340.8 nm, and the concentration of the gamma-globulin solution is calculated according to the above equation It is A1=0.196 μg/ml.
应用例2Application example 2
按照实施例的方法进行操作，不同的是，所述γ-球蛋白溶液的浓度为0.4μg/ml，测得的最大荧光强度为289nm，按照上述方程计算得出γ-球蛋白溶液的浓度为A2＝0.408μg/ml。Operate according to the method of the embodiment, the difference is that the concentration of the gamma-globulin solution is 0.4 μg/ml, and the maximum fluorescence intensity recorded is 289 nm, and the concentration of the gamma-globulin solution calculated according to the above equation is A2 = 0.408 μg/ml.
应用例3Application example 3
按照实施例的方法进行操作，不同的是，所述γ-球蛋白溶液的浓度为0.25μg/ml，测得的最大荧光强度为343.6nm，按照上述方程计算得出γ-球蛋白溶液的浓度为A3＝0.257μg/ml。Operate according to the method of the embodiment, the difference is that the concentration of the gamma-globulin solution is 0.25 μg/ml, and the measured maximum fluorescence intensity is 343.6 nm, and the concentration of the gamma-globulin solution is calculated according to the above equation It was A3=0.257 μg/ml.
应用例4Application example 4
按照实施例的方法进行操作，不同的是，所述γ-球蛋白溶液的浓度为0.5μg/ml，测得的最大荧光强度为284.3nm，按照上述方程计算得出γ-球蛋白溶液的浓度为A4＝0.516μg/ml。Operate according to the method of the embodiment, the difference is that the concentration of the gamma-globulin solution is 0.5 μg/ml, and the maximum fluorescence intensity measured is 284.3 nm, and the concentration of the gamma-globulin solution is calculated according to the above equation It was A4=0.516 μg/ml.
通过上述可以看出，通过本发明的方法测定的γ-球蛋白溶液的浓度与γ-球蛋白溶液的实际浓度值差距很小，因而在检测γ-球蛋白溶液的浓度时具有较高的灵敏度，且操作方法的稳定性良好，可重复性高，而且通过这种方式检测γ-球蛋白溶液的浓度操作方法简单，能快速测得γ-球蛋白溶液的浓度，大大降低了其他蛋白质对检测的干扰。As can be seen from the above, the concentration of the gamma-globulin solution measured by the method of the present invention has a very small gap with the actual concentration of the gamma-globulin solution, thus having a higher sensitivity when detecting the concentration of the gamma-globulin solution , and the operation method has good stability and high repeatability, and the operation method of detecting the concentration of γ-globulin solution in this way is simple, and the concentration of γ-globulin solution can be quickly measured, which greatly reduces the impact of other proteins on detection. interference.
以上详细描述了本发明的优选实施方式，但是，本发明并不限于上述实施方式中的具体细节，在本发明的技术构思范围内，可以对本发明的技术方案进行多种简单变型，这些简单变型均属于本发明的保护范围。The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solutions of the present invention. These simple modifications All belong to the protection scope of the present invention.
另外需要说明的是，在上述具体实施方式中所描述的各个具体技术特征，在不矛盾的情况下，可以通过任何合适的方式进行组合，为了避免不必要的重复，本发明对各种可能的组合方式不再另行说明。In addition, it should be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable way if there is no contradiction. The combination method will not be described separately.
此外，本发明的各种不同的实施方式之间也可以进行任意组合，只要其不违背本发明的思想，其同样应当视为本发明所公开的内容。In addition, various combinations of different embodiments of the present invention can also be combined arbitrarily, as long as they do not violate the idea of the present invention, they should also be regarded as the disclosed content of the present invention.
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Effective date of registration: 20200521 Address after: No. a16, building 25, Hupan green home community, No. 1358, Zuo'an street, Songbei District, Harbin City, Heilongjiang Province Patentee after: Heilongjiang hanpukang Pharmaceutical Co., Ltd Address before: 241002 Anhui Province, Wuhu District of Yijiang City Jiuhua Road No. 189 Technology Service Department Patentee before: ANHUI NORMAL University |