CN108801961B - Biotin detection method - Google Patents
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- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 title claims abstract description 154
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Abstract
Description
技术领域technical field
本发明涉及一种生物素的检测方法，尤其涉及一种可快速、准确、灵敏的检测生物素的方法，属于生物传感领域。The invention relates to a method for detecting biotin, in particular to a method for detecting biotin rapidly, accurately and sensitively, and belongs to the field of biosensing.
背景技术Background technique
生物素作为B族类维生素，参与机体的多种新陈代谢活动，被广泛应用于食品、医药、饲料、发酵等领域，对工农业生产和人们的日常生活产生了重要的影响。通过检测生物素的含量可以对有关生物素的工农业生产加以指导，实现产品的最优化，维护人们的健康。目前，国内外关于生物素的检测方法主要有微生物法、高效液相色谱法、酶联免疫分析法、光谱法和电化学法等。然而，微生物法准确度差且整个操作流程费时、费力；高效液相色谱法所需仪器和试剂昂贵且样品处理过程较为复杂，对研究人员的技术要求较高；基于生物素和亲和素特异性结合的酶联免疫法和光谱法虽然操作简单、分析速度快且准确度高，但是这类方法所用的试剂亲和素或链霉亲和素市场造价较高，难以在生产上进行推广应用。因此，研发一种便宜、简单、快速、准确、灵敏的生物素检测方法具有重要的意义。As a B vitamin, biotin participates in various metabolic activities of the body, and is widely used in food, medicine, feed, fermentation and other fields, and has an important impact on industrial and agricultural production and people's daily life. By detecting the content of biotin, the industrial and agricultural production of biotin can be instructed, the optimization of products can be realized, and people's health can be maintained. At present, the detection methods of biotin at home and abroad mainly include microbiological method, high performance liquid chromatography, enzyme-linked immunoassay, spectroscopic method and electrochemical method. However, the microbiological method has poor accuracy and the entire operation process is time-consuming and labor-intensive; high-performance liquid chromatography requires expensive instruments and reagents, and the sample processing process is complex, which requires high technical requirements for researchers; based on the specificity of biotin and avidin Although the combined enzyme-linked immunosorbent assay and spectroscopic method are simple in operation, fast in analysis and high in accuracy, the reagents avidin or streptavidin used in such methods have high market costs and are difficult to popularize and apply in production. Therefore, it is of great significance to develop a cheap, simple, rapid, accurate and sensitive method for biotin detection.
发明内容SUMMARY OF THE INVENTION
针对上述现有技术，本发明提供了一种快速、准确、灵敏且成本低的生物素的检测方法。In view of the above prior art, the present invention provides a fast, accurate, sensitive and low-cost biotin detection method.
本发明是通过以下技术方案实现的：The present invention is achieved through the following technical solutions:
一种生物素的检测方法，包括以下步骤：A method for detecting biotin, comprising the following steps:
一种生物素的检测方法，其特征在于：包括以下步骤：A kind of detection method of biotin, it is characterized in that: comprise the following steps:
a.取0.1～2mL的CH3(CH2)15(CH3)3N-AuCl4复合物，加入到3mL0.01M的PBS缓冲溶液中，再加入标准生物素形成不同浓度的标准生物素溶液，混合均匀后加入比色皿中，记录不同浓度的标准生物素溶液在267nm波长处的吸光度值，以267nm波长处吸光度A的变化值和生物素的浓度绘制标准曲线；a. Take 0.1-2 mL of CH 3 (CH 2 ) 15 (CH 3 ) 3 N-AuCl 4 complex, add it to 3 mL of 0.01M PBS buffer solution, and then add standard biotin to form standard biotin solutions of different concentrations , after mixing evenly, add it to the cuvette, record the absorbance values of standard biotin solutions of different concentrations at the wavelength of 267 nm, and draw the standard curve with the change value of the absorbance A at the wavelength of 267 nm and the concentration of biotin;
b.取0.1～2mL的CH3(CH2)15(CH3)3N-AuCl4复合物，加入到3mL0.01M的PBS缓冲溶液中，再加入待检测的生物素样品，混合均匀后加入比色皿中，记录267nm波长处的吸光度，通过标准工作曲线，计算得到待检测的生物素样品的浓度。b. Take 0.1-2 mL of CH 3 (CH 2 ) 15 (CH 3 ) 3 N-AuCl 4 complex, add it to 3 mL of 0.01M PBS buffer solution, then add the biotin sample to be detected, mix well and add In the cuvette, record the absorbance at the wavelength of 267 nm, and calculate the concentration of the biotin sample to be detected through the standard working curve.
或者：包括以下步骤：OR: Include the following steps:
a.取0.1～2mL的CH3(CH2)15(CH3)3N-AuCl4复合物，加入到3mL0.01M的PBS缓冲溶液中，混合均匀后加入比色皿中，得到500～200nm波长范围的吸收光谱，作为基线；a. Take 0.1-2 mL of CH 3 (CH 2 ) 15 (CH 3 ) 3 N-AuCl 4 complex, add it to 3 mL of 0.01M PBS buffer solution, mix well and add it to the cuvette to obtain 500-200 nm The absorption spectrum of the wavelength range, as the baseline;
b.取0.1～2mL的CH3(CH2)15(CH3)3N-AuCl4复合物，加入到3mL0.01M的PBS缓冲溶液中，再加入标准生物素形成不同浓度的标准生物素溶液，混合均匀后加入比色皿中，得到不同浓度的生物素的吸收光谱；b. Take 0.1-2 mL of CH 3 (CH 2 ) 15 (CH 3 ) 3 N-AuCl 4 complex, add it to 3 mL of 0.01M PBS buffer solution, and then add standard biotin to form standard biotin solutions of different concentrations , mixed evenly and added to the cuvette to obtain the absorption spectra of different concentrations of biotin;
c.记录不同浓度的标准生物素溶液在267nm波长处的吸光度值，以267nm波长处吸光度A的变化值和生物素的浓度绘制标准曲线；c. Record the absorbance values of standard biotin solutions of different concentrations at the wavelength of 267 nm, and draw a standard curve with the change value of absorbance A at the wavelength of 267 nm and the concentration of biotin;
d.取0.1～2mL的CH3(CH2)15(CH3)3N-AuCl4复合物，加入到3mL0.01M的PBS缓冲溶液中，再加入待检测的生物素样品，混合均匀后加入比色皿中，得到500～200nm波长范围的吸收光谱，记录267nm波长处的吸光度，通过标准工作曲线，常规方法计算得到待检测的生物素样品的浓度。d. Take 0.1-2 mL of CH 3 (CH 2 ) 15 (CH 3 ) 3 N-AuCl 4 complex, add it to 3 mL of 0.01M PBS buffer solution, then add the biotin sample to be detected, mix well and add In the cuvette, obtain the absorption spectrum in the wavelength range of 500-200 nm, record the absorbance at the wavelength of 267 nm, and calculate the concentration of the biotin sample to be detected through the standard working curve and conventional methods.
所述的CH3(CH2)15(CH3)3N-AuCl4复合物，是由氯金酸和阳离子表面活性剂十六烷基三甲基溴化铵(CTAB)反应得到的：取10～1000mg的CTAB，加水50ml，加热搅拌溶解后，室温条件下加入浓度为0.01～5mg/L的氯金酸溶液，搅拌0.5～10小时，即可得到橘黄色的CH3(CH2)15(CH3)3N-AuCl4复合物。Described CH 3 (CH 2 ) 15 (CH 3 ) 3 N-AuCl 4 complex is obtained by reacting chloroauric acid and cationic surfactant cetyl trimethyl ammonium bromide (CTAB): 10-1000 mg of CTAB, add 50 ml of water, heat and stir to dissolve, add chloroauric acid solution with a concentration of 0.01-5 mg/L at room temperature, and stir for 0.5-10 hours to obtain orange CH 3 (CH 2 ) 15 (CH3) 3N - AuCl4 complex.
本发明的生物素的检测方法，其检测原理为：氯金酸与十六烷基三甲基溴化铵可形成CH3(CH2)15(CH3)3N-AuCl4复合物，加入生物素样品可替代AuCl4 -的结合，形成CH3(CH2)15(CH3)3N-生物素，从而导致吸光光谱发生改变，吸光度发生改变。利用紫外可见分光光度计测定加入不同浓度生物素后吸光度的变化值(267nm波长处的吸光度值)，以267nm波长处的吸光度A的变化值对生物素的浓度C绘制标准工作曲线，对照标准工作曲线即得未知样品中生物素的浓度。The detection method of biotin of the present invention, the detection principle is as follows: chloroauric acid and cetyltrimethylammonium bromide can form CH 3 (CH 2 ) 15 (CH 3 ) 3 N-AuCl 4 complex, add The biotin sample can replace the binding of AuCl 4 - to form CH 3 (CH 2 ) 15 (CH 3 ) 3 N-biotin, resulting in a change in the absorbance spectrum and a change in absorbance. Utilize UV-Vis spectrophotometer to measure the change value of absorbance after adding different concentrations of biotin (absorbance value at 267nm wavelength), draw a standard working curve to the concentration C of biotin with the change value of absorbance A at 267nm wavelength, and compare the standard work The curve is the concentration of biotin in the unknown sample.
本发明的生物素的检测方法，具有以下优点：The detection method of biotin of the present invention has the following advantages:
1.本发明直接利用氯金酸和CTAB的复合物，无需复杂的合成工艺，因此操作简单，误差较小。1. The present invention directly utilizes the complex of chloroauric acid and CTAB without complicated synthesis process, so the operation is simple and the error is less.
2.本发明无需借助亲和素或链霉亲和素等价格昂贵的试剂，大大节约了检测成本。2. The present invention does not need expensive reagents such as avidin or streptavidin, which greatly saves the detection cost.
3.本发明不需要昂贵的分析仪器，方法简单，检测速度快，灵敏度高，线性范围宽。3. The present invention does not need expensive analytical instruments, the method is simple, the detection speed is fast, the sensitivity is high, and the linear range is wide.
4.本发明利用紫外区间的光谱进行分析，避免了因发酵液本身颜色而产生的干扰。4. The present invention utilizes the spectrum in the ultraviolet range for analysis, avoiding the interference caused by the color of the fermentation broth itself.
本发明所引述的所有文献，它们的全部内容通过引用并入本文，并且如果这些文献所表达的含义与本发明不一致时，以本发明的表述为准。此外，本发明使用的各种术语和短语具有本领域技术人员公知的一般含义。All documents cited in the present invention, their entire contents are incorporated herein by reference, and if the meaning expressed by these documents is inconsistent with the present invention, the expression of the present invention shall prevail. Also, various terms and phrases used herein have their ordinary meanings that are commonly known to those skilled in the art.
附图说明Description of drawings
图1：含有不同量CTAB的CH3(CH2)15(CH3)3N-AuCl4复合物的吸收光谱。Figure 1: Absorption spectra of CH3 ( CH2 ) 15 ( CH3 ) 3N - AuCl4 complexes containing different amounts of CTAB.
图2：不同浓度生物素的响应曲线。Figure 2: Response curves of different concentrations of biotin.
图3：生物素的标准工作曲线(左图为低浓度范围的，右图为高浓度范围的)。Figure 3: Standard working curve for biotin (left graph is in the low concentration range, right graph is in the high concentration range).
图4：本发明的检测方法的选择性测试结果。Figure 4: Selectivity test results of the detection method of the present invention.
具体实施方式Detailed ways
下面结合实施例对本发明作进一步的说明。然而，本发明的范围并不限于下述实施例。本领域的专业人员能够理解，在不背离本发明的精神和范围的前提下，可以对本发明进行各种变化和修饰。The present invention will be further described below in conjunction with the examples. However, the scope of the present invention is not limited to the following examples. It will be understood by those skilled in the art that various changes and modifications can be made in the present invention without departing from the spirit and scope of the inventions.
下述实施例中所涉及的仪器、试剂、材料等，若无特别说明，均为现有技术中已有的常规仪器、试剂、材料等，可通过正规商业途径获得。下述实施例中所涉及的实验方法，检测方法等，若无特别说明，均为现有技术中已有的常规实验方法，检测方法等。The instruments, reagents, materials, etc. involved in the following examples, unless otherwise specified, are all conventional instruments, reagents, materials, etc. existing in the prior art, and can be obtained through regular commercial channels. The experimental methods, detection methods, etc. involved in the following examples, unless otherwise specified, are all conventional experimental methods, detection methods, etc. in the prior art.
实施例1测试缓冲溶液中生物素的浓度Example 1 Testing the concentration of biotin in buffer solution
测定步骤如下：The measurement steps are as follows:
a.以CH3(CH2)15(CH3)3N-AuCl4复合物为识别分子，0.01M的PBS为缓冲溶液，紫外可见分光光度计记录吸收光谱。取0.5mL的CH3(CH2)15(CH3)3N-AuCl4复合物，加入到3mL0.01M的PBS缓冲溶液中，混合均匀后加入比色皿中，得到500～200nm波长范围的吸收光谱(如图1所示)，记录267nm波长处的吸光度，作为基线。a. Using CH 3 (CH 2 ) 15 (CH 3 ) 3 N-AuCl 4 complex as recognition molecule, 0.01M PBS as buffer solution, record the absorption spectrum with UV-Vis spectrophotometer. Take 0.5mL of CH 3 (CH 2 ) 15 (CH 3 ) 3 N-AuCl 4 complex, add it to 3mL of 0.01M PBS buffer solution, mix it evenly and add it to the cuvette to obtain 500～200nm wavelength range Absorbance spectra (shown in Figure 1), and absorbance at a wavelength of 267 nm were recorded as the baseline.
CH3(CH2)15(CH3)3N-AuCl4复合物的制备过程：取50mg的CTAB，加水50ml，加热搅拌溶解后，室温条件下加入0.1mg/L的氯金酸溶液，搅拌1小时，即可得到橘黄色的CH3(CH2)15(CH3)3N-AuCl4复合物。Preparation process of CH 3 (CH 2 ) 15 (CH 3 ) 3 N-AuCl 4 complex: take 50 mg of CTAB, add 50 ml of water, heat and stir to dissolve, add 0.1 mg/L chloroauric acid solution at room temperature, and stir After 1 hour, an orange CH 3 (CH 2 ) 15 (CH 3 ) 3 N-AuCl 4 complex can be obtained.
b.取0.5mL的CH3(CH2)15(CH3)3N-AuCl4复合物，加入到3mL0.01M的PBS缓冲溶液中，再加入生物素得到不同浓度的标准生物素溶液(5～400μg/L)，混合均匀后加入比色皿中，得到不同浓度生物素的吸收光谱，如图2所示。b. Take 0.5 mL of CH 3 (CH 2 ) 15 (CH 3 ) 3 N-AuCl 4 complex, add it to 3 mL of 0.01M PBS buffer solution, and then add biotin to obtain standard biotin solutions of different concentrations (5 ~400μg/L), mixed evenly and added to the cuvette to obtain the absorption spectra of different concentrations of biotin, as shown in Figure 2.
c.分别记录267nm波长处的吸光度值，以267nm波长处吸光度A的变化值和生物素的浓度绘制标准曲线，如图3所示。标准曲线也可以根据步骤a和步骤b的曲线的计算出来。c. Record the absorbance values at the wavelength of 267 nm respectively, and draw a standard curve with the change value of the absorbance A at the wavelength of 267 nm and the concentration of biotin, as shown in FIG. 3 . The standard curve can also be calculated from the curves of step a and step b.
性能测试：测定该方法对葡萄糖、乳糖、麦芽糖、蔗糖、钠离子、钾离子、镁离子、氯离子等的干扰。Performance test: determine the interference of this method on glucose, lactose, maltose, sucrose, sodium ion, potassium ion, magnesium ion, chloride ion, etc.
取0.5mL的CH3(CH2)15(CH3)3N-AuCl4复合物，加入到3mL0.01M的PBS缓冲溶液中，再分别加入生物素、葡萄糖、乳糖、麦芽糖、蔗糖、钠离子、钾离子、镁离子、氯离子，使其最终浓度均为100μg/L，混合均匀后分别加入比色皿中，得到这些待测物的吸收光谱，如图4所示。从图4可以看出100μg/L的生物素在267nm处的吸光度较大，而葡萄糖、乳糖、麦芽糖等在267nm处的吸光度很小，可以忽略，因此这些离子不会对生物素检测产生影响，说明该方法具有较好的选择性，因此本法有望应用于发酵液或细胞液中生物素的检测。Take 0.5mL of CH 3 (CH 2 ) 15 (CH 3 ) 3 N-AuCl 4 complex, add it to 3mL of 0.01M PBS buffer solution, and then add biotin, glucose, lactose, maltose, sucrose, sodium ions respectively , potassium ion, magnesium ion, and chloride ion, so that the final concentration is 100 μg/L. After mixing evenly, they were added to the cuvette respectively to obtain the absorption spectra of these analytes, as shown in Figure 4. It can be seen from Figure 4 that the absorbance of 100μg/L biotin at 267nm is large, while the absorbance of glucose, lactose, maltose, etc. at 267nm is very small and can be ignored, so these ions will not affect the detection of biotin, It shows that the method has good selectivity, so this method is expected to be applied to the detection of biotin in fermentation broth or cell fluid.
实施例2Example 2
采用标准加入法测定一低浓度未知样品中生物素的量。取0.01M PBS缓冲溶液配置了两个加标试样，浓度分别为20μg/L和200μg/L，依照实施例1的测定方法，记录267nm波长处的吸光度值A，对照标准工作曲线(如图3)，计算出相应的浓度。A standard addition method was used to determine the amount of biotin in a low-concentration unknown sample. Take 0.01M PBS buffer solution and configure two spiked samples with concentrations of 20 μg/L and 200 μg/L, respectively. According to the measurement method in Example 1, record the absorbance value A at the wavelength of 267 nm, and compare it to the standard working curve (as shown in the figure). 3), calculate the corresponding concentration.
实施例3测试饲料中生物素的含量Example 3 The content of biotin in the test feed
测定步骤如下：The measurement steps are as follows:
a.取1g某饲料，溶于100ml0.01M的PBS缓冲溶液中，过滤除去不溶物。a. Take 1g of a certain feed, dissolve it in 100ml of 0.01M PBS buffer solution, and filter to remove insoluble matter.
b.取0.5mL的CH3(CH2)15(CH3)3N-AuCl4复合物，加入到3mL0.01M的PBS缓冲溶液中，再加入含有生物素的饲料样品，混合均匀后加入比色皿中，得到500～200nm波长范围的吸收光谱，记录267nm波长处的吸光度，通过样品溶液267nm波长处的吸光度A，对照标准工作曲线，得到待测饲料中生物素的含量。b. Take 0.5 mL of CH 3 (CH 2 ) 15 (CH 3 ) 3 N-AuCl 4 complex, add it to 3 mL of 0.01M PBS buffer solution, and then add biotin-containing feed samples, mix evenly, and add the ratio of In the color dish, obtain the absorption spectrum in the wavelength range of 500-200 nm, record the absorbance at the wavelength of 267 nm, and compare the standard working curve with the absorbance A at the wavelength of 267 nm of the sample solution to obtain the content of biotin in the feed to be tested.
实施例4测试发酵液中生物素的含量Example 4 Testing the content of biotin in fermentation broth
测定步骤如下：The measurement steps are as follows:
a.取某发酵液，加0.01M的PBS缓冲溶液稀释10倍，过滤除去不溶物，放置待测。a. Take a fermentation broth, add 0.01M PBS buffer solution to dilute 10 times, filter to remove insoluble matter, and place it for testing.
b.取0.5mL的CH3(CH2)15(CH3)3N-AuCl4复合物，加入到3mL0.01M的PBS缓冲溶液中，再加入含有生物素的样品，混合均匀后加入比色皿中，得到500～200nm波长范围的吸收光谱，记录267nm波长处的吸光度，通过样品溶液267nm波长处的吸光度A和背景溶液267nm波长处吸光度A的差值与标准工作曲线对照，得到发酵液中生物素的含量。b. Take 0.5 mL of CH 3 (CH 2 ) 15 (CH 3 ) 3 N-AuCl 4 complex, add it to 3 mL of 0.01M PBS buffer solution, then add the sample containing biotin, mix well and add colorimetric In the dish, the absorption spectrum in the wavelength range of 500 to 200 nm is obtained, the absorbance at the wavelength of 267 nm is recorded, and the difference between the absorbance A at the wavelength of 267 nm of the sample solution and the absorbance A at the wavelength of 267 nm of the background solution is compared with the standard working curve to obtain the fermentation broth. biotin content.
给本领域技术人员提供上述实施例，以完全公开和描述如何实施和使用所主张的实施方案，而不是用于限制本文公开的范围。对于本领域技术人员而言显而易见的修饰将在所附权利要求的范围内。The foregoing examples are provided to those skilled in the art to fully disclose and describe how to make and use the claimed embodiments, and are not intended to limit the scope of the disclosure herein. Modifications obvious to those skilled in the art are intended to be within the scope of the appended claims.
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