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CN111189823B - A TMB chromogenic system and its application in detecting reducing substances - Google Patents

A TMB chromogenic system and its application in detecting reducing substances Download PDF

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CN111189823B
CN111189823B CN202010114473.XA CN202010114473A CN111189823B CN 111189823 B CN111189823 B CN 111189823B CN 202010114473 A CN202010114473 A CN 202010114473A CN 111189823 B CN111189823 B CN 111189823B
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刘伟
张柳
侯越
吕聪聪
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Shaanxi Normal University
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Abstract

本发明公开了一种TMB显色体系及其检测还原性物质的应用，所述TMB显色体系由pH值为3.0～7.0的醋酸‑醋酸钠缓冲溶液、0.08～1.5mg/mL的Fe配合物水溶液、0.4～2.0mmol/L的TMB溶液组成，其中Fe配合物为Fe(Ⅲ)的邻菲啰啉或吡啶配合物。在无需过氧化氢存在的条件下，本发明显色体系中的Fe配合物氧化TMB产生蓝色氧化产物ox‑TMB，在652nm处产生最大紫外吸收。当向该体系中加入还原性物质谷胱甘肽、抗坏血酸等时，会与氧化产物ox‑TMB发生还原反应，使体系颜色变浅，且变浅的程度与还原性物质的浓度呈现正相关。因此，可实现这些还原性物质的比色检测。本发明显色体系无需强氧化剂的使用，检测时无需加热，具有操作简单、显色快速、灵敏度高、污染小、检出限低等优点。

Figure 202010114473

The present invention discloses a TMB color developing system and its application for detecting reducing substances. It is composed of an aqueous solution and a 0.4-2.0 mmol/L TMB solution, wherein the Fe complex is the o-phenanthroline or pyridine complex of Fe(III). Without the presence of hydrogen peroxide, the Fe complex in the color developing system of the present invention oxidizes TMB to produce a blue oxidation product ox-TMB, which produces a maximum ultraviolet absorption at 652 nm. When the reducing substances glutathione, ascorbic acid, etc. are added to the system, a reduction reaction will occur with the oxidation product ox-TMB, so that the color of the system becomes light, and the degree of lightening is positively correlated with the concentration of the reducing substances. Therefore, colorimetric detection of these reducing substances can be achieved. The color developing system of the invention does not need the use of strong oxidants, does not need to be heated during detection, and has the advantages of simple operation, rapid color development, high sensitivity, little pollution, low detection limit and the like.

Figure 202010114473

Description

TMB color development system and application thereof in detection of reducing substances
Technical Field
The invention belongs to the technical field of color comparison, and particularly relates to a novel TMB color development system and application of the color development system in detection of reducing substances.
Background
A reducing substance such as Glutathione (GSH) is a tripeptide composed of glycine, glutamic acid, and cysteine, and is present almost in every cell. Glutathione has important function on the body, and has the functions of maintaining the normal immune system in the body, resisting oxidation, detoxifying and the like. Glutathione levels are associated with health and disease. Therefore, it is of great value to find a method for quickly and simply detecting low-concentration glutathione. Ascorbic Acid (AA), also known as vitamin C, is a vitamin that is most needed by humans, is also an essential nutrient for higher primates, participates in many important biosynthetic processes, is one of the important vitamins for maintaining normal physiological functions of the body, and is also an antioxidant and preservative. Therefore, the development and design of a method for detecting the low-concentration ascorbic acid have important theoretical value and application value.
In recent years, many analytical methods for detecting glutathione, ascorbic acid, and the like have been developed, and for example, various methods such as mass spectrometry, fluorescence, electrochemical methods, colorimetric methods, high performance liquid chromatography, and high performance capillary electrophoresis have been developed. These methods, although highly sensitive, still have some drawbacks, among which the chromatographic operations are complex and time consuming; the fluorescence method has large background interference influence and large error; the colorimetric method is simple and rapid to operate, and the ultraviolet spectrophotometer instrument is low in price, simple and convenient to operate and rapid in time. Therefore, a new colorimetric method for detecting the reduced substances has important significance and value.
Disclosure of Invention
The invention aims to change the traditional TMB-H2O2The chemiluminescence system provides a new TMB color development system without the participation of strong oxidant such as hydrogen peroxide, and provides a new application for the color development system.
Aiming at the purposes, the TMB color development system adopted by the invention consists of acetic acid-sodium acetate buffer solution with the pH value of 3.0-7.0, Fe complex aqueous solution with the concentration of 0.08-1.5 mg/mL and TMB solution with the concentration of 0.4-2.0 mmol/L.
The Fe complex is an o-phenanthroline complex of Fe (III) shown in a formula A or a pyridine complex of Fe (III) shown in a formula B:
Figure BDA0002391036820000021
in the TMB color development system, the concentration of the Fe complex aqueous solution is preferably 0.8-1.5 mg/mL, the concentration of the TMB solution is 0.8-1.2 mmol/L, and the pH value of the acetic acid-sodium acetate buffer solution is preferably 4.0-6.0, wherein the TMB solution is prepared by dissolving 3,3',5,5' -tetramethyl benzidine with ethanol and then adding deionized water.
The invention relates to an application of a TMB color development system in detection of a reducing substance, wherein the reducing substance is any one of glutathione, ascorbic acid and cysteine. The specific detection method comprises the following steps: adding an Fe complex aqueous solution into an acetic acid-sodium acetate buffer solution, then adding a TMB solution, then adding reducing substance standard sample solutions with different concentrations, incubating at room temperature for 5-20 minutes, and detecting the absorbance of a system at 652nm by an ultraviolet absorption spectrometer; wherein, when the reducing substance is glutathione or cysteine, a linear relation between the absorbance change value and the concentration of the reducing substance is constructed, and when the reducing substance is ascorbic acid, a linear relation between the absorbance and the concentration of the reducing substance is constructed; and detecting the reducing substances with unknown concentration by utilizing the linear relation.
In the application, the volume ratio of the acetic acid-sodium acetate buffer solution to the TMB solution and the Fe complex aqueous solution is preferably 20:1: 5-20: 1: 10.
In the color development system, superoxide anion free radicals and iron-oxygen intermediates are generated in a solution due to the activity of oxidase of an o-phenanthroline complex of Fe (III) or a pyridine complex of Fe (III), and 3,3',5,5' -Tetramethylbenzidine (TMB) can be oxidized to generate a blue oxidation product ox-TMB without the presence of hydrogen peroxide, so that maximum ultraviolet absorption is generated at 652 nm. When a trace amount of reducing substance is added into the system, the reducing substance and the oxidation product ox-TMB undergo a reduction reaction, so that the color of the system is lightened, and the lightening degree is in positive correlation with the concentration of the reducing substance. Thus, colorimetric detection of the reduced matter can be realized.
Compared with the prior art, the invention has the following beneficial effects:
1. the TMB color development system does not need strong oxidant such as hydrogen peroxide, and avoids the defects of instability, influence of temperature environment, harm and the like of the hydrogen peroxide.
2. The TMB color development system is used for detecting glutathione, ascorbic acid and the like which are reducing substances, does not need heating, and has the advantages of simple operation, quick color development, high sensitivity, small pollution, low detection limit and the like.
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FIG. 1 is a schematic diagram of detection of glutathione.
FIG. 2 is a graph showing the ultraviolet absorption spectra of glutathione at different concentrations in example 1.
FIG. 3 is a graph showing the relationship between the concentration of glutathione varied in example 1 and the change in absorbance (. DELTA.A) at 652 nm.
FIG. 4 is a linear relationship between glutathione concentrations (0.4. mu. mol/L to 1.0. mu. mol/L) and the change in absorbance (. DELTA.A) at 652nm in example 1.
FIG. 5 is a linear relationship between glutathione concentrations (1.0. mu. mol/L to 100. mu. mol/L) and the change in absorbance (. DELTA.A) at 652nm in example 1.
FIG. 6 is a linear relationship between the absorbance and the concentration of ascorbic acid in example 5.
FIG. 7 shows the results of selectivity of glutathione and resistance to metal ion interference.
FIG. 8 shows the results of selectivity for glutathione and resistance to amino acid and glucose interference.
Detailed description of the invention
The invention will be further described in detail with reference to the following figures and examples, but the scope of the invention is not limited to these examples.
The formulation method of the TMB solution in the following examples was: dissolving 0.024g of 3,3',5,5' -Tetramethylbenzidine (TMB) in 0.5mL of ethanol water solution with the volume concentration of 95%, ultrasonically shaking for 5 minutes to prepare a 20.0mmol/L TMB ethanol solution, and diluting the 20.0mmol/L TMB ethanol solution to the target concentration by using deionized water.
Example 1
The TMB color development system of this embodiment consists of an acetic acid-sodium acetate buffer solution with a pH value of 5.0, an o-phenanthroline complex aqueous solution of Fe (iii) of 0.8mg/mL, and a TMB solution of 1.0 mmol/L.
The application of the TMB color development system in glutathione detection is shown in figure 1, and the specific method is as follows:
adding 20 mu L of 0.8mg/mL o-phenanthroline complex water solution of Fe (III) into 400 mu L p at room temperatureAdding 100 mu L of 1.0mmol/L TMB solution into acetic acid-sodium acetate buffer solution with the H value of 5.0 to obtain a TMB color development system; to the obtained TMB color development system, 150. mu.L of glutathione aqueous solutions of different concentrations (0. mu. mol/L, 0.4. mu. mol/L, 0.6. mu. mol/L, 0.8. mu. mol/L, 1.0. mu. mol/L, 4. mu. mol/L, 6. mu. mol/L, 8. mu. mol/L, 10. mu. mol/L, 20. mu. mol/L, 40. mu. mol/L, 60. mu. mol/L, 80. mu. mol/L, 100. mu. mol/L) were added, respectively, mixed, and then incubated at room temperature for 10 minutes. The ultraviolet absorption spectrum of the system was detected by an ultraviolet-visible spectrophotometer (see fig. 2), and a linear relationship between the change in absorbance (Δ a) at 652nm and the glutathione concentration (C) shown in fig. 3 was obtained. To be able to obtain a better linearity we use piecewise linearity. As shown in FIG. 4, when the glutathione concentration is 0.4. mu. mol/L to 1.0. mu. mol/L, the linear equation is that. DELTA.A is 0.03875C +0.085, R20.9840; as shown in FIG. 5, the glutathione concentration is in the range of 1.0. mu. mol/L to 100. mu. mol/L, and the linear equation is that Δ A is 0.0061C +0.038, R20.9947. According to the 3. sigma. rule, the detection limit of glutathione was 0.1. mu. mol/L.
Example 2
The TMB color development system of this embodiment consists of an acetic acid-sodium acetate buffer solution with a pH value of 4.0, an o-phenanthroline complex aqueous solution of Fe (iii) of 1.2mg/mL, and a TMB solution of 0.8 mmol/L.
The method for detecting glutathione using the above-mentioned TMB color development system was the same as in example 1.
Example 3
The TMB color development system of this embodiment consists of an acetic acid-sodium acetate buffer solution with a pH value of 6.0, an o-phenanthroline complex aqueous solution of Fe (iii) of 1.5mg/mL, and a TMB solution of 1.2 mmol/L.
The method for detecting glutathione using the above-mentioned TMB color development system was the same as in example 1.
Example 4
The TMB color development system of this example consisted of an acetic acid-sodium acetate buffer solution with a pH of 5.0, an aqueous solution of 0.8mg/mL of Fe (III) pyridine complex, and a 1.0mmol/L TMB solution.
The method for detecting glutathione using the above-mentioned TMB color development system was the same as in example 1.
Example 5
The TMB color development system of this embodiment consists of an acetic acid-sodium acetate buffer solution with a pH value of 5.0, an o-phenanthroline complex aqueous solution of Fe (iii) of 0.8mg/mL, and a TMB solution of 1.0 mmol/L.
The application of the TMB color development system in ascorbic acid detection is as follows:
under the condition of room temperature, adding 20 mu L of 0.8mg/mL o-phenanthroline complex water solution of Fe (III) into 400 mu L of acetic acid-sodium acetate buffer solution with the pH value of 5.0, and then adding 100 mu L of 1.0mmol/L TMB solution to obtain a TMB developing system; to the obtained TMB color development system, 150. mu.L of ascorbic acid aqueous solutions (0. mu. mol/L, 10. mu. mol/L, 20. mu. mol/L, 40. mu. mol/L, 60. mu. mol/L, 80. mu. mol/L) were added, mixed, and incubated at room temperature for 10 minutes. The linear relationship between the absorbance at 652nm and the ascorbic acid at different concentrations was obtained by measuring the uv absorption spectrum of the system by uv-vis spectroscopy (see fig. 6).
To demonstrate the beneficial effects of the present invention, the inventors conducted the following tests using the TMB color system assay system of example 1:
1. detection of glutathione Selectivity
To evaluate the selectivity of detecting Glutathione (GSH), various metal ions, amino acids, and glucose interfering substances were included and detected. Adding 100 μ L of 0.1mmol/L glutathione water solution into TMB color development system, or respectively adding 100 μ L of 2mmol/L metal ion such as Na+、K+、Ba2+、Mg2+、Cr3+、Zn2+、Co2+、Cu2+、Ca2+、Cd2+、Mn2+Or adding 100. mu.L of 2mmol/L amino acids such as phenylalanine (Phe), threonine (Thr), serine (Ser), alanine (Ala), glutamic acid (Glu), arginine (Arg), valine (Val), methionine (Met), leucine (Leu), proline (Pro), tyrosine (Tyr), lysine (Lys) and glucose, mixing, and incubating at room temperature for 10 minutes. The detection system showed a UV absorption peak at 652nm, which is shown in FIGS. 7 and 8. The results show that the delta A value with glutathione is significantly higher compared to other interfering substances.The chromogenic system of the invention has better selectivity to glutathione.
2. Measuring glutathione in tablets
In order to prove the reliability of the practical application of the chromogenic system for detecting glutathione, glutathione in tablets is detected, and the result is shown in table 1.
TABLE 1
Figure BDA0002391036820000051
As can be seen from Table 1, the recovery rate of glutathione is between 98.3% and 105.0%, and the relative standard deviation RSD value is between 1.3% and 3.8%. The developing system of the invention is proved to have good reliability for detecting the glutathione in the actual sample.

Claims (7)

1.TMB显色体系在还原性物质检测中的应用，所述的还原性物质为谷胱甘肽、抗坏血酸、半胱氨酸中任意一种；TMB显色体系，其特征在于：所述TMB显色体系由pH值为3.0～7.0的醋酸-醋酸钠缓冲溶液、0.08～1.5 mg/mL的Fe配合物水溶液、0.4～2.0 mmol/L的TMB溶液组成；1. the application of TMB color developing system in the detection of reducing substance, and described reducing substance is any one in glutathione, ascorbic acid, cysteine; TMB color developing system is characterized in that: described TMB The color developing system is composed of acetic acid-sodium acetate buffer solution with pH value of 3.0-7.0, Fe complex aqueous solution of 0.08-1.5 mg/mL, and TMB solution of 0.4-2.0 mmol/L; 上述Fe配合物为是式A所示的Fe(Ⅲ)的吡啶配合物或式B所示的Fe(Ⅲ)的邻菲啰啉配合物：The above-mentioned Fe complex is a pyridine complex of Fe(III) represented by formula A or an o-phenanthroline complex of Fe(III) represented by formula B:
Figure DEST_PATH_IMAGE001
Figure DEST_PATH_IMAGE003
。
Figure DEST_PATH_IMAGE001
Figure DEST_PATH_IMAGE003
. 2.根据权利要求1所述的TMB显色体系在还原性物质检测中的应用，其特征在于：将Fe配合物水溶液加入到醋酸-醋酸钠缓冲溶液中，然后加入TMB溶液，再加入不同浓度的还原性物质标准样品溶液，室温孵育5～20分钟后，通过紫外吸收光谱仪检测体系在652 nm处的吸光度；其中，还原性物质为谷胱甘肽或半胱氨酸时，构建吸光度变化值与还原性物质浓度的线性关系，还原性物质为抗坏血酸时，构建吸光度与还原性物质浓度的线性关系；利用此线性关系检测未知浓度的还原性物质。2. the application of TMB color developing system according to claim 1 in the detection of reducing substances, it is characterized in that: the Fe complex aqueous solution is joined in acetic acid-sodium acetate buffer solution, then add TMB solution, then add different concentrations After incubating for 5-20 minutes at room temperature, the absorbance of the system at 652 nm was detected by ultraviolet absorption spectrometer; among them, when the reducing substance was glutathione or cysteine, the absorbance change value was constructed. Linear relationship with the concentration of reducing substances. When the reducing substance is ascorbic acid, a linear relationship between absorbance and the concentration of reducing substances is constructed; this linear relationship is used to detect reducing substances of unknown concentration. 3.根据权利要求2所述的TMB显色体系在还原性物质检测中的应用，其特征在于：所述醋酸-醋酸钠缓冲溶液与TMB溶液、Fe配合物水溶液的体积比为20:1:5～20:1:10。3. the application of TMB color developing system according to claim 2 in the detection of reducing substances, it is characterized in that: the volume ratio of described acetic acid-sodium acetate buffer solution and TMB solution, Fe complex aqueous solution is 20:1: 5～20:1:10. 4.根据权利要求1所述的TMB显色体系在还原性物质检测中的应用，其特征在于：所述Fe配合物水溶液的浓度为0.8～1.5 mg/mL。4. The application of the TMB color developing system according to claim 1 in the detection of reducing substances, wherein the concentration of the Fe complex aqueous solution is 0.8-1.5 mg/mL. 5.根据权利要求1所述的TMB显色体系在还原性物质检测中的应用，其特征在于：所述TMB溶液的浓度为0.8～1.2 mmol/L。5 . The application of the TMB color developing system according to claim 1 in the detection of reducing substances, wherein the concentration of the TMB solution is 0.8-1.2 mmol/L. 6 . 6.根据权利要求1所述的TMB显色体系在还原性物质检测中的应用，其特征在于：所述TMB溶液是将3,3',5,5'-四甲基联苯胺用乙醇溶解后加去离子水制得。6. the application of TMB color developing system according to claim 1 in reducing substance detection, it is characterized in that: described TMB solution is to dissolve 3,3',5,5'-tetramethylbenzidine with ethanol After adding deionized water obtained. 7.根据权利要求1所述的TMB显色体系在还原性物质检测中的应用，其特征在于：所述醋酸-醋酸钠缓冲溶液的pH值为4.0～6.0。7 . The application of the TMB color developing system according to claim 1 in the detection of reducing substances, wherein the pH value of the acetic acid-sodium acetate buffer solution is 4.0 to 6.0. 8 .
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