CN111189823A - TMB color development system and application thereof in detection of reducing substances - Google Patents
TMB color development system and application thereof in detection of reducing substances Download PDFInfo
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- G—PHYSICS
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- 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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- 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
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- 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
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- 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
- G01N2021/786—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 with auxiliary heating for reaction
Abstract
The invention discloses a TMB color development system and application thereof in detection of reducing substances, wherein the TMB color development system consists of an acetic acid-sodium acetate buffer solution with the pH value of 3.0-7.0, a Fe complex water solution of 0.08-1.5 mg/mL and a TMB solution of 0.4-2.0 mmol/L, wherein the Fe complex is phenanthroline or pyridine complex of Fe (III). Under the condition of no hydrogen peroxide, the Fe complex in the color development system oxidizes TMB to generate a blue oxidation product ox-TMB, and generates maximum ultraviolet absorption at 652 nm. When glutathione, ascorbic acid and the like which are reducing substances are added into the system, reduction reaction is carried out between the glutathione, ascorbic acid and an oxidation product ox-TMB, so that the color of the system is lightened, and the lightening degree is in positive correlation with the concentration of the reducing substances. Thus, colorimetric detection of these reduced substances can be realized. The color development system does not need strong oxidant, does not need heating during detection, and has the advantages of simple operation, quick color development, high sensitivity, small pollution, low detection limit and the like.
Description
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:
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.
Drawings
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:
under the condition of room temperature, adding 20 mu L of 0.8mg/mL o-phenanthroline complex water solution of Fe (III) into an acetic acid-sodium acetate buffer solution with the value of 400 mu LpH 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 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 an acetic acid-sodium acetate buffer solution with the value of 400 mu LpH 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
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 (8)
1. A TMB color development system characterized by: the TMB color development system consists of an acetic acid-sodium acetate buffer solution with the pH value of 3.0-7.0, a Fe complex aqueous solution of 0.08-1.5 mg/mL and a TMB solution 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:
2. the TMB coloring system of claim 1, wherein: the concentration of the Fe complex aqueous solution is 0.8-1.5 mg/mL.
3. The TMB coloring system of claim 1, wherein: the concentration of the TMB solution is 0.8-1.2 mmol/L.
4. The TMB coloring system according to claim 1 or 3, wherein: the TMB solution is prepared by dissolving 3,3',5,5' -tetramethyl benzidine with ethanol and then adding deionized water.
5. The TMB coloring system of claim 1, wherein: the pH value of the acetic acid-sodium acetate buffer solution is 4.0-6.0.
6. The use of the TMB color development system of claim 1 in the detection of a reduced substance, wherein the reduced substance is any one of glutathione, ascorbic acid and cysteine.
7. The use of the TMB coloring system according to claim 6 in the detection of a reduced substance, wherein: 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.
8. The use of the TMB coloring system according to claim 7 for the detection of a reduced substance, wherein: the volume ratio of the acetic acid-sodium acetate buffer solution to the TMB solution and the Fe complex aqueous solution is 20:1: 5-20: 1: 10.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111795964A (en) * | 2020-07-31 | 2020-10-20 | 中国食品药品检定研究院 | Method for quantitatively detecting caprylyl hydroximic acid in cosmetics based on spectrophotometry |
| CN112630179A (en) * | 2020-12-09 | 2021-04-09 | 安徽师范大学 | Prussian blue quantum dot with oxide mimic enzyme property, preparation method thereof and method for detecting L-cysteine |
| CN112730393A (en) * | 2020-12-17 | 2021-04-30 | 广西科技大学 | Color development reagent and method for measuring ascorbic acid |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5702955A (en) * | 1995-05-22 | 1997-12-30 | Bayer Corporation | Ascorbate resistant detection of hydrogen peroxide |
| US20050095466A1 (en) * | 2003-11-05 | 2005-05-05 | St. Louis University | Immobilized enzymes in biocathodes |
| US20070023296A1 (en) * | 2005-07-26 | 2007-02-01 | Nova Biomedical Corporation | Oxygen Sensor |
| CN102029163A (en) * | 2009-09-27 | 2011-04-27 | 华中科技大学 | Catalase catalyst analog as well as preparation method and application thereof |
| CN106829873A (en) * | 2017-03-03 | 2017-06-13 | 武汉大学 | The ferrous silicic acid complex activate molecular oxygen method for producing hydrogen peroxide in situ |
| CN110343247A (en) * | 2019-06-20 | 2019-10-18 | 西南民族大学 | A kind of peroxide intends enzyme high molecule nano material and preparation method thereof |
-
2020
- 2020-02-25 CN CN202010114473.XA patent/CN111189823B/en not_active Expired - Fee Related
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5702955A (en) * | 1995-05-22 | 1997-12-30 | Bayer Corporation | Ascorbate resistant detection of hydrogen peroxide |
| US20050095466A1 (en) * | 2003-11-05 | 2005-05-05 | St. Louis University | Immobilized enzymes in biocathodes |
| US20070023296A1 (en) * | 2005-07-26 | 2007-02-01 | Nova Biomedical Corporation | Oxygen Sensor |
| CN102029163A (en) * | 2009-09-27 | 2011-04-27 | 华中科技大学 | Catalase catalyst analog as well as preparation method and application thereof |
| CN106829873A (en) * | 2017-03-03 | 2017-06-13 | 武汉大学 | The ferrous silicic acid complex activate molecular oxygen method for producing hydrogen peroxide in situ |
| CN110343247A (en) * | 2019-06-20 | 2019-10-18 | 西南民族大学 | A kind of peroxide intends enzyme high molecule nano material and preparation method thereof |
Non-Patent Citations (2)
| Title |
|---|
| Y. SEKINE ET AL.: "Flow injection determination of trace amounts of copper based on its catalytic effect on the oxidation of 3, 3′, 5,5′-tetramethylbenzidine by cumene hydroperoxide", 《MICROCHIM ACTA 》 * |
| 赵梦月等: "Fe(III)-邻菲啰啉配合物光还原的研究及其在铁分析中的应用", 《分析化学》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111795964A (en) * | 2020-07-31 | 2020-10-20 | 中国食品药品检定研究院 | Method for quantitatively detecting caprylyl hydroximic acid in cosmetics based on spectrophotometry |
| CN112630179A (en) * | 2020-12-09 | 2021-04-09 | 安徽师范大学 | Prussian blue quantum dot with oxide mimic enzyme property, preparation method thereof and method for detecting L-cysteine |
| CN112730393A (en) * | 2020-12-17 | 2021-04-30 | 广西科技大学 | Color development reagent and method for measuring ascorbic acid |
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