CN110987893B - Method for quantitatively detecting ascorbic acid - Google Patents
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- CN110987893B CN110987893B CN201911387128.7A CN201911387128A CN110987893B CN 110987893 B CN110987893 B CN 110987893B CN 201911387128 A CN201911387128 A CN 201911387128A CN 110987893 B CN110987893 B CN 110987893B
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- 235000010323 ascorbic acid Nutrition 0.000 title claims abstract description 59
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- 229940043267 rhodamine b Drugs 0.000 claims abstract description 24
- 229910001447 ferric ion Inorganic materials 0.000 claims abstract description 20
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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
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
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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
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
- G01N2021/6432—Quenching
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- Life Sciences & Earth Sciences (AREA)
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Abstract
The invention discloses a method for quantitatively detecting ascorbic acid, belongs to the field of spectrum analysis of ascorbic acid, and particularly relates to a method for detecting ascorbic acid by a graphene-rhodamine B-ferric ion ternary system. According to the method, firstly, rhodamine B and ferric ions are added into a graphene solution at the same time, an adsorption layer jointly composed of the rhodamine B and the ferric ions is formed on the surface of the graphene, and the rhodamine B is quenched by energy resonance transfer on the surface of the rhodamine B and the graphene. Then, ascorbic acid is added into the system, and the ascorbic acid can be quantitatively detected by measuring the change of fluorescence of the system. Compared with the traditional method, the method has the advantages of simplicity, high speed, good selectivity, wide linear range and the like.
Description
Technical Field
The invention belongs to the field of spectrum analysis of ascorbic acid, and particularly relates to a method for detecting ascorbic acid by a graphene-rhodamine B-ferric ion ternary system.
Background
Ascorbic acid (vitamin C) is a water-soluble vitamin that has a variety of physiological functions in the human body. Ascorbic acid is a small bioactive molecule involved in many biological reactions involving electron transport reactions, hydroxylation, and oxidative catabolism of aromatic amino acids. Ascorbic acid plays an important role in the biosynthesis of a variety of bioactive substances, including collagen, carnitine, norepinephrine, peptide hormones, and tyrosine metabolites. In addition, ascorbic acid is an essential antioxidant for the human body, and it participates in scavenging Reactive Oxygen Species (ROS) such as hydroxyl radicals, superoxide anion radicals, and singlet oxygen. Ascorbic acid has also attracted considerable attention as an anti-cancer substance. The principle is that after the ascorbic acid is injected intravenously, Fenton reaction catalyzed by Fe and Cu ions in cells is promoted, and the generated high-concentration active oxygen can kill cancer cells. It has been reported that the combination of this method with conventional chemotherapy methods can enhance the therapeutic effects of these methods and inhibit the occurrence of side effects. Hypoascorbate causes scurvy and anemia, with some psychological abnormalities (depression). In contrast, an excess of ascorbic acid may affect vitamin B12Also associated with anemia, may also lead to gastrointestinal disturbances, kidney stones and excessive absorption of iron. Therefore, the detection of ascorbic acid in biological systems is importantIt has important meaning.
In addition to the conventional titration method, colorimetric method and fluorescence method, new methods such as capillary electrophoresis, electrochemical method and quantum dot-based fluorescence detection method have been developed in recent years. The traditional method has low sensitivity and detection conditions are not suitable for biological samples, and the emerging method needs less samples, high sensitivity and mild conditions, but needs complicated sample preparation and pretreatment and lacks field applicability. In order to easily and accurately identify and measure trace levels of ascorbic acid in complex matrices, new analytical methods continue to be developed.
Graphene is a single-layer carbon sheet with a plane conjugated structure and has a series of active oxygen-containing groups such as hydroxyl, carboxyl and epoxy groups. The rhodamine has a plane conjugated system and polar groups such as amino groups and carboxyl groups, can be adsorbed to the surface of graphene with graphene through acting forces such as pi-stacking, electrostatic action and hydrogen bonds, and generates fluorescence resonance energy transfer to quench fluorescence. If a substance with stronger binding capacity with graphene is added into the system, the substance and rhodamine can be competitively adsorbed, so that rhodamine is desorbed from the surface of graphene and fluorescence is recovered. This principle can be used to perform Fluorescence analysis of DNA (Wang X, He Y, Song G. A Graphene Oxide-Rhodamine 6G Nanocomposite as Turn-on Fluorescence Probe for Selective Detection of DNA [ J ]. animal Methods, 2012, 25: 394-400.). The method utilizes a graphene-rhodamine binary system to perform fluorescence detection on DNA, and has the characteristics of rapidness, simplicity and high sensitivity, but the method is not applicable to detection of reducing organic micromolecules.
In order to overcome the defects of the existing method, the invention utilizes a graphene-rhodamine B-ferric ion ternary system to generate fluorescence response to ascorbic acid, and establishes a method for quantitatively detecting ascorbic acid.
Disclosure of Invention
The invention aims to provide a method for quantitatively detecting ascorbic acid.
The object of the invention is achieved by the following technical scheme, and the method for quantitatively detecting the ascorbic acid is characterized by comprising the following steps of:
(1) adding 30 mu L of graphene dispersion liquid into a 10 mL serum bottle, then adding rhodamine B aqueous solution and ferric ion solution, fixing the volume by using Tris-HCl buffer solution with the pH value of 6.5, and standing for later use; adsorbing rhodamine B by graphene to quench the fluorescence of the rhodamine B;
(2) adding 100 μ L of 1M L-cysteine, 100 μ L of 1M ferrous sulfate heptahydrate, 100 μ L of 1M cuprous iodide, 100 μ L of 1M dopamine, 100 μ L of 1M urea, 100 μ L of 1M bovine serum albumin and 100 μ L of 1M ascorbic acid into the solution prepared in the step (1); fluorescence spectrum measurement shows that the fluorescence intensity of a sample added with ascorbic acid is obviously increased; the fluorescence of the sample added with other substances is not obviously changed;
(3) ascorbic acid with different concentrations is respectively added into the solution prepared in the step (1), the fluorescence spectrum measurement shows that the linear relation exists between the fluorescence intensity of the sample and the concentration of the ascorbic acid, and the ascorbic acid can be quantitatively detected through the linear relation.
Specifically, firstly, rhodamine B and ferric ions are added into a graphene solution at the same time. The rhodamine B has a plane conjugated system and polar groups such as amino groups and carboxyl groups, can be adsorbed to the surface of graphene with graphene through acting forces such as pi-stacking, electrostatic action and hydrogen bonds, and generates fluorescence resonance energy transfer to cause fluorescence quenching. The graphene surface has polar groups such as hydroxyl, carboxyl, carboxylate radical and epoxy radical, and ferric ions can be adsorbed to the graphene surface through coordination and electrostatic interaction with the ferric ions. After the adsorption equilibrium is reached, an adsorption layer jointly composed of rhodamine B and ferric ions is formed on the surface of the graphene. Fluorescence quenching is caused by the adsorption of rhodamine B on the surface of graphene, and the adsorption of ferric ions enables the adsorption layer to have oxidability. Then, ascorbic acid is added into the system, and the ascorbic acid can generate oxidation-reduction reaction with ferric ions in the adsorption layer to generate dehydroascorbic acid. Due to the fact that the binding force of the dehydroascorbic acid and the graphene is strong, competitive adsorption with rhodamine B can be achieved, so that the rhodamine B is desorbed from the surface of the graphene, and fluorescence is recovered. The ascorbic acid can be quantitatively detected by measuring the change of the fluorescence of the system. The method comprises the following specific steps:
(1) adding 30 mu L of graphene dispersion liquid into a 10 mL serum bottle, then adding rhodamine B aqueous solution and ferric ion solution, fixing the volume by using Tris-HCl buffer solution with the pH value of 6.5, and standing for later use. The graphene adsorbs rhodamine B to quench the rhodamine B fluorescence, and the fluorescence of the test system is in a 'turn-off' state at the moment.
(2) And (2) respectively adding 100 mu L of 1M L-cysteine, ferrous sulfate heptahydrate, cuprous iodide, dopamine, urea, bovine serum albumin and ascorbic acid solution into the solution prepared in the step (1). Fluorescence spectrometry shows that the fluorescence intensity of a sample added with ascorbic acid is obviously increased, and the fluorescence of a test system shows a 'turn-on' state. While the fluorescence of the other samples did not change significantly. Indicating a particularly good selectivity of the system towards ascorbic acid.
(3) Ascorbic acid with different concentrations is respectively added into the solution prepared in the step (1), the fluorescence spectrum measurement shows that the linear relation exists between the fluorescence intensity of the sample and the concentration of the ascorbic acid, and the ascorbic acid can be quantitatively detected through the linear relation.
The concentration of the graphene dispersion used in step (1) was 0.4 wt%.
The ferric ions in the step (1) come from ferric nitrate.
The ratio of the rhodamine B to the ferric ion in the step (1) is 9: 5.
The standing time of the solution prepared in step (1) was 60 minutes.
The linear range of the concentration of the ascorbic acid in the step (3) is 1-1000 mu M.
The wavelength of the excitation light measured by the fluorescence spectrum in the step (2) and the step (3) is 554 nm.
Compared with the traditional method, the method has the advantages of simplicity, high speed, good selectivity, wide linear range and the like. The method is expected to be applied to the rapid detection of the ascorbic acid content in human body fluid, cell homogenate, food and other samples.
Drawings
FIG. 1 shows fluorescence emission spectra and linear ranges of a graphene-rhodamine B-ferric ion system for ascorbic acid with different concentrations (the ascorbic acid concentrations corresponding to the spectral curves from bottom to top are 1. mu.M, 10. mu.M, 50. mu.M, 100. mu.M, 300. mu.M, 500. mu.M, 700. mu.M and 1000. mu.M, respectively).
Detailed Description
Example 1
The graphene dispersion liquid used in the invention is purchased from institute of organic chemistry of Chinese academy of sciences, and the product number is TNWRGO, the thickness of the graphene is 0.55-3.74 nm, the size of a microchip is about 0.5-3 mu m, and the total oxygen content is about 3% -5%.
30 μ L of 0.4 wt% graphene dispersion was added to a 10 mL serum bottle, followed by 300 μ L of 6X 10-2 And fixing the volume of the solution by using Tris-HCl buffer solution with the pH value of 6.5, wherein the solution is prepared by using the rhodamine B aqueous solution of mol/L and 0.1mol/L ferric nitrate solution of 100 mu L, and standing the solution for 60 minutes. The graphene adsorbs rhodamine B to quench the rhodamine B fluorescence, and the fluorescence of the test system is in a 'turn-off' state at the moment.
Example 2
To the solution prepared in example 1, 100. mu.L of 1M L-cysteine, 100. mu.L of 1M ferrous sulfate heptahydrate, 100. mu.L of 1M cuprous iodide, 100. mu.L of 1M dopamine, 100. mu.L of 1M urea, 100. mu.L of 1M bovine serum albumin and 100. mu.L of 1M ascorbic acid were added, respectively, and the fluorescence of each sample obtained above was measured at an excitation wavelength of 554 nm, and it was found that the fluorescence intensity of the sample to which ascorbic acid was added was significantly increased, at which time the fluorescence of the test system exhibited a "turn-on" state. Whereas the fluorescence of the samples with the addition of the other compounds did not change significantly. Indicating that the detection method has good selectivity for ascorbic acid.
Example 3
To the solutions prepared in example 1, 0.01. mu.L, 0.1. mu.L, 0.5. mu.L, 1. mu.L, 3. mu.L, 5. mu.L, 7. mu.L, 10. mu.L, and 1M ascorbic acid were added, respectively, so that the final concentrations of ascorbic acid in the samples were 1. mu.M, 10. mu.M, 50. mu.M, 100. mu.M, 300. mu.M, 500. mu.M, 700. mu.M, and 1000. mu.M, respectively, and fluorescence spectrometry was performed at an excitation wavelength of 554 nmThe fluorescence intensity of the sample is found to have a linear relation with the concentration of the ascorbic acid, the linear interval is 1-1000 mu M, and the linear equation is FL =27899.2784+8.5478 c (R)2 = 0.9953), see in detail fig. 1. The ascorbic acid concentration of the sample to be tested can be obtained by the fluorescence emission spectrum and the linear range (the corresponding ascorbic acid concentrations from bottom to top of the spectrum curve are 1 μ M, 10 μ M, 50 μ M, 100 μ M, 300 μ M, 500 μ M, 700 μ M and 1000 μ M in sequence) of the graphene-rhodamine B-ferric ion system shown in FIG. 1 for ascorbic acid with different concentrations.
Claims (5)
1. A method for quantitatively detecting ascorbic acid is characterized by comprising the following steps:
(1) adding 30 mu L of graphene dispersion liquid into a 10 mL serum bottle, then adding rhodamine B aqueous solution and ferric ion solution, fixing the volume by using Tris-HCl buffer solution with the pH value of 6.5, and standing for later use; adsorbing rhodamine B by graphene to quench the fluorescence of the rhodamine B;
(2) adding 100 μ L of 1M L-cysteine, 100 μ L of 1M ferrous sulfate heptahydrate, 100 μ L of 1M cuprous iodide, 100 μ L of 1M dopamine, 100 μ L of 1M urea, 100 μ L of 1M bovine serum albumin and 100 μ L of 1M ascorbic acid into the solution prepared in the step (1); fluorescence spectrum measurement shows that the fluorescence intensity of a sample added with ascorbic acid is obviously increased; the fluorescence of the sample added with other substances is not obviously changed;
(3) ascorbic acid with different concentrations is respectively added into the solution prepared in the step (1), the fluorescence spectrum measurement shows that the linear relation exists between the fluorescence intensity of the sample and the concentration of the ascorbic acid, and the ascorbic acid can be quantitatively detected through the linear relation;
the concentration of the graphene dispersion liquid used in the step (1) is 0.4 wt%;
the ratio of the rhodamine B to the ferric ion in the step (1) is 9: 5.
2. The method for quantitatively detecting ascorbic acid according to claim 1, wherein the ferric ion in the step (1) is derived from ferric nitrate.
3. The method for quantitatively detecting ascorbic acid according to claim 1, wherein the solution prepared in the step (1) is left for 60 minutes.
4. The method for quantitatively detecting ascorbic acid according to claim 1, wherein the linear range of the concentration of ascorbic acid in step (3) is 1 to 1000. mu.M.
5. The method for quantitatively detecting ascorbic acid according to claim 1, wherein the wavelength of the excitation light measured by fluorescence spectroscopy in the steps (2) and (3) is 554 nm.
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| CN103604849B (en) * | 2013-05-27 | 2014-09-10 | 济南大学 | Electrochemical sensor capable of simultaneously detecting dopamine, ascorbic acid and uric acid |
| CN104852051A (en) * | 2014-02-14 | 2015-08-19 | 东丽先端材料研究开发（中国）有限公司 | Graphene powder and preparation method and lithium ion battery containing graphene powder |
| CN104267013A (en) * | 2014-06-26 | 2015-01-07 | 广西师范学院 | Method for detecting potassium dichromate and ascorbic acid by using graphene quantum dot probe |
| CN104865232B (en) * | 2015-05-26 | 2017-05-17 | 天津师范大学 | Method for selectively detecting ascorbic acid by utilizing metal-organic framework material |
| CN109668863B (en) * | 2017-10-13 | 2023-10-20 | 国家纳米科学中心 | Copper ion detection method based on graphene and click chemistry, kit and application thereof |
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