CN108489921B - Method for quickly detecting cysteine without hydrogen peroxide - Google Patents
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- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 title claims abstract description 18
- 238000000034 method Methods 0.000 title claims abstract description 18
- 235000018417 cysteine Nutrition 0.000 title claims abstract description 14
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 title claims abstract description 14
- 238000001514 detection method Methods 0.000 claims abstract description 21
- 230000008859 change Effects 0.000 claims abstract description 5
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- 238000006243 chemical reaction Methods 0.000 claims description 18
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- 238000010521 absorption reaction Methods 0.000 claims description 10
- 238000002835 absorbance Methods 0.000 claims description 9
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- AYFVYJQAPQTCCC-GBXIJSLDSA-N L-threonine Chemical compound C[C@@H](O)[C@H](N)C(O)=O AYFVYJQAPQTCCC-GBXIJSLDSA-N 0.000 description 1
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- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 1
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- 239000000729 antidote Substances 0.000 description 1
- 229910052787 antimony Inorganic materials 0.000 description 1
- WATWJIUSRGPENY-UHFFFAOYSA-N antimony atom Chemical compound [Sb] WATWJIUSRGPENY-UHFFFAOYSA-N 0.000 description 1
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 1
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Images
Classifications
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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
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- 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
Abstract
The invention discloses a method for rapidly detecting cysteine without hydrogen peroxide, which is mainly based on that newly synthesized aspartic acid-cerium nano material (Ce-Asp) has oxidase-like activity, oxidation reaction can occur in the presence of an organic substrate 3, 3 ', 5' -Tetramethylbenzidine (TMB), the TMB can be oxidized from colorless to blue, and the oxidation reaction can be inhibited under the condition of adding cysteine, so that the color of the TMB can not change. The method provided by the invention is very simple, strong in specificity, good in stability and fast in detection speed, can be used for developing related kits and test strips, can meet the on-site fast and accurate detection requirements, and can be applied to the fields of food safety detection, disease analysis, environmental analysis and the like.
Description
Technical Field
The invention relates to the field of chemical analysis, in particular to a rapid detection method of amino acid. The invention belongs to the technical field of detection,
background
Cysteine is one of essential amino acids in human body, and is polar alpha-amino acid containing sulfhydryl in aliphatic group. Cysteine is an amino acid antidote, participates in the reduction of liver cells and the metabolism of phospholipid, protects the liver cells from being damaged, promotes the recovery of liver function, and has vigorous pharmacological action. It is mainly used for treating radiopharmaceuticals poisoning, heavy metal poisoning, antimony poisoning, etc., and can also be used for treating hepatitis, toxic hepatitis, and seropathy, and preventing liver necrosis. Currently, a variety of analytical techniques have been reported for cysteine detection, including spectroscopy, electrochemical methods, high performance liquid chromatography, inductively coupled plasma mass spectrometry, and the like (Langmuir the AcsJo μ rnal of S μ surfaces & Colloids, 29(16), 5085-92.), but most of these are time consuming, have limited selectivity, generally require expensive instrumentation, and are not suitable for rapid analysis under field conditions. Besides, some methods for detecting Cys (Analyst, 2015, 140, 5251-5256) by catalyzing TMB color development are adopted, but all the methods need to be carried out in the presence of hydrogen peroxide, and the hydrogen peroxide is particularly unstable and has a certain influence on the accuracy of the detection result.
Disclosure of Invention
Aiming at the problems, a novel aspartic acid-cerium nano material is synthesized, and a novel method which is free of hydrogen peroxide and label-free and can be used for quickly detecting cysteine is developed. The method has the advantages of simple operation process, low cost, good reproducibility of the detection result and capability of ensuring the accuracy of the detection result.
In order to achieve the purpose, the technical scheme of the invention is as follows:
the realization of a rapid detection method with high precision, high sensitivity and simplicity on a target object is an important development direction of analytical chemistry. The invention provides a simple and rapid cysteine detection method. The method comprises the steps of mixing Ce-Asp and Cys with different concentrations, incubating at room temperature for 10min, adding TMB, reacting for 10min, inhibiting oxidation of the TMB until color change and ultraviolet absorption value are also inhibited, linearly correlating the color change strength and the absorption value with the concentration of the Cys, and measuring absorbance by an ultraviolet spectrophotometer to realize rapid detection of the Cys.
The invention specifically comprises the following steps:
(1) first using Asp and Ce (NO)3)3The preparation method of the Ce-Asp nanocomposite comprises the following basic steps: 1.0mM NaOH and 1.0mM Asp were added to 6mL of alcohol and shaken for 10 minutes, followed by 1.0mM Ce (NO)3)3Dissolving in 1.0mL of deionized water, Ce (NO) was added3)3Adding the aqueous solution into a mixture of NaOH and Asp, shaking for 20min, centrifuging at 6000rpm for 6min, collecting precipitate, and washing with absolute ethanol for three times;
(2) then adding 0-10 mu M cysteine Cys solution with different concentrations into 0.6mM Ce-Asp nanocomposite solution prepared in the step (1), incubating for 10min, then adding 150 mu M TMB, reacting for 10min, stopping the reaction by 9.2mM sulfuric acid, and measuring the absorbance value at 451nm by an ultraviolet spectrophotometer;
(3) TMB, Ce-Asp reacts with Cys with different concentrations, the absorption spectrum of a reaction system is measured by an ultraviolet spectrophotometer after the reaction is stopped by sulfuric acid, the highest light absorption value intensity at 451nm is taken as an output detection signal, a linear relation exists between the absorption value of a sensing system after the Cys is added and the concentration of the Cys, then the ultraviolet absorption value of the TMB at 451nm is taken as a vertical coordinate, the concentration of the Cys is taken as a horizontal coordinate for processing to be a linear relation, and a fitting curve and a linear regression equation are obtained according to the linear relation: y-0.1374 x +1.56574, R0.9987, with a detection limit of 80 nM.
The method of the invention is a new method for rapidly detecting cysteine. Compared with the prior art, the detection method for cysteine Cys provided by the invention has the following advantages: most of the previously developed colorimetric detection methods need hydrogen peroxide, however, hydrogen peroxide is easy to decompose and has unstable properties, and the existing preparation is usually needed, so that the application of related methods in on-site rapid detection is limited. The invention utilizes the combined action of Cys, TMB and Ce-Asp, and the developed detection method has the advantages of simple operation, no mark, high sensitivity, simplicity, rapidness, strong specificity and low cost.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention, illustrate embodiments of the invention and together with the description serve to explain the invention without limiting the invention to its fullest extent. In the drawings:
FIG. 1 is an X-ray diffraction (XRD) spectrum of Ce-Asp, a material according to an embodiment of the present invention.
Fig. 2 provides data relating to oxidation of materials and acid inhibition reactions as described in the inventive examples.
FIG. 3 provides relevant data for the TMB reaction mechanism described in the inventive examples of the present invention.
FIG. 4 provides data on the effect of different pH buffers used to oxidize TMB with Ce-Asp as described in the inventive examples.
FIG. 5 provides data on the change in TMB absorbance with Cys concentration as described in the inventive examples.
FIG. 6 is a linear fit curve and equation of absorbance values at 451nm wavelength for TMB absorbance intensity as a function of Cys concentration as described in the inventive examples of the present invention.
FIG. 7 provides data for experiments to detect Cys-specific interference as described in the inventive examples.
Detailed Description
In order to make the aforementioned features of the present invention and the optimized conditions in the invention clearer and easier to understand, embodiments of the present invention will be described in further detail with reference to the accompanying drawings.
The reaction system of the following 3, 3 ', 5' -Tetramethylbenzidine (TMB), cerium nitrate-aspartic acid nanomaterial (Ce-Asp) and Cys was 200. mu.L (including TMB, Ce-Asp and Cys of different concentrations).
Example 1
First, 1mMol NaOH and 1mMol Asp were added to 6mL of alcohol and shaken for 10 minutes. Next, 1 mmoleCe (NO) was added3)3Dissolved in 1mL of deionized water. Then, Ce (NO) is added3)3The aqueous solution was added to a mixture of NaOH and Asp and shaken for 10 min. After the reaction, the precipitate was collected by centrifugation (6000rpm, 6min) and washed three times with anhydrous ethanol. The same method is used for preparing Ce (NO) with different proportions3)3And Asp to synthesize the aspartic acid-cerium nano material. The XRD spectrum was then determined. (a) Asp; (b)1:5 Ce-Asp; (c)1:3 Ce-Asp; (d)1:1 Ce-Asp; (e) ce (NO)3)3。(as shown in FIG. 1)
Example 2
Oxidation of TMB to blue and termination of the ultraviolet absorption spectrum of the oxidation reaction, (a)150 μ M TMB; (b) reaction of 150. mu.M TMB with 0.6mM Ce-Asp; (c) after reaction of 150. mu.M TMB with 0.6mM Ce-Asp, 200. mu. M H was added2SO4The reaction was terminated. (as shown in FIG. 2)
Example 3
TMB oxidation principle uv absorption spectroscopy. (a)150 μ M TMB; (b)0.6 mMCe-Asp; (c)150 μ M TMB mixed with 10 μ MCys; (d)0.6mMCe-Asp mixed with 10. mu. MCys; (e)150 μ M TMB mixed with 0.6 mMCe-Asp; (f)150 μ M TMB, 0.6mMCe-Asp mixed with 10 μ MCys. After all the reactions are terminated by concentrated sulfuric acid, an ultraviolet absorption spectrum is measured by an ultraviolet spectrophotometer, and the wavelength range is 300nm-600 nm. (as shown in FIG. 3)
Example 4
The effect of different pH values on the oxidation reaction was determined. 0.6mM Ce-Asp was incubated with 150. mu.M TMB in PB buffer at pH3, 4.5, 4, 5, 5.5, 6, 6.5, 7, 7.5, 8, 9 for 10min, and the UV absorbance at 451nm was measured for TMB. The maximum absorption peak at a wavelength of 451nm is taken as the ordinate, and the pH value is taken as the abscissa to make a dot line graph. (as shown in FIG. 4)
Example 5
TMB oxidation varied with the addition of different concentrations of Cys. 0.6mMCe-Asp and 0-10 μ M Cys are mixed and incubated for 10min, then 150 μ M TMB is added for reaction for 10min, the reaction is stopped by adding concentrated sulfuric acid, and the measurement wavelength range is 300nm-600nm ultraviolet absorption spectrum. (as shown in FIG. 5)
Example 6
0.6mMCe-Asp was mixed with 150. mu.M TMB and 0-10. mu.M Cys and reacted for 10min, and the absorbance of TMB at 451nm was measured, and the maximum absorbance of TMB at a wavelength of 451nm was plotted on the ordinate and Cys at various concentrations on the abscissa and linear fit was performed to obtain the following linear equation:
y＝-0.1374x+1.56574(R20.9973 formula (1)
Example 7
The assay detects the specificity of the Cys reaction. Blank substances, such as cysteine, proline, tryptophan, histidine, serine, glycine, threonine, glutamic acid, arginine, alanine, methionine, phenylalanine, glutamine, lysine, tyrosine and the like are sequentially arranged from left to right to serve as interference substances detected in the experiment, and the specificity of the method is researched. The concentrations of the interfering substances are 100 mu M, the interfering substances are respectively incubated with 0.6mM Ce-Asp for 10min, then 150 mu M TMB is added for mixing reaction for 10min, and an ultraviolet absorption value is measured by an ultraviolet spectrophotometer. As shown in FIG. 6, only 10. mu. MCys can obviously inhibit the absorption intensity of TMB, and other interfering amino acids do not obviously inhibit the absorption intensity of TMB, which indicates that the method has strong specificity, high sensitivity and very simple method for Cys detection. (as shown in FIG. 7)
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that are within the spirit and principle of the present invention should be included in the scope of the present invention.
Claims (1)
1. A method for quickly detecting cysteine without hydrogen peroxide is characterized by comprising the following steps: Ce-Asp, TMB and Cys are mixed, and the Cys concentration is detected by observing color change, which comprises the following steps:
(1) first using Asp and Ce (NO)3)3The preparation method of the Ce-Asp nanocomposite comprises the following basic steps: 1.0mM NaOH and 1.0mM Asp were added to 6mL of alcohol and shaken for 10 minutes, followed by 1.0mM Ce (NO)3)3Dissolving in 1.0mL of deionized water, Ce (NO) was added3)3Adding the aqueous solution into a mixture of NaOH and Asp, shaking for 20min, centrifuging at 6000rpm for 6min, collecting precipitate, and washing with absolute ethanol for three times;
(2) adding Cys solution with different concentrations of 0-10 mu M into the Ce-Asp nanocomposite solution prepared in the step (1) with the concentration of 0.6mM, incubating for 10min, adding TMB with the concentration of 150 mu M, reacting for 10min, stopping the reaction by using 9.2mM sulfuric acid, and measuring the absorbance value at 451nm by using an ultraviolet spectrophotometer;
(3) TMB, Ce-Asp reacts with Cys with different concentrations, the absorption spectrum of a reaction system is measured by an ultraviolet spectrophotometer after the reaction is stopped by sulfuric acid, the highest light absorption value intensity at 451nm is taken as an output detection signal, a linear relation exists between the absorption value of a sensing system after the Cys is added and the concentration of the Cys, then the ultraviolet absorption value of the TMB at 451nm is taken as a vertical coordinate, the concentration of the Cys is taken as a horizontal coordinate for processing to be a linear relation, and a fitting curve and a linear regression equation are obtained according to the linear relation: y-0.1374 x +1.56574, R0.9987, with a detection limit of 80 nM.
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| CN104777117B (en) * | 2015-04-16 | 2017-10-20 | 福建医科大学 | The method that cysteine is determined based on stannic oxide/graphene nano platinum composite |
| CN104792980B (en) * | 2015-04-29 | 2016-07-27 | 西安交通大学 | A kind of porous silica cerium nanometer rods composite construction and based on the preparation method of enzymatic solution of this structure and enzyme immunoassay application |
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