CN110907446A - Rapid detection method of glutathione - Google Patents
Rapid detection method of glutathione Download PDFInfo
- Publication number
- CN110907446A CN110907446A CN201911275295.2A CN201911275295A CN110907446A CN 110907446 A CN110907446 A CN 110907446A CN 201911275295 A CN201911275295 A CN 201911275295A CN 110907446 A CN110907446 A CN 110907446A
- Authority
- CN
- China
- Prior art keywords
- solution
- glutathione
- concentration
- absorbance
- detected
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 title claims abstract description 213
- 229960003180 glutathione Drugs 0.000 title claims abstract description 106
- 108010024636 Glutathione Proteins 0.000 title claims abstract description 104
- 238000001514 detection method Methods 0.000 title claims abstract description 35
- 238000002835 absorbance Methods 0.000 claims abstract description 29
- 229920001661 Chitosan Polymers 0.000 claims abstract description 26
- 238000006243 chemical reaction Methods 0.000 claims abstract description 26
- 238000000034 method Methods 0.000 claims abstract description 19
- 238000002156 mixing Methods 0.000 claims abstract description 9
- UAIUNKRWKOVEES-UHFFFAOYSA-N 3,3',5,5'-tetramethylbenzidine Chemical compound CC1=C(N)C(C)=CC(C=2C=C(C)C(N)=C(C)C=2)=C1 UAIUNKRWKOVEES-UHFFFAOYSA-N 0.000 claims abstract description 7
- 239000000243 solution Substances 0.000 claims description 104
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 6
- 238000012764 semi-quantitative analysis Methods 0.000 claims description 4
- 238000010521 absorption reaction Methods 0.000 claims description 2
- 239000007864 aqueous solution Substances 0.000 claims description 2
- 239000007788 liquid Substances 0.000 claims description 2
- 230000035484 reaction time Effects 0.000 claims description 2
- 239000007787 solid Substances 0.000 claims description 2
- 238000000870 ultraviolet spectroscopy Methods 0.000 claims description 2
- 238000002474 experimental method Methods 0.000 description 14
- 239000003086 colorant Substances 0.000 description 6
- 238000007254 oxidation reaction Methods 0.000 description 6
- 238000004458 analytical method Methods 0.000 description 5
- 239000011259 mixed solution Substances 0.000 description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 5
- 238000011534 incubation Methods 0.000 description 4
- 230000003647 oxidation Effects 0.000 description 4
- 230000001590 oxidative effect Effects 0.000 description 4
- 238000006555 catalytic reaction Methods 0.000 description 3
- 238000004737 colorimetric analysis Methods 0.000 description 3
- 150000002500 ions Chemical class 0.000 description 3
- 229930091371 Fructose Natural products 0.000 description 2
- 239000005715 Fructose Substances 0.000 description 2
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 description 2
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 2
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 2
- 102000003992 Peroxidases Human genes 0.000 description 2
- 238000000862 absorption spectrum Methods 0.000 description 2
- 239000003054 catalyst Substances 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 239000008103 glucose Substances 0.000 description 2
- -1 imine salt Chemical class 0.000 description 2
- 238000012803 optimization experiment Methods 0.000 description 2
- 108040007629 peroxidase activity proteins Proteins 0.000 description 2
- 238000006722 reduction reaction Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 238000002371 ultraviolet--visible spectrum Methods 0.000 description 2
- 230000000007 visual effect Effects 0.000 description 2
- YRNWIFYIFSBPAU-UHFFFAOYSA-N 4-[4-(dimethylamino)phenyl]-n,n-dimethylaniline Chemical compound C1=CC(N(C)C)=CC=C1C1=CC=C(N(C)C)C=C1 YRNWIFYIFSBPAU-UHFFFAOYSA-N 0.000 description 1
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 description 1
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 1
- 206010067125 Liver injury Diseases 0.000 description 1
- 206010034010 Parkinsonism Diseases 0.000 description 1
- 238000001069 Raman spectroscopy Methods 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 210000001124 body fluid Anatomy 0.000 description 1
- 239000010839 body fluid Substances 0.000 description 1
- 238000005251 capillar electrophoresis Methods 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 238000007398 colorimetric assay Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000009833 condensation Methods 0.000 description 1
- 230000005494 condensation Effects 0.000 description 1
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 1
- 235000018417 cysteine Nutrition 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 238000001784 detoxification Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000002848 electrochemical method Methods 0.000 description 1
- 238000005562 fading Methods 0.000 description 1
- 238000000684 flow cytometry Methods 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 238000002795 fluorescence method Methods 0.000 description 1
- 235000013922 glutamic acid Nutrition 0.000 description 1
- 239000004220 glutamic acid Substances 0.000 description 1
- 231100000753 hepatic injury Toxicity 0.000 description 1
- 208000006454 hepatitis Diseases 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 230000007365 immunoregulation Effects 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 230000010354 integration Effects 0.000 description 1
- 208000018191 liver inflammation Diseases 0.000 description 1
- 229910021645 metal ion Inorganic materials 0.000 description 1
- 230000003278 mimic effect Effects 0.000 description 1
- 231100000956 nontoxicity Toxicity 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 125000003396 thiol group Chemical group [H]S* 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
Images
Classifications
-
- 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
- G01N21/75—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
-
- 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
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N31/00—Investigating or analysing non-biological materials by the use of the chemical methods specified in the subgroup; Apparatus specially adapted for such methods
- G01N31/10—Investigating or analysing non-biological materials by the use of the chemical methods specified in the subgroup; Apparatus specially adapted for such methods using catalysis
Abstract
The invention discloses a rapid detection method of glutathione, which specifically comprises the steps of mixing chitosan solution and H2O2The solution and the 3, 3 ', 5, 5' -tetramethylbenzidine solution are mixed and reacted, glutathione solutions with different concentration gradients are respectively added into a mixed reaction system for continuous reaction, and the color of the solution after the glutathione with different concentration gradients is added is observed by naked eyes and photographed by a camera after the reaction is finished, so that the concentration of the glutathione is semi-quantitatively detected according to the color of the solution to be detected; or the absorbance of the solution can be measured by an ultraviolet spectrophotometer, a linear relation between the absorbance and the concentration of the glutathione is constructed, and the concentration of the glutathione in the solution to be detected can be quickly detected by utilizing the linear relation; the method has simple operation, and can realize quick and sensitive semiquantitative sum of glutathioneQuantitative detection and the like, and has good application prospect.
Description
Technical Field
The invention relates to the technical field of analysis and detection, in particular to a rapid detection method of glutathione.
Background
Glutathione (GSH) is a sulfhydryl-containing tripeptide, which is formed by the condensation of glutamic acid, cysteine and glycine and is distributed in human and animal tissue fluids and body fluids. Glutathione plays an important role in the aspects of body immunoregulation, integration detoxification, cell proliferation, apoptosis and the like, and the occurrence of some diseases, such as HIV, Parkinson's syndrome, liver injury, inflammation and the like, can cause the content level of glutathione in vivo to be abnormal. Therefore, the establishment of a sensitive glutathione analysis method has important application value for biomedical research, clinical medical examination and the like.
At present, the glutathione detection method mainly comprises an electrochemical method, a fluorescence method, a high performance liquid chromatography method, a high performance capillary electrophoresis method, a flow cytometry method, an apparent enhanced Raman spectroscopy method and the like. Some of the methods are complicated in operation steps and long in time consumption, or rely on expensive instruments and are high in analysis cost. Therefore, the establishment of a new simple, rapid and sensitive glutathione analysis method has important significance.
Chinese patent application No. 2018114342572 discloses a method for detecting glutathione, which is characterized in that Ce (IV) ions can enable TMB to generate oxidation reaction to generate blue TMB imine salt, when a trace amount of glutathione is added into a Ce (IV)/TMB system, the glutathione and the TMB imine salt generate reduction reaction, so that the blue color of the system is lightened, and the lightening degree is in direct proportion to the concentration of the glutathione, thereby establishing the method for detecting the glutathione. The technical scheme has certain similarity with the method. However, the present invention has the following advantages over this method: (1) because Ce (IV) ions are highly oxidative, excessive Ce (IV) ions are easy to directly oxidize glutathione, and analytical determination is influenced; the application is based on the catalysis of H by chitosan2O2Detection method for constructing glutathione by oxidizing TMB (tetramethylbenzidine), wherein chitosan is non-oxidizing and has oxidizing H2O2Because the glutathione is converted into oxygen and water under the catalysis of the chitosan and is removed, the direct oxidation of the glutathione is avoided. (2) Compared with Ce (IV)The chitosan used in the method has good biocompatibility and nontoxicity, so that the whole detection method is more environment-friendly.
Disclosure of Invention
The invention aims to provide a rapid detection method for glutathione, aiming at the problems of complicated operation steps, long time consumption, dependence on expensive instruments, high analysis cost and the like of the existing detection method for glutathione.
In order to realize the purpose, the invention is realized by the following technical scheme:
the invention relates to a rapid detection method of glutathione, which comprises the following steps: mixing chitosan solution with H2O2The solution and the 3, 3 ', 5, 5' -tetramethylbenzidine solution are mixed and reacted, glutathione solutions with different concentration gradients are respectively added into a mixed reaction system for continuous reaction, the absorbance of the solution is measured by an ultraviolet spectrophotometer after the reaction is finished, the linear relation between the absorbance and the glutathione concentration is constructed, and the concentration of glutathione in the solution to be detected can be quickly detected by utilizing the linear relation; or the color of the solution after glutathione with different concentration gradients is added can be observed by naked eyes and photographed by a camera, and then the photographed picture is measured by using Image J software, so that the average value of the red, green and blue colors of different solution pictures can be obtained, and the semi-quantitative analysis of the concentration of the glutathione in the liquid to be measured is realized.
Preferably, the chitosan solution is prepared by dissolving chitosan solid with molecular weight of 50-190kDa in 1% acetic acid aqueous solution.
Preferably, the final concentration of the chitosan solution in the mixed reaction system is 0.5mg/mL, H2O2The final concentration of the solution was 0.125mM, and the final concentration of the 3, 3 ', 5, 5' -tetramethylbenzidine solution was 0.625 mM.
Preferably, the concentration gradient of glutathione in the invention is 0 [ mu ] M, 1.25 [ mu ] M, 10 [ mu ] M, 20 [ mu ] M, 30 [ mu ] M, 40 [ mu ] M, 50 [ mu ] M, 60 [ mu ] M, 80 [ mu ] M, 100 [ mu ] M, 125 [ mu ] M and 200 [ mu ] M.
Preferably, the ultraviolet spectrophotometer described in the present invention measures the absorbance of the solution at 652 nm.
Preferably, the linear equation of the absorbance and the glutathione concentration in the invention is △ a = 0.0057C +0.012, and the linear correlation coefficient R is 0.9953.
Preferably, the reaction temperature of the mixed reaction system is 37 ℃ and the reaction time is 30 min.
Further, the detection method of the invention specifically comprises the following steps: taking a chitosan solution with the concentration of 200 muL being 1.0mg/mL and H with the concentration of 50 muL being 1.0mM2O2Mixing the solution with 50 muL of 3, 3 ', 5, 5' -tetramethylbenzidine solution with the concentration of 5.0mM, reacting for 30min at 37 ℃, then respectively adding 100 muL of glutathione solutions with different concentration gradients, continuing to react for 30min after uniformly mixing, and carrying out rapid colorimetric analysis by observing the color of the reacted solution by naked eyes or carrying out photo comparison on the solution by using a camera, thereby realizing semi-quantitative detection on the concentration of glutathione in the solution to be detected; the absorbance of the solution system at 652nm can be measured by using an ultraviolet-visible spectrophotometer to obtain a linear relation between the final concentration of glutathione and the absorption intensity, and the concentration of glutathione in the solution to be detected can be quantitatively detected by using the linear relation.
The detection principle of the rapid detection method of glutathione is shown in figure 1, and chitosan has the property similar to peroxidase and is applied to hydrogen peroxide (H)2O2) In the presence of the catalyst, 3 ', 5, 5' -Tetramethylbenzidine (TMB) is catalyzed to generate oxidation reaction to generate blue oxidation state TMB (Ox-TMB); when the chitosan and H react2O2After a certain amount of glutathione is added into the mixed solution system of the glutathione and the TMB, the glutathione and the Ox-TMB are subjected to reduction reaction, so that the blue color of the solution is lightened, and the lightening degree and the concentration of the glutathione are in a positive correlation relationship, so that a rapid and visual glutathione detection method is established through visual observation or camera photographing comparison. The photographed pictures are measured by using Image J software, so that the average value (namely RGB value) of the red, green and blue colors of different solution pictures can be obtained, and the semi-quantitative analysis of the glutathione can be carried out according to the average value. Further by violetAnd measuring the absorbance of the solution by an external visible absorption spectrum, constructing a linear relation between the absorbance and the concentration of the glutathione, and realizing high-sensitivity glutathione detection.
Compared with the prior art, the detection method provided by the invention has the advantages of simple operation, capability of realizing rapid and sensitive semi-quantitative and quantitative detection of glutathione and the like, and has good application prospect.
Drawings
FIG. 1 is a schematic diagram of the detection of the present invention;
FIG. 2 is a photograph of solutions obtained by adding glutathione at different concentration gradients;
FIG. 3 is a bar graph of Δ RGB values of the color of solutions obtained by adding glutathione at different concentration gradients;
FIG. 4 is a graph of the UV-VIS absorption spectra of solutions obtained by adding glutathione at different concentration gradients;
FIG. 5 is a graph of the difference in absorbance △ A versus the concentration C of the glutathione solution and a linear fit curve;
FIG. 6 is a graph of the UV-VIS absorption spectra of various solution systems;
FIG. 7 is H2O2Concentration optimized experimental result graph;
FIG. 8 is a photograph of glutathione solutions obtained by adding different classes of analogs;
FIG. 9 is a bar graph of absorbance values of glutathione solutions measured with the addition of different species.
Detailed Description
Example 1
In the detection method of glutathione in this embodiment, a chitosan solution with a concentration of 200 μ L of 1.0mg/mL and H with a concentration of 50 μ L of 1.0mM are taken2O2The solution was mixed with 50 μ L of a 5.0mM TMB solution, reacted at 37 ℃ for 30min, and 12 experiments were performed in parallel. Then, 100 muL of glutathione solutions with initial concentrations of 0 muM, 1.25 muM, 10 muM, 20 muM, 30 muM, 40 muM, 50 muM, 60 muM, 80 muM, 100 muM, 125 muM and 200 muM are added into 12 groups of solutions respectively, and reaction is continued for 30min under the condition of 37 ℃ after mixing. After the reaction was complete, it was clearly observedAnd as the concentration of the glutathione solution increases, the blue color of the solution gradually becomes lighter, and referring to fig. 2, the shade degree of the color of the solution is related to the concentration of the glutathione, so that the rapid colorimetric detection of the glutathione solution can be carried out, and the semi-quantitative detection of the concentration of the glutathione can be realized according to the difference of the colors of the solutions. We also measured the photographed pictures using Image J software to obtain the average red, green and blue colors (i.e., RGB values) of the different solution pictures. After the background when glutathione is zero is subtracted, a histogram of Δ RGB values and corresponding glutathione concentrations is obtained, as shown in fig. 3, from which a semi-quantitative analysis of glutathione can be performed. Wherein Δ RGB = RGB-RGB0RGB is the average value of the red, green and blue colors of the solution obtained after glutathione with different concentrations is added, and the average value is a signal; RGB (Red, Green, blue) color filter0The average value of the red, green and blue colors of the solution obtained after adding pure water is the background.
In addition, in this example, the ultraviolet-visible absorption spectra of the solutions after the reaction was completed were measured by an ultraviolet spectrophotometer, and as shown in fig. 4, the absorbance of the solution gradually decreased as the concentration of glutathione increased. And by the difference in absorbance Δ a, where Δ a = a0- A，A0The absorbance at 652nm when the concentration of the glutathione solution is 0, A is the absorbance at 652nm after the glutathione solutions with different concentrations are added, and the linear fitting of the absorbance difference Delta A and the concentration C of the glutathione solution finds that the absorbance difference Delta A and the concentration C have good linear relation when the concentration of the glutathione solution is in the range of 1.25 to 100 mu M, the obtained linear equation is △ A = 0.0057C +0.012, and the linear correlation coefficient R is 0.9953, which is shown in figure 5.
Example 2
To investigate the catalysis of H based on chitosan2O2Feasibility of a colorimetric assay for oxidized TMB-colored glutathione, the following three sets of experiments were designed for this example, see Table 1 below. Wherein the initial concentration of chitosan solution is 1.0mg/mL, H2O2The initial concentrations of the solutions were all 1.0mM, and the initial concentrations of the TMB solutions were all 5.0 mM. Adding the two groups 1 and 2After TMB reacts for 30min, adding deionized water and continuing incubation for 30 min; and after the group 3 is added with the TMB and reacts for 30min, adding a glutathione solution with the concentration of 0.4mM and continuing incubation for 30min, wherein the reaction temperature of each group of experiments is 37 ℃.
TABLE 1 feasibility study protocol of chitosan mimic peroxidase for glutathione colorimetric analysis
The experimental results are shown in fig. 6: in experiment set 1, after the reaction was complete, the mixed solution was colorless (a), indicating that H was present in the absence of chitosan2O2TMB cannot be oxidized to develop color by itself; in the experiment of the 2 nd group, the solution is obviously blue after the reaction is finished, and the chitosan is proved to be capable of catalyzing H2O2Oxidizing TMB to produce a blue oxidation state TMB (Ox-TMB) compound (b); in experiment set 3, first, chitosan solution and H were added2O2And (c) after the solution and the TMB solution are mixed, carrying out color development reaction to change the solution into blue, then adding a glutathione solution with a certain concentration, and finally fading the blue color of the mixed solution to be colorless. The above experimental results show that the chitosan-based catalyst H2O2And the oxidation of TMB color reaction can realize rapid glutathione colorimetric analysis.
Example 3
This embodiment takes into account H2O2Also exhibits a certain degree of oxidizability, an excess of H2O2Has obvious effect on the detection of glutathione, so the detection method has no toxic side effect on H2O2The concentration is optimized, and the optimization experiment specifically comprises the following steps:
four sets of experiments numbered 1-4 were set, with each set containing two experiments for background and signal, respectively. The background protocol for each group was 200. mu.L of chitosan solution and 50. mu.L of H2O2And 50 mu L of TMB solution is reacted for 30min at 37 ℃, and then 100 mu L of water is added for further incubation for 30 min. The signal term protocol for each set was 200. mu.L of chitosan solution and 50. mu.L of H2O2And reacting 50 mu L of TMB solution at 37 ℃ for 30min, and then adding 100 mu L of glutathione solution for further incubation for 30 min. The initial concentrations of the chitosan solution, the TMB solution and the glutathione solution used in the experiment were all 1.0mg/mL, 5.0mM and 0.8mM respectively. Experiment Nos. 1-4H2O2The initial concentration of the solution was 0.5mM, 1.0mM, 2.0mM, 5.0mM in this order, see Table 2 below. And respectively measuring the absorbance of the reacted solution at 652nm by using an ultraviolet spectrophotometer, and comparing the absorbance difference of the background term and the signal term in each group.
TABLE 2H2O2Optimization experiment of solution concentration
The results of the experiment are shown in FIG. 7, where H was added at an initial concentration of 1.0mM (final concentration of 0.125 mM)2O2In the experiment group 2 of the solution, the difference between the absorbance of the background term and the absorbance of the signal term is the largest, namely the sensitivity detection of glutathione is the most beneficial. Therefore, H used in the assay for glutathione2O2The optimal final concentration of the solution was determined to be 0.125 mM.
Example 4
The present example also examines the selectivity of glutathione detection, and the specific scheme is as follows:
in order to explore the response of the detection method of the invention to different substances, the selectivity of the detection method of the invention to glutathione is systematically examined, common metal ions and carbohydrate substances are used as analogies, and the following selective examination experiment is designed. Collecting 200 μ L of 1.0mg/mL chitosan solution and 50 μ L of 1.0mM H2O2The solution was mixed with 50. mu.L of a 5.0mM TMB solution, reacted at 37 ℃ for 30min, and 10 experiments were carried out in parallel. Then, 100. mu.L of water and glucose (glucose), fructose (fructose), Mg (Mg) at concentrations of 1mM were added to each group of solutions, respectively2+、Cu2+、K+、Na+、Ca2+、Ba2+And Glutathione Solution (GSH), mixing at 37 ℃The reaction was continued for 30min under C. After the reaction was completed, as shown in fig. 8, it was clearly observed that only the mixed solution to which glutathione was added was transparent and colorless, while the other control solutions were blue, indicating that the detection method had good selectivity for glutathione. Further, an ultraviolet spectrophotometer is used for respectively measuring the absorbance of the solution at 652nm, the obtained result is shown in fig. 9, the absorbance of the mixed solution after the glutathione is added for reaction is obviously smaller than the absorbance of other solutions, and the method is also verified to have better selectivity for glutathione detection.
Claims (8)
1. A rapid detection method of glutathione is characterized in that the detection method specifically comprises the following steps: mixing chitosan solution with H2O2The solution and the 3, 3 ', 5, 5' -tetramethylbenzidine solution are mixed and reacted, glutathione solutions with different concentration gradients are respectively added into a mixed reaction system for continuous reaction, and the color of the solution after the glutathione with different concentration gradients is added is observed by naked eyes and photographed by a camera after the reaction is finished, so that the concentration of the glutathione is semi-quantitatively detected according to the color of the solution to be detected; or the absorbance of the solution can be measured by an ultraviolet spectrophotometer, a linear relation between the absorbance and the concentration of the glutathione is constructed, and the concentration of the glutathione in the solution to be detected can be quickly detected by utilizing the linear relation.
2. The method for rapidly detecting glutathione according to claim 1, wherein the kit comprises: the chitosan solution is prepared by dissolving chitosan solid with molecular weight of 50-190kDa in acetic acid aqueous solution with mass fraction of 1%.
3. The method for rapidly detecting glutathione according to claim 1, wherein the kit comprises: the final concentration of the chitosan solution in the mixed reaction system is 0.5mg/mL, H2O2The final concentration of the solution was 0.125mM, and the final concentration of the 3, 3 ', 5, 5' -tetramethylbenzidine solution was 0.625 mM.
4. The method for rapidly detecting glutathione according to claim 1, wherein the kit comprises: the concentration gradient of the glutathione is 0 [ mu ] M, 1.25 [ mu ] M, 10 [ mu ] M, 20 [ mu ] M, 30 [ mu ] M, 40 [ mu ] M, 50 [ mu ] M, 60 [ mu ] M, 80 [ mu ] M, 100 [ mu ] M, 125 [ mu ] M and 200 [ mu ] M.
5. The method for rapidly detecting glutathione according to claim 1, wherein the kit comprises: the UV spectrophotometer measures the absorbance of the solution at 652 nm.
6. The method for rapidly detecting glutathione according to claim 1, wherein the linear equation of absorbance and glutathione concentration is △ A = 0.0057C +0.012, and the linear correlation coefficient R is 0.9953.
7. The method for rapidly detecting glutathione according to claim 1, wherein the kit comprises: the reaction temperature of the mixed reaction system is 37 ℃, and the reaction time is 30 min.
8. The method for rapidly detecting glutathione according to any one of claims 1 to 7, wherein the kit comprises: the detection method specifically comprises the steps of taking 200 microliter of chitosan solution with the concentration of 1.0mg/mL and 50 microliter of H with the concentration of 1.0mM2O2Mixing the solution with 50 muL of 3, 3 ', 5, 5' -tetramethylbenzidine solution with the concentration of 5.0mM, reacting for 30min at 37 ℃, then respectively adding 100 muL of glutathione solutions with different concentration gradients, continuing to react for 30min after uniformly mixing, observing the color of the reacted solution through naked eyes or comparing the solution by taking pictures by using a camera, and determining the pictures taken by using Image J software to obtain the red, green and blue color average values of different solution pictures, thereby realizing semi-quantitative analysis of the concentration of glutathione in the liquid to be detected; the absorbance of the solution system at 652nm can be measured by using an ultraviolet-visible spectrophotometer to obtain a linear relation between the final concentration of glutathione and the absorption intensity, and the concentration of glutathione in the solution to be detected can be quantitatively detected by using the linear relation.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911275295.2A CN110907446A (en) | 2019-12-12 | 2019-12-12 | Rapid detection method of glutathione |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911275295.2A CN110907446A (en) | 2019-12-12 | 2019-12-12 | Rapid detection method of glutathione |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN110907446A true CN110907446A (en) | 2020-03-24 |
Family
ID=69825035
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201911275295.2A Pending CN110907446A (en) | 2019-12-12 | 2019-12-12 | Rapid detection method of glutathione |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110907446A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111487242A (en) * | 2020-04-27 | 2020-08-04 | 天津工业大学 | Hydrogen peroxide detection method based on iron porphyrin two-dimensional MOFs enzyme catalysis |
| CN114606329A (en) * | 2022-04-20 | 2022-06-10 | 青岛国际旅行卫生保健中心(青岛海关口岸门诊部) | Visual detection kit for salmonella |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107159883A (en) * | 2017-05-01 | 2017-09-15 | 福建医科大学 | Chitosan nano platinum particle simulates oxidizing ferment |
| CN108037090A (en) * | 2017-11-30 | 2018-05-15 | 盐城工学院 | A kind of method using chitosan-gold nanoparticle detection mercury ion |
| AU2018101303A4 (en) * | 2018-09-06 | 2018-10-18 | Hu, Shicheng Mr | Synthesis of Rh-Cu Nanozyme and Application for the Detection of Ascorbic Acid and Tannic Acid |
| CN109211820A (en) * | 2018-11-28 | 2019-01-15 | 安徽师范大学 | A kind of detection method of glutathione |
| AU2019100369A4 (en) * | 2019-04-05 | 2019-05-16 | Siqi Guo | Utility of PtRu alloy nanozyme in detection of ascorbic acid |
-
2019
- 2019-12-12 CN CN201911275295.2A patent/CN110907446A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107159883A (en) * | 2017-05-01 | 2017-09-15 | 福建医科大学 | Chitosan nano platinum particle simulates oxidizing ferment |
| CN108037090A (en) * | 2017-11-30 | 2018-05-15 | 盐城工学院 | A kind of method using chitosan-gold nanoparticle detection mercury ion |
| AU2018101303A4 (en) * | 2018-09-06 | 2018-10-18 | Hu, Shicheng Mr | Synthesis of Rh-Cu Nanozyme and Application for the Detection of Ascorbic Acid and Tannic Acid |
| CN109211820A (en) * | 2018-11-28 | 2019-01-15 | 安徽师范大学 | A kind of detection method of glutathione |
| AU2019100369A4 (en) * | 2019-04-05 | 2019-05-16 | Siqi Guo | Utility of PtRu alloy nanozyme in detection of ascorbic acid |
Non-Patent Citations (4)
| Title |
|---|
| HUAN JIANG等: "Peroxidase-like activity of chitosan stabilized silver nanoparticles for visual and colorimetric detection of glucose", 《ANALYST》 * |
| K.V.RAGAVAN 等: "Chitosan as a peroxidase mimic: Paper based sensor for the detection of hydrogen peroxide", 《SENSORS AND ACTUATORS B: CHEMICAL》 * |
| QINGMEI ZHONG等: "Synthesis of catalytically active carbon quantum dots and its application for colorimetric detection of glutathione", 《SENSORS AND ACTUATORS B: CHEMICAL》 * |
| 庞妍娇等: "基于木瓜蛋白酶过氧化物酶活性检测谷胱甘肽", 《西南大学学报(自然科学版)》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111487242A (en) * | 2020-04-27 | 2020-08-04 | 天津工业大学 | Hydrogen peroxide detection method based on iron porphyrin two-dimensional MOFs enzyme catalysis |
| CN114606329A (en) * | 2022-04-20 | 2022-06-10 | 青岛国际旅行卫生保健中心(青岛海关口岸门诊部) | Visual detection kit for salmonella |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| FI102192B (en) | Methods to determine the glucose content of whole blood and cuvette and photometer for carrying out the method | |
| CN103805170B (en) | A kind of for identifying specificity fluorescent probe and the application thereof of hydrogen sulfide | |
| CN101329279B (en) | Method for rapidly testing cysteine in water solution | |
| CN110907446A (en) | Rapid detection method of glutathione | |
| Zeng et al. | A colorimetric and ratiometric fluorescent probe for quantitative detection of GSH at physiologically relevant levels | |
| CN108746660B (en) | Preparation method of copper-gold-platinum composite nano material and application of copper-gold-platinum composite nano material in cysteine detection | |
| CN112033943B (en) | Arginine detection method based on quantum dot-copper ion fluorescent substrate sensor | |
| Fereja et al. | Highly sensitive and selective non-enzymatic glucose detection based on indigo carmine/hemin/H 2 O 2 chemiluminescence | |
| Sivasankaran et al. | Selective recognition of creatinine–development of a colorimetric sensor | |
| CN112730393A (en) | Color development reagent and method for measuring ascorbic acid | |
| JP2013541009A (en) | Sample detection method | |
| CN104330393A (en) | Method for determining glucose by using gold nano-cluster as fluorescence probe | |
| Zloczower et al. | Relationship of flow rate, uric acid, peroxidase, and superoxide dismutase activity levels with complications in diabetic patients: can saliva be used to diagnose diabetes? | |
| CN104483275B (en) | Detection method of biothiol | |
| CN102692411A (en) | Reagent for determining percentage of glycosylated hemoglobin | |
| CN109668866B (en) | Preparation and detection method of fluorescent probe for detecting iodide ions in water environment | |
| CN110174397A (en) | A kind of application of colorimetric probe | |
| US11041860B2 (en) | Point-of-care device for the colorimetric determination of hemoglobin and glucose-6-phosphate dehydrogenase in biological samples | |
| Feng et al. | Kinetic spectrofluorimetric determination of trace ascorbic acid based on its inhibition on the oxidation of pyronine Y by nitrite | |
| GB2559520A (en) | Concentration measurement method | |
| Liu et al. | Ratiometric fluorescence and smartphone dual-mode detection of glutathione using carbon dots coupled with Ag+-triggered oxidation of o-phenylenediamine | |
| Sanz et al. | A blood-assisted optical biosensor for automatic glucose determination | |
| Chen et al. | Determination of glucose based on the effect of photons as a substitute for glucose oxidase | |
| CN106645074B (en) | Direct fluorescence spectrum detection method for cystine content in cystine tablets | |
| Lertvachirapaiboon et al. | Colorimetric determination of urinary creatinine in proteinuria patients by chromaticity analysis of gold nanoparticle colloidal solutions |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20200324 |