CN116879283A - Kit for determining ATP - Google Patents
Kit for determining ATP Download PDFInfo
- Publication number
- CN116879283A CN116879283A CN202310887574.4A CN202310887574A CN116879283A CN 116879283 A CN116879283 A CN 116879283A CN 202310887574 A CN202310887574 A CN 202310887574A CN 116879283 A CN116879283 A CN 116879283A
- Authority
- CN
- China
- Prior art keywords
- atp
- reagent
- acid
- kit
- color
- 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.)
- Granted
Links
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 81
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims abstract description 29
- 102000019197 Superoxide Dismutase Human genes 0.000 claims abstract description 28
- 108010012715 Superoxide dismutase Proteins 0.000 claims abstract description 28
- 239000012086 standard solution Substances 0.000 claims abstract description 28
- 229960001484 edetic acid Drugs 0.000 claims abstract description 25
- 239000002253 acid Substances 0.000 claims abstract description 23
- 150000003839 salts Chemical class 0.000 claims abstract description 21
- 239000007800 oxidant agent Substances 0.000 claims abstract description 15
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical group OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 claims description 39
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 15
- 239000003795 chemical substances by application Substances 0.000 claims description 15
- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical compound OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 claims description 7
- 239000004327 boric acid Substances 0.000 claims description 7
- RMAQACBXLXPBSY-UHFFFAOYSA-N silicic acid Chemical compound O[Si](O)(O)O RMAQACBXLXPBSY-UHFFFAOYSA-N 0.000 claims description 7
- 235000012239 silicon dioxide Nutrition 0.000 claims description 7
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 6
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims description 6
- XBDQKXXYIPTUBI-UHFFFAOYSA-N dimethylselenoniopropionate Natural products CCC(O)=O XBDQKXXYIPTUBI-UHFFFAOYSA-N 0.000 claims description 6
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 claims description 6
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 claims description 3
- GRYLNZFGIOXLOG-UHFFFAOYSA-N Nitric acid Chemical compound O[N+]([O-])=O GRYLNZFGIOXLOG-UHFFFAOYSA-N 0.000 claims description 3
- 235000011054 acetic acid Nutrition 0.000 claims description 3
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims description 3
- 235000019253 formic acid Nutrition 0.000 claims description 3
- 229910017604 nitric acid Inorganic materials 0.000 claims description 3
- 235000019260 propionic acid Nutrition 0.000 claims description 3
- IUVKMZGDUIUOCP-BTNSXGMBSA-N quinbolone Chemical compound O([C@H]1CC[C@H]2[C@H]3[C@@H]([C@]4(C=CC(=O)C=C4CC3)C)CC[C@@]21C)C1=CCCC1 IUVKMZGDUIUOCP-BTNSXGMBSA-N 0.000 claims description 3
- 229910052736 halogen Inorganic materials 0.000 claims description 2
- 150000002367 halogens Chemical class 0.000 claims description 2
- 230000001590 oxidative effect Effects 0.000 abstract description 5
- ZKHQWZAMYRWXGA-KQYNXXCUSA-J ATP(4-) Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP([O-])(=O)OP([O-])(=O)OP([O-])([O-])=O)[C@@H](O)[C@H]1O ZKHQWZAMYRWXGA-KQYNXXCUSA-J 0.000 description 104
- ZKHQWZAMYRWXGA-UHFFFAOYSA-N Adenosine triphosphate Natural products C1=NC=2C(N)=NC=NC=2N1C1OC(COP(O)(=O)OP(O)(=O)OP(O)(O)=O)C(O)C1O ZKHQWZAMYRWXGA-UHFFFAOYSA-N 0.000 description 104
- 238000011161 development Methods 0.000 description 20
- 238000006243 chemical reaction Methods 0.000 description 19
- 230000000694 effects Effects 0.000 description 17
- 238000005259 measurement Methods 0.000 description 16
- 238000002835 absorbance Methods 0.000 description 11
- 238000001514 detection method Methods 0.000 description 9
- 238000002360 preparation method Methods 0.000 description 7
- 238000000034 method Methods 0.000 description 6
- 239000011259 mixed solution Substances 0.000 description 6
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 5
- 239000007788 liquid Substances 0.000 description 5
- 239000007853 buffer solution Substances 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- 229940124274 edetate disodium Drugs 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 230000031700 light absorption Effects 0.000 description 3
- 230000035945 sensitivity Effects 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 238000000870 ultraviolet spectroscopy Methods 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- GEYOCULIXLDCMW-UHFFFAOYSA-N 1,2-phenylenediamine Chemical compound NC1=CC=CC=C1N GEYOCULIXLDCMW-UHFFFAOYSA-N 0.000 description 2
- KRHYYFGTRYWZRS-UHFFFAOYSA-N Fluorane Chemical compound F KRHYYFGTRYWZRS-UHFFFAOYSA-N 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 239000011248 coating agent Substances 0.000 description 2
- 238000000576 coating method Methods 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 239000012153 distilled water Substances 0.000 description 2
- 230000007774 longterm Effects 0.000 description 2
- 229920002521 macromolecule Polymers 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- BMYNFMYTOJXKLE-UHFFFAOYSA-N 3-azaniumyl-2-hydroxypropanoate Chemical compound NCC(O)C(O)=O BMYNFMYTOJXKLE-UHFFFAOYSA-N 0.000 description 1
- GFFGJBXGBJISGV-UHFFFAOYSA-N Adenine Chemical compound NC1=NC=NC2=C1N=CN2 GFFGJBXGBJISGV-UHFFFAOYSA-N 0.000 description 1
- 108091023037 Aptamer Proteins 0.000 description 1
- 101000836247 Aquifex pyrophilus Superoxide dismutase [Fe] Proteins 0.000 description 1
- HMFHBZSHGGEWLO-SOOFDHNKSA-N D-ribofuranose Chemical compound OC[C@H]1OC(O)[C@H](O)[C@@H]1O HMFHBZSHGGEWLO-SOOFDHNKSA-N 0.000 description 1
- 230000004543 DNA replication Effects 0.000 description 1
- 208000013016 Hypoglycemia Diseases 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 208000018737 Parkinson disease Diseases 0.000 description 1
- PYMYPHUHKUWMLA-LMVFSUKVSA-N Ribose Natural products OC[C@@H](O)[C@@H](O)[C@@H](O)C=O PYMYPHUHKUWMLA-LMVFSUKVSA-N 0.000 description 1
- 230000005856 abnormality Effects 0.000 description 1
- HMFHBZSHGGEWLO-UHFFFAOYSA-N alpha-D-Furanose-Ribose Natural products OCC1OC(O)C(O)C1O HMFHBZSHGGEWLO-UHFFFAOYSA-N 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 230000003197 catalytic effect Effects 0.000 description 1
- 230000032823 cell division Effects 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 239000008139 complexing agent Substances 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 239000010931 gold Substances 0.000 description 1
- 229910052737 gold Inorganic materials 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- XMBWDFGMSWQBCA-UHFFFAOYSA-N hydrogen iodide Chemical compound I XMBWDFGMSWQBCA-UHFFFAOYSA-N 0.000 description 1
- 229940071870 hydroiodic acid Drugs 0.000 description 1
- GPRLSGONYQIRFK-UHFFFAOYSA-N hydron Chemical compound [H+] GPRLSGONYQIRFK-UHFFFAOYSA-N 0.000 description 1
- 230000002218 hypoglycaemic effect Effects 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 208000028867 ischemia Diseases 0.000 description 1
- 231100000053 low toxicity Toxicity 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 150000003013 phosphoric acid derivatives Chemical class 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 239000013589 supplement Substances 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
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
Landscapes
- Physics & Mathematics (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Plasma & Fusion (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention relates to a kit for determining ATP, comprising a reagent 1 and a reagent 2, wherein the reagent 1 comprises: the reagent 2 is an ATP standard solution, and the reagent comprises an oxidant, a color reagent, a first acid, superoxide dismutase (SOD) and/or edetic acid and/or salts thereof. The kit for measuring ATP has excellent durability and can accurately measure the concentration of ATP even when left for a long period of time.
Description
Technical Field
The present invention relates to a kit for measuring ATP, and more particularly, to a kit for measuring ATP having excellent durability.
Background
The detection of biological macromolecules is of great interest to scientists in all countries, because the content and conformation of biological macromolecules in the human body are closely related to human diseases. Among the numerous negatively charged phosphate compounds, adenosine Triphosphate (ATP) (adenosine triphosphate) is a multifunctional nucleotide consisting of 1 molecule adenine, 1 molecule ribose and 3 molecules phosphate. ATP plays an important role in organisms and is an energy source for the survival and cell proliferation of all organisms. ATP is present in cells for DNA replication, transcription, cell division, intracellular biosynthesis, various enzymatic reactions, and the like. Studies have found that abnormalities in ATP often show the occurrence of, for example, ischemia, hypoglycemia, parkinson's disease, etc. Thus, the detection of ATP has become an important indicator for the diagnosis of these diseases.
The more classical means of detecting ATP are the "luciferase-luciferin" method, including high performance liquid chromatography, aptamer, and electrophoresis. Although these methods have high sensitivity, professional equipment or professional operation is generally required, and there is a high demand for not only equipment and operators but also the operating environment.
For the above problems, there has been reported a kit for detecting the ATP content in a sample by performing color development by a reaction of a color former with hydrogen peroxide, which is simple and easy to operate and has low demands on operators and operating environments.
Disclosure of Invention
Technical problem to be solved by the invention
The inventors of the present invention have studied a kit for measuring the ATP content in a sample by developing color through the reaction of the above-mentioned color developing agent with hydrogen peroxide, and have found that the kit is simple in operation but has a negative correlation in sensitivity and time, i.e., has poor durability. For example, the accuracy of the kit for determining the concentration of ATP to be detected by color development is significantly reduced after more than one month of storage. That is, for a test whose ATP concentration is identical, the measurement results obtained are different when it is measured at different times. Although the kits after long-term storage can be recalibrated by the ATP standard solution, the accuracy of the different kits varies, which results in that even if the calibration is performed, accurate measurement results cannot be obtained.
The reagent kit detects the presence and the content of ATP through the color reaction of hydrogen peroxide and a color reagent, and the invention notices that the color reagent used in the reagent kit is a stable substance after research, but the color reagent is probably due to the fact that the color reagent is in a strong oxidizing environment for a long time, after a period of time, part of the structure of the color reagent is oxidized, so that the color development effect is greatly reduced, and the accuracy of determining the ATP concentration of a sample to be detected based on the color development effect is reduced, so that the ATP concentration in the sample to be detected is difficult to accurately determine.
Accordingly, an object of the present invention is to provide a kit for detecting ATP, which has excellent durability and can maintain ATP detection accuracy for a long period of time.
Technical proposal for solving the technical problems
Scheme 1A kit for determining ATP, characterized by comprising reagent 1,
the reagent 1 comprises: an oxidizing agent, a color developing agent, a first acid, superoxide dismutase (SOD), and/or edetic acid and/or salts thereof.
Scheme 2. The kit for determining ATP according to scheme 1 is characterized in that the superoxide dismutase is Cu/Zn-SOD.
The kit for determining ATP according to scheme 1, wherein the reagent 1 further comprises a second acid selected from boric acid and silicic acid.
The kit for measuring ATP according to scheme 4, wherein the content of boric acid or silicic acid in the reagent 1 is 0.1 to 3 wt%.
The kit for measuring ATP according to scheme 5, wherein the edetic acid is contained in the reagent 1 in an amount of 0.01 to 0.1% by weight.
Scheme 6. The kit for determining ATP according to scheme 1, characterized in that the oxidizing agent is hydrogen peroxide.
The kit for measuring ATP according to claim 1, wherein the first acid is at least one selected from the group consisting of halogen acid, sulfuric acid, phosphoric acid, nitric acid, formic acid, acetic acid, and propionic acid.
The kit for measuring ATP according to claim 1, wherein the superoxide dismutase is contained in the reagent 1 in an amount of 0.1 to 5% by weight.
Scheme 9 the kit for determining ATP according to scheme 1, further comprising reagent 2, wherein reagent 2 is an ATP standard solution.
Technical effects of the invention
The kit for determining ATP of the present invention comprises a reagent 1, wherein the reagent 1 comprises: an oxidizing agent, a color developing agent, a first acid, superoxide dismutase (SOD), and/or edetic acid and/or salts thereof. The above problems are solved by using a solution containing edetic acid and/or a salt thereof as a base liquid of a color former and hydrogen peroxide, and an ATP detection kit is obtained which shows excellent color development effect even after a long period of time, high detection accuracy and excellent durability as when it was just prepared.
Detailed Description
After conducting a study on a kit for detecting the ATP content in a sample by developing the reaction of the above-mentioned color-developing agent with hydrogen peroxide, the inventors of the present invention have noted that, although the color-developing agents used therein are stable under ordinary conditions, since these color-developing agents are under a strong oxidizing environment formed by hydrogen peroxide in the kit, the sensitivity and accuracy of the color development thereof are degraded after a lapse of time. For the present invention, the inventors have solved the above problems by using a solution containing edetic acid and/or a salt thereof as a base liquid for a color developer and hydrogen peroxide, and have obtained an ATP detection kit which can exhibit excellent color development effects, high detection accuracy, and excellent durability even after a long period of time, as compared with those of the case of the immediately preceding preparation.
In particular, the invention relates to a kit for determining ATP comprising a reagent 1, the reagent 1 comprising: an oxidizing agent, a color developing agent, a first acid, superoxide dismutase (SOD), and/or edetic acid and/or salts thereof.
In the present invention, EDTA buffer containing edetic acid and/or a salt thereof is used as a base liquid of the reagent 1, thereby realizing that the color developing effect can be exhibited as excellent as that immediately after the preparation even if the color developing agent is kept for a long period of time.
The color developer used in the present invention is not particularly limited as long as it can undergo a color reaction with an oxidizing agent in the presence of a first acid, and 3, 5-Tetramethylbenzidine (TMB), o-phenylenediamine, or the like can be used. These developers may undergo a color development reaction with an oxidizing agent (e.g., hydrogen peroxide) in the presence of a first acid to turn blue-green. The first acid plays a catalyst-like role in this reaction, however, the presence of ATP may inhibit this "catalytic activity" of the first acid, such that the above-described color reaction does not proceed completely. The inhibition of the color reaction by ATP at different concentrations is also different, so that the ATP content of the sample can be measured from the color change of the color reaction.
Specifically, the higher the ATP concentration, the weaker the color reaction, and the lower the ATP concentration or the absence of ATP, the stronger the color reaction. Accordingly, the ATP content can be quantitatively detected by using an ultraviolet-visible spectrophotometer.
The inventors of the present invention noted that the above-mentioned color-developing agent is in contact with an oxidizing agent in a kit, in a strongly oxidizing environment. After a period of time, the intensity of the chromogenic reaction is reduced, so that the chromogenic effect is greatly reduced, and the accuracy of determining the ATP concentration of the sample to be detected based on the chromogenic effect is reduced. In view of the above problems, the inventors have found that the above problems can be solved by using a liquid containing edetic acid and/or a salt thereof as an environmental base liquid. In the present invention, the mechanism for solving this problem is not completely clarified, and the inventors of the present invention speculate about this mechanism as follows.
Edetic acid and/or its salts may act as complexing agents, so that the developer that has undergone a color reaction is encapsulated by ions of edetic acid and/or its salts, so that it is isolated from the remaining oxidizing agent in the environment, thus ensuring a long-term stable maintenance of the color reaction. In addition, edetic acid and/or salts thereof may form a buffer in solution, which provides a better stable pH environment for edetic acid and/or salts thereof to form a coating state, thus enabling the coating to be more durable and tight.
The ATP detection kit of the present invention includes reagent 1. Wherein the reagent 1 comprises: an oxidizing agent, a color developing agent, a first acid, superoxide dismutase (SOD), and/or edetic acid and/or salts thereof.
In the reagent 1, the oxidizing agent is hydrogen peroxide, and the concentration of hydrogen peroxide in the reagent 1 is not particularly limited as long as it can react with the color-developing agent, and is preferably 0.1 mM-10 mM, more preferably 1 mM-5 mM. Other oxidizing agents may be used in the present invention as long as they are capable of undergoing a color reaction with the color developer.
In the reagent 1, the color former may be a color former commonly used, and 3, 5-Tetramethylbenzidine (TMB), o-phenylenediamine, or the like may be used, and 3, 5-Tetramethylbenzidine (TMB) having low toxicity is preferably used. The concentration of the color former in the reagent 1 is not particularly limited, but is preferably 0.1 mM-10 mM, more preferably 1 mM-5 mM.
In reagent 1, the first acid may be at least one of a hydrohalic acid, sulfuric acid, phosphoric acid, nitric acid, formic acid, acetic acid, and propionic acid, wherein the hydrohalic acid may be hydrochloric acid, hydrobromic acid, hydrofluoric acid, hydroiodic acid. The concentration of the first acid in the reagent 1 is 0.01 mM-1M, preferably 0.05 mM-0.5 mM, calculated as hydrogen ion. Hydrochloric acid or acetic acid is preferable from the viewpoint of easy availability.
Reagent 1 further contains superoxide dismutase (SOD). In the present invention, color development is achieved due to the reaction of hydrogen peroxide with a color developer. However, hydrogen peroxide is easily volatilized, thereby affecting the color development effect. The present inventors have found that the concentration of hydrogen peroxide can be maintained for a long period of time after adding superoxide dismutase to the reagent 1, thereby maintaining an excellent color development effect. The mechanism by which the above effects are achieved by adding superoxide dismutase is not clear. The inventors speculate that superoxide dismutase does not appear to guarantee that hydrogen peroxide does not decompose or volatilize, but in the presence of superoxide dismutase it appears to be possible to supplement hydrogen peroxide.
The superoxide dismutase may be Cu/Zn-SOD, mn-SOD or Fe-SOD. However, superoxide dismutase itself has a color, and may affect color development. Therefore, the type of superoxide dismutase can be selected according to the color of the color developer. Preferably, when 3, 5-Tetramethylbenzidine (TMB) is selected as the color-developer, cu/Zn-SOD is preferably used. Because 3, 5-Tetramethylbenzidine (TMB) exhibits a bluish green color when developed, and Cu/Zn-SOD itself is also a bluish green color similar to the developed bluish green color. Thus, the color development is not affected, but the color development effect is enhanced.
The content of the superoxide dismutase in the reagent 1 is 0.1-5 wt%. When the concentration of superoxide dismutase is above the upper limit, the concentration of hydrogen peroxide is further increased, and the concentration of hydrogen peroxide can be maintained for a long time (more than one year); when the concentration is less than the lower limit, the effect on the color development effect is small, and the excellent color development effect can be ensured. The concentration is preferably 1 to 5% by weight, more preferably 2% by weight.
The reagent 1 of the present invention further comprises edetic acid and/or a salt thereof. The edetic acid and/or its salt may be edetic acid or edetate disodium (EDTA-2 Na), and from the viewpoint of excellent solubility, edetate disodium (EDTA-2 Na) is preferably used. The concentration of the edetic acid and/or the salt thereof in the reagent 1 can be 0.01-0.1 wt%, and the edetic acid and/or the salt thereof in the concentration range can realize better wrapping protection effect on the color developing agent, thereby further prolonging the time of the color developing agent not influenced by the strong oxidizing environment. Further, the preferable concentration may be 0.05 to 0.1% by weight.
In addition, edetic acid and/or salts thereof may form a buffer solution upon dissolution in water. The pH of the buffer solution may be 6.5 to 8, preferably 6.7 to 7.8.
In the present invention, reagent 1 may further contain a second acid selected from boric acid and silicic acid. The present inventors have found that the addition of boric acid or silicic acid to a reagent contributes to maintaining the stability of a buffer solution formed of edetic acid, in particular, the stability of pH of a buffer solution formed of edetic acid, thereby enabling the effect of maintaining color development for a long period of time to be achieved.
In the present invention, the kit may further include a reagent 2, and the reagent 2 is an ATP standard solution. The kit may not include reagent 2. The reagent 2 may be disposed immediately before the measurement of the sample to be measured.
Immediately before the measurement of the sample to be measured, the standard solutions of ATP may be prepared, or standard solutions having different ATP concentrations may be prepared separately, or standard solutions having a single concentration may be prepared and diluted to obtain standard solutions having different ATP concentrations.
The prepared ATP standard solutions are used for contacting with the reagent 1 before the sample to be detected is measured, so that the corresponding curves of the ATP concentration and the absorbance value are drawn according to the color development changes of the standard solutions with different ATP concentrations after the standard solutions are contacted with the reagent 1.
It should be noted that the specific design of the ATP concentration of the ATP standard solution should be designed according to the concentration of the sample to be tested. Around the possible ATP concentration of the sample to be measured, a plurality of standard ATP standard samples are densely designed around the possible ATP concentration, thereby improving the accuracy of the measurement of ATP in the sample to be measured. For example, the ATP concentration of the sample to be measured is estimated to be about 12. Mu.M, then one example of the concentrations of these ATP standard solutions can be 0. Mu.M, 5. Mu.M, 7. Mu.M, 8. Mu.M, 10. Mu.M, 11. Mu.M, 12. Mu.M, 13. Mu.M, 14. Mu.M, 16. Mu.M, 20. Mu.M. That is, a plurality of ATP standard solutions were densely designed around 12. Mu.M.
Next, a method for measuring the ATP concentration of a sample to be measured using the ATP kit of the present invention will be described.
Step one, estimating the ATP concentration of the sample to be tested.
ATP concentration is estimated initially based on the source, type, etc. of the sample. In estimating the ATP concentration of the sample to be measured, it is naturally best if the estimation is relatively accurate, but if it is difficult to estimate, it is not necessary to estimate the ATP concentration with particular accuracy, and only a rough interval is known. The minimum limit for estimating the ATP concentration of the sample to be measured is not to exceed the ATP concentration range of the ATP standard solution in reagent 2. The measurement can be performed so long as the ATP concentration of the ATP standard solution in the reagent 2 is not exceeded.
Step two, reagent 2 was prepared and the concentration of the ATP standard solution was designed.
An ATP solution of known concentration was prepared as reagent 2. And in the vicinity of the estimated value of the ATP concentration of the sample, a plurality of standard solutions having different ATP concentrations are densely designed. In the present embodiment, the concentration of the ATP standard solution in the reagent 2 is constant, and the ATP standard solution having different concentrations can be obtained by diluting the reagent 2.
Step three, obtaining the color development result (absorbance) of the ATP standard solution
The plurality of ATP standard solutions with different ATP concentrations are respectively mixed with the reagent 1 to obtain mixed solutions of the ATP standard solutions and the reagent 1, and color development results under various ATP concentrations are obtained.
And measuring the absorbance of each mixed solution in the visible light band by using an ultraviolet-visible spectrophotometer. The function of ATP concentration and absorbance can be fitted to the values of the respective absorbances as needed.
Step four, obtaining the absorbance value of the sample to be detected and obtaining the ATP concentration through comparison
And mixing the sample to be tested with the reagent 1 to obtain a mixed solution to be tested. And measuring the absorbance value of the mixed solution to be measured in the visible light band by adopting an ultraviolet and visible light spectrophotometer. By comparing the absorbance with the absorbance of each ATP standard solution measured in step three, the ATP concentration of the sample to be measured can be obtained. In addition, the absorbance value of the sample to be measured can be substituted into the fitting function, so that the accurate value of the ATP concentration can be obtained.
The following is a brief explanation of the conditions of the above measurement.
The ambient temperature for the measurement is not particularly limited as long as the color reaction can normally occur, and is preferably in the temperature range of 5 to 50 ℃, and particularly preferably at room temperature. The pressure measured above is typically atmospheric pressure. No additional pressurization or depressurization is required. In the above measurement, the time for mixing and reacting the ATP standard solution or the sample to be measured with the reagent is not particularly limited, and the ATP concentration of the sample to be measured may be slightly different depending on the sample to be measured, and generally the reaction time may be in the range of 30 minutes to 3 hours. Within such a time frame, a sufficient reaction can be achieved.
In the present invention, the accuracy of the measurement is ensured in the case of performing the measurement in a simple manner by adopting a specific ATP standard solution design method. That is, before measurement, the ATP concentration of the sample to be measured is estimated, and then the ATP standard sample with the corresponding concentration is densely designed near the estimated ATP concentration value, so that the measurement resolution near the estimated ATP concentration value is increased, and the fitting function can represent the real concentration, so that the ATP concentration in the sample to be measured can be measured more accurately. Thus, even in a sample having a very small ATP concentration, the measurement can be accurately performed without losing accuracy due to the simple method.
Examples
The technical scheme of the invention is further described in detail through specific examples.
Embodiments of the present invention generally relate to verifying the durability of an ATP detection kit of the present invention.
Method for measuring durability
Reagent 1 having the same composition was used.
Experiment 1: when the preparation is just finished, measuring a sample to be tested to obtain a light absorption value;
experiment 2: after the reagent 1 is stored for one month at normal temperature and normal pressure, the sample to be tested is measured, and the light absorption value is obtained;
experiment 3: after the reagent 1 is stored for three months at normal temperature and pressure, the sample to be tested is measured, and the light absorption value is obtained.
Examples 1 to 9 and comparative examples 1 to 3
(1) Sample to be measured
Samples with known ATP concentrations were used as test samples. The ATP concentration in the sample to be tested was 5. Mu.M. ATP is produced (food grade) by shanxi Kang He pharmaceutical limited.
(2) Preparation of reagent 1
The following products were selected according to the ingredients shown in table 1.
Oxidizing agent: 3% hydrogen peroxide solution (Shandong Dezhou medical instruments Co., ltd.)
Color-developing agent: 3, 5-tetramethylbenzidine powder (Fuzhou Feijung Biotechnology Co., ltd.)
First acid: 10% dilute hydrochloric acid (Shandong Dezhou medical instruments Co., ltd.)
First acid: 5% acetic acid (Suzhou Jinbang chemical Co., ltd.)
Superoxide dismutase: cu/Zn-SOD (Shanxi Zhonghong pharmaceutical Co., ltd.)
Edetic acid and/or salts thereof: edetate disodium (Suzhou gold Pond chemical Co., ltd.).
Boric acid: (Shandong Yi Cheng chemical technology Co., ltd.)
Silicic acid: (Shandong Yi Cheng chemical technology Co., ltd.)
The above components were added to distilled water at concentration ratios shown in table 1, and stirred slowly to obtain reagent 1.
(3) Preparation of reagent 2
ATP was dissolved in distilled water to prepare a 1mM ATP standard solution as reagent 2.
It should be noted that the purpose of this example is to test the durability of reagent 1, so reagent 2 is not tested, and reagent preparation is only a formal step.
(4) Determination of a sample to be tested
Within 8 hours of the preparation of reagent 1, 0.5mL of the sample to be measured was mixed with reagent 1 (0.5 mL), and the volume of the mixed solution was 1mL.
The absorbance of the mixed solution of the sample to be measured was measured at a wavelength of 420nm using an ultraviolet-visible spectrophotometer (Jinan Alebao instruments and equipment Co., ltd. UV-3100).
The reagent 1 was sealed and left at normal temperature and pressure for one month, and the sample to be measured was measured with the reagent 1 left for one month in the same manner as described above.
The reagent 1 was sealed and left at room temperature and pressure for three months, and the sample to be measured was measured with the reagent 1 left for one month in the same manner as described above.
The measurement results are shown in tables 1 to 2 below.
Industrial applicability
The present invention relates to a kit for measuring ATP, which has excellent durability and can ensure measurement accuracy even when left for a long period of time, and can be widely used in various fields such as biomedical treatment.
Claims (9)
1. A kit for determining ATP, characterized in that the kit comprises a reagent 1,
the reagent 1 comprises: an oxidizing agent, a color developing agent, a first acid, superoxide dismutase (SOD), and/or edetic acid and/or salts thereof.
2. The kit for determining ATP of claim 1, wherein the superoxide dismutase is Cu/Zn-SOD.
3. The kit for determining ATP of claim 1, wherein the reagent 1 further comprises a second acid selected from boric acid or silicic acid.
4. The kit for measuring ATP as claimed in claim 3, wherein the boric acid or silicic acid is contained in the reagent 1 in an amount of 0.1 to 3% by weight.
5. The kit for measuring ATP as claimed in claim 1, wherein the edetic acid and/or its salt is contained in the reagent 1 in an amount of 0.01 to 0.1% by weight.
6. The kit for determining ATP of claim 1, wherein the oxidizing agent is hydrogen peroxide.
7. The kit for determining ATP according to claim 1, wherein the first acid is at least one selected from the group consisting of halogen acid, sulfuric acid, phosphoric acid, nitric acid, formic acid, acetic acid, and propionic acid.
8. The kit for measuring ATP as claimed in claim 1, wherein the superoxide dismutase is contained in the reagent 1 in an amount of 0.1 to 5% by weight.
9. The kit for determining ATP of claim 1, further comprising reagent 2, wherein the reagent 2 is an ATP standard solution.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310887574.4A CN116879283B (en) | 2023-07-19 | 2023-07-19 | Kit for determining ATP |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310887574.4A CN116879283B (en) | 2023-07-19 | 2023-07-19 | Kit for determining ATP |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN116879283A true CN116879283A (en) | 2023-10-13 |
| CN116879283B CN116879283B (en) | 2024-09-13 |
Family
ID=88260073
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310887574.4A Active CN116879283B (en) | 2023-07-19 | 2023-07-19 | Kit for determining ATP |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116879283B (en) |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20040101922A1 (en) * | 2002-09-06 | 2004-05-27 | Promega Corporation | Method for detecting transferase enzymatic activity |
| US20060111442A1 (en) * | 2004-11-20 | 2006-05-25 | Mr. Stanley Antosh | Use of methyl pyruvate to increase cellular energy production downstream of glycolysis for the PARP-1 ablation of HIV without necrotic cell death caused by continuous, chronic PARP-1 activation through the concomitant depletion of ATP and NAD. |
| JP2019073476A (en) * | 2017-10-17 | 2019-05-16 | 国立大学法人北海道大学 | Agent for atp production promotion |
| WO2021205083A2 (en) * | 2020-04-11 | 2021-10-14 | Finindsutria Srl | Composition for the prevention or treatment of covid-19 |
| WO2022047779A1 (en) * | 2020-09-07 | 2022-03-10 | 音芙医药科技(上海)有限公司 | Use of nicotinamide mononucleotide in preparation of reagent for improving level of na+-k+-atp enzyme in sarcolemma of injured skin |
| CN116183890A (en) * | 2022-12-02 | 2023-05-30 | 浙江今复康生物科技有限公司 | Airway hydrogen peroxide detection method and kit |
-
2023
- 2023-07-19 CN CN202310887574.4A patent/CN116879283B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20040101922A1 (en) * | 2002-09-06 | 2004-05-27 | Promega Corporation | Method for detecting transferase enzymatic activity |
| US20060111442A1 (en) * | 2004-11-20 | 2006-05-25 | Mr. Stanley Antosh | Use of methyl pyruvate to increase cellular energy production downstream of glycolysis for the PARP-1 ablation of HIV without necrotic cell death caused by continuous, chronic PARP-1 activation through the concomitant depletion of ATP and NAD. |
| JP2019073476A (en) * | 2017-10-17 | 2019-05-16 | 国立大学法人北海道大学 | Agent for atp production promotion |
| WO2021205083A2 (en) * | 2020-04-11 | 2021-10-14 | Finindsutria Srl | Composition for the prevention or treatment of covid-19 |
| WO2022047779A1 (en) * | 2020-09-07 | 2022-03-10 | 音芙医药科技(上海)有限公司 | Use of nicotinamide mononucleotide in preparation of reagent for improving level of na+-k+-atp enzyme in sarcolemma of injured skin |
| CN116183890A (en) * | 2022-12-02 | 2023-05-30 | 浙江今复康生物科技有限公司 | Airway hydrogen peroxide detection method and kit |
Non-Patent Citations (2)
| Title |
|---|
| 丁振若: "《现代检验医学》", 31 January 2006, 人民军医出版社 , pages: 954 - 656 * |
| 文佳慧: "基于ATP类过氧化物酶活性的分析应用及清除活性氧的研究", 《优秀硕士论文》, 15 January 2023 (2023-01-15) * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN116879283B (en) | 2024-09-13 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Hadwan | New method for assessment of serum catalase activity | |
| Campanella et al. | New biosensor for superoxide radical used to evidence molecules of biomedical and pharmaceutical interest having radical scavenging properties | |
| Mahmoud | Iron ion-selective electrodes for direct potentiometry and potentiotitrimetry in pharmaceuticals | |
| Hamza et al. | New spectrophotometric method for the assessment of catalase enzyme activity in biological tissues | |
| Safavi et al. | Highly selective and sensitive kinetic spectrophotometric determination of vanadium (IV) in the presence of vanadium (V) | |
| CN105241830A (en) | Glycolated serum albumin detection reagent and application thereof | |
| CN102128919B (en) | Composition and use thereof | |
| CN116879283B (en) | Kit for determining ATP | |
| Shekhovtsova et al. | Determination of mercury at the picogram per milliliter level using immobilized horseradish peroxidase | |
| CN112255219A (en) | 1, 5-sorbitan determination kit, and preparation method and application thereof | |
| CN106092924A (en) | A kind of test kit measuring free fatty and preparation method thereof | |
| EP2460888B1 (en) | Method and kit for measurement of dehydrogenase or substrate for the dehydrogenase | |
| KR101942784B1 (en) | Method and Test Kit For Dectecting Concentration of Hydrogen Peroxide | |
| CN112595710B (en) | Rapid detection kit and detection method for iodide ions in serum sample | |
| Yagi et al. | Glycative stress and anti-aging: 2. The Evaluation of Glycative Stress: Measurement of blood glucose, glycated proteins and intermediates | |
| JP4577863B2 (en) | Composition for measuring calcium ion and measuring method | |
| KR101346666B1 (en) | nitrate concentration detecting agent and the kit using thereof | |
| CN102323265B (en) | Method and kit for detecting potassium ions | |
| Othman et al. | Spectrophotometric Determination of Catecholamine Containing Drugs Using Calcon Dye | |
| Patel et al. | A reagent-free phosphate chemiresistive sensor using carbon nanotubes functionalized with crystal violet | |
| Majeed et al. | Spectrophotometric determination of hydrazine sulphate using Fe (ii)-2, 2'-bipyridyl-application to various water samples | |
| CN111239126A (en) | Detection reagent combination for phosphate molybdenum antimony resistance method determination and preparation method thereof | |
| CN111351777A (en) | Method for rapidly determining soluble copper ions in water | |
| Al-Shukry et al. | Spectrophotometric Determination of Uric Acid in Samples of Urine | |
| CN107607524A (en) | Ammonia determines kit |
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 | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |