CN111999349A - Detection method or kit for hydrogen peroxide - Google Patents
Detection method or kit for hydrogen peroxide Download PDFInfo
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Abstract
The invention designs a method or a kit for detecting hydrogen peroxide, belonging to the technical field of medical inspection. The components of the kit comprise a stable free radical compound. The detection method or the kit can effectively eliminate or reduce the interference of some common interference substances existing in the sample on the detection result, and improve the reliability of the detection result.
Description
Technical Field
The invention relates to the technical field of medical examination. In particular, the present invention relates to a method or kit for detecting hydrogen peroxide.
Background
In medical tests, there are many substances whose detection involves detection of hydrogen peroxide, such as detection of glycerides, detection of fatty acids, detection of glycosylated proteins, detection of 2-imino-1-methylimidazolin-4-one, detection of adenosine deaminase activity, and the like.
In the specific implementation process, the content or activity of the substance is generally calculated by carrying out a series of enzyme-catalyzed oxidations on the substance to generate hydrogen peroxide, and then detecting the amount or generation rate of the generated hydrogen peroxide by an electrode method or an oxidase method which is called Trinder's for short.
However, in practical applications, hydrogen peroxide is unstable and susceptible to interference from other substances present in the sample. As a common example, after a patient takes or injects a medicine containing 2, 5-dihydroxybenzenesulfonate or dopa, the existence of 2, 5-dihydroxybenzenesulfonate or dopa in the blood of the patient can cause the measurement result of hydrogen peroxide depended on in the measurement process to be seriously lower, thereby misjudging the disease condition of the patient and causing serious consequences.
In addition, through experiments, a plurality of compounds, such as tocopherol, gentisic acid, gallic acid, catechin, and MEDOPA, are found to interfere with the detection of hydrogen peroxide, and result misjudgment is caused.
Therefore, there is a need in the art for a suitable technique that can eliminate or reduce interference of 2, 5-dihydroxybenzenesulfonate or dopa-like drugs or compounds in a sample during the detection of hydrogen peroxide, and ensure reliable detection results of hydrogen peroxide.
Disclosure of Invention
The invention provides a method or a kit for detecting hydrogen peroxide, which is characterized by comprising a step of contacting a test sample with a stable free radical compound so as to eliminate or reduce the interference of aromatic hydroxyl compounds in the sample.
In clinical medical examination, there are a lot of literature reports and mature solutions for the interference of conventional substances carried in patient samples, such as bilirubin, chyle, ascorbic acid, etc., on the detection items involving oxidase and hydrogen peroxide intermediates.
However, interference with specific drugs in the sample, such as 2, 5-dihydroxybenzenesulfonic acid and methyldopa, may destroy or competitively react with hydrogen peroxide to falsely lower the measured value, leading to misjudgment of the patient's condition with serious consequences. Currently, there is still a lack of effective means in the art to eliminate or reduce interference of substances such as 2, 5-dihydroxybenzenesulfonic acid and methyldopa in a sample with a test item involving hydrogen peroxide as an intermediate.
In the invention, a large number of screening experiments show that the method for contacting the sample with the stable free radical compound has a very good effect, and can very effectively eliminate or reduce the interference of 2, 5-dihydroxybenzenesulfonic acid and methyldopa on detection. For example, when a sample contains high-concentration calcium 2, 5-dihydroxybenzenesulfonate, the method described by the invention can reduce the interference degree to be within 10% of the relative deviation, and even can reduce the interference degree to be within 5% of the relative deviation under the conventional dosage concentration, thereby achieving the effect of eliminating the interference.
There are various stable radicals, such as nitroxide radicals represented by 4-hydroxy-2, 2, 6, 6-tetramethylpiperidinyloxy; 4-oxo-2, 5-cyclohexadiene-p-tolyloxy radicals represented by 2, 6-di-tert-butyl- (3, 5-di-tert-butyl-4-oxo-2, 5-cyclohexadiene) -p-tolyloxy radicals; thiazole thione oxygen free radicals represented by 3-hydroxy-4-methyl-2 (3H) -thiazole thione, picrylhydrazino free radicals represented by 2, 2-di (4-tert-octylphenyl) -1-picrylhydrazino free radical and 1, 1-diphenyl-2-picrylhydrazine free radical, and various substituted 2-oxo [1.4.2] oxathiazolo [2, 3-a ] pyridine free radicals.
The most varied of these are the nitroxide free radicals, which are the most widely used. Nitroxides are collectively characterized as comprising an (N-O. cndot.) structure with a single spin electron. The common nitroxides include piperidinoalkane nitroxide, pyrrolidine nitroxide, oxazolidinyl, imidazolium nitroxide, etc., and are mainly distinguished according to the type of heterocycle in which the nitrogen atom is located. There are hundreds of different specific nitroxide radicals, and further, according to organic chemistry knowledge, it is possible to design a wider variety of substituents and nitroxide compounds of specific structures.
In the case of piperidinoalkyloxy radicals, it is common to have a hydrocarbyl group attached to the carbons at the 2-and 6-positions, most often 2 methyl groups attached to each of the carbons at the 2-and 6-positions. Also, there are combinations of connecting ethyl, or propyl, or other hydrocarbyl and substituted hydrocarbyl groups and with hydrogen atoms. Various aliphatic hydrocarbon chains, aromatic hydrocarbons, various substituted hydrocarbons such as halogenated hydrocarbons, fatty acid chains, ketone-containing chains, and the like, and various combinations thereof, including various combinations with methyl groups or hydrogen atoms, can also be devised based on existing chemical knowledge. This is a common technique of organic chemistry and is not described in great detail here.
The carbon para (4) to the piperidinoalkyloxy group may have two hydrogen atoms, but more than one of the hydrogen atoms may be substituted with a substituent. Common substituents are, for example, hydroxyl group, oxygen atom, amino group, carboxyl chain, mercapto group, halogen atom, alkyl group, aralkyl group, aryl group, substituted oxy group, substituted amino group, substituted thio group, cyano group, isothiocyanato group and the like. Among them, hydroxyl, cyano, carboxyl, keto, methoxy, acetylamino, cyano, isothiocyanato, methacryloyloxy, 2-chloroacetamido and the like are more common.
Pyrrolidine nitroxides are also similar to piperidine nitroxides in that the carbons at the 2-and 5-positions are often substituted, more commonly hydrocarbyl or substituted hydrocarbyl, and most commonly methyl. In the 3-or 4-position of the pyrrole ring, various types of substituents are also frequently present. In a preferred embodiment of the present invention, 3-carboxy-2, 2, 5, 5-tetramethylpyrrolidin-1-yloxy radical is exemplified by a 2-and 5-carbon having two methyl groups, respectively, and a 3-carbon having a carboxy group attached thereto. Other types of structures are also similar and can be designed based on known organic chemistry knowledge and are not described in great detail herein.
Oxazolidinyloxy radicals are also similar to pyrrolidinionyloxy radicals, and the carbons at the 2 and 5 positions are often substituted, more commonly also hydrocarbyl or substituted hydrocarbyl or combinations thereof, and their structural schematic can be referred to in the present invention as preferred, for example, 2- (14-carboxytetradecyl) -2-ethyl-4, 4-dimethyl-3-oxazolidinyloxy, or 2- (10-carboxydecyl) -2-hexyl-4, 4-dimethyl-3-oxazolidinyloxy. Other types of structures are also similar, including substituents attached to the carbon atom at the 3-position, are known from the knowledge of well-known organic chemistry and are not described herein.
The hydrogens on the carbons at the 2-, 4-, 5-positions of the imidazoline nitroxide radical are generally also substituted, and the carbons at the 4-and 5-positions are often also substituted with hydrocarbyl groups, in particular methyl groups, the structural schematic of which can be referred to in the present invention as a preferred example of a 2- (4-nitrophenyl) -4, 4, 5, 5-tetramethylimidazoline-3-oxide-1-oxyl radical. Other types of structures are also similar and are known from knowledge of well known organic chemistry and are not described herein.
In the research of the present invention, taking the detection of 2-imino-1-methylimidazolin-4-one in blood as an example, the detection process of the prior art generally comprises the steps of hydrolyzing and oxidizing 2-imino-1-methylimidazolin-4-one by three tool enzymes to generate hydrogen peroxide, and then detecting the amount of the generated hydrogen peroxide by using Trinder's reaction, thereby calculating the content of 2-imino-1-methylimidazolin-4-one in the sample by the amount of the hydrogen peroxide. These procedures are well known in the art, and the manufacturer, amount of the enzyme, type of buffer used, pH, preservative and surfactant screening are well known in the art, and there are many reports and not many descriptions.
The detection of the hydrogen peroxide produced therein can be carried out by means of electrochemical electrodes, but the most widely used is the principle of the reaction generally designated as Trinder's. In the Trinder's reaction, hydrogen peroxide and 4-aminoantipyrine or an analogue thereof and a chromogen, which generally includes phenol and derivatives thereof, and various chromogens such as ADOS, ADPS, ALPS, DAOS, HDAOS, MADB, MAOS, TODB, TOOS, TOPS and the like, may be reacted to form a colored compound by the action of peroxidase. The sensitivity and stability of different chromogens are different, and the appropriate dosage of the chromogens also belongs to the common knowledge in the industry. The amount of each component is also within the general knowledge of the industry, such as 0.1-10 mM of 4-aminoantipyrine, 0.1-30 mM of chromogen, and buffers including phosphate buffer, glycine buffer, Good' S buffer, Tris buffer, etc., which are not discussed herein.
When 2-imino-1-methylimidazolin-4-one is converted to hydrogen peroxide, since hydrogen peroxide is a very active substance, it reacts with many substances, particularly reducing substances. The previously mentioned substances such as 2, 5-dihydroxybenzenesulfonic acid and methyldopa may be falsely low in measured values of 2-imino-1-methylimidazolin-4-one due to destructive or competitive reaction with hydrogen peroxide.
When the 2-imino-1-methylimidazoline-4-one is detected by an oxidase method in the prior art, the actual measurement result of the concentration of the 2-imino-1-methylimidazoline-4-one determined by the oxidase method is about 50% lower when the content of 2, 5-dihydroxybenzenesulfonic acid in a sample is 100 mg/L. In a preferred embodiment of the present invention, if a stable free radical, in one embodiment, 4-carboxy-2, 2, 6, 6-tetramethylpiperidine oxynitride is used to contact the sample, the interference of 2, 5-dihydroxybenzenesulfonic acid in the sample on the measurement result can be reduced to less than 10%, and excellent anti-interference effect can be shown.
In another similar preferred embodiment of detecting 2, 6, 8-trioxypurine based on the principle of oxidase method, when the content of 2, 5-dihydroxybenzenesulfonic acid in a sample is 100mg/L in the conventional technique for detecting 2, 6, 8-trioxypurine, the measured result of the concentration of 2, 6, 8-trioxypurine measured by the oxidase method is about 40% lower. In a preferred embodiment of the present invention, if a stable free radical, in one embodiment, 4-carboxy-2, 2, 6, 6-tetramethylpiperidine oxynitride is used to contact the sample, the interference of 2, 5-dihydroxybenzenesulfonic acid in the sample on the measurement result can be reduced to less than 10%, and a good anti-interference effect can be shown.
In another similar preferred embodiment of the method of detecting glyceride by using the principle of oxidase, when the content of 2, 5-dihydroxybenzenesulfonic acid in a sample is 100mg/L in the conventional method of detecting glyceride, the measured result of the method of oxidase for measuring the concentration of glyceride is about 20% lower. In a preferred embodiment of the present invention, if a stable free radical, in one embodiment, 4-carboxy-2, 2, 6, 6-tetramethylpiperidine oxynitride is used to contact the sample, the interference of 2, 5-dihydroxybenzenesulfonic acid in the sample on the measurement result can be reduced to less than 5%, and a good anti-interference effect is shown.
In another similar preferred embodiment of detecting glycosylated protein by the principle of the oxidase method, when the content of 2, 5-dihydroxybenzenesulfonic acid in the sample is 100mg/L in the detection of glycosylated protein by the conventional technique, the measured result of the oxidase method for determining the concentration of glycosylated protein is lower than approximately 60%. In a preferred embodiment of the present invention, if a stable free radical, in one embodiment, 4-carboxy-2, 2, 6, 6-tetramethylpiperidine oxynitride is used to contact the sample, the interference of 2, 5-dihydroxybenzenesulfonic acid in the sample on the measurement result can be reduced to less than 15%, and a good anti-interference effect can be shown.
In another similar preferred embodiment of the method of detecting fatty acids by the oxidase principle, when the content of 2, 5-dihydroxybenzenesulfonic acid in the sample is 100mg/L in the conventional method for detecting fatty acids, the measured result of the method of detecting fatty acid concentration by the oxidase method is about 30% lower. In a preferred embodiment of the present invention, if a stable free radical, in one embodiment, 4-carboxy-2, 2, 6, 6-tetramethylpiperidine oxynitride is used to contact the sample, the interference of 2, 5-dihydroxybenzenesulfonic acid in the sample on the measurement result can be reduced to less than 10%, and a good anti-interference effect can be shown.
In another similar preferred embodiment of the method of detecting glucose based on the principle of oxidase, when glucose is detected by the conventional technique, the measured result of the method of oxidase for measuring glucose concentration is about 20% lower when the 2, 5-dihydroxybenzenesulfonic acid content in the sample is 100 mg/L. In a preferred embodiment of the present invention, if a stable free radical, in one embodiment, 4-carboxy-2, 2, 6, 6-tetramethylpiperidine oxynitride is used to contact the sample, the interference of 2, 5-dihydroxybenzenesulfonic acid in the sample on the measurement result can be reduced to less than 5%, and a good anti-interference effect is shown.
In another similar preferred embodiment of cholesterol determination based on the principle of oxidase, when cholesterol is determined by conventional techniques, the measured result of cholesterol concentration determination by the above-mentioned oxidase method is about 20% lower when the content of 2, 5-dihydroxybenzenesulfonic acid in the sample is 100 mg/L. In a preferred embodiment of the present invention, if a stable free radical, in one embodiment, 4-carboxy-2, 2, 6, 6-tetramethylpiperidine oxynitride is used to contact the sample, the interference of 2, 5-dihydroxybenzenesulfonic acid in the sample on the measurement result can be reduced to less than 5%, and a good anti-interference effect is shown.
In another similar preferred embodiment for detecting adenosine deaminase using the principle of oxidase, the 2, 5-dihydroxybenzenesulfonic acid content in the sample is 100mg/L in the conventional method for detecting adenosine deaminase, which is found to be approximately 40% lower in the case of the oxidase method. In a preferred embodiment of the present invention, if a stable free radical, in one embodiment, 4-carboxy-2, 2, 6, 6-tetramethylpiperidine oxynitride is used to contact the sample, the interference of 2, 5-dihydroxybenzenesulfonic acid in the sample on the measurement result can be reduced to less than 10%, and a good anti-interference effect can be shown.
In another similar preferred embodiment of the method of detecting 5 ' nucleotidase using the principle of oxidase, when the content of 2, 5-dihydroxybenzenesulfonic acid in the sample is 100mg/L in the detection of 5 ' nucleotidase by the conventional technique, the result of the measurement of 5 ' nucleotidase activity by the above-mentioned oxidase method is about 40% lower. In a preferred embodiment of the present invention, if a stable free radical, in one embodiment, 4-carboxy-2, 2, 6, 6-tetramethylpiperidine oxynitride is used to contact the sample, the interference of 2, 5-dihydroxybenzenesulfonic acid in the sample on the measurement result can be reduced to less than 10%, and a good anti-interference effect can be shown.
Even for the detection of pure hydrogen peroxide in a sample, similar results to those described above are obtained, for example, when a sample of hydrogen peroxide prepared in an aqueous solution is detected, and the Trinder's reaction is used for the specific detection. When the 2, 5-dihydroxybenzenesulfonic acid is added into a sample, the measured result is lower by about 40 percent when the conventional technology is adopted for detection, and in a preferred embodiment of the invention, if a stable free radical is used simultaneously, and in one embodiment, 4-carboxyl-2, 2, 6, 6-tetramethylpiperidine oxynitride is used for contacting the sample, the interference of the 2, 5-dihydroxybenzenesulfonic acid in the sample on the measured result can be reduced to be within 15 percent, and a good anti-interference effect is shown.
Taking the detection of 2, 6, 8-trioxypurine in a sample as an example, or detecting 2, 6, 8-trioxypurine according to the principle of the oxidase method, when the 2, 6, 8-trioxypurine is detected by the conventional technology, the actual measurement result of the concentration of 2, 6, 8-trioxypurine measured by the oxidase method is about 30% lower when the content of 2, 5-dihydroxybenzenesulfonic acid in the sample is 30 mg/L. In a preferred embodiment of the present invention, if a stable free radical, in one embodiment, a pyrrolidine nitroxide free radical type 3-carboxy-2, 2, 5, 5-tetramethylpyrrolidine-1-oxyl free radical is contacted with a sample, the interference of 2, 5-dihydroxybenzenesulfonic acid in the sample on the measurement result can be reduced to less than 15%, and a good anti-interference effect can be shown.
Similarly, taking the detection of 2, 6, 8-trioxypurine in a sample as an example, or detecting 2, 6, 8-trioxypurine according to the above-mentioned principle of the oxidase method, when the content of 2, 5-dihydroxybenzenesulfonic acid in the sample is 30mg/L in the detection of 2, 6, 8-trioxypurine by the conventional technique, the measured result of the concentration of 2, 6, 8-trioxypurine by the above-mentioned oxidase method is nearly 30% lower. In a preferred embodiment of the present invention, if a stable free radical, in one embodiment, 2- (14-carboxytetradecyl) -2-ethyl-4, 4-dimethyl-3-oxazolidinyloxy free radical of oxazolidinyloxy free radical type is used to contact with the sample, the interference of 2, 5-dihydroxybenzenesulfonic acid in the sample to the measurement result can be reduced to less than 15%, and good anti-interference effect can be shown.
Similarly, taking the detection of 2, 6, 8-trioxypurine in a sample as an example, or detecting 2, 6, 8-trioxypurine according to the above-mentioned principle of the oxidase method, when the content of 2, 5-dihydroxybenzenesulfonic acid in the sample is 30mg/L in the detection of 2, 6, 8-trioxypurine by the conventional technique, the measured result of the concentration of 2, 6, 8-trioxypurine by the above-mentioned oxidase method is nearly 30% lower. In a preferred embodiment of the present invention, if a stable free radical, in one embodiment, a 2- (4-nitrophenyl) -4, 4, 5, 5-tetramethylimidazoline-3-oxide-1-oxyl free radical of imidazoline nitroxide free radical type is used to contact with a sample, the interference of 2, 5-dihydroxybenzenesulfonic acid in the sample on the measurement result can be reduced to less than 15%, and a good anti-interference effect can be shown.
Similarly, taking the detection of 2, 6, 8-trioxypurine in a sample as an example, or detecting 2, 6, 8-trioxypurine according to the above-mentioned principle of the oxidase method, when the content of 2, 5-dihydroxybenzenesulfonic acid in the sample is 30mg/L in the detection of 2, 6, 8-trioxypurine by the conventional technique, the measured result of the concentration of 2, 6, 8-trioxypurine by the above-mentioned oxidase method is nearly 30% lower. In a preferred embodiment of the present invention, if a stable free radical, in one embodiment, a kalimen oxygen radical of 4-oxo-2, 5-cyclohexadiene-p-tolyloxy free radical type, is used to contact with the sample, the interference of 2, 5-dihydroxybenzenesulfonic acid in the sample to the measurement result can be reduced to less than 15%, and a good anti-interference effect is shown.
Similarly, taking the detection of 2, 6, 8-trioxypurine in a sample as an example, or detecting 2, 6, 8-trioxypurine according to the above-mentioned principle of the oxidase method, when the content of 2, 5-dihydroxybenzenesulfonic acid in the sample is 30mg/L in the detection of 2, 6, 8-trioxypurine by the conventional technique, the measured result of the concentration of 2, 6, 8-trioxypurine by the above-mentioned oxidase method is nearly 30% lower. In a preferred embodiment of the present invention, if stable free radicals, in one embodiment 3-hydroxy-4-methyl-2 (3H) -thiazolidinethione of the thiazolidinethione oxygen radical type, are used to contact with the sample, the interference of 2, 5-dihydroxybenzenesulfonic acid in the sample on the measurement result can be reduced to less than 15%, and a good anti-interference effect can be shown.
Similarly, taking the detection of 2, 6, 8-trioxypurine in a sample as an example, or detecting 2, 6, 8-trioxypurine according to the above-mentioned principle of the oxidase method, when the content of 2, 5-dihydroxybenzenesulfonic acid in the sample is 30mg/L in the detection of 2, 6, 8-trioxypurine by the conventional technique, the measured result of the concentration of 2, 6, 8-trioxypurine by the above-mentioned oxidase method is nearly 30% lower. In a preferred embodiment of the present invention, if a stable free radical, in one embodiment, 1-diphenyl-2-picrylhydrazyl free radical of picrylhydrazyl free radical is used to contact with the sample, the interference of 2, 5-dihydroxybenzenesulfonic acid in the sample on the measurement result can be reduced to less than 15%, and a good anti-interference effect is shown.
Similarly, taking the detection of 2, 6, 8-trioxypurine in a sample as an example, or detecting 2, 6, 8-trioxypurine according to the above-mentioned principle of the oxidase method, when the content of 2, 5-dihydroxybenzenesulfonic acid in the sample is 30mg/L in the detection of 2, 6, 8-trioxypurine by the conventional technique, the measured result of the concentration of 2, 6, 8-trioxypurine by the above-mentioned oxidase method is nearly 30% lower. In a preferred embodiment of the present invention, when a stable radical, in one embodiment, a radical represented by 2-oxo [1.4.2] oxathiazolo [2, 3-a ] pyridines is used for contact, the interference of 2, 5-dihydroxybenzenesulfonic acid in a sample with a measurement result can be reduced to within 15%, and a good anti-interference effect can be exhibited.
Surprisingly advantageous results can be achieved when a plurality of stable free radicals are used in combination, for example by increasing the amount of inexpensive free radicals and reducing the amount of expensive free radicals, or by achieving better interference suppression.
For example, in an embodiment of the present invention, also taking the detection of 2, 6, 8-trioxypurine in a sample as an example, or detecting 2, 6, 8-trioxypurine according to the above-mentioned principle of the oxidase method, when the 2, 6, 8-trioxypurine is detected by the conventional technique, the measured result of the concentration of 2, 6, 8-trioxypurine by the above-mentioned oxidase method is lower by nearly 30% when the content of 2, 5-dihydroxybenzenesulfonic acid in the sample is 30 mg/L. In a preferred embodiment of the present invention, if the 4-carboxy-2, 2, 6, 6-tetramethylpiperidinyloxy compound is combined with another radical, for example, with a kalimen radical, the interference of 2, 5-dihydroxybenzenesulfonic acid in the sample on the measurement result can be reduced to less than 5%, and a better anti-interference effect can be exhibited.
For example, in another preferred embodiment of the present invention, if the 4-carboxy-2, 2, 6, 6-tetramethylpiperidinyloxy compound is combined with another radical, for example, with 1, 1-diphenyl-2-picrylhydrazyl radical, the amount of the 4-carboxy-2, 2, 6, 6-tetramethylpiperidinyloxy compound can be reduced, thereby saving the cost.
Similarly, in the present study, we have tried to combine stable free radicals with another conventional oxidizing agent and found that the amount of stable free radicals can be greatly reduced to achieve the same effect, and in a preferred embodiment of the present invention, when 4-carboxy-2, 2, 6, 6-tetramethylpiperidinyloxy compound is combined with sodium iodate, the amount of 4-carboxy-2, 2, 6, 6-tetramethylpiperidinyloxy compound can be greatly reduced.
In the above examples, the specific amount of the most suitable stable free radical required for each case may vary, but it is obvious that for a specific application, a gradient screening experiment is performed on the amount of the stable free radical and the suitable amount can be found, which belongs to the conventional experimental technique and does not require creative labor. Generally, the amount of the stable free radical may be between 0.001% and 10%, and in a preferred embodiment, the amount is between 0.05% and 2%, depending on the specific application environment.
For the combination of the stable free radical or the combination of the stable free radical and the conventional oxidant, the concentration gradient test is carried out according to the conventional experimental technology, and the appropriate dosage under the specific implementation condition can be quickly found out, which belongs to the conventional experimental technology and does not need creative labor.
The specific mechanism by which stable free radicals can counteract 2, 5-dihydroxybenzenesulfonic interference is not known at present, but presumably is linked to their unique oxidative nature involved in the radical reaction.
In this study, we tested a number of drugs interfering with the detection using the techniques described herein and found that various salts of 2, 5-dihydroxybenzenesulfonic acid, such as calcium salts, diethylamine salts, various dopa-like substances, including carbidopa, levodopa, and medopa, interfere with the detection of hydrogen peroxide using conventional techniques. Further research shows that the substances have common structural characteristics and are all connected with hydroxyl on a benzene ring. In view of this finding, we tested more compounds with hydroxyl groups attached to the phenyl ring, such as tocopherols, sulfophenols, gentisic acid, gallic acid, catechuic acid, resveratrol, dihydroxynaphthalene, and the results were all found to interfere with the results of hydrogen peroxide detection using conventional Trinder's method.
It has been found in a larger number of experiments that the detection of hydrogen peroxide is also disturbed when the benzene ring of the above-mentioned compounds is replaced by an aromatic heterocycle, such as 2, 6-dihydroxypyridine-4-carboxylic acid, 5-hydroxyindole, 3, 4-dihydroxyfuran-2, 5-dicarboxylic acid, 2, 5-dihydroxy-3-thienylethanesulfonic acid, 5, 6-dihydroxypyrimidine-4-carboxylic acid, and the like.
Combining the above research results, a common rule can be found: that is, when a hydroxyl group is bonded to a benzene ring, a naphthalene ring, another aromatic ring, or an aromatic heterocyclic ring, particularly when a plurality of hydroxyl groups are bonded, the result of hydrogen peroxide detection may be interfered. Of course, numerous organic compounds that meet the above characteristics can be cited based on the knowledge of well-known organic chemistry.
In this study, it was surprisingly found through experimental verification that when the method proposed by the present invention is used to contact a sample with a stable free radical compound, the interference of these substances can be effectively reduced.
Therefore, the method provided by the invention can eliminate or reduce the interference of 2, 5-dihydroxybenzenesulfonic acid or salts thereof or dopa compounds in a sample on the measurement result by using the stable free radical compound. It is also possible to eliminate or reduce interference of the measurement result with a compound containing a hydroxyl group attached to an aromatic ring or an aromatic heterocyclic ring in a sample.
Based on the above studies, we have further conducted further studies, and further found that some organic compounds of m-diketones, such as 1, 3-cyclohexanedione, mesotrione also interfere with the detection of hydrogen peroxide, such as that of conventional Trinder's method. Surprisingly, when the method provided by the invention is used for contacting the sample with the stable free radical compound, the interference of the stable free radical compound can be effectively eliminated or reduced. It is presumed that the mechanism of the reaction is that the structure of the m-diketone may be isomerized to an enol structure, and the hydroxyl group bonded to the double bond appears to have a certain reducibility, thereby disrupting the detection of hydrogen peroxide. The proposed method of the present invention unexpectedly provides the ability to cancel or mitigate such interference
Based on the method provided by the invention, in a preferable example, taking piperidine nitrogen oxide free radicals as an example, a kit for detecting 2-imino-1-methylimidazoline-4-ketone based on an oxidase method is prepared, the performance of the kit is verified, and the anti-interference capability of some conventional interfering substances is tested. In the experimental tests, it was very surprising to find that the kit using the method described in the present invention also has the ability to interfere with various other interferents, including the ability to interfere with hemoglobin, the ability to interfere with reduced glutathione, and the ability to interfere with ascorbic acid.
Hemoglobin is an iron-containing porphyrin protein, and also commonly used proteins containing iron porphyrin protein include myoglobin, cytochrome C, and the like. Iron porphyrins are oxidative and may be capable of spontaneously generating reactive oxygen species, thereby interfering with the detection of hydrogen peroxide. Therefore, the Trinder's reaction can be initiated without hydrogen peroxide to generate a colored compound, thereby interfering with the detection result. It is presumed that the above-mentioned stable radical substance may quench active oxygen and thus has an effect of inhibiting interference of iron-containing porphyrin protein.
The reducing property of the sulfhydryl of the compound containing active sulfhydryl, such as common reduced glutathione, cysteine and the like, can destroy hydrogen peroxide, so that the substances can interfere with the detection of 2-imino-1-methylimidazolin-4-one by the conventional technology, but the interference degree of the sulfhydryl compounds with the detection result can be effectively reduced by using the stable free radical compound to contact with a sample by using the method provided by the invention.
Ascorbic acid is a very common interfering substance in the Trinder's reaction, and the interference elimination of ascorbic acid is generally completed by adopting a cheap and mature ascorbic acid oxidase method. However, the method provided by the invention also has the effect of eliminating the interference of the ascorbic acid, and provides an additional solution.
Similar results were also shown in the above test experiments, instead of directly measuring the samples containing hydrogen peroxide.
Therefore, in the invention, for the detection of hydrogen peroxide, the method or the kit described in the invention can solve the interference of compounds with hydroxyl groups connected to aromatic rings, such as 2, 5-dihydroxybenzenesulfonic acid, methyldopa and the like, which are not solved by mature effective means at present, and also has the ability of additionally having the interference of substances, such as an anti-sulfhydryl compound, iron-containing porphyrin protein, ascorbic acid and the like, which is also a significant difference and a remarkable improvement of the invention from the prior art.
It is logically presumed that, in clinical examination, if an interfering substance such as 2, 5-dihydroxybenzenesulfonic acid contained in a sample interferes with the detection of hydrogen peroxide, it inevitably interferes with the items for indirectly measuring the content of other substances by detecting hydrogen peroxide with the generation of hydrogen peroxide as an intermediate. This phenomenon has been confirmed in literature reports.
In summary, the technical route described in the present invention is summarized as follows:
namely, the present invention provides a method or a kit for detecting hydrogen peroxide, characterized in that: use comprising the step of contacting a test sample with a stable free radical compound for eliminating or mitigating interference of aromatic hydroxyl compounds in the sample.
And kits comprising the stable free radical compounds.
The aromatic hydroxyl compound is 2, 5-dihydroxybenzenesulfonic acid or salt thereof, and/or dopa compound or salt thereof.
The stable free radical compounds described above include, but are not limited to: nitroxide free radicals, 4-oxo-2, 5-cyclohexadiene-p-tolyloxy radicals, thiazolidinethione oxygen radicals, picrazineyl free radicals, 2-oxo [1.4.2] oxathiazolo [2, 3-a ] pyridines.
The stable free radical compounds described above are particularly preferred nitroxide free radicals, including but not limited to: one or more of piperidine alkyl nitroxide free radical, pyrrolidine nitroxide free radical, oxazolidine nitroxide free radical and imidazoline nitroxide free radical.
When a plurality of stable free radical compounds are used in combination, a more excellent effect can be obtained by including at least one piperidinoalkyloxy radical compound.
When a piperidinoalkane nitroxide compound is employed, wherein the substituents attached to the carbons at positions 2 and 6 of the piperidinoalkane nitroxide compound are hydrocarbyl groups and/or modified hydrocarbyl groups and/or hydrogen atoms or combinations thereof. In one preferred embodiment, 2 methyl groups are attached to both the 2-and 6-carbon positions. In addition, the carbon at the 4-position of the piperidine alkyl nitroxide radical compound is connected with a substituent, and the substituent is one of hydroxyl, oxygen atom, amino, carboxyl, alkyl, sulfydryl, halogen atom, alkyl, aralkyl, aryl, substituted oxy, substituted amino, substituted alkyl, substituted sulfydryl, cyano and isothiocyanate.
The above detection method or kit may also be used in combination with additional oxides, including but not limited to: hydrogen peroxide, persulfate, halogen oxide, metal oxide, oxidizing metal salt and dessimidine oxidant.
The aromatic hydroxy compound is a compound in which at least one hydroxy group is bonded to a benzene ring and/or an aromatic heterocyclic ring.
The use of the above detection method or kit further comprises a combination of at least one or more of the following: eliminating or reducing the interference of hydroxyl groups connected to conjugated double bonds in a sample on a measurement result; eliminating or reducing interference of iron-containing porphyrin protein in a sample on the determination result; eliminating or reducing the interference of ascorbic acid on the measurement result; eliminating or reducing the interference of sulfhydryl compounds on the determination result.
The detection method or the kit can also comprise a component and a step for coupling and oxidizing the 2-imino-1-methylimidazoline-4-ketone to generate hydrogen peroxide, and the component and the step are used for detecting the content of the 2-imino-1-methylimidazoline-4-ketone in the sample.
Similarly, the above-mentioned detection method or kit may further comprise any one of the following uses:
coupling components and steps for oxidizing glycosylated protein to generate hydrogen peroxide, for detecting the content of glycosylated protein in the sample; or components and steps coupled with the oxidation of fatty acids to produce hydrogen peroxide, for detecting the content of fatty acids in the sample; or coupling components and steps for oxidizing 2, 6, 8-trihydroxy purine to generate hydrogen peroxide, and detecting the content of the 2, 6, 8-trihydroxy purine in the sample; or components and steps coupled with the oxidation of the sugar or alcohol to generate hydrogen peroxide, for detecting the content of the sugar or alcohol in the sample; or components and steps coupled with the oxidation of the glyceride to generate hydrogen peroxide, for detecting the content of the glyceride in the sample; or a component and a step for oxidizing cholesterol to generate hydrogen peroxide are coupled, and the component and the step are used for detecting the content of cholesterol in the sample; or coupled with components and steps for converting an adenosine deaminase catalytic substrate to produce hydrogen peroxide, for detecting the activity of adenosine deaminase in a sample; or a component and a step of converting the 5 '-nucleotidase catalytic substrate into hydrogen peroxide are coupled for detecting the activity of the 5' -nucleotidase in the sample.
It should be clear that it can be logically deduced that the method and technique provided by the present invention can also be applied to other items not specifically listed above mediated by the detection of hydrogen peroxide.
Drawings
FIG. 1 is a graph showing the difference in the effect (relative deviation) of the method described in the present invention and the conventional method on eliminating the calcium interference of 2, 5-dihydroxybenzenesulfonate.
Detailed Description
The implementation of the method or kit of the invention will now be further described in some specific examples. It should be noted that the following applications are only specific descriptions for the present invention to help understanding the technology of the present invention, but not to limit the present invention, and according to the known technology, the skilled person in the art should be able to adjust the concentration of each component, substitute the analog, increase or decrease the auxiliary component, adjust the pH of the buffer solution, etc. in the following examples, all fall within the protection scope of the present invention.
Example 1: observing the anti-interference effect of the stable free radical compound which is a 4-carboxyl-2, 2, 6, 6-tetramethyl piperidine nitroxide compound.
First, a detection kit (control group) for detecting 2-imino-1-methylimidazolin-4-one according to the conventional technique was prepared as shown in Table 1A:
note: TOOS is N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methylaniline sodium salt.
Then, a detection kit (investigation group) for detecting 2-imino-1-methylimidazolin-4-one according to the technique described in the present invention was prepared as shown in Table 1B,
the detection steps of the kit are as follows: reacting the sample with a reagent R1 for 3-5 min, adding a reagent R2, measuring the absorbance value after reacting for 5min, and calculating the content of 2-imino-1-methylimidazoline-4-one in the sample according to the measured absorbance value.
Specific detection instruments and detection parameters are shown in table 1C below:
the test samples were prepared as follows:
the same sample was divided into 6 groups, and different concentrations of interferents (calcium 2, 5-dihydroxybenzenesulfonate) were sequentially added, the specific addition concentrations are shown in table 1D below.
The samples were then assayed under the above assay conditions using a control reagent (conventional technique) and a test reagent (technique described in the present invention), and the results of detection of the target analyte (2-imino-1-methylimidazolin-4-one) in each sample and the relative deviation from the assay results of the control sample containing no interfering substance are shown in Table 1D below.
The test result shows that the calcium 2, 5-dihydroxybenzenesulfonate salt has very serious interference on the measurement result, but the technical research group described by the invention can greatly eliminate the interference of the interferents on the detection result.
Plotting the relative deviation data in table 1D as in fig. 1 allows for a more intuitive comparison of the differences between the control and the study groups. The method has remarkable effect on eliminating the interference of the calcium 2, 5-dihydroxybenzenesulfonate.
Example 2: observing the anti-interference effect of the stable free radical compound which is a 4-cyano-2, 2, 6, 6-tetramethyl piperidine nitroxide compound.
The control group reagent of this example is the same as example 1;
the 2-imino-1-methylimidazolin-4-one assay kit (panel) according to the technology described in the present invention was then formulated as shown in Table 2B,
the detection procedure and sample preparation method were the same as in example 1 above.
The results of the measurements are shown in table 2D below:
as can be seen from the table, good anti-interference effect can be obtained when the 4-position of the nitroxide compound is other substituent such as cyano by the technique described in the present invention.
Example 3: and observing the anti-interference effect of the stable free radical compound which is a 4-hydroxy-2, 6-diisopropyl piperidine oxynitride compound.
The control group reagent of this example is the same as example 1;
the 2-imino-1-methylimidazolin-4-one assay kit (panel) according to the technology described in the present invention was then formulated as shown in Table 3B,
the detection procedure and sample preparation method were the same as in example 1 above.
The results of the measurements are shown in table 3D below:
as can be seen from the table, good anti-interference effects can be obtained when the positions 2 and 6 of the nitroxide radical compound are other substituents such as isopropyl after the technology described in the present invention is adopted.
Example 4: the effect of the stable radical compound simultaneously with the sample contacting step and the step of generating and detecting hydrogen peroxide was observed.
The control group reagent of this example is the same as example 1;
the 2-imino-1-methylimidazolin-4-one assay kit (panel) according to the technology described in the present invention was then prepared as shown in Table 4B,
the sample preparation method is the same as that of the above specific example 1, and the detection conditions of the control group are the same as those of example 1;
the measurement conditions of the investigation group are shown in table 4C below.
The results of the measurements are shown in table 4D below:
it can be seen from the table that the three steps of the measurement, the step of contacting the stable radical compound with the sample, the step of generating hydrogen peroxide, and the step of detecting hydrogen peroxide, can still achieve good anti-interference effect when the technology described in the present invention is applied.
Example 5: the effect of the stable free radical compound to eliminate or reduce methyl dopa interference was observed.
The control group reagent and the study group reagent in this example are the same as those in example 1;
the test procedure and sample formulation method were similar to those described above in example 1, except that the interfering substance was methyldopa and the concentrations of the formulated interferents are shown in Table 5D below.
The results of the measurements are shown in table 5D below:
as can be seen from the table, the interference effect of the stable free radical compound to eliminate or reduce methyldopa is also very significant when the technology described in the present invention is applied.
Example 6: the effect of the stable free radical compound to eliminate or mitigate interference from 1, 3-cyclohexanone was observed.
The control group reagent and the study group reagent in this example are the same as those in example 1;
the test procedure and sample formulation method were similar to those described above for example 1, except that the interfering substance was 1, 3-cyclohexanone and the formulated interfering concentrations are shown in Table 6D below.
The results of the measurements are shown in table 6D below:
as can be seen from the table, the interfering effect of the stable free radical compounds on the mitigation of the above interferents is also very significant when the techniques described herein are employed.
Example 7: the effect of the stable free radical compound to eliminate or mitigate interference with reduced glutathione was observed.
The control group reagent and the study group reagent in this example are the same as those in example 1;
the detection procedure and sample preparation method were similar to those described in example 1 above, except that the interfering substance was reduced glutathione and the prepared interfering concentrations were as shown in Table 7D below.
The results of the measurements are shown in table 7D below:
as can be seen from the table, the effect of the stable free radical compound on the reduction of the interference of the above interferents is also very significant when the technique described in the present invention is applied.
Example 8: the effect of the stable free radical compound to eliminate or mitigate hemoglobin interference was observed.
The control group reagent and the study group reagent in this example are the same as those in example 1;
the test procedure and sample preparation method were similar to those described above in example 1, except that the interfering substance was hemoglobin and the prepared interfering concentrations were as shown in Table 8D below.
The results of the measurements are shown in Table 8D below:
as can be seen from the table, the effect of the stable free radical compound on the reduction of the interference of the above interferents is also very significant when the technique described in the present invention is applied.
Example 9: the effect of the combination of stable free radical compounds on the interference resistance was observed.
The control group reagent of this example is the same as example 1;
the 2-imino-1-methylimidazolin-4-one assay kit (investigational group) of the technology described in this invention was prepared as follows: ,
the detection procedure and sample preparation method were the same as in example 1 above.
The results of the measurements are shown in table 9D below:
it can be seen from the table that when the technique described in the present invention is used, a better anti-interference effect can be obtained when a combination of two stable free radical compounds is used.
Example 10: and observing the anti-interference effect of the stable free radical compound which is 3-carboxyl-2, 2, 5, 5-tetramethyl pyrrolidine-1-oxyl free radical.
The control group reagent of this example is the same as example 1;
the 2-imino-1-methylimidazolin-4-one assay kit (panel) according to the technology described in the present invention was then prepared as shown in Table 10B,
the detection procedure and sample preparation method were the same as in example 1 above.
The results of the measurements are shown in Table 10D below:
it can be seen from the table that when the stable free radical compound is pyrrolidine nitroxide radical, the technology described in the present invention can also be used to obtain good anti-interference effect.
Example 11: the anti-interference effect of the stable free radical compound 2- (14-carboxytetradecyl) -2-ethyl-4, 4-dimethyl-3-oxazolidinyl oxygen free radical is observed.
The control group reagent of this example is the same as example 1;
the 2-imino-1-methylimidazolin-4-one assay kit (panel) according to the technology described in the present invention was then prepared as shown in Table 11B,
the detection procedure and sample preparation method were the same as in example 1 above.
The results of the measurements are shown in Table 11D below:
it can be seen from the table that when the stable free radical compound is oxazolidine nitroxide radical, the technology described in the present invention can also be used to obtain good anti-interference effect.
Example 12: the anti-interference effect of the stable free radical compound 2- (4-nitrophenyl) -4, 4, 5, 5-tetramethylimidazoline-3-oxide-1-oxyl free radical is observed.
The control group reagent of this example is the same as example 1;
the 2-imino-1-methylimidazolin-4-one assay kit (panel) according to the technology described in the present invention was then prepared as shown in Table 12B,
the detection procedure and sample preparation method were the same as in example 1 above.
The results of the measurements are shown in table 12D below:
it can be seen from the table that when the stable free radical compound is oxazolidine nitroxide radical, the technology described in the present invention can also be used to obtain good anti-interference effect.
Example 13: the anti-interference effect of the stable free radical compound when used in combination with a conventional oxidant was observed.
The control group reagent of this example is the same as example 1;
the 2-imino-1-methylimidazolin-4-one assay kit (panel) according to the technology described in the present invention was then prepared as shown in Table 13B,
the detection procedure and sample preparation method were the same as in example 1 above.
The results of the measurements are shown in table 13D below:
it can be seen from the table that when using the technique described in the present invention, when a stable free radical compound is used in combination with a conventional oxide such as sodium iodate, the amount of free radical compound used can be reduced and the desired interference suppression effect can be achieved.
Example 14: the effect of directly measuring hydrogen peroxide in a sample was observed.
First, a detection kit for detecting hydrogen peroxide according to the conventional technique (control group) was prepared as shown in table 14A:
the hydrogen peroxide detection kit (panel) according to the technology described in the present invention was then prepared as shown in Table 14B,
the detection steps of the kit are as follows: reacting the sample with the reagent R1 for about 1min, adding the reagent R2, measuring the absorbance value after reacting for 5min, and calculating the content of the hydrogen peroxide in the sample according to the measured absorbance value.
The specific detection parameters and detector are shown in table 14C below:
the test samples were prepared as follows: the hydrogen peroxide solution was diluted to about 300. mu. mol/L with physiological saline as a base sample.
The same basic sample is divided into 6 groups, interferents (2, 5-dihydroxy benzene sulfonic acid calcium salt) with different concentrations are sequentially added, the specific adding concentration is shown in the following table 14D, and the interferents are immediately sampled and detected after being added into the sample.
The samples were then assayed under the above assay conditions using the control reagent (conventional technique) and the test reagent (technique described in the present invention), and the results of the assay of the target analyte hydrogen peroxide in each sample and the relative deviation from the assay of the control sample containing no interfering substance are shown in Table 14D below.
The test result shows that when the direct determination target substance is hydrogen peroxide, the calcium 2, 5-dihydroxybenzenesulfonate salt has very serious interference on the determination result, but when the technical research group described by the invention is adopted, the interference of the interference substance on the detection result can be greatly eliminated.
Example 15: the anti-interference effect of the stable free radical compound as a Garvey oxygen free radical was observed.
First, a detection kit for detecting 2, 6, 8-trioxypurine (control group) according to the conventional technique was prepared as shown in table 15A:
then, 2, 6, 8-trioxypurine detection kits (investigation group) according to the technique described in the present invention were prepared as shown in Table 15B,
the detection steps of the kit are as follows: reacting the sample with the reagent R1 for about 5min, adding the reagent R2, measuring the absorbance value after reacting for 5min, and calculating the content of the hydrogen peroxide in the sample according to the measured absorbance value.
The specific detection parameters and detector are shown in table 15C below:
the test samples were prepared as follows: the same serum basic sample containing 2, 6, 8-trioxypurine was divided into 6 groups, and different concentrations of interferents (calcium 2, 5-dihydroxybenzenesulfonate) were sequentially added, specifically, the concentrations are shown in table 14D below.
The samples were then assayed under the above assay conditions using the control reagent (conventional technique) and the investigator reagent (technique described in the present invention), and the results of the assay for the target analyte 2, 6, 8-trioxypurine in each sample and the relative deviation from the assay results of the control sample containing no interfering substance are shown in Table 15D below.
It can be seen from the table that when the stable free radical compound is a california free radical using the technique described in the present invention, a good anti-interference effect can be obtained.
Example 16: the anti-interference effect of the stable free radical compound 3-hydroxy-4-methyl-2 (3H) -thiazolidinethione is observed.
The control group reagent of this example is the same as example 15;
a2, 6, 8-trioxypurine assay kit (panel) according to the technique described in the present invention was then prepared as shown in Table 16B,
the detection procedure and sample preparation method were the same as those described in example 15
The results of the measurements are shown in Table 16D below:
it can be seen from the table that when the stable free radical compound is 3-hydroxy-4-methyl-2 (3H) -thiazolethione by the technology described in the present invention, good anti-interference effect can be obtained.
Example 17: the effect of the stable free radical compound as a picrylhydrazyl free radical was observed.
The control group reagent of this example is the same as example 15;
a2, 6, 8-trioxypurine assay kit (panel) according to the technique described in the present invention was then prepared as shown in Table 17B,
the detection procedure and sample preparation method were the same as those described in example 15
The results of the measurements are shown in Table 17D below:
it can be seen from the table that when the stable free radical compound is 1, 1-diphenyl-2-picrylhydrazyl free radical by using the technology described in the present invention, good anti-interference effect can be obtained.
Example 18: the anti-interference effect of 2-oxo [1.4.2] oxathiazolo [2, 3-a ] pyridine as a stable free radical compound was observed.
The control group reagent of this example is the same as example 15;
a2, 6, 8-trioxypurine assay kit (panel) according to the technique described in the present invention was then prepared as shown in Table 18B,
the procedure for detection and sample preparation were the same as in example 15 above.
The results of the measurements are shown in Table 18D below:
as can be seen from the table, when the stable radical compound is 2-oxo [1.4.2] oxathiazolo [2, 3-a ] pyridine by the technique described in the present invention, a good anti-interference effect can be obtained.
Example 19: and observing the anti-interference effect of the combination of the stable free radical compounds.
The control group reagent of this example is the same as example 15;
a2, 6, 8-trioxypurine assay kit (panel) according to the technique described in the present invention was then prepared as shown in Table 19B,
the detection procedure and sample preparation method were the same as those described in example 15
The results of the measurements are shown in Table 19D below:
it can be seen from the table that when the technology described in the present invention is adopted, and a combination of a plurality of stable free radicals is adopted, the cost can be effectively reduced, and a good anti-interference effect can be achieved.
Claims (14)
1. A method or a kit for detecting hydrogen peroxide, characterized in that: use comprising the step of contacting a test sample with a stable free radical compound for eliminating or mitigating interference of aromatic hydroxyl compounds in the sample.
2. The detection method or kit according to claim 1, characterized in that: the stable free radical compound is contained in the kit components.
3. The detection method or kit according to claim 1, characterized in that: the aromatic hydroxyl compound is 2, 5-dihydroxybenzenesulfonic acid or salt thereof, and/or dopa compound or salt thereof.
4. The detection method or kit according to claim 1, characterized in that: the stable free radical compounds include, but are not limited to: one or more of piperidine alkyl nitroxide free radical, pyrrolidine nitroxide free radical, oxazolidine nitroxide free radical and imidazoline nitroxide free radical.
5. The detection method or kit according to claim 1, characterized in that: the stable free radical compounds include, but are not limited to: nitroxide free radicals, 4-oxo-2, 5-cyclohexadiene-p-tolyloxy radicals, thiazolidinethione oxygen radicals, picrazineyl free radicals, 2-oxo [1.4.2] oxathiazolo [2, 3-a ] pyridines.
6. The detection method or kit according to claim 5, characterized in that: wherein the stable free radical compound comprises at least one piperidinoalkane nitroxide radical compound.
7. The detection method or kit according to claim 6, characterized in that: wherein the substituents attached to the carbons at positions 2 and 6 of the piperidinoalkyloxy compound are hydrocarbyl and/or modified hydrocarbyl and/or a hydrogen atom or a combination thereof.
8. The detection method or kit according to claim 7, characterized in that: wherein 2 methyl groups are attached to both the 2-and 6-carbon positions of the piperidinoalkane nitroxide compound.
9. The detection method or kit according to claim 7, characterized in that: wherein, the carbon at the 4-position of the piperidine alkyl nitroxide free radical compound is connected with a substituent, and the substituent is one of hydroxyl, oxygen atom, amino, carboxyl, alkyl, sulfydryl, halogen atom, alkyl, aralkyl, aryl, substituted oxy, substituted amino, substituted alkyl, substituted sulfydryl, cyano and isothiocyanic group.
10. The detection method or kit according to claim 5, characterized in that: further comprising additional oxides including, but not limited to: hydrogen peroxide, persulfate, halogen oxide, metal oxide, oxidizing metal salt and dessimidine oxidant.
11. The detection method or kit according to claim 5, characterized in that: the aromatic hydroxyl compound is a compound which is connected with at least one hydroxyl on a benzene ring and/or an aromatic heterocyclic ring.
12. The detection method or kit according to claim 5, characterized in that: the use thereof is a combination which also comprises at least one or more of the following: eliminating or reducing the interference of hydroxyl groups connected to conjugated double bonds in a sample on a measurement result; eliminating or reducing interference of iron-containing porphyrin protein in a sample on the determination result; eliminating or reducing the interference of ascorbic acid on the measurement result; eliminating or reducing the interference of sulfhydryl compounds on the determination result.
13. The detection method or kit according to any one of claims 1 to 12, characterized in that: the method also comprises a component and a step for generating hydrogen peroxide by oxidizing the 2-imino-1-methylimidazoline-4-ketone through coupling, and is used for detecting the content of the 2-imino-1-methylimidazoline-4-ketone in the sample.
14. The detection method or kit according to any one of claims 1 to 12, characterized by further comprising one of the following uses:
coupling components and steps for oxidizing glycosylated protein to generate hydrogen peroxide, for detecting the content of glycosylated protein in the sample;
or components and steps coupled with the oxidation of fatty acids to produce hydrogen peroxide, for detecting the content of fatty acids in the sample;
or coupling components and steps for oxidizing 2, 6, 8-trihydroxy purine to generate hydrogen peroxide, and detecting the content of the 2, 6, 8-trihydroxy purine in the sample;
or components and steps coupled with the oxidation of the sugar or alcohol to generate hydrogen peroxide, for detecting the content of the sugar or alcohol in the sample;
or components and steps coupled with the oxidation of the glyceride to generate hydrogen peroxide, for detecting the content of the glyceride in the sample;
or a component and a step for oxidizing cholesterol to generate hydrogen peroxide are coupled, and the component and the step are used for detecting the content of cholesterol in the sample;
or coupled with components and steps for converting an adenosine deaminase catalytic substrate to produce hydrogen peroxide, for detecting the activity of adenosine deaminase in a sample;
or a component and a step of converting the 5 '-nucleotidase catalytic substrate into hydrogen peroxide are coupled for detecting the activity of the 5' -nucleotidase in the sample.
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