CN111999352A - Detection method or kit for hydrogen peroxide - Google Patents
Detection method or kit for hydrogen peroxide Download PDFInfo
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- CN111999352A CN111999352A CN201910485515.8A CN201910485515A CN111999352A CN 111999352 A CN111999352 A CN 111999352A CN 201910485515 A CN201910485515 A CN 201910485515A CN 111999352 A CN111999352 A CN 111999352A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/28—Electrolytic cell components
- G01N27/30—Electrodes, e.g. test electrodes; Half-cells
- G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
Abstract
The invention designs a method or a kit for detecting hydrogen peroxide, belonging to the technical field of medical inspection. The kit components comprise gentisic acid-1, 2-dioxygenase. 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 above substances are generally oxidized through a series of enzyme catalysis to generate hydrogen peroxide, and then the amount or the generation rate of the generated hydrogen peroxide is detected by an electrode method or an oxidase method which is called as Trinder's for short, so as to calculate the content or the activity of the above substances.
However, in practical applications, hydrogen peroxide is unstable and susceptible to interference from other substances present in the sample. A common example is that 2, 5-dihydroxybenzenesulfonic acid present in the blood of a patient after the patient has taken or injected a drug containing 2, 5-dihydroxybenzenesulfonate causes the measurement of hydrogen peroxide relied on in the measurement process to be severely low, thereby misjudging the disease condition of the patient and causing serious consequences.
Therefore, there is a need in the art for a technique that can eliminate or reduce the interference of 2, 5-dihydroxybenzenesulfonate drugs in a sample during the detection of hydrogen peroxide, and ensure the reliability of the detection result of hydrogen peroxide, thereby making the determination result of the above substances accurate and reliable.
Disclosure of Invention
The invention provides a method or a kit for detecting hydrogen peroxide, which is characterized by comprising the step of contacting a test sample with gentisic acid-1, 2-dioxygenase so as to eliminate or reduce interference of 2, 5-dihydroxybenzenesulfonic acid 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 from some specific drugs in the sample, such as 2, 5-dihydroxybenzenesulfonic acid, may destroy or competitively react with hydrogen peroxide to falsely lower the measurement, 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 mitigate interference of 2, 5-dihydroxybenzenesulfonic acid in a sample with detection items involving hydrogen peroxide as an intermediate.
2, 5-dihydroxybenzenesulfonic acid has a reducing property, and is presumed to react with hydrogen peroxide to consume the hydrogen peroxide. According to conventional wisdom, it is suspected that it is possible to avoid the destruction of hydrogen peroxide by 2, 5-dihydroxybenzenesulfonic acid by oxidizing the 2, 5-dihydroxybenzenesulfonic acid by the addition of an oxidizing agent. However, in the present invention, we have tested a large number of oxidizing agents, including peroxides, persulfates, halogen oxides such as sodium hypochlorite, sodium iodate, etc., metal oxides such as potassium dichromate, potassium permanganate, oxidizing transition metal salts, desselatin oxidizing agents, etc., and have not found oxidizing agents that effectively eliminate the destructive effect of 2, 5-dihydroxybenzenesulfonic acid on hydrogen peroxide, and many of them interfere with normal detection, causing new interference.
In this regard, in the present study, we have tested a number of enzymes including monophenol monooxygenase, catechol oxidase, laccase, catechol 2, 3-dioxygenase, catechol 1, 2-dioxygenase, toluene dioxygenase, benzoate dioxygenase, naphthalene dioxygenase, gentisic acid 1, 2-dioxygenase. Surprisingly, the gentisic acid-1, 2-dioxygenase has a good effect, the interference degree of 2, 5-dihydroxybenzenesulfonic acid can be reduced to be within 15% of the relative deviation, even can be reduced to be within 5% of the relative deviation under the conventional dosage concentration, and the interference of 2, 5-dihydroxybenzenesulfonic acid on detection is effectively eliminated.
Gentisic acid-1, 2-dioxygenase can catalyze the ring opening of gentisic acid to maleic acid-monoacyl pyruvic acid. The 2, 5-dihydroxybenzenesulfonic acid structure is very close to gentisic acid, and the difference is that the 1-position carbon on the benzene ring of gentisic acid is connected with a methyl group, and the 1-position carbon on the benzene ring of 2, 5-dihydroxybenzenesulfonic acid is connected with a sulfo group. It is likely that the similarity in the spatial structure of the two would allow gentisic acid-1, 2-dioxygenase to also use 2, 5-dihydroxybenzenesulfonic acid as a substrate.
The distribution of gentisic acid-1, 2-dioxygenase is very broad and is present in a large number of organisms, in particular in some microorganisms, such as e.g. Pseudomonas ospermatis, Pseudomonas testosteroni, Pseudomonas acidovorans, Pseudomonas putida, Ralstonia solanacearum, halophilic archaea, Klebsiella pneumoniae, Rhodococcus, Haloferax volcanii, Cochlamys, Bacillus, Thiobacillus and the like.
The amino acid sequences of gentisic acid-1, 2-dioxygenase from different organisms are not completely the same, and the homology between some species is even less than 40%, but the functions of the gentisic acid-1, 2-dioxygenase have the common part and can catalyze the ring opening of gentisic acid to maleic acid-monoacyl pyruvic acid.
Gentisic acid-1, 2-dioxygenase from different organism sources also differs in its ability to reduce interference of 2, 5-dihydroxybenzenesulfonic acid with hydrogen peroxide detection. For example, in a preferred embodiment, gentisic acid-1, 2-dioxygenase from pseudomonas alcaligenes can reduce the interference of 2, 5-dihydroxybenzenesulfonic acid in a sample on a measurement result to be within 10 percent, and shows a good anti-interference effect. In another embodiment, the gentisic acid-1, 2-dioxygenase from the Ralstonia solanacearum can only reduce the interference of the 2, 5-dihydroxybenzenesulfonic acid in the sample to the measurement result to 10-15%, and the interference resisting effect is slightly poor. And the dosage of gentisic acid-1, 2-dioxygenase from Klebsiella pneumoniae needs more to achieve similar anti-interference effect.
According to the known technology, it can be reasonably concluded that the anti-interference effect possibly caused by the difference of affinity and catalytic ability of gentisic acid-1, 2-dioxygenase from different sources to 2, 5-dihydroxybenzenesulfonic acid is different, and the difference is related to the primary structure and spatial structure of the enzyme. According to the prior art, the enzyme can be obviously improved and screened by biological engineering or genetic engineering technologies such as mutation, recombination and the like, the reaction activity of gentisic acid-1, 2-dioxygenase on 2, 5-dihydroxybenzenesulfonic acid is enhanced, and the effect of eliminating or reducing the interference of 2, 5-dihydroxybenzenesulfonic acid on hydrogen peroxide detection is improved. In a preferred embodiment of the invention, a mutant strain P261HM is obtained by carrying out mutagenesis on Pseudomonas alcaligenes, and the reaction capability of gentisic acid-1, 2-dioxygenase to 2, 5-dihydroxybenzenesulfonic acid is improved by nearly four times, and a good interference reduction effect can be obtained by using less amount. In another preferred embodiment of the present invention, the gentisic acid-1, 2-dioxygenase gene sequence extracted from klebsiella pneumoniae is subjected to site-directed mutagenesis by genetic engineering technology to obtain a mutant strain H18CP, wherein the reaction capability of gentisic acid-1, 2-dioxygenase to 2, 5-dihydroxybenzenesulfonic acid is improved by about three times, and a good interference mitigation effect can be obtained with a smaller amount.
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 steps are well known in the art, and the manufacturer, amount of the tool enzyme used, the type of buffer used, pH, preservative and surfactant screening are well known in the industry, and numerous reports are reported and are not described herein.
The detection of hydrogen peroxide can be carried out electrochemically, but the most widely used method is the method generally designated as Trinder's reaction. In the Trinder's reaction, under the action of peroxidase, hydrogen peroxide and 4-aminoantipyrine or an analogue thereof can react with a chromogen to produce a colored compound. Commonly used chromogens include phenol and its derivatives, and various chromogens, abbreviated as ADOS, ADPS, ALPS, DAOS, HDAOS, MADB, MAOS, TODB, TOOS, TOPS, and the like. The sensitivity and stability of different chromogens are different, and the appropriate dosage of the chromogens 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, it reacts with many substances, particularly reducing substances, because hydrogen peroxide is a very active substance. Substances such as the above-mentioned 2, 5-dihydroxybenzenesulfonic acid may cause a low measured value of 2-imino-1-methylimidazolin-4-one due to destructive or competitive reaction with hydrogen peroxide.
When the 2-imino-1-methylimidazolin-4-one is detected by the oxidase method in the prior art, the actual measurement result of the 2-imino-1-methylimidazolin-4-one concentration determined by the oxidase method is about 50% lower when the content of 2, 5-dihydroxybenzenesulfonic acid in a sample is 50 mg/L. In a preferred embodiment of the invention, gentisic acid-1, 2-dioxygenase is selected to contact with the sample, so that the interference of 2, 5-dihydroxybenzenesulfonic acid in the sample on the measurement result can be reduced to be within 10%, and an excellent anti-interference effect is shown.
In another similar preferred embodiment of the method of detecting 2, 6, 8-trioxypurine using the principle of oxidase, when the 2, 6, 8-trioxypurine is detected by the conventional technique and the content of 2, 5-dihydroxybenzenesulfonic acid in a sample is 50mg/L, the measured result of the method of oxidase for detecting 2, 6, 8-trioxypurine concentration is lower than 30%. In a preferred embodiment of the invention, gentisic acid-1, 2-dioxygenase is selected to contact with the sample, so that the interference of 2, 5-dihydroxybenzenesulfonic acid in the sample on the measurement result can be reduced to be within 15%, and a good anti-interference effect is shown.
In another similar preferred embodiment of the method of detecting glycerides using the oxidase principle, the measured concentration of glycerides measured by the above-mentioned oxidase method is about 20% lower when the content of 2, 5-dihydroxybenzenesulfonic acid in the sample is 100mg/L in the detection of glycerides using the conventional technique. In a preferred embodiment of the invention, gentisic acid-1, 2-dioxygenase is selected to contact with the sample, so that the interference of 2, 5-dihydroxybenzenesulfonic acid in the sample on the measurement result can be reduced to be within 10%, and a good anti-interference effect is shown.
In another similar preferred embodiment of the method of detecting glycosylated protein using the principle of the oxidase method, when the content of 2, 5-dihydroxybenzenesulfonic acid in the sample is 50mg/L in the detection of glycosylated protein using the conventional technique, the result of the measurement of the concentration of glycosylated protein using the oxidase method is lower by approximately 40%. In a preferred embodiment of the invention, gentisic acid-1, 2-dioxygenase is selected to contact with the sample, so that the interference of 2, 5-dihydroxybenzenesulfonic acid in the sample on the measurement result can be reduced to be within 15%, and a good anti-interference effect is shown.
In another similar preferred embodiment for testing fatty acids using the principles of the oxidase method, the concentration of 2, 5-dihydroxybenzenesulfonic acid in the sample is approximately 30% lower than that measured by the oxidase method when the content of 2, 5-dihydroxybenzenesulfonic acid is 100 mg/L. In a preferred embodiment of the invention, gentisic acid-1, 2-dioxygenase is selected to contact with the sample, so that the interference of 2, 5-dihydroxybenzenesulfonic acid in the sample on the measurement result can be reduced to be within 10%, and a good anti-interference effect is shown.
In another similar preferred embodiment of the method of detecting glucose using the principle of oxidase, when the glucose is detected by the conventional technique, the measured result of the method of oxidase for measuring the 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 invention, gentisic acid-1, 2-dioxygenase is selected to contact with the sample, so that the interference of 2, 5-dihydroxybenzenesulfonic acid in the sample on the measurement result can be reduced to be within 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 invention, gentisic acid-1, 2-dioxygenase is selected to contact with the sample, so that the interference of 2, 5-dihydroxybenzenesulfonic acid in the sample on the measurement result can be reduced to be within 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 of the sample is 100mg/L when adenosine deaminase is detected using conventional techniques, as measured by the oxidase method, which is nearly 40% lower. In a preferred embodiment of the invention, gentisic acid-1, 2-dioxygenase is selected to contact with the sample, so that the interference of 2, 5-dihydroxybenzenesulfonic acid in the sample on the measurement result can be reduced to be within 10%, and a good anti-interference effect is 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 a sample is 100mg/L in the detection of 5 ' -nucleotidase using the conventional technique, the result of the measurement of 5 ' -nucleotidase activity using the above-mentioned oxidase method is about 40% lower. In a preferred embodiment of the invention, gentisic acid-1, 2-dioxygenase is selected to contact with the sample, so that the interference of 2, 5-dihydroxybenzenesulfonic acid in the sample on the measurement result can be reduced to be within 10%, and a good anti-interference effect is shown.
The above results are similar to the above results even in the case of the detection of pure hydrogen peroxide in a sample, for example, the detection of a hydrogen peroxide sample prepared in an aqueous solution is carried out by using the Trinder's reaction. When the 2, 5-dihydroxybenzenesulfonic acid is added into a sample for detection by adopting a conventional technology, the measured result is lower by about 40 percent, and in a preferred embodiment of the invention, if gentisic acid-1, 2-dioxygenase is simultaneously contacted with 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.
The above-described preferred kits are based on the principle of detection and the conventional amounts of the components, such as cholesterol oxidase kits, and the amounts of the components in the kit, including the names of buffers R1 and R2, amounts, pH, choice of surfactants, manufacturers of cholesterol esterase and cholesterol oxidase, amounts, detection parameters, etc. are reported in many ways and are well known in the art, and will not be discussed in great detail herein.
For the appropriate amount of gentisic acid-1, 2-dioxygenase, according to the conventional technology, a series of gradient experiments are designed, and tests are carried out aiming at specific detection items, so that the appropriate amount of gentisic acid-1, 2-dioxygenase in each item can be determined. In a preferred embodiment of the invention, when the final target object to be detected is glucose and the dosage of gentisic acid-1, 2-dioxygenase is 0.1 KU/L-10 KU/L, a good effect of resisting 2, 5-dihydroxybenzenesulfonic acid interference can be achieved; however, in another preferred embodiment of the invention, when the final target substance to be detected is 2-imino-1-methylimidazoline 4-one, the gentisic acid-1, 2-dioxygenase can be used in an amount of 10KU/L to 100KU/L, so that a good effect of resisting 2, 5-dihydroxybenzenesulfonic acid interference can be achieved. Such concentration gradient determination experiments are conventional experimental techniques, require no creative effort, and are not discussed herein.
In this study, we tested more gentisic acid structural analogues to the techniques mentioned in the present invention and found that various salts of 2, 5-dihydroxybenzenesulfonic acid, such as calcium salt, diethylamine salt, and 2-methyl-4-hydroxyphenol, salicylic acid, 3, 6-dichlorogentisic acid, 3-chlorogentisic acid also interfere with the detection of hydrogen peroxide when using conventional technical methods. In the present study, it was surprisingly found through experimental verification that the method proposed by the present invention, in which gentisic acid-1, 2-dioxygenase is contacted with a sample, can also effectively reduce the interference of these substances.
Therefore, the method provided by the invention, which adopts the gentisic acid-1, 2-dioxygenase compound, can not only eliminate or reduce the interference of 2, 5-dihydroxybenzenesulfonic acid or salts thereof in a sample on the determination result. Interference of compounds with similar gentisic acid structures in the sample with the assay results can also be eliminated or reduced.
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. The phenomenon has been documented and confirmed by experiments carried out in this study.
In summary, the technical route described in the present invention is summarized as follows:
the invention provides a detection method or a kit of hydrogen peroxide, which is characterized in that: comprising the step of contacting a test sample with gentisic acid-1, 2-dioxygenase, said gentisic acid-1, 2-dioxygenase having activity in catalyzing the conversion of gentisic acid to maleic acid-monoacylpyruvic acid.
The invention provides the detection method or the kit for hydrogen peroxide, and the reagent components of the detection method or the kit comprise gentisic acid-1, 2-dioxygenase.
The gentisic acid-1, 2-dioxygenase is derived from microorganisms, and/or fungi, and/or plants; or from a microorganism with induced mutations; or from a microorganism that has been bioengineered mutated and/or recombinant.
Particularly preferred is an organism derived from one or more of Pseudomonas, Ralstonia solanacearum, halophilic archaea, Klebsiella pneumoniae, Rhodococcus, Halobacterium volcanii, Ustilago, Bacillus, Thiobacillus, Moraxella Ostersii, recombinant Escherichia coli, and recombinant yeast.
The invention provides a detection method or a kit, which comprises an application for eliminating or reducing interference of 2, 5-dihydroxybenzenesulfonic acid or salts thereof in a sample, and also comprises an application for eliminating or reducing interference of gentisic acid structural analogues in the sample.
The technology provided by the invention can be used in a plurality of detection items comprising the step of detecting hydrogen peroxide: for example, components and steps coupled with oxidation of 2-imino-1-methylimidazolin-4-one to produce hydrogen peroxide, for detecting the content of 2-imino-1-methylimidazolin-4-one in a sample; or coupling components and steps for oxidizing the glycosylated protein to generate hydrogen peroxide, and detecting the content of the glycosylated protein in the sample; or a component and step coupled with the oxidation of fatty acid to produce hydrogen peroxide, for detecting the content of fatty acid 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 is to be understood that it is logical to infer that the methods and techniques provided herein can also be applied to other non-enumerated items that mediate the detection of hydrogen peroxide.
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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: assaying the activity of gentisic acid-1, 2-dioxygenase.
The gentisic acid-1, 2-dioxygenase catalyzes the ring opening and splitting of gentisic acid into maleic acid-monoacyl pyruvic acid, wherein the maximum absorption peak of the maleic acid-monoacyl pyruvic acid is 330nm, the molar extinction coefficient is 10200mol/L cm, and the activity of the gentisic acid-1, 2-dioxygenase can be calculated by detecting the absorbance change value at 330 nm.
When the specific measurement is carried out, an 752S ultraviolet spectrophotometer is adopted, the measurement wavelength is set to be 330nm, the optical path of the cuvette is 1cm, and the temperature is 25 ℃. The working solution was determined to be a 50mmol/L solution of dipotassium hydrogen phosphate/potassium dihydrogen phosphate, gentisic acid concentration 100mmol/L, pH 7.3.
During determination, the gentisic acid-1, 2-dioxygenase to be determined is dissolved by normal saline to prepare enzyme solution, then 0.1mL of the enzyme solution is added into 2mL of determination working solution, the solution is placed into a spectrophotometer, the absorbance change value delta A/min is detected, and the enzyme activity is calculated according to the molar extinction coefficient of the maleic acid-monoacyl pyruvic acid. The enzyme activity unit (U) is defined as: the conversion produced 1. mu. mol of maleic-monoacylpyruvic acid per minute.
Example 2: mutagenesis of pseudomonas and preparation of gentisic acid-1, 2-dioxygenase
The basic culture medium mainly comprises the following components: 10g/L of peptone, 5g/L of yeast extract, 10g/L of sodium chloride, 1g/L of magnesium chloride and pH 7.0.
Screening a culture medium: the basal medium gradually reduces the amount of peptone and yeast extract, and adds gentisic acid and 2, 5-dihydroxybenzenesulfonic acid as supplementary carbon sources.
And (3) mutagenesis process: the pseudomonas alcaligenes separated from the sludge is cultured in a basal culture medium, then ultraviolet mutagenesis is carried out, screening is carried out by using a screening culture medium, and mutagenesis screening is repeatedly carried out until a mutant strain P261HM is obtained in the embodiment, wherein the expression level of gentisic acid-1, 2-dioxygenase is improved by about fourfold compared with a wild strain, and the reaction capacity to 2, 5-dihydroxy benzene sulfonic acid is improved by four fold.
Recombinant expression: sequencing gentisic acid-1, 2-dioxygenase gene of the mutant strain, adding his-tag, cloning into plasmid, expressing in Escherichia coli to obtain secretory gentisic acid-1, 2-dioxygenase, and purifying with nickel column to obtain purified gentisic acid-1, 2-dioxygenase.
Example 3: observing the interference of 2, 5-dihydroxybenzenesulfonic acid on the determination of hydrogen peroxide and the effect of eliminating the interference of gentisic acid-1, 2-dioxygenase
First, a detection kit for detecting hydrogen peroxide according to the conventional technique (control group) was prepared as shown in table 3A:
note: TOOS is N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methylaniline sodium salt.
A hydrogen peroxide detection kit (panel) according to the technology described herein was then prepared as shown in Table 3B, wherein gentisic acid-1, 2-dioxygenase was derived from Pseudomonas alcaligenes.
The detection steps of the kit are as follows: and (3) 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 3C below:
the test samples were prepared as follows: the hydrogen peroxide solution was diluted to about 400. 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 3D, and the interferents are immediately sampled and detected after being added into the sample.
The samples were measured under the above-mentioned test conditions using the control reagent (conventional technique) and the test reagent (technique described in the present invention), and the results of the measurement of the target analyte hydrogen peroxide in each sample and the relative deviation from the results of the measurement of the control sample containing no interfering substance are shown in Table 3D below.
The test result shows that when the target substance is hydrogen peroxide, the calcium 2, 5-dihydroxybenzenesulfonate salt has very serious interference on the measurement result, but the investigation group adopting the technology described by the invention can greatly eliminate the influence of the interference substance on the detection result.
Example 4: observing the effect of gentisic acid-1, 2-dioxygenase on reducing the interference of 2, 5-dihydroxy benzene sulfonic acid calcium in the determination of 2, 6, 8-trioxypurine
First, a detection kit (control group) for detecting 2, 6, 8-trioxypurine according to the conventional technique was prepared as shown in table 4A:
a2, 6, 8-trioxypurine assay kit (panel) according to the technique described herein was then prepared as shown in Table 4B, wherein gentisic acid-1, 2-dioxygenase was derived from Pseudomonas alcaligenes.
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 2, 6, 8-trioxypurine in the sample according to the measured absorbance value.
The specific detection parameters and detector are shown in table 4C 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 4D below.
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 for the target analyte 2, 6, 8-trioxypurine in each sample and the relative deviation from the assay results of the base sample containing no interfering substance are shown in Table 4D below.
As can be seen from the table, when the technique described in the present invention is used, gentisic acid-1, 2-dioxygenase from Pseudomonas alcaligenes is selected to contact with the sample, so that a good anti-interference effect can be obtained.
Example 5: observation of the Effect of gentisic acid-1, 2-dioxygenase from Klebsiella pneumoniae on mitigating calcium 2, 5-dihydroxybenzenesulfonate interference
The control group reagent of this example is the same as example 4;
a2, 6, 8-trioxypurine detection kit (panel) according to the technique described in the present invention was then formulated as shown in Table 5B, wherein gentisic acid-1, 2-dioxygenase was from Klebsiella pneumoniae.
The detection procedure and sample preparation method were the same as in example 4 above.
The results of the measurements are shown in table 5D below:
it can be seen from the table that the selection of gentisic acid-1, 2-dioxygenase from Klebsiella pneumoniae also allows good anti-interference effects to be achieved when using the techniques described in the present invention.
Example 6: observation of Effect of gentisic acid-1, 2-dioxygenase derived from Ralstonia solanacearum on alleviation of calcium 2, 5-dihydroxybenzenesulfonate interference
The control group reagent of this example is the same as example 4;
a2, 6, 8-trioxypurine detection kit (panel) according to the technique described in the present invention was then prepared as shown in Table 6B, wherein gentisic acid-1, 2-dioxygenase was derived from Ralstonia solanacearum.
The detection procedure and sample preparation method were the same as in example 4 above.
The results of the measurements are shown in table 6D below:
it can be seen from the table that good anti-interference effects can also be obtained with gentisic acid-1, 2-dioxygenase from Ralstonia solanacearum when using the technology described in the present invention.
Example 7: observing the effect of gentisic acid-1, 2-dioxygenase in eliminating or reducing gentisic acid interference
The control group reagent and the study group reagent in this example are the same as those in example 4;
the test procedure and sample formulation were similar to those described above in example 4, except that the interfering substance was gentisic acid and the concentrations of the prepared interfering substances are shown in Table 7D below.
The results of the measurements are shown in table 7D below:
it can be seen from the table that the effect of gentisic acid-1, 2-dioxygenase on mitigating gentisic acid interference is also very significant when the technique described in the present invention is applied.
Example 8: observation of the Effect of gentisic acid-1, 2-dioxygenase on eliminating or reducing the interference of calcium 2, 5-Dihydroxybenzenesulfonate in the 2-imino-1-methylimidazolin-4-one assay
First, a detection kit for detecting 2-imino-1-methylimidazolin-4-one (control group) according to the conventional technique was prepared as shown in Table 8A:
a2-imino-1-methylimidazolin-4-one assay kit (panel) according to the technology described in the present invention was then formulated as Table 8B, wherein gentisic acid-1, 2-dioxygenase was derived from Pseudomonas alcaligenes.
The detection steps of the kit are as follows: reacting the sample with the reagent R1 for 3-5 min, adding the reagent R2, reacting for 5min, measuring the absorbance value, and calculating the content of the 2-imino-1-methylimidazoline-4-one in the sample according to the measured absorbance value.
The specific detection parameters and detector are shown in table 8C below:
the test samples were prepared as follows:
the same sample was divided into 6 groups, and different concentrations of an interfering substance (calcium 2, 5-dihydroxybenzenesulfonate) were sequentially added, the specific addition concentrations being shown in table 8D below.
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 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 8D 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 well eliminate the interference of the interferent on the detection result.
Example 9: observation of the Effect of gentisic acid-1, 2-dioxygenase on Elimination or alleviation of calcium interference from glycosylated Albumin assay 2, 5-Dihydroxybenzenesulfonate
First, a detection kit for detecting glycosylated albumin according to the conventional technique (control group) was prepared as shown in table 9A:
a glycosylated albumin assay kit (panel) according to the techniques described herein was then prepared as shown in Table 9B, wherein the gentisic acid-1, 2-dioxygenase was derived from Pseudomonas alcaligenes.
The detection steps of the kit are as follows: and (3) reacting the sample with the reagent R1 for 3-5 min, adding the reagent R2, reacting for 5min, measuring the absorbance value, and calculating the content of the glycosylated albumin in the sample according to the measured absorbance value.
The specific detection parameters and detector are shown in table 9C below:
the test samples were prepared as follows:
the same serum basic 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 9D below.
The samples were then assayed under the above assay conditions using the control reagent (conventional technique) and the study reagent (technique described in the present invention), and the results of detection of the target analyte (glycated albumin) in each sample and the relative deviation from the assay results of the base sample containing no interfering substance are shown in Table 9D 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 well eliminate the interference of the interferent on the detection result.
Claims (10)
1. A method or a kit for detecting hydrogen peroxide, characterized in that: comprising the step of contacting a test sample with gentisic acid-1, 2-dioxygenase, said gentisic acid-1, 2-dioxygenase having activity in catalyzing the conversion of gentisic acid to maleic acid-monoacylpyruvic acid.
2. The detection method or kit according to claim 1, characterized in that: the reagent component contains gentisic acid-1, 2-dioxygenase.
3. The detection method or kit according to claim 1, characterized in that: the gentisic acid-1, 2-dioxygenase is derived from a microorganism, and/or a fungus, and/or a plant.
4. The detection method or kit according to claim 1, characterized in that: the gentisic acid-1, 2-dioxygenase is derived from a microorganism subjected to induced mutation.
5. The detection method or kit according to claim 1, characterized in that: the gentisic acid-1, 2-dioxygenase is derived from a bioengineered mutated and/or recombinant microorganism.
6. The detection method or kit according to claim 1, characterized in that: the gentisic acid-1, 2-dioxygenase is one or more of pseudomonas, ralstonia solanacearum, halophilic archaea, klebsiella pneumoniae, rhodococcus, volcanic halibut, holotrichia, bacillus, thiobacillus, ormula oshima, recombinant escherichia coli and recombinant yeast.
7. The detection method or kit according to claim 1, characterized in that: including use for eliminating or mitigating interference of 2, 5-dihydroxybenzenesulfonic acid or a salt thereof in a sample.
8. The detection method or kit according to claim 1, characterized in that: including use for eliminating or mitigating interference of gentisic acid structural analogues in a sample.
9. The detection method or kit according to any one of claims 1 to 8, characterized by further comprising one of the following uses:
coupling components and steps for oxidizing 2-imino-1-methylimidazolidin-4-one to produce hydrogen peroxide for detecting the content of 2-imino-1-methylimidazolidin-4-one in a sample.
10. The detection method or kit according to any one of claims 1 to 8, characterized by further comprising the following:
coupling components and steps for oxidizing glycosylated protein to generate hydrogen peroxide, for detecting the content of glycosylated protein in the sample;
or a component and step coupled with the oxidation of fatty acid to produce hydrogen peroxide, for detecting the content of fatty acid 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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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108486143A (en) * | 2018-03-29 | 2018-09-04 | 河南农业大学 | A kind of fungal rna interference vector pBHt2-CHSA Intron, construction method and application |
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