CN116622802A - Nitric oxide detection reagent, detection method and application thereof - Google Patents
Nitric oxide detection reagent, detection method and application thereof Download PDFInfo
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- CN116622802A CN116622802A CN202310800239.6A CN202310800239A CN116622802A CN 116622802 A CN116622802 A CN 116622802A CN 202310800239 A CN202310800239 A CN 202310800239A CN 116622802 A CN116622802 A CN 116622802A
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/902—Oxidoreductases (1.)
- G01N2333/90245—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/902—Oxidoreductases (1.)
- G01N2333/906—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.7)
- G01N2333/90688—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.7) acting on other nitrogen compounds as donors (1.7)
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Immunology (AREA)
- Physics & Mathematics (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention provides a nitric oxide detection reagent, a detection method and application thereof. The nitric oxide detection reagent provided by the invention comprises reduced coenzyme and/or derivatives thereof, nitrate reductase, nitrite dismutase and nitric oxide dioxygenase. The detection reagent provided by the invention is applied to nitric oxide detection and has the characteristics of convenience, rapidness, automation, strong interference resistance and high sensitivity.
Description
Technical Field
The invention relates to a detection reagent for nitric oxide and application thereof.
Background
Nitric Oxide (NO) is a messenger and effector molecule that is widely found in living organisms and is also an active nitrogen (active nitrogen species, ANS). Nitric oxide is known to have important roles in physiology, pathology, pharmacology, etc. In recent years, some foreign studies have shown that nitric oxide radicals play an important role in aging memory impairment. Other scholars have found in studies of the cerebral cortex of alzheimer's patients that nitric oxide radicals have important biological functions, which are endothelial cell relaxants (EDRF), which inhibit platelet aggregation, but have a highly reactive and cytotoxic side, which is both protective and possibly damaging to the cardiovascular system, and this double side is very typical in the cardiovascular system. Among them, the relationship between myocardial ischemia reperfusion injury and nitric oxide radicals has attracted a great deal of attention. Not only is a physiological and biochemical method adopted for research and analysis, but also an ESR technology is adopted for researching nitric oxide free radicals generated in the ischemia reperfusion process. Numerous studies have shown that vascular endothelial dysfunction is closely related to hypertension. The reduction of nitric oxide free radicals and the formation and development of hypertension cases lay a foundation. In addition, NO also has the functions of immunoregulation, antiulcer, sexual function regulation and the like.
There are various methods for measuring NO such as chemiluminescence, spectrophotometry, fluorescence, etc.
Chemiluminescence is considered as a standard method for measuring NO. Classical chemiluminescence of NO is based on the combination of NO and ozone (O 3 ) Reaction to form excited nitrogen dioxide (NO) 2 *)，NO 2 * The energy is released to emit light during the return to the ground state. The method has high sensitivity and detection limit of 5pmol/L. However, this method has the disadvantages: NO and O 3 The reaction of (2) must be carried out in a gaseous state, nitric oxide in solution must be stripped off with an inert gas, the operation is complicated, and the reaction is alsoThere are those which are susceptible to interference by ammonia gas, hydrogen sulfide, olefins, dimethylsulfoxide (DMSO), etc., and the reaction conditions are not easy to control, etc. Later improvements were made to the oxidizing agent, replacing ozone with hydrogen peroxide, i.e., luminol process. NO can be H 2 O 2 Intense oxidation to form ONOO - And ONOO (oxide-nitride-oxide) - Luminol can be oxidized and emits very intense light in solution. Because only NO can excite this photochemical reaction, NO 2 - And NO 3 - The method can not be used, so that the method can be used for measuring NO in solution in real time and has the highest detection sensitivity, and the detection limit can reach 10 < -13 > mol/L. Because NO in human blood is easily oxidized into NO 2 - Even NO 3 - Therefore, this method is not suitable for blood sample detection.
The principle of the fluorescent method for measuring NO is NO 2 - React with DAN (2, 3-diaminophthalate) under acidic condition to generate fluorescent substance 1- (H) -naphthalzole, which has high fluorescence efficiency under alkaline condition (pH above 10), and is excited by 365nm light, and the fluorescence intensity is measured at 450nm for quantitative determination. Although the method has higher sensitivity, the detection needs special instruments, takes longer time and is not suitable for clinical large-scale screening.
Electrochemical detection and NO microsensors, although capable of real-time in-situ detection in vivo, are expensive in equipment and not widely used in clinical applications.
NO is easily oxidized in vivo or in aqueous solution to generate NO 2 - NO under acidic conditions 2 - Causing the Griess reaction to occur to produce the diazonium compound. Its concentration and NO 2 - The concentrations have a linear relationship. The compound can be quantitatively measured at 540-560 nm, namely a spectrophotometry, but the method requires centrifugation in the operation process, the treatment capacity is not easy to amplify, the process is time-consuming and labor-consuming, and the operation steps are tedious and the large-scale clinical application is limited.
In summary, a detection method which is simple to operate and can be applied to large-scale clinic is urgently needed.
Disclosure of Invention
Aiming at the technical problems that the NO detection method in the prior art is complex in operation and not suitable for large-scale clinical application, the invention provides a nitric oxide detection reagent.
The detection reagent of the invention utilizes the substrate specificity of the enzyme to amplify the target substance (the detected object) according to the enzyme circulation method, so as to improve the detection accuracy and simplify the detection steps.
The enzyme cycle method is a measurement method in which a target substance (a test substance) is amplified by utilizing the substrate specificity of an enzyme, and a certain reactant is continuously consumed in the process, and the test substance is cyclically generated after being consumed, and a certain reactant and the test substance which consume the corresponding amount of the substance are reacted once per cycle to generate the test substance which corresponds to the amount of the substance. The method only circulates the target substance, reduces the interference of other substances existing in the sample on the measurement, and therefore, the method does not need to pretreat the sample or extract the target substance, and does not need special equipment, thereby being a measurement technology with wide prospects. Because the specificity of the enzymatic method is good, the detection is simple and convenient, the reaction is mild, no pollution is caused, and the sensitivity is high (10) -6 mol/L) can satisfy the measurement of most substances in body fluid, and therefore, has been widely used in clinical chemistry measurement.
The invention provides a novel enzyme circulation method for measuring nitric oxide in a body fluid sample, which is very simple and convenient to operate and has excellent reaction sensitivity. And the tool enzyme, nitrate reductase and nitrite dismutase in the method for measuring the nitric oxide are adopted for the first time, and the nitric oxide dioxygenase is used for measuring the increment of enzyme circulation. In addition, the invention also discloses an enzyme circulation method based on the three tool enzymes and a nitric oxide diagnostic reagent prepared by the method for the first time, and the reagent can be applied to a clinical automatic analyzer widely used at present, thereby meeting the requirement of large-scale screening.
The invention provides a nitric oxide detection reagent, which comprises reduced coenzyme and/or derivatives thereof, nitrate reductase, nitrite dismutase and nitric oxide dioxygenase.
Preferably, it comprises:
nitrate reductase, in the presence of reduced coenzyme and/or its derivative, nitrate ion in the sample undergoes reduction reaction to generate nitrite ion;
nitrite dismutase, which can react nitrite generated by the reaction and in a sample to generate nitric oxide and nitrate ions under the condition of H < + >;
Nitric oxide dioxygenase, in the presence of oxygen and reduced coenzyme and/or derivatives thereof, reacts with the nitric oxide to form nitrate ions.
Preferably, the reduced coenzyme is selected from one or both of reduced coenzyme I (NADH) or reduced coenzyme II (NADPH);
preferably, the reduced coenzyme derivative is selected from one or both of thio-reduced coenzyme I (thio-NADH) or thio-reduced coenzyme II (thio-NADPH).
Preferably, the nitric oxide dioxygenase is capable of reacting the nitric oxide in the presence of reduced coenzyme and/or derivatives thereof and oxygen to form nitrate ions.
Preferably, the nitrate reductase causes nitrate ions in a sample to be tested to react as shown in the following formula (1) in the presence of reduced coenzyme and/or derivatives thereof:
preferably, the nitrite dismutase reacts with the nitrite ion as shown in the following formula (2):
preferably, the nitric oxide dioxygenase, nitric oxide and reduced coenzyme and/or derivative thereof undergo a reaction represented by the following formula (3):
during the reaction, the reduced coenzyme and/or derivative thereof provides a hydrogen atom and an electron required for the reactions of the above formula (1) and formula (3).
Preferably, the detection reagent further comprises a buffer;
preferably, the buffer is a phosphate buffer.
Preferably, the detection reagent comprises a reagent R1 and a reagent R2,
wherein the reagent R1 comprises: a. reduced coenzyme solution or b.buffer containing reduced coenzyme,
reagent R2 comprises nitrate reductase, nitrite dismutase and nitric oxide dioxygenase.
Preferably, the mass percentage of reduced coenzyme in the reagent R1 is 0.01-0.05%, preferably 0.02-0.03%, more preferably 0.025%.
Preferably, the buffer solution in the reagent R1 is one or more than two of Tris-HCl buffer solution, HEPES buffer solution and phosphate buffer solution, and is preferably phosphate buffer solution.
Preferably, the reagent R1 also contains a surfactant and a first preservative;
preferably, the surfactant is selected from one or a combination of more than two of tween-20,Brij 35,Triton X-100, preferably, the mass percentage of the surfactant in the reagent R1 is 0.01-1%, preferably 0.01-0.5%, more preferably 0.1%;
preferably, the first preservative is selected from one or a combination of more than two of PC300, sodium azide, gentamicin and thimerosal, preferably, the mass percentage of the first preservative in the reagent R1 is 0.01-0.5%, preferably 0.01-0.2%, and more preferably 0.1%.
Preferably, the content of nitrate reductase in the reagent R2 is 1-9KU/L, preferably 3-5KU/L;
preferably, the content of nitrite dismutase in the reagent R2 is 3-11KU/L, preferably 5-7KU/L;
preferably, the nitric oxide dioxygenase is present in the reagent R2 in an amount of 2-10KU/L, preferably 4-6KU/L.
Preferably, the reagent R2 further contains one or more than two of a second buffer solution, mannitol, metal ions or a second preservative;
preferably, the second buffer is selected from one of phosphate buffer, tris-HCl buffer and HEPES buffer;
preferably, the mannitol in the reagent R2 is 0.01-5%, preferably 0.01-1%, more preferably 0.1% by mass;
preferably, the metal ion is selected from Fe 2+ 、Mg 2+ 、Ca 2+ One or a combination of two or more of them; preferably, the molar concentration of metal ions in the reagent R2 is between 0.1 and 10mM, preferably between 1 and 5mM;
preferably, the second preservative is selected from one or more than two of PC300, sodium azide, gentamicin and thimerosal, preferably, the mass percentage of the second preservative in the reagent R2 is 0.01-0.5%, preferably 0.01-0.2%, and more preferably 0.1%.
Preferably, the detection reagent also contains a calibrator, wherein the calibrator is sodium nitrate aqueous solution, and the concentration point is 0,12.5,25,50,100,200 mu mol/L.
The present invention also provides a nitric oxide detection kit comprising:
the unit containing nitrate reductase and reduced coenzyme and/or derivatives thereof can lead nitrate ions in the sample to be detected to generate nitrite ions through reduction reaction;
a unit containing nitrite dismutase, capable of reacting said nitrite ion to produce nitric oxide and nitrate ions;
a unit comprising nitric oxide dioxygenase and reduced coenzyme and/or a derivative thereof, capable of reacting said nitric oxide in the presence of reduced coenzyme and/or a derivative thereof to form nitrate ions.
The invention also provides a method for detecting the content of nitric oxide, which comprises the following enzyme cycling reaction:
preferably, the sample is detected using a detection reagent comprising said detection reagent.
Preferably, detecting the consumption rate of reduced coenzyme to obtain the nitric oxide content in the sample;
preferably, the reduced coenzyme consumption rate is detected spectrophotometrically.
Preferably, the detection method comprises the steps of: mixing the sample with reduced coenzyme, adding nitrate reductase, nitrite dismutase and nitric oxide dioxygenase, and detecting the consumption rate of the reduced coenzyme to obtain the content of nitric oxide in the sample;
Preferably, the sample is mixed with a buffer solution containing reduced coenzyme, nitrate reductase, nitrite dismutase and nitric oxide dioxygenase are added, and the consumption rate of the reduced coenzyme is detected, so that the content of nitric oxide in the sample is obtained.
Preferably, the nitric oxide content in the sample is calculated using a rate method or an endpoint method, preferably a rate method.
Preferably, the sample is a cell lysate, a tissue or cell culture fluid, serum, plasma, urine, an aqueous solution.
Preferably, the detection method comprises mixing the reagent R1 with the sample to be detected, and then adding the reagent R2 for detection;
preferably, the volume ratio of the reagent R1, the reagent R2 and the sample to be tested is 200-300:30-100:1-20, preferably 200:60:10.
Preferably, the detection method further comprises mixing the reagent R1 with a calibrator, and then adding the reagent R2 for detection, so as to prepare a standard curve.
The invention also provides application of the detection reagent or the detection method in detecting the nitric oxide content.
The invention provides an enzymology determination method and a reagent thereof adopting a cyclic increment technology, which are used for determining the content of nitric oxide in a body fluid sample. The circulating increment measuring system mainly comprises nitrate reductase, nitrite dismutase and nitric oxide dioxygenase. Nitrate reductase has been widely used in Griess reagent at present; nitrite dismutase and nitric oxide dioxygenase are less commonly used in enzyme circulation systems.
The present invention provides a diagnostic reagent for measuring nitric oxide, comprising nitrate reductase, nitrite dismutase and nitric oxide dioxygenase, and, in addition to the above three tool enzymes, reduced coenzyme and/or derivatives thereof, since in practice the derivatives of reduced coenzyme can form reduced coenzyme I/II by some reactions, thereby an enzymatic cycling reaction takes place which consumes reduced coenzyme I/II.
Nitric oxide is typically oxidized to nitrate or nitrite in vivo or in aqueous solution, nitrate is reduced to nitrite by nitrate reductase (EC 1.7.1.1) in diagnostic reagents (EC number is a set of numbered classifications made by the enzyme Committee (Enzyme Commission) for enzymes based on the chemical reaction catalyzed by each enzyme. Nitric oxide is oxidized to nitrate/nitrite under the action of nitric oxide dioxygenase (EC 1.14.12.17) while consuming reduced coenzyme I/II. Therefore, nitrate in the sample is subjected to repeated circulation reaction, the reduced coenzyme I/II is continuously consumed, the consumption rate of the reduced coenzyme I/II is in direct proportion to the content of nitrate/nitrite in the sample, and the purpose of measuring the content of nitric oxide in the body fluid sample can be achieved by measuring the consumption rate of the reduced coenzyme I/II.
The invention provides a method for measuring nitric oxide, which mainly comprises the following chemical reaction equations:
nitric oxide is easily oxidized in vivo or in aqueous solution to generate nitrite or nitrate, so nitrate in the solution needs to be reduced into nitrite by nitrate reductase, and then nitrite dismutase and nitric oxide dioxygenase are adopted for carrying out the two-step enzyme cycle reaction.
The invention creates an enzyme circulation reaction consisting of nitrate reductase, nitrite dismutase and nitric oxide dioxygenase for the first time, and is applied to the determination of nitric oxide. Based on the characteristic of the enzyme method circulation increment measurement, the enzyme use amount of the circulation reaction system is not required to be very high. Of course, increasing the amount of enzyme does not affect the application of the method of the invention, but increases the cost of manufacturing the reagent. Optimizing the amount of enzyme and substrate used may facilitate the cycling reaction to proceed in the desired direction, as described with reference to chemical equations (1) and (3).
The nitric oxide detection reagent provided by the invention is applied to nitric oxide detection, has the characteristics of simple operation method, excellent response sensitivity, convenience, rapidness, automation, strong interference resistance and high sensitivity. The reagent can be applied to the clinical automatic analyzer widely used at present, thereby meeting the requirement of large-scale screening. The invention provides a novel enzyme circulation method for measuring nitric oxide in a body fluid sample, which adopts tool enzymes, nitrate reductase, nitrite dismutase and nitric oxide dioxygenase in the method for measuring the nitric oxide for the first time for measuring the increment of enzyme circulation.
The detection reagent provided by the invention has large detection flux and large detection quantity in unit time, and can be applied to nitric oxide detection on a large scale.
Drawings
FIG. 1 shows the Griess method and the reagent correlation standard curve of the present invention in example 3.
Detailed Description
The invention provides an enzymology determination reagent adopting a cyclic increment technology and a method for detecting nitric oxide by using the same, and the method is used for determining the content of nitric oxide in a sample. The cyclic increment measurement system comprises nitrate reductase, nitrite dismutase and nitric oxide dioxygenase.
The detection reagent provided by the invention can detect the content of nitric oxide in cell lysate, tissue or cell culture solution, serum, plasma, urine and aqueous solution.
The nitric oxide measuring reagent provided by the invention can be made into a double-component reagent, or can be made into a single reagent or a three-reagent. The double reagent is stored for a long time in two parts, wherein the reagent R1 comprises buffer solution and reduced coenzyme and/or derivatives thereof, and the reagent R2 is enzyme solution and comprises nitrate reductase, nitrite dismutase and nitric oxide dioxygenase.
In a specific embodiment of the present invention, the reduced coenzyme and/or derivative thereof is selected from one or a combination of two or more of NADH, NADPH, thio-NADH or thio-NADPH.
In a specific embodiment of the invention, after the detection reagent provided by the invention is mixed with a sample, the content of nitric oxide in the sample is determined by adopting a method for determining the consumption rate of the reduced coenzyme I/II.
Specifically, in an embodiment of the present invention, a method for detecting nitric oxide includes the steps of: (1) Reacting a detection reagent with a sample, reducing nitrate in the sample into nitrite by using nitrate reductase, and simultaneously consuming reduced coenzyme I or II, (2) generating nitrate and nitric oxide by using nitrite dismutase from nitrite in the solution obtained in the step (1); (3) Oxidizing nitric oxide in the solution obtained in the step (2) into nitrate radical by nitric oxide dioxygenase, and simultaneously consuming reduced coenzyme I or II; (4) The steps (1) - (3) are cyclically carried out, and the content of nitric oxide in the sample is obtained by measuring the consumption rate of the reduced coenzyme I or II.
In one embodiment of the invention, the consumption rate of the reduced coenzyme is determined by spectrophotometry, and in one embodiment of the invention, the rate of decrease of the light absorption value at 340nm is monitored, and the nitric oxide content in the sample is calculated.
In particular embodiments of the invention, the Nitrate Reductase (NR), nitrite dismutase (NP) and Nitric Oxide Dioxygenase (NOD) are derived from commercial or homemade sources.
In one specific embodiment of the present invention, the Nitrate Reductase (NR), nitrite dismutase (NP) and Nitric Oxide Dioxygenase (NOD) are purified by fermentation with genetically engineered bacteria.
The preparation method of the genetically engineered bacterium comprises the following steps: primer synthesis, gene cloning, plasmid construction, plasmid identification, plasmid transformation, DNA sequencing and the like.
Specifically, the main steps of gene cloning are: synthesizing gene fragments, amplifying by a PCR method to obtain fragment PCR products, and recombining into a target vector (pET-28 a (+)) by a multistage recombination method to obtain the full-length construction.
The gene sequences and amino acid sequences of NR, NP and NOD enzymes, and the template and primer sequences used in the PCR amplification are as follows:
the target gene sequence of the Nitrate Reductase (NR) is shown as SEQ ID NO. 1:
the amino acid sequence of the Nitrate Reductase (NR) is shown in SEQ ID NO. 2:
the sequences of PCR primers used in constructing the Nitrate Reductase (NR) plasmid are shown in Table 1-1 below.
TABLE 1-1
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Templates and primers used in constructing the Nitrate Reductase (NR) plasmid are shown in tables 1-2 below.
TABLE 1-2
The target gene sequence of the nitrite dismutase (NP) is shown as SEQ ID NO. 3:
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the amino acid sequence of nitrite dismutase (NP) is shown as SEQ ID NO. 4:
The sequences of PCR primers used in constructing the nitrite dismutase (NP) plasmid are shown in tables 1-3 below.
Tables 1 to 3
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Templates and primers used in constructing nitrite dismutase (NP) plasmids are shown in tables 1-4 below.
Tables 1 to 4
The target gene sequence of the Nitric Oxide Dioxygenase (NOD) is shown in SEQ ID NO. 5:
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the amino acid sequence of the Nitric Oxide Dioxygenase (NOD) is shown in SEQ ID NO. 6:
the sequences of PCR primers used in constructing the Nitric Oxide Dioxygenase (NOD) plasmid are shown in tables 1-5 below.
Tables 1 to 5
Templates and primers used in constructing Nitric Oxide Dioxygenase (NOD) plasmids are shown in tables 1-6 below.
Tables 1 to 6
Specifically, the identification method of the cloning plasmid comprises the following steps:
(1) The PCR reaction used a 20. Mu.L system: primer 0.5. Mu.L, template bacteria solution 2. Mu.L, polymerase buffer 0.5. Mu.L, buffer 3. Mu.L, ddH 2 O14. Mu.L. Cycle parameters: pre-denaturation at 96℃for 3min;95℃15S,58℃15S,72℃20s,23 cycles, 72℃final extension 1min.
(2) Positive clones are screened by a bacterial liquid PCR method, and positive bacterial liquid obtained is subjected to shaking bacterial extraction plasmid at 37 ℃ and sequencing.
(3) And sequencing and comparing the correct plasmids, and carrying out double enzyme digestion to obtain two fragments comprising the target gene and the vector.
Specifically, the method of plasmid transformation is as follows
(1) Plasmid was pipetted at a plasmid concentration of about 100ng/ul by 1-3ul into about 100 ul competent cells, gently swirled to mix, and placed on ice for 3 min. Proper few plasmids with higher plasmid concentration and some plasmids with lower plasmid concentration can be added more
(2) The water bath at 42 ℃ is not required to shake for 90 seconds
(3) Placing in ice bath for about 3 min
(4) 500-800. Mu.l of LB medium at 37℃were added to each tube, and the mixture was gently shaken for 40 minutes at 200rpm in a shaker at 37 ℃.
Verification of recombinants (DNA sequencing step)
(1) Preparation of agar plates (2) containing the corresponding resistances 100. Mu.l of the bacterial solution were then spread on agar plates containing the corresponding resistances, bacteria were gently smeared on the surfaces of the plates with a sterile glass spreader, and the plates were incubated at 37℃for 15 minutes
(3) Colonies can appear when the inverted plate is cultured for 12-16 hours at 37 DEG C
(4) The plate is picked up, the bacteria are shaken for 14 hours at 37 ℃ at 250 revolutions per minute, PCR identification is carried out by using bacterial liquid, and the positive clone bacterial liquid is sent to sequencing.
All three genetically engineered bacteria are prepared according to the method.
Specifically, the purification mode of NP is as follows: expression in E.coli was incubated at 37℃and 150rmp to OD 0.6. The IPTG concentration was 0.4mM, and the cells were collected by centrifugation at 150rpm at 30℃for 15-18 hours. 100mM Tris-HCl, pH 7.4,20mM EDTA,10% sucrose, lysis, high pressure homogenization disruption, centrifugation to collect the precipitate, washing twice with Triton X-100-containing buffer and washing twice again with Triton X100-free buffer. The inclusion bodies were dissolved in 20mL of 30mM Tris-HCl, pH 7.4,1mM EDTA,6M guanidine hydrochloride, 5mM DTT. Centrifuging at 10000g for 40min; the denatured proteins were refolded, diluted with 30mM Tris-HCl, pH 7.4,0.8M NaCl,1mM EDTA,10mM DTT, and 15mM PMSF buffer at 0deg.C over a period of 80 minutes, increased by 50-fold volumes, and stirred overnight at 4deg.C. After centrifugation, the enzyme protein solution was concentrated by ultrafiltration.
The enzyme protein solution was identified by conventional SDS-polyacrylamide gel electrophoresis. By comparing the molecular weight with the Marker, it is possible to determine whether the target protein is the target protein. Wherein NR has a molecular weight of 109.08KD, NP has a molecular weight of 25.68KD, and NOD has a molecular weight of 48.6KD.
NR and NOD are also expressed by an escherichia coli system, and a conventional Ni column affinity purification method is a conventional operation.
In one embodiment of the invention, the reagent R1 and reagent R2 formulations are used to reduce nitrate and the rate of oxidation of reduced coenzyme in a fixed period of time is determined using an enzymatic cycling reaction consisting of nitrate reductase, nitrite dismutase and nitric oxide dioxygenase.
In one embodiment of the present invention, reagent R1 comprises a phosphate buffer, reduced coenzyme (NADH/NADPH), a surfactant, and a preservative.
In one embodiment of the invention, the reagent R2 contains Tris-HCl buffer solution, nitrate reductase, nitrite dismutase, nitric oxide dioxygenase, mannitol, metal ions and preservative. Reagent R1, reagent R2 and calibrator are combined to form complete enzyme cycle assay reagent.
In one embodiment provided by the invention, the preparation method of the reagent R1 comprises the following steps: 2.584g of Na is weighed respectively 2 HPO 4 ·12H 2 O and NaH 2 PO 4 ·2H 2 Dissolving 7.068g of O in 1000mL of purified water, sequentially weighing 0.25g of NADH, 1mL of Tween-20 and 300 mL of PC, adding into the phosphate buffer solution, fully dissolving, filtering by using a 0.22 mu m membrane, and standing at 2-8 ℃ for later use;
in one embodiment provided by the invention, the reagent R2 preparation method comprises the following steps: 2.42g Tris was weighed into 1000mL purified water, pH was adjusted to 7.4 with concentrated HCl, and 1g mannitol, feCl was weighed 2 126.75mg、NaN 3 1mL of the buffer solution is added, 1-9KU, optimal 3-5KU, 3-11KU of nitrite dismutase, optimal 5-7KU, 2-10KU of nitric oxide dioxygenase and optimal 4-6KU are sequentially weighed after being fully dissolved, dissolved into the solution one by one, slowly stirred until being fully dissolved, filtered by a 0.45 mu m membrane, and kept at 2-8 ℃ for standby;
in one embodiment of the invention, the sample is measured by using a Hitachi 7080 biochemical analyzer, wherein R1 to R2 is 200-300 to 30-100 to 1-20 (or the dosage is increased or reduced in the same ratio and is in the proper dosage range of the analyzer), the temperature is 37 ℃, the measurement wavelength is 340nm, the R1 is added with the sample or the calibrator and then incubated for 300 seconds at the measurement temperature, then R2 is added, the delay is 60-120 seconds, the interference of the consumable reduced coenzyme substances in the sample and the side reaction of other nitrite dismutase and nitric oxide dioxygenase are eliminated, the measurement time is 180 seconds, and the reading selects at least 2 effective points in the measurement time.
The raw material information used in the examples of the present invention is shown in tables 1 to 7:
tables 1 to 7
| Name of the name | Vendor' s |
| Na 2 HPO 4 ·12H 2 O | Sinopharm Group Chemical Reagent Co., Ltd. |
| NaH 2 PO 4 ·2H 2 O | Sinopharm Group Chemical Reagent Co., Ltd. |
| NADH | BEIJING J&K SCIENTIFIC Ltd. |
| Tween-20 | Sinopharm Group Chemical Reagent Co., Ltd. |
| PC300 | Sigma-Aldrich |
| Tris | Amresco |
| Mannitol | Sinopharm Group Chemical Reagent Co., Ltd. |
Examples
The following examples are given by way of illustration of specific embodiments of the present invention and alterations and modifications of materials, steps, etc. may be made by persons skilled in the art, and it should be understood that such modifications and substitutions are also intended to fall within the scope of the claims and specification of the present invention.
Example 1 enzyme dosage screening
Reagent R1 is prepared: 2.584g of Na is weighed respectively 2 HPO 4 ·12H 2 O and NaH 2 PO 4 ·2H 2 Dissolving 7.068g of O in 1000mL of purified water, sequentially weighing 0.25g of NADH, 1mL of Tween-20 and 300 mL of PC, adding into the phosphate buffer solution, fully dissolving, filtering by using a 0.22 mu m membrane, and standing at 2-8 ℃ for later use;
reagent R2 is prepared: 2.42g Tris is weighed and dissolved in 1000mL purified water, the pH is regulated to 7.4 by concentrated hydrochloric acid, and 1g mannitol and NaN are weighed 3 1mL of the buffer solution is added, nitrate reductase, nitrite dismutase and nitric oxide dioxygenase are sequentially weighed after being fully dissolved, dissolved into the solution one by one, slowly stirred until being fully dissolved, filtered by a 0.45 mu m membrane, and kept at 2-8 ℃ for standby;
the circulating enzyme method detection reagent also contains a calibrator. The calibrator is sodium nitrate aqueous solution, and the concentration point is 0,12.5,25,50,100,200 mu mol/L. The preparation method comprises the following steps: 17mg of sodium nitrate is weighed and dissolved in 1000mL of purified water, and a 200 mu mol/L concentration point calibrator is obtained after full dissolution; mixing 500mL of 200 mu mol/L calibrator with purified water at a concentration of 1:1 to obtain 100 mu mol/L calibrator; and the like, 50, 25 and 12.5 mu mol/L concentration point calibration products are obtained.
Sample measurement, namely, using a Hitachi 7080 biochemical analyzer, wherein the ratio of R1 to R2 to the sample is 200:60:10, the temperature is 37 ℃, the measurement wavelength is 340nm, the reagent R1 is incubated for 300 seconds at the measurement temperature after adding a calibrator, then R2 is added, the delay is 60-120 seconds, the interference of consumable reduced coenzyme substances in the sample and the side reaction of other nitrite dismutase and nitric oxide dioxygenase are eliminated, the measurement time is 180 seconds, and at least 2 effective points are selected in the measurement time in the reading.
1.1 nitrate reductase dosage screening
According to the preparation method of R2, other components except enzyme are sequentially added, after the components are fully dissolved, nitrite dismutase is added to 12KU/L, nitric oxide dioxygenase is added to 10KU/L, nitrate reductase is respectively added to 1KU/L, 3KU/L, 5KU/L, 7KU/L and 9KU/L, reaction rates are tested, and data are shown in Table 2.
TABLE 2
As shown by the results in Table 2, the nitrate reductase dosage of 3-5KU/L is optimal.
1.2 screening of the amount of nitrite dismutase
According to the preparation method of R2, other components except enzyme are sequentially added, 5g/L of nitrate reductase and 10g/L of nitric oxide dioxygenase are added after the components are fully dissolved, 3KU/L, 5KU/L, 7KU/L, 9KU/L and 11KU/L of nitrite dismutase are respectively added, the reaction rate is tested, and the data are shown in Table 3.
TABLE 3 Table 3
The results in Table 3 show that the nitrite dismutase dosage is 5-7KU/L, and the rate is optimal.
1.3 nitric oxide dioxygenase dosage screening
According to the preparation method of R2, other components except enzyme are sequentially added, nitrate reductase 5KU/L and nitrite dismutase 7KU/L are added after the components are fully dissolved, nitric oxide dioxygenase 2KU/L, 4KU/L, 6KU/L, 8KU/L and 10KU/L are respectively added, the reaction rate is tested, and the data are shown in Table 4.
TABLE 4 Table 4
As shown by the results in Table 4, the rate of nitric oxide dioxygenase at 4-6KU/L was optimal.
Example 2 nitric oxide detection reagent
Example 2.1
Following the formulation method in example 1, reagent R2 and calibrator were formulated as follows:
reagent R1: 1000mL of 50mM Tris-HCl buffer containing 0.01wt% NADH, 0.5wt% Tween-20, and 0.01wt% sodium azide;
reagent R2: 1000mL of 20mM Tris-HCl buffer containing NR 5KU/L, NP 7KU/L, NOD 6KU/L, feCl 2 0.1mM, mannitol 1wt%, PC 300.01 wt%;
calibration material: an aqueous sodium nitrate solution was prepared at a concentration point of 0,12.5,25,50,100,200. Mu. Mol/L.
Example 2.2
The R1, R2 reagents and calibrator were prepared according to the formulation in example 1 as follows:
Reagent R1: 1000mL of 50mM HEPES buffer containing 0.05wt% NADH, 35.01 wt% Brij, and 0.5wt% gentamicin;
reagent R2: 1000mL of 20mM HEPES buffer containing NR 5KU/L, NP7KU/L, NOD 6KU/L, caCl 2 1mM, 5% mannitol, 0.5% gentamicin;
calibration material: an aqueous sodium nitrate solution was prepared at a concentration point of 0,12.5,25,50,100,200. Mu. Mol/L.
Example 2.3
The R1, R2 reagents and calibrator were prepared according to the formulation in example 1 as follows:
reagent R1: 1000mL of 50mM phosphate buffer containing 0.02wt% NADH, 0.1wt% Triton X-100, and 0.05wt% gentamicin;
reagent R2: 1000mL of 20mM phosphate buffer containing NR 5KU/L, NP7KU/L, NOD 6KU/L, mgCl 2 5mM, mannitol 0.01wt%, merthiolate 0.1wt%;
calibration material: an aqueous sodium nitrate solution was prepared at a concentration point of 0,12.5,25,50,100,200. Mu. Mol/L.
Example 2.4
The R1, R2 reagents and calibrator were prepared according to the formulation in example 1 as follows:
reagent R1: 1000mL of 50mM Tris-HCl buffer containing 0.03wt% NADH, 0.2wt% Tween-20, and 0.2wt% PC;
reagent R2: 1000mL of 20mM phosphate buffer containing NR 5KU/L, NP7KU/L, NOD 6KU/L, feCl 2 1mM, mannitol 0.1wt%, PC 300.3 wt%;
calibration material: an aqueous sodium nitrate solution was prepared at a concentration point of 0,12.5,25,50,100,200. Mu. Mol/L.
Example 2.5
The R1, R2 reagents and calibrator were prepared according to the formulation in example 1 as follows:
reagent R1: 1000mL of 50mM phosphate buffer containing 0.025wt% NADH, 0.1wt% Tween-20, and 0.1wt% PC;
reagent R2: 1000mL of 20mM Tris-HCl buffer containing NR 5KU/L, NP 7KU/L, NOD 6KU/L, feCl 2 1mM, mannitol 0.1wt%, sodium azide 0.1wt%;
calibration material: an aqueous sodium nitrate solution was prepared at a concentration point of 0,12.5,25,50,100,200. Mu. Mol/L.
Example 2.6
The R1, R2 reagents and calibrator were prepared according to the formulation in example 1 as follows:
reagent R1: 1000mL of 50mM phosphate buffer containing 0.04wt% NADH, 0.05wt% Tween-20, and 0.3wt% Thiomerosal;
reagent R2: 1000mL of 20mM phosphate buffer containing NR 5KU/L, NP 7KU/L, NOD 6KU/L, feCl 25 mM, mannitol 3wt%, PC 300.5 wt%;
calibration material: an aqueous sodium nitrate solution was prepared at a concentration point of 0,12.5,25,50,100,200. Mu. Mol/L.
The absorbance values of the reagents obtained in the above examples were measured, the test method is shown in example 1, and the test results are shown in Table 5.
TABLE 5
As can be seen from Table 5, example 2.5 has the highest response value and is the optimal formulation.
EXAMPLE 3 correlation of the reagent of the present invention with Griess-method nitric oxide detection reagent
The reagent of the invention and the Griess method nitric oxide detection reagent are used for simultaneously measuring 40 parts of human serum, the result of the Griess method nitric oxide detection reagent is taken as an abscissa, the result of the enzyme cycle method of the invention is taken as an ordinate to carry out regression analysis, and the correlation between the reagent of the invention and the Griess method nitric oxide detection reagent is detected.
The specific detection method comprises the following steps: the following Griess method was used to measure the nitric oxide content of 40 serum fractions using a Hitachi 7080 Biochemical apparatus:
griess method: mixing, standing at room temperature for 10 min, zeroing with distilled water, measuring optical path at length of 540 nm and 1.0cm, and measuring absorbance value of each tube with biochemical analyzer.
Enzyme cycling method:
the preparation method of the reagent R1 comprises the following steps: 1.292g of Na is weighed respectively 2 HPO 4 ·12H 2 O and 3.534g NaH 2 PO 4 ·2H 2 O is dissolved in 500mL of purified water, 0.125g of NADH, 200.5mL of Tween-200.5mL of PC and 0.5mL of PC are sequentially weighed and added into the phosphate buffer solution, and after the O is fully dissolved, the solution is filtered by a 0.22 mu m membrane and is placed at 2-8 ℃ for standby;
The preparation method of the reagent R2 comprises the following steps: 0.605g Tris was weighed into 250mL purified water, pH was adjusted to 7.4 with concentrated HCl, mannitol 0.25g FeCl was weighed 2 31.69mg、NaN 3 Adding 0.25g of the buffer solution, fully dissolving, sequentially weighing 1.25KU of nitrate reductase, 1.75KU of nitrite dismutase and 1.5KU of nitric oxide dioxygenase, dissolving into the solution one by one, slowly stirring until the solution is completely dissolved, filtering by using a 0.45 mu m membrane, and standing at 2-8 ℃ for later use;
using a Hitachi 7080 biochemical analyzer, wherein the ratio of R1 to R2 to the sample is 200:60:10, the temperature is 37 ℃, the measurement wavelength is 340nm, the reagent R1 is incubated at the measurement temperature for 300 seconds after adding the calibrator, then R2 is added, the reaction is delayed for 60-120 seconds, the interference of consumable reduced coenzyme substances in the sample and the side reaction of other nitrite dismutase and nitric oxide dioxygenase are eliminated, the measurement time is 180 seconds, at least 2 effective points are selected in the measurement time during the reading, and the obtained data are shown in the following table 6.
TABLE 6
The Griess reagent and the reagent of the invention are respectively subjected to standard curve measurement according to Griess instruction and the test method of the method, 40 parts of serum are measured simultaneously, and the correlation of the two reagents is compared according to the measurement result. The results are shown in Table 7 and FIG. 1.
TABLE 7
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As shown in table 7 and fig. 1, regression analysis was performed with the result of the Griess method nitric oxide detection reagent measurement as the abscissa and the result of the enzymatic cycling method of the present invention as the ordinate, and the correlation equation was y=0.9512 x+5.5082, and the correlation coefficient was 0.9784. The statistical treatment result shows that the nitric oxide determination reagent has good correlation with the clinical sample measurement value of the Griess method kit.
Example 4 interference resistance
The anti-interference performance of the reagent refers to the ability of the detection result of the detected object not to be interfered by other components, namely the ability of the reagent to control the deviation between the detection result and the actual numerical value within an acceptable range even in the environment where the interfering substance exists. It is generally considered that + -10% is an acceptable range.
Very high value special samples are generally not readily available, and therefore specific concentrations of interfering substances are typically added to normal serum during development to simulate a special sample.
Control samples: serum 450. Mu.L, double distilled water 50. Mu.L, total 500. Mu.L.
The interference samples are prepared by respectively adding bilirubin, hemoglobin, triglyceride and anti-cycloblood acid with different concentrations into serum samples, and the specific preparation methods of the interference samples are shown in the following table 8.
TABLE 8
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The test is carried out by using the circulating enzyme method reagent:
the preparation method of the reagent R1 comprises the following steps: 1.292g of Na is weighed respectively 2 HPO 4 ·12H 2 O and 3.534g NaH 2 PO 4 ·2H 2 O is dissolved in 500mL of purified water, 0.125g of NADH, 200.5mL of Tween-200.5mL of PC and 0.5mL of PC are sequentially weighed and added into the phosphate buffer solution, and after the O is fully dissolved, the solution is filtered by a 0.22 mu m membrane and is placed at 2-8 ℃ for standby;
the preparation method of the reagent R2 comprises the following steps: 0.605g Tris was weighed into 250mL purified water, pH was adjusted to 7.4 with concentrated HCl, mannitol 0.25g FeCl was weighed 2 31.69mg、NaN 3 Adding 0.25g of the buffer solution, fully dissolving, sequentially weighing 1.25KU of nitrate reductase, 1.75KU of nitrite dismutase and 1.5KU of nitric oxide dioxygenase, and dissolving one by oneSlowly stirring the solution until the solution is completely dissolved, filtering the solution by using a 0.45 mu m membrane, and standing the solution at the temperature of 2-8 ℃ for later use;
the samples were tested using a Hitachi 7080 Biochemical apparatus at a ratio of reagent R1 to reagent R2 to sample of 200:60:10, and the data are shown in Table 9.
TABLE 9
| Interfering substances | Measurement value 1 | Measurement value 2 | Mean value of | Deviation of |
| Control | 35.42 | 35.75 | 35.59 | / |
| Bilirubin (mu mol/L) | ||||
| 200 | 35.76 | 34.98 | 35.37 | -0.60％ |
| 400 | 34.19 | 35.84 | 35.02 | -1.60％ |
| 600 | 34.22 | 34.27 | 34.24 | -3.77％ |
| 800 | 33.63 | 33.26 | 33.45 | -6.01％ |
| 1000 | 31.23 | 31.78 | 31.50 | -11.47％ |
| Hemoglobin (mg/dL) | ||||
| 50 | 36.07 | 36.70 | 36.38 | 2.23％ |
| 100 | 35.66 | 35.50 | 35.58 | -0.04％ |
| 150 | 33.51 | 32.19 | 32.85 | -7.71％ |
| 200 | 31.20 | 32.22 | 31.71 | -10.90％ |
| Triglyceride (mmol/L) | ||||
| 3 | 35.52 | 35.01 | 35.27 | -0.90％ |
| 6 | 37.40 | 34.59 | 35.99 | 1.13％ |
| 9 | 36.41 | 37.72 | 37.07 | 4.15％ |
| 12 | 38.71 | 38.48 | 38.59 | 8.44％ |
| 15 | 39.71 | 39.36 | 39.54 | 11.09％ |
| Ascorbic acid (mg/dL) | ||||
| 10 | 35.40 | 36.88 | 36.14 | 1.54％ |
| 30 | 34.09 | 34.39 | 34.24 | -3.79％ |
| 50 | 32.91 | 32.61 | 32.76 | -7.96％ |
| 70 | 30.91 | 29.22 | 30.06 | -15.53％ |
As can be seen from Table 9, bilirubin, hemoglobin, triglyceride and anti-cyclosanguinic acid are added into a sample to be tested, and the nitrate and nitrite contents in the sample to be tested are detected by using the detection reagent provided by the invention to obtain the nitric oxide content, and the result is not significantly affected, which indicates that the detection reagent provided by the invention is not affected by the substances when the sample contains not more than 800umol/L of bilirubin, 150mg/dL of hemoglobin, 12mmol/L of triglyceride and 50mg/dL of anti-cyclosanguinic acid.
Example 5 real-time stability
Three batches of reagents (batches 20211022, 20211103, 20211118, respectively) were prepared as follows:
the preparation method of the reagent R1 comprises the following steps: 1.292g of Na is weighed respectively 2 HPO 4 ·12H 2 O and 3.534g NaH 2 PO 4 ·2H 2 O was dissolved in 500mL of purified water, and 0.125g of NADH was weighed in order,Tween-200.5mL and PC 300.5 mL are added into the phosphate buffer solution, and after being fully dissolved, the solution is filtered by a 0.22 mu m film and is placed at 2-8 ℃ for standby;
the preparation method of the reagent R2 comprises the following steps: 0.605g Tris was weighed into 250mL purified water, pH was adjusted to 7.4 with concentrated HCl, mannitol 0.25g FeCl was weighed 2 31.69mg、NaN 3 Adding 0.25g of the buffer solution, fully dissolving, sequentially weighing 1.25KU of nitrate reductase, 1.75KU of nitrite dismutase and 1.5KU of nitric oxide dioxygenase, dissolving into the solution one by one, slowly stirring until the solution is completely dissolved, filtering by using a 0.45 mu m membrane, and standing at 2-8 ℃ for later use;
calibration material: the concentration point is 0,12.5,25,50,100,200 mu mol/L sodium nitrate aqueous solution.
Three batches of reagents (20211022, 20211103 and 20211118 respectively) were prepared according to the above method, and were placed at 2-8 ℃ and were taken out for testing at 0 day, 1 month, 3 months, 5 months, 7 months, 9 months, 12 months and 15 months, respectively.
The volume ratio of R1 to R2 to calibrator is 250 to 60 to 10, the concentration of calibrator is as shown in tables 8-10, the temperature is 37 ℃, the measurement wavelength is 340nm, R1 is added with calibrator and then incubated for 300 seconds at the measurement temperature, then R2 is added, the measurement time is 180 seconds, and at least 2 effective points are selected in the measurement time in the reading. The data are shown in tables 10-12.
Table 10 20211022 batch real-time stability
Table 11 20211103 batch real-time stability
Table 12 20211118 batch real-time stability
As can be seen from the data in tables 10-12, the nitric oxide cyclase method reagent can be stably stored for 12 months under the light-shielding condition of 2-8 ℃. The real-time stability of the nitric oxide detection reagent by the circulating enzyme method is good.
The method for measuring the above-mentioned reagent is the same as the method conventionally used in the art, such as: the content of the object to be measured can be calculated by referring to a calibrator, drawing a standard concentration curve, or the like by using a rate method or an end point method.
The preparation method of the above-described example reagents is merely for illustrating the principle of the present invention and its application, and the present invention is in no way limited to the above-described exemplary application range; in addition, those skilled in the art to which the present invention pertains may formulate various assay reagents similar to those described herein in accordance with the principles and methods of the present invention without departing from the spirit and scope of the invention.
Claims (25)
1. A nitric oxide detection reagent, characterized in that the detection reagent comprises reduced coenzyme and/or derivatives thereof, nitrate reductase, nitrite dismutase and nitric oxide dioxygenase.
2. The nitric oxide detection reagent according to claim 1, comprising:
Nitrate reductase, in the presence of reduced coenzyme and/or its derivative, nitrate ion in the sample undergoes reduction reaction to generate nitrite ion;
nitrite dismutase, which can react nitrite generated by the reaction and in a sample to generate nitric oxide and nitrate ions under the condition of H < + >;
nitric oxide dioxygenase, in the presence of oxygen and reduced coenzyme and/or derivatives thereof, reacts with the nitric oxide to form nitrate ions.
3. The detection reagent according to claim 1 or 2, wherein the reduced coenzyme is selected from one or both of reduced coenzyme I (NADH) or reduced coenzyme II (NADPH);
preferably, the reduced coenzyme derivative is selected from one or both of thio-reduced coenzyme I (thio-NADH) or thio-reduced coenzyme II (thio-NADPH).
4. The detection reagent according to claim 2, wherein the nitric oxide dioxygenase is capable of reacting the nitric oxide in the presence of reduced coenzyme and/or a derivative thereof and oxygen to form nitrate ions.
5. The reagent according to claim 2, wherein the nitrate reductase causes nitrate ions in the sample to be measured to react in the presence of the reduced coenzyme and/or the derivative thereof as shown in the following formula (1):
6. The reagent according to any one of claims 2 to 5, wherein the nitrite dismutase reacts with the nitrite ion as shown in the following formula (2):
7. the reagent according to any one of claims 2 to 6, wherein the nitric oxide dioxygenase, nitric oxide and reduced coenzyme and/or derivative thereof react with each other as shown in the following formula (3):
8. the detection reagent of any one of claims 1 to 7, wherein the detection reagent further comprises a buffer;
preferably, the buffer is a phosphate buffer.
9. The detection reagent according to any one of claims 1 to 8, wherein the detection reagent comprises a reagent R1 and a reagent R2,
wherein the reagent R1 comprises: a. reduced coenzyme solution or b.buffer containing reduced coenzyme,
reagent R2 comprises nitrate reductase, nitrite dismutase and nitric oxide dioxygenase.
10. The detection reagent according to claim 9, wherein the mass percentage of reduced coenzyme in the reagent R1 is 0.01 to 0.05%, preferably 0.02 to 0.03%, more preferably 0.025%.
11. The detection reagent according to claim 9 or 10, wherein the buffer in the reagent R1 is one or a combination of two or more of Tris-HCl buffer, HEPES buffer, phosphate buffer, preferably phosphate buffer.
12. The detection reagent according to any one of claims 9 to 11, wherein the reagent R1 further comprises a surfactant and a first preservative;
preferably, the surfactant is selected from one or a combination of more than two of tween-20,Brij 35,Triton X-100, preferably, the mass percentage of the surfactant in the reagent R1 is 0.01-1%, preferably 0.01-0.5%, more preferably 0.1%;
preferably, the first preservative is selected from one or a combination of more than two of PC300, sodium azide, gentamicin and thimerosal, preferably, the mass percentage of the first preservative in the reagent R1 is 0.01-0.5%, preferably 0.01-0.2%, and more preferably 0.1%.
13. The detection reagent according to any one of claims 9 to 12, wherein the content of nitrate reductase in the reagent R2 is 1 to 9KU/L, preferably 3 to 5KU/L;
preferably, the content of nitrite dismutase in the reagent R2 is 3-11KU/L, preferably 5-7KU/L;
preferably, the nitric oxide dioxygenase is present in the reagent R2 in an amount of 2-10KU/L, preferably 4-6KU/L.
14. The detection reagent according to any one of claims 9 to 13, wherein the reagent R2 further comprises one or more of a second buffer, mannitol, a metal ion, and a second preservative;
Preferably, the second buffer is selected from one of phosphate buffer, tris-HCl buffer and HEPES buffer;
preferably, the mannitol in the reagent R2 is 0.01-5%, preferably 0.01-1%, more preferably 0.1% by mass;
preferably, the metal ion is selected from Fe 2+ 、Mg 2+ 、Ca 2+ One or a combination of two or more of them; preferably, the molar concentration of metal ions in the reagent R2 is between 0.1 and 10mM, preferably between 1 and 5mM;
preferably, the second preservative is selected from one or more than two of PC300, sodium azide, gentamicin and thimerosal, preferably, the mass percentage of the second preservative in the reagent R2 is 0.01-0.5%, preferably 0.01-0.2%, and more preferably 0.1%.
15. The reagent according to any one of claims 1 to 14, wherein the reagent further comprises a calibrator, the calibrator being an aqueous solution of sodium nitrate, and the concentration point being 0,12.5,25,50,100,200. Mu. Mol/L.
16. A nitric oxide detection kit, comprising:
the unit containing nitrate reductase and reduced coenzyme and/or derivatives thereof can lead nitrate ions in the sample to be detected to generate nitrite ions through reduction reaction;
A unit containing nitrite dismutase, capable of reacting said nitrite ion to produce nitric oxide and nitrate ions;
a unit comprising nitric oxide dioxygenase and reduced coenzyme and/or a derivative thereof, capable of reacting said nitric oxide in the presence of reduced coenzyme and/or a derivative thereof to form nitrate ions.
17. A method for detecting the content of nitric oxide, which is characterized by comprising the following enzyme cycling reaction:
18. the method according to claim 17, wherein the sample is detected using a detection reagent according to any one of claims 1 to 11.
19. The method according to claim 17 or 18, wherein the reduced coenzyme consumption rate is detected to obtain the nitric oxide content in the sample;
preferably, the reduced coenzyme consumption rate is detected spectrophotometrically.
20. The method of detection according to claim 18 or 19, characterized in that the method of detection comprises the steps of: mixing the sample with reduced coenzyme solution, adding nitrate reductase, nitrite dismutase and nitric oxide dioxygenase, and detecting the consumption rate of the reduced coenzyme to obtain the content of nitric oxide in the sample;
Preferably, the sample is mixed with a buffer solution containing reduced coenzyme, nitrate reductase, nitrite dismutase and nitric oxide dioxygenase are added, and the consumption rate of the reduced coenzyme is detected, so that the content of nitric oxide in the sample is obtained.
21. The method according to any of the claims 17-20, wherein the nitric oxide content of the sample is calculated using a rate method or an endpoint method, preferably a rate method.
22. The method of any one of claims 12-16, wherein the sample is a cell lysate, a tissue or cell culture fluid, serum, plasma, urine, or an aqueous solution.
23. The method according to any one of claims 17 to 21, wherein the method comprises mixing the reagent R1 with the sample to be tested and then adding the reagent R2 for detection;
preferably, the volume ratio of the reagent R1, the reagent R2 and the sample to be tested is 200-300:30-100:1-20, preferably 200:60:10.
24. The method of any one of claims 17 to 22, further comprising mixing reagent R1 with a calibrator, and then adding reagent R2 for detection, thereby preparing a standard curve.
25. Use of the detection reagent of any one of claims 1-15 or the detection method of claims 17-24 for detecting nitric oxide content.
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|---|---|---|---|---|
| US5885842A (en) * | 1996-11-08 | 1999-03-23 | Medinox, Inc. | Methods for the detection of nitric oxide in fluid media |
| CN102539730A (en) * | 2010-12-13 | 2012-07-04 | 苏州艾杰生物科技有限公司 | Ammonia (ammonia ion) measurement method and ammonia (ammonia ion) diagnosis/measurement kit |
| CN206114533U (en) * | 2016-11-01 | 2017-04-19 | 上海碧云天生物技术有限公司 | Nitric oxide detect reagent box |
| CN109557238A (en) * | 2018-12-24 | 2019-04-02 | 湖南新大陆生物技术有限公司 | A kind of nitric oxide detection method and its application |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5885842A (en) * | 1996-11-08 | 1999-03-23 | Medinox, Inc. | Methods for the detection of nitric oxide in fluid media |
| CN102539730A (en) * | 2010-12-13 | 2012-07-04 | 苏州艾杰生物科技有限公司 | Ammonia (ammonia ion) measurement method and ammonia (ammonia ion) diagnosis/measurement kit |
| CN206114533U (en) * | 2016-11-01 | 2017-04-19 | 上海碧云天生物技术有限公司 | Nitric oxide detect reagent box |
| CN109557238A (en) * | 2018-12-24 | 2019-04-02 | 湖南新大陆生物技术有限公司 | A kind of nitric oxide detection method and its application |
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