Nitric oxide detection kit with good stability and preparation method thereof

Abstract

The invention relates to the technical field of nitric oxide detection kits, and discloses a nitric oxide detection kit with good stability and a preparation method thereof, the nitric oxide detection kit comprises a reagent R1 and a reagent R2, the reagent R1 comprises potassium dihydrogen phosphate; disodium hydrogen phosphate; eDTA (ethylene diamine tetraacetic acid); 2-aminohydroquinone hydrochloride and a preservative; the reagent R2 comprises a buffer solution, MBTH and a preservative; according to the nitric oxide detection kit, 2-aminohydroquinone hydrochloride is added into a reagent R1 to replace dopamine hydrochloride, a monopotassium phosphate-disodium hydrogen phosphate buffer solution with high ion concentration is adopted, a citric acid-sodium citrate buffer solution is adopted as a reagent R2, a polycyclic phenol stabilizer is added at the same time, and therefore the nitric oxide detection kit is obtained. The stability of the reagent and the bottle opening stability are improved, and meanwhile, the thermal stability of the reagent is improved.

Description

Technical Field

[0001] The invention relates to the technical field of nitric oxide detection kits, in particular to a nitric oxide detection kit with good stability and a preparation method thereof. Background Art

[0002] Nitric oxide (NO) is a small molecule free radical with high lipophilicity and membrane penetration ability. It is an important effector and messenger molecule in the body and has an important regulatory effect on the contraction and relaxation of microcirculation. Nitric oxide is produced by L-arginine (L-arginine) under the action of nitric oxide synthase (NOS). It can be used as a good endothelial cell relaxing factor (EDRF) and a new type of biotransmitter. In addition to its activities of dilating blood vessels, lowering blood pressure, and relaxing smooth muscles, it also has biological activities such as signal transmission, enzyme activity regulation, immune regulation, and anti-(causing) oxidative damage. At the same time, nitric oxide is also a toxic molecule. Excessive nitric oxide can cause damage to the body when inflammation and immune response occur in the body. Therefore, nitric oxide detection is of great significance for clinical auxiliary diagnosis of diseases, follow-up analysis and judgment of the disease, dynamic tracking of clinical treatment efficacy, and prognosis evaluation.

[0003] Detecting nitric oxide can help researchers understand the pathogenesis of diseases and can also effectively help doctors provide appropriate clinical treatment methods. The basic principle of the current nitric oxide test kit is: add dopamine hydrochloride to the R1 reagent, add phenol reagent (3-methyl-2-benzothiazolinone hydrazone hydrochloride, MBTH) to the R2 reagent, and the nitric oxide in the serum sample is converted to nitrite (NO2 - ) and nitrate (NO3 - ) exists in the form of. Under strong acidic conditions, the catechol structure on dopamine hydrochloride can convert nitrate (NO3 - ) is reduced to nitrite (NO2 - ), and nitrite can react with aromatic primary amines in a strong acid medium to form diazonium salts, which react with phenol reagents to form purple-red complexes with a maximum absorption peak at a wavelength of 545nm. The rate of change of its absorbance is related to the nitrite (NO2 - ) and nitrate (NO3 - ) concentration, read the absorbance value there, and calculate the concentration of nitrite based on the absorbance value. Since the concentration of nitric oxide has a certain linear relationship with the concentration of nitrite, the concentration of nitric oxide can be further calculated.

[0004] However, dopamine hydrochloride has poor stability and is prone to self-oxidation reactions that produce color changes, thereby changing the color changes of the normal reaction. Therefore, the stability of the nitric oxide test kit has always been a difficult problem to solve. Summary of the invention

[0005] The invention provides a nitric oxide detection kit with good stability. 2-aminohydroquinone hydrochloride is added to an R1 reagent instead of dopamine hydrochloride, a potassium dihydrogen phosphate-disodium hydrogen phosphate buffer with a high ion concentration is adopted, a citric acid-sodium citrate buffer is adopted for an R2 reagent, and a polycyclic phenol stabilizer is added at the same time, so that the thermal stability of the reagent is improved while the stability of the reagent itself and the stability of opening a bottle are improved.

[0006] To achieve the above object, the present invention provides the following technical solutions:

[0007] In a first aspect, the present invention provides a nitric oxide detection kit with good stability, wherein the nitric oxide detection kit comprises a reagent R1 and a reagent R2;

[0008] Wherein, the reagent R1 comprises: potassium dihydrogen phosphate, disodium hydrogen phosphate, EDTA, 2-aminohydroquinone hydrochloride and a preservative; the reagent R2 comprises: a buffer solution, MBTH and a preservative.

[0009] In the present invention, the reaction principle of the nitric oxide detection kit is as follows: under strong acidic conditions, the hydroquinone structure on the 2-aminohydroquinone hydrochloride of the R1 reagent converts the nitrate (NO3 - ) is reduced to nitrite (NO2 - ), while nitrite (NO2 - ) reacts with an aromatic primary amine on 2-aminohydroquinone hydrochloride in a strong acid medium to form a diazonium salt, which reacts with a phenol reagent (MBTH) to form a purple-red complex with a maximum absorption peak at a wavelength of 545nm. The absorbance value at this location is read and nitrite (NO2 - ) concentration, and then calculate the concentration of nitric oxide (NO). In the kit, 2-aminohydroquinone hydrochloride is used instead of dopamine hydrochloride to overcome the instability of dopamine hydrochloride. The structure of hydroquinone is much better than that of catechol in terms of stability, and 2-aminohydroquinone hydrochloride contains one amino group, which can reduce nitrate to nitrite and provide the function of aromatic primary amine in a strong acid medium to generate diazonium salt.

[0010] At the same time, in order to further improve the stability of reagent R1 after opening the bottle, phosphate buffer is selected as the buffer system in reagent R1, and is used in combination with 2-aminohydroquinone hydrochloride to achieve a more ideal effect of stability after opening the bottle.

[0011] As a preferred technical solution of the present invention, the reagent R2 also includes a polycyclic phenol stabilizer.

[0012] Preferably, the polycyclic phenol stabilizer is selected from any one or a combination of at least two of plant ferritin, bovine serum albumin, trehalose or mannitol, preferably plant ferritin.

[0013] Preferably, the preservatives in the reagents R1 and R2 are independently selected from any one of sodium azide, gentamicin, thimerosal or isothiazolinone, or a combination of at least two thereof, preferably Proclin 300.

[0014] As a preferred technical solution of the present invention, the buffer of the reagent R2 is selected from any one or a combination of at least two of citric acid-sodium citrate buffer, acetic acid-sodium acetate buffer, phosphate buffer, borate buffer or MES hydrochloric acid buffer, preferably citric acid-sodium citrate buffer.

[0015] As a preferred technical solution of the present invention, the concentration of potassium dihydrogen phosphate in the reagent R1 is 40-300 mM, for example, it can be 44 mM, 88 mM, 176 mM, 204 mM, 264 mM, 296 mM, preferably 150-300 mM.

[0016] Preferably, the concentration of disodium hydrogen phosphate in the reagent R1 is 1-100 mM, for example, 4 mM, 6 mM, 12 mM, 24 mM, 36 mM, 48 mM, 60 mM, 72 mM, 84 mM, 96 mM, preferably 20-100 mM.

[0017] In the present invention, as a preferred solution, the ion concentration of the buffer solution in the reagent R1 can be selected to be as high as possible, and the total molar concentration of potassium dihydrogen phosphate and sodium dihydrogen phosphate is not less than 100mM, preferably 150mM-400mM.

[0018] Preferably, the concentration of EDTA in the reagent R1 is 0.5-1.5 g / L, for example, it can be 0.6 g / L, 0.7 g / L, 0.8 g / L, 0.9 g / L, 1.0 g / L, 1.1 g / L, 1.2 g / L, 1.3 g / L, 1.4 g / L or 1.5 g / L, etc.

[0019] Preferably, the concentration of 2-aminohydroquinone hydrochloride in the reagent R1 is 50-100 mM, for example, 52 mM, 55 mM, 58 mM, 60 mM, 65 mM, 70 mM, 75 mM, 80 mM, 85 mM, 90 mM, 95 mM, 96 mM or 100 mM.

[0020] Preferably, the concentration of the preservative in the reagent R1 is 0.5-1.5 g / L, for example, it can be 0.6 g / L, 0.7 g / L, 0.8 g / L, 0.9 g / L, 1.0 g / L, 1.1 g / L, 1.2 g / L, 1.3 g / L, 1.4 g / L or 1.5 g / L, etc.

[0021] Preferably, the pH of the reagent R1 is 5.5-6.5, for example, it can be 5.5, 5.6, 5.8, 6.0, 6.1, 6.2, 6.3, 6.4 or 6.5.

[0022] As a preferred technical solution of the present invention, the concentration of citric acid in the reagent R2 is 50-160 mM, for example, it can be 77 mM, 98 mM, 116 mM, 134 mM, 155 mM or 160 mM.

[0023] Preferably, the concentration of sodium citrate in the reagent R2 is 15-60 mM, for example, 16 mM, 23 mM, 34 mM, 45 mM or 52 mM.

[0024] Preferably, the concentration of MBTH in the reagent R2 is 10-30 mM, for example, 10 mM, 12 mM, 15 mM, 18 mM, 20 mM, 22 mM, 25 mM, 28 mM or 30 mM.

[0025] Preferably, the concentration of the polycyclic phenol stabilizer in the reagent R2 is 0.5-1.5 g / L, for example, it can be 0.6 g / L, 0.7 g / L, 0.8 g / L, 0.9 g / L, 1.0 g / L, 1.1 g / L, 1.2 g / L, 1.3 g / L, 1.4 g / L or 1.5 g / L, etc.

[0026] Preferably, the concentration of the preservative in the reagent R2 is 0.5-1.5 g / L, for example, it can be 0.6 g / L, 0.7 g / L, 0.8 g / L, 0.9 g / L, 1.0 g / L, 1.1 g / L, 1.2 g / L, 1.3 g / L, 1.4 g / L or 1.5 g / L, etc.

[0027] Preferably, the pH of the reagent R2 is 3.0-4.0, for example, it can be 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9 or 4.0.

[0028] As a preferred technical solution of the present invention, the nitric oxide detection kit comprises reagent R1 and reagent R2;

[0029] Wherein, the reagent R1 includes: potassium dihydrogen phosphate, 150-300mM; disodium hydrogen phosphate, 20-100mM; EDTA, 0.5-1.5g / L; 2-aminohydroquinone hydrochloride, 50-100mM; preservative, 0.5-1.5g / L, pH is 5.5-6.5;

[0030] Reagent R2 includes: citric acid, 50-160 mM; sodium citrate, 15-60 mM; MBTH, 10-30 mM; plant ferritin, 0.5-1.5 g / L; preservative, 0.5-1.5 g / L, and pH is 3.0-4.0.

[0031] In the present invention, the kit may further include a calibration substance (STD) and a quality control substance (QC). The calibration substance and the quality control substance include NaCl, a preservative and sodium nitrate, respectively.

[0032] For example, the components and corresponding contents of the calibration substance (STD) may be: NaCl, 9 g / L; Proclin 300, 1 g / L; sodium nitrate, 17 mg / L.

[0033] The ingredients and corresponding contents of the quality control product (QC) may be: NaCl, 9 g / L; Proclin 300, 1 g / L; sodium nitrate, 4.25 mg / L.

[0034] In a second aspect, the present invention further provides a method for preparing a nitric oxide detection kit, the preparation method comprising the following steps:

[0035] Prepare reagent R1: weigh the volume of purified water for the pre-prepared solution, then add accurately weighed potassium dihydrogen phosphate, disodium hydrogen phosphate, EDTA, 2-aminohydroquinone hydrochloride and preservative, stir, adjust the pH value of the solution after fully dissolving, and obtain the reagent R1;

[0036] Prepare reagent R2: weigh the volume of purified water for the pre-prepared solution, then add accurately weighed buffer components, MBTH, preservatives and optional polycyclic phenol stabilizers, stir, adjust the pH value of the solution after sufficient dissolution, and mix well to obtain the reagent R2.

[0037] As a preferred technical solution of the present invention, the storage temperature of the reagents R1 and R2 is 2-8°C, for example, it can be 2°C, 3°C, 4°C, 5°C, 6°C, 7°C or 8°C.

[0038] In the present invention, the kit may further include a calibrator and a quality control product. The method for preparing the calibrator and the quality control product is as follows: weigh each component according to the composition of the calibrator / quality control product, weigh the purified water of the pre-prepared solution volume, then add NaCl, sodium nitrate and a preservative, stir, mix well and store at 2 to 8°C.

[0039] In a third aspect, the present invention provides a method for using the nitric oxide detection kit according to the first aspect in non-disease diagnosis, wherein the nitric oxide detection kit is used in conjunction with a biochemical analyzer, and the specific steps include:

[0040] After the sample to be tested is mixed with reagent R1 and reagent R2 for reaction, the absorbance of the solution is detected, and the concentration of nitric oxide in the sample to be tested is obtained according to the change in the absorbance.

[0041] In a fourth aspect, the present invention further provides a use of a nitric oxide detection kit with good stability as described in the first aspect in the detection of nitric oxide content.

[0042] Compared with the prior art, the present invention has the following beneficial technical effects:

[0043] (1) The nitric oxide detection kit provided by the present invention has a reagent R1 with a phosphate buffer as a buffer system, comprising 2-aminohydroquinone hydrochloride, EDTA and a preservative, and the obtained reagent has a high opening stability. At the same time, experimental studies have found that a high concentration of phosphate buffer is better matched with 2-aminohydroquinone hydrochloride. The use of a high ion concentration phosphate buffer in reagent R1 can ensure that the sample to be tested is mixed with reagents R1 and R2, and still has a strong buffering capacity, reducing the effect of CO2 in the air on the pH of the reagent when opening the bottle. The deviation of the obtained kit is still less than 10% 15 days after opening the bottle, thereby improving the opening stability of the reagent.

[0044] (2) The nitric oxide detection kit provided by the present invention also improves the components of reagent R2. Not only is a polycyclic phenol stabilizer added to reagent R2, preferably plant ferritin, but a citric acid-sodium citrate buffer is also used. That is, in order to match the changes in the components of reagent R1, the components and parameters in reagent R2 need to be adjusted and selected. After selecting the polycyclic phenol stabilizer and the citric acid-sodium citrate buffer, the stability of MBTH in reagent R2 is significantly improved; therefore, the use of 2-aminohydroquinone hydrochloride in the reagent R1 of the kit, combined with an appropriate buffer system, and the selection of a suitable polycyclic phenol stabilizer can significantly improve the opening stability and thermal stability of the nitric oxide detection kit. DETAILED DESCRIPTION

[0045] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments. In the absence of conflict, the embodiments in this application and the features in the embodiments can be combined with each other. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work belong to the scope of protection of the present invention.

[0046] The reagents used in the following examples can all be purchased from conventional manufacturers in the field; the experimental methods used are all conventional experimental methods known to those skilled in the art.

[0047] The present invention provides a nitric oxide detection kit with good stability, comprising:

[0048] The components and corresponding contents of reagent R1 are: potassium dihydrogen phosphate, 264mM; disodium hydrogen phosphate, 36mM; EDTA, 1g / L; 2-aminohydroquinone hydrochloride, 80mM; Proclin300, 1g / L; pH is 6.0;

[0049] The components and corresponding contents of reagent R2 are: citric acid, 116 mM; sodium citrate, 34 mM; MBTH, 20 mM; plant ferritin, 1 g / L; Proclin 300, 1 g / L; pH is 3.5;

[0050] The components and corresponding contents of the calibration substance (STD) are: NaCl, 9 g / L; Proclin300, 1 g / L; Sodium nitrate, 17 mg / L;

[0051] The ingredients and corresponding contents of the quality control (QC) are: NaCl, 9g / L; Proclin300, 1g / L; Sodium nitrate, 4.25mg / L.

[0052] The present invention also provides a detection method of a nitric oxide detection kit, which specifically comprises:

[0053] Test instrument: HITACHI 7180;

[0054] The test parameters are as follows:

[0055] Method: rate method; temperature: 37℃; detection wavelength: 545nm; sample volume: 10μL; reagent R1: 180μL; reagent R2: 45μL; reaction direction: forward; incubation time: 5min; reading point: 22-30; calibration method: two-point calibration.

[0056] Test plan 1: Testing the stability of the reagent after opening the bottle

[0057] In order to improve the stability of the reagent after opening the bottle, the present invention implements the following experimental scheme, specifically:

[0058] Open bottle stability test method and evaluation method: Place the reagent in an open bottle at 2-8°C for 15 days, regularly test the quality control product provided, repeat 3 times, calculate the deviation between the mean value and the target value of each measurement, and a deviation of less than 10% meets the technical requirements.

[0059] Experiment 1:

[0060] This experiment is used to verify the stability of the reagent R1 after adding 2-aminohydroquinone hydrochloride.

[0061] 2-aminohydroquinone hydrochloride was added to the R1 reagent of Example 1, and dopamine hydrochloride was added to the R1 reagent of Comparative Example 1. The specific components and corresponding contents are shown in Table 1-1;

[0062] Table 1-1

[0063]

[0064] Similarly, 2-aminohydroquinone hydrochloride was added to the R1 reagent of Example 2, and dopamine hydrochloride was added to the R1 reagent of Comparative Example 2. The specific components and corresponding contents are shown in Table 1-2;

[0065] Table 1-2

[0066]

[0067]

[0068] The results of the evaluation of the bottle opening stability of Example 1, Comparative Example 1, Example 2 and Comparative Example 2 are shown in Tables 1-3 below;

[0069] Table 1-3

[0070]

[0071]

[0072] Combining Example 1 and Comparative Example 1, and Example 2 and Comparative Example 2, it can be seen that the reagent R1 using 2-aminohydroquinone hydrochloride instead of dopamine hydrochloride has better opening stability.

[0073] In the actual preparation process of the kit, if the type of buffer, pH value and other components are not adjusted, and only dopamine hydrochloride is replaced with 2-aminohydroquinone hydrochloride, its opening stability still cannot achieve the effect described in Experiment 1. Therefore, during the preparation process, the inventors also made specific selections and adjustments to other components and parameters in the reagents, and finally obtained the optimal effect under the selection of various parameters and reagent types.

[0074] Experiment 2:

[0075] This experiment is used to demonstrate the selection of the buffer system in reagent R1 and the effect of the specific pH value on the stability of the reagent.

[0076] 2-aminohydroquinone hydrochloride was added to the R1 reagent in Example 3, different concentrations of the R1 reagents in Examples 4-6 were selected, different buffer systems were selected in Example 7, the buffer in the R1 reagent in Comparative Example 3 was adjusted to MES buffer, and dopamine hydrochloride was added to the R1 reagent in Comparative Example 4;

[0077] The specific ingredients and corresponding contents are shown in Table 2-1:

[0078] Table 2-1

[0079]

[0080]

[0081]

[0082] The evaluation results of the bottle opening stability of Examples 3-6 and Comparative Examples 3-4 are shown in Table 2-2 below;

[0083] Table 2-2

[0084]

[0085]

[0086] By comparing Example 3 and Comparative Example 4, it can be seen that the reagent R1 using 2-aminohydroquinone hydrochloride instead of dopamine hydrochloride has better opening stability;

[0087] By comparing Example 3 and Examples 4-6, it can be seen that the reagent R1 has the best opening stability when the total buffer concentration is 300 mM, and Example 4 is also within the required range but the effect is inferior to that of Example 3; at the same time, in combination with Example 3 and Comparative Example 3, it can be seen that when the buffer system is selected, the buffer system formed by potassium dihydrogen phosphate and disodium hydrogen phosphate can achieve better opening stability;

[0088] That is to say, 2-aminohydroquinone hydrochloride has the best stability in the buffer system formed by potassium dihydrogen phosphate and disodium hydrogen phosphate.

[0089] Test plan 2: Improve thermal acceleration stability

[0090] In order to improve the thermal acceleration stability of the reagent, the present invention implements the following experimental scheme, specifically:

[0091] Thermal accelerated stability test method and evaluation method: Place the reagent at 37°C for 10 days, test the calibrator every day, repeat 3 times, calculate the deviation between the mean value and the target value of each measurement, and a deviation of less than 10% meets the technical requirements.

[0092] Experiment 3:

[0093] In Examples 3, 7 and 8, different pH values ​​were selected for reagent R2, i.e., the total concentration of the buffer solution was kept at 150 mM to adjust different pH values. In Example 9, different buffer systems were selected in the R2 reagent, and the specific components and corresponding contents are shown in Table 3-1;

[0094] Table 3-1

[0095]

[0096]

[0097] The results of the thermal acceleration stability evaluation of Example 3 and Examples 7-9 are shown in Table 3-2 below;

[0098] Table 3-2

[0099] Thermal acceleration days Example 3 Example 7 Example 8 Example 9 0 0.0％ 0.0％ 0.0％ 0.0％ 1 4.5％ -2.3％ -3.1％ -4.9％ 2 -1.5％ 0.6％ -0.4％ -6.3％ 3 -1.3％ -7.2％ -6.1％ -6.9％ 4 1.5％ -1.4％ -2.0％ -7.2％ 5 3.4％ -9.2％ 2.4％ -8.8％ 6 -0.9％ -4.3％ -0.8％ -9.1％ 7 -4.1％ -2.5％ -3.5％ -8.7％ 8 -6.0％ -5.1％ -8.2％ -9.5％ 9 -2.3％ -7.4％ -5.7％ -10.7％ 10 -5.2％ -9.4％ -9.8％ -12.6％

[0100] By comparing Example 3 and Examples 7-8, it can be seen that the thermal acceleration stability of reagent R2 is best when the pH is around 3.5; in combination with Example 3 and Example 9, it can be seen that if it is replaced with other buffer systems (such as acetic acid-sodium acetate buffer, MES buffer or boric acid buffer, etc.), even if the pH is still maintained at 3.5, its thermal acceleration stability is still poor.

[0101] Experiment 4:

[0102] In Example 3, Example 10 and Example 11, different concentrations of reagent R2 were selected, but the pH of the buffer solution remained unchanged. The specific components and corresponding contents are shown in Table 4-1;

[0103] Table 4-1

[0104]

[0105]

[0106] The results of the thermal acceleration stability evaluation of Example 3, Example 10 and Example 11 are shown in Table 4-2 below;

[0107] Table 4-2

[0108] Thermal acceleration days Example 3 Example 10 Embodiment 11 0 0.0％ 0.0％ 0.0％ 1 4.5％ -3.4％ 3.1％ 2 -1.5％ -0.5％ -1.9％ 3 -1.3％ 2.5％ 4.2％ 4 1.5％ -0.5％ -0.9％ 5 3.4％ -4.5％ -5.9％ 6 -0.9％ -4.8％ -8.5％ 7 -4.1％ -1.6％ -6.8％ 8 -6.0％ -4.5％ -3.5％ 9 -2.3％ -7.3％ -6.8％ 10 -5.2％ -5.5％ -10.6％

[0109] By comparing Example 3, Example 10 and Example 11, it can be seen that the thermal acceleration stability of reagent R2 is best when the buffer concentration is 150 mM.

[0110] Experiment 5:

[0111] In Example 3 and Examples 12-15, different stabilizers are selected for reagent R2, and the specific components and corresponding contents are shown in Table 5-1;

[0112] Table 5-1

[0113]

[0114]

[0115] The results of the thermal acceleration stability evaluation of Example 3 and Examples 12-15 are shown in Table 5-2 below;

[0116] Table 5-2

[0117]

[0118]

[0119] By comparing Example 3 and Examples 12-15, it can be seen that the thermal acceleration stability of reagent R2 is the best when plant ferritin is used as the stabilizer. When no polycyclic phenol stabilizer is added or the added stabilizer is bovine serum albumin, trehalose or mannitol, the thermal acceleration stability of the obtained reagent is poor.

[0120] In summary, the 2-aminohydroquinone hydrochloride described in the reagent R1 of the present invention cooperates with the potassium phosphate-sodium hydrogen phosphate buffer and is used together to improve the stability of the reagent after opening the bottle, and the high-concentration phosphate buffer has a better effect;

[0121] The polycyclic phenol stabilizer in reagent R2 is preferably plant ferritin, and the buffer system is selected from citric acid-sodium citrate buffer, which can improve the stability of MBTH in reagent R2, thereby obtaining a nitric oxide detection kit with better stability.

Claims

1. A nitric oxide detection kit with good stability, characterized in that: The nitric oxide detection kit comprises reagent R1 and reagent R2; Wherein, the reagent R1 comprises: potassium dihydrogen phosphate, disodium hydrogen phosphate, EDTA, 2-aminohydroquinone hydrochloride and a preservative; Reagent R2 includes: buffer, MBTH and preservative.

2. A nitric oxide detection kit with good stability according to claim 1, characterized in that: The reagent R2 also includes a polycyclic phenol stabilizer; The polycyclic phenol stabilizer is selected from any one or a combination of at least two of plant ferritin, bovine serum albumin, trehalose or mannitol; The preservatives in the reagents R1 and R2 are independently selected from any one of sodium azide, gentamicin, thimerosal or isothiazolinone, or a combination of at least two thereof; The buffer of the reagent R2 is selected from any one of citric acid-sodium citrate buffer, acetic acid-sodium acetate buffer, phosphate buffer, borate buffer or MES hydrochloric acid buffer, or a combination of at least two thereof.

3. A nitric oxide detection kit with good stability according to claim 2, characterized in that: The polycyclic phenol stabilizer is plant ferritin; The buffer of the reagent R2 is citric acid-sodium citrate buffer.

4. A nitric oxide detection kit with good stability according to claim 1, characterized in that: The concentration of potassium dihydrogen phosphate in the reagent R1 is 40-300 mM; The concentration of disodium hydrogen phosphate in the reagent R1 is 1-100 mM; The concentration of EDTA in the reagent R1 is 0.5-1.5 g / L; The concentration of 2-aminohydroquinone hydrochloride in the reagent R1 is 50-100 mM; The concentration of the preservative in the reagent R1 is 0.5-1.5 g / L; The pH of the reagent R1 is 5.5-6.

5.

5. A nitric oxide detection kit with good stability according to claim 3, characterized in that: The concentration of citric acid in the reagent R2 is 50-160 mM; The concentration of sodium citrate in the reagent R2 is 15-60 mM; The concentration of MBTH in the reagent R2 is 10-30 mM; The concentration of the polycyclic phenol stabilizer in the reagent R2 is 0.5-1.5 g / L; The concentration of the preservative in the reagent R2 is 0.5-1.5 g / L; The pH of the reagent R2 is 3.0-4.

0.

6. A nitric oxide detection kit with good stability according to claim 1, characterized in that: The nitric oxide detection kit comprises reagent R1 and reagent R2; Wherein, the reagent R1 includes: potassium dihydrogen phosphate, 150-300mM; disodium hydrogen phosphate, 20-100mM; EDTA, 0.5-1.5g / L; 2-aminohydroquinone hydrochloride, 50-100mM; preservative, 0.5-1.5g / L, pH is 5.5-6.5; Reagent R2 includes: citric acid, 50-160 mM; sodium citrate, 15-60 mM; MBTH, 10-30 mM; plant ferritin, 0.5-1.5 g / L; preservative, 0.5-1.5 g / L, and pH is 3.0-4.

0.

7. A method for preparing a nitric oxide detection kit according to any one of claims 1 to 6, characterized in that: The preparation method comprises the following steps: Prepare reagent R1: weigh the volume of purified water for the pre-prepared solution, then add accurately weighed potassium dihydrogen phosphate, disodium hydrogen phosphate, EDTA, 2-aminohydroquinone hydrochloride and preservative, stir, adjust the pH value of the solution after fully dissolving, and obtain the reagent R1; Prepare reagent R2: weigh the volume of purified water for the pre-prepared solution, then add accurately weighed buffer components, MBTH, preservatives and optional polycyclic phenol stabilizers, stir, adjust the pH value of the solution after sufficient dissolution, and mix well to obtain the reagent R2.

8. The method for preparing the nitric oxide detection kit according to claim 7, characterized in that: The storage temperature of the reagent R1 is 2-8°C; the storage temperature of the reagent R2 is 2-8°C.

9. A method for using the stable nitric oxide detection kit according to any one of claims 1 to 6 in non-disease diagnosis, characterized in that: The nitric oxide detection kit is used in conjunction with a biochemical analyzer, and the specific steps include: After the sample to be tested is mixed with reagent R1 and reagent R2 for reaction, the absorbance of the solution is detected, and the concentration of nitric oxide in the sample to be tested is obtained according to the change in the absorbance.

10. Use of the nitric oxide detection kit with good stability as claimed in any one of claims 1 to 6 in the detection of nitric oxide content.

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