Stabilizer and stabilizing method for a color developer

Abstract

The application discloses a color developing agent stabilizer and a stabilizing method. The application provides a color developing agent stabilizer, which comprises a reducing substance and a weak acid buffer. The pH of the weak acid buffer is 3.8-6.2. The reducing substance is one or more of sodium sulfite, sodium bisulfite, sodium thiosulfate or 1-mercapto glycerol. The color developing agent is one or both of a phenothiazine color developing agent or a triphenylmethane color developing agent. The color developing agent can be stably stored by using the stabilizer. The method for stably storing the color developing agent is to dissolve the color developing agent in the stabilizer. The color developing agent can be stably stored in the stabilizer for two weeks, and the stabilizer does not have an inhibiting effect on the subsequent use of the color developing agent. The application not only solves the problems of low stability and spontaneous color development of the color developing reagent, but also avoids the influence of the stabilizer on the performance of the color developing agent, and provides a new method for storing the color developing agent.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of chemical detection, and particularly relates to a stabilizer and a stabilizing method for a chromogenic agent. BACKGROUND

[0002] The changes of various components in various body fluids of human body have great relationship with disease occurrence, and the quantitative or qualitative changes of certain components in the body fluids, such as glycosylated hemoglobin, glycosylated albumin, uric acid, urea, glucose, triglyceride and other trace components in blood, are usually required for the risk prediction, auxiliary diagnosis and judgment of therapeutic effect monitoring of disease occurrence. The qualitative and quantitative analysis of these trace components plays a great role in the auxiliary diagnosis of diseases.

[0003] At present, there are various quantitative and qualitative analysis methods for various trace components in human body fluids, and the enzyme-coupled colorimetric method is widely used in clinical determination. Even if the target component reacts with the specific oxidase to generate hydrogen peroxide, the peroxidase (POD) and the oxidized chromogenic agent as the chromogenic component are used to induce the hydrogen peroxide into a chromogenic system, and then the amount of the target component is obtained by quantifying the chromogenic degree through colorimetry. The well-known chromogenic system is the Trinder reagent in which 4-aminoantipyrine (4-AAP) or 3-methyl-2-benzothiazoline hydrazone (MBTH) is oxidatively condensed with a chromophore such as a phenol derivative, an aniline derivative, etc. to generate a pigment. However, the sensitivity of the chromogenic system using the oxidized chromogenic agent is low for the quantitative analysis of trace components, and is easily affected by hemoglobin and bilirubin in the sample. In order to solve the problems of the typical Trinder reagent, a new type of hydrogen peroxide chromogenic reagent has attracted wide attention, but the stability of the new type of hydrogen peroxide chromogenic reagent is low, and the spontaneous chromogenic problem exists, which limits the application of the new type of hydrogen peroxide chromogenic reagent.

[0004] The prior art discloses a stabilizer for a chromogenic agent and its application, and the stabilizer can well stabilize, for example, 3,3',5,5'-tetramethylbenzidine (TMB) chromogenic agent. However, the stabilizer contains azo dyes, which can increase the overall blank of the reagent and is not conducive to practical application. Therefore, it is necessary to develop a new stabilizing method and stabilizer for a chromogenic agent. SUMMARY

[0005] The technical problem to be solved by the present application is to overcome the shortcomings of the existing hydrogen peroxide chromogenic agent stabilizing method, and to provide a stabilizer and a stabilizing method for a chromogenic agent.

[0006] The object of the present application is to provide a stabilizer for a chromogenic agent.

[0007] The object of the present application is also to provide the use of a composition in the preparation of a stabilizer for a chromogenic agent.

[0008] The application also provides the use of the stabilizer in the enzyme-coupled colorimetric method or a kit for preparing the enzyme-coupled colorimetric method.

[0009] The application also provides a method for stabilizing a color developing agent.

[0010] The application also provides a kit.

[0011] The above-mentioned objects of the application are achieved by the following technical means:

[0012] A stabilizer for a color developing agent, the stabilizer comprising a reducing substance and a weakly acidic buffer, the weakly acidic buffer having a pH of 3.8-6.2; the reducing substance being one or more of sodium sulfite, sodium bisulfite, sodium thiosulfate or 1-mercapto glycerol; the color developing agent being one or both of a phenothiazine type color developing agent or a triphenylmethane type color developing agent.

[0013] Preferably, the weakly acidic buffer has a pH of 4-6.

[0014] Further preferably, the weakly acidic buffer has a pH of 5.

[0015] Preferably, the reducing substance is one or both of sodium sulfite or sodium thiosulfate.

[0016] Further preferably, the color developing agent is a phenothiazine type color developing agent, and the reducing substance is sodium thiosulfate; or the color developing agent is a triphenylmethane type color developing agent, and the reducing substance is sodium sulfite.

[0017] Further preferably, the color developing agent is a phenothiazine type color developing agent, the weakly acidic buffer has a pH of 5-6, and the reducing substance is sodium thiosulfate; or the color developing agent is a triphenylmethane type color developing agent, the weakly acidic buffer has a pH of 4-5, and the reducing substance is sodium sulfite.

[0018] Preferably, the concentration of the reducing substance is 0.01-20 mM.

[0019] Further preferably, the concentration of the reducing substance is 1-10 mM.

[0020] Further preferably, the concentration of the reducing substance is 10 mM.

[0021] More preferably, the color developing agent is a phenothiazine type color developing agent, the weakly acidic buffer has a pH of 5, and the reducing substance is 10 mM sodium thiosulfate; or the color developing agent is a triphenylmethane type color developing agent, the weakly acidic buffer has a pH of 5, and the reducing substance is 10 mM sodium sulfite.

[0022] Preferably, the weak acid buffer is one or more of citric acid buffer, phosphate buffer, 4-morpholine ethanesulfonic acid buffer (MES buffer), acetic acid buffer or imidazole buffer.

[0023] Further preferably, the weak acid buffer is one or two of citric acid buffer, 4-morpholine ethanesulfonic acid buffer (MES buffer).

[0024] Preferably, the phenothiazine color reagent is one or two of 10-(carboxymethylaminocarbonyl)-3,7-bis(dimethylamino)phenothiazine sodium salt (DA-67) or 3,7-bis(dimethylamino)-N-ethyl-10H-phenothiazine-10-carboxamide (MCDP).

[0025] Preferably, the triphenylmethane color reagent is one or two of leucomalachite green or crystal violet.

[0026] Use of a composition in the preparation of a stabilizer for a color reagent, the composition being a stabilizer for a color reagent as described above; the stabilizer comprising a reducing substance and a weak acid buffer; the color reagent being one or two of a phenothiazine color reagent or a triphenylmethane color reagent.

[0027] Use of the stabilizer in the detection of an enzyme-coupled colorimetric method or in the preparation of a kit for the detection of an enzyme-coupled colorimetric method is also within the scope of the present application.

[0028] A kit comprising reagent R2, the reagent R2 comprising the color reagent and the stabilizer; the kit being used for the detection of an enzyme-coupled colorimetric method.

[0029] Preferably, the color reagent is a phenothiazine color reagent or a triphenylmethane color reagent.

[0030] The kit further comprises reagent R1, the reagent R1 comprising one or more of a buffer, a peroxidase, a preservative, an oxidase, a synthetic enzyme or adenosine triphosphate (ATP).

[0031] Preferably, the kit further comprises a pretreatment solution, the pretreatment solution comprising a surfactant and sodium nitrite.

[0032] Further preferably, the surfactant is Triton X-100, Tween-20 or Triton X-405.

[0033] Further preferably, the surfactant is Triton X-100.

[0034] Preferably, the oxidase in the reagent R1 is fructosyl amino acid oxidase or acyl-CoA oxidase.

[0035] Preferably, the synthetic enzyme in the reagent Rl is acyl-CoA synthetase.

[0036] Preferably, the reagent R2 comprises a stabilizer containing sodium thiosulfate, a buffer solution with pH of 5-6, and a phenothiazine color reagent.

[0037] Further preferably, the reagent R2 comprises a stabilizer containing sodium thiosulfate, a buffer solution with pH of 5, and a phenothiazine color reagent.

[0038] Preferably, the reagent R2 comprises a stabilizer containing sodium sulfite, a buffer solution with pH of 4-5, and a triphenylmethane color reagent.

[0039] Further preferably, the reagent R2 comprises a stabilizer containing sodium sulfite, a buffer solution with pH of 5, and a triphenylmethane color reagent.

[0040] Preferably, the reagent R2 further comprises a preservative, a surfactant, and coenzyme A.

[0041] Preferably, the preservative is Proclin-300 or sodium benzoate.

[0042] Preferably, the surfactant is linear secondary alcohol polyoxyethylene ether (Tergitol TM 15-S-9) or Genapol X 080.

[0043] Further preferably, the surfactant is linear secondary alcohol polyoxyethylene ether (Tergitol TM 15-S-9).

[0044] As a specific embodiment, a kit for detecting glycated hemoglobin in blood sample by enzyme-coupled colorimetry comprises: a pretreatment solution, a reagent Rl, and a reagent R2; the pretreatment solution is Triton X-100 and sodium nitrite; the reagent Rl is Tris-HCl buffer solution, peroxidase, fructose-based amino acid oxidase, and sodium azide; the reagent R2 is 4-morpholine ethanesulfonic acid buffer solution (MES buffer solution), 10-(carboxymethylaminocarbonyl)-3,7-bis(dimethylamino) phenothiazine sodium salt (DA-67), sodium thiosulfate, and Proclin-300.

[0045] The method for using the kit for detecting glycated hemoglobin in blood sample comprises: adding anticoagulant disodium EDTA to the whole blood sample, centrifuging to separate layers, taking red blood cells from the lowermost layer, mixing with the pretreatment solution to prepare a pretreated sample. Then mix the pretreated sample with the reagent Rl, incubate at 37°C for 5 min, measure the absorbance A1, then add the reagent R2, mix, incubate at 37°C for 5 min, and measure the absorbance A2.

[0046] As a specific embodiment, a kit for detecting free fatty acid in blood sample by enzyme coupling colorimetry comprises: reagent R1 and reagent R2, the reagent R1 is HEPES buffer, acyl-CoA synthetase, acyl-CoA oxidase, ATP, peroxidase and sodium azide; the reagent R2 is citric acid buffer, linear secondary alcohol polyoxyethylene ether (Tergitol TM 15-S-9), coenzyme A, leuco malachite green, sodium sulfite and sodium benzoate.

[0047] The method for using the kit for detecting free fatty acid in blood sample is as follows: mixing serum with reagent R2, incubating at 37 DEG C for 5 min, measuring absorbance A1 at 600 nm, then mixing with 45 muL of reagent R1, incubating at 37 DEG C for 5 min, and measuring absorbance A2 at 600 nm.

[0048] A method for stabilizing a color developing agent, wherein the color developing agent is dissolved in the stabilizer, so that the spontaneous color development of the color developing agent is inhibited, and the color developing agent is stably stored.

[0049] The stabilizer comprises a reducing substance and a weak acid buffer, the pH of the weak acid buffer is 3.8-6.2, the reducing substance is one or more of sodium sulfite, sodium bisulfite, sodium thiosulfate or 1-mercapto glycerol, and the color developing agent is one or both of a phenothiazine color developing agent and a triphenylmethane color developing agent.

[0050] The color developing agent is stored by using the stabilizer containing the reducing substance, so that the spontaneous color development of the color developing agent is inhibited, and the property of the color developing agent is not affected.

[0051] Compared with the prior art, the present application has the following beneficial effects:

[0052] (1) The present application uses a weak acid buffer or a weak acid buffer containing a reducing substance as a stabilizer to store a phenothiazine color developing agent or a triphenylmethane color developing agent, so that the spontaneous color development of the color developing agent is inhibited, the storage time of the color developing agent is prolonged, and the color developing ability of the color developing agent is maintained; meanwhile, the redox reaction of the color developing agent is not disturbed, and the color developing agent can be stably stored.

[0053] (2) The color developing agent can be stably stored by using the stabilizer, and the method for stably storing the color developing agent is to dissolve the color developing agent in the stabilizer; the color developing agent can be stably stored in the stabilizer for 2 weeks, and the stabilizer does not inhibit the subsequent use of the color developing agent; not only the problems of low stability and easy spontaneous color development of the color developing reagent are solved, but also the influence of the stabilizer on the performance of the color developing agent is avoided, thereby providing a new method for storing the color developing agent. BRIEF DESCRIPTION OF DRAWINGS

[0054] Figure 1 A correlation regression equation graph was fitted using the results of Comparative Test 1 as the X axis and the results of Test 1 as the Y axis.

[0055] Figure 2 A correlation regression equation graph was fitted using the results of Comparative Test 1 as the X axis and the results of Test 2 as the Y axis.

[0056] Figure 3 A correlation regression equation graph was fitted using the results of Comparative Test 1 as the X axis and the results of Test 3 as the Y axis.

[0057] Figure 4 A correlation regression equation graph was fitted using the results of Comparative Test 2 as the X axis and the results of Test 4 as the Y axis.

[0058] Figure 5 A correlation regression equation graph was fitted using the results of Comparative Test 2 as the X axis and the results of Test 5 as the Y axis.

[0059] Figure 6 A correlation regression equation graph was fitted using the results of Comparative Test 2 as the X axis and the results of Test 6 as the Y axis. DETAILED DESCRIPTION

[0060] The present application will be further described in conjunction with specific examples, but the examples do not limit the present application in any form. Unless otherwise specified, the reagents, methods and equipment used in the present application are conventional reagents, methods and equipment in the art.

[0061] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0062] The preservation solution described in the present application is the stabilizer.

[0063] Tris buffer (pH = 8.00): 1.21 g of tris-hydroxymethyl aminomethane was dissolved in 50 mL of purified water, and the pH was adjusted to 8.00 using hydrochloric acid, and then purified water was added to a final volume of 100 mL.

[0064] Phosphate buffer (pH = 7.00): 1.20 g of anhydrous sodium dihydrogen phosphate was dissolved in 50 mL of purified water, and the pH was adjusted to 7.00 using sodium hydroxide, and then purified water was added to a final volume of 100 mL.

[0065] MES buffer (pH = 6.00): 1.95 g of 2-(N-morpholino) ethanesulfonic acid was dissolved in 50 mL of purified water, and the pH was adjusted to 6.00 using sodium hydroxide, and then purified water was added to a final volume of 100 mL.

[0066] Imidazole buffer (pH = 6.00): Tris 0.68 g, dissolved in 50 mL of purified water, and adjusted to pH 6.00 using hydrochloric acid, and then diluted to 100 mL with purified water.

[0067] Citric acid buffer (pH = 5.00): Citric acid monohydrate 2.10 g, dissolved in 50 mL of purified water, and adjusted to pH 5.00 using sodium hydroxide, and then diluted to 100 mL with purified water.

[0068] MES buffer (pH = 5.00): 2-(N-morpholino)ethanesulfonic acid 1.95 g, dissolved in 50 mL of purified water, and adjusted to pH 5.00 using sodium hydroxide, and then diluted to 100 mL with purified water.

[0069] Acetic acid buffer (pH = 4.00): Sodium acetate trihydrate 1.36 g, dissolved in 50 mL of purified water, and adjusted to pH 4.00 using hydrochloric acid, and then diluted to 100 mL with purified water.

[0070] Potassium hydrogen phthalate buffer (pH = 4.00): Potassium hydrogen phthalate 2.04 g, dissolved in 50 mL of purified water, and adjusted to pH 4.00 using sodium hydroxide, and then diluted to 100 mL with purified water.

[0071] Example 1 Stability of DA-67 under different buffer storage conditions

[0072] 1. Method

[0073] The color reagent 10-(carboxymethylaminocarbonyl)-3,7-bis(dimethylamino)phenothiazine sodium salt (DA-67) was dissolved in different buffer storage solutions, respectively, and the final concentration of the color reagent DA-67 was 50 μM. After being stored at 37°C for 2 weeks, a portion of the solution was taken out every 2 days, and the absorbance at 660 nm was measured using a UV spectrophotometer.

[0074] 2. Results

[0075] The changes in the absorbance of the color reagent DA-67 under different storage conditions of Nos. 1 to 8 are shown in Table 1.

[0076] Table 1 Changes in the absorbance of the color reagent DA-67 under different storage conditions of Nos. 1 to 8

[0077]

[0078]

[0079] From Table 1, it can be seen that the absorbance of the chromogenic agent DA-67 has obvious differences in different buffer storage solutions at 37℃ for 2 weeks. With the increase of storage days, the change trend of the chromogenic agent DA-67 is roughly the same. The absorbance of DA-67 in Tris-HCl buffer solution with pH of 8.00 and 100mM increases by 1.7792; the absorbance of DA-67 in phosphate buffer solution with pH of 7 and 100mM increases by 1.537; and the absorbance in the buffer solution with pH of 4-6 only increases by 1.1848-1.2633; the absorbance in the buffer solution with pH of 5.00 only increases by 1.1848 or 1.1973, the change of absorbance is the smallest, which indicates that the change of the absorbance of DA-67 in the acidic environment (buffer solution with pH of 4-6) is obviously smaller than that in the alkaline environment, compared with the alkaline buffer solution, the weak acid buffer solution with pH of 4-6 can obviously slow down the spontaneous coloration of the chromogenic agent, inhibit the spontaneous coloration of the chromogenic agent to a certain extent, and stabilize the chromogenic agent. The weak acid buffer solution with pH of 5 has the best effect on inhibiting the spontaneous coloration of the chromogenic agent.

[0080] Example 2 Stability of DA-67 under different reducing substance storage conditions

[0081] 1. Method

[0082] The chromogenic agent 10-(carboxymethylaminocarbonyl)-3,7-bis(dimethylamino) phenothiazine sodium salt (DA-67) was dissolved in different reducing substance storage solutions, and the final concentration of the chromogenic agent DA-67 was 50μM. At 37℃, a part of the solution was taken out every 2 days, and the absorbance at 660nm wavelength was measured by ultraviolet spectrophotometer.

[0083] 2. Results

[0084] The change of the absorbance of the chromogenic agent DA-67 under different storage conditions of No. 9-12 is shown in Table 2.

[0085] Table 2 Change of absorbance of chromogenic agent DA-67 under different storage conditions of No. 9-12

[0086]

[0087]

[0088] As shown in Table 2, the change trends of the absorbance of the color developer DA-67 in different reducing substance storage solutions were roughly the same when stored at 37°C for 2 weeks. In different reducing substance storage solutions, the absorbance of DA-67 in the storage solution of the citric acid buffer solution with 3 mM sodium thiosulfate and pH 5 only increased by 0.6873, the change of the absorbance was the smallest, and the inhibiting effect on the spontaneous color development was better than that of the reducing substances sodium sulfite, sodium bisulfite and 1-mercapto glycerol.

[0089] When the storage solution was changed to other buffer solutions with pH 5, the inhibiting effect on the spontaneous color development of the storage solution containing sodium thiosulfate was also better than that of the reducing substances sodium sulfite, sodium bisulfite and 1-mercapto glycerol.

[0090] Example 3 Stability of DA-67 under different concentrations of sodium thiosulfate storage conditions

[0091] 1. Method

[0092] The color developer 10-(carboxymethylaminocarbonyl)-3,7-bis(dimethylamino) phenothiazine sodium salt (DA-67) was respectively dissolved in different concentrations of sodium thiosulfate storage solutions, and the final concentration of the color developer DA-67 was 50 μM. After being stored at 37°C for 2 weeks, a part of the solution was taken out every 2 days, and the absorbance at 660 nm wavelength was determined by using a UV spectrophotometer.

[0093] 2. Results

[0094] The change of the absorbance of the color developer DA-67 under different storage conditions of Nos. 13-16 is shown in Table 3.

[0095] Table 3 Change of the absorbance of the color developer DA-67 under different storage conditions of Nos. 13-16

[0096]

[0097]

[0098] As shown in Table 3, the change trends of the absorbance of the color developer DA-67 in different concentrations of sodium thiosulfate storage solutions were roughly the same when stored at 37°C for 2 weeks. In different concentrations of sodium thiosulfate storage solutions, the absorbance of DA-67 in the storage solution of the citric acid buffer solution with 10 mM sodium thiosulfate and pH 5 only increased by 0.4242, the change of the absorbance was the smallest, and the inhibiting effect on the spontaneous color development was the best. Therefore, the storage of DA-67 in the citric acid buffer solution containing 10 mM sodium thiosulfate and with pH 5 has the best stability effect.

[0099] When the storage solution was changed to other buffer solutions with pH 5, DA-67 was also stored in the buffer solution containing 10 mM sodium thiosulfate, and the stability effect was the best.

[0100] Stability of leucomalachite green in different buffer solution storage conditions

[0101] 1. Method

[0102] Leucomalachite green was dissolved in different buffer solutions, and the final concentration of leucomalachite green was 10 μM. The absorbance at 660 nm was measured by UV spectrophotometer every two days for two weeks at 37 °C.

[0103] 2. Results

[0104] The changes of absorbance of leucomalachite green in different storage conditions are shown in Table 4.

[0105] Table 4 Changes of absorbance of leucomalachite green in different storage conditions

[0106]

[0107] As shown in Table 4, the absorbance of leucomalachite green in different buffer solutions at 37 °C for two weeks has obvious differences. With the increase of storage days, the absorbance of leucomalachite green in Tris-HCl buffer solution with pH 8 increased by 0.3743; the absorbance in phosphate buffer solution with pH 7.00 increased by 0.3509; while the absorbance in buffer solutions with pH 4-6 only increased by 0.224-0.279, and the absorbance in buffer solution with pH 5 was only 0.224 or 0.2289, with the smallest change in absorbance. This indicates that the degree of spontaneous color development of leucomalachite green under weak acidic conditions (buffer solutions with pH 4-6) is lower than that under neutral or alkaline conditions, and the inhibitory effect on spontaneous color development is the best at pH 5.

[0108] Stability of leucomalachite green in different reducing substance storage conditions

[0109] 1. Method

[0110] Leucomalachite green was dissolved in different reducing substance solutions, and the final concentration of leucomalachite green was 10 μM. The absorbance at 660 nm was measured by UV spectrophotometer every two days for two weeks at 37 °C.

[0111] 2. Results

[0112] The changes of absorbance of leucomalachite green in different storage conditions are shown in Table 5.

[0113] Table 5 Changes in absorbance of color reagent leucomalachite green under different storage conditions

[0114]

[0115] As shown in Table 5, the absorbance of color reagent leucomalachite green stored in different reducing agent storage solutions at 37°C for 2 weeks has obvious differences. With the increase of storage days, the absorbance of color reagent leucomalachite green in the MES buffer solution containing 3 mM sodium sulfite at pH 5 only increases by 0.1825, and the change in absorbance is the smallest, and the inhibitory effect on spontaneous coloration is better than that of reducing agents sodium thiosulfate, sodium bisulfite and 1-mercapto glycerol.

[0116] When the storage solution is changed to other buffer solutions at pH 5, the inhibitory effect of the storage solution containing sodium sulfite on spontaneous coloration is also better than that of reducing agents sodium thiosulfate, sodium bisulfite and 1-mercapto glycerol.

[0117] Example 6 Stability of leucomalachite green under different concentrations of sodium sulfite storage conditions

[0118] 1. Method

[0119] The color reagent leucomalachite green was dissolved in different concentrations of sodium sulfite storage solutions, and the final concentration of the color reagent leucomalachite green was 10 μM. After being stored at 37°C for 2 weeks, a part of the solution was taken out every 2 days, and the absorbance at 660 nm was measured by ultraviolet spectrophotometer.

[0120] 2. Results

[0121] The changes in absorbance of color reagent leucomalachite green under different storage conditions are shown in Table 6.

[0122] Table 6 Changes in absorbance of color reagent leucomalachite green under different storage conditions

[0123]

[0124] As shown in Table 6, the absorbance of color reagent leucomalachite green stored in different concentrations of sodium sulfite storage solutions at 37°C for 2 weeks has obvious differences. With the increase of storage days, the absorbance of color reagent leucomalachite green in the MES buffer solution containing 10 mM sodium sulfite at pH 5 only increases by 0.1329, and the change in absorbance is the smallest, and the inhibitory effect on spontaneous coloration is the best. Therefore, the storage of leucomalachite green in the MES buffer solution containing 10 mM sodium sulfite at pH 5 has the best stability.

[0125] The storage solution was changed to other buffer solutions with pH 5, and the leucomalachite green was also stored in the buffer containing 10 mM sodium sulfite, and the storage effect was optimal.

[0126] Example 7 Stability of leucomalachite green under different buffer storage conditions

[0127] 1. Method

[0128] The chromogenic agent leucomalachite green was dissolved in different buffer storage solutions, and the final concentration of the chromogenic agent leucomalachite green was 10 μM. After being stored at 37°C for 2 weeks, a portion of the solution was taken out every 2 days, and the absorbance at 590 nm was measured by ultraviolet spectrophotometry.

[0129] 2. Results

[0130] The changes in the absorbance of the chromogenic agent leucomalachite green under different storage conditions are shown in Table 7.

[0131] Table 7 Changes in the absorbance of the chromogenic agent leucomalachite green under different storage conditions

[0132]

[0133] As can be seen from Table 7, the absorbance of the chromogenic agent leucomalachite green stored in different buffer storage solutions at 37°C for 2 weeks has obvious differences. With the increase in the storage days, the absorbance of the chromogenic agent leucomalachite green in the Tris-HCl buffer with pH 8 increased by 0.423; the absorbance in the phosphate buffer with pH 7.00 increased by 0.3859; the absorbance in the buffers with pH 4-6 only increased by 0.2528-0.3219, and the absorbance in the buffer with pH 5 only increased by 0.2528 or 0.2553, and the change in the absorbance was the smallest. It is indicated that the degree of spontaneous color development of leucomalachite green under weak acidic conditions (buffer with pH 4-6) is lower than that under neutral or alkaline conditions, and the inhibitory effect on spontaneous color development is the best when the pH is 5.

[0134] Example 8 Stability of leucomalachite green under different reducing substance storage conditions

[0135] 1. Method

[0136] The chromogenic agent leucomalachite green was dissolved in different reducing substance storage solutions. The final concentration of the chromogenic agent leucomalachite green was 10 μM. After being stored at 37°C for 2 weeks, a portion of the solution was taken out every 2 days, and the absorbance at 590 nm was measured by ultraviolet spectrophotometry.

[0137] 2. Results

[0138] The changes of the absorbance of the color reagent leuco crystal violet under different storage conditions of No. 41-44 are shown in Table 8.

[0139] Table 8 The changes of the absorbance of the color reagent leuco crystal violet under different storage conditions of No. 41-44

[0140]

[0141] As can be seen from Table 8, the absorbance of the color reagent leuco crystal violet stored in different reducing substance storage solutions at 37℃ for 2 weeks has obvious differences. With the increase of the storage days, the absorbance of the color reagent leuco crystal violet in the MES buffer solution containing 3mM sodium sulfite with pH of 5 only increases by 0.2276, the change of the absorbance is the smallest, and the inhibiting spontaneous coloration effect is better than that of the reducing substances sodium thiosulfate, sodium bisulfite and 1-mercapto glycerol.

[0142] When the storage solution is changed into other buffer solutions with pH of 5, the inhibiting spontaneous coloration effect of the storage solution containing sodium sulfite is also better than that of the reducing substances sodium thiosulfate, sodium bisulfite and 1-mercapto glycerol.

[0143] Example 9 Stability of leuco crystal violet under different concentrations of sodium sulfite storage conditions

[0144] 1. Method

[0145] The color reagent leuco crystal violet is respectively dissolved in different concentrations of sodium sulfite storage solutions, and the final concentration of the color reagent leuco crystal violet is 10μM. At 37℃, a part of the solution is taken out every 2 days, and the absorbance at 590nm wavelength is measured by using the ultraviolet spectrophotometer.

[0146] 2. Results

[0147] The changes of the absorbance of the color reagent leuco crystal violet under different storage conditions of No. 45-48 are shown in Table 9.

[0148] Table 9 The changes of the absorbance of the color reagent leuco crystal violet under different storage conditions of No. 45-48

[0149]

[0150] As can be seen from Table 9, the absorbance of the color reagent leuco crystal violet stored in different concentrations of sodium sulfite storage solutions at 37℃ for 2 weeks has obvious differences. With the increase of the storage days, the absorbance of the color reagent leuco crystal violet in No. 39, i.e. the MES buffer solution containing 10mM sodium sulfite with pH of 5 only increases by 0.1688, the change of the absorbance is the smallest, and the inhibiting spontaneous coloration effect is the best. Therefore, it is known that the storage of leuco crystal violet in the MES buffer solution containing 10mM sodium sulfite with pH of 5 has the best stability effect.

[0151] The storage solution was changed to other buffer solutions with pH 5, and leuco crystal violet was also stored in a buffer containing 10 mM sodium sulfite, which had the best stability.

[0152] Stability of Comparative Example DA-67 under different storage conditions

[0153] 1. Method

[0154] Leuco developer 10-(carboxymethylaminocarbonyl)-3,7-bis(dimethylamino) phenothiazine sodium salt (DA-67) was dissolved in different storage solutions, and the final concentration of the developer DA-67 was 40 μM. After being stored at 37°C for 2 weeks, a portion of the solution was taken out every 2 days, and the absorbance at 660 nm was measured using a UV spectrophotometer.

[0155] 2. Results

[0156] The changes in the absorbance of the developer DA-67 under different storage conditions in Examples 49-52 are shown in Table 10.

[0157] Table 10 Changes in the absorbance of the developer DA-67 under different storage conditions in Examples 49-52

[0158]

[0159] As can be seen from Table 10, the absorbance of the developer DA-67 stored in different storage solutions at 37°C for 2 weeks had significant differences. The absorbance of the developer DA-67 in the storage solutions containing cetyltrimethylammonium bromide and benzalkonium chloride increased by 1.2424 and 1.1663, respectively, which was significantly higher than that in the storage solutions containing the reducing substances 1-mercapto glycerol and sodium thiosulfate, indicating that the spontaneous color development of the developer DA-67 in the weak acid buffer containing the reducing substances 1-mercapto glycerol and sodium thiosulfate was significantly lower than that in the weak acid buffer containing quaternary ammonium, and that the weak acid buffer storage solution containing the reducing substances 1-mercapto glycerol and sodium thiosulfate had a better stability effect on the developer.

[0160] Application Example 1 Application of the developer

[0161] 1. Method

[0162] The developer was stored using a weak acid buffer storage solution containing a reducing substance, and the developer was used to determine glycated hemoglobin.

[0163] The components of the glycated hemoglobin determination reagent are shown in Table 11.

[0164] Table 11 Components of the glycated hemoglobin determination reagent

[0165]

[0166] Glycated hemoglobin determination method:

[0167] Add anticoagulant EDTA disodium to the whole blood sample, centrifugal stratification, and take 25 μL red blood cells from the lowermost layer and mix with 500 μL pretreatment solution to prepare a pretreatment sample. Mix 12 μL of the pretreatment sample with 180 μL of reagent R1, incubate at 37°C for 5 min, measure the absorbance A1 at 660 nm, then add 60 μL of reagent R2 and mix, incubate at 37°C for 5 min, then measure the absorbance A2 at 660 nm, and calculate the difference A2-A1.

[0168] The above operation method and machine parameters are used to calibrate and standardize the Hitachi 7180 automatic biochemical analyzer to determine the linear relationship between the glycated hemoglobin concentration and the difference A2-A1, and the concentration of glycated hemoglobin is calculated according to the linear relationship between glycated hemoglobin and the difference A2-A1 and the difference A2-A1 of the EDTA disodium whole blood sample.

[0169] After the stabilizer containing the reducing agent coexists with the chromogenic agent for one week, test 1 uses sodium thiosulfate as the reducing material in reagent R2; test 2 uses vitamin C as the reducing material in reagent R2; test 3 uses sodium borohydride as the reducing material in reagent R2; and the enzyme-based glycated hemoglobin detection kit purchased from Juxing Medical Technology (China) Co., Ltd. is used as comparative test 1, which is tested according to the instructions of the kit; the reagents of tests 1-3 and comparative test 1 are used to determine the glycated hemoglobin concentration of 30 EDTA disodium whole blood samples.

[0170] The results of comparative test 1 are used as the X-axis, and the results of tests 1-3 are used as the Y-axis to fit the correlation regression equation respectively to determine the influence of different reducing materials in reagent R2 on the determination results.

[0171] 2. Results

[0172] (1) Glycated hemoglobin concentration determination results of 30 EDTA disodium whole blood samples

[0173] The glycated hemoglobin concentration determination results of 30 EDTA disodium whole blood samples measured by tests 1-3 and comparative test 1 are shown in Table 12.

[0174] Table 12 Glycated hemoglobin concentration determination results of 30 EDTA disodium whole blood samples

[0175]

[0176]

[0177] (2) Correlation of the results of tests 1 to 3 with the results of comparative test 1

[0178] The results of comparative test 1 were taken as the X axis, and the results of tests 1 to 3 were taken as the Y axis to fit a correlation regression equation graph, and the correlation results of tests 1 to 3 were as shown in Figures 1-3 Figure 1 It can be seen that the correlation coefficient R 2 of the results of test 1 with the fitting of comparative test 1 was 0.9839, and the correlation was good, while Figure 2 and Figure 3 showed that the correlation coefficient R 2 of the results of tests 2 and 3 with the fitting of comparative test 1 was 0.2679 / 0.3786, and the correlation was very poor, indicating that when the reducing substance vitamin C or sodium borohydride was used as the kit reagent R2, the determination of glycated hemoglobin was affected by inhibition, and the determination accuracy was poor, while when the reducing substance sodium thiosulfate of the present application was used as the kit reagent R2, there was no inhibition reaction, and the determination results had good correlation with the commercial reagent, and the determination accuracy was good.

[0179] Application of the chromogenic agent of application example 2

[0180] 1. Method

[0181] The chromogenic agent was preserved by using a weakly acidic buffer solution containing a reducing substance, and the free fatty acid was determined by using the chromogenic agent.

[0182] The components of the free fatty acid determination reagent are as shown in Table 13.

[0183] Table 13 Components of the free fatty acid determination reagent

[0184]

[0185]

[0186] Free fatty acid determination method:

[0187] 3 μL of serum was mixed with 180 μL of reagent R2, incubated at 37°C for 5 min, the absorbance A1 at 600 nm was determined, then 45 μL of reagent R1 was added and mixed, incubated at 37°C for 5 min, and the absorbance A2 at 600 nm was determined, and the difference A2-A1 was calculated.

[0188] The above operation method and machine parameters were used to calibrate and standardize the Hitachi 7180 automatic biochemical analyzer to determine the linear relationship between the free fatty acid concentration and the difference A2-A1, and the concentration of free fatty acid was calculated according to the linear relationship between the free fatty acid and the difference A2-A1 and the difference A2-A1 of the serum sample. ​

[0189] After coexisting the stabilizer containing the reducing agent with the colorimetric reagent for one week, test 4 was conducted with sodium sulfite as the reducing agent in reagent R2; test 5 was conducted with vitamin C as the reducing agent in reagent R2; test 6 was conducted with sodium borohydride as the reducing agent in reagent R2; and test 2 was conducted with the free fatty acid assay kit purchased from Desay Diagnostics Systems (Shanghai) Co., Ltd. The test 2 was conducted according to the instructions of the kit. The free fatty acids of 40 serum samples were measured using the reagents of tests 4-6 and test 2, respectively.

[0190] Using the results of comparative experiment 2 as the X-axis and the results of experiments 4-6 as the Y-axis, correlation regression equations were fitted to determine the influence of different reducing substances in reagent R2 on the measurement results.

[0191] 2. Results

[0192] (1) Results of free fatty acid concentration determination in 40 serum samples.

[0193] The results of free fatty acid concentration determination in 40 serum samples from experiments 4-6 and comparative experiment 2 are shown in Table 14.

[0194] Table 14 Results of free fatty acid concentration determination in 40 serum samples

[0195]

[0196]

[0197]

[0198] (2) Correlation between the measurement results of experiments 4-6 and the measurement results of comparative experiment 2

[0199] The results of Experiment 2 were used as the X-axis, and the results of Experiments 4-6 were used as the Y-axis to fit correlation regression equations. The correlation results fitted in Experiments 4-6 are shown below. Figures 4-6 As shown. From Figure 4 It can be seen that the correlation coefficient R between the measurement results of Experiment 4 and the goodness of fit of the comparison experiment 2 is [missing information]. 2 The correlation was 0.9805, indicating a good correlation. Figure 5 and Figure 6 The correlation coefficient R between the measurement results of Experiments 5 and 6 and the control experiment 2 is shown. 2The correlation is 0.8242 / 0.8566, which is poor, indicating that when the reducing substance vitamin C or sodium borohydride is used as the kit reagent R2, the determination of free fatty acid is affected by inhibition, and the determination accuracy is poor. When the reducing substance sodium sulfite of the application is used as the kit reagent R2, there is no inhibition reaction, and the determination result has good correlation with the commercial reagent, and the determination accuracy is good.

[0200] The above embodiments are the preferred embodiments of the application, but the embodiments of the application are not limited to the above embodiments, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the application should be equivalent replacement methods, and are all included in the protection scope of the application.

Claims

1. A method for stabilizing a colorimetric agent, characterized in that, The colorimetric agent is dissolved in the stabilizer; The stabilizer consists of a reducing agent and a weakly acidic buffer solution; The colorimetric reagent is 10-(carboxymethylaminocarbonyl)-3,7-bis(dimethylamino)phenthiazine sodium salt, the reducing agent is 10 mM sodium thiosulfate, and the weakly acidic buffer is 100 mM citrate buffer with pH 5. If the colorimetric agent is leucomalle green or crystal violet, then the reducing agent is 10mM sodium sulfite, and the weakly acidic buffer is 100mM MES buffer with a pH of 5.

2. The use of a composition in a stabilizer for preparing a color developer, characterized in that, The composition is the stabilizer described in claim 1; The stabilizer consists of a reducing agent and a weakly acidic buffer solution; The colorimetric reagent is 10-(carboxymethylaminocarbonyl)-3,7-bis(dimethylamino)phenthiazine sodium salt, the reducing agent is 10 mM sodium thiosulfate, and the weakly acidic buffer is 100 mM citrate buffer with pH 5. If the colorimetric agent is leucomalle green or crystal violet, then the reducing agent is 10mM sodium sulfite, and the weakly acidic buffer is 100mM MES buffer with a pH of 5.

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