Lactic acid detection kit and preparation method thereof

Abstract

The present invention discloses a lactic acid detection kit and a preparation method thereof, which belongs to the technical field of detection reagents. The kit comprises 3 mL of R1 reagent, 1 mL of R2 reagent and 1 mL of standard product. The R1 reagent contains bovine serum albumin, nanosilver-trehalose complex and PEG4000. The bovine serum albumin can prevent enzyme decomposition and nonspecific adsorption, chelate a small amount of dissociated Ag, and + , preventing competition with H2O2 for the binding sites of horseradish peroxidase and ensuring the stable generation of quinoneimine; PEG4000 plays a dispersing role, assisting the nanosilver-trehalose complex to form a protective film on the surface of lactate oxidase and horseradish peroxidase, protecting the protein structure of the two enzymes, and can capture electrons from the surrounding environment, increasing the electron transfer efficiency of the active centers of the enzymes during the catalytic process of the two enzymes, thereby further increasing the activity of the two enzymes and improving the sensitivity and accuracy of the kit.

Description

Technical Field

[0001] The present invention belongs to the technical field of detection reagents, and in particular relates to a lactic acid detection kit and a preparation method thereof. Background Art

[0002] Lactic acid is a carboxylic acid containing a hydroxyl group, with the molecular formula C3H6O3. During cell culture, the accumulation of lactic acid lowers the pH of the culture system, thereby affecting the activity of intracellular enzymes, inhibiting normal cell growth and metabolism, and leading to cell acidification and even death. Therefore, during cell culture, monitoring and timely regulation of cytosolic lactate are key factors in maintaining cell survival. Direct, non-contact, and real-time monitoring of lactate metabolic concentrations is crucial for cell culture.

[0003] Currently, lactate content in cell culture is primarily measured using fully automated biochemical analyzers or ultraviolet spectrophotometry. Detection principles primarily include lactate oxidase, lactate dehydrogenase, and immobilized enzyme electrodes. Due to interference from numerous coexisting components in the sample and the sensitivity of the detection kit, low lactate levels may not be detected in a timely manner, hindering accurate monitoring of lactate levels during cell culture. Therefore, a highly sensitive lactate detection kit is needed. Summary of the Invention

[0004] The object of the present invention is to provide a lactic acid detection kit and a preparation method thereof, so as to solve the problems in the background technology.

[0005] The purpose of the present invention can be achieved through the following technical solutions:

[0006] A lactic acid detection kit includes 3 mL of R1 reagent, 1 mL of R2 reagent, and 1 mL of a standard.

[0007] The sample to be tested was mixed with the R1 reagent and incubated at 37°C for 3 minutes. Then, the R2 reagent was added and mixed, and the mixture was incubated at 37°C for 4 minutes. The sample was detected by an automatic biochemical analyzer, and the absorbance value A1 was recorded. Then, the sample was incubated for another 1 minute under the same conditions, and the absorbance value A2 was recorded. The absorbance value of the sample to be tested was calculated (absorbance value of the sample to be tested = A2-A1).

[0008] The standard was incubated with R1 and R2 reagents in the same manner and the absorbance of the standard was calculated. The lactic acid concentration in the test sample was then calculated (lactic acid concentration = (absorbance of the test sample / absorbance of the standard) × 2.22, in mmol / L).

[0009] The reaction principle is: lactate and O2 produce pyruvate and H2O2 under the action of lactate oxidase, and H2O2, 4-aminoantipyrine and 4-chlorophenol produce quinone imine, water and HCl under the catalysis of horseradish peroxidase.

[0010] R1 reagent: lactate oxidase 2000-3000 U / L, enzyme-bound stabilizer 15-16 wt%, 4-aminoantipyrine 0.8-0.82 wt%, sodium azide 0.8-1.2 wt%, Triton X-100 0.8-1.2 wt%, and the balance is 0.5 M Tris buffer.

[0011] R2 reagent: horseradish peroxidase 2000 U / L, 4-chlorophenol 6.9-6.95 wt%, sodium azide 0.8-1.2 wt%, and the balance is phosphate buffer.

[0012] Standard products: L-lithium lactate 2.1wt%, concentrated sulfuric acid 0.046wt%, balance ddH2O.

[0013] The preparation method of the lactate detection kit comprises the following steps: preparing 0.5M Tris buffer and phosphate buffer; taking a 5mL reagent tube, adding 0.5M Tris buffer thereto, then adding 4-aminoantipyrine, sodium azide, TritonX-100 and an enzyme binding stabilizer, mixing evenly, then adding lactate oxidase, and pipetting evenly to obtain R1 reagent;

[0014] Take a 3 mL reagent tube, add phosphate buffer, then add 4-chlorophenol and sodium azide, mix well, then add horseradish peroxidase, and pipette to mix evenly to obtain R2 reagent;

[0015] Dissolve lithium lactate with ddH2O, then add concentrated sulfuric acid, and finally dilute to volume with ddH2O according to a preset ratio to obtain a standard solution. Pipette 1 mL of the standard solution into a 3 mL reagent tube to obtain a standard product.

[0016] Furthermore, the preparation method of 0.5M Tris buffer is as follows: 3.03g of Tris is placed in a flask, then added to 400mL of ddH2O, stirred and dissolved at room temperature, and then 37% hydrochloric acid is added dropwise to adjust the pH value to 7.5-8, and then the volume is adjusted to 500mL with ddH2O. After stirring and mixing, it is sterilized, filtered, and stored at room temperature.

[0017] Furthermore, the preparation method of the enzyme-bound stabilizer is:

[0018] Dissolve trehalose in ddH2O, then add a 2 mM AgNO3 solution, mix, and ultrasonically disperse for 5-10 minutes to obtain a mixed solution. Irradiate the mixed solution under a 500 W xenon lamp for 2-3 hours, centrifuge at 10,000-13,000 rpm for 20-30 minutes, collect the precipitate, wash it 2-3 times with ddH2O, and dry it to obtain a nanosilver-trehalose complex.

[0019] Bovine serum albumin, PEG4000 and nanosilver-trehalose complex were dissolved in 0.5M Tris buffer to obtain an enzyme binding stabilizer.

[0020] Furthermore, the usage ratio of trehalose, ddH2O, and 2 mM AgNO3 solution is 1 g:50 mL:50 mL.

[0021] Furthermore, the usage ratio of bovine serum albumin, PEG4000, nanosilver-trehalose complex and 0.5 M Tris buffer is 1 g:5 g:5 g:29 mL.

[0022] Furthermore, the phosphate buffer solution was prepared by adding 1.36 g of potassium dihydrogen phosphate to 79 mL of 0.1 mol / L sodium hydroxide solution, adding ddH2O to make the volume to 200 mL, sterilizing, filtering, and storing at room temperature.

[0023] Beneficial effects of the present invention:

[0024] The enzyme binding stabilizer in the R1 reagent of the present invention contains bovine serum albumin, nanosilver-trehalose complex and PEG4000; bovine serum albumin can prevent the decomposition and non-specific adsorption of the enzyme, and can also chelate a small amount of Ag dissociated from the nanosilver-trehalose complex. + , to prevent Ag + It competes with H2O2 for the binding sites of horseradish peroxidase, ensuring the stable generation of quinoneimine; PEG4000 plays a dispersing role, assisting the nanosilver-trehalose complex to form a protective film on the surface of lactate oxidase and horseradish peroxidase, protecting the protein structure of the two enzymes, increasing the antiseptic effect, and can capture electrons from the surrounding environment, increasing the electron transfer efficiency of the active centers of the enzymes during the catalytic process of the two enzymes, thereby further increasing the activity of the two enzymes, shortening the detection time, improving the sensitivity and accuracy of the kit, reducing the batch difference, and facilitating the monitoring of lactic acid content during cell culture. DETAILED DESCRIPTION

[0025] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts shall fall within the scope of protection of the present invention.

[0026] Example 1

[0027] Prepare phosphate buffer: add 1.36 g potassium dihydrogen phosphate to 79 mL of 0.1 mol / L sodium hydroxide solution, add ddH2O to make up to 200 mL, sterilize, filter, and store at room temperature.

[0028] Prepare 0.5 M Tris buffer: Place 3.03 g of Tris in a flask, then add 400 mL of ddH2O. Stir at room temperature to dissolve, then add 37% hydrochloric acid dropwise to adjust the pH to 7.5. Then, make up to 500 mL with ddH2O. Stir thoroughly, sterilize, filter, and store at room temperature.

[0029] Example 2

[0030] Prepare phosphate buffer: add 1.36 g potassium dihydrogen phosphate to 79 mL of 0.1 mol / L sodium hydroxide solution, add ddH2O to make up to 200 mL, sterilize, filter, and store at room temperature.

[0031] Prepare 0.5 M Tris buffer: Place 3.03 g of Tris in a flask, then add 400 mL of ddH2O. Stir at room temperature to dissolve, then add 37% hydrochloric acid dropwise to adjust the pH to 7.8. Then, make up to 500 mL with ddH2O. Stir thoroughly, sterilize, filter, and store at room temperature.

[0032] Example 3

[0033] Prepare phosphate buffer: add 1.36 g potassium dihydrogen phosphate to 79 mL of 0.1 mol / L sodium hydroxide solution, add ddH2O to make up to 200 mL, sterilize, filter, and store at room temperature.

[0034] Prepare 0.5 M Tris buffer: Place 3.03 g of Tris in a flask, then add 400 mL of ddH2O. Stir at room temperature to dissolve, then add 37% hydrochloric acid dropwise to adjust the pH to 8. Then, make up to 500 mL with ddH2O. Stir thoroughly, sterilize, filter, and store at room temperature.

[0035] Example 4

[0036] The preparation of the enzyme-bound stabilizer comprises the following steps:

[0037] 10 g of trehalose was dissolved in 500 mL of ddH2O, and then 500 mL of 2 mM AgNO3 solution was added and mixed, followed by ultrasonic dispersion for 5 minutes to obtain a mixed solution. The mixed solution was irradiated under a 500 W xenon lamp for 2 hours, centrifuged at 10,000 rpm for 20 minutes, and the precipitate was collected, washed twice with ddH2O, and dried to obtain a nanosilver-trehalose complex.

[0038] 1 g of bovine serum albumin, 5 g of PEG4000 and 5 g of nanosilver-trehalose complex were dissolved in 29 mL of the 0.5 M Tris buffer prepared in Example 1 to obtain an enzyme binding stabilizer.

[0039] Example 5

[0040] The preparation of the enzyme-bound stabilizer comprises the following steps:

[0041] 10 g of trehalose was dissolved in 500 mL of ddH2O, and then 500 mL of 2 mM AgNO3 solution was added and mixed, followed by ultrasonic dispersion for 8 minutes to obtain a mixed solution. The mixed solution was irradiated under a 500 W xenon lamp for 2.5 hours, centrifuged at 12000 r / min for 25 minutes, and the precipitate was collected, washed twice with ddH2O, and dried to obtain a nanosilver-trehalose complex.

[0042] 1 g of bovine serum albumin, 5 g of PEG4000 and 5 g of nanosilver-trehalose complex were dissolved in 29 mL of the 0.5 M Tris buffer prepared in Example 2 to obtain an enzyme binding stabilizer.

[0043] Example 6

[0044] The preparation of the enzyme-bound stabilizer comprises the following steps:

[0045] 10 g of trehalose was dissolved in 500 mL of ddH2O, and then 500 mL of 2 mM AgNO3 solution was added and mixed, followed by ultrasonic dispersion for 10 min to obtain a mixed solution. The mixed solution was irradiated under a 500 W xenon lamp for 3 h, centrifuged at 13000 r / min for 30 min, and the precipitate was collected, washed three times with ddH2O, and dried to obtain a nanosilver-trehalose complex.

[0046] 1 g of bovine serum albumin, 5 g of PEG4000 and 5 g of nanosilver-trehalose complex were dissolved in 29 mL of the 0.5 M Tris buffer prepared in Example 3 to obtain an enzyme binding stabilizer.

[0047] Example 7

[0048] A lactic acid detection kit was prepared. The components of the lactic acid detection kit are shown in Table 1:

[0049] Table 1

[0050]

[0051] Example 8

[0052] A lactic acid detection kit was prepared. The components of the lactic acid detection kit are shown in Table 2:

[0053] Table 2

[0054]

[0055]

[0056] Example 9

[0057] A lactic acid detection kit was prepared. The components of the lactic acid detection kit are shown in Table 3:

[0058] Table 3

[0059]

[0060] Comparative Example 1: Based on Example 8, the nanosilver-trehalose complex contained in the enzyme binding stabilizer in the R1 reagent was replaced with trehalose of the same mass, and the other components and contents remained unchanged to prepare a lactic acid detection kit of the same specifications.

[0061] Comparative Example 2: Based on Example 8, no enzyme binding stabilizer was added to the R1 reagent, the vacant volume was supplemented with 0.5 M Tris buffer prepared in Example 2, and the other components and contents remained unchanged to prepare a lactic acid detection kit of the same specifications.

[0062] Sample treatment: A commercially available lactic acid standard solution with a concentration of 1 mmol / L was taken and divided into 5 equal parts. Four of the lactic acid standards were diluted with ddH2O to volumes of 2-5 times, 10 times, and 50 times the original volume, respectively, to obtain samples with concentrations of 1 mmol / L, 0.5 mmol / L, 0.33 mmol / L, 0.25 mmol / L, 0.2 mmol / L, 0.1 mmol / L, and 0.02 mmol / L, respectively, and labeled as sample 1 to sample 7.

[0063] Experimental method: The above 7 samples were respectively detected using the lactic acid detection kits prepared in Examples 7 to 9, Comparative Example 1, and Comparative Example 2, and a commercially available lactic acid detection kit. The specific steps were as follows: different samples were mixed with the R1 reagent and incubated at 37°C for 3 minutes, and then the R2 reagent was added and mixed and incubated at 37°C for 4 minutes. The samples were detected using a fully automatic biochemical analyzer, and the absorbance value A1 was recorded. The samples were then incubated under the same conditions for 1 minute, and the absorbance value A2 was recorded. The absorbance value of the sample to be tested was calculated (absorbance value of the sample to be tested = A2-A1).

[0064] The standard was incubated with reagents R1 and R2 using the same method and the absorbance of the standard was calculated. The lactic acid concentration in the test sample was then calculated (lactic acid concentration = (absorbance of test sample / absorbance of standard) × 2.22, unit: mmol / L). The results are shown in Table 4:

[0065] Table 4

[0066] project Example 7 Example 8 Example 9 Comparative Example 1 Comparative Example 2 Commercially available Sample 1 1.00 1.00 0.99 0.96 0.95 0.96 Sample 2 0.50 0.50 0.49 0.46 0.44 0.46 Sample 3 0.32 0.32 0.32 0.30 0.27 0.31 Sample 4 0.24 0.25 0.24 0.21 0.18 0.23 Sample 5 0.20 0.19 0.19 0.13 0.10 0.17 Sample 6 0.09 0.1 0.09 0.05 0 0.07 Sample 7 0.02 0.02 0.02 0 0 0

[0067] As can be seen from Table 4, the lactic acid detection kits prepared in Examples 7 to 9 have higher sensitivity and can detect lactic acid samples with lower concentrations under the same incubation conditions.

[0068] It should be noted that, in this document, terms such as "comprises", "includes" or any other variations thereof are intended to cover non-exclusive inclusion, so that a process, method, article or apparatus that includes a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements that are inherent to such process, method, article or apparatus.

[0069] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A lactic acid detection kit, characterized in that Including R1 reagent, R2 reagent and standard; The R1 reagent includes 2000-3000 U / L of lactate oxidase, 15-16 wt% of an enzyme-bound stabilizer, 0.8-0.82 wt% of 4-aminoantipyrine, 0.8-1.2 wt% of sodium azide, 0.8-1.2 wt% of Triton X-100, and the balance is 0.5 M Tris buffer; The R2 reagent includes 2000 U / L horseradish peroxidase, 6.9-6.95 wt% of 4-chlorophenol, 0.8-1.2 wt% of sodium azide, and the balance is phosphate buffer; The standard comprises 2.1 wt % of lithium L-lactate, 0.046 wt % of concentrated sulfuric acid, and the balance ddH2O; The preparation method of the enzyme binding stabilizer is as follows: bovine serum albumin, PEG4000 and nanosilver-trehalose complex are dissolved in 0.5M Tris buffer to obtain the enzyme binding stabilizer; The dosage ratio of bovine serum albumin, PEG4000, nanosilver-trehalose complex, and 0.5 M Tris buffer was 1 g:5 g:5 g:29 mL; The preparation method of the nanosilver-trehalose complex comprises: dissolving trehalose with ddH2O, then adding an AgNO3 solution, mixing, and ultrasonically dispersing for 5-10 minutes to obtain a mixed solution; irradiating the mixed solution under a 500W xenon lamp for 2-3 hours, centrifuging at 10,000-13,000 rpm for 20-30 minutes, collecting the precipitate, washing it with ddH2O for 2-3 times, and drying it to obtain the nanosilver-trehalose complex; The usage ratio of trehalose, ddH2O, and AgNO3 solution is 1g:50mL:50mL.

2. A lactic acid detection kit according to claim 1, characterized in that, The preparation method of 0.5M Tris buffer is as follows: take Tris and place it in a flask, then add ddH2O, stir and dissolve at room temperature, then add hydrochloric acid dropwise to adjust the pH value to 7.5-8, then make up to volume with ddH2O, stir and mix, sterilize, filter, and obtain 0.5M Tris buffer and store at room temperature.

3. A lactic acid detection kit according to claim 1, characterized in that, The preparation method of phosphate buffer is as follows: potassium dihydrogen phosphate is added to sodium hydroxide solution, and ddH2O is added to make the volume to 200 mL. The phosphate buffer is sterilized and filtered to obtain the phosphate buffer and stored at room temperature.

4. The method for preparing a lactic acid detection kit according to any one of claims 1 to 3, wherein: The steps include: Take a 5 mL reagent tube and add 0.5 M Tris buffer, then add 4-aminoantipyrine, sodium azide, Triton X-100, and an enzyme binding stabilizer. Mix well and then add lactate oxidase. Pipette evenly to obtain reagent R1. Take a 3 mL reagent tube, add phosphate buffer, then add 4-chlorophenol and sodium azide, mix well, then add horseradish peroxidase, and pipette to mix evenly to obtain R2 reagent; Dissolve lithium lactate with ddH2O, then add concentrated sulfuric acid, and finally dilute to volume with ddH2O according to a preset ratio to obtain a standard solution. Pipette 1 mL of the standard solution into a 3 mL reagent tube to obtain a standard product.

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