CN113846142A - Strong alkaline biochemical reagent, preparation method and application - Google Patents
Strong alkaline biochemical reagent, preparation method and application Download PDFInfo
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- CN113846142A CN113846142A CN202111301777.8A CN202111301777A CN113846142A CN 113846142 A CN113846142 A CN 113846142A CN 202111301777 A CN202111301777 A CN 202111301777A CN 113846142 A CN113846142 A CN 113846142A
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
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- C12Q1/42—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving phosphatase
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- G01N2333/916—Hydrolases (3) acting on ester bonds (3.1), e.g. phosphatases (3.1.3), phospholipases C or phospholipases D (3.1.4)
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Abstract
The embodiment of the application belongs to the technical field of medical examination and determination, and relates to a strong-alkaline biochemical reagent, a preparation method and application thereof, wherein the strong-alkaline biochemical reagent comprises 50-2000 mM of strong-alkaline buffer solution and 10-30 v/v% of volatile water-soluble organic solvent. The application effectively prevents the situation that the measurement sensitivity is reduced due to the change of pH when the reagent is used in the open bottle of the conventional biochemical reagent.
Description
Technical Field
The application relates to the technical field of medical examination and determination, in particular to a strong alkaline biochemical reagent, a preparation method and application.
Background
The use time of the biochemical reagent on an instrument loader can be as long as 1-2 weeks, the biochemical reagent containing the strong alkaline buffer solution can absorb carbon dioxide in the external environment when the instrument loader is opened, so that the measurement sensitivity is gradually reduced, and during the period, frequent calibration is needed to ensure the stability and reliability of the measurement result. How to effectively reduce or prevent the biochemical reagent containing the high-alkaline buffer solution from absorbing external carbon dioxide becomes a difficult problem.
The existing solution is to add a substance which is difficult to dissolve in water into the reagent, and the substance which is difficult to dissolve in water can spread on the liquid surface of the reagent to block the contact of the reagent and air, so as to block the reagent from absorbing carbon dioxide. However, when the instrument sucks the reagent, the insoluble substance is inevitably sucked, and the measurement accuracy is influenced because the insoluble substance and the reactant reagent form heterogeneous phase.
Disclosure of Invention
The embodiment of the application aims to provide a strong-alkaline biochemical reagent, a preparation method and application, and the situation that the measurement sensitivity is reduced due to pH change when the reagent is used after being opened is effectively prevented.
In order to solve the above technical problem, an embodiment of the present application provides a method for stabilizing a strongly basic reagent, which adopts the following technical scheme:
a strongly alkaline biochemical reagent comprises a strongly alkaline buffer solution of 50 mM-2000 mM and a volatile water-soluble organic solvent of 10-30 v/v%.
Further, the strongly basic buffer is N-methyl-D-glucosamine buffer, 2-amino-2-methyl-1-propanol buffer, 2-aminoethanol, 3- (cyclohexylamino) -2-hydroxy-1-propanesulfonic acid or 3- (cyclohexylamino) -1-propanesulfonic acid.
Further, the volatile water-soluble organic solvent is methanol, ethanol, acetonitrile or tetrahydrofuran.
Further, the volatile water-soluble organic solvent is methanol or ethanol.
The application also discloses a preparation method of the strong-alkaline biochemical reagent, which comprises the following steps:
preparing a strong alkaline buffer solution: weighing corresponding buffer components and biochemical reagent components, and adding purified water to fully dissolve to prepare strong-base buffer solution with the concentration of 50 mM-2000 mM;
adding a volatile water-soluble organic solvent and fully mixing uniformly, wherein the concentration of the organic solvent is 10-30 v/v%;
and (5) debugging the reagent to a preset pH value, and adding purified water to a constant volume.
Further, the strongly basic buffer is N-methyl-D-glucosamine buffer, 2-amino-2-methyl-1-propanol buffer, 2-aminoethanol, 3- (cyclohexylamino) -2-hydroxy-1-propanesulfonic acid or 3- (cyclohexylamino) -1-propanesulfonic acid.
Further, the volatile water-soluble organic solvent is methanol, ethanol, acetonitrile or tetrahydrofuran.
Further, the volatile water-soluble organic solvent is methanol or ethanol.
In addition, the strong alkaline biochemical reagent is applied to a calcium determination reagent for determining calcium ions in human body fluid.
The strong alkaline biochemical reagent is also applied to an alkaline phosphatase determination reagent for determining phosphatase in human body fluid.
Compared with the prior art, the embodiment of the application mainly has the following beneficial effects:
this application is applied to conventional biochemical reagent's use with the strong alkaline reagent who contains the organic solvent of volatile water-solubility, can effectively slow down the rate that this conventional biochemical reagent uncork in-process absorbed carbon dioxide, and then realizes restraining this conventional biochemical reagent's pH value and descend, stabilizes this conventional biochemical reagent. In addition, the water-soluble volatile organic solvent is added into the strong alkaline reagent, can be fully dissolved in the strong alkaline reagent and the conventional biochemical reagent, is a homogeneous system, and does not influence the test process in actual operation.
Drawings
In order to more clearly illustrate the solution of the present application, the drawings needed for describing the embodiments of the present application will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present application, and that other drawings can be obtained by those skilled in the art without inventive effort.
FIG. 1 is a flow chart of a method of preparing a strongly basic biochemical reagent according to the present application;
FIG. 2 is a graphical representation of the trend of the calcium assay reagent on-board-opener test results of the present application;
FIG. 3 is a graph showing the trend of the results of the on-board alkaline phosphatase assay reagent of the present application.
Detailed Description
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs; the terminology used in the description of the application herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application; the terms "including" and "having," and any variations thereof, in the description and claims of this application and the description of the above figures are intended to cover non-exclusive inclusions. The terms "first," "second," and the like in the description and claims of this application or in the above-described drawings are used for distinguishing between different objects and not for describing a particular order.
Reference herein to "an embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment can be included in at least one embodiment of the application. The appearances of the phrase in various places in the specification are not necessarily all referring to the same embodiment, nor are separate or alternative embodiments mutually exclusive of other embodiments. It is explicitly and implicitly understood by one skilled in the art that the embodiments described herein can be combined with other embodiments.
The following examples are presented to facilitate a better understanding of the present application and are not intended to limit the present application. The experimental procedures in the following examples are conventional unless otherwise specified. The experimental materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified.
In order to make the technical solutions better understood by those skilled in the art, the technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the accompanying drawings.
In some conventional biochemical reactions, the reaction must be carried out while being maintained under strongly alkaline conditions, such as a calcium ion assay reagent (OCPC method) and an alkaline phosphatase assay reagent (AMP method). In these reagents, a strongly basic buffer is often used to maintain the alkaline condition of the reaction, and when the reagent is used in an open bottle, external carbon dioxide continuously enters the reagent, so that the pH of the strongly basic buffer is reduced, and the measurement sensitivity of the reagent is also reduced.
The application provides a strong-alkaline biochemical reagent which can be applied to a calcium determination reagent for determining calcium ions in human body fluid and an alkaline phosphatase determination reagent for determining phosphatase in human body fluid.
1. The strong alkaline biochemical reagent comprises 50 mM-2000 mM strong alkaline buffer solution and 10-30 v/v% volatile water-soluble organic solvent. Among these, examples of the strongly basic buffer include N-methyl-D-glucosamine buffer, 2-amino-2-methyl-1-propanol buffer, 2-aminoethanol, 3- (cyclohexylamino) -2-hydroxy-1-propanesulfonic acid, and 3- (cyclohexylamino) -1-propanesulfonic acid. The volatile water-soluble organic solvent is methanol, ethanol, acetonitrile or tetrahydrofuran. Methanol or ethanol is preferred.
It should be noted that: the selection of the volatile water-soluble organic solvent can be used according to actual conditions, and through the screening of different concentrations, the requirement that the substance to be detected is not interfered in the determination and the reagent is effectively prevented from absorbing carbon dioxide can be taken as the basis for selection.
The application can reduce the rate of carbon dioxide absorption of the reagent by adding one to two volatile water-soluble organic solvents to the biochemical reagent containing the strong alkaline buffer solution (namely the strong alkaline biochemical reagent).
2. The general components and concentrations of the above strongly basic biochemical reagents are as follows:
secondly, the preparation method of the strong alkaline biochemical reagent is shown in fig. 1, and fig. 1 is a flow chart of the preparation method of the strong alkaline biochemical reagent of the present application. The specific process is as follows:
s1: weighing corresponding buffer components and biochemical reagent components, and adding purified water to fully dissolve to prepare strong-base buffer solution with the concentration of 50 mM-2000 mM;
s2: adding a volatile water-soluble organic solvent and fully mixing uniformly, wherein the concentration of the organic solvent is 10-30 v/v%;
s3: and (5) debugging the reagent to a preset pH value, and adding purified water to a constant volume.
Thirdly, the strong alkaline biochemical reagent can be applied to conventional biochemical reagents, such as a calcium determination reagent for determining calcium ions in human body fluid and an alkaline phosphatase determination reagent for determining phosphatase in human body fluid. The volatile water-soluble organic solvent in the strong alkaline biochemical reagent effectively slows down the absorption of carbon dioxide by the calcium determination reagent and the alkaline phosphatase determination reagent, the calcium determination reagent and the alkaline phosphatase determination reagent are matched with a full-automatic biochemical analyzer to determine the components in the body fluid of a human body, and the specific experimental data are as follows:
1. the calcium determination reagent comprises the following components in percentage by concentration:
wherein the final concentrations of ethanol in the strongly basic biochemical reagent added to the first calcium ion measuring reagent (R1) were 0%, 10%, 20%, and 30%, respectively.
The measurements were performed according to the following procedure. Using an automatic analyzer (Hitachi 7180 type), 3.0. mu.L of a sample and 160. mu.L of the first calcium ion measuring reagent (R1) were incubated at 37 ℃ for 5 minutes, and 80. mu.L of the second calcium ion measuring reagent (R2) was added, followed by further incubation for 5 minutes. The amount of change in absorbance at the main wavelength of 570nm and the sub-wavelength of 660nm was measured 5 minutes and 10 minutes after the sample and the reagent were mixed, respectively. After calibration with physiological saline and standard solution, the reagent bottles were kept open in the reagent storage of the autoanalyzer, and the test quality control was performed on days 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20, respectively, with detailed results as shown in table 1 and fig. 2, fig. 2 is a graph showing the trend of the test results of the calcium determination reagent in the open-bottle and on-carrier test, with the abscissa being the number of days and the ordinate being the result of the calcium determination reagent in the open-bottle and on-carrier test.
TABLE 1 calcium determination reagent test data on-board machine for bottle opening
As is clear from table 1 and fig. 1, the on-board bottle opening stability decreased rapidly compared to the calcium ion measurement reagent without ethanol, while the calcium ion measurement reagent containing ethanol had improved stability.
2. The alkaline phosphatase determination reagent comprises the following components in percentage by weight:
the final concentrations of methanol in the strongly basic biochemical reagent added to the first phosphatase measurement reagent (R1') were 0%, 20%, and 30%, respectively.
The measurements were performed according to the following procedure. Using an automatic analyzer (Hitachi 7180 type), 2.0. mu.L of a sample and 160. mu.L of a first phosphatase measuring reagent (R1 ') were incubated at 37 ℃ for 5 minutes, 40. mu.L of a second phosphatase measuring reagent (R2') was added, and then the change rates at the main wavelength of 405nm and the sub-wavelength of 700nm were monitored over 5 minutes. After calibration with physiological saline and a calibration solution, the reagent bottles containing the alkaline phosphatase measurement reagent were kept open in the reagent storage of the autoanalyzer, and the quality control was performed on days 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20, respectively, as shown in table 2 and fig. 3, and fig. 3 is a graph showing the trend of the results of the on-board test of the alkaline phosphatase measurement reagent, with the abscissa in days and the ordinate in the on-board test of the alkaline phosphatase measurement reagent.
TABLE 2 alkaline phosphatase determination reagent on-board test data
The results showed that the on-board bottle opening stability decreased rapidly compared to the alkaline phosphatase measurement reagent without methanol addition, while the alkaline phosphatase measurement reagent stability was significantly improved for the methanol-containing alkaline phosphatase measurement reagent.
3. The present application also provides another biochemical reagent of the strongly basic buffer, referred to as a target strongly basic reagent, formulated as follows:
wherein the final concentrations of ethanol in the strongly basic biochemical reagent added to the target strongly basic reagent (R1') were 0%, 20%, and 30%, respectively.
The reagent bottles containing the target strongly basic reagent were kept open in the reagent storage of the autoanalyzer, and the change in pH of the target strongly basic reagent was measured on days 0, 2, 4, 6, 8, 10, 12 and 14, and the detailed results are shown in Table 3.
TABLE 3 determination of the pH of strongly basic reagent on-board conveyor
The result shows that the target strong alkaline reagent containing ethanol is always in an open bottle state, the pH drop rate is obviously lower than that of the target strong alkaline reagent without the ethanol, and the fact that a certain amount of ethanol is added into the target strong alkaline reagent can effectively slow down the rate of the target strong alkaline reagent for absorbing carbon dioxide.
This application is applied to conventional biochemical reagent's use with the strong alkaline reagent who contains the organic solvent of volatile water-solubility, can effectively slow down the rate that this conventional biochemical reagent uncork in-process absorbed carbon dioxide, and then realizes restraining this conventional biochemical reagent's pH value and descend, stabilizes this conventional biochemical reagent. In addition, the water-soluble volatile organic solvent is added into the strong alkaline reagent, can be fully dissolved in the strong alkaline reagent and the conventional biochemical reagent, is a homogeneous system, and does not influence the test process in actual operation.
The foregoing description discloses the product, use, method of use, and advantages of the present invention, and it should be noted that the above-mentioned embodiments are only examples for clarity of disclosure and are not intended to limit the claims of this patent. It will be appreciated by those skilled in the art that changes may be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims.
It should be understood that, although the steps in the flowcharts of the figures are shown in order as indicated by the arrows, the steps are not necessarily performed in order as indicated by the arrows. The steps are not performed in the exact order shown and may be performed in other orders unless explicitly stated herein. Moreover, at least a portion of the steps in the flow chart of the figure may include multiple sub-steps or multiple stages, which are not necessarily performed at the same time, but may be performed at different times, which are not necessarily performed in sequence, but may be performed alternately or alternately with other steps or at least a portion of the sub-steps or stages of other steps.
It is to be understood that the above-described embodiments are merely illustrative of some, but not restrictive, of the broad invention, and that the appended drawings illustrate preferred embodiments of the invention and do not limit the scope of the invention. This application is capable of embodiments in many different forms and is provided for the purpose of enabling a thorough understanding of the disclosure of the application. Although the present application has been described in detail with reference to the foregoing embodiments, it will be apparent to one skilled in the art that the present application may be practiced without modification or with equivalents of some of the features described in the foregoing embodiments. All equivalent structures made by using the contents of the specification and the drawings of the present application are directly or indirectly applied to other related technical fields and are within the protection scope of the present application.
Claims (10)
1. A strongly alkaline biochemical reagent is characterized by comprising 50 mM-2000 mM strongly alkaline buffer solution and 10-30 v/v% volatile water-soluble organic solvent.
2. The strongly basic biochemical reagent according to claim 1, wherein the strongly basic buffer is N-methyl-D-glucosamine buffer, 2-amino-2-methyl-1-propanol buffer, 2-aminoethanol, 3- (cyclohexylamino) -2-hydroxy-1-propanesulfonic acid or 3- (cyclohexylamino) -1-propanesulfonic acid.
3. The strongly basic biochemical reagent according to claim 1, wherein the volatile water-soluble organic solvent is methanol, ethanol, acetonitrile or tetrahydrofuran.
4. The strongly basic biochemical reagent according to claim 1, wherein the volatile water-soluble organic solvent is methanol or ethanol.
5. The preparation method of the strong-alkaline biochemical reagent is characterized by comprising the following steps of:
preparing a strong alkaline buffer solution: weighing corresponding buffer components and biochemical reagent components, and adding purified water to fully dissolve to prepare strong-base buffer solution with the concentration of 50 mM-2000 mM;
adding a volatile water-soluble organic solvent and fully mixing uniformly, wherein the concentration of the organic solvent is 10-30 v/v%;
and (5) debugging the reagent to a preset pH value, and adding purified water to a constant volume.
6. The method of stabilizing highly basic reagents according to claim 5, wherein said highly basic buffer is N-methyl-D-glucosamine buffer, 2-amino-2-methyl-1-propanol buffer, 2-aminoethanol, 3- (cyclohexylamino) -2-hydroxy-1-propanesulfonic acid or 3- (cyclohexylamino) -1-propanesulfonic acid.
7. The method of stabilizing highly basic reagents according to claim 5, wherein said volatile water-soluble organic solvent is methanol, ethanol, acetonitrile or tetrahydrofuran.
8. The method of stabilizing highly basic reagents according to claim 5, wherein said volatile water-soluble organic solvent is methanol or ethanol.
9. The strongly basic biochemical reagent according to any one of claims 1 to 4, which is used as a calcium assay reagent for assaying calcium ions in human body fluids.
10. The use of a strongly basic biochemical reagent according to any one of claims 1 to 4 as an alkaline phosphatase assay reagent for the determination of phosphatase in human body fluids.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114672538A (en) * | 2022-05-09 | 2022-06-28 | 潍坊三维生物工程集团有限公司 | Method for improving bottle opening stability of alkaline detection reagent |
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| CN103562368A (en) * | 2011-03-04 | 2014-02-05 | 美国消毒器公司 | Broad spectrum disinfectant |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114672538A (en) * | 2022-05-09 | 2022-06-28 | 潍坊三维生物工程集团有限公司 | Method for improving bottle opening stability of alkaline detection reagent |
| CN114672538B (en) * | 2022-05-09 | 2024-05-10 | 潍坊三维生物工程集团有限公司 | Method for improving stability of alkaline detection reagent in bottle opening |
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