CN110715923A - alpha-D-glucose detection kit - Google Patents
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- CN110715923A CN110715923A CN201911148954.6A CN201911148954A CN110715923A CN 110715923 A CN110715923 A CN 110715923A CN 201911148954 A CN201911148954 A CN 201911148954A CN 110715923 A CN110715923 A CN 110715923A
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
- G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
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Abstract
The invention discloses an alpha-D-glucose detection kit, belonging to a method for testing materials by testing the color change of reaction results by using visible light. The technical scheme of the invention is as follows: the reagent II only contains an effective component of mutarotase, the glucose chromogen and the tool enzyme are in the reagent I, and because red quinonimine generated by beta-D-glucose and red quinonimine generated by alpha-D-glucose as beta-D-glucose in body fluid are sequentially colored, the red quinonimine generated by the reaction of the reagent I is used as blank, and the content of alpha-D-glucose is calculated by the red quinonimine generated by the reaction of the reagent II.
Description
Technical Field
The present invention pertains to a method of assay comprising an enzyme; or a method for testing materials by using visible light and generating color change through the result of test reaction, in particular to a method for quickly and accurately detecting alpha-D-glucose in body fluid by using a biochemical analyzer.
Background
D-glucose is characterized in that-OH on a C atom is on the right, is also called dextrose, has an open chain structure and a ring structure, has alpha-and beta-isomers, exists in free states in grapes, honey, sweet fruits, animal blood, spinal fluid, lymph fluid and the like, is widely existed in nature as a polysaccharide component and a glycoside form, and is obtained by hydrolyzing starch. The physical properties are white crystalline powder, relative density is 1.544(25 ℃), melting point of alpha-D-glucose is 146 ℃, [ alpha ]]D20 is +113.4 DEG, the melting point of beta-D-glucose is 148-150 DEG, [ alpha ]]D20 was +19.2 °. When the two solutions are placedOf which [ alpha ]]D20 was finally +52.5 °. Soluble in water, slightly soluble in ethanol, and approximately 70% as sweet as sucrose. The chemical properties are as follows: d-glucose has the chemical property of common aldose, and can generate gluconic acid, glucaric acid or glucuronic acid under the action of an oxidant; under the action of a reducing agent, sorbitol is generated; under the action of weak base, glucose and other two kinds of six-carbon sugar (fructose and mannose) with similar structures can be mutually converted through enol. The molecular formula of glucose is C6H12O6The aldohexose has five hydroxyl groups and one aldehyde group in the molecule. Wherein C-2, C-3, C-4 and C-5 are different chiral carbon atoms, there are 16 optically active isomers, and D-glucose is one of them. Glucose exists in a cyclic hemiacetal structure, after cyclization, the original carbonyl carbon atom (C-1) is changed into a chiral carbon atom, and the arrangement mode of a hemiacetal hydroxyl group newly formed on the C-1 in space has two possibilities. The hemiacetal hydroxyl group is called α -form on the same side of the carbon chain as the hydroxyl group (hydroxyl group on C-5) that determines the monosaccharide configuration, and β -form on the opposite side. The alpha and beta forms are diastereomers. They differ in their C-1 configuration and are therefore also called anomers (anomers). The phenomenon of glucose rotation, which is different in both melting point and specific optical rotation, is caused by the specific optical rotation change during the formation of an equilibrium system of an open chain structure and a cyclic structure. In the solution, alpha-D-glucose can be converted into an open chain structure and then converted into beta-D-glucose from the open chain structure, and similarly, the beta-D-glucose can be converted into the open chain structure and then converted into the alpha-D-glucose. After a period of time, the three isomers reach equilibrium, a tautomeric equilibrium system is formed, and the specific optical rotation of the tautomeric equilibrium system is not changed. Not only the grape has the phenomenon of despinification, but also monosaccharides which can form a ring structure can generate the phenomenon of despinification. the-OH of the alpha-D-glucopyranose except the C-1 is connected to the alpha bond, and the-OH and the-CH on the other three carbons2OH is all attached to the e bond, and-OH and all relatively large radicals (-OH, -CH) on β -D glucopyranose C-12OH) are attached to the e bond, and the beta conformation is more stable, as shown in figure 1. Thus, at equilibrium in solution, the beta form is 64% and the alpha form is only 36%.
In factThe glucose oxidase method, the hexokinase method and the glucose dehydrogenase method are commonly used in laboratory glucose determination methods, the total glucose content is determined in the methods and comprises the sum of alpha-D-glucose and beta-D-glucose, wherein the most commonly used method in the laboratory is the glucose oxidase method, mutarotase is contained in a determination reagent, the mutarotase converts the alpha-D-glucose into the beta-D-glucose, and the glucose oxidase oxidizes the beta-D-glucose into D-glucose acid and H-glucose acid2O2The hexokinase method can phosphorylate alpha-D-glucose and beta-D-glucose at the same time, and the glucose dehydrogenase can dehydrogenize alpha-D-glucose and beta-D-glucose at the same time.
In the existing detection methods, Lili and reported a method for determining alpha-D-glucoside in body fluid, and the patent application number is CN 2019110300700; methods for measuring sucrose in serum, patent application No. 201810318573.7; a method for measuring glucose oxidase in serum by using a double reagent is disclosed in patent application No. 201510168446. In the glucose measurement method, the measured glucose result includes the sum of alpha-D-glucose and beta-D-glucose, and the alpha-D-glucose measurement method has not been reported, and the vast majority of the alpha-D-glucose in the blood of a human body is beta-D-glucose, and the alpha-D-glucose mainly exists in glucose solution which is from food and is just prepared shortly after eating.
The content of the invention is as follows: the reagent II only contains an effective component of mutarotase, glucose chromogen and tool enzyme are in the reagent I, red quinonimine generated by beta-D-glucose in body fluid and red quinonimine generated by alpha-D-glucose after converting beta-D-glucose are colored in sequence, the red quinonimine generated by the reaction of the reagent I is used as blank, and the content of alpha-D-glucose is calculated by the red quinonimine generated by the reaction of the reagent II.
The reaction formula is as follows:
the method is not influenced by beta-D-glucose in body fluid during detection, and the beta-D-glucose in the body fluid can be reacted after being added with the reagent I, so that the beta-D-glucose can not participate in the subsequent reaction. After the reagent II is added, the alpha-D-glucose begins to react, so that the influence of the beta-D-glucose in body fluid is eliminated, and the detection data can truly reflect the content of the alpha-D-glucose. The determination method of the invention can be automated, reduce the detection cost and time, and improve the detection efficiency.
The technical scheme adopted by the invention is as follows: the reagent I contains main components such as glucose oxidase, 4-aminoantipyrine, 2, 4-dichlorophenol, peroxidase and the like and a proper amount of preservative; the reagent II only contains mutarotase and a proper amount of preservative; the determination method comprises the following steps: the body fluid (blood or urine) and a reagent I are subjected to warm bath at 37 ℃ for 3-5 minutes, beta-D-glucose in the body fluid reacts with the reagent I to generate D-gluconic acid and hydrogen peroxide, the hydrogen peroxide reacts with 4-aminoantipyrine and 2, 4-dichlorophenol under the catalysis of peroxidase to generate red quinonimine and water, after the reagent II is added, alpha-D-glucoside in the body fluid generates beta-D-glucose under the action of mutarotase, the beta-D-glucose reacts with the reagent I to generate D-gluconic acid and hydrogen peroxide, the hydrogen peroxide reacts with 4-aminoantipyrine and 2, 4-dichlorophenol under the catalysis of peroxidase to generate red quinonimine and water, the detection is carried out at the wavelength of 500-540 nm, and the red quinonimine generated by the reaction of the reagent I is used as a reaction blank, calculating the content of alpha-D-glucose in the body fluid by using red quinonimine generated by the reaction of the reagent II.
Description of the drawings:
FIG. 1 is a schematic diagram showing the structure and interconversion of alpha-D-glucose and beta-D-glucose
FIG. 2 is a real-time reaction curve of the alpha-D-glucose determination reaction
The specific implementation mode is as follows:
the present invention will be described in further detail by way of examples.
Example 1
The composition of the reagent is as follows:
a. reagent I: each liter of 0.25mmol/L potassium phosphate buffer (pH 7.5, 25 ℃) contains 1.0KU of ascorbic acid oxidase, 12KU of glucose oxidase, 1.2KU of peroxidase, 0.60mmol of 4-aminoantipyrine, 1.6mmol of 2, 4-dichlorophenol and 1.8978. mu.L of Proclin-300200. mu.L.
b. And (2) reagent II: per liter of 0.2mmol/L potassium phosphate buffer (pH 7.0, 25 ℃) contains 4.0KU of mutarotase, Proclin-300200. mu.L.
c. Standard solution: 11.0mmol/L glucose solution.
Wherein, the ascorbic acid oxidase can eliminate the interference reaction of vitamin C and hydrogen peroxide, and Proclin-300 is a liquid high-efficiency preservative.
Example 2.
2, 4-dichlorophenol is changed into 4-chlorophenol in the reagent I. The content is unchanged.
Example 3
Measurement procedure
The two-reagent method: on a Beckmann AU5800 full-automatic biochemical analyzer in America, 3 mul of a sample is automatically added into 300 mul of a reagent I by the analyzer to be mixed uniformly, incubation is carried out for 3 minutes at 37 ℃, 60 mul of a reagent II is added to be mixed uniformly, incubation is carried out for 5.1 minutes at 37 ℃, and the full-automatic analyzer detects at the wavelength of 500 nm. The instrument automatically calculated the alpha-D-glucose results, see Table 1 for details.
TABLE 1 test conditions for the automated analyzer of the present invention
Reaction ODalpha-D-glucoseCalculated value = OD2OD1×[（SV+R1V1）/ (SV+R1V1+R2V2)]
α -D-glucose concentration = FXODalpha-D-glucose
Wherein ODalpha-D-glucoseIs the absorbance produced by alpha-D-glucose. OD1Is the absorbance, OD, measured after the sample is added with the reagent I for reaction2Is the absorbance measured after the sample is added into the reagent II for reaction, SV is the volume of the serum sample, R1V1Is the volume of reagent I, R2V2Is the volume of reagent II. F is the correction factor.
OD after addition of reagent II1Has a dilution ratio of [ (SV + R)1V1）/ (SV+R1V1+R2V2)]The absorbance after adding the reagent II is OD1×[（SV+R1V1）/(SV+R1V1+R2V2)]. The absorbance of the quinoneimine produced from alpha-D-glucose is therefore: ODalpha-D-glucose= OD2- OD1×[（SV+R1V1）/(SV+R1V1+R2V2)]I.e. ODalpha-D-glucose= OD2–(3+300)/(3+300+60)×OD1. As long as OD is measured1And OD2The concentration of alpha-D-glucose can be calculated as shown in FIG. 2.
Example 3
The performance parameters of the process of the invention are illustrated below by methodological verification.
1. The determination method comprises the steps of 3 mul of sample, 300 mul of reagent I and 60 mul of reagent II. The sample and the reagent I are subjected to warm bath for 3 minutes at 37 ℃, the reagent II is added for reaction for 5.1 minutes, and the end point method at the wavelength of 500nm is used for measurement.
2. The instrument comprises the following steps: an AU5800 full-automatic biochemical analyzer of Beckmann USA.
3. The method has the following performance parameters: the precision in the batch is less than 3.2 percent, the precision between the batches is less than 5.0 percent, the linear range is 0.18 to 11.0mmol/L, the reported range is 0.80 to 20.5mmol/L, the blank absorbance is less than 0.11(0.5cm,37 ℃, 505nm), and the absorbance change of the sample with the sensitivity of 5.50mmol/L is more than or equal to 0.56. Hemolysis is less than 300mg/dl, chyle is less than 1.20%, ascorbic acid is less than 20mg/dl, and total bilirubin is less than 30 mg/dl, which has no interference to the measurement result.
alpha-D-glucose generates equimolar beta-D-glucose through mutarotase action, and the content of the alpha-D-glucose is converted according to a measured beta-D-glucose result by a novel reagent combination mode. Therefore, the invention can measure the alpha-D-glucose in serum by changing the components of the reagents I and II.
Claims (5)
1. The invention relates to an alpha-D-glucose detection kit, which is characterized in that a reagent II only contains an effective component of mutarotase, a glucose chromogen and a tool enzyme are in the reagent I, red quinonimine generated by beta-D-glucose in body fluid and red quinonimine generated by converting alpha-D-glucose into beta-D-glucose are sequentially colored, the red quinonimine generated by the reaction of the reagent I is used as a blank, and the content of alpha-D-glucose is calculated by the red quinonimine generated by the reaction of the reagent II.
2. The kit for detecting alpha-D-glucose according to claim 1, wherein the reagent I: 0.25mmol/L potassium phosphate buffer (pH 7.5, 25 ℃) contains 0.6-1.4 KU of ascorbic acid oxidase, 8-16 KU of glucose oxidase, 8-1.6 KU of peroxidase, 0.2-1.0 mmol of 4-aminoantipyrine, 1.0-2.0 mmol of 2, 4-dichlorophenol and 100-300 muL of Proclin-3000, and reagent II: 0.25mmol/L potassium phosphate buffer (pH 7.0, 25 ℃) contains 2.0 to 6.0KU of mutarotase and 100 to 300. mu.L of Proclin-300.
3. The kit for detecting alpha-D-glucose as claimed in claim 1, wherein the preservatives in the reagent I and the reagent II are selected from Proclin-300.
4. The kit for detecting alpha-D-glucose as claimed in claim 1, wherein the pH of Tris-HCl buffer solution in reagent I and reagent II is 7.5 ± 0.2.
5. The alpha-D-glucose assay kit according to claim 1, wherein the volume ratio of each substance used for the assay is: the ratio of the sample to the reagent I to the reagent II is 1: 80-120: 10-30.
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| US5278047A (en) * | 1989-04-25 | 1994-01-11 | Lilja Jan E | Method of analysis, reagent composition and use thereof for glucose determination |
| US5866349A (en) * | 1989-04-25 | 1999-02-02 | Lilja; Jan Evert | Method for determination of glucose in whole blood and cuvette and photometer for carrying out said method |
| US20040028608A1 (en) * | 2000-08-01 | 2004-02-12 | Saul Steven J | Hygiene monitoring |
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| CN104237147A (en) * | 2014-09-26 | 2014-12-24 | 天津市宝坻区人民医院 | Method for measuring lactose in body fluid |
| CN205404417U (en) * | 2016-02-24 | 2016-07-27 | 天津市宝坻区人民医院 | Glucose detect reagent box |
| CN107884401A (en) * | 2017-11-13 | 2018-04-06 | 天津市宝坻区人民医院 | Eliminate the glucose oxidase assay method of piarhemia interference |
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2019
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Patent Citations (12)
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| US5278047A (en) * | 1989-04-25 | 1994-01-11 | Lilja Jan E | Method of analysis, reagent composition and use thereof for glucose determination |
| US5866349A (en) * | 1989-04-25 | 1999-02-02 | Lilja; Jan Evert | Method for determination of glucose in whole blood and cuvette and photometer for carrying out said method |
| US20040028608A1 (en) * | 2000-08-01 | 2004-02-12 | Saul Steven J | Hygiene monitoring |
| US7264962B1 (en) * | 2005-03-14 | 2007-09-04 | Sandia Corporation | Enzymatic cascade bioreactor |
| CN101655493A (en) * | 2008-08-20 | 2010-02-24 | 中国科学院成都有机化学有限公司 | Colorimetric analysis method for measuring content of glucose and activity of glucose oxidase |
| CN101386882A (en) * | 2008-09-28 | 2009-03-18 | 天津市宝坻区人民医院 | Kit for detecting glucose by hexokinase method and preparation method |
| CN103088108A (en) * | 2012-11-16 | 2013-05-08 | 李立和 | Kit for detecting glucose by using glucose dehydrogenase method and preparation method |
| CN104237147A (en) * | 2014-09-26 | 2014-12-24 | 天津市宝坻区人民医院 | Method for measuring lactose in body fluid |
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| CN107884401A (en) * | 2017-11-13 | 2018-04-06 | 天津市宝坻区人民医院 | Eliminate the glucose oxidase assay method of piarhemia interference |
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