CN116297435A - Acetoacetic acid content detection kit, application method thereof and detection device - Google Patents
Acetoacetic acid content detection kit, application method thereof and detection device Download PDFInfo
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- CN116297435A CN116297435A CN202310542247.5A CN202310542247A CN116297435A CN 116297435 A CN116297435 A CN 116297435A CN 202310542247 A CN202310542247 A CN 202310542247A CN 116297435 A CN116297435 A CN 116297435A
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- WDJHALXBUFZDSR-UHFFFAOYSA-N Acetoacetic acid Natural products CC(=O)CC(O)=O WDJHALXBUFZDSR-UHFFFAOYSA-N 0.000 title claims abstract description 95
- WOWBFOBYOAGEEA-UHFFFAOYSA-N diafenthiuron Chemical compound CC(C)C1=C(NC(=S)NC(C)(C)C)C(C(C)C)=CC(OC=2C=CC=CC=2)=C1 WOWBFOBYOAGEEA-UHFFFAOYSA-N 0.000 title claims abstract description 72
- 238000000034 method Methods 0.000 title claims abstract description 32
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 159
- 239000003795 chemical substances by application Substances 0.000 claims abstract description 32
- WHBMMWSBFZVSSR-UHFFFAOYSA-N 3-hydroxybutyric acid Chemical compound CC(O)CC(O)=O WHBMMWSBFZVSSR-UHFFFAOYSA-N 0.000 claims abstract description 28
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- BOLDJAUMGUJJKM-LSDHHAIUSA-N renifolin D Natural products CC(=C)[C@@H]1Cc2c(O)c(O)ccc2[C@H]1CC(=O)c3ccc(O)cc3O BOLDJAUMGUJJKM-LSDHHAIUSA-N 0.000 description 5
- 238000013112 stability test Methods 0.000 description 4
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 3
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- JUJBNYBVVQSIOU-UHFFFAOYSA-M sodium;4-[2-(4-iodophenyl)-3-(4-nitrophenyl)tetrazol-2-ium-5-yl]benzene-1,3-disulfonate Chemical compound [Na+].C1=CC([N+](=O)[O-])=CC=C1N1[N+](C=2C=CC(I)=CC=2)=NC(C=2C(=CC(=CC=2)S([O-])(=O)=O)S([O-])(=O)=O)=N1 JUJBNYBVVQSIOU-UHFFFAOYSA-M 0.000 description 2
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- G—PHYSICS
- G01—MEASURING; TESTING
- 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
- G01—MEASURING; TESTING
- 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
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
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- G—PHYSICS
- G01—MEASURING; TESTING
- 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
- G01N2021/775—Indicator and selective membrane
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- General Physics & Mathematics (AREA)
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Abstract
The invention discloses an acetoacetic acid content detection kit, a using method and a detection device thereof. Wherein, acetoacetic acid content detection kit includes R1 reagent, R2 reagent, R3 reagent and R4 reagent, and wherein, R1 reagent includes: beta-hydroxybutyrate dehydrogenase, reduced coenzyme I and standard; the R2 reagent includes: a color developing agent 1 and a color developing agent 2, wherein the color developing agent 1 is WST-8; the color-developing agent 2 is at least one of phenazine methosulfate, 2, 6-dichlorophenol indophenol, 1,2, 3-trichloropropane and fullerene phenylbutyrate; the R3 reagent includes: a buffer; the R4 reagent includes: at least one of acetoacetic acid and lithium acetoacetate. The acetoacetic acid (AcAc) content detection kit provided by the invention can avoid the condition that the measured value is smaller due to the fact that the detection ultraviolet wavelength is easily influenced by ultraviolet absorption interference objects in a sample, and has more accurate and reliable measurement results and wider linear range.
Description
Technical Field
The invention relates to the field of biochemical detection, in particular to an acetoacetic acid content detection kit, a using method and a detection device thereof.
Background
Acetoacetic acid (AcAc) is one of the important components of ketone bodies, accounting for about 20 percent of the total amount of normal ketone bodies, is produced by oxidation of fatty acid, and is a strong organic acid. The normal content of acetoacetate is harmless to human body, but diabetics can use a large amount of fat due to the reduction of sugar utilization or the metabolic disorder of sugar during starvation, the amount of acetoacetate can be accumulated, and the excessive acetoacetate can cause harm to human body.
Acetoacetate can be converted to either acetone or β -hydroxybutyrate. The detection methods for mainly detecting the content of the acetoacetic acid at present comprise a dye tetrazole colorimetric method and a spectroscopic method. The dye tetrazolium colorimetric method has the characteristics of simple and convenient operation and visual result, but only can carry out qualitative and semi-quantitative analysis, insoluble formazan is generated by a reaction product, and precipitation is generated when the concentration is too high, so that the measured value is larger; although the spectroscopic method can quantitatively determine the acetoacetic acid content, the maximum absorption peak is detected at the ultraviolet absorption wavelength, and the measurement value is easily small due to the influence of ultraviolet absorption interference substances in the sample.
Disclosure of Invention
The invention aims to provide an acetoacetic acid content detection kit, a using method and a detection device thereof, so as to improve the accuracy of acetoacetic acid content detection.
In order to achieve the above object, according to one aspect of the present invention, there is provided an acetoacetic acid content detection kit. The acetoacetic acid content detection kit comprises an R1 reagent, an R2 reagent, an R3 reagent and an R4 reagent, wherein the R1 reagent comprises: beta-hydroxybutyrate dehydrogenase, reduced coenzyme I and standard; the R2 reagent includes: a color developing agent 1 and a color developing agent 2, wherein the color developing agent 1 is WST-8; the color-developing agent 2 is at least one of phenazine methosulfate, 2, 6-dichlorophenol indophenol, 1,2, 3-trichloropropane and fullerene phenylbutyrate; the R3 reagent includes: at least one of Tris-HCl buffer solution, hepes buffer solution, boric acid buffer solution and PBS buffer solution, wherein the pH value of the buffer solution is 5.5-7.0; the R4 reagent includes: at least one of acetoacetic acid and lithium acetoacetate is used as a standard substance, and the concentration is 0-2000 mu mol/L.
Further, in the R1 reagent, the enzyme activity of the beta-hydroxybutyrate dehydrogenase is 20-250 KU/L.
Further, in the R1 reagent, the concentration of the reduced coenzyme I is 0.5-3.5 mg/mL.
Further, in the R1 reagent, the standard substance is at least one of acetoacetic acid and lithium acetoacetate; the concentration of the standard substance is 0-2000 mu mol/L.
Further, the R1 reagent further comprises a first buffer solution, wherein the first buffer solution is at least one of Tris-HCl buffer solution, hepes buffer solution, boric acid buffer solution and PBS buffer solution; the pH of the first buffer solution is 5.5-7.0; the R2 reagent further comprises a second buffer solution, wherein the second buffer solution is at least one of Tris-HCl buffer solution, hepes buffer solution, boric acid buffer solution and PBS buffer solution; the pH of the second buffer solution is 5.5-7.0.
Further, in the R2 reagent, the concentration of the color developing agent 1 is 0.2-10 mmol/L; the concentration of the color developing agent 2 is 0.02-5 mmol/L; the volume ratio of the color developing agent 1 to the color developing agent 2 is 5-20:1.
Further, the R1 reagent further comprises: at least one of a surfactant, a stabilizer, and a preservative; the R2 reagent further comprises: at least one of a surfactant, a stabilizer, and a preservative.
Further, in the R1 reagent, the amount of the surfactant is 0.5-10 g/L, the amount of the stabilizer is 0.01-0.5 g/L, and the amount of the preservative is 0.1-0.5 g/L; in the R2 reagent, the amount of the surfactant is 0.5-5 g/L, the amount of the stabilizer is 5-10 g/L, and the amount of the preservative is 0.01-0.1 g/L.
Further, the surfactant includes at least one of tween-80, tween-20, glycerin and sodium dodecyl sulfate; the stabilizer comprises at least one of mercaptoethanol, dithiothreitol, trehalose, glutathione and dodecyl dimethyl betaine; the preservative comprises at least one of erythromycin, proclin series preservative and phenolic preservative.
According to another aspect of the present invention, there is provided an acetoacetic acid content detection device. The acetoacetic acid content detection device comprises any one of the acetoacetic acid content detection kit and a spectrophotometer or a microplate reader, wherein the spectrophotometer or the microplate reader is used for detecting absorbance at 450nm.
According to still another aspect of the present invention, there is provided a method for using an acetoacetic acid content detection kit, comprising: mixing the R1 reagent with distilled water, and then placing the mixture at 30-45 ℃ to react for 5-15 min; taking out, cooling to room temperature, adding an R2 reagent, uniformly mixing, placing at 30-45 ℃ for reaction for 15-25 min, and reading absorbance of the mixture, and marking the mixture as a blank A; mixing the R1 reagent and the R4 reagent, and then placing the mixture at 30-45 ℃ for reaction for 5-15 min; taking out, cooling to room temperature, adding an R2 reagent, fully and uniformly mixing, placing at 30-45 ℃ for reaction for 15-25 min, and reading absorbance of the mixture, and marking the absorbance as A standard; calculating the absorbance change of the R4 reagent: Δa standard = a blank-a standard; drawing a standard curve by taking the concentration of the R4 reagent as an abscissa x and the delta A standard as an ordinate y to obtain a linear regression equation y=kx+b; mixing the R1 reagent with a sample to be tested, and then placing the mixture at 30-45 ℃ for reaction for 5-15 min; taking out, cooling to room temperature, adding an R2 reagent, uniformly mixing, placing at 30-45 ℃ for reaction for 15-25 min, and reading absorbance of the mixture, and marking the absorbance as A for measurement; mixing the R3 reagent with a sample to be tested, and then placing the mixture at 30-45 ℃ for reaction for 5-15 min; taking out, cooling to room temperature, adding an R2 reagent, uniformly mixing, placing at 30-45 ℃ for reaction for 15-25 min, and reading absorbance of the mixture, and marking the absorbance as A control; calculating the absorbance change of the sample to be measured: Δaassay = ablank- (aassay-a control); substituting the relation curve y=kx+b of the concentration and absorbance change of the standard substance into the absorbance change delta A of the sample to be detected, and calculating the concentration of the beta-hydroxybutyric acid in the sample to be detected.
Further, the sample to be tested is supernatant fluid after centrifugation of serum, plasma or urine samples; preferably, the volume ratio of the R1 reagent or the R3 reagent, the sample to be tested and the R2 reagent is 15-20:1:2; preferably, the absorbance is detected at a wavelength of 450nm.
By applying the technical scheme of the invention, the acetoacetic acid in the sample to be detected is reacted with the reduced coenzyme I under the catalysis of acetoacetate dehydrogenase to generate beta-hydroxybutyric acid and NAD, and the NADH is reacted with the color developing agent (WST-8) to generate purple soluble formazan, and the content of the acetoacetic acid can be calculated because the degree of color change is related to the concentration of the acetoacetic acid in the sample. The acetoacetic acid (AcAc) content detection kit provided by the invention can avoid the condition that the measured value is smaller due to the fact that the detection ultraviolet wavelength is easily influenced by ultraviolet absorption interference objects in a sample, and has more accurate and reliable measurement results and wider linear range.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention. In the drawings:
FIG. 1 is a graph showing a linear range of acetoacetate (AcAc) content detection kit in a concentration range of 0 to 2000. Mu. Mol/L in example 1 of the present invention;
FIG. 2 shows the results of the bottle opening stability test at 4℃for the kit of example 1 of the present invention;
FIG. 3 shows the results of the stability test of the kit of example 1 of the present invention without opening the bottle.
Detailed Description
It should be noted that, in the case of no conflict, the embodiments and features in the embodiments may be combined with each other. The invention will be described in detail below with reference to the drawings in connection with embodiments.
Aiming at the problem of inaccurate detection in the acetoacetic acid content detection described in the background art, the invention provides the following technical method.
According to an exemplary embodiment of the present invention, there is provided an acetoacetate content detection kit. The acetoacetic acid content detection kit comprises an R1 reagent, an R2 reagent, an R3 reagent and an R4 reagent, wherein the R1 reagent comprises: beta-hydroxybutyrate dehydrogenase, reduced coenzyme I and standard; the R2 reagent includes: a color developing agent 1 and a color developing agent 2, wherein the color developing agent 1 is WST-8; the color-developing agent 2 is at least one of phenazine methosulfate, 2, 6-dichlorophenol indophenol, 1,2, 3-trichloropropane and fullerene phenylbutyrate; the R3 reagent includes: at least one of Tris-HCl buffer solution, hepes buffer solution, boric acid buffer solution and PBS buffer solution, wherein the pH value of the buffer solution is 5.5-7.0; the R4 reagent includes: at least one of acetoacetic acid and lithium acetoacetate, and the concentration of the standard substance is 0-2000 mu mol/L.
By applying the technical scheme of the invention, the acetoacetic acid in the sample to be detected is reacted with the reduced coenzyme I under the catalysis of acetoacetate dehydrogenase to generate beta-hydroxybutyric acid and NAD, and the NADH is reacted with the color developing agent (WST-8) to generate purple soluble formazan, and the content of the acetoacetic acid can be calculated because the degree of color change is related to the concentration of the acetoacetic acid in the sample. The acetoacetic acid (AcAc) content detection kit provided by the invention can avoid the condition that the measured value is smaller due to the fact that the ultraviolet wavelength is easy to be influenced by ultraviolet absorption interference substances in a sample, and the measured result is more accurate and reliable.
WST-8 is a water-soluble tetrazolium salt developed by the Japan institute of Corneil chemistry (Dojindo) and works on the principle: in the presence of an electron coupling reagent, the electron coupling reagent can be reduced by dehydrogenase in mitochondria to generate orange yellow formazan with high water solubility, and the electron coupling reagent has characteristic absorption peak at the wavelength of 450nm. Compared with WST-1, WST-8 has higher detection sensitivity, the generated formazan is easier to dissolve, the linear range is wider, and the experimental result is more stable.
In order to further improve the accuracy, precision or stability of the detection of the kit, in one embodiment of the present invention, the applicant further optimizes the reagents in the kit, preferably, in the R1 reagent, the enzyme activity of the β -hydroxybutyrate dehydrogenase is 20-250KU/L; in the R1 reagent, the concentration of the reduced coenzyme I is 0.5-3.5 mg/mL; the standard substance is at least one of acetoacetic acid and lithium acetoacetate; the concentration of the standard substance is 0-2000 mu mol/L.
According to an exemplary embodiment of the present invention, the R1 reagent further comprises a first buffer, the first buffer being at least one of Tris-HCl buffer, hepes buffer, boric acid buffer, and PBS buffer; the pH of the first buffer solution is 5.5-7.0; the R2 reagent further comprises a second buffer solution, wherein the second buffer solution is at least one of Tris-HCl buffer solution, hepes buffer solution, boric acid buffer solution and PBS buffer solution; the pH of the second buffer solution is 5.5-7.0. In the R2 reagent, the concentration of the color developing agent 1 is 0.2-10 mmol/L; the concentration of the color developing agent 2 is 0.02-5 mmol/L; the volume ratio of the color developing agent 1 to the color developing agent 2 is 5-20:1. The components mutually support and influence each other as an organic whole, so that the detection kit has very good accuracy, precision and stability.
To further increase the stability of the kit, according to an exemplary embodiment of the present invention, the R1 reagent further comprises: at least one of a surfactant, a stabilizer, and a preservative; the R2 reagent further comprises: at least one of a surfactant, a stabilizer, and a preservative. Preferably, in the R1 reagent, the amount of the surfactant is 0.5-10 g/L, the amount of the stabilizer is 0.01-0.5 g/L, and the amount of the preservative is 0.1-0.5 g/L; in the R2 reagent, the amount of the surfactant is 0.5-5 g/L, the amount of the stabilizer is 5-10 g/L, and the amount of the preservative is 0.01-0.1 g/L. More preferably, the surfactant comprises at least one of tween-80, tween-20, glycerol and sodium dodecyl sulfate; the stabilizer comprises at least one of mercaptoethanol, dithiothreitol, trehalose, glutathione and dodecyl dimethyl betaine; the preservative comprises at least one of erythromycin, proclin series preservative and phenolic preservative. The R3 agent and the R4 agent may also include at least one of a surfactant, a stabilizer, and a preservative according to actual needs.
According to an exemplary embodiment of the present invention, there is provided an acetoacetic acid content detection device. The acetoacetic acid content detection device comprises any acetoacetic acid content detection kit and a spectrophotometer or a microplate reader, wherein the spectrophotometer or the microplate reader is used for detecting absorbance at 450nm. The acetoacetic acid (AcAc) content detection system provided by the invention can avoid the condition that the measured value is smaller due to the fact that the ultraviolet wavelength is easy to be influenced by ultraviolet absorption interference substances in a sample, and the measured result is more accurate and reliable.
According to an exemplary embodiment of the present invention, a method of using an acetoacetate content detection kit is provided. The using method comprises the following steps: mixing the R1 reagent with distilled water, and then placing the mixture at 30-45 ℃ to react for 5-15 min; taking out, cooling to room temperature, adding an R2 reagent, uniformly mixing, placing at 30-45 ℃ for reaction for 15-25 min, and reading absorbance of the mixture, and marking the mixture as a blank A; mixing the R1 reagent and the R4 reagent, and then placing the mixture at 30-45 ℃ for reaction for 5-15 min; taking out, cooling to room temperature, adding an R2 reagent, fully and uniformly mixing, placing at 30-45 ℃ for reaction for 15-25 min, and reading absorbance of the mixture, and marking the absorbance as A standard; calculating the absorbance change of the R4 reagent: Δa standard = a blank-a standard; drawing a standard curve by taking the concentration of the R4 reagent as an abscissa x and the delta A standard as an ordinate y to obtain a linear regression equation y=kx+b; mixing the R1 reagent with a sample to be tested, and then placing the mixture at 30-45 ℃ for reaction for 5-15 min; taking out, cooling to room temperature, adding an R2 reagent, uniformly mixing, placing at 30-45 ℃ for reaction for 15-25 min, and reading absorbance of the mixture, and marking the absorbance as A for measurement; mixing the R3 reagent with a sample to be tested, and then placing the mixture at 30-45 ℃ for reaction for 5-15 min; taking out, cooling to room temperature, adding an R2 reagent, uniformly mixing, placing at 30-45 ℃ for reaction for 15-25 min, and reading absorbance of the mixture, and marking the absorbance as A control; calculating the absorbance change of the sample to be measured: Δaassay = ablank- (aassay-a control); substituting the relation curve y=kx+b of the concentration and absorbance change of the standard substance into the absorbance change delta A of the sample to be detected, and calculating the concentration of the beta-hydroxybutyric acid in the sample to be detected.
According to an exemplary embodiment of the present invention, the concentration of β -hydroxybutyric acid is calculated as follows:
(1) Calculation according to protein concentration
AcAc content (μmol/mg prot) =x×v-sample ≡ (V-sample×cpr) ++ 108.02 ×1000= 9.258 x++cpr
(2) Calculation according to sample mass
AcAc content (μmol/g mass) =x×v samples ≡ (w×v samples ≡v samples total) ≡ 108.02 ×1000= 9.258 x+.
(3) Counting according to the number of cells or bacteria
AcAc content (μmol/104 cell) =xV samples (cell x V samples ++V samples total)/(108.02 x 1000= 9.258x ++cell)
(4) Calculation according to serum (plasma) volume
AcAc content (μmol/mL) =x×v samples ≡v samples ≡ 108.02 = 9.258x
V sample: sample volume was added to the reaction, 100 μl=0.1 mL; cpr: sample protein concentration, mg/mL; w: sample mass, g; cell: total number of cells or bacteria, 104; sample V total: adding the volume of the extracting solution, 1 mL;108.02: acAc molecular weight.
By applying the application method of the invention, the detection of the kit of the invention can be ensured to be more accurate.
The kit provided by the invention has wide adaptability to the sample to be detected, for example, the sample to be detected is supernatant obtained after centrifugation of serum, plasma or urine samples.
Preferably, the volume ratio of the R1 reagent or the R3 reagent, the sample to be tested and the R2 reagent is 15-20:1:2.
The advantageous effects of the present invention will be further described below with reference to examples.
The specific experimental procedures or conditions are not noted in the examples and may be performed in accordance with the operations or conditions of conventional experimental procedures described in the literature in this field. The materials or instruments used are all conventional products commercially available, including but not limited to those used in the examples of the present application.
Example 1
An acetoacetic acid (AcAc) content detection kit comprises the following components:
r1 reagent:
beta-hydroxybutyrate dehydrogenase 50KU/L
Reduced coenzyme I (NADH) 0.8mg/mL
Tris-HCl buffer 0.2mol/L
Tween-80.2 g/L
DTT 0.3mL/L
ProClin300 0.2g/L
R2 reagent:
WST-8 0.5mmol/L
phenazine methosulfate 0.2mmol/L
Tris-HCl buffer 0.2mol/L
Tween-80.5 g/L
DTT 0.2mL/L
ProClin300 0.05g/L
R3 reagent:
Tris-HCl buffer 0.2mol/L
R4 reagent:
Comparative example 1
An acetoacetic acid (AcAc) content detection kit comprises the following components:
r1 reagent:
beta-hydroxybutyrate dehydrogenase 10KU/L
Reduced coenzyme I (NADH) 0.25mg/mL
Tris-HCl buffer 0.2mol/L
Tween-80.2 g/L
DTT 0.3mL/L
ProClin300 0.2g/L
R2 reagent:
WST-8 0.2mmol/L
phenazine methosulfate 0.2mmol/L
Tris-HCl buffer 0.2mol/L
Tween-80.5 g/L
DTT 0.2mL/L
ProCli300 0.05g/L
R3 reagent:
Tris-HCl buffer 0.2mol/L
R4 reagent:
Comparative example 2
An acetoacetic acid (AcAc) content detection kit comprises the following components:
r1 reagent:
beta-hydroxybutyrate dehydrogenase 80KU/L
Reduced coenzyme I (NADH) 1.5mg/mL
Phosphate buffer 0.2mol/L
Tween-80.2 g/L
DTT 0.3mL/L
ProClin300 0.2g/L
R2 reagent:
WST-8 20mmol/L
phenazine methosulfate 0.2mmol/L
Phosphate buffer 0.2mol/L
Tween-80.5 g/L
DTT 0.2mL/L
ProClin300 0.05g/L
R3 reagent:
phosphate buffer 0.2mol/L
R4 reagent:
Comparative example 3
An acetoacetic acid (AcAc) content detection kit comprises the following components:
r1 reagent:
beta-hydroxybutyrate dehydrogenase 30KU/L
Reduced coenzyme I (NADH) 0.8mg/mL
Tris-HCl buffer 0.2mol/L
Tween-80.5 g/L
DTT 0.3mL/L
ProClin300 0.2g/L
R2 reagent:
WST-8 0.2mmol/L
phenazine methosulfate 0.2mmol/L
Tris-HCl buffer 0.2mol/L
Tween-80.5 g/L
DTT 0.2mL/L
ProCli300 0.05g/L
R3 reagent:
Tris-HCl buffer 0.2mol/L
R4 reagent:
Comparative example 4
An acetoacetic acid (AcAc) content detection kit comprises the following components:
r1 reagent:
beta-hydroxybutyrate dehydrogenase 80KU/L
Reduced coenzyme I (NADH) 1.5mg/mL
Phosphate buffer 0.2mol/L
Tween-80.2 g/L
DTT 0.3mL/L
ProClin300 0.2g/L
R2 reagent:
WST-8 1.5mmol/L
phenazine methosulfate 0.5mmol/L
Phosphate buffer 0.5mol/L
Tween-80.5 g/L
DTT 0.2mL/L
ProClin300 0.05g/L
R3 reagent:
phosphate buffer 0.2mol/L
R4 reagent:
Comparative example 5
An acetoacetic acid (AcAc) content detection kit comprises the following components:
beta-hydroxybutyrate dehydrogenase 50KU/L
Reduced coenzyme I (NADH) 0.8mg/mL
Tris-HCl buffer 0.2mol/L
Glycerol 1.2g/L
DTT 0.3mL/L
ProClin150 0.2g/L
R2 reagent:
dye tetrazole 0.5mmol/L
Phenazine methosulfate 0.2mmol/L
Tris-HCl buffer 0.2mol/L
Glycerol 0.5g/L
DTT 0.2mL/L
ProClin150 0.05g/L
R3 reagent:
Tris-HCl buffer 0.2mol/L
R4 reagent:
Comparative example 6
An acetoacetic acid (AcAc) content detection kit comprises the following components:
r1 reagent:
beta-hydroxybutyrate dehydrogenase 50KU/L
Phosphate buffer 0.2mol/L
Tween-80.5 g/L
Sucrose 0.5mL/L
ProClin150 0.1g/L
R2 reagent:
reduced coenzyme I (NADH) 0.8mg/mL
Tween-80.5 g/L
Sucrose 0.2mL/L
ProClin150 0.05g/L
R3 reagent:
phosphate buffer 0.2mol/L
R4 reagent:
Comparative example 7
An acetoacetic acid (AcAc) content detection kit comprises the following components:
r1 reagent:
beta-hydroxybutyrate dehydrogenase 50KU/L
Reduced coenzyme I (NADH) 0.8mg/mL
Tirs-HCl buffer 0.2mol/L
Tween-80.2 g/L
DTT 0.3mL/L
ProClin300 0.2g/L
R2 reagent:
WST-1 0.5mmol/L
phenazine methosulfate 0.2mmol/L
Tirs-HCl buffer 0.2mol/L
Tween-80.5 g/L
DTT 0.2mL/L
ProClin300 0.05g/L
R3 reagent:
Tirs-HCl buffer 0.2mol/L
R4 reagent:
The preparation method of the acetoacetate (AcAc) content detection kit provided in example 1 and comparative examples 1 to 7 is as follows:
(1) Preparing an R1 reagent: weighing other components except the buffer solution in the R1 reagent according to the pre-prepared concentration, adding the components into the prepared buffer solution, and fully and uniformly mixing;
(2) Preparing an R2 reagent: weighing other components except the buffer solution in the R1 reagent according to the pre-prepared concentration, adding the components into the prepared buffer solution, and fully and uniformly mixing;
(3) And sequentially performing semi-finished product inspection, split charging, assembly (labeling, boxing, instruction manual packaging and plastic packaging), finished product inspection and warehousing.
The methods of using the acetoacetate (AcAc) content detection kits provided in example 1 and comparative examples 1-7 were as follows:
the specific operation method is shown in table 1, and specifically comprises the following steps:
(1) Mixing 20 mu L of distilled water with 180 mu L R1 reagent, and reacting at 37 ℃ for 10min; taking out, cooling to room temperature, adding 10 mu L R2 reagent, mixing, placing at 37 ℃ for reaction for 20min, and reading absorbance, and marking as A blank.
(2) Mixing 20 mu LR4 reagent and 180 mu LR1 reagent, and reacting at 37 ℃ for 10min; taking out, cooling to room temperature, adding 10 mu LR2 reagent, mixing, placing at 37 ℃ for reaction for 20min, and reading absorbance, and marking as A standard.
(3) Calculating the absorbance change Δa standard = a blank-a standard for R4 reagent; drawing a standard curve by taking the concentration of the R4 reagent as an abscissa (x) and the delta A standard as an ordinate (y) to obtain a linear regression equation y=kx+b;
(4) Mixing 20 mu L of a sample to be tested with 180 mu LR1 reagent, and placing the mixture at 37 ℃ for reaction for 10min; taking out, cooling to room temperature, adding 10 mu L R2 reagent, mixing, placing at 37 ℃ for reaction for 20min, and reading absorbance, and recording as A measurement.
(5) Mixing 20 mu L of a sample to be tested with 180 mu LR3 reagent, and placing the mixture at 37 ℃ for reaction for 10min; taking out, cooling to room temperature, adding 10 mu LR2 reagent, mixing, placing at 37 ℃ for reaction for 20min, and reading absorbance, and marking as A control. Calculating the absorbance change Δaassay=ablank- (aassay-a control) of the sample to be measured;
(6) The enzyme-labeled instrument is zeroed by distilled water, and the detection wavelength is 450nm.
(7) According to the operation method, a relation curve of the concentration and absorbance change of the standard substance is manufactured by adopting two-point linear calibration, and the relation curve is substituted into the absorbance change delta A of the sample to be measured, so that the concentration of the acetoacetic acid in the sample to be measured can be calculated.
TABLE 1 use of acetoacetate (AcAc) content detection kit
Experimental example 1 Linear Range experiment
Using the acetoacetic acid (AcAc) content detection kits provided in example 1 and comparative examples 1 to 4 of the present invention, standard substance concentrations in standard substances of different concentrations were respectively measured using acetoacetic acid standard substances (i.e., R4 reagent) at concentrations of 2000. Mu. Mol/L, 1500. Mu. Mol/L, 1000. Mu. Mol/L, 500. Mu. Mol/L, 250. Mu. Mol/L, 125. Mu. Mol/L, 62.5. Mu. Mol/L according to the methods of use provided in Table 1, and the measurement results are shown in Table 2.
TABLE 2 Linear Range experimental detection results
As can be seen from Table 2, the acetoacetic acid (AcAc) content detection kit provided in example 1 is in the concentration range of 0-2000. Mu. Mol/L, the acetoacetic acid (AcAc) content detection kit provided in example 1 has better correlation between the detection value and the theoretical value in the concentration range of 0-2000. Mu. Mol/L, and the linear range diagram of example 1 is shown in FIG. 1; the acetoacetic acid (AcAc) content detection kit provided in comparative example 1 has better correlation between the detection value and the theoretical value in the concentration range of 0-500 mu mol/L; the acetoacetic acid (AcAc) content detection kit provided in comparative example 2 has poor correlation between the detection value and the theoretical value in the concentration range of 0-2000 mu mol/L; the acetoacetic acid (AcAc) content detection kit provided in the comparative example 3 has better correlation between the detection value and the theoretical value in the concentration range of 0-500 mu mol/L, but has narrower linear range; the acetoacetic acid (AcAc) content detection kit provided in comparative example 4 has poor correlation between the detection value and the theoretical value in the concentration range of 0-125 mu mol/L. Therefore, the acetoacetic acid (AcAc) content detection kit provided by the invention has a wide linear range (0-2000 mu mol/L), and the correlation between the detection value and the theoretical value is good, so that the kit is suitable for scientific research detection requirements.
Experimental example 2 limit of detection experiment
Using the method of example 1 of the present invention, the test was conducted using water as a blank sample, and the acetoacetic acid content was measured and repeated 10 times in parallel, and the test results are shown in Table 3.
Table 3 detection limit experimental detection results
As can be seen from Table 3, according to the use method provided in Table 1, the detection was performed using water as a blank sample, the minimum detection limit was 40.41. Mu. Mol/L, and the acetoacetate (AcAc) content detection kit provided by the invention has higher sensitivity.
Experimental example 3 accuracy experiment
Using the methods of use provided in Table 1 in example 1 of the present invention and comparative examples 3 to 5, the acetoacetate content was measured on each of the 4 serum samples, and the measurement was repeated 3 times in parallel, and the measurement results are shown in Table 4.
TABLE 4 accuracy test results
As can be seen from table 4, according to the methods of use provided in table 1, the acetoacetate (AcAc) content detection kit provided in example 1 and comparative examples 5-7, respectively, were used to detect 4 serum samples, comparative example 5 was higher than the target value, comparative example 6 was lower than the target value, comparative example 7 was closer to the target value, the detection result of the method of example 1 (i.e., the present invention) was closest to the target value, and the acetoacetate (AcAc) content detection kit provided in the present invention was higher in accuracy compared with the four methods.
Experimental example 4 labeling experiment
Using the acetoacetic acid (AcAc) content detection kit provided in example 1 of the present invention, standard solutions of high, medium and low concentrations (i.e., R4 reagents of different concentrations) in a linear range were added to 3 different samples to be tested, respectively, and the measurement was repeated 2 times in parallel according to the use method provided in Table 1, and the detection results are shown in Table 4.
Table 5 test results of the labeling experiments
As can be seen from Table 5, according to the use method provided in Table 1, the acetoacetic acid (AcAc) content detection kit provided in example 1 is characterized in that standard solutions with high, medium and low concentrations (i.e., R4 reagents with different concentrations) in a linear range are respectively added into 3 different samples to be detected, and the measurement is repeated for 2 times in parallel, wherein the standard adding recovery rate is between 95% and 100%, and the requirements are 80% to 120%, so that the accuracy of the acetoacetic acid (AcAc) content detection kit provided by the invention is high. According to the method for measuring the comparative examples 1-4, the standard adding recovery rate is between 90% and 110%, and the accuracy of the acetoacetic acid (AcAc) content detection kit provided by the comparative examples 1-4 is higher.
Experimental example 5 stability test
Using the kit provided in example 1 of the present invention, the stability to opening and the stability to non-opening at 4℃were examined, respectively, according to the methods of use provided in Table 1. Once every three days, continuously detecting for 31 days, and detecting the stability of the bottle opening, wherein the detection result is shown in figure 2; the results of the unopened stability test are shown in FIG. 3.
From fig. 2, it can be seen that example 1 of the present invention remains substantially stable for 31 days.
As can be seen from FIG. 3, the fluctuation of the present invention in example 1 is small and the stability is maintained.
Experimental example 6 repeatability experiment
The same serum samples were tested using the kit provided in example 1 of the present invention according to the methods provided in table 1, and the test results are shown in table 6, repeated 10 times in parallel.
TABLE 6 repeatability test results
As can be seen from Table 6, the kit provided in example 1 was used to test the same serum sample by the method provided in Table 1, and the measurement was repeated 10 times in parallel, with the coefficient of variation CV% of 0.79 and 1.10, respectively, being less than 5%, and the results of the measurement according to the method for comparative examples 1 to 3 being less than 5%, respectively. The repeatability of the acetoacetic acid (AcAc) content detection kit provided in the embodiment 1 of the invention is better.
From the above description, it can be seen that the above embodiments of the present invention achieve the following technical effects:
1) The acetoacetic acid (AcAc) content detection kit provided by the invention can avoid the condition that the measured value is smaller due to the fact that the detection ultraviolet wavelength is easily influenced by ultraviolet absorption interference substances in a sample, and the measurement result is more accurate and reliable;
2) The acetoacetic acid (AcAc) content detection kit provided by the invention has the advantages of high accuracy, good sensitivity and high repeatability of detection results, can be kept stable at 4 ℃, is more favorable for practical application and is also favorable for industrial production.
3) According to the application method of the acetoacetic acid (AcAc) content detection kit provided by the invention, a sample to be detected is sequentially mixed with the R1 reagent and the R2 reagent, and meanwhile, the measuring tube and the control tube are arranged, so that interference values of the sample and the reagent can be offset, the concentration of the acetoacetic acid in the sample to be detected can be calculated by calculating the absorbance change delta A of the sample to be detected and substituting the relationship curve of the concentration of the standard substance and the absorbance change delta A of the sample to be detected, and the method is simple, convenient and quick to operate and suitable for scientific research detection requirements.
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (14)
1. The acetoacetic acid content detection kit is characterized by comprising an R1 reagent, an R2 reagent, an R3 reagent and an R4 reagent, wherein,
the R1 reagent comprises: beta-hydroxybutyrate dehydrogenase, reduced coenzyme I and standard;
the R2 reagent includes: a color developing agent 1 and a color developing agent 2, wherein the color developing agent 1 is WST-8; the color-developing agent 2 is at least one of phenazine methosulfate, 2, 6-dichlorophenol indophenol, 1,2, 3-trichloropropane and fullerene phenylbutyrate;
the R3 reagent includes: at least one of Tris-HCl buffer solution, hepes buffer solution, boric acid buffer solution and PBS buffer solution, wherein the pH value of the buffer solution is 5.5-7.0;
the R4 reagent includes: at least one of acetoacetic acid and lithium acetoacetate is used as a standard substance, and the concentration is 0-2000 mu mol/L.
2. The acetoacetate content detection kit according to claim 1, wherein the enzyme activity of the beta-hydroxybutyrate dehydrogenase in the R1 reagent is 20-250 KU/L.
3. The acetoacetate content detection kit according to claim 1, wherein the concentration of reduced coenzyme I in the R1 reagent is 0.5 to 3.5mg/mL.
4. The acetoacetic acid content detection kit according to claim 1, wherein in said R1 reagent, the standard substance is at least one of acetoacetic acid and lithium acetoacetate; the concentration of the standard substance is 0-2000 mu mol/L.
5. The acetoacetate content detection kit according to claim 1, wherein said R1 reagent further comprises a first buffer, said first buffer being at least one of Tris-HCl buffer, hepes buffer, boric acid buffer and PBS buffer; the pH of the first buffer solution is 5.5-7.0; the R2 reagent further comprises a second buffer solution, wherein the second buffer solution is at least one of Tris-HCl buffer solution, hepes buffer solution, boric acid buffer solution and PBS buffer solution; the pH of the second buffer solution is 5.5-7.0.
6. The acetoacetic acid content detection kit according to claim 1, wherein in the R2 reagent, the concentration of the color-developing agent 1 is 0.2-10 mmol/L; the concentration of the color developing agent 2 is 0.02-5 mmol/L; the volume ratio of the color developing agent 1 to the color developing agent 2 is 5-20:1.
7. The acetoacetate content detection kit according to any one of claims 1 to 6, wherein said R1 reagent further comprises: at least one of a surfactant, a stabilizer, and a preservative; the R2 reagent further comprises: at least one of a surfactant, a stabilizer, and a preservative.
8. The acetoacetic acid content detection kit according to claim 7, wherein in the R1 reagent, the amount of the surfactant is 0.5-10 g/L, the amount of the stabilizer is 0.01-0.5 g/L, and the amount of the preservative is 0.1-0.5 g/L; in the R2 reagent, the amount of the surfactant is 0.5-5 g/L, the amount of the stabilizer is 5-10 g/L, and the amount of the preservative is 0.01-0.1 g/L.
9. The acetoacetic acid content detection kit according to claim 7, wherein said surfactant comprises at least one of tween-80, tween-20, glycerol and sodium dodecyl sulfate; the stabilizer comprises at least one of mercaptoethanol, dithiothreitol, trehalose, glutathione and dodecyl dimethyl betaine; the preservative comprises at least one of erythromycin, proclin series preservatives and phenolic preservatives.
10. An acetoacetate content detection device, characterized by comprising an acetoacetate content detection kit according to any one of claims 1 to 9 and a spectrophotometer or a microplate reader configured to detect absorbance at 450nm.
11. Use of the acetoacetate content detection kit according to any one of claims 1 to 9, characterized by comprising:
mixing the R1 reagent with distilled water, and then placing the mixture at 30-45 ℃ to react for 5-15 min; taking out, cooling to room temperature, adding an R2 reagent, uniformly mixing, placing at 30-45 ℃ for reaction for 15-25 min, and reading absorbance of the mixture, and marking the mixture as a blank A;
mixing the R1 reagent and the R4 reagent, and then placing the mixture at 30-45 ℃ for reaction for 5-15 min; taking out, cooling to room temperature, adding an R2 reagent, fully and uniformly mixing, placing at 30-45 ℃ for reaction for 15-25 min, and reading absorbance of the mixture, and marking the absorbance as A standard;
calculating the absorbance change of the R4 reagent: Δa standard = a blank-a standard; drawing a standard curve by taking the concentration of the R4 reagent as an abscissa x and the delta A standard as an ordinate y to obtain a linear regression equation y=kx+b;
mixing the R1 reagent with a sample to be tested, and then placing the mixture at 30-45 ℃ for reaction for 5-15 min; taking out, cooling to room temperature, adding an R2 reagent, uniformly mixing, placing at 30-45 ℃ for reaction for 15-25 min, and reading absorbance of the mixture, and marking the absorbance as A for measurement;
mixing the R3 reagent with a sample to be tested, and then placing the mixture at 30-45 ℃ for reaction for 5-15 min; taking out, cooling to room temperature, adding an R2 reagent, uniformly mixing, placing at 30-45 ℃ for reaction for 15-25 min, and reading absorbance of the mixture, and marking the absorbance as A control;
calculating the absorbance change of the sample to be detected: Δaassay = ablank- (aassay-a control);
substituting the absorbance change delta A of the sample to be detected into the absorbance change delta A of the sample to be detected according to the relation curve y=kx+b of the concentration and absorbance change of the standard substance, and calculating the concentration of the beta-hydroxybutyric acid in the sample to be detected.
12. The method of claim 11, wherein the sample to be tested is a supernatant of a serum, plasma or urine sample after centrifugation.
13. The method of claim 11, wherein the volume ratio of the R1 reagent or the R3 reagent, the sample to be tested, and the R2 reagent is 15-20:1:2.
14. The method of claim 11, wherein the absorbance is detected at a wavelength of 450nm.
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