CN116297428A - Method for detecting alkaline phosphatase and application thereof - Google Patents
Method for detecting alkaline phosphatase and application thereof Download PDFInfo
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- CN116297428A CN116297428A CN202310232558.1A CN202310232558A CN116297428A CN 116297428 A CN116297428 A CN 116297428A CN 202310232558 A CN202310232558 A CN 202310232558A CN 116297428 A CN116297428 A CN 116297428A
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Abstract
The invention relates to the technical field of biological analysis and detection, in particular to a method for detecting alkaline phosphatase, which comprises the following steps: s1, preparing alkaline phosphatase solutions with different concentrations; s2, mixing and incubating the alkaline phosphatase solution and L-ascorbic acid-2-phosphate; s3, adding a copper sulfate solution; s4, shooting a color picture by using a mobile phone and obtaining RGB values of reaction products; s5, drawing a standard working curve of RGB values and alkaline phosphatase concentration; s6, repeating the steps S2-S4 on the alkaline phosphatase sample to be detected, and comparing the alkaline phosphatase sample with a standard working curve to obtain the alkaline phosphatase activity. Through the technical method, the invention solves the problems of long time consumption, complex experimental operation and labor consumption of the existing detection method.
Description
Technical Field
The invention relates to the technical field of biological analysis and detection, in particular to a method for detecting alkaline phosphatase and application thereof.
Background
Alkaline phosphatase is a common bioactive enzyme widely distributed in human organs, tissues and body fluids, and can participate in the process of catalyzing the dephosphorylation of phosphate substrates such as carbohydrates, proteins, nucleic acids and the like; as a clinical diagnosis biomarker, the abnormal elevation of the biological marker is closely related to diseases such as rickets, leukemia, uremia, liver cancer, liver cirrhosis, diabetes and the like. In general, alkaline phosphatase levels vary from healthy to diseased population, and thus, activity assays are important references and evaluation for clinical diagnosis and analysis.
Meanwhile, alkaline phosphatase is often used as an enzyme-labeled molecule in immunoassay, genetic analysis, tissue staining and other biological assays due to high catalytic efficiency, good stability, easy modification and wide substrate specificity, and has wide application prospect in the development of an enzyme-linked immunosorbent assay platform.
The p-nitrophenyl phosphate is used as an enzyme substrate to detect the activity of alkaline phosphatase, and the method is widely applied to the fields of clinical diagnosis and the like due to simplicity and high efficiency. However, this substrate is quite sensitive to light and dephosphorylation can be achieved without enzymes, which can easily lead to false positive results. Therefore, various analytical methods such as chemiluminescence, isotope labeling, surface enhanced Raman scattering, chromatography, etc. have been widely studied and used for monitoring the concentration and content of alkaline phosphatase in recent years. However, the complex procedures, special instrument requirements and the need for specialized operators have limited practical application in alkaline phosphatase assays.
Disclosure of Invention
The invention aims to solve the technical problems of long time consumption, complex experimental operation and labor consumption of the existing detection method.
The technical scheme of the invention is as follows:
a method of detecting alkaline phosphatase comprising the steps of:
s1, preparing alkaline phosphatase solutions with different concentrations;
s2, mixing and incubating the alkaline phosphatase solution and L-ascorbic acid-2-phosphate;
s3, adding a copper sulfate solution;
s4, shooting a color picture by using a mobile phone and obtaining RGB values of reaction products;
s5, drawing a standard working curve of RGB values and alkaline phosphatase concentration;
s6, repeating the steps S2-S4 on the alkaline phosphatase sample to be detected, and comparing the alkaline phosphatase sample with a standard working curve to obtain the alkaline phosphatase activity.
As a further technical scheme, in the step S1, the alkaline phosphatase solution with different concentrations specifically comprises the following concentrations: 0.01U/L, 0.025U/L, 0.05U/L, 0.1U/L, 0.25U/L, 0.5U/L, 1U/L, 2.5U/L, 5U/L, 7.5U/L.
As a further embodiment, in the step S2, the alkaline phosphatase solution is added in an amount of 25. Mu.L.
As a further embodiment, in the step S2, the concentration of L-ascorbic acid-2-phosphoric acid is 50mM and the addition amount is 25. Mu.L.
As a further technical scheme, in the step S2, the incubation temperature is 37 ℃ and the incubation time is 1 hour.
As a further technical scheme, in the step S3, the concentration of the added copper sulfate solution is 0.5M, and the addition amount is 50 mu L.
As a further technical scheme, the interval between the steps S3 and S4 is not more than 2 minutes.
A method for detecting alkaline phosphatase for use in detecting an alkaline phosphatase inhibitor, comprising the steps of:
a1, preparing alkaline phosphatase inhibitors with different concentrations;
a2, mixing and incubating the alkaline phosphatase inhibitor and alkaline phosphatase;
a3, adding L-ascorbic acid-2-phosphoric acid for continuous incubation;
a4, adding a copper sulfate solution and water;
a5, shooting a color picture by using a mobile phone and obtaining RGB values of a reaction product;
a6, drawing a standard working curve of RGB values and alkaline phosphatase inhibitor concentration;
a7, repeating the steps A2-A4 on the sample to be detected, and comparing with a standard working curve to obtain the activity of the alkaline phosphatase inhibitor.
As a further embodiment, the alkaline phosphatase inhibitor comprises dimethoate.
As a further technical scheme, in the step A1, alkaline phosphatase inhibitors with different concentrations are specifically: 0.5mM, 1mM, 2mM, 10mM, 20mM.
The technical scheme of the invention has the beneficial effects that:
1. aiming at alkaline phosphatase detection, the invention provides a method for detecting alkaline phosphatase activity by using portable equipment such as a mobile phone, wherein a colorimetric method can directly utilize naked eyes or a smart phone to evaluate the enzymolysis condition of a substrate, so that time-consuming, complex and laborious experimental operation procedures are avoided, the convenience of alkaline phosphatase detection is greatly improved, the activity of alkaline phosphatase is detected more simply, conveniently and effectively, and the false positive problem in the traditional method can be avoided.
2. The detection method of the invention has convenient operation, uses less reagent, and does not need to add other chromogenic solution (such as TMB-H 2 O 2 Color development solution), and the activity of alkaline phosphatase can be quantitatively detected by only adding L-ascorbic acid-2-phosphate and copper sulfate without adding a buffer solution (such as a buffer solution of sodium phenanthroline disulfonate).
3. Alkaline phosphatase in the range of 0.05U/L to 8U/L and Dimethoate in the range of 0.5mM to 20mM can be detected by using the present invention.
Drawings
The drawings of exemplary embodiments of the invention are shown by way of example in the following.
FIG. 1 is a schematic representation of the detection of alkaline phosphatase activity according to the present invention;
FIG. 2 is a graph showing the linear range of alkaline phosphatase detection according to the present invention;
FIG. 3 is a graph of the linear range of the present invention for music fruit detection.
Detailed Description
The present invention will be described in further detail below.
Example 1
A method of detecting alkaline phosphatase comprising the steps of:
s1, preparing alkaline phosphatase solutions with different concentrations: 0.01U/L, 0.025U/L, 0.05U/L, 0.1U/L, 0.25U/L, 0.5U/L, 1U/L, 2.5U/L, 5U/L, 7.5U/L.
S2, 25 mu L of alkaline phosphatase and 25 mu L of 50mM L-ascorbic acid-2-phosphate are contained in each 96-well plate, and incubated for 1h at 37 ℃;
s3, adding 50 mu LCuSO 4 (0.5M)；
S4, shooting a color picture of the 96-well plate by using a smart phone within 2min and obtaining RGB values of reaction products;
s5, drawing a standard working curve of RGB values and alkaline phosphatase concentration;
s6, repeating the steps S2-S4 on the alkaline phosphatase sample to be detected, and comparing the alkaline phosphatase sample with a standard working curve to obtain the alkaline phosphatase activity.
Linear equation of RGB values versus alkaline phosphatase concentration:
y＝0.365x+0.0093，R 2 ＝0.981。
wherein y represents the B/G ratio and x represents the alkaline phosphatase concentration.
In this example, the alkaline phosphatase activity signal was amplified by detecting the residual amount of phosphorylated ascorbic acid. When alkaline phosphatase activity was detected, incubation time was 1 hour at 37℃to ensure adequate hydrolysis of phosphorylated ascorbic acid. In this example, the amount of L-ascorbic acid-2-phosphate added was controlled so that it could satisfy both the requirement of color development and the requirement that alkaline phosphatase be hydrolyzed.
Example 2
A method of detecting an alkaline phosphatase inhibitor comprising the steps of:
a1, preparing dimethoate with different concentrations: 0.5mM, 1mM, 2mM, 10mM, 20mM;
a2, mixing 25 mu L of alkaline phosphatase with 25 mu L of Dimethoate with different concentrations, and incubating at 37 ℃ for 30 minutes;
a3, adding 50 mu LL-ascorbic acid-2-phosphoric acid for further incubation for 1 hour;
a4, then 0.5MCuSO is added 4 50 μl and deionized water 50 μl;
a5, shooting a color picture by using a mobile phone and obtaining RGB values of a reaction product;
a6, drawing a standard working curve of RGB values and alkaline phosphatase inhibitor concentration;
a7, repeating the steps A2-A4 on the sample to be detected, and comparing with a standard working curve to obtain the activity of the alkaline phosphatase inhibitor.
Linear equation of RGB values versus alkaline phosphatase inhibitor concentration:
y＝0.01017x+0.02586，R 2 ＝0.9466；
wherein y represents the B/G ratio and x represents the alkaline phosphatase inhibitor concentration.
In this example, the remaining alkaline phosphatase activity was tested according to the method for detecting alkaline phosphatase activity, and alkaline phosphatase inhibitor activity was detected using the RGB values of the reaction product obtained by the cell phone application in the smart phone.
It will be understood that the invention has been described in terms of several embodiments, and that various changes and equivalents may be made to these features and embodiments by those skilled in the art without departing from the spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation or material to the teachings of the invention without departing from the essential scope thereof. Therefore, it is intended that the invention not be limited to the particular embodiment disclosed, but that the invention will include all embodiments falling within the scope of the appended claims.
Claims (10)
1. A method for detecting alkaline phosphatase, comprising the steps of:
s1, preparing alkaline phosphatase solutions with different concentrations;
s2, mixing and incubating the alkaline phosphatase solution and L-ascorbic acid-2-phosphate;
s3, adding a copper sulfate solution;
s4, shooting a color picture by using a mobile phone and obtaining RGB values of reaction products;
s5, drawing a standard working curve of RGB values and alkaline phosphatase concentration;
s6, repeating the steps S2-S4 on the alkaline phosphatase sample to be detected, and comparing the alkaline phosphatase sample with a standard working curve to obtain the alkaline phosphatase activity.
2. The method for detecting alkaline phosphatase according to claim 1, wherein in the step S1, the alkaline phosphatase solution having different concentrations is specifically: 0.01U/L, 0.025U/L, 0.05U/L, 0.1U/L, 0.25U/L, 0.5U/L, 1U/L, 2.5U/L, 5U/L, 7.5U/L.
3. The method for detecting alkaline phosphatase according to claim 1, wherein in the step S2, the alkaline phosphatase solution is added in an amount of 25 μl.
4. The method for detecting alkaline phosphatase according to claim 1, wherein in the step S2, the concentration of L-ascorbic acid-2-phosphate is 50mM and the amount of the L-ascorbic acid-2-phosphate added is 25. Mu.L.
5. The method for detecting alkaline phosphatase according to claim 1, wherein in step S2, the incubation temperature is 37 ℃ and the incubation time is 1 hour.
6. The method for detecting alkaline phosphatase according to claim 1, wherein in the step S3, the copper sulfate solution is added in an amount of 50 μl at a concentration of 0.5M.
7. The method for detecting alkaline phosphatase according to claim 1, wherein the interval between steps S3 and S4 is not more than 2 minutes.
8. Use of a method for detecting alkaline phosphatase according to any of claims 1-7 in the detection of alkaline phosphatase inhibitors, comprising the steps of:
a1, preparing alkaline phosphatase inhibitors with different concentrations;
a2, mixing and incubating the alkaline phosphatase inhibitor and alkaline phosphatase;
a3, adding L-ascorbic acid-2-phosphoric acid for continuous incubation;
a4, adding a copper sulfate solution and water;
a5, shooting a color picture by using a mobile phone and obtaining RGB values of a reaction product;
a6, drawing a standard working curve of RGB values and alkaline phosphatase inhibitor concentration;
a7, repeating the steps A2-A4 on the sample to be detected, and comparing with a standard working curve to obtain the activity of the alkaline phosphatase inhibitor.
9. The use of a method for detecting alkaline phosphatase according to claim 8 in the detection of alkaline phosphatase inhibitors, wherein the alkaline phosphatase inhibitors comprise dimethoate.
10. The method for detecting alkaline phosphatase according to claim 8, wherein in step A1, alkaline phosphatase inhibitors of different concentrations are specifically: 0.5mM, 1mM, 2mM, 10mM, 20mM.
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| Application Number | Priority Date | Filing Date | Title |
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| CN202310232558.1A CN116297428A (en) | 2023-03-03 | 2023-03-03 | Method for detecting alkaline phosphatase and application thereof |
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| CN202310232558.1A CN116297428A (en) | 2023-03-03 | 2023-03-03 | Method for detecting alkaline phosphatase and application thereof |
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