CN110057879A - A kind of instant detection method of alkaline phosphatase activities - Google Patents
A kind of instant detection method of alkaline phosphatase activities Download PDFInfo
- Publication number
- CN110057879A CN110057879A CN201910314817.9A CN201910314817A CN110057879A CN 110057879 A CN110057879 A CN 110057879A CN 201910314817 A CN201910314817 A CN 201910314817A CN 110057879 A CN110057879 A CN 110057879A
- Authority
- CN
- China
- Prior art keywords
- alkaline phosphatase
- electrode
- ion detection
- pyrophosphoric acid
- detection electrode
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/28—Electrolytic cell components
- G01N27/30—Electrodes, e.g. test electrodes; Half-cells
- G01N27/333—Ion-selective electrodes or membranes
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Physics & Mathematics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Electrochemistry (AREA)
- Molecular Biology (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a kind of instant detection methods of alkaline phosphatase activities.Using the alkaline phosphatase analyte in sample solution after being catalyzed pyrophosphoric acid hydrolysis, the Cu of subsequent addition2+It can be by Cu due to not being complexed by pyrophosphoric acid2+The mechanism of the highly sensitive detection of selective enumeration method electrode.The size of the electrode response signal and the size of sample solution activity change of Alkaline phosphatase are positively correlated, i.e. the real-time and quantification detection of completion alkaline phosphatase activities.The method of the present invention is simple and easy, operator without professional training can also carry out experiment, and quantifiable signal reading is carried out without using Large-scale professionals analysis instruments such as any microplate reader, luminoscope, electrochemical workstations, analysis cost is greatly reduced, is had broad application prospects.
Description
Technical field
The invention belongs to biochemical analysis and biosensor technique field, and in particular to a kind of alkaline phosphatase activities are examined immediately
Survey method.
Background technique
Alkaline phosphatase, also referred to as alkaline phosphatase, can catalytic phosphatase ester bond under alkaline condition hydrolysis.Lactation is dynamic
In object, the alkaline phosphatase activities in liver, bile duct, kidney, bone and placenta are relatively high.Common alkaline phosphatase includes intestines
Road alkaline phosphatase, non-tissue specificity alkaline phosphatase and P-ALP.Alkaline phosphatase activities are too high or too low
It is related to a variety of diseases.For example, the activity of the colon cancer cell alkaline phosphatase of differentiation can be increased significantly, it is taken as colon cancer
The qualitative and quantitative index of cell differentiation.The raising of serum neutral and alkali phosphate is referred to as high-alkali acid phosphatase mass formed by blood stasis,
It is considered and the liver and gallbladder diseases such as pernicious bile duct obstruction, Biliary Cirrhosis, primary sclerosing cholangitis, hepatic lymphoma and hepatosarcoma
Disease is closely related.Serum activity change of Alkaline phosphatase is too low also related to some diseases.Therefore, biological sample alkaline phosphatase
Active quantitative detection has highly important medical significance.Current existing alkaline phosphatase activities detection method mainly by
The analysis instruments such as microplate reader, luminoscope, electrochemical workstation realize quantitative detection.However, these methods require operator
Higher professional skill, especially used instrument price is costly and bulky, leads to global analysis higher cost,
It can not realize field assay and immediately detection (Point-of-Care Testing).
Summary of the invention
In view of the deficiencies of the prior art, it is an object of the present invention to provide a kind of instant detection methods of alkaline phosphatase activities.
Thinking of the invention: various chemistry and biological respinse are carried out in the solution, and is examined using cheap ion selectivity
It surveys electrode and carries out quantifiable signal reading.Chemistry and the biological respinse carried out in solution includes alkaline phosphatase specific catalytic coke phosphorus
Bivalent cupric ion (Cu is specifically complexed in acid hydrolytic reaction and remaining pyrophosphoric acid2+) reaction.And then utilize cheap Cu2+Selection
Property detecting electrode it is highly sensitive detection not by pyrophosphoric acid be complexed free Cu2+.The response signal of detecting electrode is proportional to free Cu2+
Quantity, and the latter is proportional to the active size of sample alkaline phosphatase.In other words, it is simply measured by selective enumeration method electrode
Survey the Cu that dissociates in end reaction solution2+Concentration can carry out the quantitative detections of alkaline phosphatase activities.Therefore, by making
The analysis instrument of the Large expensives such as microplate reader, luminoscope, electrochemical workstation is replaced with the extremely low selective enumeration method electrode of price,
Analysis cost can be significantly reduced.If using battery powered pen type electrode can also be achieved alkaline phosphatase activities field assay and
Immediately detection.
Specific steps are as follows:
Step 1, by alkaline phosphatase sample solution and pyrophosphate solution hybrid reaction, pyrophosphoric acid molecule is by alkaline phosphatase spy
The opposite sex is hydrolyzed to phosphate anion.
Then Cu is added into step 1 gained mixed solution in step 22+Solution carries out Cu2+Pyrophosphoric acid complex reaction.
Step 3 recycles the free Cu not being complexed in mixed solution obtained by ion detection determination of electrode step 22+'s
Concentration, the electrode obtained response signal size and the size of alkaline phosphatase sample solution activity change of Alkaline phosphatase are positively correlated,
Complete the real-time and quantification detection of alkaline phosphatase activities.
Hydrolysis in the step 1 is completed by simple solution hybrid manipulation, not by alkaline phosphatase enzyme hydrolysis
Pyrophosphoric acid be capable of the Cu that subsequent addition is complexed in specificity2+。
Complex reaction in the step 2 is completed by simple solution hybrid manipulation, not by pyrophosphoric acid specificity network
The free Cu closed2+Can be by ion detection electrode quantitative detection, and ion detection electrode is to pyrophosphoric acid-Cu2+Cu in complex compound2 +Do not generate response signal.
The ion detection electrode is Cu2+Selective enumeration method electrode is battery powered pen type ion detection electrode, battery
The hand-held ion detection electrode of power supply, battery powered desk-top ion detection electrode and the desk-top ion detection electricity powered online
One of pole.
Compared with existing alkaline phosphatase activities detection method, of the invention is had the prominent advantages that: 1) analytic process letter
Single easy, the operator without professional training can also carry out experiment；2) determined using the extremely low ion detection electrode of price
Signal-obtaining is measured, analysis cost can be greatly reduced；If 3) can also be achieved alkalinity using battery powered pen type ion detection electrode
The field assay and real-time and quantification of phosphatase activity detect；4) various body fluid sample alkaline phosphatases can be directly applied to
Active level detection, has broad application prospects in fields such as clinical diagnosis, medical researches.
Detailed description of the invention
Fig. 1 is the instant detection method schematic illustration of alkaline phosphatase activities in Example 1 and Example 2 of the present invention.
Marked in the figure: 1- test tubes；2- buffer solution；3- alkaline phosphatase；4- pyrophosphoric acid；5- phosphate radical；6-Cu2+；
7- pen type Cu2+Detecting electrode.
Fig. 2 is to detect 5 U/mL using the instant detection method of alkaline phosphatase activities of the invention in the embodiment of the present invention 1
Cu obtained by alkaline phosphatase (enzyme) sample solution2+Selective enumeration method electrode response signal (Electrode signal) and sky
The comparison figure of white sample (blank) the electrode obtained response signal.
Fig. 3 is dense using the instant detection method detection activity of alkaline phosphatase activities of the invention in the embodiment of the present invention 2
Spend Cu obtained by the alkaline phosphatase sample solution that range is 0.012 ~ 5000 mU/mL2+Selective enumeration method electrode response signal
Working curve between (Electrode signal) and alkaline phosphatase activities Log value (Log (enzyeme activity)).
Specific embodiment
Following embodiment will be further described the present invention, but not thereby limiting the invention.
Embodiment 1:
5 U/mL alkaline phosphatase (enzyme) samples are detected using the instant detection method of alkaline phosphatase activities in the present invention
Solution and blank sample (blank, not the buffer of analyte-containing).
As shown in Figure 1, specific implementation process is as follows: step 1 is added 30 in the plastics test tubes of 1.5 mL
5 U/mL alkaline phosphatase sample solution of μ L is (by containing the 20 mM tris-HCl buffer preparations of 50 mM NaCl, pH
7.2) 30 μ L, 800 μM of pyrophosphate solutions then, are added (to be matched by the 20 mM tris-HCl buffer solutions containing 50 mM NaCl
System, pH 7.2) it is mixed with, it is placed at 37 DEG C and reacts 60 min；Step 2 continuously adds 30 μ L, 400 μM of Cu2+
(copper nitrate) aqueous solution reacts 15 min under room temperature；Step 3 uses pen type Cu2+It is molten that detecting electrode measures above-mentioned reaction mixing
Dissociate Cu in liquid2+Concentration.At this time from the size of sample the electrode obtained response signal value (Electrode signal) and alkalinity
The size of phosphatase sample solution activity change of Alkaline phosphatase is positively correlated, that is, completes the quantitative detection of alkaline phosphatase activities.
According to identical step, analysis margin sample (blank), i.e. 20 mM tris-HCl buffer solutions (contain 50 mM
NaCl, pH 7.2), and use pen type Cu2+Detecting electrode measures the Cu in final reaction mixed solution2+Concentration, i.e. blank electrode
Response signal value.Figure it is seen that with the 5 resulting electrode response signal values of U/mL alkaline phosphatase sample solution of detection
(Electrode signal, ~ 320 μM) is compared, and the detection resulting electrode response signal value of blank sample (~ 3 μM) almost can be with
It ignores.This is because the alkaline phosphatase in sample solution is after being catalyzed pyrophosphoric acid hydrolysis, almost without free
Pyrophosphoric acid removes complexing Cu2+.To the Cu that largely dissociates2+It can be by Cu2+Detecting electrode detects.Contrast and experiment table in Fig. 2
Bright, the instant detection method of alkaline phosphatase activities in the present invention is practical.
Embodiment 2:
It the use of the instant detection method detection active concentration range of alkaline phosphatase activities in the present invention is 0.012 ~ 5000 mU/
The alkaline phosphatase sample solution of mL.
As shown in Figure 1, detecting the specific steps of each alkaline phosphatase sample solution in the present embodiment are as follows: step 1,
The a certain active concentration alkaline phosphatase sample solution of 30 μ L is added in the plastics test tubes of one 1.5 mL (by containing 50 mM
The 20 mM tris-HCl buffer preparations of NaCl, pH 7.2), 30 μ L, 800 μM of pyrophosphate solutions are then added (by containing
The 20 mM tris-HCl buffer preparations of 50 mM NaCl, pH 7.2) it is mixed with, it is placed at 37 DEG C and reacts 60
min；Step 2 continuously adds 30 μ L, 400 μM of Cu2+(copper nitrate) aqueous solution reacts 15 min under room temperature；Step 3 makes
With pen type Cu2+Detecting electrode measures the Cu that dissociates in above-mentioned reaction mixture2+Concentration.All alkaline phosphatase enzyme samples are molten
Log value mapping (Log of the electrode response signal value (Electrode signal) that liquid obtains to alkaline phosphatase activities concentration
(enzyeme activity)) (Fig. 3), that is, complete the quantitative detection of alkaline phosphatase activities.
From the figure 3, it may be seen that with the reduction of alkaline phosphatase activities concentration, corresponding electrode response signal value (Electrode
Signal it) gradually decreases.This is because the pyrophosphoric acid not being hydrolyzed is then when sample solution activity change of Alkaline phosphatase is lower
Reservation is relatively chiefly used in that more Cu is complexed2+.Free Cu2+Surplus it is less, cause the response signal of electrode lower.In addition, figure
3 displays measure resulting electrode response signal value (Electrode signal) and alkaline phosphatase using the method for the present embodiment
Good line is presented in the Log value (Log (enzyeme activity)) of concentration of enzymatic activity within the scope of 0.32 ~ 1000 mU/mL
Sexual intercourse.
Claims (1)
1. a kind of instant detection method of alkaline phosphatase activities, the specific steps are that:
Step 1, by alkaline phosphatase sample solution and pyrophosphate solution hybrid reaction, pyrophosphoric acid molecule is by alkaline phosphatase spy
The opposite sex is hydrolyzed to phosphate anion；
Then Cu is added into step 1 gained mixed solution in step 22+Solution carries out Cu2+Pyrophosphoric acid complex reaction；
Step 3 recycles the free Cu not being complexed in mixed solution obtained by ion detection determination of electrode step 22+Concentration,
The electrode obtained response signal size and the size of alkaline phosphatase sample solution activity change of Alkaline phosphatase are positively correlated, that is, complete
The real-time and quantification of alkaline phosphatase activities detects；
Hydrolysis in the step 1 is completed by simple solution hybrid manipulation, not by the coke of alkaline phosphatase enzyme hydrolysis
Phosphoric acid is capable of the Cu of the subsequent addition of specificity complexing2+；
Complex reaction in the step 2 is completed by simple solution hybrid manipulation, is not complexed by pyrophosphoric acid specificity
Free Cu2+Can be by ion detection electrode quantitative detection, and ion detection electrode is to pyrophosphoric acid-Cu2+Cu in complex compound2+No
Generate response signal；
The ion detection electrode is Cu2+Selective enumeration method electrode is battery powered pen type ion detection electrode, battery power supply
Hand-held ion detection electrode, in battery powered desk-top ion detection electrode and the desk-top ion detection electrode powered online
One kind.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910314817.9A CN110057879A (en) | 2019-04-18 | 2019-04-18 | A kind of instant detection method of alkaline phosphatase activities |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910314817.9A CN110057879A (en) | 2019-04-18 | 2019-04-18 | A kind of instant detection method of alkaline phosphatase activities |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN110057879A true CN110057879A (en) | 2019-07-26 |
Family
ID=67319644
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910314817.9A Pending CN110057879A (en) | 2019-04-18 | 2019-04-18 | A kind of instant detection method of alkaline phosphatase activities |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110057879A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113433103A (en) * | 2021-06-29 | 2021-09-24 | 中国农业大学 | Method for detecting 2,4-dichlorophenoxyacetic acid |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS59143600A (en) * | 1983-02-07 | 1984-08-17 | Agency Of Ind Science & Technol | Method for measuring activity of acidic and alkaline phosphatase |
| US20140311908A1 (en) * | 2013-04-22 | 2014-10-23 | Uwm Research Foundation, Inc. | Native protein electrophoresis and methods of use |
| CN104391029A (en) * | 2014-11-17 | 2015-03-04 | 中国科学院化学研究所 | Electrochemical method used for measuring activity of alkaline phosphatase |
| CN107976427A (en) * | 2017-11-08 | 2018-05-01 | 安徽师范大学 | A kind of biological sensor, preparation method and its detection application to copper ion, pyrophosphate and alkaline phosphatase |
-
2019
- 2019-04-18 CN CN201910314817.9A patent/CN110057879A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS59143600A (en) * | 1983-02-07 | 1984-08-17 | Agency Of Ind Science & Technol | Method for measuring activity of acidic and alkaline phosphatase |
| US20140311908A1 (en) * | 2013-04-22 | 2014-10-23 | Uwm Research Foundation, Inc. | Native protein electrophoresis and methods of use |
| CN104391029A (en) * | 2014-11-17 | 2015-03-04 | 中国科学院化学研究所 | Electrochemical method used for measuring activity of alkaline phosphatase |
| CN107976427A (en) * | 2017-11-08 | 2018-05-01 | 安徽师范大学 | A kind of biological sensor, preparation method and its detection application to copper ion, pyrophosphate and alkaline phosphatase |
Non-Patent Citations (2)
| Title |
|---|
| JUNYONG SUN 等: "Ratiometric detection of copper ions and alkaline phosphatase activity based on semiconducting polymer dots assembled with rhodamine B hydrazide", 《BIOSENSORS AND BIOELECTRONICS》 * |
| WEN LIU等: "Potentiometric Characterization of Telomerase Activity Using a Copper(ii)-pyrophosphate Complex with a Copper ion-selective Electrode", 《ANALYTICAL LETTERS》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113433103A (en) * | 2021-06-29 | 2021-09-24 | 中国农业大学 | Method for detecting 2,4-dichlorophenoxyacetic acid |
| CN113433103B (en) * | 2021-06-29 | 2022-10-04 | 中国农业大学 | Method for detecting 2,4-dichlorophenoxyacetic acid |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106596532B (en) | A kind of detection method of simple alkaline phosphatase activities | |
| CN105784796B (en) | A kind of sensitive determination method of the aptamer sensor based on gold/molybdenum disulfide/graphene nanocomposite material to lysozyme | |
| CN104020198B (en) | A kind of signal amplification technique electrochemical sensor detects the method for DNA | |
| CN102297962B (en) | Kit for detecting alkaline phosphatase | |
| CN109576342B (en) | Fluorescence chemical method for detecting alkaline phosphatase and application | |
| CN106950380B (en) | A kind of gastric cancer monitoring reagent box and its application method | |
| CN100999758A (en) | Alpha-L-fucosidosidase active tested process and diagnostic reagent of alpha-L-fucosidosidase | |
| CN107976427A (en) | A kind of biological sensor, preparation method and its detection application to copper ion, pyrophosphate and alkaline phosphatase | |
| US20200309725A1 (en) | Portable microbial load detection | |
| CN104596959A (en) | Method for detecting potassium ion concentration based on DNA enzymes | |
| CN110057879A (en) | A kind of instant detection method of alkaline phosphatase activities | |
| Davis | The kinetics of the reaction of human erythrocyte carbonic anhydrase. I. Basic mechanism and the effect of electrolytes on enzyme activity | |
| CN104931489A (en) | Quantitative analysis method for measuring rapid test strip on basis of iodine-starch variable-color distance | |
| CN104483472A (en) | Diagnostic reagent for cervical cancer cell detection and preparation method thereof | |
| CN103940808A (en) | Preparation method and application of electrochemical luminescence biosensor with dual-signal amplification | |
| CN104049090B (en) | A kind of test kit detecting alanylamino peptase | |
| CN101226152A (en) | Automatic analysis method and liquid stabilising agent for blood serum total ferro combining ability | |
| CN104677897A (en) | PH and urea determination method based on nanogold catalytic colored system | |
| CN102747133B (en) | Adenosine deaminase (ADA) detection reagent kit and preparation method thereof | |
| CN109557060B (en) | Based on NH2Method for visually detecting alkaline phosphatase activity in serum by using (E) -Cu-MOF | |
| CN104807995B (en) | High-sensitivity continuous-detection-method GLDH (Glutamic Dehydrogenase) detection reagent | |
| CN101974612A (en) | Kit for detecting activity of acid phosphatase in seminal plasma and detection method | |
| CN113655006B (en) | Urinary system knot Dan Chengdan risk factor detection and test system | |
| Shang et al. | A turn-on fluorescent strategy for alkaline phosphatase detection based on enzyme-assisted signal amplification | |
| CN1995975A (en) | Method for detecting N-acetyl-beta-amino glucosaccharase activity and diagnosis kit therefor |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20190726 |
|
| WD01 | Invention patent application deemed withdrawn after publication |