CN109613088A - A kind of phosphatase activity detection method that black phosphorus mediates the electrochemical signals of copper ion aggregation to convert - Google Patents
A kind of phosphatase activity detection method that black phosphorus mediates the electrochemical signals of copper ion aggregation to convert Download PDFInfo
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- CN109613088A CN109613088A CN201811564153.3A CN201811564153A CN109613088A CN 109613088 A CN109613088 A CN 109613088A CN 201811564153 A CN201811564153 A CN 201811564153A CN 109613088 A CN109613088 A CN 109613088A
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Abstract
The invention belongs to phosphatase activity detection fields, the phosphatase activity detection method of the electrochemical signals conversion of copper ion aggregation is mediated more particularly to a kind of black phosphorus, the following steps are included: by pyrophosphoric acid, active inorganic pyrophosphatase hybrid reaction to be measured, then mantoquita is added, the reaction was continued, reaction solution to be measured is obtained, the electrochemical signals of copper ion are then detected by black phosphorus, obtain electrochemical response signal.Black phosphorus mediates the phosphatase activity sensing technology of the electrochemical signals conversion of copper ion aggregation for detecting PPase Activity in the present invention.This method is simple to operation, operates in homogeneous phase solution, has used the natural substrate PPi consistent with physiological condition and has combined black phosphorus nano material to the specific adsorption property of copper ion, the Sensitive Detection to pyrophosphatase may be implemented.
Description
Technical field
The invention belongs to phosphatase activity detection fields, and in particular to a kind of black phosphorus mediates the electrochemistry letter of copper ion aggregation
Number conversion phosphatase activity detection method.
Background technique
Enzyme is the important molecule device for determining biosystem chemical conversion mode.Almost in all organisms, enzymatic
Reaction all plays very important effect, even if being that organism is normal there is also thousands of kinds of enzymatic reactions in individual cells
Metabolism and breeding provide resource and energy.Inorganic pyrophosphatase (PPase, a kind of generally existing hydrolase), it can be special
It is catalyzed a molecule pyrophosphate (P2O7 4-, PPi) and it is converted into two phosphate molecule (PO4 3-, Pi).PPase enzymatic PPi hydrolysis
Reaction is a kind of high-energy discharge reaction, and therefore, it can be used for the undesirable biochemical reaction of some energy, to push or supplement this
A little reactions.In addition, PPase is also reported and some biological respinses (such as Fatty synthesis and degradation, phosphorus metabolism, carbohydrate generation
Thank, DNA synthesis, calcium uptake, bon e formation) and clinical disease (such as adenocarcinoma of lung, hyperthyroidism, colorectal cancer) relationship.
The method of traditional detection PPase is mainly by analyzing the Pi discharged, or uses radioactivity mark
The PPi of note simulates natural substrate, but these methods are time-consuming, and arduously, sensitivity is low.Afterwards again it has been proposed that with colorimetric/fluorescence analysis
The activity of PPase is detected, but these methods often relate to labelling strategies.
Summary of the invention
The purpose of the invention is to overcome shortcoming and defect of the existing technology, and provide a kind of black phosphorus mediate copper from
The phosphatase activity detection method of the electrochemical signals conversion of son aggregation.
The technical solution used in the present invention is as follows: a kind of phosphorus that black phosphorus mediates the electrochemical signals of copper ion aggregation to convert
Phytase activity detection method, comprising the following steps: by pyrophosphoric acid, active inorganic pyrophosphatase hybrid reaction to be measured, then plus
Enter mantoquita, the reaction was continued, obtains reaction solution to be measured, and the electrochemical signals of copper ion are then detected by black phosphorus, obtain electrochemistry
Response signal.
Since black phosphorus nano material has extremely strong specific adsorption to act on copper ion, we have proposed a kind of bases
In half phase electrochemical strategy of black phosphorus-enzyme hydrolysis induction copper ion release for detecting PPase Activity.When object coke
In the absence of phosphatase, strong interaction between pyrophosphoric acid and copper ion, prevent the copper ion in reaction solution is from being adsorbed on
Drop coating has on the glass-carbon electrode of black phosphorus nanometer sheet, therefore, detects extremely low copper ion electrochemical signals；Conversely, working as pyrophosphoric acid
In the presence of enzyme, pyrophosphatase can be hydrolyzed with catalysis substrate pyrophosphoric acid, it is suppressed that the formation of pyrophosphoric acid-copper ion compound, reaction
Copper ion in liquid can effectively be adsorbed on the glassy carbon electrode surface that drop coating has black phosphorus nanometer sheet, detect extremely strong copper ion
Electrochemical signals.Positive is presented in this method mentioned in the present invention, the electrochemical signals of copper ion and the concentration of pyrophosphatase
It closes, and can effectively monitor external factor to the depression effect of PPase Activity.This method is easy to operate, without label and
Complicated operation, high to the selectivity of PPase Activity detection, high sensitivity is expected to be applied to clinical diagnosis and pyrophosphatase
Relevant disease and probe into mechanism of action of the pyrophosphatase in biosystem.
It is detected by the electrochemical signals that black phosphorus detects copper ion using the glass-carbon electrode of drop coating black phosphorus nano material, it is described
The glass-carbon electrode of drop coating black phosphorus nano material is obtained by following procedure: black phosphorus nanometer sheet is placed in ultrasound 1-3h in Ultrasound Instrument,
Then plus then 5-15 V voltage, electrolysis black phosphorus nanometer sheet solution 15-45min are placed in ultrasound 1-3h in Ultrasound Instrument again, obtain black
Phosphorus solution takes black phosphorus solution drop on glass-carbon electrode, obtains the glass-carbon electrode of drop coating black phosphorus nano material after dry.It is molten to black phosphorus
It is removing black phosphorus nanometer sheet that liquid, which carries out ultrasound-electrolysis-ultrasound purpose,
The reaction solution to be measured is added drop-wise to the glassy carbon electrode surface of drop coating black phosphorus nano material, at least 4h is reacted, then washes
Dereaction liquid, drying, is placed in containing KNO3Buffer in survey square wave voltammetry, potential window is selected as -0.3 V-0.2 V,
Current potential jump is 4 mV, and amplitude is 25 mV, and frequency is 25 Hz.
The process of the reaction solution reaction to be measured is as follows: mixing 10 μ L, 2 mM pyrophosphoric acid and 50 μ L inorganic pyrophosphatases,
86 μ L buffers are added, at least reaction 30min, mantoquita is then added, reacts at least 5 min.
Reaction carries out at 25 DEG C.At 25 DEG C, pyrophosphatase working active highest.
The buffer is HEPES buffer(N-2- hydroxyethyl piperazine-N ' -2-ethanesulfonic acid solution).
The mantoquita is copper sulphate, and mantoquita may be other solvable mantoquitas.
(1) beneficial effects of the present invention are as follows: the method use the natural substrate PPi consistent with physiological condition, this
Mean that this method can provide the substantial activity level of PPase under physiological condition.
(2) this method is simple to operation, without label, reacts in homogeneous phase solution.
(3) this method combines black phosphorus nano material to the specific adsorption property of copper ion, develops a kind of novel sensing
Device realizes the detection to PPase, easy to operate, high sensitivity.
In summary: black phosphorus mediates the phosphatase activity of the electrochemical signals conversion of copper ion aggregation to sense skill in the present invention
Art is for detecting PPase Activity.This method is simple to operation, operates in homogeneous phase solution, has used and physiological condition phase one
The natural substrate PPi of cause simultaneously combines black phosphorus nano material to the specific adsorption property of copper ion, may be implemented to pyrophosphoric acid
The Sensitive Detection of enzyme.
Detailed description of the invention
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below
There is attached drawing needed in technical description to be briefly described, it should be apparent that, the accompanying drawings in the following description is only this
Some embodiments of invention, for those of ordinary skill in the art, without any creative labor, according to
These attached drawings obtain other attached drawings and still fall within scope of the invention.
Fig. 1 is the original of the phosphatase activity sensing technology for the electrochemical signals conversion that black phosphorus of the present invention mediates copper ion aggregation
Reason figure；
Fig. 2 is the phosphatase activity sensing technology for the electrochemical signals conversion that black phosphorus of the present invention mediates copper ion aggregation for special
Property detection PPase Activity；
Fig. 3 is the phosphatase activity sensing technology for the electrochemical signals conversion that black phosphorus of the present invention mediates copper ion aggregation for detecting
PPase Activity, under different pyrophosphatase concentration, the variation of electrochemical response signal；
Fig. 4 is the phosphatase activity sensing technology for the electrochemical signals conversion that black phosphorus of the present invention mediates copper ion aggregation for detecting
PPase Activity, the linear relationship of electrochemical response signal and pyrophosphatase log concentration value；
Fig. 5 is the phosphatase activity sensing technology for the electrochemical signals conversion that black phosphorus of the present invention mediates copper ion aggregation for examining
The inhibitor for surveying pyrophosphatase, when pyrophosphatase concentration is 10 mU/mL, with inhibitor Na3PO4The continuous increase of concentration,
Electrochemical response signal gradually weakens.
Specific embodiment
To make the object, technical solutions and advantages of the present invention clearer, the present invention is made into one below in conjunction with attached drawing
Step ground detailed description.
Embodiment 1
The preparation of the glass-carbon electrode (GCE) of drop coating black phosphorus nano material: black phosphorus nanometer sheet is placed in 2 h of ultrasound in Ultrasound Instrument, so
Afterwards plus then 10 V voltages, electrolysis 30 min of black phosphorus nanometer sheet solution are placed in 2 h of ultrasound in Ultrasound Instrument, it is therefore an objective to make black phosphorus again
Nanometer sheet is removed.Then, black phosphorus solution (6 μ g) is dripped on the surface GCE, is placed in drying box, obtains drop coating black phosphorus nanometer
The glass-carbon electrode of material.
Embodiment 2
(1) preparation containing variety classes enzyme reaction solution:
10 μ L, 2 mM PPi and 50 μ LPPase(1.5 μ L, 1 mU/ μ L PPase and 44.5 μ L water), 86 μ L HEPES
Buffer, 55 min of water-bath at 25 DEG C.
Guarantee that other conditions are constant, 50 μ LPPase in the above process are changed to 50 μ L(11.55 μ L 1299 respectively
MU/mL GOx and 38.45 μ L water) glucose oxidase (GOx) so that its ultimate density be 100 mU/mL and 50 μ L
(3.75 μ L 4 mU/ μ L CpG MTase, 15 μ L 10 × NEBuffer, 0.75 200 × S- of μ L
Adenosylmethionine, 30.5 μ L water) CpG transmethylase (CpG Methyltransferase) so that its
Ultimate density is 100 mU/mL.Its data measured respectively corresponds GOx the and CpG MTase histogram in Fig. 2.
Guarantee that other conditions are constant, 50 μ LPPase in the above process are changed to the PPase of 1.5 μ L 1mU/mL, 11.55
μ L 1299 mU/mL GOx, 3.75 μ L 4 mU/ μ L CpG MTase, 15 μ L 10 × NEBuffer, 0.75 μ L 200
× S-adenosylmethionine, the mixed liquor of 17.45 μ L water, so that the concentration of PPase, GOx and CpG MTase are distinguished
For 10 mU/mL, 100 mU/mL and 100 mU/mL.Mix histogram in its data corresponding diagram 2 measured.
Guarantee that other conditions are constant, 50 μ LPPase in the above process are changed to the water of 50 μ L, the data measured are corresponding
Control histogram in Fig. 2.
(2) solution is stated then up adds 8 μ L 2 mM CuSO4,25 DEG C of 10 min of water-bath.Take 30 μ L reaction solutions
The glassy carbon electrode surface of the drop coating black phosphorus nano material obtained in embodiment 1 is dripped, 5 h are reacted.Dereaction liquid is washed with deionized water,
It is dried with nitrogen with soft.
(3) above-mentioned electrode is placed in PH7.2 HEPES buffer(and contains 50 mM KNO3) solution in survey square wave volt-ampere
Method, potential window are selected as -0.3 V-0.2 V, and current potential jump is 4 mV, and amplitude is 25 mV, and frequency is 25 Hz, on the left side 56 mV
It is right characteristic peak occur.
As shown in Figure 2, black phosphorus mediates the phosphatase activity of the electrochemical signals conversion of copper ion aggregation to sense in the present invention
Technology can be used for specific detection PPase Activity.
Embodiment 3
The enzyme solution of 2 mM PPi of (1) 10 μ L and 50 μ LPPase(various concentrations), add 86 μ L HEPES buffer
(n-2-Hydroxyethylpiperazine-N'-2-ethanesulfonic acid), 55 min of water-bath at 25 DEG C.
(2) solution is stated then up adds 8 μ L, 2 mM CuSO4, 25 DEG C of 10 min of water-bath.Take 30 μ L reaction solutions
The glassy carbon electrode surface of the drop coating black phosphorus nano material obtained in embodiment 1 is dripped, 5 h are reacted.Dereaction liquid is washed with deionized water,
It is dried with nitrogen with soft.
(3) above-mentioned electrode is placed in PH7.2 HEPES buffer(and contains 50 mM KNO3) solution in survey square wave volt-ampere
Method, potential window are selected as -0.3 V-0.2 V, and current potential jump is 4 mV, and amplitude is 25 mV, and frequency is 25 Hz, on the left side 56 mV
It is right characteristic peak occur.
The phenomenon that SWAV curve measured changes with PPase concentration difference figure, as shown in Figure 3.
The variation relation of the logarithm of electrochemical response signal and PPase concentration is as shown in figure 4, with PPase concentration
Increase, more and more PPi become Pi in hydrolysis liquid, measure the electrochemical response signal enhancing of copper ion.PPase concentration
When changing in the concentration range of 0.01 mU/mL to 10 mU/mL, the logarithm of electrochemical response signal and PPase concentration it
Between have a good linear relationship, lowest detection lower limit is 0.01 mU/mL.
Embodiment 4
(1) contain PPase and inhibitor Na3PO4The preparation of reaction solution
10 μ L, 2 mM PPi and PPase(1.5 μ L, 1 mU/ μ L PPase, ultimate density are 10 mU/mL), it adds not
40 mM Na of same volume3PO4(concentration is respectively 0,0.2 mM, 0.4 mM, 0.6 mM, 1 mM, 2 mM, 4 mM) is added
86 μ L HEPES buffer, react 55 min at 25 DEG C respectively.
(2) solution is stated then up adds 8 μ L, 2 mM CuSO4, 25 DEG C of 10 min of water-bath.Take 30 μ L reaction solutions
The glassy carbon electrode surface of the drop coating black phosphorus nano material obtained in embodiment 1 is dripped, 5 h are reacted.Dereaction liquid is washed with deionized water,
It is dried with nitrogen with soft.
(3) above-mentioned electrode is placed in PH7.2 HEPES buffer(and contains 50 mM KNO3) solution in survey square wave volt-ampere
Method, potential window are selected as -0.3 V-0.2 V, and current potential jump is 4 mV, and amplitude is 25 mV, and frequency is 25 Hz, on the left side 56 mV
It is right characteristic peak occur.
As shown in Figure 5, with pyrophosphatase enzyme inhibitors Na3PO4The increase of concentration, the electrochemical response signal of copper ion by
It is decrescence weak.Therefore, black phosphorus can be mediated the phosphatase activity sensing technology of the electrochemical signals conversion of copper ion aggregation by we
Monitor the inhibitor of pyrophosphatase.
The above disclosure is only the preferred embodiments of the present invention, cannot limit the right model of the present invention with this certainly
It encloses, therefore equivalent changes made in accordance with the claims of the present invention, is still within the scope of the present invention.
Claims (7)
1. the phosphatase activity detection method that a kind of black phosphorus mediates the electrochemical signals conversion of copper ion aggregation, it is characterised in that packet
It includes following steps: by pyrophosphoric acid, active inorganic pyrophosphatase hybrid reaction to be measured, mantoquita is then added, the reaction was continued, obtains
Then reaction solution to be measured is detected the electrochemical signals of copper ion by black phosphorus, obtains electrochemical response signal.
2. the phosphatase activity detection side that black phosphorus according to claim 1 mediates the electrochemical signals conversion of copper ion aggregation
Method, which is characterized in that examined by the electrochemical signals that black phosphorus detects copper ion using the glass-carbon electrode of drop coating black phosphorus nano material
It surveys, the glass-carbon electrode of the drop coating black phosphorus nano material is obtained by following procedure: black phosphorus nanometer sheet being placed in Ultrasound Instrument and is surpassed
Sound 1-3h, then plus then 5-15 V voltage, electrolysis black phosphorus nanometer sheet solution 15-45min are placed in ultrasound 1- in Ultrasound Instrument again
3h obtains black phosphorus solution, takes black phosphorus solution drop on glass-carbon electrode, and the glass carbon electricity of drop coating black phosphorus nano material is obtained after dry
Pole.
3. the phosphatase activity detection side that black phosphorus according to claim 2 mediates the electrochemical signals conversion of copper ion aggregation
Method, it is characterised in that: the reaction solution to be measured is added drop-wise to the glassy carbon electrode surface of drop coating black phosphorus nano material, reaction is at least
Then 4h washes away reaction solution, drying is placed in the buffer containing KNO3 and surveys square wave voltammetry, potential window is selected as -0.3
V-0.2 V, current potential jump are 4 mV, and amplitude is 25 mV, and frequency is 25 Hz.
4. the phosphoric acid enzyme activity that black phosphorus according to claim 1-3 mediates the electrochemical signals conversion of copper ion aggregation
Property detection method, it is characterised in that the process of the reaction solution to be measured reaction is as follows: mixing 10 μ L, 2 mM pyrophosphoric acid and 50 μ L
Inorganic pyrophosphatase adds 86 μ L buffers, at least reaction 30min, and mantoquita is then added, reacts at least 5 min.
5. the phosphatase activity detection side that black phosphorus according to claim 4 mediates the electrochemical signals conversion of copper ion aggregation
Method, it is characterised in that: reaction carries out at 25 DEG C.
6. the phosphatase activity detection side that black phosphorus according to claim 4 mediates the electrochemical signals conversion of copper ion aggregation
Method, it is characterised in that: the buffer is HEPES buffer.
7. the phosphatase activity detection side that black phosphorus according to claim 4 mediates the electrochemical signals conversion of copper ion aggregation
Method, it is characterised in that: the mantoquita is copper sulphate.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107462619A (en) * | 2017-07-25 | 2017-12-12 | 海南师范大学 | A kind of preparation of the protein electrochemistry senser element based on black phosphorus modified electrode and electro-catalysis application |
| CN108445065A (en) * | 2018-03-06 | 2018-08-24 | 海南师范大学 | A kind of preparation method and its electrocatalysis characteristic research of black phosphorus alkene hemin modified electrode |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107462619A (en) * | 2017-07-25 | 2017-12-12 | 海南师范大学 | A kind of preparation of the protein electrochemistry senser element based on black phosphorus modified electrode and electro-catalysis application |
| CN108445065A (en) * | 2018-03-06 | 2018-08-24 | 海南师范大学 | A kind of preparation method and its electrocatalysis characteristic research of black phosphorus alkene hemin modified electrode |
Non-Patent Citations (2)
| Title |
|---|
| WANSONG CHEN ET AL.: "Black Phosphorus Nanosheets as a Neuroprotective Nanomedicine for Neurodegenerative Disorder Therapy", 《ADVANCED MATERIALS》 * |
| YOUXIU LIN ET AL.: "Magnetic Graphene Nanosheet-Based Microfluidic Device for Homogeneous Real-Time Electronic Monitoring of Pyrophosphatase Activity Using Enzymatic Hydrolysate-Induced Release of Copper Ion", 《ANALYTICAL CHEMISTRY》 * |
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Application publication date: 20190412 |