CN108267494A - Based on class nucleic acid coacetylase-Cu（II）The preparation method and application of the pyrophosphatase electrochemica biological sensor of coordination polymer - Google Patents
Based on class nucleic acid coacetylase-Cu（II）The preparation method and application of the pyrophosphatase electrochemica biological sensor of coordination polymer Download PDFInfo
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Abstract
The invention discloses the preparation method and applications of the pyrophosphatase electrochemica biological sensor based on class nucleic acid coacetylase Cu (II) coordination polymer, are as follows：(1) Cu is taken2+Solution, coacetylase aqueous solution acutely vibrate to obtain CoA Cu (II) CP；(2) PPi solution, Cu are taken successively2+Solution, Mg2+Solution, Tris HCl buffer solutions (pH 7.4), in EP pipes, acutely oscillation is uniformly mixed PPase, adds in CoA；(3) PPase is taken in EP pipes, adds in NaF, and acutely concussion is uniformly mixed, and adds PPi solution, Cu2+Solution, Mg2+Solution, Tris HCl buffer solutions (pH 7.4), acutely oscillation are uniformly mixed, and add in CoA；(4) graphene dispersion is in acetate buffer solution, graphene is electrodeposited into bare glassy carbon electrode with cyclic voltammetry and obtains GO/GCE, solution drop coating in (1) or (2) or (3) is taken on GO/GCE, being stored at room temperature up to electrochemical sensor CP/GO/GCE.For detecting PPase activity and its micromolecular inhibitor, advantage be the good, high sensitivity of specificity, detection speed be fast, result accurately and reliably, it is at low cost.
Description
Technical field
The present invention relates to a kind of electrochemica biological sensor and its detection methods, auxiliary based on class nucleic acid more particularly, to one kind
The preparation method and applications of the pyrophosphatase electrochemica biological sensor of enzyme A-Cu (II) coordination polymer, belong to functional material
With biosensor technique field.
Background technology
Pyrophosphatase (PPase) is a kind of with the hydrolase that pyrophosphate (PPi) is substrate, is acted on bis phosphoric acid key
Acid anhydrides can be catalyzed a molecule pyrophosphate and be converted into two molecule phosphate ions.PPase is widely present in nature, participates in closing
Into the hydrolysis of the pyrophosphate formed in the multiple metabolic pathways such as carbohydrate, nucleic acid and protein.Moreover, pyrophosphatase exists
Lipid metaboli (including fat synthesis with decomposing), calcium uptake and bon e formation and DNA play an important role in synthesizing.Clinically finding should
Enzyme low expression is superior to the therapeutic effect of cancer patient and life cycle high expressors, therefore pyrophosphoric acid in patient tissue or cell
The screening of the Activity determination and its inhibitor of enzyme, to the auxiliary diagnosis of tumour, antidiastole, observation of curative effect, state of illness monitoring and
Prognostic evaluation has very high value.
At present, the research of fluorescence sense system is concentrated mainly on about the detection of pyrophosphatase, there is certain sensitivity
And accuracy, but the part that comes with some shortcomings：Expensive equipment, complicated for operation, technology requirement is high, therefore, active demand exploitation
Sensitive, accurate, quick, easy PPase Activity detection method.Electrochemica biological sensor is by powerful point of electrochemistry
Analysis function is combined with the specificity of biometric identification process, it is therefore an objective to generate one and analyte concentration by biological respinse
Relevant electric signal.For this purpose, it using a biospecific reagents immobilization or is maintained on appropriate electrode, the electrode
Bio-identification signal is quantitatively converted into electric current or potential response.Electrochemica biological sensor is because having quick, stable, selectivity
By force, favorable reproducibility, it is easily operated, step is simple the advantages that be widely used.So far, the relevant electrochemical student of pyrophosphatase
Object sensing Study of An is very rare.
Coacetylase (CoA) is mainly made of as a kind of biological micromolecule containing sulfydryl, structure three parts：3 '-phosphoric acid
Adenine structure and class cysteine structure, centre are connected by pantothenic acid group.Two functional groups at CoA both ends：One is
Adenine structure, the other is mercapto groups.This special construction causes CoA to be likely to become the weight of biochemistry correlative study
Want substance, if it is possible to while biochemistry detection is carried out using two functional groups at CoA both ends, its detection will certainly be increased
Specificity and selectivity.Based on the sulfydryl of CoA ends, by itself and coin race metal ion hybrid reaction, pass through sulfydryl and gold
Belong to the repetitive unit that the effect of ion can form many CoA- metal ions, synthesize a kind of special coordination polymer, because
The class adenine structure of CoA ends so that have many adenine groups on the skeleton of the coordination polymer, have similar RNA's
Structure, the role that can play the part of RNA to a certain extent participate in the structure of bio-sensing system.Our seminars are to coenzyme
The coordination polymer that A and silver ion or gold ion are formed has carried out further investigated, but about the coordination of coacetylase and copper ion
The research of polymer is very few, non-due to the good electrochemical catalysis performance of such RNA coordination polymer itself and bio-compatibility
Often it is suitable for developing electrochemica biological sensor.
Graphene (GO) is a kind of cellular flat film formed by carbon atom with sp2 hybrid forms, is to find so far
Most thin two-dimensional material, have more large specific surface area, the edge site, surface energy height, the good, good biocompatibility of catalytic performance etc. excellent
Point, the extensive use in electrochemica biological analysis field.First, graphene can accelerate electrode surface electron transmission speed,
Effectively increase electrochemical signals, greatly improve the sensitivity of sensor；Secondly, the delocalization body pi-conjugated greatly in graphene skeleton
System by Non-covalent binding act on the compound π containing pi-electron-pi-conjugated, biomolecule can be effectively fixed.The invention
The coordination polymer of the class RNA of middle formation can just be fixed on the surface of Graphene electrodes by π-πconjugation, be pyrophosphatase
The structure of electrochemica biological sensor lays the foundation.
The present invention is based on the specific groups (sulfydryl and adenine) at coacetylase both ends, pass through d10Coin race metal ion
" metallophilicity " interaction, Metal metal interaction and sulfydryl-metal interaction, using coacetylase and
Copper ion synthesizes the coordination polymer material (being abbreviated as CoA-Cu (II) CP) of a type RNA, constructs the unmarked, Gao Ling of one kind
Quick, highly selective electrochemica biological sensor can detect PPase Activity and its micromolecular inhibitor is screened,
Have no nucleoid acidic group in the report of the pyrophosphatase electrochemica biological sensor of coacetylase-copper ion coordination polymer material at present.
Invention content
The technical problems to be solved by the invention are to provide that a good species specificity, high sensitivity, detection speed is fast, result is accurate
The really preparation method and application of the pyrophosphatase electrochemica biological sensor of class nucleic acid CoA-Cu (II) CP reliable, at low cost,
By the electrochemical catalysis to o-phenylenediamine (OPD), so as to fulfill the detection of PPase Activity and its screening of inhibitor.
Technical solution is used by the present invention solves above-mentioned technical problem：Based on class nucleic acid coacetylase-Cu (II) polycomplexation
The preparation method and application of the pyrophosphatase electrochemica biological sensor of object is closed, is as follows：
(1) preparation of CoA-Cu (II) CP
The Cu of 8.0~12.0 a concentration of 0.8~10.0mM of μ L is taken successively2+Solution, 8.0~12.0 μ L a concentration of 1.6~
20.0mM coacetylase aqueous solutions, add distilled water to be made into the solution of 80.0~100.0 μ L, solution is acutely vibrated 5~15min to get
To CoA-Cu (II) CP.
(2) PPase Enzyme assays
A. the PPi solution of 5.0~10.0 a concentration of 0.1~10.0mM of μ L is taken successively, 8.0~12.0 μ L a concentration of 0.8~
10.0mM Cu2+Solution, the Mg of 5.0~10.0 a concentration of 100.0~200.0 μM of μ L2+Solution, 10.0~15.0 μ L are a concentration of
The Tris-HCl buffer solutions (pH 7.4) of 20.0~40.0mM, the PPase of 5.0~10.0 a concentration of 0.001~100.0U/L of μ L
In EP pipes, acutely vibrating 2~5min is uniformly mixed solution, and above-mentioned solution is incubated 0.5~1.5h at 20~40 DEG C, after
The EP pipes are placed in 85~90 DEG C of water-baths inactivates enzyme, is cooled to room temperature, control enzyme final concentration ranging from 0~2U/L.
B. the CoA of 8.0~12.0 a concentration of 1.6~20.0mM of μ L is added in into a, distilled water is added to be made into 80.0~100.0 μ
Solution is acutely vibrated 5~15min by the solution of L, and it is spare to be placed on 4 DEG C of refrigerator cold-storages.
(3) detection of PPase enzyme inhibitors NaF
A. the PPase of 5.0~10.0 a concentration of 0.001~100.0U/L of μ L is taken in EP pipes, and it is dense to add in 1.0~10.0 μ L
The NaF for 0.01~1mM is spent, 1~3min is acutely shaken and is uniformly mixed, ranging from 0~120 μM of inhibitor NaF final concentrations；
B. the PPi solution of 5.0~10.0 a concentration of 0.1~10.0mM of μ L, 8.0~12.0 μ L concentration are sequentially added into a
Cu for 0.8~10.0mM2+Solution, the Mg of 5.0~10.0 a concentration of 100.0~200.0 μM of μ L2+Solution, 10.0~15.0 μ L
For the Tris-HCl buffer solutions (pH 7.4) of a concentration of 20.0~40.0mM in EP pipes, acutely vibrating 2~5min mixes solution
Uniformly, above-mentioned solution is incubated 0.5~1.5h at 20~40 DEG C, after the EP pipes be placed in 85~90 DEG C of water-baths inactivate enzyme,
It is cooled to room temperature；
C. the CoA of 8.0~12.0 a concentration of 1.6~20.0mM of μ L is added in into b, distilled water is added to be made into 80.0~100.0 μ
Solution is acutely vibrated 5~15min by the solution of L, and it is spare to be placed on 4 DEG C of refrigerator cold-storages.
(4) preparation of electrochemical sensor
A., 4.0~9.0mg graphenes are dissolved in the acetic acid of the pH 5.0~6.0 of a concentration of 0.1~0.3M of 2.0~10.0mL
In buffer solution, 2~5h of ultrasonic disperse in ultrasonic cleaning machine obtains graphene dispersing solution；
B. (0.05 μm) polishing 2 of alundum (Al2O3) powder is used on chamois leather first by glass-carbon electrode (GCE, a diameter of 3mm)
Electrode is placed in ultrasonic cleaning machine with 2~5min is cleaned by ultrasonic in redistilled water by~5min after polishing, obtains naked glass carbon electricity
Pole；Using cyclic voltammetry, potential range is -1.5~0.5V, sweeps speed as 10mV/s, graphene oxide is electrodeposited into naked glass
Carbon electrode obtains the glass-carbon electrode (being labeled as GO/GCE) of graphene modified；
C. take solution drop coating in 2.0~7.0 μ L (1) or (2) or (3) on GO/GCE, stand 0.2 at room temperature~
1.0h is to get to our required electrochemical sensors (being labeled as CP/GO/GCE).
Using the above-mentioned electrochemica biological sensor based on class nucleic acid coacetylase-Cu (II) CP to the electro catalytic activity of OPD into
Row detection so as to fulfill the screening of PPase Activity determinations and its inhibitor, using square wave voltammetry (SWV), sets potential range
For -0.4~0.1V, detection CP/GO/GCE modified electrodes are in the PBS buffer solution of 100.0mM, pH 7.2 to 0.25mg/mL
The SWV responses of OPD, obtain the corresponding reduction peak current sizes of PPase of various concentrations a series of, probe into current-responsive with
Relationship between PPase concentration.
Inventive principle：The present invention is a kind of electrochemica biological sensor, and pyrophosphate can form complex compound with Cu (II),
And PPase can be catalyzed a molecule pyrophosphate and be converted into two molecule phosphate ions, using this point, add in PPase,
PPase can hydrolyze pyrophosphate and inhibit the copper complex formazan formation of divalent, lead to the presence of largely free Cu (II) in solution, change
Reaction is learned to can continue to occur.Based on the sulfydryl of CoA ends, by itself and Cu (II) hybrid reaction dissociated in solution, pass through mercapto
The effect of base and Cu (II) can form the repetitive unit of many CoA-Cu (II), synthesize CoA-Cu (II) CP, are prepared for one kind
Efficiently the electrochemica biological sensor of PPase activity is detected by monitoring OPD electro catalytic activities signal.Graphene is one
Kind two-dimensional sheet structure, has larger surface area, good electric conductivity and biocompatibility, can pass through with CoA-Cu (II) CP
π-pi-conjugated effect stable bond securely modifies the glassy carbon electrode surface covered in graphene, so as to enhance the stabilization of sensor
Property.This patent utilizes the synergistic effect of GO and CoA-Cu (II) CP, constructs a kind of simple and quick, highly sensitive, highly selective
PPase Activity determinations and its analysis method of micromolecular inhibitor screening.
Compared with prior art, the advantage of the invention is that：The present invention is constructed based on class nucleic acid coacetylase-Cu (II) CP
Electrochemica biological sensor and screened for PPase Activity determinations and its micromolecular inhibitor.First, following for speed is swept using low
Ring voltammetry modifies graphene uniform in bare glassy carbon electrode surface, and by setting sweep parameter with the stone in coordination electrode
Black alkene thickness, obtains GO/GCE.Secondly, CoA-Cu (II) CP is assembled on GO/GCE at room temperature, because the polymer has
Special class RNA structures using CoA-Cu (II) CP and the π-πconjugation of graphene, make CoA-Cu (II) CP stablize modification
In electrode surface, sensor is successfully prepared.Subsequently, based on the special electro catalytic activities of CoA-Cu (II) CP, square wave volt-ampere is utilized
Method (SWV) detection sensor is to the electrochemical response of various concentration PPase and its micromolecular inhibitor.Obviously, in enzyme concentration one
Determine in range, target concentration is bigger, and current-responsive is more apparent.The experimental results showed that the size of electric current and the concentration of object
It is in a linear relationship in a certain range, realize the detection to object.It the advantage is that：
(1) it is highly sensitive.The present invention is first to scan graphene using cyclic voltammetry, and graphene uniform is made to deposit to electricity
Pole surface, and because of electrostatic adsorption, graphene, which can be stablized, to be adsorbed in electrode surface, greatly accelerates electron transmission.Simultaneously
CoA-Cu (II) CP and graphene are adsorbed onto graphene surface by π-πconjugation so that sensor is more stable, detection spirit
Sensitivity higher；Based on the special electro catalytic activities of CoA-Cu (II) CP so that the sensitivity of the sensor further improves.Experiment
The current-responsive for obtaining sensor is y=13.06x+0.93, R to the linearly related equation of PPase concentration2=0.9997, detection
0.0001U/L is limited to, thus illustrates that sensor can realize highly sensitive detection to PPase.
(2) high specific.Other common enzyme such as papains (Papain), acetylcholinesterase (AChE), choline
Oxidizing ferment (ChOx), lysozyme (LZM), fibrin ferment (TB), alkaline phosphatase (ALP) is noiseless to this detection architecture.
(3) result is accurate.By detection of the electrochemica biological sensor to OPD catalytic activity, so as to realize pair
The detection of PPase and its micromolecular inhibitor, pass through experimental phenomena and testing result, it can be deduced that the current-responsive of sensor with
The correlativity of PPase concentration and its micromolecular inhibitor.
(4) prepare with detection method reagent dosage is few, detection speed is fast, at low cost.The present invention only need to consume a small amount of material
Pyrophosphatase electrochemica biological sensor can be prepared with reagent, by the detection of the catalytic activity to OPD, so as to fulfill right
The detection of PPase and its micromolecular inhibitor.In conclusion the present invention is can be formed to be complexed with Cu (II) based on pyrophosphate
Object, and PPase is added in, PPase can hydrolyze pyrophosphate and inhibit the copper complex formazan formation of divalent, and chemical reaction can continue to send out
It is raw.Based on the sulfydryl of CoA ends, by itself and Cu (II) hybrid reaction dissociated after PPase hydrolysis, pass through sulfydryl and Cu
(II) effect can form the repetitive unit of many CoA-Cu (II), synthesize CoA-Cu (II) CP, and pass through the coordination polymerization
The π-πconjugation of object and graphene makes such nucleic acid coordination polymer stablize modification in electrode surface, electrochemica biological is made
Sensor, for the detection to OPD catalytic activity, so as to fulfill the detection to PPase.On the other hand, micromolecular inhibitor
(NaF) there is stronger inhibiting effect to PPase activity, cause the Cu (II) to dissociate in solution less, reduce response signal,
Realize the analysis detection of various concentration NaF.The sensor have high sensitivity, selectivity are good, easy to operate, analysis is quick,
The advantages that easily operated, has a good application prospect.
Description of the drawings
Fig. 1 is the electrochemical response comparison diagram of CoA-Cu (II) CP modified electrodes and other electrodes；
Fig. 2 is inventive sensor to whetheing there is the electrochemical response figure of the reaction system of OPD；
Fig. 3 is the feasibility Experiment figure to PPase analysis detections of inventive sensor；
Fig. 4 is electrochemical response figure of the inventive sensor to the PPase of various concentration；
Fig. 5 is calibration graph of the inventive sensor to the current-responsive of the PPase of various concentration to concentration；
Fig. 6 is selective lab diagram of the inventive sensor to PPase；
Fig. 7 is interference lab diagram of the inventive sensor to PPase；
Fig. 8 is electrochemical response figure of the inventive sensor to PPase in complex system；
Fig. 9 is sensibility of the inventive sensor to PPase inhibitor (NaF)；
Figure 10 is inhibiting effect of the NaF to PPase of various concentration.
Specific embodiment
The present invention is described in further detail below in conjunction with attached drawing embodiment.
First, specific embodiment
Embodiment 1
The preparation method of pyrophosphatase electrochemica biological sensor based on class nucleic acid coacetylase-Cu (II) coordination polymer
And application, it is as follows：
(1) preparation of CoA-Cu (II) CP
The Cu of 10.0 a concentration of 1.0mM of μ L is taken successively2+Solution, a concentration of 2.0mM coacetylases aqueous solutions of 10.0 μ L, adds distillation
Water is made into the solution of 100.0 μ L, and solution is acutely vibrated to 15min to get to CoA-Cu (II) CP.
(2) PPase Enzyme assays
A. the PPi solution of 10.0 a concentration of 2.0mM of μ L, the Cu of 10.0 a concentration of 1.0mM of μ L are taken successively2+Solution, 10.0 μ L
A concentration of 100.0 μM of Mg2+Solution, the Tris-HCl buffer solutions (pH7.4) of 10.0 a concentration of 20.0mM of μ L, 10.0 μ L differences are dense
The PPase of degree in EP pipes, acutely vibrate 2min be uniformly mixed solution, above-mentioned solution is incubated 1.0h at 37 DEG C, after will
The EP pipes, which are placed in 85 DEG C of water-baths, inactivates enzyme, is cooled to room temperature, control enzyme concentration ranging from 0~2U/L.
B. the CoA of 10.0 a concentration of 2.0mM of μ L, the H of 40 μ L are added in into a2O is uniformly mixed, and solution is acutely vibrated
15min, and it is spare to be placed on 4 DEG C of refrigerator cold-storages.
(3) detection of PPase enzyme inhibitors NaF
A. the PPase of 5.0 a concentration of 10.0U/L of μ L is taken in EP pipes, is added in the NaF of 5.0 μ L various concentrations, is acutely shaken
2min is uniformly mixed, ranging from 0~120 μM of inhibitor NaF final concentrations；
B. into a 10.0 a concentration of 2.0mM of μ L PPi solution, the Cu of 10.0 a concentration of 1.0mM of μ L2+Solution, 10.0 μ L are dense
Spend the Mg for 100.0 μM2+Solution, the Tris-HCl buffer solutions (pH7.4) of 10.0 a concentration of 20.0mM of μ L acutely shake in EP pipes
Swinging 2min is uniformly mixed solution, and above-mentioned solution is incubated 1.0h at 37 DEG C, after the EP pipes be placed in 85 DEG C of water-baths lose enzyme
It is living, it is cooled to room temperature；
C. the CoA of 10.0 a concentration of 2.0mM of μ L, the H of 40 μ L are added in into b2O is uniformly mixed, and solution is acutely vibrated
15min, and it is spare to be placed on 4 DEG C of refrigerator cold-storages.
(4) preparation of electrochemical sensor
A. 5.0mg graphenes are dissolved in the acetate buffer solution of pH 5.0 of a concentration of 0.2M of 5.0mL, in ultrasonic cleaning machine
Middle ultrasonic disperse 2h, obtains graphene dispersing solution；
B. (0.05 μm) polishing of alundum (Al2O3) powder is used on chamois leather first by glass-carbon electrode (GCE, a diameter of 3mm)
Electrode after polishing is placed in ultrasonic cleaning machine with 2min is cleaned by ultrasonic in redistilled water, obtains bare glassy carbon electrode by 2min；Profit
With cyclic voltammetry, potential range is -1.5~0.5V, sweeps speed as 10mV/s, graphene oxide is electrodeposited into bare glassy carbon electrode
Obtain the glass-carbon electrode (being labeled as GO/GCE) of graphene modified；
C. solution drop coating in 5.0 μ L (1) or (2) or (3) is taken on GO/GCE, standing 0.5h at room temperature to get to institute
The electrochemical sensor (being labeled as CP/GO/GCE) needed.
Embodiment 2
The preparation method of pyrophosphatase electrochemica biological sensor based on class nucleic acid coacetylase-Cu (II) coordination polymer
And application, it is as follows：
(1) preparation of CoA-Cu (II) CP
The Cu of 8.0 a concentration of 8.0mM of μ L is taken successively2+Solution, a concentration of 16.0mM coacetylases aqueous solutions of 8.0 μ L, adds distilled water
The solution of 80.0 μ L is made into, solution is acutely vibrated to 8min to get to CoA-Cu (II) CP.
(2) PPase Enzyme assays
A. the PPi solution of 5.0 a concentration of 5.0mM of μ L, the Cu of 8.0 a concentration of 8.0mM of μ L are taken successively2+Solution, 8.0 μ L concentration
For 180.0 μM of Mg2+Solution, the Tris-HCl buffer solutions (pH 7.4) of 13.0 a concentration of 25.0mM of μ L, 8.0 μ L various concentrations
For PPase in EP pipes, acutely vibrating 2min is uniformly mixed solution, and above-mentioned solution is incubated 1.1h at 30 DEG C, after by the EP
Pipe, which is placed in 87 DEG C of water-baths, inactivates enzyme, is cooled to room temperature, control enzyme concentration ranging from 0~2U/L；
B. the CoA of 8.0 a concentration of 16.0mM of μ L, the H of 30 μ L are added in into a2O is uniformly mixed, and solution is acutely vibrated
8min, and it is spare to be placed on 4 DEG C of refrigerator cold-storages.
(3) detection of PPase enzyme inhibitors NaF
A. the PPase of 4.0 a concentration of 10.0U/L of μ L is taken in EP pipes, is added in the NaF of 6.0 μ L various concentrations, is acutely shaken
3min is uniformly mixed, ranging from 0~120 μM of inhibitor NaF final concentrations；
B. the PPi solution of 5.0 a concentration of 5.0mM of μ L, the Cu of 8.0 a concentration of 8.0mM of μ L are sequentially added into a2+Solution,
The Mg of 8.0 a concentration of 180.0 μM of μ L2+Solution, the Tris-HCl buffer solutions (pH 7.4) of 13.0 a concentration of 25.0mM of μ L are managed in EP
In, acutely vibrating 2min is uniformly mixed solution, and above-mentioned solution is incubated 1.1h at 30 DEG C, after the EP pipes are placed in 87 DEG C of water
Bath inactivates enzyme, is cooled to room temperature；
C. the CoA of 8.0 a concentration of 16.0mM of μ L, the H of 28.0 μ L are added in into b2O is uniformly mixed, and solution is acutely vibrated
8min, and it is spare to be placed on 4 DEG C of refrigerator cold-storages.
(4) preparation of electrochemical sensor
A. 8.0mg graphenes are dissolved in the acetate buffer solution of pH 5.8 of a concentration of 0.1M of 4.0mL, in ultrasonic cleaning machine
Middle ultrasonic disperse 2h, obtains graphene dispersing solution；
B. (0.05 μm) polishing of alundum (Al2O3) powder is used on chamois leather first by glass-carbon electrode (GCE, a diameter of 3mm)
Electrode after polishing is placed in ultrasonic cleaning machine with 4min is cleaned by ultrasonic in redistilled water, obtains bare glassy carbon electrode by 4min；Profit
With cyclic voltammetry, potential range is -1.5~0.5V, sweeps speed as 10mV/s, graphene oxide is electrodeposited into bare glassy carbon electrode
Obtain the glass-carbon electrode (being labeled as GO/GCE) of graphene modified；
C. take solution drop coating in 3.5 μ L (1) or (2) or (3) on GO/GCE, stand at room temperature 0.2~1.0h to get
To required electrochemical sensor (being labeled as CP/GO/GCE).
Embodiment 3
The preparation method of pyrophosphatase electrochemica biological sensor based on class nucleic acid coacetylase-Cu (II) coordination polymer
And application, it is as follows：
(1) preparation of CoA-Cu (II) CP
The Cu of 12.0 a concentration of 6.0mM of μ L is taken successively2+Solution, a concentration of 12.0mM coacetylases aqueous solutions of 12.0 μ L, adds distillation
Water is made into the solution of 90.0 μ L, and solution is acutely vibrated to 12min to get to CoA-Cu (II) CP.
(2) PPase Enzyme assays
A. the PPi solution of 8.0 a concentration of 6.0mM of μ L, the Cu of 12.0 a concentration of 6.0mM of μ L are taken successively2+Solution, 6.0 μ L are dense
Spend the Mg for 120.0 μM2+Solution, the Tris-HCl buffer solutions (pH 7.4) of 12.0 a concentration of 35.0mM of μ L, 6.0 μ L various concentrations
PPase in EP pipes, acutely vibrate 3min be uniformly mixed solution, above-mentioned solution is incubated 1.3h at 35 DEG C, after should
EP pipes, which are placed in 89 DEG C of water-baths, inactivates enzyme, is cooled to room temperature, control enzyme concentration ranging from 0~2U/L.
B. the CoA of 12.0 a concentration of 12.0mM of μ L, the H of 34.0 μ L are added in into a2O is uniformly mixed, and solution is acutely vibrated
12min, and it is spare to be placed on 4 DEG C of refrigerator cold-storages.
(3) detection of PPase enzyme inhibitors NaF
A. the PPase of 3.0 a concentration of 40.0U/L of μ L is taken in EP pipes, is added in the NaF of 7.0 μ L various concentrations, is acutely shaken
2min is uniformly mixed, ranging from 0~120 μM of inhibitor NaF final concentrations；
B. the PPi solution of 8.0 a concentration of 6.0mM of μ L, the Cu of 12.0 a concentration of 6.0mM of μ L are sequentially added into a2+Solution,
The Mg of 6.0 a concentration of 120.0 μM of μ L2+Solution, the Tris-HCl buffer solutions (pH7.4) of 12.0 a concentration of 35.0mM of μ L are managed in EP
In, acutely vibrating 3min is uniformly mixed solution, and above-mentioned solution is incubated 1.3h at 35 DEG C, after the EP pipes are placed in 89 DEG C of water
Bath inactivates enzyme, is cooled to room temperature；
C. the CoA of 12.0 a concentration of 12.0mM of μ L, the H of 30.0 μ L are added in into b2O is uniformly mixed, and solution is acutely vibrated
12min, and it is spare to be placed on 4 DEG C of refrigerator cold-storages.
(4) preparation of electrochemical sensor
A. 4.0mg graphenes are dissolved in the acetate buffer solution of pH 6.0 of a concentration of 0.3M of 6.0mL, in ultrasonic cleaning machine
Middle ultrasonic disperse 3h, obtains graphene dispersing solution；
B. (0.05 μm) polishing of alundum (Al2O3) powder is used on chamois leather first by glass-carbon electrode (GCE, a diameter of 3mm)
Electrode after polishing is placed in ultrasonic cleaning machine with 5min is cleaned by ultrasonic in redistilled water, obtains bare glassy carbon electrode by 3min；Profit
With cyclic voltammetry, potential range is -1.5~0.5V, sweeps speed as 10mV/s, graphene oxide is electrodeposited into bare glassy carbon electrode
Obtain the glass-carbon electrode (being labeled as GO/GCE) of graphene modified；
C. solution drop coating in 4.0 μ L (1) or (2) or (3) is taken on GO/GCE, standing 0.8h at room temperature to get to institute
The electrochemical sensor (being labeled as CP/GO/GCE) needed.
2nd, feasibility Experiment
1st, proof CoA-Cu (II) the CP electro catalytic activities that electrochemica biological sensor is prepared using specific embodiment 1 are tested
(the sensor during CoA and Cu (II) synthesis CoA-Cu (II) CP prepares electrochemical sensor CP/GO/GCE
Specific preparation process is with described in above-described embodiment 1) while have studied only CoA and only Cu (II) modification GO/GCE gained modified electrodes
To the electrochemical response of the PBS solution of the OPD containing 0.25mg/mL.Such as Fig. 1, experimental phenomena shows that only CP/GO/GCE is to containing
The PBS solution of 0.25mg/mL OPD has an apparent response signal, and CoA/GO/GCE, Cu (II)/GO/GCE and GO/GCE pairs
The response signal of 0.25mg/mL OPD can be ignored.Prove CoA-Cu (II) CP to 0.25mg/mL OPD electro-catalysis abilities compared with
By force.
2nd, electrochemica biological sensor is prepared to whetheing there is the response of 0.25mg/mL OPD using specific embodiment 1
CoA-Cu (II) CP is synthesized using specific embodiment 1, prepares electrochemica biological sensor CP/GO/GCE, detects CP/
GO/GCE illustrates nothing to the electrochemical response of the PBS (100mM, pH 7.2) with or without 0.25mg/mL OPD, phenomenon such as Fig. 2
In the presence of OPD, CP/GO/GCE is almost nil to the electrochemical response of solution, and CP/GO/GCE is apparent to OPD electrochemical responses.
3rd, the feasibility Experiment of electrochemica biological sensor is prepared using specific embodiment 1
It (specific building-up process is with described in above-described embodiment 1) while is had studied during CoA-Cu (II) CP is synthesized
Lack the influence of PPi, Cu (II), PPase to synthesis CoA-Cu (II) CP.It keeps synthesis condition constant, is detected in three kinds of variables
In system, obtained synthetic solvent volume is 100 μ L, compares the electrode of four kinds of solution modifications containing 0.25mg/mL OPD
PBS (100mM, pH 7.2) in, to the electrochemical response of 0.25mg/mL OPD.Such as Fig. 3, experimental phenomena shows only PPi, Cu
(II), PPase three exists and during without PPi, and gained reaction system modified electrode has the PBS solution of the OPD containing 0.25mg/mL
Apparent response signal, the response signal of other two modified electrode can be ignored.On the one hand, it was demonstrated that in the presence of no PPi, Cu
(II) CoA-Cu (II) CP can be smoothly synthesized with CoA, on the other hand, it was demonstrated that the phosphatic hydrolysis ability of PPase focusings, thus
It proves the experiment in theory and is technically feasible.
3rd, PPase Activity determinations application
1st, the method for electrochemica biological sensor detection PPase prepared using specific embodiment 1
Using square wave voltammetry, setting potential range is -0.4~0.1V, CP/GO/ prepared by detection above-described embodiment 1
GCE is to the electrochemical response of various concentration PPase.According to experimental result, a series of PPase for obtaining various concentrations is corresponding
OPD reduction peak current sizes, establish the quantitative relationship between current-responsive and PPase concentration, according to quantitative pass between the two
System determines the concentration of PPase in sample to be tested.
2nd, sensitivity experiment
Experimental design explanation：Using square wave voltammetry, setting potential range is -0.4~0.1V, detects above-described embodiment 1
The CP/GO/GCE of preparation is to the electrochemical response of various concentration PPase, PPase detection final concentrations ranging from 0.0~2.0U/L.
For experimental result as shown in figure 4, explanation increases with PPase concentration, the electrochemical response of CP/GO/GCE is more apparent；
Shown in Fig. 5, for the current-responsive of sensor to the detection range of PPase final concentrations in 0~2.0U/L, linearly related equation is y=
13.06x+0.93 R2=0.9997, range of linearity 0.001-1.5U/L, detection are limited to 0.0001U/L, illustrate sensor pair
Highly sensitive detection can be achieved in PPase activity.
3rd, specificity experiments
Selectivity experiment and the concentration of PPase and other enzymes in interference--free experiments are 1.0U/L, used other enzymes
Abbreviation it is as follows：Papain (Papain), acetylcholinesterase (AChE), choline oxidase (ChOx), lysozyme
(LZM), fibrin ferment (TB), alkaline phosphatase (ALP)
(1) selectivity experiment
When synthesizing CoA-Cu (II) CP, PPase is replaced using different types of enzyme, other conditions are constant, acquired solution
Drop coating is to GO/GCE surfaces, and then using square wave voltammetry, setting potential range is -0.4~0.1V, and detection modified electrode is dense
It spends to the electrochemical response of 0.25mg/mL OPD in PBS (pH 7.2) solution for 100mM, the results are shown in Figure 6, with PPase
Comparison, sensor is very small to the electrochemical response of other enzymes, substantially close to blank signal, illustrates that sensor has PPase
There is outstanding selectivity.
(2) interference--free experiments
When synthesizing CoA-Cu (II) CP, change condition, combined using different types of enzyme with PPase (respectively
PPase, PPase+AChE, PPase+ChOx, PPase+LZM, PPase+TB, PPase+Papain, PPase+ALP), it prepares not
The electrochemica biological sensor of same CP/GO/GCE, then using square wave voltammetry, setting potential range is -0.4~0.1V,
CP/GO/GCE is detected in PBS (pH 7.2) solution of a concentration of 100mM to the electrochemical response of 0.25mg/mLOPD, as a result
Such as Fig. 7, compare sensor to the current-responsive in the presence of six interference systems and only PPase, observe the size of peak current with
Peak current in the presence of only PPase illustrates that other enzymes are noiseless to the detection of PPase substantially without difference, realizes special
Property detection.
4th, complex system
Using square wave voltammetry, setting potential range is -0.4~0.1V, has separately designed 5%, 10%, 30% serum
And urine system synthesis CoA-Cu (II) CP, different CP/GO/GCE is prepared, in the PBS containing OPD (0.25mg/mL)
(100mM, pH 7.2) carries out Electrochemical Detection.(such as Fig. 8) is learnt according to experimental result, can also be realized in complex system pair
The detection of PPase, to realize in the future, PPase detections are laid a good foundation in cell and tissue.
5th, the detection of PPase inhibitor (NaF)
Using square wave voltammetry, setting potential range is -0.4~0.1V, and enzyme inhibitor reaction system modified electrode is dense
It spends in PBS (pH 7.2) solution for 100mM to the electrochemical response of 0.25mg/mL OPD, is learnt according to experimental result (as schemed
9), with the increase of inhibitor NaF concentration, corresponding current-responsive is weaker, illustrates inhibiting effect of the NaF to PPase activity
It is stronger.In addition, obtaining (such as Figure 10) according to experimental phenomena, when NaF concentration is smaller, corresponding current value variation is apparent；But
It is as NaF concentration increases, corresponding current value variation is less apparent, and the NaF for illustrating small concentration can inhibit PPase's
Activity, inhibiting effect effect is good, and the 503nhibiting concentration of NaF inhibitor is 5.17 μM.
Certainly, above description is not limitation of the present invention, and the present invention is also not limited to the example above.The art
The variations, modifications, additions or substitutions that those of ordinary skill makes in the essential scope of the present invention, should also belong to protection of the present invention
Range.
Claims (8)
1. the preparation and application of the pyrophosphatase electrochemica biological sensor based on class nucleic acid coacetylase-Cu (II) coordination polymer,
It is obtained it is characterized in that, the electrochemical sensor is built by the following method：
First, bare glassy carbon electrode is modified using graphene oxide, obtains GO/GCE；Secondly, CoA- is synthesized using coacetylase and Cu (II)
Cu(II)CP；Again, the detection reaction solution of PPase Activity determinations reaction solution or PPase inhibitor NaF are prepared, then adds coenzyme
A synthesizes CoA-Cu (II) CP；Finally, the CoA-Cu of gained (II) CP is distinguished into drop coating in GO/GCE surfaces, obtains CP/GO/
GCE sensors.
2. electrochemica biological sensor as described in claim 1, it is characterized in that, the preparation method of CoA-Cu (II) CP is：According to
The secondary Cu for taking 8.0~12.0 a concentration of 0.8~10.0mM of μ L2+Solution, a concentration of 1.6~20.0mM coacetylases water of 8.0~12.0 μ L
Solution, adds distilled water to be made into the solution of 80.0~100.0 μ L, and solution is acutely vibrated 5~15min, and it is cold to be placed on 4 DEG C of refrigerators
It hides spare.
3. electrochemica biological sensor as described in claim 1, it is characterized in that, the preparation of PPase Enzyme assay reaction solutions
Method is：A. the PPi solution of 5.0~10.0 a concentration of 0.1~10.0mM of μ L is taken successively, 8.0~12.0 μ L a concentration of 0.8~
10.0mM Cu2+Solution, the Mg of 5.0~10.0 a concentration of 100.0~200.0 μM of μ L2+Solution, 10.0~15.0 μ L are a concentration of
The Tris-HCl buffer solutions (pH 7.4) of 20.0~40.0mM, the PPase of 5.0~10.0 a concentration of 0.001~100.0U/L of μ L
In EP pipes, acutely vibrating 2~5min is uniformly mixed solution, and above-mentioned solution is incubated 1.0~1.5h at 20~40 DEG C, after
The EP pipes are placed in 85~90 DEG C of water-baths inactivates enzyme, is cooled to room temperature, control enzyme final concentration ranging from 0~2U/L；B. into a
Add in the CoA, the H of 30~50 μ L of 8.0~12.0 a concentration of 1.6~20.0mM of μ L2O is uniformly mixed, solution acutely vibrates to 5~
15min, and it is spare to be placed on 4 DEG C of refrigerator cold-storages.
4. electrochemica biological sensor as described in claim 1, it is characterized in that, the detection reaction solution of PPase enzyme inhibitors NaF
Preparation method be：A. the PPase of 5.0~10.0 a concentration of 0.001~100.0U/L of μ L is taken in EP pipes, addition 1.0~
The NaF of 10.0 a concentration of 0.01~1mM of μ L acutely shakes 1~3min and is uniformly mixed, and inhibitor NaF final concentrations ranging from 0~
120μM；B. the PPi solution of 5.0~10.0 a concentration of 0.1~10.0mM of μ L is sequentially added into a, 8.0~12.0 μ L are a concentration of
The Cu of 0.8~10.0mM2+Solution, the Mg of 5.0~10.0 a concentration of 100.0~200.0 μM of μ L2+Solution, 10.0~15.0 μ L are dense
The Tris-HCl buffer solutions (pH 7.4) for 20.0~40.0mM are spent in EP pipes, and acutely vibrating 2~5min makes solution mixing equal
It is even, above-mentioned solution is incubated 1.0~1.5h at 20~40 DEG C, after the EP pipes be placed in 85~90 DEG C of water-baths inactivate enzyme, it is cold
But to room temperature；C. the CoA, the H of 30~50 μ L of 8.0~12.0 a concentration of 1.6~20.0mM of μ L is added in into b2O is uniformly mixed,
Solution is acutely vibrated into 5~15min, and it is spare to be placed on 4 DEG C of refrigerator cold-storages.
5. the electrochemica biological sensor as described in Claims 1 to 4, it is characterized in that, the preparation method of electrochemical sensor is：
A., 4.0~9.0mg graphenes are dissolved in the acetate buffer solution of the pH 5.0~6.0 of a concentration of 0.1~0.3M of 2.0~10.0mL
In, 2~5h of ultrasonic disperse in ultrasonic cleaning machine obtains graphene dispersing solution；B. first by glass-carbon electrode (GCE, it is a diameter of
3mm) electrode is placed in ultrasonic cleaning machine after polishing with (0.05 μm) 2~5min of polishing of alundum (Al2O3) powder on chamois leather
With 2~5min is cleaned by ultrasonic in redistilled water, bare glassy carbon electrode is obtained；Using cyclic voltammetry, potential range for -1.5~
0.5V sweeps speed as 10mV/s, and graphene oxide is electrodeposited into bare glassy carbon electrode obtains the glass-carbon electrode (mark of graphene modified
For GO/GCE)；C. solution drop coating described in 2.0~7.0 μ L claims 2-4 is taken on GO/GCE, stand 0.2 at room temperature~
1.0h is to get to required electrochemical sensor (being labeled as CP/GO/GCE).
6. the electrochemica biological sensor as described in Claims 1 to 5, it is characterized in that described based on class nucleic acid coacetylase-Cu
(II) electrochemica biological sensor of CP detects its catalytic action to OPD, so as to detect the method for PPase activity, feature
It is：Using square wave voltammetry, setting potential range is -0.4~0.1V, and CP/GO/GCE is in a concentration of 100mM, pH for detection
To the electrochemical response of 0.25mg/mL OPD in 7.2 PBS buffer solution, so as to fulfill the detection to PPase activity.
7. the electrochemica biological sensor based on class nucleic acid coacetylase-Cu (II) coordination polymer as described in Claims 1 to 5
To the detection method of various concentration PPase, it is characterised in that：Using square wave voltammetry, setting potential range is -0.4~0.1V,
CP/GO/GCE is detected to the electrochemical response of various concentration PPase, obtains a series of corresponding reduction of PPase of various concentrations
Peak current size establishes the quantitative relationship between current-responsive and PPase concentration, according to quantitative relationship between the two, determines
The correlation of PPase and corresponding reduction peak current.
8. the electrochemica biological sensor based on class nucleic acid coacetylase-Cu (II) coordination polymer as described in Claims 1 to 5
The method for detecting the micromolecular inhibitor NaF of various concentration, it is characterised in that：Using square wave voltammetry, setting potential range for-
0.4~0.1V, CP/GO/GCE is to the electrochemical response of PPase enzyme inhibitors for detection, establishes PPase enzyme inhibitor concentrations and enzyme
Relationship between activity.
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| CN110672696A (en) * | 2019-09-26 | 2020-01-10 | 宁波大学 | Novel electrochemical method for detecting copper ions, phosphate ions and alkaline phosphatase and application thereof |
| CN114609210A (en) * | 2022-03-01 | 2022-06-10 | 宁波大学 | Rapid scanning sensing method based on copper ion in-situ electrochemical signal output and biological application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110672696A (en) * | 2019-09-26 | 2020-01-10 | 宁波大学 | Novel electrochemical method for detecting copper ions, phosphate ions and alkaline phosphatase and application thereof |
| CN114609210A (en) * | 2022-03-01 | 2022-06-10 | 宁波大学 | Rapid scanning sensing method based on copper ion in-situ electrochemical signal output and biological application thereof |
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