CN110143584A - A kind of superoxide dismutase biomimetic material and its preparation method and application - Google Patents
A kind of superoxide dismutase biomimetic material and its preparation method and application Download PDFInfo
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- B—PERFORMING OPERATIONS; TRANSPORTING
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- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
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- C01B32/00—Carbon; Compounds thereof
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- C01B32/00—Carbon; Compounds thereof
- C01B32/15—Nano-sized carbon materials
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- C01B32/174—Derivatisation; Solubilisation; Dispersion in solvents
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- C01B32/00—Carbon; Compounds thereof
- C01B32/15—Nano-sized carbon materials
- C01B32/182—Graphene
- C01B32/194—After-treatment
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- 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
- G01N27/308—Electrodes, e.g. test electrodes; Half-cells at least partially made of carbon
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- 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
- G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
- G01N27/3275—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction
- G01N27/3278—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction involving nanosized elements, e.g. nanogaps or nanoparticles
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- 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
- G01N27/416—Systems
Abstract
The present invention relates to a kind of superoxide dismutase biomimetic materials and its preparation method and application, belong to field of material technology, the biomimetic material the preparation method is as follows: nano-carbon material is dispersed in water, then phosphate radical donor is added, after ultrasonic disperse, transition metal salt is added, centrifuging and taking precipitates after 1-5h is stirred to react at 20-30 DEG C, it is dried in vacuo after washing.Not only there is excellent selectivity, very short response time, lower detection limit by electrochemical sensor prepared by raw material of the biomimetic material, also there is high reaction sensitivity, compared to the sensor of traditional material preparation, in Real_time quantitative detection ultra-oxygen anion free radical, higher performance is shown, there can be important application prospect in terms of the ultra-oxygen anion free radical that real-time detection living cells discharges in situ.The material preparation process is simple and convenient, the cost of raw material is cheap, is convenient for commercial applications.
Description
Technical field
The invention belongs to field of material technology, and in particular to a kind of superoxide dismutase biomimetic material and preparation method thereof
And application.
Background technique
Ultra-oxygen anion free radical (superoxide radical, O2 ·-) it is intracellular different activities oxygen radical (ROS)
Initial radical in chain generates other oxygen radicals by series reaction.O2 ·-Fluctuation of concentration and many biology mistakes
The occurrence and development of journey and disease are closely related.When its concentration is normal, can participate in cell signal transduction, control cell Proliferation,
Differentiation and apoptosis simultaneously promote a variety of have the related factor of cell biocompatibility to start；And work as intracellular dynamic equilibrium and be broken,
O2 ·-When concentration is got higher, then it can cause response to oxidative stress, the lipid peroxidation for leading to cell membrane, cause DNA oxidative damage, be situated between
Protein denaturation etc. in guided cell further results in the canceration of cell, the occurrence and development of tumour and cardiovascular disease
Deng.Therefore, to the O of living cells release2 ·-Real-time quantitative detection in situ is carried out, can not only more comprehensively understand it in cell physiological
Effect in activity is more conducive to us and discloses the mechanism of disease associated therewith, to provide under pathology cognition
Reliable disease diagnosis.However, O2 ·-Cell release concentration it is very low and active high, it is very difficult to its real-time detection in situ.?
In many detection methods, electrochemical method shows fast response, high sensitivity, operates the advantages that simple, at low cost, is very suitable to
Under the premise of avoiding damaging living cells metabolism and relevant physiological activity, for discharging O in real time to living cells2 ·-
Concentration Testing.Therefore, design synthesis has highly sensitive, highly selective, low detection limit, low-cost O2 ·-Electrochemical student
Object sensor becomes one of the key points and difficulties studied at present.
So far, for O2 ·-Electrochemical sensor can be divided mainly into O2 ·-Biological enzyme electrochemical sensor and O2 ·-It is non-
Two class of enzyme electrochemical sensor.O2 ·-Biological enzyme electrochemical sensor refers to the O based on cytochrome c (cyt c) or SOD2 ·-Electricity
Chemical sensor.O2 ·-Non-enzymatic electrochemical sensor refers to using non-enzymatic material the binding site or active sites for simulating native enzyme
The O for putting and preparing2 ·-Electrochemical sensor.And natural biologic enzyme leads to it in the presence of the influence such as temperature, humidity and pH is highly prone to
The problem of loss of catalytic activity, and its cost is relatively high.These disadvantages all constrain O2 ·-Biological enzyme electrochemical sensor is answered
It is detected for actual sample.Thus, there is no the O of problems2 ·-Before non-enzymatic electrochemical sensor has bigger market application
Scape.
Currently, about O2 ·-The research of analogue enztme electrochemical sensor is reported, the substitution material of simulation native enzyme is concentrated mainly on
On material.Superoxide dismutase is that one kind is distributed widely in the intracorporal oxidoreducing enzyme of biology, and active site contains metal,
Therefore also referred to as metalloproteinases.As oxidoreducing enzyme, the oxidoreducing enzyme of electron transmission, structure and effect machine are especially participated in
System is general complex.Common three classes different metal center SOD can be by O2 ·-It is disproportionated into O2And H2O2, they are O2 ·-It is special
One property enzyme, disproportionated reaction speed are exceedingly fast, and are most effective detection O2 ·-Biological enzyme.It is bionical accordingly, with respect to superoxide dismutase
The invention of material most application potential.
Summary of the invention
In view of this, one of the objects of the present invention is to provide a kind of preparation sides of superoxide dismutase biomimetic material
Method；The second purpose is to provide a kind of superoxide dismutase biomimetic material；The third purpose is to provide a kind of electrochemical sensing
Device；The fourth purpose is to provide application of the electrochemical sensor in detection living cells release ultra-oxygen anion free radical.
In order to achieve the above objectives, the invention provides the following technical scheme:
1, a kind of preparation method of superoxide dismutase biomimetic material, the method are as follows:
Nano-carbon material is dispersed in water, then addition phosphate radical donor, after ultrasonic disperse, adds transition metal
Salt, centrifuging and taking precipitates after 1-5h is stirred to react at 20-30 DEG C, will be dried in vacuo after the washing of precipitate；The nano-sized carbon
The mass ratio of material, phosphate radical donor and transition metal salt is 1-20:2-10:1-10.
Preferably, it is 60-100W that the ultrasonic disperse, which is specially in ultrasonic power, and supersonic frequency is the condition of 20-40Hz
Lower ultrasound 10-60min.
Preferably, the centrifugation is specially to be centrifuged 5-10min with the speed of 5000-12000r/min.
Preferably, the vacuum drying is specially the dry 12-48h at 50-80 DEG C.
Preferably, the nano-carbon material is one in graphene, carbon nanotube, carbon nano-fiber or biomass carbon material
Kind；The phosphate radical donor is potassium dihydrogen phosphate, disodium hydrogen phosphate, diammonium hydrogen phosphate, sodium dihydrogen phosphate, ammonium dihydrogen phosphate, plant
One of acid or DNA；The transition metal salt is one of cobalt salt, nickel salt, manganese salt, mantoquita or molysite.
2, the superoxide dismutase biomimetic material prepared by the method.
3, a kind of electrochemical sensor, including electrochemical workstation, working electrode, to electrode, reference electrode, electrolytic cell and
Electrolyte, the working electrode surface are coated with the superoxide dismutase biomimetic material.
Preferably, the working electrode is prepared as follows:
Superoxide dismutase biomimetic material is dispersed in water with the matched proportion density of 0.5-5mg/mL, obtains electrode modification
The electrode modification solution is coated on electrode by solution, is coated with bonding agent after dry, is dried again.
Preferably, dry twice is specially the dry 3-10h at 20-30 DEG C.
Preferably, the bonding agent is Nafion solution, and the mass fraction of Nafion is in the Nafion solution
0.1%.
4, a kind of electrochemical sensor is applied in detection living cells release ultra-oxygen anion free radical.
The beneficial effects of the present invention are: the present invention provides a kind of superoxide dismutase biomimetic material and its preparation sides
Method and application, the present invention are supplied when preparing superoxide dismutase biomimetic material by reasonable set nano-carbon material, phosphate radical
The mass ratio of body and transition metal salt three, while the type of transition metal salt is reasonably selected, make the super oxygen finally to prepare
Object mutase biomimetic material be raw material preparation electrochemical sensor not only have excellent selectivity, the very short response time,
Lower detection limit, also has high reaction sensitivity.Wherein, leading for biomimetic material can be improved in the addition of nano-carbon material
Electrically, its whole electron transmission performance is promoted, suitable transition metal ions is selected and controls its load capacity, can be very good
The selectivity of biomimetic material is promoted, phosphate radical donor, which is added, can load a greater amount of transition metal ions, to make bionical material
Material has more detection active sites.With the sensor of biomimetic material preparation compared to sensor prepared by traditional material,
In Real_time quantitative detection ultra-oxygen anion free radical, higher performance is shown, it being capable of real-time detection living cells in situ
There is important application prospect in terms of the ultra-oxygen anion free radical of release.The material preparation process is simple and convenient, the cost of raw material
It is cheap, it is convenient for commercial applications.
Other advantages, target and feature of the invention will be illustrated in the following description to a certain extent, and
And to a certain extent, based on will be apparent to those skilled in the art to investigating hereafter, Huo Zheke
To be instructed from the practice of the present invention.Target of the invention and other advantages can be realized by following specification and
It obtains.
Detailed description of the invention
To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention is made below in conjunction with attached drawing excellent
The detailed description of choosing, in which:
Fig. 1 is the FESEM figure of the superoxide dismutase biomimetic material prepared in embodiment 1；
Fig. 2 is the TEM figure of the superoxide dismutase biomimetic material prepared in embodiment 1；
Fig. 3 is the contact angle test figure of the superoxide dismutase biomimetic material prepared in embodiment 1；
Fig. 4 is the FESEM figure of the superoxide dismutase biomimetic material prepared in embodiment 2；
Fig. 5 is the TEM figure of the superoxide dismutase biomimetic material prepared in embodiment 2；
Fig. 6 is the FESEM figure of the superoxide dismutase biomimetic material prepared in embodiment 3；
Fig. 7 is the TEM figure of the superoxide dismutase biomimetic material prepared in embodiment 3；
Fig. 8 is in the case where voltage range is -0.2-0.8V, and the sensor constructed in embodiment 1 is to O2 ·-Cyclic voltammetric response
Test result figure；
Fig. 9 is that the sensor constructed in embodiment 1 sweeps the CV curve graph under speed in difference；(wherein a is the sensing in Fig. 9
Device sweeps the CV curve graph under speed in difference, and b is that the variation of oxidative peak current and scanning speed shows linear relationship in Fig. 9
Figure)
Figure 10 is under the crest voltage (0.60V) of cyclic voltammetry curve, and the sensor constructed in embodiment 1 is to O2 ·-'s
Chrono-amperometric response test result figure；(a is continuously to add under the fixation current potential of 0.60V into electrolyte in Figure 10
5nmol·L-1O2 ·-When, the working electrode constructed in embodiment 1 is relative to Hg/Hg2Cl2The i-t response diagram of reference electrode, Figure 10
Middle b be in embodiment 1 sensor that constructs to O2 ·-Response time figure, c is under the fixation current potential of 0.60V, to electricity in Figure 10
25nmolL is continuously added in solution liquid-1O2 ·-When, the working electrode constructed in embodiment 1 is relative to Hg/Hg2Cl2Reference electrode
I-t response diagram, d is the steady-state current and O that the sensor constructed in embodiment 1 detects in Figure 102 ·-It is linear between concentration
Relational graph)
Figure 11 be in embodiment 1 sensor that constructs to disturbance ingredient selectivity test result figure；
Figure 12 is the O that the sensor real-time detection A549 cell constructed in embodiment 1 discharges under zym stimulation2 ·-When it
(a is the optical microscope image of A549 cell to curve graph in Figure 12；B is the sensing under the fixation current potential of 0.60V in Figure 12
Device discharges O to A549 cell2 ·-I-t response diagram).
Specific embodiment
Illustrate embodiments of the present invention below by way of specific specific example, those skilled in the art can be by this specification
Other advantages and efficacy of the present invention can be easily understood for disclosed content.The present invention can also pass through in addition different specific realities
The mode of applying is embodied or practiced, the various details in this specification can also based on different viewpoints and application, without departing from
Various modifications or alterations are carried out under spirit of the invention.
Embodiment 1
It prepares superoxide dismutase biomimetic material and coats the working electrode of the material, and construct superoxide anion freedom
Based electrochemical sensor
(1) it disperses multi-walled carbon nanotube (MWCNTs) in deionized water, phytic acid is then added under continuous stirring
It (PA), is that 80W adds manganese sulfate, stir at 25 DEG C after supersonic frequency is the condition ultrasound 30min of 40Hz in ultrasonic power
Precipitating is taken after being centrifuged 10min after reaction 1h with the speed of 10000r/min, it is true at 60 DEG C after precipitating is washed with deionized water
Sky is dry for 24 hours, and superoxide dismutase biomimetic material is made；Wherein, the mass ratio of multi-walled carbon nanotube, phytic acid and manganese sulfate is
1:3:1.5；
(2) water is dispersed with the matched proportion density of 2mg/mL by the superoxide dismutase biomimetic material prepared in step (1)
In, electrode modification solution is obtained, which is coated on glass-carbon electrode, is coated with after dry 5h at 26 DEG C
The Nafion solution that Nafion mass fraction is 0.1%, the dry 5h at 26 DEG C, is made surface and is coated with superoxides discrimination again
Change the working electrode of enzyme biomimetic material；
(3) surface obtained in step (2) is coated with to the working electrode and electrification of superoxide dismutase biomimetic material
Learn work station, to electrode (platinum electrode), reference electrode (Hg/HgCl2Electrode), (concentration is for electrolytic cell and electrolyte
The phosphate buffer solution of 0.01mol/L, pH=7.4) it is assembled into ultra-oxygen anion free radical electrochemical sensor together.
Fig. 1 is the FESEM figure of the superoxide dismutase biomimetic material prepared in embodiment 1, as shown in Figure 1, spherical Mn-
PA homoepitaxial is on the surface MWCNTs.
Fig. 2 is the TEM figure of the superoxide dismutase biomimetic material prepared in embodiment 1, as shown in Figure 2, spherical Mn-PA
Partial size be 80nm or so.
Fig. 3 is the contact angle test figure of the superoxide dismutase biomimetic material prepared in embodiment 1, from the figure 3, it may be seen that should
Biomimetic material has hydrophily, and contact angle is 45.54 °, has capture O2 ·-Ability.
Embodiment 2
It prepares superoxide dismutase biomimetic material and coats the working electrode of the material, and construct superoxide anion freedom
Based electrochemical sensor
(1) it disperses carbon nano-fiber (CNF) in deionized water, DNA is then added under continuous stirring, in ultrasonic function
Rate is 100W, after supersonic frequency is the condition ultrasound 10min of 20Hz, adds manganese nitrate, be stirred to react at 20 DEG C after 5h with
Precipitating is taken after the speed centrifugation 5min of 12000r/min, 48h is dried in vacuo at 50 DEG C after precipitating is washed with deionized water, makes
Obtain superoxide dismutase biomimetic material；Wherein, the mass ratio of carbon nano-fiber, DNA and manganese nitrate is 10:10:6；
(2) water is dispersed with the matched proportion density of 5mg/mL by the superoxide dismutase biomimetic material prepared in step (1)
In, electrode modification solution is obtained, which is coated on glass-carbon electrode, is coated with after dry 3h at 30 DEG C
The Nafion solution that Nafion mass fraction is 0.1%, the dry 3h at 30 DEG C, is made surface and is coated with superoxides discrimination again
Change the working electrode of enzyme biomimetic material；
(3) surface obtained in step (2) is coated with to the working electrode and electrification of superoxide dismutase biomimetic material
Learn work station, to electrode (platinum electrode), reference electrode (Hg/HgCl2Electrode), (concentration is for electrolytic cell and electrolyte
The phosphate buffer solution of 0.01mol/L, pH=7.4) it is assembled into ultra-oxygen anion free radical electrochemical sensor together.
Fig. 4 is the FESEM figure of the superoxide dismutase biomimetic material prepared in embodiment 2, as shown in Figure 4, spherical Mn-
DNA homoepitaxial is on CNF.
Fig. 5 is the TEM figure of the superoxide dismutase biomimetic material prepared in embodiment 2, as shown in Figure 5, spherical Mn-
The partial size of DNA is 70nm or so.
Embodiment 3
It prepares superoxide dismutase biomimetic material and coats the working electrode of the material, and construct superoxide anion freedom
Based electrochemical sensor
(1) it disperses graphene in deionized water, potassium dihydrogen phosphate is then added under continuous stirring, in ultrasonic power
Cobaltous sulfate is added, is stirred to react at 30 DEG C after 3h with 5000r/ after supersonic frequency is the condition ultrasound 60min of 30Hz for 60W
Precipitating is taken after the speed centrifugation 10min of min, 12h is dried in vacuo at 80 DEG C after precipitating is washed with deionized water, super oxygen is made
Compound mutase biomimetic material；Wherein, the mass ratio of graphene, potassium dihydrogen phosphate and cobaltous sulfate is 20:5:10；
(2) it disperses the superoxide dismutase biomimetic material prepared in step (1) in the matched proportion density of 0.5mg/mL
In water, electrode modification solution is obtained, which is coated on glass-carbon electrode, applied again after dry 10h at 20 DEG C
The Nafion solution that Nafion mass fraction is 0.1% is covered, the dry 10h at 20 DEG C, is made surface and is coated with super oxygen again
The working electrode of object mutase biomimetic material；
(3) surface obtained in step (2) is coated with to the working electrode and electrification of superoxide dismutase biomimetic material
Learn work station, to electrode (platinum electrode), reference electrode (Hg/HgCl2Electrode), (concentration is for electrolytic cell and electrolyte
The phosphate buffer solution of 0.01mol/L, pH=7.4) it is assembled into ultra-oxygen anion free radical electrochemical sensor together.
Fig. 6 is the FESEM figure of the superoxide dismutase biomimetic material prepared in embodiment 3, it will be appreciated from fig. 6 that sheet phosphorus
Sour cobalt homoepitaxial is in two-dimensional graphene.
Fig. 7 is the TEM figure of the superoxide dismutase biomimetic material prepared in embodiment 3, as shown in Figure 7, the sheet phosphorus
The scale of sour cobalt is micron order.
Embodiment 4
500nmolL will be contained-1O2 ·-PBS solution be added in the electrolyte of the sensor constructed in embodiment 1,
The sensor is tested to O in the case where voltage range is -0.2-0.8V2 ·-Cyclic voltammetric response, while it is slow to phosphoric acid with the sensor
The cyclic voltammetric response of solution is rushed as blank control.As a result as shown in figure 8, as shown in Figure 8, containing 500nmolL- 1O2 ·-PBS solution in, oxidation peak current ratio be free of O2 ·-PBS in oxidation peak current obviously increase, illustrate the sensor pair
O2 ·-There is apparent electrochemical catalytic oxidation ability.
Embodiment 5
1 μm of olL will be contained-1O2 ·-PBS solution be added in the electrolyte of the sensor constructed in embodiment 1, benefit
Speed (1mVs is swept in difference with cyclic voltammetry-1、10mV·s-1、20mV·s-1、30mV·s-1、40mV·s-1、50mV·s-1、60mV·s-1) under be scanned, as a result as shown in figure 9, wherein a is that the sensor in difference sweeps CV curves under speed in Fig. 9
Scheme, b is the linear relationship chart of oxidative peak current and scanning speed in Fig. 9, and as shown in Figure 9, oxidation peak current is with scanning speed
Increase and increase, and in a linear relationship, linear equation is y=0.2476x+1.4599 (R2=0.9943), show to sense
Biomimetic material in device on working electrode is to O2 ·-Catalysis be a kind of surface-controlled process.
Embodiment 6
The sensor constructed in testing example 1 under the crest voltage (0.6V) of cyclic voltammetry curve is to O2 ·-Timing
The O of various concentration is added in the electrolyte for the sensor that when test continuously constructs into embodiment 1 for current-responsive2 ·-Solution, when
Between between be divided into 50s, the relation curve of recording responses time and current value is to get the sensor to O2 ·-Peace times response diagram, as a result
As shown in Figure 10, wherein a is that 5nmolL is continuously added into electrolyte under the fixation current potential of 0.60V in Figure 10-1O2 ·-
When, the working electrode constructed in embodiment 1 is relative to Hg/Hg2Cl2The i-t response diagram of reference electrode, b is embodiment 1 in Figure 10
The sensor of middle building is to O2 ·-Response time figure, c is continuously to add under the fixation current potential of 0.60V into electrolyte in Figure 10
Add 25nmolL-1O2 ·-When, the working electrode constructed in embodiment 1 is relative to Hg/Hg2Cl2The i-t response diagram of reference electrode,
D is the steady-state current and O that the sensor constructed in embodiment 1 detects in Figure 102 ·-Linear relationship chart between concentration；By scheming
10 it is found that response current with O2 ·-The increase of concentration and increase (as shown in c in a and Figure 10 in Figure 10), injection O2 ·-Afterwards,
The response of sensor is very fast, and forms steady-state current (as shown in b in Figure 10) in 2.15 seconds, in 1.25nmol
L-1To 175nmolL-1In the range of, response current and O2 ·-The linear equation of concentration can indicate are as follows: I (μ A)=0.053C
(nmol·L-1)+0.669(0<I(μA)<10)(R2=0.998), sensitivity is 757.16 μ A (μm olL-1·cm2)-1, inspection
Survey is limited to 1.25n molL-1(signal-to-noise ratio S/N=3) (as shown in d in Figure 10).
Embodiment 7
In the electrolyte for the sensor that the solution of different material is added sequentially to construct in embodiment 1, the sensing is tested
Device responds the chrono-amperometric of disturbance composition, test voltage 0.6V, every 50s respectively successively continuously into sensor
Electrolyte in 25nmolL is added-1O2 ·-、5μmol·L-1NaNO3, KCl, AA, UA, DA and 1 μm of olL-1H2O2, obtain
To the sensor to the ampere response curve of disturbance ingredient selectivity test, as a result as shown in figure 11, as shown in Figure 11,5 μ
mol·L-1Na+、K+、NO3 -、Cl-, AA and UA will not detect 25nmolL to the sensor-1O2 ·-Cause to interfere, 5 μ
mol·L-1DA and 1 μm of olL-1H2O2It can cause visible inapparent response, all be unlikely to O2 ·-Detection generate interference,
Illustrate the sensor to O2 ·-With good specificity.
Embodiment 8
The sensor constructed in embodiment 1 is used for A549 cell detection, cell density is 1 × 104A/mL, specially
Under the fixation current potential of 0.60V, by chronoamperometry at following three kinds real-time detection A549 cell under zym stimulation
The O of release2 ·-: (1) to cell infusion 0.2mgmL-1zym；(2) to cell infusion 0.2mgmL-1Zym and 300UmL- 1The mixed liquor of SOD；(3) 0.2mgmL is added to not being added in the electrolyte of cell-1zym.As a result as shown in figure 12, wherein
Wherein in Figure 12 a be A549 cell optical microscope image, b is the sensor pair under the fixation current potential of 0.60V in Figure 12
A549 cell discharges O2 ·-I-t response diagram, as shown in Figure 12, when analyze current potential be 0.6V when, be added 0.2mgmL-1Zym promotees
Cell is set to discharge O2 ·-, detect biggish current-responsive (as shown in curve I in b in Figure 12), and 0.2mgmL is added-1zym
And 300UmL-1The mixed liquor of SOD does not cause significant curent change (as shown in curve II in b in Figure 12), shows cell
The O of release2 ·-It is consumed by SOD, under same test conditions, 0.2mg is added in there is no the electrolyte of A549 cell
mL-1Zym equally can't detect significant curent change (as shown in curve III in b in Figure 12).Therefore, it can be verified that curve I exhibition
The current-responsive (0.66 μ A) shown is the O discharged under zym stimulation by A549 cell2 ·-By imitative on working electrode in sensor
Green material capture simultaneously occurs oxidation reaction on its surface and generates.Go out 2.0mgmL according to normal linearity equation calculation-1zym
The O for stimulating A549 cell to discharge2 ·-Concentration is 1.32nmolL-1, and then know the O of each cell release2 ·-Concentration is
0.66fmol。
Finally, it is stated that the above examples are only used to illustrate the technical scheme of the present invention and are not limiting, although referring to compared with
Good embodiment describes the invention in detail, those skilled in the art should understand that, it can be to skill of the invention
Art scheme is modified or replaced equivalently, and without departing from the objective and range of the technical program, should all be covered in the present invention
Scope of the claims in.
Claims (10)
1. a kind of preparation method of superoxide dismutase biomimetic material, which is characterized in that the method is as follows:
Nano-carbon material is dispersed in water, then addition phosphate radical donor, after ultrasonic disperse, adds transition metal salt, 20-
Centrifuging and taking precipitates after being stirred to react 1-5h at 30 DEG C, will be dried in vacuo after the washing of precipitate；The nano-carbon material,
The mass ratio of phosphate radical donor and transition metal salt is 1-20:2-10:1-10.
2. the method as described in claim 1, which is characterized in that it is 60-100W that the ultrasonic disperse, which is specially in ultrasonic power,
Supersonic frequency is ultrasound 10-60min under conditions of 20-40Hz.
3. the method as described in claim 1, which is characterized in that the centrifugation is specially with the speed of 5000-12000r/min
It is centrifuged 5-10min.
4. the method as described in claim 1, which is characterized in that the vacuum drying is specially the dry 12- at 50-80 DEG C
48h。
5. method according to any of claims 1-4, which is characterized in that the nano-carbon material is graphene, carbon nanometer
One of pipe, carbon nano-fiber or biomass carbon material；The phosphate radical donor is potassium dihydrogen phosphate, disodium hydrogen phosphate, phosphorus
One of sour hydrogen diammonium, sodium dihydrogen phosphate, ammonium dihydrogen phosphate, phytic acid or DNA；The transition metal salt is cobalt salt, nickel salt, manganese
One of salt, mantoquita or molysite.
6. by the superoxide dismutase biomimetic material of the described in any item method preparations of claim 1-5.
7. a kind of electrochemical sensor, including electrochemical workstation, working electrode, to electrode, reference electrode, electrolytic cell and electrolysis
Liquid, which is characterized in that the working electrode surface is coated with superoxide dismutase biomimetic material as claimed in claim 6.
8. a kind of electrochemical sensor as claimed in claim 7, which is characterized in that the working electrode is made as follows
It is standby:
Superoxide dismutase biomimetic material is dispersed in water with the matched proportion density of 0.5-5mg/mL, it is molten to obtain electrode modification
The electrode modification solution is coated on electrode by liquid, is coated with bonding agent after dry, is dried again.
9. a kind of electrochemical sensor as claimed in claim 8, which is characterized in that dry twice is specially at 20-30 DEG C
Dry 3-10h.
10. a kind of electrochemical sensor as claimed in claim 7 is applied in detection living cells release ultra-oxygen anion free radical.
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Application publication date: 20190820 |