CN112573512A - Preparation method of biomimetic enzyme with phosphate group embedded into heteroatom-doped graphene layer, biomimetic enzyme and application - Google Patents
Preparation method of biomimetic enzyme with phosphate group embedded into heteroatom-doped graphene layer, biomimetic enzyme and application Download PDFInfo
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- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 title claims abstract description 68
- 102000004190 Enzymes Human genes 0.000 title claims abstract description 67
- 108090000790 Enzymes Proteins 0.000 title claims abstract description 67
- 229910021389 graphene Inorganic materials 0.000 title claims abstract description 64
- 238000002360 preparation method Methods 0.000 title claims abstract description 16
- 230000003592 biomimetic effect Effects 0.000 title claims description 31
- 125000002467 phosphate group Chemical group [H]OP(=O)(O[H])O[*] 0.000 title claims description 5
- 239000011664 nicotinic acid Substances 0.000 claims abstract description 35
- 229910019142 PO4 Inorganic materials 0.000 claims abstract description 29
- 238000006243 chemical reaction Methods 0.000 claims abstract description 28
- 238000003756 stirring Methods 0.000 claims abstract description 23
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims abstract description 19
- 239000010452 phosphate Substances 0.000 claims abstract description 19
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 17
- 238000001035 drying Methods 0.000 claims abstract description 15
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- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 5
- QAOWNCQODCNURD-UHFFFAOYSA-L Sulfate Chemical compound [O-]S([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 claims abstract description 5
- 239000003513 alkali Substances 0.000 claims abstract description 3
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- 238000005406 washing Methods 0.000 claims description 17
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- FVAUCKIRQBBSSJ-UHFFFAOYSA-M sodium iodide Chemical compound [Na+].[I-] FVAUCKIRQBBSSJ-UHFFFAOYSA-M 0.000 claims description 12
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- 229910010272 inorganic material Inorganic materials 0.000 claims description 8
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- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 6
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- NLKNQRATVPKPDG-UHFFFAOYSA-M potassium iodide Chemical compound [K+].[I-] NLKNQRATVPKPDG-UHFFFAOYSA-M 0.000 claims description 6
- VWDWKYIASSYTQR-UHFFFAOYSA-N sodium nitrate Chemical compound [Na+].[O-][N+]([O-])=O VWDWKYIASSYTQR-UHFFFAOYSA-N 0.000 claims description 6
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- 239000008367 deionised water Substances 0.000 claims description 5
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- OSGAYBCDTDRGGQ-UHFFFAOYSA-L calcium sulfate Chemical compound [Ca+2].[O-]S([O-])(=O)=O OSGAYBCDTDRGGQ-UHFFFAOYSA-L 0.000 claims description 4
- KTVIXTQDYHMGHF-UHFFFAOYSA-L cobalt(2+) sulfate Chemical compound [Co+2].[O-]S([O-])(=O)=O KTVIXTQDYHMGHF-UHFFFAOYSA-L 0.000 claims description 4
- 239000012286 potassium permanganate Substances 0.000 claims description 4
- 150000003839 salts Chemical class 0.000 claims description 4
- JHJLBTNAGRQEKS-UHFFFAOYSA-M sodium bromide Chemical compound [Na+].[Br-] JHJLBTNAGRQEKS-UHFFFAOYSA-M 0.000 claims description 4
- 235000009518 sodium iodide Nutrition 0.000 claims description 4
- LWIHDJKSTIGBAC-UHFFFAOYSA-K tripotassium phosphate Chemical compound [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 claims description 4
- 239000008280 blood Substances 0.000 claims description 3
- 210000004369 blood Anatomy 0.000 claims description 3
- SKJCKYVIQGBWTN-UHFFFAOYSA-N (4-hydroxyphenyl) methanesulfonate Chemical compound CS(=O)(=O)OC1=CC=C(O)C=C1 SKJCKYVIQGBWTN-UHFFFAOYSA-N 0.000 claims description 2
- JKFYKCYQEWQPTM-UHFFFAOYSA-N 2-azaniumyl-2-(4-fluorophenyl)acetate Chemical compound OC(=O)C(N)C1=CC=C(F)C=C1 JKFYKCYQEWQPTM-UHFFFAOYSA-N 0.000 claims description 2
- 229910021612 Silver iodide Inorganic materials 0.000 claims description 2
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 claims description 2
- 229910000361 cobalt sulfate Inorganic materials 0.000 claims description 2
- 229940044175 cobalt sulfate Drugs 0.000 claims description 2
- 229910000365 copper sulfate Inorganic materials 0.000 claims description 2
- ARUVKPQLZAKDPS-UHFFFAOYSA-L copper(II) sulfate Chemical compound [Cu+2].[O-][S+2]([O-])([O-])[O-] ARUVKPQLZAKDPS-UHFFFAOYSA-L 0.000 claims description 2
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 2
- 229910000396 dipotassium phosphate Inorganic materials 0.000 claims description 2
- 235000019797 dipotassium phosphate Nutrition 0.000 claims description 2
- 239000011790 ferrous sulphate Substances 0.000 claims description 2
- 235000003891 ferrous sulphate Nutrition 0.000 claims description 2
- XMBWDFGMSWQBCA-UHFFFAOYSA-N hydrogen iodide Chemical compound I XMBWDFGMSWQBCA-UHFFFAOYSA-N 0.000 claims description 2
- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 claims description 2
- 229910000359 iron(II) sulfate Inorganic materials 0.000 claims description 2
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 2
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 2
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims description 2
- 229910000160 potassium phosphate Inorganic materials 0.000 claims description 2
- 235000011009 potassium phosphates Nutrition 0.000 claims description 2
- 229940045105 silver iodide Drugs 0.000 claims description 2
- 235000010344 sodium nitrate Nutrition 0.000 claims description 2
- 239000004317 sodium nitrate Substances 0.000 claims description 2
- 229910052938 sodium sulfate Inorganic materials 0.000 claims description 2
- 235000011152 sodium sulphate Nutrition 0.000 claims description 2
- MQRWPMGRGIILKQ-UHFFFAOYSA-N sodium telluride Chemical compound [Na][Te][Na] MQRWPMGRGIILKQ-UHFFFAOYSA-N 0.000 claims description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N sulfuric acid Substances OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims description 2
- ADOCBEYRYZSHOI-UHFFFAOYSA-N [Ag].[At] Chemical compound [Ag].[At] ADOCBEYRYZSHOI-UHFFFAOYSA-N 0.000 claims 1
- 230000004044 response Effects 0.000 abstract description 22
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- CPSYWNLKRDURMG-UHFFFAOYSA-L hydron;manganese(2+);phosphate Chemical compound [Mn+2].OP([O-])([O-])=O CPSYWNLKRDURMG-UHFFFAOYSA-L 0.000 description 1
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- WBJZTOZJJYAKHQ-UHFFFAOYSA-K iron(3+) phosphate Chemical compound [Fe+3].[O-]P([O-])([O-])=O WBJZTOZJJYAKHQ-UHFFFAOYSA-K 0.000 description 1
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- XUIIKFGFIJCVMT-UHFFFAOYSA-N thyroxine-binding globulin Natural products IC1=CC(CC([NH3+])C([O-])=O)=CC(I)=C1OC1=CC(I)=C(O)C(I)=C1 XUIIKFGFIJCVMT-UHFFFAOYSA-N 0.000 description 1
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-
- C—CHEMISTRY; METALLURGY
- C01—INORGANIC CHEMISTRY
- C01B—NON-METALLIC ELEMENTS; COMPOUNDS THEREOF; METALLOIDS OR COMPOUNDS THEREOF NOT COVERED BY SUBCLASS C01C
- C01B32/00—Carbon; Compounds thereof
- C01B32/15—Nano-sized carbon materials
- C01B32/182—Graphene
- C01B32/194—After-treatment
-
- C—CHEMISTRY; METALLURGY
- C01—INORGANIC CHEMISTRY
- C01B—NON-METALLIC ELEMENTS; COMPOUNDS THEREOF; METALLOIDS OR COMPOUNDS THEREOF NOT COVERED BY SUBCLASS C01C
- C01B32/00—Carbon; Compounds thereof
- C01B32/15—Nano-sized carbon materials
- C01B32/182—Graphene
- C01B32/198—Graphene oxide
-
- 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
- G01N27/3271—Amperometric enzyme electrodes for analytes in body fluids, e.g. glucose in blood
-
- C—CHEMISTRY; METALLURGY
- C01—INORGANIC CHEMISTRY
- C01B—NON-METALLIC ELEMENTS; COMPOUNDS THEREOF; METALLOIDS OR COMPOUNDS THEREOF NOT COVERED BY SUBCLASS C01C
- C01B2204/00—Structure or properties of graphene
- C01B2204/20—Graphene characterized by its properties
- C01B2204/22—Electronic properties
Abstract
The invention discloses a preparation method of a bionic enzyme with phosphate embedded into a heteroatom-doped graphene layer, the bionic enzyme and application, and particularly relates to the bionic enzyme with phosphate embedded into the heteroatom-doped graphene layer, which is prepared by dispersing heteroatom-doped graphene into a sulfate solution, adding phosphate, adding alkali to adjust the pH value of the solution to be neutral, reacting under stirring to enable phosphate to be embedded into the heteroatom-doped graphene layer, cleaning with water and absolute ethyl alcohol after the reaction is finished, and drying to obtain the bionic enzyme with phosphate embedded into the heteroatom-doped graphene layer; phosphate can be allowed to enter by utilizing the interlayer spacing size of the heteroatom-doped graphene, so that the reaction active sites of the bionic enzyme are increased, the electron transfer is accelerated, and the electrochemical sensor constructed by taking the prepared bionic enzyme as a raw material has the advantages of quick response, high sensitivity, low detection limit and good selectivity; the method shows higher performance when the superoxide anion free radicals are quantitatively detected in real time, and has important application prospect in the aspect of detecting the superoxide anion free radicals released by living cells in real time.
Description
Technical Field
The invention relates to the field of materials, in particular to a preparation method of a biomimetic enzyme with phosphate groups embedded into heteroatom-doped graphene layers, and also relates to the biomimetic enzyme prepared by the method and application.
Background
Superoxide anion (O)2 .-) Is the product of the reduction of oxygen molecules by a single electron, and is the first free radical formed by cells in the oxygen metabolism process, and all other Reactive Oxygen Species (ROS) are derived from O2 .-Derived from the above-mentioned raw materials. O is2 .-The concentration fluctuation of (A) is closely related to the occurrence and development of various biological processes and diseases. Under normal physiological conditions, O2 .-The concentration in the cell can be controlled in a lower range, relatively stable dynamic balance is kept, the normal growth and metabolism of the cell can be assisted, and the special physiological effect is realized. The physiological action mainly comprises the participation of anti-infection immunity; help clear cells that are faded, mutated, and senescent; involved in the synthesis of prostaglandins, thyroxine and prothrombin; and the medicine is involved in the detoxification of medicines and poisons, and the like. At moderate level, O2.-The decrease or increase of the intracellular concentration can cause transient changes of the cells, including the reduction of the reproductive capacity and the reduction of the defense capacity. At the same time, the cells will also initiate self-repair and regulation mechanisms without irreversible damage. But when the cells produce excess O2 .-When used, it causes a series of toxic and side effects, irreversible oxidative damage to cells and effects on specific signal pathways, including causing inactivation of free radicals, damage to deoxyribonucleic acid (DNA), gene mutation, damage to amino acids and proteins, and damage to other biomolecules. The influence of these toxic and side effects on the body further causes physiological changes, including aging, neuronal degenerative diseases, cardiovascular diseases, cancer, etc. of the body. Thus, O released to living cells2.-The quantitative detection can not only more comprehensively understand the role of the cell in the physiological activities, but also help us to disclose the occurrence mechanism of the related diseases, thereby providing reliable diseases under pathological cognitionAnd (6) diagnosing diseases.
However, O2.-The released concentration of the cells is very low, the activity is very high, and the qualitative and quantitative detection of the cells is very difficult. Among the detection methods, the electrochemical method has the advantages of fast response, high sensitivity, simple operation, low cost and the like, and is very suitable for releasing O in real time to living cells on the premise of avoiding damaging the metabolism and related physiological activities of the living cells2.-The concentration of (4) is detected. Therefore, the designed synthesis has high sensitivity, high selectivity, low detection limit and low cost2.-Electrochemical biosensors have become one of the major and difficult points of current research. Conventional O2 .-The sensitive element of the electrochemical sensor mainly depends on natural biological enzyme, and the biological enzyme has the problems of easy influence of temperature, humidity, pH and the like to cause the loss of catalytic activity, and the cost is relatively high. Therefore, novel O-based biomimetic enzymes were developed2.-The electrochemical sensor has more practical significance.
Since superoxide dismutase (SOD) is O2.-Specific enzyme of (4), O constructed based on SOD2.-The electrochemical sensor can show better anti-interference capability than other biological enzymes, but is expensive, low in yield and easy to inactivate, so that research and development of the bionic enzyme capable of effectively replacing SOD become a key research problem of multidisciplinary intersection. In recent years, scientists find that the construction of electrochemical sensors based on bionic enzymes can be realized by simulating the binding sites or active sites of natural enzymes, in particular to novel special nanostructure bionic enzymes combined with nanotechnology, which can catalyze the reaction of enzyme substrates under physiological conditions and have the catalytic efficiency and enzymatic reaction kinetic properties as the natural enzymes.
According to the related research progress at home and abroad, the bionic enzyme-based O can be realized by simulating a binding site or an active site of SOD2 ·-And (5) constructing an electrochemical sensor. Among them, transition metals widely exist in the active center of SOD, and are the most commonly used materials for designing biomimetic enzymes. Phosphate is widely present in the body of an organism,considered as a relatively stable class of biocompatible materials, the PO thereof4 3-The compound contains three lone-pair electrons and is easy to form a complex with positively charged metal ions. But can utilize transition metal phosphate to detect O due to the rich transition metal active center2 ·-Is reported at present.
Disclosure of Invention
In view of the above, an object of the present invention is to provide a method for preparing a biomimetic enzyme with phosphate-intercalated heteroatom-doped graphene layer, in which a single-layer carbon-atom sheet-like nanomaterial graphene bonded by covalent bonds has a large specific surface area, can effectively increase the transmission of electrons between an electrode and a reactant, and can be modified by compounding with an inorganic compound, thereby achieving excellent properties that cannot be achieved by a single material due to the synergistic effect between the materials, and then the phosphate-intercalated heteroatom-doped graphene layer complex is applied to living cells to release O when stimulated by a drug2 ·-Detecting; the second purpose of the invention is to provide the bionic enzyme prepared by the preparation method; the third purpose of the invention is to provide an electrochemical sensor based on the bionic enzyme; the fourth purpose of the invention is to provide the application of the electrochemical sensor of the bionic enzyme in detecting the content of superoxide anion free radicals in living cells or blood; the fifth purpose of the invention is to provide the application of the bionic enzyme in preparing an electrochemical bionic sensor or wearable equipment as printing slurry.
In order to achieve the purpose, the invention provides the following technical scheme:
1. a preparation method of a bionic enzyme with phosphate embedded into a heteroatom-doped graphene layer comprises the following steps of dispersing heteroatom-doped graphene with heteroatom-doped increased graphene layer intervals into a sulfate solution, adding phosphate, adding alkali to adjust the pH value of the solution to be neutral, then reacting under stirring to enable the phosphate to be embedded into the heteroatom-doped graphene layer, washing with water and absolute ethyl alcohol after reaction is finished, and drying to obtain the bionic enzyme with phosphate embedded into the heteroatom-doped graphene layer.
Preferably, the heteroatom-doped graphene is halogen atom-doped graphene or tellurium atom-doped graphene.
In the invention, the sulfate is one or more of sodium sulfate, cobalt sulfate, calcium sulfate, copper sulfate, barium sulfate or ferrous sulfate; the phosphate is one or more of potassium dihydrogen phosphate, potassium hydrogen phosphate or potassium phosphate.
In the present invention, the reaction is stirred at room temperature for at least 2 hours.
In the invention, the drying is drying at the temperature of 60 ℃.
Preferably, the preparation method of the atom-doped graphene comprises the following steps: uniformly dispersing graphene oxide in water, then dispersing the graphene oxide in a solution added with an inorganic compound containing heteroatoms, fully reacting to enable the heteroatoms of the inorganic compound to be embedded between graphene oxide layers, washing a product with water to remove residual salt after reaction, and freeze-drying to obtain the heteroatom-doped graphene. In this process, the inorganic compound is added in an excess amount so that the hetero atoms are intercalated between graphene oxide layers under reaction conditions of 180 ℃ for 12 hours.
More preferably, the graphene oxide is prepared by the following steps: graphite powder and sodium nitrate are mixed according to the mass ratio of 1: 0.6, adding the mixture into a round-bottom flask containing 98% concentrated sulfuric acid by mass, carrying out ultrasonic treatment for 20min, and stirring in an ice-water bath for 30 min; slowly adding potassium permanganate into the mixed solution, stirring at 40 ℃ for 2 hours, adding deionized water into the mixed solution, stirring at 90 ℃ for 1 hour, and continuously slowly adding deionized water; after the addition, stirring the mixed solution at room temperature for 10 hours, slowly adding 30% hydrogen peroxide into the mixed solution, stirring for 1 hour, washing with 5% hydrochloric acid for 8-10 times, washing with deionized water, and finally freeze-drying to obtain the graphene oxide. During the washing process, the washing liquid can be washed until the washing liquid is neutral, or the pH value of the washing liquid is 5, so that the influence of the pH value of the graphene oxide on the subsequent result is small.
In the invention, the inorganic compound is one or more of sodium bromide, potassium iodide, sodium iodide, silver iodide, lead iodide, sodium telluride, zinc telluride or silver astatide.
2. The biomimetic enzyme prepared by the preparation method.
3. The bionic enzyme-based electrochemical sensor is characterized in that the bionic enzyme is coated on the surface of a working electrode of the electrochemical sensor.
4. The bionic enzyme electrochemical sensor is applied to detecting the content of superoxide anion free radicals in living cells or blood.
5. The bionic enzyme is applied to printing and preparing an electrochemical bionic sensor or wearable equipment as printing slurry.
The invention has the beneficial effects that: the invention discloses a preparation method of a biomimetic enzyme with a phosphate group embedded into a heteroatom-doped graphene layer, which is characterized in that atoms are doped on graphene oxide through reaction with inorganic salt on the graphene oxide, so that the interval between graphene layers is enlarged, and the interlayer spacing of the graphene after heteroatom doping can allow phosphate to enter, so that reaction active sites are increased, the reaction area is enlarged, the electron transfer is accelerated, the conductivity of the biomimetic material is improved, and the selectivity of the biomimetic material can be well enhanced by selecting appropriate active center metal and the content thereof.
In addition, through reasonably setting the atom species of the doped graphene, reasonably preparing the phosphate with good specificity and optimizing the proportion of the two, the finally prepared bionic enzyme sensitive element is used as a raw material to construct the electrochemical sensor which not only has short response time, high reaction sensitivity and low detection limit, but also has excellent selectivity. Compared with a sensor prepared from a traditional material, the sensor prepared from the bionic enzyme material shows higher performance when the superoxide anion free radicals are quantitatively detected in real time, and has an important application prospect in the aspect of detecting the superoxide anion free radicals released by living cells in real time. The material has outstanding performance and convenient material acquisition, and is beneficial to commercial application.
Drawings
In order to make the object, technical scheme and beneficial effect of the invention more clear, the invention provides the following drawings for explanation:
fig. 1 is a FESEM image of atom-doped graphene prepared in example 1;
fig. 2 is a TEM image of the atom-doped graphene prepared in example 1;
FIG. 3 is a FESEM photograph of the biomimetic enzyme prepared in example 1;
FIG. 4 is a TEM image of the biomimetic enzyme prepared in example 1;
FIG. 5 is a contact angle of the biomimetic enzyme prepared in example 1;
FIG. 6 is an SEM photograph of the superoxide dismutase biomimetic material prepared in example 2;
fig. 7 is a FESEM image of reduced graphene oxide prepared in example 3;
FIG. 8 is a graph of sensor pair O constructed in example 1 at a voltage range of 0.3-1.0V2 ·-A graph of the cyclic voltammetry response test results;
FIG. 9 is a graph of sensor pair O constructed in example 2 at a voltage range of 0.3-1.0V2 ·-A graph of the cyclic voltammetry response test results;
FIG. 10 is a graph of sensor pair O constructed in example 3 at a voltage range of 0.3-1.0V2 ·-A graph of the cyclic voltammetry response test results;
FIG. 11 is a sensor pair O constructed as in example 12 ·-Timed Current response test results of (1) -relative to Hg/Hg2Cl2I-t response plots for the reference electrode;
FIG. 12 is a sensor pair O constructed in example 12 ·-Current vs. O in steady state in FIG. 112 ·-A linear plot between concentrations;
FIG. 13 is a sensor pair O constructed as in example 12 ·-A response time map of (a);
FIG. 14 is a graph showing the results of the selectivity test of the sensor constructed in example 1 for different interfering components;
FIG. 15 shows the real-time detection of O released from DU145 cells stimulated by zymosan (Zym) by the sensor constructed in example 12 ·-It curve (in FIG. 15, a is an optical microscope image of DU145 cell; in FIG. 15, b is a fixed potential of 0.60V, the sensor releases O to DU145 cell2 ·-I-t response map of).
Detailed Description
The present invention is further described with reference to the following drawings and specific examples so that those skilled in the art can better understand the present invention and can practice the present invention, but the examples are not intended to limit the present invention.
Example 1
Biomimetic enzyme materials (Co)3(PO4)2The method for synthesizing the/I-rGO) comprises the following specific steps:
(1) the preparation method of the atom-doped graphene (I-rGO) comprises the following steps:
firstly, respectively mixing 2g of graphite powder and 1.2g of NaNO3Put into a 500mL three-neck round-bottom flask, and then 76mL H2SO4Slowly injecting; then, putting the mixed solution into an ultrasonic instrument with the power of 80W for ultrasonic treatment for 15min, and then putting the mixed solution into an ice water bath for stirring for 30 min; then, 8.6g of KMnO was slowly added to the mixture4Continuously stirring for 2 hours in an ice water bath to react;
secondly, slowly heating the obtained reaction liquid to 40 ℃, and continuously stirring for 2 hours at 40 ℃; then, 160mL of DIW was slowly added to the reaction solution, and the mixture was heated to 90 ℃ and stirred at 90 ℃ for 1 hour to effect a reaction;
thirdly, slowly dripping 120mL of DIW into the obtained reaction liquid, and stirring for 10 hours at room temperature to carry out reaction; subsequently, 30mL of 30% H was added thereto2O2The solution was stirred for 1h to remove excess KMnO4And MnO formed in the reaction2；
Fourthly, washing the reaction solution for many times by using 8 percent HCl solution to remove impurities which are insoluble in water; washing the product for several times by using DIW to remove acid and water-soluble impurities in the product until finally discharged washing liquid is neutral; carrying out freeze drying to obtain graphene oxide;
fifthly, mixing the product with DIW according to the proportion of 1: 8, placing the mixed solution in an ultrasonic instrument with the power of 80W for ultrasonic treatment for 5h to finally obtain a Graphene Oxide (GO) turbid liquid uniformly dispersed in DIW; next, 0.01mol (1.5g) of NaI was dissolved in28mL of DIW, then the GO suspension (12mL, 4.2 mg. L) prepared above-1) Dispersed in a solution of NaI as described above. After mixing well, the mixture was transferred to a stainless steel autoclave lined with polytetrafluoroethylene (50mL) and reacted at 180 ℃ for 12 hours. Then, the product is washed with DIW for several times to remove residual salts, and then the product is freeze-dried, so that the iodine atom doped graphene (I-rGO) is obtained.
FIG. 1 is a FESEM image of I-rGO prepared in example 1, and from FIG. 1, it can be seen that I-rGO has a corrugated structure.
FIG. 2 is a TEM image of I-rGO prepared in example 1, and from FIG. 2, it can be seen that I-rGO is sheet corrugated.
(2) Target biomimetic enzyme material (Co for short)3(PO4)2I-rGO) preparation method: dispersing atom-doped graphene (I-rGO) in 7.5mmol of CoSO4·7H2Adding 5mmol KH into O solution2PO4(ii) a 1.0mol L of water are added dropwise with continuous stirring-1KOH adjusts the pH value of the solution to be neutral; then, stirring was continued at room temperature for 2 hours to effect a reaction. Then, carrying out centrifugal cleaning on the product for multiple times by using DIW and absolute ethyl alcohol respectively, and drying at 60 ℃ to obtain the target bionic enzyme Co embedded between the atom-doped graphene layers3(PO4)2/I-rGO。
FIG. 3 is a FESEM image of the biomimetic enzyme prepared, and it can be seen from FIG. 3 that Co3(PO4)2Into the multilayer structure of I-rGO.
FIG. 4 is a TEM image of the prepared biomimetic enzyme, as can be seen from FIG. 4, Co3(PO4)2After entering the multilayer structure of I-rGO, the two are tightly attached.
FIG. 5 shows the contact angle of the produced biomimetic enzyme, Co is shown in FIG. 53(PO4)2The theta values of/I-rGO and the slide surface CA are 67.57 degrees and less than 90 degrees, which indicates that the material is hydrophilic and releases O in the captured cells2 ·-Has certain application potential.
Constructing an electrochemical sensor: co of different concentrations to be prepared by ultrasound3(PO4)2Uniformly dispersing the/I-rGO nano-composite aqueous solution, dropwise adding 4.0 mu L of the aqueous solution on the clean GCE surface, drying in the air at room temperature, dropwise adding 2.0 mu L of 0.1 wt% Nafion solution, and drying in the air to obtain Co3(PO4)2I-rGO modified working electrode. The working electrode was connected to an electrochemical workstation, a counter electrode (platinum wire electrode), a reference electrode (Hg/HgCl)2Electrodes), an electrolytic cell and an electrolyte (phosphate buffer solution with the concentration of 0.01mol/L and the pH value of 7.4) are assembled together to form the superoxide anion radical electrochemical sensor.
The iodine atom doped graphene in the present embodiment may be replaced with, but is not limited to, one or more of bromine, antimony, astatine; the phosphate may be replaced by, but is not limited to, one or more of manganese phosphate, nickel phosphate, iron phosphate; the solution pH may be replaced, but is not limited to, with an acidic or basic.
Example 2
The superoxide dismutase bionic enzyme material comprises the following specific steps:
first, 5mmol KH was added2PO4And 7.5mmol CoSO4·7H2O was dissolved in 50mL of DIW and 1.0mol L was added dropwise with constant stirring-1KOH adjusted the pH of the solution to neutral. Then, stirring was continued at room temperature for 2 hours to effect a reaction. Then, respectively using DIW and absolute ethyl alcohol to centrifugally clean the product for multiple times, and drying at 60 ℃ to obtain a target product Co3(PO4)2(Co for short)3(PO4)2Biomimetic enzyme material).
Coating with Co3(PO4)2Working electrode of bionic enzyme material:
co to be prepared3(PO4)2Dispersing a bionic enzyme material in water according to the proportion concentration of 1mg/mL to obtain an electrode modification solution, coating the electrode modification solution on a glassy carbon electrode, drying at 26 ℃ for 5 hours, coating a Nafion solution with the Nafion mass fraction of 0.1 wt%, and drying at 26 ℃ for 5 hours again to obtain the working electrode with the surface coated with the superoxide dismutase bionic material.
Constructing a superoxide anion radical electrochemical sensor:
the prepared working electrode with the surface coated with the superoxide dismutase bionic material, an electrochemical workstation, a counter electrode (platinum wire electrode) and a reference electrode (Hg/HgCl)2Electrodes), an electrolytic cell and an electrolyte (phosphate buffer solution with the concentration of 0.01mol/L and the pH value of 7.4) are assembled together to form the superoxide anion radical electrochemical sensor.
FIG. 6 is an SEM photograph of the SOD biomimetic material prepared in example 2, wherein Co is shown in FIG. 63(PO4)2And (4) synthesizing a bionic enzyme material.
Example 3
The method for reducing the graphene oxide material comprises the following specific steps:
firstly, respectively mixing 2g of graphite powder and 1.2g of NaNO3Put into a 500mL three-neck round-bottom flask, and then 76mL H2SO4Slowly injecting; then, putting the mixed solution into an ultrasonic instrument with the power of 80W for ultrasonic treatment for 15min, and then putting the mixed solution into an ice water bath for stirring for 30 min; then, 8.6g of KMnO was slowly added to the mixture4Continuously stirring for 2 hours in an ice water bath to react;
secondly, slowly heating the obtained reaction liquid to 40 ℃, and continuously stirring for 2 hours at 40 ℃; then, 160mL of DIW was slowly added to the reaction solution, and the mixture was heated to 90 ℃ and stirred at 90 ℃ for 1 hour to effect a reaction;
③ dropping 120mL DIW slowly into the reaction solution, stirring at room temperature for 10H for reaction, and then adding 30mL of 30% H2O2The solution was stirred for 1h to remove excess KMnO4And MnO formed in the reaction2；
And fourthly, washing the reaction solution for many times by using 8 percent HCl solution to remove impurities which are insoluble in water. The product was washed several times with DIW to remove acid and water-soluble impurities from the product until the final wash was neutral.
Fifthly, mixing the product with DIW according to the proportion of 1: 8, placing the mixed solution in an ultrasonic instrument with the power of 80W for ultrasonic treatment for 5h, and finally obtaining GO turbid liquid uniformly dispersed in DIW. Then the GO prepared above is suspendedLiquid (12mL, 4.2mg L)-1) Transferring the mixture into a stainless steel high-pressure reaction kettle with a polytetrafluoroethylene lining (50mL) and reacting for 12 hours at 180 ℃; and washing the product with DIW for several times to remove residual salt, and then freeze-drying the product to obtain the reduced graphene oxide material (short for rGO).
Fig. 7 is a FESEM image of the reduced graphene oxide material prepared, and as can be seen from fig. 7, some agglomeration occurred.
Working electrode coated with reduced graphene oxide material:
dispersing the prepared reduced graphene oxide material in DIW at the concentration of 1mg/mL to obtain an electrode modification solution, coating the electrode modification solution on a glassy carbon electrode, drying at 26 ℃ for 5h, coating a Nafion solution with the Nafion mass fraction of 0.1 wt%, and drying at 26 ℃ for 5h again to prepare a working electrode with the surface coated with the reduced graphene oxide material.
Constructing a superoxide anion radical electrochemical sensor:
the prepared working electrode with the surface coated with the reduced graphene oxide material is used as a working electrode, an electrochemical workstation, a counter electrode (platinum wire electrode) and a reference electrode (Hg/HgCl)2Electrodes), an electrolytic cell and an electrolyte (phosphate buffer solution with the concentration of 0.01mol/L and the pH value of 7.4) are assembled together to form the superoxide anion radical electrochemical sensor.
Example 4
Will contain 20. mu. mol. L-1O2 ·-Was added to the electrolyte of the sensor constructed in example 1, and the sensor was tested for O at a voltage ranging from 0.3 to 1.0V2 ·-While the cyclic voltammetric response of the sensor to PBS was used as a blank control. As shown in FIG. 8, it is understood from FIG. 8 that the content of the compound (D) is 20. mu. mol. L-1O2 ·-In PBS (5), the oxidation peak current ratio does not contain O2 ·-The oxidation peak current in PBS of (1) was significantly increased, indicating that the sensor is paired with O2 ·-Has obvious electrochemical catalytic oxidation capability.
Example 5
Will contain 20. mu. mol. L-1O2 ·-Was added to the electrolyte of the sensor constructed in example 2, and the sensor was tested for O at a voltage ranging from 0.3 to 1.0V2 ·-While the cyclic voltammetric response of the sensor to PBS was used as a blank control. As shown in FIG. 9, it is understood from FIG. 9 that the content of the compound (D) is 20. mu. mol. L-1O2 ·-In PBS (5), the oxidation peak current ratio does not contain O2 ·-The oxidation peak current in PBS of (1) was significantly increased, indicating that the sensor is paired with O2 ·-Has obvious electrochemical catalytic oxidation capacity, but the half-wave potential is larger than that of the example 4, and the peak current value is changed less than that of the example 4, which shows that the electrochemical catalytic oxidation capacity to O is higher2 ·-Is weaker than in example 4.
Example 6
Will contain 20. mu. mol. L-1O2 ·-Was added to the electrolyte of the sensor constructed in example 2, and the sensor was tested for O at a voltage ranging from 0.3 to 1.0V2 ·-While the cyclic voltammetric response of the sensor to PBS was used as a blank control. As shown in FIG. 10, it is understood from FIG. 10 that the content of the compound (D) is 20. mu. mol. L- 1O2 ·-In PBS, there was almost no change in oxidation peak current, indicating that the sensor is sensitive to O2 ·-Has an electrochemical catalytic oxidation capacity of almost 0.
Example 6
Test of sensor pair O constructed in example 12 ·-In the test, O was continuously added to the electrolyte of the sensor constructed in example 1 at different concentrations2 ·-The solution is kept for 50s, and the relation curve of response time and current value is recorded, thus obtaining the sensor pair O2 ·-The results are shown in FIG. 11, when different concentrations of O are continuously added to the electrolyte2 ·-The working electrode constructed in example 1 was aligned to Hg/Hg2Cl2I-t response plot of reference electrode, FIG. 12Steady state current and O detected for the sensor constructed in example 12 ·-Linear relationship between concentrations. As can be seen from FIG. 12, the response current follows O2 ·-Increases in concentration in response to current and O2 ·-The linear equation for concentration can be expressed as: i (μ a) ═ 0.0124C (nmol · L)-1)+0.3457(R20.998), the sensitivity was 177.14 μ a · (μmol · L)-1·cm2)-1The detection limit is 1.67 nmol.L-1(signal-to-noise ratio S/N-3).
FIG. 13 is a sensor pair O constructed as in example 12 ·-FIG. 13 shows the response time chart of (2) in the case of O injection2 ·-The sensor then responds rapidly, forming a steady state current in 2.99 seconds.
Example 7
Solutions of different substances were sequentially added to the electrolyte of the sensor constructed in example 1, the sensor was tested for chronoamperometric response to different interfering components, and 20nmol · L was sequentially added to the electrolyte of the sensor at a fixed potential of 0.60V, each in sequence and every 50s-1O of (A) to (B)2 ·-、5μmol·L-1AA, UA, DA and 1. mu. mol. L of-1H of (A) to (B)2O2The amperometric response curves of the sensor for selectivity tests of different interfering components were obtained, and the results are shown in FIG. 14, from which it can be seen that 5. mu. mol. L-1AA, UA, DA and 1. mu. mol. L of-1H of (A) to (B)2O2The sensor was not detected at 20 nmol.L-1O of (A) to (B)2 ·-Cause interference, accounting for the sensor pair O2 ·-Has good specificity.
Example 8
The sensor constructed in example 1 was used for the detection of DU145 cells at a cell density of 1X 105one/mL, specifically, real-time detection of O released by DU145 cells under zym stimulation in three conditions by chronoamperometry2 ·-: (1) injection of 0.2 mg/mL into cells-1zym, respectively; (2) injection of 0.2 mg/mL into cells-1zym and 300 U.mL-1A mixed solution of SOD;(3) 0.2 mg/mL of the electrolyte solution to which no cells were added-1zym are provided. The results are shown in FIG. 15, where a in FIG. 15 is the optical microscope image of DU145 cells, and b in FIG. 15 the sensor releases O to DU145 cells2 ·-FIG. 15 shows that when 0.2 mg/mL of the polymer is added-1zym promote the release of O from cells2 ·-A larger current response was detected (as shown by curve I in b of FIG. 15), and 0.2 mg. multidot.mL was added-1zym and 300 U.mL-1The SOD mixture did not cause significant current changes (as shown by curve II in b in FIG. 15), indicating that O was released by the cells2 ·-The electrolyte, which had been consumed by SOD, was added to the electrolyte in the absence of DU145 cells at 0.2 mg/mL under the same test conditions-1zym also no significant current change was detected (as shown by curve iii in b in fig. 15). Thus, it can be confirmed that the current response shown in curve I is O released by DU145 cells under zym stimulation2 ·-Is captured by the bionic material on the working electrode in the sensor and is generated by oxidation reaction on the surface of the bionic material. Further, 2.0 mg/mL can be calculated according to the standard linear equation-1zym stimulation of O released by DU145 cells2 ·-And (4) concentration.
The above-mentioned embodiments are merely preferred embodiments for fully illustrating the present invention, and the scope of the present invention is not limited thereto. The equivalent substitution or change made by the technical personnel in the technical field on the basis of the invention is all within the protection scope of the invention. The protection scope of the invention is subject to the claims.
Claims (11)
1. The preparation method of the bionic enzyme with the phosphate group embedded into the heteroatom-doped graphene layer is characterized by comprising the following steps of: dispersing heteroatom-doped graphene with heteroatom doping and graphene layer spacing increased into a sulfate solution, adding phosphate, continuously adding alkali to adjust the pH value of the solution to be neutral, then reacting under stirring to enable phosphate to be embedded into the heteroatom-doped graphene layer, washing with water and absolute ethyl alcohol after the reaction is finished, and drying to obtain the bionic enzyme with the phosphate embedded into the heteroatom-doped graphene layer.
2. The method for preparing a biomimetic enzyme according to claim 1, wherein: the sulfate is one or more of sodium sulfate, cobalt sulfate, calcium sulfate, copper sulfate, barium sulfate or ferrous sulfate; the phosphate is one or more of potassium dihydrogen phosphate, potassium hydrogen phosphate or potassium phosphate.
3. The method for preparing a biomimetic enzyme according to claim 1, wherein: the reaction was stirred at room temperature for at least 2 hours.
4. The method for preparing a biomimetic enzyme according to claim 1, wherein: the drying is drying at the temperature of 60 ℃.
5. The method for preparing a biomimetic enzyme according to any one of claims 1-4, wherein: the preparation method of the atom-doped graphene comprises the following steps: uniformly dispersing graphene oxide in water, then dispersing the graphene oxide in a solution added with an inorganic compound containing heteroatoms, fully reacting to enable the heteroatoms of the inorganic compound to be embedded between graphene oxide layers, washing a product with water to remove residual salt after reaction, and freeze-drying to obtain the heteroatom-doped graphene.
6. The method for preparing a biomimetic enzyme according to claim 5, wherein: the preparation method of the graphene oxide comprises the following specific steps: graphite powder and sodium nitrate are mixed according to the mass ratio of 1: 0.6, adding the mixture into a round-bottom flask containing 98% concentrated sulfuric acid by mass, carrying out ultrasonic treatment for 20min, and stirring in an ice-water bath for 30 min; slowly adding potassium permanganate into the mixed solution, stirring at 40 ℃ for 2 hours, adding deionized water into the mixed solution, stirring at 90 ℃ for 1 hour, and continuously slowly adding deionized water; after the addition, stirring the mixed solution at room temperature for 10h, slowly adding 30% by mass of hydrogen peroxide into the mixed solution, stirring for 1h, washing with 5% by mass of hydrochloric acid for 8-10 times, washing with water, and finally freeze-drying to obtain the graphene oxide.
7. The method for preparing a biomimetic enzyme according to claim 5, wherein: the inorganic compound is one or more of sodium bromide, potassium iodide, sodium iodide, silver iodide, lead iodide, sodium telluride, zinc telluride or astatine silver.
8. A biomimetic enzyme prepared by the preparation method of any one of claims 1 to 7.
9. An electrochemical sensor based on the biomimetic enzyme according to claim 8, wherein: the electrochemical sensor has a working electrode surface coated with the biomimetic enzyme according to claim 8.
10. Use of an electrochemical sensor of a biomimetic enzyme according to claim 9 for detecting the content of superoxide anion radicals in living cells or blood.
11. Use of the biomimetic enzyme according to claim 8 as a printing paste for the preparation of an electrochemical biomimetic sensor or wearable device.
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| 邹卓: "从纳米尺度调控仿生酶催化超氧阴离子自由基的电化学氧化行为", 《中国博士学位论文全文数据库 基础科学辑》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116818846A (en) * | 2023-06-21 | 2023-09-29 | 深圳市诺安智能股份有限公司 | Semiconductor gas sensing material and preparation method thereof |
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