阅读说明：本技术 纳米颗粒仿生酶敏感元件的制备方法及其产品和应用 (Preparation method of nano-particle bionic enzyme sensitive element, product and application thereof ) 是由 李长明 邹卓 杨鸿斌 于 2020-12-24 设计创作，主要内容包括：本发明公开了纳米颗粒仿生酶敏感元件的制备方法及其产品和应用,将二维纳米材料分散于溶剂中,加入含有过渡金属源的乙酸盐在一定温度下水浴一定时间,冷冻干燥去除溶剂；将得到的产物与六元杂环化合物和物质氨基酸研磨成均匀混合物,然后再惰性气氛中热解,冷却得到纳米颗粒仿生酶敏感元件；将制得的敏感元件制成传感器后不仅具有极短的响应时间、极高的反应灵敏度、较低的检测限,还具有优异的选择性；能够在实时检测活细胞释放的超氧阴离子自由基方面有重要的应用前景,该材料性能突出、取材方便,利于商业化应用。(The invention discloses a preparation method of a nano-particle bionic enzyme sensitive element and a product and application thereof.A two-dimensional nano material is dispersed in a solvent, acetate containing a transition metal source is added for water bath for a certain time at a certain temperature, and the solvent is removed by freeze drying; grinding the obtained product, a six-membered heterocyclic compound and amino acid serving as a substance into a uniform mixture, then pyrolyzing the mixture in an inert atmosphere, and cooling the mixture to obtain a nanoparticle bionic enzyme sensitive element; the prepared sensitive element is made into a sensor, so that the sensor not only has extremely short response time, extremely high reaction sensitivity and lower detection limit, but also has excellent selectivity; the material has important application prospect in the aspect of detecting superoxide anion free radicals released by living cells in real time, has outstanding performance and convenient material acquisition, and is beneficial to commercial application.)

1. The preparation method of the nano-particle bionic enzyme sensitive element is characterized by comprising the following steps of: dispersing the two-dimensional nano material in a solvent, adding acetate containing a transition metal source to uniformly adsorb metal ions and the two-dimensional nano material, and freeze-drying to remove the solvent; and uniformly mixing the obtained product with a hexatomic heterocyclic compound and substance amino acid to obtain a mixture, then performing pyrolysis in an inert atmosphere to form a carbon structure, and cooling to obtain the nanoparticle bionic enzyme sensitive element.

2. The preparation method of the nanoparticle biomimetic enzyme sensitive element according to claim 1, characterized in that: the two-dimensional nano material is one or more of graphene, graphene oxide, carbon nano tubes or carbon nano fibers; the acetate containing the transition metal source is acetate containing cobalt, nickel, manganese, copper or iron source; the six-membered heterocyclic compound is one or more of melamine, pyridine, pyrazine, pyrimidine or pyridazine; the biomass amino acid is one or more of serine, cysteine or alanine.

3. The preparation method of the nanoparticle biomimetic enzyme sensitive element according to claim 1, characterized in that: the mass ratio of the two-dimensional nano material, the acetate containing the transition metal source and the six-membered heterocyclic compound is 400: 1: 200.

4. the preparation method of the nanoparticle biomimetic enzyme sensitive element according to claim 1, characterized in that: the homogeneous adsorption was carried out in a water bath at 80 ℃ for 2 hours.

5. The preparation method of the nanoparticle biomimetic enzyme sensitive element according to claim 1, characterized in that: the pyrolysis is carried out at 900 ℃ for 2 hours.

6. The nanoparticle biomimetic enzyme sensitive element prepared by the method of any one of claims 1 to 5, characterized in that: the nano-particle bionic enzyme sensitive element is formed by fixing transition metal atoms at a defect structure of a nano material through doping of hetero atoms.

7. The use of the nanoparticle biomimetic enzyme sensor according to claim 6 in the preparation of an electrochemical sensor for detecting bioactive molecules released by living cells.

8. Use according to claim 7, characterized in that: the bioactive molecule is superoxide anion free radical, dopamine, hydrogen peroxide or nitric oxide.

9. The electrochemical sensor of nanoparticle biomimetic enzyme sensitive element according to claim 6, wherein: the surface of a working electrode of the electrochemical sensor is coated with the nanoparticle biomimetic enzyme sensitive element according to claim 6.

10. The electrochemical sensor of the nanoparticle biomimetic enzyme sensitive element according to claim 8, wherein: the coating is to disperse the nano-particle bionic enzyme sensitive element into water according to the proportioning concentration of 0.5-5mg/mL to obtain an electrode modification solution, coat the electrode modification solution on an electrode, coat an adhesive after drying, and dry again.

11. Use of an electrochemical sensor according to claim 9 or 10 for detecting the release of bioactive molecules from living cells.

Technical Field

The invention relates to the technical field of materials, in particular to a preparation method of a nano-particle bionic enzyme sensitive element, a product prepared by the method, application of the product and an electrochemical sensor prepared by the nano-particle bionic enzyme sensitive element.

Background

Superoxide anion (O)₂ ^.-) Is that the oxygen molecule accepts a single electronThe products of the reduction, which are also the first formed free radicals of the cell during oxygen metabolism, 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 disease diagnosis under pathological cognition.

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, is very suitable for avoiding the damage to the metabolism of living cells and related physiological activities,for releasing O in real time on 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.

The bionic enzyme sensitive element with high performance is designed to realize O₂ ^.-Key factors for higher sensitivity and selectivity of electrochemical sensors. It is reported that the metal is dispersed on the carbon nano material, which is an effective bionic enzyme preparation method. Therefore, it is reasonable to conclude that the heteroatom-doped carbon nanomaterial modified by the metal nanoparticles improves O₂ ^·-Has great potential in oxidation catalytic reaction. However, since such materials typically have high surface energies, how to construct chemically stable such materials remains a challenge. More importantly, the electronic structure of the metal nanoparticle modified atom doped carbon nanomaterial is elusive, which hinders the understanding of such materials. Therefore, there is a need to develop a novel nanoparticleBionic enzyme sensitive element, electrochemical sensor constructed by using same and application of sensor to release of O from living cells stimulated by drugs₂ ^·-In the detection of (1).

Disclosure of Invention

In view of the above, an object of the present invention is to provide a method for preparing a nanoparticle biomimetic enzyme sensor; the second purpose of the invention is to provide a nano-particle bionic enzyme sensitive element prepared by the method; the third purpose of the invention is to provide the application of the nano-particle bionic enzyme sensitive element in the preparation of an electrochemical sensor for detecting superoxide anion free radical released by living cells; the fourth purpose of the invention is to provide an electrochemical sensor based on the nano-particle bionic enzyme sensitive element; the fifth purpose of the invention is to provide the application of the electrochemical sensor in detecting the release of superoxide anion free radicals from living cells.

In order to achieve the purpose, the invention provides the following technical scheme:

1. dispersing a two-dimensional nano material in a solvent, adding acetate containing a transition metal source to uniformly adsorb metal ions and the two-dimensional nano material, and freeze-drying to remove the solvent; and uniformly mixing the obtained product with a hexatomic heterocyclic compound and substance amino acid to obtain a mixture, then performing pyrolysis in an inert atmosphere to form a carbon structure, and cooling to obtain the nanoparticle bionic enzyme sensitive element.

In the invention, the solvent can be a polar solvent or a non-dosage solvent, and is preferably deionized water; the freeze drying of the invention can not only remove the solvent quickly, but also is beneficial to forming a porous structure, and the obtained product can be uniformly mixed with the hexatomic heterocyclic compound and the substance amino acid by adopting grinding or ball milling, and other uniformly mixed modes.

In the invention, the two-dimensional nano material is one or more of graphene, graphene oxide, carbon nano tube or carbon nano fiber; the acetate containing the transition metal source is acetate containing cobalt, nickel, manganese, copper or iron source; the six-membered heterocyclic compound is one or more of melamine, pyridine, pyrazine, pyrimidine or pyridazine; the biomass amino acid is one or more of serine, cysteine or alanine.

In the invention, the mass ratio of the two-dimensional nano material, the acetate containing the transition metal source and the six-membered heterocyclic compound is 400: 1: 200.

in the present invention, the uniform adsorption is preferably carried out under water bath conditions, preferably, water bath at 80 ℃ for 2 hours.

In the present invention, the pyrolysis is carried out at 900 ℃ for 2 hours.

2. The nanoparticle biomimetic enzyme sensitive element prepared by the method is characterized in that transition metal atoms are fixed at a defect structure of a nano material by doping of hetero atoms.

3. The application of the nano-particle bionic enzyme sensitive element in preparing an electrochemical sensor for detecting bioactive molecules released by living cells.

Preferably, the bioactive molecule is a superoxide anion radical, dopamine, hydrogen peroxide or nitric oxide. Different substances can be detected at different points according to different metals. Metal gold, iron and the like can be used for detecting hydrogen peroxide, manganese, copper and the like can be used for detecting dopamine, and iron can also be used for detecting nitric oxide; iron, cobalt, nickel, manganese, copper, etc. can detect superoxide anions.

4. The surface of a working electrode of the electrochemical sensor is coated with the nano-particle bionic enzyme sensitive element.

Preferably, the coating is to disperse the nanoparticle biomimetic enzyme sensitive element in water according to the proportioning concentration of 0.5-5mg/mL to obtain an electrode modification solution, coat the electrode modification solution on an electrode, coat an adhesive after drying, and dry again.

5. The application of the electrochemical sensor in detecting the release of bioactive molecules from living cells.

The invention has the beneficial effects that: the preparation method of the nano-particle bionic enzyme sensitive element optimizes the transition metal atom types according to substances or molecules to be detected, thereby screening the optimal transition metal active center to obtain the high-sensitivity bionic enzyme material. Taking superoxide anion as an example, a high-sensitivity nanoparticle bionic enzyme sensitive element and a preparation method and application thereof are provided; according to the preparation method disclosed by the invention, in the preparation of the nanoparticle bionic enzyme sensitive element, the quality of the two-dimensional nano material, the transition metal source-containing acetate and the hexahydric heterocyclic compound is reasonably set, and the type of the transition metal is reasonably selected, so that an electrochemical sensor constructed by taking the finally prepared nanoparticle bionic enzyme sensitive element as a raw material has the advantages of extremely short response time, extremely high reaction sensitivity, lower detection limit and excellent selectivity. The carbon nanomaterial is modified by a catalyst, so that the carbon nanomaterial is more suitable for loading a larger amount of uniform metal nanoparticles, and the biomimetic material has more detection active sites. Compared with a sensor prepared from a traditional material, the sensor prepared from the nano-particle bionic enzyme material has 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. The material has outstanding performance and convenient material acquisition, and is beneficial to commercial application.

Additional advantages, objects, and features of the invention will be set forth in part in the description which follows and in part will become apparent to those having ordinary skill in the art upon examination of the following or may be learned from practice of the invention. The objectives and other advantages of the invention may be realized and attained by the means of the instrumentalities and combinations particularly pointed out hereinafter.

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 TEM image of the nanoparticle biomimetic enzyme sensor prepared in example 1;

FIG. 2 is a HRTEM image of the nanoparticle biomimetic enzyme sensor prepared in example 1;

FIG. 3 is an electron diffraction pattern of the nanoparticle biomimetic enzyme sensitive element prepared in example 1;

FIG. 4 is a graph representing the contact angle of the nanoparticle biomimetic enzyme sensor prepared in example 1;

FIG. 5 is a TEM image of the superoxide dismutase biomimetic material prepared in example 2;

FIG. 6 is a HRTEM image of the superoxide dismutase biomimetic material prepared in example 2;

FIG. 7 is an SEM photograph of the superoxide dismutase biomimetic material 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-0.2-0.8V₂ ^·-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, ab is the release of O from the sensor to DU145 cells at a fixed potential of 0.90V₂ ^·-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

In the synthesis of the nano-particle bionic enzyme material (Co-NSG), the specific steps are as follows:

(1) 100mg of Graphite Oxide (GO) was first dispersed in 30mL of deionized water (DIW) by sonication, and 2.5mg of C was added₄H₆CoO₄·4(H₂O); the mixture was stirred in a water bath at 80 ℃ for 2 hours and freeze dried for 24 hours to remove DIW. Subsequently, the product obtained is admixed with 500mg of C₃H₆N₆And 3 g.L^-1C₃H₇NO₂S were ground together to a homogeneous mixture and then pyrolysed for 2 hours at 900 ℃ under an argon atmosphere. And cooling to room temperature to obtain the bionic enzyme material (Co-NSG nano-particle bionic enzyme material for short).

Coating the working electrode of the bionic enzyme sensitive element:

dispersing the prepared Co-NSG nano-particle bionic enzyme material into water according to the proportioning concentration of 1mg/mL to obtain an electrode modification solution, coating 4.0 mu L of the electrode modification solution on a glassy carbon electrode, drying at 26 ℃ for 5h, coating 2.0 mu L of Nafion solution with the Nafion mass fraction of 0.1 wt%, and drying at 26 ℃ for 5h again to obtain the working electrode with the surface coated with the nano-particle bionic enzyme sensitive element.

Constructing a superoxide anion radical electrochemical sensor:

the prepared working electrode with the surface coated with the nano-particle bionic enzyme sensitive element, 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. 1 is a TEM image of the nanoparticle biomimetic enzyme sensor prepared in example 1, and it can be seen from fig. 1 that the heteroatom-doped graphene-based biomimetic enzyme loaded with metallic cobalt nanoparticles is synthesized, and the average diameter of the Co nanoparticles is measured to be about 60 nm.

Fig. 2 is an HRTEM of the nanoparticle biomimetic enzyme sensor prepared in example 1, and it can be seen from fig. 2 that cobalt nanoparticles are well dispersed on heteroatom-doped graphene.

Fig. 3 is an electron diffraction diagram of the nanoparticle biomimetic enzyme sensitive element prepared in example 1, and it can be known from fig. 3 that cobalt nanoparticles are well fixed on heteroatom-doped graphene.

Fig. 4 is a contact angle characterization diagram of the nanoparticle biomimetic enzyme sensor prepared in example 1, and it can be seen from fig. 4 that the contact angle of the biomimetic enzyme is 77.51 ° and is less than 90 °, which proves that the material has good cell application potential.

In the present embodiment, the graphene oxide may be replaced by, but not limited to, a reduced graphene oxide, a carbon nanotube, a carbon nanofiber, and other carbon nanomaterials; cobalt acetate tetrahydrate may be replaced by, but is not limited to, an acetate salt of a transition metal source such as cobalt, nickel, manganese, copper or iron; melamine can be replaced by, but not limited to, a six-membered heterocyclic compound such as pyridine, pyrazine, pyrimidine and pyridazine; amino acids in the half of the wing can be replaced by, but not limited to, biomass amino acids such as serine and alanine.

Example 2

The superoxide dismutase bionic enzyme material comprises the following specific steps:

mixing 100mg GO with 500mg C₃H₆N₆And 3 g.L^-1C₃H₇NO₂S were ground together to a homogeneous mixture and pyrolysed for 2 hours at 1000 ℃ under an argon atmosphere. Cooling to room temperature to obtain bionic enzyme material (NSG bionic enzyme material for short);

the working electrode coated with the NSG biomimetic enzyme material comprises:

dispersing the prepared NSG bionic enzyme material in water according to the proportioning 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. 5 is a TEM image of the superoxide dismutase biomimetic material prepared in example 2, and it can be seen from fig. 5 that the heteroatom-doped graphene biomimetic material is synthesized.

Fig. 6 is an HRTEM of the superoxide dismutase biomimetic material prepared in example 2, and it can be seen from fig. 6 that the heteroatom-doped graphene biomimetic material is amorphous.

Example 3

The superoxide dismutase bionic enzyme material comprises the following specific steps:

preparing 45mL of phosphate buffer solution, adding 0.1014g of manganese sulfate into 2mL of deionized water, uniformly mixing the two solutions, stirring and reacting for 8h at 26 ℃, centrifuging for 10min at the speed of 5000r/min, taking a precipitate, washing the precipitate with deionized water, and drying in vacuum for 12h at 80 ℃ to obtain the superoxide dismutase bionic enzyme material.

Coating the working electrode of the bionic enzyme sensitive element:

dispersing the prepared superoxide dismutase bionic material in water according to the proportioning concentration of 1.5mg/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 prepare a working electrode with the surface coated with the superoxide dismutase bionic material;

constructing a superoxide anion radical electrochemical sensor:

the prepared surfaceWorking electrode coated with superoxide dismutase bionic material, electrochemical workstation, counter electrode (platinum wire electrode) and 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. 7 is a FESEM image of the superoxide dismutase biomimetic material prepared in example 3, and it can be seen from fig. 7 that the sheet manganese phosphate biomimetic material is synthesized with micron-scale dimensions.

Example 4

Will contain 8. 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 clear from FIG. 8 that the content of L is 8. mu. mol^-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 8. 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 8. mu. mol. L^-1O₂ ^·-In PBS (5), in a volume of 8. mu. mol. L^-1O₂ ^·-In PBS, no significant oxidation peak was observed, indicating that the sensor did not have a control for O₂ ^·-Obvious electrochemical catalytic oxidation capability.

Example 6

Will contain 8. mu. mol. L^-1O₂ ^·-Phosphate Buffer Solution (PBS) of (1) was added to example 3Testing the sensor pair O in the electrolyte of the constructed sensor under the voltage range of 0.3-1.0V₂ ^·-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 8. mu. mol. L^-1O₂ ^·-In PBS (5), the oxidation peak current ratio does not contain O₂ ^·-The oxidation peak current in PBS of (1) is slightly increased, indicating that the sensor is paired with O₂ ^·-Has electrochemical catalytic oxidation capability, but has not good performance as the sensor in the example 1.

Example 7

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. 12 is a plot of steady state current versus O detected by 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) ═ 0.044C (nmol · L)^-1)+0.409(R²0.998), the sensitivity was 628.86 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 response of the sensor is then very rapid and a steady state current is established within 1.35 seconds.

Example 8

Adding solutions of different substances in sequenceTo the electrolyte of the sensor constructed in example 1, the response of the sensor to the chronoamperometric currents of different interfering components was tested, and 50nmol · L were added successively to the electrolyte of the sensor every 50s, respectively^-1O of (A) to (B)₂ ^·-、3μmol·L^-1Glucose, AA, UA, DA and 1. mu. mol. L of^-1H of (A) to (B)₂O₂The amperometric response curve of the sensor for selectivity test of different interfering components was obtained, and the result is shown in FIG. 14, from which it can be seen that 3. mu. mol. L^-1Glucose, AA, UA, DA and 1. mu. mol. L of^-1H of (A) to (B)₂O₂Will not detect 50 nmol.L for the sensor^-1O of (A) to (B)₂ ^·-Cause interference, accounting for the sensor pair O₂ ^·-Has good specificity.

Example 9

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 released by DU145 cells under zym stimulationO₂ ^·-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.