CN111398392A - Preparation method of electrochemical immunosensor for detecting dibutyl phthalate based on metal ion dependent DNA enzyme - Google Patents
Preparation method of electrochemical immunosensor for detecting dibutyl phthalate based on metal ion dependent DNA enzyme Download PDFInfo
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Abstract
The invention relates to a preparation method of an electrochemical immunosensor for detecting dibutyl phthalate based on metal ion dependent DNA enzyme, which comprises the following steps: preparing a functionalized two-dimensional carbon-based material/metal organic framework/thorny gold nanoparticle composite material by adopting a hydrothermal synthesis method, a seed mediated growth method and a physical adsorption method; synthesizing a signal molecule/gold nano particle/mesoporous metal oxide nanosphere/DNA-1 signal label by a chemical bonding method; the electrochemical sensor for detecting dibutyl phthalate is obtained by taking a functionalized two-dimensional carbon-based material/a metal organic framework/a thorny gold nanoparticle composite material/a metal ion-dependent DNA enzyme/gold electrode as a working electrode, a platinum wire electrode as a counter electrode and saturated silver chloride as a reference electrode; compared with the traditional electrochemical sensor, the electrochemical sensor has the advantages of high response speed, high sensitivity, good selectivity and high accuracy.
Description
Technical Field
The invention relates to a preparation method of an electrochemical immunosensor for detecting dibutyl phthalate based on metal ion-dependent DNase, in particular to a preparation method of a functionalized two-dimensional carbon-based material/metal organic framework/thorny gold nanoparticle composite material/metal ion-dependent DNase/gold electrode.
Background
With the continuous development of economy and the continuous improvement of living standard of people, food safety is becoming the focus of much attention due to the relation to the health, life safety and social economy of people. Phthalates are a class of organic compounds widely used as plasticizers in a variety of industrial and consumer products, such as plastic containers and food packaging. Dibutyl phthalate is a typical and commonly used plasticizer among all the phthalate esters. Since dibutyl phthalate is only physically bound to the polymer, it is easily released from the plastic material and into the surrounding environment. Thus, dibutyl phthalate can migrate into food during manufacture, use and disposal. Meanwhile, dibutyl phthalate can cause carcinogenesis and mutation when entering a human body, and can also influence the normal function of androgen, so that reproductive and developmental toxicity is caused. The maximum residual quantity of dibutyl phthalate in food and food additives specified by the Ministry of health of China is 0.3 mg.kg-1At present, common technologies for detecting dibutyl phthalate comprise high performance liquid chromatography (HP L C), capillary electrophoresis-mass spectrometry (CE-MS), enzyme-linked immunoassay (E L ISA) and the like, however, the methods need complicated and expensive equipment and professional operators, and are not favorable for rapid real-time detection.Therefore, it is necessary to establish a simple, rapid, accurate and sensitive method suitable for field detection to quantitatively detect the dibutyl phthalate. The electrochemical analysis technology has the advantages of high response speed, convenient use, easy combination with other testing and control technologies, easy miniaturization of instruments and the like, provides possibility for realizing analysis and detection, and is widely applied and researched in the field of food safety detection. Aiming at phthalate which is a small molecular target, direct determination cannot generate sensitive signal change, a competition method based on the combination point provided by an antibody which is fixed on a solid phase and is competitive by a detected antigen and a labeled antigen together is more suitable for detecting small molecules, simultaneously, the analysis process is relatively simple and rapid, but the sensitivity of an electrochemical immunosensor of a single recognition-signal response system is generally not high, high-sensitivity biosensors constructed by a plurality of researchers at present utilize the catalytic activities of proteases, exonucleases, endonucleases and other biological enzymes, but have the defects of easy pollution, sensitivity to environmental conditions, high cost and the like, DNase is deoxyribonucleic acid with the catalytic activity similar to that of the proteases, is more stable than the proteases, has low synthesis cost, and metal-dependent DNase has unique self-shearing activity, the catalytic reaction can be subjected to multi-circle circulation to initiate shearing reaction of a plurality of substrates, so that signal circulation amplification is realized, the sensitivity is obviously improved, and meanwhile, the fixation of bioactive components, the maintenance of activity and the stability are also the keys for constructing the efficient electrochemical immunosensor; in practical application, the electrochemical sensor based on a single recognition element antibody still has the problem that indexes such as sensitivity, stability and linear response are not ideal enough, and in order to further improve the response sensitivity of the sensor, a nano material is often widely applied to the construction of a biosensor as a signal amplification strategy, however, the simple carbon nano material has the defects of poor solubility, poor dispersibility and incapability of fixing biomolecules such as antibodies, so that the material performance cannot be fully exerted, and therefore, the designed and synthesized functionalized two-dimensional carbon-based material/metal organic framework/polycarpon nano particle composite material not only has the characteristics of high conductivity and good load capacity, but also has the characteristics of catalytic enhancementThe function of a signal becomes a key for constructing an electrode electrochemical immunosensor, at present, synthesis of a functionalized two-dimensional carbon-based material/metal organic framework/thorny gold nanoparticle composite material is not reported, the functionalized two-dimensional carbon-based material/metal organic framework/thorny gold nanoparticle composite material is used as an electrode modification material, meanwhile, signal molecules/gold nanoparticles/mesoporous metal oxide nanospheres/DNA-1 are used as signal labels, and dibutyl phthalate detection by using the electrochemical sensor is not reported.
Disclosure of Invention
The invention relates to a preparation method of an electrochemical immunosensor for detecting dibutyl phthalate based on metal ion dependent DNA enzyme.
A preparation method of an electrochemical immunosensor for detecting dibutyl phthalate based on metal ion dependent DNA enzyme comprises the following steps:
the preparation method of the functionalized two-dimensional carbon-based material/metal organic framework/thorny gold nanoparticle composite material comprises the steps of treating a two-dimensional carbon-based material by a hydrothermal synthesis method, weighing 10-20 mg of the two-dimensional carbon-based material, adding the two-dimensional carbon-based material into 25-50M L.0.01-0.02M buffer solution with the pH value of 7.0-9.0, carrying out ultrasonic treatment for 1-3 h, then weighing 5-10 mg of hydroxylamino functionalized material monomer, adding the hydroxylamino functionalized material monomer into a two-dimensional carbon-based material dispersion solution, degassing the solution by using high-purity neutral gas for 10-20 min, carrying out ultrasonic treatment on the solution for 10-20 min under the ice bath condition, reacting for 24-30 h at 50-70 ℃ to obtain a black suspension of the hydroxylamino functionalized two-dimensional carbon-based material, finally, centrifuging for 3-5 times at the rotation speed of 8000-10000 rpm to collect a product, drying to constant weight at 60-70 ℃, synthesizing an aminated metal organic framework by a solvothermal method, weighing 0.1-0.3 g of polyvinylpyrrolidone, dissolving in 4.5-13.5M L, weighing a hexahydrate constant weight of a triethylamine compound, drying at the rotation speed of 150-100 mg-10-70 ℃, carrying out ultrasonic treatment on a triethylamine and drying at the temperature of 30-20 ℃ to obtain a homogeneous amino-20 μ M solution, and drying at the mixture of the mixture at 25-20 μ M of the mixture, and drying at the mixture of the;preparing thorny gold nanoparticles by adopting a seed mediated growth method and a citrate reduction method, measuring 2-4M L.01-0.03M chloroauric acid solution, adding 6-8M L mass percent of 5-8% sodium citrate solution, measuring 500-800 mu L solution, adding the solution into 2-4M L aqueous solution, adjusting the pH value to 6.0-8.0 by using 6-8M L mass percent of 5-8% sodium citrate solution, adding 0.3-0.5M L.0.01-0.03M chloroauric acid solution into the mixture under slight shaking, changing the color of the solution from light pink to light purple, centrifuging at the rotating speed of 5000-8000 rpm for 7-10 min, collecting thorny gold nanoparticles, washing with water for 3-5 times, dispersing into the water, preparing a composite material by adopting a physical adsorption method, measuring 4-8 mg of the functionalized two-dimensional carbon-based material, dissolving the thorny gold nanoparticles into 2-4M L distilled water, adding 500-L mu M thorny gold nanoparticles and L-1000 mu M thorny gold nanoparticles, and preparing a composite material by adopting a physical adsorption method, weighing 4-8 mg of the functionalized two-L-1Placing the mixed solution on a shaking table, shaking for 12-14 h, and drying to obtain a functionalized two-dimensional carbon-based material/metal organic framework/thorny gold nanoparticle composite material;
preparing a signal molecule/gold nanoparticle/mesoporous metal oxide nanosphere/DNA-1 signal label, namely synthesizing mesoporous metal oxide nanospheres by adopting a one-step solvothermal method, adding 2-4 g of anhydrous metal chloride into 40-80M L ethylene glycol and dissolving, adding 6-10 g of anhydrous sodium acetate and 20-35M L amino functionalized solution into the solution and violently stirring for 30-60 min, pouring the mixed solution into a reaction kettle, heating to 180-250 ℃ for 8-12 h, cooling to 20-25 ℃, washing precipitates to be neutral by using a large amount of ultrapure water, finally drying the product at 50-70 ℃, synthesizing gold nanoparticles/mesoporous metal oxide nanospheres by adopting a chemical bonding method, weighing 5-10 mg of mesoporous metal oxide nanospheres into 5-10M L distilled water, adding L-15 mM chloroauric acid solution and 2-5M L mass percent of reducing agent into the solution under violent stirring, adding 5-10 mM L mM of mesoporous chloroauric acid solution and 2-5M of 1-3 mM of mesoporous metal oxide nanosphere into the solution, collecting a signal molecule/gold nanoparticle nanosphere after the signal molecule nanosphere is stirred for 10 h, and then adding the signal molecule nanosphere/DNA nanosphere, and obtaining a signal label, namely, after the signal label is stirred by adopting a shaking table, adding 5-10 mM DNA molecule/gold nanoparticle label, stirring, the gold nanoparticle label is stirred at a shaking table, the gold nanoparticle label is obtained by adopting a shaking table, the shaking table;
the preparation method comprises the following steps of (1) preparing the functionalized two-dimensional carbon-based material/metal organic framework/thorny gold nanoparticle composite material/metal ion dependent DNA enzyme/gold electrode: using 0.02-0.05 mu m Al2O3Polishing a gold electrode with the diameter of 1-4 mm by using powder, then sequentially carrying out ultrasonic treatment in ethanol and double distilled water, placing the polished gold electrode in a strong oxidation solution for 20-60 min, washing and drying the polished gold electrode by using distilled water, dropwise adding 2-5 mu L functionalized two-dimensional carbon-based material/metal organic framework/thorny gold nanoparticle composite material suspension onto the surface of the gold electrode, drying the gold electrode at 20-24 ℃, and sequentially carrying out 3-5 mu L0.05, 0.05-1 mu g.m L-1Adding dibutyl phthalate antibody solution and 5-10 mu L signal molecules/gold nanoparticles/mesoporous metal oxide nanospheres/DNA-1 dropwise onto an electrode, incubating for 1-2.5 h at 35-38 ℃, transferring 3-10 mu L0.1-0.5 mM of sealing agent dropwise onto the electrode, incubating for 30-50 min at 35-38 ℃, and firstly, in vitro, adding 200-1000 mu L5-10 mu g-m L-1Adding a DBP-BSA conjugate into 200-1000 mu L2-6 mu M amino modified metal ion dependent DNase DNA-2, adding 50-100 mu L0.1.1-0.25% of glutaraldehyde, reacting for 8-10 h at 35-38 ℃ to obtain a DBP-BSA-DNA-2 solution, adding a mixture of 3-5 mu L dibutyl phthalate standard solution and 3-5 mu L DBP-BSA-DNA-2, incubating for 30-60 min at 35-38 ℃, adding 3-10 mu L5-10 mu g M L-1The metal ion solution is reacted for 30-60 min at 35-38 ℃, and the functionalized two-dimensional carbon-based material/metal organic framework/thorny gold nanoparticle composite material/metal ion dependent DNase/gold electrode is obtained.
The electrochemical sensor is characterized in that a functionalized two-dimensional carbon-based material/metal organic framework/thorny gold nanoparticle composite material/metal ion-dependent DNA enzyme/gold electrode is used as a working electrode, a platinum wire electrode is used as a counter electrode, saturated silver chloride is used as a reference electrode, and the content of dibutyl phthalate in a sample is obtained through the change of signal molecule response signals, so that the electrochemical sensor for detecting dibutyl phthalate is obtained.
The two-dimensional carbon-based material is one or more of reduced graphene oxide, carbon nitride and porous carbon.
The metal organic framework is one or more of a nickel-based metal organic framework, a cobalt-based metal organic framework and a lanthanum-based metal organic framework.
The buffer solution is one or more of Tris-hydrochloric acid buffer solution, phosphate buffer solution and sodium acetate buffer solution.
The hydroxylamino functional material is one or more of polydopamine, polyether amine and amino polyethylene glycol.
The neutral gas is one or more of nitrogen, argon and helium.
The dispersant is one or more of ethylenediamine, N-dimethylformamide and isopropanol.
The hexahydrated nitric acid metal compound is one or more of hexahydrated nickel nitrate, hexahydrated cobalt nitrate and hexahydrated lanthanum nitrate.
The signal molecule is one or more of thionine, methylene blue and toluidine blue.
The anhydrous metal chloride is one or more of anhydrous ferrous chloride, anhydrous copper chloride and anhydrous magnesium chloride.
The amino functionalized solution is one or more of ethylenediamine, N-dimethylaniline and triethylamine.
The reducing agent is one or more of L-ascorbic acid, sodium borohydride and hydrazine hydrate.
The metal ion is Pb2+、Mg2+、Ag+One or more of (a).
The strong oxidation solution is one or more of a mixture of concentrated sulfuric acid and 30% hydrogen peroxide in a volume ratio of 7:3, a potassium dichromate solution and a nitric acid solution.
The blocking agent is one or more of 6-mercaptohexanol, bovine serum albumin and hexanethiol.
In the metal ion dependence-based DNase sensor, a functionalized two-dimensional carbon-based material/metal organic framework/thorny gold nanoparticle composite material is used as a substrate material, a signal molecule/gold nanoparticle/mesoporous metal oxide nanosphere/DNA-1 is used as a signal label, and compared with other electrochemical sensors for dibutyl phthalate detection, the prepared novel electrochemical sensor has the advantages of high response speed, high sensitivity, good selectivity and high accuracy.
Detailed Description
The invention is described below with reference to specific examples:
example 1
The method comprises the following specific steps:
(1) the method comprises the steps of treating reduced graphene oxide by a hydrothermal synthesis method, weighing 10 mg of reduced graphene oxide, adding the weighed 10 mg of reduced graphene oxide into 25 m L phosphate buffer solution with the value of 0.01 MpH being 7.0, carrying out ultrasonic treatment for 1 h, weighing 5 mg of polydopamine monomer, adding the polydopamine monomer into graphene oxide dispersion liquid, degassing the solution by high-purity nitrogen for 10 min, carrying out ultrasonic treatment on the solution for 10 min under an ice bath condition, reacting for 24 h at 50 ℃ to obtain black suspension of polydopamine functionalized reduced graphene oxide, centrifuging for 3 times at the rotation speed of 8000rpm, collecting a product, drying at the temperature of 60 ℃ to constant weight, synthesizing an aminated nickel-based metal organic framework by a solvothermal method, weighing 0.1 g of polyvinylpyrrolidone, dissolving in 4.5 m L N and N-dimethylformamide, weighing 100 mg of nickel nitrate hexahydrate, 30 mg of 2-amino terephthalic acid and 40 mu L triethylamine, adding the mixture into the solution, carrying out ultrasonic treatment for 20min, placing the solution into an autoclave, reacting for 18 h at the temperature of 105 ℃, cooling to 20 ℃, washing at the constant rotation speed, drying at the temperature of 8000 mg of the mixture of the triethylamine, drying at the temperature of gold particles, and drying at the temperature of gold particles by a centrifugal method, and drying at the temperature of gold reduction methodTaking 2M L0.01.01M chloroauric acid solution, adding 6M L mass percent of 5% sodium citrate solution, weighing 500 mu L of the solution, adding the solution into 2M L aqueous solution, adjusting the pH value to 6.0 by using 6M L mass percent of 5% sodium citrate solution, adding 0.3M L0.01.01M chloroauric acid solution into the mixture under slight shaking, changing the color of the solution from light pink to light purple, centrifuging at the rotating speed of 5000 rpm for 7 min to collect the obtained thorny gold nanoparticles, washing with water for 3-5 times, dispersing into water, preparing a composite material by adopting a physical adsorption method, weighing 4 mg of the functionalized two-dimensional carbon-based material, dissolving the functionalized two-dimensional carbon-based material in 2M L distilled water, adding 500 mu L thorny gold nanoparticle solution and 500 mu L2 mg.m L mg-1Placing the mixed solution on a shaking table, shaking for 12 h, and drying to obtain the functionalized reduced graphene oxide/nickel metal organic framework/thorny gold nanoparticle composite material;
(2) synthesizing mesoporous ferroferric oxide nanospheres by adopting a one-step solvothermal method, weighing 2 g of anhydrous ferrous chloride, adding the anhydrous ferrous chloride into 40M L ethylene glycol, dissolving, weighing 6 g of anhydrous sodium acetate and 20M L ethylene diamine, adding the anhydrous sodium acetate and the 20M L ethylene diamine into the solution, violently stirring for 30min, pouring the mixed solution into a reaction kettle, heating to 180 ℃ for 8 hours, cooling to 20 ℃, washing precipitates to be neutral by using a large amount of ultrapure water, finally drying at 50 ℃, synthesizing gold nanoparticles/mesoporous ferroferric oxide nanospheres by adopting a chemical bonding method, weighing 5 mg of the mesoporous ferroferric oxide nanospheres, dispersing the mesoporous ferroferric oxide nanospheres into 5M L distilled water, adding 10M L mM chloroauric acid solution and 2M L mass percent of 1% of L-ascorbic acid into the solution under violent stirring, centrifuging at the rotating speed of 8000rpm for 5min, collecting the product, washing for 3 times by using ethanol, synthesizing thionine/mesoporous ferroferric oxide nanospheres/DNA-1 signal labels by adopting the chemical bonding method, weighing 1M L.3 mM gold nanoparticles modified gold solution, adding the thionine nanospheres into 3M/mesoporous ferroferric oxide nanospheres under the solution, drying at the rotating speed of 365 ℃ under the thionine nanospheres, collecting the product of the mesoporous ferroferric nanospheres under the thionine nanospheres under the centrifugal nano nanospheres under the centrifugal speed of 365M and drying, and drying at the temperature2+In the substrate strand DNA-1 of the dependent DNase, the reaction was carried out for 8 hours with shaking on a shaker, and finally, 100. mu.M was addedL0.1.1 percent bovine serum albumin solution is subjected to oscillation reaction for 1 hour to obtain a thionine/gold nano-particle/mesoporous ferroferric oxide nanosphere/DNA-1 signal label;
(3) the functionalized reduced graphene oxide/nickel-based metal organic framework/thorny gold nanoparticle composite material/Pb2+Preparation of dependent dnase/gold electrodes: using 0.02 mu m Al2O3Polishing a gold electrode with the diameter of 3 mm by using powder, sequentially carrying out ultrasonic treatment in ethanol and double distilled water, placing the polished gold electrode into a mixture of concentrated sulfuric acid and 30% hydrogen peroxide with the volume ratio of 7:3 for 20min, washing and drying the polished gold electrode by using distilled water, dripping 2 mu L suspension of the functionalized reduced graphene oxide/nickel-based metal organic framework/multi-thorn gold nanoparticle composite material onto the surface of the gold electrode, drying the gold electrode at 20 ℃, and then adding 3 mu L0.05.05 mu g.m L-1Adding dibutyl phthalate antibody solution and 5 mu L thionine/gold nanoparticles/mesoporous ferroferric oxide nanospheres/DNA-1 dropwise onto an electrode, incubating for 1 h at 37 ℃, transferring 3 mu L0.1 mM of 6-mercaptohexanol dropwise onto the electrode, incubating for 30min at 37 ℃, and firstly incubating 200 mu L5 mu g m L in vitro-1DBP-BSA conjugate added to 200. mu. L2. mu.M amino-modified Pb2+Adding 50 mu L mass percent of 0.1% glutaraldehyde into the dependent DNase DNA-2, reacting for 8 hours at 37 ℃ to obtain a DBP-BSA-DNA-2 solution, adding a mixture of 3 mu L dibutyl phthalate standard solution and 3 mu L DBP-BSA-DNA-2, incubating for 30 minutes at 37 ℃, and adding 3 mu L5 mu g m L-1Pb of2+The solution reacts for 30min at 37 ℃ to obtain the functionalized reduced graphene oxide/nickel-based metal organic framework/thorny gold nanoparticle composite material/Pb2+Dependent dnase/gold electrodes.
(4) Functionalized reduced graphene oxide/nickel-based metal organic framework/thorny gold nanoparticle composite material/Pb2+The ion-dependent DNA enzyme/gold electrode is used as a working electrode, the platinum wire electrode is used as a counter electrode, the saturated silver chloride is used as a reference electrode, and the content of the dibutyl phthalate in the sample is obtained through the change of thionine response signals, so that the electrochemical sensor for detecting the dibutyl phthalate is obtained.
Prepared electrochemical sensorThe device has high accuracy and wide linear range (5 × 10)-6~5×10-12mol·L-1) And the lower detection limit is low (5 × 10)-13mol·L-1) The characteristics of (1). Meanwhile, the detection result of actual samples (such as white spirit and edible oil) shows that the prepared sensor has very good practical application value.
The above examples are intended to illustrate the invention, but not to limit it. Many modifications and variations of the present invention are possible in light of the above teachings. Within the scope of the appended claims, the present invention may be realized in other ways than those described above, and it is within the scope of the claims to select other reagent materials, adjust incubation time, etc.
Claims (4)
1. A preparation method of an electrochemical immunosensor for detecting dibutyl phthalate based on metal ion dependent DNA enzyme is characterized by comprising the following steps:
(1) the preparation method of the functionalized two-dimensional carbon-based material/metal organic framework/thorny gold nanoparticle composite material comprises the steps of treating a two-dimensional carbon-based material by a hydrothermal synthesis method, weighing 10-20 mg of the two-dimensional carbon-based material, adding the two-dimensional carbon-based material into 25-50M L.0.01-0.02M buffer solution with the pH value of 7.0-9.0, carrying out ultrasonic treatment for 1-3 h, then weighing 5-10 mg of hydroxylamino functionalized material monomer, adding the hydroxylamino functionalized material monomer into a two-dimensional carbon-based material dispersion solution, degassing the solution by using high-purity neutral gas for 10-20 min, carrying out ultrasonic treatment on the solution for 10-20 min under the ice bath condition, reacting for 24-30 h at 50-70 ℃ to obtain a black suspension of hydroxylamino functionalized two-dimensional carbon-based material, finally centrifuging for 3-5 times at the rotation speed of 8000-10000 rpm to collect a product, drying to constant weight at 60-70 ℃, synthesizing an aminated metal organic framework by a solvothermal method, weighing 0.1-0.3 g of polyvinylpyrrolidone 4.5-13.5M L mg of the aminated metal organic framework, weighing a hexahydrate metal compound, adding the metal organic framework into a nitric acid solution and a nitric acid mixture, carrying out ultrasonic treatment at the temperature of 50-20 μ M mixture, adding the mixture to a nitric acid,centrifuging at 8000-10000 rpm, washing with a dispersant and ethanol for 3-5 times to collect a product, finally drying at 60-70 ℃ to constant weight, preparing the spinodal gold nanoparticles by adopting a seed-mediated growth method and a citrate reduction method, measuring 2-4M L0.01-0.03M chloroauric acid solution, adding 6-8M L by mass percent of 5-8% of sodium citrate solution, measuring 500-800 mu L of the solution, adding the solution into 2-4M L by mass percent, adjusting the pH value to 6.0-8.0 by using 6-8M L by mass percent of 5-8% of sodium citrate solution, slightly shaking, adding 0.3-0.5M L by mass percent of 0.01-0.03M chloroauric acid solution into the mixture, changing the color of the solution from light pink to light purple, centrifuging at 5000-8000 rpm for 7-10 min to collect the spinodal gold nanoparticles, washing with water for 3-5 times, dispersing into L mg of a physical distillation method, and adding the spinodal gold nanoparticles into 358-1000 mg-1000 mu of a composite material, dissolving the spinodal gold nanoparticles in 5000-30-500 mu M358 mg-500 mu M carbon-2M carbon-35M solution, and weighing-1Placing the mixed solution on a shaking table, shaking for 12-14 h, and drying to obtain the functionalized two-dimensional carbon-based material/metal organic framework/thorny gold nanoparticle composite material;
(2) preparing a signal molecule/gold nanoparticle/mesoporous metal oxide nanosphere/DNA-1 signal label, namely synthesizing mesoporous metal oxide nanospheres by adopting a one-step solvothermal method, weighing 2-4 g of anhydrous metal chloride, adding the anhydrous metal chloride into 40-80M L ethylene glycol, dissolving, weighing 6-10 g of anhydrous sodium acetate and 20-35M L amino functionalized solution, adding the anhydrous sodium acetate and 20-35M L amino functionalized solution into the solution, violently stirring for 30-60 min, pouring the mixed solution into a reaction kettle, heating to 180-250 ℃ for 8-12 h, cooling to 20-25 ℃, washing precipitates to be neutral by using a large amount of ultrapure water, finally drying the product at 50-70 ℃, synthesizing gold nanoparticles/mesoporous metal oxide nanospheres by adopting a chemical bonding method, weighing 5-10 mg of mesoporous metal oxide nanospheres, dispersing the mesoporous metal oxide nanospheres in 5-10M L distilled water, adding L-15 mM chloroauric acid solution and 2-5M L mass percent reducing agent into the solution at 50-70 ℃ under violent stirring, centrifuging at a rotational speed of 5-10M of 1205 rpm, collecting a signal molecule/gold nanoparticle signal molecule/nanosphere after the gold nanosphere is stirred for 10M, and adding a signal molecule/gold nanoparticle signal molecule, and collecting a signal molecule/DNA molecule nano-10 mM signal label solution, and after the gold nanoparticle is stirred for 10 mM, and the gold nanoparticle label, and the gold nanoparticle is stirred under a reaction, and the gold nanoparticle label, and the gold nanoparticle is stirred for 10 mM signal label, and the gold nanoparticle is stirred for collecting a signal label, and the gold nanoparticle is stirred under the gold nanoparticle label, and the gold nanoparticle is obtained by adopting a signal label, and the gold nanoparticle is;
(3) preparing a functionalized two-dimensional carbon-based material/metal organic framework/thorny gold nanoparticle composite material/metal ion dependent DNA enzyme/gold electrode: using 0.02-0.05 mu m Al2O3Polishing a gold electrode with the diameter of 1-4 mm by using powder, then sequentially carrying out ultrasonic treatment in ethanol and double distilled water, placing the polished gold electrode in a strong oxidation solution for 20-60 min, washing and drying the polished gold electrode by using distilled water, dropwise adding 2-5 mu L functionalized two-dimensional carbon-based material/metal organic framework/thorny gold nanoparticle composite material suspension onto the surface of the gold electrode, drying the gold electrode at 20-24 ℃, and sequentially carrying out 3-5 mu L0.05, 0.05-1 mu g.m L-1Adding dibutyl phthalate antibody solution and 5-10 mu L signal molecules/gold nanoparticles/mesoporous metal oxide nanospheres/DNA-1 dropwise onto an electrode, incubating for 1-2.5 h at 35-38 ℃, transferring 3-10 mu L0.1-0.5 mM of sealing agent dropwise onto the electrode, incubating for 30-50 min at 35-38 ℃, and firstly, in vitro, adding 200-1000 mu L5-10 mu g-m L-1Adding a DBP-BSA conjugate into 200-1000 mu L2-6 mu M amino modified metal ion dependent DNase DNA-2, adding 50-100 mu L0.1.1-0.25% of glutaraldehyde, reacting for 8-10 h at 35-38 ℃ to obtain a DBP-BSA-DNA-2 solution, adding a mixture of 3-5 mu L dibutyl phthalate standard solution and 3-5 mu L DBP-BSA-DNA-2, incubating for 30-60 min at 35-38 ℃, adding 3-10 mu L5-10 mu g M L-1The metal ion solution is reacted for 30-60 min at 35-38 ℃ to obtain the functionalized two-dimensional carbon-based material/metal organic framework/thorny gold nanoparticle composite material/metal ion dependenceDnase/gold electrodes;
(4) the electrochemical sensor is characterized in that a functionalized two-dimensional carbon-based material/metal organic framework/thorny gold nanoparticle composite material/metal ion-dependent DNA enzyme/gold electrode is used as a working electrode, a platinum wire electrode is used as a counter electrode, saturated silver chloride is used as a reference electrode, and the content of dibutyl phthalate in a sample is obtained through the change of signal molecule response signals, so that the electrochemical sensor for detecting dibutyl phthalate is obtained.
2. The method for preparing the electrochemical immunosensor for detecting dibutyl phthalate based on the metal ion dependent dnase, according to claim 1, wherein in the step (1), the two-dimensional carbon-based material is one or more of reduced graphene oxide, carbon nitride, and porous carbon; the metal organic framework is one or more of a nickel-based metal organic framework, a cobalt-based metal organic framework and a lanthanum-based metal organic framework; the buffer solution is one or more of Tris-hydrochloric acid buffer solution, phosphate buffer solution and sodium acetate buffer solution; the hydroxylamino functional material is one or more of polydopamine, polyether amine and amino polyethylene glycol; the neutral gas is one or more of nitrogen, argon and helium; the dispersing agent is one or more of ethylenediamine, N-dimethylformamide and isopropanol; the hexahydrated nitric acid metal compound is one or more of hexahydrated nickel nitrate, hexahydrated cobalt nitrate and hexahydrated lanthanum nitrate.
3. The method for preparing an electrochemical immunosensor for detecting dibutyl phthalate based on metal ion dependent dnase according to claim 1, wherein in the step (2), the signal molecule is one or more of thionine, methylene blue and toluidine blue; the anhydrous metal chloride is one or more of anhydrous ferrous chloride, anhydrous copper chloride and anhydrous magnesium chloride; the amino functionalized solution is one or more of ethylenediamine, N-dimethylaniline and triethylamine; the above-mentionedThe reducing agent is one or more of L-ascorbic acid, sodium borohydride and hydrazine hydrate, and the metal ion is Pb2+、Mg2+、Ag+One or more of; the buffer solution is one or more of Tris-hydrochloric acid buffer solution, phosphate buffer solution and sodium acetate buffer solution.
4. The method for preparing an electrochemical immunosensor for detecting dibutyl phthalate based on metal ion dependent dnase according to claim 1, wherein in the step (3), the strong oxidation solution is one or more of a mixture of concentrated sulfuric acid and 30% hydrogen peroxide in a volume ratio of 7:3, a potassium dichromate solution, and a nitric acid solution; the blocking agent is one or more of 6-mercaptohexanol, bovine serum albumin and hexanethiol; the metal ion is Pb2+、Mg2+、Ag+One or more of (a).
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