CN109115751B - TiO against vomitoxin2Construction of-B integrated hydrogen peroxide-free electrochemiluminescence sensor - Google Patents

TiO against vomitoxin2Construction of-B integrated hydrogen peroxide-free electrochemiluminescence sensor Download PDF

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CN109115751B
CN109115751B CN201810929822.6A CN201810929822A CN109115751B CN 109115751 B CN109115751 B CN 109115751B CN 201810929822 A CN201810929822 A CN 201810929822A CN 109115751 B CN109115751 B CN 109115751B
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戴宏
任卉竹
房丹丹
陈妍洁
衣欢
郑祥钦
彭亦如
颜建英
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Fujian Normal University
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Abstract

The invention discloses TiO aiming at vomitoxin2Construction of-B Integrated Hydrogen peroxide-free electrochemiluminescence sensor based on TiO2the-B is a high-efficiency integrated electrochemiluminescence probe prepared by a nano carrier to realize high-sensitivity detection on vomitoxin, and is characterized in that the TiO2the-B nano carrier has a large specific surface area, can load a large amount of sensitizer fluorocoumarin silicon phthalocyanine and electrochemiluminescence reagent luminol, and successfully prepares an integrated electrochemiluminescence probe. The probe can obtain high-intensity electrochemiluminescence signals in a hydrogen peroxide-free solution. In addition, mesoporous nano MnO having excellent conductivity and biocompatibility2The composite material is used as a sensor substrate, and finally, the hydrogen peroxide-free electrochemiluminescence immunosensor with high sensitivity, good stability and low detection limit is successfully constructed, so that the high-sensitivity and high-stability detection of vomitoxin is realized.

Description

TiO against vomitoxin2Construction of-B integrated hydrogen peroxide-free electrochemiluminescence sensor
Technical Field
The invention belongs to the technical field of novel functional materials and biosensing detection, and particularly relates to TiO specific to vomitoxin2Construction and application of-B integrated hydrogen peroxide-free electrochemiluminescence sensor.
Background
The immunosensor has the advantages of high sensitivity, good selectivity, simple operation and easy miniaturizationThe kit has the advantages of continuous and rapid detection and analysis, and the like, and is widely applied to the fields of clinical diagnosis, drug analysis, environmental monitoring and the like. In the invention, TiO is used2the-B is a carrier, the large specific surface area of the-B can effectively load a large amount of reaction accelerators and luminescent reagents, and finally, a high-efficiency integrated electrochemiluminescence probe is successfully prepared, and the high-sensitivity detection on vomitoxin can be realized under the condition of no hydrogen peroxide.
TiO2the-B nano material has the advantages of large specific surface area, good biocompatibility, strong light scattering and light capturing capability and the like, and can be applied to the preparation of electrochemiluminescence immunosensors. In the invention, TiO is used2the-B nano material is used as a carrier to prepare a high-quality integrated electrochemiluminescence probe, a high-strength electrochemiluminescence signal can be obtained without adding a co-reaction reagent hydrogen peroxide, and the high-sensitivity and high-stability detection of vomitoxin (DON) is finally realized by combining a classical sandwich type immunosensing method. The superiority is embodied in TiO2the-B nano material has large specific surface area, can bear a large amount of sensitizer fluorocoumarin silicon phthalocyanine (F-couSiPcs) and luminescent reagent luminol (luminol), and finally constructs TiO aiming at vomitoxin2-B an integrated hydrogen peroxide-free electrochemiluminescence sensor.
Vomitoxin (DON), one of the members of the mycotoxin family, is a secondary metabolite produced by certain fungi, originally found in wheat, barley and corn, and is capable of disrupting neural functions, including neuroendocrine signaling, growth hormone signaling and central neuronal networks, which, as a protein synthesis inhibitor, affects the synthesis of intestinal epithelial proteins and barrier integrity. The invention takes DON as a detection target object, gives full play to the excellence of the prepared integrated electrochemiluminescence probe, and realizes trace detection of DON by utilizing the specific combination between antigen and antibody. More significantly, the resulting H-free202The integrated electrochemiluminescence immune sensing platform has the advantages of strong specificity, high sensitivity, good stability, low detection limit and the like, and can be used for vomitingThe detection of the toxin (DON) also has very important application value and practical significance in the aspect of clinical application.
Disclosure of Invention
One of the objects of the present invention is TiO2the-B nano material is used as an effective carrier to prepare an integrated electrochemiluminescence probe, so that the hydrogen peroxide-free electrochemiluminescence immunosensor with simplicity, rapidness, good stability and high sensitivity is constructed.
The second purpose of the invention is to apply the electrochemiluminescence immunosensor to the high-sensitivity detection of vomitoxin.
In order to realize the purpose of the invention, the invention adopts the following technical scheme:
1. TiO for vomitoxin2The construction method of the integrated hydrogen peroxide-free electrochemiluminescence sensor is characterized by comprising the following steps of:
(1) firstly, mechanically polishing and polishing a Glassy Carbon Electrode (GCE) on chamois leather paved with aluminum oxide powder, washing residual powder on the surface by using secondary water, then moving the chamois leather into an ultrasonic water bath for cleaning until the chamois leather is cleaned, and finally, thoroughly washing the chamois leather by using ethanol, dilute acid and water in sequence;
(2) taking 1 mL of mesoporous nano manganese dioxide (mp MnO) with the concentration of 3 mg/mL2) Mixing with 1 mL of 5 mg/mL Hyaluronic Acid (HA) under ultrasonic action, mechanically oscillating the obtained homogeneous solution at room temperature for 4 h, centrifuging, washing, and redispersing to obtain HA @ mp MnO2A complex solution; 5 mu L of HA @ mp MnO with the concentration of 3 mg/ml is dripped2The compound suspension is placed on the surface of a clean glassy carbon electrode, dried under an infrared lamp and cooled to room temperature to prepare HA @ mp MnO2Modifying the glassy carbon electrode;
(3) at 0.1 mol/L of lithium perchlorate (LiClO)4) Dissolving 0.01 mol/L3, 4-Ethylenedioxythiophene (EDOT) monomer in acetonitrile (acetonitrile) solution as solvent, and continuously scanning 6 times on the electrode prepared in the step (2) by using cyclic voltammetry under a potential window of-0.5 to 1.0V, and finally obtaining the electrode with the concentration of PEDOT @ PEDHA @ mp MnO2Modifying the glassy carbon electrode;
(4) taking 5 μ L of concentrateDropwise adding a mixture of (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride) (EDC) and N-hydroxysuccinimide (NHS) with the degree ratio of 2:1 onto the modified electrode prepared in the step (3) to react at room temperature for 40 min, drying under an infrared lamp, cooling to room temperature, and dropwise adding 5 mu L of a 50-concentration solutionug/mL vomitoxin antibody (Ab)1) The solution was incubated at room temperature for 50 min, followed by washing away the physisorbed Ab with deionized water1To prepare Ab1@PEDOT@HA@mp MnO2Modifying the glassy carbon electrode;
(5) taking 5 mg/mL TiO with the volume ratio of 2:12Mixing the solution B with 1.0 wt.% BSA at room temperature, shaking for 5 h, washing, centrifuging, and redispersing to obtain TiO2-B @ BSA complex solution; then, 100. mu.L of 1.0X 10 was added to the above mixed solution-6 The preparation method comprises the steps of enabling mol/L of fluoro coumarin silicon phthalocyanine (F-couSiPcs) to be gently shaken at room temperature for 1 h, and obtaining TiO after centrifugation, washing and redispersion2-B @ BSA @ F-couSiPcs complex solution; then, 100. mu.L of 1.0X 10 was added to the above solution in order-2mixing and oscillating 50 mu L of 5 wt.% glutaraldehyde solution in mol/L luminol at room temperature for 4 h, centrifuging, washing and redispersing to obtain TiO2-B @ BSA @ F-couSiPcs @ luminol complex solution, and finally 100. mu.L of 50uG/mL vomitoxin IgG antibody (Ab)2) Shaking the solution at room temperature for 50 min, centrifuging, washing, and redispersing to obtain integrated electrochemiluminescence probe (TiO)2-B@BSA@ F-couSiPcs@luminol@Ab2) The complex solution is stored in a refrigerator at 4 ℃ for standby;
(6) immersing the modified electrode obtained in the step (4) into vomitoxin (DON) standard solutions with different concentrations, incubating the electrode at room temperature for 40 mim, and washing the surface of the electrode with deionized water to prepare DON/Ab1@PEDOT@HA@mp MnO2Modifying a glassy carbon electrode, and then taking 5 mu L of the integrated electrochemiluminescence probe (TiO) prepared in the step (5)2-B@BSA@ F-couSiPcs@luminol@Ab2) Complex solution was added dropwise to DON/Ab1@PEDOT@HA@mp MnO2Incubating on the modified glassy carbon electrode for 50 min at room temperature, washing the surface of the electrode with deionized water to obtain TiO2-B@BSA@ F-couSiPcs@luminol@Ab2/DON/Ab1@PEDOT@HA@mp MnO2And modifying the glassy carbon electrode.
1. The mesoporous nano manganese dioxide (mp MnO)2) Prepared by the following method: based on a cationic surfactant soft template method, 2 g of 1-hexadecyl trimethyl ammonium bromide (CTAB) is dissolved in 200 ml of absolute ethyl alcohol, and then KMnO is sequentially added into the mixed solution in a dropwise manner under the condition of strong stirring4And manganese acetate Mn (CH)3COO)2Keeping the mixture for several hours under the stirring condition, centrifuging and washing the obtained dark brown precipitate for several times, and finally drying the precipitate for 8 hours at the temperature of 80 ℃ to obtain the mesoporous nano manganese dioxide (mp MnO)2）。
2. The above TiO compound2-B nanomaterial is prepared by the following method: 4 g of anatase TiO2Uniformly dispersing in 60M KOH aqueous solution, stirring at room temperature for 10 min, transferring the obtained suspension into a 100 ml stainless steel autoclave with a tetrafluoroethylene lining, keeping the temperature at 170 ℃ for 72 min, cooling to room temperature, washing the obtained precipitate with diluted HAc solution until the pH value reaches 7.0, centrifuging, and drying at 60 ℃ for 12 h to obtain a final product; then dispersing 600 mg of precursor titanate nanowire into 100 ml of 16M HAc solution, transferring the mixed solution into a 200 ml tetrafluoroethylene-lined stainless steel autoclave, heating for 24 h at 180 ℃, centrifuging, respectively thoroughly washing with distilled water and ethanol, and drying at 60 ℃ overnight to obtain the final target product TiO2–B。
3. The above-mentioned integrated electrochemiluminescence probe (TiO)2-B@BSA@ F-couSiPcs@luminol@Ab2) The complex solution was prepared by the following method: 1) 5 mg/mL TiO 2:1 by volume2Mixing the solution B with 1.0 wt.% BSA at room temperature, shaking for 5 h, washing, centrifuging, and redispersing to obtain TiO2-B @ BSA complex solution; 2) to the above mixed solution was added 100. mu.L of 1.0X 10-6 The preparation method comprises the steps of enabling mol/L of fluoro coumarin silicon phthalocyanine (F-couSiPcs) to be gently shaken at room temperature for 1 h, and obtaining TiO after centrifugation, washing and redispersion2-B @ BSA @ F-couSiPcs complex solution; 3) in turn to the above-mentioned complexTo the solution was added 100. mu.L of 1.0X 10-2mixing and oscillating 50 mu L of 5 wt.% glutaraldehyde solution in mol/L luminol at room temperature for 4 h, centrifuging, washing and redispersing to obtain TiO2-B @ BSA @ F-couSiPcs @ luminol complex solution, and finally 100. mu.L of 50uG/mL vomitoxin IgG antibody (Ab)2) Shaking the solution at room temperature for 50 min, centrifuging, washing, and dispersing in deionized water to obtain TiO2-B@BSA@ F-couSiPcs@luminol@Ab2The complex solution was stored in a 4 ℃ freezer for use.
The fluorocoumarin silicon phthalocyanine (F-couSiPcs) is synthesized by the following steps: 1) dispersing 8 g of 1, 4-dihydroxybenzene in 28 ml of concentrated sulfuric acid, continuously stirring at 0 ℃ for 30 min, then sequentially adding 20 g of ethyl trifluoroacetate and 40 ml of concentrated sulfuric acid into the mixed solution, stirring at room temperature for 12 h, transferring the crude product into ice water for precipitating more products, filtering and washing by using ultrapure water for multiple times, and drying in vacuum at 60 ℃ to obtain light yellow crystal 6-hydroxy-4-trifluoromethyl-1, 2-benzopyran (fluoro-coumarin); 2) respectively adding 0.46g of the prepared fluoro-coumarin, 0.42 g of silicon dichloride, 100 ml of toluene and 3.0 ml of pyridine into a 200 ml round bottom flask, refluxing the mixture, keeping the mixture for 48 hours under magnetic stirring at 110 ℃, cooling to room temperature, and further purifying by silica gel chromatography to obtain a crude product; 3) drying the obtained product under a rotary evaporator to obtain the fluoro coumarin silicon phthalocyanine (F-couSiPcs).
4. Detection of vomitoxin (DON):
(1) measuring by using an electrochemical workstation and adopting a three-electrode system, taking the prepared sandwich type immunosensor as a working electrode, Ag/AgCl as a reference electrode and a platinum wire electrode as an auxiliary electrode, and testing in 0.1 mol/mL PBS (phosphate buffer solution) with pH of 8.0;
(2) detecting vomitoxin (DON) standard solutions with different concentrations by adopting a potential range of-0.5-1V and a potential window with a scanning speed of 0.05V/s and an electrochemiluminescence device photomultiplier 800V, acquiring ECL signal intensity of 0.5V through the electrochemiluminescence device, and drawing a working curve according to the relation between the ECL signal intensity and the vomitoxin (DON) standard solution concentration;
(3) the sample solution to be detected replaces a vomitoxin (DON) standard solution for detection, and the detection result can be obtained through a working curve.
The invention has the following remarkable advantages:
(1) the double antibody sandwich method, a commonly used analysis method, has been gradually applied in the fields of fluorescence, electrochemiluminescence, photoelectricity, electrochemistry, etc. because the amounts of the solid phase antibody and the labeled antibody in the reaction system are excessive relative to the antigen to be detected, and thus the formation amount of the complex is proportional to the content of the antigen to be detected. Compared with other analysis technologies, the electrochemiluminescence has the advantages of high sensitivity, simplicity, quick reaction and the like, and the combination of the electrochemiluminescence and a sandwich type analysis technology provides a wider application prospect for the development of biosensors.
(2) TiO with large specific surface area and higher porosity2the-B nano material serving as a biosensor probe platform can bear a large amount of sensitizer and luminescent reagent, so that an efficient integrated electrochemiluminescence probe is successfully prepared, and the electrochemiluminescence efficiency is greatly improved.
(3) Hyaluronic Acid (HA) HAs a large number of hydrophilic groups, so that the stability of the biosensor is improved, and an vomitoxin antibody (Ab) can be bound through covalent interaction1) In addition, poly (3, 4-ethylenedioxythiophene) (PEDOT) formed by electropolymerization has good conductivity, and an electrochemiluminescence signal is further increased.
(4) The invention combines the advantages of the integrated probe and the sensing substrate, realizes high-sensitivity detection on the target object under the condition of no hydrogen peroxide solution, and avoids the harm to the body of an operator caused by using hydrogen peroxide and the denaturation of antigens, antibodies, the target object and the like caused by the hydrogen peroxide.
Drawings
FIG. 1 is 5 comparative views of mesoporous nano-manganese dioxide, and A in FIG. 1 is mesoporous nano-manganese dioxide (mp MnO)2) Transmission Electron Micrograph (TEM) of (A), B in FIG. 1 is mesoporous nano manganese dioxide (mp MnO)2) High power scanning electron microscopy (HRREM), C, D, E in FIG. 1 being TiO respectively2-Transmission Electron Micrograph (TEM), Selected Area Electron Diffraction (SAED), X-ray diffraction (XRD) pattern of B.
FIG. 2 is a graph of the electrochemiluminescence response signal of an immunosensing electrode plotted linearly with vomitoxin (DON) standard solution concentration.
Detailed Description
The present invention is further illustrated by the following examples, but the scope of the present invention is not limited to the following examples.
Example 1
TiO for vomitoxin2-B construction process of integrated hydrogen peroxide-free electrochemiluminescence sensor:
(1) firstly, mechanically polishing and polishing a Glassy Carbon Electrode (GCE) on chamois leather paved with aluminum oxide powder, washing residual powder on the surface by using secondary water, then moving the chamois leather into an ultrasonic water bath for cleaning until the chamois leather is cleaned, and finally, thoroughly washing the chamois leather by using ethanol, dilute acid and water in sequence;
(2) taking 1 mL of mesoporous nano manganese dioxide (mp MnO) with the concentration of 3 mg/mL2) Mixing with 1 mL of 5 mg/mL Hyaluronic Acid (HA) under ultrasonic action, mechanically oscillating the obtained homogeneous solution at room temperature for 4 h, centrifuging, washing, and redispersing to obtain HA @ mp MnO2A complex solution; 5 mu L of HA @ mp MnO with the concentration of 3 mg/ml is dripped2The compound suspension is placed on the surface of a clean glassy carbon electrode, dried under an infrared lamp and cooled to room temperature to prepare HA @ mp MnO2Modifying the glassy carbon electrode;
(3) at 0.1 mol/L of lithium perchlorate (LiClO)4) Dissolving 0.01 mol/L3, 4-Ethylenedioxythiophene (EDOT) monomer in acetonitrile (acetonitrile) solution as solvent, and continuously scanning 6 times on the electrode prepared in the step (2) by using cyclic voltammetry under a potential window of-0.5 to 1.0V, and finally obtaining the electrode with the concentration of PEDOT @ PEDHA @ mp MnO2Modifying the glassy carbon electrode;
(4) 5 μ L of a 2:1 mixture of (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride) (EDC) and N-hydroxysuccinimide (NHS) was added dropwise to stepReacting the modified electrode prepared in the step (3) at room temperature for 40 min, drying under an infrared lamp, cooling to room temperature, and dropwise adding 5 mu L of 50-concentration solutionug/mL vomitoxin antibody (Ab)1) The solution was incubated at room temperature for 50 min, followed by washing away the physisorbed Ab with deionized water1To prepare Ab1@PEDOT@HA@mp MnO2Modifying the glassy carbon electrode;
(5) taking 5 mg/mL TiO with the volume ratio of 2:12Mixing the solution B with 1.0 wt.% BSA at room temperature, shaking for 5 h, washing, centrifuging, and redispersing to obtain TiO2-B @ BSA complex solution; then, 100. mu.L of 1.0X 10 was added to the above mixed solution-6 The preparation method comprises the steps of enabling mol/L of fluoro coumarin silicon phthalocyanine (F-couSiPcs) to be gently shaken at room temperature for 1 h, and obtaining TiO after centrifugation, washing and redispersion2-B @ BSA @ F-couSiPcs complex solution; then, 100. mu.L of 1.0X 10 was added to the above solution in order-2mixing and oscillating 50 mu L of 5 wt.% glutaraldehyde solution in mol/L luminol at room temperature for 4 h, centrifuging, washing and redispersing to obtain TiO2-B @ BSA @ F-couSiPcs @ luminol complex solution, and finally 100. mu.L of 50uG/mL vomitoxin IgG antibody (Ab)2) Shaking the solution at room temperature for 50 min, centrifuging, washing, and redispersing to obtain integrated electrochemiluminescence probe (TiO)2-B@BSA@ F-couSiPcs@luminol@Ab2) The complex solution is stored in a refrigerator at 4 ℃ for standby;
(6) immersing the modified electrode obtained in the step (4) into vomitoxin (DON) standard solutions with different concentrations, incubating the electrode at room temperature for 40 mim, and washing the surface of the electrode with deionized water to prepare DON/Ab1@PEDOT@HA@mp MnO2Modifying a glassy carbon electrode, and then taking 5 mu L of the integrated electrochemiluminescence probe (TiO) prepared in the step (5)2-B@BSA@ F-couSiPcs@luminol@Ab2) Complex solution was added dropwise to DON/Ab1@PEDOT@HA@mp MnO2Incubating on the modified glassy carbon electrode for 50 min at room temperature, washing the surface of the electrode with deionized water to obtain TiO2-B@BSA@ F-couSiPcs@luminol@Ab2/DON/Ab1@PEDOT@HA@mp MnO2And modifying the glassy carbon electrode.
Example 2
Mesoporous nano manganese dioxide (mp MnO) used in example 12) The preparation of (1): based on a cationic surfactant soft template method, 2 g of 1-hexadecyl trimethyl ammonium bromide (CTAB) is dissolved in 200 ml of absolute ethyl alcohol, and then KMnO is sequentially added into the mixed solution in a dropwise manner under the condition of strong stirring4And manganese acetate Mn (CH)3COO)2Keeping the mixture for several hours under the stirring condition, centrifuging and washing the dark brown precipitate for several times, and finally drying the precipitate for 8 hours at the temperature of 80 ℃ to obtain the mesoporous nano manganese dioxide (mp MnO)2) In FIG. 1, A is mesoporous nano manganese dioxide (mp MnO)2) Transmission Electron Micrograph (TEM) of (A), B is mesoporous nano manganese dioxide (mp MnO)2) High power scanning electron microscopy (HRREM), C, D, E respectively being TiO2-Transmission Electron Micrograph (TEM), Selected Area Electron Diffraction (SAED), X-ray diffraction (XRD) pattern of B.
Example 3
TiO used in example 12-B preparation of nanomaterials: 4 g of anatase TiO2Uniformly dispersing in 60M KOH aqueous solution, stirring at room temperature for 10 min, transferring the obtained suspension into a 100 ml stainless steel autoclave with a tetrafluoroethylene lining, keeping the temperature at 170 ℃ for 72 min, cooling to room temperature, washing the obtained precipitate with diluted HAc solution until the pH value reaches 7.0, centrifuging, and drying at 60 ℃ for 12 h to obtain a final product; then dispersing 600 mg of precursor titanate nanowire into 100 ml of 16M HAc solution, transferring the mixed solution into a 200 ml tetrafluoroethylene-lined stainless steel autoclave, heating for 24 h at 180 ℃, centrifuging, respectively thoroughly washing with distilled water and ethanol, and drying at 60 ℃ overnight to obtain the final target product TiO2–B。
Example 4
Integrated electrochemiluminescence Probe (TiO) used in example 1 above2-B@BSA@ F-couSiPcs@luminol@Ab2) Preparation of the complex: 1) 5 mg/mL TiO 2:1 by volume2Mixing the solution B with 1.0 wt.% BSA at room temperature, shaking for 5 h, washing, centrifuging, and redispersing to obtain TiO2-B @ BSA complex solution; 2) to the above mixed solution was added 100. mu.L of 1.0X 10-6 The preparation method comprises the steps of enabling mol/L of fluoro coumarin silicon phthalocyanine (F-couSiPcs) to be gently shaken at room temperature for 1 h, and obtaining TiO after centrifugation, washing and redispersion2-B @ BSA @ F-couSiPcs complex solution; 3) to the above complex solution was added 100. mu.L of 1.0X 10 in sequence-2mixing and oscillating 50 mu L of 5 wt.% glutaraldehyde solution in mol/L luminol at room temperature for 4 h, centrifuging, washing and redispersing to obtain TiO2-B @ BSA @ F-couSiPcs @ luminol complex solution, and finally 100. mu.L of 50uG/mL vomitoxin IgG antibody (Ab)2) Shaking the solution at room temperature for 50 min, centrifuging, washing, and dispersing in deionized water to obtain TiO2-B@BSA@ F-couSiPcs@luminol@Ab2The complex solution was stored in a 4 ℃ freezer for use.
Example 5
Vomitoxin antibody (Ab) according to the present invention1) And vomitoxin IgG antibody (Ab)2) The fluorocoumarin silicon phthalocyanine (F-couSiPcs) as the sensitizer used in example 1, purchased from the tin-free biosensing technology company, Jiangsu, China, was synthesized by the following steps: firstly, 8 g of 1, 4-dihydroxybenzene is dispersed in 28 ml of concentrated sulfuric acid, the mixture is continuously stirred for 30 min at the temperature of 0 ℃, then 20 g of ethyl trifluoroacetate and 40 ml of concentrated sulfuric acid are sequentially added into the mixed solution, the mixture is stirred for 12 h at the room temperature, in order to precipitate more products, the crude products are transferred into ice water, and are filtered and washed by ultrapure water for multiple times, and after vacuum drying at the temperature of 60 ℃, light yellow crystals of 6-hydroxy-4-trifluoromethyl-1, 2-benzopyran (fluorine-coumarin) are obtained. Subsequently, 0.46g of the above-prepared fluoro-coumarin, 0.42 g of silicon dichloride, 100 ml of toluene and 3.0 ml of pyridine were added to a 200 ml round bottomed flask, respectively, and then the mixture was refluxed and kept under magnetic stirring at 110 ℃ for 48 hours, cooled to room temperature, and then further purified by silica gel chromatography to obtain a crude product. Finally, the dark blue product F-CouSiPc is successfully prepared after drying under a rotary evaporator.
Example 6
Detection of vomitoxin (DON):
(1) an electrochemical workstation is used for determination by adopting a three-electrode system, the sandwich type immunosensor prepared in example 1 is used as a working electrode, Ag/AgCl is used as a reference electrode, a platinum wire electrode is used as an auxiliary electrode, and the test is carried out in 0.1 mol/mL PBS (phosphate buffer solution) with pH of 8.0;
(2) detecting vomitoxin (DON) standard solutions with different concentrations by adopting a potential range of-0.5-1V and a potential window with a scanning speed of 0.05V/s and an electrochemiluminescence device photomultiplier 800V, acquiring ECL signal intensity of 0.5V by the electrochemiluminescence device, and drawing a working curve according to a graph shown in figure 2 by the relation between the ECL signal intensity and the vomitoxin (DON) standard solution concentration;
(3) the sample solution to be detected replaces a vomitoxin (DON) standard solution for detection, and the detection result can be obtained through a working curve.

Claims (6)

1. TiO for vomitoxin2The construction method of the integrated hydrogen peroxide-free electrochemiluminescence sensor is characterized by comprising the following steps of:
(1) firstly, mechanically polishing and polishing a Glassy Carbon Electrode (GCE) on chamois leather paved with aluminum oxide powder, washing residual powder on the surface by using secondary water, then moving the chamois leather into an ultrasonic water bath for cleaning until the chamois leather is cleaned, and finally, thoroughly washing the chamois leather by using ethanol, dilute acid and water in sequence;
(2) taking 1 mL of mesoporous nano manganese dioxide (mp MnO) with the concentration of 3 mg/mL2) Mixing with 1 mL of 5 mg/mL Hyaluronic Acid (HA) under ultrasonic action, mechanically oscillating the obtained homogeneous solution at room temperature for 4 h, centrifuging, washing, and redispersing to obtain HA @ mp MnO2A complex solution; 5 mu L of HA @ mp MnO with the concentration of 3 mg/ml is dripped2The compound suspension is placed on the surface of a clean glassy carbon electrode, dried under an infrared lamp and cooled to room temperature to prepare HA @ mp MnO2Modifying the glassy carbon electrode;
(3) at 0.1 mol/L of lithium perchlorate (LiClO)4) Dissolving 0.01 mol/L3, 4-Ethylenedioxythiophene (EDOT) monomer in acetonitrile (acetonitrile) solution as solvent, and performing cyclic voltage application under a potential window of-0.5-1.0VThe Ann method scans the electrode prepared in step (2) for 6 times in succession, and finally obtains the electrode as PEDOT @ HA @ mp MnO2Modifying the glassy carbon electrode;
(4) dripping 5 μ L of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) mixture with the concentration ratio of 2:1 onto the modified electrode prepared in the step (3), reacting at room temperature for 40 min, drying under an infrared lamp, cooling to room temperature, dripping 5 μ L of 50-concentration 50ug/mL vomitoxin antibody (Ab)1) The solution was incubated at room temperature for 50 min, followed by washing away the physisorbed vomitoxin antibody (Ab) with deionized water1) To prepare Ab1@PEDOT@HA@mp MnO2Modifying the glassy carbon electrode;
(5) taking 5 mg/mL TiO with the volume ratio of 2:12Mixing the solution B with 1.0 wt.% BSA at room temperature, shaking for 5 h, washing, centrifuging, and redispersing to obtain TiO2-B @ BSA complex solution; then, 100. mu.L of 1.0X 10 was added to the above mixed solution-6 The preparation method comprises the steps of enabling mol/L of fluoro coumarin silicon phthalocyanine (F-couSiPcs) to be gently shaken at room temperature for 1 h, and obtaining TiO after centrifugation, washing and redispersion2-B @ BSA @ F-couSiPcs complex solution; then, 100. mu.L of 1.0X 10 was added to the above solution in order-2mixing and oscillating 50 mu L of 5 wt.% glutaraldehyde solution in mol/L luminol at room temperature for 4 h, centrifuging, washing and redispersing to obtain TiO2-B @ BSA @ F-couSiPcs @ luminol complex solution, and finally 100. mu.L of 50uG/mL vomitoxin IgG antibody (Ab)2) The solution is vibrated for 50 min at room temperature, and the integrated electrochemiluminescence probe TiO is prepared by centrifugation, washing and redispersion2-B@BSA@ F-couSiPcs@luminol@Ab2The complex solution is stored in a refrigerator at 4 ℃ for standby;
(6) immersing the modified electrode obtained in the step (4) into vomitoxin (DON) standard solutions with different concentrations, incubating the electrode at room temperature for 40 mim, and washing the surface of the electrode with deionized water to prepare DON/Ab1@PEDOT@HA@mp MnO2Modifying a glassy carbon electrode, and then taking 5 mu L of the integrated electrochemiluminescence probe TiO prepared in the step (5)2-B@BSA@ F-couSiPcs@luminol@Ab2Dropping the compound solutionTo DON/Ab1@PEDOT@HA@mp MnO2Incubating on the modified glassy carbon electrode for 50 min at room temperature, washing the surface of the electrode with deionized water to obtain TiO2-B@BSA@ F-couSiPcs@luminol@Ab2/DON/Ab1@PEDOT@HA@mp MnO2And modifying the glassy carbon electrode.
2. The method of claim 1, wherein the mesoporous nano manganese dioxide (mp MnO)2) Prepared by the following method: based on a cationic surfactant soft template method, 2 g of 1-hexadecyl trimethyl ammonium bromide (CTAB) is dissolved in 200 ml of absolute ethyl alcohol, and then KMnO is sequentially added into the mixed solution in a dropwise manner under the condition of strong stirring4And manganese acetate Mn (CH)3COO)2Keeping the mixture for several hours under the stirring condition, centrifuging and washing the dark brown precipitate for several times, and finally drying the precipitate for 8 hours at the temperature of 80 ℃ to obtain the mesoporous nano manganese dioxide (mp MnO)2）。
3. The method of claim 1, wherein the TiO is selected from the group consisting of2-B nanomaterial is prepared by the following method: 4 g of anatase TiO2 Uniformly dispersing in 60M KOH aqueous solution, stirring for 10 min, transferring the obtained suspension into a 100 ml stainless steel autoclave with a tetrafluoroethylene lining, keeping the temperature at 170 ℃ for 72 min, cooling to room temperature, washing the obtained precipitate with diluted HAc solution until the pH value reaches 7.0, centrifuging, and drying at 60 ℃ for 12 h to obtain a final product; dispersing 600 mg of precursor titanate nanowire into 100 ml of 16M HAc solution, transferring the mixed solution into a 200 ml tetrafluoroethylene-lined stainless steel autoclave, heating for 24 h at 180 ℃, centrifuging, respectively washing with distilled water and ethanol thoroughly, and drying at 60 ℃ overnight to obtain the final target product TiO2-B nanomaterial.
4. The method of claim 1, wherein said fluorocoumarin silicon phthalocyanine (F-couSiPcs) is synthesized by: 1) dispersing 8 g of 1, 4-dihydroxybenzene in 28 ml of concentrated sulfuric acid, continuously stirring at 0 ℃ for 30 min, then sequentially adding 20 g of ethyl trifluoroacetate and 40 ml of concentrated sulfuric acid into the mixed solution, stirring at room temperature for 12 h, transferring the crude product into ice water for precipitating more products, filtering and washing by using ultrapure water for multiple times, and drying in vacuum at 60 ℃ to obtain light yellow crystal 6-hydroxy-4-trifluoromethyl-1, 2-benzopyran (fluoro-coumarin); 2) respectively adding 0.46g of the prepared 6-hydroxy-4-trifluoromethyl-1, 2-benzopyran (fluoro-coumarin) in a 200 ml round bottom flask, 0.42 g of silicon dichloride, 100 ml of toluene and 3.0 ml of pyridine, refluxing the mixture, keeping the mixture at 110 ℃ for 48 hours under magnetic stirring, cooling to room temperature, and further purifying by silica gel chromatography to obtain a crude product; 3) drying the obtained product under a rotary evaporator to obtain the fluoro coumarin silicon phthalocyanine (F-couSiPcs).
5. A TiO directed against emetic toxin prepared by the process of any one of claims 1 to 42-B an integrated hydrogen peroxide-free electrochemiluminescence sensor.
6. The TiO 2 against vomitoxin of claim 52-B an integrated hydrogen peroxide free electrochemiluminescence sensor for vomitoxin (DON) detection, comprising the steps of:
(1) performing measurement by using an electrochemical workstation and adopting a three-electrode system, taking the integrated hydrogen peroxide-free electrochemiluminescence sensor as claimed in claim 5 as a working electrode, Ag/AgCl as a reference electrode and a platinum wire electrode as an auxiliary electrode, and performing the measurement in 0.1 mol/mL PBS (phosphate buffer solution) with pH of 8.0;
(2) detecting vomitoxin (DON) standard solutions with different concentrations by adopting a potential range of-0.5-1V and a potential window with a scanning speed of 0.05V/s and an electrochemiluminescence device photomultiplier 800V, acquiring ECL signal intensity of 0.5V through the electrochemiluminescence device, and drawing a working curve according to the relation between the ECL signal intensity and the vomitoxin (DON) standard solution concentration;
(3) the sample solution to be detected replaces a vomitoxin (DON) standard solution for detection, and the detection result can be obtained through a working curve.
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