CN106645094B - Electrochemical luminescence sensor for detecting concanavalin A by Fe3O4 immobilized Ru (bpy)32+ - Google Patents
Electrochemical luminescence sensor for detecting concanavalin A by Fe3O4 immobilized Ru (bpy)32+ Download PDFInfo
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- SZVJSHCCFOBDDC-UHFFFAOYSA-N ferrosoferric oxide Chemical compound O=[Fe]O[Fe]O[Fe]=O SZVJSHCCFOBDDC-UHFFFAOYSA-N 0.000 title claims abstract description 94
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- 238000004020 luminiscence type Methods 0.000 title claims abstract description 17
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- BOTDANWDWHJENH-UHFFFAOYSA-N Tetraethyl orthosilicate Chemical compound CCO[Si](OCC)(OCC)OCC BOTDANWDWHJENH-UHFFFAOYSA-N 0.000 claims description 5
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- TWNQGVIAIRXVLR-UHFFFAOYSA-N oxo(oxoalumanyloxy)alumane Chemical compound O=[Al]O[Al]=O TWNQGVIAIRXVLR-UHFFFAOYSA-N 0.000 claims description 5
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- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Natural products NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 claims description 3
- MQRWBMAEBQOWAF-UHFFFAOYSA-N acetic acid;nickel Chemical compound [Ni].CC(O)=O.CC(O)=O MQRWBMAEBQOWAF-UHFFFAOYSA-N 0.000 claims description 3
- 229940011182 cobalt acetate Drugs 0.000 claims description 3
- QAHREYKOYSIQPH-UHFFFAOYSA-L cobalt(II) acetate Chemical compound [Co+2].CC([O-])=O.CC([O-])=O QAHREYKOYSIQPH-UHFFFAOYSA-L 0.000 claims description 3
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 claims description 3
- 229910000397 disodium phosphate Inorganic materials 0.000 claims description 3
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- USHAGKDGDHPEEY-UHFFFAOYSA-L potassium persulfate Chemical compound [K+].[K+].[O-]S(=O)(=O)OOS([O-])(=O)=O USHAGKDGDHPEEY-UHFFFAOYSA-L 0.000 claims description 3
- HKZLPVFGJNLROG-UHFFFAOYSA-M silver monochloride Chemical compound [Cl-].[Ag+] HKZLPVFGJNLROG-UHFFFAOYSA-M 0.000 claims description 3
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- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 12
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- 101000582927 Photorhabdus laumondii subsp. laumondii (strain DSM 15139 / CIP 105565 / TT01) Lectin A Proteins 0.000 description 2
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- 108010022007 Canavalia lectin Proteins 0.000 description 1
- WQZGKKKJIJFFOK-QTVWNMPRSA-N D-mannopyranose Chemical compound OC[C@H]1OC(O)[C@@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-QTVWNMPRSA-N 0.000 description 1
- 102000003886 Glycoproteins Human genes 0.000 description 1
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- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
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- 125000002791 glucosyl group Chemical group C1([C@H](O)[C@@H](O)[C@H](O)[C@H](O1)CO)* 0.000 description 1
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/66—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light electrically excited, e.g. electroluminescence
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- Health & Medical Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
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- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
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Abstract
The invention relates to an electrochemiluminescence sensor for detecting concanavalin A by Fe3O4 immobilized Ru (bpy)32 +. In the invention, a lamellar chitosan/Ru (bpy)32 +/silicon dioxide coated Fe3O4 nano material prepared from water/oil microemulsion is used as a luminescent material, and the material has stable and efficient optical performance and is beneficial to preparing a stable electrochemical luminescence sensor. In order to sensitively detect concanavalin a, glucose-functionalized dicobalt tetrasulfide-carboxylated graphene is used as a quencher for reducing the electrochemiluminescence intensity of the luminescent material. The concanavalin A can specifically recognize glucose, and based on the recognition, concanavalin A with different concentrations can be combined with different amounts of glucose functionalized cobaltosic nickel sulfide-carboxylated graphene, so that the electrochemical luminescence intensity of the sensor is different. The linear range of the invention for detecting the phaseolus vulgaris agglutinin A is 0.5 pg/mL-100 ng/mL, and the detection limit is 0.18 pg/mL.
Description
Technical Field
the invention relates to an electrochemiluminescence sensor for detecting concanavalin A by Fe3O4 immobilized Ru (bpy)32 +. Specifically, Fe3O4 immobilized Ru (bpy)32+ is used as a luminescent material, glucose functionalized cobaltosic nickel sulfide-carboxylated graphene is used as a quencher, and the electrochemical luminescence sensor for detecting the concanavalin A is prepared, and belongs to the technical field of electrochemical luminescence detection.
Background
Canavalia gladiata lectin A is a legume protein extracted from Canavalia gladiata. When the pH is below 5.5, concanavalin a exists in the form of a dimer; at a pH between 5.8 and 7.0, concanavalin A is present as a tetramer; under neutral conditions, the subunits of concanavalin a have four binding sites. The concanavalin A can specifically recognize various saccharides, such as mannose, glucose, glycoprotein, etc. Based on this, concanavalin a can be used as a protein model for molecular recognition or research of biological processes, which is very important for clinical diagnosis and development of drugs, such as detection of tumor cells and leukemia. Also, concanavalin a can proliferate and activate mature T cells by specifically recognizing the glucose functional group of cell membrane surface glycoproteins. Therefore, in order to study carbohydrate-protein binding, canavalin a is used as a target, and it is important to design a sensitive method for detecting canavalin a.
the existing methods for detecting the concanavalin A mainly comprise fluorescence, surface plasmon resonance, electrochemistry, ultraviolet visualization and the like. Compared with these methods, electrochemiluminescence has advantages of both electrochemical and chemiluminescent methods, such as high sensitivity, low detection limit, and easy control. Electrochemical luminescence is that after an electroactive substance generated by direct oxidation on the surface of an electrode undergoes an electron transfer reaction to form an excited state, excited state energy is released in the form of light. Therefore, the invention designs a quenching type electrochemical luminescence analysis method for detecting the concanavalin A.
in the invention, a lamellar chitosan/Ru (bpy)32 +/silicon dioxide coated Fe3O4 nano material is prepared as a luminescent material by using water/oil microemulsion for the first time. Through comparison of the existing documents, Dang project group discloses a preparation method of an electrochemiluminescence sensor, which adopts water/oil microemulsion to prepare spherical chitosan/Ru (bpy)32 +/silicon dioxide nano material (anal. chem. 2014, 86, 8943-8950). Compared with the literature, the method adds the magnetic nano material into the method, not only can save time in the separation process, but also can increase the conductivity of the composite material through the conductivity of the magnetic nano material, thereby improving the electrochemical luminescence intensity of the luminescent material and improving the sensitivity of the sensor. In addition, the lamellar composite material prepared by the invention has a large specific surface area, and can be used for immobilizing a large amount of concanavalin A. In order to sensitively detect the concanavalin A, glucose functionalized cobaltosic nickel sulfide-carboxylated graphene is used as a quencher to reduce the electrochemiluminescence intensity of the chitosan/Ru (bpy)32 +/silicon dioxide coated Fe3O4 nano material. The concanavalin A can specifically recognize glucose, and based on the recognition, different concentrations of concanavalin A can be combined with different amounts of glucose functionalized cobaltosic nickel sulfide-carboxylated graphene, so that the electrochemiluminescence intensity of the chitosan/Ru (bpy)32 +/silicon dioxide coated Fe3O4 nano material is changed. Therefore, the biosensor designed by the invention not only can sensitively detect the concanavalin A, but also provides a new method for detecting other analytes. At present, a method for detecting the concanavalin A based on cobaltosic nickel tetrasulfide-carboxylated graphene quenching Ru (bpy)32+ is not reported.
disclosure of Invention
One of the purposes of the invention is to prepare Ru (bpy)32+ immobilized by a magnetic nano material Fe3O4, enhance the electrochemical luminescence property of a luminophor on the surface of an electrode and improve the sensitivity of a sensor.
the second purpose of the invention is to prepare cobaltosic sulfide nickel-carboxylated graphene by a hydrothermal method, and realize the quenching of electrochemiluminescence by the charge transfer between NiCo2S4 and Ru (bpy)32 +.
the third purpose of the invention is to fix glucose on the surface of cobaltosic sulfide nickel-carboxylated graphene by a covalent crosslinking method, realize the preparation of a biosensor based on the specific recognition of the sword bean agglutinin A on saccharides, and achieve the purpose of detecting the sword bean agglutinin A
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
1. a preparation method of an electrochemiluminescence sensor for detecting concanavalin A by using Fe3O4 immobilized Ru (bpy)32+ is characterized by comprising the following steps:
(1) Polishing a glassy carbon electrode with the diameter of 4 mm by using aluminum oxide polishing powder with the particle size of 1.0 mu m, 0.3 mu m and 0.05 mu m in sequence, ultrasonically cleaning by using ethanol, and washing by using ultrapure water;
(2) Dripping 6 mu L of chitosan/Ru (bpy)32 +/silica coated Fe3O4 nano-material chitosan solution onto the surface of an electrode, and storing at room temperature until the solution is dry;
(3) dripping 5 mu L of glutaraldehyde solution with volume fraction of 0.5% on the surface of the glassy carbon electrode, preserving at room temperature until the solution is dried, and cleaning with ultrapure water;
(4) dripping 10 μ L of concanavalin A with different concentrations on different electrode surfaces, preserving in a refrigerator at 4 deg.C until it is dried, and cleaning with ultrapure water;
(5) Dripping 3 μ L of bovine serum albumin with mass fraction of 1%, sealing nonspecific active sites, storing in a refrigerator at 4 deg.C until dried, and cleaning with ultrapure water;
(6) 6 mu L of 1-7 mg/mL glucose functionalized cobaltosic nickel sulfide-carboxylated graphene is dropwise coated on the surface of an electrode, stored in a refrigerator at 4 ℃ until being dried, and cleaned by ultrapure water to obtain the Fe3O4 immobilized Ru (bpy)32+ electrochemiluminescence sensor for detecting the concanavalin A, and the electrochemiluminescence sensor is stored in the refrigerator at 4 ℃.
2. The invention relates to a preparation method of an electrochemiluminescence sensor for detecting concanavalin A by Fe3O4 immobilized Ru (bpy)32+, wherein chitosan/Ru (bpy)32 +/silicon dioxide wraps a chitosan solution of Fe3O4 nano-materials, and the preparation method is characterized by comprising the following steps:
mixing 1.77 mL of emulsifier TX-100, 7.5 mL of cyclohexane and 1.8 mL of n-butanol, adding 1 mL of 50 mg/mL of Fe3O4 aqueous solution, and stirring for 1 h; adding 3-10 mL of 2.5 mmol/L Ru (bpy)32+ and 1 mL of chitosan with the mass fraction of 0.5% into the solution, and continuously stirring for 2 h; then, adding 150 mu L of tetraethyl orthosilicate and 1 mL of ammonia water, and continuously reacting for 24 h; finally, adding acetone to terminate the emulsification reaction, carrying out magnetic separation, washing with water and absolute ethyl alcohol to obtain a chitosan/Ru (bpy)32 +/silicon dioxide coated Fe3O4 nano material, and dispersing the nano material in a chitosan solution with the mass fraction of 0.5%;
The chitosan solution with the mass fraction of 0.5% is prepared by adding 0.5 g of chitosan into 100 mL of acetic acid with the volume fraction of 1% and stirring for 2 h.
3. The invention discloses a preparation method of an electrochemiluminescence sensor for detecting concanavalin A by using Fe3O4 immobilized Ru (bpy)32+, which is used for preparing glucose functionalized cobaltosic nickel sulfide-carboxylated graphene and is characterized by comprising the following steps of:
(1) Preparation of dicobalt tetrasulfide-carboxylated graphene
0.0747 g of cobalt acetate and 0.0373 g of nickel acetate are dispersed in 30 mL of ethylene glycol containing 50 mg of carboxylated graphene oxide, the mixture is stirred for 2 hours at the temperature of 80 ℃, then 0.0685 g of thiourea is added into the solution, the solution is transferred to a reaction kettle, the reaction is carried out for 6 hours at the temperature of 200 ℃, and then the cobaltosic nickel tetrasulfide-carboxylated graphene is obtained after centrifugation, washing and drying;
(2) preparation of glucose functionalized cobaltosic nickel tetrasulfide-carboxylated graphene
Dispersing 40 mg of cobaltosic nickel tetrasulfide-carboxylated graphene into 10 mL of N, N' -carbonyldiimidazole aqueous solution with the concentration of 100 mg/mL, stirring for 12 h at room temperature, adding 100 mg of glucose, continuing to react for 12 h, centrifuging and washing to obtain the glucose functionalized cobaltosic nickel tetrasulfide-carboxylated graphene.
4. the electrochemical luminescence sensor for detecting the concanavalin A, which is prepared by the preparation method of the invention and is provided with Fe3O4 immobilized Ru (bpy)32+, is used for detecting the concanavalin A and is characterized by comprising the following specific steps:
(1) connecting a reference electrode-Ag/AgCl electrode, a counter electrode-platinum wire electrode and the prepared electrochemical luminescence sensor as a working electrode in a cassette of a chemiluminescence detector, connecting an electrochemical workstation and the chemiluminescence detector together, setting the high voltage of a photomultiplier to be 750V, and setting the scanning voltage to be-1.8-0V;
(2) detecting the electrochemical luminescence signal intensity generated by the concanavalin A with different concentrations by an electrochemical luminescence method by using PBS buffer solution containing 20-120 mmol/L potassium persulfate and 0.1 mol/L potassium chloride;
the PBS buffer solution with the pH = 6.5-8.5 is prepared from 1/15 mol/L Na2HPO4 and 1/15 mol/L KH2PO 4;
(3) And drawing a working curve according to the linear relation between the obtained electrochemiluminescence intensity value and the concentration of the concanavalin A.
advantageous results of the invention
(1) the Fe3O4 immobilized Ru (bpy)32+ is prepared by adopting a water/oil emulsion method to obtain a chitosan/Ru (bpy)32 +/silica-coated Fe3O4 nano material, so that the Ru (bpy)32+ can stably exist on the surface of an electrode, and the conductivity of the mixed material can be increased through the conductivity of Fe3O4, so that the electrochemical luminescence intensity of the luminescent material is increased.
(2) the prepared chitosan/Ru (bpy)32 +/silicon dioxide coated Fe3O4 nano material has a sheet structure and a large specific surface area, amino groups on chitosan in the mixed material are activated by glutaraldehyde, a large amount of concanavalin A can be combined, and the quenching efficiency of glucose functionalized cobaltosic sulfide nickel-carboxylated graphene is amplified.
(3) the electrochemical luminescence sensor prepared by the invention is used for detecting the concanavalin A, has the advantages of small using amount of luminescent substances and short response time, and can realize simple, quick and high-sensitivity detection. The linear range of the invention for detecting the phaseolus vulgaris agglutinin A is 0.5 pg/mL-100 ng/mL, and the detection limit is 0.18 pg/mL.
Detailed Description
the invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.
Example 1 preparation of Chitosan/Ru (bpy)32 +/silica-coated Fe3O4 nanomaterial Chitosan solution
1.77 mL of emulsifier TX-100, 7.5 mL of cyclohexane and 1.8 mL of n-butanol were mixed, and 1 mL of a 50 mg/mL aqueous solution of Fe3O4 was added and stirred for 1 hour. 3 mL of 2.5 mmol/L Ru (bpy)32+ and 1 mL of 0.5% chitosan were added to the solution and stirring was continued for 2 h. Then, 150. mu.L of tetraethyl orthosilicate and 1 mL of aqueous ammonia were added, and the reaction was continued for 24 hours. Finally, adding acetone to terminate the emulsification reaction, carrying out magnetic separation, washing with water and absolute ethyl alcohol to obtain a chitosan/Ru (bpy)32 +/silicon dioxide coated Fe3O4 nano material, and dispersing the nano material in a 0.5% chitosan solution;
the 0.5% chitosan solution is prepared by adding 0.5 g chitosan into 100 mL acetic acid with volume fraction of 1%, and stirring for 2 h.
Example 2 preparation of Chitosan solution of Chitosan/Ru (bpy)32 +/silica-coated Fe3O4 nanomaterial
1.77 mL of emulsifier TX-100, 7.5 mL of cyclohexane and 1.8 mL of n-butanol were mixed, and 1 mL of a 50 mg/mL aqueous solution of Fe3O4 was added and stirred for 1 hour. 5 mL of 2.5 mmol/L Ru (bpy)32+ and 1 mL of 0.5% chitosan were added to the solution and stirring was continued for 2 h. Then, 150. mu.L of tetraethyl orthosilicate and 1 mL of aqueous ammonia were added, and the reaction was continued for 24 hours. Finally, adding acetone to terminate the emulsification reaction, carrying out magnetic separation, washing with water and absolute ethyl alcohol to obtain a chitosan/Ru (bpy)32 +/silicon dioxide coated Fe3O4 nano material, and dispersing the nano material in a 0.5% chitosan solution;
The 0.5% chitosan solution is prepared by adding 0.5 g chitosan into 100 mL acetic acid with volume fraction of 1%, and stirring for 2 h.
example 3 preparation of Chitosan/Ru (bpy)32 +/silica-coated Fe3O4 nanomaterial Chitosan solution
1.77 mL of emulsifier TX-100, 7.5 mL of cyclohexane and 1.8 mL of n-butanol were mixed, and 1 mL of a 50 mg/mL aqueous solution of Fe3O4 was added and stirred for 1 hour. 10 mL of 2.5 mmol/L Ru (bpy)32+ and 1 mL of 0.5% chitosan were added to the solution and stirring was continued for 2 h. Then, 150. mu.L of tetraethyl orthosilicate and 1 mL of aqueous ammonia were added, and the reaction was continued for 24 hours. Finally, adding acetone to terminate the emulsification reaction, carrying out magnetic separation, washing with water and absolute ethyl alcohol to obtain a chitosan/Ru (bpy)32 +/silicon dioxide coated Fe3O4 nano material, and dispersing the nano material in a 0.5% chitosan solution;
the 0.5% chitosan solution is prepared by adding 0.5 g chitosan into 100 mL acetic acid with volume fraction of 1%, and stirring for 2 h.
Example 4 preparation of glucose functionalized dicobalt-nickel tetrasulfide-carboxylated graphene
(1) preparation of dicobalt tetrasulfide-carboxylated graphene
Dispersing 0.0747 g of cobalt acetate and 0.0373 g of nickel acetate in 30 mL of ethylene glycol containing 50 mg of carboxylated graphene oxide, stirring at 80 ℃ for 2h, adding 0.0685 g of thiourea into the solution, transferring to a reaction kettle, reacting at 200 ℃ for 6 h, centrifuging, washing and drying to obtain cobaltosic nickel tetrasulfide-carboxylated graphene;
(2) preparation of glucose functionalized cobaltosic nickel tetrasulfide-carboxylated graphene
Dispersing 40 mg of cobaltosic nickel tetrasulfide-carboxylated graphene into 10 mL of N, N' -carbonyldiimidazole aqueous solution with the concentration of 100 mg/mL, stirring for 12 h at room temperature, adding 100 mg of glucose, continuing to react for 12 h, centrifuging and washing to obtain the glucose functionalized cobaltosic nickel tetrasulfide-carboxylated graphene.
Example 5 a concanavalin a electrochemiluminescence sensor was prepared.
(1) Polishing a glassy carbon electrode with the diameter of 4 mm by using aluminum oxide polishing powder with the particle size of 1.0 mu m, 0.3 mu m and 0.05 mu m in sequence, ultrasonically cleaning by using ethanol, and washing by using ultrapure water;
(2) Dripping 6 mu L of chitosan solution of 1mg/mL of chitosan/Ru (bpy)32 +/silicon dioxide coated Fe3O4 nano material on the surface of an electrode, and storing at room temperature until the solution is dried;
(3) dripping 5 mu L of glutaraldehyde solution with the volume fraction of 0.5% on the surface of the glassy carbon electrode, preserving at room temperature until the solution is dried, and cleaning with ultrapure water;
(4) dripping 10 μ L of concanavalin A with different concentrations on the surface of different electrodes, preserving in a refrigerator at 4 deg.C until it is dried, and cleaning with ultrapure water;
(5) Dripping 3 μ L of bovine serum albumin with mass fraction of 1%, sealing nonspecific active sites, storing in a refrigerator at 4 deg.C until dried, and cleaning with ultrapure water;
(6) 6 mu L of 2 mg/mL glucose functionalized cobaltosic nickel tetrasulfide-carboxylated graphene solution is dropwise coated on the surface of an electrode, stored in a refrigerator at 4 ℃ until being dried, and cleaned by ultrapure water, so that the electrochemiluminescence sensor for detecting the concanavalin A, which is supported by Ru (bpy)32+ on Fe3O4, is prepared and stored in the refrigerator at 4 ℃.
example 6 a concanavalin a electrochemiluminescence sensor was prepared.
(1) polishing a glassy carbon electrode with the diameter of 4 mm by using aluminum oxide polishing powder with the particle size of 1.0 mu m, 0.3 mu m and 0.05 mu m in sequence, ultrasonically cleaning by using ethanol, and washing by using ultrapure water;
(2) Dripping 6 mu L of chitosan solution of 3 mg/mL of chitosan/Ru (bpy)32 +/silicon dioxide coated Fe3O4 nano material on the surface of an electrode, and storing at room temperature until the solution is dried;
(3) dripping 5 mu L of glutaraldehyde solution with the volume fraction of 0.5% on the surface of the glassy carbon electrode, preserving at room temperature until the solution is dried, and cleaning with ultrapure water;
(4) dripping 10 μ L of concanavalin A with different concentrations on the surface of different electrodes, preserving in a refrigerator at 4 deg.C until it is dried, and cleaning with ultrapure water;
(5) Dripping 3 μ L of bovine serum albumin with mass fraction of 1%, sealing nonspecific active sites, storing in a refrigerator at 4 deg.C until dried, and cleaning with ultrapure water;
(6) 6 mu L of 3 mg/mL glucose functionalized cobaltosic nickel tetrasulfide-carboxylated graphene solution is dropwise coated on the surface of an electrode, stored in a refrigerator at 4 ℃ until being dried, and cleaned by ultrapure water, so that the electrochemiluminescence sensor for detecting the concanavalin A, which is supported by Ru (bpy)32+ on Fe3O4, is prepared and stored in the refrigerator at 4 ℃.
example 7 preparation of Canavalia lectin A electrochemiluminescence sensor
(1) polishing a glassy carbon electrode with the diameter of 4 mm by using aluminum oxide polishing powder with the particle size of 1.0 mu m, 0.3 mu m and 0.05 mu m in sequence, ultrasonically cleaning by using ethanol, and washing by using ultrapure water;
(2) Dripping 6 mu L of chitosan solution of 5 mg/mL chitosan/Ru (bpy)32 +/silicon dioxide coated Fe3O4 nano material on the surface of an electrode, and storing at room temperature until the solution is dried;
(3) Dripping 5 mu L of glutaraldehyde solution with the volume fraction of 0.5% on the surface of the glassy carbon electrode, preserving at room temperature until the solution is dried, and cleaning with ultrapure water;
(4) Dripping 10 μ L of concanavalin A with different concentrations on the surface of different electrodes, preserving in a refrigerator at 4 deg.C until it is dried, and cleaning with ultrapure water;
(5) dripping 3 μ L of bovine serum albumin with mass fraction of 1%, sealing nonspecific active sites, storing in a refrigerator at 4 deg.C until dried, and cleaning with ultrapure water;
(6) 6 mu L of 7 mg/mL glucose functionalized cobaltosic nickel sulfide-carboxylated graphene solution is dripped on the surface of an electrode, stored in a refrigerator at 4 ℃ until being dried, and cleaned by ultrapure water, so that the electrochemiluminescence sensor for detecting the concanavalin A, which is supported by Ru (bpy)32+ on Fe3O4, is prepared and stored in the refrigerator at 4 ℃.
Example 8 detection method of Canavalia ensiformis lectin A
(1) connecting a reference electrode-Ag/AgCl electrode, a counter electrode-platinum wire electrode and the prepared electrochemical luminescence sensor as a working electrode in a cassette of a chemiluminescence detector, connecting an electrochemical workstation and the chemiluminescence detector together, setting the high voltage of a photomultiplier to be 750V, and setting the scanning voltage to be-1.8-0V;
(2) detecting the intensity of an electrochemiluminescence signal generated by the concanavalin A with different concentrations by an electrochemiluminescence method by using a PBS (phosphate buffer solution) containing 100 mmol/L potassium persulfate and 0.1 mol/L potassium chloride;
The PBS buffer, pH =8.0, was formulated with 1/15 mol/L Na2HPO4 and 1/15 mol/L KH2PO 4;
(3) and drawing a working curve according to the linear relation between the obtained electrochemiluminescence intensity value and the concentration of the concanavalin A.
Example 9 detection of concanavalin A in Bovine Serum Albumin (BSA)
(1) Bovine Serum Albumin (BSA) 1 mL, 0.1 mg/mL, various concentrations of concanavalin A were added thereto, and the average recovery of concanavalin A in the sample was determined by standard addition methods, and the results are shown in Table 1.
TABLE 1 detection results of concanavalin A in samples
the detection results in table 1 show that the recovery rate of the detection result of concanavalin A in sample BSA is 95.0-105%, which indicates that the method can be applied to the detection of practical biological samples, and the method has high precision and accurate and reliable results.
Claims (4)
1. a preparation method of an electrochemiluminescence sensor for detecting concanavalin A by using Fe3O4 immobilized Ru (bpy)32+ is characterized by comprising the following steps:
(1) polishing a glassy carbon electrode with the diameter of 4 mm by using aluminum oxide polishing powder with the particle size of 1.0 mu m, 0.3 mu m and 0.05 mu m in sequence, ultrasonically cleaning by using ethanol, and washing by using ultrapure water;
(2) Dripping 6 mu L of chitosan/Ru (bpy)32 +/silica coated Fe3O4 nano-material chitosan solution onto the surface of an electrode, and storing at room temperature until the solution is dry;
(3) Dripping 5 mu L of glutaraldehyde solution with volume fraction of 0.5% on the surface of the glassy carbon electrode, preserving at room temperature until the solution is dried, and cleaning with ultrapure water;
(4) Dripping 10 μ L of concanavalin A with different concentrations on different electrode surfaces, preserving in a refrigerator at 4 deg.C until it is dried, and cleaning with ultrapure water;
(5) Dripping 3 μ L of bovine serum albumin with mass fraction of 1%, sealing nonspecific active sites, storing in a refrigerator at 4 deg.C until dried, and cleaning with ultrapure water;
(6) 6 mu L of 1-7 mg/mL glucose functionalized cobaltosic nickel sulfide-carboxylated graphene is dropwise coated on the surface of an electrode, stored in a refrigerator at 4 ℃ until being dried, and cleaned by ultrapure water to obtain the Fe3O4 immobilized Ru (bpy)32+ electrochemiluminescence sensor for detecting the concanavalin A, and the electrochemiluminescence sensor is stored in the refrigerator at 4 ℃.
2. The method for preparing the electrochemiluminescence sensor for detecting the concanavalin A, wherein the electrochemiluminescence sensor comprises Fe3O4 immobilized Ru (bpy)32+, chitosan/Ru (bpy)32 +/silica coated Fe3O4 nano-material chitosan solution, and the preparation method comprises the following steps:
mixing 1.77 mL of emulsifier TX-100, 7.5 mL of cyclohexane and 1.8 mL of n-butanol, adding 1 mL of 50 mg/mL of Fe3O4 aqueous solution, and stirring for 1 h; adding 3-10 mL of 2.5 mmol/L Ru (bpy)32+ and 1 mL of chitosan with the mass fraction of 0.5% into the solution, and continuously stirring for 2 h; then, adding 150 mu L of tetraethyl orthosilicate and 1 mL of ammonia water, and continuously reacting for 24 h; finally, acetone is added to terminate the emulsification reaction, and the chitosan/Ru (bpy)32 +/silicon dioxide coated Fe3O4 nano-material is obtained by magnetic separation and washing with water and absolute ethyl alcohol and is dispersed in a chitosan solution with the mass fraction of 0.5%.
3. The method for preparing the electrochemiluminescence sensor for detecting the concanavalin A, which is immobilized on Ru (bpy)32+ of Fe3O4, according to claim 1, wherein the glucose functionalized cobaltosic nickel sulfide-carboxylated graphene is prepared by the following steps:
(1) preparation of dicobalt tetrasulfide-carboxylated graphene
0.0747 g of cobalt acetate and 0.0373 g of nickel acetate are dispersed in 30 mL of ethylene glycol containing 50 mg of carboxylated graphene oxide, the mixture is stirred for 2 hours at the temperature of 80 ℃, then 0.0685 g of thiourea is added into the solution, the solution is transferred to a reaction kettle, the reaction is carried out for 6 hours at the temperature of 200 ℃, and then the cobaltosic nickel tetrasulfide-carboxylated graphene is obtained after centrifugation, washing and drying;
(2) Preparation of glucose functionalized cobaltosic nickel tetrasulfide-carboxylated graphene
Dispersing 40 mg of cobaltosic nickel tetrasulfide-carboxylated graphene into 10 mL of N, N' -carbonyldiimidazole aqueous solution with the concentration of 100 mg/mL, stirring for 12 h at room temperature, adding 100 mg of glucose, continuing to react for 12 h, centrifuging and washing to obtain the glucose functionalized cobaltosic nickel tetrasulfide-carboxylated graphene.
4. the electrochemiluminescence sensor for detecting the concanavalin A, prepared by the preparation method of claim 1, of Fe3O4 immobilized Ru (bpy)32+, is used for detecting the concanavalin A, and is characterized by comprising the following specific steps:
(1) connecting a reference electrode-Ag/AgCl electrode, a counter electrode-platinum wire electrode and the prepared electrochemical luminescence sensor as a working electrode in a cassette of a chemiluminescence detector, connecting an electrochemical workstation and the chemiluminescence detector together, setting the high voltage of a photomultiplier to be 750V, and setting the scanning voltage to be-1.8-0V;
(2) Detecting the electrochemical luminescence signal intensity generated by the concanavalin A with different concentrations by an electrochemical luminescence method by using PBS buffer solution containing 20-120 mmol/L potassium persulfate and 0.1 mol/L potassium chloride;
the PBS buffer solution with the pH = 6.5-8.5 is prepared from 1/15 mol/L Na2HPO4 and 1/15 mol/L KH2PO 4;
(3) And drawing a working curve according to the linear relation between the obtained electrochemiluminescence intensity value and the concentration of the concanavalin A.
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