CN114720525B - Parathion immunosensor and preparation method and application thereof - Google Patents
Parathion immunosensor and preparation method and application thereof Download PDFInfo
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- CN114720525B CN114720525B CN202210283368.8A CN202210283368A CN114720525B CN 114720525 B CN114720525 B CN 114720525B CN 202210283368 A CN202210283368 A CN 202210283368A CN 114720525 B CN114720525 B CN 114720525B
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- parathion
- polyvinyl alcohol
- citric acid
- nanofiber membrane
- electrode
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- LCCNCVORNKJIRZ-UHFFFAOYSA-N parathion Chemical compound CCOP(=S)(OCC)OC1=CC=C([N+]([O-])=O)C=C1 LCCNCVORNKJIRZ-UHFFFAOYSA-N 0.000 title claims abstract description 132
- 238000002360 preparation method Methods 0.000 title claims abstract description 24
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 claims abstract description 355
- 239000004372 Polyvinyl alcohol Substances 0.000 claims abstract description 132
- 229920002451 polyvinyl alcohol Polymers 0.000 claims abstract description 132
- 239000012528 membrane Substances 0.000 claims abstract description 93
- 239000002121 nanofiber Substances 0.000 claims abstract description 92
- 238000004132 cross linking Methods 0.000 claims abstract description 46
- 238000000034 method Methods 0.000 claims abstract description 33
- 238000010041 electrostatic spinning Methods 0.000 claims abstract description 7
- 229940092253 ovalbumin Drugs 0.000 claims description 12
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 9
- 238000007789 sealing Methods 0.000 claims description 7
- 230000003213 activating effect Effects 0.000 claims description 6
- 239000011248 coating agent Substances 0.000 claims description 5
- 238000000576 coating method Methods 0.000 claims description 5
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- 239000007864 aqueous solution Substances 0.000 abstract description 2
- 239000000243 solution Substances 0.000 description 66
- PXRKCOCTEMYUEG-UHFFFAOYSA-N 5-aminoisoindole-1,3-dione Chemical compound NC1=CC=C2C(=O)NC(=O)C2=C1 PXRKCOCTEMYUEG-UHFFFAOYSA-N 0.000 description 29
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- QIGBRXMKCJKVMJ-UHFFFAOYSA-N Hydroquinone Chemical compound OC1=CC=C(O)C=C1 QIGBRXMKCJKVMJ-UHFFFAOYSA-N 0.000 description 16
- 230000000052 comparative effect Effects 0.000 description 16
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- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 6
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- 238000005266 casting Methods 0.000 description 6
- 239000000575 pesticide Substances 0.000 description 6
- 239000012086 standard solution Substances 0.000 description 6
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 5
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 description 5
- 229940098773 bovine serum albumin Drugs 0.000 description 5
- 239000007853 buffer solution Substances 0.000 description 5
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- BXNANOICGRISHX-UHFFFAOYSA-N coumaphos Chemical compound CC1=C(Cl)C(=O)OC2=CC(OP(=S)(OCC)OCC)=CC=C21 BXNANOICGRISHX-UHFFFAOYSA-N 0.000 description 5
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- LMDZBCPBFSXMTL-UHFFFAOYSA-N 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide Chemical compound CCN=C=NCCCN(C)C LMDZBCPBFSXMTL-UHFFFAOYSA-N 0.000 description 4
- 240000008067 Cucumis sativus Species 0.000 description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- BQCADISMDOOEFD-UHFFFAOYSA-N Silver Chemical compound [Ag] BQCADISMDOOEFD-UHFFFAOYSA-N 0.000 description 4
- 230000005684 electric field Effects 0.000 description 4
- BULVZWIRKLYCBC-UHFFFAOYSA-N phorate Chemical compound CCOP(=S)(OCC)SCSCC BULVZWIRKLYCBC-UHFFFAOYSA-N 0.000 description 4
- ATROHALUCMTWTB-OWBHPGMISA-N phoxim Chemical compound CCOP(=S)(OCC)O\N=C(\C#N)C1=CC=CC=C1 ATROHALUCMTWTB-OWBHPGMISA-N 0.000 description 4
- 229950001664 phoxim Drugs 0.000 description 4
- 238000007650 screen-printing Methods 0.000 description 4
- 229910052709 silver Inorganic materials 0.000 description 4
- 239000004332 silver Substances 0.000 description 4
- AMFGTOFWMRQMEM-UHFFFAOYSA-N triazophos Chemical compound N1=C(OP(=S)(OCC)OCC)N=CN1C1=CC=CC=C1 AMFGTOFWMRQMEM-UHFFFAOYSA-N 0.000 description 4
- 235000010799 Cucumis sativus var sativus Nutrition 0.000 description 3
- NQTADLQHYWFPDB-UHFFFAOYSA-N N-Hydroxysuccinimide Chemical compound ON1C(=O)CCC1=O NQTADLQHYWFPDB-UHFFFAOYSA-N 0.000 description 3
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 3
- 230000027455 binding Effects 0.000 description 3
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- IVLXQGJVBGMLRR-UHFFFAOYSA-N 2-aminoacetic acid;hydron;chloride Chemical compound Cl.NCC(O)=O IVLXQGJVBGMLRR-UHFFFAOYSA-N 0.000 description 2
- 240000007124 Brassica oleracea Species 0.000 description 2
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- 235000011301 Brassica oleracea var capitata Nutrition 0.000 description 2
- 235000001169 Brassica oleracea var oleracea Nutrition 0.000 description 2
- 235000010149 Brassica rapa subsp chinensis Nutrition 0.000 description 2
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- 241000499436 Brassica rapa subsp. pekinensis Species 0.000 description 2
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 description 2
- 238000002965 ELISA Methods 0.000 description 2
- 241000872931 Myoporum sandwicense Species 0.000 description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
- 239000008351 acetate buffer Substances 0.000 description 2
- 230000009471 action Effects 0.000 description 2
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical class N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 2
- 239000000427 antigen Substances 0.000 description 2
- 108091007433 antigens Proteins 0.000 description 2
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- 238000005859 coupling reaction Methods 0.000 description 2
- 230000009260 cross reactivity Effects 0.000 description 2
- 239000012153 distilled water Substances 0.000 description 2
- 238000002848 electrochemical method Methods 0.000 description 2
- 238000003487 electrochemical reaction Methods 0.000 description 2
- 238000001523 electrospinning Methods 0.000 description 2
- 238000011049 filling Methods 0.000 description 2
- 238000011010 flushing procedure Methods 0.000 description 2
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- 108010025899 gelatin film Proteins 0.000 description 2
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- 239000000741 silica gel Substances 0.000 description 2
- 229910002027 silica gel Inorganic materials 0.000 description 2
- JQWHASGSAFIOCM-UHFFFAOYSA-M sodium periodate Chemical compound [Na+].[O-]I(=O)(=O)=O JQWHASGSAFIOCM-UHFFFAOYSA-M 0.000 description 2
- 230000008961 swelling Effects 0.000 description 2
- 102000003914 Cholinesterases Human genes 0.000 description 1
- 108090000322 Cholinesterases Proteins 0.000 description 1
- 235000009849 Cucumis sativus Nutrition 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 241000282414 Homo sapiens Species 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 244000269722 Thea sinensis Species 0.000 description 1
- 206010070863 Toxicity to various agents Diseases 0.000 description 1
- 241000607479 Yersinia pestis Species 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
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- WGCNASOHLSPBMP-UHFFFAOYSA-N hydroxyacetaldehyde Natural products OCC=O WGCNASOHLSPBMP-UHFFFAOYSA-N 0.000 description 1
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- 229910001220 stainless steel Inorganic materials 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
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
-
- D—TEXTILES; PAPER
- D01—NATURAL OR MAN-MADE THREADS OR FIBRES; SPINNING
- D01F—CHEMICAL FEATURES IN THE MANUFACTURE OF ARTIFICIAL FILAMENTS, THREADS, FIBRES, BRISTLES OR RIBBONS; APPARATUS SPECIALLY ADAPTED FOR THE MANUFACTURE OF CARBON FILAMENTS
- D01F1/00—General methods for the manufacture of artificial filaments or the like
- D01F1/02—Addition of substances to the spinning solution or to the melt
- D01F1/10—Other agents for modifying properties
-
- D—TEXTILES; PAPER
- D01—NATURAL OR MAN-MADE THREADS OR FIBRES; SPINNING
- D01F—CHEMICAL FEATURES IN THE MANUFACTURE OF ARTIFICIAL FILAMENTS, THREADS, FIBRES, BRISTLES OR RIBBONS; APPARATUS SPECIALLY ADAPTED FOR THE MANUFACTURE OF CARBON FILAMENTS
- D01F6/00—Monocomponent artificial filaments or the like of synthetic polymers; Manufacture thereof
- D01F6/44—Monocomponent artificial filaments or the like of synthetic polymers; Manufacture thereof from mixtures of polymers obtained by reactions only involving carbon-to-carbon unsaturated bonds as major constituent with other polymers or low-molecular-weight compounds
- D01F6/50—Monocomponent artificial filaments or the like of synthetic polymers; Manufacture thereof from mixtures of polymers obtained by reactions only involving carbon-to-carbon unsaturated bonds as major constituent with other polymers or low-molecular-weight compounds of polyalcohols, polyacetals or polyketals
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Pathology (AREA)
- Biomedical Technology (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Physics & Mathematics (AREA)
- General Chemical & Material Sciences (AREA)
- Urology & Nephrology (AREA)
- Textile Engineering (AREA)
- Biochemistry (AREA)
- Hematology (AREA)
- Cell Biology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Electrochemistry (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Manufacturing & Machinery (AREA)
- Manufacture Of Macromolecular Shaped Articles (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention belongs to the technical field of nanofiber membranes, and particularly relates to a polyvinyl alcohol/citric acid nanofiber membrane as well as a preparation method and application thereof. According to the invention, polyvinyl alcohol and citric acid are adopted to prepare the polyvinyl alcohol/citric acid nanofiber membrane through an electrostatic spinning method and a thermal crosslinking reaction, the fiber diameter in the membrane is uniformly distributed, the stable fiber morphology can be maintained, the hydrophobicity of the membrane can be greatly improved, the aim of detecting parathion in an aqueous solution of the membrane is fulfilled, the electrochemical response speed is accelerated, and an electrochemical response signal is amplified; meanwhile, after the membrane is prepared into the parathion electrochemical immunosensor, the detection range is wide, the specificity is good, the sensitivity is high, the stability and the repeatability are good, the minimum detection limit is greatly reduced, the operation is simple and convenient, the cost is low, and the membrane can be widely applied to quantitative detection of parathion in vegetables and fruits.
Description
Technical Field
The invention belongs to the technical field of nanofiber membranes. More particularly, relates to a polyvinyl alcohol/citric acid nanofiber membrane and a preparation method and application thereof.
Background
Parathion pesticide is widely applied to pest control in the agricultural planting process, but the parathion pesticide has acute toxicity to human beings and animals, can inhibit cholinesterase activity or destroy endocrine systems, thereby causing toxic symptoms such as nerve dysfunction, language disorder and the like. In 2002, china has clearly prohibited or limited the use of parathion pesticides on vegetables, fruit trees, tea and Chinese herbal medicines, but poisoning events caused by parathion pesticides still occur, so that an effective and rapid parathion detection method is necessary to be established so as to ensure diet safety.
The existing detection methods of parathion pesticide residues mainly comprise conventional detection methods such as a Gas Chromatography (GC) method, a High Performance Liquid Chromatography (HPLC) method and the like. The conventional detection method has higher sensitivity and accuracy, can simultaneously analyze various pesticide residues, has accurate qualitative and quantitative analysis, but the required instrument is huge and expensive, requires professional technical personnel to operate, can only detect in a laboratory, and is difficult to meet the requirements of rapid detection of a sample site, the practical requirements of rapid detection, low cost and the like.
In order to solve the problems, researchers have shifted the eyes to the electrochemical immunosensor, which is an immunosensor combining a strong-specificity immunoassay method with a high-sensitivity electrochemical sensor, does not need expensive large-scale instruments, has the advantages of high sensitivity, high selectivity, small sample consumption, simple operation, high analysis speed and the like, and has good application prospect in the field of pesticide residue detection. For example, chinese patent application discloses a label-free immunosensor for parathion pesticide detection, which has a detection limit of 0.073 mug/ml and a detection range of 1-10 mug/ml. Therefore, the detection limit of the existing immunosensor for detecting parathion needs to be further reduced, and the detection range needs to be further improved.
Disclosure of Invention
The invention aims to overcome the defects of high detection limit and narrow detection range of the existing parathion pesticide detection sensor, and provides the parathion electrochemical immunosensor which can greatly reduce the detection limit of parathion and has the advantages of wide detection range, high sensitivity, good stability and good repeatability.
The invention aims to provide a preparation method of a polyvinyl alcohol/citric acid nanofiber membrane.
It is another object of the present invention to provide a polyvinyl alcohol/citric acid nanofiber membrane.
Another object of the invention is to provide a method for manufacturing an electrochemical immunosensor for parathion.
Another object of the invention is to provide a polyvinyl alcohol/citric acid nanofiber membrane and the use of an electrochemical immunosensor for parathion detection.
The above object of the present invention is achieved by the following technical solutions:
a preparation method of a polyvinyl alcohol/citric acid nanofiber membrane comprises the following steps:
and adding polyvinyl alcohol into water, heating until the polyvinyl alcohol is completely dissolved, cooling, adding citric acid, preparing the nanofiber membrane by an electrostatic spinning method, and thermally crosslinking the nanofiber membrane at 130-150 ℃ for 0.5-6 h to obtain the polyvinyl alcohol/citric acid nanofiber membrane.
Preferably, the concentration of the polyvinyl alcohol is 8 to 20wt%.
Preferably, the mass of the citric acid is 5-25% of that of the polyvinyl alcohol.
Preferably, the temperature of the thermal crosslinking is 140 to 145 ℃.
Preferably, the time of thermal crosslinking is 0.5 to 4 hours.
Preferably, the step of the electrospinning method is as follows: slowly filling a polyvinyl alcohol/citric acid (PVA/CA) solution into a syringe to avoid bubble generation, fixing the syringe filled with the PVA/CA solution on a push injection clamping groove, connecting an injection pump of an electrostatic spinning machine with a positive electrode of a high-voltage power supply, connecting a receiving plate with a negative electrode, using a 20-gauge stainless needle, controlling the injection rate by the injection pump, switching on the power supply, stretching the PVA/CA solution at the needle under the action of a high-voltage electric field, and spraying the stretched PVA/CA solution onto the receiving plate to form the nanofiber membrane.
Preferably, the process parameters of the electrospinning method are respectively as follows: spinning voltage: 15-20 kV, the injection rate of the injector is 0.3-0.4 mL/h, the receiving distance is 12-18 cm, the ambient temperature is 25+/-2 ℃, the relative humidity is 55+/-2%, and the time is 3-5 h.
Specifically, the electrostatic spinning method comprises the following technological parameters: spinning voltage: the injection rate of the injector is 0.35mL/h at 18kV, the receiving distance is 15cm, the ambient temperature is 25+/-2 ℃, the relative humidity is 55+/-2%, and the time is 4h.
The invention further provides a polyvinyl alcohol/citric acid nanofiber membrane which is prepared by the method.
The invention further provides a parathion electrochemical immunosensor, which comprises an electrode, parathion hapten-ovalbumin conjugate and a polyvinyl alcohol/citric acid nanofiber membrane, wherein the electrode is used as a substrate, the polyvinyl alcohol/citric acid nanofiber membrane is used for modifying the electrode, and the parathion hapten-ovalbumin conjugate is fixed on the modified electrode.
The porous structure formed among the nanofibers is beneficial to diffusion of substances (hydroquinone and hydrogen peroxide) participating in electrochemical reaction to an interface, improves the electron transfer rate, and realizes faster response of the biosensor.
Preferably, the electrode is a screen printed electrode that integrates a working electrode, a reference electrode and an auxiliary electrode.
Preferably, the parathion hapten-ovalbumin conjugate is composed of parathion hapten H 1 And coupling with ovalbumin OVA.
The invention further provides a preparation method of the parathion electrochemical immunosensor, which comprises the following steps:
and (3) adhering the polyvinyl alcohol/citric acid nanofiber membrane to a working electrode area of the pretreated electrode by using conductive adhesive, standing, adding an activating solution, activating for 1-3 hours at 2-6 ℃, adding parathion hapten-ovalbumin conjugate, coating completely at 2-6 ℃, adding a sealing solution, and reacting completely at 2-6 ℃ to obtain the electrode.
Preferably, the final concentration of parathion hapten-ovalbumin conjugate is 10 -3 ～1mg/mL。
Preferably, the activating solution is one or both of 1-ethyl- (3-dimethylaminopropyl) carbodiimide (EDC) or N-hydroxysuccinimide (NHS).
Preferably, the adding mode of the activating liquid and the sealing liquid is dripping.
Preferably, the blocking solution comprises a 1-3% bovine serum albumin solution.
Preferably, the coating time is 30-150 min.
Preferably, the reaction time is 1 to 3 hours.
Preferably, the blocking solution is added dropwise, and the reaction is carried out for 2 hours at 4 ℃.
Preferably, the standing time is 20 to 40 minutes.
Preferably, the pretreatment method of the electrode comprises the following steps: sequentially immersing the electrode in 50% ethanol solution and distilled water for 1-2 min, airing, and then putting the electrode into a solution containing 5mM Fe (CN) 6 3-/4- And testing the performance of the electrode in 0.1mol/L KCl solution, wherein the scanning speed is 100mV/s, the scanning voltage range is-0.6V, continuously scanning until the cyclic voltammogram is stable, and airing again to obtain the electrode.
The invention further protects application of the polyvinyl alcohol/citric acid nanofiber membrane and the parathion electrochemical immunosensor in detecting parathion.
Preferably, the method for detecting parathion comprises the following steps:
s1, preparing an enzyme-labeled antibody: coupling horseradish peroxidase with an anti-parathion nano antibody VHH9 by a sodium periodate method to obtain the anti-parathion nano antibody;
s2, preparing parathion standard solutions with the concentrations of 0, 0.006, 0.024, 0.098, 0.39, 1.56, 6.25, 25 and 100ng/mL respectively, uniformly mixing an equal volume of parathion standard solution and the solution of the enzyme-labeled antibody obtained in the step S1, dropwise adding the solution on a parathion electrochemical immunosensor, incubating for a period of time, flushing the solution with PBS, immersing the parathion electrochemical immunosensor in 1/15mol/L phosphoric acid buffer solution containing 0.5mmol/L hydroquinone respectively, adding 30% hydrogen peroxide, performing cyclic voltammetry scanning at a scanning voltage range of-0.6V and a scanning speed of 100mV/S, and measuring the change of a reduction peak current response value of a cyclic voltammetry curve before and after the hydrogen peroxide is added, wherein the concentration of the parathion standard solution is taken as a horizontal coordinate, and the change of the current response value corresponding to each concentration is taken as a vertical coordinate to obtain a standard curve. And substituting the change value of the current response in the sample liquid to be detected into a standard curve to quantitatively determine the concentration of parathion in the sample liquid to be detected.
Specifically, the preparation method of the enzyme-labeled antibody in the step S1 is as follows:
dissolving Horse Radish Peroxidase (HRP) in acetate buffer, adding NaIO dropwise 4 Stirring the solution at 2-6 ℃ for 20-40 min, adding glycol solution dropwise for reacting for 20-40 min, adding VHH9 antibody dropwise after the reaction is complete, stirring at 2-6 ℃ for 20-40 min, dialyzing overnight with carbonate buffer solution, and adding NaBH dropwise 4 Stirring the solution for 1-3 h at 2-6 ℃ in a dark place, adding an equal volume of saturated ammonium sulfate solution, reacting for 20-40 min at 2-6 ℃, standing for 0.5-1 h, centrifuging at 2-6 ℃, dissolving the precipitate in PBS, dialyzing with PBS overnight, changing the dialysate the next day, dialyzing for 12h, collecting the supernatant of the dialysate as an enzyme-labeled antibody, mixing, packaging, and storing at-20 ℃ for later use.
During detection, adding a dropping liquid (an equal volume of enzyme-labeled antibody solution and a sample liquid to be detected) to incubate on a working electrode area of the parathion electrochemical immunosensor for a period of time, wherein parathion hapten-ovalbumin conjugate fixed on the surface of the electrode and parathion in the sample liquid to be detected can compete for binding the enzyme-labeled antibody, and a part of the enzyme-labeled antibody is combined with free parathion; and (3) a part of enzyme-labeled antibody is combined with parathion hapten-ovalbumin conjugate on the parathion electrochemical immunosensor, PBS is used for washing to remove unbound enzyme-labeled antibody, parathion and a complex of the enzyme-labeled antibody and free parathion, the parathion electrochemical immunosensor is immersed in 1/15mol/L phosphoric acid buffer solution containing 0.5mmol/L hydroquinone, 30% hydrogen peroxide is added, cyclic voltammetry scanning test is carried out on the parathion electrochemical immunosensor, and the concentration of parathion in a sample solution to be detected is quantitatively determined through the change of front and back reduction peak current values in a cyclic voltammetry curve. The redox peaks generated by cyclic voltammograms are derived from horseradish peroxidase (HRP) versus H 2 O 2 The reactions that occur during the detection are as follows:
(1)HRP(Fe 3+ )+H 2 O 2 compound (I) +H 2 O
(2) Compound (I) +HQ→compound (II) +Q
(3) Compound (II) +HQ→HRP (Fe) 3+ )+Q+H 2 O
(4) Q+H++2e- & gtHQ (electrode reaction)
Wherein HQ and Q are the oxidation states of hydroquinone and hydroquinone, respectively. At H 2 O 2 In the presence of HRP can be oxidized to compound (I), which can oxidize HQ to Q by a double electron transfer process, accordingly, HQ is greatly reduced, Q is greatly increased, equation (4) is the final electrochemical reaction on the electrode, the reduction peak current increases with increasing Q, so that H can be added 2 O 2 The concentration of parathion was quantitatively determined by measuring the magnitude of the change in the front and rear peak current values.
Preferably, in step S2, the concentration of the enzyme-labeled antibody solution is 1.8mg/mL, and the enzyme-labeled antibody solution is diluted by 50-800 times for use.
Preferably, in step S2, the incubation time is 20 to 60min.
Preferably, in step S2, the concentration of hydrogen peroxide is 0.5-2.5 mmol/L.
The invention has the following beneficial effects:
according to the invention, polyvinyl alcohol and citric acid are adopted to prepare the polyvinyl alcohol/citric acid nanofiber membrane through an electrostatic spinning method and a thermal crosslinking reaction, the fiber diameter in the membrane is uniformly distributed, the stable fiber morphology can be maintained, the hydrophobicity of the membrane can be greatly improved, the aim of detecting parathion in an aqueous solution of the membrane is fulfilled, the electrochemical response speed is accelerated, and an electrochemical response signal is amplified; meanwhile, after the membrane is prepared into the parathion electrochemical immunosensor, the detection range is wide, the specificity is good, the sensitivity is high, the stability and the repeatability are good, the minimum detection limit is greatly reduced, the operation is simple and convenient, the cost is low, and the membrane can be widely applied to quantitative detection of parathion in vegetables and fruits.
Drawings
FIG. 1 is an SEM image of each step of a polyvinyl alcohol/citric acid nanofiber membrane and fiber diameter distributionFIG. 1a is an SEM image and fiber diameter distribution diagram of a nanofiber membrane, FIG. 1b is an SEM image and fiber diameter distribution diagram of a polyvinyl alcohol/citric acid nanofiber membrane, FIG. 1c is an SEM image and fiber diameter distribution diagram of an activated polyvinyl alcohol/citric acid nanofiber membrane, FIG. 1d is a bond H 1 SEM images and fiber diameter distribution diagrams of polyvinyl alcohol/citric acid nanofiber membrane after OVA, SEM images and fiber diameter distribution diagrams of polyvinyl alcohol/citric acid nanofiber membrane after dropping a blocking solution reaction, and SEM images and fiber diameter distribution diagrams of polyvinyl alcohol/citric acid nanofiber membrane after VHH9-HRP incubation, respectively, in fig. 1 e.
FIG. 2 is a cyclic voltammogram of an electrode at each step in the preparation process for the parathion electrochemical immunosensor of example 1 and the electrochemical immunosensor of comparative example 1.
FIG. 3 is an SEM image of a polyvinyl alcohol/citric acid nanofiber membrane prepared from different concentrations of polyvinyl alcohol and different concentrations of citric acid, and FIG. 3a is an SEM image of a polyvinyl alcohol concentration of 14wt% and a fiber diameter distribution diagram; FIG. 3b is an SEM image and fiber diameter distribution plot at a polyvinyl alcohol concentration of 16 wt%; FIG. 3c is an SEM image and fiber diameter distribution plot at a polyvinyl alcohol concentration of 18wt%, wherein the amount of citric acid added is 15% of the polyvinyl alcohol.
FIG. 4 is an SEM image of a polyvinyl alcohol/citric acid nanofiber membrane prepared by different addition amounts of citric acid, and the addition amount of citric acid in FIG. 4a is 10% of that of the polyvinyl alcohol, and the distribution diagram of the fiber diameter; FIG. 4b is a SEM image and fiber diameter distribution chart of citric acid added at 15% of polyvinyl alcohol; in FIG. 4c, the amount of citric acid added is 20% of that of the polyvinyl alcohol, and the concentration of the polyvinyl alcohol is 16% by weight.
Fig. 5 is a graph of test results of polyvinyl alcohol/citric acid nanofiber membranes prepared at different thermal crosslinking temperatures, fig. 5a is a SEM graph of polyvinyl alcohol/citric acid nanofiber membranes prepared at 125 ℃ thermal crosslinking temperatures, fig. 5b is a SEM graph of polyvinyl alcohol/citric acid nanofiber membranes prepared at 145 ℃ thermal crosslinking temperatures, fig. 5c is a SEM graph of polyvinyl alcohol/citric acid nanofiber membranes prepared at 165 ℃ thermal crosslinking temperatures, fig. 5d is a SEM graph of polyvinyl alcohol/citric acid nanofiber membranes prepared at 125 ℃ thermal crosslinking temperatures after being immersed in PBS, fig. 5e is a SEM graph of polyvinyl alcohol/citric acid nanofiber membranes prepared at 145 ℃ thermal crosslinking temperatures after being immersed in PBS, fig. 5f is a SEM graph of polyvinyl alcohol/citric acid nanofiber membranes prepared at 165 ℃ thermal crosslinking temperatures after being immersed in PBS, and fig. 5g is a graph of contact angle measurement results of polyvinyl alcohol/citric acid nanofiber membranes prepared at different thermal crosslinking temperatures.
Fig. 6 is a graph of test results of a polyvinyl alcohol/citric acid nanofiber membrane prepared at different thermal crosslinking times, fig. 6a is a SEM graph of a polyvinyl alcohol/citric acid nanofiber membrane prepared at a thermal crosslinking time of 0.5h, fig. 6b is a SEM graph of a polyvinyl alcohol/citric acid nanofiber membrane prepared at a thermal crosslinking time of 1.0h, fig. 6c is a SEM graph of a polyvinyl alcohol/citric acid nanofiber membrane prepared at a thermal crosslinking time of 2.0h, fig. 6d is a SEM graph of a polyvinyl alcohol/citric acid nanofiber membrane prepared at a thermal crosslinking time of 4.0h, fig. 6e is a SEM graph of a polyvinyl alcohol/citric acid nanofiber membrane prepared at a thermal crosslinking time of 0.5h after immersing in PBS, fig. 6f is a SEM graph of a polyvinyl alcohol/citric acid nanofiber membrane prepared at a thermal crosslinking time of 1.0h after immersing in PBS, fig. 6g is a SEM graph of a contact angle of a polyvinyl alcohol/citric acid nanofiber membrane prepared at a thermal crosslinking time of 2.0h after immersing in PBS, and fig. 6i is a graph of a graph after a thermal crosslinking time of a polyvinyl alcohol/citric acid nanofiber membrane prepared at a thermal crosslinking time of 4h after immersing in PBS.
Fig. 7 is an ultraviolet scan of the enzyme-labeled antibody VHH9-HRP, parathion nanobody VHH9, and horseradish peroxidase HRP.
FIG. 8 is a graph showing the effect of ELISA on the titer and inhibition ratio of enzyme-labeled antibodies.
Fig. 9 is a standard curve for parathion detection by an electrochemical immunosensor for parathion.
Fig. 10 is a schematic diagram of the principle of the parathion electrochemical immunosensor for detecting parathion.
Fig. 11 is a graph of the linear relationship between parathion electrochemical immunosensory and UPLC detection.
FIG. 12 is a graph showing comparison of the cross-over rates of detection of compounds by parathion electrochemical immunosensor.
Fig. 13 is a graph of the effect of the electrochemical immunosensor for parathion on detecting parathion reusability.
Fig. 14 is a graph showing the effect of an electrochemical immunosensor for parathion on detecting parathion stability.
Detailed Description
The invention is further illustrated in the following drawings and specific examples, which are not intended to limit the invention in any way. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art.
Reagents and materials used in the following examples are commercially available unless otherwise specified.
Example 1 preparation of parathion electrochemical immunosensor
S1, adding 3.2g of polyvinyl alcohol (PVA) into 20mL of distilled water, heating and stirring at 80 ℃ for about 4 hours until the PVA is completely dissolved, cooling, adding 0.48g of Citric Acid (CA), stirring at room temperature until the CA is completely dissolved to obtain 16% PVA/15% CA solution, slowly filling the 16% PVA/15% CA solution into a 10mL syringe to avoid bubble generation, fixing the syringe filled with the 16% PVA/15% CA solution on a push injection clamping groove, connecting an injection pump of an electrostatic spinning machine with an anode of a high-voltage power supply, connecting a receiving plate with a cathode, using a No. 20 stainless steel needle to sample, controlling the sample injection rate by the injection pump, switching on the power supply, stretching the 16% PVA/15% CA solution at the needle under the action of a high-voltage electric field, and injecting the 16% PVA/15% CA solution onto the receiving plate to form a nanofiber membrane;
the technological parameters are respectively as follows: spinning voltage: 18kV, the injection rate of the injector is 0.35mL/h, the receiving distance is 15cm, the ambient temperature is 25+/-2 ℃, the relative humidity is 55+/-2%, the time is 4h, and after spinning is finished, the nanofiber membrane is thermally crosslinked for 2h at 145 ℃ and then is placed in a dryer to obtain the polyvinyl alcohol/citric acid nanofiber membrane;
s2, sequentially immersing screen printing electrodes (SPCE) in 50% ethanol solution and distillingUltrasonic treatment in water for 1-2 min, air drying, and adding SPCE into water containing 5mM Fe (CN) 6 3- / 4- Testing the performance of the screen printing electrode in 0.1mol/L KCl solution, wherein the scanning speed is 100mV/s, the scanning voltage range is-0.6V, and continuously scanning until the cyclic voltammetry curve is stable, and airing again for later use;
a3 mM polyvinyl alcohol/citric acid nanofiber membrane (NFM) was adhered to the working electrode area of the screen-printed electrode with Silver Conductive Paste (SCP), left at room temperature for 30min, 100. Mu.L of EDC/NHS solution (1 mM) was added dropwise to the surface of the NFM, activated at 4℃for 2H (the purpose of activating-COOH on the NFM for subsequent reaction), rinsed 5 times with PBS, and 10. Mu.L of parathion hapten-ovalbumin conjugate (H) 1 -OVA), coating at 4℃for 2H, washing unbound H with PBS 1 -OVA, 100 μl of blocking solution containing 1% BSA was added dropwise, reacted at 4 ℃ for 2h (to block unbound active groups on NFM surface), rinsed 5 times with PBS and dried to prepare parathion electrochemical immunosensor.
Structural characterization:
for the prepared nanofiber membrane, the polyvinyl alcohol/citric acid nanofiber membrane, the activated polyvinyl alcohol/citric acid nanofiber membrane and the combination H 1 And carrying out SEM characterization on the polyvinyl alcohol/citric acid nanofiber membrane after OVA, the polyvinyl alcohol/citric acid nanofiber membrane after dripping the sealing liquid for reaction, and the polyvinyl alcohol/citric acid nanofiber membrane after dripping VHH9-HRP for 40min of incubation, and carrying out statistics on the fiber diameter according to an SEM graph to obtain a fiber diameter distribution diagram.
As shown in fig. 1: the nanofiber membrane (figure 1 a) has almost unchanged fiber morphology in the process of preparing the polyvinyl alcohol/citric acid nanofiber membrane (figure 1 b) after thermal crosslinking, the fiber of the polyvinyl alcohol/citric acid nanofiber membrane slightly swells (figure 1 c) after being immersed in EDC/NHS solution for 2H of activation, and H is added 1 After the OVA solution is coated for 2 hours (figure 1 d) and the dripping sealing liquid is reacted for 2 hours (figure 1 e), the water-absorbing swelling diameter of the fiber becomes thicker, some fibers are adhered, the nano fiber adhesion condition becomes serious after the dripping VHH9-HRP is incubated for 40min, but the fiber morphology (figure 1 f) can still be kept, which indicates that the nano fiber membrane is in the following stateThe parathion electrochemical immunosensor can provide high surface area and high porosity in the preparation process, so that the electron transfer rate is quickened, and the sensitivity of the electrochemical sensor is improved.
Electrochemical characterization:
placing screen printing electrode, silver conductive adhesive modified electrode, polyvinyl alcohol/citric acid nanofiber membrane modified electrode, H1-OVA combined electrode, sealing liquid reaction dropwise and enzyme-labeled antibody (VHH 9-HRP) dropwise into a solution containing 5mM Fe (CN) 6 3-/4- And 0.1mol/KCl, the scanning speed is 100mV/s, the scanning voltage range is-0.6V, and the cyclic voltammogram of each electrode is obtained.
As shown in fig. 2, the cyclic voltammogram of the screen printed electrode (Bare SPCE) (curve a) exhibited a symmetrical redox peak with a reduction peak current value of 139 μa; then, the reduction peak current value increased to 347 μA (curve b) after the electrode was modified with the silver conductive paste, probably because the silver conductive paste has conductivity to promote electron transfer at the electrode interface; after the polyvinyl alcohol/citric acid nanofiber membrane is modified on the electrode (curve c), the reduction peak current value is obviously increased to 1230 mu A, and the unique porous structure of the polyvinyl alcohol/citric acid nanofiber membrane accelerates the transfer of electrons to the surface of the electrode and amplifies the electrochemical response signal; sequentially adding H to the electrodes 1 Reduction peak current values after OVA and blocking solution (1% bovine serum albumin) decreased to 919. Mu.A (curve d) and 817. Mu.A (curve e) due to H 1 -OVA and bovine serum albumin are non-conductive biomacromolecules that block electron transfer, indicating successful binding of antigen and bovine serum albumin to polyvinyl alcohol/citric acid nanofiber membrane modified electrodes; the electrode after dropping VHH9-HRP had its original peak current value decreased to 746. Mu.A (curve f) because of H 1 The complex formed by specific binding of OVA and VHH9-HRP impedes electron transfer, and these results indicate successful production of parathion electrochemical immunosensor.
Example 2 preparation of electrochemical immunosensor
The difference from example 1 is that:
s1, heat crosslinking is carried out for 4 hours.
The method, reagents and amounts of addition in the remaining steps were the same as in example 1.
Comparative example 1 preparation of electrochemical immunosensor
The difference from example 1 is that:
s1, pouring a 16% PVA/15% CA solution into a silica gel film tool, and placing the silica gel film tool in a fume hood for 24 hours to volatilize water to obtain a polyvinyl alcohol/citric acid casting film (PVA/CA CM).
The method, reagents and amounts of addition in the remaining steps were the same as in example 1.
Electrochemical characterization:
placing an electrode modified by a polyvinyl alcohol/citric acid casting film (PVA/CA CM) into a film containing 5mM Fe (CN) 6 3-/4- And 0.1mol/KCl, the scanning speed is 100mV/s, the scanning voltage range is-0.6V, and the cyclic voltammetry curve of the electrode modified by the polyvinyl alcohol/citric acid casting film (PVA/CA CM) is obtained.
As shown in fig. 2, the electrode modified with the polyvinyl alcohol/citric acid casting film (PVA/CA CM) prepared in comparative example 1 has a reduction peak current value lower than that of the screen printing electrode (curve g), the reduction peak current value of the polyvinyl alcohol/citric acid nanofiber film modified with example 1 is 17 times that of the electrode modified with the polyvinyl alcohol/citric acid casting film (PVA/CA CM) prepared in comparative example 1, which indicates that the casting film without porous structure prepared in comparative example 1 may hinder electron transfer,
comparative example 2 preparation of electrochemical immunosensor
The difference from example 1 is that:
s1, the addition amount of the polyvinyl alcohol is 2.8g, and the concentration of the polyvinyl alcohol is 14%, so that 14% PVA/15% CA solution is obtained.
The method, reagents and amounts of addition in the remaining steps were the same as in example 1.
Comparative example 3 preparation of electrochemical immunosensor
The difference from example 1 is that:
s1, the addition amount of the polyvinyl alcohol is 3.6g, and the concentration of the polyvinyl alcohol is 18%, so as to obtain 18% PVA/15% CA solution.
The method, reagents and amounts of addition in the remaining steps were the same as in example 1.
Comparative example 4 preparation of electrochemical immunosensor
The difference from example 1 is that:
s1, the addition amount of citric acid is 0.32g, and the concentration of the citric acid is 10%, so that 16% PVA/10% CA solution is obtained.
The method, reagents and amounts of addition in the remaining steps were the same as in example 1.
Comparative example 5 preparation of electrochemical immunosensor
The difference from example 1 is that:
s1, the addition amount of citric acid is 0.36g, and the concentration of the citric acid is 20%, so that 16% PVA/20% CA solution is obtained.
The method, reagents and amounts of addition in the remaining steps were the same as in example 1.
The viscosity and conductivity of the PVA/CA solutions prepared in example 1 and comparative examples 1 to 5 were measured, and the polyvinyl alcohol/citric acid films obtained in example 1 and comparative examples 1 to 5 were SEM-characterized, and fiber diameter distribution maps were obtained by counting fiber diameters according to SEM images.
As shown in table 1, when the CA concentration was constant, the viscosity of the PVA/CA solution increased with increasing PVA content, from 1.534pa·s to 3.851pa·s, while the conductivity did not change much. When the viscosity of the PVA/CA solution is too large or too small, unbalanced surface tension of jet flow and electric field force are caused, so that the jet flow is unstable, and finally the diameter distribution of the obtained nanofiber is uneven; when the CA concentration was increased from 10% to 20%, the conductivity of the PVA/CA solution was increased from 1.340ms/cm to 2.013ms/cm because citric acid was a weak electrolyte.
Table 1: viscosity and conductivity (n=3) of spinning solutions of different concentrations of polyvinyl alcohol and citric acid
As shown in FIG. 3, spindle-like fibers were not present at a PVA concentration of 16wt%, and the nanofiber diameters were relatively uniform, with the fiber diameters concentrated at 220nm to 410nm (FIG. 3 b). At PVA concentrations of 14wt% and 18wt%, part of the fibers were spindle-shaped (FIGS. 3a, 3 c), and the fiber diameter distribution range was widened.
As shown in fig. 4, when the CA content was increased from 10% (fig. 4 a) to 15% (fig. 4 b), the average diameter of the nanofibers decreased. Studies have shown that increasing the conductivity of the solution can reduce the diameter of the fiber, mainly due to the surface charge distribution around the electrospun jet and the change in tangential electric field along the surface of the jet. When the CA content increased to 20% (fig. 4 c), the morphology of the nanofibers was degraded, resulting in spindle-like fibers.
Comparative example 6 preparation of electrochemical immunosensor
The difference from example 1 is that:
s1, the temperature of thermal crosslinking is 125 ℃.
The method, reagents and amounts of addition in the remaining steps were the same as in example 1.
Comparative example 7 preparation of electrochemical immunosensor
The difference from example 1 is that:
s1, the temperature of thermal crosslinking is 165 ℃.
The method, reagents and amounts of addition in the remaining steps were the same as in example 1.
Comparative example 8 preparation of electrochemical immunosensor
The difference from example 1 is that:
s1, heat crosslinking is carried out for 0.5h.
The method, reagents and amounts of addition in the remaining steps were the same as in example 1.
Comparative example 9 preparation of electrochemical immunosensor
The difference from example 1 is that:
s1, heat crosslinking is carried out for 1h.
The method, reagents and amounts of addition in the remaining steps were the same as in example 1.
Structural characterization:
the polyvinyl alcohol/citric acid nanofiber membranes prepared in examples 1 and 2 and comparative examples 6 to 9 were subjected to SEM characterization and contact angle measurement, and the polyvinyl alcohol/citric acid nanofiber membranes washed with PBS were subjected to SEM characterization.
As shown in fig. 5: it can be seen that the morphology of the resulting polyvinyl alcohol/citric acid nanofibers is not very different at different thermal crosslinking temperatures (fig. 5 a-5 c), the resulting polyvinyl alcohol/citric acid nanofiber membranes at 125 ℃ have severe swelling and blocking of the fibers after immersion in PBS (fig. 5 d), and when the thermal crosslinking temperature is increased to 145 ℃ (fig. 5 e) and 165 ℃ (fig. 5 f), the polyvinyl alcohol/citric acid nanofiber membranes only slightly swell in PBS solution, probably because the crosslinking reaction between polyvinyl alcohol and citric acid is more complete at higher temperatures, resulting in better water stability of the polyvinyl alcohol/citric acid nanofiber membranes.
The contact angle of the polyvinyl alcohol/citric acid nanofiber membrane increased with increasing thermal crosslinking temperature, i.e. the hydrophobicity of the polyvinyl alcohol/citric acid nanofiber membrane increased, consistent with SEM results (fig. 5 g). But too high a temperature may result in a decrease in tensile strength and a color change of the film. After thermal crosslinking at 165 ℃ for 2 hours, the electrospun film turns slightly yellow, the discoloration is mainly due to unsaturated acid formation caused by citric acid dehydration, and the film is discolored. Therefore, the optimum crosslinking temperature is 145 ℃.
As shown in fig. 6: the fiber morphology of the polyvinyl alcohol/citric acid nanofiber membrane prepared by thermal crosslinking at 145 ℃ is not different (fig. 6 a-6 d), the fiber of the polyvinyl alcohol/citric acid nanofiber membrane prepared by thermal crosslinking at 0.5h (fig. 6 e) and 1h (fig. 6 f) is severely swelled and adhered after being immersed in PBS, and the fiber of the polyvinyl alcohol/citric acid nanofiber membrane prepared by thermal crosslinking at 2h (fig. 6 g) and 4h (fig. 6 h) is only slightly swelled in PBS solution. It is possible that the thermal crosslinking reaction is incomplete due to the too short thermal crosslinking time, and increasing the thermal crosslinking time can increase the crosslinking degree and the hydrophobicity of the nanofibers. The contact angle of the polyvinyl alcohol/citric acid nanofiber membrane increased from 30.36 ° to 57.73 ° (fig. 6 i) as the thermal crosslinking time increased, i.e., the hydrophobicity of the nanofiber membrane increased, consistent with SEM results. Therefore, the optimal crosslinking time is 2h.
Example 3 sensitivity and accuracy study of parathion electrochemical immunosensor detection of parathion
Preparation of enzyme-labeled antibody:
transforming the gene of the anti-parathion nano antibody VHH9 into E.coli BL21 (DE 3), and obtaining the anti-parathion nano antibody VHH9 after expression and purification;
12mg of horseradish peroxidase (HRP) was weighed and dissolved in 4mL of acetate buffer (0.2 moL/L, pH 5.6), and freshly prepared 2mL of NaIO was added dropwise 4 Stirring the solution (0.1 moL/L) at 4deg.C for 30min, adding dropwise 2mL of ethylene glycol solution (0.16 moL/L), reacting at room temperature for 30min, adding dropwise 6mg of VHH9 antibody, stirring at 4deg.C for 30min, dialyzing overnight with carbonate buffer (0.05 moL/L, pH 9.6), taking out the dialysate, and adding dropwise 0.4mL of 5mg/mL of NaBH 4 Stirring the solution for 2 hours at 4 ℃ in a dark place, adding an equal volume of saturated ammonium sulfate solution, reacting for 30 minutes at 4 ℃ and standing for 1 hour, centrifuging for 20 minutes at 4000r/min at 4 ℃, discarding the supernatant, draining, dissolving the precipitate in 2mL of PBS (0.01 mol/L, pH 7.4), placing into a dialysis bag, dialyzing with PBS (0.01 mol/L, pH 7.4) for overnight, changing the dialyzate the next day for 12 hours, collecting the supernatant of the dialyzate, namely the enzyme-labeled antibody VHH9-HRP, mixing, packaging, and storing at-20 ℃ for later use.
The ultraviolet scan of the enzyme-labeled antibody, parathion nanobody VHH9 and horseradish peroxidase is shown in fig. 7: the peak positions of VHH9-HRP were significantly shifted relative to VHH9 and HRP. The titers and inhibition rates of enzyme-labeled antibodies obtained by ELISA are shown in fig. 8: the titer of the enzyme-labeled antibody can reach 1:6400, the inhibition rate was 91.66%, and the preparation of the enzyme-labeled antibody VHH9-HRP was successful.
Standard curve:
parathion standard solutions with concentrations of 0, 0.006, 0.024, 0.098, 0.39, 1.56, 6.25, 25 and 100ng/mL were prepared, and 10. Mu.L of a dropping solution (equal volume) was added dropwiseEnzyme-labeled antibody solution and parathion standard solution) were incubated at 4 ℃ for 40min in the working electrode area of the parathion electrochemical immunosensor, and the coating antigen immobilized on the electrode surface and parathion in the drop solution simultaneously competed for binding to VHH9-HRP. After PBS washing, the parathion electrochemical immunosensor was immersed in 10mL of 1/15mol/L phosphate buffer containing 0.5mmol/L hydroquinone, 13.77. Mu.L 30% hydrogen peroxide was added, and cyclic voltammetry scanning was performed at a voltage of-0.6V to +0.6V, and the standard curve was shown in FIG. 9, with the curve equation of ΔI (μA) = (126.83 + -0.45) + (33.23+ -0.29) LogC Parathion (ng/L), the detection range is 0.01-100 ng/mL, and the detection limit is 0.00226ng/mL.
Wherein Δi is the change in the peak current response of cyclic voltammogram reduction before and after hydrogen peroxide addition.
Detection of parathion in actual samples:
fresh cucumber, orange and cabbage are purchased from local markets, washed with water, dried in the air, purified by a dispersion solid-phase extraction method, and added with parathion solutions with different concentrations (0, 1, 5 and 10 ng/mL) to obtain the sample liquid to be detected. The sample solution to be detected is added to the parathion electrochemical immunosensor prepared in the embodiment 1, and the parathion concentration in the sample solution to be detected is detected according to the following steps: uniformly mixing an equal volume of sample liquid to be detected and an enzyme-labeled antibody, taking 10 mu L of the mixture, dripping the mixture on a working electrode area of a parathion electrochemical immunosensor for 40min, flushing the mixture by PBS, immersing the electrode in 10mL of 1/15mol/L phosphoric acid buffer solution containing 0.5mmol/L hydroquinone, adding 13.77 mu L of 30% hydrogen peroxide, and measuring the concentration of parathion in the sample liquid to be detected by comparing the change of the peak current value before and after the hydrogen peroxide with the curve of the sensor (the detection principle schematic diagram is shown in figure 10), and repeating the test for three times. Meanwhile, ultra-high performance liquid chromatography (UPLC) is also adopted to detect and add parathion vegetable samples with different concentrations (0, 1, 5 and 10 ng/mL).
The detection results are shown in table 2, the average standard adding recovery rate of the parathion electrochemical immunosensor of the embodiment 1 for detecting the cucumbers, the oranges and the cabbages is 96.20-114.61%, and the calculation formula of the average standard adding recovery rate is as follows:the coefficient of variation (CV, ratio of standard deviation to average number of detection) is 1.06% -5.28%, the average standard adding recovery rate of cucumber, orange and celery cabbage detected by UPLC is 76.30% -105.60%, the average standard adding recovery rate meets the quantitative methodology requirement within the range of 80% -120%, the coefficient of variation is 1.25% -11.80%, the smaller the coefficient of variation value (less than 15%), the more concentrated the data, the more stable the data, and the better the precision of the test method. The results of the above parathion electrochemical immunosensor and UPLC detection were compared linearly as shown in fig. 11: the correlation between the embodiment and the detection result of the UPLC is good, and the correlation coefficient of the embodiment and the detection result of the UPLC is 0.9964 (the measured results of the embodiment and the UPLC are close to each other), which indicates that the sensor has good accuracy when being used for detecting an actual sample.
In addition, two samples added with low-concentration parathion (0.05 and 0.10 ng/mL) are detected by using parathion electrochemical immunosensor, as shown in table 2, the average standard adding recovery rate of cucumber, orange and celery cabbage is 89.15% -116.13%, and the variation coefficient is 1.83% -10.92%, which shows that the sensor has higher sensitivity when being used for detecting actual samples.
Table 2: results of parathion electrochemical immunosensor and ultra performance liquid chromatography detection of parathion in vegetables (n=3)
Example 4 specificity study of parathion electrochemical immunosensor detection of parathion
The compounds triazophos (triazophos), quiniphos (quinaphos), coumaphos (coumaphos), phorate (phorate) and phoxim (phoxim) similar to parathion (parathion) were selected and the effect of the above compound solutions and their mixture solutions (each at a concentration of 1 ng/mL) on parathion electrochemical immunosensor was investigated.
The detection method comprises the following steps: to the parathion electrochemical immunosensor prepared in example 1, each of compound sample solutions having a similar structure to parathion (parathion) and a mixture solution thereof were added at a concentration of 1ng/mL, and a change (Δi) in a peak current response value of a cyclic voltammogram before and after the addition of hydrogen peroxide was measured as follows: after uniformly mixing an equal volume of sample solution and an enzyme-labeled antibody, 10 mu L of the sample solution is dripped on a working electrode area of a parathion electrochemical immunosensor, the electrode is immersed in 10mL of a mixed solution containing 0.5mmol/L hydroquinone and 1/15mol/L phosphate buffer solution, 13.7 mu L of 30% hydrogen peroxide is added to obtain delta I of each sample solution, and the test is repeated three times.
The specificity of the immunosensor is expressed by cross-reactivity (CR), as follows:
wherein DeltaI [blank] Is the change of the current response value of a blank control group without parathion solution, delta I [Interfernce] Is that the current response value of each compound solution of parathion electrochemical immunosensor is changed, delta I is detected under the condition of the same concentration (1 ng/mL) [parathion] The current response value of the parathion electrochemical immunosensor in the parathion standard solution with the detection concentration of 1ng/mL is changed. The crossing rate of each compound solution was 100% for parathion, and 11.31%, 9.46% and 5.31% for triazophos (triazophos), quiniphos (coumaphos) and coumaphos (coumaphos), respectively, and 1.15% and 2.54% for phorate (phorate) and phoxim (phoxim), respectively, as shown in fig. 12, indicating that the prepared immunosensor had higher specificity.
Example 5 detection of parathion electrochemical immunosensor reusability study of parathion
Firstly, parathion sample solutions with concentrations of 0.1, 1 and 10ng/mL are detected by an electrochemical immunosensor of parathion, and after detection, the solution is immersed in a 0.2M glycine-hydrochloric acid buffer solution with pH value of 2.8 for 5 minutes to break H 1 -OVA with VHH9Binding and then washing with PBS (ph=7.4) gave a regenerated sensor, which was again tested on parathion samples of the same concentration. The above steps are repeated and the regenerated sensor is used for detection, and the result is shown in fig. 13: the electrochemical response value (DeltaI) of the first detection is 100%, the activity is maintained to be more than 90% after the second regeneration, the activity is maintained to be about 85% after the third regeneration, and the sensor after the fourth regeneration is reduced to about 75% (the activity is reduced to 66% after the 2 regenerations of the electrochemical immunosensor prepared by EL-Moghazy et al). The results show that the parathion electrochemical immunosensor prepared by the method has good reusability. The reduced activity of the sensor after multiple regenerations during detection may be due to denaturation of bovine serum albumin or destruction of the structure of the polyvinyl alcohol/citric acid nanofiber membrane during the sensor's soaking and washing in the acidic glycine-hydrochloric acid buffer.
Example 6 stability study of parathion electrochemical immunosensor detection of parathion
The stability of the sensor has an important influence on the practical application of the sensor, and the stability of the sensor is represented by periodically detecting the response value of the sensor to the current signal. The parathion electrochemical immunosensor is sealed by a sealing bag and placed in a refrigerator (4 ℃), and three parathion electrochemical immunosensors are taken out every other week to detect sample solutions with parathion concentration of 1ng/mL and 0.1ng/mL respectively.
As shown in FIG. 14, the current response value of the parathion electrochemical immunosensor is basically unchanged after the parathion electrochemical immunosensor is stored for three weeks in a refrigerator (4 ℃), the current response value of the parathion electrochemical immunosensor is still kept at about 90% after the parathion electrochemical immunosensor is stored for six weeks, and the current response value of the parathion electrochemical immunosensor is kept at about 85% after the parathion electrochemical immunosensor is stored for nine weeks, so that the sensor has good stability.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
Claims (7)
1. The parathion electrochemical immunosensor is characterized by comprising an electrode, parathion hapten-ovalbumin conjugate and a polyvinyl alcohol/citric acid nanofiber membrane, wherein the electrode is taken as a substrate, the polyvinyl alcohol/citric acid nanofiber membrane is used for modifying the electrode, and the parathion hapten-ovalbumin conjugate is fixed on the modified electrode;
the preparation method of the polyvinyl alcohol/citric acid nanofiber membrane comprises the following steps:
and adding polyvinyl alcohol into water, heating until the polyvinyl alcohol is completely dissolved, cooling, adding citric acid, preparing a nanofiber membrane by an electrostatic spinning method, and thermally crosslinking the nanofiber membrane at 105-185 ℃ for 0.5-6 hours to obtain the polyvinyl alcohol/citric acid nanofiber membrane.
2. The parathion electrochemical immunosensor of claim 1, wherein the concentration of the polyvinyl alcohol is 8-20 wt%.
3. The parathion electrochemical immunosensor of claim 1, wherein the mass of the citric acid is 5-25% of the mass of the polyvinyl alcohol.
4. The parathion electrochemical immunosensor of claim 1, wherein the temperature of the thermal cross-linking is 125-165 ℃.
5. The method for preparing the parathion electrochemical immunosensor according to any one of claims 1 to 4, which is characterized by comprising the following steps:
and (3) adhering the polyvinyl alcohol/citric acid nanofiber membrane to a working electrode area of the pretreated electrode by using conductive adhesive, standing, adding an activating solution, activating for 1-3 hours at 2-6 ℃, adding parathion hapten-ovalbumin conjugate, coating completely at 2-6 ℃, adding a sealing solution, and reacting completely at 2-6 ℃ to obtain the electrode.
6. The process according to claim 5, whereinThe final concentration of parathion hapten-ovalbumin conjugate is 10 -3 ~1 mg/mL。
7. Use of the parathion electrochemical immunosensor of any one of claims 1 to 4 for detecting parathion.
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| US11801328B2 (en) * | 2017-07-28 | 2023-10-31 | Universitá Degli Studi Di Pavia | Electrospun nanofibers and membrane |
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