CN118010998B - A chemiluminescent immunosensor for detecting zearalenone and its application - Google Patents
A chemiluminescent immunosensor for detecting zearalenone and its application Download PDFInfo
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- CN118010998B CN118010998B CN202410134063.XA CN202410134063A CN118010998B CN 118010998 B CN118010998 B CN 118010998B CN 202410134063 A CN202410134063 A CN 202410134063A CN 118010998 B CN118010998 B CN 118010998B
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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
- 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
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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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
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/76—Chemiluminescence; Bioluminescence
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- 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
- G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
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- 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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- 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
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54346—Nanoparticles
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- Urology & Nephrology (AREA)
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- General Health & Medical Sciences (AREA)
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- Biotechnology (AREA)
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Abstract
The invention discloses a chemiluminescent immunosensor for detecting zearalenone and application thereof, wherein the chemiluminescent immunosensor comprises a chemiluminescent probe and a chemiluminescent immunosensor substrate, and the chemiluminescent probe is prepared by incubating a CuCo 2O4 nanomaterial solution with a ZEN antibody to obtain the chemiluminescent immunosensor; the preparation method of the chemiluminescent immunosensor substrate comprises the steps of activating carboxyl resin beads to obtain activated resin beads, and incubating the activated resin beads with ZEN coating antigen to obtain the chemiluminescent immunosensor substrate for detecting zearalenone. The chemiluminescent immunosensor for detecting zearalenone provided by the invention has excellent sensitivity, stability and reproducibility, is used for detecting the content of ZEN in foods such as corn and wheat by adopting a flow injection chemiluminescent immunoassay method, realizes quick and ultrasensitive detection of ZEN, has the advantages of simplicity in operation, cost effectiveness, high sensitivity and the like, and has good practical value and practical significance.
Description
Technical Field
The invention relates to the technical field of chemiluminescent immunosensors, in particular to a chemiluminescent immunosensor for detecting zearalenone and application thereof.
Background
Zearalenone (ZEN) is a mycotoxin produced by fusarium fungi and contamination of food and feed with Zearalenone has evolved into an important global problem. ZEN has toxic effects, mainly in terms of reproductive toxicity, hepatotoxicity, immune toxicity, genetic toxicity, carcinogenicity, etc. In addition, the chemical structure of ZEN is very stable and not easily destroyed even at high temperature, so ZEN has been a major goal of monitoring quality and safety of cereal foods and feeds. To date, detection of ZEN has typically been high performance liquid chromatography, gas chromatography-mass spectrometry, liquid chromatography-mass spectrometry, and enzyme-linked immunosorbent assay (ELISA). Although chromatography is simple to operate and rapid in detection, expensive instrumentation, large amounts of organic solvents, and poor selectivity and sensitivity are required. Furthermore, although conventional ELISA is a sensitive detection method, in most cases, a professional is required to perform the operation in a laboratory, and the subsequent steps are numerous and very time-consuming. Against these background, there is an urgent need to develop a method with higher selectivity, higher sensitivity and higher automation to cope with trace detection of ZEN in foods.
Disclosure of Invention
In order to solve the technical problems, one object of the present invention is to provide a chemiluminescent immunosensor for detecting zearalenone, wherein a Co 2O4 nanomaterial based on a DES solvent forms an immune probe with an antibody through a Co-N bond, a carboxyl modified resin bead forms an immunosensor substrate with a coating antigen through an amide bond, and according to the competitive immune principle, the immunosensor presents a quantity difference due to the fact that an analyte and the coating antigen compete for specific binding sites on the antibody together, so that different Chemiluminescent (CL) signals are generated, and detection of ZEN in an actual sample is finally realized.
Another object of the present invention is to provide an application of the chemiluminescent immunosensor for detecting zearalenone in detecting ZEN, which uses a flow injection chemiluminescent immunoassay method of CuCo 2O4 nanomaterial based on DES solvent.
The technical scheme of the invention for detecting ZEN combines a chemiluminescent reaction with an immunoassay technology based on a chemiluminescent immunoassay (CLIA) method, becomes a novel method for carrying out high-specificity detection on a detected object based on a chemiluminescent signal, and has been widely applied to clinical diagnosis, food safety and environmental analysis. On the basis, the novel detection method (FI-CLIA) combining the flow injection and the chemiluminescent immunoassay method has the advantages of simplicity in operation, easiness in automation, strong specificity, high sensitivity, wide linear range and the like.
The above object of the present invention is achieved by the following technical scheme:
A chemiluminescent immunosensor for detecting zearalenone comprises a chemiluminescent probe and a chemiluminescent immunosensor substrate, wherein the chemiluminescent probe is prepared by incubating a CuCo 2O4 nano-material solution with a ZEN antibody to obtain the chemiluminescent immunosensor;
The chemiluminescent immunosensor substrate is prepared by activating carboxyl resin beads to obtain activated resin beads, and incubating the activated resin beads with ZEN coating antigen to obtain the chemiluminescent immunosensor substrate.
The invention provides a chemiluminescent immunosensor based on CuCo 2O4 nano materials and carboxyl resin beads, wherein the CuCo 2O4 nano materials in a chemiluminescent probe are used as nano enzymes, the chemiluminescent immunosensor has excellent catalytic performance and good chemical stability, and the chemiluminescent immunosensor substrate is used for loading more coating antigens by taking the carboxyl resin beads with good biocompatibility and larger specific surface area as a solid phase carrier, so that the chemiluminescent immunosensor is high in loading efficiency and stable in property.
Stringent physiological conditions, high difficulty of preparation requirements, and unstable enzyme activity are inherent vulnerabilities of the native enzyme. In order to find alternatives, chemists have been striving to develop nanomaterials that can fully mimic the natural enzymes, commonly known as "nanoezymes". With the rapid development of nanoscience, more and more new nano-materials are found to have excellent peroxidase properties, and these classes of peroxidases not only exhibit catalytic properties similar to those of natural enzymes, but also have higher stability and higher specific surface area. In recent years, nano-enzymes with peroxidase activity have been developed remarkably, such as noble metals, metal oxides, bimetallic oxides, metal sulfides, metal organic frameworks and carbon-based nano-materials all exhibit better peroxidase activity. The CuCo 2O4 nano material is adopted as the nano enzyme, and the bimetallic oxide nano enzyme can improve the current situation of lower catalytic performance of single metal ion, and has wide application in the aspects of tumor treatment, cancer imaging, biosensing and the like.
Further, the CuCo 2O4 nano material is prepared by dissolving copper salt and cobalt salt in Deep Eutectic Solvent (DES) to obtain mixed solution, adding water into the mixed solution, and drying to obtain the CuCo 2O4 nano material.
Deep Eutectic Solvents (DES) are a new class of green solvents, and eutectic mixtures can be prepared simply by mixing Hydrogen Bond Acceptors (HBA) with Hydrogen Bond Donors (HBD) at a temperature of 70-80 ℃. They exhibit better biocompatibility, biodegradability and lower toxicity than other solvents. To date, DES has found many applications in biosensor development, where they can be combined with different materials to improve the sensing performance of the biosensor device.
Further, the copper salt is Cu (NO 3)2·3H2 O) and the cobalt salt is Co (NO 3)2·6H2 O).
Further, the copper salt and the cobalt salt are dissolved in DES at the temperature of 70-80 ℃ for 1-3 hours.
Further, the water is ultrapure water.
In a specific embodiment, adding water into the mixed solution to obtain a blue substance, centrifugally washing the blue substance, and drying to obtain the CuCo 2O4 nano material.
Specifically, the washing is performed with ultrapure water and ethanol.
Specifically, the drying condition is vacuum drying, the temperature is 60-70 ℃ and the time is 10-12 hours.
Further, the DES is formulated from a base and an alcohol compound.
The preparation method of the DES comprises the steps of adding alkali into an alcohol compound to obtain a mixed solution, and stirring the mixed solution through a constant-temperature magnetic stirrer to obtain the Deep Eutectic Solvent (DES).
Further, the stirring temperature is 60-80 ℃, and the stirring time is 20-30 min.
Further, the base is potassium hydroxide (KOH) and the alcohol compound is 1, 2-propanediol.
Further, the concentration of the CuCo 2O4 nano material solution is 1.4-1.6 mg/mL.
Further, the mass ratio of the CuCo 2O4 nano-material to the ZEN antibody is (200-300): 1, preferably 250:1.
Further, the mass ratio of the carboxyl resin beads to the ZEN coating antigen is (150-250): 1, preferably 213:1.
Further, the zearalenone antibody is a monoclonal antibody secreted by zearalenone hybridoma cell line ZEN-2D 2.
Further, the zearalenone coating antigen is prepared from zearalenone hapten, and the zearalenone hapten has a structural formula of
Further, coupling the zearalenone hapten with Ovalbumin (OVA) to obtain the zearalenone coating antigen.
Further, the activation method is to add an activating reagent, wherein the activating reagent is 1-ethyl- (3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS).
Further, the concentration of the 1-ethyl- (3-dimethylaminopropyl) carbodiimide is 8-9 mg/mL.
Further, the concentration of the N-hydroxysuccinimide is 8-9 mg/mL.
Further, the activation time is 2-3 h.
The chemiluminescent immunosensor for detecting zearalenone provided by the invention not only introduces carboxyl resin beads with low price, good biocompatibility and larger specific surface area as a solid phase carrier to load more coating antigens, but also introduces CuCo 2O4 nano-materials synthesized based on a novel green solvent-deep eutectic solvent (with excellent biocompatibility, biodegradability and low toxicity) as antibody carrier label-removing antibodies for the first time, and the preparation method of the constructed chemiluminescent immunosensor is simple, has better accuracy, selectivity and sensitivity, and detects ZEN in food by using the chemiluminescent immunosensor for the first time.
The invention also protects application of the chemiluminescent immunosensor for detecting zearalenone in detecting zearalenone.
The invention adopts a nano material with peroxidase-like activity to replace natural enzyme molecules to carry out catalytic amplification on a Luminol-H 2O2 chemiluminescence system, and provides a novel flow injection chemiluminescence immunosensor based on non-natural enzyme catalysis for detecting a real sample.
Compared with the ZEN detection method which has appeared at present, the analysis method has strong controllability in time, space and luminous intensity, and simultaneously has the advantages of simple operation, cost effectiveness and high sensitivity, provides a brand new method for detecting the ZEN, realizes quick and ultrasensitive detection of the ZEN, and has good practical value and practical significance.
Further, detecting ZEN using flow injection chemiluminescent immunoassay comprises the steps of:
S1, mixing a chemiluminescent probe with ZEN standard solutions with different concentrations, and incubating to obtain a compound solution;
S2, adding a chemiluminescent immunosensor substrate into the compound solution, and reacting to obtain an immunosensor composition;
S3, placing the immunosensor composition in a detector, adding 3-amino-phthalhydrazide, hydrogen peroxide and a buffer solution, recording a luminous intensity-time curve, and establishing a relation between the luminous intensity of the immunosensor composition and the concentration of the ZEN standard solution to obtain a linear regression equation;
s4, repeating the steps S1-S3 on the solution to be detected, and obtaining the concentration of ZEN in the solution to be detected through the luminous intensity of the immunosensor composition according to the linear regression equation.
When the chemiluminescent immunosensor for detecting zearalenone provided by the invention detects ZEN, ZEN solution competes with ZEN coating antigen for a limited amount of ZEN antibody. When ZEN is not added, the antigen-antibody is directly linked at this time, and there is no competition reaction, so ZEN-coated antigen can be well bound to the antibody, thereby showing a strong CL signal. When the ZEN standard solution concentration is applied, the coating antigen competes with the ZEN standard solution for limited binding sites on the antibody, resulting in a decrease in the immune probes attached to the immunosensor substrate, at which point CL signal decreases. Because of the strong specific recognition capability of antigen and antibody, the CL immunosensor has excellent sensitivity, stability and reproducibility, and has important significance for detection of ZEN and similar small molecules.
The invention has the beneficial effects that:
1. The chemiluminescent immunosensor for detecting zearalenone provided by the invention not only introduces carboxyl resin beads with low price, good biocompatibility and larger specific surface area as a solid phase carrier to load more coating antigens, but also introduces CuCo 2O4 nano-materials synthesized based on a novel green solvent-deep eutectic solvent (with excellent biocompatibility, biodegradability and low toxicity) as antibody carrier label-removing antibodies for the first time, and the preparation method of the constructed chemiluminescent immunosensor is simple, has better accuracy, selectivity and sensitivity, and detects ZEN in food by using the chemiluminescent immunosensor for the first time.
2. Compared with the ZEN detection method which has appeared at present, the analysis method has strong controllability in time, space and luminous intensity, and simultaneously has the advantages of simple operation, cost effectiveness and high sensitivity, provides a brand new method for detecting the ZEN, realizes quick and ultrasensitive detection of the ZEN, and has good practical value and practical significance.
Drawings
FIG. 1 is a schematic diagram of the mechanism of detecting ZEN by the construction and flow injection chemiluminescent immunoassay of a chemiluminescent immunosensor for detecting zearalenone.
Fig. 2 is a Scanning Electron Microscope (SEM) image and a Transmission Electron Microscope (TEM) image of CuCo 2O4, wherein A, B is an SEM image of CuCo 2O4 and C, D is a TEM image of CuCo 2O4.
FIG. 3 is an SEM image of a carboxyl-resin bead and a ZEN-coated antigen-loaded carboxyl-resin bead, wherein A is an SEM image of a carboxyl-resin bead and B is an SEM image of a ZEN-coated antigen-loaded carboxyl-resin bead.
FIG. 4 is a graph of CL light emission intensity versus time and a graph of linear regression for ZEN standard solutions of different concentrations, wherein A is a graph of CL light emission intensity versus time for ZEN standard solutions of different concentrations, the concentrations of ZEN standard solutions corresponding to each adjacent three peaks are (a)0.0001ng·mL-1,(b)0.005ng·mL-1,(c)0.01ng·mL-1,(d)0.1ng·mL-1,(e)0.5ng·mL-1,(f)10ng·mL-1 and (g) 150ng mL -1 in sequence from left to right, and B is a graph of linear regression for the logarithm of CL light emission intensity versus ZEN concentration for ZEN standard solutions of different concentrations.
FIG. 5 is a concentration optimization graph of CuCo 2O4, ZEN coated antigen and ZEN antibody, wherein A is a concentration optimization graph of CuCo 2O4, B is a concentration optimization graph of ZEN coated antigen, and C is a concentration optimization graph of ZEN antibody.
FIG. 6 is a graph of a Michaelis-Menten model of CuCo 2O4 with TMB or H 2O2 as substrate, wherein A, C is a graph of a Michaelis-Menten graph of CuCo 2O4 with TMB as substrate and corresponding Lineweaver-Burk graph, and B, D is a graph of a Michaelis-Menten graph of CuCo 2O4 with H 2O2 as substrate and corresponding Lineweaver-Burk graph.
Detailed Description
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.
The experimental methods used in the following examples are conventional methods unless otherwise specified, and materials, reagents, etc. used, unless otherwise specified, are commercially available.
In the following examples of the present invention, both the ZEN coating antigen and the ZEN monoclonal antibody were given to the university of sulzhou Deng Anping professor task group. Wherein the ZEN monoclonal antibody is a monoclonal antibody secreted by a ZEN hybridoma cell line ZEN-2D2 (preserved in China center for type culture collection, with the preservation number of CCTCC No. C202383 and the preservation address of eight ways of university of Wuhan in mountain area of Wuhan, hubei province). The preparation of the antibody firstly determines proper modification sites in the ZEN molecular structure according to hapten molecular design theory and ZEN molecular structure characteristics, synthesizes two ZEN modifications with different connecting arm lengths and carboxyl end groups, is connected with carrier protein, and then prepares the high-sensitivity and high-specificity anti-ZEN monoclonal antibody through the steps of animal immunization, fusion, screening and the like by applying hybridoma antibody preparation technology. The IC 50 value of ELISA method for measuring ZEN established based on the antibody is 0.43 ng.mL -1, and the cross reaction rate with aflatoxin and patulin is less than 0.1 percent.
Wherein, the zearalenone coating antigen is obtained by coupling zearalenone hapten and Ovalbumin (OVA), and the reaction formula is as follows:
In the following examples of the present invention, carboxyl resin beads were provided by Nanjing wheat Coffey high efficiency isolation carrier Co.
A chemiluminescent immunosensor for detecting zearalenone comprises a chemiluminescent probe and a chemiluminescent immunosensor substrate, wherein the chemiluminescent probe is prepared by the following steps of incubating a CuCo 2O4 nano-material solution with a ZEN antibody (ZEN-Ab) to obtain the chemiluminescent immunosensor;
The CuCo 2O4 nano material is prepared by dissolving copper salt and cobalt salt in Deep Eutectic Solvent (DES) to obtain mixed solution, adding water into the mixed solution, and drying to obtain the CuCo 2O4 nano material.
The chemiluminescent immunosensor substrate is prepared by activating carboxyl resin beads to obtain activated resin beads, and incubating the activated resin beads with ZEN coating antigen (ZEN-Ae) to obtain the chemiluminescent immunosensor substrate.
The invention aims at overcoming the defects of the existing detection method of ZEN, and aims to construct a CL immunosensor method of CuCo 2O4 nano-enzyme based on DES solvent, which is applied to detection of the content of ZEN in foods such as corn, wheat and the like and provides a brand new method for detection of ZEN.
In one embodiment of the invention, the copper salt is Cu (NO 3)2·3H2 O and the cobalt salt is Co (NO 3)2·6H2 O).
In one embodiment of the invention, the DES is formulated from KOH and 1, 2-propanediol.
In one embodiment of the invention, copper and cobalt salts are dissolved in DES at a temperature of 70-80 ℃ for 1-3 hours.
In one embodiment of the invention, the concentration of the CuCo 2O4 nanomaterial solution is 1.4-1.6 mg/mL.
In one embodiment of the invention, the mass ratio of the CuCo 2O4 nanomaterial to the ZEN antibody is (200-300): 1, preferably 250:1.
In one embodiment of the invention, the mass ratio of the carboxyl resin beads to the ZEN coating antigen is (150-250): 1, preferably 213:1.
On the basis, the ZEN is detected by adopting a flow injection chemiluminescence immunoassay method, and the method comprises the following steps of:
S1, mixing a chemiluminescent probe with ZEN standard solutions with different concentrations, and incubating to obtain a compound solution;
S2, adding a chemiluminescent immunosensor substrate into the compound solution, and reacting to obtain an immunosensor composition;
S3, placing the immunosensor composition in a detector, adding 3-amino-phthalhydrazide, hydrogen peroxide and a buffer solution, recording a luminous intensity-time curve, and establishing a relation between the luminous intensity of the immunosensor composition and the concentration of the ZEN standard solution to obtain a linear regression equation;
s4, repeating the steps S1-S3 on the solution to be detected, and obtaining the concentration of ZEN in the solution to be detected through the luminous intensity of the immunosensor composition according to the linear regression equation.
Fig. 1 is a schematic diagram of the mechanism of the chemiluminescent immunosensor construction and the detection of ZEN by flow injection chemiluminescent immunoassay, and as can be seen from fig. 1, the ZEN solution competes with the ZEN-coated antigen for a limited amount of ZEN antibodies. When ZEN is not added, the antigen-antibody is directly linked at this time, and there is no competition reaction, so ZEN-coated antigen can be well bound to the antibody, thereby showing a strong CL signal. When the ZEN standard solution concentration is applied, the coating antigen competes with the ZEN standard solution for limited binding sites on the antibody, resulting in a decrease in the immune probes attached to the immunosensor substrate, at which point CL signal decreases.
Fig. 2 is a Scanning Electron Microscope (SEM) image and a Transmission Electron Microscope (TEM) image of CuCo 2O4, wherein A, B is an SEM image of CuCo 2O4 and C, D is a TEM image of CuCo 2O4.
FIG. 3 is an SEM image of a carboxyl-resin bead and a ZEN-coated antigen-loaded carboxyl-resin bead, wherein A is an SEM image of a carboxyl-resin bead and B is an SEM image of a ZEN-coated antigen-loaded carboxyl-resin bead.
Example 1
A chemiluminescent immunosensor for detecting zearalenone, comprising a chemiluminescent probe (CuCo 2O4 -Ab) and a chemiluminescent immunosensor substrate, wherein the chemiluminescent probe is prepared by the following method:
Preparing a CuCo 2O4 nanometer material solution with a certain concentration, slowly adding a ZEN antibody stock solution with a certain concentration into the CuCo 2O4 nanometer material solution (the mass ratio of the CuCo 2O4 nanometer material to the ZEN antibody is 250:1), slightly and uniformly mixing to obtain a mixed solution, and placing the mixed solution in a refrigerator at a temperature of 4 ℃ for incubation overnight. The next day, 5% Bovine Serum Albumin (BSA) was added to the mixed solution, after 2 hours, unbound ZEN antibody and BSA were removed by centrifugation at 4500rpm for 5min, and finally 0.01M PBS was added thereto, and after mixing, stored in a refrigerator at 4 ℃ for later use.
The CuCo 2O4 nano material is prepared by adding 0.34g KOH into 16.16mL 1, 2-propanediol to obtain a mixed solution, and stirring the mixed solution by a constant temperature magnetic stirrer at 60 ℃ for 25min to obtain DES. Then, cu (NO 3)2·3H2 O and Co (NO 3)2·6H2 O) are dispersed in 5mL DES according to a molar ratio of 1:2 to obtain a mixed solution, then the mixed solution is stirred for 2h at 70 ℃ through an oil bath pot, 5mL deionized water is added into the mixed solution after the reaction is finished to obtain a blue substance, finally, the blue substance is centrifugally washed by deionized water and ethanol for several times, and dried for 10h at 60 ℃ to obtain the CuCo 2O4 nanomaterial.
The chemiluminescent immunosensor substrate is prepared by the following method:
Carboxyl resin beads were first dissolved in an aqueous ethanol mixture (V Absolute ethyl alcohol :V Water and its preparation method = 1:4) and placed in a refrigerator at 4 ℃ for subsequent use. The carboxyl resin beads, 0.17g, were placed in a 5mL centrifuge tube, and the carboxyl resin beads were first activated in the absence of light on a shaker with 8mg mL -1 EDC and 8mg mL -1 NHS. After 2h, the activated resin beads were washed multiple times with 0.01M PBS. Afterwards, the ZEN-coated antigen stock solution was diluted to a certain concentration with 0.01M PBS solution, and added into a centrifuge tube containing washed carboxyl resin beads (mass ratio of carboxyl resin beads to ZEN-coated antigen: 213:1), and finally incubated overnight in a 4 ℃ refrigerator. The next day, the resin beads modified with ZEN-coated antigen were washed once with 0.01M PBS, 1ml of 2% Casein (Casein) was added to each tube, and after shaking for 2 hours, washed 3 times with PBST and PBS, respectively, and then 0.01M PBS buffer was added to the centrifuge tube and kept in a 4 ℃ refrigerator for later use.
Example 2
The chemiluminescent immunosensor for zearalenone detection of example 1 was used for ZEN detection using flow injection chemiluminescent immunoassay, comprising the steps of:
s1, centrifuging 110 mu L of CuCo 2O4 -Ab solution to remove supernatant, then taking a lower precipitate, adding the lower precipitate into 200 mu L of ZEN standard solution with different concentrations of ((a)0.0001ng·mL-1,(b)0.005ng·mL-1,(c)0.01ng·mL-1,(d)0.1ng·mL-1,(e)0.5ng·mL-1,(f)10ng·mL-1 and (g) 150 ng-mL -1), and oscillating at medium speed for 1h to obtain a compound solution;
S2, adding chemiluminescent immunosensor substrates into the complex solution, wherein both ZEN-Ae and ZEN can be combined with ZEN-Ab in the process. After 1h of reaction, cuCo 2O4 -Ab which is not combined with ZEN-Ae and excessive ZEN are washed by 0.01M PBS to obtain an immune sensor composition;
S3, placing the immunosensor composition in a detector, simultaneously injecting a chemiluminescent substrate solution (0.01M PBS buffer solution serving as a carrier liquid) containing 0.1M Luminol (Luminol, also known as luminescent ammonia, with a chemical name of 3-amino-phthalhydrazide, CAS number 521-31-3) and 8mM H 2O2, positioning an immunosensor by controlling the speed of a peristaltic pump, and selecting the pump speed of the peristaltic pump 1 (P1) to be 2 mL/min -1 and the pump speed of the peristaltic pump 2 (P2) to be 0.5 mL/min -1. The intensity of the chemiluminescent signal detected by a photomultiplier tube (PMT) (-800V, ×2) is linearly inversely related to the concentration of the ZEN standard solution, the signal is converted by a computer to be displayed as an amplified electrical signal, a luminescence intensity-time curve is recorded, and a linear relationship between the CL luminescence intensity of the immunosensor composition and the concentration vs. value of the ZEN standard solution is established to obtain a linear regression equation I CL＝8117.3-1141.8logC(R2 = 0.9921.
The detection range of the chemiluminescent immunosensor for detecting zearalenone provided by the invention is 0.0001-100 ng.mL -1, and when the signal-to-noise ratio is 3, the detection Limit (LOD) is 0.036 pg.mL -1.
FIG. 4 is a graph of CL light emission intensity versus time and a graph of linear regression for ZEN standard solutions of different concentrations, wherein A is a graph of CL light emission intensity versus time for ZEN standard solutions of different concentrations, the concentrations of ZEN standard solutions corresponding to each adjacent three peaks are (a)0.0001ng·mL-1,(b)0.005ng·mL-1,(c)0.01ng·mL-1,(d)0.1ng·mL-1,(e)0.5ng·mL-1,(f)10ng·mL-1 and (g) 150ng mL -1 in sequence from left to right, and B is a graph of linear regression for the logarithm of CL light emission intensity versus ZEN concentration for ZEN standard solutions of different concentrations.
FIG. 5 is a concentration optimization graph of CuCo 2O4, ZEN coated antigen and ZEN antibody, wherein A is a concentration optimization graph of CuCo 2O4, B is a concentration optimization graph of ZEN coated antigen, and C is a concentration optimization graph of ZEN antibody. As can be seen from FIG. 5A, the ΔCL intensity gradually increases with increasing concentration of CuCo 2O4, and when the concentration exceeds 1.5mg/mL, the ΔCL intensity drops sharply. This is because, as the concentration of CuCo 2O4 increases, the effective concentration of the immuno-probe increases, the binding efficiency of the immuno-probe to the sensor substrate increases, and the catalytic amplification effect of CuCo 2O4 on the luminel-H 2O2 system increases, but when the concentration of nanomaterial reaches a certain value, the nanomaterial aggregates, and as the aggregation degree increases, the coupling efficiency of the ZEN-coated antigen and the ZEN antibody eventually decreases. As can be seen from fig. 5B, the Δcl value gradually increases as the ZEN coating antigen concentration increases. When the ZEN coating antigen concentration reached 8 μg mL -1, Δcl reached a maximum, indicating that 8 μg mL -1 is the optimal parameter for the ZEN coating antigen concentration. As can be seen from fig. 5C, ZEN antibody concentration is also critical, and as ZEN antibody concentration increases, the Δcl value tends to increase and then decrease. At lower ZEN antibody concentrations, the CuCo 2O4 material has lower antibody loading rates, less number of immuno-probes formed, and lower chemiluminescent signals. Furthermore, it is not the higher the ZEN antibody concentration, the better, because higher ZEN antibody concentration, while increasing the antibody loading rate, will also increase the background signal, ultimately affecting the performance of the sensor.
Example 3
The peroxidase activity of CuCo 2O4 was evaluated using steady state kinetic measurements with typical chromogenic substrates 3, 5-tetramethyl-benzidine (TMB) and H 2O2.
By varying the concentrations of TMB and H 2O2, apparent steady state kinetic parameters following a typical Michaelis-Menton model curve (Michaelis-Menten curve) and corresponding Lineweaver-Burk plot were determined, as shown in FIG. 6. The Michaelis constants (K m) and maximum reaction rate (V max) of CuCo 2O4 and horseradish peroxidase (HRP) were achieved by double reciprocal mapping (Lineweaver-Burk plot), and the results are shown in Table 1:
Table 1 CuCo 2O4 and steady state kinetic parameter comparison of HRP (K m,Vmax)
In general, the smaller the K m value, the higher the affinity between the catalyst and the substrate. As can be seen from Table 1, the K m value of CuCo 2O4 obtained with TMB as substrate was lower than that of HRP, demonstrating that CuCo 2O4 has a higher affinity for substrate TMB than that of HRP. The K m value of CuCo 2O4 obtained by taking H 2O2 as a substrate is higher than that of HRP, and it is speculated that more H 2O2 is consumed by the reaction of CuCo 2O4 and H 2O2 under the same condition, so that the CuCo 2O4 nano material can catalyze H 2O2, and powerful support is provided for the CuCo 2O4 material to play a role in catalytic amplification in a Luminol-H 2O2 system.
Test example 1
In order to evaluate the detection capability of the chemiluminescent immunosensor for detecting zearalenone, disclosed by the invention, a labeling recovery experiment on an actual sample is carried out, and samples containing corn and wheat with 0.1 ng/mL -1、0.2ng·mL-1 and 0.5 ng/mL -1 are prepared respectively. Wheat flour and corn flour are selected as real samples for detection, and the actual application capability of the sensor is evaluated through recovery rate. The final experimental results are shown in Table 2, the recovery rate of wheat flour and corn flour is 90.49% -112.5%, and the error is in an acceptable range.
TABLE 2 labelling recovery experiments on ZEN in real samples
N.d. =undetected.
ZEN is a typical estrogen metabolite with a nonsteroidal molecular structure that acts mainly on the reproductive system and causes hyperstimulation in livestock and poultry. Often contaminating agricultural and cereal products, eating contaminated products can cause various toxic effects to humans and animals, and therefore accurate and quantitative analysis of ZEN in agricultural products is essential for food safety and animal husbandry.
The invention introduces carboxyl resin beads with low price, good biocompatibility and larger specific surface area as solid phase carriers to load more coating antigens in the flow injection chemiluminescence immunoassay method, and also introduces CuCo 2O4 nano materials synthesized based on a novel green solvent-deep eutectic solvent (with excellent biocompatibility, biodegradability and low toxicity) as antibody carriers to label antibodies for the first time. The chemiluminescent immunosensor constructed on the basis has the advantages of simple preparation method, better accuracy, selectivity and sensitivity, and is used for detecting the ZEN in the feed for the first time, under the optimal experimental condition, the detection range of the ZEN is 0.0001-100 ng.mL -1, and the detection Limit (LOD) is 0.036 pg.mL -1 (S/N=3). As a novel green solvent, the combination of DES and nano materials provides some new ideas for biosensing, which is beneficial to further intensive research, and the sensor also provides a referent idea for quantitative detection of other small molecules.
Compared with the prior art, the method has the advantages of high controllability in time, space, luminous intensity and the like, capability of realizing quick and ultrasensitive detection of the ZEN, simplicity in operation, cost effectiveness, high sensitivity and the like, and good practical value and practical significance.
The above-described embodiments are merely preferred embodiments for fully explaining the present invention, and the scope of the present invention is not limited thereto. Equivalent substitutions and modifications will occur to those skilled in the art based on the present invention, and are intended to be within the scope of the present invention. The protection scope of the invention is subject to the claims.
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