CN112326955B - Fixing and coupling protein composite material based on copperas monohydrate, and preparation method and application thereof - Google Patents
Fixing and coupling protein composite material based on copperas monohydrate, and preparation method and application thereof Download PDFInfo
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- 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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- G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
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- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
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- 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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- 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
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/581—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)
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- 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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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- G01N2333/32—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Bacillus (G)
- G01N2333/325—Bacillus thuringiensis crystal protein (delta-endotoxin)
Abstract
The invention relates to a high-efficiency protein fixing and coupling composite material based on copperas monohydrate, and a preparation method and application thereof. The synthesis and protein fixation and coupling of the composite material designed in the invention can be realized through one-step reaction. The synthesis conditions were simple, green, mild and without complicated purification and operating steps. Meanwhile, a large amount of horseradish peroxidase and a proper amount of streptavidin fixed on the composite material have the functions of signal amplification and biological recognition respectively, and can be applied to enzyme-linked immunosorbent assay for detecting transgenic protein Cry1Ab or other biological analytes.
Description
Technical Field
The invention belongs to the field of nano materials and biological analysis and detection, and particularly relates to a high-efficiency fixing and coupling protein composite material based on copperas monohydrate, and a preparation method and application thereof.
Background
In an enzyme-linked immunosorbent assay (ELISA), a signal protein (e.g., an enzyme) is coupled with a recognition protein (e.g., streptavidin, an antibody, etc.) in advance, and then the coupled signal protein is used for antigen-antibody reaction, and then the enzyme and a substrate generate color reaction, and a standard curve is established through the relationship between the intensity of a color light absorption value and the concentration of a substance to be detected, so that the standard curve is used for quantitatively determining a certain analyte, such as enzyme-linked immunosorbent assay (ELISA). There are two areas to be improved in the traditional ELISA method. For one, in coupling between proteins, a chemical crosslinking method, i.e., a chemical method in which two proteins are linked together by forming a covalent bond using a crosslinking agent, is often used. The cross-linking agent is generally organic, and the related reaction conditions are harsh, the operation is complex, the purification steps are multiple, and the time consumption is long. Such as 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS). Secondly, in the conventional ELISA method, the number of enzyme molecules bound to the analyte is relatively small, and the signal intensity generated by the enzyme catalysis is limited to a certain extent. Researchers have generally sought to increase the number of enzyme molecules bound to the analyte to achieve signal amplification and thus increase the sensitivity of the assay.
Due to the ultrahigh specific surface area of the nano material, the nano material can be used as a carrier for fixing a large number of enzyme molecules (signal proteins) to realize signal amplification, and simultaneously, a certain amount of streptavidin, antibodies and the like (recognition proteins) can be fixed for biological recognition. However, there are two problems with the use of nanomaterials to immobilize proteins. Firstly, the problems of complexity in the selection and operation of nano materials, such as nano gold, carbon nano materials, metal oxide materials (titanium, aluminum, zirconium, etc.), silicon nano materials, polymers, etc., exist, and although these materials can well fix proteins, the fixation of proteins generally needs to be performed after the synthesis of nano materials is completed. That is, the synthesis of the nanomaterial and the fixation of the protein are performed stepwise, which adds complexity to the operation. Secondly, the immobilization efficiency of the protein on the nano material is high. There are generally two types of securing means: one is physical adsorption and one is chemical bond fixation. The physical adsorption is to adsorb the protein on the surface of the nano material by utilizing the acting forces such as hydrogen bond, pi-pi accumulation, van der waals force, static electricity, hydrophobic interaction and the like through the physical mixing of the nano material and the protein. Chemical bond fixation can connect protein and a nano material carrier by using a covalent bond, so that the fixation can be firmer, but the steps are complicated, the reagents are various, and certain protein activity loss can be caused. Therefore, how to simplify the fixing mode and the whole operation, the reduction of protein activity is not caused while the efficient fixing is realized, and the method is a core problem of applying the nano material to enzyme-labeled immunoassay and is also a current bottleneck problem.
Disclosure of Invention
In order to solve the existing problems, the invention provides a high-efficiency protein fixing and coupling composite material based on ceruloplasmin and a preparation method thereof, and the synthesis and protein fixing and coupling of the designed composite material can be realized through one-step reaction. The synthesis conditions are simple, green and mild, no complex purification and operation steps are needed, and the protein is efficiently fixed without loss of protein activity.
The other purpose is that a large amount of horseradish peroxidase and a proper amount of streptavidin which are immobilized on the obtained composite material have the functions of signal amplification and biological recognition respectively, and can be applied to enzyme-linked immunosorbent assay for detecting transgenic protein Cry1Ab or other biological analytes.
In order to achieve the purpose, the following technical scheme is adopted in the application: the preparation method of the protein composite material for fixing and coupling based on the copperas monohydrate comprises the following steps:
s1 horseradish peroxidase and streptavidin are dissolved in the buffer solution;
s2, adding a copper sulfate solution into the solution obtained in the step S1 for reaction, and then centrifugally separating and washing the product to obtain the fixed and coupled protein composite material based on the copperas monohydrate.
The preparation and reaction principle of the invention is as follows: the copper sulfate solution can generate blue copperas monohydrate in a boric acid-potassium chloride buffer solution with the pH value of 8.1, and copper ions of the copper sulfate solution can be coordinated with the amino groups on the horseradish peroxidase and the streptavidin to form a coordination covalent bond. The copper ions play a role of a connector in the whole reaction, one side of the copper ions is firmly connected with protein through a coordination covalent bond, and the other side of the copper ions forms ceruloplasmin in the reaction liquid. Therefore, a large amount of horseradish peroxidase and streptavidin can be firmly fixed together with the copperas monohydrate and are co-precipitated in the reaction solution to form the hybrid material compounded by two proteins and the copperas monohydrate. Meanwhile, two different functional proteins are creatively added, so that the copperas monohydrate not only can be used as a carrier to firmly fix the proteins, but also can be used as a connector to couple two proteins with different functions (horseradish peroxidase is used for signal amplification, and streptavidin is used for biological recognition). Not only simplifies the operation, but also achieves the aim of dual-function integration and can be used for enzyme-labeled immunoassay.
The obtained composite material is in the shape of hollow tube bundle, and the inorganic component in the composite material is ceruloplasmin monohydrate, and the chemical formula is Cu4(SO4)(OH)6·H2O, length 450-550 nm. The invention utilizes the ceruloplasmin monohydrate to fix and couple the protein, is a universal method, the fixed and coupled protein is not limited to horseradish peroxidase and streptavidin, and other arbitrary proteins can be fixed and coupled according to different experimental purposes.
According to the scheme, the mass ratio of the horseradish peroxidase to the streptavidin is 1-20: 1.
According to the scheme, the buffer solution is a boric acid-potassium chloride buffer solution with the pH value of 8.1 and the concentration of 0.03 mol/L.
According to the scheme, the concentration of the copper sulfate is 0.01-5mol/L, and the volume ratio of the copper sulfate to the solution obtained in the step S1 is 1-1000: 1.
According to the scheme, the reaction temperature of the step S2 is room temperature, and the reaction time is 5-60 minutes.
The obtained composite material is dissolved in 1mL of phosphate buffer solution and then needs to be diluted by 5-20 times, and then is applied to enzyme-linked immunosorbent assay for detecting transgenic protein, the enzyme-linked immunosorbent assay adopts a double-antibody sandwich method, and Cry1Ab is detected as the transgenic protein.
The invention has the beneficial effects that:
firstly, the operation is simple. The operation only needs one-step reaction, and the synthesis of the nano material and the fixation and coupling of the protein can be completed;
second, the protein is efficiently immobilized and coupled without reducing the activity of the protein. The designed and synthesized composite material not only can firmly fix a large number of protein molecules, but also can stabilize and even improve the activity of the protein (see example 1), two proteins with different functions (horseradish peroxidase is used for signal amplification, and streptavidin is used for biological recognition) are added in the reaction at the same time, and the enzyme and the recognition protein are coupled by utilizing the material. The synthesis of the material, the fixation and coupling of the protein are integrated, and the signal amplification and recognition functions are integrated, so that the complex reaction and purification steps are avoided, and the obtained composite material can be used for enzyme-labeled immunoassay;
thirdly, the reaction conditions are mild, green and less time-consuming. The synthesis of the nano material and the fixation and coupling of the protein are carried out under the mild conditions of normal temperature, normal pressure and pH, no organic reagent is needed, the preparation can be completed within half an hour, the operation feasibility is strong, and the method is easy to enlarge and popularize in a market.
Drawings
FIG. 1 is a scanning electron microscope image of a composite material in an embodiment of the invention;
FIG. 2 is an X-ray diffractometer analysis of a composite material in an example of the invention;
FIG. 3 is an energy dispersive X-ray spectrometer analysis of a composite material in an embodiment of the invention;
FIG. 4 is a graph showing fluorescence intensities of streptavidin-FITC and horseradish peroxidase-Cy 5 before and after reaction when a composite material was synthesized in the example of the present invention;
FIG. 5 is a comparison of the activity of immobilized and non-immobilized horseradish peroxidase in examples of the invention;
FIG. 6 is a graph showing the results of applying the composite material of the invention to ELISA for detecting transgenic protein Cry1Ab in the embodiment of the invention versus a linear relationship;
FIG. 7 is a graph of the results of detecting the transgenic protein Cry1Ab by a common ELISA-linear relationship.
Detailed Description
In order to better understand the present invention, the following examples are further provided to illustrate the present invention, but the present invention is not limited to the following examples.
Example 1:
firstly, preparing a composite material, comprising the following steps:
(1) 0.006mg of streptavidin and 0.024mg of horseradish peroxidase were dissolved in 990. mu.L of 0.03mol/L boric acid-potassium chloride buffer (adjusted to pH8.1 with sodium hydroxide).
(2) And (2) adding 10 mu L of 0.5mol/L copper sulfate solution into the mixed solution obtained in the step (1), mixing uniformly, slightly shaking, reacting for 30 minutes at room temperature to obtain the streptavidin-horseradish peroxidase-copperas monohydrate composite material, centrifuging and collecting the composite material, washing for 3 times by deionized water, and dissolving in 1mL phosphate buffer.
FIG. 1 is an image of the composite material obtained in step (2) under an electron scanning microscope, and the result shows that the composite material is in the shape of a hollow tube bundle with a length of 450-550 nm.
The structure of the composite material obtained in step (2) was determined by X-ray diffractometry, as shown in FIG. 2, using Cu 1410 with card number 01-083-4(SO4)(OH)6·H2The O standard sample is completely coincided, and the inorganic component in the composite material is Cu4(SO4)(OH)6·H2O (bluestone monohydrate).
The elemental composition of the composite material obtained in step (2) was determined by an energy dispersive X-ray spectrometer, and as shown in fig. 3, carbon elements were detected in the composite material in addition to copper, sulfur and oxygen elements. In the preparation process of the material, only the streptavidin and the horseradish peroxidase have carbon elements, which indicates that the streptavidin or the horseradish peroxidase is fixed in the composite material.
After FITC and Cy5 fluorescence labeling of streptavidin and horseradish peroxidase, respectively (labeling method is performed with reference to FITC and Cy5 product instructions (Sigma-Aldrich Co.)), 10. mu.L of the supernatant of the solution obtained in step (1) and step (2) was taken according to the procedure (1)) and (2) for the synthesis of the material, and the fluorescence intensities of streptavidin-FITC and horseradish peroxidase-Cy 5 in the supernatant were measured, respectively. The fluorescence intensity may reflect the relative content changes of streptavidin and horseradish peroxidase in the supernatant. As shown in FIG. 4, in step (1), i.e., before the reaction, the measured fluorescence intensities of streptavidin-FITC and horseradish peroxidase-Cy 5 reflect the initial concentrations of streptavidin and horseradish peroxidase before the material synthesis, and after adding copper sulfate and reacting for 30 minutes in step (2), part of streptavidin or horseradish peroxidase participates in the reaction and is immobilized on the ceruloplasmin monohydrate. Therefore, after the synthesis reaction in the step (2) is finished, the fluorescence intensity of the streptavidin-FITC and the horseradish peroxidase-Cy 5 in the supernatant is measured to be greatly reduced, which shows that most of the streptavidin and the horseradish peroxidase are simultaneously fixed on the ceruloplasmin. Meanwhile, a standard curve is established through the relation between the fluorescence intensity and the concentration of streptavidin-FITC or horseradish peroxidase-Cy 5 in the supernatant, and the immobilization rates of the streptavidin and the horseradish peroxidase are respectively 95.6% and 91.8% through calculation. Indicating that the method possesses very high efficiency of immobilizing and coupling protein.
Enzyme activity assays were performed on horseradish peroxidase immobilized on ceruloplasmin monohydrate and on non-immobilized horseradish peroxidase, respectively (using a horseradish peroxidase enzyme activity assay kit, according to the method given in the kit, purchased from Sigma-Aldrich). The non-immobilized horseradish peroxidase was sampled in the following amounts and patterns: and (3) adding 10 mu L of ultrapure water into the mixed solution in the step (1) to complement the total volume to 1mL, and taking 5 mu L of the mixed solution to perform enzyme activity determination within 30 minutes after uniform mixing. The fixed horseradish peroxidase was sampled in the following amounts and patterns: after the reaction in the step (2) is carried out for 30 minutes, the reaction liquid and the product are fully and uniformly mixed, and 5 mu L of the mixed liquid is taken for enzyme activity determination within 30 minutes before centrifugal collection and washing. As shown in fig. 5, horseradish peroxidase showed higher enzyme catalytic activity after immobilization, relative to non-immobilized horseradish peroxidase. Before the reaction in the step (1), the activity of the non-immobilized horseradish peroxidase is measured to be 5.62U/mL, and after the reaction in the step (2), the activity of the horseradish peroxidase is 17.51U/mL. The enzyme activity is improved by 3.11 times. Indicating that the horseradish peroxidase has no loss of activity after being fixed on the ceruloplasmin monohydrate. The method is proved to be capable of stabilizing and even improving the activity of the protein.
Secondly, the composite material prepared in this example is applied as follows:
(1) mu.g/mLCry 1Ab murine monoclonal antibody was added to the ELISA plate at 100. mu.L/well and incubated overnight at 4 ℃. The plates were washed three times with PBST to remove unbound monoclonal antibody.
(2) The plate was incubated with 1% Bovine Serum Albumin (BSA), 300. mu.L/well, to prevent nonspecific adsorption, at 37 ℃ for 30 minutes, and the plate was washed three times with PBST. The transgenic protein Cry1Ab solutions at different concentrations were added to the well plates at 100. mu.L/well, incubated at 37 ℃ for 45 minutes, and the plates were washed three times with PBST.
(3) 200ng/mL biotin-labeled Cry1Ab rabbit polyclonal antibody was added to an ELISA plate at 100. mu.L/well, incubated at 37 ℃ for 45 min, and the plate was washed three times with PBST.
(4) The prepared composite material is diluted by 15 times by phosphate buffer solution, 100 mu L/hole and incubated for 20 minutes at 37 ℃ (in the common ELISA, the step replaces the composite material with 100ng/mL streptavidin labeled horseradish peroxidase, and other steps are the same.) because the streptavidin is fixed in the composite material, when Cry1Ab exists in a hole plate, the streptavidin on the composite material and biotin on rabbit polyclonal antibody which is combined on Cry1Ab have specific recognition and combination, and a large amount of horseradish peroxidase is simultaneously fixed in the composite material, compared with the common ELISA method, the number of enzyme molecules of the horseradish peroxidase which are combined on a single Cry1Ab protein molecule is more, and the signal amplification effect is achieved), the hole plate is washed by PBST for four times, and the unbound composite material is removed. Substrate developer TMB solution, 100. mu.L/well, was added and incubated at 37 ℃ for 15 minutes. The reaction was stopped with a 2M sulfuric acid solution, 100. mu.L/well.
(5) The absorbance of each well was measured at a wavelength of 450nm using a microplate reader. A standard curve was established by the relationship between absorbance and different concentrations of the transgenic protein Cry1 Ab.
As shown in fig. 6, the composite material prepared in this example was applied to ELISA test Cry1Ab and calculated by Origin software, and the linear regression equation of the method was Y0.02133 × X +0.23349 (R0.991), the linear range was 0.1-80ng/mL, and the detection limit was 0.008 ng/mL. As shown in fig. 7, the linear regression equation for the detection of Cry1Ab (using commercial ELISA kit) by the conventional ELISA method was Y0.01523X +0.11817 (R0.992), the linear range was 1-32ng/mL, and the detection limit was 0.11 ng/mL. The result shows that the composite material obtained by the embodiment is applied to ELISA detection of the transgenic protein Cry1Ab, the detection sensitivity can be obviously improved, and a wider linear range is provided.
Example 2:
firstly, preparing a composite material, comprising the following steps:
(1) 0.010mg of streptavidin and 0.010mg of horseradish peroxidase were dissolved in 500. mu.L of 0.03mol/L boric acid-potassium chloride buffer (adjusted to pH8.1 with sodium hydroxide).
(2) And (2) adding 500 mu L of 0.01mol/L copper sulfate solution into the mixed solution obtained in the step (1), mixing uniformly, slightly shaking, reacting for 5 minutes at room temperature to obtain the streptavidin-horseradish peroxidase-copperas monohydrate composite material, centrifuging and collecting the composite material, washing for 3 times by deionized water, and dissolving in 1mL phosphate buffer.
Taking the composite material obtained in this example as an example, the streptavidin immobilization rate and the horseradish peroxidase immobilization rate were calculated to be 90.8% and 92.4%, respectively, according to the operation method of example 1. After the horseradish peroxidase is fixed on the ceruloplasmin monohydrate, the enzyme activity is 15.88U/mL, and the enzyme activity is 2.83 times higher than that of the non-fixed horseradish peroxidase.
Secondly, the application of the composite material prepared in this example is performed according to the procedure of example 1, wherein the composite material prepared in step (4) is diluted 5 times with phosphate buffer, and other steps are the same.
The composite material prepared in the embodiment is applied to ELISA detection Cry1Ab and is calculated by Origin software, and the linear regression equation of the method is that Y is 0.01876X +0.3132(R is 0.994), the linear range is 0.2-50ng/mL, and the detection limit is 0.025 ng/mL.
Example 3:
firstly, preparing a composite material, comprising the following steps:
(1) 0.005mg of streptavidin and 0.100mg of horseradish peroxidase were dissolved in 1000. mu.L of 0.03mol/L boric acid-potassium chloride buffer (adjusted to pH8.1 with sodium hydroxide).
(2) And (2) adding 1 mu L of 5mol/L copper sulfate solution into the mixed solution obtained in the step (1), mixing uniformly, slightly shaking, reacting for 60 minutes at room temperature to obtain the streptavidin-horseradish peroxidase-copperas monohydrate composite material, centrifuging and collecting the composite material, washing for 3 times by using deionized water, and dissolving in 1mL phosphate buffer solution.
Taking the composite material obtained in this example as an example, the streptavidin immobilization rate and the horseradish peroxidase immobilization rate were calculated to be 96.8% and 80.4% respectively according to the operation method of example 1. After the horseradish peroxidase is fixed on the ceruloplasmin monohydrate, the enzyme activity is 18.39U/mL, and the enzyme activity is 3.27 times higher than that of the non-fixed horseradish peroxidase.
Secondly, the application of the composite material prepared in this example is performed according to the procedure of example 1, wherein the composite material prepared in step (4) is diluted 20 times with phosphate buffer, and other steps are the same.
The composite material prepared in the embodiment is applied to ELISA detection Cry1Ab and is calculated by Origin software, and the linear regression equation of the method is that Y is 0.01643X +0.28071(R is 0.996), the linear range is 0.8-36ng/mL, and the detection limit is 0.038 ng/mL.
Example 4:
firstly, preparing a composite material, comprising the following steps:
(1) 0.005mg of streptavidin and 0.035mg of horseradish peroxidase were dissolved in 1000. mu.L of 0.03mol/L boric acid-potassium chloride buffer (adjusted to pH8.1 with sodium hydroxide).
(2) And (2) adding 2.5 mu L of 2mol/L copper sulfate solution into the mixed solution obtained in the step (1), mixing uniformly, slightly shaking, reacting for 40 minutes at room temperature to obtain the streptavidin-horseradish peroxidase-copperas monohydrate composite material, centrifuging and collecting the composite material, washing for 3 times by deionized water, and dissolving in 1mL phosphate buffer.
Taking the composite material obtained in this example as an example, the streptavidin immobilization rate and the horseradish peroxidase immobilization rate were calculated to be 96.1% and 89.7% respectively according to the operation method of example 1. After the horseradish peroxidase is fixed on the ceruloplasmin monohydrate, the enzyme activity is 17.96U/mL, and the enzyme activity is 3.19 times higher than that of the non-fixed horseradish peroxidase.
Secondly, the application of the composite material prepared in this example is performed according to the procedure of example 1, wherein the composite material prepared in step (4) is diluted 10 times with phosphate buffer, and other steps are the same.
The composite material prepared in the embodiment is applied to ELISA detection Cry1Ab and is calculated by Origin software, and the linear regression equation of the method is that Y is 0.01917X +0.24982(R is 0.995), the linear range is 0.2-80ng/mL, and the detection limit is 0.016 ng/mL.
Claims (8)
1. The preparation method of the protein composite material for fixing and coupling based on the copperas monohydrate comprises the following steps:
s1 horseradish peroxidase and streptavidin are dissolved in the buffer solution; the buffer solution is a boric acid-potassium chloride buffer solution with the pH value of 8.1 and the concentration of 0.03 mol/L;
s2, adding a copper sulfate solution into the solution obtained in the step S1 for reaction, and then centrifugally separating and washing the product to obtain the fixed and coupled protein composite material based on the copperas monohydrate.
2. The method of claim 1, wherein: the mass ratio of the horseradish peroxidase to the streptavidin is 1-20: 1.
3. The method of claim 1, wherein: the concentration of the copper sulfate is 0.01-5mol/L, and the volume ratio of the copper sulfate to the solution obtained in the step S1 is 1-1000: 1.
4. The method of claim 1, wherein: the reaction temperature of step S2 is room temperature and the reaction time is 5 to 60 minutes.
5. The method of claim 1, wherein the composite material is in the form of hollow tube bundle, and the inorganic component is blue copperas monohydrate, which has a chemical formula ofCu4(SO4)(OH)6·H2O, the length of which is 450-550 nm.
6. Use of the composite material of claim 5 as a signal amplification and recognition agent in an enzyme-linked immunosorbent assay (ELISA) for the detection of transgenic proteins.
7. Use according to claim 6, characterized in that: the composite material is dissolved in 1mL of phosphate buffer solution and then needs to be diluted by 5-20 times, and then is applied to enzyme-linked immunosorbent assay for detecting transgenic protein, wherein the enzyme-linked immunosorbent assay adopts a double-antibody sandwich method.
8. Use according to claim 6, characterized in that: the transgenic protein is Cry1 Ab.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1643006A2 (en) * | 2004-09-29 | 2006-04-05 | Outokumpu Copper Products Oy | Method for the fabrication of a multi-layered patina and said multi-layaered patina |
| CN104133066A (en) * | 2014-01-04 | 2014-11-05 | 常州碳宇纳米科技有限公司 | Multifunctional vector capable of carrying out instant positioning of protein |
| CN105403696A (en) * | 2015-12-11 | 2016-03-16 | 扬州大学 | Label-free chemiluminescent immunosensor based on nanometer mimic enzyme, and preparation and analysis methods thereof |
| WO2019229495A1 (en) * | 2018-05-29 | 2019-12-05 | Copperprotek Spa. | Microstructured multicomposite copper microparticle with antibacterial and/or biocidal activity that comprises 5 different types of copper compounds |
| CN110878296A (en) * | 2019-10-16 | 2020-03-13 | 武汉伊莱瑞特生物科技股份有限公司 | High-sensitivity HRP enzyme and preparation method and application thereof |
-
2020
- 2020-11-09 CN CN202011236292.0A patent/CN112326955B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1643006A2 (en) * | 2004-09-29 | 2006-04-05 | Outokumpu Copper Products Oy | Method for the fabrication of a multi-layered patina and said multi-layaered patina |
| CN104133066A (en) * | 2014-01-04 | 2014-11-05 | 常州碳宇纳米科技有限公司 | Multifunctional vector capable of carrying out instant positioning of protein |
| CN105403696A (en) * | 2015-12-11 | 2016-03-16 | 扬州大学 | Label-free chemiluminescent immunosensor based on nanometer mimic enzyme, and preparation and analysis methods thereof |
| WO2019229495A1 (en) * | 2018-05-29 | 2019-12-05 | Copperprotek Spa. | Microstructured multicomposite copper microparticle with antibacterial and/or biocidal activity that comprises 5 different types of copper compounds |
| CN110878296A (en) * | 2019-10-16 | 2020-03-13 | 武汉伊莱瑞特生物科技股份有限公司 | High-sensitivity HRP enzyme and preparation method and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| Cyanobacterial mineralisation of posnjakite (Cu4(SO4)(OH)6·H2O) inCu-rich acid mine drainage at Yanqul, northern Oman;Bernhard Pracejus 等;《Chemie der Erde》;20171231;第77卷;第535-544页 * |
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