CN116990276A - Protein molecule detection method - Google Patents
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/40—Concentrating samples
- G01N1/405—Concentrating samples by adsorption or absorption
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- 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
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- 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
- G01N27/416—Systems
- G01N27/447—Systems using electrophoresis
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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
- G01N27/416—Systems
- G01N27/447—Systems using electrophoresis
- G01N27/44704—Details; Accessories
- G01N27/44747—Composition of gel or of carrier mixture
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Abstract
The invention relates to a protein molecule detection method, which comprises the following steps: preparing reagents and preparing magnetic molecular imprinting nano particles, utilizing the magnetic molecular imprinting nano particles to realize specific enrichment of target proteins, weighing 5.0g of glycine, 3.0g of tris (hydroxymethyl) aminomethane and 1.0g of sodium dodecyl sulfate, adding a proper amount of double distilled water for dissolution, adjusting the pH to 8.3 by using 1mol/l hydrochloric acid, adding double distilled water to 1000ml, and storing at 4 ℃; dripping a sample to be detected of the insect protein molecules onto the SOI-based porous silicon, drying the SOI-based porous silicon dripped with the sample to be detected of the insect protein molecules until no water is visible to naked eyes, and measuring fluorescence of the dried SOI-based porous silicon dripped with the sample to be detected of the insect protein molecules by using an ultraviolet-visible fluorescence spectroscope, wherein the excitation wavelength is 320-450 nm. The invention has simple process, low cost and strong controllability, can realize quantitative detection of the object to be detected according to the change of the spectrum data, and has simpler detection flow and convenient and quick operation.
Description
Technical Field
The invention relates to the technical field of protein molecule detection, in particular to a protein molecule detection method.
Background
Proteins, which are important components of human cells and tissues, are involved in almost all life processes and are closely related to the occurrence and development of various diseases. Proteins have been widely used as disease markers for clinical diagnosis and disease assessment of major diseases such as cancer, cardiovascular and infectious diseases. Therefore, the development of accurate detection research of proteins has very important scientific research value and clinical application value in early diagnosis, treatment and prevention and control of serious diseases, and the expression of the proteins is detected semi-quantitatively by ELISA or immunoblotting in scientific research at present, and the two technologies of ELISA and immunoblotting require special instruments and equipment, so that the experimental operation is complex, the detection period is long, and the invention provides a new solution to the problems.
Disclosure of Invention
(one) solving the technical problems
Aiming at the defects of the prior art, the invention provides a protein molecule detection method to solve the technical problem of the structure proposed in the background art.
(II) technical scheme
In order to achieve the above purpose, the present invention provides the following technical solutions: a method for detecting a protein molecule comprising the steps of:
step one: preparing reagents and preparing magnetic molecular imprinting nano particles, utilizing the magnetic molecular imprinting nano particles to realize specific enrichment of target proteins, weighing 5.0g of glycine, 3.0g of tris (hydroxymethyl) aminomethane and 1.0g of sodium dodecyl sulfate, adding a proper amount of double distilled water for dissolution, adjusting the pH to 8.3 by using 1mol/l hydrochloric acid, adding double distilled water to 1000ml, and storing at 4 ℃;
step two: dripping a sample to be detected of the insect protein molecules onto the SOI-based porous silicon, drying the SOI-based porous silicon dripped with the sample to be detected of the insect protein molecules until no water is visible to naked eyes, and measuring fluorescence of the dried SOI-based porous silicon dripped with the sample to be detected of the insect protein molecules by using an ultraviolet-visible fluorescence spectroscope, wherein the excitation wavelength is 320-450 nm;
step three: combining electroactive molecules or ions on the surfaces of nano particles to perform electric signal conversion, preparing a test solution, taking 5.0g of fresh air capsule content, grinding into fine powder in a mortar, adding 6ml of 1MTris-HCl buffer solution to dissolve, grinding uniformly, standing for 5min, centrifuging for 20min at 4000r/min, sucking 900 μl of supernatant, adding 300 μl of 4 x protein loading buffer solution, mixing, boiling water bath for 5min, centrifuging for 20min at 12000r/min, cooling, sucking supernatant, subpackaging for 20 μl per tube, and storing at-20 ℃;
step four: according to fluorescence variables obtained by detecting samples to be detected of the different concentrations of the artemia salina molecules, a linear regression equation between the fluorescence reduction degree and the concentration of the artemia salina molecules is established, and then the concentration of the artemia salina molecules under different fluorescence reduction degrees can be obtained by the linear regression equation; wherein: the bag worm protein molecule is P38 antigen;
step five: and (3) installing an electrophoresis tank and preparing gel, then carrying out electrophoresis on the gel, finally carrying out protein molecular weight measurement, calculating relative mobility according to the migration distance of the sample/the migration distance of the indicator, carrying out a standard curve on the relative mobility according to the logarithm of the standard protein molecular weight, and searching the curve according to the relative mobility of the sample to be detected, so as to calculate the molecular weight of the sample to be detected.
In a further preferred aspect, after the electrophoresis is finished, opening an electrophoresis tank, taking out gel, cutting off concentrated gel, reserving separation gel and marking the front edge of bromophenol blue, placing the gel in a fixing solution for fixing for half an hour, taking the gel, placing the gel in a proper amount of coomassie brilliant blue staining solution, ensuring that the staining solution can fully cover the gel, placing the gel on a horizontal shaking table for slowly shaking, and staining at room temperature for 1.5-2.5 hours; pouring out the dyeing liquid, adding a proper amount of coomassie brilliant blue decolorization liquid, ensuring that the coomassie brilliant blue decolorization liquid can fully cover gel, slowly shaking on a horizontal shaking table, and decolorizing at room temperature for 3.5-4.5 hours; changing the coomassie brilliant blue decolorization liquid for 2-4 times until the blue background is completely removed and the dyeing effect of the protein strips reaches the expectation, and after decolorization, storing the gel in water containing 20% of glycerol for photographing; and (5) analyzing the integral value of the optical density of the shot photo.
In a further preferred embodiment, in the third step, the content of the electroactive material is detected on the surface of the magnetic electrode by an electrochemical method, so as to realize indirect electrochemical detection of the target protein; and finally, outputting and analyzing the detection result.
In a further preferred embodiment, in the first step, the protein molecules are dissolved in a solution containing the magnetic nanomaterial and the polymeric monomer, and the magnetic molecularly imprinted nanomaterial is prepared by binding the protein molecules to the surfaces of the magnetic nanoparticles through a silylation reaction and washing to remove the protein molecules.
In a further preferred aspect, the magnetic molecularly imprinted nanomaterial is dispersed in a solution to be detected to perform specific enrichment of target proteins, and after enrichment is completed, separation and washing are simply realized by using a magnet; through the biological coupling effect between the electroactive molecule and the protein molecule, the electroactive molecule is combined to the surface of the magnetic molecular imprinting nano material to perform electric signal conversion, and the material is adsorbed on the surface of the magnetic electrode to perform electrochemical detection of the electroactive substance content, so that indirect electrochemical detection of the protein molecule is realized.
In a further preferred embodiment, the electroactive metal ions are combined to the surface of the magnetic molecularly imprinted nanomaterial by metal coordination, so as to perform electrical signal conversion of protein molecules, and the nanomaterial is adsorbed to the surface of the magnetic electrode, so as to perform electrochemical detection of the metal ion content, and further realize indirect electrochemical detection of the protein molecules.
In a further preferred embodiment, the SOI-based porous silicon is obtained by the following method: cutting an SOI monocrystalline silicon wafer with the crystal orientation of < 100 >, the resistivity of 3 omega cm to 3.5 omega cm and the thickness of 500 mu m to 550 mu m into square with the thickness of 2cm multiplied by 2cm, thoroughly cleaning the square for 10min in an ultrasonic instrument by acetone, absolute ethyl alcohol and deionized water in sequence, putting the cleaned SOI monocrystalline silicon wafer into a corrosion groove, and soaking the SOI monocrystalline silicon wafer in a corrosion solution with the concentration of HF, wherein the volume ratio of C2H5OH is 1:3, carrying out electrochemical corrosion for 600s under the condition that the corrosion current intensity is 35mA/cm < 2 >, repeatedly flushing the SOI monocrystalline silicon wafer subjected to electrochemical corrosion with deionized water, drying in nitrogen, and finally oxidizing the SOI monocrystalline silicon wafer in air for 72 hours to obtain the SOI porous silicon.
In a further preferred embodiment, the excitation wavelength of the ultraviolet-visible fluorescence spectrometer is 370nm.
(III) beneficial effects
Compared with the prior art, the invention provides a protein molecule detection method, which has the following beneficial effects:
the method has the advantages that the process is simple, the cost is low, the controllability is strong, the quantitative detection work of the object to be detected can be realized according to the change of spectral data, compared with protein quantitative methods such as immunoblotting and ELISA, the detection flow is simpler, the operation is convenient and quick, the detection time is short and is 3 to 4 hours, the advantages are provided for researching and developing a quantitative echinococcosis diagnosis technology based on the SOI technology, the electrochemical detection of the non-electroactive protein molecules is realized simply by utilizing an electric signal conversion technology, the method has good universality, the application of a magnetic molecular imprinting technology greatly enhances the chemical stability of electrode materials, the protein detection cost is reduced, the detection specificity is improved, in addition, the method for detecting the water-soluble protein molecular weight of the fresh air capsule combines an advanced biological technology with a new idea of the fingerprint spectrum by applying a SDS-PAGE electrophoresis technology, and provides a basis for establishing the electrophoresis characteristic spectrum of the preparation in the future.
Drawings
FIG. 1 is a flow chart showing the whole detection process of a protein molecule detection method of the present invention.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Embodiment one:
referring to fig. 1, a protein molecule detection method includes the following steps:
step one: preparing reagents and preparing magnetic molecular imprinting nano particles, utilizing the magnetic molecular imprinting nano particles to realize specific enrichment of target proteins, weighing 5.0g of glycine, 3.0g of tris (hydroxymethyl) aminomethane and 1.0g of sodium dodecyl sulfate, adding a proper amount of double distilled water for dissolution, adjusting the pH to 8.3 by using 1mol/l hydrochloric acid, adding double distilled water to 1000ml, and storing at 4 ℃;
step two: dripping a sample to be detected of the insect protein molecules onto the SOI-based porous silicon, drying the SOI-based porous silicon dripped with the sample to be detected of the insect protein molecules until no water is visible to naked eyes, and measuring fluorescence of the dried SOI-based porous silicon dripped with the sample to be detected of the insect protein molecules by using an ultraviolet-visible fluorescence spectroscope, wherein the excitation wavelength is 320-450 nm;
step three: combining electroactive molecules or ions on the surfaces of nano particles to perform electric signal conversion, preparing a test solution, taking 5.0g of fresh air capsule content, grinding into fine powder in a mortar, adding 6ml of 1MTris-HCl buffer solution to dissolve, grinding uniformly, standing for 5min, centrifuging for 20min at 4000r/min, sucking 900 μl of supernatant, adding 300 μl of 4 x protein loading buffer solution, mixing, boiling water bath for 5min, centrifuging for 20min at 12000r/min, cooling, sucking supernatant, subpackaging for 20 μl per tube, and storing at-20 ℃;
step four: according to fluorescence variables obtained by detecting samples to be detected of the different concentrations of the artemia salina molecules, a linear regression equation between the fluorescence reduction degree and the concentration of the artemia salina molecules is established, and then the concentration of the artemia salina molecules under different fluorescence reduction degrees can be obtained by the linear regression equation; wherein: the bag worm protein molecule is P38 antigen;
step five: and (3) installing an electrophoresis tank and preparing gel, then carrying out electrophoresis on the gel, finally carrying out protein molecular weight measurement, calculating relative mobility according to the migration distance of the sample/the migration distance of the indicator, carrying out a standard curve on the relative mobility according to the logarithm of the standard protein molecular weight, and searching the curve according to the relative mobility of the sample to be detected, so as to calculate the molecular weight of the sample to be detected.
In this embodiment, after the electrophoresis is completed, opening the electrophoresis tank, taking out the gel, cutting off the concentrated gel, reserving the separation gel and marking the front edge of bromophenol blue, placing the gel in the fixing solution for half an hour, placing the gel in a proper amount of coomassie brilliant blue staining solution, ensuring that the staining solution can fully cover the gel, placing the gel on a horizontal shaking table for slow shaking, and staining at room temperature for 1.5-2.5 hours; pouring out the dyeing liquid, adding a proper amount of coomassie brilliant blue decolorization liquid, ensuring that the coomassie brilliant blue decolorization liquid can fully cover gel, slowly shaking on a horizontal shaking table, and decolorizing at room temperature for 3.5-4.5 hours; changing the coomassie brilliant blue decolorization liquid for 2-4 times until the blue background is completely removed and the dyeing effect of the protein strips reaches the expectation, and after decolorization, storing the gel in water containing 20% of glycerol for photographing; and (5) analyzing the integral value of the optical density of the shot photo.
Example 2:
a method for detecting a protein molecule comprising the steps of:
step one: preparing reagents and preparing magnetic molecular imprinting nano particles, utilizing the magnetic molecular imprinting nano particles to realize specific enrichment of target proteins, weighing 5.0g of glycine, 3.0g of tris (hydroxymethyl) aminomethane and 1.0g of sodium dodecyl sulfate, adding a proper amount of double distilled water for dissolution, adjusting the pH to 8.3 by using 1mol/l hydrochloric acid, adding double distilled water to 1000ml, and storing at 4 ℃;
step two: dripping a sample to be detected of the insect protein molecules onto the SOI-based porous silicon, drying the SOI-based porous silicon dripped with the sample to be detected of the insect protein molecules until no water is visible to naked eyes, and measuring fluorescence of the dried SOI-based porous silicon dripped with the sample to be detected of the insect protein molecules by using an ultraviolet-visible fluorescence spectroscope, wherein the excitation wavelength is 320-450 nm;
step three: combining electroactive molecules or ions on the surfaces of nano particles to perform electric signal conversion, preparing a test solution, taking 5.0g of fresh air capsule content, grinding into fine powder in a mortar, adding 6ml of 1MTris-HCl buffer solution to dissolve, grinding uniformly, standing for 5min, centrifuging for 20min at 4000r/min, sucking 900 μl of supernatant, adding 300 μl of 4 x protein loading buffer solution, mixing, boiling water bath for 5min, centrifuging for 20min at 12000r/min, cooling, sucking supernatant, subpackaging for 20 μl per tube, and storing at-20 ℃;
step four: according to fluorescence variables obtained by detecting samples to be detected of the different concentrations of the artemia salina molecules, a linear regression equation between the fluorescence reduction degree and the concentration of the artemia salina molecules is established, and then the concentration of the artemia salina molecules under different fluorescence reduction degrees can be obtained by the linear regression equation; wherein: the bag worm protein molecule is P38 antigen;
step five: and (3) installing an electrophoresis tank and preparing gel, then carrying out electrophoresis on the gel, finally carrying out protein molecular weight measurement, calculating relative mobility according to the migration distance of the sample/the migration distance of the indicator, carrying out a standard curve on the relative mobility according to the logarithm of the standard protein molecular weight, and searching the curve according to the relative mobility of the sample to be detected, so as to calculate the molecular weight of the sample to be detected.
In the embodiment, in the third step, the content of the electroactive material is detected on the surface of the magnetic electrode by an electrochemical method, so as to realize indirect electrochemical detection of the target protein; and finally, outputting and analyzing the detection result.
In this embodiment, in the first step, the protein molecules are dissolved in a solution containing the magnetic nanomaterial and the polymeric monomer, and the magnetic molecularly imprinted nanomaterial is prepared by binding the protein molecules to the surfaces of the magnetic nanoparticles through a silylation reaction, and washing to remove the protein molecules.
In the embodiment, the magnetic molecular imprinting nano material is dispersed in the liquid to be detected, the specificity enrichment of the target protein is carried out, and the magnet is utilized to simply realize separation and washing after the enrichment is completed;
3) Through the biological coupling effect between the electroactive molecule and the protein molecule, the electroactive molecule is combined to the surface of the magnetic molecular imprinting nano material to perform electric signal conversion, and the material is adsorbed on the surface of the magnetic electrode to perform electrochemical detection of the electroactive substance content, so that indirect electrochemical detection of the protein molecule is realized.
In the embodiment, through metal coordination, electroactive metal ions are combined to the surface of the magnetic molecular imprinting nano material to perform electric signal conversion of protein molecules, the nano material is adsorbed on the surface of the magnetic electrode to perform electrochemical detection of the metal ion content, and then indirect electrochemical detection of the protein molecules is realized; the SOI-based porous silicon is obtained by the following method: cutting an SOI monocrystalline silicon wafer with the crystal orientation of < 100 >, the resistivity of 3 omega cm to 3.5 omega cm and the thickness of 500 mu m to 550 mu m into square with the thickness of 2cm multiplied by 2cm, thoroughly cleaning the square for 10min in an ultrasonic instrument by acetone, absolute ethyl alcohol and deionized water in sequence, putting the cleaned SOI monocrystalline silicon wafer into a corrosion groove, and soaking the SOI monocrystalline silicon wafer in a corrosion solution with the concentration of HF, wherein the volume ratio of C2H5OH is 1:3, carrying out electrochemical corrosion for 600s under the condition that the corrosion current intensity is 35mA/cm < 2 >, repeatedly flushing the SOI monocrystalline silicon wafer subjected to electrochemical corrosion with deionized water, drying in nitrogen, and oxidizing in air for 72 hours to obtain SOI porous silicon; the excitation wavelength of the ultraviolet-visible fluorescence spectrometer is 370nm.
Example 3:
a method for detecting a protein molecule comprising the steps of:
step one: preparing reagents and preparing magnetic molecular imprinting nano particles, utilizing the magnetic molecular imprinting nano particles to realize specific enrichment of target proteins, weighing 5.0g of glycine, 3.0g of tris (hydroxymethyl) aminomethane and 1.0g of sodium dodecyl sulfate, adding a proper amount of double distilled water for dissolution, adjusting the pH to 8.3 by using 1mol/l hydrochloric acid, adding double distilled water to 1000ml, and storing at 4 ℃;
step two: dripping a sample to be detected of the insect protein molecules onto the SOI-based porous silicon, drying the SOI-based porous silicon dripped with the sample to be detected of the insect protein molecules until no water is visible to naked eyes, and measuring fluorescence of the dried SOI-based porous silicon dripped with the sample to be detected of the insect protein molecules by using an ultraviolet-visible fluorescence spectroscope, wherein the excitation wavelength is 320-450 nm;
step three: combining electroactive molecules or ions on the surfaces of nano particles to perform electric signal conversion, preparing a test solution, taking 5.0g of fresh air capsule content, grinding into fine powder in a mortar, adding 6ml of 1MTris-HCl buffer solution to dissolve, grinding uniformly, standing for 5min, centrifuging for 20min at 4000r/min, sucking 900 μl of supernatant, adding 300 μl of 4 x protein loading buffer solution, mixing, boiling water bath for 5min, centrifuging for 20min at 12000r/min, cooling, sucking supernatant, subpackaging for 20 μl per tube, and storing at-20 ℃;
step four: according to fluorescence variables obtained by detecting samples to be detected of the different concentrations of the artemia salina molecules, a linear regression equation between the fluorescence reduction degree and the concentration of the artemia salina molecules is established, and then the concentration of the artemia salina molecules under different fluorescence reduction degrees can be obtained by the linear regression equation; wherein: the bag worm protein molecule is P38 antigen;
step five: and (3) installing an electrophoresis tank and preparing gel, then carrying out electrophoresis on the gel, finally carrying out protein molecular weight measurement, calculating relative mobility according to the migration distance of the sample/the migration distance of the indicator, carrying out a standard curve on the relative mobility according to the logarithm of the standard protein molecular weight, and searching the curve according to the relative mobility of the sample to be detected, so as to calculate the molecular weight of the sample to be detected.
The foregoing is only a preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art, who is within the scope of the present invention, should make equivalent substitutions or modifications according to the technical scheme of the present invention and the inventive concept thereof, and should be covered by the scope of the present invention.
Claims (8)
1. A method for detecting a protein molecule, comprising the steps of:
step one: preparing reagents and preparing magnetic molecular imprinting nano particles, utilizing the magnetic molecular imprinting nano particles to realize specific enrichment of target proteins, weighing 5.0g of glycine, 3.0g of tris (hydroxymethyl) aminomethane and 1.0g of sodium dodecyl sulfate, adding a proper amount of double distilled water for dissolution, adjusting the pH to 8.3 by using 1mol/l hydrochloric acid, adding double distilled water to 1000ml, and storing at 4 ℃;
step two: dripping a sample to be detected of the insect protein molecules onto the SOI-based porous silicon, drying the SOI-based porous silicon dripped with the sample to be detected of the insect protein molecules until no water is visible to naked eyes, and measuring fluorescence of the dried SOI-based porous silicon dripped with the sample to be detected of the insect protein molecules by using an ultraviolet-visible fluorescence spectroscope, wherein the excitation wavelength is 320-450 nm;
step three: combining electroactive molecules or ions on the surfaces of nano particles to perform electric signal conversion, preparing a test solution, taking 5.0g of fresh air capsule content, grinding into fine powder in a mortar, adding 6ml of 1MTris-HCl buffer solution to dissolve, grinding uniformly, standing for 5min, centrifuging for 20min at 4000r/min, sucking 900 μl of supernatant, adding 300 μl of 4 x protein loading buffer solution, mixing, boiling water bath for 5min, centrifuging for 20min at 12000r/min, cooling, sucking supernatant, subpackaging for 20 μl per tube, and storing at-20 ℃;
step four: according to fluorescence variables obtained by detecting samples to be detected of the different concentrations of the artemia salina molecules, a linear regression equation between the fluorescence reduction degree and the concentration of the artemia salina molecules is established, and then the concentration of the artemia salina molecules under different fluorescence reduction degrees can be obtained by the linear regression equation; wherein: the bag worm protein molecule is P38 antigen;
step five: and (3) installing an electrophoresis tank and preparing gel, then carrying out electrophoresis on the gel, finally carrying out protein molecular weight measurement, calculating relative mobility according to the migration distance of the sample/the migration distance of the indicator, carrying out a standard curve on the relative mobility according to the logarithm of the standard protein molecular weight, and searching the curve according to the relative mobility of the sample to be detected, so as to calculate the molecular weight of the sample to be detected.
2. The method for detecting protein molecules according to claim 1, wherein after the electrophoresis is finished, opening an electrophoresis tank, taking out gel, cutting off concentrated gel, reserving separation gel and marking the front edge of bromophenol blue, placing the gel in a fixing solution for fixing for half an hour, taking the gel, placing the gel in a proper amount of coomassie brilliant blue staining solution, ensuring that the staining solution can fully cover the gel, placing the gel on a horizontal shaking table for slowly shaking, and staining at room temperature for 1.5-2.5 hours; pouring out the dyeing liquid, adding a proper amount of coomassie brilliant blue decolorization liquid, ensuring that the coomassie brilliant blue decolorization liquid can fully cover gel, slowly shaking on a horizontal shaking table, and decolorizing at room temperature for 3.5-4.5 hours; changing the coomassie brilliant blue decolorization liquid for 2-4 times until the blue background is completely removed and the dyeing effect of the protein strips reaches the expectation, and after decolorization, storing the gel in water containing 20% of glycerol for photographing; and (5) analyzing the integral value of the optical density of the shot photo.
3. The method for detecting protein molecules according to claim 1, wherein in the third step, the content of the electroactive substance is detected on the surface of the magnetic electrode by an electrochemical method, so as to realize indirect electrochemical detection of the target protein; and finally, outputting and analyzing the detection result.
4. The method according to claim 3, wherein in the first step, the protein molecules are dissolved in a solution containing the magnetic nanomaterial and the polymer monomer, and the magnetic molecularly imprinted nanomaterial is prepared by binding the protein molecules to the surfaces of the magnetic nanoparticles through a silylation reaction and washing to remove the protein molecules.
5. The method for detecting protein molecules according to claim 3, wherein the magnetic molecular imprinting nano material is dispersed in the liquid to be detected, the specific enrichment of the target protein is carried out, and the separation and the washing are simply realized by using a magnet after the enrichment is completed; through the biological coupling effect between the electroactive molecule and the protein molecule, the electroactive molecule is combined to the surface of the magnetic molecular imprinting nano material to perform electric signal conversion, and the material is adsorbed on the surface of the magnetic electrode to perform electrochemical detection of the electroactive substance content, so that indirect electrochemical detection of the protein molecule is realized.
6. The method for detecting protein molecules according to claim 1, wherein electroactive metal ions are combined to the surface of the magnetic molecularly imprinted nanomaterial by metal coordination, so as to perform electrical signal conversion of protein molecules, and the nanomaterial is adsorbed to the surface of the magnetic electrode, so as to perform electrochemical detection of metal ion content, and further realize indirect electrochemical detection of protein molecules.
7. A method for detecting a protein molecule according to claim 3, wherein said SOI-based porous silicon is obtained by: cutting an SOI monocrystalline silicon wafer with the crystal orientation of < 100 >, the resistivity of 3 omega cm to 3.5 omega cm and the thickness of 500 mu m to 550 mu m into square with the thickness of 2cm multiplied by 2cm, thoroughly cleaning the square for 10min in an ultrasonic instrument by acetone, absolute ethyl alcohol and deionized water in sequence, putting the cleaned SOI monocrystalline silicon wafer into a corrosion groove, and soaking the SOI monocrystalline silicon wafer in a corrosion solution with the concentration of HF, wherein the volume ratio of C2H5OH is 1:3, carrying out electrochemical corrosion for 600s under the condition that the corrosion current intensity is 35mA/cm < 2 >, repeatedly flushing the SOI monocrystalline silicon wafer subjected to electrochemical corrosion with deionized water, drying in nitrogen, and finally oxidizing the SOI monocrystalline silicon wafer in air for 72 hours to obtain the SOI porous silicon.
8. The method according to claim 7, wherein the excitation wavelength of the ultraviolet-visible fluorescence spectrometer is 370nm.
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