CN102183562A - Unmarked current-mode immunosensor and manufacturing method and application thereof - Google Patents
Unmarked current-mode immunosensor and manufacturing method and application thereof Download PDFInfo
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Abstract
The invention relates to an unmarked current-mode immunosensor; a work electrode surface of the immunosensor is covered with four layers of films as follows: the first layer is a redox layer formed by the mixture of a chitosan, a potassium ferricyanide and a metallic nano-material on the electrode surface; the second layer is a protective layer formed by the mixture of a perfluorinated proton exchange resin and the metallic nano-material on the redox layer; the third layer is a polyethyleneimine layer formed on the surface of the protective layer; and the fourth layer is the polyethyleneimine layer that is processed by using glutaradehyde and capable of chemically adsorbing antibody. The potassium ferricyanide is used as a redox probe material and the chitosan is used as a fixed matrix; and therefore, the reproducibility of the current-mode sensor is effectively improved.
Description
Technical Field
The invention relates to a current type immunosensor taking potassium ferricyanide as an oxidation-reduction probe substance and chitosan as a fixed matrix, belonging to the field of electrochemical sensors.
Background
Compared with traditional radioimmunoassay, enzyme-linked immunoassay and chemiluminescence immunoassay, the electrochemical immunosensor has the advantages of relatively simple pretreatment process, short analysis time, low detection limit, low requirements on instruments and the like, and thus has received wide attention (journal of clinical examination, 2003, 03, 181. journal of chinese medical physics, 2006, 2, 132.). Various types of electrochemical immunosensors have been developed, such as amperometric, voltage, capacitance, and impedance based sensors (anal. chem.2001, 73, 3219.electrochem. commun.2004, 6, 1222.sens. activators B1999, 57, 201.anal. chem.2002, 74, 4814.) that perform immunoassays by causing a change in the electrochemical signal of the sensor after an antibody-antigen reaction.
The current type immunosensor for enhancing detection sensitivity by utilizing nano materials is mainly divided into a marked type and a non-marked type. The labeled amperometric immunosensor is prepared by immobilizing antibodies or antigen molecules on the surface of an electrode, and indirectly measuring the content of antigens to be measured by measuring electrochemical signals of electrochemical active substances generated by the action of enzyme labeled on the antibodies and a substrate after the sensor is incubated in a solution containing the antigens to be measured and enzyme labeled antibodies. Horseradish peroxidase, alkaline phosphatase, glucose oxidase, and the like are often used as a labeling enzyme for the antibody (i.e., enzyme-labeled antibody). The existing labeled amperometric immunosensor needs to add an electroactive substance with redox capability into a solution to be measured as a mediator or a non-immunological reagent such as an enzymatic substrate of the enzyme-labeled antibody, and the reagent influences the stability of the measurement result.
The non-labeling type current immunosensor is characterized in that a layer of redox probe substance film with electrochemical activity is formed on the surface of an electrode, then antibody molecules are connected to the surface of the redox probe substance film, and then immune recognition is carried out on an antigen to be detected and electrochemical immune detection is carried out. The non-labeling type electrochemical immunosensor does not need an electroactive substance with redox capability as a mediator, and does not need the intervention of an enzymatic substrate of an enzyme-labeled antibody in the labeling type current immunosensor, so that the non-labeling type electrochemical immunosensor has the advantages of simplicity, rapidness and the like, and the problem of pollution of the mediator to the surface of an electrode in the labeling type current immunosensor is avoided. Nevertheless, the unlabeled amperometric immunosensor is linked to the antibody mainly by electrostatic interaction, and it is difficult to ensure a consistent amount of antibody immobilized each time, thus affecting the reproducibility of the sensor.
In order to overcome the defects of the existing non-labeled amperometric immunosensor, the invention firstly utilizes chitosan to directly fix a redox probe substance on the surface of an electrode, a redox probe substance film is formed on the electrode, and an antibody is fixed on the redox probe substance film through chemical modification, so that the novel non-labeled amperometric immunosensor is prepared.
Disclosure of Invention
The invention aims to provide a non-labeled current-mode immunosensor, so as to overcome the defect of poor reproducibility of the immunosensor in the prior art.
The invention is realized by the following technical scheme:
a non-labeling type current immunosensor is characterized in that the surface of a working electrode is covered with 4 layers of films: the first layer is an oxidation-reduction layer formed on the surface of the electrode by a mixture of chitosan, potassium ferricyanide and a metal nano material; the second layer is a protective layer formed by a mixture of perfluorinated proton exchange resin and a metal nano material on the surface of the redox layer; the third layer is a polyethylene imine layer formed on the surface of the protective layer; the fourth layer is a polyethyleneimine layer which is treated with glutaraldehyde and then chemisorbed with an antibody.
The antibody can be immune antibodies commonly used in the field, such as immunoglobulin and its family, carcinoembryonic antibody and other cancer marker antibodies and the like.
Wherein, in the first layer, the metal nano material is gold nano particles or silver nano particles.
The particle size of the metal nano material is 10-60 nm.
The perfluorinated proton exchange resin is a perfluorinated sulfonic acid exchange resin (nafion).
And combining the working electrode, the counter electrode and the reference electrode to form an immunosensor to detect the antigen.
The antigen may be an immunological antigen commonly used in the art, such as immunoglobulins and families thereof, carcinoembryonic antigen, and other cancer marker antigens, and the like.
In an embodiment of the invention, the counter electrode is a large area platinum sheet electrode.
The reference electrode is a saturated potassium chloride Ag/AgCl electrode or a saturated calomel electrode.
It is another object of the present invention to provide a method for preparing the non-labeled amperometric immunosensor, wherein the preparation of the working electrode comprises the steps of:
1) mixing a chitosan aqueous solution and a potassium ferricyanide aqueous solution, adding a metal nano material aqueous solution, coating the obtained mixture on the surface of a working electrode, and airing at normal temperature or drying in an oven at 50-80 ℃;
2) mixing an ethanol solution of perfluorinated sulfonic acid exchange resin with an ethanol solution of a metal nano material, coating the obtained mixture on the working electrode treated in the step 1), and airing at normal temperature or drying in an oven at 50-80 ℃;
3) immersing the working electrode treated in the step 2) into a polyethyleneimine water solution, taking out, washing with water, and drying with nitrogen to form a polyethyleneimine layer;
4) immersing the working electrode treated in the step 3) into a glutaraldehyde aqueous solution to form an aldehyde-rich surface;
5) immersing the working electrode treated in the step 4) into an antibody aqueous solution, taking out the working electrode, and sealing the working electrode by using a bovine serum albumin aqueous solution.
Wherein,
in the step 1), the concentration of the chitosan aqueous solution is 0.5-2 wt%, the concentration of the potassium ferricyanide aqueous solution is 1-10mM, and the absorbance of the metal nano material aqueous solution at the wavelength of the maximum visible absorption peak is 1-5; the volume ratio of the chitosan water solution, the potassium ferricyanide water solution and the metal nano material water solution is 0.05-0.2: 0.01-0.1: 0.02-0.1;
in the step 2), the concentration of the ethanol solution of the perfluorinated proton exchange resin is 1-5 wt%, the absorbance of the ethanol solution of the metal nano material under the wavelength of the maximum visible absorption peak is 1-5, and the volume ratio of the ethanol solution of the perfluorinated proton exchange resin to the ethanol solution of the metal nano material is 0.05-0.2: 0.02-0.1;
in the step 3), the concentration of the polyethyleneimine water solution is 10-50 mg/mL; the working electrode is immersed in the polyethyleneimine water solution for 5-30 min;
in the step 4), the concentration of the glutaraldehyde aqueous solution is 1.0-5.0 mg/mL; the working electrode is immersed in the glutaraldehyde water solution for 5-30 min;
in the step 5), the concentration of the antibody aqueous solution is 100-300 ug/mL; the time for immersing the working electrode into the antibody water solution is 2-4 hours;
the concentration of the bovine serum albumin aqueous solution is 5-20 mg/mL; the time for the working electrode to be immersed in the bovine serum albumin aqueous solution is 20-40 min.
Another object of the present invention is to provide a method for using the non-labeled amperometric immunosensor, which comprises:
1) working curves for the antigen were determined.
Preparing an antigen standard aqueous solution with the concentration range of 0-1000 ng/mL, wherein the used antigen needs to correspond to the antibody adsorbed in the working electrode. Incubating the working electrode in an antigen standard aqueous solution for 30-70min at 30-40 ℃, taking out, washing with secondary distilled water, taking the incubated working electrode as a working electrode of a sensor, taking a large-area platinum sheet electrode as a counter electrode, taking a saturated calomel electrode or a saturated potassium chloride Ag/AgCl electrode as a reference electrode, and detecting in a phosphoric acid buffer solution or a Tris-HCl buffer solution with the pH value of 6.5-7.3 by adopting a cyclic voltammetry method, a square wave voltammetry method and a differential pulse voltammetry method to obtain a determination working curve of the antigen to be detected;
2) the antigen to be detected is measured.
Incubating an immunosensor in an aqueous solution containing an antigen to be detected for 30-70min at 30-40 ℃, taking out, washing with secondary distilled water, taking the incubated non-labeled current type immunosensor as a working electrode, a large-area platinum sheet electrode as a counter electrode, a saturated calomel electrode or a saturated potassium chloride Ag/AgCl electrode as a reference electrode, detecting in a phosphoric acid buffer solution or a Tris-HCl buffer solution with the pH value of 6.5-7.3 by adopting a cyclic voltammetry method, a square wave voltammetry method or a differential pulse voltammetry method, and contrasting the detection result with a measurement working curve of the antigen to find out the corresponding concentration.
The immunosensor provided by the invention has the advantages of simple and convenient preparation process, low cost, excellent reproducibility, high detection sensitivity and the like, and can be widely used for various immunoassay and detection.
Drawings
FIG. 1 is a standard curve for carcinoembryonic antigen (CEA) measurement;
FIG. 2 is a graph showing a standard curve for measurement of human IgG.
Detailed Description
The present invention is further described below in conjunction with examples, which are to be understood as being illustrative only and in no way limiting to the scope of the invention.
Example 1
Firstly, preparing a non-labeling type current mode immunosensor for CEA detection
Selecting a disc-shaped plane glassy carbon electrode, taking CEA as a detection antigen, taking a CEA antibody as an immobilized antibody:
1) mixing 0.1mL of 1% chitosan aqueous solution with 0.05mL of 5mM potassium ferricyanide aqueous solution, adding 0.05mL of gold nanoparticle aqueous solution with the particle size of 25nm and the absorbance of 5 at the wavelength of the maximum visible absorption peak, uniformly mixing, brushing the mixture on the surface of an electrode, and drying the electrode in an oven at 50 ℃;
2) mixing 0.1mL of 1.25% nafion ethanol solution and 0.05mL of gold nanoparticle ethanol solution with the particle size of 25nm and the absorbance of 5 at the wavelength of the maximum visible absorption peak, coating the obtained mixture on the surface of the electrode treated in the step 1), and drying in an oven at 50 ℃;
3) immersing the modified electrode obtained in the step 2) into a polyethyleneimine water solution (50mg/mL) for 15min, taking out, washing with water, and drying with nitrogen to form a polyethyleneimine layer;
4) immersing the modified electrode obtained in the step 3) into 2.5mg/mL glutaraldehyde aqueous solution for 15min to form an aldehyde-rich surface;
5) and (3) immersing the modified electrode obtained in the step 4) into a CEA antibody aqueous solution of 200 mug/mL for incubation for 3 hours, taking out the modified electrode, and then putting the modified electrode into a bovine serum albumin aqueous solution of 10mg/mL for 30 minutes to obtain the non-labeled amperometric immunosensor capable of detecting CEA.
II, detecting CEA:
1) working curve for determining CEA
Preparing a CEA standard aqueous solution with the concentration range of 0-1000 ng/mL, respectively incubating the prepared CEA antibody modified non-labeling type current type immunosensor in the CEA standard aqueous solutions with different concentrations for 30min at 30 ℃, taking out, washing with secondary distilled water, taking the obtained product as a working electrode, taking a large-area platinum sheet electrode as a counter electrode, taking a saturated potassium chloride Ag/AgCl electrode as a reference electrode, and detecting in a phosphoric acid buffer solution with the pH value of 7.0 by adopting a square wave voltammetry to obtain a CEA determination working curve, wherein the working curve is shown in FIG. 1.
2) Determination of CEA
Incubating a CEA antibody modified non-labeled amperometric immunosensor in an aqueous solution containing an antigen to be detected for 30min at 35 ℃, taking out, washing with secondary distilled water, taking the washed sensor as a working electrode, a large-area platinum sheet electrode as a counter electrode, a saturated potassium chloride Ag/AgCl electrode as a reference electrode, detecting in a phosphoric acid buffer solution with the pH value of 7.0 by adopting a square wave voltammetry method, and comparing the detection result with the determination working curve of the CEA in the step 1) to find out the corresponding concentration.
The results showed that the detection limit for CEA was 3 pg/mL.
Example 2
Preparation of non-labeled amperometric immunosensor for human IgG detection
Selecting a disc-shaped plane gold electrode, taking human IgG as a detection object and anti-human IgG as an immobilized antibody:
1) mixing 0.2mL of 2% chitosan aqueous solution with 0.1mL of 10mM potassium ferricyanide aqueous solution, adding 0.1mL of aqueous solution of silver nanoparticles with the particle size of 20nm and the absorbance of 1 at the wavelength of the maximum visible absorption peak, uniformly mixing, coating the obtained mixture on the surface of an electrode, and drying in an oven at 80 ℃;
2) mixing 0.2mL of 1% nafion aqueous solution with 0.1mL of ethanol solution of silver nanoparticles with the particle size of 20nm and the absorbance of 1 at the wavelength of the maximum visible absorption peak, coating the obtained mixture on the surface of the electrode treated in the step 1), and drying in an oven at 80 ℃;
3) immersing the modified electrode obtained in the step 2) into a polyethyleneimine aqueous solution (with the concentration of 20mg/mL) for 30min, taking out, washing with water, and drying with nitrogen to form a polyethyleneimine layer;
4) immersing the modified electrode obtained in the step 3) into 5mg/mL glutaraldehyde aqueous solution for 30min to form an aldehyde group-rich surface;
5) and (3) immersing the modified electrode obtained in the step 4) into a 300 mu g/mL aqueous solution of human IgG antibody, incubating for 3 hours, taking out, and then putting into a 20mg/mL aqueous solution of bovine serum albumin for 30min to obtain the non-labeled amperometric immunosensor capable of detecting human IgG.
II, detecting human IgG:
1) working curve for determination of human IgG
Preparing a standard human IgG solution with a concentration range of 0-1000 ng/mL, incubating the prepared non-labeled amperometric immunosensor capable of detecting human IgG in the standard human IgG solutions with different concentrations for 35min at 40 ℃, taking out, washing with secondary distilled water, taking the washed sensor as a working electrode, a large-area platinum sheet electrode as a counter electrode, a saturated calomel electrode as a reference electrode, and detecting in a phosphoric acid buffer solution with the pH value of 7.0 by adopting a cyclic voltammetry method to obtain a measurement working curve of the human IgG, wherein the measurement working curve is shown in FIG. 2;
2) determination of human IgG
Incubating a non-labeled amperometric immunosensor capable of detecting human IgG in an aqueous solution containing an antigen to be detected for 30min at 35 ℃, taking out, washing with secondary distilled water, taking the secondary distilled water as a working electrode, a large-area platinum sheet electrode as a counter electrode, a saturated calomel electrode as a reference electrode, detecting in a phosphate buffer solution with the pH of 7.0 by adopting a cyclic voltammetry method, and comparing the detection result with the measurement working curve of the human IgG in the step 1) to find out the corresponding concentration.
The results showed that the limit of detection of human IgG was 10 pg/mL.
According to the invention, the redox probe substance is directly fixed on the surface of the electrode by utilizing chitosan, the redox probe substance film is formed on the electrode, and the antibody is modified on the redox probe substance film, so that the non-labeled current-mode immunosensor provided by the invention is obtained, and the defects of complexity and time consumption in the preparation process, poor repeatability, high sensor preparation cost and the like of the conventional non-labeled current-mode immunosensor are overcome.
The advantages of the invention are mainly shown in:
1) the preparation process of the redox probe substance film in the non-labeled immunosensor is simple;
2) the chemical bond is adopted for fixing when the antibody is fixed, so that the amount of the antibody fixed each time tends to be consistent, and the problems of poor reproducibility and the like caused by physical adsorption used in the conventional fixed antibody are solved;
3) the non-labeled amperometric immunosensor disclosed by the invention has extremely high detection sensitivity on corresponding antigens.
The non-labeled current mode immunosensor provided by the invention has the advantages of simpler preparation process, lower cost, better reproducibility and more sensitive detection, and can be widely used for various immunoassay and detection.
The foregoing is merely a preferred embodiment of the invention, which is intended to be illustrative and not limiting. It will be understood by those skilled in the art that various changes, modifications and equivalents may be made therein without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (10)
1.A non-labeling type current immunosensor is characterized in that the surface of a working electrode is covered with 4 layers of films: the first layer is an oxidation-reduction layer formed on the surface of the electrode by a mixture of chitosan, potassium ferricyanide and metal nano materials; the second layer is a protective layer formed by a mixture of perfluorinated proton exchange resin and a metal nano material on the surface of the redox layer; the third layer is a polyethylene imine layer formed on the surface of the protective layer; the fourth layer is a polyethyleneimine layer which is treated with glutaraldehyde and then chemisorbed with an antibody.
2. The unlabeled amperometric immunosensor according to claim 1,
the metal nano material is gold nano particles or silver nano particles.
3. The unlabeled amperometric immunosensor according to claim 2, wherein the metal nanomaterial has a particle size of 10-60 nm.
4. The unlabeled amperometric immunosensor according to claim 1 or 2, wherein the perfluorinated proton exchange resin is a perfluorosulfonic acid exchange resin.
5. A method of making the unlabeled amperometric immunosensor of the preceding claims, wherein the working electrode is prepared by the steps of:
1) mixing a chitosan aqueous solution and a potassium ferricyanide aqueous solution, adding a metal nano material aqueous solution, coating the obtained mixture on the surface of a working electrode, and airing at normal temperature or drying in an oven at 50-80 ℃;
2) mixing the ethanol solution of perfluorinated proton exchange resin with the ethanol solution of metal nano material, coating the obtained mixture on the working electrode treated in the step 1), and airing at normal temperature or drying in an oven at 50-80 ℃;
3) immersing the working electrode treated in the step 2) into a polyethyleneimine water solution, taking out, washing with water, and drying with nitrogen to form a polyethyleneimine layer;
4) immersing the working electrode treated in the step 3) into a glutaraldehyde aqueous solution to form an aldehyde-rich surface;
5) immersing the working electrode treated in the step 4) into an antibody aqueous solution, taking out the working electrode, and sealing the working electrode by using a bovine serum albumin aqueous solution.
6. The method for producing a non-labeled amperometric immunosensor according to claim 5,
in the step 1), the concentration of the chitosan aqueous solution is 0.5-2 wt%, the concentration of the potassium ferricyanide aqueous solution is 1-10mM, and the absorbance of the metal nano material aqueous solution at the wavelength of the maximum visible absorption peak is 1-5; the volume ratio of the chitosan water solution, the potassium ferricyanide water solution and the metal nano material water solution is 0.05-0.2: 0.01-0.1: 0.02-0.1.
7. The method for producing a non-labeled amperometric immunosensor according to claim 5,
in the step 2), the concentration of the ethanol solution of the perfluorinated proton exchange resin is 1-5 wt%, the absorbance of the ethanol solution of the metal nano material under the wavelength of the maximum visible absorption peak is 1-5, and the volume ratio of the ethanol solution of the perfluorinated proton exchange resin to the ethanol solution of the metal nano material is 0.05-0.2: 0.02-0.1.
8. The method for producing a non-labeled amperometric immunosensor according to claim 5,
in the step 3), the concentration of the polyethyleneimine water solution is 10-50 mg/mL; the working electrode is immersed in the polyethyleneimine water solution for 5-30 min;
in the step 4), the concentration of the glutaraldehyde aqueous solution is 1.0-5.0 mg/mL; the working electrode is immersed in the glutaraldehyde water solution for 5-30 min;
in the step 5), the concentration of the antibody aqueous solution is 100-300 ug/mL; the time for immersing the working electrode into the antibody water solution is 2-4 hours;
the concentration of the bovine serum albumin aqueous solution is 5-20 mg/mL; the time for the working electrode to be immersed in the bovine serum albumin aqueous solution is 20-40 min.
9. A method of using the unlabeled amperometric immunosensor of any one of claims 1-4,
processing of the working electrode: incubating the working electrode in an antigen standard aqueous solution for 30-70min at 30-40 ℃, taking out, and washing with secondary distilled water; followed by
And detecting the antigen by the electrochemical method of the processed working electrode in a buffer solution.
10. The method of using the unlabeled amperometric immunosensor according to claim 9, wherein the buffer solution is a phosphate buffer solution or a Tris-HCl buffer solution with a pH of 6.5 to 7.3; the electrochemical method is square wave voltammetry or cyclic voltammetry.
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Cited By (4)
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| CN102818893A (en) * | 2012-08-28 | 2012-12-12 | 济南大学 | Preparation and application of Au@Pd core-shell material constructed lung cancer tumor marker immunosensor |
| CN104114075A (en) * | 2012-02-13 | 2014-10-22 | 皇家飞利浦有限公司 | Photonic probe apparatus with integrated tissue marking facility |
| CN105842438A (en) * | 2016-03-28 | 2016-08-10 | 南京邮电大学 | Preparation method of prussian blue cube / molybdenum disulfide nano composite material |
| CN114414642A (en) * | 2022-01-07 | 2022-04-29 | 重庆医科大学 | Electrochemical sensor for detecting human papillomavirus type 16E 6 oncoprotein |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104114075A (en) * | 2012-02-13 | 2014-10-22 | 皇家飞利浦有限公司 | Photonic probe apparatus with integrated tissue marking facility |
| CN104114075B (en) * | 2012-02-13 | 2016-08-31 | 皇家飞利浦有限公司 | There is the photon probe unit of integrated organization marking arrangement |
| CN102818893A (en) * | 2012-08-28 | 2012-12-12 | 济南大学 | Preparation and application of Au@Pd core-shell material constructed lung cancer tumor marker immunosensor |
| CN105842438A (en) * | 2016-03-28 | 2016-08-10 | 南京邮电大学 | Preparation method of prussian blue cube / molybdenum disulfide nano composite material |
| CN114414642A (en) * | 2022-01-07 | 2022-04-29 | 重庆医科大学 | Electrochemical sensor for detecting human papillomavirus type 16E 6 oncoprotein |
| CN114414642B (en) * | 2022-01-07 | 2023-06-02 | 重庆医科大学 | Electrochemical sensor for detecting human papilloma virus 16 type E6 oncoprotein |
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| CN102183562B (en) | 2013-06-19 |
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