CN102183562A - Unmarked current-mode immunosensor and manufacturing method and application thereof - Google Patents

Abstract

The invention relates to a non-labeled current type immunosensor. The surface of the working electrode of the immunosensor of the present invention is covered with four layers of film: the first layer is a redox layer formed on the electrode surface by a mixture of chitosan, potassium ferricyanide and metal nanomaterials; the second layer is a perfluorinated proton exchange layer. A protective layer formed on the surface of the redox layer by a mixture of resin and metal nanomaterials; the third layer is a polyethyleneimine layer formed on the surface of the protective layer; Polyethyleneimine layer for re-chemisorbed antibody after treatment. The immunosensor of the present invention uses potassium ferricyanide as a redox probe substance and chitosan as a fixed matrix, thereby effectively improving the reproducibility of the current type immunosensor.

Description

A non-labeled amperometric immunosensor and its preparation method and application

technical field

The invention relates to an amperometric immunosensor which uses potassium ferricyanide as a redox probe substance and chitosan as a fixed substrate, and belongs to the field of electrochemical sensors.

Background technique

Compared with traditional radioimmunoassays, enzyme-linked immunoassays, and chemiluminescence immunoassays, electrochemical immunosensors have the advantages of relatively simple pretreatment process, short analysis time, low detection limit, and low requirements for instruments. , which has received extensive attention (Journal of Clinical Laboratory, 2003, 03, 181. Chinese Journal of Medical Physics, 2006, 2, 132.). People have developed various types of electrochemical immunosensors, such as current-type, voltage-type, capacitive-type and impedance-type sensors (Anal.Chem.2001,73,3219.Electrochem.Commun.2004,6,1222.Sens. Actuators B 1999, 57, 201. Anal. Chem. 2002, 74, 4814.), these sensors all realize immunodetection by changing the electrochemical signal of the sensor after the antibody antigen reaction.

Amperometric immunosensors that use nanomaterials to enhance detection sensitivity are mainly divided into two types: labeled and non-labeled. The labeled amperometric immunosensor immobilizes the antibody or antigen molecule on the surface of the electrode. After the sensor is incubated in the solution containing the antigen to be tested and the enzyme-labeled antibody, the electric current generated by the interaction between the enzyme labeled on the antibody and the substrate is measured. The electrochemical signal of the chemically active substance indirectly measures the content of the antigen to be tested. Horseradish peroxidase, alkaline phosphatase, glucose oxidase, etc. are often used as labeled enzymes for antibodies (ie, enzyme-labeled antibodies). The existing labeled amperometric immunosensors need to add electroactive substances with redox ability as mediators or non-immune reagents such as the enzymatic substrates of the above-mentioned enzyme-labeled antibodies to the solution to be tested. These reagents will affect the stability of the measurement results. sex.

The non-labeled amperometric immunosensor is formed by first forming a layer of electrochemically active redox probe material film on the surface of the electrode, and then connecting antibody molecules on the surface of the redox probe material film, and then immunorecognizing the antigen to be tested and Perform electrochemical immunoassay. The unlabeled electrochemical immunosensor does not require the electroactive substance with redox ability as a mediator, nor does it require the intervention of the enzymatic substrate of the enzyme-labeled antibody in the labeled amperometric immunosensor, so the unlabeled electrochemical immunosensor The chemical immunosensor has the advantages of simplicity and rapidity, and at the same time avoids the problem of contamination of the electrode surface by the media in the labeled amperometric immunosensor. However, the non-labeled amperometric immunosensor mainly connects antibodies through electrostatic interaction, and it is difficult to ensure the same amount of antibody immobilization each time, thus affecting the reproducibility of the sensor.

In order to overcome the shortcomings of the existing non-labeled amperometric immunosensors, the present invention uses chitosan to directly immobilize the redox probe substance on the electrode surface for the first time, forms a redox probe substance film on the electrode, and passes the antibody through The chemical modification is immobilized on the redox probe substance membrane, and a new type of non-labeled current type immunosensor is prepared.

Contents of the invention

The purpose of the present invention is to provide a non-labeled current type immunosensor, so as to solve the defect of poor reproducibility of the prior art immunosensor.

The present invention is achieved through the following technical solutions:

A non-marking type current type immunosensor is characterized in that the surface of the working electrode is covered with 4 layers of film: the first layer is a redox layer formed on the electrode surface by a mixture of chitosan, potassium ferricyanide and metal nanomaterials; The second layer is a protective layer formed on the surface of the redox layer by a mixture of perfluorinated proton exchange resin and metal nanomaterials; the third layer is a polyethyleneimine layer formed on the surface of the protective layer; the fourth layer is The third layer was treated with glutaraldehyde and then the polyethylenimine layer chemisorbed the antibody.

The antibody may be an immune antibody commonly used in the art, such as immunoglobulin and its family, carcinoembryonic antibody, and other cancer marker antibodies.

Wherein, in the first layer, the metal nanomaterial is gold nanoparticles or silver nanoparticles.

The particle size of the metal nanomaterial is 10-60nm.

The perfluorinated proton exchange resin is perfluorosulfonic acid exchange resin (nafion).

The working electrode, the counter electrode and the reference electrode constitute an immunosensor to detect the antigen.

The antigen may be an immune antigen commonly used in the field, such as immunoglobulin and its family, carcinoembryonic antigen, and other cancer marker antigens.

In an embodiment of the present 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.

Another object of the present invention is to provide a method for preparing the non-labeled amperometric immunosensor, wherein the preparation of the working electrode comprises the following steps:

1) Mix chitosan aqueous solution with potassium ferricyanide aqueous solution, add metal nanomaterial aqueous solution, paint the resulting mixture on the surface of the working electrode, and dry at room temperature or in an oven at 50-80°C;

2) Mix the ethanol solution of the perfluorosulfonic acid exchange resin with the ethanol solution of the metal nanomaterial, paint the resulting mixture on the working electrode treated in step 1), and dry it at room temperature or in an oven at 50- Dry at 80°C;

3) immerse the working electrode treated in step 2) in the polyethyleneimine aqueous solution, take it out, wash it with water and dry it with nitrogen to form a polyethyleneimine layer;

4) immersing the working electrode treated in step 3) in an aqueous glutaraldehyde solution to form an aldehyde-rich surface;

5) Immerse the working electrode treated in step 4) in the antibody aqueous solution, take it out and seal it with bovine serum albumin aqueous solution.

in,

In step 1), the concentration of the chitosan aqueous solution is 0.5-2wt%, the concentration of the potassium ferricyanide aqueous solution is 1-10mM, and the absorbance of the metal nanomaterial aqueous solution at the maximum visible absorption peak wavelength is 1-5; The volume ratio of polysaccharide aqueous solution: potassium ferricyanide aqueous solution: metal nanomaterial aqueous solution is 0.05-0.2: 0.01-0.1: 0.02-0.1;

In step 2), the concentration of the ethanol solution of the perfluorinated proton exchange resin is 1-5wt%, the absorbance of the ethanol solution of the metal nanomaterial at the maximum visible absorption peak wavelength is 1-5, and the perfluorinated proton exchange resin The ethanol solution: the volume ratio of the ethanol solution of metal nanomaterials is 0.05-0.2: 0.02-0.1;

In step 3), the concentration of the polyethyleneimine aqueous solution is 10-50mg/mL; the time for the working electrode to be immersed in the polyethyleneimine aqueous solution is 5-30min;

In step 4), the concentration of the glutaraldehyde aqueous solution is 1.0-5.0mg/mL; the time for the working electrode to be immersed in the glutaraldehyde aqueous solution is 5-30min;

In step 5), the concentration of the antibody aqueous solution is 100-300ug/mL; the time for the working electrode to be immersed in the antibody aqueous solution is 2-4 hours;

The concentration of the bovine serum albumin aqueous solution is 5-20mg/mL; the time for the working electrode to be immersed in the bovine serum albumin aqueous solution is 20-40min.

Another object of the present invention is to provide a method for using the non-labeled amperometric immunosensor, specifically:

1) Determine the working curve of the antigen.

Prepare an antigen standard aqueous solution with a concentration range of 0-1000ng/mL, and the antigen used must correspond to the antibody adsorbed in the working electrode. At 30-40°C, incubate the working electrode of the present invention in an antigen standard aqueous solution for 30-70 minutes, take it out, wash it with twice distilled water, use the incubated working electrode as the working electrode of the sensor, and use the large-area platinum plate electrode As a counter electrode, a saturated calomel electrode or a saturated potassium chloride Ag/AgCl electrode is used as a reference electrode, and cyclic voltammetry and square wave voltammetry are used in a phosphate buffer solution or a Tris-HCl buffer solution with a pH of 6.5 to 7.3 method, differential pulse voltammetry to detect, and obtain the assay working curve of the antigen to be tested;

2) Determination of the antigen to be tested.

Incubate the immunosensor in the aqueous solution containing the antigen to be tested at 30-40°C for 30-70min, take it out, wash it with double distilled water, use the incubated non-labeled amperometric immunosensor as the working electrode, and use the large-area platinum The sheet electrode is used as the counter electrode, and the saturated calomel electrode or the saturated potassium chloride Ag/AgCl electrode is used as the reference electrode, and the cyclic voltammetry and square wave Voltammetry or differential pulse voltammetry is used for detection, and the detection result is compared with the determination working curve of the antigen to find out the corresponding concentration.

The immunosensor of the invention has the advantages of simple preparation process, low cost, excellent reproducibility, high detection sensitivity, etc., and can be widely used in various immunoassays and detections.

Description of drawings

Fig. 1 is carcinoembryonic antigen (CEA) assay standard curve;

Figure 2 is a standard curve for the determination of human IgG.

Detailed ways

The present invention will be further described below in conjunction with the examples. It should be understood that these examples are only for the purpose of illustration, and in no way limit the protection scope of the present invention.

Example 1

1. Preparation of non-labeled amperometric immunosensor for CEA detection

Choose a disc-shaped planar glassy carbon electrode, use CEA as the detection antigen, and use CEA antibody as the immobilized antibody:

1) Mix 0.1mL of 1% chitosan aqueous solution with 0.05mL of 5mM potassium ferricyanide aqueous solution, and add 0.05mL of gold nanoparticles with a particle diameter of 25nm and an absorbance of 5 at the maximum visible absorption peak wavelength Aqueous solution, mixed evenly, brushed on the surface of the electrode, and dried in an oven at 50°C;

2) Mix 0.1 mL of 1.25% nafion ethanol solution with 0.05 mL of ethanol solution of gold nanoparticles with a particle size of 25 nm and an absorbance of 5 at the wavelength of the maximum visible absorption peak, and paint the resulting mixture to step 1) for processing The final electrode surface was dried in an oven at 50°C;

3) Immerse the modified electrode obtained in step 2) in polyethyleneimine aqueous solution (50 mg/mL) for 15 minutes, take it out, wash it with water and dry it with nitrogen to form a polyethyleneimine layer;

4) Immerse the modified electrode obtained in step 3) in a 2.5 mg/mL glutaraldehyde aqueous solution for 15 minutes to form a surface rich in aldehyde groups;

5) Immerse the modified electrode obtained in step 4) in 200 μg/mL CEA antibody aqueous solution and incubate for 3 hours, take it out and put it in 10 mg/mL bovine serum albumin aqueous solution for 30 minutes to obtain a non-labeled amperometric immunoassay that can detect CEA sensor.

Second, the detection of CEA:

1) Determination of the working curve of CEA

Prepare CEA standard aqueous solution with a concentration range of 0-1000ng/mL, and incubate the prepared CEA antibody-modified non-labeled amperometric immunosensor in different concentrations of CEA standard aqueous solution at 30°C for 30 min, take it out, and use two Washing with sub-distilled water, using it as the working electrode, using the large-area platinum sheet electrode as the counter electrode, and using the saturated potassium chloride Ag/AgCl electrode as the reference electrode, the square wave voltammetry was carried out in a phosphate buffer solution with a pH of 7.0. Detect, obtain the measurement working curve of CEA, as shown in Figure 1.

2) Determination of CEA

Incubate the non-labeled amperometric immunosensor modified by CEA antibody in the aqueous solution containing the antigen to be tested for 30min at 35°C, take it out, wash it with twice distilled water, use it as the working electrode, and use the large-area platinum sheet electrode as the counter Electrode, with saturated potassium chloride Ag/AgCl electrode as reference electrode, in the phosphate buffer solution that pH is 7.0 adopts square wave voltammetry to detect, the measurement work curve of the CEA of detection result and step 1) is contrasted, Find out the corresponding concentration.

The results showed that the detection limit of CEA was 3pg/mL.

Example 2

1. Preparation of a non-labeled amperometric immunosensor for human IgG detection

Choose a disc-shaped planar gold electrode, take human IgG as the detection object, and use anti-human IgG as the immobilized antibody:

1) the 2% chitosan aqueous solution of 0.2mL is mixed with the 10mM potassium ferricyanide aqueous solution of 0.1mL, and the particle diameter that adds 0.1mL is that 20nm is the silver nanoparticle that absorbance is 1 under the maximum visible absorption peak wavelength Aqueous solution, after mixing evenly, paint the resulting mixture on the surface of the electrode, and dry it in an oven at 80°C;

2) Mix 0.2 mL of 1% nafion aqueous solution with 0.1 mL of an ethanol solution of silver nanoparticles with a particle size of 20 nm and an absorbance of 1 at the wavelength of the maximum visible absorption peak, and paint the resulting mixture to step 1) after the treatment The surface of the electrode was dried in an oven at 80°C;

3) Immerse the modified electrode obtained in step 2) in polyethyleneimine aqueous solution (concentration: 20 mg/mL) for 30 minutes, take it out, wash it with water and dry it with nitrogen to form a polyethyleneimine layer;

4) Immerse the modified electrode obtained in step 3) in a 5 mg/mL glutaraldehyde aqueous solution for 30 minutes to form a surface rich in aldehyde groups;

5) Immerse the modified electrode obtained in step 4) in 300 μg/mL human IgG antibody aqueous solution and incubate for 3 hours, take it out and put it in 20 mg/mL bovine serum albumin aqueous solution for 30 minutes to obtain a non-labeled current that can detect human IgG type immunosensor.

Second, the detection of human IgG:

1) Determination of the working curve of human IgG

Prepare a human IgG standard aqueous solution with a concentration range of 0-1000ng/mL, and incubate the prepared non-labeled amperometric immunosensor capable of detecting human IgG in human IgG standard aqueous solutions of different concentrations for 35 minutes at 40°C. Take it out, wash it with twice distilled water, use it as the working electrode, use the large-area platinum sheet electrode as the counter electrode, and use the saturated calomel electrode as the reference electrode, and use cyclic voltammetry to detect it in a phosphate buffer solution with a pH of 7.0 , obtain the determination work curve of human IgG, as shown in Figure 2;

2) Determination of human IgG

At 35°C, incubate the non-labeled amperometric immunosensor capable of detecting human IgG in an aqueous solution containing the antigen to be tested for 30 min, take it out, wash it with double distilled water, use it as a working electrode, and use a large-area platinum electrode as a working electrode. Counter electrode, with saturated calomel electrode as reference electrode, in the phosphate buffer solution that pH is 7.0 adopts cyclic voltammetry to detect, the detection result is compared with the measurement work curve of the people IgG of step 1), finds out corresponding concentration.

The results showed that the detection limit of human IgG was 10pg/mL.

The present invention utilizes chitosan to directly immobilize the redox probe substance on the surface of the electrode, forms a redox probe substance film on the electrode, and modifies the antibody on the redox probe substance film to obtain the non-marked type of the present invention. The amperometric immunosensor overcomes the defects of the existing non-labeled amperometric immunosensor, such as cumbersome and time-consuming preparation process, insufficient reproducibility, and high cost of preparing the sensor.

The advantages of the present invention are mainly manifested in:

1) The preparation process of the redox probe substance film in the non-labeled immunosensor of the present invention is simple;

2) Chemical bonds are used to immobilize antibodies, so that the amount of antibodies immobilized each time tends to be consistent, which solves the problems of poor reproducibility caused by physical adsorption usually used in immobilizing antibodies;

3) The non-labeled amperometric immunosensor of the present invention has extremely high detection sensitivity for corresponding antigens.

The preparation process of the non-labeled current type immunosensor of the present invention is simpler, the cost is lower, the reproducibility is better, the detection is more sensitive, and it can be widely used in various immunoassays and detections.

The above descriptions are only preferred embodiments of the present invention, and are only illustrative rather than restrictive to the present invention. Those skilled in the art understand that many changes, modifications, and even equivalents can be made within the spirit and scope defined by the claims of the present invention, but all will fall within the protection scope of the present invention.

Claims (10)

1. A non-labeled amperometric immunosensor, characterized in that the surface of the working electrode is covered with 4 layers of film: the first layer is a redox layer formed on the electrode surface by a mixture of chitosan, potassium ferricyanide and metal nanomaterials The second layer is a protective layer formed on the surface of the redox layer by a perfluorinated proton exchange resin and metal nanomaterial mixture; the third layer is a polyethyleneimine layer formed on the surface of the protective layer; the fourth layer is The third layer was treated with glutaraldehyde and then the polyethylenimine layer chemisorbed the antibody. 2. The non-labeled amperometric immunosensor according to claim 1, characterized in that, The metal nanomaterials are gold nanoparticles or silver nanoparticles. 3. The non-labeled amperometric immunosensor according to claim 2, characterized in that the particle diameter of the metal nanomaterial is 10-60 nm. 4. The non-labeled amperometric immunosensor according to claim 1 or 2, characterized in that the perfluorinated proton exchange resin is a perfluorosulfonic acid exchange resin. 5. A method for preparing the non-labeled amperometric immunosensor of the above claim, characterized in that the preparation of the working electrode comprises the following steps: 1) Mix chitosan aqueous solution with potassium ferricyanide aqueous solution, add metal nanomaterial aqueous solution, paint the resulting mixture on the surface of the working electrode, and dry at room temperature or in an oven at 50-80°C; 2) Mix the ethanol solution of the perfluorinated proton exchange resin with the ethanol solution of the metal nanomaterial, paint the resulting mixture on the working electrode treated in step 1), and dry it at normal temperature or in an oven at 50- Dry at 80°C; 3) immerse the working electrode treated in step 2) in the polyethyleneimine aqueous solution, take it out, wash it with water and dry it with nitrogen to form a polyethyleneimine layer; 4) immersing the working electrode treated in step 3) in an aqueous glutaraldehyde solution to form an aldehyde-rich surface; 5) Immerse the working electrode treated in step 4) in the antibody aqueous solution, take it out and seal it with bovine serum albumin aqueous solution. 6. the preparation method of non-labeled amperometric immunosensor according to claim 5, is characterized in that, In step 1), the concentration of the chitosan aqueous solution is 0.5-2wt%, the concentration of the potassium ferricyanide aqueous solution is 1-10mM, and the absorbance of the metal nanomaterial aqueous solution at the maximum visible absorption peak wavelength is 1-5; The volume ratio of polysaccharide aqueous solution:potassium ferricyanide aqueous solution:metal nanomaterial aqueous solution is 0.05-0.2:0.01-0.1:0.02-0.1. 7. the preparation method of non-labeled amperometric immunosensor according to claim 5, is characterized in that, In step 2), the concentration of the ethanol solution of the perfluorinated proton exchange resin is 1-5wt%, the absorbance of the ethanol solution of the metal nanomaterial at the maximum visible absorption peak wavelength is 1-5, and the perfluorinated proton exchange resin The ethanol solution: the volume ratio of the ethanol solution of the metal nanomaterial is 0.05-0.2:0.02-0.1. 8. the preparation method of non-labeled amperometric immunosensor according to claim 5, is characterized in that, In step 3), the concentration of the polyethyleneimine aqueous solution is 10-50mg/mL; the time for the working electrode to be immersed in the polyethyleneimine aqueous solution is 5-30min; In step 4), the concentration of the glutaraldehyde aqueous solution is 1.0-5.0mg/mL; the time for the working electrode to be immersed in the glutaraldehyde aqueous solution is 5-30min; In step 5), the concentration of the antibody aqueous solution is 100-300ug/mL; the time for the working electrode to be immersed in the antibody aqueous solution is 2-4 hours; The concentration of the bovine serum albumin aqueous solution is 5-20mg/mL; the time for the working electrode to be immersed in the bovine serum albumin aqueous solution is 20-40min. 9. A method for using the non-labeled amperometric immunosensor according to any one of claims 1-4, characterized in that, Working electrode treatment: at 30-40°C, incubate the working electrode in the antigen standard aqueous solution for 30-70min, take it out, and wash it with twice distilled water; then The processed working electrode is used to detect the antigen electrochemically in the buffer solution. 10. The method for using the non-labeled 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 For square wave voltammetry or cyclic voltammetry.

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