CN1226614C - Nano immunological biosensor - Google Patents

Abstract

In the present invention, a sulfhydryl compound, such as p-aminothiophenol, mercaptoethylamine, etc., is chemically modified on the surface of a gold electrode. The characteristic that a sulfhydryl end of a sulfhydryl compound can be combined with a gold electrode in the mode of a chemical bond is used; consequently, the sulfhydryl end is fixed on the gold electrode, and an amino group at the other end of the sulfhydryl compound is exposed on the electrode surface. Under certain conditions, the exposed amino group can be combined with gold nanometer crystal by a chemical bond, and consequently, the gold nanometer crystal is fixed on the surface of the gold electrode. The surface of the gold nanometer crystal has charges so that strong adsorption action on antibodies is generated in the neutral or physiology pH environment; consequently, the antibodies are firmly combined on the surface of the gold nanometer crystal, which means that the antibodies are fixed on the surface of the gold electrode. As a result, a nanometer immunity biosensor is prepared.

Description

Preparation method of nanometer immune biosensor

technical field

The invention relates to a preparation method of a nanometer immune biosensor.

Background technique

Using chemical methods to detect the interaction between antigen and antibody is the development direction of many researches. Biosensors that combine high-sensitivity sensing technology with specific immune responses to monitor antigen and antibody responses are called immunosensors, and are used in medical diagnosis and treatment, environmental pollution monitoring, and food analysis. The working principle of the immunosensor is similar to that of the traditional immunoassay, which is a solid-phase immunoassay, that is, the antigen or antibody is immobilized on the surface of a solid support to detect the antibody or antigen in the sample. During the past 15 years of development in the field of immunodiagnostics, more interest has been placed on the binding kinetics of antibodies immobilized on the surface of solid supports. Biological samples are immobilized on the electrode surface, and the biological interaction is converted into electrical signals to achieve the purpose of detecting biological activities. How to immobilize biological samples on the electrode surface has extremely important practical application value for the construction of biosensors, immunosensors, and bioelectronic devices. The lifetime of biosensors is often determined by the combination of antibodies and transducers. In previous work, Richad et al. (Richad, C.; Korri-Youssoufi, H.; Yassar, A. Synt. Met., 2001, 121, 1261.) synthesized a conjugated polymer as an antibody and transducer This conjugated polymer can also be directly chemically substituted, for example, it can be connected with ferrocene, crown ether, DNA group, etc. through a substitution reaction. Ben-Dov etc. (Ben-Dov, I., Willner, I., Zisman, E.Anal.Chem.1997,69,3506) adopt maleimide monolayer film to immobilize antibody, but this sensor It is disposable and cannot be reused.

Contents of the invention

The purpose of the invention is to propose a method for preparing a nano-immune biosensor.

The invention chemically modifies the mercapto compound on the surface of the gold electrode, such as mercaptoaniline, mercaptoethylamine and the like. The mercapto end of the mercapto compound can be chemically bonded to the gold electrode, and the mercapto end is fixed on the gold electrode while the amino group at the other end is exposed on the surface of the electrode. Under certain conditions, the exposed amino groups can chemically bond with the gold nanocrystals, thereby fixing the gold nanocrystals on the surface of the gold electrode. The surface of the gold nanocrystal is charged, and it has a strong adsorption effect with the antibody in a neutral or physiological pH environment, so that the antibody is firmly bound to the surface of the gold nanocrystal, so that the antibody is fixed on the surface of the gold electrode and made into a nanocrystal. Immunobiosensors.

The preparation of nanometer immune biosensor comprises the following steps:

The immobilization of a gold nanocrystal on the surface of a gold electrode:

1) Immerse the cleaned gold electrode in a solution containing a mercapto compound, soak it at room temperature for 1 to 10 hours, take it out, wash it with a large amount of high-purity water and dry it with high-purity nitrogen;

2) The electrode is placed in a gold nanocrystal solution with a pH value of 3 to 10, assembled in the dark for 1 to 20 hours, and cleaned with high-purity water after taking it out;

Immobilization of secondary antibodies on gold nanocrystal modified electrodes:

3) Put the electrode modified with gold nanocrystals into 0.5ml phosphate buffer solution containing 0.1mg/ml antibody, the pH is 6-9, and soak overnight at 4°C and 37°C respectively;

4) After taking out the electrode soaked overnight, use 0.02M phosphate buffer solution with a pH of 7.4 to wash off the antibodies that are not stable enough to bind to the gold nanocrystals, and then put it into 0.5mg/ml bovine serum albumin (BSA) to act 60 minutes, and washed with 0.02M phosphate buffer solution with a pH of 7.4.

Three immunoassays:

Put the electrodes into different concentrations of antigens, act at 37°C for 30 minutes, take them out and wash them with 0.02M phosphate buffer solution with a pH of 7.4, and detect them by electrochemical impedance spectroscopy. The results show that this method has good sensitivity. When detecting HBV surface antigen, the concentration linear range is 0.5-200μg/1, and the detection limit is 50ng/1. Compared with other methods, the sensitivity is greatly improved.

The preparation method of the nano-immune biosensor prepared by the invention is simple, and the gold nano-crystal is used to immobilize the antibody, and the activity of the antibody is kept to the greatest extent. Moreover, this sensor can be used repeatedly, and it is the product of the combination of nanotechnology and immune technology. At the same time, high sensitivity can be obtained by using electrochemical detection method.

Description of drawings

Fig. 1 is a schematic diagram of using 4-mercaptoaniline to immobilize gold nanocrystals on the surface of a gold electrode. It can be seen in the figure that the surface of the gold nanocrystal is charged, and it can have a strong adsorption with the antibody in a neutral or physiological pH environment, so that the antibody is firmly bound to the surface of the gold nanocrystal, so that the antibody is fixed on the surface of the gold electrode . Different concentrations of antigens were detected with gold electrodes immobilized with antibodies.

Accompanying drawing 2 is the graph that detects the HBsAg of different concentrations with the sensor of the present invention, detects with AC impedance, the result shows that this method has good sensitivity, and the concentration linear range of detecting HBsAg is 0.5～200 μ g /l, the detection limit is 50ng/1.

Detailed ways

Example 1:

The immobilization of a gold nanocrystal on the surface of a gold electrode:

1) Immerse the cleaned gold electrode in 0.1M 4-mercaptoaniline solution for 1 hour at room temperature, take it out and clean it with absolute ethanol to clean the 4-mercaptoaniline physically adsorbed on the surface of the electrode. Finally, rinse with a large amount of high-purity water and blow dry with high-purity nitrogen.

2) Put the electrode into a gold nanocrystal solution with a pH value of 3, assemble it in the dark for 1 hour, and clean it with high-purity water after taking it out.

Immobilization of secondary antibodies on gold nanocrystal modified electrodes:

1) Put the electrode modified with gold nanocrystals into 0.5ml phosphate buffer solution containing 0.1mg/ml antibody, pH is 6. And after mixing thoroughly for 20 minutes in a micro-vortex mixer, soak overnight at 4°C.

2) After taking it out, use 0.02M pH of 7.4 phosphate buffer solution to wash off the antibody that is not stable enough to bind to the gold nanocrystal, put it into 0.5mg/ml bovine serum albumin (BSA) for 60 minutes, and use 0.02M pH to 7.4 Rinse with phosphate buffer solution.

Three immunoassays:

Put the electrodes into different concentrations of antigens, act at 37°C for 30 minutes, take them out and wash them with 0.02M phosphate buffer solution with a pH of 7.4. Detection was performed by electrochemical impedance spectroscopy. The concentration linear range for detection of hepatitis B virus surface antigen is 0.5-2 μg/1. The results showed that antibody activity was affected and very little antibody was immobilized.

Example 2:

The immobilization of a gold nanocrystal on the surface of a gold electrode:

1) Immerse the cleaned gold electrode in 0.1M 4-mercaptoaniline solution for 4 hours at room temperature, take it out and clean it with absolute ethanol to clean the 4-mercaptoaniline physically adsorbed on the electrode surface. Finally, rinse with a large amount of high-purity water and blow dry with high-purity nitrogen.

2) Put the electrode into a gold nanocrystal solution with a pH value of 10, assemble it in the dark for 5 hours, and clean it with high-purity water after taking it out.

Immobilization of secondary antibodies on gold nanocrystal modified electrodes:

1) Put the electrode modified with gold nanocrystals into 0.5ml phosphate buffer solution containing 0.1mg/ml antibody, pH is 6. And after fully mixing for 20 minutes in a micro-vortex mixer, soak overnight at 37°C.

2) After taking it out, use 0.02M pH of 7.4 phosphate buffer solution to wash off the antibody that is not stable enough to bind to the gold nanocrystal, put it into 0.5mg/ml bovine serum albumin (BSA) for 60 minutes, and use 0.02M pH to 7.4 Rinse with phosphate buffer solution.

Three immunoassays:

Put the electrodes into different concentrations of antigens, act at 37°C for 30 minutes, take them out and wash them with 0.02M phosphate buffer solution with a pH of 7.4. Detection was performed by electrochemical impedance spectroscopy. The concentration linear range for detection of hepatitis B virus surface antigen is 0.5-1 μg/1.

Example 3:

The immobilization of a gold nanocrystal on the surface of a gold electrode:

1) Immerse the cleaned gold electrode in 0.1M 4-mercaptoaniline solution for 6 hours at room temperature, take it out and clean it with absolute ethanol to clean the 4-mercaptoaniline physically adsorbed on the electrode surface. Finally, rinse with a large amount of high-purity water and blow dry with high-purity nitrogen.

2) Put the electrode into a gold nanocrystal solution with a pH value of 8, assemble it in the dark for 10 hours, and clean it with high-purity water after taking it out.

Immobilization of secondary antibodies on gold nanocrystal modified electrodes:

1) The electrode modified with gold nanocrystals is placed in 0.5 ml of phosphate buffer solution containing 0.1 mg/ml antibody, and the pH is 7.4. And after fully mixing for 20 minutes in a micro-vortex mixer, soak overnight at 37°C.

2) After taking it out, use 0.02M pH of 7.4 phosphate buffer solution to wash off the antibody that is not stable enough to bind to the gold nanocrystal, put it into 0.5mg/ml bovine serum albumin (BSA) for 60 minutes, and use 0.02M pH to 7.4 Rinse with phosphate buffer solution.

Three immunoassays:

Put the electrodes into different concentrations of antigens, act at 37°C for 30 minutes, take them out and wash them with 0.02M phosphate buffer solution with a pH of 7.4. Detection was performed by electrochemical impedance spectroscopy. The concentration linear range for detection of hepatitis B virus surface antigen is 1-5 μg/l.

Example 4:

The immobilization of a gold nanocrystal on the surface of a gold electrode:

1) Immerse the cleaned gold electrode in 0.1M 4-mercaptoaniline solution for 4 hours at room temperature, take it out and clean it with absolute ethanol to clean the 4-mercaptoaniline physically adsorbed on the electrode surface. Finally, rinse with a large amount of high-purity water and blow dry with high-purity nitrogen.

2) Put the electrode into a gold nanocrystal solution with a pH value of 3.5, assemble it in the dark for 5 hours, and clean it with high-purity water after taking it out.

Immobilization of secondary antibodies on gold nanocrystal modified electrodes:

1) Put the electrode modified with gold nanocrystals into 0.5ml phosphate buffer solution containing 0.1mg/ml antibody, the pH is 8. And after mixing thoroughly for 20 minutes in a micro-vortex mixer, soak overnight at 4°C.

2) After taking it out, use 0.02M pH of 7.4 phosphate buffer solution to wash off the antibody that is not stable enough to bind to the gold nanocrystal, put it into 0.5mg/ml bovine serum albumin (BSA) for 60 minutes, and use 0.02M pH to 7.4 Rinse with phosphate buffer solution.

Three immunoassays:

Put the electrodes into different concentrations of antigens, act at 37°C for 30 minutes, take them out and wash them with 0.02M phosphate buffer solution with a pH of 7.4. Detection was performed by electrochemical impedance spectroscopy. The concentration linear range for detection of hepatitis B virus surface antigen is 1-20μg/1. The results showed that the amount of antibody immobilization was not large.

Example 5:

The immobilization of a gold nanocrystal on the surface of a gold electrode:

1) Immerse the cleaned gold electrode in 0.1M 4-mercaptoaniline solution for 10 hours at room temperature, take it out and clean it with absolute ethanol to clean the 4-mercaptoaniline physically adsorbed on the surface of the electrode. Finally, rinse with a large amount of high-purity water and blow dry with high-purity nitrogen.

2) Put the electrode into a gold nanocrystal solution with a pH value of 3.5, assemble it in the dark for 5 hours, and clean it with high-purity water after taking it out.

Immobilization of secondary antibodies on gold nanocrystal modified electrodes:

1) Put the electrode modified with gold nanocrystals into 0.5ml phosphate buffer solution containing 0.1mg/ml antibody, the pH is 9. And after fully mixing for 20 minutes in a micro-vortex mixer, soak overnight at 37°C.

2) After taking it out, use 0.02M pH of 7.4 phosphate buffer solution to wash off the antibody that is not stable enough to bind to the gold nanocrystal, put it into 0.5mg/ml bovine serum albumin (BSA) for 60 minutes, and use 0.02M pH to 7.4 Rinse with phosphate buffer solution.

Three immunoassays:

Put the electrodes into different concentrations of antigens, act at 37°C for 30 minutes, take them out and wash them with 0.02M phosphate buffer solution with a pH of 7.4. Detection was performed by electrochemical impedance spectroscopy. The concentration linear range of detecting hepatitis B virus surface antigen is 1～10μg/l, the result shows that the amount of antibody immobilization is very small

Embodiment 6:

The immobilization of a gold nanocrystal on the surface of a gold electrode:

1) Immerse the cleaned gold electrode in 0.1M 4-mercaptoaniline solution for 4 hours at room temperature, take it out and clean it with absolute ethanol to clean the 4-mercaptoaniline physically adsorbed on the surface of the electrode. Finally, rinse with a large amount of high-purity water and blow dry with high-purity nitrogen.

2) Put the electrode into a gold nanocrystal solution with a pH value of 3.5, assemble it in the dark for 5 hours, and clean it with high-purity water after taking it out.

Immobilization of secondary antibodies on gold nanocrystal modified electrodes:

1) The electrode modified with gold nanocrystals is placed in 0.5 ml of phosphate buffer solution containing 0.1 mg/ml antibody, and the pH is 7. And after mixing thoroughly for 20 minutes in a micro-vortex mixer, soak overnight at 4°C.

2) After taking it out, use 0.02M pH of 7.4 phosphate buffer solution to wash off the antibody that is not stable enough to bind to the gold nanocrystal, put it into 0.5mg/ml bovine serum albumin (BSA) for 60 minutes, and use 0.02M pH to 7.4 Rinse with phosphate buffer solution.

Three immunoassays:

Put the electrodes into different concentrations of antigens, act at 37°C for 30 minutes, take them out and wash them with 0.02M phosphate buffer solution with a pH of 7.4. Detection was performed by electrochemical impedance spectroscopy. The concentration linear range for detection of hepatitis B virus surface antigen is 0.5-100μg/l, and the detection limit is 100ng/l.

Embodiment 7:

The immobilization of a gold nanocrystal on the surface of a gold electrode:

1) Immerse the cleaned gold electrode in 0.1M 4-mercaptoaniline solution for 4 hours at room temperature, take it out and clean it with absolute ethanol to clean the 4-mercaptoaniline physically adsorbed on the electrode surface. Finally, rinse with a large amount of high-purity water and blow dry with high-purity nitrogen.

2) Put the electrode into a gold nanocrystal solution with a pH value of 3.5, assemble it in the dark for 5 hours, and clean it with high-purity water after taking it out.

Immobilization of secondary antibodies on gold nanocrystal modified electrodes:

1) The electrode decorated with gold nanocrystals is placed in 0.5ml of phosphate buffer solution containing 0.1mg/ml antibody, the pH is 7.4. And after mixing thoroughly for 20 minutes in a micro-vortex mixer, soak overnight at 4°C.

2) After taking it out, use 0.02M pH of 7.4 phosphate buffer solution to wash off the antibody that is not stable enough to bind to the gold nanocrystal, put it into 0.5mg/ml bovine serum albumin (BSA) for 60 minutes, and use 0.02M pH to 7.4 Rinse with phosphate buffer solution.

Three immunoassays:

Put the electrodes into different concentrations of antigens, act at 37°C for 30 minutes, take them out and wash them with 0.02M phosphate buffer solution with a pH of 7.4. Detection was performed by electrochemical impedance spectroscopy. The results show that this method has good sensitivity, the concentration linear range of detecting HBV surface antigen is 0.5～200μg/l, and the detection limit is 50ng/l.

Claims (2)

1. A preparation method for a nano-immune biosensor, comprising the following steps: the fixing of a gold nanocrystal on the gold electrode surface: 1) Immerse the cleaned gold electrode in a solution containing mercapto compounds, soak at room temperature for 1 to 10 hours, take it out, clean it with a large amount of high-purity water and dry it with high-purity nitrogen; 2) Put the electrode into a gold nanocrystal solution with a pH value of 3-10, assemble it in the dark for 1-20 hours, and clean it with high-purity water after taking it out; Immobilization of secondary antibodies on gold nanocrystal modified electrodes: 1) Put the electrode modified with gold nanocrystals into 0.5ml phosphate buffer solution containing 0.1mg/ml antibody, the pH is 6-9, and soak overnight at 4°C and 37°C respectively; 2) After taking out the electrode soaked overnight, use 0.02M phosphate buffer solution with a pH of 7.4 to wash off the antibody that is not stable enough to bind to the gold nanocrystal, and put it into 0.5mg/ml bovine serum albumin for 60 minutes. And wash with 0.02M phosphate buffer solution with pH 7.4. 2. The preparation method of the nano-immune biosensor according to claim 1, characterized in that the mercapto compound is mercaptoaniline or mercaptoethylamine.

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