CN109975377B - Preparation method of tungsten oxide and graphene oxide-gold nanoparticle sensor - Google Patents

Abstract

The invention discloses a preparation method of a sandwich-type signal amplification electrochemical biosensor based on tungsten oxide (WO ₃ ) nanoflowers and graphene oxide-gold nanoparticles (GO-AuNPs). The flower-like WO ₃ structure has a large specific surface area and can load more detection substances; AuNPs are loaded on the flower-like WO ₃ to further increase the electrochemical signal; in addition, the secondary antibody is modified with large Graphene oxide structure and AuNPs structure with specific surface area and good conductivity, GO-AuNPs as a signal carrier for signal amplification, which significantly reduces the detection line, thus constructing an electrochemical sensor for prostate antigen detection through layer-by-layer modification.

Description

Preparation method of tungsten oxide and graphene oxide-gold nanoparticle sensor

Technical Field

The invention relates to the field of quantitative detection of prostate specific antigens, more specifically based on tungsten oxide (WO)₃) A preparation method of a sandwich type signal amplification electrochemical biosensor of nanoflowers and graphene oxide-gold nanoparticles (GO-AuNPs) is provided.

Background

The tumor marker is synthesized and secreted by tumor cells through gene expression, or is a substance abnormally produced by the body due to the reaction to the tumor, and has important significance in early diagnosis of the tumor. Early diagnosis of cancer requires the use of highly selective, highly sensitive, rapid analytical detection methods for its tumor markers. Therefore, a new simple, rapid, sensitive and selective method for detecting the malignant tumor biomarker is established, and the method has very important significance for early discovery and treatment effect evaluation of malignant tumors.

The working principle of the electrochemical sensing immunosensor mainly utilizes the specific recognition function of an antigen-antibody, a generated sensing signal is converted into an outputtable electrochemical signal through a signal converter, and the signal and the concentration of a target analyte present a certain proportional relation, so that the quantitative analysis of the target substance can be realized. In recent years, electrochemical immunosensors have attracted attention of many researchers, and have been widely used in the fields of clinical medicine, environmental pollutant detection, food analysis, biotechnology, and the like because of the advantages of high specificity, simple operation, high sensitivity, high selectivity, high analysis speed, and the like of immune response.

Disclosure of Invention

The invention aims to construct a sandwich type signal amplification electrochemical biosensor for detecting prostate specific antigen.

In order to solve the technical problem, the invention is realized by the following measures: based on tungsten oxide (WO)₃) The construction of the sandwich type signal amplification electrochemical biosensor of the nanoflower and the graphene oxide-gold nanoparticles (GO-AuNPs) is characterized by comprising the following steps:

(1) 1.0g of WCl₆Dissolving in 50 mL of absolute ethyl alcohol, and heating for 4h in a water bath kettle at 160 ℃; naturally cooling the obtained product to room temperature, washing the product with absolute ethyl alcohol and ultrapure water for three times respectively, and drying the product at the temperature of 60 ℃ for 12 hours;

(2) adding 0.5 g of the composition of step (1) to 100 mL of distilled water; subsequently, 0.66 mL of 1% HAuCl was added₄·4H₂O is dropped into the suspension and stirred vigorously at room temperature; after stirring for 4h, 1.2 mL of HAuCl was added to the suspension₄·4H₂O, continuously stirring the reaction mixture for 12 h, and centrifuging the obtained solution for 2 min at the rotating speed of 8000rmp to obtain WO₃-AuNPs solution;

(3) 25 mL of HAuCl with a concentration of 0.48 mmol₄The solution was mixed with 1.25 mL of 1mg mL^-1Mixing the GO suspension, and stirring at room temperature for 30 min; heating the solution to 80 ℃, slowly adding 470L sodium citrate with the concentration of 85 mmol into the solution, continuously stirring for 60 min, cooling to room temperature, centrifuging the obtained solution at the rotating speed of 5000rmp for 5 min, and washing with ultrapure water;

(4) 50 μ L of 20 μ g mL⁻¹1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and 50. mu.L of 10 mg mL⁻¹Adding the N-hydroxysuccinimide solution into the solution synthesized in the step (3)In the solution, 1 mL of the solution was added at a concentration of 10. mu.g mL⁻¹A secondary antibody of (i), Ab₂Adding into the solution, and incubating at 4 deg.C for 2 h; removal of uncomplexed Ab by 3 washes with pH 7.4 phosphate buffer₂；

(5) The conductive glass is Indium Tin Oxide (ITO), is cut into strips of 4.0 multiplied by 0.5 cm, is ultrasonically cleaned for 5 min by acetone solution, secondary distilled water and absolute ethyl alcohol in sequence, and is dried under nitrogen for standby; 20 μ L of WO prepared above₃The AuNPs solution was coated on the ITO electrode surface, dried at room temperature, and then 6. mu.L of 10. mu.g mL⁻¹The primary antibody Ab1 was incubated at 4 ℃ for 16 h and washed thoroughly 3 times with pH 7.4 phosphate buffer; continuously dripping 20 mu L of 3% bovine serum albumin to block the non-specific binding sites, thoroughly washing the non-specific binding sites for 3 times by using a phosphate buffer solution with pH 7.4, dripping 20 mu L of prostate antigens with different concentrations onto the surface of the electrode, incubating for 30min at room temperature, and washing for 3 times by using a phosphate buffer solution with pH 7.4; continuously dropwise adding 20 mu L of Ab2-GO-AuNPs synthesized in the step (4); then 6. mu.L of the solution was added to a concentration of 0.1 mg mL^-1Dropping diethyl pyrocarbonate and streptomycin modified alkaline phosphoric acid on the electrode, and standing at room temperature for 50 min;

(6) and (3) taking the modified electrode treated in the step (5) as a working electrode, taking a counter electrode as a platinum wire electrode and a reference electrode as an Ag/AgCl electrode, adding 1.0 mL of ascorbyl phosphate with the concentration of 5 mmol into 100 mL of phosphate buffer solution system with the pH value of 7.4, and measuring by a pulse voltammetry method.

The invention has the beneficial effects that:

(1) the method has the advantages of low cost, simple experimental operation and easy control of reaction conditions.

（2）WO₃AuNPs are used as a detection platform, GO-AuNPs are used as a signal carrier to amplify signals, and the detection line is obviously reduced.

(3) Synthetic WO₃The AuNPs composite material has a flower-like structure, has a relatively large specific surface area, and can increase the area of antibody growth.

(4) GO serving as a modifier on the secondary antibody has large specific surface area and excellent charge transfer capability, so that the number of loaded secondary antibody markers AuNPs can be increased, and electrochemical signals can be further increased.

Detailed Description

In order to further understand the invention, the technical scheme is implemented by combining the embodiment, and the specific implementation mode is given:

(1) 1.0g of WCl₆Dissolving in 50 mL of absolute ethyl alcohol, and heating for 4h in a water bath kettle at 160 ℃; naturally cooling the obtained product to room temperature, washing the product with absolute ethyl alcohol and ultrapure water for three times respectively, and drying the product at the temperature of 60 ℃ for 12 hours;

(2) adding 0.5 g of the composition of step (1) to 100 mL of distilled water; subsequently, 0.66 mL of 1% HAuCl was added₄·4H₂O is dropped into the suspension and stirred vigorously at room temperature; after stirring for 4h, 1.2 mL of HAuCl was added to the suspension₄·4H₂O, continuously stirring the reaction mixture for 12 h, and centrifuging the obtained solution for 2 min at the rotating speed of 8000rmp to obtain WO₃-AuNPs solution;

(3) 25 mL of HAuCl with a concentration of 0.48 mmol₄The solution was mixed with 1.25 mL of 1mg mL^-1Mixing the GO suspension, and stirring at room temperature for 30 min; heating the solution to 80 ℃, slowly adding 470L of sodium citrate with the concentration of 85 mmol into the solution, continuously stirring for 60 min, cooling to room temperature, centrifuging the obtained solution at the rotating speed of 5000rmp for 5 min, and washing with ultrapure water;

(4) 50 μ L of 20 μ g mL⁻¹1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and 50. mu.L of 10 mg mL⁻¹Was added to the solution synthesized in step (3), and 1 mL of a solution having a concentration of 10. mu.g mL was added⁻¹A secondary antibody of (i), Ab₂Adding into the solution, and incubating at 4 deg.C for 2 h; removal of uncomplexed Ab by 3 washes with pH 7.4 phosphate buffer₂；

(5) The conductive glass is Indium Tin Oxide (ITO), and is cut into 4.0 × 0.5 cm strips, sequentially treated with acetone solution, redistilled water and anhydrous ethanolUltrasonically cleaning for 5 min, and drying under nitrogen for later use; 20 μ L of WO prepared above₃The AuNPs solution was coated on the ITO electrode surface, dried at room temperature, and then 6. mu.L of 10. mu.g mL⁻¹Ab1 at 4 ℃ for 16 h, washed thoroughly 3 times with phosphate buffer pH 7.4; continuously dripping 20 mu L of 3% bovine serum albumin to block the non-specific binding sites, thoroughly washing with phosphate buffer solution with pH of 7.4 for 3 times, and adding 20 mu L of 0.005 ng-mL^-1To 50 ng.mL^-1Dripping prostate antigens with different concentrations onto the surface of the electrode, incubating for 30min at room temperature, and washing for 3 times with phosphate buffer solution with pH of 7.4; continuously dropwise adding 20 mu L of Ab2-GO-AuNPs synthesized in the step (4); then 6. mu.L of the solution was added to a concentration of 0.1 mg mL^-1Dropping diethyl pyrocarbonate and streptomycin modified alkaline phosphoric acid on the electrode, and standing at room temperature for 50 min;

(6) taking the modified electrode processed in the step (5) as a working electrode, taking a counter electrode as a platinum wire electrode and a reference electrode as an Ag/AgCl electrode, adding 1.0 mL of ascorbyl phosphate with the concentration of 5 mmol into 100 mL of phosphate buffer solution system with the pH value of 7.4, and measuring by a pulse voltammetry method to obtain a linear equation: i = -4.21 log (c) -64.24, correlation coefficient is 0.996, detection limit is 0.05 pg.mL^-1And the high-sensitivity detection of the prostate specific antigen is realized.

Claims (1)

1. The preparation method of the tungsten oxide and graphene oxide-gold nanoparticle sensor is characterized by comprising the following steps:

(1) synthetic tungsten oxide (WO)₃): 1.0g of WCl₆Dissolving in 50 mL of absolute ethyl alcohol, and heating for 4h in a water bath kettle at 160 ℃; naturally cooling the obtained product to room temperature, washing with absolute ethyl alcohol and ultrapure water for three times respectively, and drying at 60 ℃ for 12 h;

(2) synthesis of WO₃-AuNPs: adding 0.5 g of the composition of step (1) to 100 mL of distilled water; subsequently, 0.66 mL1% HAuCl was added₄·4H₂Dropping O, and stirring vigorously at room temperature; after stirring for 4h, 1.2 mL of HAuCl was added to the suspension₄·4H₂O, continue toThe reaction mixture was stirred for 12 h and the resulting solution was centrifuged at 8000rmp for 2 min to give WO₃-AuNPs solution;

(3) synthesizing graphene oxide-gold nanoparticles (GO-AuNPs): 25 mL of HAuCl with a concentration of 0.48 mmol₄The solution was mixed with 1.25 mL of 1mg mL^-1Mixing the GO suspension, and stirring at room temperature for 30 min; heating the solution to 80 ℃, slowly adding 470L sodium citrate with the concentration of 85 mmol into the solution, continuously stirring for 60 min, cooling to room temperature, centrifuging the obtained solution at the rotating speed of 5000rmp for 5 min, and washing with ultrapure water;

(4) synthesis of Ab₂-GO-AuNPs: 50 μ L of 20 μ g mL⁻¹1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and 50. mu.L of 10 mg mL⁻¹Was added to the solution synthesized in step (3), and 1 mL of a solution having a concentration of 10. mu.g mL was added⁻¹A secondary antibody of (i), Ab₂Adding into the solution, and incubating at 4 deg.C for 2 h; removal of uncomplexed Ab by 3 washes with pH 7.4 phosphate buffer₂；

(5) Construction of the electrochemical sensor: the conductive glass is Indium Tin Oxide (ITO), is cut into strips of 4.0 multiplied by 0.5 cm, is ultrasonically cleaned for 5 min by acetone solution, secondary distilled water and absolute ethyl alcohol in sequence, and is dried under nitrogen for standby; 20 μ L of WO prepared above₃The AuNPs solution was coated on the ITO electrode surface, dried at room temperature, and then 6. mu.L of 10. mu.g mL⁻¹The primary antibody Ab1 was incubated at 4 ℃ for 16 h and washed thoroughly 3 times with pH 7.4 phosphate buffer; continuously dripping 20 mu L of 3% bovine serum albumin to block the non-specific binding sites, thoroughly washing the non-specific binding sites for 3 times by using a phosphate buffer solution with pH 7.4, dripping 20 mu L of prostate antigens with different concentrations onto the surface of the electrode, incubating for 30min at room temperature, and washing for 3 times by using a phosphate buffer solution with pH 7.4; continuously dropwise adding 20 mu L of Ab2-GO-AuNPs synthesized in the step (4); then 6. mu.L of the solution was added to a concentration of 0.1 mg mL^-1Dropping diethyl pyrocarbonate and streptomycin modified alkaline phosphoric acid on the electrode, and standing at room temperature for 50 min;

(6) electrochemical detection of a sandwich-type signal amplification biosensor: and (3) taking the modified electrode treated in the step (5) as a working electrode, taking a counter electrode as a platinum wire electrode and a reference electrode as an Ag/AgCl electrode, adding 1.0 mL of ascorbyl phosphate with the concentration of 5 mmol into 100 mL of phosphate buffer solution system with the pH value of 7.4, and measuring by a pulse voltammetry method.