CN108375623A - The preparation method and applications of the electrochemical immunosensor of food-borne pathogens are detected based on quick scan anode Stripping Voltammetry technology - Google Patents

Abstract

The invention discloses a preparation method and application of an electrochemical immunosensor for detecting food-borne pathogenic bacteria based on a fast-scanning anode stripping voltammetry technique, which is characterized by the following _steps : (1) adding aminated _Fe3O4 nanoparticle solution After reacting in glutaraldehyde solution, adding food-borne pathogenic bacteria capture antibody reaction after magnetic separation and cleaning, and obtaining the capture unit solution after magnetic separation and cleaning; (2) the composite nanomaterial AgNPs@g‑C ₃ N ₄ Obtain the signal unit after combining with the antibody of food-borne pathogenic bacteria; (3) Add capture unit, food-borne pathogen sample solution, and signal unit to the surface of the magnetic glassy carbon electrode successively to obtain an electrochemical immunosensor, and its application In order to determine the concentration of food-borne pathogens in unknown samples according to the quantitative relationship between the anodic dissolution peak current i _p and the concentration of food-borne pathogens, the advantages of high sensitivity, high specificity, reliable detection results, simple steps and detection high speed.

Description

Electrochemical detection of food-borne pathogens based on fast-scan anodic stripping voltammetry Preparation method and application of immunosensor

technical field

The invention relates to a detection method of food-borne pathogenic bacteria, in particular to a preparation method and application of an electrochemical immunosensor for detecting food-borne pathogenic bacteria based on a rapid scanning anode stripping voltammetry technique.

Background technique

Foodborne pathogens refer to pathogenic bacteria that can cause food poisoning or use food as a medium of transmission. Food is easily contaminated by food-borne pathogenic bacteria in the process of collection, processing, transportation, etc., resulting in frequent food poisoning and disease outbreaks, and the annual economic losses caused by food-borne pathogenic bacteria in my country are as high as $17 billion. Common food-borne pathogens include: Vibrio parahaemolyticus, Vibrio vulnificus, Staphylococcus aureus, Escherichia coli, Salmonella, etc. The traditional method for detecting food-borne pathogens is the biochemical culture identification method. It is cumbersome, the detection cycle is long, time-consuming and labor-intensive. With the rapid development of molecular biology techniques, methods such as polymerase chain reaction (PCR), DNA hybridization and loop-mediated isothermal amplification (LAMP), and biochips have also been applied to the detection of foodborne pathogens, and relatively good results have been obtained. Good accuracy and sensitivity, but there are many problems in practical applications: high false positive probability, expensive equipment, high detection cost, complicated detection steps, long detection time, etc. Therefore, it is an urgent need to develop sensitive, accurate, simple and rapid detection methods for foodborne pathogens.

Anodic stripping voltammetry is a commonly used electrochemical analysis method. It is an electrochemical analysis method that applies an oxidation scanning voltage to the working electrode to oxidize and dissolve the metal on the working electrode to generate an oxidation current. Quantitative analysis is performed according to the current of the oxidation process. . Usually, before the anode is dissolved, a reduction voltage is applied to the working electrode, so that the metal ions to be tested are partially reduced to metals on the surface of the working electrode to achieve enrichment and concentration, so as to improve the detection sensitivity. At present, there are few reports on the use of functionalized composite nanomaterials to improve the detection sensitivity of anodic stripping voltammetry. In addition, according to the basic theory of electrochemistry, the greater the voltage scanning speed during anodic dissolution, the greater the number of dissolved metal ions per unit time, the greater the oxidation current, and the higher the detection sensitivity. However, due to the difficulty in circuit design, this Class studies are rare.

Electrochemical immunosensor combines electrochemical sensing technology with immunoassay technology, which not only has the advantages of sensitivity, simplicity, speed, and economy of electrochemical sensors, but also has the characteristics of specificity and accuracy of immunoassay. In recent years, various nanomaterials, especially two-dimensional nanomaterials such as graphene and graphitic carbon nitride (gC ₃ N ₄ ), have been widely used in sensor construction. At present, there are no relevant research reports on the preparation method of electrochemical immunosensors for detecting foodborne pathogenic bacteria based on rapid scanning anodic stripping voltammetry at home and abroad.

Contents of the invention

The technical problem to be solved by the present invention is to provide a high-sensitivity, high-specificity, reliable detection result, simple steps and fast detection speed electrochemical immunosensor based on fast scanning anode stripping voltammetry technology to detect food-borne pathogens Preparation method and its application.

The technical scheme adopted by the present invention to solve the above-mentioned technical problems is: a method for preparing an electrochemical immunosensor for detecting food-borne pathogenic bacteria based on fast-scanning anode stripping voltammetry, comprising the following steps:

(1) Preparation of capture unit (cAb@Fe ₃ O ₄ )

a. Dissolve 0.1-0.3 g FeCl ₂ •4H ₂ O and 0.6-0.9 g FeCl ₃ •6H ₂ O in 40-50 mL of deaerated secondary water, stir to mix evenly under nitrogen protection, and add 28 wt% ammonia solution until the pH of the reaction solution was 10, heated up to 80 °C and maintained at this temperature for 2 h, after the reaction was completed, cooled to room temperature under the protection of nitrogen, washed with water until neutral, and adjusted to 50 mL to obtain Fe ₃ O ₄ nanoparticle solution;

b. Mix 40-50 mL Fe ₃ O ₄ nanoparticle solution with 0.4-0.6 mL 3-aminopropyltriethoxysilane (APTES), sonicate for 0.5 h, stir at room temperature for 5-8 h, and magnetically separate , washed, and redispersed in 50 mL of water to obtain aminated Fe ₃ O ₄ nanoparticle solution;

c. Add 0.1 to 0.3 mL of 2.5 wt% glutaraldehyde solution to 1 to 3 mL of aminated Fe ₃ O ₄ nanoparticle solution, react at room temperature for 1 to 3 h, and add 1 to 3 mL of 10-50 μg/mL of food-borne pathogen capture antibody (cAb), reacted at room temperature for 1-3 h, after magnetic separation and washing, dispersed in 10-20 mL of phosphate buffer solution with pH=7.5-8.2 , the capture unit (cAb@Fe ₃ O ₄ ) solution was obtained;

(2) Preparation of signaling unit (sAb-AgNPs@gC ₃ N ₄ )

a. Weigh 5-10 g of melamine powder, calcinate in a muffle furnace at 500-600 °C for 3-5 h, and vacuum dry overnight to obtain carbon nitride powder. Take 0.8-1.5 g of carbon nitride powder Add to 80-120 mL 4-6 M _HNO3 solution, reflux at 120-150 ℃ for 16-20 h, cool to room temperature, centrifuge at 12000 rpm, wash with water until the solution pH=7, and continuously sonicate the obtained precipitate for 16 h , to obtain graphitic carbon nitride (gC ₃ N ₄ ) after vacuum drying;

b. Add 1-3 mL 10 mM AgNO ₃ solution in 30-60 mL water, heat to boiling, then add 1-3 mL 1wt% trisodium citrate solution and 300-500 μL 3 mM NaBH ₄ solution, stir vigorously under boiling state After cooling to room temperature for 1 h, centrifuge, wash, and disperse into 40-60 mL of water to obtain a silver nanoparticle (AgNPs) solution;

c. Add 30 to 70 mg of graphitic carbon nitride to 10 to 70 mL of nano silver particle solution, stir at room temperature for 16 to 24 h, wash and centrifuge, discard the supernatant, and disperse into 40 to 60 mL of water to obtain a composite Nanomaterial AgNPs@gC ₃ N ₄ solution;

d. Take 200 μL of composite nanomaterial AgNPs@gC ₃ N ₄ solution, add 120-150 μL containing 10-100 mmol/L 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride ( EDC) and 1-10 mmol/L N-hydroxysuccinimide (NHS), adjust the pH to 4.0-6.0 with 0.8-1.2 M hydrochloric acid, incubate at 35 °C for 1-2 h, centrifuge, wash, and settling in water To 200-300 μL, adjust the pH to 7.0-9.0 with 0.1-0.2 M NaOH solution, add 80-120 μL of 0.01-1 μg/mL food-borne pathogenic bacteria antibody (sAb) and incubate for 3-5 h, then Add 80-100 µL of 2wt% bovine serum albumin (BSA) solution and incubate for 1-2 h to seal the active site, wash, centrifuge, and disperse into 5-10 mL of water to obtain the signal unit sAb-AgNPs@gC ₃ N ₄ ;

(3) Assembly of electrochemical immunosensor

a. The magnetic glassy carbon electrode (MGCE) was sequentially polished to the mirror surface with 1.0, 0.3, 0.05 μm Al ₂ O ₃ , and then ultrasonically cleaned with 50 vt% ethanol solution, 50 vt % nitric acid aqueous solution and distilled water;

b. Add 5-10 μL capture unit dropwise to the surface of the magnetic glassy carbon electrode, then add 5-10 μL food-borne pathogenic bacteria sample solution dropwise, incubate at room temperature for 30-50 min, wash with water, then add 5-10 μL signal unit, After incubating at room temperature for 30-50 min, wash with water to obtain the electrochemical immunosensor.

The food-borne pathogens include Vibrio parahaemolyticus, Vibrio vulnificus, Staphylococcus aureus, Escherichia coli and Salmonella.

The method for detecting food-borne pathogenic bacteria using the electrochemical immunosensor comprises the following steps

Adopting the electrochemical immunosensor described in claim 1 as the working electrode, the platinum wire electrode as the counter electrode, and the 0.1 M KCl solution as the supporting electrolyte solution, adopting a voltage scanning speed of 100 V/s to perform fast scanning anode stripping voltammetry analysis, Measure the anodic dissolution peak current ip (μA); measure the magnitude of the anodic dissolution peak current ip _{corresponding to a series of different concentrations of food-borne pathogenic bacteria, and establish the relationship between ip and} the concentration of food- _borne pathogenic bacteria (cfu/mL) The quantitative relationship between them; according to this quantitative relationship, the concentration of foodborne pathogenic bacteria in unknown samples can be determined.

The principle of the present invention is to construct an electrochemical immunosensor through functionalized biological nanomaterials, and to realize the sensitive, accurate, simple and rapid detection of food-borne pathogenic bacteria by using rapid scanning anodic stripping voltammetry as the detection technology. In the first step, the capture unit (cAb@Fe ₃ O ₄ ) is composed of food-borne pathogenic bacteria capture antibody (cAb) immobilized on the surface of nano-Fe ₃ O ₄ nanoparticles. Fe ₃ O ₄ nanoparticles are magnetic and extremely It is easy to be adsorbed on the surface of magnetic glassy carbon electrodes, and cAb can specifically capture food-borne pathogenic bacteria. In the second step, food-borne pathogenic bacteria are specifically captured by the capture unit. In the third step, the signal unit (sAb-AgNPs@gC ₃ N ₄ ) is composed of food-borne pathogenic antibody (sAb) and silver nanoparticles immobilized on the surface of gC ₃ N ₄ at the same time, and the silver nanoparticles can be used as a fast scanning The electrochemical beacon of anodic stripping voltammetry, during anodic stripping, nano-silver particles are oxidized to silver ions, generating an oxidation current; gC ₃ N ₄ has a huge surface area, which can load a large number of nano-silver particles, greatly improving the detection sensitivity; sAb can Recognize food-borne pathogenic bacteria and immunocombined with them to form an immune complex of capture unit-food-borne pathogenic bacteria-signal unit, which is bound to the surface of the electrode. The fourth step is to detect food-borne pathogenic bacteria by fast scanning anodic stripping voltammetry. The higher the concentration of food-borne pathogenic bacteria, the more signal units are combined, corresponding to the electrochemical beacon nano The more silver particles, the greater the oxidation current of nano-silver particles during anodic dissolution. According to this principle, the quantitative detection of food-borne pathogenic bacteria can be carried out; moreover, the rapid scanning technology is used to increase the voltage scanning speed, and the unit time The more the amount of metal ions leached out, the oxidation current during the anodic leaching of nano-silver particles can be significantly improved, so the detection sensitivity can be further improved.

Compared with existing methods, the present invention has the advantages of:

(1) High sensitivity. The method _of the present invention has high sensitivity, and the detection limit _of the method of the present invention is about 100 times higher than that of the existing method. The material has good thermal stability, chemical stability, biocompatibility and certain conductivity, and its huge surface area can load a large number of electrochemical beacon nano-silver particles to greatly enhance its conductivity, which means that a Food-borne pathogenic bacteria correspond to a signal unit, but correspond to a large number of electrochemical beacon nano-silver particles loaded on it to realize the first-level amplification of electrochemical signals; the second is to use fast scanning technology to increase the voltage scanning speed, It can greatly increase the oxidation current when the nano-silver particles are stripped out at the anode, and realize the secondary amplification of the electrochemical signal.

(2) High specificity. The method of the present invention has high specificity, and other bacteria other than the target object do not interfere with the detection system, because the construction of the immune sensor of the present invention is based on the specific recognition of antigen-antibody.

(3) The test results are reliable. The recoveries were all between 90% and 110%.

(4) The steps are simple and the detection is fast. After synthesizing the capture unit and the signal unit, it only needs to be completed in four steps: placing the capture unit on the electrode surface, adding food-borne pathogenic bacteria samples, adding the signal unit, and electrochemical detection; except for the time necessary for the antigen-antibody immune combination In addition, this method requires almost no additional time, and the detection is completed quickly.

(5) This method is universal, and the detection of different foodborne pathogens can be realized only by changing the type of antibody.

In summary, the present invention prepared an electrochemical immunosensor based on fast scanning anodic stripping voltammetry to detect food-borne pathogens. The composite nanomaterial AgNPs@gC ₃ N ₄ was used to construct the electrochemical immunosensor, and gC A large number of AgNPs loaded on the surface of ₃ N ₄ were used as electrochemical beacons, and fast scanning anodic stripping voltammetry was used as the detection method to detect foodborne pathogens. The results show that the method of the present invention has the advantages of high sensitivity, good specificity, accurate and reliable results, low cost, rapidity, simple and rapid detection process, etc., and has good application prospects.

Description of drawings

Figure 1 is a flow chart of the preparation of the capture unit (cAb@Fe ₃ O ₄ ) of the present invention;

Fig. 2 is a flow chart of the preparation of the signal unit (sAb-AgNPs@gC ₃ N ₄ ) of the present invention;

Fig. 3 is the schematic diagram of the electrochemical immunosensor detection of food-borne pathogenic bacteria detected by fast scanning anode stripping voltammetry of the present invention;

Fig. 4 is the comparison chart of fast scanning anodic stripping method of the present invention and common scanning anodic stripping method;

Fig. 5 is the linear relationship diagram of the anodic stripping peak current value and the concentration logarithm of Vibrio vulnificus of different concentrations;

Fig. 6 is a graph showing the linear relationship between the anodic dissolution peak current value of different concentrations of Vibrio parahaemolyticus and the logarithm of the concentration of Vibrio vulnificus.

Detailed ways

The present invention will be further described in detail below in conjunction with the accompanying drawings and embodiments.

Specific embodiment one

A method for preparing an electrochemical immunosensor for detecting food-borne pathogenic bacteria based on a fast-scanning anode stripping voltammetry technique, comprising the following steps:

(1) Preparation of capture unit (cAb@Fe ₃ O ₄ ) (the preparation process is shown in Figure 1)

a. Dissolve 0.1-0.3 g FeCl ₂ •4H ₂ O and 0.6-0.9 g FeCl ₃ •6H ₂ O in 40-50 mL of deaerated secondary water, stir to mix evenly under nitrogen protection, and add 28 wt% ammonia solution until the pH of the reaction solution was 10, heated up to 80 °C and maintained at this temperature for 2 h, after the reaction was completed, cooled to room temperature under the protection of nitrogen, washed with water until neutral, and adjusted to 50 mL to obtain Fe ₃ O ₄ nanoparticle solution;

b. Mix 40-50 mL Fe ₃ O ₄ nanoparticle solution with 0.4-0.6 mL 3-aminopropyltriethoxysilane (APTES), sonicate for 0.5 h, stir at room temperature for 5-8 h, and magnetically separate , washed, and redispersed in 50 mL of water to obtain aminated Fe ₃ O ₄ nanoparticle solution;

c. Add 0.1 to 0.3 mL of 2.5wt% glutaraldehyde solution to 1 to 3 mL of aminated Fe ₃ O ₄ nanoparticle solution, react at room temperature for 1 to 3 h, and add 1 to 3 mL of 10-50 μg/mL of food-borne pathogen capture antibody (cAb), reacted at room temperature for 1-3 h, after magnetic separation and washing, dispersed in 10-20 mL of phosphate buffer solution with pH=7.5-8.2 , the capture unit (cAb@Fe ₃ O ₄ ) solution was obtained;

(2) Preparation of signaling unit (sAb-AgNPs@gC ₃ N ₄ ) (the preparation process is shown in Figure 2)

a. Weigh 5-10 g of melamine powder, calcinate in a muffle furnace at 500-600 °C for 3-5 h, and vacuum dry overnight to obtain carbon nitride powder. Take 0.8-1.5 g of carbon nitride powder Add to 80-120 mL 4-6 M _HNO3 solution, reflux at 120-150 ℃ for 16-20 h, cool to room temperature, centrifuge at 12000 rpm, wash with water until the solution pH=7, and continuously sonicate the obtained precipitate for 16 h , to obtain graphitic carbon nitride (gC ₃ N ₄ ) after vacuum drying;

b. Add 1-3 mL 10 mM AgNO ₃ solution in 30-60 mL water, heat to boiling, then add 1-3 mL 1wt% trisodium citrate solution and 300-500 μL 3 mM NaBH ₄ solution, stir vigorously under boiling state After cooling to room temperature for 1 h, centrifuge, wash, and disperse into 40-60 mL of water to obtain a silver nanoparticle (AgNPs) solution;

c. Add 30 to 70 mg of graphitic carbon nitride to 10 to 70 mL of nano silver particle solution, stir at room temperature for 16 to 24 h, wash and centrifuge, discard the supernatant, and disperse into 40 to 60 mL of water to obtain a composite Nanomaterial AgNPs@gC ₃ N ₄ solution;

d. Take 200 μL of composite nanomaterial AgNPs@gC ₃ N ₄ solution, add 120-150 μL containing 10-100 mmol/L 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride ( EDC) and 1-10 mmol/L N-hydroxysuccinimide (NHS), adjust the pH to 4.0-6.0 with 0.8-1.2 M hydrochloric acid, incubate at 35 °C for 1-2 h, centrifuge, wash, and settling in water To 200-300 μL, adjust the pH to 7.0-9.0 with 0.1-0.2 M NaOH solution, add 80-120 μL of 0.01-1 μg/mL food-borne pathogenic bacteria antibody (sAb) and incubate for 3-5 h, then Add 80-100 µL of 2 wt% bovine serum albumin (BSA) solution and incubate for 1-2 h to seal the active site, wash, centrifuge, and disperse into 5-10 mL of water to obtain the signal unit sAb-AgNPs@gC ₃ N ₄ ;

(3) Assembly of electrochemical immunosensor

a. The magnetic glassy carbon electrode (MGCE) was sequentially polished to the mirror surface with 1.0, 0.3, 0.05 μm Al ₂ O ₃ , and then ultrasonically cleaned with 50 vt% ethanol solution, 50 vt % nitric acid aqueous solution and distilled water;

b. Add 5-10 μL capture unit dropwise to the surface of the magnetic glassy carbon electrode, then add 5-10 μL food-borne pathogenic bacteria sample solution dropwise, incubate at room temperature for 30-50 min, wash with water, then add 5-10 μL signal unit, After incubating at room temperature for 30-50 min, wash with water to obtain the electrochemical immunosensor.

The aforementioned foodborne pathogens include Vibrio parahaemolyticus, Vibrio vulnificus, Staphylococcus aureus, Escherichia coli and Salmonella.

Specific embodiment two

The method for detecting food-borne pathogenic bacteria using the electrochemical immunosensor prepared in specific embodiment 1, the principle is as shown in Figure 3, including the following steps

The electrochemical immunosensor prepared in the above-mentioned specific example 1 is used as the working electrode, the platinum wire electrode is used as the counter electrode, and the 0.1M KCl solution is used as the supporting electrolyte solution. The fast-scanning anode stripping voltammetry analysis is carried out at a voltage scanning speed of 100 V/s. , measure the anodic dissolution peak current ip (μA); measure the size of the anodic dissolution peak current _ip corresponding to a series of different concentrations of food _{-borne pathogenic bacteria, and establish the relationship between ip} and the concentration of food- borne _pathogenic bacteria (cfu/mL ) between the quantitative relationship; according to the quantitative relationship, the concentration of foodborne pathogenic bacteria in the unknown sample can be determined.

As can be seen from Figure 4, the rapid scanning anodic stripping method of the present invention is compared with the ordinary scanning anodic stripping method. When other conditions are the same, the stripping peak current using the fast scanning anodic stripping method is more than a hundred times that of the ordinary scanning anodic stripping method.

Specific embodiment three

The preparation and detection application of an electrochemical immunosensor for detecting Vibrio vulnificus based on a fast-scanning anode stripping voltammetry technique specifically includes the following steps:

(1) Preparation of capture unit (cAb@Fe ₃ O ₄ ) (the preparation process is shown in Figure 1)

a. Dissolve 0.2 g FeCl ₂ •4H ₂ O and 0.8 g FeCl ₃ •6H ₂ O in 45 mL of deoxygenated secondary water, stir under nitrogen protection to mix well, add 28wt% ammonia solution dropwise until the reaction liquid pH = 10, heat up to 80 °C and maintain this temperature for 2 h. After the reaction, cool to room temperature under nitrogen protection, wash with water until neutral, and set the volume to 50 mL to obtain Fe ₃ O ₄ nanoparticle solution;

b. Mix 45 mL Fe ₃ O ₄ nanoparticle solution with 0.5 mL 3-aminopropyltriethoxysilane (APTES), sonicate for 0.5 h, stir at room temperature for 7 h, magnetically separate, wash, and redisperse in 50 mL of water to obtain aminated Fe ₃ O ₄ nanoparticle solution;

c. Add 0.2 mL of 2.5wt% glutaraldehyde solution to 2 mL of aminated Fe ₃ O ₄ nanoparticle solution, react at room temperature for 2 h, and add 2 mL of 10-50 μg/mL trauma arc after magnetic separation and washing Bacterial capture antibody (cAb), reacted at room temperature for 1-3 h, and after magnetic separation and washing, dispersed in 15 mL of phosphate buffer solution with pH=7.5-8.2 to obtain the capture unit (cAb@Fe ₃ O ₄ ) solution;

(2) Preparation of signaling unit (sAb-AgNPs@gC ₃ N ₄ ) (the preparation process is shown in Figure 2)

a. Weigh 7 g of melamine powder, calcinate it in a muffle furnace at 550 °C for 4 h, and dry it under vacuum overnight to obtain carbon nitride powder. Take 1.2 g of carbon nitride powder and add it to 100 mL of 5 M HNO ₃ The solution was refluxed at 135 °C for 18 h, cooled to room temperature, centrifuged at 12000 rpm, washed with water until the pH of the solution was 7, the resulting precipitate was continuously ultrasonicated for 16 h, and dried in vacuum to obtain graphitic carbon nitride (gC ₃ N ₄ );

b. Add 2 mL 10 mM AgNO ₃ solution in 45 mL water, heat to boiling, then add 2 mL 1wt% trisodium citrate solution and 400 μL 3 mM NaBH ₄ solution, stir vigorously for 1 h under boiling state, cool to room temperature , centrifuged, washed, and dispersed in 50 mL of water to obtain a silver nanoparticle (AgNPs) solution;

c. Add 50 mg graphitic carbon nitride to 40 mL nano-silver particle solution, stir at room temperature for 20 h, wash and centrifuge, discard the supernatant, and disperse into 50 mL water to obtain the composite nanomaterial AgNPs@gC ₃ N ₄ solution;

d. Take 200 μL composite nanomaterial AgNPs@gC ₃ N ₄ solution, add 135 μL containing 50 mmol/L 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and 5 mmol/L N-hydroxysuccinimide (NHS) mixed solution, 0.8-1.2 M hydrochloric acid to adjust the pH to 4.0-6.0, incubate at 35 ℃ for 1-2 h, centrifuge, wash, dilute to 250 µL with water, Use 0.1-0.2 M NaOH solution to adjust the pH to 7.0-9.0, add 100 μL 0.1 μg/mL Vibrio vulnificus antibody (sAb) and incubate for 4 hours, then add 90 μL 2wt% bovine serum albumin (BSA) solution and incubate for 1 ~2 h to seal the active site, wash, centrifuge, and disperse into 7 mL of water to obtain the signal unit sAb-AgNPs@gC ₃ N ₄ ;

(3) Assembly of electrochemical immunosensor

a. The magnetic glassy carbon electrode (MGCE) was sequentially polished to the mirror surface with 1.0, 0.3, 0.05 μm Al ₂ O ₃ , and then ultrasonically cleaned with 50 vt% ethanol solution, 50 vt % nitric acid aqueous solution and distilled water;

b. Add 8 μL capture unit dropwise to the surface of the magnetic glassy carbon electrode, then add 8 μL Vibrio vulnificus sample solution dropwise, incubate at room temperature for 40 minutes, wash with water, then add 8 μL signal unit, incubate at room temperature for 40 minutes, wash with water, and get Electrochemical immunosensors;

(4) Quantitative analysis of Vibrio vulnificus

The electrochemical immunosensor prepared in the above-mentioned specific example 1 is used as the working electrode, the platinum wire electrode is used as the counter electrode, and the 0.1M KCl solution is used as the supporting electrolyte solution. The fast-scanning anode stripping voltammetry analysis is carried out at a voltage scanning speed of 100 V/s. , measure the anodic dissolution peak current ip (μA); measure the size of _{the anodic dissolution peak current ip} corresponding to a series of different concentrations of food-borne _{pathogenic bacteria, and establish the relationship between ip} and the concentration of Vibrio vulnificus (cfu/mL ₎ The quantitative relationship; according to the quantitative relationship, the concentration of Vibrio vulnificus in the unknown sample can be determined.

As shown in Figure 5, there is a linear relationship between the anodic dissolution peak current i _p and the logarithm of the concentration of Vibrio vulnificus solution, the linear range is 4-10 ⁴ cfu/mL, and the linear equation is: y=-51.28+132*logx , the correlation coefficient R ² =0.994, and the detection limit is 1cfu/mL. Good linearity, can be used for unknown sample detection.

Specific embodiment four

The preparation and detection application of an electrochemical immunosensor for detecting Vibrio parahaemolyticus based on a fast-scanning anode stripping voltammetry technique specifically includes the following steps:

(1) Preparation of capture unit (cAb@Fe ₃ O ₄ )

a. Dissolve 0.1 g FeCl ₂ • 4H ₂ O and 0.6 g FeCl ₃ • 6H ₂ O in 40 mL of deoxygenated secondary water, stir under nitrogen protection to mix evenly, add 28wt% ammonia solution drop by drop until the reaction liquid pH = 10, heat up to 80 °C and maintain this temperature for 2 h. After the reaction, cool to room temperature under nitrogen protection, wash with water until neutral, and set the volume to 50 mL to obtain Fe ₃ O ₄ nanoparticle solution;

b. Mix 40 mL Fe ₃ O ₄ nanoparticle solution with 0.4 mL 3-aminopropyltriethoxysilane (APTES), sonicate for 0.5 h, stir at room temperature for 5 h, magnetically separate, wash, and redisperse in 50 mL of water to obtain aminated Fe ₃ O ₄ nanoparticle solution;

c. Add 0.1 mL 2.5wt% glutaraldehyde solution to 1 mL aminated Fe ₃ O ₄ nanoparticle solution, react at room temperature for 1 h, add 1 mL 50 μg/mL parahemolytic arc after magnetic separation and washing Bacterial capture antibody (cAb), after reacting at room temperature for 1 hour, after magnetic separation and washing, dispersed in 10 mL of phosphate buffer solution with pH=7.5-8.2 to obtain capture unit (cAb@Fe ₃ O ₄ ) solution;

(2) Preparation of signaling unit (sAb-AgNPs@gC ₃ N ₄ )

a. Weigh 5 g of melamine powder, calcinate in a muffle furnace at 500 °C for 5 h, and vacuum dry overnight to obtain carbon nitride powder. Add 0.8 g of carbon nitride powder to 80 mL of 6 M HNO ₃ The solution was refluxed at 120 °C for 20 h, cooled to room temperature, centrifuged at 12000 rpm, washed with water until the pH of the solution was 7, the resulting precipitate was continuously ultrasonicated for 16 h, and dried in vacuum to obtain graphitic carbon nitride (gC ₃ N ₄ );

b. Add 1 mL 10 mM AgNO ₃ solution in 30 mL water, heat to boiling, then add 1 mL 1wt% trisodium citrate solution and 300 μL 3 mM NaBH ₄ solution, stir vigorously for 1 h under boiling state, cool to room temperature , centrifuged, washed, and dispersed in 40 mL of water to obtain a silver nanoparticle (AgNPs) solution;

c. Add 30 mg graphitic carbon nitride to 10 mL nano-silver particle solution, stir at room temperature for 16 h, wash and centrifuge, discard the supernatant, and disperse into 40 mL water to obtain the composite nanomaterial AgNPs@gC ₃ N ₄ solution;

d. Take 200 μL composite nanomaterial AgNPs@gC ₃ N ₄ solution, add 120 μL containing 10 mmol/L 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and 1 mmol/L N-hydroxysuccinimide (NHS) mixed solution, 0.8-1.2 M hydrochloric acid to adjust the pH to 4.0-6.0, incubate at 35 °C for 1-2 h, centrifuge, wash, dilute to 200 µL with water, Use 0.1-0.2 M NaOH solution to adjust the pH to 7.0-9.0, add 80 μL 1 μg/mL Vibrio parahaemolyticus antibody (sAb) and incubate for 3 hours, then add 80 μL 2wt% bovine serum albumin (BSA) solution and incubate for 1 ~2 h to seal the active site, wash, centrifuge, and disperse into 5 mL of water to obtain the signal unit sAb-AgNPs@gC ₃ N ₄ ;

(3) Assembly of electrochemical immunosensor

a. The magnetic glassy carbon electrode (MGCE) was sequentially polished to the mirror surface with 1.0, 0.3, 0.05 μm Al ₂ O ₃ , and then ultrasonically cleaned with 50 vt% ethanol solution, 50 vt % nitric acid aqueous solution and distilled water;

b. Add 5 μL capture unit dropwise to the surface of the magnetic glassy carbon electrode, then add dropwise 5 μL Vibrio parahaemolyticus sample solution, incubate at room temperature for 30-50 min, wash with water, then add 5 μL signal unit, incubate at room temperature for 30 min, rinse with water After cleaning, the electrochemical immunosensor is obtained;

(4) Quantitative analysis of Vibrio parahaemolyticus

The electrochemical immunosensor prepared in the above-mentioned specific example 1 is used as the working electrode, the platinum wire electrode is used as the counter electrode, and the 0.1M KCl solution is used as the supporting electrolyte solution. The fast-scanning anode stripping voltammetry analysis is carried out at a voltage scanning speed of 100 V/s. , measure the anodic dissolution peak current ip (μA); measure the magnitude of _{the anodic dissolution peak current ip} corresponding to a _series of different concentrations of food-borne _{pathogenic bacteria, and establish the relationship between ip} and the concentration of Vibrio parahaemolyticus (cfu/mL) The quantitative relationship among them; according to the quantitative relationship, the concentration of Vibrio parahaemolyticus in the unknown sample can be determined.

As shown in Figure 6, there is a linear relationship between the anodic dissolution peak current i _p and the logarithm of the concentration of Vibrio parahaemolyticus solution, the linear range is 40-10 ⁵ cfu/mL, and the linear equation is: y=-192+125 *logx, correlation coefficient R ² =0.992, detection limit 10 cfu/mL. Good linearity, can be used for unknown sample detection.

Specific embodiment five

With above-mentioned specific embodiment three, its difference is: the food-borne pathogenic bacterium that detects is Staphylococcus aureus, concrete steps are as follows:

(1) Preparation of capture unit

a. Dissolve 0.3 g FeCl ₂ •4H ₂ O and 0.9 g FeCl ₃ •6H ₂ O in 50 mL of deoxygenated secondary water, stir under nitrogen protection to mix evenly, add 28wt% ammonia solution drop by drop until the reaction liquid pH = 10, heat up to 80 °C and maintain this temperature for 2 h. After the reaction, cool to room temperature under nitrogen protection, wash with water until neutral, and set the volume to 50 mL to obtain Fe ₃ O ₄ nanoparticle solution;

b. Mix 50 mL Fe ₃ O ₄ nanoparticle solution with 0.6 mL 3-aminopropyltriethoxysilane, sonicate for 0.5 h, stir at room temperature for 8 h, magnetically separate, wash, and redisperse in 50 mL water , to obtain aminated Fe ₃ O ₄ nanoparticle solution;

c. Add 0.3 mL 2.5wt% glutaraldehyde solution to 3 mL aminated Fe ₃ O ₄ nanoparticle solution, react at room temperature for 3 h, add 3 mL 10 μg/mL Staphylococcus aureus after magnetic separation and washing After the capture antibody was reacted at room temperature for 3 h, after magnetic separation and washing, it was dispersed in 20 mL of phosphate buffer solution with pH=7.5-8.2 to obtain the capture unit solution;

(2) Preparation of signaling unit (sAb-AgNPs@gC ₃ N ₄ )

a. Weigh 10 g of melamine powder, calcinate in a muffle furnace at 600 °C for 3 h, and vacuum dry overnight to obtain carbon nitride powder. Take 1.5 g of carbon nitride powder and add it to 120 mL of 4 M HNO ₃ The solution was refluxed at 150 °C for 16 h, cooled to room temperature, centrifuged at 12000 rpm, washed with water until the pH of the solution was 7, and the resulting precipitate was continuously ultrasonicated for 16 h, and dried in vacuum to obtain graphitic carbon nitride (gC ₃ N ₄ );

b. Add 3 mL 10 mM AgNO ₃ solution in 60 mL water, heat to boiling, then add 3 mL 1wt% trisodium citrate solution and 500 μL 3 mM NaBH ₄ solution, stir vigorously for 1 h under boiling state, cool to room temperature , centrifuged, washed, and dispersed in 60 mL of water to obtain a silver nanoparticle (AgNPs) solution;

c. Add 70 mg graphitic carbon nitride to 70 mL nano-silver particle solution, stir at room temperature for 24 h, wash and centrifuge, discard the supernatant, and disperse into 60 mL water to obtain the composite nanomaterial AgNPs@gC ₃ N ₄ solution;

d. Take 200 μL composite nanomaterial AgNPs@gC ₃ N ₄ solution, add 150 μL containing 100 mmol/L 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and Mix solution of 10 mmol/L N-hydroxysuccinimide, adjust pH to 4.0-6.0 with 0.8-1.2 M hydrochloric acid, incubate at 35 ℃ for 1-2 h, centrifuge, wash, dilute to 300 µL with water, wash with 0.1- Adjust the pH to 7.0-9.0 with 0.2 M NaOH solution, add 120 μL 0.01 μg/mL Staphylococcus aureus antibody (sAb) and incubate for 5 h, then add 100 μL 2wt% bovine serum albumin (BSA) solution and incubate for 2 h to block The active site was washed, centrifuged, and dispersed in 10 mL of water to obtain the signal unit sAb-AgNPs@gC ₃ N ₄ ;

(3) Assembly of electrochemical immunosensor

a. The magnetic glassy carbon electrode (MGCE) was sequentially polished to the mirror surface with 1.0, 0.3, 0.05 μm Al ₂ O ₃ , and then ultrasonically cleaned with 50 vt% ethanol solution, 50 vt % nitric acid aqueous solution and distilled water;

b. Add 10 μL capture unit dropwise to the surface of the magnetic glassy carbon electrode, then add 10 μL Staphylococcus aureus sample solution dropwise, incubate at room temperature for 50 min, wash with water, then add 10 μL signal unit, incubate at room temperature for 50 min, then wash with water, that is Electrochemical immunosensor;

(4) Quantitative analysis of Staphylococcus aureus

The electrochemical immunosensor prepared in the above-mentioned specific example 1 is used as the working electrode, the platinum wire electrode is used as the counter electrode, and the 0.1M KCl solution is used as the supporting electrolyte solution. The fast-scanning anode stripping voltammetry analysis is carried out at a voltage scanning speed of 100 V/s. , measure the anodic dissolution peak current ip (μA); measure the magnitude _{of the anodic dissolution peak current ip} corresponding to a series of different concentrations of food-borne pathogenic bacteria, and establish the relationship between ip and the concentration of Staphylococcus aureus (cfu/mL ₎ The quantitative relationship between; according to the quantitative relationship, the concentration of Staphylococcus aureus in the unknown sample can be determined.

Specific embodiment six

The same as the third specific example above, the difference is that the detected food-borne pathogenic bacteria are Escherichia coli.

Specific embodiment seven

The same as the third specific embodiment above, the difference is that the food-borne pathogenic bacteria detected are Salmonella.

Embodiment 8

In order to examine the accuracy and practical application value of this method, the standard recovery method was adopted, that is, a certain concentration of food-borne pathogenic bacteria was added to tap water. As can be seen from Table 1, the detection results using the method of the present invention show that the relative standard deviation (RSD) is less than 5.9%, the recovery rate is 93.6-105.4%, and the results are satisfactory. It shows that the detection results of the present invention for various food-borne pathogenic bacteria in water samples are accurate and reliable.

Table 1 Detection results of various foodborne pathogens in tap water ( n = 5)

The above results show that the present invention has successfully constructed an electrochemical immunosensor based on fast scanning anode stripping voltammetry to detect food-borne pathogens. The sensor can detect food-borne pathogens with high sensitivity and selectivity. Simple, accurate and reliable results. By changing the antibodies in the immunosensor, high sensitivity and high specificity detection of different pathogenic bacteria can be realized.

Of course, the above descriptions are not intended to limit the present invention, and the present invention is not limited to the above examples. Changes, modifications, additions or substitutions made by those skilled in the art within the essential scope of the present invention shall also belong to the protection scope of the present invention.

Claims (3)

1. prepared by a kind of electrochemical immunosensor being detected food-borne pathogens based on quick scan anode Stripping Voltammetry technology Method, it is characterised in that include the following steps：

（1）The preparation of capturing unit

A. by 0.1~0.3 g FeCl₂•4H₂O and 0.6~0.9 g FeCl₃•6H₂O is dissolved in the secondary water of 40~50 mL deoxygenations In, stirred under nitrogen atmosphere makes it be uniformly mixed, and 28wt% ammonia spirits are added dropwise up to pH=10 of reaction solution, are warming up to 80 DEG C and maintain 2 h of this thermotonus to be cooled to room temperature under nitrogen protection after reaction, water is cleaned to neutrality, is settled to 50 ML is to get Fe₃O₄Nanoparticles solution；

B. by 40~50 mL Fe₃O₄Nanoparticles solution is mixed with 0.4~0.6 mL 3- aminopropyl triethoxysilanes, is surpassed After 0.5 h of sound, 5~8 h are stirred at room temperature, through Magneto separate, cleaning, are dispersed in again in 50 mL water to get amination Fe₃O₄It receives Rice grain solution；

C. in 1~3 mL aminations Fe₃O₄The glutaraldehyde solution of 0.1~0.3 mL 2.5wt% is added in nanoparticles solution, 1~3 h is reacted at room temperature, and the food-borne pathogens capture that 1~3 mL, 10~50 μ g/mL are added after Magneto separate, cleaning is anti- Body after Magneto separate, cleaning, is dispersed in the phosphate-buffered of 10~20 pH=7.5~8.2 mL after reacting 1~3 h at room temperature To get capturing unit solution in solution；

（2）The preparation of signal element

A. it weighs 5~10 g melamine powders, calcines 3~5 h in Muffle furnace at 500~600 DEG C, be dried in vacuum overnight Afterwards, you can obtain nitridation carbon dust, 0.8~1.5 g nitridation carbon dusts is taken to be added to 80~120 mL, 4~6 M HNO₃Solution In, flow back 16~20 h at 120~150 DEG C, and after being cooled to room temperature, 12000 rpm centrifugations are washed to pH value of solution=7, by gained 16 h of sediment continuous ultrasound, up to graphite phase carbon nitride after vacuum drying；

B. 1~3 mL, 10 mM AgNO are added in 30~60 mL water₃Solution is heated to boiling, and adds 1~3 mL 3 mM NaBH of 1wt% citric acid three sodium solutions and 300~500 μ L₄Solution is vigorously stirred 1 h under fluidized state, is cooled to room Wen Hou is centrifuged, and cleaning is distributed in 40~60 mL water to get nano silver solution；

C. in 10~70 mL nano silver solutions be added 30~70 mg graphite phase carbon nitrides, at room temperature stir 16~ 24 h, washing centrifugation, abandon supernatant, are distributed in 40~60 mL water to get composite nano materials AgNPs@g-C₃N₄Solution；

D. 200 μ L composite nano materials AgNPs@g-C are taken₃N₄Solution is added 120~150 μ L and contains 10~100 mmol/L 1- Ethyl-（3- dimethylaminopropyls）The mixing of carbodiimide hydrochloride and 1~10 mmol/L n-hydroxysuccinimides is molten Liquid, 0.8~1.2 M hydrochloric acid tune pH are that 1~2 h is incubated at 4.0~6.0,35 DEG C, are centrifuged, washing, water is settled to 200~300 μ L are 7.0~9.0 with 0.1~0.2 M NaOH solution tune pH, 80~120 μ L, 0.01~1 food-borne causes of μ g/mL are added It is incubated 3~5 h after germ antibody, adds 80~100 μ L 2wt% bovine serum albumin solutions and is incubated 1~2 h to close work Property site, clean, centrifugation, be distributed in 5~10 mL water to get signal element sAb-AgNPs@g-C₃N₄；

（3）The assembling of electrochemical immunosensor

A. by magnetic glassy carbon electrode successively with 1.0,0.3,0.05 μm of Al₂O₃It is polished to minute surface, then uses the ethyl alcohol of 50vt% successively Solution, the aqueous solution of nitric acid of 50 vt % and distilled water are cleaned by ultrasonic；

B. 5~10 μ L capturing units are added dropwise in magnetropism glassy carbon electrode surface, and 5~10 μ L food-borne pathogens samples are then added dropwise Product solution, after being incubated at room temperature 30~50 min, water cleaning adds 5~10 μ L signal units, is incubated at room temperature 30~50 min Afterwards, water cleaning is to get electrochemical immunosensor.

2. the electrochemistry according to claim 1 for detecting food-borne pathogens based on quick scan anode Stripping Voltammetry technology Immunosensor preparation method, it is characterised in that：The food-borne pathogens include vibrio parahemolyticus, Vibrio vulnificus, gold Staphylococcus aureus, Escherichia coli and salmonella.

3. a kind of method detecting food-borne pathogens using electrochemical immunosensor described in claim 1, feature exist In including the following steps

Use electrochemical immunosensor described in claim 1 for working electrode, platinum electrode is to electrode, 0.1 M KCl Solution is supporting electrolyte solution, using the voltage scan rate of 100 V/s, carries out quick scan anode stripping volt ampere analysis, Measure Anodic Stripping peak currenti _p ；Measure a series of corresponding Anodic Stripping peak current of food-borne pathogens of various concentrationsi _p 's Size establishes Anodic Stripping peak currenti _p With the quantitative relationship between food-borne pathogenic bacteria concentration；According to the quantitative relationship Measure food-borne pathogenic bacteria concentration in unknown sample.