CN102565163B - Screen-printed electrode and multiple modification method thereof and method for detecting zearalenone - Google Patents

Abstract

The invention discloses a method for detecting zearalenone by a screen printing electrode. The working electrode area of the screen-printed electrode is coated with a Nafion-multi-walled carbon nanotube-colloidal gold-chitosan mixture film layer. During preparation, activate the multi-walled carbon nanotubes and mix them with Nafion; prepare colloidal gold, and use chitosan to mix Nafion-multi-walled carbon nanotubes and colloidal gold evenly; coat the mixture on the working area of the working electrode and dry at 37°C film forming. The method for detecting ZEN of the present invention is that if there is ZEN toxin in the sample to be tested, it will compete with the anti-ZEN antibody to reduce the amount of antibody combined with ZEN-OVA on the electrode, thereby reducing the amount of HRP-goat anti-mouse secondary antibody on the electrode. Also reduce, and influence _Δ I; Then determine the content of ZEN in the grain test sample according to the standard curve drawn by ZEN standard. The detection object of the invention is single and has strong pertinence, high accuracy and strong sensitivity.

Description

Method for detecting zearalenone with screen-printed electrode

technical field

The invention relates to the technical field of detection, in particular to a screen-printed electrode modified with a self-assembled film (Nafion film, carbon nanotube and colloidal gold), a preparation method thereof, and a method for detecting zearalenone. the

Background technique

Zearalenone (ZEN) is a secondary metabolite produced by some strains of Fusarium under certain humidity and temperature conditions. Researchers have detected zearalenone in grains such as wheat, barley, corn, rye, and sorghum, and also in some animal tissues or products, including milk, eggs, etc. Zearalenone toxin can cause a variety of diseases in humans and animals, mainly carcinogenic and cancer-promoting toxicity. Reproductive system diseases have an important relationship. Zearalenone toxin has the characteristics of widespread distribution, long residual time, difficult to handle, and enhanced toxicity with other toxins. After joining the WTO, the international trade volume of agricultural products and related foods has increased day by day, and the requirements for the biosafety of imported and exported products have also become higher and higher. In order to ensure the smooth listing of such products and the health of consumers, entry-exit quarantine , Customs, production enterprises, supervision departments and other departments are in urgent need of a specific, fast and simple detection method for zearalenone. the

Electrochemical sensors are devices that convert information such as physical quantities and chemical quantities sensed into electrical signals that are convenient for measurement and transmission according to certain rules. It uses immobilized bio-sensitive materials as identification elements, generates intermittent or continuous signals through physical and chemical transducers, and then is amplified or directly collected and processed by receiving devices. Because the signal strength generated by the transducer is proportional to the analyte concentration, electrochemical sensors can quantify the analyte with some degree of precision. the

With the development of electrochemical detection methods, the use of nanomaterials as electrode modifiers to increase electron transfer speed, amplify detection signals, and improve detection sensitivity has become an important field of electrochemical sensors. Among them, multi-walled carbon nanotubes are widely used in the research of electrochemical sensors due to their large surface area, good channels for electron transfer, and hole structure that can enhance electron transfer. Compared with ordinary glassy carbon electrodes, screen-printed electrodes have the following advantages in addition to good electrochemical performance: 1. The three-electrode system is integrated on a smaller substrate; 2. Disposable, no need to grind again ; 3. It can be used in large quantities, which is beneficial to large-scale on-site detection; 4. It can be modified arbitrarily and stored for a long time. the

Various methods have been established at home and abroad for the detection of zearalenone. The current detection methods for zearalenone are mainly high-performance liquid chromatography (HPLC) and enzyme-linked immunosorbent assay (ELISA). However, since zearalenone itself has neither specific UV-absorbing groups nor fluorescence However, under certain conditions, zearalenone can react with certain substances to form fluorescent derivatives, so the selection of fluorescent derivatives and derivatization methods is closely related to the accuracy and sensitivity of HPLC detection of zearalenone relation. In addition, this method requires strict pretreatment of test samples, high-performance liquid chromatography and other expensive instruments, and requires professional operators, which is not conducive to on-site routine testing. Although the ELISA method is simpler than HPLC and can be detected in large quantities, its sensitivity is limited and it is not suitable for on-site detection. The instrument used in the method is small in size, light in weight, easy to carry, suitable for on-site detection, and has the characteristics of detection sensitivity. the

Contents of the invention

The purpose of the present invention is to overcome the deficiencies of the above-mentioned prior art, and provide a screen-printed electrode, a multiple modification method and a method for detecting zearalenone. The present invention uses Nafion-multi-walled carbon nanotube/colloidal gold/chitosan mixture as the modification system of the electrode, fully utilizes the characteristics of multi-walled carbon nanotubes, colloidal gold and Nafion film, realizes effective amplification detection signal, improves Sensitivity for detection of zearalenone. Among them, multi-walled carbon nanotubes can enhance electron transfer due to their large surface area, providing good channels and hole structures for electron transfer; colloidal gold can enhance electron transfer after being attached to multi-walled carbon nanotubes due to its spherical structure; Nafion membrane is an excellent cation exchanger. It is used as an electrode modification material with good ion selectivity. It only selectively exchanges with cations and repels neutral molecules and anions. The porous structure formed by its ion clusters consists of 1nm channels. Connected to provide a channel for cation transmission, and has chemical inertness and corrosion resistance. the

The purpose of the present invention is achieved by the following technical solutions:

The invention relates to a screen printing electrode, the working area of the working electrode of the screen printing electrode is coated with a Nafion-multi-wall carbon nanotube-colloid gold-chitosan mixture film layer. the

Preferably, the colloidal gold is 16-18 nm. the

The present invention also relates to a method for preparing the above-mentioned screen printing electrode comprising the following steps:

Step 1, activating multi-walled carbon nanotubes and mixing them with Nafion to prepare Nafion-multi-walled carbon nanotubes;

Step 2, preparing colloidal gold, using chitosan to mix Nafion-multi-walled carbon nanotubes and colloidal gold evenly;

Step 3, coating the mixture prepared in Step 2 on the working area of the working electrode of the screen-printed electrode, and drying at 37°C to form a film. the

Preferably, in step 1, the carbon nanotube activation method is: dissolving 5 mg of multi-walled carbon nanotubes in 15 ml of activation solution, ultrasonicating for 30 min, centrifuging at 9000 rpm for 5 min, removing the supernatant, adding ultrapure water to wash at 9000 rpm Centrifuge for 5 minutes, remove the supernatant, combine the residues, oven-dry at 60°C, add 5ml of water to prepare a 1mg/ml solution, and store at 4°C for later use; the volume ratio of HNO ₃ and H ₂ SO ₄ in the activation solution is 1 :3.

Preferably, in step 1, the preparation method of the Nafion-multi-walled carbon nanotubes is: dissolving 1 mg of the activated multi-walled carbon nanotubes in the Nafion solution, ultrasonically dispersing for 30 minutes, and storing at 4°C for later use ; The percentage of Nafion mass in the Nafion solution to the total volume of the solution is 0.1%. the

Preferably, in step 2, the preparation method of the colloidal gold is as follows: heating 100ml of _HAuCl4 solution to boiling, adding 1.2ml of trisodium citrate solution, boiling for 7-10min, and finally adding triple distilled water to 100ml, A colloidal gold solution is obtained; the mass of trisodium citrate in the trisodium citrate solution accounts for 1% of the total volume of the solution, and the mass of _HAuCl in the _HAuCl solution accounts for 0.01% of the total volume of the solution.

Preferably, in step 2, the mixing of the chitosan with Nafion-multiwalled carbon nanotubes and colloidal gold is specifically: adding 10 μl Nafion-multiwalled carbon nanotubes solution and 50 μl colloidal gold solution to 50 μl chitosan In the solution, ultrasonically mix; the mass of chitosan in the chitosan solution accounts for 2% of the total volume of the solution. the

The present invention also relates to a method for detecting zearalenone (ZEN) with the aforementioned screen-printed electrode, comprising the following steps:

Step 1, add 6μl ZEN-OVA dropwise to the working electrode area, and place it at 37°C for 30min;

Step 2, wash the working electrode with 0.01M PBST, and dry it, add 8 μl of BSA solution dropwise to seal the working electrode, and place it at 37°C for 30 minutes; the percentage of BSA mass in the BSA solution to the total volume of the solution is 1%;

Step 3: Take 0.75g of the sample to be tested, add 3ml of methanol solution, shake, stand still, and centrifuge, dilute 5 times with 0.01MPBS, and take 5μl for later use;

Step 4: Mix the anti-ZEN monoclonal antibody with different concentrations of the ZEN standard and the sample diluent to be tested, and incubate at 37°C for 60 minutes;

Step 5, wash the working electrode with 0.01M PBST and dry it, add dropwise 6 μl of the mixed solution of anti-ZEN monoclonal antibody and ZEN standard or diluent of the sample to be tested, and place at 37°C for 30 minutes;

Step 6: Wash the working electrode with 0.01M PBST, dry it, add 6 μl of HRP-labeled goat anti-mouse secondary antibody dropwise, and place it at 37°C for 30 minutes;

Step 7, use 0.01M PBST to wash the working electrode, and dry it, put the screen printing electrode into 10ml of 0.01M PBS buffer solution with a pH of 7.4, measure the background current, and record it as I ₀ ;

Step 8, take out the screen printing electrode, wash the working electrode with 0.01M PBST, and dry it, put it into 10ml of 0.01M PBS buffer solution with a pH of 7.4, then add hydrogen peroxide and hydroquinone to make the concentrations respectively 2mM and 0.1mM, after stirring the solution for 20min, stop stirring, measure the current, record it as I, the current change value _△ I=I－I ₀ ;

Step 9: Use Excel software to draw a standard curve with different concentrations of the ZEN standard and the corresponding _△ I, and bring the corresponding _△ I of the sample to be tested into the standard curve to obtain the ZEN concentration, and multiply it by the dilution factor, which is ZEN content in the sample to be tested.

Preferably, in step 3, the centrifugation is performed at 3000 rpm for 15 minutes. the

Preferably, in step three, the methanol solution is methanol and water in a volume ratio of 80:20. the

Compared with the prior art, the present invention has the following beneficial effects: the detection object of the present invention is single and highly targeted, with high accuracy and strong sensitivity, which is greater than that of the commonly used enzyme-linked immunosorbent assay. It can meet the requirements of grain storage and sales agencies, entry-exit, customs and other inspection departments to quickly and correctly judge the ZEN toxin content, and is convenient for grassroots promotion and application. the

Description of drawings

Fig. 1 is a schematic diagram of an embodiment of the present invention;

Among them, 1 is the reference electrode, 2 is the working electrode, 3 is the counter electrode, 4 is colloidal gold, 5 is Nafion-multi-walled carbon nanotubes, 6 is OVA-ZEN, 7 is anti-ZEN monoclonal antibody, 8 is sheep Anti-mouse HRP-conjugated secondary antibody. the

Detailed ways

The embodiments of the present invention are described in detail below in conjunction with the accompanying drawings: this embodiment is implemented on the premise of the technical solution of the present invention, and detailed implementation methods and specific operating procedures are provided, but the protection scope of the present invention is not limited to the following the described embodiment. In the following examples, the experimental methods for which specific conditions are not indicated are generally in accordance with conventional conditions, or in accordance with the conditions suggested by the manufacturer. the

Example

The schematic diagram of the embodiment of the present invention is as shown in Figure 1, and described screen printing electrode comprises reference electrode 1, working electrode 2, counter electrode 3; The working area of described working electrode 2 is coated with Nafion-multi-walled carbon nanotube 5-colloidal gold 4-chitosan composite film layer; when using the screen printing electrode to detect ZEN in the sample, first drop OVA-ZEN6 on the working electrode that has been coated with the mixture film layer, after drying Then add the mixed solution of anti-ZEN monoclonal antibody 7 and the sample extract to be tested, after drying, add the HRP-labeled goat anti-mouse secondary antibody 8, and finally add the substrate solution H ₂ O ₂ and HQ.

Prepare screen printing electrode of the present invention and the method concrete steps of detecting zearalenone with it as follows:

Step 1, activation of carbon nanotubes

Dissolve 5 mg of carbon nanotubes in 15 ml of a mixed acid solution (HNO ₃ and H ₂ SO ₄ at a volume ratio of 1:3), and mix for 30 minutes by ultrasonic. The solution was subpackaged, placed in a centrifuge, and centrifuged at 9,000 rpm for 5 minutes. After removing the supernatant, it was washed once with ultrapure water, specifically, centrifuged at 9,000 rpm for 5 minutes. After the supernatant was removed, the residues were combined and dried in an oven at 60°C. Finally, 5ml of water was added and stored at 4°C for later use.

Step 2, preparation of Nafion-multi-walled carbon nanotubes

1 mg of activated carbon nanotubes was dissolved in 0.1% (W/V) Nafion solution, ultrasonically dispersed for 30 minutes, and stored at 4°C for use. the

Step 2, preparation of colloidal gold

First heat 100ml of 0.001% (W/V) HAuCl4 solution to boiling, then quickly add 1.3ml of 1% (W/V) trisodium citrate aqueous solution, some blue at first, then light blue, blue, and then heating appears Red, boiled for 7-10 minutes to appear transparent wine red, finally add triple distilled water to 100ml. Electron microscopy is used to ensure that the prepared gold particles are as consistent and uniform in size as possible, and the diameter of the particles is 16-18nm. the

Step 3, modifying composite nanomaterials on screen-printed electrodes

Add 10 μl Nafion-multi-walled carbon nanotube solution and 50 μl colloidal gold solution to 50 μl 2% (W/V) chitosan solution. After ultrasonically mixing for 5 minutes, 6 μl was added dropwise on the working electrode of the screen-printed electrode, and dried at room temperature for 30 minutes. the

Step 4, sample processing

Take 0.75g of the sample to be tested, add 3ml of methanol solution (the volume ratio of methanol to water is 80:20), shake for 15min, let stand for 10min, centrifuge at 3000 rpm for 15min, dilute 5 times with 0.01M PBS, take 5μl for later use . the

Step five, the detection process

Add 6μl ZEN-OVA dropwise to the working electrode area, and place it at 37°C for 30min. Wash the working electrode with 0.01M PBST and dry it, add 8 μl of 1% (W/V) BSA solution dropwise to seal the working electrode, and place it at 37°C for 30 minutes. the

Mix anti-ZEN monoclonal antibody with ZEN standard (0, 0.1, 0.5, 1, 5, 10, 50, 100 ng/ml) and test sample dilution respectively, and incubate at 37°C for 60 min. Wash the working electrode with 0.01M PBST and dry it, add dropwise 6 μl of the mixed solution of anti-ZEN monoclonal antibody and ZEN standard or diluent of the sample to be tested, and place at 37°C for 30 minutes. Wash the working electrode with 0.01M PBST and dry it, add 6 μl of HRP-labeled goat anti-mouse secondary antibody dropwise, and place at 37°C for 30 minutes. Use 0.01M PBST to wash the working electrode and dry it. Put the screen printing electrode into 10ml of 0.01M PBS (pH7.4) buffer solution to measure the background current and record it as I ₀ ; take the screen printing electrode out and use Wash the working electrode with 0.01M PBST and dry it, put it into 10ml of 0.01M PBS (pH7.4) buffer solution, then add hydrogen peroxide (H ₂ O ₂ ) and hydroquinone (HQ) to make the concentrations respectively 2mM and 0.1mM, after stirring the solution for 20min, stop stirring, measure the current, record it as I, the current change value _△ I=I－I ₀ ; use Excel software to draw the standard curve between the different concentrations of the ZEN standard and the corresponding _△ I , put _{the △} I corresponding to the sample to be tested into the standard curve to obtain the ZEN concentration, and × the dilution factor (4×5), which is the ZEN content (μg/kg) in the sample to be tested.

In summary, the method for detecting ZEN toxin of the present invention is based on the fact that if there is ZEN toxin in the sample to be tested, it will compete with the anti-ZEN antibody for binding, so that the amount of antibody combined with ZEN-OVA on the electrode will be reduced, and thus the amount of antibody on the electrode will be reduced. The amount of HRP-goat anti-mouse secondary antibody also decreases, and affects the current change value △I; then determine the content of ZEN in the grain test sample according to the standard curve drawn by the ZEN standard. It can be seen that the method of this example can directly and quantitatively detect ZEN in the sample, does not require professional training, and is easy and fast to operate. the

Claims (9)

1. a method for detecting zearalenone with a screen-printed electrode, the working area of the working electrode of the screen-printed electrode is coated with a Nafion-multi-walled carbon nanotube-colloidal gold-chitosan mixture film layer , characterized in that it includes the following steps: Step 1: Add 6 μL of zearalenone-ovalbumin ZEN-OVA dropwise to the working electrode area, and place it at 37°C for 30 minutes; Step 2, wash the working electrode with 0.01M phosphate-tween buffer solution PBST, and dry it, add 8 μL of bovine serum albumin BSA solution dropwise to seal the working electrode, and place it at 37°C for 30min; the mass of BSA in the BSA solution accounts for The percentage of the total volume of the solution is 1%; Step 3: Take 0.75g of the sample to be tested, add 3mL of methanol solution, shake, stand still, and centrifuge, dilute 5 times with 0.01M PBS, and take 5μL for later use; Step 4: Mix the anti-zearalenone ZEN monoclonal antibody with different concentrations of the ZEN standard and the sample diluent to be tested, and incubate at 37°C for 60 minutes; Step 5: Wash the working electrode with 0.01M PBST and dry it, add dropwise 6 μL of the mixed solution of anti-ZEN monoclonal antibody and ZEN standard or diluent of the sample to be tested, and place at 37°C for 30 minutes; Step 6: Wash the working electrode with 0.01M PBST, dry it, add 6 μL horseradish peroxidase HRP-labeled goat anti-mouse secondary antibody dropwise, and place at 37°C for 30 minutes; Step 7, use 0.01M PBST to wash the working electrode, and dry it, put the screen printing electrode into 10mL of 0.01M phosphate buffered saline PBS buffer solution with a pH of 7.4, measure the background current, and record it as I ₀ ; Step 8, take out the screen-printed electrode, wash the working electrode with 0.01M PBST, and dry it, put it into 10 mL of 0.01M PBS buffer solution with a pH of 7.4, and then add hydrogen peroxide and hydroquinone so that the concentrations are respectively 2mM and 0.1mM, after stirring the solution for 20min, stop stirring, measure the current, record it as I, the current change value _△ I=I－I ₀ ; Step 9: Use Excel software to draw a standard curve with the different concentrations of the ZEN standard substance and the corresponding _△ I, and bring the corresponding _△ I of the sample to be tested into the standard curve to obtain the ZEN concentration, and multiply it by the dilution factor, which is ZEN content in the sample to be tested. 2. The method for detecting zearalenone with a screen-printed electrode as claimed in claim 1, wherein in step 3, the centrifugation is 3000 rpm for 15 minutes. 3. the method for detecting zearalenone with screen printing electrode as claimed in claim 2, is characterized in that, in step 3, described methanol solution is that methanol and water volume ratio are 80:20. 4. The method for detecting zearalenone with a screen-printed electrode according to claim 1, wherein the colloidal gold is 16-18nm. 5. the method for detecting zearalenone with screen printing electrode as claimed in claim 1, is characterized in that, the preparation method of described screen printing electrode comprises the steps: Step 1, activating multi-walled carbon nanotubes and mixing them with Nafion to prepare Nafion-multi-walled carbon nanotubes; Step 2, preparing colloidal gold, using chitosan to mix Nafion-multi-walled carbon nanotubes and colloidal gold evenly; Step 3, coating the mixture prepared in Step 2 on the working area of the working electrode of the screen-printed electrode, and drying at 37°C to form a film. 6. the method for detecting zearalenone with screen printing electrode as claimed in claim 5, is characterized in that, in step 1, described carbon nanotube activation method is: 5mg multi-walled carbon nanotube is dissolved in 15mL In the activation solution, after ultrasonication for 30min, centrifuge at 9000rpm for 5min, remove the supernatant, add ultrapure water to wash, centrifuge at 9000rpm for 5min, remove the supernatant, combine the residue, dry in an oven at 60°C, add 5mL of water, and prepare 1mg/mL solution, stored at 4°C for future use; the activation solution is HNO ₃ and H ₂ SO ₄ at a volume ratio of 1:3. 7. the method for detecting zearalenone with screen printing electrodes as claimed in claim 5, is characterized in that, in step 1, the preparation method of described Nafion-multi-walled carbon nanotubes is: after 1mg described activation The multi-walled carbon nanotubes were dissolved in the Nafion solution, ultrasonically dispersed for 30 minutes, and stored at 4°C for use; the Nafion mass in the Nafion solution accounted for 0.1% of the total volume of the solution. 8. the method for detecting zearalenone with screen printing electrode as claimed in claim 5, is characterized in that, in step 2, the preparation method of described colloidal gold is specifically: the HAuCl of _100mL solution is heated to boiling , add 1.3mL of trisodium citrate solution, boil for 7 to 10min, finally add triple distilled water to 100mL, and make a colloidal gold solution; in the trisodium citrate solution, the mass of trisodium citrate accounts for a percentage of the total volume of the solution of 1%, in the _HAuCl solution, the mass of _HAuCl accounts for 0.001% of the total volume of the solution. 9. the method for detecting zearalenone with screen printing electrode as claimed in claim 5, is characterized in that, in step 2, the mixing of described chitosan and Nafion-multi-walled carbon nanotubes and colloidal gold is specific For: 10 μL Nafion-multi-walled carbon nanotube solution and 50 μL colloidal gold solution were added to 50 μL chitosan solution, and ultrasonically mixed; the mass of chitosan in the chitosan solution accounted for 2% of the total volume of the solution.

Images