CN101718742B - Method for detecting atrazine - Google Patents

Abstract

The invention provides a chlorine-modified gold nano channel membrane for detecting atrazine and a method for detecting atrazine by using the gold nano channel membrane. The polycarbonate film is used as the base film, and the Au nano channel film with a diameter of 20-50 nm is prepared by chemical deposition method, and the chloride ions are self-assembled on the hole wall of the gold nano channel through the gold-chlorine covalent bond. The detection method is that the above-mentioned chlorine-modified gold nanochannel membrane is placed between the sampling pool and the permeation pool, the buffer solution containing atrazine antibody is added to the sampling pool, and the buffer solution is added to the permeation pool to maintain the two pools. The liquid surface is parallel, voltage is applied, and the basal current is measured; the buffer solution containing the sample to be tested is added to the sample pool, and the current signal is tested by the electrochemical workstation; a sudden drop in the current indicates that the sample to be tested contains atrazine. The invention has high detection efficiency and strong specificity, can detect atrazine with a concentration lower limit of 1.7×10 ^-10 M, and has good application prospects.

Description

A method for detecting atrazine

technical field

The invention relates to the field of materials and the field of sensing technology, in particular to a molecular-level membrane detection technology based on nano-channel membrane materials.

Background technique

In recent years, with the rapid development of nanotechnology, nanotechnology and many current technologies have collided and combined with each other, resulting in many emerging topics and directions. Nanochannel membrane technology combines the advantages of low energy consumption, simple operation, high sensitivity, pollution-free and recyclable membrane detection with the nanoscale manipulation of nanotechnology, and can detect target substances at the molecular level.

In the development of agriculture, pesticides and herbicides play an important role. It is estimated that the annual grain loss due to diseases, insects, and weeds accounts for about half of the world's agricultural production, and the use of pesticides and herbicides can probably regain 30% of it. , while the benefits of pesticides and herbicides are roughly four times their costs. However, due to the long-term use of pesticides and herbicides, it has caused serious pollution to the environment.

Atrazine (Atrazine, abbreviated as ATR, also known as atrazine, 2-chloro-4-ethylamine-6-isopropylamine-1,3,5-triazine, C ₈ H ₁₄ ClN ₅ ) belongs to the Azobenzene compounds are triazine herbicides with moderate water solubility (33μg/L, 22°C). They have a good control effect on most annual dicotyledonous weeds and are the most widely used herbicides in the world in the past 50 years. agent. The main problem is that it is leaching, and it is easy to be leached by rainwater and irrigation water to deeper soil layers, or enter rivers, lakes and reservoirs with surface runoff, causing pollution to groundwater and surface water, and a large amount of pollutants will be adsorbed and stored by particulate matter. Exist in sediments, re-release under appropriate conditions and become a secondary pollution source. It has a long residual period in the soil, and it is easy to cause phytotoxicity to sensitive crops such as wheat, soybean, and rice. The ecotoxicity of ATR showed that 0.1μg/L ATR aqueous solution could lead to hermaphroditism in frogs; the concentration of testosterone in male frogs decreased significantly when observed in ATR water with a concentration of 25μg/L. Long-term exposure to ATR can lead to breast cancer and ovarian cancer, and ATR has been listed as a carcinogen by the US Environmental Protection Agency. ATR can affect the endocrine system of the human body and the reproduction of animals. According to the preliminary evaluation of the US Environmental Protection Agency, ATR is an environmental endocrine disruptor. Countries such as Germany, France and Belgium have banned the use of ATR, and the United States has restricted the maximum standard content in primary drinking water to 3.0 μg/L. The maximum concentration of ATR and its metabolites in Canadian drinking water is 5.0 μg/L. my country's ATR maximum residue standard (GB16323-1996) stipulates that its content in corn and sugarcane is ≤0.05mg/kg, and the standard value of specific items in surface water Ⅰ, Ⅱ, and Ⅲ waters is ≤3.0μg/L

(GHZBI-999). However, the standards for ATR residues in groundwater and the residues of degradants in surface water/groundwater have not yet been formulated.

Due to the long-term use of ATR residues and the existence of other toxic metabolites, degradation products, and transformation products, the harm to the human body is incalculable. Internationally, the requirements for maximum pesticide residues are becoming more and more stringent, all of which put forward higher requirements for pesticide residue detection technology.

In order to control and contain this dangerous pollutant, a rapid detection method for this organic toxin is urgently needed. Some established analytical methods, such as thin-layer chromatography, gas chromatography, high-performance liquid chromatography, and gas chromatography-mass spectrometry, are widely used to detect and quantify pesticides at trace levels. However, these methods are complex and time-consuming for the detection of pesticides, the instruments are not easy to carry, and require a large amount of samples, which are limited in continuous monitoring and on-site determination. Therefore, the development of detection technology with high sensitivity and specific response to ATR and its metabolites, degradation products and transformation products is undoubtedly of great significance to national health and economic development.

This patent attempts to realize the detection of atrazine based on gold nanochannel membrane technology, which proposes a new approach for the analysis and detection of triazine herbicides, and also provides a new idea for the detection of other herbicides.

Contents of the invention

The invention aims to provide a gold nanochannel membrane for detecting atrazine.

The purpose of this method is to apply the above-mentioned gold nanochannel membrane technology to the separation and detection of atrazine, which can be better applied to the analysis and detection of pesticides.

The technical solution is a chlorine-modified gold nanochannel membrane for detecting atrazine, characterized in that the preparation method is as follows:

(a) Using the porous polycarbonate film as the base film, deposit gold on the porous polycarbonate film (PC film) with a pore size of 80nm to 150nm by chemical deposition method: wash the PC film and wash it in 0.01M to 0.05M SnCl ₂ Soak in the solution for 30-120 minutes, so that Sn ²⁺ is evenly adsorbed on _the surface of the base membrane and membrane pores; after taking out and cleaning, immerse the membrane in 0.01M-0.04M Ag(NH ₃ ) ₂ ⁺ solution for 5- 30min, after washing, immerse in 6×10 ^-3 M～9×10 ^-3 M sodium gold sulfite deposition solution with pH=9.5～10.5, electroless gold plating at 0～10℃ for ₄ ～10h; The reacted Ag will obtain the gold nanochannel array film;

(b) Modification of chloride ions: After activation and cleaning of the prepared gold nanochannel membrane, immerse in a solution containing 0.005M to 0.02M Cl ^- for 10 to 16 hours, and the chloride ions are covalently bonded to the monolayer through the gold-chlorine bond. self-assembled on gold nanochannel membranes.

In step (b), the gold nanochannel membrane is activated by immersing in sulfuric acid hydrogen peroxide mixed solution with a volume concentration of 60% to 80% for 5 minutes.

The chlorine-modified gold nano channel membrane prepared by the above method has a pore diameter of 20-50 nm.

A method for detecting atrazine, the prepared chlorine-modified gold nanochannel membrane is placed between the sampling pool and the permeation pool of the U-shaped pool, the working electrode is inserted into the sampling pool, and the reference electrode is inserted into the permeation pool. pool; add the buffer solution containing atrazine antibody to the sample pool, and add the buffer solution not containing atrazine antibody to the permeation pool to keep the liquid levels in the two pools parallel. Apply a voltage (generally 0.8-1.5V) across the electrodes to measure the base current; then add the buffer solution containing the sample to be tested into the sample pool, and conduct the current signal test through the electrochemical workstation; a sudden drop in the current indicates that the sample to be tested The sample contained atrazine.

The buffer solution is a PBS solution with a pH of 7-8 and a concentration of 0.005M-0.02M.

Under a certain electric field, when the electrolyte ions pass through the nanochannel, a stable current is generated. After the atrazine is added, it forms a complex with the antibody in the sample pool, thereby hindering the flow of the electrolyte to a certain extent, causing a corresponding current drop. And other herbicides, such as paraquat, diuron, etc., do not bind to the antibody, will not form molecules larger than the antibody, and will not produce a current drop.

In order to improve the sensitivity, the sample to be tested can be bonded with BSA, so that atrazine can be bonded with BSA to make atrazine immunogen; the preparation method is as follows:

Heat atrazine and 3-mercaptopropionic acid to reflux under alkaline conditions, remove the solvent and then acidify the precipitate to be atrazine-mercaptopropionic acid derivatives; then use dicyclohexylcarbodiimide and N-hydroxy Succinimide linked atrazine-mercaptopropionic acid derivatives to bovine serum albumin to yield atrazine immunogens.

When the atrazine immunogen is added, it forms an MPAD-BSA\antibody complex with the antibody in the sample pool, which is larger in volume than the individual antibody, thereby hindering the flow of electrolyte to a certain extent, causing corresponding current drop. And other herbicides, such as paraquat, diuron, etc., do not bind to the antibody, will not form molecules larger than the antibody, and will not produce a current drop. This allows detection of samples with low levels of atrazine.

The method of the invention detects atrazine, has high detection efficiency and strong specificity, can detect atrazine with a concentration lower limit of 1.7×10 ^-10 M, and has good application prospects.

Description of drawings

Figure 1 is a roadmap for the preparation of atrazine immunogen

Figure 2 is a schematic diagram of the detection device. The Au nanochannel membrane modified with chloride ions is placed in the middle of the U-shaped pool. After the atrazine immunogen or atrazine is combined with the antibody, a larger macromolecule is formed. The flow of the flow produces an obstruction, causing a decrease in current. Other herbicides such as paraquat and diuron do not bind to the antibody, will not form molecules larger than the antibody, and will not produce a current drop.

Figure 3 is a field emission scanning electron microscope image of the product of Example 1; (a) is a field emission scanning electron microscope image of a polycarbonate film after electroless gold plating for 7 hours; (b) is a transmission electron microscope image after dissolving the film with dichloromethane picture.

Fig. 4 is a current-time diagram measured after successively adding atrazine immunogen into the sample pool containing atrazine antibody. The illustration shows the current response before the addition of atrazine immunogen, the current can reach a steady state before the addition of atrazine immunogen, and the addition of the immunogen causes a significant decrease in the current.

Fig. 5 is a comparison of current-time when atrazine immunogen (A), atrazine (B), paraquat (C) and diuron (D) are added respectively in Example 4. When the atrazine immunogen and atrazine were added, the current suddenly decreased, and when the atrazine immunogen was added, the current drop was greater than that of atrazine alone; while the addition of paraquat, enemy Meuron does not bind to the atrazine antibody, and no larger molecules are formed, so there will be no sudden drop in current.

Detailed ways

Below in conjunction with specific example, further set forth the present invention. It should be noted that these examples are only for illustrating the present invention and do not limit the scope of the present invention. In addition, it should be understood that after reading the description of the present invention, those skilled in the art may make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

For the experimental methods that do not indicate the specific conditions in the following examples, usually follow the conventional conditions, such as the operation manual, or the conditions suggested by the manufacturer.

Embodiment 1 Preparation of gold nanochannel membrane

With the porous polycarbonate film as the base film, gold was deposited on the 100nm porous polycarbonate (PC) film by chemical deposition. Immerse the PC membrane in anhydrous methanol and oscillate ultrasonically for 5 minutes to wash away the impurities adsorbed on the base membrane; then immerse the cleaned PC membrane in 0.025 M SnCl ₂ solution for 45 minutes, and shake the solution continuously to make Sn ²⁺ evenly adsorb on the base membrane. Membrane and membrane pore surface, take out and rinse with methanol twice; under the protection of N ₂ gas, immerse the membrane in 0.029MAg(NH ₃ ) ₂ ⁺ solution for 15min, take out and wash with methanol for 3 times, and then wash with water for 3 times, the immersion concentration is 7.9×10 ^-3 M sodium gold sulfite deposition solution (pH=10.00). After electroless gold plating at 4° C. for 7 hours, it was taken out and washed with water for 4 times, and then soaked in 25% HN0 ₃ for 12 hours to remove unreacted Ag to obtain an Au nanochannel array film.

Figure 3(a) is a field emission scanning electron microscope image after electroless gold plating for 7 hours, showing that its inner diameter is about 35nm; Figure 3(b) is a transmission electron microscope image after dissolving the film Au nanochannel array with dichloromethane, showing that After electroless gold plating for 7h, well-shaped gold nanochannels can be obtained.

Embodiment 2 Modification of chloride ion

浸泡5min活化，取出用水冲洗三次，浸入O.01M的KCI水溶液中12h，氯离子即通过金-氯键自组装在Au纳米通道膜上，修饰完毕后将膜取出用甲醇清洗三次，N₂吹干，得到氯修饰的金纳米通道。回流6h。溶剂进行减压蒸发，剩余物加入25mL含5％（质量分数）NaHCO₃的水溶液，用10mLCHCl₃清洗三次，然后用6M盐酸酸化至pH为2，以促使得到的衍生物立即沉淀。The prepared Au nanochannel membrane using piranha solution

Soak for 5 minutes to activate, remove and rinse with water three times, immerse in 0.01M KCI aqueous solution for 12 hours, the chloride ions self-assemble on the Au nanochannel membrane through the gold-chloride bond, after the modification is completed, take out the membrane and wash it with methanol three times, blow with _N2 Dry to obtain chlorine-modified gold nanochannels. Reflux 6h. The solvent was evaporated under reduced pressure, and the residue was added to 25 mL of an aqueous solution containing 5% (mass fraction) NaHCO ₃ , washed three times with 10 mL of CHCl ₃ , and then acidified to pH 2 with 6M hydrochloric acid to promote the immediate precipitation of the obtained derivative.

The supernatant was decanted, and the vacuum-dried precipitate was re-dissolved with 1 mL of ethanol for further purification, and then vacuum-dried at 37° C. to obtain MPAD crystals. Mix 50 μmol of MPAD, 125 μmol of dicyclohexylcarbodiimide (DCC), 125 μmol of N-hydroxysuccinimide (NHS) and 1 mL of dimethyl sulfoxide (DMF), react at room temperature for 4 h, and then centrifuge at 10,000 r/pm for 5 min. Take 150 μL of the supernatant and add it to 850 μL of boric acid solution (pH=9.00) containing 1.47×10 ^-4 M bovine serum albumin (BSA), and react at 4°C for 22 hours. Then put the above mixture into a dialysis bag and dialyze with PBS (pH=7.40). The measurement of embodiment 4 electric current signal

The U-shaped pool is used as the device for the separation and determination of atrazine. The U-shaped pool is divided into two parts: the sample pool and the permeation pool. The Au nanochannel membrane is placed between the sample pool and the permeation pool. The effective permeation area of the membrane is 1.96cm ² , and the volumes of the injection cell and the permeation cell are 6mL, respectively. The platinum wire electrode is used as the working electrode and the reference electrode, the working electrode is inserted into the sample cell, the reference electrode is inserted into the permeation cell, the working electrode and the reference electrode are connected to the electrochemical workstation, and the current signal is tested through the electrochemical workstation. as shown in picture 2.

Add 4.0mL of 0.01M PBS solution with a pH of 7.40 and 1.0mL of PBS containing atrazine antibody (0.585mg/L) into the sampling pool of the U-shaped communication pool, and add 5mL of PBS solution into the permeation pool, Keep the liquid levels in the two pools parallel, apply a voltage of 1.0V across the reference electrode and the working electrode, and measure the substrate current at room temperature. Then add atrazine immunogen, add 2.5X10 ^-8 mol atrazine immunogen (based on atrazine content) to the sample pool every 60s, measure the current-time curve; then use the herbicide A Atrazine, paraquat, and diuron were tested sequentially instead of atrazine immunogen, and the amount added each time was also 2.5X10 ^-8 mol, and the corresponding current response was measured. The results are shown in Figures 4 and 5.

Under a certain electric field, when the electrolyte ions pass through the nanochannel, a stable current is generated. Gradual addition of atrazine immunogen and atrazine resulted in a sudden drop in current, and addition of atrazine immunogen produced a greater current drop than addition of atrazine alone.

It can be seen from Figure 4 that after the addition of atrazine immunogen, due to the formation of atrazine immunogen\antibody complexes, larger molecules are produced, which hinder the flow of electrolytes, compared with adding atrazine alone Compared with , the current is significantly reduced. The illustration in Figure 4 is the current response before the addition of the atrazine immunogen. It can be seen that the current can reach a steady state before the addition of the atrazine immunogen, and the addition of the atrazine immunogen causes a significant decrease in the current.

It can be seen from Figure 5 that the addition of atrazine immunogen (A) and atrazine (B) caused a significant current drop, while the addition of paraquat (C) and diuron (D) did not interact with atrazine Antibody binding, no larger molecules are formed, so there will be no sudden drop in current.

Claims (3)

1. a method that detects Atrazine is characterized in that, the gold nano channel membrane that chlorine is modified places the sample inlet pool in U-shaped pond and sees through between the pond, and working electrode inserts sample inlet pool, and contrast electrode inserts and sees through the pond;

Add the damping fluid that contains Atrazine antibody in sample inlet pool, add the damping fluid that does not contain Atrazine antibody in seeing through the pond, the liquid level of keeping in two ponds is parallel, applies voltage at contrast electrode and working electrode two ends, measures substrate current;

With adding the damping fluid that contains testing sample in the backward sample inlet pool, carry out the current signal test by electrochemical workstation; Generation electric current bust then illustrates and contains Atrazine in the testing sample;

The gold nano channel membrane aperture that described chlorine is modified is 20～50nm, and the preparation method is as follows:

(a) take the porous polycarbonate film as basement membrane, adopting chemical deposition is deposited gold on the porous polycarbonate film of 80nm～150nm in the aperture: with after the washing of porous polycarbonate film at 0.01M～0.05M SnCl ₂Solution soaks 30～120min, makes Sn ²⁺Be adsorbed on equably basement membrane and fenestra surface; It is rear at N to take out cleaning ₂The gas protection is descended film at 0.01M～0.04M Ag (NH ₃) ₂ ⁺Submergence 5～30min in the solution immerses 6 * 10 of pH=9.5～10.5 after the washing ^-3M～9 * 10 ^-3In the M gold sodium sulfide deposit solution, at 0～10 ℃ of lower chemical gilding 4～10h; Washing is also used HNO ₃Remove unreacted Ag, namely obtain gold nano channel array film;

(b) modification of chlorion: will contain 0.005M～0.02M Cl after the prepared gold nano channel array film activation cleaning ^-Solution in soak 10～16h, make the self assembly of chlorion covalent bond individual layer on gold nano channel array film by gold-chlorine key, obtain the gold nano channel membrane that chlorine is modified; Utilizing volumetric concentration is that 60%～80% dioxysulfate water mixed liquid soaks 2～20min gold nano channel array film is activated.

2. the described a kind of method that detects Atrazine of claim 1 is characterized in that, described damping fluid is that pH is 7～8, concentration is the PBS solution of 0.005M～0.02M.

3. the described a kind of method that detects Atrazine of claim 1 is characterized in that, detects adding sample inlet pool behind described testing sample and the bovine serum albumin(BSA) bonding again.

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