1. A method for rapidly detecting glyphosate based on a smart phone is characterized by comprising the following steps:

(1) glyphosate working curve preparation

Adding 50-100 mu L Fe into a 5mL colorimetric tube with a plug₃O₄@C₇Nano particles, glyphosate standard solution with the concentration range of 10-100 mg/L, 10mM ABTS and 40 mM H₂O₂Diluting to 4mL with pH 2.0 acetic acid-sodium acetate buffer solution, standing at room temperature for 5-10 min, and separating Fe with magnet₃O₄@C₇Pouring the nano particles and the solution into a 1 cm cuvette, placing the cuvette in a home-made visible light spectrum analysis device of an intelligent mobile phone, taking a picture to collect a picture, processing the picture by using an image processing software Adobe photoshop CC 2015.5, reading the RGB value of the picture taken by the mobile phone, converting the RGB value into a gray value Gr, establishing a quantitative relation between the gray value and the glyphosate concentration, and drawing a standard curve to obtain a regression equation;

(2) sample assay

Extraction of glyphosate

Accurately weighing 1.00 g (accurate to 0.01 g) of a sample, placing the sample in a 50 mL polyethylene centrifugal tube with a plug, adding 1 mL of 1M NaOH and 30 mL of deionized water, carrying out ultrasonic treatment for 15-30min, centrifuging at 8000 rpm for 5 min, and transferring the supernatant into another centrifugal tube to obtain a yellow extracting solution to be purified;

purification

The purification process is subjected to two-position precipitation treatment, and comprises the following specific steps: taking 2 mL of the extracting solution, adding 300 mu L of 0.33M aluminum nitrate, uniformly mixing, adding 300 mu L of 1M NaOH, leading the solution to become turbid and generate colored precipitate, carrying out vortex for 30-60 seconds, centrifuging at 4000 rpm for 5 min, leading the lower layer to be colored precipitate, leading the upper layer solution to be clear and transparent, and transferring the supernatant into another centrifugal tube; adding 1 mL of deionized water into the precipitate, adding 300 mu L of 1M HCl, swirling for 30-60 seconds until the precipitate is dissolved, adding 300 mu L of 1M NaOH, swirling for 30-60 seconds, centrifuging at 4000 rpm for 5 min, wherein the lower layer is a precipitate, the upper layer is clear and transparent, and taking the supernatant and combining the supernatant obtained in the first treatment to obtain a sample purification solution;

glyphosate assay in samples

Adding 50-100 mu L Fe into a 5mL colorimetric tube with a plug₃O₄@C₇Nanoparticles, sample purification solution, 10mM ABTS, 50 μ L (30%) H₂O₂Diluting to 4mL with pH 2.0 acetic acid-sodium acetate buffer solution, standing at room temperature for 5-10 min, and separating Fe with magnet₃O₄@C₇And (3) pouring the nano particles and the solution into a 1 cm cuvette, placing the cuvette in a home-made visible light spectrum analysis device of a smart phone, taking a picture to collect a picture, reading the RGB value of the picture taken by the smart phone by using Image processing software Image J, converting the RGB value into a gray value Gr, substituting the gray value Gr into the regression equation in the step (1), and calculating the content of the glyphosate in the sample.

2. The method for rapidly detecting glyphosate based on smart phone as claimed in claim 1, wherein Fe₃O₄@C₇The preparation method comprises the following steps:

weighing 2.05 g of ferrous ammonium sulfate and 1.41 g of ferric trichloride, dissolving in 50 mL of deionized water, transferring the mixed solution into a 250 mL three-neck flask, mechanically stirring under the protection of nitrogen, heating in a water bath, and adding 5mL of acetonitrile solution containing 100mg of heptanoic acid when the reaction solution is heated to 80 ℃;

after the mixture was vigorously stirred for 5 min, 5mL of ammonia (w/v, 28%) was added, followed by the slow addition of 1g of heptanoic acid;

finally, stirring the reaction solution at 80 ℃ for 30min, and cooling to room temperature;

precipitating the obtained suspension with methanol, collecting with neodymium iron boron (Nd-Fe-B) ferromagnetic magnet, washing with deionized water/methanol for 4-6 times to remove excessive heptanoic acid, and making into heptanoic acid coated Fe₃O₄Drying at 60 deg.C under vacuum for 12 h.

3. The method for rapidly detecting glyphosate based on the smart phone as claimed in claim 1, wherein: the gray value is converted by the following formula: gr = 0.299R + 0.587G + 0.114B.

4. The method for rapidly detecting glyphosate based on the smart phone as claimed in claim 1, wherein: the self-made visible light spectrum analysis device of the smart phone is a paper box or a wood box, white paper is used as an inner liner in the self-made visible light spectrum analysis device, a cuvette fixing groove is formed in the bottom of the self-made visible light spectrum analysis device, the front size of the self-made visible light spectrum analysis device is consistent with the size of the smart phone, a flash lamp mode is started to serve as a light source during photographing, and a camera aligns to a cuvette solution to photograph.

5. The method for rapidly detecting glyphosate based on the smart phone as claimed in claim 1, wherein: the detection sample comprises tea, tobacco, soil and an environmental water sample.

Background

Glyphosate (also known as glyphosate, glyphosate, also known as glyphosate, glyphosate, glyphosate glycine, has a chemical name of N- (phosphomethyl) glycine, developed by amandon, usa, is a systemic, conductive, broad-spectrum, biocidal herbicide. The glyphosate can remove annual or perennial malignant weeds, is widely applied to many fields, particularly the agricultural field, due to the characteristics of high efficiency, low toxicity, low price and the like, and is now a herbicide with the largest global production and use amount. The maximum residue limit of glyphosate in tea leaves is 1mg/kg specified in the maximum residue limit of pesticides in national food standards for food safety issued by China (GB 2763-2019). The glyphosate belongs to amino acid herbicides, has strong polarity, is insoluble in common organic solvents, lacks of chromophoric and fluorescent groups, has strong binding capacity with organic matters in plants, and makes direct analysis of the herbicides difficult. The existing glyphosate detection method comprises a liquid chromatography-mass spectrometry method and an ion chromatography method, and has the defects of complex detection, long time and large-scale instrument and equipment and professional personnel.

The nano enzyme is a mimic enzyme which has the unique performance of a nano material and also has a catalytic function, is a novel mimic enzyme, has the advantages which can not be achieved by other traditional mimic enzymes, can be researched and utilized by people according to the characteristics of the nano material mimic enzyme, and has a larger application prospect; fe₃O₄The nano material is the earliest discovered simulated nano enzyme, and Fe is obtained by modification₃O₄The simulated nano enzyme has higher enzyme activity and application.

Digital Image Colorimetry (DIC) is a novel colorimetric analysis method, mainly comprising the steps of photographing a detection area by using a smart phone, and converting the detection area in an Image into a color value of an object to be detected through a color model, so that quantitative detection of the object is realized. The RGB-based (wherein R represents red, G represents green and B represents blue) detection method is a method for determining the content of a component to be detected by comparing or measuring the RGB value of the color of a generated colored substance solution based on the color reaction of a generated colored compound, has the characteristics of rapidness, accuracy, simplicity, convenience, stability and the like, and can play an important role in rapid qualitative and quantitative detection on site.

Disclosure of Invention

Aiming at the defects of the prior art for detecting the glyphosate by the glyphosate , the invention provides a method for quickly detecting the glyphosate based on a smart phone, and the method specifically inhibits the heptanoic acid coated ferroferric oxide (Fe) based on the glyphosate₃O₄@C₇) The peroxidase mimic catalytic activity, and the visualized detection of the glyphosate is realized by distinguishing the change of the color intensity of the established nano enzyme, in particular to Fe₃O₄@C₇2,2' -biazobis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) is catalytically oxidized by nano enzyme in H₂O₂In the presence of the glyphosate, blue-green color is generated, the catalytic oxidation reaction of enzyme is inhibited in the presence of the glyphosate, the color becomes lighter along with the increase of the concentration of the glyphosate, an image is obtained by photographing with a smart phone, the content of the glyphosate is determined by measuring the color RGB value of the generated colored substance solution, so that a new glyphosate detection method is established, the detection limit is 0.1mg/kg, the reaction is completed within 10 minutes, the system stability exceeds 30 minutes, other pesticides do not interfere the reaction, and the detection method has specificity; the method is applied to detection and analysis of glyphosate in a sample, and the result conforms to the related national standard determination method; the method has the characteristics of simple operation, high sensitivity, high speed and the like.

The invention establishes a method for rapidly detecting glyphosate as follows:

(1) glyphosate working curve preparation

Adding 50-100 mu L Fe into a 5mL colorimetric tube with a plug₃O₄@C₇Nano particles, glyphosate standard solution with the concentration range of 10-100 mg/L, 10mM ABTS and 40 mM H₂O₂Diluting to 4mL with pH 2.0 acetic acid-sodium acetate buffer solution, standing at room temperature for 5-10 min, and separating Fe with magnet₃O₄@C₇Pouring the nanoparticles and the solution into a 1 cm cuvette, placing the cuvette in a self-made visible light spectrum analysis device of a smart phone, taking a picture to collect pictures, processing the pictures by using image processing software Adobe photoshop CC 2015.5, and reading RG of the pictures taken by the smart phoneB value is converted into gray value Gr, a quantitative relation between the gray value and the glyphosate concentration is established, a standard curve is drawn, and a regression equation is obtained;

(2) sample assay

Extraction of glyphosate

Accurately weighing 1.00 g (accurate to 0.01 g) of sample, placing in a 50 mL polyethylene centrifuge tube with a plug, adding 1 mL of 1M NaOH and 30 mL of deionized water, performing ultrasonic treatment for 15-30min, centrifuging at 8000 rpm for 5 min, transferring the supernatant into another centrifuge tube to obtain yellow extract, and purifying.

Purification

The purification process is subjected to two-position precipitation treatment, and comprises the following specific steps: taking 2 mL of the extracting solution, adding 300 mu L of 0.33M aluminum nitrate, uniformly mixing, adding 300 mu L of 1M NaOH, leading the solution to become turbid and generate precipitates, carrying out vortex for 30-60 seconds, centrifuging at 4000 rpm for 5 min, leading the lower layer to be yellow precipitate, leading the upper layer solution to be clear and transparent, and transferring the supernatant into another centrifugal tube; adding 1 mL of deionized water into the yellow precipitate, adding 300 mu L of 1M HCl, swirling for 30-60 seconds until the precipitate is dissolved, adding 300 mu L of 1M NaOH, swirling for 30-60 seconds, centrifuging at 4000 rpm for 5 min, wherein the lower layer is a yellow precipitate, the upper layer is clear and transparent, and the supernatant is combined with the supernatant of the first treatment to obtain a sample purification solution.

Glyphosate assay in samples

Adding 50-100 mu L Fe into a 5mL colorimetric tube with a plug₃O₄@C₇Nanoparticles, sample purification solution, 10mM ABTS, 40 mM H₂O₂Diluting to 4mL with pH 2.0 acetic acid-sodium acetate buffer solution, standing at room temperature for 5-10 min, and separating Fe with magnet₃O₄@C₇Nano particleAnd (3) pouring the solution into a 1 cm cuvette, placing the cuvette in a home-made visible light spectrum analysis device of a smart phone, taking a picture to collect a picture, reading the RGB value of the picture taken by the smart phone by using an Image processing software Image J, converting the RGB value into a gray value Gr, substituting the gray value Gr into the regression equation in the step (1), and calculating the content of the glyphosate in the sample.

Fe₃O₄@C₇The preparation method comprises the following steps: 2.05 g of ferrous ammonium sulfate and 1.41 g of ferric chloride are weighed and dissolved in 50 mL of deionized water, the mixed solution is transferred to a 250 mL three-neck flask, mechanical stirring and water bath heating are carried out under the protection of nitrogen, and when the reaction solution is heated to 80 ℃, 5mL of acetonitrile solution containing 100mg of heptanoic acid is added. After the mixture was vigorously stirred for 5 min, 5mL of aqueous ammonia (w/v, 28%) was added, followed by the slow addition of 1g of heptanoic acid. Finally, the reaction solution was stirred at 80 ℃ for 30min and then cooled to room temperature. Precipitating the obtained suspension with methanol, collecting with neodymium iron boron (Nd-Fe-B) ferromagnetic magnet, washing with deionized water/methanol for 4-6 times to remove excessive heptanoic acid, and making into heptanoic acid coated Fe₃O₄Drying at 60 deg.C under vacuum for 12 h.

The gray value is converted by the following formula: gr = 0.299R + 0.587G + 0.114B.

The self-made visible light spectrum analysis device of the smart phone is a paper box or a wood box, white paper is used as an inner liner in the self-made visible light spectrum analysis device, a cuvette fixing groove is formed in the bottom of the self-made visible light spectrum analysis device, the front size of the self-made visible light spectrum analysis device is consistent with the size of the smart phone, a flash lamp mode is started to serve as a light source during photographing, and a camera aligns to a cuvette solution to photograph.

The detection sample comprises tea, tobacco, soil and an environmental water sample.

The invention has the advantages that:

1. the invention utilizes the heptanoic acid to coat the ferroferric oxide (Fe)₃O₄@C₇) The catalytic activity of the pseudoperoxidase is simulated in H₂O₂In the presence of the glyphosate, 2' -dinitrobis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) is catalytically oxidized to generate blue-green, the catalytic oxidation reaction of enzyme is inhibited in the presence of glyphosate, the color becomes lighter along with the increase of the concentration of the glyphosate, and the colorimetric detection is carried out by using a smart phone to realize the grass color detectionThe detection of the glyphosate is rapid, reliable and simple, and the detection limit is 0.1 mg/kg.

2. The detection time of the detection method established by the invention is not more than 10 minutes, the system stability is more than 30 minutes, other pesticides do not interfere the reaction, and the detection method has specificity.

3. When the method is used for measuring a tobacco sample, because of matrix interference, the method adopts a coprecipitation method to carry out purification treatment twice, the interference is reduced, the recovery rate is improved, the standard addition recovery rate of the treated sample reaches 92.6-98.5%, meanwhile, the method has good precision and accuracy, the measurement result is about the same as that of a national standard method, and compared with the national standard method, the method has low cost and simple operation, can finish detection by a mobile phone, and has certain application prospect in the field of field monitoring and emergency monitoring.

Detailed Description

The technical solutions of the present invention will be described in further detail with reference to specific examples, but the scope of the present invention is not limited thereto.

Example 1: determination of glyphosate in tea samples

（1）Fe₃O₄@C₇Preparation: 2.05 g of ferrous ammonium sulfate and 1.41 g of ferric chloride are weighed and dissolved in 50 mL of deionized water, the mixed solution is transferred to a 250 mL three-neck flask, mechanical stirring and water bath heating are carried out under the protection of nitrogen, and when the reaction solution is heated to 80 ℃, 5mL of acetonitrile solution containing 100mg of heptanoic acid is added. After the mixture was vigorously stirred for 5 min, 5mL of aqueous ammonia (w/v, 28%) was added, followed by the slow addition of 1g of heptanoic acid. Finally, the reaction solution was stirred at 80 ℃ for 30min and then cooled to room temperature. Precipitating the obtained suspension with methanol, collecting with neodymium iron boron (Nd-Fe-B) ferromagnetic magnet, washing with deionized water/methanol for 4-6 times to remove excessive heptanoic acid, and making into heptanoic acid coated Fe₃O₄Drying at 60 deg.C under vacuum for 12 h.

(2) And (3) making a glyphosate working curve: adding 100 mu L Fe into a 5mL colorimetric tube with a plug₃O₄@C₇The concentration range of the nano particles and the glyphosate standard is 10-100 mg/LSolution, 10mM ABTS, 40 mM H₂O₂Diluting to 4mL with pH 2.0 acetic acid-sodium acetate buffer solution, standing at room temperature for 5-10 min, and separating Fe with magnet₃O₄@C₇The nano particles and the solution are poured into a 1 cm cuvette, the cuvette is placed in a home-made smart phone visible light spectrum analysis device, pictures are taken to collect, image processing software Adobe photoshop CC 2015.5 is used for processing, RGB values of the images shot by the mobile phone are read and converted into gray values Gr, a quantitative relation between the gray values and glyphosate concentration is established, and a regression equation, correlation coefficients, relative standard deviation, linear range and the like are obtained and shown in table 1.

TABLE 1 Linear equation, correlation coefficient, relative standard deviation, Linear Range

；

(3) Determination of glyphosate in tea samples

Extraction of glyphosate

Accurately weighing 1.00 g (accurate to 0.01 g) of tea sample, placing in a 50 mL polyethylene centrifuge tube with a plug, adding 1 mL of 1M NaOH and 30 mL of deionized water, performing ultrasonic treatment for 15-30min, centrifuging at 8000 rpm for 5 min, transferring the supernatant into another centrifuge tube to obtain yellow extractive solution, and purifying.

Purification

The purification process is subjected to two-position precipitation treatment, and comprises the following specific steps: taking 2 mL of the extracting solution, adding 300 mu L of 0.33M aluminum nitrate, uniformly mixing, adding 300 mu L of 1M NaOH, leading the solution to become turbid and generate precipitates, carrying out vortex for 30-60 seconds, centrifuging at 4000 rpm for 5 min, leading the lower layer to be yellow precipitate, leading the upper layer solution to be clear and transparent, and transferring the supernatant into another centrifugal tube; adding 1 mL of deionized water into the yellow precipitate, adding 300 mu L of 1M HCl, swirling for 30-60 seconds until the precipitate is dissolved, adding 300 mu L of 1M NaOH, swirling for 30-60 seconds, centrifuging at 4000 rpm for 5 min, wherein the lower layer is a yellow precipitate, the upper layer is clear and transparent, and the supernatant is combined with the supernatant of the first treatment to obtain a sample purification solution.

And (3) determining glyphosate in the tea sample: adding 50-100 mu L Fe into a 5mL colorimetric tube with a plug₃O₄@C₇2 mL of nanoparticle and sample purification solution, 10mM ABTS and 40 mM H₂O₂Diluting to 4mL with pH 2.0 acetic acid-sodium acetate buffer solution, standing at room temperature for 5-10 min, and separating Fe with magnet₃O₄@C₇And (3) pouring the nano particles and the solution into a 1 cm cuvette, placing the cuvette in a home-made visible light spectrum analysis device of a smart phone, taking a picture to collect a picture, reading the RGB value of the picture taken by the smart phone by using Image processing software Image J, converting the RGB value into a gray value Gr, substituting the gray value Gr into the regression equation in the step (1), and enabling the glyphosate sample not to be detected.

(4) Recovery and precision experiments: respectively adding 3 glyphosate standard solutions with different concentrations into a tea sample; each concentration is measured in parallel for 3 times, the standard recovery rate is calculated, and the relative standard deviation RSD is calculated, and the result is shown in a table 2; the measured standard recovery rate of glyphosate is 97.5-102.1%, RSD is 1.11-2.25%, and the method has good accuracy and precision.

TABLE 2 sample Glyphosate recovery on Standard and RSD (n = 3)

。

Example 2: determination of glyphosate content in tobacco sample

（1）Fe₃O₄@C₇Preparation: the same as example 1;

(2) and (3) making a glyphosate working curve: the same as example 1;

(3) determination of glyphosate content in tobacco sample

And (3) extracting glyphosate: the same as example 1;

purifying: the same as example 1;

and (3) determining glyphosate in a sample: the glyphosate content of the sample is 2.1 mg/kg.

Example 3: determination of glyphosate content in soil sample

（1）Fe₃O₄@C₇Preparation: the same as example 1;

(2) and (3) making a glyphosate working curve: the same as example 1;

(3) determination of glyphosate content in soil sample

And (3) extracting glyphosate: the same as example 1;

purifying: the same as example 1;

and (3) determining glyphosate in a sample: the glyphosate sample was not detected.

Example 4: determination of glyphosate content in environmental water sample

（1）Fe₃O₄@C₇Preparation: the same as example 1;

(2) and (3) making a glyphosate working curve: the same as example 1;

(3) determination of Glyphosate content in environmental Water sample without extraction and purification by measuring example 1

And detecting glyphosate in the sample, wherein the sample is not detected.

The method established by the invention is compared with a GC-MS method, the detection error is within +/-3 percent, the consistency of the detection result is better, but the method has the advantages of short time, low cost, simple and convenient operation, no need of large-scale instruments and equipment, capability of being completed by only equipping one smart phone, and stronger advantage in actual detection.