A method for colorimetric and chemiluminescence dual-mode detection of glufosinate residues in coffee

Abstract

The present invention discloses a method for dual-mode detection of glufosinate residues in coffee by colorimetry and chemiluminescence, belonging to the technical field of chemical analysis and detection. In this method, the Schiff base formed by 4-aminoantipyrine and glutaraldehyde is used as a complexing agent for Cu 2+ . Then, microwave digestion is used to prepare copper-doped carbon dots Cu-CDs nanozyme. The Cu-CDs nanozyme has excellent peroxidase-like activity, oxidizes colorless 2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) into blue-green oxidized ABTS, and is used as a catalyst for chemiluminescence, which can increase the chemiluminescence intensity of the reaction between N-(4-aminobutyl)-N-ethylisoluminol and H2O2 by nearly 100 times. Based on the inhibitory effect of glufosinate on the POD-like activity of Cu-CDs, a sensitive and rapid dual-channel GM detection technology by colorimetry and chemiluminescence has been developed. The method of the present invention has potential application value in coffee safety monitoring.

Description

Technical Field

[0001] The present invention belongs to the technical field of chemical analysis and detection, and particularly relates to a method for colorimetric and chemiluminescence dual-mode detection of glufosinate residue in coffee by Schiff base copper-doped carbon dot nanozyme. Background Art

[0002] Glufosinate (GM) is a broad-spectrum contact herbicide with a broad-spectrum non-selective effect on gramineous plants, and has the characteristics of low toxicity, high efficiency, and environmental friendliness. However, GM residues in plants can accumulate and transfer through the food chain, and human DNA is oxidized and damaged by GM, posing a risk to human health. Therefore, developing a method for detecting GM pesticides is crucial for ensuring food safety. Currently, various instrumental analysis methods such as HPLC, LC-MS / MS, GC-MS, electrochemistry, and ion chromatography have been used to detect the GM content in foods. These methods are very reliable, but there are also many drawbacks, such as high purchase and service costs, complex operation procedures, and the need for professional operators. Therefore, various sensitive and convenient technologies have been developed to achieve the detection of GM residues.

[0003] Chemiluminescence (CL) is the light radiation generated by a chemical reaction. Without an excitation light source, the background signal is eliminated, and CL is considered a feasible alternative to overcome the defects of fluorescence measurement. At the same time, CL is considered a promising method because of its simple equipment, high sensitivity, convenient operation, fast analysis speed, and easy automation and high-throughput detection. The low chemiluminescence efficiency of the classical luminol / hydrogen peroxide (H2O2) system has greatly limited its extensive development in analytical applications. Therefore, a large amount of effort has been focused on exploring new co-reactants and synthesizing functional materials with catalytic properties to improve the chemiluminescence efficiency. Summary of the Invention

[0004] The present invention provides a method for colorimetric and chemiluminescence dual-mode detection of glufosinate residue in coffee by Schiff base copper-doped carbon dot nanozyme. The method uses the Schiff base formed by 4-aminoantipyrine and glutaraldehyde as Cu 2+Chelating agent, and then microwave digestion was used to prepare copper-doped carbon dots Cu-CDs nanozyme. The Cu-CDs nanozyme has excellent peroxidase-like activity, oxidizes colorless 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) into blue-green oxidized ABTS (oxABTS), and is used as a catalyst for chemiluminescence (CL), increasing the chemiluminescence intensity of the reaction between N-(4-aminobutyl)-N-ethylisoluminol (ABEI) and H2O2 by nearly 100 times; Based on the inhibitory effect of glufosinate (GM) on the POD-like activity of Cu-CDs, a sensitive and rapid colorimetric and chemiluminescence dual-channel GM detection technique was developed. This colorimetric and chemiluminescence dual-mode method provides a simple and highly sensitive method for GM detection and has potential application value in coffee safety monitoring.

[0005] The method for dual-mode colorimetric and chemiluminescence detection of glufosinate residues in coffee by the Schiff base copper-doped carbon dots nanozyme of the present invention is as follows:

[0006] (1) After mixing 4-6 mmol of 4-aminoantipyrine and 2-4 mmol of glutaraldehyde, add 10-15 mL of a 50% ethanol solution by volume. The mixture is stirred at room temperature for 30-40 min. The stirring initially turns milky white and finally produces a white precipitate. Centrifuge, and the precipitate is washed 3-4 times with deionized water and ethanol in sequence, and then dried in a vacuum oven to obtain the Schiff base;

[0007] (2) Dissolve 4-6 mmol of CuCl2·2H2O and 0.7-1.0 g of Schiff base in 15-20 mL of a 50% ethanol solution by volume, ultrasonically treat for 10-15 min, transfer the mixed solution to a polytetrafluoroethylene high-pressure reaction kettle, place it in a microwave digestion instrument, heat with 1 KW microwave to 170-200 °C for reaction for 2-3 h, naturally cool to room temperature after the reaction is completed, remove large particle impurities with a 0.22 μm filter membrane, and then perform high-speed centrifugation. The supernatant is dried in vacuum to obtain copper-doped carbon dots Cu-CDs nanozyme;

[0008] (3) After mixing the copper-doped carbon dots Cu-CDs nanozyme solution, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) solution, and H2O2, add glufosinate solutions with different concentrations to generate a blue-green oxidation product. Add a pH 4.0 acetate buffer solution, mix well and let stand for 5-10 min, measure the absorbance at a wavelength of 417 nm, determine the linear relationship between the glufosinate concentration and the absorbance value ΔA, and obtain a regression equation, where ΔA = A - A0, and A and A0 are the absorbance values of the reaction system without adding glufosinate and adding glufosinate, respectively;

[0009] (4) After mixing the copper-doped carbon dots Cu-CDs nanozyme solution, N-(4-aminobutyl)-N-ethylisolauminol (ABEI) solution, and H2O2, different concentrations of glufosinate solution were added to generate chemiluminescent products. Then, a pH 10.0 phosphate buffer solution was added. After reacting for 4 - 6 min, the chemiluminescence intensity was measured at 458 nm using a microplate reader and ΔI was calculated. CL , to determine the linear relationship between the glufosinate concentration and ΔI CL , and obtain the regression equation, where ΔI CL = I - I0, and I and I0 are the chemiluminescence intensities of the reaction systems without and with the addition of glufosinate, respectively.

[0010] (5) The test sample solution was operated according to steps (3) and (4), ΔA and ΔI were measured respectively, and substituted into the regression equation to calculate the GM concentration in the test sample solution.

[0011] The concentration of the copper-doped carbon dots Cu-CDs nanozyme solution was 20 μg / mL, the addition amount was 10 - 20 μL, the concentration of the ABTS solution was 0.2 mmol / L, the addition amount was 10 - 20 μL, the concentration of the ABEI solution was 135 μmol / L, the addition amount was 10 - 20 μL, and the concentration of H2O2 was 0.2 mmol / L, the addition amount was 10 - 20 μL.

[0012] The advantages of the present invention are as follows:

[0013] 1. In the present invention, Schiff base is synthesized from 4-aminoantipyrine and glutaraldehyde, and the Schiff base complex formed with Cu 2+ has a stable and specific structure. The copper-doped carbon dots prepared using this complex as a precursor have excellent peroxidase-like activity, can oxidize colorless ABTS into blue-green oxidized ABTS (oxABTS), and can also be used as a catalyst for chemiluminescence (CL), increasing the chemiluminescence intensity of the reaction between N-(4-aminobutyl)-N-ethylisolauminol (ABEI) and H2O2 by nearly 100 times. Importantly, glufosinate inhibits the peroxidase-like activity of Cu-CDs, resulting in a decrease in the absorbance and chemiluminescence intensity of the colorimetric and chemiluminescent systems. Therefore, a simple, rapid, specific, sensitive, and background-free colorimetric and chemiluminescent dual-mode glufosinate detection method is established.

[0014] 2. The established colorimetric and chemiluminescence methods for detecting glufosinate have a wide linear range, which are 1.3 - 133.3 μg / L and 0.6 - 46.7 μg / L respectively, and the detection limits are 0.67 μg / L and 0.21 μg / L respectively. The established methods were successfully applied to the detection of glufosinate in coffee samples, and GM in coffee samples was successfully detected using the spiking method, with a recovery rate of 83.91% - 99.54%. The detection system provided by the present invention has high detection accuracy. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 FIG. 6 is the TEM image of the Cu-CDs nanozyme in Example 1. Figure a is a 50 nm transmission electron micrograph, and Figure b is a 10 nm TEM and high-resolution transmission electron micrograph.

[0016] FIG. 2 is the FTIR spectrum of the Cu-CDs nanozyme in Example 1.

[0017] FIG. 3 is the UV-visible absorption spectra of Cu-CDs + H2O2 + TMB (Figure a) and Cu-CDs + H2O2 + ABTS (Figure b) in Example 1 in the presence or absence of GM.

[0018] Figure 4 FIG. 16 is the Michaelis-Menten equation of the Cu-CDs nanozyme in Example 1. Figure a is with TMB as the substrate, and Figure b is with H2O2 as the substrate.

[0019] Figure 5 FIG. 20 is the Michaelis-Menten equation of the Cu-CDs + GM nanozyme in Example 1. Figure a is with TMB as the substrate, and Figure b is with H2O2 as the substrate.

[0020] Figure 6 FIG. 24 is the Michaelis-Menten equation of HRP in Example 1. Figure a is with TMB as the substrate, and Figure b is with H2O2 as the substrate.

[0021] Figure 7 FIG. 28 is the CL spectra of the reaction systems Cu-CDs + H2O2 + ABEI, potassium ferricyanide + H2O2 + ABEI (Figure a), and Cu-CDs + H2O2 + ABEI + GM (Figure b).

[0022] Figure 8 FIG. 32 is the colorimetric linear regression equation of GM in Example 1.

[0023] Figure 9 FIG. 36 is the CL linear regression equation of GM in Example 1.

[0024] Figure 10 FIG. 40 shows the results of the influence of interfering substances on the GM detection system. Figure a shows the main components in coffee, and Figure b shows organophosphorus pesticides. DETAILED DESCRIPTION OF THE INVENTION

[0025] The content of the present invention will be further illustrated by the following examples, but these examples do not limit the protection scope of the present invention. The methods in the examples are all conventional methods without special instructions, and the reagents used are all conventional commercially available reagents or reagents prepared according to conventional methods without special instructions.

[0026] Example 1: Determination of glufosinate in coffee samples

[0027] 1. After mixing 5 mmol of 4 - aminophenazone and 3 mmol of glutaraldehyde, 15 mL of an ethanol solution with a volume concentration of 50% was added. The mixture was stirred at room temperature for 30 min. The stirring initially turned milky white and finally produced a white precipitate. It was centrifuged at 4000 rpm for 10 min, and the precipitate was repeatedly washed 3 times with deionized water and ethanol in sequence, and then dried in a vacuum oven to obtain the Schiff base.

[0028] 2. 5 mmol of CuCl₂·2H₂O and 0.8 g of the Schiff base were dissolved in 20 mL of an ethanol solution with a volume concentration of 50%. It was ultrasonically treated for 15 min, and the mixed solution was transferred to a polytetrafluoroethylene high - pressure reaction kettle and placed in a microwave digestion instrument. It was heated to 180 °C at 1 kW for 2 h. After the reaction was completed, it was naturally cooled to room temperature. Large - particle impurities were removed with a 0.22 - μm filter membrane, and it was centrifuged at 10000 rpm for 10 min. The supernatant was dried in vacuo to obtain the copper - doped carbon dots Cu - CDs nanozyme. The prepared Cu - CDs nanozyme was analyzed by transmission electron microscopy (TEM). As Figure 1 shown in a, the synthesized Cu - CD nanozyme was in the shape of quasi - spherical particles, with good dispersibility and uniformity, and the average particle size was about 4.5 nm. As Figure 1 shown in b, the high - resolution TEM (HRTEM) image presented a lattice spacing of 0.25 nm, corresponding to the (110) crystal plane of graphite carbon; The FTIR spectrum of Cu - CD was as Figure 2 , and the characteristic peak at 1630 cm -1 was attributed to the C=N bond, confirming the successful synthesis of the Schiff base. The peak near 3454 cm -1 was attributed to the stretching vibration of O - H / N - H. The characteristic peaks at 2067 cm -1 and 1630 cm -1 corresponded to the stretching vibration of C=O. The characteristic peak at 582 cm -1 was attributed to the stretching vibration of Cu - O / Cu - N.

[0029] 3. Evaluation of the peroxidase - like activity of Cu - CDs nanozyme

[0030] Take 100 μL each of TMB or ABTS with a concentration of 0.2 mmol / L, add 100 μL of 2 mmol / L H2O2 and 100 μL of 20 μg / mL Cu-CDs nanozyme respectively, add 0.1 mmol / L acetate buffer solution with pH 4.0 to make up to 3 mL, shake well, let stand for 10 min, and measure the absorbance at wavelengths of 654 nm and 417 nm. Figure 3 The results show that Cu-CDs nanozyme has good peroxidase-like activity; under exactly the same conditions, add 100 μL of 20 μg / L glufosinate (GM), measure the absorbance, and the results show that GM can significantly inhibit the absorbance of the reaction system.

[0031] In this example, the Michaelis catalytic kinetic parameters were also measured, and the results are shown in Figures 4 - 6 and Table 1, where Figure 4 shows the Michaelis constants of Cu-CDs nanozyme for substrates TMB and H2O2 K m are 0.037 mmol / L and 0.037 mmol / L respectively, and the reaction rate constants are 6.696×10 -7 mol / L·s and 7.942×10 -7 mol / L·s respectively. Figure 5 shows the K m of TMB and H2O2 after adding GM are 0.331 mmol / L and 0.088 mmol / L respectively, and the reaction rate constants are 0.166×10 -7 mol / L·s and 0.308×10 - 7 mol / L·s respectively, indicating that the addition of GM greatly inhibits the affinity and reaction rate of Cu-CDs nanozyme with substrates TMB and H2O2; the Michaelis constants of horseradish peroxidase (HRP) for substrates TMB and H2O2 K m are 0.274 mmol / L and 0.029 mmol / L respectively, and the reaction rate constants are 10.080×10 -7 mol / L·s and 10.770×10 -7 mol / L·s respectively. The V max of Cu-CDs nanozyme for TMB and H2O2 reach 66% and 74% of HRP.

[0032] Table 1 Michaelis catalytic kinetic parameters

[0033] .

[0034] 4. Evaluation of the chemiluminescence performance of Cu-CDs catalysis

[0035] In a 98-well enzyme-linked immunosorbent assay (ELISA) plate, add 10 μL of ABEI with a concentration of 135 μmol / L, 20 μL of H2O2 with a concentration of 0.2 mmol / L, 10 μL of Cu-CDs nanozyme with a concentration of 20 μg / mL, and 20 μL of 0.1 mmol / L PBS buffer solution with pH 10.0. Mix well and measure the CL intensity of the system at a wavelength of 458 nm. Figure 7 a The results show that the reaction system has a strong CL peak. Compared with the classical CL oxidant potassium ferricyanide, the CL intensity increases by 100 times. Figure 7 b The results show that with the addition of GM, the CL intensity of the reaction system is greatly inhibited, providing conditions for the detection of GM.

[0036] 5. Preparation of the colorimetric working curve of glufosinate

[0037] In a 98-well ELISA plate, add 10 μL of ABTS with a concentration of 0.2 mmol / L, 10 μL of H2O2 with a concentration of 0.2 mmol / L, 10 μL of Cu-CDs nanozyme with a concentration of 20 μg / mL, then add 20 μL of GM solution with a concentration range of 1.3 - 133.3 μg / L, and 20 μL of 0.1 mmol / L acetate buffer solution with pH 4.0. Mix well and measure ΔA at a wavelength of 417 nm. ΔA = A - A0, where A and A0 are the absorbance values of the reaction system before and after adding GM, respectively. Plot a standard curve with the GM concentration as the abscissa and ΔA as the ordinate, and obtain the regression equation, correlation coefficient, relative standard deviation, linear range, etc., as shown in Table 2. The results are shown in Figure 8 ;

[0038] 6. Preparation of the CL working curve of glufosinate

[0039] In a 98-well ELISA plate, add 10 μL of ABEI with a concentration of 135 μmol / L, 10 μL of H2O2 with a concentration of 0.2 mmol / L, 10 μL of Cu-CDs nanozyme with a concentration of 20 μg / mL, then add 20 μL of GM solution with a concentration range of 0.6 - 46.7 μg / L, and 20 μL of 0.1 mmol / L PBS buffer solution with pH 10.0. Mix well and measure ΔI CL intensity, ΔI CL = I - I0, where I and I0 are the chemiluminescence intensities of the reaction system before and after adding GM, respectively. Plot a standard curve with the GM concentration as the abscissa and ΔI CL as the ordinate, and obtain the regression equation, correlation coefficient, relative standard deviation, linear range, etc., as shown in Table 2. The results are shown in Figure 9 ;

[0040] Table 2 Linear equation, correlation coefficient, relative standard deviation, linear range

[0041] 。

[0042] 7. Specificity investigation of the method

[0043] Replace GM with other interfering substances, and detect the influence of other interfering substances on the Cu-CDs nanozyme catalytic system in the above detection system. The interfering substances include the main components in coffee: caffeine, citric acid, casein, tannic acid, caffeic acid, sucrose, glucose, K + , Mg 2+ , Mn 2+ , Zn 2+ , and organophosphorus pesticides: parathion-methyl, glyphosate, parathion, profenofos, paraoxon, glufosinate (where the concentration of citric acid and tannic acid is 13 mg / L, the concentration of caffeic acid is 146.7 mg / L, the concentration of sucrose and glucose is 5 mg / L, the concentration of glyphosate is 80 μg / L, the concentration of GM is 45 μg / L, and the concentration of the remaining substances is 500 mg / L). The results are shown in Figure 10 , and it can be seen from the figure that only GM has a good inhibitory effect on the Cu-CDs nanozyme-catalyzed CL system. This result shows that the Cu-CDs nanozyme has good selective specificity for GM.

[0044] 8. Determination of glufosinate in coffee samples

[0045] (1) Treatment of coffee samples: Weigh 5.0 g (accurate to 0.01 g) of coffee powder into a 50 mL centrifuge tube, add 10.0 mL of 0.1 mol / L NH₃·H₂O, vortex extract for 2 min, ultrasonically oscillate and extract for 30 min, centrifuge at 4000 rpm for 20 min, take 4 mL of the supernatant and transfer it to a round-bottom flask, concentrate it under reduced pressure until almost dry, and make up the volume to 1 mL with 50% methanol aqueous solution to obtain the sample extract;

[0046] (2) Determination of coffee samples: Add 10 μL of ABTS with a concentration of 0.2 mmol / L, 10 μL of H₂O₂ with a concentration of 0.2 mmol / L, and 10 μL of 20 μg / mL Cu-CDs nanozyme to a 96-well microplate, then add 100 μL of the sample extract and 20 μL of 0.1 mmol / L acetate buffer solution with pH 4.0, mix well, measure ΔA at a wavelength of 417 nm, substitute ΔA into the regression equation in step 5, and GM in coffee is not detected; at the same time, add 10 μL of ABEI with a concentration of 135 μmol / L, 10 μL of H₂O₂ with a concentration of 0.2 mmol / L, and 10 μL of 20 μg / mL Cu-CDs nanozyme to a 96-well microplate, then add 100 μL of the sample extract and 20 μL of 0.1 mmol / L PBS buffer solution with pH 10.0, mix well, and measure ΔI at a wavelength of 458 nmCL Strength, substitute ΔI CL into the regression equation in Step 6, and GM was not detected in the coffee;

[0047] (3) Recovery and precision experiments: Add GM standard solutions with two different concentrations to the coffee samples respectively; measure each concentration in parallel for 3 times, calculate the spiked recovery rate, and calculate the relative standard deviation RSD. The results are shown in Table 3. The spiked recovery rate of GM is between 83.91% and 99.54%, and the RSD is between 1.88% and 6.29%. This method has good accuracy and precision;

[0048] Table 3 Spiked recovery rate and RSD of the samples (n = 3)

[0049]

[0050] The glufosinate determination method established in the present invention has the advantages of fewer treatment steps, being fast, simple, not requiring large-scale instrument equipment and professional operators for the liquid chromatography - mass spectrometry method of GB 23200.108 - 2018, short time consumption, low treatment cost, and simple operation, and has strong advantages in actual detection.

Claims

1. A method for detecting glufosinate residues in coffee by colorimetric and chemiluminescence dual-mode detection, characterized in that, The steps are as follows: (1) After mixing 4 - 6 mmol of 4 - aminophenazone and 2 - 4 mmol of glutaraldehyde, add 10 - 15 mL of 50% ethanol solution by volume. Stir the mixture at room temperature for 30 - 40 min, then centrifuge. Wash the solid with deionized water and ethanol 3 - 4 times successively, and dry it under vacuum to obtain the Schiff base; (2) Dissolve 4 - 6 mmol of CuCl₂·2H₂O and 0.7 - 1.0 g of Schiff base in 15 - 20 mL of 50% ethanol solution by volume. After ultrasonic treatment for 10 - 15 min, react at microwave and 170 - 200 °C for 2 - 3 h. Naturally cool to room temperature, remove large - particle impurities with a 0.22 - μm filter membrane, centrifuge, and vacuum - dry the supernatant to obtain copper - doped carbon dots Cu - CDs nanozyme; (3) After mixing the copper - doped carbon dots Cu - CDs nanozyme solution, 2,2'-azino - bis(3 - ethylbenzothiazoline - 6 - sulfonic acid) solution, and H₂O₂, add different concentrations of glufosinate solution and pH 4.0 acetate buffer solution. Mix well and let it stand for 5 - 10 min. Measure the absorbance at a wavelength of 417 nm to determine the linear relationship between the glufosinate concentration and the absorbance value ΔA, and obtain the regression equation, where ΔA = A - A₀, and A and A₀ are the absorbance values of the reaction systems without and with the addition of glufosinate, respectively; (4) After mixing the copper-doped carbon dots Cu-CDs nanozyme solution, N-(4-aminobutyl)-N-ethylisoluminol solution, and H2O2, different concentrations of glufosinate solution and pH 10.0 phosphate buffer solution were added. After reacting for 4 - 6 min, the chemiluminescence intensity was measured using a microplate reader at 458 nm and ΔI was calculated. CL , to determine the linear relationship between the glufosinate concentration and ΔI CL , and obtain the regression equation, where ΔI CL = I - I0, and I and I0 are the chemiluminescence intensities of the reaction systems without and with the addition of glufosinate, respectively. (5) Measure ΔA and ΔI respectively for the sample solution to be tested according to the operations in step (3) or step (4), and substitute them into the regression equation to obtain the concentration of glufosinate-ammonium in the sample solution to be tested. CL , and substitute into the regression equation to obtain the concentration of glufosinate-ammonium in the sample solution to be tested.

2. The method according to claim 1, wherein: The concentration of the doped carbon dots Cu - CDs nanozyme solution is 20 μg / mL, and the addition amount is 10 - 20 μL. The concentration of the 2,2'-azino - bis(3 - ethylbenzothiazoline - 6 - sulfonic acid) solution is 0.2 mmol / L, and the addition amount is 10 - 20 μL. The concentration of the N-(4 - aminobutyl)-N - ethylisoluminol solution is 135 μmol / L, and the addition amount is 10 - 20 μL. The concentration of H₂O₂ is 0.2 mmol / L, and the addition amount is 10 - 20 μL.

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