Nitrogen-doped carbon dots and rapid fluorescence detection method thereof for indoxacarb residues

Abstract

The invention discloses a nitrogen-doped carbon dot and a rapid fluorescence detection method thereof for indoxacarb residue, and belongs to the field of biosensor technology and food quality detection. L-theanine and ethidene diamine are used as raw materials, the nitrogen-doped carbon dots are prepared through a hydrothermal method, the N-CDs have the advantages of being stable in fluorescence emission, good in salt tolerance and excellent in photobleaching resistance, and indoxacarb can specifically quench fluorescence through the static quenching effect. By optimizing N-CDs synthesis conditions and indoxacarb detection conditions, an indoxacarb fluorescence sensing detection system with high selectivity and high sensitivity is constructed. The detection method disclosed by the invention can be suitable for detecting indoxacarb residues in environmental water samples such as tap water and river water and agricultural product matrixes such as apples and tea leaves, the standard recovery rate is good, the relative standard deviation is low, on-site rapid screening of the indoxacarb residues can be realized, and a new technical means is provided for monitoring the quality safety of the environment and agricultural products.

Description

Technical Field

[0001] This invention belongs to the fields of biosensor technology and food quality testing technology, specifically relating to a nitrogen-doped carbon dot and its rapid fluorescent detection method for indoxacarb residues. Background Technology

[0002] Indoxacarb is a highly effective and low-toxicity oxadiazine insecticide used to control lepidopteran pests in crops such as fruits, vegetables, and tea. It works by blocking sodium ion channels in the nerve cells of pests, leading to paralysis and death. Indoxacarb is relatively safe for non-target organisms such as humans and livestock, but high doses can cause poisoning symptoms in humans. Therefore, various countries have established maximum residue limits (MRLs), such as 5 mg / kg in Chinese tea and 0.01 mg / kg in Japan. These limits impose high requirements on the quality and safety control of agricultural products and international trade.

[0003] Conventional detection methods such as chromatography-mass spectrometry (GC-MS), while accurate, are cumbersome, require expensive equipment, and are time-consuming, making them unsuitable for rapid detection. Fluorescence detection technology has gained attention due to its simplicity, speed, and low cost, and nanomaterials such as carbon quantum dots (CDs) are widely used in pesticide detection. Existing fluorescence methods, such as perovskite oxides or molecularly imprinted polymer probes, have been reported, but their application in actual agricultural product matrices is limited, and their preparation is complex.

[0004] This invention uses L-theanine and ethylenediamine as raw materials to prepare nitrogen-doped carbon dots via a hydrothermal method, constructs a fluorescence sensing method for indoxacarb, optimizes conditions to improve selectivity and sensitivity, and verifies the method in actual samples such as tap water, river water, tea, and apples, aiming to provide a simple, reliable, and rapid detection technology. Summary of the Invention

[0005] The purpose of this invention is to provide a technical solution for nitrogen-doped carbon dots and a rapid fluorescent detection method for indoxacarb residues, solving the problems of existing indoxacarb detection methods being cumbersome to operate, expensive to use, and unable to detect rapidly.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution: The first aspect of this invention provides a nitrogen-doped carbon dot, which is prepared by a hydrothermal method using L-theanine and anhydrous ethylenediamine as raw materials.

[0007] Further, the solution is obtained through the following steps: L-theanine and anhydrous ethylenediamine are weighed and dissolved in ultrapure water, and sonicated until fully dissolved to obtain a mixed solution; the mixed solution is transferred to a polytetrafluoroethylene high-pressure reactor and heated to react, then naturally cooled to room temperature, centrifuged and the supernatant is filtered; the filtrate is dialyzed and then freeze-dried under vacuum to obtain nitrogen-doped carbon dot solid powder.

[0008] Furthermore, the L-theanine to water mass ratio is 1:40-200, the amount of anhydrous ethylenediamine is 0.1-1 mL, and the ultrasonic time is 2-10 min.

[0009] Furthermore, the heating reaction conditions are as follows: reaction in a forced-air drying oven at 160-220℃ for 4-10 h; centrifugation conditions are as follows: 10000-15000 r / min for 10-30 min; and filtration is performed using a microporous membrane.

[0010] Furthermore, the dialysis conditions are as follows: the filtrate is placed in a dialysis bag with a molecular weight cutoff of 500-1500 Da, and dialyzed in deionized water for 12-36 h, with the dialysis medium being replaced every 3-5 h; the vacuum freeze-drying is specifically performed at -70~-90℃ and 0.01-0.2 Pa for 40-60 h.

[0011] The second aspect of the present invention provides the application of the above-mentioned nitrogen-doped carbon dots in the detection of indoxacarb.

[0012] A third aspect of the present invention provides a method for detecting indoxacarb based on the above-described nitrogen-doped carbon dot fluorescence method, comprising the following steps: S.1 Nitrogen-doped carbon dot solid powder is dispersed in ultrapure water to obtain N-CDs working solution; S.2 Add Tris-HCl buffer to the N-CDs working solution, vortex at room temperature, add indoxacarb standard solutions of different concentrations, incubate at room temperature, measure the fluorescence spectrum, and calculate the fluorescence intensity difference ΔF=F. 310 nm -F 390 nm A standard curve equation was constructed using ΔF and indoxacarb concentration. S.3 Pre-treat the water or agricultural product sample to be tested, and determine the fluorescence intensity according to the method in step S.2; calculate the actual concentration of indoxacarb based on the standard curve.

[0013] Furthermore, the Tris-HCl buffer solution described in step S.2 has a pH of 8-10, a vortexing time of 10-20 min, and an incubation time of 0.5-5 min.

[0014] Furthermore, the water sample pretreatment described in step S.3 specifically involves filtering the water sample and adding indoxacarb standard solution to prepare water samples with different spiking concentrations; the spiking levels of the water samples are 0.01, 0.1, and 1 mg / L.

[0015] Furthermore, the agricultural product pretreatment described in step S.3 specifically involves crushing the agricultural product, accurately weighing the sample, adding indoxacarb standard solution to the set spiking level, allowing it to stand, and then pretreating it using the QuEChERS method; the spiking levels of the agricultural product samples are 0.5 mg / L, 1 mg / L, and 5 mg / L.

[0016] The beneficial effects of this invention include: (1) The N-CDs prepared by this invention are rich in nitrogen and oxygen functional groups on the surface, and have excellent salt resistance and photobleaching resistance. The fluorescence intensity remains stable in the range of 0~3.0 M NaCl concentration. After continuous irradiation with ultraviolet light, the fluorescence intensity does not change significantly, which ensures the stability and reliability of the detection system in complex matrices.

[0017] (2) The N-CDs prepared by this invention are readily available and have a simple preparation process. They are synthesized in one step by hydrothermal method without the need for complex equipment, and are low in cost. Furthermore, the N-CDs have stable fluorescence properties, good salt resistance, excellent anti-photobleaching properties, and good water solubility, making them convenient for practical applications.

[0018] (3) The indoxacarb fluorescence sensing detection system constructed in this invention has good selectivity. Only indoxacarb can specifically quench the fluorescence of N-CDs. Common pesticides such as difenoconazole and glufosinate have no obvious interference to the detection system and have strong anti-interference ability.

[0019] (4) The detection method of the present invention has high sensitivity, and the linear range for the detection of indoxacarb is 0.005~1.5 mg / L, with a detection limit as low as 0.0027 mg / L, which can meet the stringent domestic and international standards for indoxacarb residue limits.

[0020] (5) The detection method of the present invention is simple to operate and fast to detect. The entire detection process only takes a few minutes and does not require expensive large instruments. It can realize rapid on-site screening of indoxacarb residues.

[0021] (6) The detection method of the present invention has a wide range of applications. It can be applied to the detection of indoxacarb residues in environmental water samples such as tap water and river water, as well as in complex matrices of agricultural products such as apples and tea. The actual sample spiked recovery rate is good, the relative standard deviation is low, and the detection results are accurate and reliable, providing a new technical means for monitoring the quality and safety of agricultural products and detecting environmental water samples. Attached Figure Description

[0022] Figure 1 In the image, 'a' is a transmission electron microscope (TEM) image of N-CDs. Figure 1 In the figure, b is the particle size distribution bar chart; N-CDs are uniformly dispersed, basically spherical, with no obvious aggregation, and the particle size distribution is 2.02 nm~4.68 nm, with an average particle size of 2.88 nm. They have good water solubility. Figure 2 The image shows the Fourier transform infrared spectrum of N-CDs. The surface of N-CDs is rich in functional groups such as OH, NH, C=O, CN, and CO, and has typical structural features of nitrogen and oxygen functional group modification and aromatic carbon core. Figure 3 In the figure, 'a' represents the XPS energy spectrum of N-CDs. Figure 3 In the image, b represents the high-resolution XPS energy dispersive spectroscopy C1s spectrum. Figure 3 In the image, 'c' represents the high-resolution XPS energy dispersive spectroscopy spectrum N1s. Figure 3 In the figure, d represents the high-resolution XPS energy spectrum O1s; the elemental composition of N-CDs is C1s 68.49%, N1s 15.02%, and O1s 11.24%, and the surface contains chemical bonds such as CC / H, CO / CN, C=O, NC, O=C, and OC, which is consistent with the FTIR analysis results. Figure 4 In this context, 'a' represents the UV-Vis absorption spectrum of N-CDs. Figure 4 In the figure, b represents the fluorescence spectrum at different excitation wavelengths; N-CDs have a characteristic absorption peak at 260 nm, the optimal excitation wavelength is 310 nm, the corresponding emission wavelength is 390 nm, and the position of the fluorescence emission peak does not shift significantly in the excitation wavelength range of 250-340 nm, showing an excitation wavelength-independent characteristic. Figure 5 In the figure, 'a' represents the effect of different concentrations of NaCl on the fluorescence intensity of N-CDs; Figure 5 In the figure, b represents the effect of continuous irradiation by a 365nm UV lamp on the fluorescence intensity of N-CDs. The fluorescence intensity of N-CDs remains stable in the concentration range of 0~3.0 M NaCl, and there is no significant change in fluorescence intensity after continuous irradiation by UV light, indicating excellent salt resistance and photobleaching resistance. Figure 6 Optimization diagram of N-CDs synthesis conditions; Figure 6 In this context, 'a' represents the reaction temperature. Figure 6 In this context, 'a' represents the reaction time. Figure 6 In this context, c represents the mass ratio of theanine to water. The optimal synthesis conditions for N-CDs are a reaction at 200℃ for 8 h and a mass ratio of theanine to water of 1:50, at which point the fluorescence intensity of N-CDs reaches its peak. Figure 7 Optimization diagram for indoxacarb detection conditions; Figure 7 In this context, 'a' represents the pH of the buffer solution. Figure 7 b in the figure represents the reaction time; the optimal conditions for indoxacarb detection are incubation at room temperature for 1 min in Tris-HCl buffer at pH 9, at which point indoxacarb has the best fluorescence quenching effect on N-CDs; among them, 3-morpholinopropanesulfonic acid (Mops) is a zwitterionic buffer reagent commonly used in biochemistry and molecular biology; Figure 8 This is a graph showing the selectivity of N-CDs to different pesticides; only indoxacarb can cause F...390 nm / F 310 nm The value changed significantly, while other interfering pesticides had no significant effect on the ratio. N-CDs showed good specific recognition ability for indoxacarb. Figure 9 The standard curve for the detection of indoxacarb shows a good linear relationship between the concentration of indoxacarb and the ΔF value in the range of 0.005~1.5 mg / L. The standard curve equation is y=631.19x+414.31, and the correlation coefficient R²=0.9925. Figure 10 In this context, 'a' represents the fluorescence lifetime of N-CDs and N-CDs + indoxacarb; Figure 10 In this context, b represents the ultraviolet absorption spectrum of the reaction system. Figure 10 c represents the fluorescence spectrum of N-CDs and indoxacarb; the fluorescence lifetime of N-CDs did not change significantly after the addition of indoxacarb, ruling out dynamic quenching; the UV absorption spectrum changed significantly, confirming that indoxacarb and N-CDs undergo complexation, which is a static quenching mechanism; Figure 11 This is a matrix standard curve for actual sample testing. The matrix of the actual sample has little impact on the detection system, and the matrix standard curve has good consistency with the standard solution curve, which can be used to calculate the concentration of indoxacarb in actual samples. Detailed Implementation

[0023] The present invention will be further described in detail below with reference to specific embodiments.

[0024] Example 1: Preparation and Characterization of N-CDs Weigh 0.20 g L-theanine and dissolve it in 20 mL of ultrapure water. Add 0.25 mL of anhydrous ethylenediamine and sonicate for 5 min until fully dissolved. Transfer the mixed solution to a polytetrafluoroethylene high-pressure reactor and react in a 200℃ forced-air drying oven for 8 h. After naturally cooling to room temperature, centrifuge at 12000 r / min for 20 min. Filter the supernatant through a 0.22 μm aqueous microporous membrane. Place the filtrate into a dialysis bag with a molecular weight cutoff of 1000 Da and dialyze in deionized water for 24 h, changing the dialysis medium every 4 h. After dialysis, freeze-dry the solution at -80℃ and 0.1 Pa for 48 h to obtain N-CDs solid powder, which is then prepared into a 10 mg / L working solution and stored at 4℃ protected from light.

[0025] The N-CDs prepared above were characterized as follows: After sonicating the N-CDs solution for 10 min, 3-5 drops were placed on an ultrathin copper mesh. After drying, the morphology was analyzed using transmission electron microscopy (TEM). The results are as follows: Figure 1 As shown, N-CDs have a spherical structure with an average particle size of 2.88 nm and no obvious aggregation.

[0026] Dry N-CDs powder and spectroscopically pure potassium bromide powder were mixed at a mass ratio of 1:100 to 1:200; the mixture was ground thoroughly multiple times, and then pressed into a transparent thin sheet using a tablet press. During testing, the background was collected first, followed by the sample's infrared spectrum (FTIR) at a resolution of 4 cm⁻¹. -1 The number of scans was 32, and the test wavenumber range was 400 / 600-4000 cm⁻¹. -1 The FTIR characterization results are as follows: Figure 2 As shown, this indicates that the surface of N-CDs is rich in functional groups such as OH, NH, and C=O.

[0027] After pressing an appropriate amount of N-CDs powder into a tablet, it was placed on a sample tray for X-ray photoelectron spectroscopy (XPS) measurement. The pressure in the sample chamber was less than 2.0 × 10⁻⁶. -7 At mbar, the sample was fed into the analysis chamber with a spot size of 400 μm, an operating voltage of 12 kV, and a filament current of 6 mA. The full-spectrum scan pass energy was 150 eV with a step size of 1 eV; the narrow-spectrum scan pass energy was 50 eV with a step size of 0.1 eV. The XPS characterization results are as follows: Figure 3 As shown, the elemental composition of N-CDs is 68.49% C1s, 15.02% N1s, and 11.24% O1s.

[0028] The fluorescence properties of CDs solutions were measured using a fluorescence spectrophotometer, such as... Figure 4 As shown, the optimal excitation wavelength of N-CDs is 310 nm, the emission wavelength is 390 nm, and they exhibit excitation wavelength independence.

[0029] like Figure 5 As shown in Figure a, by changing the NaCl concentration (0-3.0 M) to simulate different ionic intensities in the environment, the fluorescence intensity of N-CDs remained stable, indicating that N-CDs have excellent salt tolerance; Figure 5 As shown in b, after continuous irradiation with ultraviolet light, the fluorescence intensity of N-CDs did not change significantly with the extension of irradiation time, confirming that it has good anti-photobleaching properties.

[0030] Example 2: Optimization of Synthesis and Detection Conditions Synthesis conditions optimization: The effects of reaction temperatures of 160℃, 180℃, 200℃, and 220℃, reaction times of 4h, 6h, 8h, and 10h, and the mass ratios of theanine to water of 1:200, 1:100, 1:50, and 1:40 on the fluorescence intensity of N-CDs were investigated. Results are shown below. Figure 6As shown: the fluorescence intensity of N-CDs reaches its peak at 200℃. Excessive temperature will lead to excessive carbon nucleus growth and fluorescence quenching. 8h is the optimal reaction time. Too long a reaction time will increase non-radiative recombination centers. The fluorescence intensity is the highest when the theanine to water mass ratio is 1:50. Therefore, the optimal synthesis conditions are determined to be 200℃ for 8h and the theanine to water mass ratio of 1:50.

[0031] Optimization of detection conditions: The detection performance of PBS, MOPS, and Tris-HCl buffers was investigated at pH 3.0–10.0. The results are as follows: Figure 7 As shown, indoxacarb showed the best fluorescence quenching effect on N-CDs in Tris-HCl buffer at pH 9. Incubation times of 0.5, 1, 2, 3, 4, and 5 min were investigated. The results showed that the incubation reaction reached equilibrium at 1 min, with no further change in fluorescence intensity. Therefore, the optimal detection conditions were determined to be incubation at room temperature for 1 min in Tris-HCl buffer at pH 9.

[0032] The fluorescence quenching mechanism of indoxacarb on N-CDs is a static quenching effect, as shown in the results. Figure 10 As shown, indoxacarb complexes with N-CDs to form a new compound, leading to specific quenching of the fluorescence of N-CDs. Measurements of the fluorescence lifetime of N-CDs before and after indoxacarb addition revealed that the average fluorescence lifetime of N-CDs changed from 4.74 ns to 4.94 ns after indoxacarb addition, with no significant change in decay time, ruling out dynamic quenching. Simultaneously, the UV-Vis absorption spectrum of N-CDs changed significantly after indoxacarb addition, further confirming the static quenching mechanism.

[0033] Example 3: Plotting the Fluorescence Standard Curve and Calculating the Detection Limit Under optimal detection conditions, 800 μL of pH 9 Tris-HCl buffer was added to 100 μL of N-CDs working solution, and the mixture was vortexed at room temperature for 15 min. Then, 100 μL of indoxacarb standard solutions with concentrations of 0.005, 0.01, 0.05, 0.1, 0.5, 1, and 1.5 mg / L were added, and the mixture was incubated at room temperature for 1 min. Fluorescence spectra were then measured, and the fluorescence intensity at 310 nm and 390 nm was recorded. The ΔF value was calculated. A linear fit was performed with ΔF as the ordinate and indoxacarb concentration as the abscissa. The results are shown below. Figure 9As shown, the standard curve equation was obtained as y = 631.19x + 414.31, with R² = 0.9925. The limit of detection was calculated using the signal-to-noise ratio 3σ / S, where σ is the standard deviation of 10 parallel determinations of the blank sample, and S is the slope of the standard curve. The calculated limit of detection was 0.0027 mg / L.

[0034] Example 4: Selectivity Analysis Under the same experimental conditions, 10 mg / L of indoxacarb, difenoconazole, glufosinate, glyphosate, chlorpyrifos, imidacloprid, acetamiprid, thiamethoxam, thiamethoxam, and azoxystrobin were added to the detection system, respectively. After incubation at room temperature for 1 min, F was measured. 390 nm / F 310 nm Value. Result as follows Figure 8 As shown, only indoxacarb can cause a significant decrease in this ratio, while other pesticides have no significant effect on this ratio, indicating that the detection method of the present invention has good selectivity and anti-interference ability against indoxacarb.

[0035] Example 5: Actual Sample Testing Water sample testing: Tap water and river water were filtered through a 0.22 μm filter membrane, and spiked to concentrations of 0.01, 0.1, and 1 mg / L respectively. The samples were then tested according to the method of this invention. The results are as follows: Figure 11 As shown in Table 1, the recovery rate of spiked tap water was 83.6%–100.9%, with an RSD of less than 15%; the recovery rate of spiked river water was 95%–100.5%, with an RSD of less than 15%, indicating that this method is suitable for the detection of indoxacarb in environmental water samples.

[0036] Agricultural product sample testing: Apples and black tea were pulverized and spiked to concentrations of 0.5, 1, and 5 mg / L, respectively. After pretreatment using the QuEChERS method, the samples were tested according to the method of this invention. The results are as follows: Figure 11 As shown in Table 1, the spiked recoveries for apples were 75.18–112.97%, with RSDs below 10%; the spiked recoveries for black tea were 88.08–115.14%, with RSDs below 10%, indicating that this method is suitable for the detection of indoxacarb in complex matrices of agricultural products.

[0037] Table 1. Detection of indoxacarb in actual samples .

Claims

1. A nitrogen-doped carbon dot, characterized in that, It was prepared by hydrothermal method using L-theanine and anhydrous ethylenediamine as raw materials.

2. The nitrogen-doped carbon dot as described in claim 1, characterized in that, Specifically, the following steps are taken: L-theanine and anhydrous ethylenediamine are weighed and dissolved in ultrapure water, and sonicated until fully dissolved to obtain a mixed solution; the mixed solution is transferred to a polytetrafluoroethylene high-pressure reactor and heated to react, then naturally cooled to room temperature, centrifuged and the supernatant is filtered; the filtrate is dialyzed and then freeze-dried under vacuum to obtain nitrogen-doped carbon dot solid powder.

3. The nitrogen-doped carbon dot as described in claim 2, characterized in that, The L-theanine to water mass ratio is 1:40-200, and the amount of anhydrous ethylenediamine is 0.1-1 mL; the ultrasonic time is 2-10 min.

4. The nitrogen-doped carbon dot as described in claim 2, characterized in that, The heating reaction conditions are as follows: reaction in a forced-air drying oven at 160-220℃ for 4-10 h; centrifugation conditions are as follows: 10000-15000 r / min for 10-30 min; filtration is performed using a microporous membrane.

5. A nitrogen-doped carbon dot as described in claim 2, characterized in that, The dialysis conditions are as follows: the filtrate is placed in a dialysis bag with a molecular weight cutoff of 500-1500 Da, and dialyzed in deionized water for 12-36 h, with the dialysis medium being replaced every 3-5 h; the vacuum freeze-drying is specifically performed at -70~-90℃ and 0.01-0.2 Pa for 40-60 h.

6. The application of a nitrogen-doped carbon dot as described in any one of claims 1-5 in the detection of indoxacarb.

7. A method for detecting indoxacarb using a nitrogen-doped carbon dot fluorescence method according to any one of claims 1-5, characterized in that, Includes the following steps: S.1 Nitrogen-doped carbon dot solid powder is dispersed in ultrapure water to obtain N-CDs working solution; S.2 Add Tris-HCl buffer to the N-CDs working solution, vortex at room temperature, add indoxacarb standard solutions of different concentrations, incubate at room temperature, measure the fluorescence spectrum, and calculate the fluorescence intensity difference ΔF=F. 310 nm -F 390 nm A standard curve equation was constructed using ΔF and indoxacarb concentration. S.3 Pre-treat the water or agricultural product sample to be tested, and determine the fluorescence intensity according to the method in step S.2; calculate the actual concentration of indoxacarb based on the standard curve.

8. The method as described in claim 7, characterized in that, The Tris-HCl buffer solution described in step S.2 has a pH of 8-10, a vortexing time of 10-20 min, and an incubation time of 0.5-5 min.

9. The method as described in claim 7, characterized in that, The water sample pretreatment described in step S.3 specifically involves filtering the water sample and adding indoxacarb standard solution to prepare water samples with different spiking concentrations; the spiking levels of the water samples are 0.01, 0.1 and 1 mg / L.

10. The method as described in claim 7, characterized in that, The agricultural product pretreatment described in step S.3 specifically involves crushing the agricultural product, accurately weighing the sample, adding indoxacarb standard solution to the set spiking level, allowing it to stand, and then pretreating it using the QuEChERS method; the spiking levels of the agricultural product samples are 0.5 mg / L, 1 mg / L, and 5 mg / L.

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