A method for constructing a dual-mode detection of neonicotinoid pesticide residues based on waste tobacco-based biomass carbon dots

Abstract

The present invention relates to the technical field of environmental and food pollution detection, and particularly relates to a method for constructing a dual-mode detection of neonicotinoid pesticide residues based on waste tobacco-based biomass carbon dots. In the present invention, waste tobacco by-products (such as leaves and stems) are used as raw materials to synthesize waste tobacco-based biomass carbon dots (CDs), which have the activities of oxidase-like (OD) and peroxidase-like (POD). On this basis, taking imidacloprid (IMI) as a neonicotinoid pesticide model, a highly efficient, low-cost, green and environmentally friendly detection of neonicotinoid pesticide residues in medicated and edible homologous samples such as wolfberry, honeysuckle, and notoginseng is established based on the dual enzyme-like activities of OD-like and POD-like of waste tobacco-based biomass carbon dots, driven by two detection modes: ultraviolet spectral absorbance and fluorescence spectral fluorescence intensity. The present invention combines the advantages of colorimetry and fluorimetry, with a dual-ratio built-in reference, effectively avoiding the false positive phenomenon caused by external signal interference, while also enhancing the sensitivity and specificity of the detection and avoiding the occurrence of false negatives.

Description

Technical Field

[0001] The present invention belongs to the technical field of environmental and food pollution detection, and particularly relates to a method for constructing a dual-mode detection of neonicotinoid pesticide residues based on waste tobacco-based biomass carbon dots. Background Art

[0002] Neonicotinoid pesticides are a type of pesticide with high insecticidal activity and are the fourth generation of insecticides after organophosphorus, carbamate, and pyrethroid insecticides. As a broad-spectrum insecticide, neonicotinoid pesticides are widely used in agriculture. However, with the large-scale input and use of neonicotinoid pesticides, the resistance of organisms has increased, and some neonicotinoid pesticides show acute or sub-lethal toxicity to bees and other non-target organisms. In addition, neonicotinoid insecticides have good water solubility, permeability, and persistent activity, with a long half-life in the soil, posing a threat to the quality and safety of agricultural products. Therefore, in order to ensure biological health, many countries have successively established maximum residue limits for neonicotinoid pesticides. For example, the European Union has established maximum residue limits (10 - 200 μg / kg) for neonicotinoid pesticides and their metabolites in honey and pollen. Currently, methods for detecting neonicotinoid pesticides include high-performance liquid chromatography (HPLC), gas chromatography (GC), liquid chromatography-mass spectrometry (LC-MS), enzyme-linked immunosorbent assay, and electrochemistry. However, these analytical methods require expensive instruments, professional personnel for operation, and long time consumption. So far, the dual-mode detection of neonicotinoid pesticides reported mainly relies on antibody or nucleic acid aptamer-labeled noble metal nanomaterials, with deficiencies such as complex preparation processes, high costs, and poor reproducibility. Therefore, it is necessary to develop a rapid and efficient method to detect neonicotinoid pesticides in food and medicine homologous substances.

[0003] Tobacco is widely planted in the north and south of China as an economic crop, with the planting area ranking first in the world. It is worth noting that a large amount of waste tobacco by-products (such as leaves and stems) are generated during tobacco planting and processing, accounting for about 25% of the total tobacco output, causing serious resource waste and environmental pollution. It can be imagined that if waste tobacco by-products can be effectively utilized, their added value will be greatly improved. Considering this, a large number of studies have shown that the high-value utilization of waste plant by-products into multifunctional biomass-based carbon dots (CDs) is a promising method to solve resource waste, because biomass-based CDs usually have wide applications in biological / chemical sensing, food safety detection, environmental pollution analysis and degradation, disease diagnosis and treatment, etc. Therefore, using tobacco waste as raw material to develop multifunctional biomass CDs for rapid detection of food and medicine homologous substances may be a new way for the high-value utilization of tobacco waste. Summary of the Invention

[0004] Technical Solution

[0005] The present invention designs a kind of waste tobacco-based biomass CDs with dual enzyme-like activities. These CDs are economical and environmentally friendly, and can rapidly and efficiently detect the residues of neonicotinoid pesticides. The waste tobacco-based biomass CDs detect neonicotinoid pesticides by dual ratio and dual mode, and the dual ratio internal reference can increase the accuracy and sensitivity of detection. Therefore, using these biomass CDs nanoenzymes to detect neonicotinoid pesticides in medicated diet homologous substances is of great significance for protecting biological health.

[0006] The biomass carbon dots (CDs) in the present invention have the activity of OD enzyme-like and the activity of POD enzyme-like.

[0007] In the first aspect of the present invention, the preparation method of the biomass CDs of the present invention includes the following steps:

[0008] (1) Mix tobacco leaf powder with antioxidant and formamide, and dissolve by ultrasonic wave;

[0009] (2) Mix the dissolved sample with a vortex mixer, then transfer it to a polytetrafluoroethylene reaction kettle, react at high temperature in an oven, and naturally cool to room temperature;

[0010] (3) Filter the product of step (2) with a 0.22 μm organic filter membrane to remove large particles and precipitates, and then transfer the obtained dark brown filtrate to a dialysis bag for dialysis;

[0011] (4) Filter the dialysis-cleaned solution with a 0.22 μm water-based membrane, and freeze-dry to obtain CDs.

[0012] In one embodiment, the antioxidant in step (1) is glutathione; the ultrasonic time is 30.0 min;

[0013] In one embodiment, the high-temperature reaction in the oven in step (2) is to react at 180.0 °C in the oven for 8.0 h.

[0014] In one embodiment, the dialysis in step (3) is to use a 500 Da dialysis bag, dialyze for 7 days, and change the water every 24.0 h.

[0015] In one embodiment, the present invention discloses a preparation method of biomass CDs, which includes the following steps:

[0016] (1) Weigh 0.25 g of waste tobacco leaf powder and 0.30 g of glutathione respectively, add 10.0 mL of formamide and mix, and dissolve by ultrasonic wave for 30.0 min;

[0017] (2) Mix the sample dissolved in step (1) with a vortex mixer, then transfer it to a polytetrafluoroethylene reaction kettle, react at 180.0 °C in the oven for 8.0 h, and naturally cool to room temperature;

[0018] (3) Filter the product of step (2) by suction filtration through a 0.22 μm organic filter membrane to remove large particles and precipitates. Then transfer the obtained dark brown filtrate into a 500 Da dialysis bag and dialyze for 7 days, changing the water every 24.0 h;

[0019] (4) Filter the dialyzed solution by suction filtration through a 0.22 μm aqueous membrane and freeze-dry to obtain CDs.

[0020] In the second aspect of the present invention, the present invention provides a method for constructing a dual-mode detection of neonicotinoid pesticide residues based on waste tobacco-based biomass CDs, and the method includes the following steps:

[0021] (1) Absorbance detection of standard samples: Measure the absorbance ratios at 430 nm and 650 nm after adding IMI standard products with different concentration gradients in the OD-like system and the POD-like system respectively: For the OD-like system: Add different concentrations of IMI to ACP (acid phosphatase) and treat for 10.0 min, then add AAP (L-ascorbic acid-2-phosphate trisodium salt), CDs, OPD, and NaAC-HAC buffer and mix well. After the reaction ends, measure the absorbance ratio ODA 430nm / ODA 650nm ; For the POD-like system: Add different concentrations of IMI to ACP and treat for 10.0 min, then add AAP, CDs, H 2 O 2 , OPD, and NaAC-HAC buffer and mix well. After the reaction ends, measure the absorbance ratio PODA 430nm / PODA 650nm ;

[0022] (2) Fluorescence intensity detection of standard samples: Measure the fluorescence intensity signal ratios at 550 nm and 650 nm excited at 425 nm for different concentration gradients of IMI in the OD-like system and the POD-like system respectively: OD-like system: Add different concentrations of IMI to ACP and treat for 10.0 min, then add AAP, CDs, OPD, and NaAC-HAC buffer and mix well. After the reaction ends, measure the fluorescence intensity signal values at 550 nm and 650 nm with a fluorescence spectrophotometer at an excitation wavelength of 425 nm to obtain the fluorescence intensity signal ratio ODF 550nm / ODF 650nm ; POD-like system: Add different concentrations of IMI to ACP and treat for 10.0 min, then add AAP, CDs, H 2 O 2, Mix well OPD and NaAC-HAC buffer. After the reaction ends, measure the fluorescence intensity signal values at 550 nm and 650 nm using a fluorescence spectrophotometer at an excitation wavelength of 425 nm to obtain the fluorescence intensity signal ratio PODF 550nm / PODF 650nm ;

[0023] (3) Use the absorbance ratio measured with IMI standards of different concentration gradients and the concentration of the standards to construct a ratio ultraviolet linear regression equation: ODA 430nm / ODA 650nm = XC IMI + y, or PODA 430nm / PODA 650nm = XC IMI + y, where C IMI is the concentration of the standard;

[0024] Use the fluorescence intensity signal ratio measured with IMI standards of different concentration gradients and the concentration of the standards to construct a ratio fluorescence signal linear regression equation ODF 550nm / ODF 650nm = XC IMI + y, or PODF 550nm / PODF 650nm = XC IMI + y, where C IMI is the concentration of the standard;

[0025] (4) Take the sample to be tested, repeat steps (1) and (2) to obtain the absorbance ratio of the sample to be tested and the fluorescence intensity signal ratio of the sample to be tested, and substitute them into the corresponding linear equations respectively to obtain the concentration of IMI in the sample to be tested.

[0026] In one embodiment, in the above detection steps (1) and (2), in the ODA-like reaction system: the pH value of the NaAC-HAC buffer is 5.0; the concentration of OPD is 10.0 mmol / L; the concentration of CDs is 200.0 μg / mL; the concentration of ACP is 2.5 mmol / L; the concentration of AAP is 5.0 mmol / L; the total volume of the mixed solution is 200.0 μL; the reaction temperature is 40.0 °C; the reaction time is 20.0 min. In the POD-like system: the pH value of the NaAC-HAC buffer is 5.0; the concentration of OPD is 10.0 mmol / L; the concentration of CDs is 200.0 μg / mL; the concentration of ACP is 6.0 mmol / L; the concentration of AAP is 26.0 mmol / L; the concentration of H 2 O 2 is 10.0 mmol / L; the total volume of the mixed solution is 200.0 μL; the reaction temperature is 40.0 °C; the reaction time is 20.0 min.

[0027] In one embodiment, in the above step (3), the ultraviolet linear regression equation of the OD-like system is ODA 430nm / ODA 650nm = 0.0778C IMI + 5.08026; the ultraviolet linear regression equation of the POD-like system is PODA 430nm / PODA 650nm = 0.04751C IMI + 4.9511; the linear regression equation of the OD ratio fluorescence signal is ODF 550nm / ODF 650nm = 0.00605C IMI + 0.16165; the linear regression equation of the POD ratio fluorescence signal is PODF 550nm / PODF 650nm = 0.00561C IMI + 0.37037.

[0028] The waste tobacco-based biomass CDs prepared by the present invention have two absorption peaks at 420 nm and 650 nm. At the same time, there are oxidase-like and peroxidase-like activities that can oxidize colorless and non-absorbing o-phenylenediamine (OPD) into a dark yellow oxidation product 2,3-diaminophenazine (DAP) and generate an absorbance value A at 430 nm. However, ACP hydrolyzes AAP to generate L-ascorbic acid (AA), and AA has strong reducibility, significantly inhibiting the generation of DAP and causing the colorimetric signal at 430 nm to decrease. After adding IMI to the reaction system, since IMI inhibits the activity of ACP, the generation of AA is hindered, so that the color reaction is not inhibited and the colorimetric signal at 430 nm is restored.

[0029] The biomass carbon dots prepared by the present invention have a fluorescence signal at 650 nm under 425 nm excitation light. At the same time, there are oxidase-like and peroxidase activities that can oxidize colorless and non-fluorescent OPD into dark yellow DAP that emits yellow fluorescence at 550 nm. However, ACP hydrolyzes AAP to generate AA, and AA has strong reducibility, significantly inhibiting the generation of DAP. The fluorescence signal at 550 nm decreases, and the red fluorescence of the CDs at 650 nm is quenched through the inner filter effect. After adding IMI to the reaction system, due to the inhibitory effect of IMI on the activity of ACP enzyme, the generation of AA is hindered, so that the color reaction is not inhibited and the fluorescence signal at 550 nm is restored, thereby constructing a ratio fluorescence signal.

[0030] Beneficial effects

[0031] (1) The fluorescence analysis technique for detecting neonicotinoid pesticides based on CDs provided by the present invention mainly designs a method for detecting neonicotinoid pesticides by regulating the color reaction of OPD. AA generated by ACP catalyzing AAP has strong reducibility and can effectively hinder the color reaction. However, neonicotinoid pesticides can inhibit the activity of ACP, hinder the generation of AA, protect this color reaction from inhibition, and thus realize the detection of neonicotinoid pesticides. In addition, the invention uses dual-ratio detection with an internal reference signal, which can avoid interference from the external environment and increase the accuracy and sensitivity of detection.

[0032] (2) The present invention synthesizes CDs using waste tobacco leaves, which meets the requirements of the development of green economy, and combines them with the colorimetric substrate o-phenylenediamine (OPD) to develop a method for dual-mode detection of neonicotinoid pesticides using waste tobacco-based biomass CDs. This method is low-cost, simple to operate, and time-consuming, showing great potential for quantitative detection of neonicotinoid pesticides. Description of the Drawings

[0033] Figure 1 Schematic design for constructing a dual-mode detection of neonicotinoid pesticide residues based on waste tobacco-based biomass CDs.

[0034] Figure 2 Characterization of CDs. (A) TEM image of CDs and (B-F) XPS images (mainly composed of C, O, N, S elements).

[0035] Figure 3 Verification of the peroxidase-like and catalase-like activities of CDs.

[0036] Figure 4 and Figure 5 Double-reciprocal plot of TMB concentration vs. reaction rate for the catalytic kinetics test of CDs.

[0037] Figure 6 Feasibility of detecting imidacloprid (IMI) in the CDs + ACP + AAP + OPD + IMI (5A, 5B) and CDs + ACP + AAP + OPD + H 2 O 2 + IMI (5C, 5D) systems.

[0038] Figure 7 Optimization of the optimal conditions for sensing system detection based on the CDs + ACP + AAP + OPD + IMI system.

[0039] Figure 8 Optimization of the optimal conditions for sensing system detection based on the CDs + ACP + AAP + OPD + H 2 O 2 + IMI system.

[0040] Figure 9 For the OD-like system, detect the color and fluorescence changes of 0.0 - 60.0 μmol / L IMI and the standard curves of ODA 430nm / ODA 650nm and ODF 550nm / ODF 650nm

[0041] Figure 10 For the POD-like system, detect the color and fluorescence changes of 0.0 - 80.0 μmol / L IMI and the standard curves of PODA 430nm / PODA 650nm and PODF 550nm / PODF 650nm

[0042] Figure 11 For the selectivity and anti-interference evaluation of the CDs + ACP + AAP + OPD + IMI system. 1 - 15 in 10A and 10B are respectively IMI; dinotefuran; acetamiprid; blank; carbaryl; cypermethrin; Ca 2+ ; Mg 2+ ; PO 4 2- ; NH 4 + ; Na + ; CO 3 2- ; sucrose; maltose; glucose.

[0043] Figure 12 For the selectivity and anti-interference evaluation of the CDs + ACP + AAP + OPD + H 2 O 2 + IMI system. 1 - 15 in 11A and 11B are respectively IMI; dinotefuran; acetamiprid; blank; carbaryl; cypermethrin; Ca 2+ ; Mg 2+ ; PO 4 2- ; NH 4 + ; Na + ; CO 3 2- ; sucrose; maltose. Specific embodiments

[0044] The present invention will be further described below in conjunction with specific embodiments.

[0045] Reagents and materials:

[0046] ​​Acid phosphatase (ACP) was purchased from Shanghai Macklin Biochemical Co., Ltd.; L-ascorbic acid-2-phosphate trisodium salt (AAP), o-phenylenediamine (OPD), imidacloprid (IMI) were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; NaAC-HAC buffer was purchased from Shanghai Yuanye Bio-Technology Co., Ltd., H 2 O 2 was purchased from Sinopharm Chemical Reagent Co., Ltd. All water used in the experimental process was ultrapure water.

[0047] Example 1: Synthesis of waste tobacco-based biomass carbon dots (CDs)

[0048] (1) Weigh 0.25 g of waste tobacco leaf powder and 0.30 g of glutathione respectively, add them to 10.0 mL of formamide and mix, ultrasonically dissolve for 30.0 min, mix the sample with a vortex mixer, then transfer it to a polytetrafluoroethylene reaction kettle, react at 180.0 °C in an oven for 8.0 h, and naturally cool to room temperature; filter it with a 0.22 μm organic filter membrane to remove large particles and precipitates. Then transfer the obtained dark brown filtrate to a 500 Da dialysis bag, dialyze for 7 days, change the water every 24.0 h, filter the dialyzed solution with a 0.22 μm water-based membrane, and freeze-dry to obtain CDs, which are stored in a desiccator for subsequent tests.

[0049] Example 2: Verification of oxidase and peroxidase activities of CDs

[0050] Take the following five groups of reactions: (1) CDs (2) TMB (3) H 2 O 2 (4) H 2 O 2 + TMB (5) CDs + TMB (6) CDs + TMB + H 2 O 2 . After full reaction, transfer it to a cuvette, measure the absorbance value at 650 nm with a UV-visible spectrophotometer, and compare the catalytic ability of the nanozyme. As shown in A and B in Figure 3 , only the absorbance values of CDs + TMB and CD + TMB + H 2 O 2 are larger, indicating that the synthesized CDs have good oxidase and peroxidase activities.

[0051] In the above verification steps, the concentration of CDs added is 200.0 μg / mL; the concentration of TMB is 10.0 mmol / L; the pH of the added NaAC-HAC buffer is 4.5; the concentration of H 2 O 2 is 10.0 mmol / L; the total volume of the mixed solution is 200.0 μL; the reaction temperature is 40.0 °C; the mixing and full reaction time is 20.0 min.

[0052] Example 3: Catalytic Kinetics Test of CDs

[0053] OD-like system: In the NaAC-HAC buffer solution containing CDs, by changing the concentration of TMB (0.0–0.7 mmol / L), a steady-state kinetics experiment was carried out ( Figure 4 in A). The double-reciprocal plot of TMB concentration vs. reaction rate was calculated by the following Michaelis-Menten equation:

[0054]

[0055] where V represents the initial reaction rate, [S] is the substrate concentration, K m is the Michaelis constant, and V max represents the maximum reaction rate. The results are shown in Figure 4 B. The K m value of CDs for TMB is 0.537, indicating the good catalytic activity of the CDs prepared by the present invention.

[0056] In the above verification steps, the concentration of CDs added was 200.0 μg / mL; the pH of the added NaAC-HAC buffer solution was 4.5; the total volume of the mixed solution was 200.0 μL; the reaction temperature was 40.0 °C; the mixing was uniform and the sufficient reaction time was 20.0 min.

[0057] POD-like system: In the NaAC-HAC buffer solution containing CDs, by changing the concentration of H 2 O 2 (0.0–0.8 mmol / L)( Figure 5 in A) and the concentration of TMB (0.0–0.8 mmol / L)( Figure 5 in C), a steady-state kinetics experiment was carried out. The double-reciprocal plots of TMB concentration and H 2 O 2 vs. reaction rate were calculated by the following Michaelis-Menten equation:

[0058]

[0059] where V represents the initial reaction rate, [S] is the substrate concentration, K m is the Michaelis constant, and V max represents the maximum reaction rate. The results are shown in Figure 5 B. The K 2 O 2 value of CDs for H m is 2.38, and as shown in Figure 5 D, the K mThe value is 0.378, indicating the good catalytic activity of the CDs prepared by the present invention.

[0060] Example 4: Feasibility verification of detecting neonicotinoid pesticides in a dual-enzyme activity waste tobacco-based biomass CDs dual-mode system. Verification of the feasibility of the ODA system: CDs + ACP + AAP + OPD system for detecting IMI

[0061] Using OPD as the chromogenic agent and NaAC-HAC as the buffer. Take the following three groups of reactions: (1) CDs + OPD; (2) CDs + OPD + ACP + AAP; (3) CDs + OPD + ACP + AAP + IMI. After full reaction, the results are shown in Figure 6 Figure A, and measure the absorbance ratio ODA at 430 nm and 650 nm with a UV spectrophotometer 430nm / ODA 650nm and measure the fluorescence intensity signal ratio ODF at 550 nm and 650 nm with a fluorescence spectrometer 550nm / ODF 650nm and compare their magnitudes. The results are shown in Figure 6 Figure B, indicating that ACP + AAP can inhibit the absorbance intensity of CDs + OPD, and the absorbance of CDs + OPD + ACP + AAP is restored after adding IMI. ACP + AAP can inhibit the fluorescence intensity of CDs + OPD, making the CDs + OPD + ACP + AAP system emit red fluorescence at 650 nm. After adding IMI, IMI will hinder the formation of AA by ACP + AAP, and activate the oxidase activity of CDs through competitive effects, oxidizing OPD to DAP to restore the dual-color ratio fluorescence. Both the absorbance and fluorescence intensity change significantly, demonstrating the feasibility of the detection system proposed by the present invention.

[0062] In the above verification steps, the concentration of CDs added is 200.0 μg / mL; the concentration of OPD is 10.0 mmol / L; the concentration of IMI is 60.0 μmol / L; the concentration of ACP is 2.5 mmol / L; the concentration of AAP is 5.0 mmol / L; the pH of the added NaAC-HAC buffer is 5.0; the total volume of the mixed solution is 200.0 μL; the reaction temperature is 40.0 °C; the mixing and full reaction time is 20.0 min.

[0063] POD-like system: CDs + ACP + AAP + OPD + H 2 O 2 System for feasibility verification of detecting IMI

[0064] Using OPD as the chromogenic agent and NaAC-HAC as the buffer. Take the following three groups of reactions: (1) CDs + OPD + H 2 O 2(2) CDs + OPD + H 2 O 2 + ACP + AAP (3) CDs + OPD + H 2 O 2 + ACP + AAP + IMI. After complete reaction, the results are shown in Figure 6 C. Measure the absorbance ratio PODA at 430 nm and 650 nm using a UV - visible spectrophotometer 430nm / PODA 650nm and measure the fluorescence intensity signal ratio PODF at 550 nm and 650 nm using a fluorescence spectrometer 550nm / PODF 650nm and compare their magnitudes. It shows that ACP + AAP can inhibit the absorbance intensity of CDs + OPD + H 2 O 2 and the absorbance of CDs + OPD + H 2 O 2 + ACP + AAP is restored after adding IMI. The results are shown in Figure 6 D. ACP + AAP can inhibit the fluorescence intensity of CDs + OPD, making the CDs + OPD + ACP + AAP system emit only red fluorescence at 650 nm. After adding IMI, IMI will hinder the formation of AA by ACP + AAP and activate the peroxidase activity of CDs through competitive effect, oxidizing OPD to DAP to restore the dual - color ratio fluorescence. Both the absorbance and fluorescence intensity change significantly, demonstrating the feasibility of the detection system proposed in this invention.

[0065] In the above verification steps, the concentration of CDs added is 200.0 μg / mL; the concentration of OPD is 10.0 mmol / L; the concentration of IMI is 80.0 μmol / L; the concentration of ACP is 6.0 mmol / L; the concentration of AAP is 26.0 mmol / L; the pH of the added NaAC - HAC buffer is 5.0; the total volume of the mixed solution is 200.0 μL; the reaction temperature is 40.0 °C; the mixing and complete reaction time is 20.0 min.

[0066] Example 5: Optimization of the Optimal Conditions for Detecting Neonicotinoid Pesticides by a Dual - Enzyme - Activity Waste - Tobacco - Based Biomass CDs Dual - Mode System

[0067] OD - like system: Optimization of the Optimal Conditions for Detecting IMI by the CDs + ACP + AAP + OPD System

[0068] To optimize the analytical performance of the CDs+ACP+AAP+OPD system, the experimental parameters of pH value, temperature, reaction time, concentration of ACP, and concentration of AAP in the detection system were studied in detail. First, the performance of the system in the pH range of 3.5–6.0 was explored, and NaAC-HAC was selected as the reaction buffer. The results are as Figure 7 shown in A. When the pH value was in the range of 3.5 to 5.0, the absorbance value of the reaction system at 430 nm increased with the increase of the pH value of the buffer system. When the pH value exceeded 5.0, the absorbance value decreased with its increase. Therefore, 5.0 was selected as the optimal pH value of the system. Next, the performance of the system in the temperature range of 25.0–50.0 °C was explored. The results are as Figure 7 shown in B. When the temperature value was in the range of 25.0 °C to 40.0 °C, the absorbance value of the reaction system at 430 nm increased with the increase of the reaction temperature. When the temperature exceeded 40.0 °C and increased to 50.0 °C, the absorbance value decreased with its increase. Therefore, 40.0 °C was selected as the optimal temperature of the system. The reaction time was optimized, and the results are as Figure 7 shown in C. Within 0.0 to 20.0 min, the absorbance value of the reaction system at 430 nm increased with the increase of the reaction time and basically no longer changed after 20.0 min. Therefore, 20.0 min was selected as the optimal reaction time of the system. As Figure 7 shown in D&7E, the concentration of ACP was optimized. When ACP was in the range of 0.0 to 2.5 mmol / L, the absorbance decreased with its increase and basically no longer changed after 2.5 mmol / L. Therefore, 2.5 mmol / L was selected for subsequent experiments. Similarly, the optimal concentration of AAP was 5.0 mmol / L.

[0069] POD-like system: CDs+ACP+AAP+OPD+H 2 O 2 Optimization of the optimal conditions for detecting IMI in the system

[0070] To optimize the analytical performance of the CDs+ACP+AAP+OPD+H 2 O 2 system, the experimental parameters of pH value, temperature, reaction time, concentration of ACP, and concentration of AAP in the detection system were studied in detail. First, the performance of the system in the pH range of 3.5–6.0 was explored, and NaAC-HAC was selected as the reaction buffer. The results are as Figure 8As shown in A, when the pH value is in the range of 3.5 to 5.0, the absorbance value of the reaction system at 430 nm increases with the increase of the pH value of the buffer system. When the pH value exceeds 5.0, the absorbance value decreases with its increase. Therefore, 5.0 is selected as the optimal pH value of the system. Next, the performance of the system in the temperature range of 25.0–50.0 °C was explored. The results are as Figure 8 shown in B. When the temperature value is in the range of 25.0 °C to 40.0 °C, the absorbance value of the reaction system at 430 nm increases with the increase of the reaction temperature. When the temperature exceeds 40.0 °C and increases to 50.0 °C, the absorbance value decreases with its increase. Therefore, 40.0 °C is selected as the optimal temperature of the system. The reaction time was optimized, and the results are as Figure 8 shown in C. Within 0.0 to 20.0 min, the absorbance value of the reaction system at 430 nm increases with the increase of the reaction time and basically does not change after 20.0 min. Therefore, 20.0 min is selected as the optimal reaction time of the system. Then the concentration of ACP was optimized. As Figure 8 shown in (D, E), when ACP is in the range of 0.0 to 6.0 mmol / L, the absorbance decreases with its increase and basically does not change after 6.0 mmol / L. Therefore, 6.0 mmol / L is selected for subsequent experiments. Similarly, the optimal concentration of AAP is 26.0 mmol / L.

[0071] Example 6: Detection of neonicotinoid pesticide standards by a dual-class enzyme activity waste tobacco-based biomass CDs dual-mode system

[0072] OD-like system: Detection of IMI standards by the CDs + ACP + AAP + OPD system

[0073] Solutions of different concentrations of IMI (including 0.0, 10.0, 20.0, 30.0, 40.0, 50.0 and 60.0 μmol / L) were fully reacted with 200.0 μg / mL of CDs and 10.0 mmol / L of OPD solution in NaAC-HAC buffer (pH = 5.0) at 40.0 °C for 20.0 min.

[0074] After the reaction, the absorbance value at 430 nm was measured with a UV spectrophotometer. As the concentration of IMI increases, the absorbance value at 430 nm increases, and the absorbance ratio ODA 430 nm / ODA 650nm increases. Therefore, with the absorbance ratio ODA 430nm / ODA 650nm as the ordinate and the concentration of IMI as the abscissa, linear equation fitting was performed using Origin software. As Figure 9As shown in (A, B), it is obtained that the ratio of the concentration of IMI to the absorbance is linear in the range of 0.0 - 60.0 μmol / L, and the regression equation of the absorbance ratio is ODA 430nm / ODA 650nm = 0.0778C IMI + 5.08026, the correlation coefficient is 0.99188, and the detection limit is 0.66 μmol / L.

[0075] Under the same reaction conditions, the fluorescence intensity signals at 550 nm and 650 nm were measured with a fluorescence spectrophotometer under an excitation light of 425 nm. As the concentration of IMI increased, the ratio of the fluorescence intensities at 550 nm and 650 nm increased. Therefore, with the ratio of fluorescence intensities ODF 550nm / ODF 650nm as the ordinate and the concentration of IMI as the abscissa, linear equation fitting was performed using Origin software. As Figure 9 shown in (C, D), it is obtained that the concentration of IMI is linear with the fluorescence intensity ODF 550nm / ODF 650nm in the range of 0.0–60.0 μmol / L, and the regression equation of the fluorescence intensity ratio ODF 550nm / ODF 650nm is F 550nm / F 650nm = 0.00605C IMI + 0.16165, the correlation coefficient is 0.9975, and the detection limit is 0.024 μmol / L.

[0076] POD-like system: CDs + ACP + AAP + OPD + H 2 O 2 system for detecting IMI standard

[0077] Solutions of IMI at different concentrations (including 0.0, 10.0, 20.0, 30.0, 40.0, 50.0, 60.0, 70.0, and 80.0 μmol / L) were reacted with 200.0 μg / mL of CDs and 10.0 mmol / L of OPD solution in NaAC-HAC buffer (pH = 5.0) at 40.0 °C for 20.0 min.

[0078] After the reaction, the absorbance value at 430 nm was measured with a UV spectrophotometer. As the concentration of IMI increased, the absorbance value at 430 nm increased, and the ratio of absorbance PODA 430nm / PODA 650nm increased. Therefore, with the ratio of absorbance PODA 430nm / PODA 650nm as the ordinate and the concentration of IMI as the abscissa, linear equation fitting was performed using Origin software. AsFigure 10 As shown in (A, B), it is obtained that the ratio of the concentration of IMI to the absorbance is linear in the range of 0.0 - 80.0 μmol / L, and the regression equation of the absorbance ratio is PODA 430nm / PODA 650nm = 0.04751C IMI + 4.9511, the correlation coefficient is 0.99807, and the detection limit is 0.46 μmol / L.

[0079] Under the same reaction conditions, the fluorescence intensity signals at 550 nm and 650 nm were measured with a fluorescence spectrophotometer under 425 nm excitation light. As the concentration of IMI increased, the ratio of the fluorescence intensity at 550 nm to that at 650 nm increased. Therefore, with the fluorescence intensity ratio PODF 550nm / PODF 650nm as the ordinate and the concentration of IMI as the abscissa, the Origin software was used for linear equation fitting. As Figure 10 shown in (C, D), it is obtained that the concentration of IMI and the fluorescence intensity PODF 550nm / PODF 650nm are linear in the range of 0.0–80.0 μmol / L, and the regression equation of the fluorescence intensity ratio PODF 550nm / PODF 650nm is PODF 550nm / PODF 650nm = 0.00561C IMI + 0.37037, the correlation coefficient is 0.99175, and the detection limit is 0.016 μmol / L.

[0080] Example 7: Selective OD-like system for detecting neonicotinoid pesticides by a dual-class enzyme activity waste tobacco-based biomass CDs dual-mode detection system: Selectivity of CDs + ACP + AAP + OPD system for IMI

[0081] To evaluate the selectivity of the CDs + ACP + AAP + OPD system for IMI, some representative substances were used as proof-of-concept, including blank; carbaryl; cypermethrin; Ca 2+ ; Mg 2+ ; PO 4 2- ; NH 4 + ; Na + ; CO 3 2-; Sucrose; Maltose; Glucose; Other neonicotinoid pesticides (such as: Dinotefuran and Acetamiprid). At room temperature, react CDs, OPD, ACP, and AAP with the above substances respectively in NaAC-HAC buffer solution; after mixing evenly, react for 20.0 min and transfer to a cuvette. Measure the absorbance ratio ODA 430nm / ODA 650nm , and the fluorescence intensity signal ratio ODF at 550 nm and 650 nm under excitation at a wavelength of 425 nm 550nm / ODF 650nm . The results are as shown in Figure 11 (A, B), only the absorbance ratios A of Dinotefuran, Acetamiprid, and IMI increase significantly, and the fluorescence intensity signal ratio ODF 550nm / ODF 650nm increases. It indicates that the detection system has good selectivity for IMI.

[0082] In the above verification steps, the concentration of CDs added is 200.0 μg / mL; the concentration of OPD is 10.0 mmol / L; the concentration of ACP is 2.5 mmol / L; the concentration of AAP is 5.0 mmol / L; the concentration of each selective substance is also 60.0 μmol / L; the pH of the added NaAC-HAC buffer solution is 5.0; the total volume of the mixed solution is 200.0 μL; the reaction temperature is 40.0 °C; the mixing and full reaction time is 20.0 min.

[0083] POD-like system: CDs + ACP + AAP + OPD + H 2 O 2 Selectivity of the system for detecting IMI

[0084] To evaluate the selectivity of the CDs + ACP + AAP + OPD + H 2 O 2 system for IMI, some representative substances are used for proof-of-concept, including blank; Carbaryl; Cypermethrin; Ca 2+ ; Mg 2+ ; PO 4 2- ; NH 4 + ; Na + ; CO 3 2- ; Sucrose; Maltose; Glucose; Other neonicotinoid pesticides (such as: Dinotefuran and Acetamiprid). At room temperature, react CDs, OPD, ACP, AAP, and H 2 O 2 with the above substances respectively in NaAC-HAC buffer solution; after mixing evenly, react for 20.0 min and transfer to a cuvette. Measure the absorbance ratio PODA430nm / PODA 650nm , and the fluorescence intensity signal ratio PODF at 550 nm and 650 nm under excitation at a wavelength of 425 nm 550nm / PODF 650nm . The results are as shown in Figure 12 (A, B) below. Only the absorbance ratio A of dinotefuran, acetamiprid, and IMI increased significantly, and the fluorescence intensity signal ratio PODF 550nm / PODF 650nm increased. This indicates that the detection system has good selectivity for neonicotinoid pesticides.

[0085] In the above verification steps, the concentration of CDs added was 200.0 μg / mL; the concentration of OPD was 10.0 mmol / L; the concentration of ACP was 6.0 mmol / L; the concentration of AAP was 26.0 mmol / L; the concentration of each selective substance was also 80.0 μmol / L; the pH of the added NaAC-HAC buffer solution was 5.0; the total volume of the mixed solution was 200.0 μL; the reaction temperature was 40.0 °C; the reaction time after thorough mixing was 20.0 min.

[0086] Example 8: Detection of neonicotinoid pesticides in actual samples by a dual-enzyme activity waste tobacco-based biomass CDs dual-mode detection system

[0087] OD-like system: To verify the sensing performance of the dual-enzyme activity waste tobacco-based biomass carbon dots dual-detection system for the detection of IMI in actual samples, goji berries, honeysuckle, and notoginseng were selected as representatives of the actual samples. Among them, the goji berries, honeysuckle, and notoginseng in this work were randomly purchased from a supermarket in Hefei, China. Different concentrations of IMI were dropped on the surface of the actual samples to be tested, air-dried, soaked in deionized water for one hour, and the soaking solution was taken for detection. At room temperature, CDs, OPD, ACP, and AAP were reacted with the above mixed solution in NaAC-HAC buffer (pH = 5.0); after thorough mixing, the reaction was carried out for 20.0 min and then transferred to a cuvette. The absorbance ratio and fluorescence intensity signal ratio were measured respectively. The obtained absorbance ratio and fluorescence intensity signal ratio were substituted into the corresponding linear equations, and the IMI results measured in the OD-like detection mode are shown in Table 1.

[0088] POD-like system: To verify the sensing performance of the dual-enzyme activity waste tobacco-based biomass carbon dots dual-mode ratio detection system for the detection of IMI in actual samples, goji berries, honeysuckle, and notoginseng were selected as representatives of the actual samples. Among them, the goji berries, honeysuckle, and notoginseng in this work were randomly purchased from a supermarket in Hefei, China. Different concentrations of IMI were dropped on the surface of the actual samples to be tested, air-dried, soaked in deionized water for one hour, and the soaking solution was taken for detection. At room temperature, CDs, OPD, H 2 O2 , ACP and AAP react with the above mixed solution in NaAC-HAC buffer (pH = 5.0) respectively; after mixing evenly, the reaction is carried out for 20.0 min and then transferred to a cuvette. The absorbance ratio and the fluorescence intensity signal ratio are measured respectively. The obtained absorbance ratio and fluorescence intensity signal ratio are substituted into the corresponding linear equation, and the IMI results measured in the POD-like detection mode are shown in Table 1.

[0089] Table 1 shows the detection of IMI in actual samples by the CDs dual enzyme activity dual-mode system. The actual samples are wolfberry, honeysuckle, and notoginseng respectively.

[0090]

[0091] It can be seen from the results that this reaction system can be applied to the detection of actual samples, and the recovery rate is between 97.25% and 104.90%, and the relative deviation is less than 3.

[0092] In summary, the present invention develops a new colorimetric-fluorescent dual-mode label-free detection method for neonicotinoid pesticide residues based on the oxidase (OD) and peroxidase (POD) dual enzyme activities of waste tobacco-based biomass CDs. This method has the characteristics of low cost and environmental friendliness. In particular, a colorimetric and fluorescence dual ratio detection mechanism is proposed, which can avoid the interference of the external environment through an internal reference, and increases the accuracy and sensitivity of detection under complex sample matrices.

[0093] The above content further elaborates on the present invention in combination with specific embodiments. It cannot be determined that the specific implementation of the present invention is only limited to these descriptions. For those of ordinary skill in the technical field to which the present invention belongs, without departing from the concept of the present invention, several simple deductions or substitutions can still be made, which should all be regarded as belonging to the protection scope determined by the claims submitted for the present invention.

Claims

1. A method for constructing a dual-mode detection method for neonicotinoid pesticide residues based on waste tobacco-based biomass carbon dots CDs, the method comprising the following steps: (1) Absorbance detection of standard samples: In the oxidase OD detection system and the peroxidase POD detection system, the absorbance ratio at 430 nm and 650 nm was measured after adding different concentration gradients of IMI standard. For the OD system: add different concentrations of IMI to acid phosphatase ACP and treat for 10.0 min, then add L-ascorbic acid-2-phosphate trisodium salt AAP, CDs, OPD and NaAC-HAC buffer and mix well. After the reaction, use a UV spectrophotometer to measure the absorbance ratio of 430 nm and 650 nm (ODA). 430nm / ODA 650nm ; For the POD-like system: add different concentrations of IMI to ACP for 10.0 min, then add AAP, CDs, H2O2, OPD and NaAC-HAC buffer and mix well. After the reaction, use a UV spectrophotometer to measure the absorbance ratio of 430 nm and 650 nm (PODA) 430nm / PODA 650nm ; (2) Fluorescence intensity detection of standard samples: The fluorescence intensity signal ratio of IMI at different concentration gradients at 550 nm and 650 nm under 425 nm excitation wavelength was measured in OD-like system and POD-like system respectively; OD-like system: IMI at different concentrations was added to ACP for 10.0 min, and then AAP, CDs, OPD and NaAC-HAC buffer were added and mixed. After the reaction, the fluorescence intensity signal values ​​at 550 nm and 650 nm were measured using a fluorescence spectrophotometer under 425 nm excitation wavelength to obtain the fluorescence intensity signal ratio (ODF). 550nm / ODF 650nm ; POD-like system: add different concentrations of IMI to ACP for 10.0 min, then add AAP, CDs, H2O2, OPD and NaAC-HAC buffer and mix well. After the reaction, measure the fluorescence intensity signal value at 550 nm and 650 nm with a fluorescence spectrophotometer at an excitation wavelength of 425 nm to obtain the fluorescence intensity signal ratio PODF 550nm / PODF 650nm ; (3) Using the absorbance ratios of IMI standards at different concentration gradients and the concentration of the standards, a ratio UV regression equation was constructed: ODA 430nm / ODA 650nm =XC IMI + y, or PODA 430nm / PODA 650nm =XC IMI + y , where C IMI is the concentration of the standard; The ratio fluorescence signal linear regression equation (ODF) was constructed using the fluorescence intensity signal ratios measured by IMI standards with different concentration gradients and the concentration of the standards. 550nm / ODF 650nm =XC IMI +y, or PODF 550nm / PODF 650nm =XC IMI +y, where C IMI is the concentration of the standard; Take the sample to be tested, repeat steps (1) and (2) to obtain the absorbance ratio of the sample to be tested and the fluorescence intensity signal ratio of the sample to be tested, substitute them into the corresponding linear equations, and obtain the concentration of IMI in the sample to be tested.

2. The method according to claim 1, characterized in that In the OD-like system: the pH value of the NaAC-HAC buffer is 5.0, the concentration of OPD is 10.0 mmol / L, the concentration of CDs is 200.0 μg / mL, the concentration of ACP is 2.5 mmol / L, the concentration of AAP is 5.0 mmol / L, the total amount of the mixed solution is 200.0 μL, the reaction temperature is 40.0°C, and the total reaction time is 20.0 min; in the POD-like system: the pH value of the NaAC-HAC buffer is 5.0, the concentration of OPD is 10.0 mmol / L, the concentration of CDs is 200.0 μg / mL, the concentration of ACP is 6.0 mmol / L, the concentration of AAP is 26.0 mmol / L, the concentration of H2O2 is 10.0 mmol / L, the total amount of the mixed solution is 200.0 μL, the reaction temperature is 40.0°C, and the total reaction time is 20.0 min.

3. The method according to claim 2, characterized in that in, The regression equation of ultraviolet resistance of OD-like system is ODA 430nm / ODA 650nm = 0.0778C IMI +5.08026; The regression equation for ultraviolet resistance of POD-like system is PODA 430nm / PODA 650nm = 0.04751C IMI +4.9511; the linear regression equation of the OD ratio fluorescence signal is ODF 550nm / ODF 650nm = 0.00605C IMI +0.16165; the linear regression equation of POD-like ratio fluorescence signal is PODF 550nm / PODF 650nm = 0.00561C IMI +0.37037.

4. The method according to claim 1, characterized in that: The waste tobacco-based biomass carbon dots CDs are prepared by the following method: Weigh 0.25 g of waste tobacco powder and 0.30 g of glutathione, add 10.0 mL of formamide, mix, and dissolve by ultrasonic for 30.0 min; The sample dissolved in step (1) was mixed with a vortexer, then transferred to a tetrafluoroethylene reactor, reacted in an oven at 180.0°C for 8.0 h, and naturally cooled to room temperature; The product of step (2) was filtered through a 0.22 mm organic filter membrane to remove large particles and precipitates, and then the obtained dark brown filtrate was transferred to a 500 Da dialysis bag and dialyzed for 7 days, with the water changed every 24.0 h; (4) The dialyzed solution was filtered through a 0.22 mm aqueous membrane and freeze-dried to obtain CDs.

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