Method for detecting neonicotinoid pesticide residues by constructing dual modes based on waste tobacco-based biomass carbon dots

Abstract

The invention belongs to the technical field of environment and food pollution detection, and particularly relates to a method for detecting neonicotinoid pesticide residues by constructing a dual mode based on waste tobacco-based biomass carbon dots. According to the method, waste tobacco byproducts (such as leaves and stems) are used as raw materials to synthesize the waste tobacco-based biomass carbon dots (CDs), and the carbon dots have oxidase-like (OD) and peroxidase-like (POD) activities. On the basis, imidacloprid (IMI) is used as a neonicotinoid pesticide model; two detection modes of ultraviolet spectrum absorbance and fluorescence spectrum fluorescence intensity driven by OD-like and POD-like enzyme activity based on the waste tobacco-based biomass carbon dots are established for efficient, low-cost and environment-friendly detection of neonicotinoid pesticide residues in medicinal and edible samples such as Chinese wolfberry, honeysuckle and pseudo-ginseng. The advantages of a colorimetric method and a fluorescence method are integrated, double-ratio built-in reference is adopted, the false positive phenomenon caused by external signal interference is effectively avoided, meanwhile, the detection sensitivity and specificity are enhanced, and false negative is avoided.

Description

Technical Field

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

[0002] Neonicotinoid pesticides are a class of pesticides with high insecticidal activity. They 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 investment and use of neonicotinoid pesticides, the resistance of organisms has increased, and some neonicotinoid pesticides have shown acute or sublethal toxicity to bees and other non-target organisms. In addition, neonicotinoid pesticides have good water solubility, permeability and long-lasting activity, and a long half-life in the soil, posing a threat to the quality and safety of agricultural products. Therefore, in order to protect the health of organisms, many countries have successively formulated maximum residue limits for neonicotinoid pesticides. For example, the European Union has established maximum residue limits (10-200μg / kg) for neonicotinoid pesticides and their metabolites in honey and pollen. At present, the methods used for the detection of neonicotinoid pesticides include high performance liquid chromatography (HPLC), gas chromatography (GC), liquid chromatography-mass spectrometry (LC-MS), enzyme-linked immunosorbent assay and electrochemical method. However, these analytical methods are expensive, require professional operation, and are time-consuming. The dual-mode detection of neonicotinoid pesticides reported so far mainly relies on antibody or nucleic acid aptamer labeling precious metal nano-strategies, which have the disadvantages of complex preparation process, high cost, and poor reproducibility. Therefore, it is necessary to develop a fast and efficient method to detect neonicotinoid pesticides in food and medicine congeners.

[0003] As an economic crop, tobacco is widely planted in the north and south of China, and its planting area ranks first in the world. It is worth noting that a large amount of waste tobacco byproducts (such as leaves and stems) are generated during tobacco planting and processing, accounting for about 25% of the total tobacco production, causing serious resource waste and environmental pollution. It is conceivable that if waste tobacco byproducts can be effectively utilized, their added value will be greatly improved. With this in mind, a large number of studies have demonstrated that the high-value utilization of waste plant byproducts into multifunctional biomass-based carbon dots (CDs) is a promising way to solve resource waste, because biomass-based CDs are generally widely used in bio / chemical sensing, food safety testing, environmental pollution analysis and degradation, disease diagnosis and treatment, etc. Therefore, using tobacco waste as raw materials to develop multifunctional biomass CDs for rapid detection of medicinal and edible homologs may be a new way to high-value utilization of tobacco waste. Summary of the invention

[0004] Technical Solution

[0005] The present invention designs a waste tobacco-based biomass CDs with dual-type enzyme activity. The CDs are economical and environmentally friendly, and can quickly and efficiently detect neonicotinoid pesticide residues. The waste tobacco-based biomass CDs dual-ratio dual-mode detect neonicotinoid pesticides, and the dual-ratio built-in reference can increase the accuracy and sensitivity of the detection. Therefore, the use of the biomass CDs nanozyme to detect neonicotinoid pesticides in medicinal and edible homologues is of great significance for protecting biological health.

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

[0007] In a first aspect of the present invention, the method for preparing biomass CDs of the present invention comprises the following steps:

[0008] (1) mixing tobacco leaf powder, antioxidant and formamide, and dissolving by ultrasonication;

[0009] (2) The dissolved sample is mixed with a vortexer, then transferred to a tetrafluoroethylene reactor, reacted at high temperature in an oven, and naturally cooled to room temperature;

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

[0011] (4) The dialyzed solution was filtered using a 0.22 μm water membrane and freeze-dried 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 carried out at 180.0° C. for 8.0 h in the oven.

[0014] In one embodiment, the dialysis in step (3) is performed using a 500Da dialysis bag for 7 days, with water being replaced every 24.0 hours.

[0015] In one embodiment, the present invention discloses a method for preparing biomass CDs comprising the following steps:

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

[0017] (2) 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;

[0018] (3) The product of step (2) was filtered with a 0.22 μm organic filter membrane to remove large particles and precipitates. The obtained dark brown filtrate was then transferred to a 500 Da dialysis bag and dialyzed for 7 days, with the water changed every 24.0 hours;

[0019] (4) The dialyzed solution was filtered using a 0.22 μm water membrane and freeze-dried to obtain CDs.

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

[0021] (1) Absorbance detection of standard samples: The absorbance ratio at 430 nm and 650 nm after adding different concentration gradients of IMI standard in OD-like system and POD-like system was measured respectively: For OD-like system: different concentrations of IMI were added to ACP (acid phosphatase) for 10.0 min, and then AAP (L-ascorbic acid-2-phosphate trisodium salt), CDs, OPD and NaAC-HAC buffer were added and mixed. After the reaction was completed, the absorbance ratio at 430 nm and 650 nm was measured using a UV spectrophotometer (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, H2O2, OPD and NaAC-HAC buffer and mix well. After the reaction, use a UV spectrophotometer to measure the absorbance ratio of 430nm and 650nm PODA 430nm / PODA 650nm ;

[0022] (2) Fluorescence intensity detection of standard samples: The fluorescence intensity signal ratio of IMI at different concentration gradients at 550nm and 650nm under 425nm wavelength excitation was measured in OD-like system and POD-like system respectively: OD-like system: Add different concentrations of IMI to ACP for 10.0min, then add AAP, CDs, OPD and NaAC-HAC buffer and mix well. After the reaction, measure the fluorescence intensity signal value at 550nm and 650nm with a fluorescence spectrophotometer under 425nm 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 is completed, the fluorescence intensity signal values ​​at 550nm and 650nm are measured with a fluorescence spectrophotometer at an excitation wavelength of 425nm to obtain the fluorescence intensity signal ratio PODF 550nm / PODF 650nm ;

[0023] (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;

[0024] 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;

[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, substitute them into the corresponding linear equations, and obtain the concentration of IMI in the sample to be tested.

[0026] In one embodiment, in the above detection step (1) and detection step (2), in the OD-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 amount of the mixed solution is 200.0 μL; the reaction temperature is 40.0°C; and the reaction time is 20.0 min. 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 reaction time is 20.0 min.

[0027] In one embodiment, in the above step (3), the regression equation of the ultraviolet property of the 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.

[0028] The waste tobacco-based biomass CDs prepared by the present invention have two absorption peaks at 420nm and 650nm, and the presence of oxidase-like and peroxidase-like activities can oxidize colorless and non-absorbing o-phenylenediamine (OPD) into a dark yellow oxidation product 2,3-diaminophenazine (DAP) and generate an absorption value A at 430nm. However, ACP is used to hydrolyze AAP to generate L-ascorbic acid (AA), and AA has a strong reducing property, which significantly inhibits the generation of DAP, and the colorimetric signal at 430nm decreases. After IMI is added to the reaction system, IMI inhibits the activity of ACP and hinders the generation of AA, so that the color development reaction is not inhibited, and the colorimetric signal at 430nm is restored.

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

[0030] Beneficial Effects

[0031] (1) The fluorescence analysis technology for detecting neonicotinoid pesticides based on CDs provided by the present invention is mainly designed to detect neonicotinoid pesticides by adjusting the color reaction of OPD. AA generated by ACP catalyzing AAP has strong reducing properties and can effectively hinder the color reaction. However, neonicotinoid pesticides can inhibit the activity of ACP, hinder the generation of AA, and protect the color reaction from being inhibited, thereby realizing the detection of neonicotinoid pesticides. In addition, the invention uses dual ratio detection with a built-in reference signal, which can avoid interference from the external environment and increase the accuracy and sensitivity of the detection.

[0032] (2) The present invention utilizes waste tobacco leaves to synthesize CDs, which meets the development requirements of the green economy, and combines them with the chromogenic substrate o-phenylenediamine (OPD) to develop a dual-mode method for detecting neonicotinoid pesticides using waste tobacco-based biomass CDs. This method is low-cost, simple to operate, and time-saving, showing great potential for quantitative detection of neonicotinoid pesticides. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

[0035] Figure 3 To verify the oxidase and peroxidase activities of CDs.

[0036] Figure 4 and Figure 5 The double-reverse plot of TMB concentration and reaction rate for the catalytic kinetic test of CDs.

[0037] Figure 6 The feasibility of IMI was tested for the CDs+ACP+AAP+OPD+IMI (5A, 5B) and CDs+ACP+AAP+OPD+H2O2+IMI (5C, 5D) systems.

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

[0039] Figure 8 To optimize the optimal conditions for detection based on the CDs+ACP+AAP+OPD+H2O2+IMI system sensing system.

[0040] Fig. 9Detect color and fluorescence changes of 0.0-60.0μmol / L IMI and ODA for OD-like system 430nm / ODA 650nm and ODF 550nm / ODF 650nm The standard curve of

[0041] Fig.10 Detect the color and fluorescence changes of 0.0-80.0μmol / L IMI and PODA for POD-like system 430nm / PODA 650nm and PODF 550nm / PODF 650nm The standard curve of

[0042] Fig.11 The selectivity and anti-interference evaluation of CDs+ACP+AAP+OPD+IMI system. 1-15 in 10A and 10B are IMI; dinotefuran; acetamiprid; blank; carbaryl; cypermethrin; Ca 2+ Mg 2+ PO4 2- ; NH4 + ;Na + ;CO3 2- ; sucrose; maltose; glucose.

[0043] Fig.12 The selectivity and anti-interference evaluation of CDs+ACP+AAP+OPD+H2O2+IMI system. 1-15 in 11A and 11B are IMI; dinotefuran; acetamiprid; blank; carbaryl; cypermethrin; Ca 2+ Mg 2+ PO4 2- ; NH4 + ;Na + ;CO3 2- ; sucrose; maltose. DETAILED DESCRIPTION

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

[0045] Reagents and Materials:

[0046] Acid phosphatase (ACP) was purchased from Shanghai MacLean Biotechnology Co., Ltd.; L-ascorbic acid-2-phosphate trisodium salt (AAP), o-phenylenediamine (OPD), and imidacloprid (IMI) were purchased from Shanghai Aladdin Biotechnology Co., Ltd.; NaAC-HAC buffer was purchased from Shanghai Yuanye Biotechnology Co., Ltd., and H2O2 was purchased from Sinopharm Chemical Reagent Co., Ltd. All water used in the experiment was ultrapure water.

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

[0048] (1) Weigh 0.25g of waste tobacco powder and 0.30g of glutathione respectively, add 10.0mL of formamide, dissolve by ultrasound for 30.0min, mix the sample with a vortexer, and then transfer it to a tetrafluoroethylene reactor, react at 180.0℃ in an oven for 8.0h, cool naturally to room temperature, and filter with a 0.22μm organic filter membrane to remove large particles and precipitates. Then transfer the obtained dark brown filtrate to a 500Da dialysis bag and dialyze for 7 days, changing the water every 24.0h. Filter the dialyzed solution with a 0.22μm water membrane, freeze-dry to obtain CDs, and store them in a desiccator for subsequent testing.

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

[0050] Take the following five groups of reactions: (1) CDs (2) TMB (3) H2O2 (4) H2O2 + TMB (5) CDs + TMB (6) CDs + TMB + H2O2. After sufficient reaction, transfer to a cuvette, measure the absorbance at 650nm with a UV spectrophotometer, and compare the catalytic ability of the nanozyme. Figure 3 As shown in A and B, only CDs+TMB and CD+TMB+H2O2 have larger absorbance values, indicating that the synthesized CDs have good oxidase and peroxidase activities.

[0051] In the above verification steps, the concentration of CDs added was 200.0 μg / mL; the concentration of TMB was 10.0 mmol / L; the pH of the added NaAC-HAC buffer was 4.5; the concentration of H2O2 was 10.0 mmol / L; the total mixed solution was 200.0 μL; the reaction temperature was 40.0°C; and the mixing and sufficient reaction time was 20.0 min.

[0052] Example 3: Catalytic kinetics test of CDs

[0053] OD-like system: In NaAC-HAC buffer containing CDs, steady-state kinetic experiments were performed by changing the concentration of TMB (0.0–0.7 mmol / L) ( Figure 4 (A) The double-reverse plot of TMB concentration and reaction rate was calculated using the following Michaelis-Menten formula:

[0054]

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

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

[0057] POD-like system: In NaAC-HAC buffer containing CDs, the concentration of H2O2 (0.0–0.8 mmol / L) ( Figure 5 A) and TMB concentration (0.0–0.8 mmol / L) ( Figure 5 C), a steady-state kinetic experiment was performed. The double reverse graph of TMB concentration and H2O2 and reaction rate was calculated by the following Michaelis-Menten formula:

[0058]

[0059] Where V is the initial reaction rate, [S] is the substrate concentration, and K m is the Michaelis constant, V max represents the maximum reaction rate. Figure 5 B shows the K of CDs to H2O2 m The value is 2.38, such as Figure 5 D shows the K of CDs for TMB m The value is 0.378, indicating that the CDs prepared in the present invention have good catalytic activity.

[0060] Example 4: Feasibility verification of dual enzyme activity waste tobacco-based biomass CDs dual-mode system for detecting neonicotinoid pesticides OD-like system: Feasibility verification of CDs+ACP+AAP+OPD system for detecting IMI

[0061] Use OPD as the colorimetric reagent 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 sufficient reaction, the results are shown in Figure 6 In A, the absorbance ratio (ODA) at 430nm and 650nm was measured using a UV spectrophotometer. 430nm / ODA 650nm The fluorescence intensity signal ratio (ODF) at 550nm and 650nm was measured by fluorescence spectrometer. 550nm / ODF 650nm , and compare their sizes. See the results in Figure 6In B, it is shown 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, so that the CDs+OPD+ACP+AAP system emits red fluorescence at 650nm. After adding IMI, IMI will hinder the generation of AA by ACP+AAP, activate the oxidase activity of CDs through the competitive effect, oxidize OPD to DAP and restore the dual-color ratio fluorescence. Both the absorbance and fluorescence intensity change significantly. It shows the feasibility of the detection system proposed by the present invention.

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

[0063] POD-like system: Feasibility verification of CDs+ACP+AAP+OPD+H2O2 system for detecting IMI

[0064] Use OPD as the colorimetric agent and NaAC-HAC as the buffer. Take the following three groups of reactions: (1) CDs+OPD+H2O2 (2) CDs+OPD+H2O2+ACP+AAP (3) CDs+OPD+H2O2+ACP+AAP+IMI. After sufficient reaction, the results are shown in Figure 6 C, use UV spectrophotometer to measure the absorbance ratio PODA at 430nm and 650nm 430nm / PODA 650nm The fluorescence intensity signal ratio PODF at 550nm and 650nm was measured by fluorescence spectrometer 550nm / PODF 650nm , and compare their sizes. This shows that ACP+AAP can inhibit the absorbance intensity of CDs+OPD+H2O2, and the absorbance of CDs+OPD+H2O2+ACP+AAP can be restored after adding IMI. Figure 6 D, ACP + AAP can inhibit the fluorescence intensity of CDs + OPD, so that the CDs + OPD + ACP + AAP system only emits red fluorescence at 650nm. After adding IMI, IMI will hinder the generation of AA by ACP + AAP, activate the peroxidase activity of CDs through the competitive effect, oxidize OPD to DAP and restore the dual-color ratio fluorescence. Both the absorbance and fluorescence intensity change significantly, showing the feasibility of the detection system proposed by the present invention.

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

[0066] Example 5: Optimization of the optimal conditions for detecting neonicotinoid pesticides using a dual-enzyme activity waste tobacco-based biomass CDs dual-mode system

[0067] OD-like system: Optimization of the optimal conditions for detecting IMI in the CDs+ACP+AAP+OPD system

[0068] In order to optimize the analytical performance of the CDs+ACP+AAP+OPD system, the experimental parameters of pH value, temperature, reaction time, ACP concentration and AAP concentration in the detection system were studied in detail. First, the performance of the system in the pH range of 3.5–6.0 was investigated, and NaAC-HAC was selected as the reaction buffer. The results are shown in Figure 2. Figure 7 As shown in Figure A, when the pH value is in the range of 3.5 to 5.0, the absorbance value of the reaction system at 430nm increases with the increase of the pH value of the buffer system. When the pH value exceeds 5.0, the absorbance value decreases as it increases, so 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℃ was explored. The results are shown in Figure 7 As shown in Figure B, when the temperature is in the range of 25.0℃ to 40.0℃, the absorbance value of the reaction system at 430nm increases with the increase of reaction temperature. When the temperature exceeds 40.0℃ and increases to 50.0℃, the absorbance value decreases as it increases, so 40.0℃ is selected as the optimal temperature of the system. The reaction time was optimized, and the results are shown in Figure 7 In Figure C, from 0.0 to 20.0 min, the absorbance value of the reaction system at 430 nm increases with the increase of reaction time, and basically does not change after 20.0 min, so 20.0 min is selected as the optimal reaction time of the system. Figure 7 As 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 as it increased, and basically did not change 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] Quasi-POD system: Optimization of optimal conditions for detecting IMI in CDs+ACP+AAP+OPD+H2O2 system

[0070] In order to optimize the analytical performance of the CDs+ACP+AAP+OPD+H2O2 system, the experimental parameters of pH value, temperature, reaction time, ACP concentration and AAP concentration in the detection system were studied in detail. First, the performance of the system in the pH range of 3.5–6.0 was investigated, and NaAC-HAC was selected as the buffer for the reaction. The results are shown in Figure 8 As shown in Figure A, when the pH value is in the range of 3.5 to 5.0, the absorbance value of the reaction system at 430nm increases with the increase of the pH value of the buffer system. When the pH value exceeds 5.0, the absorbance value decreases as it increases, so 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℃ was explored. The results are shown in Figure 8 As shown in Figure B, when the temperature is in the range of 25.0℃ to 40.0℃, the absorbance value of the reaction system at 430nm increases with the increase of reaction temperature. When the temperature exceeds 40.0℃ and increases to 50.0℃, the absorbance value decreases as it increases, so 40.0℃ is selected as the optimal temperature of the system. The reaction time was optimized, and the results are shown in Figure 8 As shown in Figure C, from 0.0 to 20.0 min, the absorbance value of the reaction system at 430 nm increases with the increase of 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 is optimized, as shown in Figure C. Figure 8 As shown in (D, E), when ACP is between 0.0 and 6.0 mmol / L, the absorbance decreases as it increases, 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: Dual-enzyme activity of waste tobacco-based biomass CDs dual-mode system for detecting neonicotinoid pesticide standards

[0072] OD-like system: CDs+ACP+AAP+OPD system to detect IMI standards

[0073] IMI solutions of different concentrations (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 at 430nm was measured with a UV spectrophotometer. As the concentration of IMI increased, the absorbance at 430nm increased, and the absorbance ratio ODA 430 nm / ODA 650nm Therefore, the absorbance ratio ODA 430nm / ODA 650nm The vertical axis is the concentration of IMI, and the linear equation is fitted using Origin software. Fig. 9 As shown in (A, B), the concentration of IMI and the absorbance ratio are 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 was 0.99188, and the detection limit was 0.66μmol / L.

[0075] Under the same reaction conditions, the fluorescence intensity signals at 550nm and 650nm were measured by a fluorescence spectrophotometer under 425nm excitation light. As the concentration of IMI increased, the ratio of fluorescence intensity at 550nm to 650nm increased. Therefore, the fluorescence intensity ratio ODF 550nm / ODF 650nm The vertical axis is the concentration of IMI, and the linear equation is fitted using Origin software. Fig. 9 The concentration and fluorescence intensity ODF of IMI are shown in (C, D) 550nm / ODF 650nm The fluorescence intensity ratio ODF was linear in the range of 0.0–60.0 μmol / L. 550nm / ODF 650nm The regression equation is F 550nm / F 650nm =0.00605C IMI +0.16165, the correlation coefficient was 0.9975, and the detection limit was 0.024 μmol / L.

[0076] POD-like system: CDs+ACP+AAP+OPD+H2O2 system to detect IMI standards

[0077] IMI solutions of 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 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.

[0078] After the reaction, the absorbance at 430nm was measured with a UV spectrophotometer. As the concentration of IMI increased, the absorbance at 430nm increased, and the absorbance ratio PODA 430nm / PODA 650nm Therefore, the absorbance ratio PODA 430nm / PODA 650nm The vertical axis is the concentration of IMI, and the linear equation is fitted using Origin software. Fig.10 As shown in (A, B), the concentration of IMI and the absorbance ratio are 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 was 0.99807, and the detection limit was 0.46μmol / L.

[0079] Under the same reaction conditions, the fluorescence intensity signals at 550nm and 650nm were measured by a fluorescence spectrophotometer under 425nm excitation light. As the concentration of IMI increased, the ratio of fluorescence intensity at 550nm to 650nm increased. Therefore, the fluorescence intensity ratio PODF 550nm / PODF 650nm The vertical axis is the concentration of IMI, and the linear equation is fitted using Origin software. Fig.10 The concentration and fluorescence intensity of IMI were shown in (C, D) 550nm / PODF 650nm The fluorescence intensity ratio PODF is linear in the range of 0.0–80.0 μmol / L. 550nm / PODF 650nm The regression equation is PODF 550nm / PODF 650nm =0.00561C IMI +0.37037, the correlation coefficient was 0.99175, and the detection limit was 0.016μmol / L.

[0080] Example 7: Selectivity of the dual-mode detection system of CDs in waste tobacco-based biomass for detecting neonicotinoid pesticides OD-like system: Selectivity of CDs+ACP+AAP+OPD system for IMI

[0081] In order to evaluate the selectivity of the CDs+ACP+AAP+OPD system for IMI, some representative contents were used as proof of concept, including blank; carbaryl; cypermethrin; Ca 2+ Mg 2+ PO4 2- ; NH4 +;Na + ;CO3 2- ; sucrose; maltose; glucose; other neonicotinoid pesticides (such as dinotefuran and acetamiprid). At room temperature, CDs, OPD, ACP, and AAP were reacted with the above substances in NaAC-HAC buffer respectively; after mixing evenly, the mixture was reacted for 20.0 min and then transferred to a cuvette. The absorbance ratio ODA was measured respectively. 430nm / ODA 650nm , and the fluorescence intensity signal ratio ODF at 550nm and 650nm under 425nm wavelength excitation 550nm / ODF 650nm The results are as follows Fig.11 As shown in (A, B), only the absorbance ratio A of dinotefuran, acetamiprid and IMI increased significantly, and the fluorescence intensity signal ratio ODF 550nm / ODF 650nm This indicates that the detection system has good selectivity for IMI.

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

[0083] POD-like system: Selectivity of CDs+ACP+AAP+OPD+H2O2 system for detecting IMI

[0084] In order to evaluate the selectivity of IMI based on the CDs+ACP+AAP+OPD+H2O2 system, some representative contents were used as proof of concept, including blank; carbaryl; cypermethrin; Ca 2+ Mg 2+ PO4 2- ; NH4 + ;Na + ;CO3 2- ; sucrose; maltose; glucose; other neonicotinoid pesticides (such as dinotefuran and acetamiprid). At room temperature, CDs, OPD, ACP, AAP, and H2O2 were reacted with the above substances in NaAC-HAC buffer respectively; after mixing evenly, the mixture was reacted for 20.0 min and then transferred to a cuvette. The absorbance ratio PODA was measured respectively. 430nm / PODA 650nm , and the fluorescence intensity signal ratio PODF at 550nm and 650nm under 425nm wavelength excitation550nm / PODF 650nm The results are as follows Fig.12 As shown in (A, B), only the absorbance ratio A of dinotefuran, acetamiprid and IMI increased significantly, while the fluorescence intensity signal ratio PODF 550nm / PODF 650nm This indicates that the detection system has good selectivity for neonicotinoid pesticides.

[0085] In the above verification steps, the concentration of added CDs 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 was 5.0; the total mixed solution was 200.0 μL; the reaction temperature was 40.0°C; and the mixing and sufficient reaction time was 20.0 min.

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

[0087] OD-like system: In order to verify the sensing performance of the dual-enzyme activity waste tobacco-based biomass carbon dot dual detection system for IMI detection in actual samples, wolfberry, honeysuckle and Panax notoginseng were selected as representatives of actual samples. Among them, wolfberry, honeysuckle and Panax notoginseng in this work were randomly purchased in a supermarket in Hefei, China. Different concentrations of IMI were dripped on the surface of the actual sample to be tested, air-dried and soaked in deionized water for one hour, and the soaking liquid was taken for detection. At room temperature, CDs, OPD, ACP and AAP were reacted with the above mixed solutions in NaAC-HAC buffer (pH=5.0) respectively; after mixing evenly, the reaction was transferred to the cuvette for 20.0min. 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 equation, and the IMI results measured under the OD-like detection mode are shown in Table 1.

[0088] Quasi-POD system: In order to verify the sensing performance of the dual-enzyme activity waste tobacco-based biomass carbon dot dual-mode ratio detection system for IMI detection in actual samples, wolfberry, honeysuckle and Panax notoginseng were selected as representatives of actual samples. Among them, wolfberry, honeysuckle and Panax notoginseng in this work were randomly purchased in a supermarket in Hefei, China. Different concentrations of IMI were dripped on the surface of the actual sample to be tested, air-dried and soaked in deionized water for one hour, and the soaking liquid was taken for detection. At room temperature, CDs, OPD, H2O2, ACP and AAP were reacted with the above mixed solutions in NaAC-HAC buffer (pH=5.0) respectively; after mixing evenly, the reaction was transferred to the cuvette for 20.0min. 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 equation, and the IMI results measured under the quasi-POD 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 Panax notoginseng.

[0090]

[0091] The results show that the reaction system can be applied to the detection of actual samples, and the recovery rate is between 97.25% and 104.90%, with a relative deviation of less than 3.

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

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

Claims

1. A method for preparing waste tobacco-based biomass carbon dots (CDs), characterized in that: The method comprises the following steps: (1) mixing tobacco leaf powder, antioxidant and formamide, and dissolving by ultrasonication; (2) The dissolved sample is mixed with a vortexer, then transferred to a tetrafluoroethylene reactor, reacted at high temperature in an oven, and naturally cooled to room temperature; (3) The product of step (2) was filtered through a 0.22 μm organic filter membrane to remove large particles and precipitates, and then the obtained dark brown filtrate was transferred to a dialysis bag for dialysis; (4) The dialyzed solution was filtered using a 0.22 μm water membrane and freeze-dried to obtain CDs.

2. The preparation method according to claim 1, characterized in that: The antioxidant in step (1) is glutathione; and the ultrasonic time is 30.0 min.

3. The preparation method according to claim 1, characterized in that: The high temperature reaction in the oven in step (2) is carried out at 180.0° C. for 8.0 h.

4. The preparation method according to claim 1, characterized in that: In step (3), the dialysis is performed using a 500Da dialysis bag for 7 days, with the water being changed every 24.0 hours.

5. A method for preparing waste tobacco-based biomass carbon dots (CDs), characterized in that: The following steps are involved: (1) Weigh 0.25 g of waste tobacco powder and 0.30 g of glutathione, add 10.0 mL of formamide, mix, and dissolve by ultrasonication for 30.0 min; (2) 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; (3) The product of step (2) was filtered with a 0.22 μm organic filter membrane to remove large particles and precipitates. The obtained dark brown filtrate was then transferred to a 500 Da dialysis bag and dialyzed for 7 days, with the water changed every 24.0 hours; (4) The dialyzed solution was filtered using a 0.22 μm water membrane and freeze-dried to obtain CDs.

6. A method for constructing dual-mode detection of neonicotinoid pesticide residues using waste tobacco-based biomass carbon dots prepared by the method for preparing waste tobacco-based biomass CDs according to any one of claims 1 to 5, the method comprising the following steps: (1) Absorbance detection of standard samples: The absorbance ratio at 430 nm and 650 nm after adding different concentration gradients of IMI standard was measured in the oxidase (OD) detection system and the peroxidase (POD) detection system respectively: For the OD-like system: add different concentrations of IMI to acid phosphatase (ACP) for 10.0 min, then add trisodium L-ascorbic acid-2-phosphate (AAP), CDs, OPD and NaAC-HAC buffer and mix well. After the reaction is completed, the absorbance ratio at 430 nm and 650 nm is measured using a UV spectrophotometer (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, H2O2, OPD and NaAC-HAC buffer and mix well. After the reaction, use a UV spectrophotometer to measure the absorbance ratio of 430nm and 650nm PODA 430nm / PODA 650nm ; (2) Fluorescence intensity detection of standard samples: The fluorescence intensity signal ratio of IMI at different concentration gradients at 550nm and 650nm under 425nm wavelength excitation was measured in OD-like system and POD-like system respectively: OD-like system: Add different concentrations of IMI to ACP for 10.0min, then add AAP, CDs, OPD and NaAC-HAC buffer and mix well. After the reaction, measure the fluorescence intensity signal value at 550nm and 650nm with a fluorescence spectrophotometer under 425nm 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 550nm and 650nm with a fluorescence spectrophotometer at an excitation wavelength of 425nm 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; (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, substitute them into the corresponding linear equations, and obtain the concentration of IMI in the sample to be tested.

7. The method according to claim 6, characterized in that In the OD-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 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.

8. The method according to claim 6, 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.

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