CN114371158A

CN114371158A - Preparation method of hydrogel kit for monitoring degradation of organophosphorus pesticide

Info

Publication number: CN114371158A
Application number: CN202210113048.8A
Authority: CN
Inventors: 李红霞; 邹睿琦; 孙春燕; 吕婷; 刘妮; 苏长顺; 张铁华
Original assignee: Jilin University
Current assignee: Jilin University
Priority date: 2022-01-29
Filing date: 2022-01-29
Publication date: 2022-04-19

Abstract

The invention discloses a preparation method of a hydrogel kit for monitoring degradation of organophosphorus pesticide. Embedding the fluorescent composite material into a sodium alginate hydrogel system in a micropore plate as a signal indicator to prepare the gel kit. By using AuNCs fluorescence characteristic and MnO in composite material₂And (3) constructing a ratio fluorescence method for the activity of the nanosheet on OPD oxidase-like enzyme. Binding of AChE enzymatic product TCh to MnO₂The redox reaction of the nanosheets and the efficient inhibition effect of the pesticide on AChE are achieved, a timely monitoring (POC) hydrogel kit for degradation of the organophosphorus pesticide is ingeniously constructed, color information is converted into pesticide concentration information by means of smartphone software, and the kit is applied to accurate monitoring of the residual and degradation of the oxyphosphorus in the Chinese cabbage. The ratio type hydrogel kit developed by the invention not only simplifies the sensing process, but also greatly improves the sensing efficiency and sensitivity, and provides technical support for establishing a new method for detecting organophosphorus pesticides, and more particularly for the application of POC technology in food safety monitoring.

Description

Preparation method of hydrogel kit for monitoring degradation of organophosphorus pesticide

Technical Field

The invention belongs to the fields of biosensor technology and food safety detection, and particularly relates to a gold nanocluster anchored manganese dioxide nanosheet fluorescent composite material (AuNCs @ MnO)₂NSs) preparation method, synergistic application of AuNCs fluorescence characteristic and MnO in composite material₂The fluorescent method is constructed by catalyzing o-phenylenediamine oxidation by the enzyme-like characteristic of NSs, and background interference is reduced by built-in fluorescent signal correction. Combining with acetylcholinesterase (AChE) to catalyze the generation of thiocholine (TCh) and MnO from thiocholine (ATCH)₂The redox reaction between NSs and the efficient inhibition effect of the organophosphorus pesticide on AChE skillfully construct a hydrogel kit for timely monitoring the degradation of the organophosphorus Pesticide (POC), and the kit is applied to the accurate monitoring of the residue and degradation of the oxyphosphorus in the Chinese cabbage, thereby providing technical support for the POC detection applied to the food safety monitoring.

Background

The organophosphorus pesticide is widely applied to protecting crops from being attacked by plant diseases and insect pests, weeds and diseases, and further ensures and promotes modern agricultural production and development. Although low-toxicity and low-residue organophosphorus pesticides are continuously developed and applied, the organophosphorus pesticides still have the phenomena of abuse and accidental release, so that the bioaccumulation effect of a food chain is caused, the nervous system of mammals can be damaged, and the serious threat to human health is caused. Therefore, the establishment of an effective organophosphorus pesticide detection method has important significance for food quality control and environmental management. Traditional organophosphorus pesticide detection methods include chromatography and mass spectrometry, and although the methods have high accuracy, the methods are relatively complex to operate, long in time consumption and not suitable for instant detection. Therefore, a novel method which is rapid, simple to operate and capable of detecting organophosphorus pesticide residues on site is urgently needed.

In the POC monitoring system, the immobilized carrier for loading the bioactive substances or the sensing nano-materials plays an important role in interface recognition and sensing response. The paper-based platform based on the fixed fluorescent substances (such as quantum dots, metal nanoclusters and organic dyes) has the characteristics of cost saving, easiness in operation and the like, a sensing device can be miniaturized, but the price of an instrument is relatively high, semi-quantitative and qualitative identification of a target object is carried out depending on fluorescent brightness change, and detection of trace pesticides cannot be achieved. In addition, the loaded fluorophore is unevenly distributed due to the "coffee ring" effect, and is easily aggregated under a complex matrix condition, and the stability is poor. The novel carrier such as the porous polymer network hydrogel has the advantages of stable mechanics, strong bearing capacity, good flexibility and the like, and provides a fixed microenvironment for uniform distribution of the nano material, thereby realizing accurate and stable quantitative detection of a target object. To date, hydrogels have been widely used in drug/gene delivery, cell/tissue scaffolds, and electronic devices, but their development in POC monitoring is still in the first stage. The hydrogel is utilized to provide an inert environment, and the immobilized matrix not only improves the stability of the nano material, but also allows target molecules to diffuse through the porous size; on the other hand, the nano-scale porous size of the hydrogel is used as a barrier, so that macromolecules in the external environment can be blocked, and the anti-interference capability of the system is improved. Therefore, the hydrogel system can effectively reduce the aggregation of fluorescent materials and improve the performance of POC monitoring. In addition, the brightness displayed by the monochromatic fluorescent indicator is easily interfered by the background of phytochrome and secondary metabolites, the visual quantification based on eyes is limited to semi-quantitative analysis, false positive results are easily caused, the accurate measurement still depends on complex instruments, and the application of the monochromatic fluorescent indicator in POC monitoring is greatly limited. Therefore, the integration of a portable quantitative system with a hydrogel-responsive ratiometric fluorescence measurement platform shows great application potential in the detection of organophosphorus pesticide residues.

Based on the above research background, we propose MnO anchoring gold nanoclusters₂Fluorescent composite material (AuNCs @ MnO)₂) The ratio type fluorescent hydrogel kit is embedded into Sodium Alginate (SA) hydrogel as a signal indicator and is fixed on a microporous plate, and the ratio type fluorescent hydrogel kit for accurately detecting organophosphorus pesticides on site is developed.

Disclosure of Invention

The invention adopts a co-template method to synthesize AuNCs @ MnO₂Composite materialEmbedding the composite material into Sodium Alginate (SA) hydrogel as a signal indicator, and fixing the signal indicator on a microporous plate to prepare the portable hydrogel kit. Using AuNCs @ MnO₂The mimic enzyme catalytic activity of the composite material oxidizes o-phenylenediamine (OPD) without fluorescence characteristics to generate 2, 3-Diaminophenyloxazine (DAP) with yellow fluorescence, so AuNCs (optimal emission 650nm) and DAP (optimal emission 550nm) with red fluorescence form a ratio type fluorescence signal, and the background interference is reduced by built-in fluorescence signal correction. Combining with MnO and thiocholine (TCh) generated by catalyzing the generation of thiocholine (ATCH) by acetylcholinesterase (AChE)₂The redox reaction between NSs and the efficient inhibition effect of the organophosphorus pesticide on AChE develop an organophosphorus pesticide detection method with simple operation and high sensitivity, and a stable stimulus-response hydrogel detection kit is constructed by virtue of the inert environment provided by hydrogel. The organophosphorus pesticide residue detection gel kit constructed by the invention can effectively reduce the interference caused by the environment and instruments, not only utilizes the specific catalytic performance of the biological enzyme, but also realizes the residue degradation detection of the paraoxon in the pakchoi by means of the hydrogel portable kit and the color development sensing assisted by a smart phone, and provides technical support for the application of the POC detection technology in food safety monitoring.

The purpose of the invention can be realized by the following technical scheme:

the development of a fluorescent hydrogel kit for instantly monitoring the rate of degradation of organophosphorus pesticide comprises the following steps:

A、AuNCs@MnO₂preparing a fluorescent composite material:

synthesis of AuNCs @ MnO by co-template method₂Fluorescent composite material prepared by mixing MnCl₂Solution (50mmol L)^-1) AuNCs solution and sodium hydroxide solution (1.0mol L)^-1) Mixing according to the volume ratio of 1:10:1, stirring and reacting for 3h, then purifying by using a dialysis bag (MWCO-1.0 kDa), and dialyzing for 1 day to remove small molecules.

B. Preparation of paraoxon detection gel kit

First, a Sodium Alginate (SA) solution (20mg mL)^-1)、ATCh(2mmol L^-1) And AuNCs @ MnO₂Fluorescent composite (2.5. mu.g mL)^-1) Adding the mixture into a 96-well plate according to the volume ratio of 10:1:2, fully mixing, and adding Ca (NO)₃) ₂Solution (10.0mg mL)^-1) Adding the mixed solution into the solution according to the volume ratio of 3:13 to the mixed solution to prepare the hydrogel. The obtained kit can be stored at 4 ℃ for later use.

Different concentrations of paraoxon standard solution (2.5-2000ng mL)^-1)、AChE(0.18U mL^-1) And Tris buffer (pH 8.0,100mM) at a volume ratio of 5:5:4, incubating at 37 ℃ for 30 minutes, adding the mixed solution to the well plate at a volume ratio of 7:32 to the hydrogel, and reacting at 37 ℃ for 25 minutes. Finally, OPD (0.6mmol L)^-1) The mixture was added to the well plate at a volume ratio of 2:195 to the above mixed solution, and the reaction was continued at 50 ℃ for 10 minutes. The color of the hydrogel was analyzed under natural light using ImageJ software.

C. Actual sample detection

Tap water, Songhua river water, orange juice and milk actual samples are used as detection targets to verify the actual application of the hydrogel kit. Filtering 10.0mL of water sample by using a 0.22mm membrane; diluting milk by 50 times; the orange juice is processed by a juicer and diluted by 50 times. 5, 25, 100 and 250ng mL of each^-1Adding 10mL of acetonitrile into the paraoxon standard solution, performing ultrasonic treatment for 5min, oscillating for 30min, centrifuging for 10 min at 4000rpm, and detecting by adopting the method.

The processing method of the pakchoi sample comprises the following steps: first, after the pakchoi was cultured for 10 days, one group was sprayed with paraoxon solution (5ppm) 10mL per day for 3 consecutive days. The other group was sprayed with 10mL of ultrapure water in the same manner as a control. After spraying, extracting the residual paraoxon in the leaves and roots of the pakchoi according to the extraction method. Finally, the rate-type fluorescent hydrogel kit constructed by the method is used for detecting the Chinese cabbage extract degraded for different days after spraying the pesticide.

The mechanism of the invention is as follows:

mixing AuNCs @ MnO₂Fluorescent composite material embeddingSodium Alginate (SA) hydrogel is used as a signal indicator and is fixed on a micropore plate to prepare the portable kit. Wherein AuNCs @ MnO₂Middle MnO of composite material₂NSs have both fluorescence quenching ability and mimic enzyme catalytic activity, and not only can efficiently quench the fluorescence of AuNCs, but also can oxidize OPD having no fluorescence characteristic to produce DAP having yellow fluorescence, so that a ratiometric fluorescence method is constructed from changes in double fluorescence signals formed from AuNCs having red fluorescence (optimal emission 650nm) and DAP having yellow fluorescence (optimal emission 550 nm). The AChE is used for catalyzing the thioacetylcholine ATCH to generate TCh which can convert MnO into MnO₂Reduction of NSs to Mn²⁺Thereby losing fluorescence quenching ability and enzyme-like catalytic activity, increasing the fluorescence of AuNCs at 650nm and reducing the fluorescence of DAP at 550nm in the system; when the organophosphorus pesticide exists, the AChE activity can be effectively inhibited, so that the change of fluorescence signals of AuNCs and DAP is reversed, and the color of the gel gradually changes from red to yellow along with the addition of the organophosphorus pesticide. For accurate quantification, the fluorescence color change of the portable kit is converted into digital information by using Image J software, and an applicable linear range is presented in pesticide residue detection. Moreover, the portable hydrogel kit constructed by the method is successfully applied to the degradation detection of the paraoxon in the pakchoi residues.

The ratio type hydrogel kit developed by the invention not only effectively improves the stability of signal materials and simplifies the sensing process, but also greatly improves the sensing efficiency and the detection sensitivity through the diffusion effect of matrix molecules formed by a porous structure. The quantitative detection of the paraoxon is realized by utilizing the color information by means of intelligent mobile phone software, a new method is created for degrading organophosphorus pesticide and detecting residues, and the method is more supported by the application technology of POC detection technology in food safety monitoring.

Drawings

FIG. 1: example 1, AuNCs @ MnO₂Preparation and characterization of fluorescent composite material, wherein (A) is AuNCs @ MnO₂A schematic diagram of a synthetic process of the fluorescent composite material; (B) TEM images of AuNCs @ MnO2 fluorescent composite; (C) as AuNCs @ MnO₂EDS images of the fluorescent composite; (D) as AuNCs @ MnO₂Fluorescent composite material, MnO₂NFs and AuNCs; (E) as AuNCs @ MnO₂Fluorescent composites and fluorescence spectra of AuNCs (inset images AuNCs and AuNCs @ MnO under UV and Natural Lighting)₂The picture of (a); (F) as AuNCs @ MnO₂The mimic enzyme catalytic activity of the fluorescent composite material on OPD is shown schematically; (G) as AuNCs @ MnO₂Fluorescence spectra and uv-vis absorption spectra of the fluorescent composite and OPD.

FIG. 2: as described in example 2. Wherein, (A) is a composition schematic diagram of an SA hydrogel platform; (B) color photographs under aqueous phase and hydrogel systems; (C) as AuNCs @ MnO₂SEM pictures of SA hydrogel; (D) is MnO₂The reaction rate of OPD in hydrogel and liquid phase systems; (E) as AuNCs @ MnO₂The fluorescent composite material has the stability of being stored in a hydrogel system and a liquid phase system for 10 days.

FIG. 3: the ratiometric fluorescent hydrogel kit described in example 2 was used for the detection of paraoxon. Wherein (A) is a schematic diagram of a ratiometric fluorescence analysis strategy of paraoxon; (B) the feasibility analysis of a paraoxonium ratio fluorescence system is carried out; (C) the hydrogel kit is provided with a corresponding color change in the presence of a substance.

FIG. 4: as described in example 2. Wherein, (A) is AuNCs @ MnO₂Ratiometric fluorescence spectra of the/OPD/ATCH/AChE system in the presence of different concentrations of paraoxon; (B) is system F₅₅₀/F₆₅₀The relation with the logarithm of the concentration of paraoxon; (C) the corresponding color change of the hydrogel system under the irradiation of ultraviolet light; (D) is the tone intensity digitized using Image J software; (E) the linear relation between the tone intensity and the logarithm of the pesticide concentration is obtained; (F) the correlation between the detection result of the microplate reader and the auxiliary analysis result of the software; (G) the specificity of the system to the interference substances is researched; (H) for the sensitivity study of other pesticides in the system.

FIG. 5: the detection of paraoxon in real samples is described in example 3. Wherein (A) is the recovery rate of the standard sample (0.025, 0.1, 0.25. mu.g mL-1); (B) a correlation curve of the concentration of paraoxon and the fluorescence ratio in a Chinese cabbage sample is obtained; (C) is a picture of pakchoi (leaves and roots); (D) the profile of the degradation of paraoxon in leaves and roots is shown.

FIG. 6: example 1 describes AuNCs, MnO₂NFs and AuCNs @ MnO₂Zeta potential map of the fluorescent composite.

Detailed Description

The invention is further described below with reference to the accompanying drawings.

Example 1: AuNCs @ MnO₂Preparation and characterization of fluorescent composites

Firstly, using BSA-fixed as a template to prepare AuNCs as a nano raw material, and synthesizing the AuNCs @ MnO by using a co-template method₂Fluorescent composite material prepared by mixing MnCl₂Solution (50mmol L)^-1100. mu.L), AuNCs solution (1.0mL) and sodium hydroxide solution (1.0mol L)^-1100 mul) was mixed and stirred for 3h, then purified with dialysis bag and dialyzed for 1 day to remove small molecules.

The results are shown in FIG. 1 for AuNCs @ MnO by Transmission Electron Microscopy (TEM)₂The morphology and structure of the composite material were characterized and lattice fringes of AuNCs of size 0.216nm were observed, indicating that AuNCs had been successfully anchored to MnO₂Surface (fig. 1B). From the EDX image, the presence of Au and Mn in AuNCs @ MnO was observed₂Fluorescent composite material (fig. 1C). Because of MnO₂The function of the nano-sheet is to ensure that AuNCs @ MnO is₂The zeta potential value of the composite (-43.5mV) was lower than that of AuNCs (-33.3mV) (FIG. 6). In addition, the ultraviolet-visible absorption spectrum of the fluorescent composite material shows typical MnO within the range of 300-600 nm₂Characteristic of corresponding to Mn²⁺D-D transition of (FIG. 1D). Formation of MnO₂After nanosheet, based on

The resonance energy transfer (FRET) effect, quenching the AuNCs fluorescence spectrum at 650nm with a corresponding change in fluorescence color under UV light (FIG. 1E), indicates AuNCs @ MnO₂Successful synthesis of fluorescent composites.

Example 2: ratio type fluorescent hydrogel kit for detecting paraoxon

First, to implement POC testing, AuNCs @ MnO was applied₂Fluorescent composite materialAnd fixing the mixture in a three-dimensional hydrogel carrier to prepare the test kit. First, SA solution (20mg mL)^-1，100μL)、ATCh(2mmol L ^-110 μ L) and AuNCs @ MnO₂Fluorescent composite (2.5. mu.g mL)^-120 μ L) were added to a 96-well plate, and after thorough mixing, Ca (NO) was added₃)₂Solution (10.0mg mL)^-130. mu.L) was added to the above solution to prepare a hydrogel.

Hydrogel preparation procedure As shown in FIG. 2A, the well-shaped SA hydrogel has no fluorescent background under UV lamp, the tube is fixed when it is inverted (FIG. 2B), and AuNCs @ MnO is introduced into the hydrogel₂After fluorescence of the composite, red-emitting fluorescence was observed at the bottom of the tube. SEM image in FIG. 2C shows three-dimensional fibrous structure of hydrogel, indicating AuNCs @ MnO₂The fluorescent composite was successfully implanted into the hydrogel. The performance of the hydrogel kit in terms of reaction rate and stability was next evaluated. In the hydrogel system, after adding OPD, the characteristic fluorescence peak at 550nm was gradually enhanced, consistent with that of the homogeneous solution (fig. 2D), indicating that the heterogeneous hydrogel interface did not affect the catalytic reaction rate. AuNCs @ MnO₂The fluorescence of the fluorescent composite material remained over 88% when the fluorescent composite material was stored in the hydrogel for 8 days (FIG. 2E). These findings indicate AuNCs @ MnO under SA hydrogel systems₂The fluorescent composite material has good performance.

Next, the standard solution of paraoxon (15. mu.L) and AChE (0.18U mL) at different concentrations were added^-115 μ L) and Tris buffer (pH 8.0,100mM) were incubated at 37 ℃ for 30 minutes, added to the above-mentioned well plate, and reacted at 37 ℃ for 25 minutes. Finally, OPD (0.6mmol L)^-12. mu.L) was added to the above well plate and the reaction was continued at 50 ℃ for 10 minutes. Under the irradiation of an ultraviolet lamp, the mobile phone shoots the change of the fluorescence color in the air of the hydrogel, and the color of the hydrogel is analyzed by using Image J software.

The specific detection principle and feasible analysis of the organophosphorus pesticide are shown in figure 3 by monitoring the change of the fluorescence ratio by using the ratio type fluorescent hydrogel kit. When 5.0ng mL of^-1～2000ng mL^-1With reference to a standard paraoxon solution ofIncreasing, the fluorescence intensity of AuNCs decreased, the fluorescence intensity of DAP increased, and a significant change in the fluorescence intensity ratio was formed (FIG. 4A). Fluorescence intensity ratio (FL)₅₅₀/FL₆₅₀) Has a good linear relation with the logarithm of the paraoxon concentration (R)²0.996), calibration data was linearly fitted: FL₅₅₀/FL₆₅₀＝0.259+0.738Log [paraoxon]ng mL^-1(FIG. 4B). A series of colors in the orange spectrum of the hydrogel kit can be observed by naked eyes under an ultraviolet lamp, so that the rapid semi-quantitative analysis of the organophosphorus pesticide is realized; converting the ratio fluorescence Image of the hydrogel kit into corresponding digital information by using Image J software, wherein the logarithm of the tone intensity and the concentration of paraoxon of the ratio fluorescence hydrogel kit is 5.0-500 ng mL by an Image processing method^-1Has good linear relation (R) in the range²0.966) (fig. 4E). In addition, the measurement results obtained by software-assisted data analysis have good correlation (R) with the measurement results of the microplate reader²0.951) (fig. 4F). The results show that the hydrogel kit can realize accurate monitoring of the organophosphorus pesticide in the POC detection by combining with Image J software.

Finally, in order to test the anti-interference capability of the kit, the invention detects common cations (Ca) in food samples²⁺、 Fe²⁺) The influence of sugars (glucose, lactose, sucrose), proteins (whey protein, glutathione, egg albumin) on the system. The result is shown in fig. 4G, and these influence factors do not significantly influence the change of the fluorescence ratio of the detection system, indicating that the method has good anti-interference capability. Other organophosphorus pesticides (malathion, methamidophos, chlorpyrifos, phorate and metofos) have similar phosphate framework structures and show inhibition effects on AChE, so that a significant ratiometric fluorescence reaction is obtained (fig. 4H), which confirms the universality of the hydrogel kit for organophosphorus pesticide detection.

Example 3: detection of paraoxon in real samples

To verify that the method is based on AuNCs @ MnO₂The accuracy of the composite ratio type hydrogel kit is that the oxygen phosphorus is added into the actual samples of tap water, Songhua river water, orange juice, milk and the likeAnd (5) recovering the standard and detecting. Different concentrations (0.025, 0.1 and 0.25. mu.g mL) were added^-1) The measured standard recovery rate of the paraoxon standard solution is between 98.49 and 104 percent, and the Relative Standard Deviation (RSD) of different real samples is between 1.17 and 8.78 percent (figure 5A). The proportional fluorescent hydrogel kit combined with Image J software is shown to have practicability and reliability in the POC detection of paraoxon residues.

And (3) researching the degradation of the organophosphorus pesticide in the pakchoi by using the ratio type fluorescent hydrogel kit, and linearly fitting calibration data: FL₅₅₀/FL₆₅₀＝0.375+0.670Log[paraoxon]ng mL^-1(R²0.998) (fig. 5B). The hydrogel kit is used for monitoring the change of the content of paraoxon in the leaves and the root system of the pakchoi sprayed with pesticide, a large amount of paraoxon residues are found in the leaves and the root system, and the residual level in the leaves is higher than that of the root system. The decrease in the ratio fluorescence signal over time indicates a gradual decrease in the residual levels in the leaves and roots due to biodegradation, transpiration losses and dilution of plant growth. The degradation curve of the leaf is Y-3.62 e^-0.361x(R²0.997), root system Y1.776 e^-0.422x(R²0.947) (fig. 5D).

Table 1: the ratio-type fluorescent hydrogel kit developed by the invention is applied to detect paraoxon (n ═ 3) in actual samples

Claims

1. A preparation method of a hydrogel kit for monitoring degradation of organophosphorus pesticide is characterized by comprising the following steps:

A. manganese dioxide nanosheet, MnO for short₂NSs; gold nanocluster anchoring MnO₂Nanosheet fluorescent composite material AuNCs @ MnO for short₂The preparation of (1):

adding 100 mu L of MnCl₂The solution, 1.0mL AuNCs solution and 100. mu.L sodium hydroxide solution were slowly stirred for reaction for 3h to obtain a brown yellow AuNCs @MnO₂Purifying the fluorescent composite material solution by using a dialysis bag for 1 day to remove small molecules, and placing the solution at 4 ℃ for later use;

B. preparing a paraoxon detection gel kit:

add 100. mu.L of sodium alginate solution to 96-well plate and add 10. mu.L of 2mmol L^-1ATCH and 20 μ L AuNCs @ MnO for step A₂The fluorescent composite was added to the wells and after thorough mixing, 30. mu.L of Ca (NO) was added₃)₂Adding the solution into the SA mixed solution to prepare hydrogel, and storing the obtained kit at 4 ℃ for later use;

the paraoxon standard solution with different concentrations is added into the solution in 0.18U mL^-1Mixing AChE and 100mM Tris buffer solution uniformly according to a volume ratio of 5:5:4, and incubating for 30 minutes at 37 ℃; then adding the mixed solution into the pore plate according to the volume ratio of 7:32 compared with the hydrogel, and reacting for 25 minutes at 37 ℃; finally, add 0.6mmol L^-1Adding OPD into the pore plate according to the volume ratio of 2:195 compared with the mixed solution, and continuing to react for 10 minutes at 50 ℃; shooting the change of the fluorescence color of the gel under the irradiation of an ultraviolet lamp, and analyzing the color of the hydrogel by using Image J software;

C. and (3) actual sample detection:

tap water, Songhua river water, orange juice and milk actual samples are used as detection targets to verify the actual application of the hydrogel kit, and 10.0mL of water sample is filtered by a 0.22mm membrane; diluting milk by 50 times; processing orange juice with juicer, diluting 50 times, adding 5, 25, 100 and 250ng mL^-1Adding 10mL of acetonitrile into the paraoxon standard solution, performing ultrasonic treatment for 5min, oscillating for 30min, centrifuging for 10 min at 4000rpm, and detecting by adopting the method;

the processing method of the pakchoi sample comprises the following steps: firstly, after the pakchoi is cultured for 10 days, spraying 10mL of paraoxon solution (5ppm) to one group of pakchoi every day, and continuously spraying for 3 days; spraying 10mL of ultrapure water by the same method as a control, and extracting residual paraoxon in the leaves and roots of the pakchoi according to the extraction method after spraying; finally, the rate-type fluorescent hydrogel kit constructed by the method is used for detecting the Chinese cabbage extract degraded for different days after spraying the pesticide.

2. The preparation method of the hydrogel kit for monitoring degradation of organophosphorus pesticide according to claim 1, wherein the MnCl in the step A is adopted₂Has a concentration of 50mmol L^-1The concentration of NaOH is 1mol L^-1。

3. The method for preparing the hydrogel kit for monitoring the degradation of organophosphorus pesticide according to claim 1, wherein the whole synthesis process in the step A is performed at room temperature.

4. The preparation method of the hydrogel kit for monitoring degradation of organophosphorus pesticide according to claim 1, wherein the concentration of the SA solution in the step B is 20mg mL^-1，Ca(NO₃)₂Is 10.0mg mL^-1。

5. The method for preparing the hydrogel kit for monitoring the degradation of organophosphorus pesticide according to claim 1, wherein the Tris-HCl buffer solution in the step C has a pH of 8.0 and a concentration of 100mmol L^-1。

6. The preparation method of the hydrogel kit for monitoring the degradation of organophosphorus pesticide according to claim 1, wherein in the fluorescence value measurement in the step C, the AuNCs parameters are set to have an excitation wavelength of 578nm and an emission wavelength of 650 nm; for 2, 3-diaminophenyloxazine, DAP for short, the parameters were set to an excitation wavelength of 442nm and an emission wavelength of 550 nm.

7. The preparation method of the hydrogel kit for monitoring degradation of organophosphorus pesticide as claimed in claim 1, wherein the scanning range of the ultraviolet-visible absorption spectrum detection in the step C is 700-300 nm.