Chemiluminescent probe for detecting rogor and preparation method and application thereof

Abstract

The application discloses a chemiluminescent probe for detecting rogor, a preparation method and application of the chemiluminescent probe. The chemiluminescent probe is phosphorus nitride quantum dots synthesized by a one-step hydrothermal method with hexachlorocyclotriphosphazene as a precursor. The probe can accelerate electron transfer, promote FeO4 2‑ to generate a large amount of active oxygen, thereby effectively triggering chemiluminescence. The addition of rogor can further enhance the chemiluminescence of the PNDs / FeO4 2‑ system. Based on this, a chemiluminescence method for detecting rogor is successfully constructed.

Description

Technical Field

[0001] This invention belongs to the field of chemiluminescent probe technology, and in particular relates to a chemiluminescent probe for detecting cycloquinone, its preparation method and application. Background Technology

[0002] Pesticides are widely used in agriculture to improve crop yields and agricultural product quality. However, little is known about the long-term, low-dose effects of these pesticides on public health and non-target species. Therefore, pesticide overuse is widely recognized as causing serious food and environmental pollution, and has become one of the most alarming challenges in the global public health field. Pesticide residue detection is a key approach to pesticide pollution management. Developing efficient, high-throughput, highly sensitive, and high-precision methods for pesticide residue detection is an urgent priority for food safety and environmental monitoring. Pyn is a crop fungicide primarily used to control rice blast and panicle blast. This fungicide is frequently applied to seedlings before transplanting in April and is widely used worldwide. To date, very few studies have reported on the detection of Pyn, and methods have primarily focused on liquid chromatography-mass spectrometry (LC-MS) and gas chromatography-mass spectrometry (GC-MS). However, these methods require complex sample pretreatment, long detection cycles, specialized operators, and expensive equipment. Therefore, establishing a simple and sensitive analytical technique for pyrrolidine is particularly important. Chemiluminescence (CL) is a light radiation phenomenon based on the relaxation of high-energy intermediates to the ground state. It has the advantages of simple equipment, convenient operation, fast analysis speed, easy automation and high-throughput detection.

[0003] Currently, there are no existing patents for detecting quinolones residues in crops based on chemiluminescence phenomena. This invention uses hexachlorocyclotriphosphazene as a raw material and employs a simple one-step hydrothermal method to synthesize electron-rich phosphorus nitride quantum dot nanoprobes (PNDs probes). These probes can accelerate electron transfer, promoting the detection of FeO4+ ions. 2- It generates a large amount of reactive oxygen species, thereby effectively initiating chemiluminescence. The addition of quinolones can further enhance the PNDs / FeO4 ratio. 2- The system exhibits chemiluminescence. Based on this, a chemiluminescent method for detecting quinolones was successfully constructed. Summary of the Invention

[0004] The purpose of this invention is to prepare a chemiluminescent probe for detecting quinolones and to use this probe to detect quinolones in crops. The detection method employs a peristaltic pump static injection method. This probe can accelerate electron transfer and promote the detection of FeO4. 2- The decomposition generates superoxide anions that excite pyrrolidine to produce singlet oxygen. Subsequently, the singlet oxygen aggregates to form a singlet oxygen dimer, which returns to the ground state and produces strong chemiluminescence, thus successfully constructing a chemiluminescent probe for detecting pyrrolidine.

[0005] The technical solution of the present invention: A chemiluminescent probe for detecting cycloquinone, wherein the chemiluminescent probe is a phosphorus nitride quantum dot synthesized in one step via a hydrothermal method using hexachlorocyclotriphosphazene as a precursor.

[0006] The aforementioned method for preparing the chemiluminescent probe for detecting quinolones is carried out according to the following steps: (1) Dissolve 15-25 mg of hexachlorocyclotriphosphazene as a precursor in 15-25 mL of anhydrous ethanol and sonicate for 3-10 min to obtain product A; (2) Take product A and transfer it to a 40-60 ml polytetrafluoroethylene-lined reactor. Heat the reactor at 160-200℃ for 10-14 h and then cool it to room temperature to obtain product B. (3) Take product B in a rotary evaporator and remove anhydrous ethanol by rotary evaporation at 40-60℃. Add 0.8-1.2 mL of acetone to the precipitate and centrifuge at 2500-3500 rpm for 5-15 min. Then dry in a vacuum drying oven at 70-90℃ for 4-7 h to obtain a light yellow solid. Dissolve the obtained light yellow solid in 15-25 mL of deionized water to obtain the PNDs probe.

[0007] In step (1) above, 18-22 mg of hexachlorocyclotriphosphazene was added to 18-22 mL of anhydrous ethanol as a precursor and sonicated for 4-8 min to completely dissolve the hexachlorocyclotriphosphazene to obtain product A.

[0008] Specifically, in step (1) above, 20 mg of hexachlorocyclotriphosphazene is added to 20 mL of anhydrous ethanol as a precursor and sonicated for 5 min to completely dissolve the hexachlorocyclotriphosphazene to obtain product A.

[0009] In step (2) above, product A is transferred to a 45-55 ml polytetrafluoroethylene-lined reactor, heated at 170-190°C for 11-13 h, and then cooled to room temperature to obtain product B.

[0010] Specifically, in step (2) above, product A is transferred to a 50ml polytetrafluoroethylene-lined reactor, heated at 180°C for 12 hours, and then cooled to room temperature to obtain product B.

[0011] In step (3) above, product B is placed in a rotary evaporator and anhydrous ethanol is removed by rotary evaporation at 50°C. The precipitate is then added to 1 mL of acetone and centrifuged at 3000 rpm for 10 min. After centrifugation, the precipitate is dried in a vacuum drying oven at 80°C for 5 h to obtain a pale yellow solid. The obtained pale yellow solid is dissolved in 20 mL of deionized water to obtain the PNDs probe.

[0012] The aforementioned application of the chemiluminescent probe for detecting quinolones is described above, and the probe is used for the detection of quinolones in crops.

[0013] The aforementioned application of the chemiluminescent probe for detecting quinolones involves a detection method using a peristaltic pump static injection technique.

[0014] The aforementioned detection method involves first mixing 200 µL of PNDs probe with 100 µL of cycloquinone solution in a dedicated luminescent dish, and then adding 200 µL of FeO4... 2- The solution was placed in a plastic tube, and then a peristaltic pump was started to remove the FeO4 from the disposable plastic tube. 2- The solution was rapidly injected into the luminescent dish, FeO4 2- The concentration was controlled at 0.001 mol / L, and at the same time, the chemiluminescence signal detection instrument was turned on to collect the light signal.

[0015] Compared with the prior art, the present invention has the following advantages: By utilizing the electron-rich properties of PNDs probes, the generation of superoxide anions is accelerated, which excites roxiquinone to produce a large amount of singlet oxygen. Subsequently, the singlet oxygen aggregates to form singlet oxygen dimers, which return to the ground state and generate strong chemiluminescence. The light signal of singlet oxygen at 475 nm is used to detect roxiquinone. This method has the advantages of high selectivity, low detection cost and simple operation. Attached Figure Description

[0016] Figure 1 TEM image of PNDs probe; Figure 2 HRTEM image of PNDs probe; Figure 3 XRD patterns of PNDs probes; Figure 4 Graph showing the change in chemiluminescence intensity as a function of pyrrolizidine concentration; Figure 5 Linear fitting calibration curve of quinolones. Detailed Implementation

[0017] The present invention will be further described below with reference to embodiments, but these embodiments are not intended to limit the scope of the invention.

[0018] Example 1.

[0019] Preparation method of quinolone chemiluminescent probe: (1) Add 20 mg of hexachlorocyclotriphosphazene as a precursor to 20 mL of anhydrous ethanol and sonicate for 5 min to completely dissolve the hexachlorocyclotriphosphazene to obtain product A; (2) Take product A and transfer it to a 50ml polytetrafluoroethylene-lined reactor. Heat it at 180℃ for 12 hours and then cool it to room temperature to obtain product B. (3) Take product B in a rotary evaporator and remove anhydrous ethanol by rotary evaporation at 50°C. Add 1 mL of acetone to the precipitate and centrifuge at 3000 rpm for 10 min. Then dry it in a vacuum drying oven at 80°C for 5 h to obtain a light yellow solid. Dissolve the obtained light yellow solid in 20 mL of deionized water to obtain the PNDs probe.

[0020] Example 2.

[0021] Preparation method of quinolone chemiluminescent probe: (1) Dissolve 25 mg of hexachlorocyclotriphosphazene as a precursor in 15 mL of anhydrous ethanol and sonicate for 10 min to obtain product A; (2) Take product A and transfer it to a 40ml polytetrafluoroethylene-lined reactor. Heat it at 200℃ for 14 hours and then cool it to room temperature to obtain product B. (3) Take product B in a rotary evaporator and remove anhydrous ethanol by rotary evaporation at 60°C. Add 1.2 mL of acetone to the precipitate and centrifuge at 2500 rpm for 15 min. Then dry it in a vacuum drying oven at 90°C for 4 h to obtain a light yellow solid. Dissolve the obtained light yellow solid in 25 mL of deionized water to obtain the PNDs probe.

[0022] Example 3.

[0023] Preparation method of quinolone chemiluminescent probe: (1) Dissolve 15 mg of hexachlorocyclotriphosphazene as a precursor in 25 mL of anhydrous ethanol and sonicate for 3 min to obtain product A; (2) Take product A and transfer it to a 60ml polytetrafluoroethylene-lined reactor. Heat it at 160℃ for 10h and then cool it to room temperature to obtain product B. (3) Take product B in a rotary evaporator and remove anhydrous ethanol by rotary evaporation at 60°C. Add 0.8 mL of acetone to the precipitate and centrifuge at 3500 rpm for 5 min. Then dry it in a vacuum drying oven at 70°C for 7 h to obtain a light yellow solid. Dissolve the obtained light yellow solid in 15 mL of deionized water to obtain the PNDs probe.

[0024] Example 4.

[0025] Preparation method of quinolone chemiluminescent probe: (1) Dissolve 18 mg of hexachlorocyclotriphosphazene as a precursor in 22 mL of anhydrous ethanol and sonicate for 7 min to obtain product A; (2) Take product A and transfer it to a 45ml polytetrafluoroethylene-lined reactor. Heat it at 170℃ for 12 hours and then cool it to room temperature to obtain product B. (3) Take product B in a rotary evaporator and remove anhydrous ethanol by rotary evaporation at 55°C. Add 0.9 mL of acetone to the precipitate and centrifuge at 3000 rpm for 10 min. Then dry it in a vacuum drying oven at 75°C for 6 h to obtain a light yellow solid. Dissolve the obtained light yellow solid in 18 mL of deionized water to obtain the PNDs probe.

[0026] Example 5.

[0027] Preparation method of quinolone chemiluminescent probe: (1) Dissolve 22 mg of hexachlorocyclotriphosphazene as a precursor in 18 mL of anhydrous ethanol and sonicate for 8 min to obtain product A; (2) Take product A and transfer it to a 55ml polytetrafluoroethylene-lined reactor. Heat it at 190℃ for 11 hours and then cool it to room temperature to obtain product B. (3) Take product B in a rotary evaporator and remove anhydrous ethanol by rotary evaporation at 45°C. Add 1.1 mL of acetone to the precipitate and centrifuge at 3000 rpm for 8 min. Then dry it in a vacuum drying oven at 85°C for 5 h to obtain a pale yellow solid. Dissolve the obtained pale yellow solid in 22 mL of deionized water to obtain the PNDs probe.

[0028] Example 6.

[0029] Detection method of quinolones using chemiluminescent probes: Mix 200 µL of PNDs probe with 100 µL of cyclophosphamide solution in a dedicated luminescent dish, and separately add 200 µL of FFeO4. 2- The solution was placed in a plastic tube, and then a peristaltic pump was started to remove the FeO4 from the disposable plastic tube. 2- The solution was rapidly injected into the luminescent dish, FeO4 2- The concentration was controlled at 0.001 mol / L, and at the same time, the chemiluminescence signal detection instrument was turned on to collect the light signal.

[0030] To verify the beneficial effects of this invention, the inventors conducted extensive experimental research, the process and results of which are as follows: 1. Reagents Hexachlorocyclotriphosphazene (PCT) and potassium ferrate (K2FeO4) were purchased from Shanghai Titan Technology Co., Ltd.; pyrrolizidine ether (Pyn) was purchased from Tanmo Quality Inspection Technology Co., Ltd.; acetone was purchased from Chongqing Chuandong Chemical (Group) Co., Ltd. 2 Instruments Chemiluminescence signals were detected using an ultra-weak chemiluminescence instrument (BPCL-2-TGG, Guangzhou Weiguang Technology Co., Ltd., China); TEM images of PNDs were taken using a Tecnai G2 F20 S-Twin (Thermo Fisher Scientific, USA) with an accelerating voltage set to 200 kV. X-ray diffraction (XRD) analysis was performed using an X'PertPROMPD (Panaco, Netherlands). The PTFE-lined reactor was purchased from Xi'an Hongchen Instrument Equipment Co., Ltd.

[0031] 3. PNDs probe preparation method 20 mg of hexachlorocyclotriphosphazene (PCT) was mixed with 20 mL of ethanol and then transferred to a polytetrafluoroethylene-lined autoclave. The mixture was reacted at 180 °C for 12 h. After cooling to room temperature, the ethanol was removed by rotary evaporation at 50 °C, yielding a precipitate. 1 mL of acetone was added, and the mixture was centrifuged at 3000 rpm for 10 min to obtain the precipitate. Finally, the precipitate was dried under vacuum at 70 °C for approximately 6 h to obtain a pale yellow solid. For use, the obtained product is dissolved in deionized water to form a solution containing PNDs (1 mg / mL), thus obtaining the PNDs probe.

[0032] 4. TEM image characterization of PNDs probes TEM images of PNDs probes are shown in the attached image. Figure 1 The image shown is a nanostructure. (See attached HRTEM image.) Figure 2 The lattice spacing of the material is 0.19 nm, corresponding to the (200) crystal plane in the XRD pattern. Additionally, the XRD pattern (see attached image)... Figure 3 The red curve represents the XRD pattern of PNDs, and the blue curve represents the standard card of the raw material PCT. TEM and XRD experiments show that phosphorus nitride quantum dots were successfully obtained.

[0033] 5. Methods and results of PNDs luminescent probe detection of pyrrolizidine ketones 5.1 PNDs / FeO4 2- Construction of Pyn Chemiluminescence System This invention utilizes a traditional chemiluminescence device with a peristaltic pump static injection method for detection, comprising a sample introduction system, a reaction system, and a detection system. The main function of the sample introduction system is to deliver 200 μL of FeO4 via a peristaltic pump. 2- The solution was introduced into the reaction system. A mixed solution of 200 μL PNDs luminescent probes and 100 μL Pyn was placed in the luminescent dish of the reaction system and reacted with the solution introduced by the peristaltic pump, thus forming the reaction system. The luminescence signal generated by the reaction was monitored by a BPCL ultra-low luminescence analyzer equipped with a photomultiplier tube. The operating voltage of the photomultiplier tube was set to 1000V, and the data integration time of the BPCL ultra-low luminescence analyzer was set to 0.1s.

[0034] 5.2 PNDs / FeO4 2- / Detection of Pyn using Pyn chemiluminescence system PNDs / FeO4 2- The / Pyn system was used to detect Pyn,FeO4 in wheat seedlings. 2- The concentration was controlled at 0.001 mol / L, and the concentration of PNDs was 1 mg / mL. The Pyn concentration was used for the quantitative analysis of Pyn; the injection concentration is shown in the attached figure. Figure 4 As shown, the linear range of the detection Pyn is 1.1 × 10⁻⁶. -7 -9.2×10 -7 With a concentration of mol / L, the detection limit is calculated to be 14 nM. Therefore, this probe exhibits good performance in detecting Pyn and is feasible as a chemiluminescent probe for Pyn.

[0035] 5.3 Detection of Pyn in wheat seedlings Two batches of wheat seeds were cultured simultaneously under the same conditions. The first batch was cultured in a sterile nutrient solution containing 50 mg / L quinolones, with the leaves sprayed with a 50 mg / L quinolones solution for 10 days. The second batch was cultured in a sterile nutrient solution containing 50 mg / L quinolones, with the leaves sprayed with ultrapure water for 10 days. Once the wheat plants reached 20-30 cm in height, the first batch of plants were collected, dried with paper towels, and then divided into rootstock and leaf parts, which were weighed. The plants were then thoroughly crushed and dissolved in 100 mL of ultrapure water, sonicated for 30 min, and centrifuged at 4000 rpm for 10 min to remove impurities. This process was repeated three times to extract Pyn. The external residual Pyn concentrations in the rootstock and leaves were determined to be 10.4 μg / g and 7.3 μg / g, respectively. The second batch of plants was collected, the surface Pyn thoroughly washed with ultrapure water, dried with paper towels, and then treated in the same manner as described above. The internal absorption concentrations of Pyn in the root and stem parts were determined to be 0.32 μg / g and 0.14 μg / g, respectively. After three parallel measurements, as shown in Table 1, the recoveries of Pyn ranged from 96% to 112.3%.

[0036] Calculation method: I = 455.98[Pyn] × 10 -7 +346.15, substitute the chemiluminescence intensity of the actual sample into this equation to obtain the corresponding concentration (see appendix). Figure 5 ).

[0037] Table 1. Using PNDs / FeO4 2- Determination of Pyn in wheat using luminescent systems

[0038] 6 Conclusions This invention develops a novel chemiluminescence strategy for detecting the pesticide quinclorac. PNDs catalyze FeO4 2- Decomposition produces superoxide anions (O2) •- Pyn was then reacted with superoxide anions (O2). •- Activation produces a large amount of singlet oxygen 1 O2, singlet oxygen 1 O2 dimers to become excited molecules ( 1 O2)2 * When it returns to its ground state, it releases a strong chemiluminescence at 475 nm. In this context, we developed a highly sensitive detection method for Pyn's PNDs-FeO4. 2- A chemiluminescence sensing platform was used to successfully detect the residual amount of Pyn on the external surface and the trace amount absorbed internally in the roots, stems and leaves of wheat cultures. In summary, the chemiluminescence method for detecting Pyn based on PND nanoprobes offers advantages such as high selectivity, low detection cost, and ease of operation.

Claims

1. The application of a chemiluminescent probe for detecting cycloquinone, characterized in that: The probe is used for the detection of quinolones in crops; The detection method uses a peristaltic pump static injection method. The detection method involves first mixing 200 µL of PNDs probe with 100 µL of cycloquinone solution in a dedicated luminescent dish, and then adding 200 µL of FeO4. 2- The solution was placed in a plastic tube, and then a peristaltic pump was started to remove the FeO4 from the plastic tube. 2- The solution was rapidly injected into the luminescent dish, FeO4 2- The concentration was controlled at 0.001 mol / L. At the same time, the chemiluminescence signal detection instrument was turned on to collect the light signal, the working voltage of the photomultiplier tube was set to -1000V, and the data integration time of the BPCL ultra-weak luminescence analyzer was 0.1s. The chemiluminescent probe is a phosphorus nitride quantum dot synthesized in one step via a hydrothermal method using hexachlorocyclotriphosphazene as a precursor. The preparation method is carried out according to the following steps: (1) Dissolve 15-25 mg of hexachlorocyclotriphosphazene as a precursor in 15-25 mL of anhydrous ethanol and sonicate for 3-10 min to obtain product A; (2) Take product A and transfer it to a 40-60 ml polytetrafluoroethylene-lined reactor. Heat the reactor at 160-200℃ for 10-14 h and then cool it to room temperature to obtain product B. (3) Take product B in a rotary evaporator and remove anhydrous ethanol by rotary evaporation at 40-60℃. Add 0.8-1.2 mL of acetone to the precipitate and centrifuge at 2500-3500 rpm for 5-15 min. Then dry in a vacuum drying oven at 70-90℃ for 4-7 h to obtain a light yellow solid. Dissolve the obtained light yellow solid in 15-25 mL of deionized water to obtain the PNDs probe.

2. The application as described in claim 1, characterized in that: In step (1), 18-22 mg of hexachlorocyclotriphosphazene is added as a precursor to 18-22 mL of anhydrous ethanol and sonicated for 4-8 min to completely dissolve the hexachlorocyclotriphosphazene to obtain product A.

3. The application as described in claim 2, characterized in that: In step (1), 20 mg of hexachlorocyclotriphosphazene is added to 20 mL of anhydrous ethanol as a precursor and sonicated for 5 min to completely dissolve the hexachlorocyclotriphosphazene to obtain product A.

4. The application as described in claim 1, characterized in that: In step (2), product A is transferred to a 45-55 ml polytetrafluoroethylene-lined reactor, heated at 170-190°C for 11-13 h, and then cooled to room temperature to obtain product B.

5. The application as described in claim 4, characterized in that: In step (2), product A is transferred to a 50ml polytetrafluoroethylene-lined reactor, heated at 180°C for 12 hours, and then cooled to room temperature to obtain product B.

6. The application as described in claim 1, characterized in that: In step (3), product B is placed in a rotary evaporator and anhydrous ethanol is removed by rotary evaporation at 50°C. The precipitate is then added to 1 mL of acetone and centrifuged at 3000 rpm for 10 min. After centrifugation, the precipitate is dried in a vacuum drying oven at 80°C for 5 h to obtain a pale yellow solid. The obtained pale yellow solid is dissolved in 20 mL of deionized water to obtain the PNDs probe.

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