Preparation method of singlet oxygen chemiluminescence probe and application thereof

Abstract

This invention discloses a method for preparing a chemiluminescent probe for detecting singlet oxygen. The chemiluminescent probe is made from melamine and rare earth element Eu, using Eu-modified carbon nitride nanoparticles. Utilizing the nitrogen defect property of the Eu-CNNP probe, the generation of singlet oxygen is accelerated, leading to energy transfer between the singlet oxygen dimer and the nanoprobe. The detection of singlet oxygen is achieved using the light signal from the nanoprobe at 520 nm. This method has advantages such as high selectivity, low detection cost, and simple operation.

Description

Technical Field

[0001] This invention belongs to the field of chemiluminescence probe technology, and in particular relates to a method for preparing a singlet chemiluminescence probe for detecting oxidation and its application. Background Technology

[0002] Reactive oxygen species (ROS) in the air are associated with the toxicological effects of various environmental pollutants and atmospheric particulate events. Studies have shown that they are generated by the sensitized oxidative interactions between endogenous persistent free radicals and oxygen, metals, and organoquinones. ROS include hydrogen peroxide, peroxynitrite, hydroxyl radicals, superoxide radicals, organic peroxides, and singlet oxygen. Singlet oxygen, as one of the main oxidizing agents in atmospheric particulate matter, plays a crucial role in secondary free radical photoreactions and damage to various enzyme systems. Therefore, from the perspective of environmental governance and health implications, direct and accurate monitoring of singlet oxygen in atmospheric particulate matter is of great significance.

[0003] The generation of singlet oxygen is often accompanied by the formation of other reactive oxygen species, making the detection of singlet oxygen with high selectivity and sensitivity a challenge. For nanomaterials, the presence of defects can accelerate electron transfer and catalyze chemical reactions. Existing methods for detecting singlet oxygen in atmospheric particulate matter include electron spin resonance, fluorescence, and chromatography. However, the practical application is limited by the need for additional capture reagents and complex pretreatment processes. Chemiluminescence, a detection method based on the luminescence phenomenon of molecules in chemical reactions, is considered one of the most powerful tools for detecting chemical substances due to its high sensitivity, ease of operation, and fast detection speed.

[0004] Currently, no existing patents disclose inventions using melamine and the rare earth element Eu as raw materials to monitor singlet oxygen in atmospheric particulate matter. Therefore, this invention synthesizes a nitrogen-deficient europium-functionalized graphitic carbon nitride nanoprobe (Eu-CNNPs probe) using melamine and Eu as raw materials via a solvothermal method. This probe possesses abundant nitrogen defects, which accelerate electron transfer and induce the generation of a large amount of singlet oxygen. The singlet oxygen aggregates to form singlet oxygen dimers, and then strong chemiluminescence is generated based on the energy transfer between the singlet oxygen dimers and the probe, thus successfully constructing a chemiluminescent probe for detecting singlet oxygen. Summary of the Invention

[0005] The purpose of this invention is to prepare a chemiluminescent probe for detecting singlet oxygen and to use the prepared probe to detect singlet oxygen in atmospheric particulate matter. The detection method adopts the peristaltic pump static injection method. The probe has abundant nitrogen defects, which can accelerate electron transfer and induce the generation of a large amount of singlet oxygen. Then, the singlet oxygen aggregates to form a singlet oxygen dimer. Then, based on the energy transfer between the singlet oxygen dimer and the probe, strong chemiluminescence is generated, thus successfully constructing a chemiluminescent probe for detecting singlet oxygen.

[0006] The technical solution of the present invention is: a chemiluminescent probe for detecting singlet oxidation, wherein the chemiluminescent probe is carbon nitride nanoparticles modified with rare earth element Eu using melamine and rare earth element Eu as raw materials.

[0007] The aforementioned method for preparing a singlet chemiluminescent probe for detection is carried out according to the following steps:

[0008] (1) Melamine was used as a precursor and calcined in a muffle furnace to obtain graphite carbon nitride. After grinding with 98% N-methylpyrrolidone as a solvent, product A was obtained.

[0009] (2) Take product A and transfer it to a round-bottom flask containing 98% N-methylpyrrolidone, and add rare earth element Eu to obtain product B;

[0010] (3) Take product B, add sodium hydroxide to adjust the pH value to 11, heat and reflux, and stir continuously during reflux to obtain product C;

[0011] (4) Centrifuge C, remove N-methylpyrrolidone by rotary evaporation of the supernatant, add acetone to the precipitate, wash with sonication, centrifuge again to obtain the sediment, place it in a vacuum drying oven to dry, and finally dissolve the solid in deionized water to obtain the Eu-CNNPs probe.

[0012] In step (1) above, 4-6g of melamine was placed in a muffle furnace and calcined at 550°C for 4h to obtain graphite carbon nitride. 0.1g of graphite carbon nitride was added to 2mL of 98% N-methylpyrrolidone as a solvent and ground for 10min to obtain product A.

[0013] In step (2) above, product A is transferred to a round-bottom flask containing 98 mL of 98% N-methylpyrrolidone, and 0.0006 g to 0.0599 g of rare earth element Eu is added, wherein the rare earth element Eu is europium trifluoromethanesulfonate, to obtain product B.

[0014] In step (3) above, after adding sodium hydroxide to product B to adjust the pH value to 11, heat and reflux at 140°C for 6 hours, stirring continuously during reflux, to obtain product C.

[0015] In step (4) above, product C is centrifuged at 8000 rpm for 10 min, the supernatant is taken and N-methylpyrrolidone is removed by evaporation using a rotary evaporator, the precipitate is added to 10-20 mL of acetone, washed by sonication for 2 min, centrifuged at 8000 rpm for 10 min, the precipitate obtained after centrifugation is placed in an 80℃ vacuum drying oven and dried for 2 h, the finally obtained dried solid is dissolved in 100 mL of deionized water to obtain the Eu-CNNPs probe.

[0016] The aforementioned application of the chemiluminescent probe for detecting singlet oxygen is described above, whereby the probe is used for the detection of singlet oxygen in atmospheric particulate matter.

[0017] The aforementioned application of the singlet chemiluminescent probe for detection uses a peristaltic pump static injection method.

[0018] Specifically, in the aforementioned application of the singlet chemiluminescent probe for detection, the detection method involves first mixing 800 µL of Eu-CNNPs probe with 200 µL of H2O2 solution in a dedicated luminescent dish, and then placing 200 µL of NaClO solution in a plastic tube. Subsequently, a peristaltic pump is activated to rapidly inject the NaClO solution from the disposable plastic tube into the luminescent dish, while simultaneously turning on the chemiluminescence signal detection instrument to collect the light signal.

[0019] More specifically, the aforementioned application of the chemiluminescent probe for detecting singlet oxidative stresses, and the detection method for ClO - The concentration was controlled at 8 mmol / L, and the detection characteristic wavelength was 520 nm.

[0020] Compared with the prior art, the present invention has the following advantages:

[0021] By utilizing the nitrogen defect properties of Eu-CNNPs probes, the generation of singlet oxygen is accelerated. The large amount of singlet oxygen aggregates to produce singlet oxygen dimers, which then cause energy transfer between the singlet oxygen dimers and the Eu-CNNPs probes. The singlet oxygen is detected by using the optical signal of the Eu-CNNPs probes at 520 nm. This method has the advantages of high selectivity, low detection cost and simple operation. Attached image description:

[0022] Figure 1 TEM images of Eu-CNNP probes;

[0023] Figure 2 HRTEM image of Eu-CNNP probes;

[0024] Figure 3 EDS element mapping of Eu elements in Eu-CNNP probes;

[0025] Figure 4 TEM-EDS images of Eu-CNNP probes;

[0026] Figure 5 Graph showing the variation of chemiluminescence intensity with singlet oxygen concentration;

[0027] Figure 6 Linear fitting calibration curve for singlet oxygen. Detailed Implementation

[0028] 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.

[0029] Example 1:

[0030] 5g of melamine was used as a precursor and calcined in a muffle furnace at 550℃ for 4h to obtain graphitic carbon nitride. 0.1g of graphitic carbon nitride was added to 2mL of 98% N-methylpyrrolidone as a solvent and ground for 10min. The mixture was then transferred to a round-bottom flask containing 98mL of 98% N-methylpyrrolidone, and 0.0599g of europium trifluoromethanesulfonate was added. Sodium hydroxide was added to adjust the pH to 11, and the mixture was refluxed at 140℃ for 6h with continuous stirring during reflux. After reflux, the mixture was centrifuged at 8000rpm for 10min. The supernatant was collected and N-methylpyrrolidone was removed by rotary evaporation, yielding a precipitate. 10mL of acetone was added, and the mixture was washed by sonication for 2min to disperse the precipitate in the acetone. The mixture was then centrifuged at 8000rpm for 10 minutes. After centrifugation, the resulting sediment was dried in an 80°C vacuum drying oven for 2 hours. The final dried solid was dissolved in 100 mL of deionized water to obtain the Eu-CNNPs probe.

[0031] Example 2:

[0032] 4g of melamine was used as a precursor and calcined in a muffle furnace at 550℃ for 4h to obtain graphitic carbon nitride. 0.1g of graphitic carbon nitride was added to 2mL of 98% N-methylpyrrolidone as a solvent and ground for 10min. The mixture was then transferred to a round-bottom flask containing 98mL of 98% N-methylpyrrolidone, and 0.0006g of europium trifluoromethanesulfonate was added. Sodium hydroxide was added to adjust the pH to 11, and the mixture was refluxed at 140℃ for 6h with continuous stirring. After reflux, the mixture was centrifuged at 8000rpm for 10min. The supernatant was collected and N-methylpyrrolidone was removed by rotary evaporation. The resulting precipitate was then washed with 20mL of acetone and sonicated for 2min to disperse the precipitate in the acetone. The mixture was then centrifuged at 8000rpm for 10min. After centrifugation, the resulting sediment was dried in an 80°C vacuum drying oven for 2 hours. The final dried solid was dissolved in 100 mL of deionized water to obtain the Eu-CNNPs probe.

[0033] Example 3:

[0034] 6g of melamine was used as a precursor and calcined in a muffle furnace at 550℃ for 4h to obtain graphitic carbon nitride. 0.1g of graphitic carbon nitride was added to 2mL of 98% N-methylpyrrolidone as a solvent and ground for 10min. The mixture was then transferred to a round-bottom flask containing 98mL of 98% N-methylpyrrolidone, and 0.0059g of europium trifluoromethanesulfonate was added. Sodium hydroxide was added to adjust the pH to 11, and the mixture was refluxed at 140℃ for 6h with continuous stirring. After reflux, the mixture was centrifuged at 8000rpm for 10min. The supernatant was collected and N-methylpyrrolidone was removed by rotary evaporation. The resulting precipitate was then washed with 15mL of acetone and sonicated for 2min to disperse the precipitate in the acetone. The mixture was then centrifuged at 8000rpm for 10min. After centrifugation, the resulting sediment was dried in an 80°C vacuum drying oven for 2 hours. The final dried solid was dissolved in 100 mL of deionized water to obtain the Eu-CNNPs probe.

[0035] Example 4:

[0036] 5g of melamine was used as a precursor and calcined in a muffle furnace at 550℃ for 4h to obtain graphitic carbon nitride. 0.1g of graphitic carbon nitride was added to 2mL of 98% N-methylpyrrolidone as a solvent and ground for 10min. The mixture was then transferred to a round-bottom flask containing 98mL of 98% N-methylpyrrolidone, and 0.0006g of europium trifluoromethanesulfonate was added. Sodium hydroxide was added to adjust the pH to 11, and the mixture was refluxed at 140℃ for 6h with continuous stirring. After reflux, the mixture was centrifuged at 8000rpm for 10min. The supernatant was collected and N-methylpyrrolidone was removed by rotary evaporation. The resulting precipitate was then washed with 20mL of acetone and sonicated for 2min to disperse the precipitate in the acetone. The mixture was then centrifuged at 8000rpm for 10 minutes. After centrifugation, the resulting sediment was dried in an 80°C vacuum drying oven for 2 hours. The final dried solid was dissolved in 100 mL of deionized water to obtain the Eu-CNNPs probe.

[0037] Example 5:

[0038] 5g of melamine was used as a precursor and calcined in a muffle furnace at 550℃ for 4h to obtain graphitic carbon nitride. 0.1g of graphitic carbon nitride was added to 2mL of 98% N-methylpyrrolidone as a solvent and ground for 10min. The mixture was then transferred to a round-bottom flask containing 98mL of 98% N-methylpyrrolidone, and 0.0059g of europium trifluoromethanesulfonate was added. Sodium hydroxide was added to adjust the pH to 11, and the mixture was refluxed at 140℃ for 6h with continuous stirring during reflux. After reflux, the mixture was centrifuged at 8000rpm for 10min. The supernatant was collected and N-methylpyrrolidone was removed by rotary evaporation. The resulting precipitate was then washed with 10mL of acetone and sonicated for 2min to disperse the precipitate in the acetone. The mixture was then washed and centrifuged at 8000rpm for 10 minutes. After centrifugation, the resulting sediment was dried in an 80°C vacuum drying oven for 2 hours. The final dried solid was dissolved in 100 mL of deionized water to obtain the Eu-CNNPs probe.

[0039] To verify the beneficial effects of this invention, the inventors conducted extensive experimental research, the process and results of which are as follows:

[0040] I. Detection of Cl Response Signal of Singlet Oxygen by Eu-CNNP Probes

[0041] 1. Reagents

[0042] Europium trifluoromethanesulfonate (a rare earth element, Eu), melamine, and sodium hypochlorite were purchased from Shanghai Titan Technology Co., Ltd.; 98% N-methylpyrrolidone was purchased from Tianjin Kemei Chemical Reagent Co., Ltd.; hydrogen peroxide and acetone were purchased from Chongqing Chuandong Chemical (Group) Co., Ltd.; and sodium hydroxide was purchased from Sinopharm Chemical Reagent Co., Ltd.

[0043] instrument

[0044] Chemiluminescence signals were detected using an ultra-weak chemiluminescence instrument (BPCL-2-TGG, Guangzhou Micro-Light Technology Co., Ltd., China); TEM images of Eu-CNNPs were captured using a Tecnai G2 F20 S-Twin (Thermo Fisher Scientific, USA) with an accelerating voltage set to 200 kV. X-ray energy-dispersive spectroscopy (EDS) was analyzed using a Gemini SEM 300 (Zeiss, Germany).

[0045] Probe preparation method

[0046] 0.1 g of graphitic carbon nitride and a small amount of 98% N-methylpyrrolidone (NMP) were ground together for 10 min and then transferred to a round-bottom flask containing 98% NMP. Subsequently, different amounts of rare earth element Eu (0 g, 0.0006 g, 0.0059 g, and 0.0599 g) and 0.1 g of sodium hydroxide were added to the mixture to adjust the pH to 11. The solution was heated under reflux at 140 °C for 6 hours with continuous stirring. The resulting solution was centrifuged at 8000 rpm for 10 min, and the supernatant was collected and evaporated using a rotary evaporator to remove the organic solvent NMP. The precipitate was then dispersed in acetone and washed by sonication for 2 min, followed by centrifugation at 8000 rpm for 10 min to collect the precipitate. Finally, the precipitate was dried in a vacuum drying oven at 80 °C for 2 h. The resulting dried solid was dissolved in 100 mL of deionized water to obtain the Eu-CNNP probe.

[0047] TEM image detection of Eu-CNNP probes

[0048] TEM images of Eu-CNNP probes are attached. Figure 1 The image shown is of the nanoparticle structure. (See attached HRTEM image). Figure 2 The lattice interstices of the material are found to be 0.320 nm, which is consistent with the (002) crystal plane of graphitic carbon nitride. Additionally, the EDS spectrum (attached) Figure 3-4 This indicates that europium-functionalized graphitic carbon nitride has been successfully obtained.

[0049] Methods and results of probe detection of singlet oxygen

[0050] 5.1 Eu-CNNP probes with different nitrogen vacancies

[0051] 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 introduce 200 µL of NaClO solution into the reaction system via a peristaltic pump. A mixed solution of 800 µL of Eu-CNNPs probe and 200 µL of H₂O₂ is placed in the luminescent dish of the reaction system, reacting with the solution introduced by the peristaltic pump; this constitutes the reaction system. The luminescence signal generated by the reaction is monitored by a BPCL ultra-low luminescence analyzer equipped with a photomultiplier tube. The operating voltage of the photomultiplier tube is set to -1000V, and the data integration time of the BPCL ultra-low luminescence analyzer is 0.1 s.

[0052] The CL response signals of Eu-CNNP probes with different nitrogen defect contents to singlet oxygen are shown in Appendix Table 1.

[0053] Table 1: Nitrogen defect content, CL intensity, and rare earth Eu content parameters of Eu-CNNP probes

[0054]

[0055] 5.2 Simulation of singlet oxygen in the H2O2-NaClO system

[0056] The H₂O₂-NaClO system simulates the generation of singlet oxygen, with the ClO₂ concentration controlled at 8 mmol / L and the Eu-CNNP concentration at 1.125 mg / mL. The H₂O₂ concentration is used for quantitative analysis of singlet oxygen, and the linear relationship of the injection concentration is as follows: Figure 5-6 As shown, the linear range for detecting singlet oxygen is 1.67 × 10⁻⁶. -7 Up to 1.67×10 -5 mol / L, the calculated detection limit is 1.36 × 10⁻⁶. -7 The concentration is mol / L. Therefore, this probe performs well in detecting singlet oxygen and is feasible as a chemiluminescent probe for singlet oxygen.

[0057] Detection of singlet oxygen recovery rate in atmospheric particulate matter

[0058] Sampling points were set up above the chimney opening, and samples were collected from stations in four wind directions: east, west, north, and south. The obtained atmospheric particulate filter paper was stored at -18°C. An appropriate amount of sample was immersed in ultrapure water and ultrasonically treated for 30 min. Then, the solution was treated with a 0.22 µm filter membrane and used for the recovery of singlet oxygen from atmospheric particulate matter. The concentration of Eu-CNNPs was controlled at 1.125 mg / mL, and a concentration of 2 × 10⁻⁶ mg / mL was added to the atmospheric particulate matter samples. -5 4×10 -5 and 8×10 -5 Singlet oxygen at mol / L. After three parallel measurements, as shown in Table 2, the recovery rate of singlet oxygen ranged from 91.54% to 110.85%.

[0059] Table 2: Recovery rate and determination results of singlet oxygen in atmospheric particulate matter samples

[0060]

[0061] 6. Conclusion

[0062] This invention utilizes the rare earth element Eu to regulate the nitrogen defect content of graphitic carbon nitride. As shown in Table 1, a higher Eu content results in a richer nitrogen defect content. When the Eu content is 0.0599 g, both the defect content and the chemiluminescence intensity are significantly improved. To avoid altering the original configuration of graphitic carbon nitride due to excessive Eu content, 0.0599 g of Eu is set as the optimal content. The presence of nitrogen defects accelerates the electron transfer process and generates a large amount of singlet oxygen. The singlet oxygen dimer formed by the aggregation of singlet oxygen undergoes energy transfer with the nanoprobe, thereby enabling the detection of singlet oxygen. Specifically, H2O2-NaClO is a classic singlet oxygen production system. The rare earth Eu-functionalized graphitic carbon nitride exhibits nitrogen defects, which accelerate electron transfer and catalyze the production of singlet oxygen in the H2O2-NaClO system, resulting in a large amount of singlet oxygen. Energy transfer occurs due to the effective overlap between the emission wavelength of the singlet oxygen dimer and the absorption wavelength of the Eu-CNNPs probe, and the energized Eu-CNNPs are excited into Eu-CNNPs. * Finally, Eu-CNNPs * The released energy generates a light signal at a characteristic wavelength of 520 nm. Simultaneously, singlet oxygen was recovered and detected by spiked analysis of actual atmospheric particulate matter samples. The recovery rate of singlet oxygen ranged from 91.54% to 110.85%, indicating that this chemical probe is beneficial for the detection of singlet oxygen in atmospheric particulate matter.

[0063] By leveraging the nitrogen-deficient properties of Eu-CNNP probes, the generation of singlet oxygen is accelerated, leading to energy transfer between the singlet oxygen dimer and the nanoprobe. The detection of singlet oxygen is then achieved using the optical signal from the nanoprobe at 520 nm. This method offers several advantages: firstly, the surface states of the nanomaterial can be easily modulated to achieve singlet oxygen detection; secondly, it demonstrates high feasibility for detecting singlet oxygen in real atmospheric particulate matter samples. In summary, the chemiluminescence method for detecting singlet oxygen based on Eu-CNNP nanoprobes offers advantages such as high selectivity, low detection cost, and ease of operation.

Claims

1. A singlet oxygen chemiluminescence probe for detection, characterized by: The chemiluminescence probe is prepared from melamine and rare earth element Eu, and is a carbon nitride nanoparticle modified by the rare earth element Eu.

2. The method for preparing the singlet chemiluminescent probe as described in claim 1, characterized in that: The preparation method is performed according to the following steps: (1) Melamine is used as a precursor and is calcined in a muffle furnace to obtain graphite carbon nitride, and then 98% N-methyl pyrrolidone is added as a solvent to obtain product A; (2) Product A is moved to a round-bottom flask containing 98% N-methyl pyrrolidone, and rare earth element Eu is added to obtain product B; (3) Product B is added to sodium hydroxide to adjust the pH value to 11, and then heated to reflux, and the reflux is continuously stirred to obtain product C; (4) Product C is centrifuged, the supernatant is taken, and N-methyl pyrrolidone is removed by rotary evaporation to obtain a precipitate, which is washed with acetone by ultrasonic, and then centrifuged to obtain a deposit which is dried in a vacuum drying box, and finally the obtained solid is dissolved in deionized water to obtain the Eu-CNNPs probe.

3. The preparation method of the singlet oxygen chemiluminescence probe according to claim 2, wherein in step (1), 4-6 g of melamine is used as a precursor and is calcined in a muffle furnace at 550 DEG C for 4 h to obtain graphite carbon nitride, and then 0.1 g of the graphite carbon nitride is added to 2 mL of 98% N-methyl pyrrolidone as a solvent to obtain product A after grinding for 10 min.

4. The preparation method of the singlet oxygen chemiluminescence probe according to claim 2, wherein in step (2), product A is moved to a round-bottom flask containing 98 mL of 98% N-methyl pyrrolidone, and 0.0006 g-0.0599 g of rare earth element Eu is added to obtain product B, wherein the rare earth element Eu is europium triflate. In step (3), product B is added to sodium hydroxide to adjust the pH value to 11, and then heated to reflux at 140 DEG C for 6 h, and the reflux is continuously stirred to obtain product C. In step (4), product C is centrifuged at 8000 rpm for 10 min, the supernatant is taken and N-methyl pyrrolidone is removed by rotary evaporation to obtain a precipitate, which is washed with 10-20 mL of acetone by ultrasonic, and then centrifuged at 8000 rpm for 10 min, and the obtained deposit is dried in a vacuum drying box at 80 DEG C for 2 h, and finally the obtained dried solid is dissolved in 100 mL of deionized water to obtain the Eu-CNNPs probe.

5. The method for preparing the singlet chemiluminescent probe as described in claim 2, characterized in that: The probe is used for detecting singlet oxygen in atmospheric particulate matter.

6. The method for preparing the singlet chemiluminescent probe as described in claim 2, characterized in that: The detection method is a static injection method using a peristaltic pump.

7. The application of the singlet chemiluminescent probe for detecting oxidation as described in claim 1, characterized in that: The detection method is that 800 µL of the singlet oxygen chemiluminescence probe is mixed with 200 µL of an H2O2 solution in a special luminescence dish, 200 µL of a NaClO solution is placed in a plastic tube, and then the peristaltic pump is started to quickly inject the NaClO solution in the plastic tube into the luminescence dish, and at the same time, the chemiluminescence signal detection instrument is turned on to collect the light signal.

8. The use of claim 7, wherein the singlet oxygen chemiluminescent probe is detected by the method of claim 1. ​ 9. The application of the singlet chemiluminescent probe for detecting oxidation as described in claim 8, characterized in that: ​ 10. The application of the singlet chemiluminescent probe for detecting oxidation as described in claim 9, characterized in that: The detection method of ClO - The concentration was controlled at 8 mmol / L, and the characteristic wavelength was 520 nm.

Images