In vitro detection method for reactive oxygen free radical concentration simulating the health effects of PM2.5

Abstract

The present invention discloses a method for detecting the concentration of active oxygen free radicals for simulating the health effects of PM2.5 in vitro, comprising the following steps: step (1) obtaining a sample solution; step (2) preparing a reagent A; step (3) preparing a reagent B; step (4) a detection process; and step (5) converting and calculating the characteristic function of the extinction intensity ΔI-free radical concentration c to obtain the free radical concentration in the sample solution. The present invention belongs to the field of biotechnology science and technology, utilizes a photoluminescent agent as background light, has obvious detection characteristics, high precision, can well simulate and study the effects of PM2.5 on various systems in the body, and provides a reference for evaluating the concentration of active oxygen free radicals generated by PM2.5 in the human body.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology science, and in particular to a method for detecting the concentration of active oxygen free radicals by simulating the health effects of PM2.5 in vitro. Background Art

[0002] Particulate matter (particle size ≤ 2.5 μm) Particulate matter (PM2.5) refers to particles in the ambient air with an aerodynamic equivalent diameter of ≤ 2.5 μm, also known as fine particulate matter, which is an important indicator of environmental pollutants. Transition metals and quinones in the atmosphere are important components of PM2.5, which can induce cellular oxidative stress and produce reactive oxygen free radicals. Reactive oxygen species (ROS) broadly refers to free radicals and non-free radicals derived from oxygen, including superoxide anions (O 2 - ), hydrogen peroxide (H 2 O 2 ), hydroxyl radical (OH-), ozone (O 3 ) and singlet oxygen (O 2 ), which are highly chemically reactive because they contain unpaired electrons.

[0003] In the prior art, CN106248811A discloses a method for detecting the generation of reactive oxygen free radicals in the human body by atmospheric fine particulate matter. The detection method comprises the following steps: 1) collecting atmospheric fine particulate matter as a test sample; 2) ultrasonically extracting the test sample in an artificial lung fluid, the artificial lung fluid being composed of a phosphate buffer solution, sodium benzoate (·OH free radical probe) and four antioxidants, and the pH value of the solution being between 7.2 and 7.4; 3) after the reaction is completed, the solution is acidified to a pH value of 2, and a liquid chromatograph is used to detect the addition product of ·OH and benzoic acid, p-hydroxybenzoic acid, and based on the generation of reactive oxygen free radicals, the oxidative damage effect of atmospheric fine particulate matter on the human body is evaluated. CN109839498A discloses a method for evaluating the effect of PM2.5 full chemical component particles on intracellular free radical metabolism. By exposing human lung-bronchial epithelial cells to PM2.5 particles with full chemical composition, liquid chromatography-mass spectrometry (LC-MS) was used to effectively analyze intracellular small molecule metabolites, combined with biological detection methods to quickly analyze enzyme activity, integrating changes in small molecule metabolites related to intracellular free radical metabolism and changes in the activity of rate-limiting enzymes that play a key role in energy metabolism. In summary, a new method based on molecular biology was constructed to evaluate the interference of intracellular oxygen free radical metabolism after cells were exposed to the toxic effects of exogenous pollution.

[0004] Existing technologies mostly use liquid chromatography-mass spectrometry for detection, which has a low ability to eliminate noise interference and is not conducive to high-precision detection. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides an in vitro detection method for the concentration of reactive oxygen free radicals that simulates the health effects of PM2.5. The method utilizes a photoluminescent agent as background light, has obvious detection characteristics and high precision, and can well simulate the effects of PM2.5 on various systems in the body, providing a reference for the evaluation of the concentration of reactive oxygen free radicals generated by PM2.5 in the human body.

[0006] The method for detecting the concentration of active oxygen free radicals by simulating the health effects of PM2.5 in vitro comprises the following steps:

[0007] Step (1) obtaining a sample solution: placing a sampling membrane in a test environment for 3-10 days, recovering the sampling membrane and soaking it in simulated body fluid for 1-3 days, and removing the sampling membrane to obtain a sample solution;

[0008] Step (2) preparing reagent A: adding a photoluminescent agent and a dispersant into deionized water to obtain reagent A; the concentration of the photoluminescent agent in the reagent A is 0.01-0.2 g / L, and the concentration of the dispersant is 0.001-0.002 g / L;

[0009] Step (3) preparing reagent B: under light-shielding conditions, place 0.01-0.1 g of the active agent in 100 mL of deionized water to obtain reagent B;

[0010] Step (4) Detection process: The effective detection volume of the reaction pool of the light detector is 1. 0.5 volume of reagent A is added to the reaction pool of the light detector, followed by 0.5 volume of deionized water. The light intensity is measured as I 0 ; 0.5 volume of reagent A was added to the reaction cell of the light detector, followed by 0.5 volume of reagent B, and the light intensity was measured to be 1 1 ; 0.5 volume of reagent A was added to the reaction cell of the light detector, and then 0.5 volume of sample solution was added, and the light intensity was measured to be I 2 ; Add 0.5 volume of reagent A to the reaction cell of the light detector, then add a volume of reagent B and b volume of sample solution, and measure the light intensity to be I 3 ; Calculate the extinction intensity of the reaction as ΔI=I 0 -I 3 -2a(I 0 -I 1 )-2b(I 0 -I 2 ), a+b=0.5;

[0011] Step (5) converts and calculates the characteristic function of extinction intensity ΔI-free radical concentration c to obtain the free radical concentration in the sample solution.

[0012] The sampling membrane is placed in simulated body fluid for 1-3 days, and the sample liquid obtained by taking out the sampling membrane is used to simulate the metabolic process of PM2.5 particles after entering the respiratory system, digestive system, circulatory system, etc. Therefore, different simulation liquids should be used to study the impact of different locations.

[0013] Reagent A is a suspension of photoluminescent agent. The selected photoluminescent agent is essentially a long afterglow luminescent material with two basic properties: 1. Stable emission of light waves of a specific wavelength after light treatment; 2. The characteristic wavelength is obvious, and there are multiple emission wavelengths, and the two wavelengths are far apart, and the second wavelength can be absorbed by free radical reactions. The concentration of the photoluminescent agent should not be too high. If it is too high, it will cause the background light to be too strong, resulting in too low extinction intensity value, and increase the equipment detection requirements.

[0014] The reactive agent of reagent B can be dissolved salt, organic matter, or insoluble particles. If the insoluble particles are used for this test, there are two requirements: 1. They can absorb the second wavelength of the photoluminescent agent and react with free radicals; 2. They are fine enough and can be dispersed in aqueous solution. Since water itself has a certain amount of free radicals, light-shielding conditions should be preferred when preparing reagent B. Otherwise, under natural light conditions, the reactive agent in reagent B will react with free radicals in water, affecting the final result.

[0015] Step (4) is mainly to eliminate background noise. Reagent A has stable luminescence characteristics, so it serves as a background light source. However, when reagent A is mixed with reagent B or sample solution, complex changes occur in the system. However, since the actual content of each component is relatively small, the dilution process of reagent A as the background is accompanied by system changes such as free radical reactions and active substance scattered light. Therefore, if it is an initial test sample, it is necessary to eliminate the noise when each component is introduced separately. This has never been mentioned in previous detection methods. During the experiment, this scheme found that the mixing of multiple components can be simply calculated in a weighted manner. Therefore, when reagent A is added to reagent B and sample solution, the actual background light intensity I of the system is 4 Should be I 4 =I 0 -2a(I 0 -I 1 )-2b(I 0 -I 2 ), a+b=0.5.

[0016] The characteristic function of extinction intensity ΔI-free radical concentration c can be obtained by using the standard solution, in which the extinction intensity ΔI is proportional to the free radical concentration.

[0017] Preferably, in step (1), the sampling membrane is a PE filter membrane with a pore size of 100 nm.

[0018] PE filter membrane is a hydrophobic linear polymer and is not sensitive to free radicals. Therefore, even if PE nanoparticles enter the sample liquid, it will not affect the final test results.

[0019] Preferably, in step (2), the photoluminescent agent is a rare earth doped nano-photoluminescent agent, which comprises the following preparation method:

[0020] Step (2-a): Add 1 volume of tetrabutyl titanate Ti(OBu) 4 Add to 1-5 volumes of alcohol solvent to obtain solution C, then add 0.7-1 volumes of monobasic organic acid solution with a mass concentration of 80%-90% to solution C, stir thoroughly, and then add soluble aluminum salt and soluble rare earth salt to obtain a preform;

[0021] Step (2-b): the preform is allowed to stand in air to obtain a sol, which is then dried and subjected to high temperature treatment to obtain a rare earth-doped nano-photoluminescent agent.

[0022] The rare earth doped nano photoluminescent agent prepared by the above method is essentially rare earth doped titanium aluminum oxide. Since the rare earth provides a step for the energy bands of titanium oxide and aluminum oxide after rare earth doping, the process of electron transition from the conduction band to the valence band has more transition platforms than the titanium aluminum oxide without rare earth doping, so that the emitted light has multiple wavelengths. At the same time, since the rare earth doped nano photoluminescent agent provided by the present invention also has catalytic properties, after the reagent B and the sample solution are added, the second wavelength of the rare earth doped nano photoluminescent agent provides energy for the reaction. The special structure of the rare earth doped nano photoluminescent agent reduces the reaction activation energy when the reagent B and the sample solution are added, so that it reacts quickly under the second wavelength condition.

[0023] Preferably, the alcohol solvent in step (2-a) is ethanol or propanol, the monobasic organic acid is acetic acid or propionic acid, and the soluble aluminum salt is selected from Al(NO 3 ) 3 、AlCl 3 、Al 2 (SO 4 ) 3 ; The soluble rare earth salt is one or two of the nitrates or chlorides of La, Eu, Tb or Dy.

[0024] Preferably, the molar ratio of n-butyl titanate: soluble aluminum salt: soluble rare earth salt is 1: 0.001-0.005: 0.001-0.016.

[0025] Preferably, the high temperature treatment in step (2-b) is carried out by keeping the temperature at 900-1050°C for 10-30 min in an anaerobic environment, and the heating process of the high temperature treatment is 5-10°C / min from room temperature to 600°C, and 2-3°C / min above 600°C.

[0026] To reduce the occurrence of side reactions, high-temperature treatment needs to take place in an anaerobic environment. The overall reaction is divided into three stages. The first stage is the volatilization and carbonization of organic matter, the second stage is the decomposition of soluble salts, and the third stage is the occurrence of solid-phase reaction. In the actual preparation control process, it is mainly necessary to control the heating rate and insulation time of the solid-phase reaction stage, which determines the final particle size of the rare earth-doped nanophotoluminescent agent.

[0027] Preferably, the photoluminescent agent comprises two or more emission wavelengths.

[0028] The emission wavelengths of the rare earth-doped nano-photoluminescent agent prepared in this scheme are respectively a first wavelength of 464 nm and a second wavelength of 532 nm.

[0029] Preferably, the particle size of the photoluminescent agent is 10nm-200nm.

[0030] Preferably, in step (2), the dispersant is polyvinyl pyrrolidone (PVP).

[0031] Preferably, in step (3), the reactive substance is polypropylene nanoparticles.

[0032] Preferably, the reactive agent is placed in deionized water and a dispersant having a reactive concentration of 0.1%-1% is added for dispersion treatment, wherein the dispersant is a silane dispersant.

[0033] Preferably, in step (4), the reagent A is subjected to light treatment before being added to the reaction cell of the light detector, and the light treatment is irradiation under natural light for 5-10 minutes.

[0034] Preferably, the characteristic function of extinction intensity ΔI-free radical concentration c in step (5) is ΔI=13253c+3246, R 2 =0.999.

[0035] The characteristic function of extinction intensity ΔI-free radical concentration c is obtained by fitting a large number of standard solution detection results in this scheme, and has a high confidence level.

[0036] Compared with the existing technology, the advantages of this solution are:

[0037] This scheme uses photoluminescent agents as background light, which has obvious detection characteristics and high accuracy. It can well simulate the impact of PM2.5 on various systems in the body and provide a reference for the evaluation of the concentration of active oxygen free radicals generated by PM2.5 in the human body.

[0038] In this scheme, the preferred scheme adds europium-doped nano-photoluminescent agents with catalytic reaction effects. At the same time, this scheme fully considers various influencing parameters of the sample preparation system and the reaction system, and has a high degree of credibility.

[0039] In this solution, the second wavelength of the rare earth-doped nanophotoluminescent agent can trigger the reactive agent to react with free radicals, making the detection result more accurate and efficient. DETAILED DESCRIPTION

[0040] The following is a description of the implementation of the present invention by specific embodiments. People familiar with the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0041] Example 1: A method for detecting the concentration of active oxygen free radicals by simulating the health effects of PM2.5 in vitro, comprising the following steps:

[0042] Step (1) obtaining a sample solution: placing a PE filter membrane with a thickness of 0.3 mm, a diameter of 5 cm, and a pore size of 100 nm in a dry place outdoors for 5 days, recovering the PE filter membrane and immersing it in simulated body fluid for 3 days, and taking out the sampling membrane to obtain a sample solution;

[0043] Step (2) preparing reagent A: adding a photoluminescent agent and a dispersant into deionized water to obtain reagent A; the concentration of the photoluminescent agent in the reagent A is 0.1 g / L, and the concentration of polyvinyl pyrrolidone is 0.001 g / L; the photoluminescent agent has two emission wavelengths: a first wavelength of 464 nm and a second wavelength of 532 nm, and a particle size of 10 nm-200 nm;

[0044] Step (3) preparing reagent B: under light-shielding conditions, place 0.05 g of polypropylene nanoparticles in 100 mL of deionized water, and add 0.0005 g of titanate coupling agent 201 to obtain reagent B;

[0045] Step (4) Detection process: The effective detection volume of the reaction pool of the light detector is 1. 0.5 volume of reagent A is irradiated under natural light for 10 minutes and added to the reaction pool of the light detector. Then 0.5 volume of deionized water is added. The light intensity is measured as I 0 ; 0.5 volume of reagent A was added to the reaction cell of the light detector, followed by 0.5 volume of reagent B, and the light intensity was measured to be 1 1 ; 0.5 volume of reagent A was added to the reaction cell of the light detector, and then 0.5 volume of sample solution was added, and the light intensity was measured to be I 2 ; 0.5 volume of reagent A was added to the reaction cell of the light detector, followed by 0.3 volume of reagent B and 0.2 volume of sample solution, and the light intensity was measured to be I 3; Calculate the extinction intensity of the reaction as ΔI=I 0 -I 3 -0.3(I 0 -I 1 )-0.2(I 0 -I 2 ), a+b=0.5;

[0046] Step (5) converts and calculates the characteristic function of extinction intensity ΔI-free radical concentration c to obtain the free radical concentration in the sample solution; ΔI=13253c+3246, R 2 =0.999.

[0047] The photoluminescent agent in step (1) is a rare earth doped nano-photoluminescent agent, which comprises the following preparation method:

[0048] Step (2-a): Add 1 volume of tetrabutyl titanate Ti(OBu) 4 Add to 2 volumes of ethanol to obtain solution C, then add 0.8 volumes of 85% acetic acid solution to solution C, stir thoroughly and add Al(NO 3 ) 3 and Eu(NO 3 ) 3 , the molar ratio of n-butyl titanate: soluble aluminum salt: soluble rare earth salt is 1:0.003:0.01 to obtain a preform;

[0049] Step (2-b): The preform is allowed to stand in air to obtain a sol, and after drying, it is kept at 1000°C in an anaerobic environment for 30 minutes (the heating process is 5°C / min from room temperature to 600°C, and 3°C / min above 600°C) to obtain a rare earth doped nano-photoluminescent agent.

[0050] Example 2: A method for detecting the concentration of reactive oxygen free radicals by simulating the health effects of PM2.5 in vitro, comprising the following steps:

[0051] Step (1) obtaining a sample solution: placing a PE filter membrane with a thickness of 0.3 mm, a diameter of 5 cm, and a pore size of 100 nm in a dry place outdoors for 5 days, recovering the PE filter membrane and immersing it in simulated body fluid for 3 days, and taking out the sampling membrane to obtain a sample solution;

[0052] Step (2) preparing reagent A: adding a photoluminescent agent and a dispersant into deionized water to obtain reagent A; the concentration of the photoluminescent agent in the reagent A is 0.2 g / L, and the concentration of polyvinyl pyrrolidone is 0.001 g / L; the photoluminescent agent has two emission wavelengths: a first wavelength of 464 nm and a second wavelength of 532 nm, and a particle size of 10 nm-200 nm;

[0053] Step (3) preparing reagent B: under light-shielding conditions, place 0.1 g of polypropylene nanoparticles in 100 mL of deionized water, and add 0.001 g of titanate coupling agent 201 to obtain reagent B;

[0054] Step (4) Detection process: The effective detection volume of the reaction pool of the light detector is 1. 0.5 volume of reagent A is irradiated under natural light for 10 minutes and added to the reaction pool of the light detector. Then 0.5 volume of deionized water is added. The light intensity is measured as I 0 ; 0.5 volume of reagent A was added to the reaction cell of the light detector, followed by 0.5 volume of reagent B, and the light intensity was measured to be 1 1 ; 0.5 volume of reagent A was added to the reaction cell of the light detector, and then 0.5 volume of sample solution was added, and the light intensity was measured to be I 2 ; 0.5 volume of reagent A was added to the reaction cell of the light detector, followed by 0.3 volume of reagent B and 0.2 volume of sample solution, and the light intensity was measured to be I 3 ; Calculate the extinction intensity of the reaction as ΔI=I 0 -I 3 -0.3(I 0 -I 1 )-0.2(I 0 -I 2 ), a+b=0.5;

[0055] Step (5) converts and calculates the characteristic function of extinction intensity ΔI-free radical concentration c to obtain the free radical concentration in the sample solution; ΔI=13253c+3246, R 2 =0.999.

[0056] The photoluminescent agent in step (1) is a rare earth doped nano-photoluminescent agent, which includes the following preparation method:

[0057] Step (2-a): Add 1 volume of tetrabutyl titanate Ti(OBu) 4 Add to 2 volumes of ethanol to obtain solution C, then add 0.8 volumes of acetic acid solution with a mass concentration of 85% to solution C, stir thoroughly and add Al(NO 3 ) 3 and Eu(NO 3 ) 3 , the molar ratio of n-butyl titanate: soluble aluminum salt: soluble rare earth salt is 1:0.003:0.01 to obtain a preform;

[0058] Step (2-b): The preform is allowed to stand in air to obtain a sol, and after drying, it is kept at 1000°C in an anaerobic environment for 30 minutes (the heating process is 5°C / min from room temperature to 600°C, and 3°C / min above 600°C) to obtain a rare earth doped nano-photoluminescent agent.

[0059] Example 3: A method for detecting the concentration of reactive oxygen free radicals by simulating the health effects of PM2.5 in vitro, comprising the following steps:

[0060] Step (1) obtaining a sample solution: placing a PE filter membrane with a thickness of 0.3 mm, a diameter of 5 cm, and a pore size of 100 nm in a kitchen flue for 5 days, and recovering the PE filter membrane to obtain a sample solution;

[0061] Step (2) preparing reagent A: adding a photoluminescent agent and a dispersant into deionized water to obtain reagent A; the concentration of the photoluminescent agent in the reagent A is 0.01 g / L, and the concentration of polyvinyl pyrrolidone is 0.001 g / L; the photoluminescent agent has two emission wavelengths: a first wavelength of 464 nm and a second wavelength of 532 nm, and a particle size of 10 nm to 200 nm;

[0062] Step (3) preparing reagent B: under light-shielding conditions, placing 0.01 g of polypropylene nanoparticles in 100 mL of deionized water, and adding 0.0001 g of titanate coupling agent 201 to obtain reagent B;

[0063] Step (4) Detection process: The effective detection volume of the reaction pool of the light detector is 1. 0.5 volume of reagent A is irradiated under natural light for 10 minutes and added to the reaction pool of the light detector. Then 0.5 volume of deionized water is added. The light intensity is measured as I 0 ; 0.5 volume of reagent A was added to the reaction cell of the light detector, followed by 0.5 volume of reagent B, and the light intensity was measured to be 1 1 ; 0.5 volume of reagent A was added to the reaction cell of the light detector, and then 0.5 volume of sample solution was added, and the light intensity was measured to be I 2 ; 0.5 volume of reagent A was added to the reaction cell of the light detector, followed by 0.3 volume of reagent B and 0.2 volume of sample solution, and the light intensity was measured to be I 3 ; Calculate the extinction intensity of the reaction as ΔI=I 0 -I 3 -0.3(I 0 -I 1 )-0.2(I 0 -I 2 ), a+b=0.5;

[0064] Step (5) converts and calculates the characteristic function of extinction intensity ΔI-free radical concentration c to obtain the free radical concentration in the sample solution; ΔI=13253c+3246, R 2 =0.999.

[0065] The photoluminescent agent in step (1) is a rare earth doped nano-photoluminescent agent, which includes the following preparation method:

[0066] Step (2-a): Add 1 volume of tetrabutyl titanate Ti(OBu) 4 Add to 2 volumes of ethanol to obtain solution C, then add 0.8 volumes of acetic acid solution with a mass concentration of 85% to solution C, stir thoroughly and add Al(NO 3 ) 3 and Eu(NO 3 ) 3 , the molar ratio of n-butyl titanate: soluble aluminum salt: soluble rare earth salt is 1:0.003:0.01 to obtain a preform;

[0067] Step (2-b): The preform is allowed to stand in air to obtain a sol, and after drying, it is kept at 1000°C in an anaerobic environment for 30 minutes (the heating process is 5°C / min from room temperature to 600°C, and 3°C / min above 600°C) to obtain a rare earth doped nano-photoluminescent agent.

[0068] Example 4: A method for detecting the concentration of reactive oxygen free radicals by simulating the health effects of PM2.5 in vitro, comprising the following steps:

[0069] Step (1) obtaining a sample solution: placing a PE filter membrane with a thickness of 0.3 mm, a diameter of 5 cm, and a pore size of 100 nm in a dry place outdoors for 5 days, recovering the PE filter membrane and immersing it in simulated body fluid for 3 days, and taking out the sampling membrane to obtain a sample solution;

[0070] Step (2) preparing reagent A: adding a photoluminescent agent and a dispersant into deionized water to obtain reagent A; the concentration of the photoluminescent agent in the reagent A is 0.1 g / L, and the concentration of polyvinyl pyrrolidone is 0.001 g / L; the photoluminescent agent has two emission wavelengths: a first wavelength of 464 nm and a second wavelength of 532 nm, and a particle size of 10 nm-200 nm;

[0071] Step (3) preparing reagent B: under light-shielding conditions, place 0.05 g of polypropylene nanoparticles in 100 mL of deionized water, and add 0.0005 g of titanate coupling agent 201 to obtain reagent B;

[0072] Step (4) Detection process: The effective detection volume of the reaction pool of the light detector is 1. 0.5 volume of reagent A is irradiated under natural light for 10 minutes and added to the reaction pool of the light detector. Then 0.5 volume of deionized water is added. The light intensity is measured as I 0 ; 0.5 volume of reagent A was added to the reaction cell of the light detector, followed by 0.5 volume of reagent B, and the light intensity was measured to be 1 1 ; 0.5 volume of reagent A was added to the reaction cell of the light detector, and then 0.5 volume of sample solution was added, and the light intensity was measured to be I 2 ; 0.5 volume of reagent A was added to the reaction cell of the light detector, followed by 0.3 volume of reagent B and 0.2 volume of sample solution, and the light intensity was measured to be I 3 ; Calculate the extinction intensity of the reaction as ΔI=I 0 -I 3 -0.3(I 0 -I 1 )-0.2(I 0 -I 2 ), a+b=0.5;

[0073] Step (5) converts and calculates the characteristic function of extinction intensity ΔI-free radical concentration c to obtain the free radical concentration in the sample solution; ΔI=13253c+3246, R 2 =0.999.

[0074] The photoluminescent agent in step (1) is a rare earth doped nano-photoluminescent agent, which includes the following preparation method:

[0075] Step (2-a): Add 1 volume of tetrabutyl titanate Ti(OBu) 4 Add to 2 volumes of ethanol to obtain solution C, then add 0.8 volumes of acetic acid solution with a mass concentration of 85% to solution C, stir thoroughly and add Al(NO 3 ) 3 and Eu(NO 3 ) 3 , the molar ratio of n-butyl titanate: soluble aluminum salt: soluble rare earth salt is 1:0.005:0.015 to obtain a preform;

[0076] Step (2-b): The preform is allowed to stand in air to obtain a sol, and after drying, it is kept at 900°C in an anaerobic environment for 10-30 minutes (the heating process is 5°C / min from room temperature to 600°C, and 3°C / min above 600°C) to obtain a rare earth doped nano-photoluminescent agent.

[0077] Example 5: A method for detecting the concentration of reactive oxygen free radicals by simulating the health effects of PM2.5 in vitro, comprising the following steps:

[0078] Step (1) obtaining a sample solution: placing a PE filter membrane with a thickness of 0.3 mm, a diameter of 5 cm, and a pore size of 100 nm in a dry place outdoors for 10 days, recovering the PE filter membrane and immersing it in simulated body fluid for 3 days, and taking out the sampling membrane to obtain a sample solution;

[0079] Step (2) preparing reagent A: adding a photoluminescent agent and a dispersant into deionized water to obtain reagent A; the concentration of the photoluminescent agent in the reagent A is 0.1 g / L, and the concentration of polyvinyl pyrrolidone is 0.001 g / L; the photoluminescent agent has two emission wavelengths: a first wavelength of 464 nm and a second wavelength of 532 nm, and a particle size of 10 nm-200 nm;

[0080] Step (3) preparing reagent B: under light-shielding conditions, place 0.05 g of polypropylene nanoparticles in 100 mL of deionized water, and add 0.0005 g of titanate coupling agent 201 to obtain reagent B;

[0081] Step (4) Detection process: The effective detection volume of the reaction pool of the light detector is 1. 0.5 volume of reagent A is irradiated under natural light for 10 minutes and added to the reaction pool of the light detector. Then 0.5 volume of deionized water is added. The light intensity is measured as I 0 ; 0.5 volume of reagent A was added to the reaction cell of the light detector, followed by 0.5 volume of reagent B, and the light intensity was measured to be 1 1 ; 0.5 volume of reagent A was added to the reaction cell of the light detector, and then 0.5 volume of sample solution was added, and the light intensity was measured to be I 2 ; 0.5 volume of reagent A was added to the reaction cell of the light detector, followed by 0.3 volume of reagent B and 0.2 volume of sample solution, and the light intensity was measured to be I 3 ; Calculate the extinction intensity of the reaction as ΔI=I 0 -I 3 -0.3(I 0 -I 1 )-0.2(I 0 -I 2 ), a+b=0.5;

[0082] Step (5) converts and calculates the characteristic function of extinction intensity ΔI-free radical concentration c to obtain the free radical concentration in the sample solution; ΔI=13253c+3246, R 2 =0.999.

[0083] The photoluminescent agent in step (1) is a rare earth doped nano-photoluminescent agent, which includes the following preparation method:

[0084] Step (2-a): Add 1 volume of tetrabutyl titanate Ti(OBu) 4Add to 2 volumes of ethanol to obtain solution C, then add 0.8 volumes of acetic acid solution with a mass concentration of 85% to solution C, stir thoroughly and add Al(NO 3 ) 3 and Eu(NO 3 ) 3 , the molar ratio of n-butyl titanate: soluble aluminum salt: soluble rare earth salt is 1:0.003:0.01 to obtain a preform;

[0085] Step (2-b): The preform is allowed to stand in air to obtain a sol, and after drying, it is kept at 1000°C in an anaerobic environment for 30 minutes (the heating process is 5°C / min from room temperature to 600°C, and 3°C / min above 600°C) to obtain a rare earth doped nano-photoluminescent agent.

[0086] Comparative Example 1: Liquid chromatography-mass spectrometry was used for detection, and the sample solution was prepared in the same manner as in Example 1.

[0087] Comparative Example 2: A method for detecting the concentration of active oxygen free radicals by simulating the health effects of PM2.5 in vitro, comprising the following steps:

[0088] Step (1) obtaining a sample solution: placing a PE filter membrane with a thickness of 0.3 mm, a diameter of 5 cm, and a pore size of 100 nm in a dry place outdoors for 5 days, recovering the PE filter membrane and immersing it in simulated body fluid for 3 days, and taking out the sampling membrane to obtain a sample solution;

[0089] Step (2) preparing reagent A: adding commercially available fluorescent powder and a dispersant into deionized water to obtain reagent A; the concentration of the commercially available fluorescent powder in the reagent A is 0.2 g / L, and the concentration of polyvinyl pyrrolidone is 0.001 g / L;

[0090] Step (3) preparing reagent B: under light-shielding conditions, place 0.1 g of polypropylene nanoparticles in 100 mL of deionized water, and add 0.001 g of titanate coupling agent 201 to obtain reagent B;

[0091] Step (4) Detection process: The effective detection volume of the reaction pool of the light detector is 1. 0.5 volume of reagent A is irradiated under natural light for 10 minutes and added to the reaction pool of the light detector. Then 0.5 volume of deionized water is added. The light intensity is measured as I 0 ; 0.5 volume of reagent A was added to the reaction cell of the light detector, followed by 0.5 volume of reagent B, and the light intensity was measured to be 1 1 ; 0.5 volume of reagent A was added to the reaction cell of the light detector, and then 0.5 volume of sample solution was added, and the light intensity was measured to be I 2 ; 0.5 volume of reagent A was added to the reaction cell of the light detector, followed by 0.3 volume of reagent B and 0.2 volume of sample solution, and the light intensity was measured to be I3 ; Calculate the extinction intensity of the reaction as ΔI=I 0 -I 3 -0.3(I 0 -I 1 )-0.2(I 0 -I 2 ), a+b=0.5;

[0092] Step (5) converts and calculates the characteristic function of extinction intensity ΔI-free radical concentration c to obtain the free radical concentration in the sample solution; ΔI=13253c+3246, R 2 =0.999.

[0093] Comparative Example 3: A method for detecting the concentration of active oxygen free radicals by simulating the health effects of PM2.5 in vitro, comprising the following steps:

[0094] Step (1) obtaining a sample solution: placing a PES filter membrane with a thickness of 0.3 mm, a diameter of 5 cm, and a pore size of 100 nm in an outdoor dry place for 5 days, recovering the PE filter membrane and immersing it in simulated body fluid for 3 days, and taking out the sampling membrane to obtain a sample solution;

[0095] Step (2) preparing reagent A: adding a photoluminescent agent and a dispersant into deionized water to obtain reagent A; the concentration of the photoluminescent agent in the reagent A is 0.1 g / L, and the concentration of polyvinyl pyrrolidone is 0.001 g / L; the photoluminescent agent has two emission wavelengths: a first wavelength of 464 nm and a second wavelength of 532 nm, and a particle size of 10 nm-200 nm;

[0096] Step (3) preparing reagent B: under light-shielding conditions, place 0.05 g of polypropylene nanoparticles in 100 mL of deionized water, and add 0.0005 g of titanate coupling agent 201 to obtain reagent B;

[0097] Step (4) Detection process: The effective detection volume of the reaction pool of the light detector is 1. 0.5 volume of reagent A is irradiated under natural light for 10 minutes and added to the reaction pool of the light detector. Then 0.5 volume of deionized water is added. The light intensity is measured as I 0 ; 0.5 volume of reagent A was added to the reaction cell of the light detector, followed by 0.5 volume of reagent B, and the light intensity was measured to be 1 1 ; 0.5 volume of reagent A was added to the reaction cell of the light detector, and then 0.5 volume of sample solution was added, and the light intensity was measured to be I 2 ; 0.5 volume of reagent A was added to the reaction cell of the light detector, followed by 0.3 volume of reagent B and 0.2 volume of sample solution, and the light intensity was measured to be I 3 ; Calculate the extinction intensity of the reaction as ΔI=I 0 -I3 -0.3(I 0 -I 1 )-0.2(I 0 -I 2 ), a+b=0.5;

[0098] Step (5) converts and calculates the characteristic function of extinction intensity ΔI-free radical concentration c to obtain the free radical concentration in the sample solution; ΔI=13253c+3246, R 2 =0.999.

[0099] The photoluminescent agent in step (1) is a rare earth doped nano-photoluminescent agent, which comprises the following preparation method:

[0100] Step (2-a): Add 1 volume of tetrabutyl titanate Ti(OBu) 4 Add to 2 volumes of ethanol to obtain solution C, then add 0.8 volumes of 85% acetic acid solution to solution C, stir thoroughly and add Al(NO 3 ) 3 and Eu(NO 3 ) 3 , the molar ratio of n-butyl titanate: soluble aluminum salt: soluble rare earth salt is 1:0.003:0.01 to obtain a preform;

[0101] Step (2-b): The preform is allowed to stand in air to obtain a sol, and after drying, it is kept at 1000°C in an anaerobic environment for 30 minutes (the heating process is 5°C / min from room temperature to 600°C, and 3°C / min above 600°C) to obtain a rare earth doped nano-photoluminescent agent.

[0102] Comparative Example 4: A method for detecting the concentration of active oxygen free radicals by simulating the health effects of PM2.5 in vitro, comprising the following steps:

[0103] Step (1) obtaining a sample solution: placing a PE filter membrane with a thickness of 0.3 mm, a diameter of 5 cm, and a pore size of 100 nm in a dry place outdoors for 5 days, recovering the PE filter membrane and immersing it in simulated body fluid for 3 days, and taking out the sampling membrane to obtain a sample solution;

[0104] Step (2) preparing reagent A: adding a photoluminescent agent and a dispersant into deionized water to obtain reagent A; the concentration of the photoluminescent agent in the reagent A is 0.5 g / L, and the concentration of polyvinyl pyrrolidone is 0.005 g / L; the photoluminescent agent has two emission wavelengths: a first wavelength of 464 nm and a second wavelength of 532 nm, and a particle size of 10 nm to 200 nm;

[0105] Step (3) preparing reagent B: under light-shielding conditions, placing 0.001 g of polypropylene nanoparticles in 100 mL of deionized water, and adding 0.00001 g of titanate coupling agent 201 to obtain reagent B;

[0106] Step (4) Detection process: The effective detection volume of the reaction pool of the light detector is 1. 0.5 volume of reagent A is irradiated under natural light for 10 minutes and added to the reaction pool of the light detector. Then 0.5 volume of deionized water is added. The light intensity is measured as I 0 ; 0.5 volume of reagent A was added to the reaction cell of the light detector, followed by 0.5 volume of reagent B, and the light intensity was measured to be 1 1 ; 0.5 volume of reagent A was added to the reaction cell of the light detector, and then 0.5 volume of sample solution was added, and the light intensity was measured to be I 2 ; 0.5 volume of reagent A was added to the reaction cell of the light detector, followed by 0.3 volume of reagent B and 0.2 volume of sample solution, and the light intensity was measured to be I 3 ; Calculate the extinction intensity of the reaction as ΔI=I 0 -I 3 -0.3(I 0 -I 1 )-0.2(I 0 -I 2 ), a+b=0.5;

[0107] Step (5) converts and calculates the characteristic function of extinction intensity ΔI-free radical concentration c to obtain the free radical concentration in the sample solution; ΔI=13253c+3246, R 2 =0.999.

[0108] The photoluminescent agent in step (1) is a rare earth doped nano-photoluminescent agent, which comprises the following preparation method:

[0109] Step (2-a): Add 1 volume of tetrabutyl titanate Ti(OBu) 4 Add to 2 volumes of ethanol to obtain solution C, then add 0.8 volumes of 85% acetic acid solution to solution C, stir thoroughly and add Al(NO 3 ) 3 and Eu(NO 3 ) 3 , the molar ratio of n-butyl titanate: soluble aluminum salt: soluble rare earth salt is 1:0.003:0.01 to obtain a preform;

[0110] Step (2-b): The preform is allowed to stand in air to obtain a sol, and after drying, it is kept at 1000°C in an anaerobic environment for 30 minutes (the heating process is 5°C / min from room temperature to 600°C, and 3°C / min above 600°C) to obtain a rare earth doped nano-photoluminescent agent.

[0111] Comparative Example 5: A method for detecting the concentration of active oxygen free radicals by simulating the health effects of PM2.5 in vitro, comprising the following steps:

[0112] Step (1) obtaining a sample solution: placing a PE filter membrane with a thickness of 0.3 mm, a diameter of 5 cm, and a pore size of 100 nm in a dry place outdoors for 5 days, recovering the PE filter membrane and immersing it in simulated body fluid for 3 days, and taking out the sampling membrane to obtain a sample solution;

[0113] Step (2) preparing reagent A: adding a photoluminescent agent and a dispersant into deionized water to obtain reagent A; the concentration of the photoluminescent agent in the reagent A is 0.1 g / L, and the concentration of polyvinyl pyrrolidone is 0.001 g / L; the photoluminescent agent has two emission wavelengths: a first wavelength of 464 nm and a second wavelength of 532 nm, and a particle size of 10 nm-200 nm;

[0114] Step (3) preparing reagent B: under light-shielding conditions, place 0.05 g of polypropylene nanoparticles in 100 mL of deionized water, and add 0.0005 g of titanate coupling agent 201 to obtain reagent B;

[0115] Step (4) Detection process: The effective detection volume of the reaction pool of the light detector is 1. 0.5 volume of reagent A is irradiated under natural light for 10 minutes and added to the reaction pool of the light detector. Then 0.5 volume of deionized water is added. The light intensity is measured as I 0 ; 0.5 volume of reagent A was added to the reaction cell of the light detector, followed by 0.3 volume of reagent B and 0.2 volume of sample solution, and the light intensity was measured to be I 3 ; Calculate the extinction intensity of the reaction as ΔI=I 0 -I 3 ;

[0116] Step (5) converts and calculates the characteristic function of extinction intensity ΔI-free radical concentration c to obtain the free radical concentration in the sample solution; ΔI=13253c+3246, R 2 =0.999.

[0117] The photoluminescent agent in step (1) is a rare earth doped nano-photoluminescent agent, which includes the following preparation method:

[0118] Step (2-a): Add 1 volume of tetrabutyl titanate Ti(OBu) 4Add to 2 volumes of ethanol to obtain solution C, then add 0.8 volumes of 85% acetic acid solution to solution C, stir thoroughly and add Al(NO 3 ) 3 and Eu(NO 3 ) 3 , the molar ratio of n-butyl titanate: soluble aluminum salt: soluble rare earth salt is 1:0.003:0.01 to obtain a preform;

[0119] Step (2-b): The preform is allowed to stand in air to obtain a sol, and after drying, it is kept at 1000°C in an anaerobic environment for 30 minutes (the heating process is 5°C / min from room temperature to 600°C, and 3°C / min above 600°C) to obtain a rare earth doped nano-photoluminescent agent.

[0120] The sample solutions prepared in the above-mentioned Examples 1-5 and Comparative Examples 1-5 should be able to meet the requirements of 5 tests.

[0121] The detection concentration data are shown in Table 1.

[0122] Table 1

[0123]

[0124] It can be seen from Examples 1, 2, and 4 that under the same sample preparation conditions, the detection results of this solution are relatively stable. Even if there are certain changes between reagent A and reagent B, the difference in the detection results is not large, and the 5 data points in the same group of experiments fluctuate slightly.

[0125] It can be seen from Examples 1, 3, and 5 that under different sample preparation conditions, such as Example 1 and Example 3, the PM2.5 in the kitchen flue environment is higher than that in the outdoor environment, and the free radical concentration of the sample liquid here is also higher; Example 1 and Example 5 compare the difference in outdoor placement time. The longer the placement time, the more PM2.5 absorbed by the PE filter paper, and the higher the free radical concentration of the sample liquid.

[0126] Comparative Example 1 adopts the conventional method. Although the average value of the measured results is relatively close to that of Example 1, its standard deviation is larger than that of Example 1, that is, the fluctuation of 5 repeated tests is large, indicating that the accuracy of this method is questionable.

[0127] Comparative Example 2 selected a commercially available fluorescent agent, which could not promote the reaction of free radicals, so the test result was lower than that of Example 1.

[0128] Comparative Example 3 selected PES membrane sampling. The particles remaining in the membrane are equivalent to adding more PM2.5 particles to the sample liquid, so the test result is higher.

[0129] In Comparative Example 4, an excessive amount of photoluminescent agent was added to reagent A, resulting in an excessively strong background light and a too small ratio of the reaction extinction amount, which resulted in a smaller result.

[0130] In the detection process of Comparative Example 5, weighted extinction was not performed on reagent B and the sample solution, so the detection data was too large.

[0131] The experimental process and results of the above-mentioned Examples 1-5 have fully demonstrated the feasibility of this solution. Compared with the experimental results of Comparative Examples 1-5, this solution has obvious technical advantages, namely, more stable and accurate detection results.

Claims

1. A method for detecting the concentration of active oxygen free radicals by simulating the health effects of PM2.5 in vitro, characterized in that: The following steps are involved: Step (1) obtaining a sample solution: placing a sampling membrane in a test environment for 3-10 days, recovering the sampling membrane and soaking it in simulated body fluid for 1-3 days, and removing the sampling membrane to obtain a sample solution; Step (2) preparing reagent A: adding a photoluminescent agent and a dispersant into deionized water to obtain reagent A; the concentration of the photoluminescent agent in the reagent A is 0.01-0.2 g / L, and the concentration of the dispersant is 0.001-0.002 g / L; Step (3) preparing reagent B: under light-shielding conditions, place 0.01-0.1 g of the active agent in 100 mL of deionized water to obtain reagent B; Step (4) Detection process: the total volume of liquid added to the reaction pool of the light detector is recorded as unit 1, 0.5 volume of reagent A is added to the reaction pool of the light detector, and then 0.5 volume of deionized water is added, and the light intensity is measured as I0; 0.5 volume of reagent A is added to the reaction pool of the light detector, and then 0.5 volume of reagent B is added, and the light intensity is measured as I1; 0.5 volume of reagent A was added to the reaction cell of the light detector, followed by 0.5 volume of sample solution, and the light intensity was measured to be I2; Add 0.5 volume of reagent A to the reaction pool of the light detector, then add a volume of reagent B and b volume of sample solution, and measure the light intensity to be I3; calculate the extinction intensity of the reaction to be ΔI=I0-I3-2a(I0-I1)-2b(I0-I2), a+b=0.5; Step (5) converting and calculating the characteristic function of extinction intensity ΔI-free radical concentration c to obtain the free radical concentration in the sample solution; In step (2), the photoluminescent agent is a rare earth doped nano-photoluminescent agent, which includes the following preparation method: Step (2-a): adding 1 volume of tetrabutyl titanate Ti(OBu)4 to 1-5 volumes of an alcohol solvent to obtain a solution C, then adding 0.7-1 volumes of a monobasic organic acid solution having a mass concentration of 80%-90% to the solution C, stirring thoroughly, and then adding a soluble aluminum salt and a soluble rare earth salt to obtain a preform; Step (2-b): allowing the preform to stand in air to obtain a sol, and then drying and subjecting the preform to high temperature treatment to obtain a rare earth-doped nano-photoluminescent agent; In step (2), the particle size of the photoluminescent agent is 10 nm-200 nm; the photoluminescent agent contains two or more emission wavelengths; and the dispersant is polyvinyl pyrrolidone; In step (3), the reactive agent is polypropylene nanoparticles; the reactive agent is placed in deionized water and a dispersant having a reactive concentration of 0.1% to 1% is added for dispersion treatment, and the dispersant is a silane dispersant.

2. The method for detecting the concentration of active oxygen free radicals by simulating the health effects of PM2.5 in vitro according to claim 1, characterized in that: In step (1), the sampling membrane is a PE filter membrane with a pore size of 100 nm.

3. The method for detecting the concentration of active oxygen free radicals by simulating the health effects of PM2.5 in vitro according to claim 1, characterized in that: The alcohol solvent in step (2-a) is ethanol or propanol, the monobasic organic acid is acetic acid or propionic acid, the soluble aluminum salt is selected from one of Al(NO3)3, AlCl3 or Al2(SO4)3; the soluble rare earth salt is one or two of the nitrates or chlorides of La, Eu, Tb or Dy.

4. The method for detecting the concentration of active oxygen free radicals by simulating the health effects of PM2.5 in vitro according to claim 1, characterized in that: The molar ratio of n-butyl titanate: soluble aluminum salt: soluble rare earth salt is 1: 0.001-0.005: 0.001-0.

016.

5. The method for detecting the concentration of active oxygen free radicals by simulating the health effects of PM2.5 in vitro according to claim 1, characterized in that: The high temperature treatment in step (2-b) is carried out by keeping the temperature at 900-1050°C for 10-30min in an anaerobic environment, and the heating process of the high temperature treatment is 5-10°C / min from room temperature to 600°C, and 2-3°C / min above 600°C.

6. The method for detecting the concentration of active oxygen free radicals by simulating the health effects of PM2.5 in vitro according to claim 1, characterized in that: In step (4), the reagent A needs to be exposed to light before being added to the reaction cell of the light detector, and the exposure to light is performed under natural light for 5-10 minutes.

7. The method for detecting the concentration of active oxygen free radicals by simulating the health effects of PM2.5 in vitro according to claim 1, characterized in that: The characteristic function of extinction intensity ΔI-free radical concentration c in step (5) is ΔI=13253c+3246, R 2 =0.999.

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