Method for detecting water toxicity by using CHO-k1 cell fluorescence and application thereof

Abstract

This invention discloses a method for sensitively detecting water toxicity using CHO-k1 cell fluorescence and its application, belonging to the field of water safety detection technology. The method uses CHO-k1 cells as the test carrier. After cell preparation, solid-phase extraction enrichment of water samples, and exposure to toxic substances, the method simultaneously detects cell inhibition rate and intracellular reactive oxygen species (ROS) fluorescence intensity. The total fluorescence intensity is corrected by the number of surviving cells, and the relative ROS fluorescence intensity and oxidative stress impact rate (R) of a single cell are calculated to establish a four-level toxicity grading standard, enabling quantitative evaluation of water sample toxicity. The detection sensitivity of this method is far higher than traditional cytotoxicity detection methods. It can detect sub-lethal damage from low concentrations of pollutants, and the results are accurate and reliable. The operation is standardized and applicable to rapid toxicity screening and water environment health risk assessment of various water samples, including surface water, groundwater, municipal water supply, industrial wastewater, and reclaimed water.

Description

Technical Field

[0001] This invention relates to the fields of water safety testing, environmental toxicology, and cell fluorescence detection technology. Specifically, it relates to a sensitive detection method for water toxicity based on the fluorescence response of CHO-k1 cells to reactive oxygen species and its application. It can be applied to the comprehensive rapid screening, quantitative detection, and water environment health risk classification assessment of various water samples, including surface water, groundwater, municipal water supply, reclaimed water, industrial wastewater, emergency water sources, and landscape water bodies. Background Technology

[0002] Water environment safety is a core aspect of ecological protection and public health safeguards. Various types of water bodies (surface water, groundwater, drinking water, reclaimed water, industrial wastewater, etc.) commonly contain organic pollutants, disinfection byproducts, industrial pollutants, agricultural non-point source pollutants, and emerging trace pollutants. These pollutants, whether alone or in combination, can cause water bodies to possess potential comprehensive toxicity.

[0003] Prolonged exposure to polluted water can cause irreversible damage to the human reproductive, nervous, and immune systems, as well as disrupt the aquatic ecological balance, inhibit the growth of aquatic organisms, and threaten ecosystem stability. Therefore, accurate and sensitive detection of the comprehensive toxicity of various water samples is a key technological requirement for water environment quality monitoring, pollution risk management, and ecological security assurance.

[0004] Existing water toxicity detection technologies can be mainly divided into the following categories, all of which have insurmountable technical defects: 1. Chemical instrument detection methods: such as GC-MS, LC-MS, ICP-MS, etc., can only quantitatively detect known target pollutants, cannot identify unknown pollutants in water bodies, and cannot reflect the combined toxicity and comprehensive biological effects of multiple pollutants, nor can they directly reflect the health risks of water bodies to humans and the ecosystem.

[0005] 2. Aquatic model organism detection method: This method uses fish, protozoa, and algae as test subjects. Although it can reflect the overall biological toxicity of water bodies, the test process is cumbersome, the detection cycle is long, and the labor costs are high. Individual differences are also large, which cannot meet the needs of rapid detection of water toxicity.

[0006] 3. Microbial toxicity detection methods: Represented by luminescent bacteria methods, although the detection cycle is short, the sensitivity to pollutants with mammalian cytotoxicity is low, easily affected by matrix interference such as water pH, residual chlorine, and turbidity, and the correlation with human health risks is weak, resulting in poor universality. 4. Mammalian cytotoxicity detection methods: Using mammalian cells such as CHO cells as test subjects, these methods are the closest to the detection methods for human cytotoxicity responses. Current mainstream methods are the MTT and CCK-8 colorimetric methods, which can only reflect the cytotoxicity of high-concentration pollutants through cell proliferation / death, and cannot identify sublethal oxidative stress damage caused by low-concentration pollutants. They have high detection limits and are prone to false negatives. Existing cell ROS fluorescence detection technologies do not correct for the number of viable cells in the detection results, and the total fluorescence intensity is severely affected by cell viability, failing to accurately reflect the degree of oxidative damage in individual cells. Furthermore, there is no standardized enrichment pretreatment system or toxicity grading assessment model for trace pollutants in water, making it impossible to achieve highly sensitive and stable detection of various water samples. Summary of the Invention

[0007] To solve the above technical problems, this invention provides a method for detecting water toxicity using CHO-k1 cell fluorescence sensitivity and its application. (1) It provides a water toxicity detection method with a sensitivity far higher than that of traditional cytotoxicity detection methods, which can identify sublethal oxidative stress damage caused by low concentrations of pollutants and significantly reduce false negative results; (2) It establishes a correction method based on the relative ROS fluorescence intensity of a single cell to eliminate the interference of cell survival rate differences on fluorescence detection results and improve the accuracy and reliability of detection results; (3) It develops a standardized enrichment pretreatment, 96-well plate detection system and four-level toxicity grading assessment model adapted to various water matrixes to realize quantitative evaluation and risk grading of water sample toxicity; (4) It simplifies the detection operation process, realizes the simultaneous detection of batch water samples, takes into account both detection accuracy and detection efficiency, and provides scientific and standardized technical support for water environment supervision and water quality safety assessment. Therefore, this invention uses CHO-k1 cells, which are highly sensitive to organic pollutants in water, as test carriers to develop a toxicity detection method based on cell ROS fluorescence response and combined with correction for the number of surviving cells, and establishes a matching fluorescence toxicity assessment model to solve the core defects of existing technologies, such as low sensitivity, poor accuracy of results, and lack of standardized water body adaptation system.

[0008] The first objective of this invention is to provide a method for sensitively detecting water toxicity using CHO-k1 cell fluorescence, comprising the following steps: CHO-k1 cells in the logarithmic growth phase were cultured in a culture dish; Solid-phase extraction was performed on the water sample to be tested using a mixed resin column of XAD-2 and XAD-8. After elution, nitrogen blowing and solvent replacement, the enriched sample to be tested was obtained. The enriched test sample was added to a culture dish inoculated with CHO-k1 cells, and a blank group and a negative control group were set up simultaneously. The samples were then cultured in an incubator. After the culture was completed, the cell viability in each well of the culture dish was detected by the CCK-8 assay to obtain the relative number of surviving cells N in the well; the fluorescence intensity of reactive oxygen species (ROS) in the total cells of each well was detected by the DCFH-DA fluorescent probe method. The total fluorescence intensity was corrected by the relative number of surviving cells. The relative ROS fluorescence intensity Rs of a single cell was calculated. The oxidative stress impact rate R was calculated according to the following formulas I and II. The toxicity of the water body to be tested was quantitatively evaluated and risk-classified based on the R value. The relative ROS fluorescence intensity of a single cell is Rs = F / N; Formula I; The effect rate of oxidative stress R (%) = [(Rs-Rc) / Rc] × 100%; Formula II; Fluorescent toxicity equivalent REF=C R有机污染物=100% / C R水样=100% ; Formula III; Where F represents the fluorescence intensity of reactive oxygen species (ROS) in the total cells of each well, N represents the relative number of surviving cells in the well; Rs represents the relative ROS fluorescence intensity of a single cell in the experimental group, and Rc represents the relative ROS fluorescence intensity of a single cell in the negative control group; C R有机污染物=100% C represents the concentration of organic pollutants in the standard at a 100% oxidative stress effect rate. R水样=100% The enrichment factor (REF) is the enrichment factor of the water sample at a 100% oxidative stress effect rate. A higher REF value indicates greater fluorescence toxicity. The organic pollutant is selected from phenol.

[0009] In some embodiments of the present invention, the culture medium used to culture CHO-k1 cells is F12-K medium, which is supplemented with 10 vt% fetal bovine serum, 100 U / mL penicillin G and 100 μg / mL streptomycin. The CHO-k1 cells were cultured at 37°C, 5% CO2, and saturated humidity, and passaged every 2-3 days.

[0010] In some embodiments of the present invention, the CHO-k1 cell density is (1.0~1.5)×10⁻¹⁰. 4 The culture dish used 96-well microplates; the inoculation volume per well was 200 μL, and the culture time was 12 h; the edge wells of the 96-well microplate were filled with 0.01M PBS buffer to eliminate edge effects.

[0011] In some embodiments of the present invention, the volume ratio of XAD-2 to XAD-8 is 1:1. Before the solid phase extraction enrichment, the mixed resin column is activated sequentially with ethanol and ethyl acetate. After activation, 1L of the water to be tested is introduced to complete the adsorption.

[0012] In some embodiments of the present invention, anhydrous ethanol is used as the eluent for elution. After elution, the sample is dried by nitrogen gas and then solvent-displaced using dimethyl sulfoxide to obtain an enriched sample for testing.

[0013] In some embodiments of the present invention, the conditions for exposure to the toxic substance are 37°C, 5% CO2, and a culture time of 48 h. The blank group was the test group containing DMSO, F12-K medium, cells-free and without enrichment of the test sample; The negative control group consisted of the experimental group containing DMSO, F12-K medium, CHO-k1 cells, and no enriched test sample.

[0014] In some embodiments of the present invention, the specific steps of the CCK-8 assay are as follows: 10 μL of CCK-8 reagent is added to a cell culture dish, and after reacting at 37°C in the dark for 3 h, the absorbance value at a wavelength of 450 nm is measured; the formula for calculating the cell inhibition rate and the relative number of surviving cells is as follows: Cell inhibition rate IR (%) = [(As-Ab) / (Ac-Ab)] × 100%; The relative number of surviving cells in the experimental group N = As - Ab; Where As is the absorbance value of the cell experimental group, Ac is the absorbance value of the negative control group, and Ab is the absorbance value of the blank group.

[0015] In some embodiments of the present invention, the specific steps of the DCFH-DA fluorescent probe method for detection are as follows: remove the culture medium from the microwell of the culture dish, add the DCFH-DA probe, incubate at 37°C in the dark for 1 h, remove the probe, digest with trypsin, wash with PBS, and then detect the fluorescence intensity. The detection parameters are excitation wavelength 480 nm and emission wavelength 525 nm.

[0016] In some embodiments of the present invention, the risk classification criteria are as follows: when 0 < R ≤ 10%, the water sample is judged to have a low degree of influence on cellular oxidative stress; when 10% < R ≤ 100%, the water sample is judged to have a moderate degree of influence on cellular oxidative stress; when 100% < R ≤ 500%, the water sample is judged to have a high degree of influence on cellular oxidative stress; and when R > 500%, the water sample is judged to have an extremely high degree of influence on cellular oxidative stress.

[0017] The second objective of this invention is to apply the method for sensitive detection of water toxicity using CHO-k1 cell fluorescence in water sample safety testing, rapid screening of comprehensive water toxicity, evaluation of the toxicity of organic pollutants in water samples, and health risk assessment of drinking water.

[0018] The technical solution of the present invention has the following advantages over the prior art: (1) Significantly improved detection sensitivity, effectively avoiding false negative results: This invention uses cellular oxidative stress damage as the detection target. Compared with the traditional CCK-8 cytotoxicity detection method, it can identify sub-lethal cell damage caused by low concentrations of pollutants, and improves the sensitivity of water sample toxicity detection by more than one order of magnitude. It can detect water sample toxicity with an enrichment factor as low as 3.125 times, solving the core defects of traditional methods that can only detect cell death and are prone to missing low concentrations of pollutants. (2) Accurate and reliable detection results, eliminating system interference: This invention corrects the total ROS fluorescence intensity by the number of surviving cells to obtain the relative ROS fluorescence intensity of a single cell, completely eliminating the interference of cell survival rate differences on fluorescence detection results. It can accurately and objectively reflect the degree of oxidative damage of pollutants to cells, avoiding the problem of large deviations in traditional ROS detection results. (3) Establish a standardized grading assessment system to meet the needs of water environment supervision: This invention establishes a four-level toxicity grading standard based on the oxidative stress impact rate R, and defines the fluorescence toxicity equivalent to achieve toxicity quantification and normalization. It can realize the quantitative evaluation and risk classification of water body toxicity, and provide a standardized and practical evaluation basis for water environment supervision and water quality safety assessment. (4) The whole process is adaptable to various water matrixes and has strong universality: This invention optimizes the XAD-2 / XAD-8 mixed resin solid phase extraction enrichment system, which can efficiently enrich trace organic toxic substances in water. At the same time, it optimizes the 96-well plate detection system to eliminate edge effects and realize the simultaneous detection of batch water samples. It is suitable for comprehensive toxicity screening of various water samples such as surface water, groundwater, municipal water supply, industrial wastewater, reclaimed water, and emergency water sources. It can also be used for sensitive detection of characteristic organic pollutants in water bodies, with a very wide range of applications. (5) Simple operation and high detection efficiency: The entire process of this invention can be completed within 72 hours. It only requires conventional cell culture equipment and multifunctional enzyme-linked immunosorbent assay (ELISA) reader, without the need for large-scale precision instruments. The operation steps are standardized and do not require complicated professional operations. It can be widely used in the daily monitoring and emergency screening of grassroots water environment testing institutions. Attached Figure Description

[0019] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings, wherein... Figure 1 This is a schematic diagram of the overall process of the water sample toxicity detection method of the present invention; Figure 2This is a schematic diagram of the well arrangement of the 96-well microplate used in the detection of this invention; Figure 3 This is a comparison of the cytotoxicity curves and fluorescence toxicity curves of the water samples enriched in Example 1. Figure 4 This is a comparison of the cytotoxicity fitting curve and the fluorescence toxicity fitting curve of phenol in Example 2; Figure 5 This is a bar chart comparing the cell inhibition rate and the oxidative stress effect rate at low enrichment folds in water in Example 1. Figure 6 This is a bar chart comparing the cell inhibition rate and the effect rate of oxidative stress at low phenol concentrations in Example 2. Detailed Implementation

[0020] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0021] Experimental materials and instruments 1. Test cell line: CHO-k1 cells, purchased from the American Type Culture Collection (ATCC), catalog number CCL-61; 2. Reagents and Materials: F12-K medium, fetal bovine serum, penicillin G, streptomycin, and trypsin were purchased from Abiowell Biotechnology Co., Ltd. (Changsha, China); CCK-8 reagent kit and DCFH-DA fluorescent probe were purchased from Shanghai Beyotime Biotechnology Co., Ltd.; XAD-2 resin, XAD-8 resin, phenol, anhydrous ethanol, ethyl acetate, and dimethyl sulfoxide (DMSO) were all analytical grade and purchased from Sinopharm Chemical Reagent Co., Ltd.; 0.01M PBS buffer was prepared according to laboratory standard methods. 3. Instruments and equipment: CO2 constant temperature incubator (ThermoFisher), Synergy4 multi-functional microplate reader (BioTek), biosafety cabinet, high-speed centrifuge, nitrogen blower, solid phase extraction device, cell counter.

[0022] Example 1: Toxicity Detection of Typical Water Samples This embodiment focuses on toxicity testing of water samples from typical water bodies such as municipal water supply and surface water. The specific steps are as follows: 1. Preparation of test cells: CHO-k1 cells were cultured in F12-K medium supplemented with 10% fetal bovine serum, 100 U / mL penicillin G, and 100 μg / mL streptomycin under the following conditions: 37℃, 5% CO2, and saturated humidity. Cells were passaged every 2 days. Cells in the logarithmic growth phase were washed twice with 0.01M PBS, digested with trypsin for 3 min, and then digested with complete culture medium to terminate the digestion. After centrifugation and resuspending, the cell density was adjusted to 1.5 × 10⁶ cells / mL. 4 Cells were seeded at a density of 1 / mL into black 96-well microplates, with an inoculation volume of 200 μL per well. All wells at the edge of the microplates were filled with 0.01 M PBS buffer to eliminate edge effects. The microplates were incubated at 37°C and 5% CO2 for 12 h until the cells were fully adhered.

[0023] 2. Water sample pretreatment: 1.5 mL of XAD-2 resin and 1.5 mL of XAD-8 resin were mixed in equal volumes and packed into a 6 mL solid-phase extraction column. The resin column was activated sequentially with 10 mL of anhydrous ethanol and 20 mL of ethyl acetate. After activation, 1 L of surface water was introduced at a flow rate of 5 mL / min to complete the adsorption of organic pollutants. After adsorption, the resin column was dried with nitrogen gas and eluted with 20 mL of anhydrous ethanol. After collecting all the eluent, it was dried with nitrogen gas in a 40 °C water bath and replaced with 100 μL of DMSO to obtain the water sample enriched for testing. The sample was then stored at 4 °C for later use.

[0024] 3. Exposure to Toxins: Water samples enriched with the test samples were serially diluted and added to 96-well microplates inoculated with CHO-k1 cells, with 2 μL added to each well. This resulted in enrichment factors of 100, 50, 25, 12.5, 6.25, and 3.125 times the test samples, with three parallel wells for each concentration. A blank control group and a negative control group were simultaneously established. The well arrangement of the 96-well microplates was as follows: Figure 2 As shown, the microplates were placed in a 37°C, 5% CO2 incubator for 48 hours to expose them to the virus.

[0025] 4. Parallel detection of dual indicators: After the culture was completed, 10 μL of CCK-8 reagent was added to the corresponding wells of groups 1-3. After reacting at 37℃ in the dark for 3 h, the absorbance value at 450 nm wavelength was measured using an ELISA reader, and the cell inhibition rate and relative number of surviving cells were calculated. For the four groups of fluorescence detection plates, the upper culture medium was removed, and 200 μL of DCFH-DA fluorescent probe with a final concentration of 2 μM was added to each well. After incubation at 37℃ in the dark for 1 h, the probe was removed. After digestion with trypsin for 6 min, centrifugation and washing twice with 0.01M PBS, the total ROS fluorescence intensity at 480 nm excitation wavelength and 525 nm emission wavelength was measured using an ELISA reader.

[0026] 5. Fluorescent toxicity assessment and grading: The relative ROS fluorescence intensity and oxidative stress effect rate R of a single cell are calculated according to the above formula. Toxicity grading is performed based on the R value, and the fluorescent toxicity equivalent of the water sample is calculated at the same time.

[0027] The detection results of this embodiment are as follows: Figure 3 and Figure 5 It was found that when the enrichment factor of the water sample was 100, 50, and 25 times, the cell inhibition rate increased significantly, and obvious cytotoxicity could be detected. When the enrichment factor decreased to 12.5, 6.25, and 3.125 times, the cell inhibition rate was only 0-10%, and the traditional CCK-8 method could not detect obvious toxicity. However, the oxidative stress effect rate R detected by the method of this invention reached 10%-500%, indicating that the water sample had a high degree of influence on cellular oxidative stress. The fluorescence toxicity equivalent was 11.07 mg phenol / L, which can sensitively detect the toxicity of water samples at low enrichment factors. The EC50 of the water sample cytotoxicity was calculated. 50 The corresponding enrichment factor was 40.86 times, at which the oxidative stress effect rate (R) reached 283%. In summary, the oxidative stress effect rate (R) more sensitively reflects the potential toxicity of the water sample.

[0028] Example 2: Toxicity Detection of Phenol, a Typical Organic Pollutant in Water Bodies This embodiment focuses on the toxicity detection of phenol, a typical organic pollutant in water, to verify the detection sensitivity of the method of the present invention for characteristic pollutants. The specific steps are as follows: 1. Preparation of test cells: Same as step 1 in Example 1; 2. Preparation of test samples: Phenol gradient concentration stock solutions were prepared using DMSO, with concentrations of 1×10⁻⁶. 5 1×10 4 1×10 3 500, 50, 0.5, 0.005 mg / L, refrigerate at 4℃ for later use; 3. Exposure to phenol: Phenol stock solution was added to 96-well microplates seeded with CHO-k1 cells, with a volume of 2 μL added to each well to achieve final phenol concentrations of 1000, 100, 10, 5, 0.5, 0.005, and 0.00005 mg / L, with three parallel wells for each concentration. A blank control group and a negative control group were set up simultaneously, and the cells were incubated at 37℃ in a 5% CO2 incubator for 48 h. 4. Parallel detection of dual indicators and toxicity assessment: Same as steps 4 and 5 in Example 1.

[0029] The detection results of this embodiment are provided by Figure 4 and Figure 6It was found that when the phenol concentration was 1000, 500, and 50 mg / L, both the cell inhibition rate and the fluorescence toxicity response increased significantly. However, when the phenol concentration decreased to 0.5, 0.005, and 0.00005 mg / L, the cell inhibition rate was only 0-10%, and the traditional CCK-8 assay could not detect significant toxicity. In contrast, the oxidative stress effect rate (R) detected by the method of this invention reached 50%-200%, enabling sensitive detection of the cellular oxidative damage effect of low-concentration phenol. The EC50 of phenol cytotoxicity was calculated through fitting. 50 The concentration was 503.56 mg / L, at which the oxidative stress effect rate (R) reached 343%, further verifying that the detection sensitivity of the method of the present invention is much higher than that of traditional cytotoxicity detection methods.

[0030] Comparative Example 1: Traditional CCK-8 Cytotoxicity Assay This comparative example uses the traditional CCK-8 method to test the water in Example 1. The steps are as follows: 1. Cell preparation, water sample pretreatment, and exposure to the toxin were the same as in Example 1; 2. After the culture was completed, the absorbance at 450 nm was measured using only the CCK-8 assay to calculate the cell inhibition rate. The toxicity of the water sample was evaluated solely based on the cell inhibition rate.

[0031] Comparative results: It can only detect the toxicity of water samples with an enrichment factor of ≥25 times. When the enrichment factor is <25 times, the cell inhibition rate is <10%, and the potential toxicity of water samples cannot be detected. The detection limit is high, and false negative results are easy to occur. The sensitivity is far lower than that of the method of this invention.

[0032] Comparative Example 2: ROS fluorescence detection method without cell number correction This comparative example uses uncorrected total ROS fluorescence intensity to detect the water body from Example 1, and the steps are as follows: 1. Cell preparation, water sample pretreatment, exposure to toxins, and ROS fluorescence detection are the same as in Example 1; 2. Water toxicity was evaluated using only total ROS fluorescence intensity as an indicator, without correction for the number of surviving cells.

[0033] Comparative results: At high enrichment folds, a large number of cells died, and the total ROS fluorescence intensity decreased significantly, resulting in false negative results; at low enrichment folds, the total ROS fluorescence intensity was affected by fluctuations in cell number, and the detection results had an RSD > 15%, with poor repeatability and accuracy, and could not objectively reflect the true toxicity of the water sample.

[0034] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A method for sensitively detecting water toxicity using CHO-k1 cell fluorescence, characterized in that, Includes the following steps: CHO-k1 cells in the logarithmic growth phase were cultured in a culture dish; Solid-phase extraction was performed on the water sample to be tested using a mixed resin column of XAD-2 and XAD-8. After elution, nitrogen blowing and solvent replacement, the enriched sample to be tested was obtained. The enriched test sample was added to a culture dish inoculated with CHO-k1 cells, and a blank group and a negative control group were set up simultaneously. The samples were then cultured in an incubator. After the culture was completed, the cell viability in each well of the culture dish was detected by the CCK-8 assay to obtain the relative number of surviving cells N in the well; the fluorescence intensity F of reactive oxygen species (ROS) in the total cells of each well was detected by the DCFH-DA fluorescent probe method. The total fluorescence intensity was corrected by the relative number of surviving cells. The relative ROS fluorescence intensity Rs of a single cell was calculated. The oxidative stress impact rate R was calculated according to the following formulas I and II. The toxicity of the water body to be tested was quantitatively evaluated and risk-classified based on the R value. The relative ROS fluorescence intensity of a single cell is Rs = F / N; Formula I; The effect rate of oxidative stress R (%) = [(Rs-Rc) / Rc] × 100%; Formula II; Where F represents the total fluorescence intensity of reactive oxygen species (ROS) in each well, N represents the relative number of surviving cells in the well, Rs represents the relative ROS fluorescence intensity of a single cell in the cell experimental group, and Rc represents the relative ROS fluorescence intensity of a single cell in the negative control group.

2. The method for sensitively detecting water toxicity using CHO-k1 cell fluorescence according to claim 1, characterized in that, The CHO-k1 cell density was (1.0~1.5)×10⁻¹⁰. 4 The culture dish used 96-well microplates with a density of 1 / mL.

3. The method for sensitively detecting water toxicity using CHO-k1 cell fluorescence according to claim 1, characterized in that, The volume ratio of XAD-2 to XAD-8 is 1:

1. Before the solid-phase extraction enrichment, the mixed resin column is activated sequentially with ethanol and ethyl acetate. After activation, the water to be tested is introduced to complete the adsorption.

4. The method for sensitively detecting water toxicity using CHO-k1 cell fluorescence according to claim 1, characterized in that, The elution process uses anhydrous ethanol as the eluent. After elution, the sample is dried by nitrogen gas and then replaced with dimethyl sulfoxide to obtain an enriched sample for testing.

5. The method for sensitively detecting water toxicity using CHO-k1 cell fluorescence according to claim 1, characterized in that, The conditions for the culture with the contaminated environment were 37°C, 5% CO2, and a culture time of 48 h.

6. The method for sensitively detecting water toxicity using CHO-k1 cell fluorescence according to claim 1, characterized in that, The blank group was the test group containing DMSO, F12-K medium, cells-free and without enrichment of the test sample; The negative control group consisted of the experimental group containing DMSO, F12-K medium, CHO-k1 cells, and no enriched test sample.

7. The method for sensitively detecting water toxicity using CHO-k1 cell fluorescence according to claim 1, characterized in that, The specific steps of the CCK-8 assay are as follows: CCK-8 reagent is added to the cell culture dish, and the mixture is reacted at 37°C in the dark for 3 hours. The absorbance value at a wavelength of 450 nm is then measured. The formula for calculating the cell inhibition rate and the relative number of surviving cells is as follows: Cell inhibition rate IR (%) = [(A-Ab) / (Ac-Ab)] × 100%; The relative number of surviving cells in the experimental group N = As - Ab; Where As is the absorbance value of the cell experimental group, Ac is the absorbance value of the negative control group, and Ab is the absorbance value of the blank group.

8. The method for sensitively detecting water toxicity using CHO-k1 cell fluorescence according to claim 1, characterized in that, The specific steps of the DCFH-DA fluorescent probe method are as follows: remove the culture medium from the microwell of the culture dish, add the DCFH-DA probe, incubate at 37°C in the dark for 1 h, remove the probe, digest with trypsin, wash with PBS, and then detect the fluorescence intensity. The detection parameters are excitation wavelength 480 nm and emission wavelength 525 nm.

9. The method for sensitively detecting water toxicity using CHO-k1 cell fluorescence according to claim 1, characterized in that, The risk classification criteria are as follows: when 0 < R ≤ 10%, the water sample is considered to have a low degree of influence on cellular oxidative stress; when 10% < R ≤ 100%, the water sample is considered to have a moderate degree of influence on cellular oxidative stress; when 100% < R ≤ 500%, the water sample is considered to have a high degree of influence on cellular oxidative stress; and when R > 500%, the water sample is considered to have an extremely high degree of influence on cellular oxidative stress.

10. The application of the method for sensitive detection of water toxicity using CHO-k1 cell fluorescence according to any one of claims 1 to 9 in water sample safety testing, rapid screening of comprehensive water toxicity, evaluation of the toxicity of organic pollutants in water samples, and health risk assessment of drinking water.

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