LU500609B1

LU500609B1 - Method for detecting low-concentration ros and use thereof

Info

Publication number: LU500609B1
Application number: LU500609A
Authority: LU
Inventors: Yingying Zha; Daliang Yu; Sen Hou
Original assignee: Univ Jinan
Priority date: 2020-10-16
Filing date: 2021-09-01
Publication date: 2022-06-07
Also published as: CN112326607A; LU500609A1; CN112326607B

Abstract

The present disclosure discloses a method for detecting low-concentration ROS and use thereof. The detection method disclosed by the present disclosure is used for detecting a level of reactive oxygen species (ROS) in a cell subjected to oxidative damage by utilizing a fluorescence probe DCFH-DA. The detection method comprises the following steps: (1) adding a 2',7'- dichlorodihydrofluorescein diacetate (DCFH-DA) solution into a cell subjected to oxidative damage, conducting incubation in dark, removing the solution, and washing the cell to obtain a cell sample to be detected; and (2) irradiating the cell sample to be detected with light and detecting a fluorescence signal. The detection method disclosed by the present disclosure can be used for detecting low-dose ROS in a cell and solves problems that detection of the low-dose ROS is easily affected by an optical radiation and the real level of the ROS cannot be accurately detected. The method disclosed by the present disclosure can increase fluorescence intensity of different samples, while it does not change relative fluorescence intensity, achieves an equal-ratio increase of the fluorescence intensity of the different samples and thus enables a detection numerical value to be more obvious.

Description

BL-5293 METHOD FOR DETECTING LOW-CONCENTRATION ROS AND USE THEREOF

TECHNICAL FIELD

[01] The present disclosure relates to the technical field of bioactivity detection, in particular to a method for detecting low-concentration ROS and use thereof.

BACKGROUND ART

[02] Reactive oxygen species (ROS) refer to highly reactive by-products in a form of oxygen- containing radicals and non-radicals involved in oxygen metabolism in organisms, including superoxide anions, hydroxyl radicals, hydrogen peroxide, and the like. The ROS participate in the processes such as immune responses, gene expression regulation, and signal transduction in organisms. The detection of the ROS has important significance in many scientific researches, such as characterization of oxidative stress, toxicity research of pollutants, development of antitumor drugs and the like. Excessive ROS can cause oxidative damage to biomacromolecules such as cytoplasmic membranes, proteins, nucleic acids, etc., and the oxidative damage is an important toxication mechanism for many pollutants. Therefore, detection of changes in the ROS content in organisms plays an important role in characterizing the oxidative damage suffered by the organisms.

[03] The ROS have active property, strong reactivity and short life time. Besides, various antioxidant mechanisms exist in organisms, thus the detection of the ROS is somewhat difficult. At present, the ROS in cells are mainly detected by a fluorescence probe method. The fluorescence probe method is widely applied in the detection of the ROS due to its characteristics of high sensitivity, high resolution, easiness in data processing and the like.

[04] In recent years, a DCFH-DA (2',7'-dichlorodihydrofluorescein diacetate) fluorescence probe method is widely used for detection of intracellular ROS. DCFH-DA does not contain fluorescence, but has the ability to freely penetrate cell membranes. It is hydrolyzed by an intracellular esterase to generate DCFH which is further oxidized by ROS to generate a fluorescent substance DCF (2',7'- dichlorodihydrofluorescein diacetate). The level of the ROS in cells is quantified by measuring fluorescence intensity of the DCF. However, the DCFH-DA based method has many disadvantages and the detection is often interfered by unwanted factors such as light, pH value, cytochrome c, antioxidants and the like.

[05] The fluorescence probe is reported to have photosensitivity and be easy to experience auto- oxidation to improve the fluorescence intensity of the background. The research from Marchesie et al. showed that DCF was reduced by continuous irradiation of visible light in the presence of a reducing agent, which further induced generation of superoxides, and initiated a chain reaction. As a result, DCFH was further oxidized and finally a self-amplification of a DCF fluorescence signal was 1

BL-5293 caused. The photoreaction mechanism of the DCF is shown in FIG. 1. Afzal M et al. found that the DCFH produced the fluorescent substance DCF under irradiation of light at 250, 300, 330, 400, 500, or 600 nm, thus the result was unstable. Meanwhile, Setsukinai K et al. found that excitation light of a fluorescence microscope could induce auto-oxidation of DCFH-DA to significantly enhance cell fluorescence. In addition, apoptotic cells release cytochrome C and catalyze reaction of DCFH with intracellular ROS to increase fluorescence intensity when the intracellular ROS is unchanged.

Changes in pH can affect the loading efficiency of the DCFH-DA probe. A research showed that the DCFH-DA could be hydrolyzed to generate DCFH under an alkaline condition. Since the DCFH could not penetrate through the cell membrane, it caused a reduction in content of the probe.

Intracellular antioxidants could react with the probe, and the probe was reduced to form free radicals, which affected the detection of the ROS.

[06] Therefore, to overcome the disadvantages and shortcomings of the prior art, a method for detecting low-concentration ROS is provided by using a fluorescent dye and light irradiation.

SUMMARY

[07] The principal objective of the present disclosure is to overcome the disadvantages and shortcomings in the prior arts and the present disclosure provides a method for detecting low- concentration ROS.

[08] Another objective of the present disclosure is to provide use of the detection method.

[09] The objective of the present disclosure is realized by the following technical scheme. In the present invention, a method for detecting low-concentration ROS is provided to detect the ROS level in a cell subjected to oxidative damage by using a fluorescence probe DCFH-DA and the method comprises the following steps:

[10] (1) adding a cell subjected to oxidative damage into a DCFH-DA solution, conducting incubation in dark, and washing the cell to obtain a cell sample to be detected; and

[11] (2) irradiating the cell sample to be detected with light and detecting a fluorescence signal.

[12] The cell subjected to oxidative damage in step (1) has low ROS content which cannot be detected by existing detection kits for reactive oxygen species.

[13] That the low ROS content which cannot be detected means that the detected value shows no obvious difference from a blank background value and a P value obtained by statistical analysis is greater than 0.05, indicating that there is no significant difference.

[14] The final concentration of the DCFH-DA in a system is 10 uM when an existing ROS detection kit is used to detect the ROS.

[15] The cell subjected to oxidative damage in step (1) is stimulated to produce ROS by the following method: the cell is inoculated into a 96-well plate with 15,000 cells in each well, the cell 2

BL-5293 grows in a wall-adhesion manner for 12 h until a confluence reaches 50-70%, a HO» solution or a 0° polystyrene plastic nanoparticle is used to stimulate the cell, and the cell is cultured at 37°C in dark and washed with 1xPBS buffer (0.01M, pH=7.4) to remove residual stimulants.

[16] The HzO; solution has a concentration of 0.03-0.06% by volume.

[17] The polystyrene plastic nanoparticle has a particle size of 20-100 nm and a concentration of 500 mg/L.

[18] When the H,O; solution is used to stimulate the cell, the culture is conducted in dark for 30 min.

[19] When the polystyrene plastic nanoparticle is used to stimulate the cell, the culture is conducted in dark for 4 h.

[20] The cell growth medium used in the inoculation is prepared as follows: 89 mL of a high- sugar DMEM medium, 10 mL of fetal bovine serum and 1 mL of a penicillin-streptomycin double- antibody solution (100%) are added into a container, and the pH of the cell growth medium is adjusted to 7.0.

[21] The DCFH-DA solution in step (1) is obtained by diluting DCFH-DA with a high-sugar DMEM serum-free medium (containing a 1% penicillin-streptomycin double antibody solution) and adjusting pH of the solution to 7.0.

[22] The DCFH-DA solution in step (1) is obtained by diluting with the high-sugar DMEM serum- free medium to a concentration of 2-20 uM and preferably to a concentration of 10 uM.

[23] The pH of the solution is adjusted by using a 0.1 M NaOH solution or a 12 M HCl solution.

[24] The incubation in step (1) is conducted in dark for 15-60 min and may preferably be conducted for 30 min.

[25] The cell washing in step (1) is conducted by washing with PBS buffer for 2-3 times to remove the DCFH-DA molecules that do not enter the cell.

[26] The PBS buffer is 1xPBS buffer (0.01 M, pH=7.4).

[27] The irradiation with light in step (2) is conducted by using an LED lamp or a fluorescent lamp.

[28] The irradiation with light in step (2) is conducted for 5-10 min and may preferably be conducted for 10 min.

[29] The irradiation with light in step (2) is conducted at an optical power density of 5.43-13.40 mW/cm? and may preferably be conducted at 13.40 mW/cm?.

[30] The fluorescence signal in step (2) is detected by a multifunctional microplate reader, with the excitation wavelength for detecting fluorescence signal being at 488 nm and the emission wavelength being at 525 nm.

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[31] The present disclosure also provides the use of the method for detecting low-concentration ROS in biological science and research.

[32] Compared with the prior arts, the present disclosure has the following advantages and effects.

[33] (1) In prior arts, detection of low-dose ROS by a fluorescence probe method is easily affected by optical radiation and the real level of the ROS cannot be accurately measured, while the method in the present disclosure makes up for the existing shortcomings and allows for detection of the low- dose ROS in cells.

[34] (2) The method disclosed in the present disclosure can increase fluorescence intensity of different samples at the same time, it does not change relative fluorescence intensity, which achieves an equal-ratio increase of the fluorescence intensity of the different samples and thus makes the detection results more obvious.

[35] (3) When the method of the present disclosure is used for detection, fluorescence intensity of the sample to be tested is enhanced and stabilized after light irradiation. The fluorescence intensity does not fluctuate with the number of detections during the process of detection by a microplate reader, such that a detection value is more stable.

BRIEF DESCRIPTION OF THE DRAWINGS

[36] FIG. 1 shows the mechanism for photoreaction of DCF.

[37] FIG. 2 shows the results for detecting the ROS produced by cells induced by hydrogen peroxide using the detection method of the present disclosure; where panel a is the detection result for ROS produced by cells induced by hydrogen peroxide at different concentrations; and panel b is the fluorescence ratio between the cells treated with 0.06% H,O» and with 0.03% H,O, for different light irradiation time.

[38] FIG. 3 is a detection graph of ROS in HeLa cells subjected to oxidative damage induced by

0.06% HxO; for different light irradiation time.

[39] FIG. 4 is a statistical diagram of a result for detecting ROS produced by cells induced by polystyrene plastic nanoparticles of different particle sizes by using the detection method of the present disclosure.

[40] FIG. 5 shows the results for detecting intracellular ROS by using a conventional method; where panel a shows the detection result of ROS produced by cells induced by hydrogen peroxide at different concentrations; and panel b shows a detection result of ROS produced by cells induced by polystyrene plastic nanoparticles of different particle sizes.

[41] FIG. 6 shows results of changes in fluorescence intensity of DCFH and DCF over light irradiation time under different intensity of light irradiation; where panel a is for the control group, 4

BL-5293 panel b uses an optical power density of 5.43 mW/cm?, panel c uses an optical power density of 6.80 °° mW/cm? and d uses an optical power density of 13.40 mW/cm?.

[42] FIG. 7 is a statistical graph of a fluorescence intensity ratio of DCF samples with concentrations of 0.05 uM and 0.02 uM under different light irradiation time and optical power densities.

[43] FIG. 8 shows the influence of different light irradiation intensity and light irradiation time on fluorescence stability of a DCF sample at a concentration of 0.02 uM; where panel a is for control group, panel b is for light irradiation for 10 min, panel c is for light irradiation for 20 min and d is for light irradiation for 40 min.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[44] The following parts further describe in detail the present disclosure with reference to the examples and the accompanying drawings, but implementations of the present disclosure are not limited thereto. If no specific conditions are specified, the examples will be conducted according to conventional conditions or the conditions recommended by manufacturers. All of the used reagents or instruments which are not specified with manufacturers are conventional commercially-available products.

[45] Example 1 Stimulation of HeLa cells to generate ROS

[46] (1) Stimulation of HeLa cells by H,O, to generate ROS

[47] 1) A cell growth medium was prepared with 89 mL of a high-sugar DMEM medium, 10 mL of fetal bovine serum and 1 mL of a penicillin-streptomycin double-antibody solution, and the pH of the cell growth medium was adjusted to 7.0 by using 0.1M of NaOH solution or 12M of HCI solution.

[48] 2) A human cervical cancer cell line HeLa was selected as the experimental cell line and the cells were inoculated into a 96-well plate the day before the experiment with 15,000 cells per well. The cells grew in a wall-adhesion manner for 12 h until the confluence level reached 50-70%.

[49] 3) Different volumes of H,O, were respectively added to stimulate the cells (the concentration of the H,O, in the system was 0.03% and 0.06%, respectively) and the cells were cultured in a cell incubator at 37°C in dark for 30 min.

[50] 4) After 30 min of stimulation, the solution was removed and the cells were washed with 1xPBS (0.01M, pH=7.4) buffer to remove residual H>O; to obtain HeLa cells subjected to oxidative damage.

[51] (2) Stimulation of HeLa cells by polystyrene plastic nanoparticles to generate ROS

[52] 1) A cell growth medium was prepared with 89 mL of a high-sugar DMEM medium, 10 mL of fetal bovine serum and 1 mL of a double antibody solution, and the pH of the cell growth medium was adjusted to 7.0.

BL-5293

[53] 2) A human cervical cancer cell line HeLa was selected as the experimental cell line and tha 0” cells were inoculated a 96-well plate the day before the experiment with 15,000 cells per well. The cells grew in a wall-adhesion manner for 12 h until the confluence level reached 50-70%.

[54] 3) Polystyrene plastic nanoparticles of different particle sizes (purchased from Shanghai Huge Biotechnology Co., Ltd., Model No: DS20 and DS100, particle size: 20 nm and 100 nm, and the concentration in the system was 500mg/L) to stimulate the cells and the cells were cultured in dark in a cell incubator at 37°C for 4 h.

[55] 4) After 4 h of stimulation, the solution was removed and the cells were washed with 1 x PBS (0.01M, pH=7.4) buffer to remove residual plastic nanoparticles to obtain HeLa cells subjected to oxidative damage.

[56] Example 2 Detection of ROS in HeLa cells stimulated by HO»

[57] 1) A high-sugar DMEM serum-free medium (containing a 1% penicillin-streptomycin double-antibody solution) was added into the HeLa cells subjected to oxidative damage in step (1) of example 1 to dilute a DCFH-DA solution (a final concentration was 10 uM), the pH of the solution was adjusted to 7.0 by adding dropwise 0.1 M NaOH solution or 12 M HCI solution, and the cells were incubated in dark for 30 min.

[58] 2) After 30 min of incubation, the solution was removed and the cells were washed with 1xPBS (0.01M, pH=7.4) buffer for 2-3 times to remove the DCFH-DA solution that did not enter the cells to obtain a sample to be detected.

[59] 3) The sample to be detected was irradiated with LED light for a light irradiation time duration of 5 min and 10 min respectively and at an optical power density of 13.40 mW/cm?.

[60] 4) Fluorescence signal in each well plate was detected by a multifunctional microplate reader, with the excitation wavelength for detecting fluorescence signal being at 488 nm and the emission wavelength being at 525 nm.

[61] The result is shown in FIG. 2a. Fluorescence intensity, detected by the method of the present disclosure, measured before and after hydrogen peroxide treatment of the cells had a significant difference (p<0.05), which was consistent with the report in which substantial ROS were produced by the hydrogen peroxide-treated cells. After the sample to be detected was irradiated with the LED light, fluorescence intensity of different samples was enhanced, while the relative fluorescence intensity was not changed, achieving the effect that an equal-ratio increase of the fluorescence intensity of the different samples was measured. Besides, after light irradiation, compared with a blank background value, the overall fluorescence value of the sample became larger and a detection value was more prominent. As shown in FIG. 2b, when no light irradiation was conducted, the fluorescence ratio between the cells treated with 0.06% H,O, and with 0.03% Hz:O2 was

3.1017+3.3513. After light irradiation for 5 min, the fluorescence ratio was 3.3120+0.8759; and after 6

BL-5293 light irradiation for 10 min, the fluorescence ratio was 3.2934+0.9330. It showed that the fluorescence intensity of the sample to be detected can achieve an effect of equal-ratio increase. The fluorescence ratio between the experimental groups treated with different concentrations of hydrogen peroxide and irradiated for the same time was stable, which proved that the relative ROS would not change and the light irradiation had no effect on the detection result of the ROS. FIG. 3 was a detection graph of ROS in HeLa cells subjected to oxidative damage induced by 0.06% H:O» under different light irradiation time duration.

[62] Example 3 Detection of ROS in HeLa cells stimulated by polystyrene plastic nanoparticles

[63] 1) A high-sugar DMEM serum-free medium (containing a 1% penicillin-streptomycin double-antibody solution) was added into the HeLa cells subjected to oxidative damage in step (2) of example 1 to dilute a DCFH-DA solution (final concentration was 10 uM), the pH of the solution was adjusted to 7.0 by adding dropwise 0.1M NaOH solution or 12M HCI solution, and the cells were incubated in dark for 30 min.

[64] 2) After 30 min of incubation, the solution was removed and the cells were washed with 1xPBS (0.01M, pH=7 4) buffer for 2-3 times to remove the DCFH-DA that did not enter the cells.

[65] 3) The sample to be detected was irradiated with LED light for a light irradiation time of 5 min and 10 min respectively and at an optical power density of 13.40 mW/cm?.

[66] 4) Fluorescence signal in each well plate was detected by a multifunctional microplate reader, with the excitation wavelength for detecting fluorescence signal being at 488 nm and the emission wavelength being at 525 nm.

[67] As shown in FIG. 4, fluorescence intensity generated by treating the cells with polystyrene plastic nanoparticles of different particle sizes had a significant difference (p<0.05), which was consistent with the reports in which significant ROS were produced. The fluorescence intensity of the cells in each treatment group increased significantly. Compared with the case of non-light irradiation, light irradiation can significantly increase the fluorescence signal of the cells and makes the detection value more prominent. For example, after light irradiation for 10 min, the fluorescence intensity of the cells treated with 20-nm polystyrene plastic nanoparticles (500 mg/L) was

27.85+10.76 times of the untreated group; and the fluorescence intensity of the cells treated with 100- nm polystyrene plastic nanoparticles (500 mg/L) was 7.20+2.94 times of the untreated group.

[68] Comparative example 1 Detection of ROS in HeLa cells stimulated with HO» by using conventional method

[69] 1) The HeLa cells subjected to oxidative damage in step (1) of example 1 were added to a DCFH-DA solution (final concentration was 10 uM) diluted with high-sugar DMEM serum-free medium (containing a 1% penicillin-streptomycin double antibody solution), the pH of the solution 7

BL-5293 was adjusted to 7.0 by adding dropwise the 0.1 M NaOH solution or the 12 M HCI solution, and the cells were incubated in dark for 30 min.

[70] 2) After 30 min of incubation, the solution was removed and the cells were washed with 1xPBS (0.01M, pH=7.4) buffer for 2-3 times to remove the DCFH-DA that did not enter the cells to give a sample to be detected.

[71] 3) Fluorescence signal in each well plate was detected by a multifunctional microplate reader, with the excitation wavelength for detecting fluorescence signal being at 488 nm and the emission wavelength being at 525 nm.

[72] The result is shown in FIG. Sa, the fluorescence intensity, detected by using a conventional method, generated by treating the cells with hydrogen peroxide had no significant difference (p>0.05) and the value showed bstantial fluctuation. The fluorescence intensity of the cells treated with 0.03% HO» was 1.0659+0.2487 times of the untreated group. The fluorescence intensity of the cells treated with 0.06% HO» was 1.6471+0.6566 times of the untreated group.

[73] Comparative example 2 Stimulation of HeLa cells to generate ROS by polystyrene plastic nanoparticles by using conventional method

[74] 1) The HeLa cells subjected to oxidative damage in step (2) of example 1 was add to the DCFH-DA solution (final concentration was 10 uM) diluted with high-sugar DMEM serum-free medium (containing a 1% penicillin-streptomycin double-antibody solution) was added into to dilute a, the pH of the solution was adjusted to 7.0 by adding dropwise 0.1M NaOH solution or 12M HCI solution, and the cells were incubated in dark for 30 min.

[75] 2) After 30 min of incubation, the solution was removed and the cells were washed with 1xPBS (0.01M, pH=7 4) buffer for 2-3 times to remove the DCFH-DA that did not enter the cells.

[76] 3) Fluorescence signal in each well plate was detected by a multifunctional microplate reader, with the excitation wavelength for detecting fluorescence signal being at 488 nm and the emission wavelength being at 525 nm.

[77] The result is shown in FIG. 5b, the fluorescence intensity generated by treating the cells with polystyrene plastic nanoparticles of different particle sizes had no significant difference (p>0.05) as treating with hydrogen peroxide. The fluorescence intensity of the cells treated with 20-nm polystyrene plastic nanoparticles (500 mg/L) was 0.8267+0.0459 times of the untreated group. The fluorescence intensity of the cells treated with 100-nm polystyrene plastic nanoparticles (500 mg/L) was 0.9693+0.0381 times of the untreated group.

[78] Example 4 DCF fluorescence amplification test

[79] 1) Purified water was used to prepare 1xPBS buffer and the pH was adjusted to 7.4, DMSO was used to prepare a DCFH-DA stock solution with a concentration of 10 mM, and DMSO was used to prepare a DCF stock solution with a concentration of 10 mM.

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[80] 2) The DCF stock solution was diluted 1,000 times with purified water to prepare a DOR solution with a concentration of 10 uM and the DCF solution was preserved in dark.

[81] 3) The DCFH-DA stock solution was diluted 1,000 times with 1xPBS buffer (0.01M, pH=7 4) and hydrolyzed in dark for 20 min to generate a DCFH solution which was recorded as Solution A.

[82] 4) Preparation of a sample to be detected: 1.5 uL of DCF solution was added into 1.5 mL of Solution A to prepare a 0.01-uM DCF solution which was recorded as Solution B. 8 uL of DCF solution was added into 4 mL of the Solution A to prepare a 0.02 uM DCF solution which was recorded as Solution C. 20 pL of the DCF solution was added into 4 mL of the Solution A to prepare a 0.05-uM DCF solution which was recorded as Solution D.

[83] 5) 200 uL of solution eachfrom the Solutions A, B, C, and D were added in a 96-well plate, with 3 duplicate wells for each group of the Solutions A, B, C, and D. After light irradiation for 0 min, 1 min, 3 min, 5 min, 7 min, 10 min, 15 min, 20 min, 30 min and 40 min, a multifunctional microplate reader was used to detect a fluorescence signal in each well plate. An optical power density was adjusted to 0, 5.43 mW/cm?, 6.80 mW/cm? and 13.40 mW/cm?. In addition, the fluorescence signal in non-light-irradiation control group was additionally detected at 0.5 min and 2 min.

[84] The result is shown in FIG. 6a. DCF fluorescence was unstable when there was no light irradiation. The fluorescence value had a clear upward trend over time. It may be due to that DCFH underwent an auto-oxidation to generate DCF to increase the fluorescence. FIGS. 6b, 6¢, and 6d showed that as the light irradiation time increased, the DCF fluorescence was significantly enhanced, and as the light irradiation intensity increased, a photo-oxidation reaction became more intense and finally the fluorescence intensity reached saturation. In addition, taking the 0.02-uM and 0.05-uM DCF samples were used as examples (FIG. 7), when the optical power density was 5.43 mW/cm?, a fluorescence intensity ratio ofthe two samples had a very good equal-ratio amplification effect within min of light irradiation. However, when the light irradiation time continued to be extended, the relative fluorescence intensity would change in a manner such that the equal-ratio increase effect cannot be achieved. When the light irradiation intensity increased to 6.80 mW/em” and 13.40 mW/cm?, the equal-ratio increase effect could be achieved. Therefore, after a specified time of the light irradiation, the fluorescence intensity of different DCF samples could reach an equal-ratio increase. As a result, the relative fluorescence intensity was not changed and the detection value was more prominent.

[85] In addition, it can be extrapolated from FIG. 6d that when the optical power density was high, the fluorescence of the DCF samples with different concentrations would be directly saturated in a short time. At this time, the fluorescence intensity cannot be amplified in an equal-ratio manner, thus the relative ROS level would be changed, the ROS cannot be accurately detected, and strong light could also cause cell death. If the optical power density was too low, it would take longer time to 9

BL-5293 amplify the fluorescence and long-term light irradiation would also cause cell necrosis. Therefore, 99 the light irradiation intensity should not be too high or too low.

[86] Example 5 DCF fluorescence stability test

[87] 1) Purified water was used to prepare 1xPBS buffer and the pH was adjusted to 7.4, DMSO was used to prepare a DCFH-DA stock solution with a concentration of 10 mM, and DMSO was used to prepare a DCF stock solution with a concentration of 10 mM.

[88] 2) The DCF stock solution was diluted by 1,000 folds with the purified water to prepare a DCF solution with a concentration of 10 uM and the DCF solution was preserved in dark.

[89] 3) The DCFH-DA stock solution was diluted by 1,000 folds with 1xPBS buffer (0.01M, pH=7.4) and hydrolyzed in dark for 20 min to generate a DCFH solution which was recorded as Solution A.

[90] 4) Preparation of a sample to be detected: 8 uL of the DCF solution was added into 4 mL of Solution A to prepare a 0.02 uM DCF solution which was recorded as Solution B.

[91] 5) 200 uL of solution from the Solution B were added in a 96-well plate, with 3 duplicate wells being set for each group, and a total of 4 groups of the sample were prepared (as group a, group b, group c and group d). The sample in group a was placed in dark and fluorescence intensity was measured every 5 min, the sample in group b was irradiated for 10 min and the fluorescence intensity was measured every 5 min, the sample in group c was irradiated for 20 min and the fluorescence intensity was measured every 5 min, and the sample in group d was irradiated for 40 min and the fluorescence intensity was measured every 5 min. The optical power densities of group b, group c and group d were adjusted to 5.43 mW/cm?, 6.80 mW/cm? and 13.40 mW/cm?, respectively.

[92] The result is shown in FIG. 8a. When the DCFH solution containing 0.02 uM DCF sample was not irradiated, the fluorescence intensity was not stable. As the number of detections increased, the fluorescence intensity of the samples gradually increased. As shown in FIG. 8b, when the optical power density was 5.43 mW/cm? and 6.80 mW/cm?, the fluorescence of the samples was not stable after 10 min of light irradiation, and when the optical power density was 13.40 mW/cm?, the fluorescence of the samples exhibited very good stability and no longer increased with the increase of the number of the detections. When the optical power density was 6.80 mW/cm? and 13.40 mW/cm?, the long-term light irradiation for 20 min and 40 min would cause the fluorescence intensity of the DCF samples to decrease due to photobleaching (FIG. 8c-d).

[93] In summary, after the DCF/DCFH samples were irradiated with light of different intensity, the fluorescence intensity of the DCF/DCFH samples had very good stability and did not change significantly over time. As the light irradiation intensity increased, the fluorescence stability was better. After the DCF/DCFH samples were irradiated with specific light irradiation time, the

BL-5293 fluorescence can be stabilized. The light irradiation time should not be too long, otherwise The 0%? photobleaching would tend to occur.

[94] The above examples are preferred embodiments of the present disclosure. However, the embodiments of the present disclosure are not limited by the above examples. Any change, modification, substitution, combination and simplification made without departing from the spiritual essence and principle of the present disclosure should be an equivalent replacement manner, and all are included in a protection scope of the present disclosure.

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Claims

BL-5293 CLAIMS LU500609 WHAT IS CLAIMED IS:

1. À method for detecting low-concentration reactive oxygen species (ROS), wherein the method comprises the following steps: (1) adding a DCFH-DA solution into a cell subjected to oxidative damage, conducting incubation in dark, and washing the cell to obtain a cell sample to be detected; and (2) irradiating the cell sample to be detected with light and detecting a fluorescence signal.

2. The method according to claim 1, wherein the cell subjected to oxidative damage in step (1) has low ROS content which cannot be detected by a detection kit for existing reactive oxygen species; that the low ROS content which cannot be detected means that an obtained value shows no substantial difference from a blank background value and that a P value obtained by statistical analysis is greater than 0.05, indicating that there is no significant difference; and a final concentration of the DCFH-DA in a system is 10 uM when an existing ROS detection kit is used to detect the ROS.

3. The method according to claim 1, wherein, the DCFH-DA solution in step (1) is obtained by diluting DCFH-DA with a high-sugar DMEM serum-free medium containing a 1% penicillin-streptomycin double-antibody solution and adjusting pH of the solution to 7.0; the DCFH-DA solution in step (1) is obtained by diluting with the high-sugar DMEM serum- free medium to a concentration of 2-20 uM; the incubation in step (1) is conducted in dark for 15-60 min; the irradiation with light in step (2) is conducted for 5-10 min; and the irradiation with light in step (2) is conducted at an optical power density of 5.43-13.40 mW/cm?.

4. The method according to claim 3, wherein the DCFH-DA solution in step (1) is obtained by diluting with the high-sugar DMEM serum-free medium to a concentration of 10 uM; the incubation in step (1) is conducted in dark for 30 min; the irradiation with light in step (2) is conducted for 10 min; and the irradiation with light in step (2) is conducted at an optical power density of 13.40 mW/cm?.

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5. The method according to claim 1, wherein the cell washing in step (1) is conducted by washing with PBS buffer for 2-3 times to remove the DCFH-DA solution that does not enter the cell; the PBS buffer is 1xPBS buffer; the irradiation with light in step (2) uses an LED lamp or a fluorescent lamp; and the fluorescence signal in step (2) is detected by a multifunctional microplate reader, with the excitation wavelength for detecting fluorescence signal being at 488 nm and the emission wavelength being at 525 nm.

6. The method according to claim 1, wherein the cell subjected to oxidative damage in step (1) is stimulated to produce ROS by the following method: the cell is inoculated into a 96-well plate with 15,000 cells each well, the cell grows in a wall-adhesion manner for 12 h until a confluence reaches 50-70%, a H,O, solution or a polystyrene plastic nanoparticle is used to stimulate the cell, and the cell is cultured at 37°C in dark and washed with 1xPBS buffer to remove a residual stimulant.

7. The method according to claim 6, wherein, the H,O> solution has a concentration of 0.03-0.06% by volume; the polystyrene plastic nanoparticle has a particle size of 20-100 nm; and the polystyrene plastic nanoparticle has a concentration of 500 mg/L.

8. The method according to claim 7, wherein when the H,O», solution is used to stimulate the cell, the culture is conducted in dark for 30 min; and when the polystyrene plastic nanoparticle is used to stimulate the cell, the culture is conducted in dark for 4 h.

9. The method according to claim 6, wherein a cell growth medium used in the inoculation 1s prepared as follows: 89 mL of a high-sugar DMEM medium, 10 mL of fetal bovine serum and 1 mL of a 100xpenicillin-streptomycin double antibody solution are added to a container, and pH of the cell growth medium is adjusted to 7.0.

10. Use of the method according to any one of claims 1-9 in a biological science and research.

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