US-20260126387-A1 - IN-SITU DETECTION METHOD FOR REACTIVE OXYGEN SPECIES BASED ON RATIOMETRIC FLUORESCENT PROBE

US20260126387A1US 20260126387 A1US20260126387 A1US 20260126387A1US-20260126387-A1

Abstract

The present invention discloses an in-situ detection method for reactive oxygen species, which includes the following steps: attaching a reactive oxygen species composite membrane prepared based on a ratiometric fluorescent probe to a to-be-detected region to react with reactive oxygen species; capturing a green light signal on the reactive oxygen species composite membrane using a digital camera equipped with a 520-540 nm narrow-band filter to obtain an image A; capturing a red light signal on the reactive oxygen species composite membrane using a digital camera equipped with a 640-660 nm filter to obtain an image B; acquiring a numerical value (G) of a green light channel in the image A and a numerical value (R) of a red light channel in the image B respectively, and calculating relative fluorescence intensity (FI) as a reactive oxygen species response value; and obtaining a concentration of the reactive oxygen species in situ.

Inventors

Jun Luo
Xing Liu
Longgang CHU
Cheng Gu

Assignees

NANJING UNIVERSITY

Dates

Publication Date: 20260507
Application Date: 20251231
Priority Date: 20250401

Claims (8)

1 . An in-situ detection method for reactive oxygen species, comprising the following steps: S1, attaching a reactive oxygen species composite membrane prepared based on a ratiometric fluorescent probe to a to-be-detected region to react with reactive oxygen species, wherein a reaction time is 4-70 min, and the preparation of the reactive oxygen species composite membrane comprises the following steps: step 1: dissolving 2′,7′-dichlorodihydrofluorescein in an acetonitrile solvent to obtain 5-15 mmol/L of a 2′,7′-dichlorodihydrofluorescein mother solution; dissolving Nile Red in an acetonitrile solvent to obtain 5-15 mmol/L of a Nile Red mother solution; step 2: dissolving agarose in water by heating to obtain an agarose solution; step 3: cooling the agarose solution to 50-60° C., and then adding the 2′,7′-dichlorodihydrofluorescein mother solution and the Nile Red mother solution to obtain a mixed gel solution, wherein concentrations of both 2′,7′-dichlorodihydrofluorescein and Nile Red are 40-60 μmol/L; and step 4: injecting the mixed gel solution into a template, and cooling for solidifying to obtain the reactive oxygen species composite membrane; S2, irradiating the reactive oxygen species composite membrane with a 490-510 nm LED lamp as excitation light, and capturing a green light signal on the reactive oxygen species composite membrane using a digital camera equipped with a 520-540 nm narrow-band filter to obtain an image A with fluorescence intensity of green light recorded; S3, irradiating the reactive oxygen species composite membrane with a 520-540 nm LED lamp as excitation light, and capturing a red light signal on the reactive oxygen species composite membrane using a digital camera equipped with a 640-660 nm filter to obtain an image B with fluorescence intensity of red light recorded; S4, performing digital processing on the image A and the image B using image processing software to respectively acquire a numerical value G1 of a green light channel in the image A and a numerical value R1 of a red light channel in the image B, and calculating relative fluorescence intensity (FI 1 ) as a reactive oxygen species response value via formula (1); and F ⁢ I 1 = G ⁢ 1 R ⁢ 1 ( 1 ) S5, substituting the reactive oxygen species response value (FI) obtained in S4 into a quantitative standard curve of reactive oxygen species concentration to obtain a concentration of the reactive oxygen species in situ.
2 . The in-situ detection method for reactive oxygen species according to claim 1 , wherein a mass percentage of agarose in the agarose solution is 0.5 wt %-1.5 wt %; and a thickness of the reactive oxygen species composite membrane is 1-4 mm.
3 . The in-situ detection method for reactive oxygen species according to claim 2 , wherein the establishment of the quantitative standard curve of reactive oxygen species concentration comprises the following steps: step a: reacting the reactive oxygen species composite membrane with reactive oxygen species of known concentration for 4-70 min; step b: irradiating the reactive oxygen species composite membrane with a 490-510 nm LED lamp as excitation light, and capturing a green light signal on the reactive oxygen species composite membrane using a digital camera equipped with a 520-540 nm narrow-band filter to obtain an image C with fluorescence intensity of green light recorded; step c, irradiating the reactive oxygen species composite membrane with a 520-540 nm LED lamp as excitation light, and capturing a red light signal on the reactive oxygen species composite membrane using a digital camera equipped with a 640-660 nm filter to obtain an image D with fluorescence intensity of red light recorded; step d: performing digital processing on the image C and the image D using image processing software to respectively acquire a numerical value G2 of a green light channel in the image C and a numerical value R2 of a red light channel in the image D, and calculating relative fluorescence intensity (FI 2 ) as a reactive oxygen species response value via formula (2); and F ⁢ I 2 = G ⁢ 2 R ⁢ 2 ( 2 ) step e: repeating the above steps a to d with different concentrations of reactive oxygen species to obtain corresponding reactive oxygen species response values, and taking the reactive oxygen species concentration as an abscissa X and the reactive oxygen species response value as an ordinate Y to formulate a relationship equation between X and Y: Y=aX+b to obtain the quantitative standard curve of reactive oxygen species concentration for the reactive oxygen species composite membrane.
4 . The in-situ detection method for reactive oxygen species according to claim 3 , wherein R 2 in the relationship equation: Y=aX+b is 0.980-0.996, and p<0.002.
5 . Application of the in-situ detection method for reactive oxygen species according to claim 1 in detection of reactive oxygen species.
6 . Application of the in-situ detection method for reactive oxygen species according to claim 1 in quantitative detection of reactive oxygen species in soil.
7 . The application according to claim 6 , wherein an ionic strength in the soil is equivalent to 0-260 mmol/L o NaCl.
8 . The application according to claim 7 , wherein the soil has a pH value of 2-11.

Description

TECHNICAL FIELD The present invention belongs to the technical field of environmental monitoring, and more specifically, relates to an in-situ detection method for reactive oxygen species based on a ratiometric fluorescent probe. BACKGROUND Reactive oxygen species (ROS) are important reactive substances in the environment, including hydroxyl radicals (·OH), hydrogen peroxide (H2O2), and superoxide anions (O2−), which are widely involved in biological, chemical, and physical processes. Recent studies have shown that plant roots are an important source for ROS production and play a variety of key roles in the rhizosphere environment, such as regulating the speciation transformation of heavy metals. In the prior art, detection methods for ROS mainly rely on in vitro analysis methods, such as high performance liquid chromatography (HPLC) technology based on molecular probes and electron paramagnetic resonance (EPR) technology based on unpaired electron spins. Although these methods can provide relatively accurate quantitative analysis, their applications are limited by the following aspects: (a) Limited integrity of sampling: In vitro detection techniques require sample collection and processing, and inevitable chemical and biological changes during sampling may alter sample properties, thereby affecting the accuracy of detection results. (b) Relatively low spatial resolution: Traditional detection methods have difficulty in capturing ROS distribution at the micrometer or even smaller scale, which is particularly inadequate when studying the dynamic behavior of ROS in microenvironments such as the plant rhizosphere. (c) Environmental disturbance: Fluorescent probes may be subject to background fluorescence interference in complex soil and sediment environments, leading to a decline in detection sensitivity and accuracy. In addition, the uneven distribution of fluorescent probes may also affect the results. To address these challenges, in-situ detection technology has attracted increasing attention in recent years. An in-situ detection method can directly capture the dynamic distribution of ROS without disrupting the original redox state of a sample. However, current in-situ detection methods still have significant limitations, such as insufficient stability of fluorescence signals, significant background interference, and difficulty in achieving high-resolution detection of ROS. Moreover, the spatial resolution and soil universality of the existing in-situ detection methods still need to be further improved. SUMMARY 1. Problems to be Solved Aiming at the technical problems, such as insufficient stability of fluorescence signals, significant background interference, and the need for further improvement in spatial resolution and soil universality, existing in the in-situ detection methods in the prior art, the present invention provides an in-situ detection method for reactive oxygen species based on a ratiometric fluorescent probe, which significantly improves the stability of fluorescence signals and effectively solves the problem of environmental disturbance by combining the ratiometric fluorescent probe with planar optrode technology for the first time, and further improves spatial resolution and soil universality based on the existing in-situ detection methods. 2. Technical Solution To solve the above problems, the technical solutions adopted by the present invention are as follows: [In-Situ Detection Method for Reactive Oxygen Species] A first aspect of the present invention provides an in-situ detection method for reactive oxygen species, which includes the following steps: S1, attaching a reactive oxygen species composite membrane prepared based on a ratiometric fluorescent probe to a to-be-detected region to react with reactive oxygen species;S2, irradiating the reactive oxygen species composite membrane with a 490-510 nm LED lamp as excitation light, and capturing a green light signal on the reactive oxygen species composite membrane using a digital camera equipped with a 520-540 nm narrow-band filter to obtain an image A with fluorescence intensity of green light recorded;S3, irradiating the reactive oxygen species composite membrane with a 520-540 nm LED lamp as excitation light, and capturing a red light signal on the reactive oxygen species composite membrane using a digital camera equipped with a 640-660 nm filter to obtain an image B with fluorescence intensity of red light recorded;S4, performing digital processing on the image A and the image B using image processing software to respectively acquire a numerical value (G) of a green light channel in the image A and a numerical value (R) of a red light channel in the image B, and calculating relative fluorescence intensity (FI) as a reactive oxygen species response value via formula (1); and F⁢I=GR(1)S5, substituting the reactive oxygen species response value (FI) obtained in S4 into a quantitative standard curve of reactive oxygen species concent