Synthesis and application of bifunctional fluorescent probe for simultaneously distinguishing and detecting superoxide anions and viscosity

Abstract

The invention discloses a bifunctional fluorescent probe for simultaneously distinguishing and detecting superoxide anions and viscosity. The chemical structural formula of the bifunctional probe is as follows: # imgabs0 #. According to the bifunctional fluorescent probe, a coumarin derivative and a thiazole derivative are combined through piperazine, superoxide anions are detected by taking a trifluorosulfonate group as a recognition site, and viscosity is responded through a molecular rotor strategy. When the probe reacts with superoxide anions, strong yellow fluorescence of 550 nm is emitted under the excitation wavelength of 470 nm; when the probe is in high viscosity, near infrared fluorescence of 660 nm is emitted at the excitation wavelength of 540 nm. In addition, the probe has the advantages of high sensitivity, excellent selectivity and the like when being used for detecting superoxide anions and viscosity, and has a huge application prospect in the technical fields of analytical chemistry, life science, biomedical treatment and the like.

Description

Technical Field

[0001] The present invention belongs to the technical field of analytical chemistry, and specifically relates to the synthesis and application of a dual-function fluorescent probe for simultaneously distinguishing and detecting superoxide anions and viscosity. The probe connects two fluorophores through piperazine and can rapidly and selectively detect superoxide anions and respond to viscosity from various biologically active substances. The yellow fluorescent channel selectively detects superoxide anions, and the near-infrared fluorescent channel selectively responds to viscosity. It has the advantages of large Stokes shift, high detection sensitivity, and visual detection. Background Art

[0002] Superoxide anion (O2 •− ) is one of the important reactive oxygen species (ROS) in biological systems and plays a wide range of roles in regulating cell function. However, its abnormal and excessive production may lead to excessive oxidative stress and disruption of redox balance in the body, which is closely related to the occurrence and progression of many diseases ( Anal. Chem. 2019, 91, 9, 5786–5793). In order to reveal the pathological significance of superoxide anions in these diseases, it is imperative to develop effective monitoring technologies in biological systems. Viscosity is one of the main parameters in the cell microenvironment and plays a vital role in many cell life activities, such as the transport of intracellular substances, the interaction of biomolecules, the transmission of chemical signals, and the diffusion of active metabolites ( Chem. Soc. Rev. 2014, 43, 4563-4601). In addition, changes in cell viscosity are closely related to redox balance. Increased reactive oxygen species leading to strong oxidative stress may lead to increased viscosity. Therefore, simultaneous detection of superoxide anion levels and viscosity changes is particularly important for disease monitoring.

[0003] Fluorescence imaging provides a convenient way to visualize the real-time tracking of trace metabolites and key biomolecules in organisms, and therefore plays an invaluable role in basic biological research and clinical research. Compared with other molecular imaging methods (such as magnetic resonance imaging, ultrasound imaging, and positron emission tomography), fluorescence imaging generally has high spatial and temporal resolution and high sensitivity, and is less invasive. However, most of the fluorescent probes reported for detecting superoxide anions / viscosity detect only one of them, and it is still a huge challenge to distinguish and detect superoxide anions and viscosity at the same time. Summary of the invention

[0004] In view of the above situation, in order to overcome some deficiencies of the prior art, the present invention aims to provide a dual-function fluorescent probe for simultaneously distinguishing and detecting superoxide anions and viscosity. The probe can rapidly and selectively detect superoxide anions and viscosity from various bioactive substances under specific detection conditions.

[0005] The present invention also aims to provide a method for synthesizing and applying the above-mentioned bifunctional fluorescent probe with simple preparation method, high sensitivity, low detection limit and low cost.

[0006] The specific technical solution adopted by the present invention to solve the problem is to synthesize a dual-function fluorescent probe for simultaneously distinguishing and detecting superoxide anions and viscosity, and to prepare a device for quantitatively analyzing superoxide anions and viscosity in the environment, and simultaneously distinguishing and imaging superoxide anions and viscosity in living cells. The chemical structure of the dual-function probe is as follows: .

[0007] A synthesis of a dual-function fluorescent probe for simultaneously distinguishing and detecting superoxide anions and viscosity, characterized in that the preparation method of the dual-function fluorescent probe comprises the following steps: Step 1. Synthesis of (E)-2-(1-cyano-2-(4-(diethylamino)-2-((trifluoromethyl)sulfonyl)oxy)phenyl)vinyl)benzo[d]thiazole-6-carboxylic acid 5-(Diethylamino)-2-formylphenyl trifluoromethanesulfonate and 2-(cyanomethyl)benzo[d]thiazole-6-carboxylic acid are added to ethanol, and the mixture is reacted at 50 degrees Celsius overnight. After the reaction is complete, the mixture is filtered, and the solid is spin-dried to obtain (E)-2-(1-cyano-2-(4-(diethylamino)-2-((trifluoromethyl)sulfonyl)oxy)phenyl)vinyl)benzo[d]thiazole-6-carboxylic acid; Step 2. Synthesis of tert-butyl (E)-4-(2-(1-cyano-2-(4-(diethylamino)-2-((trifluoromethyl)sulfonyl)oxy)phenyl)vinyl)benzo[d]thiazole-6-carbonyl)piperazine-1-carboxylate (E)-2-(1-cyano-2-(4-(diethylamino)-2-((trifluoromethyl)sulfonyl)oxy)phenyl)vinyl)benzo[d]thiazole-6-carboxylic acid was added to anhydrous dichloromethane, and then 4-dimethylaminopyridine (DMAP) was added, and the reaction was carried out at room temperature for 5 minutes. Then, tert-butyl piperazine-1-carboxylate was added and stirred for 5 minutes. Finally, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride was added, and the reaction was stirred at room temperature overnight. After the reaction was complete, the reaction system was spin-dried, and column chromatography was used for separation and purification to obtain (E)-4-(2-(1-cyano-2-(4-(diethylamino)-2-((trifluoromethyl)sulfonyl)oxy)phenyl)vinyl)benzo[d]thiazole-6-carbonyl)piperazine-1-carboxylic acid tert-butyl ester; Step 3. Synthesis of (E)-4-(2-(1-cyano-2-(4-(diethylamino)-2-((trifluoromethyl)sulfonyl)oxy)phenyl)vinyl)benzo[d]thiazole-6-carbonyl)piperazin-1-ium 2,2,2-trifluoroacetate Add tert-butyl (E)-4-(2-(1-cyano-2-(4-(diethylamino)-2-((trifluoromethyl)sulfonyl)oxy)phenyl)vinyl)benzo[d]thiazole-6-carbonyl)piperazine-1-carboxylate to anhydrous dichloromethane, then add trifluoroacetic acid, stir at room temperature overnight, after the reaction is complete, spin dry the reaction system, separate and purify by column chromatography to obtain (E)-4-(2-(1-cyano-2-(4-(diethylamino)-2-((trifluoromethyl)sulfonyl)oxy)phenyl)vinyl)benzo[d]thiazole-6-carbonyl)piperazine-1-ium 2,2,2-trifluoroacetate; Step 4. Synthesis of 2-((1E,3E)-1-cyano-4-(4-(dimethylamino)phenyl)buta-1,3-dien-1-yl)benzo[d]thiazole-6-carboxylic acid 2-(Cyanomethyl)benzo[d]thiazole-6-carboxylic acid and 4-dimethylaminocinnamaldehyde were added to ethanol, and the mixture was reacted at 60 degrees Celsius overnight. After the reaction was complete, the solid was filtered and dried to obtain 2-((1E,3E)-1-cyano-4-(4-(dimethylamino)phenyl)but-1,3-dien-1-yl)benzo[d]thiazole-6-carboxylic acid; Step 5. Synthesis of the bifunctional fluorescent probe 2-((1E,3E)-1-cyano-4-(4-(dimethylamino)phenyl)but-1,3-dien-1-yl)benzo[d]thiazole-6-carboxylic acid is added to anhydrous dichloromethane, and then 4-dimethylaminopyridine (DMAP) is added, and the reaction is carried out at room temperature for 5 minutes. Subsequently, (E)-4-(2-(1-cyano-2-(4-(diethylamino)-2-((trifluoromethyl)sulfonyl)oxy)phenyl)vinyl)benzo[d]thiazole-6-carbonyl)piperazin-1-ium 2,2,2-trifluoroacetate is added and stirred for 5 minutes. Finally, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is added and stirred at room temperature overnight. After the reaction is complete, the reaction system is spin-dried, and the bifunctional fluorescent probe is separated and purified by column chromatography.

[0008] The method for using the dual-function fluorescent probe for simultaneously distinguishing and detecting superoxide anions and viscosity of the present invention is as follows: unless otherwise specified, the dual-function probe is usually dissolved in dimethyl sulfoxide (DMSO) at room temperature and used for analysis and detection in an environment where the volume ratio of the organic phase to the aqueous phase is 5:5, the organic phase is dimethyl sulfoxide, and the aqueous phase is a phosphate buffer solution (PBS) with a pH of 7.4.

[0009] The specific characteristics of the dual-function fluorescent probe of the present invention for simultaneously distinguishing and detecting superoxide anions and viscosity are as follows: the dual-function fluorescent probe is dissolved in DMSO, and the dual-function probe is dissolved in an organic phase and an aqueous phase (5:5, v / v) solution. After reacting with superoxide anions for 30 minutes, it emits 550 nm yellow fluorescence at an excitation wavelength of 470 nm; the probe is tested for response viscosity in a glycerol-methanol system with different proportions, and emits 660 nm near-infrared fluorescence at an excitation wavelength of 540 nm. Therefore, it is possible to detect specific analytes with specific excitation and fluorescence emission signals, and when detecting simultaneously, the two can be well distinguished by using different excitation and fluorescence emission signals. The above-mentioned dual-function fluorescent probe realizes the simultaneous distinguishing and detecting of superoxide anions and viscosity under different detection conditions, and has no obvious response to other active oxygen, active sulfur, common amino acids, metal ions and active nitrogen. The detection limits of superoxide anions and viscosity are as low as 24.7 nM and 0.13 cP, respectively. Therefore, the dual-function fluorescent probe disclosed in the present invention can realize highly sensitive distinguishing detection of the two. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 The hydrogen nuclear magnetic resonance spectrum of the bifunctional fluorescent probe of the present invention.

[0011] Figure 2 The dual-function fluorescent probe of the present invention responds to the fluorescence spectrum of superoxide anions and viscosity.

[0012] Figure 3 Fluorescence quantitative analysis graph of the bifunctional fluorescent probe of the present invention in response to superoxide anions and viscosity.

[0013] Figure 4 The dual-function fluorescent probe of the present invention simultaneously distinguishes and images superoxide anions in cells and viscosity cell maps. DETAILED DESCRIPTION

[0014] The present invention is further described with reference to the following figures.

[0015] The synthesis route of the bifunctional fluorescent probe of the present invention is shown in the figure below:

[0016] Example 1. Synthesis of (E)-2-(1-cyano-2-(4-(diethylamino)-2-((trifluoromethyl)sulfonyl)oxy)phenyl)vinyl)benzo[d]thiazole-6-carboxylic acid 300.0 mg (922.22 µmol) of 5-(diethylamino)-2-formylphenyl trifluoromethanesulfonate and 201.3 mg (922.22 µmol) of 2-(cyanomethyl)benzo[d]thiazole-6-carboxylic acid were added to 12 mL of ethanol and reacted at 50 degrees Celsius overnight. After the reaction was complete, the mixture was filtered and the solid was spin-dried to obtain 390.0 mg of (E)-2-(1-cyano-2-(4-(diethylamino)-2-((trifluoromethyl)sulfonyl)oxy)phenyl)vinyl)benzo[d]thiazole-6-carboxylic acid with a yield of 80.47%; Example 2. Synthesis of tert-butyl (E)-4-(2-(1-cyano-2-(4-(diethylamino)-2-((trifluoromethyl)sulfonyl)oxy)phenyl)vinyl)benzo[d]thiazole-6-carbonyl)piperazine-1-carboxylate 300.0 mg (570.87 µmol) of (E)-2-(1-cyano-2-(4-(diethylamino)-2-((trifluoromethyl)sulfonyl)oxy)phenyl)vinyl)benzo[d]thiazole-6-carboxylic acid was added to anhydrous dichloromethane, and then 7 mg of 4-dimethylaminopyridine (DMAP) was added. The mixture was reacted at room temperature for 5 min. Subsequently, 159.5 mg (856.30 µmol) of tert-butyl piperazine-1-carboxylate was added and stirred for 5 min. Finally, 164.2 mg (856.30 µmol) of tert-butyl piperazine-1-carboxylate was added. The mixture was stirred at room temperature overnight for 1 h. After the reaction was complete, the reaction system was dried by rotary evaporation and purified by column chromatography to obtain tert-butyl (E)-4-(2-(1-cyano-2-(4-(diethylamino)-2-((trifluoromethyl)sulfonyl)oxy)phenyl)vinyl)benzo[d]thiazole-6-carbonyl)piperazine-1-carboxylate (315.0 mg) with a yield of 79.54%. Example 3. Synthesis of (E)-4-(2-(1-cyano-2-(4-(diethylamino)-2-((trifluoromethyl)sulfonyl)oxy)phenyl)vinyl)benzo[d]thiazole-6-carbonyl)piperazin-1-ium 2,2,2-trifluoroacetate 300.0 mg (432.4 µmol) of (E)-tert-butyl 4-(2-(1-cyano-2-(4-(diethylamino)-2-((trifluoromethyl)sulfonyl)oxy)phenyl)vinyl)benzo[d]thiazole-6-carbonyl)piperazine-1-carboxylate was added to 8 mL of anhydrous dichloromethane, and then 2 mL of trifluoroacetic acid was added. The mixture was stirred at room temperature overnight. After the reaction was complete, the reaction system was dried by spin drying and purified by column chromatography to obtain 232.0 mg of (E)-4-(2-(1-cyano-2-(4-(diethylamino)-2-((trifluoromethyl)sulfonyl)oxy)phenyl)vinyl)benzo[d]thiazole-6-carbonyl)piperazin-1-ium 2,2,2-trifluoroacetate with a yield of 75.81%; Example 4. Synthesis of 2-((1E,3E)-1-cyano-4-(4-(dimethylamino)phenyl)buta-1,3-dien-1-yl)benzo[d]thiazole-6-carboxylic acid 500.0 mg (2.29 mmol) of 2-(cyanomethyl)benzo[d]thiazole-6-carboxylic acid and 401.5 mg (2.29 mmol) of 4-dimethylaminocinnamaldehyde were added to 12 mL of ethanol and reacted at 60 degrees Celsius overnight. After the reaction was complete, the solid was filtered and dried to obtain 663.0 mg of 2-((1E,3E)-1-cyano-4-(4-(dimethylamino)phenyl)but-1,3-dien-1-yl)benzo[d]thiazole-6-carboxylic acid with a yield of 77.01%; Example 5. Synthesis of the bifunctional fluorescent probe 80.0 mg (213.08 µmol) of 2-((1E,3E)-1-cyano-4-(4-(dimethylamino)phenyl)but-1,3-dien-1-yl)benzo[d]thiazole-6-carboxylic acid was added to 8 mL of anhydrous dichloromethane, and then 3 mg of 4-dimethylaminopyridine was added. The mixture was reacted at room temperature for 5 min. Then 150.8 mg (213.08 µmol) of (E)-4-(2-(1-cyano-2-(4-(diethylamino)-2-((trifluoromethyl)sulfonyl)oxy)phenyl)vinyl)benzo[d]thiazole-6-carbonyl)piperazin-1-ium 2,2,2-trifluoroacetate was added. After stirring for 5 min, 61.3 mg (319.62 The mixture was stirred at room temperature overnight. After the reaction was complete, the reaction system was dried by rotary evaporation and purified by column chromatography to obtain 90.3 mg of the bifunctional fluorescent probe with a yield of 44.56%.

[0017] Example 6. Dual-functional fluorescent probe for simultaneous detection of superoxide anions and viscosity in vitro The spectral property experiment of the bifunctional fluorescent probe described in the present invention: the bifunctional probe is dissolved in DMSO to prepare a probe solution with a concentration of 1 mM, and a solution with a concentration of 10 mM superoxide anion and different viscosities (different ratios of methanol: glycerol) is prepared. The specific test method is: take 20 μL of 1 mM probe solution, add 20 μL of 10 mM superoxide anion solution, and finally add 980 μL DMSO and 980 μL PBS. All tests maintain the volume ratio of organic phase to aqueous phase at 5:5 (the total volume of each test sample is 2 mL). For example, when the fluorescence intensity of superoxide anion with a concentration of 100 μM is required to be tested, the sample preparation is: take 20 μL of 1 mM probe solution, 20 μL of 10 mM superoxide anion solution, and then add 980 μL DMSO and 980 μL PBS in a 2 mL sample tube. After shaking at room temperature for 30 minutes, the fluorescence emission intensity can be measured with an excitation wavelength of 470 nm. When detecting viscosity, the specific test method is: take 20 μL of 1 mM probe solution, add 1980 μL of prepared solutions of different viscosities, shake at room temperature for 30 minutes, and measure its fluorescence emission intensity with an excitation wavelength of 540 nm. This dual-function probe can distinguish and detect superoxide anions and viscosity, two biologically active substances, with different excitation wavelengths and fluorescence emission signals. It has high sensitivity, with detection limits as low as 24.7 nM and 0.13 cP, respectively, which is very suitable for imaging / quantitative analysis of superoxide anions and viscosity in living cells.

[0018] Example 6. Dual-channel fluorescence imaging analysis of endogenous superoxide anions and viscosity in macrophages (RAW264.7) RAW264.7 cells were passaged into a confocal dish cell culture medium, and after culturing for 24 hours under standard growth conditions, an appropriate amount of probe (5 μM) was added and continued to be cultured under standard growth conditions for 30 minutes, and then photographed under a confocal fluorescence microscope, and superoxide anions and viscosity in RAW264.7 cells were imaged using a 500-600 nm green channel and a 600-700 nm near-infrared fluorescence channel, respectively. The dual-functional fluorescent probe of the present invention can emit two different fluorescences in cells to simultaneously distinguish and detect superoxide anions and viscosity in cells, and successfully realizes dual-channel fluorescence imaging analysis of superoxide anions and viscosity in cells.

[0019] The present invention provides a dual-function fluorescent probe for simultaneously distinguishing and detecting superoxide anions and viscosity. The coumarin derivative and the thiazole derivative are connected by piperazine, and the superoxide anion is detected by the trifluorosulfonate group as the recognition site, and the viscosity is responded to by the molecular rotor strategy. When reacting with superoxide anions, a strong yellow fluorescence of 550 nm is emitted at an excitation wavelength of 470 nm; when acting with viscosity, a near-infrared fluorescence of 660 nm is emitted at an excitation wavelength of 540 nm, and the fluorescence phenomenon is obvious. And the product after the reaction has good water solubility, fast response speed, large Stokes shift and the like. It has great practical application value in the fields of biochemistry, fluorescence imaging analysis, etc. Although the content of the present invention has been described in detail through the above preferred embodiments, it should be recognized that the above description should not be regarded as a limitation of the present invention. After reading the above content, it will be obvious to those skilled in the art for various modifications and substitutions of the present invention. Therefore, fluorescent probes with similar technical features described herein all fall within the scope of protection of this patent.

Claims

1. A dual-function fluorescent probe for simultaneously distinguishing and detecting superoxide anions and viscosity, characterized in that: The chemical structure of the bifunctional fluorescent probe is as follows: 。 2. The synthesis of the bifunctional fluorescent probe according to claim 1, characterized in that: The synthesis method of the bifunctional fluorescent probe comprises the following steps: Step 1. Synthesis of (E)-2-(1-cyano-2-(4-(diethylamino)-2-((trifluoromethyl)sulfonyl)oxy)phenyl)vinyl)benzo[d]thiazole-6-carboxylic acid 5-(Diethylamino)-2-formylphenyl trifluoromethanesulfonate and 2-(cyanomethyl)benzo[d]thiazole-6-carboxylic acid are added to ethanol, and the mixture is reacted at 50 degrees Celsius overnight. After the reaction is complete, the mixture is filtered, and the solid is spin-dried to obtain (E)-2-(1-cyano-2-(4-(diethylamino)-2-((trifluoromethyl)sulfonyl)oxy)phenyl)vinyl)benzo[d]thiazole-6-carboxylic acid; Step 2. Synthesis of tert-butyl (E)-4-(2-(1-cyano-2-(4-(diethylamino)-2-((trifluoromethyl)sulfonyl)oxy)phenyl)vinyl)benzo[d]thiazole-6-carbonyl)piperazine-1-carboxylate (E)-2-(1-cyano-2-(4-(diethylamino)-2-((trifluoromethyl)sulfonyl)oxy)phenyl)vinyl)benzo[d]thiazole-6-carboxylic acid was added to anhydrous dichloromethane, and then 4-dimethylaminopyridine (DMAP) was added, and the reaction was carried out at room temperature for 5 minutes. Then, tert-butyl piperazine-1-carboxylate was added and stirred for 5 minutes. Finally, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride was added, and the reaction was stirred at room temperature overnight. After the reaction was complete, the reaction system was spin-dried, and column chromatography was used for separation and purification to obtain (E)-4-(2-(1-cyano-2-(4-(diethylamino)-2-((trifluoromethyl)sulfonyl)oxy)phenyl)vinyl)benzo[d]thiazole-6-carbonyl)piperazine-1-carboxylic acid tert-butyl ester; Step 3. Synthesis of (E)-4-(2-(1-cyano-2-(4-(diethylamino)-2-((trifluoromethyl)sulfonyl)oxy)phenyl)vinyl)benzo[d]thiazole-6-carbonyl)piperazin-1-ium 2,2,2-trifluoroacetate Add tert-butyl (E)-4-(2-(1-cyano-2-(4-(diethylamino)-2-((trifluoromethyl)sulfonyl)oxy)phenyl)vinyl)benzo[d]thiazole-6-carbonyl)piperazine-1-carboxylate to anhydrous dichloromethane, then add trifluoroacetic acid, stir at room temperature overnight, after the reaction is complete, spin dry the reaction system, separate and purify by column chromatography to obtain (E)-4-(2-(1-cyano-2-(4-(diethylamino)-2-((trifluoromethyl)sulfonyl)oxy)phenyl)vinyl)benzo[d]thiazole-6-carbonyl)piperazine-1-ium 2,2,2-trifluoroacetate; Step 4. Synthesis of 2-((1E,3E)-1-cyano-4-(4-(dimethylamino)phenyl)buta-1,3-dien-1-yl)benzo[d]thiazole-6-carboxylic acid 2-(Cyanomethyl)benzo[d]thiazole-6-carboxylic acid and 4-dimethylaminocinnamaldehyde were added to ethanol, and the mixture was reacted at 60 degrees Celsius overnight. After the reaction was complete, the solid was filtered and dried to obtain 2-((1E,3E)-1-cyano-4-(4-(dimethylamino)phenyl)but-1,3-dien-1-yl)benzo[d]thiazole-6-carboxylic acid; Step 5. Synthesis of the bifunctional fluorescent probe 2-((1E,3E)-1-cyano-4-(4-(dimethylamino)phenyl)but-1,3-dien-1-yl)benzo[d]thiazole-6-carboxylic acid is added to anhydrous dichloromethane, and then 4-dimethylaminopyridine (DMAP) is added, and the reaction is carried out at room temperature for 5 minutes. Subsequently, (E)-4-(2-(1-cyano-2-(4-(diethylamino)-2-((trifluoromethyl)sulfonyl)oxy)phenyl)vinyl)benzo[d]thiazole-6-carbonyl)piperazin-1-ium 2,2,2-trifluoroacetate is added and stirred for 5 minutes. Finally, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is added and stirred at room temperature overnight. After the reaction is complete, the reaction system is spin-dried, and the bifunctional fluorescent probe is separated and purified by column chromatography.

3. The method for synthesizing a bifunctional probe according to claim 2, wherein: The molar ratio of 2-((1E,3E)-1-cyano-4-(4-(dimethylamino)phenyl)buta-1,3-dien-1-yl)benzo[d]thiazole-6-carboxylic acid and (E)-4-(2-(1-cyano-2-(4-(diethylamino)-2-((trifluoromethyl)sulfonyl)oxy)phenyl)vinyl)benzo[d]thiazole-6-carbonyl)piperazin-1-ium 2,2,2-trifluoroacetate in step 5 is 1:

1.

4. The use of the bifunctional fluorescent probe according to claim 1 for preparing a device, characterized in that: The fabricated device is capable of quantitatively analyzing superoxide anions and viscosity in the environment, as well as simultaneously distinguishing and imaging superoxide anions and viscosity in cells, tissues, and living bodies.

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