Colorimetric-near-infrared dual-detection fluorescent probe for hypochlorous acid detection as well as preparation method and application of colorimetric-near-infrared dual-detection fluorescent probe

Abstract

The invention provides a colorimetric-near-infrared dual-detection fluorescent probe for hypochlorous acid detection and a preparation method and application thereof.According to the fluorescent probe, O-phenyl methyl mercaptan ester is adopted as a recognition group of HClO on an NIR fluorophore, high-sensitivity and high-selectivity NIR fluorescent response to HClO is shown, meanwhile, remarkable colorimetric change is generated, and the fluorescent probe has the advantages of being high in sensitivity, high in sensitivity and high in selectivity. The detection reliability is obviously improved, and the method can be applied to imaging of exogenous and endogenous HClO in living cells. Meanwhile, through colorimetric and near-infrared fluorescence double signals, the result is applied to detection of residual HClO in a water sample and food.

Description

Technical Field

[0001] The present invention relates to the technical field of fluorescence detection, and particularly to a colorimetric-near infrared dual-detection fluorescent probe for hypochlorous acid detection, a preparation method thereof, and an application thereof. Background Art

[0002] Hypochlorous acid (HClO) is a typical reactive oxygen species (ROS), which is synthesized in the human body through the peroxidation reaction of chloride catalyzed by myeloperoxidase (MPO). It plays a key role in resisting pathogen invasion and is crucial for maintaining health. More importantly, HClO is of extremely important significance in the food industry. It can be used as a potent food disinfectant. Perishable foods such as vegetables, fruits, eggs, fish, and raw meat can be soaked in an appropriate concentration of HClO solution before processing and storage to rapidly kill stubborn microorganisms, bacteria, fungi, and pathogens, reduce the initial microbial count, and ensure food safety. In addition, it is also an excellent fresh-keeping agent for fruits and vegetables. By soaking or spraying, it can inhibit ethylene release, delay decay, reduce cell membrane permeability, inhibit the activity of polyphenol oxidase, and postpone discoloration, thereby extending the shelf life. However, high concentrations of HClO residues in food can not only damage nutrients but also cause damage to human tissues, leading to diseases such as atherosclerosis, rheumatoid arthritis, neurological diseases, cardiovascular diseases, and cancer. Therefore, it is imperative to develop an efficient analytical method for in-situ quantitative detection of HClO in food.

[0003] Currently, a variety of hypochlorous acid (HClO) detection methods have been developed, including ion chromatography, electrochemistry, spectrophotometry, high performance liquid chromatography (HPLC), surface enhanced Raman spectroscopy (SERS), fluorescence method, and colorimetry. Among them, the fluorescence method and colorimetry are particularly prominent in the in-situ detection of hypochlorous acid in food samples because their signals can be observed by the naked eye and do not require complex equipment. In recent years, a variety of hypochlorous acid fluorescent probes have been reported, and their response mechanisms include oxidative cleavage of C═C bonds, hydrolysis of oximes, oxidation of p-methoxyphenol / p-alkoxyaniline, hydrolysis of hydrazides, electrophilic oxidation of N-heterocyclic carbene (NHC) boranes, and oxidation of sulfur atoms, etc. These probes are mainly used for detection in living cells and animals. However, for the in-situ quantitative detection of hypochlorous acid in food samples, specific probes are still lacking. To achieve effective detection, an ideal probe should have the following characteristics: (1) exhibit near-infrared (NIR, 650-900 nm) fluorescence response to hypochlorous acid to better penetrate food tissues and reduce background interference; (2) preferably produce a colorimetric response to hypochlorous acid to enhance the reliability of detection through dual signals.

[0004] Aiming at the defects of the above-mentioned prior art, a fluorescent probe capable of in-situ quantitative detection of HClO needs to be provided. SUMMARY OF THE INVENTION

[0005] As described in the background art, the prior art hypochlorous acid fluorescent probes have the technical problem of lacking specific probes for in-situ quantitative detection. In view of the above situation, the present invention is made.

[0006] One aspect of the present invention is to provide a colorimetric-NIR dual-detection fluorescent probe for hypochlorous acid detection, which has a highly sensitive and selective NIR fluorescence response to hypochlorous acid.

[0007] Another aspect of the present invention is to provide a preparation method of the above fluorescent probe.

[0008] Another aspect of the present invention is to provide a method for detecting the presence of hypochlorous acid in a sample and / or determining the content of hypochlorous acid in a sample. By using the fluorescent probe, a high selectivity for HClO detection can be obtained according to the method of the present invention.

[0009] Another aspect of the present invention is to provide the application of the above fluorescent probe in the detection of hypochlorous acid.

[0010] According to the purpose of the present application, the present invention relates to a colorimetric-NIR dual-detection fluorescent probe for hypochlorous acid detection, which has the following structural formula:

[0011]

[0012] As a further improvement of the present application, the preparation method of the above fluorescent probe includes the following steps:

[0013] Step (s1), reacting 2-hydroxyacetophenone with ethyl acetate to obtain 1-(2-hydroxyphenyl)-1,3-butanedione, denoted as Compound 1;

[0014] Step (s2), reacting Compound 1 with glacial acetic acid under acidic conditions to obtain methyl isoflavone, denoted as Compound 2;

[0015] Step (s3), mixing and reacting Compound 2, malononitrile and acetic anhydride to obtain 2-(2-methyl-4H-chromen-4-ylidene)malononitrile, denoted as Compound 3;

[0016] Step (s4), reacting cyclopentanone with phosphorus tribromide to obtain 2-bromocyclopent-1-ene-1-carbaldehyde, denoted as Compound 4;

[0017] Step (s5), reacting Compound 4 with 2-hydroxy-4-methoxybenzaldehyde to obtain 6-hydroxy-1,2-dihydrocyclopenta[b]chromene-3-carbaldehyde, denoted as Compound 5;

[0018] Step (s6), subjecting Compound 5 to a demethylation reaction to obtain Compound 6;

[0019] Step (s7): Compound 3 and compound 6 are obtained through an Aldol condensation reaction, denoted as compound YQ-OH;

[0020] Step (s8): Compound YQ-OH undergoes a nucleophilic substitution reaction with phenyl thiocarbonochloridate under alkaline conditions to obtain the fluorescent probe, denoted as compound YQ.

[0021] As a further improvement of the present application, in the step (s1), the dosage ratio of 2-hydroxyacetophenone to ethyl acetate is 0.5 g - 1.5 g: 1 mL - 3 mL.

[0022] As a further improvement of the present application, in the step (s3), the dosage ratio of compound 2, malononitrile, and acetic anhydride is 0.3 g - 1.5 g: 0.1 g - 0.5 g: 3 mL - 8 mL, the reaction time is 10 - 14 h, and the reaction temperature is 120 - 160 °C.

[0023] As a further improvement of the present application, in the step (s4), the dosage ratio of cyclopentanone to phosphorus tribromide is 1 mL - 6 mL: 2 mL - 8 mL, and the reaction time is 15 - 20 h.

[0024] As a further improvement of the present application, the demethylation reaction occurs in the presence of boron tribromide, and the dosage ratio of compound 5 to boron tribromide is 1 g: (2 g - 4 g).

[0025] As a further improvement of the present application, in the step (s8), the dosage ratio of compound YQ-OH to phenyl thiocarbonochloridate is 0.1 mmol - 0.3 mmol: 0.3 mmol - 0.5 mmol, and the reaction time is 1.5 - 4 h.

[0026] In a second aspect, the present application discloses a method for detecting the presence of hypochlorous acid in a sample and / or determining the content of hypochlorous acid in a sample, which includes: contacting the sample with the fluorescent probe defined in claim 1 under conditions suitable for converting the fluorescent probe into a fluorescent product to form a fluorescent product; and

[0027] Quantitatively detecting and judging the content of hypochlorous acid in the sample by visual colorimetry or fluorescence spectrophotometry.

[0028] In a third aspect, the present application discloses a fluorescent probe test paper obtained by immersing a filter paper or a thin layer chromatography plate in a dichloromethane solution of the above-mentioned fluorescent probe and air-drying it naturally.

[0029] In a fourth aspect, the present application discloses the application of the above-mentioned fluorescent probe in the detection and evaluation of hypochlorous acid in water samples, foods, and cells.

[0030] Beneficial effects:

[0031] (1) The present application discloses a fluorescence probe YQ for in-situ quantitative detection of hypochlorous acid (HClO) in various food samples. Using O-phenylmethanethiol ester as the recognition group for HClO, it can covalently bind to the O 14 atom of the NIR fluorophore, effectively inhibiting the ICT process between the O 14 atom and the DCM group, resulting in fluorescence quenching. When the probe YQ reacts with HClO, the recognition group in YQ is cleaved, restoring the ICT process within the fluorophore and triggering a NIR fluorescence response;

[0032] (2) The probe YQ exhibits a highly sensitive and selective NIR fluorescence response to HClO, and at the same time, a significant colorimetric change also occurs, significantly improving the reliability of detection. It can be applied to the imaging of exogenous and endogenous HClO in living cells;

[0033] (3) Meanwhile, for the in-situ quantitative detection of the HClO content in samples, the present application discloses a fluorescence probe test paper, effectively expanding the practical application of the probe YQ. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 shows the sensing mechanism of the fluorescence probe YQ for HClO;

[0035] Figure 2 shows the synthesis route map of the fluorescence probe in some embodiments;

[0036] Figure 3 . (a) Absorption spectra of the probe YQ (10 μM) at different concentrations of HClO (0 - 100 μM); (b) Linear relationship between the absorbance at 600 nm and the HClO concentration (0 - 80 μM); (c) Color changes of the probe YQ solution under visible light and ultraviolet light before and after treatment with HClO; (d) Fluorescence spectra of the probe YQ (10 μM) at different concentrations of HClO (0 - 100 μM) (λex = 610 nm); (e) Relationship between the fluorescence intensity of the probe YQ at 716 nm and the HClO concentration (0 - 100 μM); (f) Linear relationship between the fluorescence intensity at 716 nm and the HClO concentration (0 - 80 μM);

[0037] Figure 4 . (a) Influence of pH on the detection of HClO (100 μM) by the probe YQ (10 μM); (b) Fluorescence change of the probe YQ over time with (▲) and without (●) HClO (100 μM); (c) Response of the fluorescence (I716) of the probe YQ (10 μM) to different substances (100 μM): 1) blank, 2) Na + , 3) Ca 2+ , 4) NO 2- , 5) Cysteine, 6) Glycine, 7) 1 O 2 , 8) NO, 9) ·OH, 10) H 2 O 2 , 11) O 2 - , 12) ONOO - , 13) HClO; (d) Color changes of the probe YQ (10 μM) solution under different substances (under visible light and ultraviolet light).

[0038] Figure 5 . (A) Fluorescence imaging of exogenous HClO in HeLa cells using the probe YQ. (a - d) Bright - field imaging; (e - h) Near - infrared fluorescence imaging (700 - 740 nm); (i) Average intensity of the regions in Figures e - h. (B) Fluorescence imaging of endogenous HClO in PMA - stimulated RAW 264.7 cells using the probe YQ. (a - d) Bright - field imaging; (e - h) Near - infrared fluorescence imaging (700 - 740 nm); (i) Average intensity of the regions in Figures e - h;

[0039] Figure 6 . (a - d) Photos of lettuce after spraying 0, 10, 50, and 200 μM HClO for five days respectively. (e - h) Bright - field images; (i - l) Near - infrared fluorescence images of lettuce tissue sections from Figures a, b, c, and d respectively. (m) Average intensity of the regions in Figures i - l.

[0040] Figure 7 This is a schematic diagram of the portable optical data acquisition system in this application. Detailed implementation manners

[0041] To make the objectives, technical solutions, and advantages of this application clearer, the technical solutions of this application will be clearly and completely described below in conjunction with specific embodiments of this application and the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of this application, rather than all of the embodiments, and are not used to limit the scope of the present invention. Based on the embodiments in this application, all other embodiments obtained by those of ordinary skill in the art without creative efforts fall within the scope of protection of this application.

[0042] Definition

[0043] Throughout the description of this specification and the claims, "comprising" and "including" and variations of these descriptions, such as "comprises" and "comprising", mean "including but not limited to" and are not intended to exclude other components.

[0044] The terms "fluorophore", "fluorescent compound", "fluorescent substance" and "fluorescent probe" can also be used as synonyms.

[0045] The term "NIR fluorophore" refers to a compound that fluoresces in the NIR region of the spectrum (e.g., from about 680 nm to 1000 nm).

[0046] The term "ICT" is an intramolecular charge transfer phenomenon, which means that when there are two or more groups with different electron affinities in a molecule, through the charge transfer between the excited state and the ground state, the molecule shows different emission colors in different environments.

[0047] Detail

[0048] Specific embodiments will be combined below to detail the structure, preparation, and detection effect of the fluorescent probe of the present invention. In the embodiments of the present invention, unless otherwise specified, the ratios are all mass ratios.

[0049] Currently, the research direction of fluorescence analysis mainly focuses on developing highly selective fluorescent probes. One of the main problems of fluorescent probes for hypochlorous acid detection is the lack of specific probes. Therefore, the inventors expect to obtain a fluorescent probe that overcomes the above defects and exhibits a highly sensitive and selective NIR fluorescence response to HClO.

[0050] In a first aspect, the present application relates to a colorimetric-NIR dual-detection fluorescent probe for hypochlorous acid detection, which has the following structural formula:

[0051]

[0052] The sensing mechanism of probe YQ for HClO is as Figure 1 shown. In the structure of YQ, based on the 2-(2-methyl-4H-benzo[b]pyran-4-ylidene)malononitrile (DCM) moiety conjugated with the 1,2-dihydrocyclopenta[b]chromen-6-ol moiety, it serves as an NIR fluorophore ( Figure 1 , purple part). In this structure, an efficient intramolecular charge transfer (ICT) process can occur from the O 14 atom to the DCM moiety. In addition, O-phenylmethanethiol ester ( Figure 1 , within the green box) is used as the recognition group for HClO. This recognition group is covalently bonded to the O 14 atom of the NIR fluorophore, effectively inhibiting the ICT process between the O14 atom and the DCM group, resulting in fluorescence quenching. When reacting with HClO, the recognition group in the probe is cleaved, restoring the ICT process within the fluorophore and triggering an NIR fluorescence response.

[0053] Second aspect, the present application relates to a method for preparing a fluorescent probe, as Figure 1 shown, the synthesis route of probe YQ is as Figure 1 shown, specifically, it includes the following steps:

[0054] Step (s1), reacting 2-hydroxyacetophenone with ethyl acetate to obtain 1-(2-hydroxyphenyl)-1,3-butanedione, denoted as compound 1;

[0055] Step (s2), reacting compound 1 with glacial acetic acid under acidic conditions to obtain methyl isoflavone, denoted as compound 2;

[0056] Step (s3), mixing and reacting compound 2, malononitrile and acetic anhydride to obtain 2-(2-methyl-4H-chromen-4-ylidene)malononitrile, denoted as compound 3;

[0057] Step (s4), reacting cyclopentanone with phosphorus tribromide to obtain 2-bromocyclopent-1-ene-1-carbaldehyde, denoted as compound 4;

[0058] Step (s5), reacting compound 4 with 2-hydroxy-4-methoxybenzaldehyde to obtain 6-hydroxy-1,2-dihydrocyclopenta[b]chromene-3-carbaldehyde, denoted as compound 5;

[0059] Step (s6), subjecting compound 5 to a demethylation reaction to obtain compound 6;

[0060] Step (s7), obtaining through an Aldol condensation reaction of compound 3 and compound 6, denoted as compound YQ-OH;

[0061] Step (s8), subjecting compound YQ-OH to a nucleophilic substitution reaction with phenyl chloroformate under alkaline conditions to obtain the near-infrared fluorescent probe, denoted as compound YQ.

[0062] In certain embodiments, in step (s1), the specific method for obtaining compound 1 is: adding sodium hydride to a three-necked flask containing tetrahydrofuran. Dissolving 2-hydroxyacetophenone and ethyl acetate in THF to prepare a mixed solution, and adding it to the three-necked flask. Stirring and heating the mixture to reflux. After the reaction is completed, pouring the reaction solution into ice water and adjusting the pH to neutral. Extracting and drying the product, and then subjecting it to vacuum distillation to obtain it.

[0063] In certain embodiments, in step (s2), the specific method for obtaining compound 2 is: putting compound 1 into a round-bottom flask and dissolving it in a mixed solution of sulfuric acid and glacial acetic acid. Continuously stirring and heating the mixture to 120 °C. After the reaction is completed, naturally cooling it to room temperature, and adjusting the pH of the reaction solution to neutral under ice bath conditions. Extracting and drying the product. Further purifying it by vacuum distillation to obtain it.

[0064] In certain embodiments, in step (s3), the specific method for obtaining Compound 3 is as follows: Add Compound 2, malononitrile, and acetic anhydride into a round-bottom flask. Continuously stir the mixture and heat it at 140 °C for 12 hours. After the reaction is completed, remove the solvent. Subsequently, add 30 mL of deionized water to the remaining residue, and heat and stir it under reflux conditions for 0.5 hour. Extract and dry the product, and further purify it by distillation under reduced pressure to obtain the product. In a specific example, the dosage ratio of Compound 2, malononitrile, and acetic anhydride is 0.3 g - 1.5 g: 0.1 g - 0.5 g: 3 mL - 8 mL, the reaction time is 10 - 14 h, and the reaction temperature is 120 - 160 °C.

[0065] In certain embodiments, in step (s4), the specific method for obtaining Compound 4 is as follows: Under N 2 atmosphere, drop phosphorus tribromide into a three-necked flask containing DMF and chloroform, add cyclopentanone under continuous conditions, and neutralize the reaction mixture after the reaction is completed. Extract and dry the product. After distillation under reduced pressure, the product is obtained. In a specific example, the dosage ratio of cyclopentanone to phosphorus tribromide is 1 mL - 6 mL: 2 mL - 8 mL, and the reaction time is 15 - 20 h.

[0066] In certain embodiments, in step (s5), the specific method for obtaining Compound 5 is as follows: Add Compound 4, 2-hydroxy-4-methoxybenzaldehyde, CsCO 3 and DMF into a round-bottom flask. Continuously stir the mixture at room temperature for 16 hours. After the reaction is completed, filter, extract, and wash. Subsequently, further purify it by distillation under reduced pressure to obtain the product.

[0067] In certain embodiments, in step (s6), the specific method for obtaining Compound 6 is as follows: Under N 2 atmosphere, add BBr 3 to a three-necked flask containing Compound 5 and CH 2 Cl 2 . Continuously stir the reaction mixture. After the reaction is completed, neutralize the reaction solution and wash the reaction mixture. Extract the aqueous layer with an organic solvent and dry it. Subsequently, distill it under reduced pressure and further purify it to obtain the product.

[0068] In certain embodiments, in step (s7), the specific method for obtaining Compound YQ-OH is as follows: Add Compound 3, Compound 6, and ethanol into a round-bottom flask. Stir and heat under reflux for 10 minutes. Add piperidine to the mixture. Further stir and heat the mixture under reflux for 6 hours. After the reaction solution is cooled to room temperature, distill it under reduced pressure and further purify it to obtain the product.

[0069] In certain embodiments, in step (s8), the specific method for obtaining Compound YQ is as follows: In an ice-water bath, add Et 3N is added to a round-bottom flask containing phenyl thiocarbonochloridate, compound YQ-OH and anhydrous CH 2 Cl 2 . Stir the reaction for 10 minutes, and then continue stirring at room temperature for 2 hours. After the reaction is completed, distill under reduced pressure and further purify to obtain the product. In a specific example, the dosage ratio of compound YQ-OH to phenyl thiocarbonochloridate is 0.1 mmol - 0.3 mmol: 0.3 mmol - 0.5 mmol, and the reaction time is 1.5 - 4 h.

[0070] In a second aspect, the present application relates to a method for detecting the presence of hypochlorous acid in a sample and / or determining the content of hypochlorous acid in a sample, which includes: contacting the sample with the above-mentioned fluorescent probe under conditions suitable for converting the fluorescent probe into a fluorescent product to form a fluorescent product; and

[0071] judging the content of hypochlorous acid in the sample by visual colorimetry or quantitative detection with a fluorescence spectrophotometer.

[0072] In a third aspect, the present application relates to a fluorescent probe test paper, which is obtained by immersing a filter paper or a thin-layer chromatography plate in a dichloromethane solution of the above-mentioned near-infrared fluorescent probe and naturally drying it.

[0073] In a fourth aspect, the present application also discloses the application of the above-mentioned fluorescent probe in the detection and evaluation of hypochlorous acid in water samples, foods and cells. Among them, the water sample can include one of tap water, industrial wastewater, and urban sewage, and the industrial wastewater can be selected from the discharged sewage from the workshop of a silicone factory.

[0074] The present application also relates to a portable optical data acquisition system, as Figure 7 shown, including:

[0075] Test solution preparation module 1: Prepare a test sample solution containing the above-mentioned fluorescent probe;

[0076] Fluorescent signal detection module 2: Measure the fluorescence intensity of the test sample solution;

[0077] Calculation module 3: Calculate the concentration of HClO in the test sample solution.

[0078] In some embodiments, as Figure 7 shown, the fluorescent signal detection module 2 includes a dark box, a culture dish is arranged at the bottom of the dark box, a white light LED for colorimetric detection and an LED for near-infrared fluorescence detection are respectively arranged at the top of the dark box, an optical filter is arranged on the light-emitting optical path of the LED for near-infrared fluorescence detection, and a camera is arranged at the top of the dark box, wherein the wavelength range of the LED for near-infrared fluorescence detection is 550 - 575 nm.

[0079] Specifically, the white light LED for colorimetric detection and the LED for near-infrared fluorescence detection emit excitation light. After passing through an optical filter, it enters a petri dish containing the sample solution to be measured. The sample emits fluorescence, and the camera of the fluorescence signal detection module captures the colorimetric signal and the fluorescence signal. For the obtained colorimetric signal or fluorescence signal, the concentration of HClO in the sample solution to be measured is calculated by the calculation module. In a specific embodiment, the calculation module can use ImageJ software for image analysis. In a specific embodiment, the food sample to be measured can be selected from tomatoes or strawberries.

[0080] Experiment

[0081] The following examples are provided for illustrative purposes only and are not intended to limit the present invention as defined by the appended claims.

[0082] Example 1: Preparation of Probe YQ

[0083] Step (s1): Sodium hydride (0.85 g, 35.42 mmol) was added to a three-necked flask containing 15 mL of tetrahydrofuran (THF). 2-Hydroxyacetophenone (0.92 g, 6.74 mmol) and ethyl acetate (1.72 mL, 17.67 mmol) were dissolved in 5 mL of THF to prepare a mixed solution, which was slowly added to the three-necked flask by syringe. The mixture was stirred and heated to reflux. After the reaction was completed, the reaction solution was poured into ice water, and the pH of the reaction solution was adjusted to neutral with a dilute hydrochloric acid solution. It was extracted three times with ethyl acetate, and the organic layer was collected and dried with anhydrous sodium sulfate. Subsequently, the organic layer was evaporated under reduced pressure to obtain Compound 1 as a white solid (0.75 g, yield: 62.4%). Compound 1 was used directly in the next reaction without purification;

[0084] Step (s2): Compound 1 (0.8 g, 4.50 mmol) was placed in a round-bottom flask and dissolved in a mixed solution of sulfuric acid (0.4 mL) and glacial acetic acid (5.6 mL). The mixture was continuously stirred and heated to 120 °C. After the reaction was completed, when the temperature of the reaction solution naturally dropped to room temperature, the pH of the reaction solution was adjusted to neutral with saturated sodium carbonate solution under ice bath conditions. Subsequently, it was extracted three times with dichloromethane. The organic layer was collected and dried with anhydrous sodium sulfate. Then, the organic layer was evaporated under reduced pressure and further purified by silica gel column chromatography (petroleum ether:ethyl acetate = 20:1, v / v) to obtain Compound 2 as a white solid (0.64 g, yield: 88.8%);

[0085] Step (s3): Add compound 2 (0.5 g, 3.12 mmol), malononitrile (0.27 g, 4.10 mmol) and acetic anhydride (5 mL) into a round-bottom flask. Stir the mixture continuously and heat it at 140 °C for 12 h. After the reaction is completed, remove the solvent under reduced pressure. Subsequently, add 30 mL of deionized water to the remaining residue, and heat and stir it under reflux conditions for 0.5 h. Next, extract the reaction solution with dichloromethane. Collect the organic layer and dry it with anhydrous sodium sulfate. Then, evaporate the solvent under reduced pressure. Purify the product by silica gel column chromatography to obtain compound 3 (0.25 g, yield: 38.5%);

[0086] Step (s4): Under N 2 atmosphere, dropwise add PBr3 (3.7 mL, 40 mmol) to a three-necked flask containing DMF (3.4 mL, 44.25 mmol) and chloroform (20 mL) at 0 °C, and stir continuously for 45 min. Subsequently, add cyclopentanone (2.5 mL, 28 mmol) to the mixture at room temperature, and stir continuously for 16 h. After the reaction is completed, neutralize the reaction mixture by adding saturated sodium bicarbonate solution. Then extract the mixture with ethyl acetate. Collect the organic layer and dry it with anhydrous sodium sulfate. Then, evaporate the solvent under reduced pressure to obtain compound 4 (2.3 g, yield: 47%), which is directly used in the subsequent steps without further purification;

[0087] Step (s5): Add compound 4 (0.44 g, 2.5 mmol), 2-hydroxy-4-methoxybenzaldehyde (0.55 g, 3.62 mmol), CsCO 3 (0.75 g, 2.31 mmol) and DMF (10 mL) into a round-bottom flask. Stir the mixture continuously at room temperature for 16 h. After the reaction is completed, remove the insoluble solid impurities by filtration. Extract the filtrate with ethyl acetate. Wash the organic layer with saturated NaCl solution. Then, evaporate the organic layer under reduced pressure. Purify the residue by silica gel column chromatography (CH 2 Cl 2 :CH 3 OH = 20:1, v / v) to obtain compound 5 (270 mg, yield: 49%);

[0088] Step (s6): Under N 2 atmosphere, add BBr 3 (1.60 g, 6.60 mmol) to a three-necked flask containing compound 5 (0.36 g, 1.65 mmol) and CH 2 Cl 2 (20 mL) in an ice-water bath. Stir the reaction mixture for 1 h, and then continue to stir it at room temperature for 16 h. After neutralization with saturated sodium bicarbonate solution, wash the reaction mixture with brine. Wash the aqueous layer with CH2 Cl 2 Extraction. The organic extract was dried over anhydrous sodium sulfate and then the solvent was evaporated under reduced pressure. The residue was purified by silica gel column chromatography (CH 2 Cl 2 :CH 3 OH = 10:1, v / v) to give Compound 6 (250.3 mg, yield: 70.8%).

[0089] In step (s7), Compound 3 (0.085 g, 0.41 mmol), Compound 6 (0.10 g, 0.49 mmol) and ethanol (10 mL) were added to a round-bottom flask. The mixture was stirred and heated to reflux for 10 minutes. Subsequently, piperidine (10 μL, 0.01 mmol) was added to the mixture. The mixture was further stirred and heated to reflux for 6 hours. After the reaction solution was cooled to room temperature, the solvent was evaporated under reduced pressure. The residue was purified by silica gel column chromatography (CH 2 Cl 2 : petroleum ether = 4:1, v / v) to give Compound YQ-OH (0.10 g, yield: 60%);

[0090] In step (s8), in an ice-water bath, Et 3 N (15 μL, 0.11 mmol) was added to a round-bottom flask containing phenyl chloroformate (54 μL, 0.42 mmol), Compound YQ-OH (74 mg, 0.18 mmol) and anhydrous CH 2 Cl 2 (9 mL). The mixture was stirred in the ice-water bath for 10 minutes and then continued to be stirred at room temperature for 2 hours. After the reaction was completed, the solvent was removed by evaporation under reduced pressure. The product was purified by silica gel column chromatography (CH 2 Cl 2 : petroleum ether = 5:1, v / v) to give the probe YQ as a purple solid (75 mg, yield 77%).

[0091] Example 2: Preparation of the test sample solution

[0092] YQ stock solution: An appropriate amount of the fluorescent compound YQ was dissolved in DMSO to prepare a stock solution of YQ (5×10 - 4 M).

[0093] Analyte stock solution: A stock solution of the analyte (1×10-3 M) was prepared by dissolving a specific amount of the analyte in water.

[0094] Preparation of YQ test solution: Transfer 0.10 mL of YQ stock solution, 1.4 mL of DMSO and an appropriate amount of analyte stock solution to a 5.0 mL volumetric flask. Subsequently, dilute to the mark with 20 mM potassium phosphate buffer (pH = 7.4). Shake the solution well and then incubate at room temperature for 1 minute.

[0095] Preparation of hypochlorite solution: Weigh an appropriate amount of sodium hypochlorite and make up the volume with distilled water to obtain a 10 mM hypochlorous acid solution, which is then serially diluted to obtain hypochlorous acid solutions with concentrations of 10 - 0.1 mM.

[0096] Test Example 1: UV-visible spectroscopic determination of the interaction between probe YQ and hypochlorous acid

[0097] As Figure 3 (a) shows that probe YQ has a significant absorption peak at 545 nm. When HClO is added to the probe YQ solution, a new red-shifted peak appears at 600 nm. As Figure 3 (b) shows, the absorbance at 600 nm (A600) is linearly correlated with the HClO concentration (0 - 80 μM), which causes the solution to change from purple to blue under visible light. As Figure 3 (c) shows, clearly demonstrating the colorimetric response of YQ to HClO.

[0098] As Figure 3 (d) shows that probe YQ exhibits low background fluorescence (Φf = 0.006). However, as the HClO concentration increases, the fluorescence intensity at 716 nm gradually increases. Obviously, this increase in intensity is attributed to the elimination of the O - phenyl methylthioester group when compound YQ interacts with HClO, thus restoring the ICT effect within YQ. When 90 μM HClO is added, Figure 3 (e) shows that the intensity of compound YQ reaches a plateau. In addition, Figure 3 (f) shows that the intensity at 716 nm is linearly correlated with the HClO concentration (0 - 80 μM). Compound YQ can detect HClO with high sensitivity, and the detection limit reaches 74 nM (S / N = 3), which means it can detect trace amounts of HClO. In addition, under ultraviolet light, the fluorescence color of the solution changes from colorless to rose red, as Figure 3 (c) shows.

[0099] Test Example 2: Influence of the pH of the solution on the detection of hypochlorous acid by probe YQ

[0100] Figure 4 (a) shows the addition of 100 μM ClO -Before (■) and after (▲), the fluorescence intensity of compound YQ at a concentration of 10 μM at 488 nm as a function of pH. The pH range investigated was 4.2 - 10.3. As can be seen from the figure, YQ itself has basically no fluorescence in this pH range. After adding 100 μM ClO - , in the pH ≥ 6.0 range, the fluorescence intensity of the probe increased significantly. It indicates that this fluorescent probe can be used for the detection of ClO - under physiological conditions.

[0101] Test Example 3: Response time and selectivity of probe YQ for hypochlorous acid determination

[0102] Figure 4 (b)'s kinetic study shows that the fluorescence intensity (I716) of compound YQ reaches its maximum value only 35 seconds after adding HClO. At the same time, to evaluate its selective detection ability, compound YQ was exposed to a variety of related substances. As can be seen from Figure 4 (c), HClO is the only substance that significantly enhances the fluorescence of the detection solution. In addition, as can be seen from Figure 4 (d), compound YQ can visually detect HClO under visible light and ultraviolet light conditions. These findings together indicate that compound YQ has a high selectivity for HClO.

[0103] Test Example 4: Fluorescence imaging of exogenous HClO in HeLa cells using probe YQ

[0104] HeLa cells were incubated in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum in a humidified incubator at 37 °C and 5% CO 2 . The HeLa cells were stained with compound YQ (10 μM) in PBS buffer (containing 0.1% DMSO, v / v) at 37 °C for 30 minutes. After washing three times with PBS buffer, they were further incubated with HClO (0 μM, 5 μM, 10 μM, 20 μM) for 30 minutes. Then, fluorescence imaging was performed using a 20x objective on a Leica TCS SP5 II laser confocal scanning microscope. The near-infrared (NIR) fluorescence channel recorded the emission at 700 nm - 740 nm under excitation at 633 nm. As can be seen from Figure 5 A(i), the near-infrared fluorescence gradually increased, indicating that compound YQ can specifically detect exogenous HClO in living cells.

[0105] Test Example 5: Fluorescence imaging of endogenous HClO in RAW264.7 using probe YQ

[0106] RAW 264.7 cells were incubated in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum for 24 hours. RAW 264.7 cells were stained with compound YQ (10 μM) at 37 °C for 30 minutes. After washing three times with PBS buffer, the cells were stimulated with PMA (0 μM, 5 μM, 20 μM) for 2 hours. Subsequently, the cells were washed three times with PBS and imaged using a Leica TCS SP5 II laser confocal scanning microscope. During imaging, the NIR fluorescence channel recorded the emission at 700 nm - 740 nm under excitation at 633 nm.

[0107] As Figure 5 shown in B (e - g), the fluorescence intensity in the near - infrared channel gradually increased. In addition, when RAW 264.7 cells stained with YQ were pre - treated with 4 - aminobenzoic acid hydrazide (ABAH, an MPO inhibitor) and then treated with PMA, almost no fluorescence was observed inside the cells ( Figure 5 B (h)). These findings indicate that compound YQ can also selectively detect endogenous HClO in living cells.

[0108] Test Example 6: Detection of Hypochlorous Acid by Probe YQ in Food

[0109] As Figure 6 shown, different concentrations (0, 10, 50, and 200 μM) of HClO solution were sprayed onto lettuce leaves. After continuous treatment for five days, necrosis was observed in the lettuce leaves sprayed with 200 μM HClO solution. Subsequently, the lettuce leaves were sliced, stained with compound YQ, and fluorescence imaging was performed. Apparently, as Figure 6 (i - l) shown, with the increase in the concentration of the sprayed HClO solution, the fluorescence intensity of the lettuce tissue in the near - infrared channel showed an upward trend. The results proved that compound YQ can effectively monitor the HClO residue in lettuce tissue.

[0110] Although this specification is described according to the embodiments, not every embodiment only contains an independent technical solution. This narrative way of the specification is only for clarity. Those skilled in the art should regard the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

[0111] The series of detailed descriptions listed above are only specific descriptions of the feasible embodiments of the present invention, and they are not used to limit the protection scope of the present invention. Any equivalent embodiments or changes made without departing from the technical spirit of the present invention should be included in the protection scope of the present invention.

Claims

1. A colorimetric-near infrared dual detection fluorescent probe for hypochlorous acid detection, characterized in that: It has the following structural formula:

2. The method for preparing the fluorescent probe according to claim 1, characterized in that: The steps include: Step (s1), reacting 2-hydroxyacetophenone with ethyl acetate to obtain 1-(2-hydroxyphenyl)-1,3-butanedione, recorded as compound 1; Step (s2), reacting compound 1 with glacial acetic acid under acidic conditions to obtain methyl isoflavone, recorded as compound 2; Step (s3), compound 2, malononitrile and acetic anhydride are mixed and reacted to obtain 2-(2-methyl-4H-benzopyran-4-ylidene) malononitrile, which is recorded as compound 3; Step (s4), reacting cyclopentanone with phosphorus tribromide to obtain 2-bromocyclopent-1-ene-1-carbaldehyde, recorded as compound 4; Step (s5), compound 4 is reacted with 2-hydroxy-4-methoxybenzaldehyde to obtain 6-hydroxy-1,2-dihydrocyclopentane[b]chromene-3-carbaldehyde, which is referred to as compound 5; Step (s6), compound 5 is subjected to a demethylation reaction to obtain compound 6; Step (s7), compound 3 and compound 6 are subjected to Aldol condensation reaction to obtain compound YQ-OH; Step (s8), compound YQ-OH reacts with phenyl chlorothioformate under alkaline conditions to undergo a nucleophilic substitution reaction to obtain the fluorescent probe, which is recorded as compound YQ.

3. The preparation method according to claim 2, characterized in that: In the step (s1), the usage ratio of 2-hydroxyacetophenone to ethyl acetate is 0.5 g-1.5 g: 1 mL-3 mL.

4. The preparation method according to claim 2, wherein in the step (s3), the amount ratio of compound 2, malononitrile and acetic anhydride is 0.3g-1.5g:0.1g-0.5g:3mL-8mL, the reaction time is 10-14h, and the reaction temperature is 120-160°C.

5. The preparation method according to claim 2, characterized in that: In the step (s4), the dosage ratio of cyclopentanone to phosphorus tribromide is 1 mL-6 mL: 2 mL-8 mL, and the reaction time is 15-20 h.

6. The preparation method according to claim 2, characterized in that: In the step (s6), the demethylation reaction occurs in the presence of boron tribromide, and the dosage ratio of the compound 5 to boron tribromide is 1g:(2g-4g).

7. The preparation method according to claim 2, characterized in that: In the step (s8), the usage ratio of compound YQ-OH to phenyl chlorothioformate is 0.1 mmol-0.3 mmol: 0.3 mmol-0.5 mmol, and the reaction time is 1.5-4 h.

8. A method for detecting the presence of hypochlorous acid in a sample and / or determining the hypochlorous acid content in a sample, comprising: contacting the sample with the fluorescent probe defined in claim 1 under conditions suitable for converting the fluorescent probe into a fluorescent product to form a fluorescent product; and The hypochlorous acid content in the sample can be determined by visual colorimetry or quantitative detection using a fluorescence spectrophotometer.

9. A fluorescent probe test paper, characterized in that: The method is prepared by immersing filter paper or a thin layer chromatography plate in a dichloromethane solution of the fluorescent probe according to claim 1 and drying the solution naturally.

10. Use of the fluorescent probe described in claim 1 in the detection and evaluation of hypochlorous acid in water samples, foods and cells.

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