Coumarin chemiluminescent material, hydrogen sulfide chemiluminescent probe, and preparation method and application thereof

Abstract

The application discloses coumarin chemiluminescence material, hydrogen sulfide chemiluminescence probe and a preparation method and application thereof, relates to the technical field of chemiluminescence material. The structural formula of the coumarin chemiluminescence material is shown in the figure; TBS represents a tert-butyldimethylsilyl group. The coumarin chemiluminescence material provided by the application shows extremely high-intensity and short-time chemiluminescence, and the controllable activation chemiluminescence characteristics lay a foundation for developing activated chemiluminescence probes. The electron-withdrawing trifluoromethyl group introduced at the 4th position of coumarin 4 can further increase the electrophilicity of the lactone and red-shift the light wavelength. In addition, the coumarin chemiluminescence material provided by the application has a brand-new structure, the application enriches the chemiluminescence material library, and has important guiding significance for further expansion of the chemiluminescence material library.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of chemiluminescent materials, in particular to a coumarin chemiluminescent material, a hydrogen sulfide chemiluminescent probe, and a preparation method and application thereof. BACKGROUND

[0002] Chemiluminescence imaging does not require external excitation light, can effectively avoid the interference of autofluorescence, and has high sensitivity and excellent signal-to-noise ratio. Among chemiluminescent substrates, the phenolic dioxetane chemiluminescent group (Schaap dioxetane) developed by the Schaap research group has attracted widespread attention due to its controllable chemiluminescent properties. Such compounds usually contain a tert-butyldimethylsilyl protecting group at the phenolic hydroxyl site, and a phenolate dioxetane intermediate is generated by removing the protecting group through a specific analyte (such as fluoride ion). The intermediate is unstable and can spontaneously undergo a chemical excitation process to generate an excited state benzoate, thereby producing a luminescent signal.

[0003] Some recent studies have improved the chemiluminescent properties by introducing electron-withdrawing groups at the phenolic group. Although some progress has been made, the preparation and structural diversity of dioxetane chemiluminescent materials are still limited. Currently, almost all dioxetane chemiluminescent materials are based on the phenolic dioxetane skeleton developed by the Schaap research group. Therefore, there is still a major challenge in developing new core structure dioxetane chemiluminescent materials.

[0004] Therefore, the prior art still needs to be improved and developed. SUMMARY

[0005] Based on the deficiencies of the prior art described above, the purpose of the present application is to provide a coumarin chemiluminescent material, a hydrogen sulfide chemiluminescent probe, and a preparation method and application thereof, aiming to provide a dioxetane chemiluminescent material with a novel skeleton structure to enrich the library of chemiluminescent materials.

[0006] The technical solutions of the present application are as follows:

[0007] In a first aspect of the present application, a coumarin chemiluminescent material is provided, wherein the structure of the coumarin chemiluminescent material is as follows:

[0008] ;

[0009] wherein TBS represents a tert-butyldimethylsilyl group.

[0010] ​The coumarin chemiluminescent material provided by the application has controllable activated chemiluminescent characteristics, and through removing the TBS protective group, is converted into unstable phenolate negative ions and triggers a chemical excitation process to generate the excited state compound CF3-coumarin, i.e. , and the excited state relaxes to the ground state to generate a chemiluminescence phenomenon, and the coumarin chemiluminescent material provided by the application exhibits extremely high intensity and short time chemiluminescence, and the controllable activated chemiluminescent characteristics lay a foundation for the development of activated chemiluminescent probes. Further, the coumarin chemiluminescent material provided by the application further increases the electrophilicity of the lactone and red-shifts the light wavelength by introducing an electron-withdrawing trifluoromethyl group at the 4-position of coumarin. In addition, the coumarin chemiluminescent material provided by the application has a brand-new structure, and the application enriches the chemiluminescent material library and has important guiding significance for further expansion of the chemiluminescent material library.

[0011] In a second aspect, the application provides a preparation method of the coumarin chemiluminescent material provided by the application, and the preparation method comprises the following steps:

[0012] S11, reacting 7-hydroxy-4-(trifluoromethyl) coumarin with tert-butyldimethylsilyl chloride to obtain ;

[0013] S12, mixing , titanium tetrachloride (TiCl4), lithium aluminum hydride (LiAlH4), 2-adamantanone, triethylamine and a first organic solvent (such as tetrahydrofuran) and then reacting to obtain ;

[0014] S13, mixing , rose Bengal, deuterated chloroform and deuterated methanol, introducing oxygen, and reacting under irradiation of a yellow light source with a color temperature of 3000 K to obtain the coumarin chemiluminescent material.

[0015] In the prior art, as a key precursor of dioxetane chemiluminescent materials, adamantyl phenyl enol ether (AE, structural formula as ) is oxidized by singlet oxygen to form dioxetane. However, the synthesis of AE involves a complicated multi-step route, harsh reaction conditions or hazardous reagents. For example, AE is prepared by McMurry coupling reaction of methyl benzoate with 2-adamantanone, but it requires the use of titanium trichloride (TiCl3) which is difficult to purchase and can be self-igniting; for another example, AE is synthesized by Horner-Wadsworth-Emmons coupling reaction of phosphonate with 2-adamantanone, but it requires harsh reaction conditions and a complicated process for preparing phosphonate; for yet another example, AE is prepared by Stille coupling reaction of aryl halide with adamantly alcohol ether metal tin compound, but it requires multi-step reactions and toxic metal tin compound.

[0016] However, the present application starts from commercially available 7-hydroxy-4- (trifluoromethyl) coumarin, and uses tert-butyldimethylsilyl chloride to protect the phenolic hydroxyl group to obtain . Subsequently, carbon-carbon double bond is constructed by McMurry reaction using titanium tetrachloride and lithium aluminum hydride as low-valence titanium reagent to obtain . Then, [2+2] cycloaddition reaction occurs between the double bond of and singlet oxygen generated by using photosensitizer rose Bengal to sensitize oxygen under light condition, and the coumarin chemiluminescent material is obtained.

[0017] Due to electronic effect and steric effect, lactone structure has higher electrophilicity than ordinary ester group, and the present application uses lactone structure to improve reactivity (or reaction yield), wherein the McMurry reaction in step S12 only needs to use commercially available and safe titanium tetrachloride, avoiding the use of titanium trichloride which is difficult to purchase and self-igniting. In addition, the present application greatly simplifies the preparation steps of chemiluminescent material, and the preparation route only needs three steps to prepare coumarin chemiluminescent material from commercially available raw materials.

[0018] Optionally, step S11 specifically comprises:

[0019] 7-hydroxy-4- (trifluoromethyl) coumarin, tert-butyldimethylsilyl chloride, imidazole and a second organic solvent (for example, anhydrous dichloromethane) are mixed, and after reaction, obtain .

[0020] Optionally, the molar ratio of 7-hydroxy-4- (trifluoromethyl) coumarin, tert-butyldimethylsilyl chloride and imidazole is 1: (1-2): (1-2) (for example, it can be 1:1:1, 1:1.2:1.5, 1:1:2, 1:2:1 or 1:2:2, etc.).

[0021] Optionally, in step S12, The molar ratio of titanium tetrachloride, lithium aluminum hydride, 2-adamantanone and triethylamine is (1-2.5):(10-20):(10-20):(5-12):(40-50).

[0022] For example, the molar ratio of the four substances can be 2.2:10:10:5:40, 2.2:13.7:15.75:10.3:44.17, 2.2:10:20:5:40, 2.2:10:10:12:40, 2.2:10:10:5:50, 2.2:20:20:12:50, 1:10:10:5:40, 1:13.7:15.75:10.3:44.17, 1:10:20:5:40, 1:10:10:12:40, 1:10:10:5:50 or 1:20:20:12:50, etc.

[0023] Optionally, in step S13, the volume ratio of deuterated chloroform and deuterated methanol is 10:1.

[0024] In some embodiments, in step S13, oxygen is bubbled.

[0025] In a third aspect, the present application provides a hydrogen sulfide chemiluminescence probe, wherein the structure of the hydrogen sulfide chemiluminescence probe is:

[0026] .

[0027] In a fourth aspect, the present application provides a preparation method of the hydrogen sulfide chemiluminescence probe as described above, comprising the following steps:

[0028] S21, mixing , tetrabutylammonium fluoride and 2,4-dinitrofluorobenzene, to obtain ;

[0029] S22, mixing , rose Bengal, deuterated chloroform and deuterated methanol, bubbling oxygen, and placing under a yellow light source with a color temperature of 3000 K to react, to obtain the hydrogen sulfide chemiluminescence probe.

[0030] In some embodiments, in step S21, , the molar ratio of tetrabutylammonium fluoride and 2,4-dinitrofluorobenzene is 0.065:0.1:0.195;

[0031] In step S22, the volume ratio of deuterated chloroform and deuterated methanol is 10:1.

[0032] In some embodiments, in step S22, oxygen is bubbled.

[0033] In a fifth aspect, the present application provides a use of the hydrogen sulfide chemiluminescence probe as described above in the present application in detecting hydrogen sulfide, wherein the detection of hydrogen sulfide is not for the purpose of disease diagnosis.

[0034] Specifically, the hydrogen sulfide chemiluminescence probe can be used to detect extracellular hydrogen sulfide, intracellular hydrogen sulfide, hydrogen sulfide in food (such as meat food, red wine) or tap water.

[0035] In a sixth aspect, the present application provides a use of the hydrogen sulfide chemiluminescence probe as described above in the present application in preparing a hydrogen sulfide detection test paper.

[0036] Specifically, the hydrogen sulfide detection test paper comprises filter paper and the hydrogen sulfide chemiluminescence probe on the surface of the filter paper.

[0037] Beneficial effects: the coumarin chemiluminescence material provided by the present application has controllable activated chemiluminescence characteristics, which is converted into unstable phenolate negative ions by removing the TBS protective group and triggers a chemical excitation process to generate an excited state compound CF3-Coumarin, i.e. , which relaxes to the ground state to produce a chemiluminescence phenomenon, and the coumarin chemiluminescence material provided by the present application exhibits extremely high intensity and short duration of chemiluminescence, and the controllable activated chemiluminescence characteristics lay a foundation for the development of activated chemiluminescence probes. Further, the present application introduces an electron-withdrawing trifluoromethyl group at the 4-position of coumarin to further increase the electrophilicity of the lactone and red-shift the emission wavelength. In addition, the coumarin chemiluminescence material provided by the present application has a brand-new structure, and the present application enriches the chemiluminescence material library, which has important guiding significance for further expansion of the chemiluminescence material library. BRIEF DESCRIPTION OF DRAWINGS

[0038] Figure 1 FIG. 1 is a diagram of the chemiluminescence mechanism of the compound Cou-CL and the test results of the luminescence performance of the compound Cou-CL, wherein A is the chemiluminescence mechanism of the compound Cou-CL, B is the visual luminescence image of the compound Cou-CL in DMSO, C is the chemiluminescence kinetics curve of the compound Cou-CL in DMSO, and D is the chemiluminescence spectrum of the compound Cou-CL in DMSO.

[0039] Figure 2Figures of HPLC analysis results and luminescence kinetics characterization results of the reaction of compound Cou-CL with tetrabutylammonium fluoride in DMSO in Example 1, wherein A is the HPLC analysis results of the reaction of compound Cou-CL with tetrabutylammonium fluoride, B is the chemiluminescence kinetics curve of compound Cou-CL with Schaap dioxetane within the range of 0-60 minutes, C is the chemiluminescence kinetics curve of Schaap dioxetane within the range of 0-800 minutes, and D is the total luminescence kinetics curve of compound Cou-CL with Schaap dioxetane.

[0040] Figure 3 Figures of chemiluminescence performance test results of compound Cou-CL in an aqueous phase system in Example 1, wherein A is the luminescence kinetics curve of compound Cou-CL in a mixture of DMSO and PBS buffer (the volume content of DMSO in the mixture is 70%-99%), B is the luminescence kinetics curve of compound Cou-CL in a mixture of DMSO and PBS buffer (the volume content of DMSO in the mixture is 5%-60%), and C is the luminescence kinetics curve of compound Cou-CL in a mixture of DMSO and PBS buffer (the volume content of DMSO in the mixture is 5%) with or without the addition of Emerald-II chemiluminescence enhancer.

[0041] Figure 4 Figures of luminescence mechanism and performance test results of probe Cou-CL-H2S in Example 2, wherein A is the chemiluminescence mechanism of probe Cou-CL-H2S, B is the chemiluminescence kinetics curve of probe Cou-CL-H2S, C is the chemiluminescence spectrum of probe Cou-CL-H2S, and D is the linear fitting curve of the chemiluminescence intensity of probe Cou-CL-H2S and the concentration of hydrogen sulfide.

[0042] Figure 5 Figure of HPLC analysis results of the reaction of probe Cou-CL-H2S with hydrogen sulfide in Example 2.

[0043] Figure 6 Figure of selectivity test results of probe Cou-CL-H2S in Example 2.

[0044] Figure 7 Figure of screening results of surfactants and their equivalents required for probe Cou-CL-H2S to detect hydrogen sulfide in an aqueous phase system in Example 2, wherein A is the screening results of surfactants, and B is the screening results of the equivalent of surfactant Pluronic F127.

[0045] Figure 8The result figure of chemiluminescence imaging analysis of Cou-CL-H2S probe in Example 2 for extracellular and endogenous hydrogen sulfide.

[0046] Figure 9 The result figure of Cou-CL-H2S probe in Example 2 for detecting hydrogen sulfide in BALB / c mice, in which A is the chemiluminescence imaging figure, B is the chemiluminescence imaging kinetics curve figure, and C is the result figure of chemiluminescence imaging quantitative analysis at 1 min time point after injecting Cou-CL-H2S probe.

[0047] Figure 10 The result figure of Cou-CL-H2S probe in Example 2 for detecting hydrogen sulfide in mouse inflammation model, in which A is the chemiluminescence imaging figure, B is the chemiluminescence imaging kinetics curve figure, and C is the result figure of chemiluminescence imaging quantitative analysis at 1 min time point after injecting Cou-CL-H2S probe.

[0048] Figure 11 The result figure of Cou-CL-H2S probe in Example 2 for preparing into test paper for detecting hydrogen sulfide, in which A is the structural schematic diagram of hydrogen sulfide gas detection device, B is the luminescence kinetics result figure of Cou-CL-H2S test paper for detecting hydrogen sulfide gas, C is the luminescence response result figure of Cou-CL-H2S test paper to hydrogen sulfide gas and other gas interferents, and D is the linear fitting curve figure of chemiluminescence intensity of Cou-CL-H2S test paper and hydrogen sulfide gas concentration.

[0049] Figure 12 The result figure of Cou-CL-H2S test paper for detecting hydrogen sulfide gas released in the spoilage process of pork sample, in which A is the result figure of freshness degree of pork sample changing with storage time, B is the chemiluminescence response result figure of Cou-CL-H2S test paper in the spoilage process of pork sample, and C is the total volatile basic nitrogen detection result figure in the spoilage process of pork sample.

[0050] Figure 13 The result figure of Cou-CL-H2S test paper for detecting hydrogen sulfide gas released in the spoilage process of shrimp sample, in which A is the result figure of freshness degree of shrimp sample changing with storage time, B is the chemiluminescence response result figure of Cou-CL-H2S test paper in the spoilage process of shrimp sample, and C is the total volatile basic nitrogen detection result figure in the spoilage process of shrimp sample.

[0051] Figure 14The results of detecting hydrogen sulfide gas released during the spoilage of salmon meat samples using Cou-CL-H2S test strips are shown in Figure A, which shows the change in the freshness of salmon meat samples with storage time; Figure B shows the chemiluminescence response of Cou-CL-H2S test strips during the spoilage of salmon meat samples; and Figure C shows the detection results of total volatile basic nitrogen during the spoilage of salmon meat samples. Detailed Implementation

[0052] This invention provides coumarin chemiluminescent materials, hydrogen sulfide chemiluminescent probes, their preparation methods, and applications. To make the objectives, technical solutions, and effects of this invention clearer and more explicit, the invention is further described in detail below. It should be understood that the specific embodiments described herein are only for explaining the invention and are not intended to limit the invention.

[0053] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0054] The present invention will be further described below through specific embodiments.

[0055] Example 1

[0056] The synthetic route for coumarin chemiluminescent materials is as follows:

[0057] .

[0058] Following the above synthetic route, the preparation method of coumarin chemiluminescent materials includes the following steps:

[0059] Preparation of Compound 1: 7-hydroxy-4-(trifluoromethyl)coumarin (230 mg, 1 mmol, i.e., compound CF3-Coumarin) was dissolved in anhydrous dichloromethane (10 mL), followed by the sequential addition of imidazole (102 mg, 1.5 mmol) and tert-butyldimethylchlorosilane (180 mg, 1.2 mmol). The mixture was stirred at room temperature for 1 hour. The reaction was monitored by thin-layer chromatography (TLC) (developing solvent: ethyl acetate and petroleum ether, v / v). After the reaction was complete, the reaction solution was concentrated under reduced pressure using a vacuum pump and separated by rapid column chromatography (eluting solvent: ethyl acetate and petroleum ether, v / v) to obtain a white solid, namely compound 1 (326 mg, yield 95%). The 1H NMR spectrum of compound 1 is as follows: 1 H NMR (500 MHz, CDCl3) δ 7.61 (dd, J = 9.5, 2.0 Hz, 1H), 6.87 (dd,J = 4.7, 2.4 Hz, 2H), 6.64 (s, 1H), 1.02 (s, 9H), 0.29 (s, 6H). The carbon nuclear magnetic resonance data of compound 1 is 13 C NMR (126 MHz, CDC13) δ 160.35, 159.33, 156.08, 141.43, 126.34, 118.11, 112.59, 112.54, 108.26, 107.68, 25.52, 18.28, -4.38. The fluorine nuclear magnetic resonance data of compound 1 is 19 F NMR (376 MHz, CDC13) δ -64.72. The high resolution mass spectrometry data of compound 1 is HRMS calcd for C 16 H 20 F3O3Si + [M+H] + : 345.11283; found: 345.11288 (calcd for C

[0060] Preparation of compound 2: Anhydrous tetrahydrofuran (20 mL) was placed under nitrogen protection, and ice bathed for 10 minutes, until the temperature was reduced to 0 ℃, titanium tetrachloride (1.5 mL, 13.7 mmol) was slowly added, and the reaction solution changed from colorless to yellow. Then lithium aluminum hydride (598 mg, 15.75 mmol) was slowly added, and the reaction was continued for 10 minutes under ice bath condition. Subsequently, the ice bath was removed, triethylamine (4.1 mL, 44.17 mmol) was added, and heated to reflux for 1 hour to obtain the first reaction solution. Compound 1 (760 mg, 2.20 mmol) and 2-adamantanone (1.54 g, 10.3 mmol) were dissolved in anhydrous tetrahydrofuran (20 mL), and then slowly added dropwise into the first reaction solution within 30 minutes, and then heated to reflux at 70 ℃ for 2 hours. After cooling to room temperature, water (100 mL) was added to quench the reaction, and extracted with ethyl acetate (3 x 20 mL), and the organic phase was combined and washed with saturated brine (50 mL), and dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure by vacuum pump, and separated by flash column chromatography (eluent: ethyl acetate and petroleum ether at a volume ratio of 1:10) to obtain a gray-green solid, which was compound 2 (408 mg, yield 40%). The hydrogen nuclear magnetic resonance data of compound 2 is 1 H NMR (500 MHz, CDC13) δ 7.04 (dd, J = 8.5, 2.0 Hz, 1H), 6.70 (s, 1H), 6.37 (dd, J=8.5, 2.4 Hz, 1H), 6.31 (d, J = 2.4 Hz, 1H), 3.30 (s, 1H), 2.75 (s, 1H), 2.01 – 1.71 (m, 12H), 0.98 (s, 9H), 0.22 (s, 6H). The carbon NMR data for compound 2 are: 13 C10 NMR (126 MHz, CDCl3) δ 157.61, 155.49, 136.37, 130.38, 124.43, 122.16, 121.00, 117.26, 113.72, 109.85, 107.42, 39.37, 38.62, 36.98, 31.72, 29.52, 28.20, 25.63, 18.20, -4.39. The fluorine NMR data for compound 2 are as follows: 19 F NMR (376 MHz, CDCl3) δ -64.94. High-resolution mass spectrometry data for compound 2 are HRMS calcd for C 26 H 34 F3O2Si + [M+H] + :463.22747; found: 463.22763.

[0061] Synthesis of coumarin chemiluminescent material: Compound 2 (10 mg, 0.022 mmol) was dissolved in 2 mL of a mixture of deuterated chloroform and deuterated methanol (volume ratio of deuterated chloroform to deuterated methanol: 10:1). Then, a catalytic amount of Bengal rose red (0.01 mmol) was added. The mixture was placed under a yellow light source at a color temperature of 3000 K, with oxygen introduced and bubbled into the reaction. The reaction was monitored by TLC (eluent: ethyl acetate and petroleum ether, volume ratio 1:10). After the reaction was complete (approximately 3 minutes), the reaction solution was concentrated under reduced pressure using a vacuum pump and separated by preparative thin-layer chromatography on silica gel plates (eluent: ethyl acetate and petroleum ether, volume ratio 1:10) to obtain a colorless solid, namely the coumarin chemiluminescent material, compound Cou-CL (9.7 mg, yield 90%). The 1H NMR spectrum of compound Cou-CL is as follows: 1 H NMR (400 MHz, CDCl3) δ 7.31 (dd, J =8.7, 2.0 Hz, 1H), 6.70 (d, J = 2.4 Hz, 1H), 6.57 (dd, J = 8.7, 2.4 Hz, 1H), 6.53 (d, J= 1.2 Hz, 1H), 3.11 (s, 1H), 2.74 (s, 1H), 1.99 - 1.66 (m, 12H), 0.98 (s, 9H), 0.22 (s, 6H). The nuclear magnetic resonance carbon spectrum data of compound Cou-CL is 13 C NMR (101 MHz, CDC13) δ 158.79, 152.76, 129.39, 125.67, 115.25, 114.18, 108.79, 107.20, 106.50, 97.02, 36.17, 34.60, 33.78, 33.17, 32.16, 31.73, 31.12, 26.07, 25.70, 18.33, -4.27. The nuclear magnetic resonance fluorine spectrum data of compound Cou-CL is 19 F NMR (376 MHz, CDC13) δ -60.13, -64.91. The high resolution mass spectrum data of compound Cou-CL is HRMS calcd for C 26 H 33 F3NaO4Si + [M+Na] + : 517.19924; found: 517.19946.

[0062] Compound Cou-CL was tested as follows:

[0063] (1) First, in vitro characterization of compound Cou-CL was carried out, compound Cou-CL was dissolved in DMSO (dimethyl sulfoxide) to obtain solutions of compound Cou-CL with final concentrations of 10 mM, 25 μM and 50 μM, respectively, and then visual luminescence, kinetics and luminescence spectrum tests were carried out respectively to evaluate the chemiluminescence performance of compound Cou-CL in organic solvent DMSO, and the results are shown in Figure 1 .

[0064] Among them, the chemiluminescence mechanism of compound Cou-CL is shown as A in Figure 1 Due to the protection of phenolic hydroxyl group by TBS, compound Cou-CL does not exhibit chemiluminescence; after the removal of TBS protecting group by adding tetrabutylammonium fluoride (TBAF), compound Cou-CL is converted into unstable phenoxide anion and triggers the chemical excitation process to generate excited state compound CF3-Coumarin, and the excited state relaxes to the ground state to produce chemiluminescence phenomenon.

[0065] The visual luminescence image of compound Cou-CL (with a final concentration of 10 mM, i.e. the concentration in the final solution, and the final concentrations of the remaining compounds refer to the concentrations in the final solution) in DMSO is as follows: Figure 1As shown in B, no luminescence signal was observed in the visible luminescence image before the addition of tetrabutylammonium fluoride (i.e., when the time was 0 seconds); after the addition of tetrabutylammonium fluoride (final concentration of 20mM), a clear green luminescence signal could be observed immediately, reaching the maximum luminescence value in 0.32 seconds, and then the luminescence intensity rapidly decayed within 2 seconds, exhibiting a "flash" phenomenon with extremely high intensity and short duration.

[0066] The chemiluminescence kinetics curve of compound Cou-CL (final concentration 25 μM) in DMSO is shown in the figure below. Figure 1 As shown in C (RLU stands for Relative Light Unit), it also exhibits a similar characteristic of rapid decay after the "flash".

[0067] The chemiluminescence spectrum of compound Cou-CL (final concentration 50 μM) in DMSO is shown below. Figure 1 As shown in Figure D, the maximum chemiluminescence wavelength of compound Cou-CL is 514 nm, consistent with the green light observed in the visualized luminescence image, and also consistent with the maximum fluorescence wavelength (λ) of compound CF3-Coumarin. max = 510 nm) similar.

[0068] (2) In order to prove the chemiluminescence mechanism of compound Cou-CL, the reaction of compound Cou-CL with tetrabutylammonium fluoride in DMSO was analyzed by high performance liquid chromatography (HPLC), and the luminescence kinetics of compound Cou-CL were characterized.

[0069] Specifically, compound Cou-CL (final concentration 10 mM) was dissolved in DMSO and analyzed by HPLC; then, compound Cou-CL (final concentration 10 mM) and tetrabutylammonium fluoride (final concentration 20 mM) were dissolved in DMSO and analyzed by HPLC. The results are as follows: Figure 2 As shown in Figure A, in HPLC analysis, compound Cou-CL appeared as a single chromatographic peak in DMSO with a retention time of 26.63 min. After reacting with tetrabutylammonium fluoride, the chromatographic peak of compound Cou-CL in solution disappeared, and a new product chromatographic peak appeared at a retention time of 3.51 min. The retention time of this peak was consistent with that of compound CF3-Coumarin, indicating that the product of the reaction between compound Cou-CL and tetrabutylammonium fluoride is CF3-Coumarin. Therefore, the chemiluminescence spectrum of the above compound Cou-CL (as shown in Figure A) is... Figure 1 The results of HPLC analysis (as shown in D in the figure) strongly support the following: Figure 1 The chemiluminescence mechanism of compound Cou-CL, shown in A.

[0070] Compound Cou-CL and tetrabutylammonium fluoride were added to DMSO to obtain a mixture (where the final concentration of compound Cou-CL was 25 μM and the final concentration of tetrabutylammonium fluoride was 50 μM); Schaap dioxane ( Tetrabutylammonium fluoride was added to DMSO to obtain a mixture (where the final concentration of Schap dioxane was 25 μM and the final concentration of tetrabutylammonium fluoride was 50 μM). The two mixtures were tested to compare the chemiluminescence properties of compound Cou-CL and Schap dioxane in DMSO. The chemiluminescence kinetic curves of compound Cou-CL and Schap dioxane in DMSO from 0 to 60 minutes are shown in the figure below. Figure 2 As shown in Figure B, the chemiluminescence kinetics curve of Schaap dioxane from 0 to 800 minutes is shown in Figure B. Figure 2 As shown in Figure C, the total luminescence kinetics of compound Cou-CL and Schaap dioxane in DMSO is shown in Figure C. Figure 2 As shown in D in the diagram.

[0071] Depend on Figure 2 B and Figure 2 As can be seen from C, compound Cou-CL exhibits high-intensity, short-duration chemiluminescence, while Schaap dioxane exhibits weak-intensity, long-duration chemiluminescence.

[0072] In addition, the luminescent quantum yield (Φ) of Schaap dioxane was obtained. CL Using (29%) as a reference, the luminescence quantum yield of compound Cou-CL in DMSO was calculated to be approximately 0.173% through kinetic curves. Nevertheless, compared to Schaap dioxane, the maximum chemiluminescence wavelength of compound Cou-CL is redshifted by 44 nm.

[0073] Depend on Figure 2 As shown in D, the luminescence half-life of compound Cou-CL is 24 seconds, which is significantly shorter than the luminescence half-life of Schaap dioxane (T). 1 / 2 = 147 minutes), indicating that the chemical excitation rate of compound Cou-CL is 368 times that of Schaap dioxane. Therefore, the luminescence mode of compound Cou-CL is a very high-intensity, short-duration "flash" mode, while Schaap dioxane exhibits a weaker, longer-lasting "glow" mode. Compared to the weaker, longer-lasting "glow" mode, the "flash" mode, which emits a high-intensity signal in a short time, provides higher detection sensitivity and faster detection speed.

[0074] (3) Test the chemiluminescence properties of compound Cou-CL in an aqueous system.

[0075] Compounds Cou-CL and tetrabutylammonium fluoride were dissolved in mixed solutions of DMSO and PBS buffer (pH = 7.4, 10 mM, i.e., phosphate buffer) with different DMSO concentrations (99%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, and 5% by volume). The final concentrations of Cou-CL and tetrabutylammonium fluoride were 25 μM and 50 μM, respectively. Chemiluminescence kinetics were then analyzed, and the results are shown below. Figure 3 As shown in A and B in the figure, similar to Schaap dioxane, compound Cou-CL exhibits a weakening of chemiluminescence signal in PBS buffer. The higher the proportion of water, the greater the decrease in chemiluminescence intensity, which may be due to water-induced quenching of the chemically excited state.

[0076] Compound Cou-CL and tetrabutylammonium fluoride were dissolved in a 5% (v / v) mixture of DMSO and PBS buffer (pH = 7.4, 10 mM), where the final concentration of Cou-CL was 25 μM and the final concentration of tetrabutylammonium fluoride was 50 μM. Chemiluminescence kinetics were then analyzed. Simultaneously, compound Cou-CL, tetrabutylammonium fluoride, and Emerald-II chemiluminescence enhancer were dissolved in a 5% (v / v) mixture of DMSO and PBS buffer (pH = 7.4, 10 mM), where the final concentration of Cou-CL was 25 μM, the final concentration of tetrabutylammonium fluoride was 50 μM, and the volume of Emerald-II enhancer was 5% of the total volume of the mixture. Chemiluminescence kinetics were then analyzed. The results are as follows: Figure 3 As shown in C, Emerald-II chemiluminescence enhancer can enhance the chemiluminescence signal of compound Cou-CL in an aqueous system (containing 5% DMSO by volume). This is attributed to Emerald-II chemiluminescence enhancer providing a hydrophobic environment for the chemiluminescence reaction and avoiding induced quenching effects. This result makes it possible to apply compound Cou-CL to an aqueous system.

[0077] In summary, this embodiment, from the perspective of dioxane chemical synthesis, proposes using lactone structures to improve the synthetic efficiency of dioxane precursors (i.e., adamantyl phenyl enol ether compounds), and designs and synthesizes compound Cou-CL, which requires only three steps and has an overall yield of 34.2%, while avoiding the use of hazardous reagents. Compound Cou-CL exhibits extremely high intensity and short duration of chemiluminescence, and its controllable activation chemiluminescence characteristics lay the foundation for the development of activated chemiluminescent probes.

[0078] Example 2

[0079] The synthetic route for the hydrogen sulfide chemiluminescent probe (i.e., probe Cou-CL-H2S) is as follows:

[0080] .

[0081] Following the above synthetic route, the preparation method of the hydrogen sulfide chemiluminescent probe (i.e., probe Cou-CL-H2S) includes the following steps:

[0082] Synthesis of Compound 3: Compound 2 (30 mg, 0.065 mmol) was dissolved in tetrahydrofuran (2 mL), followed by the addition of a tetrahydrofuran solution of tetrabutylammonium fluoride (0.1 mmol) (0.1 mL). The mixture was stirred at room temperature (25 °C) for 1 hour. The reaction was quenched with water (10 mL) and extracted with ethyl acetate (3 × 10 mL). The combined organic phases were washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure using a vacuum pump. The concentrated liquid was dissolved in dichloromethane (2 mL), and potassium carbonate (26 mg, 0.195 mmol) and 2,4-dinitrofluorobenzene (22 μL, 0.195 mmol) were added. The mixture was stirred at room temperature for 1 hour, followed by concentration under reduced pressure using a vacuum pump. The solution was then separated by rapid column chromatography (eluent: ethyl acetate and petroleum ether in a 1:8 v / v ratio) to give a yellow solid, compound 3 (19 mg, yield 57%). The proton NMR spectrum data of compound 3 are as follows: 1 H NMR (400 MHz, CDCl3) δ 8.83 (d, J = 2.8 Hz, 1H), 8.35 (dd, J = 9.2, 2.8 Hz, 1H), 7.22 (dd, J = 8.5, 1.8 Hz, 1H), 7.16 (d, J = 9.3 Hz, 1H), 6.84 (s, 1H), 6.64 (dd, J = 8.6, 2.5Hz, 1H), 6.54 (d, J = 2.5 Hz, 1H), 3.22 (s, 1H), 2.76 (s, 1H), 2.00 – 1.70 (m, 12H). The carbon NMR data for compound 3 are: 13C10 NMR (101 MHz, CDCl3) δ 156.23, 155.36, 155.10, 142.08, 140.01, 135.97, 133.38, 128.94, 125.51, 122.18, 121.66, 120.03, 119.96, 119.56, 114.81, 113.49, 107.61, 39.49, 38.69, 36.90, 32.00, 29.85, 28.15. The fluorine NMR data for compound 3 are as follows: 19 F NMR (376 MHz, CDCl3) δ -65.17. High-resolution mass spectrometry data for compound 3 are HRMS calcd for C 26 H 22 F3N2O6 + [M+H] + :515.14245; found:515.14270.

[0083] Synthesis of probe Cou-CL-H2S: Compound 3 (10 mg, 0.019 mmol) was dissolved in 2 mL of a mixture of deuterated chloroform and deuterated methanol (volume ratio of deuterated chloroform to deuterated methanol: 10:1). A catalytic amount of Bengal rose red (0.009 mmol) was then added, and the mixture was placed under a yellow light source at a color temperature of 3000 K. Oxygen was introduced and bubbled into the reaction. The reaction was monitored by TLC (eluent: ethyl acetate and petroleum ether, volume ratio 1:8). After the reaction was complete (approximately 3 minutes), the reaction solution was concentrated under reduced pressure using a vacuum pump and separated by preparative thin-layer chromatography on silica gel plates (eluent: ethyl acetate and petroleum ether, volume ratio 1:8) to obtain a colorless solid, probe Cou-CL-H2S (9.9 mg, yield 96%). The 1H NMR spectrum of probe Cou-CL-H2S is as follows: 1 H NMR (400 MHz, CDCl3) δ 8.87 (d, J = 2.7 Hz, 1H), 8.39 (dd, J = 9.2, 2.8 Hz, 1H), 7.56 (dd, J = 8.7, 1.9 Hz, 1H), 7.19 (d, J = 9.1 Hz, 1H), 7.01 (d, J = 2.5 Hz, 1H), 6.88 (dd, J = 8.7 (2.5 Hz, 1H), 6.70 (s, 1H), 3.06 (s, 1H), 2.77 (s, 1H), 1.99–1.54 (m, 12H). The carbon NMR data of the probe Cou-CL-H2S are as follows:13 C NMR (101 MHz, CDCl3) δ 156.29, 154.63, 153.06, 142.63, 140.45, 129.09, 128.71, 126.97, 123.38, 122.30, 120.29, 116.95, 114.38, 111.55, 109.08, 106.40, 96.79, 36.02, 34.66, 33.77, 33.13, 32.08, 31.72, 30.98, 26.03, 25.91. The fluorine NMR data for the probe Cou-CL-H2S are as follows: 19 F NMR (376 MHz, CDCl3) δ -64.95. High-resolution mass spectrometry data for probe Cou-CL-H2S are HRMS calcd for C 26 H 21 F3N2NaO8 + [M+Na] + :569.11422; found:569.11456.

[0084] The following tests were performed on the probe Cou-CL-H2S:

[0085] (1) The kinetics, spectra and detection limits of the probe Cou-CL-H2S for the detection of hydrogen sulfide were characterized.

[0086] The luminescence mechanism of probe Cou-CL-H2S is shown in A of section 4. Due to the protection of the phenolic hydroxyl group by 2,4-dinitrophenyl (DNP), probe Cou-CL-H2S does not produce chemiluminescence. The addition of hydrogen sulfide can remove the DNP protecting group, generating an unstable phenolic salt anion, which triggers a chemical excitation process to generate the excited-state compound CF3-Coumarin. The chemiluminescent signal is released when the excited state relaxes to the ground state.

[0087] The probe Cou-CL-H2S (final concentration 25 μM) was added to PBS buffer solution (pH = 7.4, 10 mM, containing 95% DMSO by volume), and the chemiluminescence kinetics were tested. The probe Cou-CL-H2S (final concentration 25 μM) and hydrogen sulfide (final concentration 25 μM) were added to PBS buffer solution (pH = 7.4, 10 mM, containing 95% DMSO by volume), and the chemiluminescence kinetics were tested.

[0088] The probe Cou-CL-H2S (final concentration 50 μM) was added to PBS buffer solution (pH = 7.4, 10 mM, containing 95% DMSO by volume), and the chemiluminescence spectrum was tested. The probe Cou-CL-H2S (final concentration 50 μM) and hydrogen sulfide (final concentration 50 μM) were added to PBS buffer solution (pH = 7.4, 10 mM, containing 95% DMSO by volume), and the chemiluminescence spectrum was tested.

[0089] The results are as follows Figure 4 B and Figure 4 As shown in Figure C, no chemiluminescence signal is generated without the addition of hydrogen sulfide; after the addition of hydrogen sulfide, a strong chemiluminescence signal is observed, reaching its maximum value in just 2 seconds, followed by rapid decay, exhibiting a "flash" pattern. The maximum chemiluminescence wavelength of the probe Cou-CL-H2S after reacting with hydrogen sulfide is 530 nm, which is similar to the maximum chemiluminescence wavelength of the compound Cou-CL.

[0090] The probe Cou-CL-H2S (final concentration 25 μM) was added to PBS buffer solution (pH = 7.4, 10 mM, containing 95% DMSO by volume), and then different concentrations of hydrogen sulfide (final concentrations of 0 μM, 1.5 μM, 3.1 μM, 6.2 μM, 12.5 μM, and 25 μM) were added. The linear fitting curves of the chemiluminescence intensity of the probe Cou-CL-H2S and the hydrogen sulfide concentration (0~25 μM) were tested, and the results are shown below. Figure 4 As shown in Figure D, when the hydrogen sulfide concentration is in the range of 0~25 μM, the luminescence intensity of the probe Cou-CL-H2S shows a good linear correlation with the hydrogen sulfide concentration (R0). 2 = 0.980). Based on the linear relationship fitting curve, and calculated using the limit of detection formula (LOD = 3×S / N, where LOD represents the lowest detection limit, S represents the standard deviation of the luminescence intensity of the chemiluminescent probe under analyte-free conditions, and N represents the slope in the linear fitting curve), the detection limit of probe Cou-CL-H2S for hydrogen sulfide is 11.9 nM, indicating that probe Cou-CL-H2S has high detection sensitivity for hydrogen sulfide.

[0091] (2) To demonstrate that the luminescent response of probe Cou-CL-H2S is due to the reaction of 2,4-dinitrophenyl in the molecular structure with hydrogen sulfide, the reaction of probe Cou-CL-H2S with hydrogen sulfide was analyzed by HPLC. Specifically, probe Cou-CL-H2S (final concentration 25 μM) was added to PBS buffer (pH = 7.4, 10 mM, containing 95% DMSO by volume) for HPLC analysis; then probe Cou-CL-H2S (final concentration 25 μM) and hydrogen sulfide (final concentration 25 μM) were added to PBS buffer (pH = 7.4, 10 mM, containing 95% DMSO by volume) for HPLC analysis; compound CF3-Coumarin and compound DNP-SH ( HPLC testing was performed, and the results are as follows: Figure 5 As shown in the figure, probe Cou-CL-H2S exhibits a single chromatographic peak with a retention time of 14.85 minutes. After the addition of hydrogen sulfide, the chromatographic peak of probe Cou-CL-H2S disappears, and two new absorption peaks of the products appear at retention times of 3.42 minutes and 5.37 minutes, respectively. Their retention times are consistent with those of compounds CF3-Coumarin and DNP-SH, indicating that the products of the reaction between probe Cou-CL-H2S and hydrogen sulfide are compounds CF3-Coumarin and DNP-SH. Therefore, the chemiluminescence spectrum (e.g., Figure 4 (As shown in C) Together with the above HPLC analysis results, it proves the chemical reaction mechanism and luminescence mechanism of the probe Cou-CL-H2S in detecting hydrogen sulfide, as follows: Figure 4 As shown in A in the diagram.

[0092] (3) Selectivity test of probe Cou-CL-H2S

[0093] The probe Cou-CL-H2S (final concentration 25 μM) and different substances (final concentration 25 μM) were added to PBS buffer (pH = 7.4, 10 mM, containing 95% DMSO by volume), and chemiluminescence intensity was measured. The results are as follows: Figure 6 As shown.

[0094] Among them, the different substances are Na + K + NH4 + F – Cl – ,Br – I – NO3 – ClO4 – H2PO4 – OH– OAc – (Acetate), SCN – BF4 – HCO3 – CO3 2– HSO4 – HSO3 – SO4 2– SO3 2– S2O8 2– S2O4 2– S2O3 2– β-ME (β-mercaptoethanol), Cys (cysteine), GSH (glutathione) and H2S.

[0095] It is evident that the probe Cou-CL-H2S exhibits a significant luminescence signal for H2S, while showing virtually no luminescence signal response for the potential interfering substances being tested, indicating that the probe Cou-CL-H2S has good detection selectivity for H2S.

[0096] (4) Surfactant screening

[0097] In the in vitro characterization of the above compound Cou-CL, it was found that Emerald-II chemiluminescence enhancer could improve the luminescence intensity of compound Cou-CL in aqueous system by providing a hydrophobic environment for the chemiluminescence reaction. In order to improve the chemiluminescence intensity of probe Cou-CL-H2S when detecting hydrogen sulfide under aqueous conditions, different surfactants were screened, including cucurbituril[7], cucurbituril[8], polyoxyethylene polyoxypropylene ether (Pluronic F127), phospholipid-polyethylene glycol (DSPE-PEG), polylactic acid-glycolic acid-polyethylene glycol (PLGA-PEG), trimethyl-β-cyclodextrin (T-β-CD), Tween-80 and Emerald-II chemiluminescence enhancer.

[0098] The probe Cou-CL-H2S (final concentration 25 μM), hydrogen sulfide (final concentration 25 μM), and different surfactants were added to PBS buffer solution (pH = 7.4, 10 mM, containing 50% DMSO by volume), and chemiluminescence intensity was measured. The results are as follows: Figure 7 As shown in A, under aqueous conditions, cucurbituril[8] and Pluronic F127 can significantly enhance the chemiluminescence intensity of the probe Cou-CL-H2S when detecting hydrogen sulfide. Among them, Pluronic F127 showed the best luminescence signal enhancement effect, which may be related to the fact that Pluronic F127 has the most suitable micelle structure size.

[0099] Next, the chemiluminescence intensity of the probe Cou-CL-H2S in detecting hydrogen sulfide was tested under different equivalent conditions (10 eq, 20 eq, 40 eq, 60 eq, 80 eq, and 100 eq, where eq represents equivalent). The results are as follows: Figure 7 As shown in Figure B, the chemiluminescence intensity first increases and then decreases with increasing Pluronic F127 equivalents, reaching its maximum when the Pluronic F127 equivalent is 20. This trend may be due to insufficient Pluronic F127 concentration preventing complete micelle formation, while excessively high concentrations hinder substrate diffusion. In conclusion, these results indicate that an appropriate amount of Pluronic F127 can enhance the response signal of the Cou-CL-H2S probe to hydrogen sulfide in an aqueous system.

[0100] (5) The probe Cou-CL-H2S was used to detect endogenous and exogenous hydrogen sulfide in live cells.

[0101] This assay evaluated the potential of the probe Cou-CL-H2S to visualize exogenous and endogenous hydrogen sulfide in live cells. HCT116 cells were incubated for half an hour with different final concentrations of hydrogen sulfide (0 μM, 50 μM, 100 μM, and 150 μM) and PBS buffer (pH = 7.4, 10 mM, containing 10% DMSO and 1 mM Pluronic F127). Then, the probe Cou-CL-H2S (final concentration 50 µM) was added for chemiluminescence imaging analysis.

[0102] As a substrate for enzymatic reactions, Cys can produce endogenous hydrogen sulfide within cells through metabolism. Therefore, in this assay, HCT116 cells were incubated with Cys (final concentration 100 µM) and PBS buffer (pH = 7.4, 10 mM, containing 10% DMSO and 1 mM Pluronic F127 by volume) for 2 hours, followed by the addition of the probe Cou-CL-H2S (final concentration 50 µM) for chemiluminescence imaging analysis.

[0103] The results are as follows Figure 8As shown, the chemiluminescence intensity of probe Cou-CL-H2S increases with increasing hydrogen sulfide concentration, exhibiting a clear concentration-dependent response, indicating that probe Cou-CL-H2S can effectively detect endogenous and exogenous hydrogen sulfide within cells. Furthermore, compared to cells without Cys treatment (which have the same chemiluminescence intensity as at a hydrogen sulfide concentration of 0 µM), the chemiluminescence intensity of cells treated with Cys increased approximately 10-fold, demonstrating that probe Cou-CL-H2S can effectively detect endogenous hydrogen sulfide produced by Cys metabolism within cells.

[0104] (6) Hydrogen sulfide in BALB / c mice was detected using the probe Cou-CL-H2S.

[0105] This study investigated the applicability of the probe Cou-CL-H2S for chemiluminescence imaging of endogenous hydrogen sulfide in a mouse model. Specifically, BALB / c mice were subcutaneously injected with Cys aqueous solution (100 µL, 2 mM) in the right leg and with PBS buffer solution (100 µL, 10 mM) in the left leg. 35 min later, 100 µL of probe Cou-CL-H2S solution (50 µM concentration, containing 1 mM Pluronic F127, in a 1:9 volume ratio of DMSO and PBS buffer) was injected subcutaneously into both legs. Chemiluminescence imaging and quantitative analysis were performed at 1 min, 2 min, 3 min, 4 min, 5 min, and 6 min post-injection. The results are as follows: Figure 9 As shown (statistical analysis was performed using one-way ANOVA), represent P (Less than 0.01). It can be seen that, compared to the left leg treated with PBS buffer, the right leg treated with Cys exhibited a strong chemiluminescent signal, and the intensity of this signal first increased and then decreased with time (e.g., ...). Figure 9 (As shown in A). One minute after injecting probe Cou-CL-H2S, the chemiluminescence intensity at the Cys-treated right leg was 3.5 times that at the PBS-buffered left leg (as shown in A). Figure 9 B and Figure 9 As shown in Figure C, the ordinate represents the average chemiluminescence intensity ± standard deviation (sample size n=3). These results indicate that the probe Cou-CL-H2S can be used for rapid detection and imaging of endogenous hydrogen sulfide in mouse models.

[0106] (7) Hydrogen sulfide in a mouse inflammation model was detected using the probe Cou-CL-H2S.

[0107] Given the ability of the probe Cou-CL-H2S to image hydrogen sulfide in mice, this invention further validates its applicability for chemiluminescent imaging of hydrogen sulfide in a mouse inflammation model. Lipopolysaccharide (LPS) induces an acute inflammatory response in mice, leading to an increase in hydrogen sulfide concentration. Therefore, this test utilizes an acute inflammation model induced by LPS in mice and employs the probe Cou-CL-H2S to image endogenous hydrogen sulfide. Specifically, mice were subcutaneously injected with 100 µL of LPS aqueous solution (1 mg / mL) in the right leg and 100 µL of PBS buffer solution (10 mM) in the left leg. Five hours later, 100 µL of probe Cou-CL-H2S solution (50 µM concentration, containing 1 mM Pluronic F127, in a 1:9 volume ratio of DMSO and PBS buffer) was injected subcutaneously into both legs. Chemiluminescence imaging and quantitative analysis were performed at 1 min, 2 min, 3 min, 4 min, 5 min, and 6 min after injection. The results are as follows: Figure 10 As shown (statistical analysis was performed using one-way ANOVA), represent P Less than 0.01). Compared to the left leg treated with PBS buffer, the right leg treated with LPS showed a strong chemiluminescent signal (e.g., ...). Figure 10 (As shown in A). One minute after injecting probe Cou-CL-H2S, the chemiluminescence intensity at the LPS-treated right leg was 2.2 times that at the PBS-buffered left leg (as shown in A). Figure 10 B and Figure 10 As shown in Figure C, the ordinate represents the average chemiluminescence intensity ± standard deviation (sample size n = 3). These results indicate that the probe Cou-CL-H2S can detect endogenous hydrogen sulfide in a mouse inflammation model.

[0108] (8) The probe Cou-CL-H2S was used to detect hydrogen sulfide in food.

[0109] a. Prepare test strips from probe Cou-CL-H2S for the detection of hydrogen sulfide.

[0110] A circular filter paper is immersed in a DMSO solution containing Cou-CL-H2S, and after being removed and dried, hydrogen sulfide gas detection test paper (denoted as Cou-CL-H2S test paper) is made.

[0111] This invention designs a simple hydrogen sulfide gas detection device (such as...) Figure 11(As shown in A). The device consists of two glass bottles of different volumes. Cou-CL-H2S test paper is adsorbed to the bottom of the larger glass bottle by surface tension. The smaller glass bottle contains sodium hydrosulfide solution, which can release hydrogen sulfide gas in situ. To begin the test, the cap of the smaller glass bottle is opened, and then the larger glass bottle is quickly inverted over the smaller glass bottle before being placed in a chemiluminescence imaging device for detection.

[0112] The chemiluminescence kinetics of hydrogen sulfide gas detection using Cou-CL-H2S test paper were investigated using the signal accumulation imaging mode of a chemiluminescence imager. The results are as follows: Figure 11 As shown in Figure B, the chemiluminescence signal increases rapidly from 0 to 25 seconds and reaches saturation at 40 seconds, indicating that the Cou-CL-H2S test paper has a rapid response capability to hydrogen sulfide gas. This invention then investigated the chemiluminescence response of the Cou-CL-H2S test paper to potential gaseous interfering substances, including carbon monoxide (CO), hydrogen chloride (HCl), formaldehyde (HCOH), phosgene (ClCOCl), nitric oxide (NO), and ammonia (NH3), etc., with results as follows. Figure 11 As shown in C, the Cou-CL-H2S test paper only exhibits a significant luminescent signal response to hydrogen sulfide gas, while showing virtually no luminescent signal response to other potential interfering substances. Since ammonia and hydrogen sulfide are two major harmful gases produced during food spoilage, this invention also tested the chemiluminescent response of the Cou-CL-H2S test paper to 500 equivalents of ammonia gas. The results showed (as shown in C). Figure 11 As shown in Figure C), the Cou-CL-H2S test paper also showed no chemiluminescent signal response to high concentrations of ammonia. These results confirm the high selectivity of the Cou-CL-H2S test paper for the detection of hydrogen sulfide gas.

[0113] Finally, this invention investigated the effect of hydrogen sulfide gas concentration on the chemiluminescence signal of the Cou-CL-H2S test paper. For example... Figure 11 As shown in D, when the hydrogen sulfide gas concentration ranges from 0 to 0.45 ppm, the chemiluminescence intensity of the Cou-CL-H2S test paper exhibits a good linear correlation with the hydrogen sulfide gas concentration (R0). 2 = 0.985). Based on the linear relationship fitting curve and calculated using the detection limit formula (LOD = 3×S / N), the detection limit for hydrogen sulfide gas of the Cou-CL-H2S test paper is 6.75 ppb. This value is lower than the LOD value (50 ppb) of the standard hydrogen sulfide gas detection method of the US Environmental Protection Agency, indicating that the Cou-CL-H2S test paper has high sensitivity for the detection of hydrogen sulfide gas.

[0114] b. Detect hydrogen sulfide gas released during the spoilage of pork samples using Cou-CL-H2S test paper.

[0115] like Figure 12 As shown in Figure A, pork samples were placed in sealed containers and stored at 37 °C for 12–60 h, while the control group was stored at 0 °C. Under the 37 °C storage condition, the color of the pork samples changed from bright red to dark red with prolonged storage time, indicating that the pork samples had spoiled.

[0116] To test the hydrogen sulfide gas released from pork samples stored for different periods, simply open the sealed container lid, quickly invert the large glass bottle (with Cou-CL-H2S test paper adsorbed at the bottom) over the sealed container, and use the Cou-CL-H2S test paper to detect the hydrogen sulfide gas released from the pork sample. The results are as follows: Figure 12 As shown in Figure B, the intensity of the chemiluminescence signal of the Cou-CL-H2S test paper increases in a time-dependent manner, with a chemiluminescence signal observed at 12 h and a strong chemiluminescence signal observed between 24 and 60 h. In contrast, the chemiluminescence signal of the Cou-CL-H2S test paper is extremely weak even after 60 h of storage at 0 ℃.

[0117] Total volatile basic nitrogen (TVB-N) is the gold standard for evaluating meat freshness in the food industry. This invention tested the TVB-N of pork samples, and the results are as follows: Figure 12 As shown in Figure C, the total volatile basic nitrogen (TVNA) value remained essentially unchanged under 0 °C storage conditions; however, under 37 °C storage conditions, the TVNA value increased in a time-dependent manner. When the TVNA value exceeded 15 mg / 100 g after 48 h, the pork sample was deemed spoiled according to the National Food Safety Standard GB2707-2016. The trend of TVNA value changes in the pork sample during storage at 37 °C was largely consistent with the trend of chemiluminescence signal changes in the Cou-CL-H2S test paper. A significant increase in chemiluminescence signal intensity was observed in the Cou-CL-H2S test paper after 12 h, while the TVNA value only began to increase significantly after 48 h. These results indicate that the Cou-CL-H2S test paper can effectively detect hydrogen sulfide gas released during the spoilage of pork samples without complex sample pretreatment, and it has higher sensitivity in detecting the degree of spoilage of pork samples compared to the detection of TVNA.

[0118] c. Detect hydrogen sulfide gas released during the spoilage of shrimp meat samples using Cou-CL-H2S test strips.

[0119] like Figure 13As shown in Figure A, shrimp meat samples were placed in sealed containers and stored at 37 °C for 12–60 h, while the control group was stored at 0 °C. Under the storage condition of 37 °C, as the storage time increased, the color of the shrimp meat samples changed from dark blue-green to yellowish-red, indicating that the shrimp meat samples had spoiled.

[0120] To test the hydrogen sulfide gas released from shrimp samples stored for different periods, simply open the sealed container lid, quickly invert the large glass bottle (with Cou-CL-H2S test paper adsorbed at the bottom) over the sealed container, and use the Cou-CL-H2S test paper to detect the hydrogen sulfide gas released from the shrimp sample. The results are as follows: Figure 13 As shown in Figure B, the chemiluminescence signal intensity of the Cou-CL-H2S test paper increases in a time-dependent manner, with a chemiluminescence signal observable at 12 h and a strong chemiluminescence signal observed between 24 and 60 h. In contrast, at 0 ℃, even after 60 h of storage, the chemiluminescence signal of the Cou-CL-H2S test paper remains extremely weak. Figure 13 As shown in Figure C, the total volatile basic nitrogen (TVNA) value remained essentially unchanged under 0 °C storage conditions; however, under 37 °C storage conditions, the TVNA value increased in a time-dependent manner. When the TVNA value exceeded 30 mg / 100 g after 36 h, the shrimp sample was deemed spoiled according to the National Food Safety Standard GB2733-2015. The trend of TVNA value changes during 37 °C storage was largely consistent with the trend of chemiluminescence signal changes observed by the Cou-CL-H2S test paper. A significant increase in chemiluminescence signal intensity was observed with the Cou-CL-H2S test paper after 12 h, while the TVNA value only began to increase significantly after 24 h. These results indicate that the Cou-CL-H2S test paper can effectively detect hydrogen sulfide gas released during the spoilage of shrimp samples without complex food sample pretreatment, and it has higher sensitivity in detecting the degree of spoilage of shrimp samples compared to the detection of TVNA.

[0121] d. Detect hydrogen sulfide gas released during the spoilage of salmon meat samples using Cou-CL-H2S test strips.

[0122] like Figure 14 As shown in Figure A, salmon samples were placed in sealed containers and stored at 37 °C for 12–60 h, while the control group was stored at 0 °C. Under the 37 °C storage condition, the color of the salmon samples changed from bright red to dark yellow with prolonged storage time, indicating that the salmon samples had spoiled.

[0123] To test the hydrogen sulfide gas released from salmon samples stored for different periods, simply open the sealed container, quickly invert the large glass bottle (with Cou-CL-H2S test paper adsorbed at the bottom) over the sealed container, and use the Cou-CL-H2S test paper to detect the hydrogen sulfide gas released from the salmon sample. The results are as follows: Figure 14 As shown in Figure B, the chemiluminescence signal intensity of the Cou-CL-H2S test paper increases in a time-dependent manner, with a weak chemiluminescence signal observed at 12 h and a strong chemiluminescence signal observed between 24 and 60 h. In contrast, the chemiluminescence signal of the Cou-CL-H2S test paper remains extremely weak even after 60 h of storage at 0 ℃.

[0124] like Figure 14 As shown in Figure C, the total volatile basic nitrogen (TVNA) value remained essentially unchanged under 0 °C storage conditions; however, under 37 °C storage conditions, the TVNA value increased in a time-dependent manner. When the TVNA value exceeded 30 mg / 100 g after 48 h, the salmon was deemed spoiled according to the National Food Safety Standard GB2733-2015. The trend of TVNA value changes in salmon samples stored at 37 °C was largely consistent with the trend of chemiluminescence signal changes in the Cou-CL-H2S test paper. A significant increase in chemiluminescence signal intensity was observed in the Cou-CL-H2S test paper after 24 h, while the TVNA value only began to increase significantly between 24 and 36 h. These results indicate that the Cou-CL-H2S test paper can effectively detect hydrogen sulfide gas released during the spoilage of salmon samples without complex food sample pretreatment, and it has higher sensitivity in detecting the degree of spoilage in salmon samples compared to the detection of TVNA.

[0125] e. Detecting hydrogen sulfide in red wine and tap water using the probe Cou-CL-H2S.

[0126] Hydrogen sulfide of different concentrations was added to red wine and tap water, and the accuracy of the Cou-CL-H2S probe for detecting hydrogen sulfide was tested using the spiking method. As shown in Table 1, the recovery rate of hydrogen sulfide detection in red wine ranged from 98.2% to 101.8%, and the recovery rate in tap water ranged from 95.6% to 102.1%. The good recovery rates demonstrate that the Cou-CL-H2S probe can accurately detect hydrogen sulfide in both red wine and tap water.

[0127] Table 1. Results of hydrogen sulfide detection in red wine and tap water using probe Cou-CL-H2S

[0128]

[0129] The above results demonstrate that the probe Cou-CL-H2S exhibits a strong luminescent response to hydrogen sulfide in in vitro experiments, ultrafast response kinetics (reaching maximum luminescence in just 2 seconds), and high sensitivity (detection limit of 11.9 nM) and selectivity. This indicates that the probe Cou-CL-H2S can achieve rapid, sensitive, and highly selective chemiluminescent detection of hydrogen sulfide in vitro. Furthermore, the probe Cou-CL-H2S can perform luminescent imaging detection of hydrogen sulfide in live cells and can be used for in vivo luminescent imaging of hydrogen sulfide in a mouse acute inflammation model.

[0130] The Cou-CL-H2S test strip provides efficient, rapid (40 seconds), sensitive (LOD = 6.75 ppb), and highly selective detection of hydrogen sulfide gas. It can also be used for in-situ detection of hydrogen sulfide released during the spoilage of meat products (such as pork, shrimp, and salmon), assessing the freshness and degree of spoilage of food samples. Compared to traditional methods for detecting food freshness, the Cou-CL-H2S test strip offers the advantages of in-situ speed and high sensitivity.

[0131] It should be understood that the application of the present invention is not limited to the examples above. Those skilled in the art can make improvements or modifications based on the above description, and all such improvements and modifications should fall within the protection scope of the appended claims.

Claims

1. A coumarin chemiluminescent material, characterized by comprising: The coumarin chemiluminescent material has a structural formula as shown in the following formula (I): ； In the formula, TBS represents a tert-butyldimethylsilyl group.

2. A method for preparing the coumarin chemiluminescent material according to claim 1, characterized by, The method comprises the following steps: S11. 7-hydroxy-4-(trifluoromethyl)coumarin is reacted with tert-butyldimethylsilyl chloride to give ; S12, will Titanium tetrachloride, lithium aluminum hydride, 2-adamantanone, triethylamine, and a first organic solvent are mixed and reacted to obtain... ; S13, will The coumarin chemiluminescent material is obtained by mixing Bengal rose red, deuterated chloroform and deuterated methanol, introducing oxygen, and irradiating the mixture with a yellow light source at a color temperature of 3000 K.

3. The preparation method according to claim 2, characterized in that, The step S11 specifically comprises: mixing 7-hydroxy-4-(trifluoromethyl)coumarin, tert-butyldimethylsilyl chloride, imidazole, and a second organic solvent, and after reaction, obtaining .

4. The preparation method according to claim 3, characterized in that, The molar ratio of 7-hydroxy-4-(trifluoromethyl) coumarin, tert-butyldimethylchlorosilane and imidazole is 1:(1-2):(1-2).

5. The preparation method according to claim 2, characterized in that, In step S12, The molar ratio of titanium tetrachloride, lithium aluminum hydride, 2-adamantanone, and triethylamine is (1-2.5):(10-20):(10-20):(5-12):(40-50).

6. The preparation method according to claim 2, characterized in that, In the step S13, the volume ratio of deuterated chloroform and deuterated methanol is 10:

1.

7. A hydrogen sulfide chemiluminescent probe, characterized by, The hydrogen sulfide chemiluminescent probe has a structural formula as shown in the following formula (II): 。 8. A method of preparing the hydrogen sulfide chemiluminescent probe of claim 7, wherein, The method comprises the following steps: S21, to with tetrabutylammonium fluoride, followed by the addition of 2,4-dinitrofluorobenzene, to give ; S22, will A mixture of Bengal rose red, deuterated chloroform, and deuterated methanol was introduced, and the mixture was placed under a yellow light source with a color temperature of 3000 K to react and obtain the hydrogen sulfide chemiluminescent probe.

9. Use of the hydrogen sulfide chemiluminescent probe according to claim 7 in detection of hydrogen sulfide, wherein the detection of hydrogen sulfide is not for disease diagnosis.

10. Use of the hydrogen sulfide chemiluminescent probe according to claim 7 in preparation of a hydrogen sulfide detection test paper.

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