A fluorescent probe for hypochlorite detection with molecular glue as a skeleton, and a preparation method and application thereof

Abstract

The application discloses a fluorescent probe for hypochlorite detection with molecular glue as a skeleton, and a preparation method and application thereof. 16 H 15 N3O5S. The preparation method of the fluorescent probe is simple, and the yield is high. Meanwhile, the probe can effectively eliminate the influence of other ions and free radicals, and exhibits the characteristics of quick response, high sensitivity and high selectivity in the hypochlorite detection.

Description

Technical Field

[0001] This invention relates to a fluorescent probe for hypochlorite detection with 3-hydroxythalidomide as the molecular glue backbone, its preparation method and application, belonging to the field of analytical detection technology. Background Technology

[0002] In recent years, with increasing public concern about water quality and environmental safety, the concentration of hypochlorite (ClO) has risen. - The need for accurate hypochlorite detection is becoming increasingly urgent. Hypochlorite is widely used in water purification, medical disinfection, and food processing, but excessive or incorrect use can have negative impacts on human health and the environment. Therefore, developing a highly selective and sensitive hypochlorite detection method is of great significance.

[0003] Traditional methods for hypochlorite detection include enzyme-linked immunosorbent assay (ELISA), electrochemical methods, and spectroscopic methods. However, these methods have limitations, such as complex operating procedures, expensive and inconvenient equipment and reagent costs, and dependence on professional operators. Therefore, developing a simpler, more economical, and efficient method for hypochlorite detection has become a research hotspot.

[0004] Fluorescent probe technology, as a rapid, sensitive, and selective detection method, has been widely used in life sciences and environmental sciences. Fluorescent probes detect targets based on the specific recognition of molecules and changes in fluorescence signals. Therefore, fluorescent probes targeting hypochlorite ions have become a promising research direction.

[0005] Currently, some research reports have been published on fluorescent probes for hypochlorite. Among them, some probes based on fluorescent dyes have high sensitivity and selectivity, but they still have inherent drawbacks in practical applications, such as the fluorescence signal being greatly affected by environmental factors and poor probe stability. Therefore, further research and development of more stable and reliable fluorescent probes for hypochlorite has great practical significance and application value. Summary of the Invention

[0006] The purpose of this invention is to provide a fluorescent probe for the detection of hypochlorite, which uses the molecular glue 3-hydroxythalidomide as a backbone to achieve accurate detection of hypochlorite in complex aquatic environments.

[0007] Based on the electrophilic addition mechanism of the receptor thiocarbamate, this invention designs a novel fluorescent probe for detecting hypochlorite using the molecular glue 3-hydroxythalidomide molecule as the fluorophore. When the fluorescent probe interacts with hypochlorite ions, the solution changes from no fluorescence to yellow-green fluorescence. The change in fluorescence intensity can be used to accurately detect hypochlorite.

[0008] In this invention, the fluorescent probe for hypochlorite detection (hereinafter referred to as the fluorescent probe) has the molecular formula C0. 16 H 15 N3O5S, named O-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-4-yl)dimethylaminomethylsulfate, has the structural formula shown in Formula I: The aforementioned fluorescent probe can be prepared by reacting 3-hydroxythalidomide and dimethylaminothiocarbamoyl chloride in the presence of a base and a solvent, as shown in the following equation: Furthermore, the base can be selected from organic bases, inorganic bases, or combinations thereof. The base can be one, two, or more. The base can be a combination of organic bases, a combination of inorganic bases, or a combination of organic and inorganic bases. Optionally, for example, the inorganic base can be at least one of potassium carbonate, sodium carbonate, potassium bicarbonate, sodium bicarbonate, cesium carbonate, etc., and the organic base can be at least one of N,N-diisopropylethylamine (DIPEA), 4-dimethylaminopyridine (DMAP), pyridine, triethylamine, etc.

[0009] Preferably, the base is selected from one or more organic bases, and more preferably, the base is a composition of N,N-diisopropylethylamine (DIPEA) and 4-dimethylaminopyridine (DMAP).

[0010] Furthermore, the solvent is an organic solvent, and its function is to dissolve the raw materials and provide a medium for the reaction. The organic solvent can be a haloalkane solvent such as dichloromethane or chloroform, or an ether solvent such as tetrahydrofuran or diethyl ether. There are no special requirements for the choice of solvent, as long as it can act as a medium and does not chemically react with the reactants. The amount of solvent used can be adjusted according to actual needs.

[0011] Furthermore, the reaction needs to be carried out in an anhydrous environment to prevent hydrolysis of the raw materials during the reaction. Therefore, it is necessary to avoid the introduction of water from the raw materials. For example, the raw materials should be dried in advance, and an anhydrous drying solvent should be selected.

[0012] Furthermore, the reaction is carried out under the protection of an inert gas, which includes nitrogen, argon, etc., to prevent the organic amine from being oxidized.

[0013] Furthermore, the molar ratio of 3-hydroxythalidomide to dimethylaminothiocarbamoyl chloride is 1:(1~2.5), for example 1:1, 1:1.5, 1:2, 1:2.5, preferably 1:2.0.

[0014] Furthermore, the molar ratio of 3-hydroxythalidomide to the base is 1:(1~23), for example 1:1, 1:2, 1:4, 1:6, 1:10, 1:12, 1:15, 1:20, 1:22, 1:23.

[0015] In one specific embodiment of the present invention, the base is a composition of N,N-diisopropylethylamine (DIPEA) and 4-dimethylaminopyridine (DMAP). The amount of N,N-diisopropylethylamine (DIPEA) is 1 to 20 times the molar amount of 3-hydroxythalidomide, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 times. The amount of 4-dimethylaminopyridine (DMAP) is 1 to 3 times the molar amount of 3-hydroxythalidomide, for example, 1, 2, or 3 times.

[0016] Furthermore, the reaction is carried out at room temperature for a time of 2 to 8 hours, for example, 2 hours, 4 hours, 6 hours, or 8 hours.

[0017] Furthermore, the above method may specifically include the following steps: adding 3-hydroxythalidomide molecules, dimethylaminothiocarbamoyl chloride, solvent, and base into a reaction vessel, reacting under inert gas protection, quenching the reaction with water after reaction, and purifying the crude product by column chromatography after post-treatment to obtain the target product.

[0018] Furthermore, there are no special requirements regarding the order in which the ingredients are added, as long as they are mixed evenly.

[0019] This invention also provides the application of the fluorescent probe described above for hypochlorite detection in the detection of hypochlorite ions present in a liquid environment. This liquid environment is a liquid water environment, which can be tap water or various complex aquatic environments.

[0020] A method for detecting hypochlorite in an aquatic environment, wherein the method uses the aforementioned fluorescent probe for hypochlorite detection to detect hypochlorite in the aquatic environment.

[0021] Furthermore, in the above method, the fluorescent probe is dissolved in the detection solvent to prepare a probe solution, and then the water sample to be tested is dropped into the probe solution, mixed well, and then fluorescence detection is performed.

[0022] Furthermore, in the above method, the concentration of the fluorescent probe in the probe solution is 0.5 × 10⁻⁶. -5 -1.5×10 - 5 mol / L.

[0023] Furthermore, in the above method, the detection solvent is one or more of ethanol, tetrahydrofuran, dimethyl sulfoxide, N,N-dimethylformamide, PBS buffer, and Tris buffer, preferably a mixture of PBS buffer and ethanol or a mixture of PBS buffer and dimethyl sulfoxide. When the detection solvent is a mixture of PBS buffer and ethanol, the volume concentration of PBS buffer is 75-85%, and the volume concentration of ethanol is 15-25%. When the detection solvent is a mixture of PBS buffer and dimethyl sulfoxide, the volume concentration of PBS buffer is 75-85%, and the volume concentration of dimethyl sulfoxide is 15-25%.

[0024] Furthermore, in the above method, the volume of the water sample to be tested does not exceed 1% of the volume of the probe solution, for example, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1%.

[0025] Furthermore, in the above method, the concentration of hypochlorite is detected by the change in fluorescence intensity.

[0026] The present invention has the following advantages: 1. The fluorescent probe of this invention is based on an electrophilic addition mechanism, which can undergo a specific chemical reaction with hypochlorite ions. It has high selectivity and sensitivity, fast response, and can effectively eliminate the influence of other ions and free radicals. It can accurately detect the concentration of hypochlorite ions in complex aquatic environments.

[0027] 2. The backbone of the fluorescent probe of this invention is made of 3-hydroxythalidomide molecule, which has good biocompatibility, low toxicity, good stability and fluorescence characteristics, can maintain good signal stability and sensitivity, and is less affected by environmental factors.

[0028] 3. The fluorescent probe of this invention exhibits fluorescence at 512 nm when detecting hypochlorite, and is less susceptible to interference from biological background, making it more advantageous in biological applications.

[0029] 4. The preparation method of the fluorescent probe of the present invention is simple, rapid and has a high yield. It does not require complex equipment and reagents and is suitable for laboratory and field applications. Attached Figure Description

[0030] Figure 1 This is the 1H NMR spectrum of the fluorescent probe.

[0031] Figure 2 This is the carbon NMR spectrum of the fluorescent probe.

[0032] Figure 3 The figure shows the experimental results of the specific recognition of the fluorescent probe.

[0033] Figure 4The fluorescence response of the fluorescent probe to different concentrations of hypochlorite is shown.

[0034] Figure 5 This is a graph showing the change in fluorescence response of the fluorescent probe to hypochlorite over time.

[0035] Figure 6 These are confocal microscope images (from left to right: bright field, DAPI (nucleus localization), green channel imaging, and superimposed field). Detailed Implementation

[0036] The following specific embodiments illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0037] It should be noted that, unless otherwise specified, the following embodiments and features can be combined with each other. It should also be understood that the terminology used in the embodiments of this invention is for describing specific implementations and not for limiting the scope of protection of this invention. Test methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions or as recommended by the respective manufacturers.

[0038] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the present invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this invention, as well as the prior art known to those skilled in the art and the description of this invention, may be implemented using any prior art methods, devices, and materials similar to or equivalent to those described, used, or made of materials in the embodiments of this invention.

[0039] Preparation method of fluorescent probe The target product can be obtained by stirring 3-hydroxythalidomide, dimethylaminothiocarbamate chloride, base, and solvent at room temperature and then purifying the mixture.

[0040] Example 1 3-Hydroxythalidomide (274 mg, 1.0 mmol, 1.0 eq), N,N-diisopropylethylamine (DIPEA, 1.29 g, 10.0 mmol, 10.0 eq), and anhydrous dichloromethane (DCM) (10 mL) were added to a dry 50 mL round-bottom flask. The mixture was stirred for 10 min at room temperature under argon protection, and then 4-dimethylaminopyridine (244.2 mg, 2.0 mmol, 2.0 eq) was added. Subsequently, dimethylaminothiocarbamate chloride (247.2 mg, 2.0 mmol, 2.0 eq) was added in a single addition at room temperature. After addition, the mixture was stirred at room temperature for 6 h. The reaction was quenched with water (15 mL), and the organic phase was collected. The organic phase was dried over Na₂SO₄, filtered, and the filtrate was collected and concentrated under reduced pressure to obtain the crude product. The crude product was purified by column chromatography under the following conditions: column: SiO2 (40 g); mobile phase: ethyl acetate (EA) / petroleum ether (PE) = 1 / 5 (V / V), increased to EA / PE = 1 / 1 within 20 min; flow rate: 15 mL / min; detector: UV 254 nm. The obtained product (probe) was a white, foamy solid (298.6 mg), with a yield of 81.7%. Gas chromatography analysis showed a purity of 98.74%.

[0041] The proton and carbon NMR spectra of the obtained products are as follows: Figure 1 and Figure 2 As shown, the specific information is as follows: 1 H NMR (600 MHz, DMSO) δ 11.13 (s, 1H), 7.91 (dd, J = 8.0, 7.6 Hz, 1H), 7.82 (d, J = 7.2 Hz, 1H), 7.58 (dd, J = 8.2, 0.5 Hz, 1H), 5.11 (d, J = 13.0 Hz, 1H), 3.38 (d, J = 2.7 Hz, 6H), 2.87 – 2.83 (m, 1H), 2.62 – 2.50 (m, 2H), 2.07 –2.02 (m, 1H). 13C NMR (151 MHz, DMSO) δ 185.51, 173.21, 170.15, 166.85, 164.98,149.93, 136.72, 133.05, 131.29, 123.19, 121.57, 49.44, 43.44, 39.28, 31.38,22.45. Example 2 3-Hydroxythalidomide (274 mg, 1.0 mmol, 1.0 eq), N,N-diisopropylethylamine (DIPEA, 258 mg, 2.0 mmol, 2.0 eq), and anhydrous dichloromethane (DCM) (10 mL) were added to a dry 50 mL round-bottom flask. The mixture was stirred for 10 minutes at room temperature under argon protection, and then 4-dimethylaminopyridine (244.2 mg, 2.0 mmol, 2.0 eq) was added. Subsequently, dimethylaminothiocarbamoyl chloride (247.2 mg, 2.0 mmol, 2.0 eq) was added in a single addition at room temperature. After addition, the mixture was stirred at room temperature for 6 h. The reaction was then post-processed and purified as described in Example 1, yielding 163.3 mg of the product, with a yield of 44.1%. Gas chromatography analysis showed a purity of 97.61%.

[0042] Example 3 3-Hydroxythalidomide (274 mg, 1.0 mmol, 1.0 eq), N,N-diisopropylethylamine (DIPEA, 645.3 mg, 5.0 mmol, 5.0 eq), and anhydrous dichloromethane (DCM) (10 mL) were added to a dry 50 mL round-bottom flask. The mixture was stirred for 10 min at room temperature under argon protection, and then 4-dimethylaminopyridine (244.2 mg, 2.0 mmol, 2.0 eq) was added. Subsequently, dimethylaminothiocarbamoyl chloride (247.2 mg, 2.0 mmol, 2.0 eq) was added in a single addition at room temperature. After addition, the mixture was stirred at room temperature for 6 h. The reaction was then post-processed and purified as described in Example 1, yielding 217.7 mg of the product, with a yield of 59.1%. Gas chromatography analysis showed a purity of 98.06%.

[0043] Example 4 3-Hydroxythalidomide (274 mg, 1.0 mmol, 1.0 eq), N,N-diisopropylethylamine (DIPEA, 1.548 g, 12.0 mmol, 12.0 eq), and anhydrous dichloromethane (DCM) (10 mL) were added to a dry 50 mL round-bottom flask. The mixture was stirred for 10 minutes at room temperature under argon protection. Subsequently, dimethylaminothiocarbamoyl chloride (123.6 mg, 1.0 mmol, 1.0 eq) was added in a single batch at room temperature. After addition, the mixture was stirred at room temperature for 6 h. The reaction was then post-processed and purified as described in Example 1, yielding 64.3 mg of the product. Gas chromatography analysis showed a yield of 17.6% and a purity of 98.83%.

[0044] Example 5 3-Hydroxythalidomide (274 mg, 1.0 mmol, 1.0 eq), N,N-diisopropylethylamine (DIPEA, 1.935 g, 15.0 mmol, 15.0 eq), and anhydrous dichloromethane (DCM) (10 mL) were added to a dry 50 mL round-bottom flask. The mixture was stirred for 10 min at room temperature under argon protection, and then 4-dimethylaminopyridine (244.2 mg, 2.0 mmol, 2.0 eq) was added. Subsequently, dimethylaminothiocarbamoyl chloride (247.2 mg, 2.0 mmol, 2.0 eq) was added in a single addition at room temperature. After addition, the mixture was stirred at room temperature for 6 h. The reaction was then post-processed and purified as described in Example 1, yielding 316.7 mg of the product, with a yield of 86.7%. Gas chromatography analysis showed a purity of 98.86%.

[0045] Example 6 3-Hydroxythalidomide (274 mg, 1.0 mmol, 1.0 eq), N,N-diisopropylethylamine (DIPEA, 2.583 g, 20.0 mmol, 20.0 eq), and anhydrous dichloromethane (DCM) (10 mL) were added to a dry 50 mL round-bottom flask. The mixture was stirred for 10 min at room temperature under argon protection, and then 4-dimethylaminopyridine (244.2 mg, 2.0 mmol, 2.0 eq) was added. Subsequently, dimethylaminothiocarbamoyl chloride (247.2 mg, 2.0 mmol, 2.0 eq) was added in a single addition at room temperature. After addition, the mixture was stirred at room temperature for 6 h. The reaction was then post-processed and purified as described in Example 1, yielding 318.1 mg of the product, with a yield of 87.1%. Gas chromatography analysis showed a purity of 98.85%.

[0046] Example 7 3-Hydroxythalidomide (274 mg, 1.0 mmol, 1.0 eq) and anhydrous DCM (10 mL) were added to a dry 50 mL round-bottom flask. The mixture was stirred for 10 min at room temperature under argon protection, and then 4-dimethylaminopyridine (366.9 mg, 3.0 mmol, 3.0 eq) was added. Subsequently, dimethylaminothiocarbamoyl chloride (247.3 mg, 2.0 mmol, 2.0 eq) was added in a single dose at room temperature, and the reaction was allowed to proceed for 6 h at room temperature. The reaction was then post-processed and purified as described in Example 1, yielding 55.6 mg of the product, with a yield of 15.1%. Gas chromatography analysis showed a purity of 98.09%.

[0047] Example 8 3-Hydroxythalidomide (274 mg, 1.0 mmol, 1.0 eq), DIPEA (2.59 g, 20.0 mmol, 20.0 eq), and anhydrous DCM (10 mL) were added to a dry 50 mL round-bottom flask. The mixture was stirred for 10 minutes at room temperature under argon protection, and then 4-dimethylaminopyridine (366.3 mg, 3.0 mmol, 3.0 eq) was added. Subsequently, dimethylaminothiocarbamoyl chloride (247.0 mg, 2.0 mmol, 2.0 eq) was added in a single dose at room temperature, and the reaction was allowed to proceed for 6 h at room temperature. The reaction was then post-processed and purified as described in Example 1, yielding 277.3 mg of the product, with a yield of 75.8%. Gas chromatography analysis showed a purity of 98.73%.

[0048] Example 9 3-Hydroxythalidomide (274 mg, 1.0 mmol, 1.0 eq) and anhydrous dichloromethane (DCM) (10 mL) were added to a dry 50 mL round-bottom flask. K₂CO₃ (276 mg, 2.0 mmol, 2.0 eq) was added under argon protection at room temperature. Subsequently, dimethylaminothiocarbamoyl chloride (247.2 mg, 2.0 mmol, 2.0 eq) was added in a single dose at room temperature, and the reaction was carried out for 6 h at room temperature. The reaction was followed by post-treatment and purification as described in Example 1, yielding 67.3 mg of product, with a yield of 18.1%. Gas chromatography analysis showed a purity of 97.17%.

[0049] Example 10 3-Hydroxythalidomide (274 mg, 1.0 mmol, 1.0 eq) and anhydrous dichloromethane (DCM) (10 mL) were added to a dry 50 mL round-bottom flask. Na₂CO₃ (212.2 mg, 2.0 mmol, 2.0 eq) was added under argon protection at room temperature. Subsequently, dimethylaminothiocarbamoyl chloride (247.1 mg, 2.0 mmol, 2.0 eq) was added in a single dose at room temperature, and the reaction was carried out for 6 h at room temperature. The reaction was then post-processed and purified as described in Example 1, yielding 56.5 mg of product, with a yield of 15.2%. Gas chromatography analysis showed a purity of 97.15%.

[0050] Example 11 3-Hydroxythalidomide (274 mg, 1.0 mmol, 1.0 eq) and anhydrous dichloromethane (DCM) (10 mL) were added to a dry 50 mL round-bottom flask. K₂CO₃ (138.4 mg, 1.0 mmol, 1.0 eq) was added under argon protection at room temperature. Subsequently, dimethylaminothiocarbamate chloride (247.2 mg, 2.0 mmol, 2.0 eq) was added in a single dose at room temperature, and the reaction was carried out for 6 h at room temperature. The reaction was then post-processed and purified as described in Example 1, yielding 49.8 mg of product, with a yield of 13.4%. Gas chromatography analysis showed a purity of 97.29%.

[0051] Example 12 3-Hydroxythalidomide (274 mg, 1.0 mmol, 1.0 eq) and anhydrous dichloromethane (DCM) (10 mL) were added to a dry 50 mL round-bottom flask. K₂CO₃ (1.385 g, 10.0 mmol, 10.0 eq) was added under argon protection at room temperature. Subsequently, dimethylaminothiocarbamoyl chloride (247.1 mg, 2.0 mmol, 2.0 eq) was added in a single dose at room temperature, and the reaction was carried out for 6 h at room temperature. The reaction was followed by post-treatment and purification as described in Example 1, yielding 68.2 mg of product, with a yield of 18.4%. Gas chromatography analysis showed a purity of 97.27%.

[0052] Application examples of fluorescent probes The fluorescent probe prepared by this invention can be used to detect hypochlorite in water. The specific steps used in the detection are as follows: The fluorescent probe molecule was dissolved in the detection solvent to prepare a probe solution, with a concentration of 10 in the probe solution. -5 M. Add the sample solution to be tested (volume not exceeding 1% of the probe solution volume) dropwise into the probe solution, sonicate or shake for 10 seconds, and then perform fluorescence measurement. Determine the concentration of hypochlorite based on the change in fluorescence intensity.

[0053] Among the above-mentioned detection solvents, one or more of the following can be selected: ethanol, tetrahydrofuran, dimethyl sulfoxide, N,N-dimethylformamide, PBS buffer, and Tris buffer. Considering the final processing of the test samples, a mixture of PBS buffer solution and anhydrous ethanol solution is preferred, with a volume ratio of 8:2 and a pH of 7.4. If intracellular testing is required, cell compatibility must be considered, and a mixture of PBS buffer solution and dimethyl sulfoxide solution is preferred, with the volume ratio remaining unchanged.

[0054] Performance verification of fluorescent probes 1. Verification of specific recognition by fluorescent probes The fluorescent probe molecules prepared in Example 1 were dissolved in a mixture of PBS buffer and anhydrous ethanol (v / v = 8 / 2, pH = 7.4) to a concentration of 10. -5 M, to obtain the probe solution.

[0055] Dissolve 1 mmol of the analyte (related ionic compound or free radical species) in 50 mL of ultrapure water, and bring the volume to 100 mL at room temperature to prepare CH3COO solutions. - CO3 2− H2PO4 - SO4 2- NO3 - CN - , S 2- SO3 2- S2O8 2- , F − , Cl − , Br − , I − Li + Na + , K + Ag + Cs 2+ Mg 2+ Ca 2+ Ba 2+ Pb 2+ Al 3 + Fe 3+ Eu 3+ Fe 2+ Co 2+ Ni 2+ Cu 2+ Zn 2+ , Mn 2+ O2- •, HO•, 1 O2 and H2O2 solutions were used as the analyte solutions. 1 mmol / L ClO2 solution was added. - Dissolve in 50 ml of ultrapure water and bring to a final volume of 100 mL at room temperature to prepare ClO. - Solution.

[0056] O2 - •, HO•, 1 The method for preparing O2 solution is described in the following references: H. Zhao, S. Kalivendi, H. Zhang, J. Joseph, K. Nithipatikom, J. Vásquez-Vivar, B. Kalyanaraman, FreeRadical Biol. Med., 34 (2003) 1359-1368; K.-i. Setsukinai, Y. Urano, K. Kakinuma, HJ Majima, T. Nagano, J. Biol. Chem., 278 (2003) 3170-3175; AM Held, DJ Halko, JK Hurst, J. Am. Chem. Soc., 100 (1978) 5732-5740.

[0057] Mix the above analyte solutions (volume equal to 0.5% of the probe solution volume) or the analyte solutions with ClO - The mixture of solutions (0.5% of the total volume of the probe solution) was added dropwise to the probe solution. After sonication or shaking for 10 seconds, the fluorescence intensity at 510 nm was measured. Ultrapure water was used as a blank control. The fluorescence response results are as follows: Figure 3 As shown in the figure. It can be seen from the figure that when only ions or free radicals are added without the addition of ClO... - At that time, the system did not show obvious fluorescence change, but ClO was added to this system. - The system then exhibited a strong fluorescence response, indicating that the fluorescent probe of the present invention can only specifically recognize hypochlorite ions, demonstrating that the probe has strong anti-interference ability.

[0058] 2. Validation of the detection limit of the fluorescent probe 2.1. The concentration is 10... -5 The probe solution of M was diluted to 10 with the same solvent. -7 M.

[0059] 2.2. Following the same procedure as the fluorescent probe specificity recognition verification experiment, take 3 mL of the above probe solution and place it in a cuvette, then add different concentrations of ClO₂. - The fluorescence intensity at 510 nm was measured after the solution was sonicated or shaken for 10 seconds, and the test was repeated three times.

[0060] 2.3, based on each ClO - The results of three tests at different concentrations were processed using Origin software to obtain error bars, which were then sequentially linearly fitted to obtain a linear equation, as shown below. Figure 4 As shown, the slope of the standard equation is 11.25992.

[0061] 2.4 According to the formula for calculating the limit of detection (LOD) = 3σ / k, where σ is the standard deviation of the fluorescence intensity detection values ​​of 20 blank samples at 510 nm, σ is 2.078459, and K is the slope of the linear curve, which is 11.25992, the LOD is calculated to be 55 nM.

[0062] 3. Practical applications of fluorescent probes Four types of wastewater containing hypochlorite (wastewater 1-4) were collected from four disinfectant manufacturing companies. Probe solutions were prepared according to the method described in the fluorescent probe specificity identification experiment. The four types of wastewater were added dropwise to the probe solutions, with a volume not exceeding 0.5% of the probe solution volume. After sonication or shaking for 10 seconds, the fluorescence intensity change at 510 nm was measured. The fluorescence intensity was then calculated using the standard equation (y = 27.48484 + 11.25992x, where y is the fluorescence intensity and x is the hypochlorite concentration (10...). -7 The concentration of hypochlorite in the wastewater was calculated using the method described in this invention (M). Simultaneously, the wastewater samples from these four companies were sent to a third-party testing institution for hypochlorite detection. Furthermore, the fluorescence intensity was measured again using the method of this invention after 24 hours. The results are shown in Table 1 below.

[0063] The comparison of the detection results in the table above shows that the fluorescent probe of this invention can accurately detect the content of hypochlorite, serving both qualitative and quantitative purposes. Furthermore, the probe exhibits rapid response, reaching its peak fluorescence response value within 30 seconds, and showing almost no change in fluorescence intensity within one hour (e.g., ...). Figure 5 As shown in Table 1), even after 24 hours, it still exhibits extremely high stability.

[0064] 4. Application of fluorescent probes in biological cells To further evaluate the potential biological applications of this probe, HeLa cells were incubated with the probe solution and stimulated with exogenous hypochlorite. The HeLa cells were then observed using a laser confocal microscope (CLSM, Leica, model TCS SP8) to assess their potential for biological application. The specific steps were as follows: HeLa cells were cultured on sterile coverslips. The coverslips were removed from the culture medium, excess culture medium was aspirated, and the coverslips were placed in a humid environment. 100 μL (10 μM) of probe solution was added to one corner of the coverslip, and the mixture was gently agitated to evenly cover all cells. The cells were incubated at 37°C for 20 min. The probe solution was aspirated, and the coverslips were washed 2–3 times with culture medium, each time covering all cells with pre-warmed culture medium for 5–10 min. The culture medium was then aspirated. Subsequently, HeLa cells incubated with the probe were stimulated with 100 μL (10 μM) sodium hypochlorite solution. After 5 min, the sodium hypochlorite solution was aspirated, and the cells were washed three times with culture medium before observation under a confocal microscope. The experimental results showed that exogenous hypochlorite stimulation resulted in the formation of strong green fluorescence (e.g., ...) within the cells. Figure 6 This indicates that the probe can be applied to biological cells.

[0065] In summary, the fluorescent probe for hypochlorite detection based on molecular glue in this invention has high selectivity, sensitivity and stability, enabling accurate detection of hypochlorite in complex aquatic environments and has potential cellular applications.

Claims

1. A fluorescent probe for the detection of hypochlorite, characterized in that: It has The structural formula shown in Equation I: 。 2. A method for preparing the fluorescent probe for hypochlorite detection as described in claim 1, characterized in that: A fluorescent probe of formula I is formed by reacting 3-hydroxythalidomide and dimethylaminothiocarbamoyl chloride in the presence of a base and a solvent.

3. The preparation method according to claim 2, characterized in that: The base is at least one of an organic base, an inorganic base, or a combination thereof.

4. The preparation method according to claim 2 or 3, characterized in that: The molar ratio of 3-hydroxythalidomide to base is 1:1 to 23.

5. The preparation method according to claim 3, characterized in that: The base is at least one of organic bases.

6. The preparation method according to claim 5, characterized in that: The base is a composition of N,N-diisopropylethylamine and 4-dimethylaminopyridine.

7. The preparation method according to claim 6, characterized in that: The amount of N,N-diisopropylethylamine is 1 to 20 times the molar amount of 3-hydroxythalidomide, and the amount of 4-dimethylaminopyridine is 1 to 3 times the molar amount of 3-hydroxythalidomide.

8. The preparation method according to claim 2, characterized in that: At least one of the following conditions shall be met: Condition 1: The solvent is an organic solvent; Condition 2: The reaction is carried out under anhydrous conditions; Condition 3: The reaction is carried out under the protection of an inert gas; Condition 4: The reaction is carried out at room temperature for 2-8 hours.

9. The preparation method according to claim 2, characterized in that: The molar ratio of 3-hydroxythalidomide to dimethylaminothiocarbamoyl chloride is 1:1~2.

5.

10. The use of the fluorescent probe for hypochlorite detection as described in claim 1 for the detection of hypochlorite in a liquid environment for non-diagnostic or therapeutic purposes.

11. The application according to claim 10, characterized in that: The liquid environment is an aquatic environment.

12. A method for detecting hypochlorite in an aquatic environment for non-diagnostic or therapeutic purposes, characterized in that: The fluorescent probe for hypochlorite detection described in claim 1 is used to detect hypochlorite in the aquatic environment.

13. The method according to claim 12, characterized in that: The fluorescent probe is dissolved in the detection solvent to prepare a probe solution. Then, the water sample to be tested is dropped into the probe solution, mixed well, and then fluorescence detection is performed.

14. The method according to claim 13, characterized in that: The detection solvent is one or more of the following: ethanol, tetrahydrofuran, dimethyl sulfoxide, N,N-dimethylformamide, PBS buffer, and Tris buffer.

15. The method according to claim 14, characterized in that: The detection solvent is a mixture of PBS buffer and ethanol or a mixture of PBS buffer and dimethyl sulfoxide.

16. The method according to claim 13, characterized in that: The concentration of the fluorescent probe in the probe solution is 0.5 × 10⁻⁶. -5 -1.5×10 -5 mol / L.

17. The method according to claim 13, characterized in that: The volume of the water sample to be tested should not exceed 1% of the volume of the probe solution.

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