阅读说明：本技术 一种次氯酸根荧光探针及其制备方法和应用、次氯酸根的检测方法 (Hypochlorite fluorescent probe, preparation method and application thereof, and hypochlorite detection method ) 是由 杜宇婷 王宏亮 于 2021-07-30 设计创作，主要内容包括：本发明提供了一种次氯酸根荧光探针及其制备方法和应用、次氯酸根的检测方法,涉及生物化学材料技术领域。本发明提供的次氯酸根荧光探针对次氯酸根离子的灵敏度高、选择性好、响应速度快,检测结果准确。与现有的次氯酸荧光探针相比,本发明提供的次氯酸根荧光探针制备方法简单,检测试剂廉价,检测过程简便、快速,适宜推广应用。(The invention provides a hypochlorite fluorescent probe, a preparation method and application thereof, and a hypochlorite detection method, and relates to the technical field of biochemical materials. The hypochlorite fluorescent probe provided by the invention has the advantages of high sensitivity, good selectivity, high response speed and accurate detection result on hypochlorite ions. Compared with the existing hypochlorous acid fluorescent probe, the hypochlorite fluorescent probe provided by the invention is simple in preparation method, low in detection reagent cost, simple and rapid in detection process, and suitable for popularization and application.)

1. A hypochlorite fluorescent probe is characterized by having a structure shown in a formula I:

2. the method for preparing the hypochlorite fluorescent probe of claim 1, comprising the following steps:

mixing 4-pyridine acetonitrile and 2-bromoethanol, and carrying out nucleophilic substitution reaction to obtain a compound with a structure shown in a formula II;

mixing the compound with the structure shown in the formula II with 9-anthracenealdehyde and an alkali catalyst, and carrying out a KenaonWenger condensation reaction to obtain a hypochlorite fluorescent probe with the structure shown in the formula I;

3. the preparation method according to claim 2, wherein the molar ratio of the 4-pyridineacetonitrile to the 2-bromoethanol is 1:1 to 3.

4. The preparation method according to claim 2 or 3, wherein the temperature of the nucleophilic substitution reaction is 70-75 ℃; the time of the nucleophilic substitution reaction is 6-8 h.

5. The method according to claim 2, wherein the molar ratio of the compound having the structure represented by formula II to 9-anthracenealdehyde is 1:1 to 2.

6. The process according to claim 2 or 5, wherein the temperature of the knoevenagel condensation reaction is below 70 ℃; the time of the KenaonWenger condensation reaction is 5-6 h.

7. Use of the hypochlorite fluorescent probe according to claim 1 or the hypochlorite fluorescent probe prepared by the preparation method according to any one of claims 2 to 6 in detection of hypochlorite.

8. The hypochlorite detection method is characterized by comprising the following steps:

mixing the hypochlorite fluorescent probe as defined in claim 1 or the hypochlorite fluorescent probe prepared by the preparation method as defined in any one of claims 2 to 6 with a sample to be tested, and performing fluorescence detection.

9. The detection method according to claim 8, wherein the method for mixing the hypochlorite fluorescent probe with the sample to be detected comprises: dissolving the hypochlorite fluorescent probe in dimethyl sulfoxide to obtain a fluorescent probe stock solution; mixing the fluorescent probe stock solution with an acetonitrile/PBS buffer solution system to obtain a fluorescent probe detection solution; and adding a sample to be detected into the fluorescent probe detection solution.

10. The detection method according to claim 8 or 9, wherein the fluorescence detection comprises qualitative fluorescence detection and quantitative fluorescence detection.

Technical Field

The invention relates to the technical field of biochemical materials, in particular to a hypochlorite fluorescent probe, a preparation method and application thereof, and a hypochlorite detection method.

Background

Biologically, Reactive Oxygen Species (ROS) are involved in diseases of organisms. Hypochlorite (ClO) as an active oxygen species^-) Plays a very important role in the organism. Hypochlorite is chloride ion (Cl) catalyzed by Myeloperoxidase (MPO)^-) And hydrogen peroxide (H)₂O₂) And the generated active oxygen. Studies have shown that hypochlorite in normal levels in the body can sustain basic vital activities while acting as a defense against disease in the immune system. However, hypochlorite with abnormal concentration (1000 mg/L or more) causes bodyThe internal biomolecules oxidize, which in turn leads to various physiological diseases including arthritis, atherosclerosis, cirrhosis and cancer. Therefore, it is of great interest to develop a reliable and efficient method for hypochlorite detection and imaging.

Disclosure of Invention

The invention aims to provide a hypochlorite fluorescent probe, and a preparation method and application thereof.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides a hypochlorite fluorescent probe, which has a structure shown in a formula I:

the invention provides a preparation method of the hypochlorite fluorescent probe in the technical scheme, which comprises the following steps:

mixing 4-pyridine acetonitrile and 2-bromoethanol, and carrying out nucleophilic substitution reaction to obtain a compound with a structure shown in a formula II;

mixing the compound with the structure shown in the formula II with 9-anthracenealdehyde and an alkali catalyst, and carrying out a KenaonWenger condensation reaction to obtain a hypochlorite fluorescent probe with the structure shown in the formula I;

preferably, the molar ratio of the 4-pyridine acetonitrile to the 2-bromoethanol is 1: 1-3.

Preferably, the temperature of the nucleophilic substitution reaction is 70-75 ℃; the time of the nucleophilic substitution reaction is 6-8 h.

Preferably, the molar ratio of the compound with the structure shown in the formula II to the 9-anthracene formaldehyde is 1: 1-2.

Preferably, the temperature of the knoevenagel condensation reaction is below 70 ℃; the time of the KenaonWenger condensation reaction is 5-6 h.

The invention provides an application of the hypochlorite fluorescent probe in the technical scheme or the hypochlorite fluorescent probe prepared by the preparation method in the technical scheme in detection of hypochlorite.

The invention provides a hypochlorite detection method, which comprises the following steps:

and mixing the hypochlorite fluorescent probe in the technical scheme or the hypochlorite fluorescent probe prepared by the preparation method in the technical scheme with a sample to be detected, and performing fluorescence detection.

Preferably, the method for mixing the hypochlorite fluorescent probe and the sample to be detected comprises the following steps: dissolving the hypochlorite fluorescent probe in dimethyl sulfoxide to obtain a fluorescent probe stock solution; mixing the fluorescent probe stock solution with an acetonitrile/PBS buffer solution system to obtain a fluorescent probe detection solution; and adding a sample to be detected into the fluorescent probe detection solution.

Preferably, the fluorescence detection comprises qualitative fluorescence detection and quantitative fluorescence detection.

The invention provides a hypochlorite fluorescent probe, which has high sensitivity to hypochlorite ions, and the detection limit is 0.19 mu m; the selectivity is good, the specific response to hypochlorite ions is realized, and the response to other ions and biological mercaptan is not realized; the response speed is high, and the time is only 2 min.

The hypochlorite fluorescent probe adopted by the invention has the advantages of simple preparation method, cheap detection reagent, simple and rapid detection process and suitability for popularization and application.

Drawings

FIG. 1 is a flow chart of the process for preparing a hypochlorite fluorescent probe according to the present invention;

FIG. 2 is a graph showing the UV change of hypochlorite by a hypochlorite fluorescent probe;

FIG. 3 is a graph showing the fluorescence change of hypochlorite by adding a hypochlorite fluorescent probe;

FIG. 4 is a graph showing the change in the fluorescence of a hypochlorite fluorescent probe added to a hypochlorite solution;

FIG. 5 is a graph showing the fluorescence change of a hypochlorite fluorescent probe at different pH values;

FIG. 6 is a graph comparing the selectivity of hypochlorite fluorescence probes for different ions and biological thiols;

FIG. 7 is a graph of the response time of hypochlorite fluorescence probe to hypochlorite.

Detailed Description

The invention provides a hypochlorite fluorescent probe, which has a structure shown in a formula I:

the invention also provides a preparation method of the hypochlorite fluorescent probe in the technical scheme, which comprises the following steps:

mixing 4-pyridine acetonitrile and 2-bromoethanol, and carrying out nucleophilic substitution reaction to obtain a compound with a structure shown in a formula II;

mixing the compound with the structure shown in the formula II with 9-anthracenealdehyde and an alkali catalyst, and carrying out a KenaonWenger condensation reaction to obtain a hypochlorite fluorescent probe with the structure shown in the formula I;

the method mixes 4-pyridine acetonitrile and 2-bromoethanol to carry out nucleophilic substitution reaction, and obtains the compound with the structure shown in formula II. In the invention, the molar ratio of the 4-pyridine acetonitrile to the 2-bromoethanol is preferably 1: 1-3.

In the invention, the nucleophilic substitution reaction is preferably carried out under a reflux condition, and the temperature of the nucleophilic substitution reaction is preferably 70-75 ℃; the time of the nucleophilic substitution reaction is preferably 6-8 h, and more preferably 6 h. The invention preferably adopts TLC to track the reaction until the reaction is finished; the developing solvent adopted by the TLC tracking is preferably a mixed solvent of ethyl acetate and petroleum ether; the volume ratio of the mixed solvent of ethyl acetate and petroleum ether is preferably 1: 1.

After the nucleophilic substitution reaction, the substitution reaction system is preferably dissolved in absolute ethyl alcohol for ultrasonic treatment, and then the compound with the structure shown in formula II is obtained by sequentially carrying out solid-liquid separation, washing and drying. The invention uses ultrasonic treatment to shatter solid in the substitution reaction system. In the present invention, the method of solid-liquid separation is preferably filtration. In the present invention, the method of washing preferably comprises: and washing the solid obtained by solid-liquid separation with absolute ethyl alcohol for 3-4 times.

In the invention, the compound with the structure shown in the formula II is bromide hydrochloride of 4-pyridine acetonitrile ethanol, and the color is pink. In a specific embodiment of the present invention, the yield of the compound having the structure represented by formula II is 90.8%.

After the compound with the structure shown in the formula II is obtained, the compound with the structure shown in the formula II, 9-anthracene formaldehyde and an alkali catalyst are mixed for performing a Kenaonvell condensation reaction to obtain the hypochlorite fluorescent probe with the structure shown in the formula I. In the invention, the molar ratio of the compound with the structure shown in the formula II to 9-anthracene formaldehyde is preferably 1: 1-2. In the present invention, the compound having the structure represented by formula II is preferably dissolved in anhydrous ethanol and then mixed with 9-anthracenealdehyde and a base catalyst. In the invention, the mass of the anhydrous ethanol is preferably 5-10 times of the total mass of the compound with the structure shown in the formula II and the 9-anthracene formaldehyde.

In the present invention, the base catalyst preferably includes piperidine, sodium hydroxide, triethylamine, pyridine, sodium ethoxide, or sodium tert-butoxide, more preferably piperidine. In the invention, the addition amount of the alkali catalyst is preferably 2-3 drops.

In the present invention, the temperature of the knoevenagel condensation reaction is preferably 70 ℃ or lower, more preferably 65 ℃; the time of the Kenaonwier condensation reaction is preferably 5-6 h, and more preferably 5 h. The invention preferably adopts TLC to track the reaction until the reaction is finished; the developing solvent adopted by the TLC tracking is preferably a mixed solvent of ethyl acetate and petroleum ether; the volume ratio of the mixed solvent of ethyl acetate and petroleum ether is preferably 1: 1.

According to the invention, preferably, after the KenaonWeckel condensation reaction, the solvent in the obtained condensation reaction system is removed, then the solid product is dissolved in absolute ethyl alcohol, and the hypochlorite fluorescent probe with the structure shown in the formula I is obtained through column chromatography separation. In the present invention, the solvent in the resulting condensation reaction system is preferably removed by evaporation with a rotary evaporator. In a specific embodiment of the present invention, the hypochlorite fluorescent probe having the structure shown in formula I is a deep red solid with a yield of 70.7%.

The invention also provides the application of the hypochlorite fluorescent probe in the technical scheme or the hypochlorite fluorescent probe prepared by the preparation method in the technical scheme in detecting hypochlorite.

The invention also provides a hypochlorite detection method, which comprises the following steps:

and mixing the hypochlorite fluorescent probe in the technical scheme or the hypochlorite fluorescent probe prepared by the preparation method in the technical scheme with a sample to be detected, and performing fluorescence detection.

In the present invention, the method for mixing the hypochlorite fluorescent probe and the sample to be tested preferably comprises: dissolving the hypochlorite fluorescent probe in dimethyl sulfoxide to obtain a fluorescent probe stock solution; mixing the fluorescent probe stock solution with an acetonitrile/PBS buffer solution system to obtain a fluorescent probe detection solution; and adding a sample to be detected into the fluorescent probe detection solution.

In the present invention, the purity of the dimethyl sulfoxide is preferably spectral purity. In the invention, the concentration of the fluorescent probe stock solution is preferably 10-15 mmol/L, and more preferably 10 mmol/L.

In the invention, the pH value of the acetonitrile/PBS buffer solution system is preferably 7-7.4, and more preferably 7.4. In the present invention, the preparation method of the acetonitrile/PBS buffer solution system preferably comprises: and mixing the acetonitrile and the PBS buffer solution to obtain an acetonitrile/PBS buffer solution system. In the present invention, the solute of the PBS buffer solution preferably comprises NaCl, KCl, Na₂HPO₄·12H₂O and KH₂PO₄(ii) a The NaCl, KCl and Na₂HPO₄·12H₂O and KH₂PO₄Is preferably 16.0129: 0.4026: 5.8018: 0.4082; the solvent of the PBS buffer solution is preferably water. In the embodiment of the invention, the pH value of the PBS buffer solution is preferably adjusted to 7-7.4 by adopting a sodium hydroxide solution or a hydrochloric acid solution.

In the present invention, the volume ratio of acetonitrile to PBS buffer solution is preferably 1: 1.

In the present invention, when ultraviolet detection is employed, the concentration of the fluorescent probe in the fluorescent probe-detecting solution is preferably 50 μmol/L (high concentration fluorescent probe-detecting solution); when fluorescence detection is adopted, the concentration of the fluorescent probe in the fluorescent probe detection solution is preferably 5-10 mu mol/L, and more preferably 5 mu mol/L (low-concentration fluorescent probe detection solution).

In the invention, the concentration of hypochlorite in the sample to be detected is preferably 50-750 mu mol/L, and more preferably 100-300 mu mol/L.

In the present invention, the fluorescence detection preferably includes fluorescence qualitative detection and fluorescence quantitative detection.

In the present invention, the method for qualitative detection of fluorescence preferably comprises:

adding a solution containing hypochlorite into the high-concentration fluorescent probe detection solution, carrying out ultraviolet detection, and determining the maximum excitation wavelength of the fluorescent probe; the concentration of the fluorescent probe in the high-concentration fluorescent probe detection solution is preferably 50 mu mol/L;

performing fluorescence scanning on the low-concentration fluorescent probe detection solution to obtain a fluorescence spectrum of the fluorescent probe and the maximum emission wavelength of the fluorescent probe; the concentration of the fluorescent probe in the low-concentration fluorescent probe detection solution is preferably 5-10 mu mol/L, and more preferably 5 mu mol/L;

adding a sample to be detected into the low-concentration fluorescent probe detection solution, and performing fluorescence scanning again under the maximum excitation wavelength to obtain a fluorescence spectrum of the sample;

comparing the fluorescence spectrum of the sample with the fluorescence spectrum of the fluorescent probe, and if the fluorescence intensity at the maximum emission wavelength is weakened, indicating that the sample to be detected contains hypochlorite; if the fluorescence intensity at the maximum emission wavelength is not changed, it indicates that the sample contains no hypochlorite.

In the present invention, the concentration of the sodium hypochlorite solution is preferably 100 mmol/L.

In the present invention, the parameters of the fluorescence scan preferably include: the excitation slit is 5.0nm and the emission slit is 5.0 nm.

In a specific embodiment of the invention, the maximum excitation wavelength of the fluorescent probe is 460 nm; the maximum emission wavelength of the fluorescent probe is 608 nm.

In the present invention, the method for fluorescence quantitative detection preferably includes:

adding a solution containing hypochlorite into the high-concentration fluorescent probe detection solution, carrying out ultraviolet detection, and determining the maximum excitation wavelength of the fluorescent probe;

performing fluorescence scanning on the low-concentration fluorescent probe detection solution to obtain a fluorescence spectrum of the fluorescent probe and the maximum emission wavelength of the fluorescent probe;

adding hypochlorite with different amounts into the low-concentration fluorescent probe detection solution to obtain a standard solution with hypochlorite concentration in gradient distribution;

performing fluorescence scanning on the standard solution at the maximum excitation wavelength to obtain a standard curve of hypochlorite concentration and fluorescence intensity at the maximum emission wavelength;

adding a sample to be detected into the low-concentration fluorescent probe detection solution, and performing fluorescence scanning again at the maximum excitation wavelength to obtain the fluorescence intensity of the sample to be detected at the maximum emission wavelength;

and calculating the concentration of hypochlorite in the sample to be detected by using the fluorescence intensity of the sample to be detected according to the standard curve.

In the present invention, the parameters of the fluorescence scan are preferably the same as those in the fluorescence qualitative detection described above, and are not described herein again.

In a specific embodiment of the invention, the hypochlorite concentration and the maximum emission waveThe standard curve of fluorescence intensity at the long point is: f ═ 13.3C +5398, R²0.9945; wherein C represents hypochlorite concentration in 10^-6mol/L, F represents fluorescence intensity.

The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1

Hypochlorite fluorescent probe was prepared using the method shown in figure 1:

mixing 0.0025mol of 4-pyridine acetonitrile and 0.0025mol of 2-bromoethanol, reacting for 6 hours at 70 ℃, tracking the reaction by TLC (the volume ratio of ethyl acetate to petroleum ether in a developing agent is 1:1), after the reaction is finished, adding absolute ethyl alcohol to dissolve, crushing the solid by ultrasonic vibration, carrying out suction filtration, washing the filter cake by absolute ethyl alcohol for 3-4 times, and drying to obtain 0.43g of pink solid with the yield of 90.8%; the resulting pink solid has the structure shown in formula II;

placing 0.43g of the compound with the structure shown in the formula II in a single-neck round-bottom flask, dissolving in absolute ethyl alcohol, adding 0.53g of 9-anthracenealdehyde and 2-3 drops of piperidine, heating, controlling the temperature to be 65 ℃, reacting for 5 hours, tracking and reacting by TLC (volume ratio of ethyl acetate to petroleum ether in developing solvent is 1:1), after the reaction is finished, performing spin drying, adding absolute ethyl alcohol to dissolve, and performing column chromatography to obtain a hypochlorite fluorescent probe which is a deep red solid with a yield of 70.7%.

The nuclear magnetic resonance hydrogen spectrum result of the hypochlorite fluorescent probe prepared in the embodiment is as follows:

¹HNMR(600MHz，DMSO-d6)δ8.51(s，2H)，8.08(s，2H)，7.49(s，5H)，7.18–6.86(m，4H)，6.32(d，J＝7.7Hz，2H)，5.56(s，2H)，5.30(s，1H)，4.92(s，2H).

the hypochlorite fluorescent probe prepared in the embodiment has a structure shown in formula I:

application example 1

Hypochlorite fluorescence probes the ultraviolet change of the addition of hypochlorite.

Dissolving the hypochlorite fluorescent probe prepared in example 1 in dimethyl sulfoxide to obtain a fluorescent probe stock solution with the concentration of 10 mmol/L;

mixing NaCl, KCl and Na₂HPO₄·12H₂O and KH₂PO₄According to 16.0129: 0.4026: 5.8018: dissolving the solution in water according to the mass ratio of 0.4082, and adjusting the pH value to 7.4 by using dilute hydrochloric acid to obtain a PBS buffer solution;

mixing acetonitrile and the PBS buffer solution according to the volume ratio of 1:1 to obtain an acetonitrile/PBS buffer solution system;

adding 15 mu L of the fluorescent probe stock solution into 3mL of the acetonitrile/PBS buffer solution system to obtain a high-concentration fluorescent probe detection solution; the concentration of the fluorescent probe in the high-concentration fluorescent probe detection liquid is 50 mu mol/L;

carrying out ultraviolet detection on the high-concentration fluorescent probe detection solution to obtain an ultraviolet absorption spectrum curve of the fluorescent probe, as shown in FIG. 2;

and adding 9 mu L of sodium hypochlorite solution with the concentration of 100mmol/L into the high-concentration fluorescent probe detection solution to obtain a solution with the concentration of hypochlorite of 300 mu mol/L, and performing ultraviolet detection again under the same experimental conditions to obtain an ultraviolet absorption spectrum curve of the sample, wherein the ultraviolet absorption spectrum curve is shown in figure 2.

As can be seen from FIG. 2, the UV absorption spectrum of the solution changed with the addition of the sodium hypochlorite solution, indicating that the hypochlorite fluorescent probe indeed reacted with hypochlorite. Also, as can be seen from FIG. 2, the maximum excitation wavelength of the hypochlorite fluorescent probe is 460 nm.

Application example 2

Hypochlorite fluorescence probe changes in fluorescence upon addition of hypochlorite.

Preparing a fluorescent probe stock solution with the concentration of 10mmol/L and an acetonitrile/PBS buffer solution system according to the method of application example 1;

adding 1.5 mu L of the fluorescent probe stock solution into 3mL of the acetonitrile/PBS buffer solution system to obtain a low-concentration fluorescent probe detection solution; the concentration of the fluorescent probe in the low-concentration fluorescent probe detection liquid is 5 mu mol/L;

performing fluorescence scanning on the low-concentration fluorescent probe detection solution to obtain a fluorescence spectrum of the fluorescent probe and the maximum emission wavelength of the hypochlorite fluorescent probe; the fluorescence spectrum of the obtained fluorescent probe is shown in FIG. 3, and the maximum emission wavelength is 608 nm; the parameters of the fluorescence scan are: the excitation slit is 5.0nm, the emission slit is 5.0nm, and the maximum excitation wavelength is 460 nm;

sodium hypochlorite solutions with different volumes and concentrations of 100mmol/L were added to the low concentration fluorescent probe detection solution, and the concentrations of hypochlorite in the obtained solutions were 50. mu. mol/L, 100. mu. mol/L, 150. mu. mol/L, 200. mu. mol/L, 250. mu. mol/L, 350. mu. mol/L, 550. mu. mol/L, and 750. mu. mol/L, and fluorescence scanning was performed again at the maximum excitation wavelength to obtain the fluorescence spectra of each sample, as shown in FIG. 3.

As can be seen from FIG. 3, the fluorescence at 608nm gradually blue-shifts with the addition of the sodium hypochlorite solution and the fluorescence intensity gradually decreases, indicating that the response of the hypochlorite fluorescent probe of the present invention to hypochlorite is quenching.

Application example 3

Hypochlorite fluorescence probe add to the titration fluorescence change of hypochlorite.

Preparing a low-concentration fluorescent probe detection solution according to the method of application example 2;

performing fluorescence scanning on the low-concentration fluorescent probe detection solution to obtain a fluorescence spectrum of the fluorescent probe and the maximum emission wavelength of the fluorescent probe, wherein the maximum emission wavelength is 608 nm; the parameters of the fluorescence scan are: the excitation slit is 5.0nm, the emission slit is 5.0nm, and the maximum excitation wavelength is 460 nm;

adding hypochlorite with different amounts into the low-concentration fluorescent probe detection solution to obtain a standard solution with hypochlorite concentration in gradient distribution;

fluorescence scanning of the standard solution at the maximum excitation wavelength gave a standard curve of hypochlorite concentration versus fluorescence intensity at the maximum emission wavelength, as shown in FIG. 4, where F is-13.3C +5398, and R is²0.9945; wherein C represents hypochlorite concentration in 10^-6mol/L, F represents fluorescence intensity.

As can be seen from FIG. 4, with the addition of hypochlorite with different concentrations, the fluorescence intensity of the hypochlorite fluorescence probe has a good linear relationship with the concentration of hypochlorite, and according to the IUPAC (CDL ═ 3sd/slope) rule, the detection limit of the hypochlorite fluorescence probe to hypochlorite is calculated to be 0.19. mu. mol/L, and the sensitivity is high. The result shows that the detection limit concentration of the hypochlorite fluorescent probe can reach 10 by using a spectral analysis method^-7And (4) mol/L grade.

Application example 4

Fluorescence change of hypochlorite fluorescent probe at different pH values.

Preparing a low-concentration fluorescent probe detection solution according to the method of application example 2;

performing fluorescence scanning on the low-concentration fluorescent probe detection solution under different pH values, and testing the change of fluorescence intensity, as shown in FIG. 5; the result of adding 9. mu.L of a sodium hypochlorite solution having a concentration of 100mmol/L to the low concentration fluorescent probe detection solution to obtain a solution having a hypochlorite concentration of 300. mu. mol/L and performing fluorescence scanning again under different pH conditions is shown in FIG. 5. Wherein the ordinate of FIG. 5 is the fluorescence intensity at 608 nm. The parameters of the fluorescence scan are: the excitation slit is 5.0nm, the emission slit is 5.0nm, and the maximum excitation wavelength is 460 nm.

As can be seen from FIG. 5, the hypochlorite fluorescent probe prepared by the invention is relatively stable under different pH values; the fluorescence intensity of the hypochlorite fluorescent probe is reduced continuously under different pH values along with the addition of hypochlorite, and the hypochlorite fluorescent probe responds best at pH 7, which shows that the hypochlorite fluorescent probe prepared by the invention can respond to hypochlorite under physiological conditions.

Application example 5

Hypochlorite fluorescence probes selectivity for different ions and biological thiols.

Preparing a low-concentration fluorescent probe detection solution according to the method of application example 2;

preparing different interfering ions and biological mercaptan into interfering solution with the concentration of 100mmol/L by using distilled water;

adding different interference solutions into the low-concentration fluorescent probe detection solution, incubating the obtained mixed solution for 5min, and performing fluorescence scanning to obtain the fluorescence intensity at 608nm, as shown in FIG. 6. Wherein a in FIG. 6 is the acetonitrile/PBS buffer solution system prepared in application example 1; b is ClO^-(100. mu.M); c is Cl^-(100. mu.M); d is ClO₃ ^-(100. mu.M); e is ClO₄ ^-(100. mu.M); f is F^-(100. mu.M); g is H₂PO₄ ^-(100. mu.M); h is HPO₃ ^2-(100. mu.M); i is I^-(100. mu.M); j is NO₂ ^-(100. mu.M); k is NO₃ ^-((100. mu.M)); l is S₂O₃ ^2-(100. mu.M; M is SO)₄ ^2-(100. mu.M); n is Cys (the name of cysteine in Chinese) (10. mu.M); o is Hcy (the Chinese name is homocysteine glutathione) (100. mu.M); p is GSH (Chinese name) (100. mu.M).

The parameters of the fluorescence scan are: the excitation slit is 5.0nm, the emission slit is 5.0nm, and the maximum excitation wavelength is 460 nm.

With "b as ClO^-(100. mu.M) "as an example, indicates ClO in the mixed solution^-The concentration of (A) was 100. mu.M, and the other samples were identical in meaning and not described in detail.

As can be seen from FIG. 6, only hypochlorite ions weaken the fluorescence intensity of the fluorescent probe, while other interfering substances do not change the fluorescence intensity of the fluorescent probe. The hypochlorite fluorescent probe prepared by the invention has specific response to hypochlorite and cannot be interfered by other substances.

Application example 6

Response time of hypochlorite fluorescent probe to hypochlorite.

Preparing a low-concentration fluorescent probe detection solution according to the method of application example 2;

and adding 9 mu L of sodium hypochlorite solution with the concentration of 100mmol/L into the low-concentration fluorescent probe detection solution, wherein the concentration of hypochlorite in the obtained solution is 300 mu mol/L, performing fluorescence scanning, and continuously reducing the fluorescence intensity of the probe along with the addition of the sodium hypochlorite, and simultaneously completing the response within 2min, as shown in FIG. 7. The parameters of the fluorescence scan are: the excitation slit is 5.0nm, the emission slit is 5.0nm, and the maximum excitation wavelength is 460 nm.

As can be seen from FIG. 7, the response time of the hypochlorite fluorescent probe prepared by the present invention to hypochlorite is 2min, which shows that the hypochlorite fluorescent probe prepared by the present invention has a fast response speed to hypochlorite.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.