A naked-eye and real-time colorimetric fluorescent probe for detecting hypochlorous acid and preparation method and application thereof

Abstract

The application relates to a naked-eye and real-time colorimetric fluorescent probe for detecting hypochlorous acid and a preparation method and application thereof, and relates to a colorimetric fluorescent probe for detecting hypochlorous acid and a preparation method and application thereof. The application aims to solve the technical problems of the existing hypochlorous acid fluorescent probe, such as complex synthesis, small stokes shift, easy interference of recognition performance, poor specificity, long detection time, high detection limit and weak biological imaging capability. The structural formula of the naked-eye and real-time colorimetric fluorescent probe for detecting hypochlorous acid is as follows: the colorimetric fluorescent probe is obtained by the reaction of 4-[1-(4-methoxyphenyl)-1H-phenanthro[9,10-d]imidazol-2-yl]benzaldehyde and benzoyl hydrazine. The colorimetric fluorescent probe is used for detecting hypochlorous acid in a solution, response is completed within 5s in a pH value of 2-12, and the detection limit is as low as 57.66 nmol / L. The colorimetric fluorescent probe can be used for detecting hypochlorous acid in a water environment.

Description

TECHNICAL FIELD

[0001] The present application relates to a colorimetric fluorescent probe for detecting hypochlorous acid and a preparation method and application thereof. BACKGROUND

[0002] Reactive oxygen species (ROS) refers to the general term of substances composed of oxygen, containing oxygen and having active properties in the body or natural environment. As an important representative of reactive oxygen species (ROS), people have increasingly realized its important functions in cell homeostasis, information transmission, bactericidal effect, resistance to microbial invasion of the immune system and maintenance of cell redox balance. Endogenous hypochlorous acid is produced by peroxidation reaction assisted by myeloperoxidase (MPO) in the presence of hydrogen peroxide and chlorine, however, abnormal changes in hypochlorous acid level can lead to a series of many serious human diseases caused by excessive oxidative stress, including atherosclerosis, reperfusion injury, neurodegenerative diseases, neurodegenerative diseases, lung inflammation, and even cancer. Undoubtedly, exploring the level of hypochlorous acid in the process of drug-induced oxidative stress is urgently needed to further understand the exact biological role of oxidative stress. Therefore, there is an urgent need for an effective imaging tool to track hypochlorous acid in real time. Fluorescence imaging combined with small molecules has attracted widespread attention from scientific researchers due to its non-invasiveness, high sensitivity, good spatiotemporal resolution and other advantages. At present, typical small molecule fluorescent probes have shown great potential in monitoring microenvironment changes.

[0003] In recent years, more and more researchers have carried out research work on hypochlorous acid fluorescent probes. In 2023, Zhou Yang et al. in the article “Bimodal Probe Based on AIE and TICT Effects for Detection of Hypochlorite Anion in Living Cells and Its Bioimaging” in Organic & Biomolecular Chemistry, Vol. 21, No. 6, pp. 1270-1274, designed a fluorescent probe SWJT-12 for detecting ClO - in living cells based on the aggregation-induced emission (AIE) and twisted intramolecular charge transfer (TICT) mechanisms, with C=N bond as the reaction site. This synthetic probe can react with ClO -The reaction shows a large Stokes shift (144 nm) and low biological toxicity, but its detection limit is higher (0.28 muM). In 2022, Zhang Zhihao et al. synthesized a nitrobenzenesulfonyl hydrazine-based fluorescence-enhanced two-photon fluorescent probe for detecting hypochlorous acid in the article "A new fast-response two-photon fluorescent probe with modified groups for monitoring endogenous hypochlorous acid with a large opening signal and its application in zebrafish imaging" in Spectrochimica Acta Part A 278. The probe shows good recognition ability to hypochlorous acid, and the probe has good selectivity, high sensitivity, low cytotoxicity and good two-photon performance (about 65 muS) in living tissues. But the Stokes shift is small, about 85 nm, and it is greatly affected by the environmental pH value, so the application range is limited. In 2021, Wang Xinyu et al. designed and developed a new water-soluble optimized hypochlorous acid fluorescent probe 3-(2-cyanacrylate-ethyl)-4-hydroxy-N-n-propyl-1,8-naphthalimide containing C=C double bond and having intramolecular charge transfer (ICT) effect in "Synthesis of a hypochlorous acid fluorescent probe based on naphthalimide and its cell imaging performance research" in Analytical Chemistry, Vol. 41, Chapter 2, 719-725. The probe can complete the detection of hypochlorous acid in 10 s, the fluorescence analysis detection limit is 2.4 nmol / L, the Stokes shift is 100 nm, and the probe shows strong anti-interference, but the biological imaging ability of the probe molecule is weak.

[0004] According to the current literature reports, the hypochlorous acid fluorescent probe mainly has the following defects:

[0005] 1. The structure of the hypochlorous acid response site is relatively single;

[0006] 2. The Stokes shift of the fluorescent probe is small;

[0007] 3. It is easily disturbed by background and environmental factors, and has poor recognition specificity;

[0008] 4. The detection time is long, and the detection limit is high;

[0009] 5. The biological imaging ability is weak, and the life is short. SUMMARY

[0010] The present application aims to solve the technical problems of the existing hypochlorous acid fluorescent probe, such as complex synthesis, small Stokes shift, easily disturbed recognition performance, poor specificity, long detection time, high detection limit, and weak biological imaging ability, and provides a colorimetric fluorescent probe for naked eye and real-time detection of hypochlorous acid, and a preparation method and application thereof.

[0011] The structure formula of the colorimetric fluorescent probe for naked eye and real-time detection of hypochlorous acid according to the present application is:

[0012]

[0013] The preparation method of the naked-eye and real-time detection hypochlorous acid colorimetric fluorescent probe is carried out according to the following steps:

[0014] I. The mass ratio of 4-[1-(4-methoxyphenyl)-1H-phenanthro[9,10-d]imidazol-2-yl] benzaldehyde and benzoyl hydrazine is 1:(1-5), and 4-[1-(4-methoxyphenyl)-1H-phenanthro[9,10-d]imidazol-2-yl] benzaldehyde and benzoyl hydrazine are weighed into a reactor;

[0015] II. Organic solvent I is added to the reactor in step I, and heated to boiling reflux under stirring for 2-10 hours, and after the reaction is completed, the crude product is obtained by concentration under reduced pressure;

[0016] III. The crude product obtained in step II is recrystallized with organic solvent II, and after filtration, washing and drying, the naked-eye and real-time detection hypochlorous acid colorimetric fluorescent probe is obtained.

[0017] Further, the organic solvent I in step II is methanol, ethanol, propanol, isopropanol, butanol, benzene, chlorobenzene or xylene.

[0018] Further, the organic solvent II in step III is a mixture of ethyl acetate and petroleum ether in a volume ratio of 1:(1-5); or a mixture of ethyl acetate and dichloromethane in a volume ratio of 1:(1-5).

[0019] Further, the washing in step III is washing with methanol or ethanol.

[0020] The synthesis process of the naked-eye and real-time detection hypochlorous acid colorimetric fluorescent probe of the present application can be represented by the following formula:

[0021]

[0022] The application of the naked-eye and real-time detection hypochlorous acid colorimetric fluorescent probe is to detect hypochlorous acid in a solution.

[0023] Further, the method for naked-eye colorimetric qualitative detection of hypochlorous acid in a solution by using the naked-eye and real-time detection hypochlorous acid colorimetric fluorescent probe is carried out according to the following steps:

[0024] I. The naked-eye and real-time detection hypochlorous acid colorimetric fluorescent probe is dissolved in a water-miscible organic solvent III to prepare a probe stock solution A;

[0025] II. The probe stock solution A obtained in step I is diluted into a probe solution B using a mixed solution of methanol and PBS buffer solution;

[0026] Three, the probe solution B is irradiated by a handheld ultraviolet lamp, and the probe solution is bright green;

[0027] Four, the probe solution B obtained in step two is mixed with the solution to be tested to obtain a test solution C;

[0028] Five, after the probe solution C is placed for 30 seconds, the test solution C is irradiated by a handheld ultraviolet lamp, and if the test solution C is dark blue, it is determined that the solution to be tested contains hypochlorous acid;

[0029] Further, the concentration of the colorimetric fluorescent probe for naked-eye and real-time detection of hypochlorous acid in step one is 0.5-1 mmol·L –1 .

[0030] Further, the organic solvent III in step one is methanol, ethanol, DMF, dimethyl sulfoxide, tetrahydrofuran or acetonitrile.

[0031] Further, the volume ratio of methanol to PBS solution in step two is 7:3.

[0032] Further, the concentration of the colorimetric fluorescent probe for naked-eye and real-time detection of hypochlorous acid in the probe solution B in step two is 0.01-0.05 mmol·L –1 .

[0033] Further, the concentration of the PBS solution in step two is 0.01 mol·L –1 , pH = 7.40.

[0034] Further, the method for qualitatively detecting hypochlorous acid in a solution by fluorescence by using the colorimetric fluorescent probe for naked-eye and real-time detection of hypochlorous acid is carried out according to the following steps:

[0035] One, a colorimetric fluorescent probe for naked-eye and real-time detection of hypochlorous acid is dissolved in a water-miscible organic solvent III to prepare a probe stock solution A;

[0036] Two, the probe stock solution A obtained in step one is diluted into a probe solution B by using a mixed solution of methanol and a PBS buffer solution;

[0037] Three, the probe solution B obtained in step two is mixed with the solution to be tested to obtain a test solution C;

[0038] Four, the fluorescence emission spectrum of the probe solution B is measured with an excitation wavelength of 330 nm, and the emission intensity at an emission wavelength of 495 nm is recorded as T B ;

[0039] Five, after the test solution C is placed for 30 seconds, the fluorescence emission spectrum of the test solution C is measured, and the emission intensity at an emission wavelength of 495 nm is recorded as TC ;

[0040] VI. Comparison T B and T C , if 8T C ≤T B , it is determined that the solution to be tested contains hypochlorous acid.

[0041] Further, the concentration of the colorimetric fluorescent probe for naked-eye and real-time detection of hypochlorous acid in step one is 0.5-1 mmol·L –1 .

[0042] Further, the organic solvent III in step one is methanol, ethanol, DMF, dimethyl sulfoxide, tetrahydrofuran or acetonitrile.

[0043] Further, the volume ratio of methanol to PBS solution in step two is 7:3.

[0044] Further, the concentration of the colorimetric fluorescent probe for naked-eye and real-time detection of hypochlorous acid in probe solution B in step two is 0.01-0.05 mmol·L –1 .

[0045] Further, the concentration of the PBS solution in step two is 0.01 mol·L –1 , pH=7.40.

[0046] Further, the method for quantitatively detecting hypochlorous acid in a solution by using the colorimetric fluorescent probe for naked-eye and real-time detection of hypochlorous acid by fluorescence method is determined by standard curve method.

[0047] The colorimetric fluorescent probe solution for naked-eye and real-time detection of hypochlorous acid of the present application is bright green in color under the test system when irradiated by a handheld ultraviolet lamp. When irradiated by a handheld ultraviolet lamp in the presence of hypochlorous acid, the naked-eye color is dark blue, and the fluorescence intensity is quenched to 1 / 8 of the intensity of the probe. The change in fluorescence intensity is linearly related to the change in the concentration of hypochlorous acid, and the content of hypochlorous acid in the sample to be tested can be obtained through the corresponding fluorescence intensity. The qualitative and quantitative detection of hypochlorous acid in the sample to be tested is realized through visual effect or fluorescence spectrum.

[0048] This invention provides a colorimetric fluorescent probe for naked-eye and real-time detection of hypochlorous acid. Utilizing the oxidizing properties of hypochlorous acid, the C=N group in the fluorescent probe is oxidized. This oxidation alters the structure of the probe, resulting in both naked-eye color changes and fluorescent colorimetric changes in aqueous solution. This achieves selective recognition of hypochlorous acid. The fluorescent probe can detect hypochlorous acid in aquatic systems without interference from other similar substances in the aqueous solution, exhibiting strong anti-interference capabilities. The detection limit is as low as 57.66 nmol / L. This fluorescent probe can detect hypochlorous acid within a pH range of 2–12. The method for detecting hypochlorous acid using this colorimetric fluorescent probe is simple, with a sensitive and rapid response, completing the response within 5 seconds and maintaining stability. It has the ability to detect hypochlorous acid in aquatic environments. The synthesis method of this fluorescent probe is simple, low-cost, and involves simple steps and mild reaction conditions, making it suitable for hypochlorous acid detection in aquatic systems and possessing good bioimaging capabilities. Attached Figure Description

[0049] Figure 1 This is a colorimetric fluorescent probe for detecting hypochlorous acid in the naked eye and in real time, prepared in Example 1, and a color change diagram of hypochlorous acid in the naked eye.

[0050] Figure 2 This is the colorimetric fluorescent probe (10 μmol·L⁻¹) for naked-eye and real-time detection of hypochlorous acid prepared in Example 1. –1 V 甲醇 :V PBS =7:3, pH=7.40) for different analytes ((F - Cl - , Br - I - AcO - CO3 2- HCO3 - H2PO4 - HPO4 2- PO4 3- BrO3 - NO3 - NO2 - SO4 2- SO3 2- HSO3 - SCN - S 2- ,Cys,Hcy,GSH,Ala,Gly,H2O2,OH, 1 O2, O2 - ,t-BOOH,ONOO - and ClO - 30 μmol·L–1 The fluorescence emission spectrum of λ ex =330nm), the horizontal axis is wavelength, and the vertical axis is fluorescence intensity;

[0051] Figure 3 This is the colorimetric fluorescent probe (10 μmol·L⁻¹) for naked-eye and real-time detection of hypochlorous acid prepared in Example 1. –1 V 甲醇 :V PBS =7:3, pH=7.40) added hypochlorous acid (30 μmol·L) –1 ) and other analytes (30 μmol·L –1 Fluorescence emission intensity histogram (λ) during coexistence ex =330nm, λ em =495nm), the horizontal axis represents the analyte species (1:F - 2:Cl - 3:Br - 4:I - 5:AcO - 6:CO3 2- 7:HCO3 - 8:H2PO4 - 9:HPO4 2- 10:PO4 3- 11:BrO3 - 12:NO3 - 13:NO2 - 14:SO4 2- 15:SO3 2- 16:HSO3 - 17:SCN - 18:S 2- , 19:Cys, 20:Hcy, 21:GSH, 22:Ala, 23:Gly, 24:H2O2, 25:·OH, 26: 1 O2、27:·O2 - 28:t-BOOH, 29:ONOO - (and 30: probe), the vertical axis is fluorescence intensity, the gray bar chart is the fluorescence intensity of the fluorescent probe with other analogs added, and the black bar chart is the fluorescence intensity of the fluorescent probe with hypochlorous acid and other analogs added at the same time;

[0052] Figure 4 This is the colorimetric fluorescent probe (10 μmol·L⁻¹) for naked-eye and real-time detection of hypochlorous acid prepared in Example 1. –1 V 甲醇 :V PBS= 7:3, pH = 7.40) and the fluorescence emission intensity map (λ ex = 330 nm, λ em = 495 nm) under different pH conditions in the presence of hypochlorous acid, with the abscissa representing the pH value and the ordinate representing the fluorescence intensity;

[0053] Figure 5 is the colorimetric fluorescent probe for naked-eye and real-time detection of hypochlorous acid (10 μmol·L –1 ) prepared in Example 1, and the fluorescence emission spectrum (λ –1 = 330 nm) in the presence of different concentrations of hypochlorous acid (0-30 μmol·L ex ), with the abscissa representing the wavelength and the ordinate representing the fluorescence intensity;

[0054] Figure 6 is the colorimetric fluorescent probe for naked-eye and real-time detection of hypochlorous acid (10 μmol·L –1 ) prepared in Example 1, and the fluorescence emission spectrum (λ –1 = 330 nm, λ ex = 495 nm) over time in the presence of hypochlorous acid (0-30 μmol·L em ), with the abscissa representing time and the ordinate representing fluorescence intensity. DETAILED DESCRIPTION

[0055] The beneficial effects of the present application are verified by the following examples.

[0056] Example 1: The preparation method of the colorimetric fluorescent probe for naked-eye and real-time detection of hypochlorous acid in this example is specifically performed according to the following steps:

[0057] I. 0.428 g (1.0 mmol) of 4-[1-(4-methoxyphenyl)-1H-phenanthro[9,10-d]imidazol-2-yl]benzaldehyde and 0.136 g (1.0 mmol) of benzoyl hydrazine were weighed and added to a three-necked flask, respectively.

[0058] II. 20 mL of ethanol was added as a solvent to the three-necked flask, and the reaction was heated to boiling reflux under stirring for 4 h. After the reaction was completed, the crude product was obtained by concentration under reduced pressure.

[0059] III. The obtained crude product was recrystallized with ethyl acetate, and after suction filtration and washing with methanol, the colorimetric fluorescent probe for naked-eye and real-time detection of hypochlorous acid was obtained.

[0060] In this example, the yield of the colorimetric fluorescent probe for naked-eye and real-time detection of hypochlorous acid was 82%, and the melting point was > 300℃.

[0061] It was characterized by infrared spectroscopy, nuclear magnetic resonance spectroscopy and mass spectrometry, and the results obtained are as follows:

[0062] IR (KBr, cm -1 ): 3328, 3057, 2824, 1654, 1610, 1520, 1453, 1354, 1253, 1144, 1032, 848, 763, 530.

[0063] 1 H NMR (600 MHz, DMSO-d6) d (ppm): 11.954 (s, 1H), 8.938 (d, J = 8.4 Hz, 1H), 8.885 (d, J = 8.3 Hz, 1H), 8.710 (d, J = 7.9 Hz, 1H), 8.449 (s, 1H), 7.928 (d, J = 7.2 Hz, 2H), 7.783 (t, J = 7.4 Hz, 1H), 7.739 (d, J = 8.1 Hz, 2H), 7.687 (d, J = 8.3 Hz, 3H), 7.673 (d, J = 8.6 Hz, 2H), 7.659 (t, J = 7.2 Hz, 1H), 7.576 (s, 1H), 7.542 (t, J = 7.4 Hz, 2H), 7.400 (t, J = 7.7 Hz, 1H), 7.233 (dd, J = 18.1, 8.5 Hz, 3H), 3.921 (s, 3H).

[0064] 13 C NMR (150 MHz, DMSO-d6) d (ppm): 165.26, 160.62, 159.51, 150.49, 148.16, 137.01, 134.88, 134.36, 132.20, 131.00, 130.70, 129.77, 129.10, 129.06, 128.68, 128.20, 127.97, 127.46, 127.22, 127.17, 126.28, 125.80, 124.98, 124.16, 123.05, 122.51, 120.79, 119.43, 117.77, 116.43, 115.92, 56.09. ESI-MS m / z: [M+H] + calcd for C 36 H 26 N4O2, 547.2134; found, 547.2124.

[0065] From the above characterization results, the structure of the naked-eye and real-time detection of hypochlorous acid colorimetric fluorescent probe prepared in this embodiment is:

[0066] The colorimetric fluorescent probe for naked-eye and real-time detection of hypochlorous acid prepared in the embodiment is used to qualitatively detect hypochlorous acid in a solution by a naked-eye colorimetric method, and the following steps are performed:

[0067] I. The colorimetric fluorescent probe for naked-eye and real-time detection of hypochlorous acid is dissolved in water-miscible methanol to prepare a probe stock solution A with a concentration of 1 mmol·L –1 The probe stock solution A;

[0068] II. The probe stock solution A is prepared into a fluorescent probe solution B with a concentration of 1.0×10 -5 mol / L by using a mixed solution of methanol and PBS (the concentration of the PBS buffer solution is 0.01 mol / L, and pH is 7.4) with a volume ratio of 7:3 as a solvent;

[0069] III. Hypochlorous acid is added to the fluorescent probe solution B to obtain a test solution C, and the concentration of the hypochlorous acid is 3.0×10 -4 mol / L. In the test solution, the molar ratio of the fluorescent probe to the hypochlorous acid is 1:30;

[0070] IV. After the test solution C is placed at room temperature for 30 seconds, a handheld ultraviolet lamp is used to irradiate the probe solution B and the test solution C, respectively. The probe solution B is bright green, and the test solution C is dark blue, as shown in FIG. 1. Figure 1 .

[0071] This indicates that the emission wavelength and the emission intensity of the colorimetric fluorescent probe for naked-eye and real-time detection of hypochlorous acid change in the presence of hypochlorous acid, and the purpose of qualitatively detecting hypochlorous acid in a solution by using the colorimetric fluorescent probe for naked-eye and real-time detection of hypochlorous acid can be achieved.

[0072] The colorimetric fluorescent probe for naked-eye and real-time detection of hypochlorous acid prepared in the embodiment is used to qualitatively detect hypochlorous acid in a solution by a fluorescence method, and the following steps are performed:

[0073] I. The colorimetric fluorescent probe for naked-eye and real-time detection of hypochlorous acid is dissolved in water-miscible methanol to prepare a probe stock solution A with a concentration of 1 mmol·L –1 The probe stock solution A;

[0074] II. The probe stock solution A is prepared into a fluorescent probe solution B with a concentration of 1.0×10 -5 mol / L by using a mixed solution of methanol and PBS (the concentration of the PBS buffer solution is 0.01 mol / L, and pH is 7.4) with a volume ratio of 7:3 as a solvent;

[0075] III. Different analytes (F - , Cl - , Br - , I -, AcO - , CO3 2- , HCO3 - , H2PO4 - , HPO4 2- , PO4 3- , BrO3 - , NO3 - , NO2 - , SO4 2- , SO3 2- , HSO3 - , SCN - , S 2- , Cys, Hcy, GSH, Ala, Gly, ClO - , H2O2, ·OH, 1 O2, ·O2 - , t-BOOH and ONOO - ) to obtain the test solution C. In the test solution C, the concentration of the analyte is 3.0 x 10 -4 mol / L, and the molar ratio of the fluorescent probe to the analyte is 1:30;

[0076] IV. After the test solution C was placed at room temperature for 30 seconds, the fluorescence emission spectrum of each test solution was measured under the condition that the excitation wavelength was 330 nm and the excitation slit width was 3.0 nm, and the results are shown in Figure 2 .

[0077] As can be seen from Figure 2 , the fluorescence emission wavelength of the fluorescent probe solution B is 495 nm, and the fluorescence intensity is about 900 a.u. After adding different analytes, it can be seen that F - , Cl - , Br - , I - , AcO - , CO3 2- , HCO3 - , H2PO4 - , HPO4 2- , PO4 3- , BrO3 - , NO3 - , NO2 - , SO4 2- , SO3 2- , HSO3 - , SCN - , S 2- , Cys, Hcy, GSH, Ala, Gly, H2O2, ·OH, 1 O2, ·O2 - , t-BOOH and ONOO- The fluorescence intensity of the fluorescent probe solution was not affected, and the intensity was about 900 a.u. After the addition of hypochlorous acid, the maximum emission wavelength was blue-shifted to about 450 nm, and the fluorescence intensity was reduced to about 300 a.u. The quenching degree at 495 nm was 1 / 8 of the fluorescence intensity of the fluorescent probe solution. Therefore, it can be determined from the fluorescence emission spectrum that the fluorescent probe solution has selective recognition characteristics for hypochlorous acid.

[0078] In order to further verify the anti-interference ability of the naked-eye and real-time detection of hypochlorous acid colorimetric fluorescent probe prepared in Example 1 for recognizing hypochlorous acid, the following test was performed:

[0079] I. The naked-eye and real-time detection of hypochlorous acid colorimetric fluorescent probe was dissolved in water-miscible methanol to prepare a solution with a concentration of 1 mmol·L –1 Probe stock solution A;

[0080] II. The probe stock solution A was prepared into a fluorescent probe solution B with a concentration of 1.0×10 -5 mol / L using a mixed solution of methanol / PBS (the concentration of the PBS buffer solution was 0.01 mol / L, and the pH was 7.4) with a volume ratio of 7:3 as the solvent;

[0081] III. Other analytes (F - , Cl - , Br - , I - , AcO - , CO3 2- , HCO3 - , H2PO4 - , HPO4 2- , PO4 3- , BrO3 - , NO3 - , NO2 - , SO4 2- , SO3 2- , HSO3 - , SCN - , S 2- , Cys, Hcy, GSH, Ala, Gly, H2O2, ·OH, 1 O2, ·O2 - , t-BOOH and ONOO - ) were added to the fluorescent probe solution B, respectively, to obtain different mixed solutions; in the mixed solutions, the concentration of different other analytes was 3.0×10 -4 mol / L, and the molar ratio of the fluorescent probe to other analyte was 1:30;

[0082] 4. Let the mixed solution obtained in step 3 stand for 30 seconds, then add ClO2 separately. - The solutions were mixed thoroughly to obtain different test solutions C; the concentration of hypochlorous acid in test solution C was 3.0 × 10⁻⁶. –4 mol / L, and the concentration of other analytes is 3.0 × 10⁻⁶ mol / L. -4 mol / L. At this point, the molar ratio of the fluorescent probe / anion / hypochlorous acid is 1:30:30;

[0083] 5. After the test solution C is placed at room temperature for 30 seconds, with an excitation wavelength of 330 nm and an excitation slit width of 3.0 nm, the emission intensities of probe solution B and test solution C at 495 nm are measured using 330 nm as the excitation wavelength, and recorded as T. B and Tc1~Tc 29 The result is as follows Figure 3 As shown. From Figure 3 It can be seen that hypochlorous acid reacts with other ions such as F. - Cl - ,Br - I - AcO - CO3 2- HCO3 - H2PO4 - HPO4 2- PO4 3- BrO3 - NO3 - NO2 - SO4 2- SO3 2- HSO3 - SCN - S 2- ,Cys,Hcy,GSH,Ala,Gly,H2O2,·OH, 1 O2、·O2 - t-BOOH and ONOO - In the presence of other analytes, the fluorescence intensity of the fluorescent probe for hypochlorous acid is not affected by the presence of these other analytes. In other words, the presence of other analytes does not interfere with the recognition of hypochlorous acid by the colorimetric fluorescent probe for both naked-eye and real-time detection. Therefore, Figure 3 This demonstrates that the fluorescent probe has selective recognition properties for hypochlorous acid, and also shows that other analytes have no effect on the fluorescent probe's recognition of hypochlorous acid, indicating strong anti-interference capabilities.

[0084] To investigate the changes in the fluorescence emission spectrum of hypochlorous acid detected by the colorimetric fluorescent probe prepared in this embodiment under different pH values ​​(2-12), the following experiments were conducted:

[0085] I. The naked-eye and real-time detection of hypochlorous acid colorimetric fluorescent probe was dissolved in water-miscible methanol to prepare a probe solution A with a concentration of 1 mmol / L. –1 Probe stock solution A;

[0086] II. A naked-eye and real-time detection of hypochlorous acid colorimetric fluorescent probe prepared in Example 1 was prepared into a fluorescent probe solution B with a concentration of 1.0 x 10 -5 mol / L using a mixed solution of methanol / PBS (the concentration of PBS buffer solution was 0.01 mol / L, and pH = 7.4) with a volume ratio of 7:3 as a solvent.

[0087] III. Hypochlorous acid was added to the fluorescent probe solution to obtain a test solution C. In the test solution C, the concentration of hypochlorous acid was 3.0 x 10 -4 mol / L, and the molar ratio of the fluorescent probe to the analyte was 1:30.

[0088] III. The pH value of the test solution C in step III was adjusted using a 5% NaOH solution and a 1% HCl solution to control the pH value to be 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0 and 12.0, respectively, to obtain 11 test solutions D.

[0089] IV. After the test solution D was placed at room temperature for 30 seconds, the emission intensity of the probe solution B in step II and the test solution D with different pH values in step III at 495 nm was measured respectively under the condition that the excitation wavelength was 330 nm and the excitation slit width was 3.0 nm, and was recorded as T B and T D1 ~T D11 , respectively. The results are shown in Figure 4 . As can be seen from Figure 4 , the fluorescence intensity of the naked-eye and real-time detection of hypochlorous acid colorimetric fluorescent probe solution remains stable under different pH conditions, whether in acid, neutral or alkaline. After adding hypochlorous acid (3.0 x 10 -5 mol / L) to the fluorescent probe solution (1.0 x 10 -4 mol / L), the fluorescence intensity of the fluorescent probe solution at 495 nm can be quenched to about 1 / 8 in the pH range of 2-12. That is, the change of pH value has little effect on the fluorescence intensity of the naked-eye and real-time detection of hypochlorous acid colorimetric fluorescent probe solution and its recognition of hypochlorous acid, and the detection of hypochlorous acid can be realized in the pH range of 2-12.

[0090] The method for quantitatively detecting hypochlorous acid concentration using the colorimetric fluorescent probe for naked-eye and real-time detection prepared in Example 1 via fluorescence method is carried out according to the following steps:

[0091] 1. Dissolve the colorimetric fluorescent probe for naked-eye and real-time detection of hypochlorous acid in water-miscible methanol to prepare a solution with a concentration of 1 mmol·L⁻¹. –1 Probe stock solution A;

[0092] 2. Using a methanol / PBS mixture with a volume ratio of 7:3 (PBS buffer solution concentration of 0.01 mol / L, pH = 7.4) as the solvent, prepare probe stock solution A with a concentration of 1.0 × 10⁻⁶. -5 Fluorescent probe solution B (mol / L);

[0093] III. According to the concentration of 1–30.0 μmol·L –1 Prepare hypochlorous acid standard solutions. Take 10 mL of hypochlorous acid standard solutions of different concentrations and mix them thoroughly with probe solution B obtained in step two to obtain test solution E.

[0094] IV. After the test solutions are placed at room temperature for 30 seconds, with an excitation wavelength of 330 nm and an excitation slit width of 3.0 nm, the emission intensity of probe solution B from step II and test solution E from step III at 495 nm is measured using 330 nm as the excitation wavelength, and recorded as T. B and T E1 ~T En Plot a graph with hypochlorous acid concentration as the x-axis and emission intensity at 495 nm as the y-axis, as shown below. Figure 5 As shown. From Figure 5 It can be seen that the fluorescence intensity at 495 nm gradually quenches with increasing hypochlorous acid concentration, reaching approximately 300 a.u. at 30.00 μmol / L. This indicates that the addition of trace amounts of hypochlorous acid to the probe solution results in significant fluorescence quenching. The fitted standard curve is: y = -0.4903x + 1.7062, with a standard deviation R0. 2 =0.9962. Based on the calculation, the lowest detectable concentration of hypochlorous acid is 57.66 nmol / L, indicating that the colorimetric fluorescent probe for detecting hypochlorous acid in naked eye and in real time prepared in this embodiment has a good linear relationship.

[0095] The effect of the colorimetric fluorescent probe for detecting hypochlorous acid by naked eye and in real time prepared in Example 1 on detecting changes in hypochlorous acid over time using fluorescence method was investigated. The experiment was conducted according to the following steps:

[0096] 1. Dissolve the colorimetric fluorescent probe for naked-eye and real-time detection of hypochlorous acid in water-miscible methanol to prepare a solution with a concentration of 1 mmol·L⁻¹. –1 Probe stock solution A;

[0097] II. The probe stock solution A was prepared into a fluorescent probe solution with a concentration of 1.0 x 10 -5 mol / L by using a mixed solution of methanol / PBS (the concentration of PBS buffer solution is 0.01 mol / L, pH = 7.4) with a volume ratio of 7:3 as a solvent;

[0098] II. Hypochlorous acid was added to the fluorescent probe solution to obtain a test solution C; in the test solution C, the concentration of hypochlorous acid was 3.0 x 10 -4 mol / L, and the amount-of-substance ratio of the fluorescent probe to the analyte was 1:30;

[0099] IV. After the test solution was placed at room temperature for 30 seconds, the emission intensity of the probe solution B in step I and the test solution C in step II at 495 nm was measured respectively at an excitation wavelength of 330 nm and an excitation slit width of 3.0 nm, and was recorded as T B and T F1 ~ T Fn , respectively. The results are shown in Figure 6 From Figure 6 it can be seen that when hypochlorous acid exists, the emission intensity of the probe solution at 495 nm gradually decreases with the increase of the response time, that is, the fluorescence quenching phenomenon occurs. Moreover, when the response time reaches about 5 seconds, the fluorescence intensity is quenched to the lowest. The fluorescence intensity remains unchanged when the time continues to increase. This result shows that the colorimetric fluorescent probe for naked-eye and real-time detection of hypochlorous acid prepared in this embodiment 1 has a relatively short response time to hypochlorous acid, which can be completed within 5 seconds, and can realize the real-time monitoring of hypochlorous acid in an aqueous solution.

[0100] Embodiment 2: A method for preparing a colorimetric fluorescent probe for naked-eye and real-time detection of hypochlorous acid in this embodiment was carried out according to the following steps:

[0101] I. 0.428 g (1.0 mmol) of 4-[1-(4-methoxyphenyl)-1H-phenanthro[9,10-d]imidazol-2-yl]benzaldehyde and 0.280 g (2.0 mmol) of benzoyl hydrazine were weighed respectively and added to a three-necked flask;

[0102] II. 20 mL of ethanol was further added to the three-necked flask as a solvent, and the reaction was heated to boiling reflux under stirring for 6 h; after the reaction was completed, the reaction solution was concentrated under reduced pressure to obtain a crude product;

[0103] III. The obtained crude product was recrystallized with dichloromethane, and after being filtered and washed with methanol, the colorimetric fluorescent probe for naked-eye and real-time detection of hypochlorous acid was obtained by drying.

[0104] The yield of the colorimetric fluorescent probe for naked-eye and real-time detection of hypochlorous acid in this embodiment is 75%.

[0105] Example 3: The preparation method of the colorimetric fluorescent probe for naked-eye and real-time detection of hypochlorous acid in this embodiment is carried out in the following steps:

[0106] I. 0.428 g (1.0 mmol) of 4-[1-(4-methoxyphenyl)-1H-phenanthro[9,10-d]imidazol-2-yl]benzaldehyde and 0.410 g (3.0 mmol) of benzoyl hydrazine are weighed into a three-necked flask, respectively;

[0107] II. 20 mL of propanol is added as a solvent into the three-necked flask, and the reaction is stirred to boiling reflux for 8.5 h; after the reaction is completed, the crude product is obtained by concentration under reduced pressure;

[0108] III. The obtained crude product is recrystallized with petroleum ether, and the colorimetric fluorescent probe for naked-eye and real-time detection of hypochlorous acid is obtained after suction filtration, washing with methanol and drying.

[0109] The yield of the colorimetric fluorescent probe for naked-eye and real-time detection of hypochlorous acid in this embodiment is 73%.

[0110] Example 4: The preparation method of the colorimetric fluorescent probe for naked-eye and real-time detection of hypochlorous acid in this embodiment is carried out in the following steps:

[0111] I. 0.428 g (1.0 mmol) of 4-[1-(4-methoxyphenyl)-1H-phenanthro[9,10-d]imidazol-2-yl]benzaldehyde and 0.493 g (3.5 mmol) of benzoyl hydrazine are weighed into a three-necked flask, respectively;

[0112] II. 20 mL of benzene is added as a solvent into the three-necked flask, and the reaction is stirred to boiling reflux for 10 h; after the reaction is completed, the crude product is obtained by concentration under reduced pressure;

[0113] III. The obtained crude product is recrystallized with ethyl acetate, and the colorimetric fluorescent probe for naked-eye and real-time detection of hypochlorous acid is obtained after suction filtration, washing with ethanol and drying.

[0114] The yield of the colorimetric fluorescent probe for naked-eye and real-time detection of hypochlorous acid in this embodiment is 76%.

[0115] Example 5: The preparation method of a colorimetric fluorescent probe for naked-eye and real-time detection of hypochlorous acid in this embodiment is carried out in the following steps:

[0116] I. Take 0.431 g (1.0 mmol) of 4-[1-(4-methoxyphenyl)-1H-phenanthro[9,10-d]imidazol-2-yl]benzaldehyde and 0.343 g (2.5 mmol) of benzoylhydrazine, respectively, and add them to a three-neck flask;

[0117] II. Add 40 mL of xylene as a solvent to the three-neck flask, and heat to boiling reflux under stirring for 3.5 h while stirring; after the reaction is completed, concentrate under reduced pressure to obtain a crude product;

[0118] III. Recrystallize the obtained crude product with petroleum ether, and dry after suction filtration and washing with ethanol to obtain the colorimetric fluorescent probe for naked-eye and real-time detection of hypochlorous acid.

[0119] The yield of the colorimetric fluorescent probe for naked-eye and real-time detection of hypochlorous acid in this example is 80%.

[0120] Example 6: A preparation method of a colorimetric fluorescent probe for naked-eye and real-time detection of hypochlorous acid in this example is performed according to the following steps:

[0121] I. Take 0.428 g (1.0 mmol) of 4-[1-(4-methoxyphenyl)-1H-phenanthro[9,10-d]imidazol-2-yl]benzaldehyde and 0.709 g (5 mmol) of benzoylhydrazine, respectively, and add them to a three-neck flask;

[0122] II. Add 20 mL of isobutyl alcohol as a solvent to the three-neck flask, and heat to boiling reflux under stirring for 10 h while stirring; after the reaction is completed, concentrate under reduced pressure to obtain a crude product;

[0123] III. Recrystallize the obtained crude product with dichloromethane, and dry after suction filtration and washing with ethanol to obtain the colorimetric fluorescent probe for naked-eye and real-time detection of hypochlorous acid.

[0124] The yield of the colorimetric fluorescent probe for naked-eye and real-time detection of hypochlorous acid in this example is 79%.

[0125] Example 7: A preparation method of a colorimetric fluorescent probe for naked-eye and real-time detection of hypochlorous acid in this example is performed according to the following steps:

[0126] I. Take 0.428 g (1.0 mmol) of 4-[1-(4-methoxyphenyl)-1H-phenanthro[9,10-d]imidazol-2-yl]benzaldehyde and 0.688 g (4.5 mmol) of benzoylhydrazine, respectively, and add them to a three-neck flask;

[0127] II. Add 70 mL of chlorobenzene as a solvent to the three-neck flask, and heat to boiling reflux under stirring for 5.5 h while stirring; after the reaction is completed, concentrate under reduced pressure to obtain a crude product;

[0128] iii. The obtained crude product was recrystallized with dichloromethane, filtered, washed with methanol and dried to obtain the colorimetric fluorescent probe for naked eye and real-time detection of hypochlorous acid.

[0129] The yield of the colorimetric fluorescent probe for naked eye and real-time detection of hypochlorous acid of the present example was 83%.

Claims

1. A colorimetric fluorescent probe for naked eye and real-time detection of hypochlorous acid, characterized in that The structure of the fluorescent probe is:

2. A process for the preparation of a colorimetric fluorescent probe for naked eye and real time detection of hypochlorous acid as claimed in claim 1, wherein, The method is performed according to the following steps: I. 4-[1-(4-methoxyphenyl)-1H-phenanthro[9,10-d]imidazol-2-yl]benzaldehyde and benzoyl hydrazine are weighed in a mass ratio of 1:(1-5) and added to a reactor; II. Organic solvent I is added to the reactor in step I, and heated to boiling reflux under stirring for 2-10 hours. After the reaction is completed, the crude product is obtained by concentration under reduced pressure; III. The crude product obtained in step II is recrystallized with organic solvent II, and the naked-eye and real-time detection of hypochlorous acid colorimetric fluorescent probe is obtained after filtration, washing and drying.

3. The method for preparing a colorimetric fluorescent probe for naked-eye and real-time detection of hypochlorous acid according to claim 2, characterized in that, The organic solvent I in step II is methanol, ethanol, propanol, isopropanol, butanol, benzene, chlorobenzene or xylene.

4. The preparation method of the colorimetric fluorescent probe for detecting hypochlorous acid in real time and naked eye according to claim 2 or 3, characterized in that, The organic solvent II in step III is a mixture of ethyl acetate and petroleum ether in a volume ratio of 1:(1-5); or a mixture of ethyl acetate and dichloromethane in a volume ratio of 1:(1-5).

5. The method for preparing a colorimetric fluorescent probe for naked-eye and real-time detection of hypochlorous acid according to claim 2, characterized in that, The washing in step III is washing with methanol or ethanol.

6. The use of a naked-eye and real-time colorimetric fluorescent probe for detecting hypochlorous acid according to claim 1, characterized in that The application is the naked-eye and real-time detection of hypochlorous acid colorimetric fluorescent probe for detecting hypochlorous acid in a solution.

7. The use of a colorimetric fluorescent probe for naked-eye and real-time detection of hypochlorous acid according to claim 6, characterized in that The method for naked-eye colorimetric qualitative detection of hypochlorous acid in a solution by using the naked-eye and real-time detection of hypochlorous acid colorimetric fluorescent probe is performed according to the following steps: I. The naked-eye and real-time detection of hypochlorous acid colorimetric fluorescent probe is dissolved in water-miscible organic solvent III to prepare probe stock solution A; II. The probe stock solution A obtained in step I is diluted with a mixed solution of methanol and PBS buffer solution to prepare probe solution B; III. The probe solution B is irradiated with a handheld ultraviolet lamp, and the probe solution appears bright green; IV. The probe solution B obtained in step II is mixed with the solution to be tested to obtain test solution C; V. The test solution C is placed for 30 seconds, and then irradiated with a handheld ultraviolet lamp. If the test solution C appears dark blue, it is determined that the solution to be tested contains hypochlorous acid.

8. The use of the colorimetric fluorescent probe for naked-eye and real-time detection of hypochlorous acid according to claim 6, characterized in that, The method for qualitative detection of hypochlorous acid in a solution by using the naked-eye and real-time detection of hypochlorous acid colorimetric fluorescent probe by fluorescence method is performed according to the following steps: I. The naked-eye and real-time detection of hypochlorous acid colorimetric fluorescent probe is dissolved in water-miscible organic solvent III to prepare probe stock solution A; II. The probe stock solution A obtained in step I is diluted with a mixed solution of methanol and PBS buffer solution to prepare probe solution B; III. The probe solution B obtained in step II is mixed with the solution to be tested to obtain test solution C; Four, with 330 nm as the excitation wavelength, the fluorescence emission spectrum of the probe solution B was determined, and the emission intensity at an emission wavelength of 495 nm was recorded as T B ; V. After 30 seconds of rest of test solution C, the fluorescence emission spectrum of test solution C was measured and the emission intensity at an emission wavelength of 495 nm was recorded as T C ; VI. Comparison T B and T C , if 8T C ≤ T B , then it is determined that the solution under test contains hypochlorous acid.

9. The use of a colorimetric fluorescent probe for naked eye and real-time detection of hypochlorous acid according to claim 6, characterized in that The method for quantitative detection of hypochlorous acid in a solution by using the naked-eye and real-time detection of hypochlorous acid colorimetric fluorescent probe by fluorescence method is a standard curve method.

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