AU2020103559A4

AU2020103559A4 - Ratiometric fluorescent probe for detecting hypochlorous acid, and preparation method and use thereof

Info

Publication number: AU2020103559A4
Application number: AU2020103559A
Authority: AU
Inventors: Yunling Chen; Fangong KONG; Xiangpeng Lin; Keyin Liu
Original assignee: Qilu University of Technology
Current assignee: Qilu University of Technology
Priority date: 2020-01-13
Filing date: 2020-11-19
Publication date: 2021-02-04
Anticipated expiration: 2028-11-19
Also published as: CN111205280B; CN111205280A

Abstract

The disclosure discloses a ratiometric fluorescent probe for detecting hypochlorous acid, and a preparation method and use thereof. The ratiometric fluorescent probe for detecting ~oxyN hypochlorous acid has the following structural formula: The preparation method includes: adding compound Cy7Cl, compound Nil-OH and sodium hydride to dimethyl formamide (DMF); conducting reaction at room temperature for 20 h to 30 h; and then subjecting the reaction solution to separation and purification to give the ratiometric fluorescent probe Cy7-Nil for detecting hypochlorous acid. The fluorescent probe can be used for identifying and detecting hypochlorous acid in water systems, organic solvent systems or organisms. The fluorescent probe itself has three fluorescence peaks. A solution of the fluorescent probe in water or an organic solvent is purple, and becomes pink after the fluorescent probe reacts with hypochlorous acid. The fluorescent probe uses the ratio of fluorescence intensities as a standard for quantifying the concentration of hypochlorous acid, and is successfully used in water and organic solvents, which has characteristics of multi-channel, high sensitivity, high selectivity and the like.

Description

RATIOMETRIC FLUORESCENT PROBE FOR DETECTING HYPOCHLOROUS ACID, AND PREPARATION METHOD AND USE THEREOF TECHNICAL FIELD The disclosure belongs to the field of analysis and detection, and particularly relates to a ratiometric fluorescent probe for detecting hypochlorous acid, and a preparation method and use thereof. BACKGROUND Hypochlorous acid, a strong oxidant and active oxygen, is produced from hydrogen peroxide and chloride ions under the catalysis of peroxidase in organisms. Hypochlorous acid is considered to be the leading killer for invading pathogens and plays an important role in the innate immune system of organisms. However, hypochlorous acid, when at a high concentration, can cause a series of diseases, such as rheumatoid arthritis (RA) and cancer. Therefore, it is of great significance to achieve the real-time visual detection of hypochlorous acid for the monitoring and diagnosis of such diseases. With the improvement of modern living standards, people are paying more and more attention to health. In recent years, the importance of hypochlorous acid to human health has been increasingly concerned, so it is of vital significance to rapidly and quantitatively detect the concentration of hypochlorous acid in organisms. Therefore, it is extremely important to develop an effective method for detecting hypochlorous acid in quantitative safety detection and safety supervision of foods, clinical and environmental applications. There are many traditional detection methods, such as iodometric titration, spectrophotometry, chemiluminescence analysis and coulometry. However, most of the above methods require cumbersome operating procedures, which brings certain difficulties in practical applications. In recent years, measurement with a fluorescent probe as an excellent detection technique has attracted increasing concerns due to high selectivity, high sensitivity and real time imaging thereof and is widely used for measurement of various substances. Generally, measurement with a fluorescent probe depends on increase or decrease of fluorescence intensity. Thus, an output signal can be affected by factors such as concentration of a probe, efficiency of a device and environment. But for ratiometric fluorescent probes, the use of changes in the fluorescence intensity at two or more different wavelengths can well eliminate these factors and, moreover, near-infrared fluorescence emission can effectively reduce the autofluorescence and the scattering of light by biological tissue and blood, thereby enabling high-resolution imaging. At present, there are few fluorescent probes for detecting hypochlorous acid, and most of the reported fluorescent probes are based on the nucleophilic reaction for double bonds. These reported fluorescent probes often require a long reaction time and have low selectivity, which greatly limits the application thereof. Furthermore, it is difficult to accurately quantify the concentration of hypochlorous acid in a short time only based on the intensity of fluorescence. Therefore, it is very important for real-time visual detection of hypochlorous acid to develop a fluorescent probe with high sensitivity and short response time that can accurately quantify the concentration of hypochlorous acid. SUMMARY In view of the shortcomings of fluorescent probes in the prior art, such as long response time, poor sensitivity, low selectivity and inability to accurately quantify the concentration of hypochlorous acid, the disclosure discloses a ratiometric fluorescent probe for detecting hypochlorous acid, and a preparation method and use thereof. The fluorescent probe itself has three fluorescence peaks. A solution of the fluorescent probe in water or an organic solvent is purple, and becomes pink after the fluorescent probe reacts with hypochlorous acid. The fluorescent probe uses the ratio of fluorescence intensities as a standard for quantifying the concentration of hypochlorous acid, and is successfully used in water and organic solvents, which has characteristics of multi-channel, high sensitivity, high selectivity and the like. The disclosure is implemented by the following technical solutions: The disclosure provides a ratiometric fluorescent probe Cy7-Nil for detecting hypochlorous acid, with a structure shown as follows:

N N

The disclosure also provides a method for preparing the ratiometric fluorescent probe for detecting hypochlorous acid, including: adding compound Cy7Cl, compound Nil-OH and sodium hydride to dimethyl formamide (DMF); conducting reaction at room temperature for 20 h to 30 h; and then subjecting the reaction solution to separation and purification to give the ratiometric fluorescent probe Cy7-Nil for detecting hypochlorous acid.

NaH DMF

2N

Cy7CI Cy7-NiI Nil-OH

Further, the compound Cy7Cl, compound Nil-OH and sodium hydride are used at a molar ratio of 1:3:1. Further, the separation and purification includes: washing the reaction solution obtained from the reaction with deionized water, and drying with anhydrous magnesium sulfate; then removing the solvent by rotary distillation of the organic phase; and dissolving the solid in dichloromethane (DCM), and conducting separation by column chromatography with a mixed solvent of DCM and methanol with a volume ratio of 20:1 to give the ratiometric fluorescent probe Cy7-Nil. Further, the compound Nil-OH is prepared by the following method: adding compound 1 and 1,6-dihydroxynaphthalene at a molar ratio of 1:1.1 to DMF, and conducting reaction at 140°C for h; after the reaction is completed, washing the reaction solution with deionized water, and drying with anhydrous magnesium sulfate; then removing the organic solvent by rotary evaporation of the organic phase; and dissolving the solid in DCM, and conducting separation by column chromatography with a mixed solvent of DCM and methanol with a volume ratio of 50:1 to give the compound Nil-OH.

00 OH Oi O N

± 'j::J P.j HO + 140 0C NO H

1 Nil-OH Further, the compound Cy7Cl is prepared by the following method: adding compound 2, compound 3 and sodium acetate at a molar ratio of 1:2.6:1 to acetic anhydride, and conducting reaction at 120°C for 5 h; after the reaction is completed, washing the reaction solution with deionized water, and drying with anhydrous magnesium sulfate; then removing the solvent by rotary evaporation of the organic phase; and dissolving the solid in DCM, and conducting separation by column chromatography with a mixed solvent of DCM and methanol with a volume ratio of 150:1 to give the compound Cy7Cl.

O H NaOAc AC 2O 0 ^OH N K.O 120 C0 K 2 3 Cy7CI

The disclosure also provides the use of the ratiometric fluorescent probe for detecting hypochlorous acid in the identification and detection of hypochlorous acid. Further, the fluorescent probe is used in water systems, organic solvent systems or organisms. The fluorescent probe can identify hypochlorous acid in water systems, organic solvent systems or organisms with high selectivity. The probe itself has three fluorescence peaks (560 nm, 650 nm, and 780 nm). A solution of the fluorescent probe in water or an organic solvent is purple. After the fluorescent probe reacts with hypochlorous acid, the solution becomes pink, the fluorescence at 560 nm and 780 nm is quenched, but the fluorescence at 650 nm is enhanced, which achieves the detection of hypochlorous acid by multi-channel fluorescence change and significant color change. Beneficial effects: (1) The fluorescent probe in the disclosure itself has three fluorescence peaks (560 nm, 650 nm, and 780 nm). A solution of the fluorescent probe in water or an organic solvent is purple. After the fluorescent probe reacts with hypochlorous acid, the solution obviously becomes pink, the fluorescence at 560 nm and 780 nm is significantly quenched, but the fluorescence at 650 nm is significantly enhanced, which can achieve the detection of hypochlorous acid by multi-channel fluorescence change and significant color change. (2) The fluorescent probe in the disclosure is a ratiometric fluorescent probe, which is not interfered by factors such as the concentration of the probe, the efficiency of the instrument, and the environment during the detection process. The ratio of fluorescence intensities has a linear relationship with the concentration of hypochlorous acid. The ratiometric fluorescent probe uses the ratio of fluorescence intensities as the standard for quantifying the concentration of hypochlorous acid, and has high selectivity, accuracy and sensitivity. (3) The preparation method of the fluorescent probe for detecting hypochlorous acid in the disclosure is simple and exhibits high productivity, which is suitable for promotion and application at a large scale. BRIEF DESCRIPTION OF DRAWINGS FIG. 1 shows the 1 H NMR spectrum for the ratiometric fluorescent probe Cy7-Nil; FIG. 2 shows the mass spectrum for the ratiometric fluorescent probe Cy7-Nil; FIG. 3 shows the fluorescence spectra for the fluorescent probe at different concentrations of hypochlorous acid under a pH of 7.4, illustrating the change of the three fluorescence peaks; FIG. 4 shows the curve of fluorescence intensity at 780 nm changing with time after different concentrations of hypochlorous acid are added at a pH of 7.4, where, the excitation wavelength is 700 nm, and the fluorescence signal at 780 nm is collected; FIG. 5 is a comparison diagram illustrating the change of the fluorescence intensity ratio after different biological small molecules are added, where, the excitation wavelength is 560 nm, and 1 to 15 represent bioactive small molecules (CH 3) 3COOH, Glu, Cys, S2032-, S2-, S03 2 -, SO 4 2 -, HS0 3 2 , NO2, H 2 0 2 , Zn2+, Fe3 , Mg2+, Ca, C10-, respectively; and FIG. 6 shows the variation of the fluorescence intensity ratio with the concentration of hypochlorous acid. DETAILED DESCRIPTION Examples of the disclosure will be described in detail below. The examples are implemented on the premise of the technical solutions of the disclosure, and the detailed implementations and specific operation processes are given, but the protection scope of the disclosure is not limited to the following examples. Example 1 (1) 0.194 g of compound 1 and 0.176 g of 1,6-dihydroxynaphthalene were added to 10 ml of DMF, and the resulting mixture reacted at 140°C for 10 h; the reaction solution was washed with deionized water to remove DMF, and dried with anhydrous magnesium sulfate; then the solvent was removed by rotary distillation of the organic phase; and the solid was dissolved in DCM, and the resulting solution was subjected to column chromatography separation with a mixed solvent of DCM and methanol with a volume ratio of 50:1 to give the compound Nil-OH, with a yield of 54%. The NMR data of the compound Nil-OH are as follows: 'H NMR (400 MHz, DMSO-d6 ) 610.44 (s, 1H), 7.98 (d, J = 8.5 Hz, 1H), 7.89 (d, J = 2.2 Hz, 1H), 7.59 (d, J = 9.1 Hz, 1H), 7.10 (dd, J = 8.5, 2.3 Hz, 1H), 6.81 (d, J = 6.6 Hz, 1H), 6.66(s, 1H), 6.16 (s, 1H), 3.51 (q, J =7.0 Hz,4H),1.17(t, J= 6.9 Hz, 6H); 1 3 C NMR(100 MHz, DMSO-d 6) 6 181.99,161.04, 155.95, 153.83, 151.96, 151.09, 146.77,139.23, 134.17, 131.17, 127.85, 127.19, 124.29, 124.13, 118.76, 117.37,117.25, 110.28, 108.95, 108.62, 105.72, 104.57, 96.48, 44.84, 12.88;

OH OMF

H0 + Hm 1400C HO HO M NO H

1 Nil-OH

(2) 0.120 g of compound 2, 0.341 g of compound 3 and 0.095 g of sodium acetate were added to 24 ml of acetic anhydride, and the resulting mixture reacted at 120°C for 5 h; the reaction solution was washed with deionized water to remove acetic anhydride, and dried with anhydrous magnesium sulfate; then the solvent was removed by rotary distillation of the organic phase; and the solid was dissolved in DCM, and the resulting solution was subjected to column chromatography separation with a mixed solvent of DCM and methanol with a volume ratio of 150:1 to give the compound Cy7Cl, with a yield of 51%.

O 1 HNaOAc Ac 20 O- - H1200C

2 3 Cy 7 CI

(3) 0.05 g of compound Nil-OH and 0.02 g of Cy7C1 were added to 5 ml of DMF, and the resulting mixture reacted at room temperature for 24 h; the reaction solution was subjected to separation and purification, washed with deionized water to remove DMF, and dried with anhydrous magnesium sulfate; then the solvent was removed by rotary distillation of the organic phase; and the solid was dissolved in DCM, and the resulting solution was subjected to column chromatography separation with a mixed solvent of DCM and methanol with a volume ratio of :1 to give the ratiometric fluorescent probe Cy7-Nil, with a yield of 58%. The 'H NMR spectrum and mass spectrum for the ratiometric fluorescent probe Cy7-Nil are shown in FIG. 1 and FIG. 2, respectively. 'H NMR (400 MHz, MeOD) 6 8.31 (d, J = 9.1 Hz, 2H), 8.01 (d, J = 14.3 Hz, 2H),7.69 (d, J= 9.2 Hz, 1H), 7.52 (dd, J= 8.6, 2.1 Hz, 1H), 7.34 (t, J= 7.1Hz, 4H), 7.24 (d, J = 8.1 Hz, 2H), 7.17 (t, J = 7.4 Hz, 2H), 6.92 (dd, J =9.2, 2.2 Hz, 1H), 6.69 (d, J = 2.2 Hz, 1H), 6.30 (s, 1H), 6.21 (d, J = 14.2Hz, 2H), 4.14 (q, J = 7.0 Hz, 4H), 3.57 (q, J = 6.9 Hz, 4H), 2.84 (t, J = 5.6Hz, 4H), 1.37 - 1.23 (m, 26H). "C NMR (100 MHz, MeOD) 6 181.34, 170.46, 161.38, 160.83, 151.57, 150.53, 145.74, 140.15, 140.04, 139.66, 133.35, 129.89, 126.98, 126.90, 124.81, 123.68, 123.40, 120.55, 119.85, 115.45,109.37, 108.92, 106.54, 102.16, 98.14, 94.34, 47.43, 43.31, 37.57, 37.33, 27.85, 27.81, 25.16, 22.44, 19.61, 9.98, 9.55;

CO NaH DMF

Cy7CI Cy7-Nil Nil-OH

Example 2 Titration experiment of hypochlorous acid fluorescent probe and hypochlorous acid

A fluorescent probe solution with an initial concentration of 1 mM was added to PBS (pH = 7.4) so that the fluorescent probe had a concentration of 10 M in the solution. Then, different amounts of sodium hypochlorite with an initial concentration of 1.00 mM were added separately to give solutions with sodium hypochlorite concentrations of 0 M, 1 M, 2 M, 3 M, 4 M, 5 pM, 6 M, 8 M, 10 M, 12 M, 14 M, 16 M, 18 M, 20 M, 25 M, 30 M, 35 M, 40 [M, M, 50 M, 55 M, 60 M, 70 M, 80 M, 90 M, and 100 [M. The solution without sodium hypochlorite was set as a control. The solutions stood for 0.5 h to allow the sufficient reaction of sodium hypochlorite with the fluorescent probe. A fluorescence spectrometer was used to obtain the fluorescence spectra at different concentrations of hypochlorous acid. The probes were excited with light of 500 nm, 580 nm and 700 nm, separately. The emission wavelengths of the probe were 560 nm, 650 nm and 780 nm. The results are shown in FIG. 3. It can be seen from FIG. 3 that, when the concentration of hypochlorous acid is between 0 M to 20 M, the three fluorescence peaks all show a decrease in fluorescence intensity, especially the fluorescence peak at 780 nm is extremely sensitive to hypochlorous acid; when the concentration of hypochlorous acid reaches 20 M, the fluorescence at 780 nm is almost completely quenched; and with the increase of hypochlorous acid concentration (20 M to 100 [M), the fluorescence peak at 650 nm exhibits an increase in fluorescence. It indicates that the fluorescent probe prepared in the disclosure can conduct multi channel response to hypochlorous acid and realize the ratio detection for hypochlorous acid. Experiment for detecting the fluorescence change of the hypochlorous acid fluorescent probe over time responsive to hypochlorous acid A fluorescence spectrometer was used to obtain fluorescence spectra at different time points. The excitation wavelength of fluorescence spectra was 700 nm, the emission wavelength was 780 nm, and the detection wavelength was 780 nm. Results are shown in FIG. 4. It can be seen from FIG. 4 that, after hypochlorous acid is added, the fluorescence intensity can be stabilized within 2 s, and as the concentration of hypochlorous acid increases, the fluorescence at 780 nm gradually decreases. It indicates that the fluorescent probe prepared in the disclosure can achieve the rapid detection of hypochlorous acid. Example 3 Selectivity test of the fluorescent probe for detecting hypochlorous acid A fluorescent probe solution with an initial concentration of 1 mM was added to PBS buffer (pH = 7.4) so that the fluorescent probe had a concentration of 10 M in the solution. An excessive amount of other bioactive small molecules were added to the solution under the same test conditions as described in Example 2. The fluorescence spectra were determined after different bioactive small molecules were added, and the ratio of the fluorescence intensity at 650 nm to the fluorescence intensity at 780 nm was calculated. The excitation wavelength was 580 nm, and the emission wavelengths were 650 nm and 780 nm. The results are shown in FIG. 4. It can be seen from FIG. 4 that,1 to 15 represent bioactive small molecules (CH 3) 3 COOH, Glu, Cys, S2032-, S2-, SO 3 2-, SO42-, HS0 32-, NO2-, H2 0 2 , Zn2+, Fe3+, Mg2+, Ca2+, C10-, respectively. Only when hypochlorous acid is present, F6 5 0 nm/F780 nm is significantly increased, and other bioactive small molecules do not interfere with the detection results, indicating that the fluorescent probe prepared in the disclosure has high selectivity for hypochlorous acid. Example 4 The linear relationship of the fluorescence intensity ratio with the hypochlorous acid concentration: A fluorescent probe solution with an initial concentration of 1 mM was added to PBS (pH= 7.4) so that the fluorescent probe had a concentration of 10 M in the solution. Then, different amounts of sodium hypochlorite with an initial concentration of 1.00 mM were added separately to give solutions with sodium hypochlorite concentrations of 0 M, 1 M, 2 M, 3 M, 4 M, 5 pM, 6 M, 8 M, 10 M, 12 M, 14 M, 16 M, 18 M, 20 M, 25 M, 30 M, 35 M, 40 M, M, 50 M, 55 M, 60 M, 70 M, 80 M, 90 M, and 100 [M. A fluorescence spectrometer was used to obtain the fluorescence spectra at different concentrations of hypochlorous acid, and the ratio of the fluorescence intensity at 560 nm to the fluorescence intensity at 650 nm was calculated. The excitation wavelength was 520 nm, and the emission wavelengths were 560 nm and 650 nm. The change and linear relationship of F 5 60 nm/F 650 m with the hypochlorous acid concentration is shown in FIG. 6 (a, b, c). A fluorescence spectrometer was used to obtain the fluorescence spectra at different concentrations of hypochlorous acid, and the ratio of the fluorescence intensity at 650 nm to the fluorescence intensity at 780 nm was calculated. The excitation wavelength was 580 nm, and the emission wavelengths were 650 nm and 780 nm. The change and linear relationship of F6 5 0 nm/F 7 8 0 nm with the hypochlorous acid concentration is shown in FIG. 6 (d, e, f). It can be seen from FIG. 6 that the ratio of fluorescence intensities at the two different fluorescence peaks of the fluorescent probe Cy7-Nil can be used to accurately quantify the concentration of HC1O/C10- (FIG. 6a, FIG. 6d). According to the fitted curve, when the concentration of HC1O/C10- is between 2 M to 16 M and between 40 M to 80 M, 1560nm/1650 nm (FIG. 6b, FIG. 6c) and1650 nm/780 nm (FIG. 6e, FIG. 6f) are linearly correlated. It can be seen that Cy7-Nil can achieve the accurate detection of a concentration range of HClO/ClO-.

Claims

What is claimed is: 1. A ratiometric fluorescent probe for detecting hypochlorous acid, wherein, the ratiometric fluorescent probe Cy7-Nil for detecting hypochlorous acid has a structure shown as follows:

N N
2. A method for preparing the ratiometric fluorescent probe for detecting hypochlorous acid according to claim 1, comprising: adding compound Cy7Cl, compound Nil-OH and sodium hydride to dimethyl formamide (DMF); conducting reaction at room temperature for 20 h to 30 h; and then subjecting the reaction solution to separation and purification to give the ratiometric fluorescent probe Cy7-Nil for detecting hypochlorous acid;

O

O NaH DMF

N 0

Cy7CI Cy7-NiI Nil-OH
3. The preparation method according to claim 2, wherein, the compound Cy7Cl, compound Nil-OH and sodium hydride are used at a molar ratio of 1:3:1.
4. The preparation method according to claim 2, wherein, the separation and purification comprises: washing the reaction solution obtained from the reaction with deionized water, and drying with anhydrous magnesium sulfate; then removing the solvent by rotary distillation of the organic phase; and dissolving the solid in dichloromethane (DCM), and conducting separation by column chromatography with a mixed solvent of DCM and methanol with a volume ratio of 20:1 to give the ratiometric fluorescent probe Cy7-Nil.
5. The preparation method according to claim 2, wherein, the compound Nil-OH is prepared by the following method: adding compound 1 and 1,6-dihydroxynaphthalene at a molar ratio of 1:1.1 to DMF, and conducting reaction at 140°C for 10 h; after the reaction is completed, washing the reaction solution with deionized water, and drying with anhydrous magnesium sulfate; then removing the organic solvent by rotary evaporation of the organic phase; and dissolving the solid in DCM, and conducting separation by column chromatography with a mixed solvent of DCM and methanol with a volume ratio of 50:1 to give the compound Nil-OH;

00 OH O O N

H OHOME H0 H O'j::J 140 0C NO H

1 Nil-OH
6. The preparation method according to claim 2, wherein, the compound Cy7Cl is prepared by the following method: adding compound 2, compound 3 and sodium acetate at a molar ratio of 1:2.6:1 to acetic anhydride, and conducting reaction at 120°C for 5 h; after the reaction is completed, washing the reaction solution with deionized water, and drying with anhydrous magnesium sulfate; then removing the solvent by rotary evaporation of the organic phase; and dissolving the solid in DCM, and conducting separation by column chromatography with a mixed solvent of DCM and methanol with a volume ratio of 150:1 to give the compound Cy7Cl;

O1 HNaOAc 0 Ac 20 0 - H 120 CN

2 3 Cy7CI
7. Use of the ratiometric fluorescent probe for detecting hypochlorous acid according to claim 1 in the identification and detection of hypochlorous acid.
8. The use according to claim 7, wherein, the fluorescent probe is used in water systems, organic solvent systems or organisms.

FIG. 2 FIG. 1 DRAWINGS

Hypochlorous acid concentration Fluorescence intensity Wavelength/nm Fluorescence intensity

FIG. 3 Wavelength/nm

Wavelength/nm Fluorescence intensity Fluorescence intensity 19 Nov 2020 2020103559

Time/s

Molecule type FIG. 4

FIG. 5 Fluorescence intensity (780 nm) Fluorescence intensity ratio (650 nm/780 nm) 19 Nov 2020 2020103559

FIG. 6