Red light emission multifunctional fluorescent probe and preparation method and application thereof

Abstract

The invention discloses a red light emission multifunctional fluorescent probe as well as a preparation method and application thereof, and belongs to the technical field of fluorescent probes. The preparation method comprises the following steps: synthesizing 2-bromo-1-cyclohexene formaldehyde by taking PBr3 and cyclohexanone as raw materials; then, the product, 2-hydroxy-4-methyl benzaldehyde and Cs2CO3 are used as raw materials to synthesize 6-methoxy-2, 3-dihydro-1H-xanthene-4-formaldehyde, and the product, 2-hydroxy-4-methyl benzaldehyde and Cs2CO3 are used as raw materials to synthesize 6-methoxy-2, 3-dihydro-1H-xanthene-4- Then, the product and BBr3 are used as raw materials to synthesize 6-hydroxy-2, 3-dihydro-1H-xanthene-4-formaldehyde; then mixing the product with pyridine and malononitrile in a solvent for reaction to prepare hemicyanine; and finally reacting the hemicyanine with acetyl chloride to prepare the hemicyanine. The invention provides a red-light multifunctional fluorescent probe which can be used for detecting hypochlorous acid, cysteine and hydrazine hydrate, and realizes rapid detection of three substances including hypochlorous acid, cysteine and hydrazine hydrate at the same time.

Description

Technical Field

[0001] The present invention relates to the technical field of fluorescent probes, and in particular to a red light emitting multifunctional fluorescent probe and a preparation method and application thereof. Background Art

[0002] Traditional fluorescence imaging has high absorption or scattering in the visible absorption band, which is interfered by some components in biological tissues and can only penetrate tens of microns below the epidermis, so the penetration ability of traditional fluorescent probes is poor. Red light or near-infrared fluorescence can reach the subcutaneous part of biological tissues, so it is easier to penetrate tissues for in vivo fluorescence imaging.

[0003] Red light fluorescent probes are usually divided into inorganic molecular probes and organic molecular probes. Inorganic red light fluorescent probes are quantum dots combined with other nanoparticles to effectively eliminate tissue background in cell organisms. However, there are some heavy metals in the nanoparticles or the surface coatings have certain toxicity. In addition, the preparation cost is high, so organic red light fluorescent probes become a better choice. They show specific recognition functions for intracellular proteins, ribonucleotides, deoxyribonucleic acids, small chemical molecules, etc., and have low cytotoxicity, and are widely used in clinical research.

[0004] Biothiols, including cysteine ​​(Cys), homocysteine ​​(Hcy), and glutathione (GSH) play key roles in cell growth, metabolism, and maintaining the proper redox state in cells. Abnormal levels of these thiols are responsible for poor function of responsive enzymes and proteins, which may lead to many diseases. Cysteine ​​plays a key role in redox balance and is associated with many diseases. Cysteine ​​is an essential amino acid for protein synthesis, and its deficiency can lead to skin damage, liver damage, slowed growth, muscle atrophy, hair depigmentation, lethargy, and weakness. Therefore, it is necessary to detect cysteine ​​in the human body.

[0005] Hypochlorous acid (HClO) is a bleaching agent, oxidant, and deodorant. It is weakly acidic and strongly oxidizing. It is a very harmful gas to the human body. It can damage nails, cause hair loss, irritate the respiratory system, cause burning or pain in the respiratory system, and cause allergic reactions. However, an appropriate amount of hypochlorous acid has good antibacterial and antiviral effects. HClO in organisms can prevent pathogens from invading the body, reduce inflammation in the body, thereby reducing damage to the body, and strengthen the body's protective function to block damage from the outside world. An appropriate amount of HClO can maintain the dynamic balance inside cells and the normal operation of living organisms. However, excessive HClO can destroy the structure of proteins, cause inflammation, acute lung injury, cancer and other major diseases, and even endanger health. Therefore, people want to design probes that specifically recognize HClO.

[0006] Hydrazine (N2H4) is used as a highly active base and reducing agent in the chemical, pharmaceutical and agricultural industries. It is also involved in catalysts, corrosion inhibitors, textile dyes, pharmaceutical intermediates, etc. Due to its characteristics and its development performance research, it is also used in rocket propulsion systems and missile systems. However, hydrazine has considerable toxic effects and may cause serious environmental pollution during its manufacture, use, transportation and disposal. In fact, we classify it as a possible carcinogen with a lower threshold of 10 ppb. Hydrazine is also a neurotoxin with severe mutagenic effects, causing serious damage to the liver, lungs, kidneys and human central nervous system. Although there is no endogenous hydrazine in living cells, it is easily absorbed through oral, dermal or inhalation exposure routes, causing damage to living cells. Therefore, the selection and sensitive detection of small amounts of hydrazine are increasingly valued.

[0007] At present, the existing red light fluorescent probes can usually only realize the rapid detection of a single substance, and cannot conveniently realize the rapid detection of multiple substances such as hypochlorous acid, cysteine ​​and hydrazine hydrate. A red light fluorescent probe that can realize the simultaneous detection of hypochlorous acid, cysteine ​​and hydrazine hydrate is urgently needed to be developed. Summary of the invention

[0008] In order to solve the above technical problems, the purpose of the present invention is to provide a red light emitting multifunctional fluorescent probe and a preparation method and application thereof, so as to solve the problem of the existing lack of fluorescent probes that can quickly detect multiple substances such as hypochlorous acid, cysteine ​​and hydrazine hydrate.

[0009] The technical solution of the present invention to solve the above technical problems is as follows:

[0010] A red light emitting multifunctional fluorescent probe, the structural formula is:

[0011]

[0012] The beneficial effects of the present invention are as follows: with the addition of hypochlorous acid, the malononitrile part of the red light emitting multifunctional fluorescent probe prepared by the present invention is used as an electron-withdrawing group, and after the addition of hypochlorous acid, the cyano group is oxidized to an aldehyde group, and the hydroxyl group is oxidized to a ketone group by hypochlorous acid, resulting in a weakened electron-withdrawing ability, a blue shift of the ultraviolet spectrum, and a continuous weakening of the fluorescence. The fluorescence intensity of the fluorescent probe is significantly increased by the addition of cysteine. When the pH is 14, the fluorescence intensity is significantly weakened by the addition of hydrazine hydrate to the fluorescent probe. Thereby, the rapid identification and detection of hypochlorous acid, cysteine ​​and hydrazine hydrate are effectively achieved.

[0013] The method for preparing the above-mentioned red light emitting multifunctional fluorescent probe comprises the following steps:

[0014] (1) Synthesis of 2-bromo-1-cyclohexene carboxaldehyde: PBr3 and cyclohexanone are mixed and reacted in a solvent to obtain the product;

[0015] (2) Synthesis of 6-methoxy-2,3-dihydro-1H-xanthene-4-carboxaldehyde: 2-bromo-1-cyclohexenecarboxaldehyde, 2-hydroxy-4-methylbenzaldehyde and Cs2CO3 are mixed and reacted in a solvent to obtain a product;

[0016] (3) Synthesis of 6-hydroxy-2,3-dihydro-1H-xanthene-4-carboxaldehyde: 6-methoxy-2,3-dihydro-1H-xanthene-4-carboxaldehyde and BBr3 are mixed and reacted in a solvent to obtain a product;

[0017] (4) Synthesis of hemicyanine: 6-hydroxy-2,3-dihydro-1H-xanthene-4-carboxaldehyde, pyridine and malononitrile are mixed and reacted in a solvent to obtain a product;

[0018] (5) Preparation of fluorescent probe: Hemicyanine and acetyl chloride are mixed and reacted in a solvent to obtain the fluorescent probe.

[0019] Furthermore, in step (1), the molar ratio of PBr3 to cyclohexanone is (0.5-2):(1-5); the solvent is a mixed solvent of N'N-dimethylformamide and chloroform in a volume ratio of (0.5-2):(3-7); the reaction conditions are: first, PBr3 is added to the solvent under ice bath conditions and reacted for 30-60 minutes, and then cyclohexanone is added and reacted at 20-30°C for 15-20 hours.

[0020] Preferably, in step (1), the molar ratio of PBr3 to cyclohexanone is 1:3; the solvent is a mixed solvent of N'N-dimethylformamide and chloroform in a volume ratio of 1:5; the reaction conditions are: first, PBr3 is added to the solvent under ice bath conditions and reacted for 45 minutes, and then cyclohexanone is added and reacted at 25°C for 17 hours.

[0021] Furthermore, in step (2), the molar ratio of 2-bromo-1-cyclohexenecarboxaldehyde, 2-hydroxy-4-methylbenzaldehyde and Cs2CO3 is (0.5-2):(3-5):(50-80); the solvent is N'N-dimethylformamide; the reaction temperature is 20-30°C and the reaction time is 10-20h.

[0022] Preferably, in step (2), the molar ratio of 2-bromo-1-cyclohexenecarboxaldehyde, 2-hydroxy-4-methylbenzaldehyde and Cs2CO3 is 1:4:66; the solvent is N'N-dimethylformamide; the reaction temperature is 25°C and the reaction time is 16 hours.

[0023] Furthermore, in step (3), the molar ratio of 6-methoxy-2,3-dihydro-1H-oxanthene-4-carboxaldehyde and BBr3 is (0.5-2):(10-30); the solvent is dichloromethane; and the reaction conditions are: first react at -5-5°C for 20-40 minutes, and then react at 20-30°C for 10-20 hours.

[0024] Preferably, in step (3), the molar ratio of 6-methoxy-2,3-dihydro-1H-oxanthene-4-carboxaldehyde to BBr3 is 1:20; the solvent is dichloromethane; and the reaction conditions are: first react at 0°C for 30 minutes and then react at 25°C for 16 hours.

[0025] Furthermore, in step (4), the molar ratio of 6-hydroxy-2,3-dihydro-1H-oxanthene-4-carboxaldehyde, pyridine and malononitrile is (0.5-2):(1-5):(1-5); the solvent is anhydrous ethanol; the reaction temperature is 50-70°C and the reaction time is 10-20h.

[0026] Preferably, in step (4), the molar ratio of 6-hydroxy-2,3-dihydro-1H-oxanthene-4-carboxaldehyde, pyridine and malononitrile is 1:3:3; the solvent is anhydrous ethanol; the reaction temperature is 60° C. and the reaction time is 16 h.

[0027] Furthermore, in step (5), the molar ratio of hemicyanine to acetyl chloride is (0.5-2):(3-7); the solvent is a mixed solvent of dichloromethane and ethyl acetate in a volume ratio of (0.5-2):(0.5-2); the reaction temperature is 20-30°C and the reaction time is 5-10h.

[0028] Preferably, in step (5), the molar ratio of hemicyanine to acetyl chloride is 1:5; the solvent is a mixed solvent of dichloromethane and ethyl acetate in a volume ratio of 1:1; the reaction temperature is 25° C. and the reaction time is 7 hours.

[0029] The red light emitting multifunctional fluorescent probe is used for detecting hypochlorite, cysteine ​​and hydrazine hydrate.

[0030] The present invention has the following beneficial effects:

[0031] The present invention provides a red light multifunctional fluorescent probe that can be used for the detection of hypochlorous acid, cysteine ​​and hydrazine hydrate. The fluorescent probe can achieve rapid response to hypochlorous acid, cysteine ​​and hydrazine hydrate in a solution, produce obvious color fading phenomenon when reacting with hypochlorous acid, produce obvious color enhancement when reacting with cysteine, and can significantly reduce the fluorescence intensity at 561nm when reacting with hydrazine hydrate. The fluorescent probe can be used for the detection of three substances, hypochlorous acid, cysteine ​​and hydrazine hydrate, at the same time, wherein the detection of two substances, hypochlorous acid and cysteine, can be well applied to cells, and the detection of hydrazine hydrate can be better applied to the detection in water bodies. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 is the hydrogen spectrum of Dd-CN;

[0033] Figure 2is the fluorescence intensity of Dd-CN at different pH (5-9) (slit width 20-20);

[0034] Figure 3 is the fluorescence intensity of Dd-CN at different pH (9-14) (slit width 5-5);

[0035] Figure 4 This is the fluorescence spectrum of Dd-CN at pH = 14 in time response (slit width 5-5), and the time gradient in the figure is 10 min;

[0036] Figure 5 It is the time response diagram of Dd-CN at pH = 14;

[0037] Figure 6 This is the time-responsive fluorescence spectrum of Dd-CN to hydrazine hydrate. In the figure, the time gradient between 3-15min is 3min, and the time gradient between 15-135min is 5min;

[0038] Figure 7 This is the fluorescence intensity and time response diagram of Dd-CN to hydrazine hydrate;

[0039] Figure 8 The fluorescence spectrum of Dd-CN in the presence of hydrazine hydrate interfering substance (slit width 10-10);

[0040] Fig. 9 This is a comparison of mixed solutions of hydrazine hydrate and different interfering substances and Dd-CN under natural light and ultraviolet light. In the figure, the upper part is natural light and the lower part is irradiated by 365nm ultraviolet light;

[0041] Fig.10 The fluorescence spectrum of Dd-CN (PBS:DMSO=5:1, pH=7.4) changes with the increase of hydrazine hydrate concentration (slit width 10-10). In the figure, the concentration gradient is 0.225×10 3 μmol / L;

[0042] Fig.11 It is the fitting curve diagram of Dd-CN (PBS:DMSO=5:1, pH=7.4) changing with the increase of hydrazine hydrate concentration;

[0043] Fig.12 The UV spectrum of Dd-CN (PBS:DMSO=5:1, pH=7.4) changes with the increase of sodium hypochlorite concentration;

[0044] Fig.13 The fluorescence spectrum of Dd-CN (PBS: DMSO = 5:1, pH = 7.4) changes with the increase of sodium hypochlorite concentration. In the figure, the concentration gradient is 0.03623×103 μmol / L;

[0045] Fig.14 The graph is a functional relationship between the fluorescence intensity of Dd-CN (PBS:DMSO=5:1, pH=7.4) and the hypochlorite concentration as the concentration of sodium hypochlorite increases, and the inset is a linear fitting graph;

[0046] Fig.15 The fluorescence spectrum of Dd-CN in the presence of sodium hypochlorite interfering substances;

[0047] Fig.16 This is a comparison of the mixed solution of sodium hypochlorite and different interfering substances and Dd-CN under natural light and ultraviolet light. In the figure, the upper part is natural light, and the lower part is irradiated by 365nm ultraviolet light;

[0048] Fig.17 This is the time-dependent fluorescence spectrum of Dd-CN (PBS:DMSO=5:1, pH=7.4) to cysteine ​​(slit width 10-5), in which the time gradient is 5 min;

[0049] Fig.18 The fluorescence intensity of Dd-CN (PBS:DMSO=5:1, pH=7.4) to cysteine ​​corresponds to time;

[0050] Fig.19 The fluorescence spectrum of Dd-CN in the presence of cysteine ​​interfering substances;

[0051] Fig. 20 This is a comparison of mixed solutions of cysteine, different interfering substances and Dd-CN under natural light and ultraviolet light. In the figure, the upper part is natural light, and the lower part is irradiated by 365nm ultraviolet light;

[0052] Fig.21 The fluorescence spectra of Dd-CN (PBS: DMSO = 5:1, pH = 7.4) with different concentrations of cysteine ​​added. In the figure, the concentration gradient is 0.225×10 3 μmol / L;

[0053] Fig. 22 The graph is a functional relationship between the fluorescence intensity of Dd-CN (PBS:DMSO=5:1, pH=7.4) and the cysteine ​​concentration as the cysteine ​​concentration increases, and the inset is a linear fitting graph. DETAILED DESCRIPTION

[0054] The principles and features of the present invention are described below in conjunction with the accompanying drawings. The examples are only used to explain the present invention and are not used to limit the scope of the present invention. If specific conditions are not specified in the embodiments, they are carried out according to normal conditions or conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0055] Example:

[0056] A method for preparing a red light emitting multifunctional fluorescent probe comprises the following steps:

[0057] (1) Synthesis of Compound A (2-bromo-1-cyclohexenecarboxaldehyde):

[0058]

[0059] Mix 50 mL of dehydrated CHCl3 and (11.2 mL, 145 mmol) of dehydrated DMF. In an ice bath, slowly add (12.4 mL, 130.19 mmol) of PBr3 and react for 45 min to produce a yellow turbid liquid. Then add (5 mL, 48.24 mmol) of cyclohexanone and react at 25 °C for 17 h to obtain a brown turbid liquid. Pour the product after the reaction into saturated salt ice water, adjust the pH to 7, extract with dichloromethane, and rotary evaporate to obtain compound A.

[0060] (2) Synthesis of Compound B (6-methoxy-2,3-dihydro-1H-xanthene-4-carboxaldehyde)

[0061]

[0062] Compound A (204 mg, 1.0 mmol), 2-hydroxy-4-methylbenzaldehyde (600 mg, 3.9 mmol), and Cs2CO3 (21.6 g, 66.29 mmol) were dissolved in 50 mL of dehydrated DMF and reacted at room temperature for 16 h. The product was extracted with dichloromethane and washed with a large amount of saturated brine. The organic layer was collected and the solvent was evaporated under reduced pressure to obtain 165 mg of compound B as a solid with a yield of 76%.

[0063] (3) Synthesis of Compound C (6-Hydroxy-2,3-dihydro-1H-xanthene-4-carboxaldehyde)

[0064]

[0065] Compound B (200 mg, 0.826 mmol) and BBr3 (2 mL, 20 mmol) were dissolved in 20 mL of dehydrated CH2Cl2, reacted at 0°C for 30 min, then slowly heated up and reacted at room temperature (25°C) for 16 h to obtain a yellow-brown reaction mixture. The reaction mixture was poured into saturated salt water and adjusted with Na2CO3 until the pH = 7, extracted with dichloromethane, the organic layer was collected, the solvent was evaporated under reduced pressure, and further purified by silica gel column chromatography to obtain 68.7 mg of compound C as a solid with a yield of 35%.

[0066] (4) Synthesis of hemicyanine D

[0067]

[0068] Compound C (300 mg, 1.3 mmol), pyridine (0.5 mL, 6.21 mmol) and malononitrile (516 mg, 7.8 mmol) were dissolved in 30 mL of anhydrous ethanol and heated to reflux at 60 °C for 16 h under a nitrogen atmosphere. After the reaction was completed, the solvent was evaporated under reduced pressure and the mixture was extracted with dichloromethane. The organic layer was collected and the solvent was evaporated. The crude product HDXMM was purified and separated by column chromatography to obtain 236 mg of solid hemicyanine D with a yield of 65.4%.

[0069] (5) Synthesis of red-light-emitting multifunctional fluorescent probe Dd-CN

[0070]

[0071] 236 mg of hemicyanine D was dissolved in a mixed solvent of 10 mL of dichloromethane and 10 mL of ethyl acetate, 0.3 mL of acetyl chloride was added, and the mixture was refluxed at 25° C. for 7 h to obtain the product. The crude product Dd-CN was purified and separated by column chromatography with a yield of 45.2%.

[0072] Test example:

[0073] (1) The red light emitting multifunctional fluorescent probe Dd-CN prepared in Example 1 was dissolved in deuterated DMSO solution and 1 Determination of H-NMR spectrum.

[0074] The experimental results are as follows Figure 1 As shown, the chemical shift is about δ = 1.71-2.70ppm, and the chemical shift is about δ = 7.02-8.20ppm, and the three corresponding hydrogen signal peaks on the benzene ring appear. At δ = 7.02ppm, there is a signal peak for the para hydrogen of the cyano group connected to the double bond, and the chemical shift is about δ = 2.0-2.5ppm, and the hydrogen signal peak on the methyl group appears. The structure of the compound Dd-CN can be determined by characterizing the hydrogen spectrum of the nuclear magnetic resonance spectrum.

[0075] (2) The probe Dd-CN was dissolved in a mixed solvent of PBS and DMSO with a volume ratio of 5:1. The concentration of the probe was 1×10 -5 mol / L, the pH of the probe solution was adjusted with NaOH and HCl, and the fluorescence intensity at different pH values ​​was measured.

[0076] The experimental results are as follows Figure 2-Figure 5 shown.

[0077] Figure 2 The fluorescence spectrum shows that in the probe Dd-CN solution (slit width 20-20), the fluorescence intensity remains basically unchanged in the range of pH = 5-9.

[0078] Figure 3 The fluorescence spectrum shows that in the probe Dd-CN solution (slit width 5-5), the fluorescence intensity increases with the increase of pH in the range of pH = 9-14.

[0079] Figure 4 and Figure 5 It shows that when pH=14 (slit width 5-5), the fluorescence intensity increases continuously with time, and the rising trend of the fluorescence intensity is gentle around 300 min, indicating that the reaction is complete.

[0080] (3) A mixed solvent of PBS and DMSO with a volume ratio of 5:1 was adjusted to pH 14 and 0.7 × 10 -5 10 μL of 85% N2H4 was added to the 1.5 mol / L probe Dd-CN, and the time response fluorescence spectrum was measured.

[0081] The experimental results are as follows Figure 6 and Figure 7 As shown, the fluorescence intensity gradually weakened with the passage of time, and the rising trend of the fluorescence intensity was gentle around 135 min, indicating that the reaction was complete.

[0082] Figure 8 In the mixed solution (PBS:DMSO=5:1, pH=14), the concentration was 1.2×10 -5 mol / L probe Dd-CN, left for 5 h, and then other interfering substances at a concentration of 3 mmol / L were added, such as glutathione, cations (Mg 2+ , K + 、Na + , Ba 2 + , Ca 2+ 、Cd 2+ 、Al 3+ , Pb 2+ 、Ag + , Cu 2+ ), anions (N2H4, F- , HS - , S 2- 、CO3 2- Br - 、SO4 2- , Cl - ,I - ), ROS (H2O2, ·OH) and mix the prepared probe solution evenly according to the above concentration, and test the fluorescence spectrum of the solution.

[0083] Fig. 9 It can be seen that when only the probe Dd-CN is present, the solution appears orange. With the addition of N2H4, the solution is placed at room temperature for 135 minutes. Under natural light, it can be clearly seen that the color of the solution fades, and the colors of other interfering substances remain unchanged. When irradiated under 365nm light, it is obvious that after the addition of hydrazine hydrate, the probe Dd-CN presents a light blue light. This shows that the probe Dd-CN has good selectivity for hydrazine hydrate.

[0084] Fig.10 In order to study whether there is a linear relationship between the concentration of hydrazine hydrate and the fluorescence intensity of the probe Dd-CN after response, and thus to determine the detection limit, 3 μL of the mother solution was diluted in 3 mL of a mixed solution (PBS:DMSO=5:1, pH=14) to prepare a concentration of 1×10 -5 mol / L probe Dd-CN. After 300 min of standing, different concentrations of hydrazine hydrate were added and left overnight. The fluorescence spectrum was measured (slit width 10-10).

[0085] Fig.11 The linear fitting diagram of the fluorescence intensity of the probe Dd-CN and [N2H4] shows a good linear relationship between the fluorescence intensity and [N2H4] when the concentration of hydrazine hydrate is 0.68-5.18mmol / L. The formula is I0-I=120.53[N2H4]-29.20, [N2H4] represents the concentration of hydrazine hydrate, and the slope k=120.53 is obtained. The detection limit calculation method is: DL=3σ / K, and the detection limit is 1.5μmol / L. The probe Dd-CN has a certain sensitivity to hydrazine hydrate and a very low detection limit.

[0086] (5) To 1×10 -5 Sodium hypochlorite of different concentrations (0.0362-1.0143 μmol / L) was added to the 1.5 μmol / L probe Dd-CN (the solvent was a mixed solvent of PBS and DMSO in a volume ratio of 5:1, pH=7.4), and the UV and fluorescence spectra were measured.

[0087] The experimental results are as follows Figure 12-14 As shown in the UV spectrum, as the concentration of sodium hypochlorite increases, the fluorescence absorbance and fluorescence intensity continue to weaken. Fig.14 Fluorescence intensity of Dd-CN-ClO - The linear fitting diagram shows that the fluorescence intensity of -ClO at 0.22-0.58mmol / L - It showed a good linear relationship, and the detection limit was 1.8μmol / L.

[0088] Depend on Fig.15 It can be seen that to 1×10 -5 0.3 mmol / L of other interfering substances were added to the 1.5 mmol / L probe Dd-CN (the solvent was a mixed solvent of PBS and DMSO in a volume ratio of 5:1, pH = 7.4). It can be seen that the fluorescence of the probe solution did not change significantly after the addition of other substances, while the fluorescence intensity was significantly weakened after the addition of 0.3 mmol / L hypochlorous acid, indicating that Dd-CN has a high selectivity for hypochlorous acid.

[0089] Depend on Fig.16 It can be seen that in the presence of probe Dd-CN, the solution appears orange. With the addition of hypochlorous acid, the color of the solution fades under natural light, and the color of the probe solution with other interferences remains unchanged. Under 365nm ultraviolet light, it is obvious that the probe solution after adding hypochlorous acid presents blue light.

[0090] (6) 1×10 -5 mol / L probe Dd-CN (solvent is a mixed solvent of PBS and DMSO with a volume ratio of 5:1, pH = 7.4, slit width 10-5), 3mmol / L cysteine ​​was added, and the fluorescence spectrum was measured every 5min

[0091] The experimental results are as follows Fig.17 and Fig.18 As shown, the results showed that the fluorescence intensity at 626 nm increased with the passage of time, and the rising trend of fluorescence was gentle around 80 min, indicating that the reaction was complete.

[0092] To 1×10 -5 Different concentrations of cysteine ​​were added to the 1 mol / L probe Dd-CN (the solvent was a mixed solvent of PBS and DMSO in a volume ratio of 5:1, pH = 7.4), and the fluorescence spectrum was measured.

[0093] The experimental results are as follows Fig.19 As shown, it can be seen that the fluorescence of the probe did not change significantly when other substances were added, while the fluorescence intensity was significantly weakened after the addition of cysteine.

[0094] Depend on Fig. 20It can be seen that in the presence of only the probe Dd-CN, the solution appears orange. With the addition of cysteine, the solution color turns red under natural light, and the color remains unchanged when other interfering substances are added. Under 365nm light, it can be seen that the addition of cysteine ​​presents a deeper red light.

[0095] The experimental results are as follows Fig.21 and Fig. 22 As shown, it can be seen that with the increase of cysteine ​​concentration, the fluorescence intensity continues to rise. When the concentration is 1.80-4.05mmol / L, the fluorescence intensity has a good linear relationship with the cysteine ​​concentration, and the detection limit is 1.24μmo / L.

[0096] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A red light emitting multifunctional fluorescent probe, characterized in that: The structural formula of the fluorescent probe is:

2. The method for preparing the red light emitting multifunctional fluorescent probe according to claim 1, characterized in that: The following steps are involved: (1) Synthesis of 2-bromo-1-cyclohexene carboxaldehyde: PBr3 and cyclohexanone are mixed and reacted in a solvent to obtain the product; (2) Synthesis of 6-methoxy-2,3-dihydro-1H-xanthene-4-carboxaldehyde: 2-bromo-1-cyclohexenecarboxaldehyde, 2-hydroxy-4-methylbenzaldehyde and Cs2CO3 are mixed and reacted in a solvent to obtain a product; (3) Synthesis of 6-hydroxy-2,3-dihydro-1H-xanthene-4-carboxaldehyde: 6-methoxy-2,3-dihydro-1H-xanthene-4-carboxaldehyde and BBr3 are mixed and reacted in a solvent to obtain a product; (4) Synthesis of hemicyanine: 6-hydroxy-2,3-dihydro-1H-xanthene-4-carboxaldehyde, pyridine and malononitrile are mixed and reacted in a solvent to obtain a product; (5) Preparation of fluorescent probe: Hemicyanine and acetyl chloride are mixed and reacted in a solvent to obtain the fluorescent probe.

3. The method for preparing a red light emitting multifunctional fluorescent probe according to claim 2, characterized in that: In the step (1), the molar ratio of PBr3 to cyclohexanone is (0.5-2):(1-5); the solvent is a mixed solvent of N'N-dimethylformamide and chloroform in a volume ratio of (0.5-2):(3-7); the reaction conditions are: first, PBr3 is added to the solvent under ice bath conditions and reacted for 30-60 minutes, and then cyclohexanone is added and reacted at 20-30°C for 15-20 hours.

4. The method for preparing a red light emitting multifunctional fluorescent probe according to claim 2, characterized in that: In the step (2), the molar ratio of 2-bromo-1-cyclohexenecarboxaldehyde, 2-hydroxy-4-methylbenzaldehyde and Cs2CO3 is (0.5-2):(3-5):(50-80); the solvent is N'N-dimethylformamide; the reaction temperature is 20-30°C and the reaction time is 10-20h.

5. The method for preparing a red light emitting multifunctional fluorescent probe according to claim 2, characterized in that: In the step (3), the molar ratio of 6-methoxy-2,3-dihydro-1H-xanthene-4-carboxaldehyde to BBr3 is (0.5-2):(10-30); the solvent is dichloromethane; and the reaction conditions are: first react at -5-5°C for 20-40 minutes, and then react at 20-30°C for 10-20 hours.

6. The method for preparing a red light emitting multifunctional fluorescent probe according to claim 2, characterized in that: In the step (4), the molar ratio of 6-hydroxy-2,3-dihydro-1H-xanthene-4-carboxaldehyde, pyridine and malononitrile is (0.5-2):(1-5):(1-5); the solvent is anhydrous ethanol; the reaction temperature is 50-70° C., and the reaction time is 10-20 hours.

7. The method for preparing a red light emitting multifunctional fluorescent probe according to claim 2, characterized in that: In the step (5), the molar ratio of hemicyanine to acetyl chloride is (0.5-2):(3-7); the solvent is a mixed solvent of dichloromethane and ethyl acetate in a volume ratio of (0.5-2):(0.5-2); the reaction temperature is 20-30°C and the reaction time is 5-10h.

8. Use of the red light emitting multifunctional fluorescent probe according to claim 1 in detecting hypochlorite, cysteine ​​and hydrazine hydrate.

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