Near-infrared fluorescent compound for specifically detecting hydrazine and preparation method thereof
阅读说明：本技术 一种用于特异性检测肼的近红外荧光化合物及制备方法 (Near-infrared fluorescent compound for specifically detecting hydrazine and preparation method thereof ) 是由 王毅 朱美庆 赵宗元 凡福港 吴祥为 花日茂 于 2020-07-22 设计创作，主要内容包括：本发明公开一种新型特异性识别肼的近红外荧光化合物,(E)-2-(2-(3-(二氰基亚甲基)-5,5-二甲基环己-1-烯-1-基)乙烯基)-5-(二乙氨基)苯基4-溴丁酸,其可以特异性检测生物体内的肼；该近红外荧光化合物制备过程简单,原料易得,成本低廉,结构稳定,并且具有较好的细胞膜通透性以及较低的细胞毒性,可进入活细胞和动物组织中并与外源肼发生反应,产生可肉眼分辨的强烈红色荧光；通过紫外吸收和荧光分光光度法分析得出该近红外荧光化合物可以在各种干扰物下对肼有着优越的选择性,对常见生物分子有着很强的抗干扰能力；不仅可对外源性肼选择性识别,而且能在各种活细胞的生长环境下高灵敏度的定量检测肼,并成功应用于活细胞及斑马鱼成像中。(The invention discloses a novel near-infrared fluorescent compound for specifically recognizing hydrazine, (E) -2- (2- (3- (dicyanomethylene) -5, 5-dimethylcyclohex-1-en-1-yl) vinyl) -5- (diethylamino) phenyl 4-bromobutyric acid, which can specifically detect hydrazine in an organism; the near-infrared fluorescent compound has the advantages of simple preparation process, easily obtained raw materials, low cost, stable structure, better cell membrane permeability and lower cytotoxicity, can enter living cells and animal tissues and react with exogenous hydrazine to generate strong red fluorescence which can be distinguished by naked eyes; the near-infrared fluorescent compound is analyzed and obtained through ultraviolet absorption and a fluorescence spectrophotometry, has excellent selectivity on hydrazine under various interferents, and has strong anti-interference capability on common biomolecules; not only can selectively identify exogenous hydrazine, but also can quantitatively detect hydrazine with high sensitivity in various living cell growth environments, and can be successfully applied to living cells and zebra fish imaging.)
1. A near-infrared fluorescent compound for specifically detecting hydrazine is characterized byThe method comprises the following steps: the near-infrared fluorescent compound is (E) -2- (2- (3- (dicyanomethylene) -5, 5-dimethylcyclohex-1-en-1-yl) vinyl) -5- (diethylamino) phenyl 4-bromobutanoic acid, and the molecular formula of the near-infrared fluorescent compound is C27H32BrN3O2The chemical structural formula is shown as the formula (I):
2. the method for preparing a near-infrared fluorescent compound for specifically detecting hydrazine as claimed in claim 1, characterized by comprising the following steps:
(1) under nitrogen protection, 1.0g of (E) -2- (3- (4- (diethylamino) -2-hydroxystyryl) -5, 5-dimethylcyclohex-2-en-1-yl) malononitrile (DHDM), 1.0mL of triethylamine and 50mL of anhydrous dichloromethane are mixed, and the temperature is reduced to 0 ℃ in an ice bath;
(2) slowly dripping 0.62mg of 4-bromobutyryl chloride, and stirring and reacting at room temperature for 10-12 hours to obtain a mixture;
(3) pouring the mixture into ice water, stirring, and extracting with dichloromethane for 3 times, wherein 10mL of dichloromethane is used for each extraction;
(4) drying with anhydrous sodium sulfate, and concentrating the organic phase to obtain a crude product; the crude product was purified by column chromatography eluting with n-hexane and ethyl acetate formulated as eluent in a volume ratio of 3:1 to give 1.03g of the violet black product (E) -2- (2- (3- (dicyanomethylene) -5, 5-dimethylcyclohex-1-en-1-yl) vinyl) -5- (diethylamino) phenyl 4-bromobutyric acid DCDB in 72% yield.
3. Use of the near infrared fluorescent compound according to claim 1, characterized in that: for the detection of hydrazine specificity.
4. The method for detecting hydrazine in a solution system by using the near-infrared fluorescent compound as described in claim 1, which is characterized by comprising the following steps:
(1) preparing the near-infrared fluorescent compound into working solution with the concentration of 10 mu M by using buffer solution, wherein the buffer solution is prepared from Phosphate Buffered Saline (PBS) and dimethyl sulfoxide (DMSO) in a volume ratio of 1:1, and the pH value of the buffer solution is 7.4;
(2) 81 portions of 3mL of 10. mu.M near-infrared fluorescent compound solution were added to each of the solutions at 120. mu.L concentration of 5X 10-527 kinds of analytes to be detected in mol/L, wherein each analyte to be detected consists of 3 parallel units, 81 parts of reactants are obtained through reaction, the final concentration of the analytes in the reactants is 200 mu M, and the fluorescence intensity of the 81 parts of reactants is respectively measured after the reaction is completed;
(3) the results show that hydrazine (N)2H4) The fluorescence intensity of the near-infrared fluorescent compound working solution can be improved; the near infrared fluorescent compound can only react with hydrazine (N)2H4) And (3) carrying out fluorescence enhancement reaction, namely specifically recognizing hydrazine by the near-infrared fluorescent compound.
5. The method for detecting hydrazine on a test paper by using the near-infrared fluorescent compound as described in claim 1, which is characterized by comprising the following steps:
(1) preparing the near-infrared fluorescent compound detection test paper of claim 1, preparing the near-infrared fluorescent compound into working solution with the concentration of 10 μ M by using dichloromethane, immersing a plurality of filter papers with the same size and shape into the working solution for 30 minutes, taking out the filter papers, and airing to obtain test paper;
(2) respectively dripping 200 mu M of 27 aqueous solutions of different analytes on corresponding 27 test paper; then placing under an ultraviolet lamp with 365nm excitation wavelength for observation;
(3) the result shows that the fluorescence color of the test paper only dripped with the hydrazine solution changes from colorless to red, and other analytes do not change the test paper at all, namely the test paper can simply and quickly detect the environmental pollutant hydrazine.
6. The method for detecting hydrazine in HeLa cells by using the near-infrared fluorescent compound as described in claim 1, which is characterized by comprising the following steps:
(1) preparation of the working solution of the near-infrared fluorescent compound according to claim 1
Preparing the near-infrared fluorescent compound according to claim 1 into a working solution with a concentration of 20 μ M using a buffer solution; the volume ratio of Phosphate Buffered Saline (PBS) to dimethyl sulfoxide (DMSO) in the buffer was 1:1, the pH value of the buffer solution is 7.4;
(2) a, B, C, D four groups of experimental groups were selected;
group a is blank control: HeLa cells without any treatment were group A test substances;
group B is the hydrazine treatment control group: 200 μ L of hydrazine at a concentration of 50 μ M in a 5X 10 solution4Incubating the HeLa cells in a hole plate of the HeLa cells for 30 minutes to obtain B group detected substances for detection;
group C is near infrared fluorescent compound treatment control: 200 μ L of 20 μ M near infrared fluorescent compound was added to the mixture containing 5X 104Incubating the HeLa cells in a hole plate of the HeLa cells for 30 minutes to obtain C group detection objects for detection; group D is a near-infrared fluorescent compound and hydrazine treatment group: pretreatment with 200. mu.L of 20. mu.M near-infrared fluorescent compound containing 5X 104HeLa cells were plated for 30 minutes in a well plate and then incubated with 200. mu.L of 50. mu. M hydrazine (N)2H4) Incubating for 30 minutes to obtain D groups of detected objects for detection;
(3) respectively placing the A group of detected objects, the B group of detected objects, the C group of detected objects and the D group of detected objects under a fluorescence microscope with the excitation wavelength of 520nm for observation;
(4) the fluorescence imaging result shows that the group A detected objects, the group B detected objects and the group C detected objects do not find fluorescence; and the D group of detected substances show obvious red fluorescence; fluorescence imaging results show that the near-infrared fluorescent compound of claim 1 can enter HeLa cells and react with exogenous hydrazine to generate strong red fluorescence, so that the specificity detection of the exogenous hydrazine in the HeLa cells is realized.
7. The method for detecting the exogenous hydrazine in the zebra fish by using the near-infrared fluorescent compound as claimed in claim 1, which is characterized by comprising the following operation steps:
(1) preparation of the working solution of the near-infrared fluorescent compound according to claim 1
The near-infrared fluorescent compound according to claim 1 was formulated into a working solution at a concentration of 20 μ M using a buffer composed of Phosphate Buffered Saline (PBS) and dimethyl sulfoxide (DMSO) in a volume ratio of 1:1, the pH value of the buffer solution is 7.4;
(2) a, B, C, D four groups of experimental groups were collected
Group a is blank control: obtaining an A group of detected objects for detection by untreated zebra fish of 3 days old;
group B is the hydrazine treatment control group: incubating normal 3-day-old zebra fish with 10mL of 50-mu M hydrazine to obtain B group of detected substances for detection for 30 minutes;
group C is near infrared fluorescent compound treatment control: treating 3-day-old zebra fish with 10mL of the near-infrared fluorescent compound of claim 1 at a concentration of 20 μ M for 30 minutes to obtain group C test substances for detection;
group D is a near-infrared fluorescent compound and hydrazine treatment group: 3 days old zebra fish are incubated with 10mL of hydrazine with a concentration of 50 μ M for 30 minutes, and then treated with 20 μ M of the near-infrared fluorescent compound according to claim 1 for 30 minutes to obtain D groups of test substances for detection;
(5) respectively placing the group A detected object, the group B detected object, the group C detected object and the group D detected object under a fluorescence microscope with the excitation wavelength of 520nm for observation;
(6) the result shows that the group A detected object, the group B detected object and the group C detected object do not find fluorescence; and the D group of detected substances show obvious red fluorescence; fluorescence imaging results show that the near-infrared fluorescent compound of claim 1 can enter a zebra fish body and react with exogenous hydrazine to generate strong red fluorescence, so that specific detection of the exogenous hydrazine is realized.
8. The method for detecting hydrazine in an environmental water sample by using the near-infrared fluorescent compound as claimed in claim 1, which is characterized by comprising the following steps:
(1) preparation of the working solution of the near-infrared fluorescent compound according to claim 1
Formulating the near-infrared fluorescent compound of claim 1 into a working solution with a concentration of 10 μ M using a buffer; the buffer solution is prepared from Phosphate Buffered Saline (PBS) and dimethyl sulfoxide (DMSO) according to the volume ratio of 1:1, and the pH value of the buffer solution is 7.4;
(2) preparation of environmental water sample to be detected
When the method is used for detecting exogenous hydrazine in an environmental water sample, the environmental water samples in different areas are collected and filtered through a 200-micron water-phase filter membrane to obtain a treated environmental water sample; addition of 0.1. mu.M, 0.5. mu.M and 1.0. mu.M hydrazine to the treated environmental water sample
(3) Adding different environmental water samples to be detected into the near-infrared fluorescent compound working solution, and detecting the reaction solution by using a fluorescence spectrometer;
(4) the recovery rate of hydrazine reaches 87-109%, and the result shows that the near-infrared fluorescent compound working solution can quantitatively detect the existence of hydrazine in an environmental water sample.
9. The method for detecting hydrazine in sewage by using the near-infrared fluorescent compound as described in claim 1, which is characterized by comprising the following steps:
(1) formulating the near-infrared fluorescent compound of claim 1 into a working solution with a concentration of 10 μ M using a buffer; the buffer solution is prepared from Phosphate Buffered Saline (PBS) and dimethyl sulfoxide (DMSO) according to the volume ratio of 1:1, and the pH value of the buffer solution is 7.4;
(2) when the method is used for detecting exogenous hydrazine in a sewage sample, collecting sewage samples of different regional chemical plants, and filtering the sewage samples through a 200-micron water-phase filter membrane to obtain a sewage sample to be detected;
(3) detecting a reaction liquid of a near-infrared fluorescent compound working solution and a sewage sample by using a fluorescence spectrometer;
(4) the results indicate that the near-infrared fluorescent compound of claim 1 can quantitatively detect hydrazine in sewage with high sensitivity.
Technical Field
The invention belongs to the technical field of organic small molecule fluorescent probes, and particularly relates to a fluorescent chemical compound for specifically detecting an environmental pollutant hydrazine by using (E) -2- (3- (4- (diethylamino) -2-hydroxystyryl) -5, 5-dimethylcyclohex-2-en-1-yl) malononitrile as a fluorescent parent and an application thereof.
Background
Inorganic pollutants in the environment are widely sourced and complicated in type, and most of the inorganic pollutants enter human bodies. When the concentration of these contaminants in the environment exceeds a safe threshold, it can pose a threat to the environment and human health. Hydrazine (N)2H4) Is an important inorganic compound with certain reducibility, and is widely applied to the fields of pesticide production, aerospace, chemical industry and the like. Furthermore, hydrazine is a volatile and potentially carcinogenic environmental pollutant. Due to wide application and better water solubility, hydrazine can generate pollution to a certain degree in the environment and can generate toxic action on human bodies. Therefore, there is an urgent need to establish a reliable, accurate and efficient analytical method for detecting hydrazine in the environment.
There are many methods for detecting hydrazine, such as High Performance Liquid Chromatography (HPLC), Gas Chromatography (GC), Electrochemical Analysis (EA) and Capillary Electrophoresis (CE). However, these methods have disadvantages of time consumption and complicated operation. In contrast, fluorescent probes have attracted the interest of many researchers due to their high specificity, sensitivity and ease of manipulation. The fluorescent molecular probe can react with an analyte to be detected; thus, the microscopic reaction events can be visualized by fluorescence signals. Therefore, these characteristics give the fluorescent probe excellent properties, and can be used for environmental monitoring, life sciences, and disease diagnosis.
To date, many chemical sensors for detecting hydrazine have been reported. Due to the nucleophilicity of hydrazine, various mechanisms are used in the detection of hydrazine, such as the Gabriel reaction, addition reaction with aldehydes and ester hydrolysis. Wherein, the application of the hydrazinolysis reaction can improve the response rate of hydrazine detection. The hydrazinolysis reaction can be typically carried out on a chemical sensor which takes 4-bromobutyryl as a recognition group, and the hydrazinolysis reaction is mainly formed by modifying hydroxyl on a fluorescent group, so that the introduction of the hydroxyl into a parent structure of a fluorescent probe is a better idea.
In conclusion, the (E) -2- (3- (4- (diethylamino) -2-hydroxystyryl) -5, 5-dimethylcyclohex-2-en-1-yl) malononitrile is selected as a fluorescent parent structure, and the hydroxyl in the 4-bromobutyryl parent structure is used for modification, so that the fluorescence of the (E) -2- (3- (4- (diethylamino) -2-hydroxystyryl) -5, 5-dimethylcyclohex-2-en-1-yl) malononitrile is quenched, the sensitivity of the (E) -2- (3- (4- (diethylamino) -2-hydroxystyryl) malononitrile is improved, and a novel.
Disclosure of Invention
The first purpose of the invention is to develop a near infrared fluorescent compound capable of selectively detecting the environmental pollutant hydrazine, and the near infrared fluorescent compound can distinguish the existence of the hydrazine under the interference of other analytes.
The second purpose of the invention is to provide a preparation method of the near-infrared fluorescent compound capable of specifically detecting the environmental pollutant hydrazine.
The third purpose of the invention is to provide a method for detecting exogenous hydrazine applicable to solution, biological tissues and cells.
A near-infrared fluorescent compound for specifically detecting hydrazine is (E) -2- (2- (3- (dicyanomethylene) -5, 5-dimethylcyclohex-1-en-1-yl) ethenyl) -5- (diethylamino) phenyl 4-bromobutanoic acid, and its molecular formula is C27H32BrN3O2The chemical structural formula is shown as the formula (I):
formula (I).
The preparation operation steps of the near-infrared fluorescent compound for specifically detecting hydrazine are as follows:
(1) under nitrogen protection, 1.0g of (E) -2- (3- (4- (diethylamino) -2-hydroxystyryl) -5, 5-dimethylcyclohex-2-en-1-yl) malononitrile (DHDM), 1.0mL of triethylamine and 50mL of anhydrous dichloromethane are mixed, and the temperature is reduced to 0 ℃ in an ice bath;
(2) slowly dripping 0.62mg of 4-bromobutyryl chloride, and stirring and reacting at room temperature for 10-12 hours to obtain a mixture;
(3) pouring the mixture into ice water, stirring, and extracting with dichloromethane for 3 times, wherein 10mL of dichloromethane is used for each extraction;
(4) drying with anhydrous sodium sulfate, and concentrating the organic phase to obtain a crude product; the crude product was purified by column chromatography eluting with n-hexane and ethyl acetate formulated as eluent in a volume ratio of 3:1 to give 1.03g of the violet black product (E) -2- (2- (3- (dicyanomethylene) -5, 5-dimethylcyclohex-1-en-1-yl) vinyl) -5- (diethylamino) phenyl 4-bromobutyric acid DCDB in 72% yield.
The near-infrared fluorescent compound is used for detecting the specificity of hydrazine.
The operation steps of the near-infrared fluorescent compound for detecting hydrazine in a solution system are as follows:
(1) preparing the near-infrared fluorescent compound into working solution with the concentration of 10 mu M by using buffer solution, wherein the buffer solution is prepared from Phosphate Buffered Saline (PBS) and dimethyl sulfoxide (DMSO) in a volume ratio of 1:1, and the pH value of the buffer solution is 7.4;
(2) 81 portions of 3mL of 10. mu.M near-infrared fluorescent compound solution were added to each of the solutions at 120. mu.L concentration of
(3) the results show that hydrazine (N)2H4) The fluorescence intensity of the near-infrared fluorescent compound working solution can be improved; the near infrared fluorescent compound can only react with hydrazine (N)2H4) And (3) carrying out fluorescence enhancement reaction, namely specifically recognizing hydrazine by the near-infrared fluorescent compound.
The operation steps of the near-infrared fluorescent compound for detecting hydrazine on the test paper are as follows:
(1) preparing the near-infrared fluorescent compound detection test paper of
(2) respectively dripping 200 mu M of 27 aqueous solutions of different analytes on corresponding 27 test paper; then placing under an ultraviolet lamp with 365nm excitation wavelength for observation;
(3) the result shows that the fluorescence color of the test paper only dripped with the hydrazine solution changes from colorless to red, and other analytes do not change the test paper at all, namely the test paper can simply and quickly detect the environmental pollutant hydrazine.
The operation steps of the near-infrared fluorescent compound for detecting hydrazine in HeLa cells are as follows:
(1) preparation of the working solution of the near-infrared fluorescent compound according to
Preparing the near-infrared fluorescent compound according to claim 1 into a working solution with a concentration of 20 μ M using a buffer solution; the volume ratio of Phosphate Buffered Saline (PBS) to dimethyl sulfoxide (DMSO) in the buffer was 1:1, the pH value of the buffer solution is 7.4;
(2) a, B, C, D four groups of experimental groups were selected;
group a is blank control: HeLa cells without any treatment were group A test substances;
group B is the hydrazine treatment control group: 200 μ L of hydrazine at a concentration of 50 μ M in a
group C is near infrared fluorescent compound treatment control: 200 μ L of 20 μ M near infrared fluorescent compound was added to the
(3) respectively placing the A group of detected objects, the B group of detected objects, the C group of detected objects and the D group of detected objects under a fluorescence microscope with the excitation wavelength of 520nm for observation;
(4) the fluorescence imaging result shows that the group A detected objects, the group B detected objects and the group C detected objects do not find fluorescence; and the D group of detected substances show obvious red fluorescence; fluorescence imaging results show that the near-infrared fluorescent compound of
The operation steps of the near-infrared fluorescent compound for detecting the external hydrazine in the zebra fish are as follows:
(1) preparation of the working solution of the near-infrared fluorescent compound according to
The near-infrared fluorescent compound according to
(2) a, B, C, D four groups of experimental groups were collected
Group a is blank control: obtaining an A group of detected objects for detection by untreated zebra fish of 3 days old; group B is the hydrazine treatment control group: incubating normal 3-day-old zebra fish with 10mL of 50-mu M hydrazine to obtain B group of detected substances for detection for 30 minutes;
group C is near infrared fluorescent compound treatment control: treating 3-day-old zebra fish with 10mL of the near-infrared fluorescent compound of
group D is a near-infrared fluorescent compound and hydrazine treatment group: 3 days old zebra fish are incubated with 10mL of hydrazine with a concentration of 50 μ M for 30 minutes, and then treated with 20 μ M of the near-infrared fluorescent compound according to claim 1 for 30 minutes to obtain D groups of test substances for detection;
(3) respectively placing the group A detected object, the group B detected object, the group C detected object and the group D detected object under a fluorescence microscope with the excitation wavelength of 520nm for observation;
(4) the result shows that the group A detected object, the group B detected object and the group C detected object do not find fluorescence; and the D group of detected substances show obvious red fluorescence; fluorescence imaging results show that the near-infrared fluorescent compound of
The operation steps of the near-infrared fluorescent compound for detecting hydrazine in an environmental water sample are as follows:
(1) preparation of the working solution of the near-infrared fluorescent compound according to
Formulating the near-infrared fluorescent compound of
(2) preparation of environmental water sample to be detected
When the method is used for detecting exogenous hydrazine in an environmental water sample, the environmental water samples in different areas are collected and filtered through a 200-micron water-phase filter membrane to obtain a treated environmental water sample; addition of 0.1. mu.M, 0.5. mu.M and 1.0. mu.M hydrazine to the treated environmental water sample
(3) Adding different environmental water samples to be detected into the near-infrared fluorescent compound working solution, and detecting the reaction solution by using a fluorescence spectrometer;
(4) the recovery rate of hydrazine reaches 87-109%, and the result shows that the near-infrared fluorescent compound working solution can quantitatively detect the existence of hydrazine in an environmental water sample.
The operation steps of the near-infrared fluorescent compound for detecting hydrazine in sewage are as follows:
(1) formulating the near-infrared fluorescent compound of
(2) when the method is used for detecting exogenous hydrazine in a sewage sample, collecting sewage samples of different regional chemical plants, and filtering the sewage samples through a 200-micron water-phase filter membrane to obtain a sewage sample to be detected;
(3) detecting a reaction liquid of a near-infrared fluorescent compound working solution and a sewage sample by using a fluorescence spectrometer;
(4) the results indicate that the near-infrared fluorescent compound of
1. the near-infrared fluorescent compound provided by the invention has the advantages of simple preparation steps, easily available raw materials and easiness in synthesis, the synthesis of the near-infrared fluorescent compound is completed by only one-step acylation reaction, and the reaction conditions are mild.
2. The N atom of the hydrazine of the near-infrared fluorescent compound detection object can perform nucleophilic substitution reaction with the carbon atom near the bromine atom on the 4-bromobutyryl of the near-infrared fluorescent compound. Then another N atom of hydrazine attacks carbonyl carbon atom on the near-infrared fluorescent compound, and the addition cyclization cracking generates stable six-membered ring cyclization product tetrahydropyridazine (ESI MS [ M + H ] M]+, m/z: 101.0710) and the compound DHDM (ESI MS [ M + H ]]+, m/z: 362.2228) that produce identifiable fluorescence (FIGS. 1-2), thus eliminating interference from common analytes in solutions and organisms, and with detection limits as low as 39.6nM, which is a great advantage over most near infrared fluorescent compounds of the same type; the results in FIG. 3 show that the near infrared fluorescent compound has a good selectivity for hydrazine. As shown in FIG. 4, the fluorescence intensity of the near-infrared fluorescent compound increases with the increase of hydrazine content, and has a good linear relationship (R)20.9967), namely the near infrared fluorescent compound can quantitatively detect trace hydrazine in the water environment.
3. The near-infrared fluorescent compound has potential application value. The near-infrared fluorescent compound can rapidly and efficiently distinguish hydrazine in an aqueous solution through the preparation of a test strip, and the near-infrared fluorescent compound has good cell membrane permeability and low cytotoxicity, so that the near-infrared fluorescent compound can be successfully applied to the imaging of hydrazine in living cells and zebra fish bodies, and has good biological application potential; (1) the near-infrared fluorescent compound has potential application value. As shown in figure 5, the test strip containing the near-infrared fluorescent compound can quickly and efficiently carry out specific identification and preliminary quantitative detection on hydrazine in a water environment.
(2) The near infrared fluorescent compound logP is 3.98, belongs to a lipophilic compound, is easier to enter cells, and has better cell membrane permeability;
(3) the near-infrared fluorescent compound has low cytotoxicity. As shown in fig. 6a, in the presence of the near-infrared fluorescent compound at a concentration of 40 μ M, the survival rate of HeLa cells was still over 85%, indicating that the near-infrared fluorescent compound has low cytotoxicity;
(4) the near-infrared fluorescent compound has good biological application potential. As shown in the results of fig. 6b, the untreated HeLa cells showed significant red fluorescence after 30 minutes incubation with the near-infrared fluorescent compound and hydrazine, respectively, while no change in fluorescence was observed in the HeLa cells of the other treatment groups; as shown in FIG. 7, red fluorescence was observed under a fluorescence microscope after incubating zebrafish with near-infrared fluorescent compound (20. mu.M) and hydrazine (50. mu.M), respectively, for 30 minutes. However, no significant fluorescence change was observed when zebrafish in the other treatment groups.
Drawings
FIG. 1 is a high resolution mass spectrum of the reaction product of (E) -2- (2- (3- (dicyanomethylene) -5, 5-dimethylcyclohex-1-en-1-yl) vinyl) -5- (diethylamino) phenyl 4-bromobutyric acid with hydrazine;
FIG. 2 is a NMR chart of a reaction product of (E) -2- (2- (3- (dicyanomethylene) -5, 5-dimethylcyclohex-1-en-1-yl) vinyl) -5- (diethylamino) phenyl 4-bromobutyric acid and hydrazine;
FIG. 3 is a graph of fluorescence emission and UV spectra of (E) -2- (2- (3- (dicyanomethylene) -5, 5-dimethylcyclohex-1-en-1-yl) vinyl) -5- (diethylamino) phenyl 4-bromobutyric acid in the presence of various analytes;
FIG. 4 is a fluorescence emission spectrum of (E) -2- (2- (3- (dicyanomethylene) -5, 5-dimethylcyclohex-1-en-1-yl) vinyl) -5- (diethylamino) phenyl 4-bromobutanoic acid and hydrazine;
FIG. 5 is a graph of the UV luminescence of (E) -2- (2- (3- (dicyanomethylene) -5, 5-dimethylcyclohex-1-en-1-yl) vinyl) -5- (diethylamino) phenyl 4-bromobutyric acid test strip with different analytes;
FIG. 6 is a fluorescence micrograph of (E) -2- (2- (3- (dicyanomethylene) -5, 5-dimethylcyclohex-1-en-1-yl) vinyl) -5- (diethylamino) phenyl 4-bromobutyric acid in HeLa cells;
FIG. 7 is a fluorescent micrograph of (E) -2- (2- (3- (dicyanomethylene) -5, 5-dimethylcyclohex-1-en-1-yl) vinyl) -5- (diethylamino) phenyl 4-bromobutyric acid incubated in 3-day-old zebra fish.
Detailed Description
The invention discloses a near-infrared fluorescent compound capable of specifically detecting hydrazine and a preparation method thereof. The near-infrared fluorescent compound is characterized by consisting of two parts, wherein 4-bromobutyryl is used as a recognition group, and (E) -2- (3- (4- (diethylamino) -2-hydroxystyryl) -5, 5-dimethylcyclohex-2-en-1-yl) malononitrile is used as an information report group. 4-bromobutyryl on the reported near-infrared fluorescent compound can perform specific reaction with hydrazine in a system, so that the fluorescence of the near-infrared fluorescent compound is changed, and the specific detection of the hydrazine is realized.
The invention will now be further described with reference to the following examples
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