CN115260120B - ESIPT fluorescent compound for hydrazine specific detection and preparation method and application thereof - Google Patents
ESIPT fluorescent compound for hydrazine specific detection and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses an ESIPT fluorescent compound for hydrazine specific detection, a preparation method and application thereof, wherein the fluorescent compound is 2- (benzo [ d ] thiazole-2-yl) -1, 4-phenylene bis (4-bromobutyrate), and can specifically detect hydrazine in cells and organisms; the fluorescent dye has better cell membrane permeability and lower cytotoxicity, can enter living cells and rapidly undergo Michael addition cyclization reaction with extracellular and intracellular source hydrazine, and generates intense fluorescence which can be distinguished by naked eyes; the fluorescent compound obtained by analysis of a fluorescence spectrophotometry can have excellent selectivity on hydrazine under various interferents and has strong anti-interference capability on common biomolecules; the fluorescent compound also has the potential of quantitatively detecting hydrazine in water samples in different environments; not only can selectively identify the intracellular and extracellular endogenous hydrazine, but also can detect the hydrazine with high sensitivity in the growth environment of various living cells, and can be applied to living cells and zebra fish imaging.
Description
Technical Field
The invention relates to the technical field of small organic molecule fluorescent probes, in particular to an ESIPT fluorescent compound for hydrazine specific detection, a preparation method and application thereof.
Background
Inorganic pollutants in the environment are widely available and of complex types, most of the pollutants can enter human bodies, when the content of the pollutants exceeds a safety threshold, the pollutants possibly threatens the environment and human health, and hydrazine (N2H 4) is an important inorganic compound and has a certain reducibility, and is widely applied to the fields of pesticide production, aerospace, chemical industry and the like, however, the environment is also a highly toxic water-soluble biochemical reagent, cancerogenic, teratogenic and mutagenic effects are realized, N2H4 can cause damage to livers, respiratory systems and nervous systems through oronasal respiration and skin permeation, no clear evidence exists at present, whether the human bodies contain endogenous N2H4 or not can be shown, but part of the medicines entering the human bodies can generate N2H4, for example, isonicotinyl hydrazine is a specific drug for treating recessive and active tuberculosis and can be metabolized into hydrazine, liver is damaged, the U.S. environmental protection agency (USEPA) and health organization (WHO) have determined N2H4 as potential cancerogenic substances, the threshold (0.32 mu.M) is allowed, a highly sensitive and sensitive method for detecting N2H4 can be developed and the environmental impact can be carried out in the human body by a high-sensitive method to the human body, and the human body can be subjected to the experimental method of the environmental evaluation of N2H 4;
detection of N2H4 generally uses chromatography-mass spectrometry, surface enhanced raman spectroscopy, electrochemistry and chemiluminescence detection, which have the defects of high cost, complex sample pretreatment and operation procedures, long analysis time, expensive instrument requirement and the like, compared with fluorescence detection, which has the advantages of simple operation, high sensitivity and low cost, in particular, the method can perform noninvasive analysis on biological samples, is suitable for imaging analysis of living cells and even whole organisms, and fluorescent probes have been the preferred method for detecting certain environmental pollutants and endogenous biomolecules so far;
to date, many chemical sensors for detecting N2H4 have been reported, using several mechanisms in the detection of N2H4, such as Gabriel reaction, hydrazone formation, addition reaction with aldehyde, and ester hydrolysis reaction, wherein the use of a hydrazine hydrolysis reaction can improve the detection response rate, and since the first brominated fluorescent probe was developed by Sarkar team 2013, the rapid development of a fluorescent probe based on hydrazine reaction, as a typical hydrazine hydrolysis reaction, the hydroxyl group on the fluorescent structure may be an effective hydrazine recognition site, and more encouraging that the introduction of the hydroxyl group into the structure of the fluorescent probe is easily achieved;
in summary, 2- (benzo [ d ] thiazole-2-yl) benzene-1, 4-diphenol is selected as a fluorescent parent structure, 4-bromobutyryl ester is utilized to modify hydroxyl on 2- (benzo [ d ] thiazole-2-yl) benzene-1, 4-diphenol, self fluorescence is quenched, sensitivity is improved, and the novel fluorescent compound which can specifically identify hydrazine and can be applied to cell and living tissue imaging is hopeful to be developed;
therefore, there is a need to provide an esit fluorescent compound for hydrazine specific detection, and a preparation method and application thereof to solve the above technical problems.
Disclosure of Invention
The first technical problem to be solved by the invention is to develop a fluorescent compound capable of selectively detecting hydrazine, which has the characteristics of high specificity, high anti-interference performance and the like.
The second technical problem to be solved by the invention is to provide a preparation method of a fluorescent compound capable of selectively detecting hydrazine.
The third technical problem to be solved by the invention is to research a novel method capable of visualizing the migration distribution rule of exogenous hydrazine in cells and living bodies.
In order to achieve the above purpose, the present invention provides the following technical solutions: an esit fluorescent compound for hydrazine specific detection comprising 2- (benzo [ d ] thiazol-2-yl) -1, 4-phenylenebis (4-bromobutyrate) having the chemical structural formula shown in formula I:
the invention also provides a preparation method of the ESIPT fluorescent compound for hydrazine specific detection, which comprises the following operation steps:
(1) 2- (benzo [ d ] thiazole-2-yl) benzene-1, 4-diphenol is prepared by reacting 2-amino thiophenol, 2, 5-dihydroxybenzaldehyde and sodium thiosulfate;
(2) 2- (benzo [ d ] thiazol-2-yl) -1, 4-phenylenebis (4-bromobutyrate) is prepared from 2- (benzo [ d ] thiazol-2-yl) benzene-1, 4-diol obtained in step (1) by modification of 4-bromobutyryl ester.
Specifically, in step (1), 500mg (3.99 mmol) of 2-aminophenylsulfol and 580mg (4.2 mmol) of 2, 5-dihydroxybenzaldehyde are dissolved in 20mL of N, N-dimethylformamide, and 0.70g (4.20 mmol) of sodium thiosulfate is added under continuous stirring; the reaction mixture was refluxed for 2 hours and monitored by TLC, after completion of the reaction, cooled to room temperature, 50mL of water was added, and the solid precipitate was collected on a suction filter funnel and recrystallized from methanol to yield 702mg (72%) of 2- (benzo [ d ] thiazol-2-yl) benzene-1, 4-diol as a white solid.
Specifically, in step (2), 730mg (3 mmol) of 2- (benzo [ d ] thiazol-2-yl) benzene-1, 4-diol as above and 455mg (4.5 mmol) of triethylamine were dissolved in 60mL of methylene chloride under ice bath conditions; 668mg (3.6 mmol) of 4-bromobutyryl chloride was slowly added to the mixture using a constant pressure dropping funnel and then reacted at room temperature, the progress of the reaction was monitored by thin layer chromatography silica gel plate (TLC), and when the reaction was completed, the solvent was evaporated to dryness and the product was separated by eluting silica gel column to obtain 740mg (76%) of 2- (benzo [ d ] thiazol-2-yl) -1, 4-phenylenebis (4-bromobutyrate) as a white solid, and the eluent used for eluting silica gel column was petroleum ether and ethyl acetate in a volume ratio of 3: 1.
The invention also provides application of the ESIPT fluorescent compound for hydrazine specific detection, wherein the fluorescent compound is used as a fluorescent chemical sensor for detecting hydrazine.
Further, the ESIPT fluorescent compound for hydrazine specific detection is used for detecting hydrazine in a solution system, and the operation steps are as follows:
(a) The fluorescent compound was prepared into a fluorescent compound solution with a concentration of 10 μm by using a buffer solution of 4-hydroxyethyl piperazine ethane sulfonic acid (HEPES) and dimethyl sulfoxide (DMSO), wherein the volume ratio of HEPES to DMSO in the buffer solution is 1:9, the pH value of the buffer solution is 7.4;
(b) Adding 100 mu M of aqueous solutions of tryptophan (Try), cysteine (Cys), threonine (Thr), tyrosine (Tyr), histidine (His), glutamic acid (Glu) and aspartic acid (Asp), potassium chloride (KCl), calcium chloride (CaCl 2), sodium chloride (NaCl), magnesium chloride (MgCl 2), copper chloride (CuCl 2), barium chloride (BaCl 2), ferric chloride (FeCl 3), silver chloride (AgCl) and nickel chloride (NiCl 2) sodium hydrosulfide (NaHS) into the 10 mu M fluorescent compound solution prepared in the step (a), and measuring the fluorescence intensity after the reaction is completed;
(c) Through research on the relationship between the fluorescence intensity and the reaction time, the fluorescent compound is found to generate fluorescence enhancement only on hydrazine, namely the fluorescent compound can specifically recognize the hydrazine.
The ESIPT fluorescent compound for hydrazine specific detection is used for quantitative detection of hydrazine in an environmental water sample, and comprises the following operation steps:
(a) The fluorescent compound was prepared into a fluorescent compound solution with a concentration of 10 μm by using a buffer solution of 4-hydroxyethyl piperazine ethane sulfonic acid (HEPES) and dimethyl sulfoxide (DMSO), wherein the volume ratio of HEPES to DMSO in the buffer solution is 1:9, the pH value of the buffer solution is 7.4;
(b) Selecting different types of environmental water samples, adding hydrazine with the concentration of 0.10,0.50 and 1.0mg/L into the different environmental water samples, and carrying out an experiment of adding and recycling the hydrazine by using the fluorescent compound prepared in the step (a) by utilizing the specificity detection of the hydrazine, wherein the recycling rate of 72-111% is obtained by comparing the added concentration, so that the fluorescent compound can quantitatively detect the hydrazine in the different environmental water samples.
The ESIPT fluorescent compound for hydrazine specific detection is used for exogenous hydrazine detection in cervical cancer cells (HeLa), and the operation steps are as follows:
(a) The fluorescent compound was prepared into a 20 μm concentration fluorescent compound solution by using 4-hydroxyethyl piperazine ethane sulfonic acid (HEPES) and dimethyl sulfoxide (DMSO) buffer solution, wherein the volume ratio of HEPES to DMSO in the buffer solution is 1:9, the pH value of the buffer solution is 7.4;
(b) Five experimental groups A, B, C, D and E were set up:
group a blank control: heLa cells without any treatment;
group B hydrazine treatment control: heLa cells were incubated with 50. Mu.M hydrazine solution for 30 min;
group C probe treatment control: heLa cells were incubated with 2- (benzo [ d ] thiazol-2-yl) -1, 4-phenylenedi (4-bromobutyrate) (20. Mu.M) for 30 min;
group D experimental group 1: heLa cells were incubated with 50 μΜ hydrazine for 30 min followed by 2- (benzo [ d ] thiazol-2-yl) -1, 4-phenylenedi (4-bromobutyrate) (20 μΜ) for 30 min;
group E, experimental group 2: heLa cells were incubated with 100 μΜ hydrazine for 30 min followed by 2- (benzo [ d ] thiazol-2-yl) -1, 4-phenylenedi (4-bromobutyrate) (20 μΜ) for 30 min;
(a) The results of the A, B, C, D, E group fluorescence imaging show that 2- (benzo [ d ] thiazol-2-yl) -1, 4-phenylene bis (4-bromobutyrate) can enter cells and react with exogenous hydrazine in the cells, and the distribution of the exogenous hydrazine in the cells can be obviously seen by the cell imaging.
The ESIPT fluorescent compound for hydrazine specific detection is used for exogenous hydrazine detection in zebra fish, and comprises the following operation steps:
(a) The fluorescent compound was prepared into a 20 μm concentration fluorescent compound solution by using 4-hydroxyethyl piperazine ethane sulfonic acid (HEPES) and dimethyl sulfoxide (DMSO) buffer solution, wherein the volume ratio of HEPES to DMSO in the buffer solution is 1:9, the pH value of the buffer solution is 7.4;
(b) Four experimental groups a, B, C, D were set up:
group a blank control: untreated 3-day-old zebra fish;
group B probe treatment control: 3 day old zebra fish treated with 2- (benzo [ d ] thiazol-2-yl) -1, 4-phenylenebis (4-bromobutyrate) (30 μm);
group C hydrazine treatment control: zebra fish of 3 days of age treated with hydrazine (50. Mu.M);
group D experimental group: zebra fish of 3 days of age were treated with hydrazine for 30 minutes and then incubated with 2- (benzo [ d ] thiazol-2-yl) -1, 4-phenylenedi (4-bromobutyrate) (30 μm) for 30 minutes;
(c) The results show that zebra fish treated simultaneously with 2- (benzo [ d ] thiazol-2-yl) -1, 4-phenylenedi (4-bromobutyrate) and exogenous hydrazine at 28 ℃ showed significant fluorescence, whereas zebra fish untreated or zebra fish not treated simultaneously with 2- (benzo [ d ] thiazol-2-yl) -1, 4-phenylenedi (4-bromobutyrate) and exogenous hydrazine were not found to be fluorescent; the fluorescence imaging result shows that 2- (benzo [ d ] thiazole-2-yl) -1, 4-phenylene bis (4-bromobutyrate) can enter a zebra fish body and react with exogenous hydrazine to generate strong fluorescence, so that the detection effect is achieved;
(d) The reaction mechanism of the fluorescent compound is that one N atom of hydrazine is firstly subjected to nucleophilic substitution reaction with a carbon atom connected with a bromine atom on 2- (benzo [ d ] thiazole-2-yl) -1, 4-phenylene bis (4-bromobutyrate), and Br atoms are substituted; then, another N atom of hydrazine attacks the carbonyl C atom on 2- (benzo [ d ] thiazol-2-yl) -1, 4-phenylene bis (4-bromobutyrate), stable six-membered ring compound is generated through addition cyclization, and the other 4-bromobutyryl on 2- (benzo [ d ] thiazol-2-yl) -1, 4-phenylene bis (4-bromobutyrate) also reacts through the mechanism, so that the fluorescent group 2- (benzo [ d ] thiazol-2-yl) benzene-1, 4-diphenol is completely released to lead to fluorescence enhancement, and the aim of detecting the hydrazine is achieved.
The beneficial technical effects of the invention are as follows:
1. the fluorescent compound is synthesized in only two steps, and has mild reaction conditions and easy synthesis;
2. the fluorescent compound has strong anti-interference capability on common analytes in organisms, can effectively detect hydrazine under the growth environment of various living cells, and can effectively remove the interference of other common analytes due to the specificity of a hydrazine hydrolysis mechanism;
3. the fluorescent compound can have excellent selectivity for hydrazine under various interferents;
4. the detection limit of the fluorescent compound on Cys is as low as 0.1 mu M;
5. the fluorescent compound has better cell membrane permeability and lower cytotoxicity. The oil-water partition coefficient of the fluorescent compound logp=5.91 shows that the fluorescent compound is a lipophilic compound, i.e. can easily enter cells. As shown in fig. 5, cytotoxicity assays at different concentrations indicated that IPPA had lower cytotoxicity;
6. the hydrazine concentration in the actual water sample can be effectively detected. As shown in Table 6, hydrazine (0.10,0.50 and 1.0 mg/L) with different concentrations is added into different environmental water samples, and the recovery rate of the hydrazine is measured to be 72-111% by using the fluorescent compound, so that the fluorescent compound can be used for effectively and quantitatively detecting the hydrazine in the environmental water samples;
7. the fluorescent compound has biological application potential for detecting the intracellular exogenous hydrazine, as shown in a result of fig. 7, the zebra fish which is not treated by the 2- (benzo [ d ] thiazol-2-yl) -1, 4-phenylene bis (4-bromobutyrate) and the exogenous hydrazine simultaneously does not observe fluorescence change, the zebra fish which is treated by the 2- (benzo [ d ] thiazol-2-yl) -1, 4-phenylene bis (4-bromobutyrate) and the exogenous hydrazine simultaneously does not observe fluorescence change, after the zebra fish is treated by the 2- (benzo [ d ] thiazol-2-yl) -1, 4-phenylene bis (4-bromobutyrate) and the exogenous hydrazine simultaneously for 30 minutes, the fluorescent compound (30 mu M) is used for incubation with the exogenous hydrazine respectively, the cell imaging result shows strong green fluorescence after the zebra fish is incubated for 30 minutes, the fluorescent compound has biological application potential for detecting the intracellular exogenous hydrazine, as shown in fig. 7, the zebra fish which is not treated by the 2- (benzo [ d ] thiazol-2-yl) -1, 4-phenylene bis (4-bromobutyrate) and the exogenous hydrazine simultaneously does not observe fluorescence change, when the zebra fish is incubated with the exogenous hydrazine for 30 minutes, the zebra fish is accurately and can not display the fluorescence in the stomach, and the zebra fish can be accurately and can not display the exogenous hydrazine in the stomach.
Drawings
FIG. 1 is a nuclear magnetic resonance hydrogen spectrum of 2- (benzo [ d ] thiazol-2-yl) -1, 4-phenylenedi (4-bromobutyrate) before and after reaction with hydrazine;
FIG. 2 is a high resolution mass spectrum of the reaction product of 2- (benzo [ d ] thiazol-2-yl) -1, 4-phenylenedi (4-bromobutyrate) with hydrazine;
FIG. 3 is a graph showing fluorescence emission spectra of a reaction in which different amino acids and compounds such as common ions of human bodies are added into a working solution of 2- (benzo [ d ] thiazol-2-yl) -1, 4-phenylene bis (4-bromobutyrate);
FIG. 4 is a density pan function theoretical calculation of the reaction product of 2- (benzo [ d ] thiazol-2-yl) -1, 4-phenylenebis (4-bromobutyrate) and hydrazine;
FIG. 5 is a bar graph of the effect of varying concentrations of 2- (benzo [ d ] thiazol-2-yl) -1, 4-phenylenedi (4-bromobutyrate) on cell viability;
FIG. 6 is a fluorescent imaging of 2- (benzo [ d ] thiazol-2-yl) -1, 4-phenylenedi (4-bromobutyrate) as exogenic hydrazine in HeLa cells;
FIG. 7 is a fluorescent imaging of exogenous hydrazine in 3 day old zebra fish with 2- (benzo [ d ] thiazol-2-yl) -1, 4-phenylene bis (4-bromobutyrate);
Detailed Description
The invention discloses a fluorescent compound capable of specifically detecting hydrazine, a preparation method and application thereof, and is characterized in that the fluorescent compound consists of two parts, wherein 4-bromobutyryl is taken as a recognition group, 2- (benzo [ d ] thiazole-2-yl) benzene-1, 4-diphenol is taken as an information reporting group, and the 4-bromobutyryl on the reported fluorescent compound can react specifically with hydrazine in a system to change the fluorescence of the fluorescent compound, thereby realizing specific detection of the hydrazine.
The invention will be further described with reference to the following examples
EXAMPLE 1 preparation of fluorescent Compounds for hydrazine detection
The fluorescent compound is 2- (benzo [ d ] thiazole-2-yl) -1, 4-phenylene bis (4-bromobutyrate), and the specific preparation process is as follows:
500mg (3.99 mmol) of 2-aminophenylthiophenol and 580mg (4.2 mmol) of 2, 5-dihydroxybenzaldehyde are dissolved in 20mL of N, N-dimethylformamide, and 0.70g (4.20 mmol) of sodium thiosulfate is added with continuous stirring; the reaction mixture was refluxed for 2 hours and the reaction was monitored by TLC. After the reaction was completed, 50mL of water was added after cooling to room temperature. The solid precipitate was collected on a suction filter funnel and recrystallized from methanol to yield 702mg (72%) of 2- (benzo [ d ] thiazol-2-yl) benzene-1, 4-diol as a white solid. Nuclear magnetic resonance hydrogen spectrometry: 1H NMR (600 MHz, DMSO-d 6): delta 10.84 (t, J=3.0 Hz, 1H), 9.17 (t, J=3.1 Hz, 1H), 8.12 (dt, J=7.1, 2.6Hz, 1H), 8.00 (dd, J=7.6, 3.1Hz, 1H), 7.65-7.47 (m, 2H), 7.41 (dt, J=9.0, 5.6Hz, 1H), 6.91-6.88 (m, 1H), 6.83 (dq, J=8.7, 3.4,2.9Hz, 1H). HRMS (ESI, m/z) calculated for [ C13H9NO2S+H ] +:244.0432, found: 244.0427.
(2) 730mg (3 mmol) of 2- (benzo [ d ] thiazol-2-yl) benzene-1, 4-diol and 455mg (4.5 mmol) of triethylamine were dissolved in 60mL of dichloromethane under ice-bath conditions; 668mg (3.6 mmol) of 4-bromobutyryl chloride was slowly added to the mixture using a constant pressure dropping funnel and then reacted at room temperature, the progress of the reaction was monitored by thin layer chromatography silica gel plate (TLC), when the reaction was completed, the solvent was evaporated to dryness, and the product was separated by eluting silica gel column to obtain 740mg (76%) of 2- (benzo [ d ] thiazol-2-yl) -1, 4-phenylenebis (4-bromobutyrate) as a white solid, the eluent was prepared from petroleum ether and ethyl acetate in a volume ratio of 3: 1. Nuclear magnetic resonance hydrogen spectrum: 1H NMR (600 mhz, dmso-d 6) delta 8.17-8.13 (m, 1H), 8.13-8.00 (m, 2H), 7.57 (ddd, j=8.3, 7.2,1.3hz, 1H), 7.51-7.45 (m, 2H), 7.41 (dt, j=8.7, 2.3hz, 1H), 3.75 (td, j=6.5, 1.4hz, 1H), 3.64 (t, j=6.6 hz, 3H), 2.97 (td, j=7.2, 2.7hz, 2H), 2.79 (t, j=7.3 hz, 2H), 2.24-2.12 (m, 4H) ·nuclear magnetic resonance carbon spectrum: 13C NMR (151 mhz, dmso-d 6) delta 171.39,171.33,161.36,152.56,148.63,145.76, 135.27,127.29,126.70,126.38,123.50,122.84,122.64,44.93,44.81, 34.34,34.24,33.05,32.61,27.93,27.82. High resolution mass spectrometry: HRMS (ESI, m/z) calcd for [ C21H19Br2NO4S+H ] +:539.9474, found:539.9465.
The detection principle of the fluorescent compound 2- (benzo [ d ] thiazole-2-yl) -1, 4-phenylene bis (4-bromobutyrate) is as follows:
the mechanism for specifically detecting hydrazine of the fluorescent compound 2- (benzo [ d ] thiazole-2-yl) -1, 4-phenylene bis (4-bromobutyrate) is as follows: after addition of hydrazine, the proton signal on the 4-bromobutyryl group in 2- (benzo [ d ] thiazol-2-yl) -1, 4-phenylenedioyl bis (4-bromobutyrate) disappears from 3.3ppm to 3.8ppm, and a new proton signal appears near 8.0ppm, corresponding to the hydrogen atoms on the two phenolic hydroxyl groups on the fluorescent group 2- (benzo [ d ] thiazol-2-yl) benzene-1, 4-diol (FIG. 1), the N atom contains a lone pair electron, which results in that the hydrazine has strong nucleophilicity, when the hydrazine reacts with the fluorescent compound, one of the N atoms of the hydrazine first reacts with the bromine atom on 2- (benzo [ d ] thiazol-2-yl) -1, 4-phenylenedioyl bis (4-bromobutyrate), and the bromine atom is substituted; then, the other N atom of hydrazine attacks the carbonyl C atom on 2- (benzo [ d ] thiazol-2-yl) -1, 4-phenylene bis (4-bromobutyrate) and a stable six-membered ring compound is generated through addition cyclization, the compound with molecular weight of 101.0789 found in a high resolution mass spectrum (figure 2) verifies the assumption, the other 4-bromobutyryl group on 2- (benzo [ d ] thiazol-2-yl) -1, 4-phenylene bis (4-bromobutyrate) also reacts through the mechanism, so that the fluorescent group 2- (benzo [ d ] thiazol-2-yl) benzene-1, 4-bisphenol is completely released to enhance fluorescence, the purpose of specifically detecting hydrazine is achieved, and the appearance of the compound with molecular weight of 244.0426 in the high resolution mass spectrum also indicates the establishment of the mechanism.
To further verify the reaction mechanism of the fluorescent compound 2- (benzo [ d ] thiazol-2-yl) -1, 4-phenylenebis (4-bromobutyrate) with hydrazine, the spatial distribution and orbital energy of 2- (benzo [ d ] thiazol-2-yl) -1, 4-phenylenebis (4-bromobutyrate) and 2- (benzo [ d ] thiazol-2-yl) benzene-1, 4-diol were optimized in the gaussian 09 model in combination with density pan function theoretical calculations. As shown in FIG. 3, HOMO is uniformly distributed at the fluorophore sites, while LUMO is distributed near 4-bromobutyryl. 2- (benzo [ d ] thiazol-2-yl) -1, 4-phenylenedi (4-bromobutyrate) reacted with hydrazine, resulting in complete transfer of the HOMO orbital to the fluorophore group, resulting in complete release of the 2- (benzo [ d ] thiazol-2-yl) benzene-1, 4-diol. In addition, the energy level differences (Δe=ehomo-ELUMO) of 2- (benzo [ d ] thiazol-2-yl) -1, 4-phenylenebis (4-bromobutyrate) and 2- (benzo [ d ] thiazol-2-yl) benzene-1, 4-diol were 3.66eV and 2.45eV, respectively. 2- (benzo [ d ] thiazol-2-yl) -1, 4-phenylenebis (4-bromobutyrate) is more susceptible to electronic transitions than 2- (benzo [ d ] thiazol-2-yl) benzene-1, 4-diol. The density pan function theory calculation result is consistent with the conclusion.
Example 2 Selective detection of hydrazine in a solution System
Fluorescent compound detection solution with concentration of 10 mu M is prepared by using 4-hydroxyethyl piperazine ethane sulfonic acid (HEPES)/dimethyl sulfoxide (DMSO) buffer solution and the fluorescent compound prepared in the example 1, and hydrazine in the solution is selectively detected; the specific operation process is as follows:
10 times equivalent tryptophan (Try), cysteine (Cys), threonine (Thr), tyrosine (Tyr), histidine (His), glutamic acid (Glu) and aspartic acid (Asp), potassium chloride (KCl), calcium chloride (CaCl 2), sodium chloride (NaCl), magnesium chloride (MgCl 2), copper chloride (CuCl 2), barium chloride (BaCl 2), ferric chloride (FeCl 3), silver chloride (AgCl), nickel chloride (NiCl 2) sodium hydrosulfide (NaHS) are respectively added into the prepared 10 mu M fluorescent compound solution, after the reaction is completed, the fluorescent intensity measurement is carried out, the result of FIG. 4 shows that 2- (benzo [ d ] thiazol-2-yl) -1, 4-phenylene bis (4-bromobutyrate) has better selectivity to hydrazine, and under the interference of various analytes, the specificity detection of 2- (benzo [ d ] thiazol-2-yl) -1, 4-phenylene bis (4-bromobutyrate) to the hydrazine can be completed, namely the fluorescent compound has the characteristics of specific recognition, strong interference resistance and the like.
Example 3 application to specific detection of hydrazine in environmental Water samples
When the kit is used for quantitatively detecting hydrazine in an environmental water sample, a working solution with the concentration of 10 mu M is prepared by using a 4-hydroxyethyl piperazine ethane sulfonic acid (HEPES)/dimethyl sulfoxide (DMSO) buffer solution and the fluorescent compound;
the specific operation process is as follows: collecting environmental water samples in different water areas, and filtering the water samples for later use; by adding hydrazine with different concentrations (0.1, 0.5 and 1 mg/L) into collected water samples respectively, carrying out an addition recovery test, reacting the prepared fluorescent compound (10 mu M) with the hydrazine in different water samples for 20 minutes, and measuring the change of the fluorescence intensity by a fluorescence spectrophotometer, the results shown in the table 1 show that the addition recovery rate is in the range of 72-111%, namely 2- (benzo [ d ] thiazol-2-yl) -1, 4-phenylene bis (4-bromobutyrate) can carry out quantitative detection on the hydrazine in different environmental water samples;
TABLE 1N in Water samples of different environments 2 H 4 Additive recovery test of (2)
a The water sample is collected at a plurality of points, and the number of sampling points is not less than 5. Data are mean ± SE (bars) (n=3).
EXAMPLE 4 cytotoxicity
When the fluorescent compound is used for verifying the HeLa cytotoxicity of cervical cancer tissues, a working solution with the concentration of 10 mu M is prepared by using a 4-hydroxyethyl piperazine ethane sulfonic acid (HEPES)/dimethyl sulfoxide (DMSO) buffer solution and the fluorescent compound;
the specific operation process is as follows: heLa cells were cultured in DMEM and 10% calf serum at 37℃was added to 5% carbon dioxide, then, 2- (benzo [ d ] thiazol-2-yl) -1, 4-phenylenebis (4-bromobutyrate) (0-40. Mu.M) was added to HeLa cells (5X 104 per well) at various concentrations, and after 24h incubation, cell viability was examined by MTT assay, with increasing concentration of 2- (benzo [ d ] thiazol-2-yl) -1, 4-phenylenebis (4-bromobutyrate), heLa cells were progressively decreased in activity, and when concentration of 2- (benzo [ d ] thiazol-2-yl) -1, 4-phenylenebis (4-bromobutyrate) reached 40. Mu.M, the minimum viability of HeLa cells was still over 73% and the average viability after 24h incubation was over 75% (FIG. 5), indicating that 2- (benzo [ d ] thiazol-2-yl) -1, 4-phenylenebis (4-bromobutyrate) had lower cytotoxicity and was suitable for cell imaging.
EXAMPLE 5 HeLa intracellular fluorescence imaging
When the kit is used for detecting hydrazine in HeLa cells of cervical cancer tissues, a working solution with the concentration of 20 mu M is prepared by using a 4-hydroxyethyl piperazine ethane sulfonic acid (HEPES)/dimethyl sulfoxide (DMSO) buffer solution and the fluorescent compound;
the specific operation process is as follows: five experimental groups a, B, C, D, E were performed:
group a blank control: heLa cells without any treatment;
group B hydrazine treatment control: heLa cells were incubated with 50. Mu.M hydrazine solution for 30 min;
group C probe treatment control: heLa cells were incubated with 2- (benzo [ d ] thiazol-2-yl) -1, 4-phenylenedi (4-bromobutyrate) (20. Mu.M) for 30 min;
group D experimental group 1: heLa cells were incubated with 50 μΜ hydrazine for 30 min followed by 2- (benzo [ d ] thiazol-2-yl) -1, 4-phenylenedi (4-bromobutyrate) (20 μΜ) for 30 min;
group E, experimental group 2: heLa cells were incubated with 100 μΜ hydrazine for 30 min followed by 2- (benzo [ d ] thiazol-2-yl) -1, 4-phenylenedi (4-bromobutyrate) (20 μΜ) for 30 min; the results of the A, B, C, D, E group fluorescence imaging show that the 2- (benzo [ d ] thiazole-2-yl) -1, 4-phenylene bis (4-bromobutyrate) can enter cells and react with exogenous hydrazine in the cells, and the distribution of the exogenous hydrazine in the cells can be obviously seen by the cell imaging.
Example 6 fluorescence imaging of zebra fish
When the kit is used for in-vivo hydrazine detection of zebra fish, a working solution with the concentration of 20 mu M is prepared by using a 4-hydroxyethyl piperazine ethane sulfonic acid (HEPES)/dimethyl sulfoxide (DMSO) buffer solution and the fluorescent compound;
the specific operation process is as follows: four test groups A, B, C, and D were performed:
group a blank control: untreated 3-day-old zebra fish;
group B probe treatment control: 3 day old zebra fish treated with 2- (benzo [ d ] thiazol-2-yl) -1, 4-phenylenebis (4-bromobutyrate) (30 μm);
group C hydrazine treatment control: zebra fish of 3 days of age treated with hydrazine (50. Mu.M);
group D experimental group: zebra fish of 3 days of age were treated with hydrazine for 30 minutes and then incubated with 2- (benzo [ d ] thiazol-2-yl) -1, 4-phenylenedi (4-bromobutyrate) (30 μm) for 30 minutes;
the results show that zebra fish treated simultaneously with 2- (benzo [ d ] thiazol-2-yl) -1, 4-phenylenedi (4-bromobutyrate) and exogenous hydrazine at 28 ℃ showed significant fluorescence, whereas zebra fish untreated or zebra fish not treated simultaneously with 2- (benzo [ d ] thiazol-2-yl) -1, 4-phenylenedi (4-bromobutyrate) and exogenous hydrazine were not found to be fluorescent; the fluorescence imaging result shows that 2- (benzo [ d ] thiazole-2-yl) -1, 4-phenylene bis (4-bromobutyrate) can enter a zebra fish body and react with exogenous hydrazine to generate strong fluorescence, so that the detection effect is achieved. Furthermore, fluorescence is distributed in the yolk, stomach and digestive tract of zebra fish. Obviously, the fluorescent compound not only can be used for visualizing the exogenous hydrazine in the zebra fish, but also can accurately position the distribution condition of the exogenous hydrazine in the zebra fish.
It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein. Any reference sign in a claim should not be construed as limiting the claim concerned.
Claims (9)
1. An esit fluorescent compound for hydrazine specific detection, characterized in that the compound is: 2- (benzo [ d ] thiazol-2-yl) -1, 4-phenylenedi (4-bromobutyrate) having a chemical structural formula shown in formula I:
2. the method for preparing an esit fluorescent compound for hydrazine specific detection according to claim 1, wherein the steps of:
(1) 2- (benzo [ d ] thiazole-2-yl) benzene-1, 4-diphenol is prepared by reacting 2-amino thiophenol, 2, 5-dihydroxybenzaldehyde and sodium thiosulfate;
(2) 2- (benzo [ d ] thiazol-2-yl) -1, 4-phenylenebis (4-bromobutyrate) is prepared from 2- (benzo [ d ] thiazol-2-yl) benzene-1, 4-diol obtained in step (1) by modification of 4-bromobutyryl ester.
3. The method for preparing an esit fluorescent compound for hydrazine specific detection according to claim 2, wherein: in step (1), 500mg of 2-aminophenylthiophenol and 580mg of 2, 5-dihydroxybenzaldehyde are dissolved in 20mL of N, N-dimethylformamide, and 0.70g of sodium thiosulfate is added with continuous stirring; the reaction mixture was refluxed for 2 hours and monitored by TLC, after completion of the reaction, cooled to room temperature, 50mL of water was added, and the solid precipitate was collected on a suction filter funnel and recrystallized from methanol to obtain 2- (benzo [ d ] thiazol-2-yl) benzene-1, 4-diol as a white solid.
4. The method for preparing an esit fluorescent compound for hydrazine specific detection according to claim 2, wherein: in step (2), 730mg of 2- (benzo [ d ] thiazol-2-yl) benzene-1, 4-diol, supra, and 455mg of triethylamine were dissolved in 60mL of methylene chloride under ice-bath conditions; 668mg of 4-bromobutyryl chloride was slowly added to the mixture using a constant pressure dropping funnel and then reacted at room temperature, the progress of the reaction was monitored by thin layer chromatography silica gel plate (TLC), and when the reaction was completed, the solvent was evaporated to dryness, and the product was separated by eluting a silica gel column, to obtain 2- (benzo [ d ] thiazol-2-yl) -1, 4-phenylenebis (4-bromobutyrate) as a white solid, the eluent used for eluting the silica gel column was petroleum ether and ethyl acetate in a volume ratio of 3: 1.
5. Use of an esit fluorescent compound for hydrazine specific detection according to claim 1, wherein: the fluorescent compound is used as a fluorescent chemical sensor for detecting hydrazine.
6. Use of esit fluorescent compounds for hydrazine specific detection according to claim 5, for the detection of hydrazine in a solution system, comprising the following steps:
(a) Preparing the fluorescent compound according to claim 1 into a fluorescent compound solution with a concentration of 10 mu M by using 4-hydroxyethyl piperazine ethane sulfonic acid (HEPES) and dimethyl sulfoxide (DMSO) buffer solution, wherein the volume ratio of HEPES to DMSO in the buffer solution is 1:9, the pH value of the buffer solution is 7.4;
(b) Adding 100. Mu.M tryptophan (Try), cysteine (Cys), threonine (Thr), tyrosine (Tyr), histidine (His), glutamic acid (Glu) and aspartic acid (Asp), potassium chloride (KCl), calcium chloride (CaCl) to the 10. Mu.M fluorescent compound solution prepared in step (a), respectively 2 ) Sodium chloride (NaCl), magnesium chloride (MgCl) 2 ) Copper chloride (CuCl) 2 ) Barium chloride (BaCl) 2 ) Ferric chloride (FeCl) 3 ) Silver chloride (AgCl), nickel chloride(NiCl 2 ) An aqueous solution of sodium hydrosulfide (NaHS), and performing fluorescence intensity measurement after the reaction is completed;
(c) Through research on the relationship between the fluorescence intensity and the reaction time, the fluorescent compound is found to generate fluorescence enhancement only on hydrazine, namely the fluorescent compound can specifically recognize the hydrazine.
7. Use of an esit fluorescent compound for hydrazine specific detection according to claim 5 for quantitative detection of hydrazine in an environmental water sample, comprising the following steps:
(a) Preparing the fluorescent compound according to claim 1 into a fluorescent compound solution with a concentration of 10 mu M by using 4-hydroxyethyl piperazine ethane sulfonic acid (HEPES) and dimethyl sulfoxide (DMSO) buffer solution, wherein the volume ratio of HEPES to DMSO in the buffer solution is 1:9, the pH value of the buffer solution is 7.4;
(b) Selecting different types of environmental water samples, and adding hydrazine with the concentration of 0.10,0.50 and 1.0mg/L into the different environmental water samples; and (3) performing an experiment of adding and recovering the hydrazine by using the fluorescent compound prepared in the step (a), and comparing the added concentration with the added concentration to obtain the recovery rate of 72-111%, so that the fluorescent compound can quantitatively detect the hydrazine in water samples in different environments.
8. Use of an esit fluorescent compound for hydrazine specific detection according to claim 5, for exogenous hydrazine detection in HeLa of cervical cancer cells, comprising the following steps:
(a) A fluorescent compound according to claim 1 was prepared into a 20 μm concentration fluorescent compound solution by passing 4-hydroxyethyl piperazine ethane sulfonic acid (HEPES) and dimethyl sulfoxide (DMSO) buffer at a volume ratio of HEPES to DMSO of 1:9, the pH value of the buffer solution is 7.4;
(b) Five experimental groups A, B, C, D and E were set up:
group a blank control: heLa cells without any treatment;
group B hydrazine treatment control: heLa cells were incubated with 50. Mu.M hydrazine solution for 30 min;
group C probe treatment control: heLa cells were incubated with 20. Mu.M 2- (benzo [ d ] thiazol-2-yl) -1, 4-phenylenedi (4-bromobutyrate) for 30 min;
group D experimental group 1: heLa cells were incubated with 50 μm hydrazine for 30 min followed by 20. Mu.M 2- (benzo [ d ] thiazol-2-yl) -1, 4-phenylenedi (4-bromobutyrate) for 30 min;
group E, experimental group 2: heLa cells were incubated with 100 μm hydrazine for 30 min followed by 20. Mu.M 2- (benzo [ d ] thiazol-2-yl) -1, 4-phenylenedi (4-bromobutyrate) for 30 min;
(c) The results of the A, B, C, D, E group fluorescence imaging show that the 2- (benzo [ d ] thiazole-2-yl) -1, 4-phenylene bis (4-bromobutyrate) can enter cells and react with exogenous hydrazine in the cells, and the distribution of the exogenous hydrazine in the cells can be obviously seen by the cell imaging.
9. Use of an esit fluorescent compound for hydrazine specific detection according to claim 5, for exogenous hydrazine detection in zebra fish, comprising the following steps:
(a) A fluorescent compound according to claim 1 was prepared into a 20 μm concentration fluorescent compound solution by passing 4-hydroxyethyl piperazine ethane sulfonic acid (HEPES) and dimethyl sulfoxide (DMSO) buffer at a volume ratio of HEPES to DMSO of 1:9, the pH value of the buffer solution is 7.4;
(b) Four experimental groups a, B, C, D were set up:
group a blank control: untreated 3-day-old zebra fish;
group B probe treatment control: 3 day old zebra fish treated with 30 μm 2- (benzo [ d ] thiazol-2-yl) -1, 4-phenylenebis (4-bromobutyrate);
group C hydrazine treatment control: zebra fish of 3 days of age treated with 50 μm hydrazine;
group D experimental group: zebra fish of 3 days of age were treated with hydrazine for 30 minutes and then incubated with 30. Mu.M 2- (benzo [ d ] thiazol-2-yl) -1, 4-phenylenedi (4-bromobutyrate) for 30 minutes;
(c) The results show that zebra fish treated simultaneously with 2- (benzo [ d ] thiazol-2-yl) -1, 4-phenylenedi (4-bromobutyrate) and exogenous hydrazine at 28 ℃ showed significant fluorescence, whereas zebra fish untreated or zebra fish not treated simultaneously with 2- (benzo [ d ] thiazol-2-yl) -1, 4-phenylenedi (4-bromobutyrate) and exogenous hydrazine were not found to be fluorescent; the fluorescence imaging result shows that 2- (benzo [ d ] thiazole-2-yl) -1, 4-phenylene bis (4-bromobutyrate) can enter a zebra fish body and react with exogenous hydrazine to generate strong fluorescence, so that the detection effect is achieved;
(d) The reaction mechanism of the fluorescent compound is that one N atom of hydrazine is firstly subjected to nucleophilic substitution reaction with a carbon atom connected with a bromine atom on 2- (benzo [ d ] thiazole-2-yl) -1, 4-phenylene bis (4-bromobutyrate), and Br atoms are substituted; then, another N atom of hydrazine attacks the carbonyl C atom on 2- (benzo [ d ] thiazol-2-yl) -1, 4-phenylene bis (4-bromobutyrate), stable six-membered ring compound is generated through addition cyclization, and the other 4-bromobutyryl on 2- (benzo [ d ] thiazol-2-yl) -1, 4-phenylene bis (4-bromobutyrate) also reacts through the mechanism, so that the fluorescent group 2- (benzo [ d ] thiazol-2-yl) benzene-1, 4-diphenol is completely released to lead to fluorescence enhancement, and the aim of detecting the hydrazine is achieved.
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