CN111807993B - Near infrared fluorescent compound for specific detection of hydrazine and preparation method thereof - Google Patents
Near infrared fluorescent compound for specific detection of hydrazine and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a novel near infrared fluorescent compound 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 organisms; 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 intense red fluorescence which can be distinguished by naked eyes; the near infrared fluorescent compound obtained by ultraviolet absorption and fluorescence spectrophotometry analysis can have excellent selectivity to hydrazine under various interferents and has strong anti-interference capability to common biomolecules; not only can selectively identify exogenous hydrazine, but also can quantitatively detect hydrazine with high sensitivity in the growth environment of various living cells, and can be successfully applied to living cells and zebra fish imaging.
Description
Technical Field
The invention belongs to the technical field of small organic molecule fluorescent probes, and particularly relates to a fluorescent chemical compound for specifically detecting environmental pollutant hydrazine by taking (E) -2- (3- (4- (diethylamino) -2-hydroxystyryl) -5, 5-dimethylcyclohex-2-en-1-yl) malononitrile as a fluorescent matrix and application thereof.
Background
Inorganic pollutants in the environment are widely available and of complex types, most of which enter the human body. When the concentration of these contaminants in the environment exceeds a safety threshold, it may pose a threat to the environment and human health. Hydrazine (N) 2 H 4 ) 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 also a volatile and potentially carcinogenic environmental pollutant. Because of wide application and better water solubility, hydrazine can produce pollution to a certain extent in the environment, and can produce toxic action on human bodies. Thus, there is an urgent need to establish a reliable, accurate and efficient analytical method to detect hydrazine in an 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 drawbacks of being time-consuming and complicated to operate. In contrast, fluorescent probes have attracted interest to many researchers due to their high specificity, sensitivity and ease of handling. The fluorescent molecular probe can react with an analyte to be detected; thus, microscopic reaction events can be visualized by fluorescent signals. Thus, these properties impart excellent properties to fluorescent probes, which can be used for environmental monitoring, life sciences and disease diagnosis.
To date, a number of 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 Gabriel reactions, addition reactions with aldehydes and ester hydrolysis reactions. Wherein, the application of hydrazinolysis reaction can improve the response rate of detecting hydrazine. The chemical sensor typically capable of hydrazinolysis is a chemical sensor using 4-bromobutyryl as a recognition group, which is mainly formed by modifying a hydroxyl group on a fluorescent group, so that it is a good idea to introduce a hydroxyl group into a fluorescent probe matrix structure.
In conclusion, (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 utilized for modification, so that self fluorescence is quenched, the sensitivity is improved, and the novel near infrared fluorescent compound capable of specifically recognizing hydrazine and applicable to cell and living tissue imaging is hopeful to be developed.
Disclosure of Invention
A first object of the present invention is to develop a near infrared fluorescent compound that can selectively detect the environmental contaminant hydrazine, which can distinguish the presence of hydrazine under the interference of other analytes.
The second object of the invention is to provide a preparation method of the near infrared fluorescent compound capable of specifically detecting the hydrazine as an environmental pollutant.
The third object of the invention is to provide a method which can be applied to the detection of exogenous hydrazine in solutions, 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) vinyl) -5- (diethylamino) phenyl 4-bromobutyric acid, and has a molecular formula of C 27 H 32 BrN 3 O 2 The chemical structural formula is shown as formula (I):
the preparation operation steps of the near infrared fluorescent compound for specifically detecting hydrazine are as follows:
(1) 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 were mixed under nitrogen, and the ice bath was cooled to 0 ℃;
(2) Slowly dripping 0.62mg of 4-bromobutyryl chloride, and stirring at room temperature for reaction for 10-12 hours to obtain a mixture;
(3) The mixture was poured into ice water, stirred, and extracted 3 times with 10mL of dichloromethane 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 in a volume ratio of 3:1 to give 1.03g of the purplish black product (E) -2- (2- (3- (dicyanomethylene) -5, 5-dimethylcyclohex-1-en-1-yl) vinyl) -5- (diethylamino) phenyl 4-bromobutanoic acid DCDB in 72% yield.
The near infrared fluorescent compound is used for detecting the specificity of hydrazine.
The detection operation steps of the near infrared fluorescent compound for 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 a buffer solution, wherein the buffer solution is prepared from phosphate buffer salt solution (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 parts of a 3mL near infrared fluorescent compound solution having a concentration of 10. Mu.M was taken, and 120. Mu.L of each solution having a concentration of 5X 10 was added thereto -5 The method comprises the steps of (1) mol/L of analytes to be detected, namely 27 analytes to be detected, 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 after the reaction is completed, the fluorescence intensity of 81 parts of reactants is measured respectively;
(3) The results showed hydrazine (N) 2 H 4 ) The fluorescence intensity of the near infrared fluorescent compound working solution can be improved; the near infrared fluorescent compound can be combined with hydrazine (N) 2 H 4 ) A fluorescence enhancement reaction occurs, namely the near infrared fluorescent compound realizes specific recognition of hydrazine.
The detection operation steps of the near infrared fluorescent compound for hydrazine on the test paper are as follows:
(1) Preparing the near infrared fluorescent compound detection test paper according to claim 1, preparing the near infrared fluorescent compound into working solution with the concentration of 10 mu M by using methylene dichloride, 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 the test paper;
(2) 200 mu M of aqueous solutions of 27 different analytes are respectively dripped on corresponding 27 test papers; then placing the mixture into an ultraviolet lamp with excitation wavelength of 365nm for observation;
(3) The result shows that the fluorescent color of the test paper with the hydrazine solution added dropwise is changed from colorless to red, and other analytes do not change the test paper, namely the test paper can simply and rapidly detect the hydrazine as an environmental pollutant.
The detection operation steps of the near infrared fluorescent compound for hydrazine in HeLa cells are as follows:
(1) The process for preparing a near infrared fluorescent compound working solution as claimed in claim 1
Preparing the near infrared fluorescent compound of claim 1 into working solution with the concentration of 20 mu M by using buffer solution; the volume ratio of Phosphate Buffered Saline (PBS) to dimethyl sulfoxide (DMSO) in the buffer solution is 1:1, the pH value of the buffer solution is 7.4;
(2) Taking A, B, C, D experimental groups;
group a is a blank group: heLa cells without any treatment were the group A test subjects;
group B is a hydrazine treatment control group: 200. Mu.L of 50. Mu.M hydrazine in a solution containing 5X 10 4 Incubating HeLa cells in an orifice plate of the HeLa cells for 30 minutes to obtain a group B detected object for detection;
group C is near infrared fluorescent compound treated control group: at a concentration of 20. Mu.M, 200. Mu.L of near infrared fluorescent compound was used in a solution containing 5X 10 4 Incubating HeLa cells in an orifice plate of the HeLa cells for 30 minutes to obtain a group C detected object for detection; group D is near infrared fluorescent compound and hydrazine treatment group: pretreatment of 5X 10-containing compositions with 200. Mu.L of 20. Mu.M near infrared fluorescent compound 4 HeLa cells were plated in an orifice plate for 30 minutes and then with 200. Mu.L of 50. Mu.M hydrazine (N) 2 H 4 ) Incubating for 30 minutes to obtain the group D detected objects for detection;
(3) Placing the A group detected object, the B group detected object, the C group detected object and the D group detected object under a fluorescence microscope with 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 have no fluorescence; and the group D detected objects show obvious red fluorescence; the fluorescence imaging result shows that the near infrared fluorescent compound in the invention 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.
The detection operation steps of the near infrared fluorescent compound for the exogenous hydrazine in the zebra fish are as follows:
(1) The process for preparing a near infrared fluorescent compound working solution as claimed in claim 1
Preparing the near infrared fluorescent compound of claim 1 into working solution with concentration of 20 mu M by using buffer solution, wherein the buffer solution comprises Phosphate Buffered Saline (PBS) and dimethyl sulfoxide (DMSO) according to volume ratio of 1:1, wherein the pH value of the buffer solution is 7.4;
(2) Take A, B, C, D four experimental groups
Group a is a blank group: untreated 3-day-old zebra fish, obtaining a group A of detected objects for detection; group B is a hydrazine treatment control group: incubating the zebra fish with 10mL of hydrazine with the concentration of 50 mu M for 30 minutes, and obtaining a group B detected object for detection;
group C is near infrared fluorescent compound treated control group: the 3-day-old zebra fish is treated with 10mL of the near infrared fluorescent compound of claim 1 at a concentration of 20 mu M for 30 minutes to obtain group C detection objects for detection;
group D is near infrared fluorescent compound and hydrazine treatment group: incubation of 3 day old zebra fish with 10mL of 50 μm hydrazine for 30 min followed by treatment with 20 μm of the near infrared fluorescent compound of claim 1 for 30 min to give group D test objects for detection;
(3) 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 excitation wavelength of 520nm for observation;
(4) The results showed that the group A, B and C subjects were not found to have fluorescence; and the group D detected objects show obvious red fluorescence; the fluorescence imaging result shows that the near infrared fluorescent compound in the invention in the claim 1 can enter the zebra fish body and react with exogenous hydrazine to generate strong red fluorescence, thereby realizing specific detection of the exogenous hydrazine.
The detection operation steps of the near infrared fluorescent compound for hydrazine in an environmental water sample are as follows:
(1) The process for preparing a near infrared fluorescent compound working solution as claimed in claim 1
Preparing the near infrared fluorescent compound of claim 1 into a working solution with the concentration of 10 mu M by using a buffer solution; the buffer solution is prepared from Phosphate Buffer Solution (PBS) and dimethyl sulfoxide (DMSO) according to a volume ratio of 1:1, and the pH value of the buffer solution is 7.4;
(2) Preparation of environmental water sample to be tested
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 mu m water phase filter membrane to obtain a treated environmental water sample; adding 0.1 mu M, 0.5 mu M and 1.0 mu M hydrazine into 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 the hydrazine reaches 87-109%, and the result shows that the near infrared fluorescent compound working solution can quantitatively detect the existence of the hydrazine in an environmental water sample.
The detection operation steps of the near infrared fluorescent compound for hydrazine in sewage are as follows:
(1) Preparing the near infrared fluorescent compound of claim 1 into a working solution with the concentration of 10 mu M by using a buffer solution; the buffer solution is prepared from Phosphate Buffer Solution (PBS) and dimethyl sulfoxide (DMSO) according to a 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, the sewage samples of chemical plants in different areas are collected and filtered through a 200 mu m water phase filter membrane to obtain a sewage sample to be detected;
(3) Detecting the reaction liquid of the near infrared fluorescent compound working solution and the sewage sample by using a fluorescence spectrometer;
(4) The result shows that the near infrared fluorescent compound of claim 1 can quantitatively detect hydrazine in sewage with high sensitivity. The beneficial technical effects of the invention are as follows:
1. the near infrared fluorescent compound has the advantages of simple preparation steps, easily obtained raw materials, easy synthesis, mild reaction conditions and completion of the synthesis of the near infrared fluorescent compound only through one-step acylation reaction.
2. The N atom of the near infrared fluorescent compound detector hydrazine can undergo nucleophilic substitution reaction with a carbon atom near a bromine atom on the 4-bromobutyryl of the near infrared fluorescent compound. Then the other N atom of hydrazine attacks carbonyl carbon atom on near infrared fluorescent compound, and the stable six-membered ring cyclization product tetrahydropyridazine is generated by addition cyclization and cleavage (ESI MS [ M+H)]++, m/z:101.0710 Compound DHDM (ESI MS [ M+H)]++, m/z:362.2228 Generating identifiable fluorescence (fig. 1-2), thus eliminating interference from common analytes in solution and organisms, and with detection limits as low as 39.6nM, providing great advantages over most near infrared fluorescent compounds of the same type; the results of fig. 3 show that the near infrared fluorescent compound has better selectivity to hydrazine. As shown in FIG. 4, the fluorescence intensity of the near infrared fluorescent compound increases with increasing hydrazine content, and has a good linear relationship (R 2 =0.9967), i.e. the near infrared fluorescent compound is capable of quantitatively detecting trace amounts of hydrazine in an aqueous environment.
3. The near infrared fluorescent compound has potential application value. The near infrared fluorescent compound can rapidly and efficiently distinguish hydrazine in the aqueous solution through the preparation of the test strip, and has good cell membrane permeability and low cytotoxicity, so that the near infrared fluorescent compound can be successfully applied to the imaging of living cells and hydrazine in zebra fish bodies, and has good biological application potential; (1) The near infrared fluorescent compound has potential application value. Referring to fig. 5, the test strip containing the near infrared fluorescent compound can rapidly and efficiently perform specific recognition and preliminary quantitative detection on hydrazine in water environment.
(2) The near infrared fluorescent compound logp=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 lower cytotoxicity. The survival rate of HeLa cells is still more than 85% in the presence of the near infrared fluorescent compound with the concentration of 40 mu M, which shows that the near infrared fluorescent compound has lower cytotoxicity;
(4) The near infrared fluorescent compound has good biological application potential. As shown in the results of fig. 6, untreated HeLa cells showed significant red fluorescence after incubation with near infrared fluorescent compound and hydrazine for 30 minutes, respectively, whereas no fluorescence change was observed in HeLa cells of other treatment groups; as shown in FIG. 7, when zebra fish were incubated with near infrared fluorescent compound (20. Mu.M) and hydrazine (50. Mu.M) for 30 minutes, respectively, red fluorescence was observed under a fluorescence microscope. However, no significant fluorescence change was observed when the zebra fish of 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-bromobutanoic acid with hydrazine;
FIG. 2 is a nuclear magnetic resonance diagram of the reaction product of (E) -2- (2- (3- (dicyanomethylene) -5, 5-dimethylcyclohex-1-en-1-yl) vinyl) -5- (diethylamino) phenyl 4-bromobutanoic acid with hydrazine;
FIG. 3 is a graph showing fluorescence emission and ultraviolet spectra of a reaction in which compounds such as different analytes are added to a working solution of (E) -2- (2- (3- (dicyanomethylene) -5, 5-dimethylcyclohex-1-en-1-yl) vinyl) -5- (diethylamino) phenyl 4-bromobutyric acid;
FIG. 4 is a graph showing fluorescence emission spectra 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 UV luminescence diagram of (E) -2- (2- (3- (dicyanomethylene) -5, 5-dimethylcyclohex-1-en-1-yl) vinyl) -5- (diethylamino) phenyl 4-bromobutyric acid test strip with various analytes;
FIG. 6 is a fluorescence microscopy image 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 fluorescence microscopy image 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 comprising 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 reporting group. The 4-bromobutyryl on the reported near infrared fluorescent compound can react specifically with hydrazine in the 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 be further described with reference to the following examples
Example 1
Preparation of near infrared fluorescent compounds for hydrazine detection
The near infrared fluorescent compound is (E) -2- (2- (3- (dicyanomethylene) -5, 5-dimethylcyclohex-1-en-1-yl) vinyl) -5- (diethylamino) phenyl 4-bromobutyric acid (DCDB), and the specific preparation process is as follows:
(1) DHDM (1.0 g,2.8 mmol), triethylamine (1.0 mL) and anhydrous dichloromethane (50 mL) were mixed and added to a 100mL three-necked flask under nitrogen and cooled to 0deg.C using an ice bath.
(2) 4-Bromobutyryl chloride (0.62 mg,3.3 mmol) was then slowly added dropwise to the three-necked flask containing the mixture of step (1) under ice-bath conditions and stirred at room temperature for 10-12 hours. The progress of the reaction was monitored during the reaction using Thin Layer Chromatography (TLC).
(3) After the reaction was completed, the mixture was poured into 50mL of ice water at 0℃and stirred to mix well, and methylene chloride was added to extract 3 times, 10mL each. After drying over 5.0g of anhydrous sodium sulfate, the organic phase was concentrated to an oil.
(4) The crude product was purified by column chromatography eluting with n-hexane and ethyl acetate in a volume ratio of 3:1 to give 1.03g of (E) -2- (2- (3- (dicyanomethylene) -5, 5-dimethylcyclohex-1-en-1-yl) vinyl) -5- (diethylamino) phenyl 4-bromobutanoic acid DCDB as a dark solid in 72% yield.
(5) (E) -2- (2- (3- (dicyanomethylene) -5, 5-dimethylcyclohex-1-en-1-yl) vinyl) -5- (diethylamino) phenyl 4-bromobutanoic acid was characterized as follows:
nuclear magnetic resonance hydrogen spectrometry: 1 H NMR(d 6 -DMSO,600Hz)δ7.73(d,J＝9.0Hz,1H),7.11(d,J＝16.0Hz,1H),6.99(d,J＝16.0Hz,1H),6.75(s,1H),6.61(dd,J＝9.2,2.6Hz,1H),6.41(d,J＝2.6Hz,1H),3.63(t,J＝6.5Hz,2H),3.37(m,4H),2.84(t,J＝7.1Hz,2H),2.55(s,2H),2.44(s,2H),2.20(m,2H),1.09(m,6H),0.98(s,6H). 13 C NMR(d 6 DMSO,151 MHz) δ:171.28,170.29,157.02,151.40,150.19,131.51,128.77,125.65,121.39,115.19,114.79,113.96,110.31,105.28,105.24,74.25,44.37,42.78,40.55,39.65,38.60,34.42,32.50,32.04,27.96,27.92,12.94; high resolution mass spectrometry: HRMS (ESI, m/z) Calcd for [ C 12 H 14 N 2 +H] + 510.1751, found:510.1750; melting point: 228.9 ℃. Fourier infrared: FT-IR (KBr, cm) -1 ):3334.67,2961.19,2925.62,2866.72,2221.56,1617.10,1537.98,1497.52,1413.79,1337.27,1286.64,1190.23,1151.19,1075.33,1016.59,963.47,892.53,822.89,784.23.
The near infrared fluorescent compound is (E) -2- (2- (3- (dicyanomethylene) -5, 5-dimethylcyclohex-1-en-1-yl) vinyl) -5- (diethylamino) phenyl 4-bromobutyric acid, and the chemical structural formula is shown in formula (I):
the detection mechanism of the near infrared fluorescent compound (E) -2- (2- (3- (dicyanomethylene) -5, 5-dimethylcyclohex-1-en-1-yl) vinyl) -5- (diethylamino) phenyl 4-bromobutyric acid is as follows:
(1) According to the near infrared fluorescent compound, the (E) -2- (2- (3- (dicyanomethylene) -5, 5-dimethylcyclohex-1-en-1-yl) vinyl) -5- (diethylamino) phenyl 4-bromobutyric acid reacts with hydrazine, firstly, one N atom of the hydrazine and a C atom near Br atoms of the near infrared fluorescent compound undergo nucleophilic substitution reaction, and the Br atoms are replaced;
(2) Then, the other N atom in the hydrazine attacks the C atom of the carbonyl group on the near infrared fluorescent compound, and the addition cyclization generates a stable six-membered ring cyclization product tetrahydropyridazine (ESI-MS [ M+H)] + M/z:101.0710 Compound DHDM (ESI-MS [ M+H)] + M/z:362.2228 Generating identifiable fluorescence, and figures 1-2 show high resolution mass spectra and nuclear magnetic resonance spectra of the near infrared fluorescent compound and hydrazine reaction product;
(3) The results exclude interference of common analytes in solution and organisms and detection limits as low as 39.6nM have great advantages over most types of fluorescent compounds.
Example 2
Selective detection of hydrazine in solution systems
(1) Preparing a near infrared fluorescent compound prepared in the example 1 into a near infrared fluorescent compound solution with the concentration of 10 mu M by using a buffer solution, wherein the buffer solution is prepared from phosphate buffer salt solution (PBS) and dimethyl sulfoxide (DMSO) in a volume ratio of 1:1, and the pH value of the buffer solution is 7.4;
(2) Selectively detecting hydrazine in the solution by using 10 mu M near infrared fluorescent compound solution;
81 parts of a 3mL near infrared fluorescent compound solution having a concentration of 10. Mu.M was taken, and 120. Mu.L of each solution having a concentration of 5X 10 was added thereto -5 mol/L of 27 analytes to be detected, wherein each analyte to be detected consists of 3 parallel units to form a group, and 81 reactants are obtained; the 27 analytes to be tested were 200. Mu.M hydrazine (N 2 H 4 ) 200. Mu.M magnesium nitrate hexahydrate (Mg (NO) 3 ) 2 ·6H 2 O), 200. Mu.M Methylhydrazine (methyl hydrozine), 200. Mu.M 1,2-dimethylhydrazine (1, 2-dimethylhydrazinedihydro chloride), 200. Mu.M cysteine(Cys), 200. Mu.M Glutathione (GSH), 200. Mu.M proline (pro), 200. Mu.M aspartic acid (aspartic), 200. Mu.M tryptophan (trptophan), 200. Mu.M arginine (arginine), 200. Mu.M tyrosine (tyrosine), 200. Mu.M histidine (histidine), 200. Mu.M glutamic acid (glutamicam), 200. Mu.M lysine (lysine), 200. Mu.M threonine (threonine), 200. Mu.M glycine (glycone), 200. Mu.M potassium nitrate (KNO) 3 ) 200. Mu.M calcium nitrate tetrahydrate (Ca (NO) 3 ) 2 ·4H 2 O), 200. Mu.M sodium nitrate (NaNO) 3 ) 200. Mu.M copper nitrate trihydrate (Cu (NO) 3 ) 2 ·3H 2 O), 200. Mu.M zinc nitrate hexahydrate (Zn (NO) 3 ) 2 ·6H 2 O), 200. Mu.M ferric nitrate nonahydrate (Fe (NO) 3 ) 3 ·9H 2 O), 200. Mu.M sodium hydrosulfide (NaHS), 200. Mu.M Nickel chloride (NiCl) 2 ) 200 mu M silver nitrate (AgNO) 3 ) 200 mu M mercuric chloride (HgCl) 2 ) And 200. Mu.M cobalt chloride (CoCl) 2 )。
(3) The reaction is completed, 81 parts of reactants are obtained, and fluorescence intensity measurement is carried out on 81 parts of reactants respectively;
(4) FIGS. 3a and 3b show the results of fluorescence intensity and ultraviolet absorption intensity, respectively, showing that only the fluorescence intensity and ultraviolet absorption intensity of hydrazine are significantly enhanced, namely (E) -2- (2- (3- (dicyanomethylene) -5, 5-dimethylcyclohex-1-en-1-yl) vinyl) -5- (diethylamino) phenyl 4-bromobutyric acid has better selectivity to hydrazine; furthermore, FIG. 3c shows the results of the anti-interference ability of the near infrared fluorescent compound to hydrazine, which indicates that other interfering analytes do not affect the response of the near infrared fluorescent compound to hydrazine; in summary, the near infrared fluorescent compound can specifically identify hydrazine and has stronger anti-interference capability.
Example 3
Detection sensitivity of near infrared fluorescent compound to hydrazine in solution system
(1) Preparing a near infrared fluorescent compound prepared in the example 1 into a near infrared fluorescent compound solution with the concentration of 10 mu M by using a buffer solution, wherein the buffer solution is prepared from phosphate buffer salt solution (PBS) and dimethyl sulfoxide (DMSO) in a volume ratio of 1:1, and the pH value of the buffer solution is 7.4;
(2) Fluorescence detection of hydrazine at various concentrations (0. Mu.M, 10. Mu.M, 20. Mu.M, 30. Mu.M, 40. Mu.M, 50. Mu.M, 60. Mu.M, 70. Mu.M, 80. Mu.M, 90. Mu.M, 100. Mu.M, 110. Mu.M, 120. Mu.M, 130. Mu.M, 140. Mu.M, 150. Mu.M) was performed with a solution of near infrared fluorescent compound at 10. Mu.M;
(3) To 48 parts of the prepared near infrared fluorescent compound solution having a concentration of 10. Mu.M were added respective different concentrations (0. Mu.M, 10. Mu.M, 20. Mu.M, 30. Mu.M, 40. Mu.M, 50. Mu.M, 60. Mu.M, 70. Mu.M, 80. Mu.M, 90. Mu.M, 100. Mu.M, 110. Mu.M, 120. Mu.M, 130. Mu.M, 140. Mu.M, 150. Mu.M) of hydrazine, each concentration being provided with three sets of parallel units;
(4) After the completion of the reaction, 48 parts of the reaction product were obtained, and fluorescence intensities of the 48 parts of the reaction product were measured.
(5) As can be seen from fig. 4a, hydrazine (N 2 H 4 ) The fluorescence intensity of the near infrared fluorescent compound solution can be improved, and along with the continuous increase of the concentration of hydrazine, the fluorescence intensity of the near infrared fluorescent compound solution is improved; FIG. 4b shows that the fluorescence intensity of near infrared fluorescent compound solution has a good linear relationship with the concentration of hydrazine (R 2 =0.9967), the detection limit of the near infrared fluorescent compound on hydrazine is calculated to be as low as 39.6nM, i.e. the near infrared fluorescent compound can quantitatively detect the existence of trace hydrazine in the solution.
Example 4
Near infrared fluorescent compound test paper color development
When the method is used for detecting hydrazine on filter paper, working solution with the concentration of 20 mu M is prepared by using dichloromethane and near infrared fluorescent compound; the specific operation process is as follows:
(1) Preparing detection test paper of the near infrared fluorescent compound, preparing the near infrared fluorescent compound into working solution with the concentration of 10 mu M by using methylene dichloride, immersing a plurality of pieces of filter paper with the same size and shape into the working solution of the near infrared fluorescent compound for 30 minutes, taking out the filter paper and airing;
(2) 200. Mu.M hydrazine (N) 2 H 4 ) 200. Mu.M magnesium nitrate hexahydrate (Mg (NO) 3 ) 2 ·6H 2 O), 200. Mu.M methyl hydrazine (methyl hydrazine), 200. Mu.M 1,2-dimethylhydrazine (1, 2-dimethylhydrazine), 200. Mu.M cysteineCys), 200. Mu.M Glutathione (GSH), 200. Mu.M proline (pro), 200. Mu.M aspartic acid (aspartic), 200. Mu.M tryptophan (trptophan), 200. Mu.M arginine (arginine), 200. Mu.M tyrosine (tyrosine), 200. Mu.M histidine (histidine), 200. Mu.M glutamic acid (glutamicam), 200. Mu.M lysine (lysine), 200. Mu.M threonine (threonine), 200. Mu.M glycine (glycone), 200. Mu.M potassium nitrate (KNO) 3 ) 200. Mu.M calcium nitrate tetrahydrate (Ca (NO) 3 ) 2 ·4H 2 O), 200. Mu.M sodium nitrate (NaNO) 3 ) 200. Mu.M copper nitrate trihydrate (Cu (NO) 3 ) 2 ·3H 2 O), 200. Mu.M zinc nitrate hexahydrate (Zn (NO) 3 ) 2 ·6H 2 O), 200. Mu.M ferric nitrate nonahydrate (Fe (NO) 3 ) 3 ·9H 2 O), 200. Mu.M sodium hydrosulfide (NaHS), 200. Mu.M Nickel chloride (NiCl) 2 ) 200 mu M silver nitrate (AgNO) 3 ) 200 mu M mercuric chloride (HgCl) 2 ) And 200. Mu.M cobalt chloride (CoCl) 2 ) Respectively dripping the water solution on the prepared near infrared fluorescent compound filter paper;
(3) Observing under ultraviolet lamp with excitation wavelength of 365 nm;
(4) Solutions of hydrazine (0. Mu.M, 1. Mu.M, 5. Mu.M, 10. Mu.M, 50. Mu.M, 100. Mu.M, 200. Mu.M) with different concentrations are respectively dripped on the prepared near infrared fluorescent compound filter papers;
(5) Observing the change of the test strip under the naked eye and an ultraviolet lamp with the excitation wavelength of 365 nm;
(6) As can be seen from fig. 5a, the fluorescent color of the test strip changes to red fluorescence in the presence of hydrazine, i.e. the environmental pollutant hydrazine is detected; in the presence of hydrazine at different concentrations, the test strip shows different degrees of color change under the naked eye and ultraviolet lamp, see fig. 5b; the result shows that the near infrared fluorescent compound test strip can rapidly detect hydrazine in the solution, and can primarily quantify the hydrazine in the solution; in conclusion, the near infrared fluorescent compound test strip has a great application prospect in the field of rapid detection.
Example 5
Quantitative detection of hydrazine applied to environmental water sample
(1) When the near infrared fluorescent compound is used for detecting hydrazine in an environmental water sample, the near infrared fluorescent compound of claim 1 is prepared into a working solution with the concentration of 10 mu M by using a buffer solution; the buffer solution is prepared from Phosphate Buffer Solution (PBS) and dimethyl sulfoxide (DMSO) according to a 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 an environmental water sample, the environmental water samples in different areas are collected and filtered through a 200 mu m water phase filter membrane to obtain a treated environmental water sample;
(3) And respectively adding 0.1,0.5 and 1.0 mu M of hydrazine into the treated environmental water sample, and detecting the hydrazine in the environmental water sample by utilizing the near infrared fluorescent compound working solution.
Table 1 shows the recovery of hydrazine in an actual water sample by adding (E) -2- (2- (3- (dicyanomethylene) -5, 5-dimethylcyclohex-1-en-1-yl) vinyl) -5- (diethylamino) phenyl 4-bromobutyric acid;
* the water sample is collected at a plurality of points, and the sampling point is not less than 5. Data are mean ± standard deviation (n=3). Loq=0.10 μm as can be seen from table 1, the recovery of hydrazine reached 87-109% and the near infrared fluorescent compound working solution was able to quantitatively detect the presence of hydrazine in environmental water samples.
Example 6
Detection of hydrazine applied to sewage
(1) When the near infrared fluorescent compound is used for detecting hydrazine in a sewage sample, the near infrared fluorescent compound of claim 1 is prepared into a working solution with the concentration of 10 mu M by using a buffer solution; the buffer solution is prepared from Phosphate Buffer Solution (PBS) and dimethyl sulfoxide (DMSO) according to a volume ratio of 1:1, and the pH value of the buffer solution is 7.4;
(2) When the method is used for detecting hydrazine in a sewage sample, sewage of chemical plants in different areas is collected and filtered through a 200 mu m water phase filter membrane to obtain a sewage sample to be detected;
(3) And detecting hydrazine in the sewage sample by utilizing the near infrared fluorescent compound working solution.
Table 2 shows the detection of hydrazine in a sewage sample by (E) -2- (2- (3- (dicyanomethylene) -5, 5-dimethylcyclohex-1-en-1-yl) vinyl) -5- (diethylamino) phenyl 4-bromobutyric acid;
* the water sample is collected at a plurality of points, and the sampling point is not less than 5. Data are mean ± standard deviation (n=3). Loq=0.10 μm as can be seen from table 2, the near infrared fluorescent compound is capable of quantitatively detecting trace hydrazine in sewage with high sensitivity.
Example 7
HeLa intracellular fluorescence imaging
(1) For hydrazine detection in HeLa cells, preparing the near infrared fluorescent compound as claimed in claim 1 into working solution with the concentration of 20 mu M by using a buffer solution; the volume ratio of Phosphate Buffered Saline (PBS) to dimethyl sulfoxide (DMSO) in the buffer solution is 1:1, the pH value of the buffer solution is 7.4;
(2) Four experimental groups of A, B, C, D were taken,
group a is a blank group: heLa cells without any treatment.
Group B is a hydrazine treatment control group: 200. Mu.L of 50. Mu.M hydrazine in a solution containing 5X 10 4 Incubating HeLa cells in an orifice plate of the HeLa cells for 30 minutes to obtain a group B detected object for detection;
group C is near infrared fluorescent compound treated control group: with 200. Mu.L of 20. Mu.M (E) -2- (2- (3- (dicyanomethylene) -5, 5-dimethylcyclohex-1-en-1-yl) vinyl) -5- (diethylamino) phenyl 4-bromobutanoic acid in a concentration of 5X 10 4 Incubating HeLa cells in an orifice plate of the HeLa cells for 30 minutes to obtain a group C detected object for detection;
group D is near infrared fluorescent compound and hydrazine treatment group: pretreatment with 200. Mu.L of 20. Mu.M (E) -2- (2- (3- (dicyanomethylene) -5, 5-dimethylcyclohex-1-en-1-yl) vinyl) -5- (diethylamino) phenyl 4-bromobutanoic acidContaining 5X 10 4 The well plate of HeLa cells was incubated with 200. Mu.L of 50. Mu.m hydrazine for 30 min to obtain group D test objects;
(3) 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 excitation wavelength of 520nm for observation;
(4) The results of fluorescence imaging of the group A, B, C and D test objects are shown in FIG. 6, which shows that HeLa cells treated with (E) -2- (2- (3- (dicyanomethylene) -5, 5-dimethylcyclohex-1-en-1-yl) vinyl) -5- (diethylamino) phenyl 4-bromobutyric acid and hydrazine simultaneously have no fluorescence; whereas zebra fish treated with (E) -2- (2- (3- (dicyanomethylene) -5, 5-dimethylcyclohex-1-en-1-yl) vinyl) -5- (diethylamino) phenyl 4-bromobutyric acid and hydrazine showed a clear red fluorescence; the fluorescence imaging result shows that (E) -2- (2- (3- (dicyanomethylene) -5, 5-dimethylcyclohex-1-en-1-yl) vinyl) -5- (diethylamino) phenyl 4-bromobutyric acid 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.
Example 8
Fluorescence imaging of zebra fish
(1) When the near infrared fluorescent compound is used for detecting the exogenous hydrazine in the zebra fish, the near infrared fluorescent compound is prepared into a working solution with the concentration of 20 mu M by using a buffer solution, wherein the buffer solution comprises Phosphate Buffered Saline (PBS) and dimethyl sulfoxide (DMSO) according to the volume ratio of 1:1, the pH value of the buffer solution is 7.4.
(2) Four experimental groups of A, B, C, D were taken,
group a is a blank group: untreated zebra fish of 3 days old, obtaining group A detected objects;
group B is a hydrazine treatment control group: incubating the zebra fish with 5mL of hydrazine with the concentration of 50 mu M for 30 minutes, and obtaining a group B detected object for detection;
group C is near infrared fluorescent compound treated control group: group C test objects for detection were obtained by treating 3-day-old zebra fish with 10mL of (E) -2- (2- (3- (dicyanomethylene) -5, 5-dimethylcyclohex-1-en-1-yl) vinyl) -5- (diethylamino) phenyl 4-bromobutyric acid at a concentration of 20. Mu.M for 30 minutes;
group D is near infrared fluorescent compound and hydrazine treatment group: 3-day-old zebra fish were incubated with 10mL of 50. Mu.M hydrazine for 30 minutes with normal-developing 3-day-old zebra fish, and then treated with 20. Mu.M (E) -2- (2- (3- (dicyanomethylene) -5, 5-dimethylcyclohex-1-en-1-yl) vinyl) -5- (diethylamino) phenyl 4-bromobutyric acid for 30 minutes to give group B test objects for detection;
(3) 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 excitation wavelength of 520nm for observation;
(4) The fluorescence imaging results of zebra fish are shown in fig. 7, and it can be seen from fig. 7a-C that HeLa cells treated with group a, group B and group C substances simultaneously with (E) -2- (2- (3- (dicyanomethylene) -5, 5-dimethylcyclohex-1-en-1-yl) vinyl) -5- (diethylamino) phenyl 4-bromobutyric acid and hydrazine were not found to have fluorescence; while FIG. 7D shows that zebra fish treated with (E) -2- (2- (3- (dicyanomethylene) -5, 5-dimethylcyclohex-1-en-1-yl) vinyl) -5- (diethylamino) phenyl 4-bromobutyric acid and hydrazine show a clear red fluorescence; the fluorescence imaging result shows that (E) -2- (2- (3- (dicyanomethylene) -5, 5-dimethylcyclohex-1-en-1-yl) vinyl) -5- (diethylamino) phenyl 4-bromobutyric acid 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.
Claims (7)
1. A near infrared fluorescent compound for specifically detecting hydrazine, characterized in that: the near infrared fluorescent compound is (E) -2- (2- (3- (dicyanomethylene) -5, 5-dimethylcyclohex-1-en-1-yl) vinyl) -5- (diethylamino) phenyl 4-bromobutyrate, and the molecular formula is C 27 H 32 BrN 3 O 2 The chemical structural formula is shown as formula (I):
formula (I);
the preparation operation steps of the near infrared fluorescent compound for specifically detecting hydrazine are as follows:
(1) 1.0g of (E) -2- (3- (4- (diethylamino) -2-hydroxystyryl) -5, 5-dimethylcyclohex-2-en-1-yl) malononitrile, 1.0mL of triethylamine and 50mL of anhydrous dichloromethane are mixed under the protection of nitrogen, and the temperature is reduced to 0 ℃ in an ice bath;
(2) Slowly dripping 0.62mg of 4-bromobutyryl chloride, and stirring at room temperature for reaction for 10-12 hours to obtain a mixture;
(3) The mixture was poured into ice water, stirred, and extracted 3 times with 10mL of dichloromethane 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 in a volume ratio of 3:1 to give 1.03g of the purplish black product (E) -2- (2- (3- (dicyanomethylene) -5, 5-dimethylcyclohex-1-en-1-yl) vinyl) -5- (diethylamino) phenyl 4-bromobutyrate in 72% yield.
2. The method for detecting hydrazine in a solution system by using the near infrared fluorescent compound as claimed in claim 1, which is characterized by comprising the following operation 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 buffer salt solution and dimethyl sulfoxide in the volume ratio of 1:1, and the pH value of the buffer solution is 7.4;
(2) 81 parts of a 3mL near infrared fluorescent compound solution having a concentration of 10. Mu.M was taken, and 120. Mu.L of each solution having a concentration of 5X 10 was added thereto -5 The method comprises the steps of (1) mol/L of analytes to be detected, namely 27 analytes to be detected, 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 after the reaction is completed, the fluorescence intensity of 81 parts of reactants is measured respectively;
(3) The result shows that the hydrazine can improve the fluorescence intensity of the near infrared fluorescent compound working solution; the near infrared fluorescent compound can only perform fluorescence enhancement reaction with hydrazine, namely the near infrared fluorescent compound can realize specific recognition of the hydrazine.
3. The method for detecting hydrazine on test paper by using the near infrared fluorescent compound as claimed in claim 1, which is characterized by comprising the following operation steps:
(1) Preparing the near infrared fluorescent compound detection test paper according to claim 1, preparing the near infrared fluorescent compound into working solution with the concentration of 10 mu M by using methylene dichloride, 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 the test paper;
(2) 200 mu M of aqueous solutions of 27 different analytes are respectively dripped on corresponding 27 test papers; then placing the mixture into an ultraviolet lamp with excitation wavelength of 365nm for observation;
(3) The result shows that the fluorescent color of the test paper with the hydrazine solution added dropwise is changed from colorless to red, and other analytes do not change the test paper, namely the test paper can simply and rapidly detect the hydrazine as an environmental pollutant.
4. The method for detecting hydrazine in HeLa cells by using the near infrared fluorescent compound according to claim 1, wherein the method comprises the following steps:
(1) The process for preparing a near infrared fluorescent compound working solution as claimed in claim 1
Preparing the near infrared fluorescent compound of claim 1 into working solution with the concentration of 20 mu M by using buffer solution; the volume ratio of the phosphate buffer salt solution to the dimethyl sulfoxide in the buffer solution is 1:1, the pH value of the buffer solution is 7.4;
(2) Taking A, B, C, D experimental groups;
group a is a blank group: heLa cells without any treatment were the group A test subjects;
group B is a hydrazine treatment control group: 200. Mu.L of 50. Mu.M hydrazine in a solution containing 5X 10 4 Incubating HeLa cells in an orifice plate of the HeLa cells for 30 minutes to obtain a group B detected object for detection;
group C is near infrared fluorescenceCompound-treated control: at a concentration of 20. Mu.M, 200. Mu.L of near infrared fluorescent compound was used in a solution containing 5X 10 4 Incubating HeLa cells in an orifice plate of the HeLa cells for 30 minutes to obtain a group C detected object for detection; group D is near infrared fluorescent compound and hydrazine treatment group: pretreatment of 5X 10-containing compositions with 200. Mu.L of 20. Mu.M near infrared fluorescent compound 4 The well plate of HeLa cells was incubated with 200. Mu.L of 50. Mu.m hydrazine for 30 min to obtain group D test objects;
(3) Placing the A group detected object, the B group detected object, the C group detected object and the D group detected object under a fluorescence microscope with 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 have no fluorescence; and the group D detected objects show obvious red fluorescence; the fluorescence imaging result shows that the near infrared fluorescent compound in the invention 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.
5. The method for detecting the external 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) The process for preparing a near infrared fluorescent compound working solution as claimed in claim 1
Preparing the near infrared fluorescent compound of claim 1 into a working solution with the concentration of 20 mu M by using a buffer solution, wherein the buffer solution comprises phosphate buffer salt solution and dimethyl sulfoxide according to the volume ratio of 1:1, wherein the pH value of the buffer solution is 7.4;
(2) Take A, B, C, D four experimental groups
Group a is a blank group: untreated 3-day-old zebra fish, obtaining a group A of detected objects for detection; group B is a hydrazine treatment control group: incubating the zebra fish with 10mL of hydrazine with the concentration of 50 mu M for 30 minutes, and obtaining a group B detected object for detection;
group C is near infrared fluorescent compound treated control group: the 3-day-old zebra fish is treated with 10mL of the near infrared fluorescent compound of claim 1 at a concentration of 20 mu M for 30 minutes to obtain group C detection objects for detection;
group D is near infrared fluorescent compound and hydrazine treatment group: incubation of 3 day old zebra fish with 10mL of 50 μm hydrazine for 30 min followed by treatment with 20 μm of the near infrared fluorescent compound of claim 1 for 30 min to give group D test objects for detection;
(5) 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 excitation wavelength of 520nm for observation;
(6) The results showed that the group A, B and C subjects were not found to have fluorescence; and the group D detected objects show obvious red fluorescence; the fluorescence imaging result shows that the near infrared fluorescent compound in the invention in the claim 1 can enter the zebra fish body and react with exogenous hydrazine to generate strong red fluorescence, thereby realizing specific detection of the exogenous hydrazine.
6. 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 operation steps:
(1) The process for preparing a near infrared fluorescent compound working solution as claimed in claim 1
Preparing the near infrared fluorescent compound of claim 1 into a working solution with the concentration of 10 mu M by using a buffer solution; the buffer solution is prepared from phosphate buffer salt solution and dimethyl sulfoxide according to a volume ratio of 1:1, and the pH value of the buffer solution is 7.4;
(2) Preparation of environmental water sample to be tested
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 mu m water phase filter membrane to obtain a treated environmental water sample; adding 0.1 mu M, 0.5 mu M and 1.0 mu M hydrazine into 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 the hydrazine reaches 87-109%, and the result shows that the near infrared fluorescent compound working solution can quantitatively detect the existence of the hydrazine in an environmental water sample.
7. The method for detecting hydrazine in sewage by using the near infrared fluorescent compound as claimed in claim 1, which is characterized by comprising the following operation steps:
(1) Preparing the near infrared fluorescent compound of claim 1 into a working solution with the concentration of 10 mu M by using a buffer solution; the buffer solution is prepared from phosphate buffer salt solution and dimethyl sulfoxide according to a 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, the sewage samples of chemical plants in different areas are collected and filtered through a 200 mu m water phase filter membrane to obtain a sewage sample to be detected;
(3) Detecting the reaction liquid of the near infrared fluorescent compound working solution and the sewage sample by using a fluorescence spectrometer;
(4) The result shows that the near infrared fluorescent compound of claim 1 can quantitatively detect hydrazine in sewage with high sensitivity.
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