A fluorescent reagent for on-site detection of hydrazine

Abstract

This invention provides a fluorescent reagent for the on-site detection of hydrazine. The reagent, named 5-bromo-2-(8-(4-tert-butylphenyl)-5-phenylpyrimidino[4,5-d]pyridazin-2-yl)phenol, is combined with an organic solvent to form the detection reagent. Upon addition of hydrazine, the fluorescence color changes from colorless to blue-green, exhibiting a lit fluorescent signal. Under the same operation, ethylenediamine shows no response. This reagent has the advantages of simple operation, high sensitivity, good specificity, and easy visualization, and has broad application prospects for the on-site detection of hydrazine in complex environments.

Description

Technical Field

[0001] This invention belongs to the field of analytical chemistry and explosives detection technology, and specifically relates to a fluorescent reagent for on-site detection of hydrazine. Background Technology

[0002] The high specificity and ultrasensitivity of the detection of hazardous chemicals is a fundamental and crucial topic in analytical chemistry. It is not only the core driving force for the advancement of its methodology, but also a key application area that closely integrates the discipline with real-world issues such as safety, health, and environmental sustainability.

[0003] Hydrazine (N₂H₄), a typical hazardous chemical, has been widely used as a key precursor or reagent in industries such as pharmaceuticals, pesticides, dye synthesis, and aerospace. Its high volatility and recognized mutagenicity can cause irreversible damage to the nervous system, liver, and kidneys after acute exposure, posing a significant threat to human health and ecosystems. Therefore, developing precise hydrazine detection methods with both high specificity and sensitivity is crucial for ensuring environmental safety, human health, and public safety. Currently, various commercial detection methods have been developed using advanced instruments and sensing technologies such as high-performance liquid chromatography (HPLC), gas chromatography-mass spectrometry (GC-MS), electrochemical methods, fluorescence spectroscopy, and colorimetry. Among these, small-molecule fluorescent probes have attracted considerable attention due to their advantages, including specific recognition sites, high sensitivity, visualized response signals, and ease of operation. Based on the nucleophilicity of hydrazine, researchers have developed various small-molecule fluorescent probes containing potentiophilic sites (such as carbon-carbon double bonds, carbonyl groups, and ester groups). However, these probes are often limited by insufficient specificity—structurally similar aliphatic primary amines (such as ethylenediamine, EDA) can induce the same fluorescence response through nucleophilic addition reactions. This cross-reactivity has become a major obstacle to the accurate detection of hydrazine, making the development of probes that maintain high selectivity even at high EDA concentrations an urgent need to be addressed.

[0004] Currently, only a few studies attempt to distinguish between hydrazine and ethylenediamine, mainly involving amphiphilic potential design or fine-tuning of the reactivity of recognition sites. Both of these methods require sophisticated molecular structure engineering. Our team's previous research used phthalaldehyde and thiols as sensing reagents, achieving specific on / off detection of ethylenediamine through a characteristic six-membered ring formation mechanism, while hydrazine showed no response under these conditions. Introducing this unique six-membered ring construction strategy into hydrazine probe design holds promise for establishing a novel specific detection method. Building on this, effectively improving sensitivity through rational molecular design remains the primary key challenge for advancing precise hydrazine detection methods. Currently, most sensitivity enhancement studies focus on the structural regulation of small-molecule fluorescent probes, optimizing detection performance by improving the reactivity of recognition sites and enhancing probe-analyte interactions. A common strategy is to introduce electron-withdrawing groups (R-substituents) at the ortho position of the recognition site, promoting nucleophilic attack of hydrazine by increasing the positive charge density of the electrophilic carbon atom. However, with probe structure optimization becoming a standardized and mature method, how to innovate strategies to further improve detection sensitivity has become a key challenge. Summary of the Invention

[0005] The purpose of this invention is to provide a fluorescent reagent for the on-site detection of hydrazine. The reagent, named 5-bromo-2-(8-(4-tert-butylphenyl)-5-phenylpyrimidino[4,5-d]pyridazin-2-yl)phenol, is formulated with an organic solvent to form a fluorescent detection reagent. Upon addition of hydrazine, the fluorescence color changes from colorless to blue-green, exhibiting a bright fluorescent signal. Under the same operating conditions, ethylenediamine shows no response. This reagent has the advantages of simple operation, high sensitivity, good specificity, and easy visualization, and has broad application prospects, making it suitable for on-site detection of hydrazine in complex environments.

[0006] The present invention discloses a fluorescent reagent for on-site detection of hydrazine, the reagent being named 5-bromo-2-(8-(4-tert-butylphenyl)-5-phenylpyrimidino[4,5-d]pyridazin-2-yl)phenol, with the following structural formula (I):

[0007] (I) It is prepared by reacting 4-tert-butylbenzaldehyde, 3-(dimethylamino)-1-phenyl-2-propen-1-one and 3-amino-5-bromobenzo[D]isoxazole.

[0008] The method for preparing a fluorescent reagent for on-site detection of hydrazine comprises the following steps: Synthetic organic probes: a. 4-tert-butylbenzaldehyde and iodine catalyst were dissolved in 4 mL of dimethyl sulfoxide organic solvent at a molar ratio of 1:1.6 and reacted at 100℃ for 1 h. Then, 3-(dimethylamino)-1-phenyl-2-propen-1-one and 3-amino-5-bromobenzo[D]isoxazole were added at a molar ratio of 1:1 and the reaction was continued at 100℃ for 10 h to obtain a mixed solution. b. Cool the mixture obtained in step a to room temperature and quench it with saturated sodium thiosulfate solution. Dilute with water and extract three times with ethyl acetate. Combine the organic phases, wash with saturated sodium chloride solution, dry with anhydrous sodium sulfate, remove the solvent under reduced pressure, and purify the residue by silica gel column chromatography to obtain a yellow solid probe 5-bromo-2-(8-(4-tert-butylphenyl)-5-phenylpyrimidino[4,5-d]pyridazin-2-yl)phenol; Preparation of detection reagents; c. Dissolve the solid probe 5-bromo-2-(8-(4-tert-butylphenyl)-5-phenylpyrimidino[4,5-d]pyridazin-2-yl)phenol obtained in step b in dimethyl sulfoxide, sonicate to dissolve, and prepare a 0.1-10 mM 5-bromo-2-(8-(4-tert-butylphenyl)-5-phenylpyrimidino[4,5-d]pyridazin-2-yl)phenol solution to obtain the detection reagent; Prepare a standard solution of hydrazine: d. Prepare a standard solution of hydrazine with a concentration of 0-10 mM using deionized water as the solution; Preparation of alkaline buffers: e. Prepare a 5 M standard solution of NaOH using deionized water as the solution; Fluorescence detection of hydrazine: f. Take 20 μL of the detection reagent obtained in step c, dilute to 1800 μL with dimethyl sulfoxide solvent, add 20 μL of the NaOH standard solution prepared in step e (pH=7.5), and then add 180 μL of the hydrazine solutions of various concentrations prepared in step d to the detection reagent. After the reaction is complete, measure the fluorescence spectrum under 365 nm excitation light. As the concentration of hydrazine solution increases, the fluorescence color changes from colorless to blue-green. Linearly fit the fluorescence intensity at a wavelength of 489 nm to obtain a standard curve, which is used for the quantitative analysis of hydrazine content in the sample.

[0009] This invention discloses a fluorescent reagent for on-site detection of hydrazine. This reagent is based on a dual-control strategy of electrophilicity of the probe reaction site and nucleophilicity of the analyte. On one hand, the electrophilicity of the recognition site is adjusted by controlling the electron-withdrawing ability of the R substituent. Three substituents with different electron-withdrawing abilities (−Br>−H>−OCH3) are controlled, resulting in different maximum positive electrostatic potential values ​​at the carbonyl group of the 1,4-diketone unit in the probe. The -Br group probe has the largest electrostatic potential, indicating stronger electrophilicity of the carbonyl group and facilitating nucleophilic attack of hydrazine on the carbonyl group. On the other hand, the nucleophilicity of hydrazine is controlled by adjusting the solution pH. Acidic, neutral, and alkaline probe solutions are prepared, and hydrazine exists in different forms in different environments. In an acidic environment, hydrazine exists as N2H5. + It exists in a neutral environment as hydrazine (N2H4). When the pH is further adjusted to alkaline conditions, the N–H bond in hydrazine undergoes deprotonation to generate N2H3⁻. Its nitrogen atom carries a significantly enhanced negative charge, thereby significantly improving nucleophilicity and promoting efficient nucleophilic attack.

[0010] R-group (-Br,-H,-OMe) regulated fluorescent probe molecules were designed and synthesized: 5-benzoyl-2-(4-bromo-2-hydroxyphenyl)pyrimidin-4-yl](4-tert-butylphenyl) ketone; 5-benzoyl-2-(2-hydroxyphenyl)pyrimidin-4-yl](4-tert-butylphenyl) ketone; 5-benzoyl-2-(2-hydroxy-4-methoxyphenyl)pyrimidin-4-yl](4-tert-butylphenyl) ketone. The detection reagent in this method consists of 5-benzoyl-2-(4-bromo-2-hydroxyphenyl)pyrimidin-4-yl](4-tert-butylphenyl) ketone and an organic reagent. The nucleophilic reaction between the dicarbonyl group in the fluorescent reagent and hydrazine produces a fluorescence change from colorless to blue-green, with a detection limit as high as 5.77 nM. It is particularly effective in distinguishing ethylenediamine, a structural analog of hydrazine. It can be used for rapid on-site visual classification and identification of hydrazine, and features simple operation, high sensitivity, and easy visual observation, thus having broad application prospects.

[0011] This invention discloses a fluorescent reagent for on-site detection of hydrazine. After application, the fluorescence color changes from colorless to bluish-green, with a detection limit of 5.77 nM, and exhibits good interference resistance to analogues. The color change of fluorescence is highly beneficial for identification. The specific detection method is as follows: Use a pipette to add 1.8 mL of 0.1 mM test reagent to a reagent bottle. Then use a pipette to add 200 μL of hydrazine with concentrations of 10, 30, 50, 70, 90, 100, 200, 300 and 400 µM to the reagent bottle. Take a picture under a 365 nm UV lamp and record the result. The naked-eye detection limit of the test reagent is 10 µM. Alternatively, 1.8 mL of the test reagent can be pipetted into a quartz cuvette, and then 200 μL of hydrazine solutions with concentrations of 10, 30, 50, 70, 90, 100, 200, 300, and 400 µM can be added. Fluorescence emission spectra can be scanned using a fluorescence spectrometer to measure the fluorescence emission spectra of the test reagent after measuring the concentrations of hydrazine. Then, a linear equation in one variable can be fitted with the ratio of the maximum fluorescence emission peak intensity to the newly appearing peak as the ordinate and the concentration of hydrazine as the abscissa to determine the fluorescence detection limit of the test reagent as 5.77 nM.

[0012] Compared with existing technologies, this reagent has advantages such as simple preparation, low cost, mild reaction, sensitive reaction, and obvious visual response signal. It provides an excellent technical means for the on-site detection of hydrazine and has broad application prospects. At the same time, it provides a good research foundation for the field of explosives detection. Attached Figure Description

[0013] Figure 1 The present invention screens the optimal detection reagents for hydrazine using fluorescence spectra and visualization images; where a is the fluorescence spectrum and visualization image of the BrPMtBu detection reagent for hydrazine; b is the fluorescence spectrum and visualization image of the PMtBu detection reagent for hydrazine; and c is the fluorescence spectrum and visualization image of the OMePMtBu detection reagent for hydrazine. Figure 2 The diagram shows the optimal solvent effect of the detection reagent in this invention for screening the response of the detection reagent to hydrazine; where a is the fluorescence spectrum of the detection reagent without N2H4; and b is the fluorescence spectrum of the detection reagent to N2H4. Figure 3 This is a graph showing the optimal pH response of the detection reagent to hydrazine in this invention; where a is the fluorescence spectrum of the detection reagent without N2H4; and b is the fluorescence spectrum of the detection reagent to N2H4. Figure 4 The diagram shows the optimal concentration effect of the detection reagent for hydrazine response in this invention; where a is the fluorescence spectrum of the detection reagent without N2H4; and b is the fluorescence spectrum of the detection reagent with N2H4. Figure 5 The graph shows the fluorescence changes of the detection reagent of the present invention before and after the reaction with a series of concentrations of hydrazine and the linear relationship between the fluorescence and the concentration; where a is the optical response image of the probe detecting a series of concentrations of hydrazine; b is the fluorescence spectrum of the probe detecting a series of concentrations of hydrazine; and c is the linear correlation between the emission intensity at 489 nm and the hydrazine concentration. Figure 6 This is the specific fluorescence emission spectrum of the detection reagent of the present invention for hydrazine and other common interfering substances; Figure 7 This is the fluorescence emission spectrum of the detection reagent of the present invention against hydrazine and other common interfering substances; Figure 8 These are visualization images showing the specificity and interference of the detection reagent of the present invention with hydrazine structural analogs, solvents, and redox agents; where i represents selectivity and ii represents interference. Detailed Implementation

[0014] The present invention will be further described below with reference to specific embodiments, but the present invention is not limited to these embodiments. Example 1

[0015] Synthetic organic probes: a. 4-tert-butylbenzaldehyde and iodine catalyst were dissolved in 4 mL of dimethyl sulfoxide organic solvent at a molar ratio of 1:1.6 and reacted at 100℃ for 1 h. Then, 3-(dimethylamino)-1-phenyl-2-propen-1-one and 3-amino-5-bromobenzo[D]isoxazole were added at a molar ratio of 1:1 and the reaction was continued at 100℃ for 10 h to obtain a mixed solution. b. Cool the mixture obtained in step a to room temperature and quench it with saturated sodium thiosulfate solution. Dilute with water and extract three times with ethyl acetate. Combine the organic phases, wash with saturated sodium chloride solution, dry with anhydrous sodium sulfate, remove the solvent under reduced pressure, and purify the residue by silica gel column chromatography to obtain a yellow solid probe 5-bromo-2-(8-(4-tert-butylphenyl)-5-phenylpyrimidino[4,5-d]pyridazin-2-yl)phenol; Preparation of detection reagents; c. Dissolve the solid probe 5-bromo-2-(8-(4-tert-butylphenyl)-5-phenylpyrimidino[4,5-d]pyridazin-2-yl)phenol obtained in step b in dimethyl sulfoxide, and sonicate to prepare a 1-10 mM 5-bromo-2-(8-(4-tert-butylphenyl)-5-phenylpyrimidino[4,5-d]pyridazin-2-yl)phenol solution, which is the detection reagent; Prepare a standard solution of hydrazine: d. Prepare standard solutions of hydrazine with a concentration of 0.1-10 mM using deionized water as the solution; Preparation of alkaline buffers: e. Prepare a 5 M standard solution of NaOH using deionized water as the solution; Fluorescence detection of hydrazine: f. Take 20 μL of the detection reagent obtained in step c, and dilute to 1800 μL with dimethyl sulfoxide solvent. Take 20 μL of the NaOH standard solution prepared in step e, and then take 180 μL of the hydrazine solutions of various concentrations prepared in step d. Add them to the detection reagent respectively. After the reaction is complete, measure the fluorescence spectrum under 365 nm excitation light. As the concentration of hydrazine solution increases, the fluorescence color changes from colorless to blue-green. Linearly fit the fluorescence intensity at a wavelength of 489 nm to obtain a standard curve, which is used for the quantitative analysis of hydrazine content in the sample to be tested. Example 2

[0016] The test reagents were prepared according to Example 1: Fluorescence detection of hydrazine (N2H4): Take 1800 μL of the 5-bromo-2-(8-(4-tert-butylphenyl)-5-phenylpyrimidino[4,5-d]pyridazin-2-yl)phenol solution obtained in Example 1, and adjust the pH of the fluorescent detection reagent to 7.5 with 20 μL of the standard NaOH solution prepared in step e. After mixing evenly, add 200 μL of the 1 mM hydrazine (N2H4) solution prepared in step d to the detection reagent. After the reaction is complete, measure the fluorescence emission spectrum under 365 nm ultraviolet excitation light and calculate the difference between the maximum value of the fluorescence emission peak after the reaction and the maximum value of the fluorescence emission peak before the reaction. Example 3

[0017] The test reagents were prepared according to Example 1: Fluorescence detection of hydrazine (N2H4): 1800 μL of the 0.1 mM 5-bromo-2-(8-(4-tert-butylphenyl)-5-phenylpyrimidino[4,5-d]pyridazin-2-yl)phenol solution obtained in Example 1 was mixed with 20 μL of the NaOH standard solution prepared in step e. After mixing thoroughly, 180 μL of the 1 mM hydrazine (N2H4) solution prepared in step d was added to the detection reagent. After the reaction was complete, the fluorescence emission spectrum was measured and photographed under 365 nm ultraviolet excitation light. The difference between the maximum value of the fluorescence emission peak after the reaction and the maximum value of the fluorescence emission peak before the reaction was calculated.

[0018] Example 4

[0019] The test reagents were prepared according to Example 1: Fluorescence detection of hydrazine (N2H4): The detection reagent 5-bromo-2-(8-(4-tert-butylphenyl)-5-phenylpyrimidino[4,5-d]pyridazin-2-yl)phenol obtained in Example 1 was diluted to 1800 μL with dimethyl sulfoxide solvent using 20 μL of the fluorescent detection reagent. 20 μL of the NaOH standard solution prepared in step e was added, and the mixture was thoroughly mixed. Then, 180 μL of the 1 mM hydrazine (N2H4) solution prepared in step d was added to the detection reagent. After sufficient reaction, the fluorescence spectrum was measured under 365 nm excitation light, and an optical photograph was taken. Furthermore, using a fluorescence spectrometer, the fluorescence emission peak at 489 nm was enhanced when the excitation wavelength was selected as 365 nm. The fluorescence intensity at 489 nm was linearly fitted to obtain a standard curve, which was used for the quantitative analysis of hydrazine content in the sample. As the concentration of N2H4 solution increased, the fluorescence color changed from colorless to blue-green, and finally stabilized, indicating the presence of hydrazine in the detection system. The corresponding fluorescence color change and spectrum are shown below. Figure 5 As shown. Example 5

[0020] The test reagents were prepared according to Example 1: Prepare a standard solution of N2H4: The standard N2H4 solution was diluted with deionized water to a concentration of 0-10 mM. Preparation of alkaline buffers: A standard NaOH solution with a concentration of 5 M was prepared using deionized water. Preparation of hydrazine solutions: Blank solution and hydrazine solutions are all dimethyl sulfoxide solutions; Other substance concentration configurations: The selection of analytes includes amine compounds such as ethylenediamine, urea, and alanine; oxidizing and reducing agents such as sodium bromate, sodium iodate, sodium nitrite, potassium iodide, sodium iodide, thiourea, aniline, and cysteine; and other compounds such as sodium chloride, ammonium chloride, sodium bicarbonate, sodium thiosulfate, 3,5-bis(trifluoromethyl)benzylthiophenol, and triphenylamine. Fluorescence detection of hydrazine: 1.8 mL of 0.5 mM 5-bromo-2-(8-(4-tert-butylphenyl)-5-phenylpyrimidino[4,5-d]pyridazin-2-yl)phenol, obtained in Example 1, was added to a test bottle. 200 µL of 5 mM / L hydrazine solution and 200 µL of 5 mM / L solutions of the remaining substances were added separately. When hydrazine was observed to be present, fluorescence spectroscopy revealed that the fluorescence emission peak intensity at 489 nm increased to its maximum value when only synthetic hydrazine was present. No change in fluorescence emission peak intensity was observed when other substances were present. A broken line was plotted based on the emission peak intensity at 489 nm in the fluorescence spectrum, as shown in Figure 6.

[0021] The above results demonstrate that this test reagent has excellent visual differentiation and detection capabilities for hydrazine and amine compounds, oxidants and reductants, and other compounds when they coexist. Example 6

[0022] The test reagents were prepared according to Example 1: Fluorescence detection of hydrazine: 1.8 mL of the 0.5 mM detection reagent 5-bromo-2-(8-(4-tert-butylphenyl)-5-phenylpyrimidino[4,5-d]pyridazin-2-yl)phenol obtained in Example 1 was added to the test bottle, along with 100 µL of 5 mM / L hydrazine solution and 100 µL of 5 mM / L solution of the remaining substances. When hydrazine was observed in all cases, fluorescence spectroscopy showed that when only the synthetic hydrazine was present, the fluorescence emission peak intensity at 489 nm increased, reaching the maximum value. A broken line was plotted based on the emission peak intensity at 489 nm in the fluorescence spectrum, as shown in Figure 7.

[0023] The above results demonstrate that this test reagent has excellent visual differentiation and detection capabilities for hydrazine and amine compounds, oxidants and reductants, and other compounds when they coexist. Example 7

[0024] The test reagents were prepared according to Example 1: The 1.8 mL of 0.5 mM 5-bromo-2-(8-(4-tert-butylphenyl)-5-phenylpyrimidino[4,5-d]pyridazin-2-yl)phenol obtained in Example 1 was added to a test bottle, followed by the addition of 200 µL of 5 mM / L hydrazine solution and 200 µL of the remaining solutions. Under 365 nm UV light, the presence of hydrazine was observed with the naked eye, and the fluorescence color of the test reagent solution changed from colorless to bluish-green. The fluorescence color of the test reagent solution remained unchanged in the presence of the other substances. Add 1.8 mL of the 0.5 mM detection reagent obtained in step c to the test vial, along with 100 µL of 5 mM / L hydrazine solution and 100 µL of 5 mM / L solution of the remaining substances. Under a 365 nm UV lamp, the presence of hydrazine was observed with the naked eye, and the fluorescence color of the detection reagent solution was bluish-green. Figure 8 As shown.

[0025] The above results indicate that the detection of hydrazine by this reagent is not affected by amine compounds, oxidizing agents, reducing agents, or other compounds.

Claims

1. A fluorescent reagent for on-site detection of hydrazine, characterized in that, The reagent is named 5-bromo-2-(8-(4-tert-butylphenyl)-5-phenylpyrimidino[4,5-d]pyridazin-2-yl)phenol, and its structural formula (I) is: （Ⅰ） It is prepared by reacting 4-tert-butylbenzaldehyde, 3-(dimethylamino)-1-phenyl-2-propen-1-one and 3-amino-5-bromobenzo[D]isoxazole.

2. The method for preparing a fluorescent reagent for on-site detection of hydrazine as described in claim 1, characterized in that, The method is performed according to the following steps: Synthetic organic probes: a. 4-tert-butylbenzaldehyde and iodine catalyst were dissolved in 4 mL of dimethyl sulfoxide organic solvent at a molar ratio of 1:1.6 and reacted at 100℃ for 1 h. Then, 3-(dimethylamino)-1-phenyl-2-propen-1-one and 3-amino-5-bromobenzo[D]isoxazole were added at a molar ratio of 1:1 and the reaction was continued at 100℃ for 10 h to obtain a mixed solution. b. Cool the mixture obtained in step a to room temperature and quench it with saturated sodium thiosulfate solution. Dilute with water and extract three times with ethyl acetate. Combine the organic phases, wash with saturated sodium chloride solution, dry with anhydrous sodium sulfate, remove the solvent under reduced pressure, and purify the residue by silica gel column chromatography to obtain a yellow solid probe 5-bromo-2-(8-(4-tert-butylphenyl)-5-phenylpyrimidino[4,5-d]pyridazin-2-yl)phenol; Preparation of detection reagents; c. Dissolve the solid probe 5-bromo-2-(8-(4-tert-butylphenyl)-5-phenylpyrimidino[4,5-d]pyridazin-2-yl)phenol obtained in step b in dimethyl sulfoxide, sonicate to dissolve, and prepare a 0.1-10 mM 5-bromo-2-(8-(4-tert-butylphenyl)-5-phenylpyrimidino[4,5-d]pyridazin-2-yl)phenol solution to obtain the detection reagent; Prepare a standard solution of hydrazine: d. Prepare a standard solution of hydrazine with a concentration of 0-10 mM using deionized water as the solution; Preparation of alkaline buffers: e. Prepare a 5 M standard solution of NaOH using deionized water as the solution; Fluorescence detection of hydrazine: f. Take 20 μL of the detection reagent obtained in step c, dilute to 1800 μL with dimethyl sulfoxide solvent, add 20 μL of the NaOH standard solution prepared in step e (pH=7.5), and then add 180 μL of the hydrazine solutions of various concentrations prepared in step d to the detection reagent. After the reaction is complete, measure the fluorescence spectrum under 365 nm excitation light. As the concentration of hydrazine solution increases, the fluorescence color changes from colorless to blue-green. Linearly fit the fluorescence intensity at a wavelength of 489 nm to obtain a standard curve, which is used for the quantitative analysis of hydrazine content in the sample.

Images