An oxazine compound, a preparation method and application thereof, and a fluorescence detection method for copper ions

Abstract

The application provides an oxazine compound and a preparation method and application thereof and a fluorescence detection method of copper ions, relates to the technical field of analysis and detection. The oxazine compound provided by the application has strong selective combination capacity for copper ions, greatly reduces background interference, can be used for efficiently detecting copper ions, and can realize higher detection sensitivity and accuracy of copper ions in biological and environmental samples, provides a more reliable tool for rapid detection of copper ions, and is especially suitable for application in environmental water quality monitoring and biological samples.

Description

Technical Field

[0001] This invention relates to the field of analytical detection technology, specifically to an oxazine compound, its preparation method and application, and a fluorescence detection method for copper ions. Background Technology

[0002] Copper ions (Cu) 2+ Copper is an important soft transition metal, widely involved in various physiological processes in the human body, such as electron transport, enzymatic reactions, and neurotransmitter synthesis. Although its content in the body is lower than that of iron and zinc, it is crucial for life activities. 2+ The physiological functions involved include energy metabolism, immune responses, and the normal functioning of the nervous system. However, Cu 2+ Excessive accumulation of Cu can also pose serious health risks. Short-term exposure to high concentrations of Cu... 2+ In the environment, it typically manifests as gastrointestinal discomfort, such as nausea and vomiting; while long-term exposure can lead to damage to organs such as the liver and kidneys, and even cause severe poisoning. Studies have shown that Cu... 2+ Interactions with free radicals can lead to cell damage, impair protein function, and trigger oxidative stress. Therefore, international organizations such as the World Health Organization (WHO) and the U.S. Environmental Protection Agency (EPA) have already addressed the issue of Cu in drinking water. 2+ The concentration has a maximum permissible concentration (MAL). The WHO recommends that Cu in drinking water... 2+ The concentration should not exceed 2.0 mg / L, while the EPA sets this value at 1.3 mg / L. Excessive Cu 2+ Cu not only poses a threat to human health but also has a negative impact on aquatic ecosystems. Therefore, real-time and accurate monitoring of Cu in water is crucial. 2+ Concentration is of particular importance.

[0003] Currently, Cu detection 2+ The main methods include atomic absorption spectroscopy (AAS), atomic emission spectroscopy (AES), atomic fluorescence spectroscopy (AFS), inductively coupled plasma mass spectrometry (ICP-MS), and fluorescent probe sensing technology. Among these, fluorescent probe sensing technology has significant advantages such as low cost, ease of operation, high sensitivity, and fast response speed. Therefore, fluorescent probes can not only accurately detect target metal ions at low concentrations, but also perform quantitative analysis through optical imaging technology, facilitating rapid on-site detection and real-time monitoring, and showing great application potential in environmental monitoring, food safety testing, and life science research.

[0004] Significant progress has been made in the research of fluorescent probes over the past few decades. As early as 1997, the first fluorescent probe for detecting Cu was designed and synthesized. 2+A fluorescent probe containing a rhodamine B hydrazine derivative. Studies have found that rhodamine hydrazine reacts with Cu... 2+ The resulting coordination complex leads to ring-opening of the spironolactone ring. Further hydrolysis in a neutral HEPES buffer solution produces strong fluorescence emission accompanied by a change in solution color. This finding provides a basis for Cu 2+ The detection method offers a new approach. However, the structure of rhodamine has certain limitations, leading to false positive signals from the fluorescent probe during detection, which affects the accuracy of the analytical results. Therefore, developing a fluorescent probe capable of accurately detecting copper ions is of great significance. Summary of the Invention

[0005] In view of this, the purpose of this invention is to provide an oxazine compound, its preparation method and application, and a fluorescence detection method for copper ions. The oxazine compound (denoted as Ox1) provided by this invention is used for the fluorescence detection of copper ions, exhibiting low background interference, high detection accuracy, and high sensitivity.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] This invention provides an oxazine compound having the structure shown in Formula I:

[0008] Formula I;

[0009] Wherein, R is a C1~C6 alkyl group.

[0010] Preferably, R is methyl, ethyl, propyl, butyl, pentyl, or hexyl.

[0011] The present invention also provides a method for preparing the oxazine compounds described in the above technical solution, comprising the following steps: mixing compound 1, compound 2, an organic base and a polar organic solvent, and carrying out a substitution reaction to obtain the oxazine compounds;

[0012] .

[0013] Preferably, the molar ratio of compound 1 to compound 2 is 1:1 to 2.

[0014] Preferably, the organic base includes an organic amine;

[0015] The molar ratio of compound 1 to organic base is 1:2~5.

[0016] Preferably, the polar organic solvent includes one or more of the following: halogenated hydrocarbons, nitrile solvents, furan solvents, sulfoxide solvents, and amide solvents.

[0017] Preferably, the substitution reaction is carried out at a temperature of 25~60℃ for a time of 0.5~4h, and the substitution reaction is carried out under a protective atmosphere.

[0018] This invention also provides the application of the oxazine compounds described in the above technical solution in the detection of copper ions.

[0019] This invention also provides a method for fluorescence detection of copper ions, comprising the following steps:

[0020] The copper ion solution to be tested, the fluorescent probe solution, and the solvent are mixed to obtain the test solution; the fluorescent probe in the fluorescent probe solution includes the oxazine compound described in the above technical solution;

[0021] The test solution was subjected to fluorescence emission spectroscopy to obtain the detection results of copper ions.

[0022] Preferably, the solvent comprises a phosphate buffer solution;

[0023] The conditions for fluorescence emission spectroscopy detection include: excitation wavelength of 615~655nm, excitation slit width of 5~10nm, emission slit width of 5~10nm, and photomultiplier tube operating voltage of 300~700V.

[0024] The oxazine compounds with the structure shown in Formula I provided by this invention are based on oxazine 1 (structure shown in Formula II), and incorporate a copper ion recognition group for sensitive response and a flexible p-ester benzyl alcohol chain, achieving functional modification of the oxazine 1 fluorophore. The oxazine compounds provided by this invention have a non-conjugated molecular structure, exhibiting extremely low background fluorescence emission intensity when used for copper ion detection, with minimal background interference, high detection accuracy, and high sensitivity. Unlike most fluorescent probes for copper ion detection based on intramolecular charge transfer (ICT) mechanisms, the relationship between the oxazine 1 fluorophore and the oxazine compound is based on a transition between conjugated and non-conjugated structures. This molecular structure design theoretically leads to effective fluorescence quenching without requiring additional explanation of the molecular mechanism. After the introduction of copper ions, the copper ions catalyze the cleavage of the oxazine compound, causing the linking group to self-destruct and form the oxazine 1 fluorophore, which emits a strong near-infrared fluorescence signal.

[0025] Formula II

[0026] The oxazine compounds provided by this invention have a strong selective binding ability to copper ions, which greatly reduces background interference and can be used for efficient detection of copper ions. Furthermore, they can achieve higher detection sensitivity and accuracy of copper ions in biological and environmental samples, providing a more reliable tool for rapid detection of copper ions. They have great application prospects in environmental water quality monitoring and detection of copper ions in biological samples.

[0027] The method for preparing oxazine compounds provided by this invention is simple in process and operation, has high yield and low production cost, and is suitable for industrial production.

[0028] The fluorescence detection method for copper ions provided by this invention uses an oxazine compound with the structure shown in Formula I as a fluorescent probe, which has low background interference and can achieve accurate, highly sensitive and rapid detection of copper ions. Attached Figure Description

[0029] Figure 1 The image shows the detection results of copper ions in Example 2, where (A) shows the results of different concentrations of Cu under 635nm excitation light. 2+ The fluorescence spectrum of the test solution is shown in (B), which shows the fluorescence emission intensity of the test solution at 685 nm under 635 nm excitation light and the fluorescence intensity of Cu. 2+ The linear relationship of concentration (0~0.8μM), (C) is the concentration of the test solution (Cu) under irradiation with 635nm excitation light. 2+ The trend of fluorescence intensity change over time for Ox1 solution (10 μM) at a concentration of 0.8 μM, where (D) represents the fluorescence intensity of Ox1 solution (10 μM), Ox1 solution (10 μM), and Cu. 2+ The photostability of the (0.8 μM) reaction system and oxazine 1 (10 μM, Oxazine 1, PBS, pH=7.4) under continuous irradiation with 635 nm excitation light. Detailed Implementation

[0030] This invention provides an oxazine compound having the structure shown in Formula I:

[0031] Formula I;

[0032] Wherein, R is a C1~C6 alkyl group, which in specific embodiments can be methyl, ethyl, propyl, butyl, pentyl or hexyl.

[0033] The present invention also provides a method for preparing the oxazine compounds described in the above technical solution, comprising the following steps: mixing compound 1, compound 2, an organic base and a polar organic solvent, and carrying out a substitution reaction to obtain the oxazine compounds;

[0034] .

[0035] Unless otherwise specified, the materials and equipment used in this invention are all commercially available products in the field.

[0036] In this invention, the molar ratio of compound 1 to compound 2 is preferably 1:1 to 2, and in specific embodiments it can be 1:1, 1:1.05, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9 or 1:2.

[0037] In this invention, the organic base preferably comprises an organic amine, which preferably comprises one or more of 4-dimethylaminopyridine (DMAP), triethylamine (TEA), pyridine, 4-pyrrolylpyridine (PPY), 4-methylaminopyridine (MAP), 4-aminopyridine (4-AP), and 2-dimethylaminopyridine (2-DMAP). In specific embodiments, it can be a mixture of 4-dimethylaminopyridine and triethylamine, wherein the molar ratio of 4-dimethylaminopyridine to triethylamine in the mixture is preferably 1:10 to 20, and in specific embodiments, it can be 1:10, 1:12, 1:14, 1:14.4, 1:15, 1:18, or 1:20. In this invention, the molar ratio of compound 1 to the organic base is preferably 1:2 to 5, and in specific embodiments, it can be 1:2, 1:2.5, 1:2.8, 1:3, 1:3.5, 1:4, 1:4.5, or 1:5.

[0038] In this invention, the polar organic solvent preferably includes one or more of halogenated hydrocarbons, nitrile solvents, furan solvents, sulfoxide solvents, and amide solvents. In specific embodiments, it can be one or more of dichloromethane, trichloromethane, acetonitrile, tetrahydrofuran, dimethyl sulfoxide (DMSO), and dimethylformamide (DMF). In this invention, the molar ratio of compound 1 to the volume of the polar organic solvent is preferably 0.1 mmol: 1~5 mL. In specific embodiments, it can be 0.1 mmol: 1 mL, 0.1 mmol: 2 mL, 0.1 mmol: 3 mL, 0.1 mmol: 4 mL, or 0.1 mmol: 5 mL.

[0039] In this invention, the temperature of the substitution reaction is preferably 25~60℃, and in specific embodiments it can be 25℃, 30℃, 35℃, 40℃, 45℃, 50℃, 55℃ or 60℃; the time of the substitution reaction is preferably 0.5~4h, and in specific embodiments it can be 0.5h, 1h, 1.5h, 2h, 3h or 4h; the substitution reaction is preferably carried out under a protective atmosphere, which preferably includes nitrogen, argon or helium.

[0040] After the substitution reaction is completed, the present invention preferably further includes: concentrating the reaction solution obtained from the substitution reaction and then purifying it by silica gel column chromatography to obtain the oxazine compound. The present invention does not have a specific limitation on the concentration method; any concentration method well known to those skilled in the art that can remove the solvent is acceptable, such as vacuum distillation. In the present invention, the eluent used for the silica gel column chromatography purification is preferably petroleum ether and ethyl acetate, and the volume ratio of petroleum ether to ethyl acetate is preferably 0.5~2:1, which can be 0.5:1, 1:1, 1.5:1, or 2:1 in specific embodiments.

[0041] This invention also provides the application of the oxazine compounds described in the above-mentioned technical solution in the detection of copper ions. By optimizing the probe molecular structure, this invention designs oxazine compounds (fluorescent probes based on oxazine 1) that can be used for efficient detection of copper ions. These oxazine compounds not only exhibit lower interference in background signals but also show significant improvements in sensitivity and stability, providing a more reliable tool for rapid, accurate, and highly sensitive detection of copper ions, especially suitable for environmental water quality monitoring and the detection of copper ions in biological samples.

[0042] This invention also provides a method for fluorescence detection of copper ions, comprising the following steps:

[0043] The copper ion solution to be tested, the fluorescent probe solution, and the solvent are mixed to obtain the test solution; the fluorescent probe in the fluorescent probe solution includes the oxazine compound described in the above technical solution;

[0044] The test solution was subjected to fluorescence emission spectroscopy to obtain the detection results of copper ions.

[0045] The present invention mixes a copper ion solution to be tested, a fluorescent probe solution, and a solvent to obtain a test solution; the fluorescent probe in the fluorescent probe solution includes the oxazine compounds described in the above technical solution.

[0046] In this invention, the solvent in the copper ion solution to be tested preferably includes water.

[0047] In this invention, the solvent preferably includes a phosphate buffer solution, the pH of which is preferably 7.2 to 7.6, and in specific embodiments may be 7.2, 7.4 or 7.6; the concentration of which is preferably 5 to 20 mM (mmol / L), and in specific embodiments may be 5 mM, 10 mM, 15 mM or 20 mM.

[0048] In this invention, the solvent in the fluorescent probe solution preferably includes dimethyl sulfoxide and / or dimethylformamide; the concentration of the fluorescent probe solution is preferably 0.5~1.5mM, and in specific embodiments it can be 0.5mM, 1mM or 1.5mM; the fluorescent probe solution is preferably stored in a light-protected environment at -30~-50℃ before use, and in specific embodiments it can be stored in a light-protected environment at -30℃, -35℃, -40℃, -45℃ or -50℃.

[0049] In this invention, the copper ion concentration in the test solution is preferably 0~0.8 μM (μmol / L), and in specific embodiments it can be 0 μM, 0.001 μM, 0.005 μM, 0.01 μM, 0.05 μM, 0.1 μM, 0.2 μM, 0.3 μM, 0.4 μM, 0.5 μM, 0.6 μM, 0.7 μM or 0.8 μM. In this invention, when the copper ion concentration in the test solution is >8 μM, it is preferably diluted to a concentration ≤0.8 μM. The diluent used for dilution preferably includes water, phosphate buffer solution or Tris-HCl ammonium buffer solution; the pH value of the phosphate buffer solution and the Tris-HCl ammonium buffer solution is independently preferably 6~8, and in specific embodiments it can be 6, 6.5, 7, 7.2, 7.4, 7.6, 7.8 or 8; the concentration of the phosphate buffer solution is preferably... The concentration is 10~100mM, and in specific embodiments it can be 10mM, 20mM, 30mM, 40mM, 50mM, 60mM, 70mM, 80mM, 90mM or 100mM; the concentration of the Tris-HCl ammonium buffer solution is preferably 10~100mM, and in specific embodiments it can be 10mM, 20mM, 30mM, 40mM, 50mM, 60mM, 70mM, 80mM, 90mM or 100mM.

[0050] In this invention, the molar ratio of copper ions to fluorescent probe in the test solution is preferably 0~0.08:0.1, and in specific embodiments it can be 0:1, 0.001:1, 0.005:1, 0.008:1, 0.01:1, 0.02:1, 0.03:100, 0.04:1, 0.05:1, 0.06:1, 0.07:1 or 0.08:1.

[0051] After obtaining the test solution, the present invention performs fluorescence emission spectroscopy on the test solution to obtain the detection result of copper ions.

[0052] In this invention, the preferred conditions for fluorescence emission spectroscopy detection include: an excitation wavelength of 615-655 nm, which in specific embodiments can be 615 nm, 620 nm, 625 nm, 630 nm, 635 nm, 640 nm, 645 nm, 650 nm, or 655 nm; an excitation slit width of 5-10 nm, which in specific embodiments can be 5 nm or 10 nm; an emission slit width of 5-10 nm, which in specific embodiments can be 5 nm or 10 nm; and a photomultiplier tube operating voltage (PMT voltage) of 300-700 V, which in specific embodiments can be 300 V, 350 V, 400 V, 450 V, 500 V, 550 V, 600 V, 650 V, or 700 V; and recording the emission spectrum in the range of 660-800 nm during the fluorescence emission spectroscopy detection process.

[0053] In this invention, the detection results preferably include quantitative detection results and / or qualitative detection results.

[0054] In this invention, an oxazine compound with R being methyl (denoted as Ox1) is used as an example. The reaction mechanism of the oxazine compound for detecting copper ions is as follows: Although the methylene blue fluorophore is widely used in fluorescent probes, its quantum yield is low, and it generates strongly oxidizing singlet oxygen under excitation light, affecting the determination of metal ions. In recent years, the commercially available dye oxazine 1 fluorophore has shown advantages due to its stable structure and superior fluorescence brightness. This invention selects oxazine 1, which has superior performance and better application effects, as a raw material. Na2S2O4 is added to the reaction system to break the rigid conjugated structure of oxazine 1, and a Cu for sensitive response is introduced. 2+ The functionalization modification of the oxazine 1 fluorophore, including the recognition group and the flexible chain of p-ester benzyl alcohol, enriches the fluorophore. The formation of the non-conjugated molecular structure results in the extremely low background fluorescence emission intensity of probe Ox1, a highlight of the Ox1 structural design. This contrasts with most ICT-based Cu detection methods. 2+ The fluorescent probe differs from the others; the transition between the oxazine 1 fluorophore and the probe Ox1 is based on a conjugated and non-conjugated structure. This design of the probe molecular structure theoretically leads to efficient fluorescence quenching without requiring further explanation of the molecular mechanism. As shown in equation (1), the introduction of Cu... 2+ After that, Cu 2+ Catalytic cleavage of Ox1 involves the cleavage of the linker group, which then disappears, forming an oxazine 1 fluorophore that emits a strong near-infrared fluorescence signal. In this invention, if the test solution produces a near-infrared fluorescence signal, it indicates the presence of copper ions.

[0055] Equation (1).

[0056] In this invention, the method for obtaining the quantitative detection results is preferably the standard curve method. In this invention, the standard curve used in the standard curve method uses copper ion concentration as the independent variable and fluorescence intensity as the dependent variable.

[0057] In this invention, the method for plotting the standard curve preferably includes the following steps: preparing a series of standard solutions, performing fluorescence emission spectroscopy detection on the series of standard solutions according to the aforementioned fluorescence emission spectroscopy detection conditions, and plotting a standard curve with copper ion concentration as the independent variable and fluorescence intensity as the dependent variable. In this invention, the series of standard solutions includes copper ions, oxazine compounds, and a solvent. The solvent preferably includes dimethyl sulfoxide (DMSO) and phosphate buffer solution. The copper ion concentrations in the series of standard solutions are preferably 0 μM, 0.05 μM, 0.15 μM, 0.25 μM, 0.38 μM, 0.58 μM, 0.7 μM, 0.75 μM, and 0.8 μM, respectively. The concentration of the oxazine compounds in the series of standard solutions is preferably 10 μM. In this invention, the pH value of the phosphate buffer solution is preferably 6-8, and in specific embodiments, it can be 6, 6.5, 7, 7.2, 7.4, 7.6, 7.8, or 8. The concentration of the phosphate buffer solution is preferably 10-100 mM. In this invention, the preferred method for preparing the series of standard solutions includes the following steps: preparing an oxazine compound stock solution; adding the oxazine compound stock solution to a phosphate buffer solution, adding different volumes of copper ion stock solution, and adjusting the volume of the phosphate buffer solution to obtain the series of standard solutions. In this invention, the concentration of the oxazine compound stock solution is preferably 1 mM, and the solvent in the oxazine compound stock solution is preferably dimethyl sulfoxide. In this invention, the concentration of copper ions in the copper ion stock solution is preferably 0.1 mM, and the solvent in the copper ion stock solution is preferably water. In this invention, the fluorescence intensity is preferably the fluorescence intensity at 685 nm.

[0058] To further illustrate the present invention, the following detailed descriptions, in conjunction with embodiments, illustrate the oxazine compounds, their preparation methods and applications, and the fluorescence detection method for copper ions provided by the present invention. However, these descriptions should not be construed as limiting the scope of protection of the present invention.

[0059] Example 1

[0060]

[0061] Synthesis of probe Ox1: Compound 1 (0.36 mmol) and 4-acetoxybenzyl alcohol (0.375 mmol) were mixed in 18 mL of dichloromethane under a nitrogen atmosphere, and triethylamine (2 mL) and 4-dimethylaminopyridine (1.0 mmol) were added. The mixture was stirred at 45 °C for 1 h, and the solvent was removed by vacuum distillation. The mixture was purified by silica gel column chromatography (eluent: petroleum ether: ethyl acetate volume ratio = 1:1) to give an oxazine compound (Ox1, light blue solid, 37 mg, yield 20%, purity 98%). 1 H NMR (300 MHz, DMSO-) d 6 ) δ 7.43 (d, J = 8.5 Hz , 2H), 7.30 (s, 1H), 7.15 – 7.10 (m,2H), 6.67 – 6.62 (m, 2H), 6.42 – 6.38 (m, 1H), 6.32 (d, J = 2.8 Hz , 2H), 5.20(s, 1H), 3.28 (d, J = 5.8 Hz , 5H), 3.25 (s, 4H), 2.26 (s, 3H), 1.04 (s, 12H). 13 C-NMR (75 MHz, DMSO- d 6 ) δ 179.38, 170.59, 153.67, 151.22, 146.28, 137.54,132.41, 128.81, 125.09, 120.13, 106.38, 99.34, 67.42, 57.72, 44.58, 29.67,25.62, 12.46. HR-MS (m / z): Calculated for [C 33 H 35 N3O5] + : 518.2650, found:518.2654.

[0062] Example 2

[0063] Detection of copper ions

[0064] Experimental group: Ox1 solid was dissolved in 1.930 mL of DMSO to obtain a 1 mM Ox1 stock solution, which was stored in a -40°C refrigerator protected from light. 10 μL of the Ox1 stock solution was added to phosphate buffered saline (PBS, pH 7.4, 10 mM), followed by different volumes of 0.1 mM Cu. 2+The aqueous solution was diluted to a total volume of 1000 μL using phosphate buffered saline (PBS, pH 7.4, 10 mM). The final Cu concentration in the test solution was... 2+ The concentrations were 0 μM, 0.05 μM, 0.15 μM, 0.25 μM, 0.38 μM, 0.58 μM, 0.7 μM, 0.75 μM, and 0.8 μM, respectively, with Ox1 concentration at 10 μM. Cu 2+ A standard curve was plotted with concentration on the x-axis and fluorescence intensity at 685 nm on the y-axis.

[0065] Control group: The only difference from the experimental group is that Cu was used... 2+ Replace the aqueous solution with water.

[0066] The fluorescence emission spectrum of the test solution was measured using a quartz cuvette with an optical path length of 1 cm. The excitation wavelength was set to 635 nm, the excitation and emission slit widths were 5 nm and 10 nm, respectively, the PMT voltage was 700 V, and the emission spectrum in the range of 660–800 nm was recorded.

[0067] Figure 1 The image shows the detection results of copper ions, where (A) represents the results of different concentrations of Cu under 635nm excitation light. 2+ Fluorescence spectrum of the test solution (from top to bottom Cu) 2+ The concentrations decrease sequentially. (B) represents the fluorescence emission intensity of the test solution at 685 nm under 635 nm excitation light and the relationship between the concentrations and Cu. 2+ Linearity of concentration (0~0.8μM) (standard curve), (C) represents the concentration of the test solution (Cu) under irradiation with 635nm excitation light. 2+ The trend of fluorescence intensity change over time for Ox1 solution (10 μM) at a concentration of 0.8 μM, where (D) represents the fluorescence intensity of Ox1 solution (10 μM), Ox1 solution (10 μM), and Cu. 2+ The photostability of the (0.8 μM) reaction system and oxazine 1 (10 μM, Oxazine 1, PBS, pH=7.4) under continuous irradiation with 635 nm excitation light.

[0068] like Figure 1 As shown in (A), the interaction between probe Ox1 and Cu was first investigated. 2+ Fluorescence spectral responses before and after the reaction. Cu was added to probe Ox1. 2+ After the reaction, it does not contain Cu. 2+ The Ox1 system exhibits extremely low fluorescence emission intensity at 685 nm. A 0.1 mM Cu solution was gradually added to an Ox1-containing solution. 2+ At this time, the system color gradually changed from colorless to light blue, and the fluorescence emission intensity at 685 nm significantly increased, increasing by up to approximately 23 times. This good fluorescence enhancement indicates the interaction between Ox1 and Cu.2+ The effective reaction between them. This phenomenon is related to the substrate Ox1 in Cu. 2+ This is consistent with the explanation that after degradation, it gradually transforms into the oxazine 1 fluorophore.

[0069] The fluorescence intensity of the system and Cu were further investigated. 2+ Quantitative relationships between contents, such as Figure 1 As shown in (B), different concentrations of Cu were added. 2+ The corresponding fluorescence intensity values ​​of the system were measured. The results show that the fluorescence intensity values ​​of the system are related to Cu... 2+ The concentration (0–0.8 μM) showed a linear correlation. The detection limit for copper ions calculated using the 3σ / k formula was 0.015 μM.

[0070] Then, the relationship between Ox1 and Cu was investigated. 2+ The change in fluorescence intensity of the system with reaction time during the reaction process is studied to determine the optimal reaction time. For example... Figure 1 As shown in (C), the final Cu in the reaction system 2+ The concentration was 0.8 μM, with Cu 2+ The substrate Ox1 was cleaved to generate more and more oxazine 1 fluorophores, and the fluorescence intensity of the system at 685 nm increased significantly with time (λ). ex The fluorescence intensity of the probe Ox1 (635 nm) peaked at 2 minutes and remained almost stable thereafter, indicating that the probe Ox1 reacts with Cu. 2+ It responds quickly, and can react completely in 2 minutes.

[0071] Good photostability is an important foundation for subsequent spectroscopic experiments, such as Figure 1 As shown in Figure (D), the photostability of the system was tested. Under continuous irradiation with 635 nm excitation light, Ox1, Ox1 and Cu... 2+ The changes in fluorescence intensity at 685 nm between the mixed system (test solution) and oxazine 1 are almost negligible, indicating that the synthesized Ox1, the reaction system, and the commercially available oxazine 1 fluorophore can all resist photodecomposition and maintain their stable molecular structure.

[0072] In conclusion, this fully demonstrates the relationship between Ox1 and Cu. 2+ The effective binding ability and interaction between them further clarify their wide application in precise quantitative detection.

[0073] Example 3

[0074] Detection of copper ions in environmental water

[0075] The only difference between the detection method and that in the experimental group in Example 2 is that Cu 2+The aqueous solution should be replaced with an environmental water sample (river water, lake water, industrial wastewater, urban drainage, or reservoir water). Add 10 μL of the Ox1 stock solution prepared in Example 2 to phosphate buffer solution (PBS, pH=7.4, 10 mM), then add 10 μL of the environmental water sample, and dilute to a final volume of 1000 μL with phosphate buffer solution (PBS, pH=7.4, 10 mM) to obtain the test solution.

[0076] The AAS method (GB / T 7475-1987) was used as a comparative experiment.

[0077] The detection results of copper ions are shown in Table 1.

[0078] Table 1 Cu in environmental water samples 2+ Concentration analysis results (n=5)

[0079]

[0080] As shown in Table 1, probe Ox1 was successfully applied to the removal of copper ions (Cu) from drinking water. 2+ The detection results were not significantly different from those obtained by the AAS method (GB / T7475-1987). Compared with the AAS method, this invention uses oxazine compounds as fluorescent probes to detect Cu. 2+ The method (fluorescence method) has significant advantages such as low cost and simple operation. Oxazine compounds are Cu in drinking water. 2+ Rapid testing offers an effective and economical alternative, with significant potential for application, especially in field surveillance and large-scale screening.

[0081] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. An oxazin compound, characterized by, A compound having a structure shown in formula I: Formula I; wherein R is C1-C6 alkyl.

2. The oxazin compound according to claim 1, characterized by The R is methyl, ethyl, propyl, butyl, pentyl or hexyl.

3. A process for the preparation of the oxazinane compounds according to claim 1 or 2, characterized in that, The method comprises the following steps: Mixing compound 1, compound 2, organic base and polar organic solvent to carry out substitution reaction to obtain the oxazine compound; 。 4. The production method according to claim 3, characterized by, The molar ratio of the compound 1 and the compound 2 is 1:1-2.

5. The preparation method according to claim 3, characterized in that, The organic base comprises organic amine; The molar ratio of the compound 1 and the organic base is 1:2-5.

6. The preparation method according to claim 3, characterized in that, The polar organic solvent comprises one or more of halogenated hydrocarbon, nitrile solvent, furan solvent, sulfoxide solvent and amide solvent.

7. The method according to any one of claims 3 to 6, wherein the method further comprises the step of: The temperature of the substitution reaction is 25-60℃, and the time is 0.5-4h; the substitution reaction is carried out under a protective atmosphere.

8. Use of the oxazine compound of claim 1 or 2 in the preparation of a fluorescent probe for detecting copper ions.

9. A method for fluorescent detection of copper ions in non-disease diagnosis and treatment, characterized by, The method comprises the following steps: Mixing the solution of copper ions to be detected, the solution of the fluorescent probe and a solvent to obtain a to-be-detected solution; The fluorescent probe in the solution of the fluorescent probe comprises the oxazine compound of claim 1 or 2; The to-be-detected solution is subjected to fluorescence emission spectrum detection to obtain a detection result of copper ions.

10. The detection method according to claim 9, characterized in that, The solvent comprises a phosphate buffer solution; The conditions of the fluorescence emission spectrum detection comprise: excitation wavelength is 615-655nm, excitation slit width is 5-10nm, emission slit width is 5-10nm, and the working voltage of a photomultiplier tube is 300-700V.

Images