A fluorescent probe compound DM-2 for detecting Cu 2+ and a preparation method and application method thereof

Abstract

The application relates to the technical field of fluorescent probe, in particular to a fluorescent probe compound for detecting Cu 2+ and a preparation method and application method thereof. The application provides a fluorescent probe compound DM-2 for detecting Cu 2+ and a preparation method and application method thereof; the probe DM-2 emits weak green fluorescence in a weak alkaline HEPES buffer solution; after Cu 2+ is added, the fluorescence intensity at 600 nm is rapidly increased, and reaches stability in 10 min; the main advantages of the probe are as follows: first, the probe DM-2 has the properties of a ratio type probe and has a large stokes shift; second, after the probe reacts with Cu 2+ , the solution color changes from colorless to yellow, and the fluorescence color changes from green to orange, so that visual detection and analysis of Cu 2+ can be realized; and third, the fluorescent probe has simple synthesis steps, high yield and good stability.

Description

Technical Field

[0001] The present invention relates to the technical field of fluorescent probes, in particular to a method for detecting Cu 2+ The invention discloses a fluorescent probe compound DM-2 and a preparation method and an application method thereof. Background Art

[0002] Copper is the third most abundant essential trace element in the human body (after iron and zinc) and plays a vital role in various physiological processes. 2+ It is essential for our physiological and pathological activities, including redox processes, enzyme catalysis reactions and oxygen transport. 2+ It can lead to various serious diseases, including spinal cord disease, anemia and coronary heart disease. In addition, excessive cellular copper is also associated with neurological diseases such as Parkinson's, Alzheimer's and Wilson's. 2+ There are strict regulations on the daily intake of Cu. For example, the National Research Council of the United States recommends that adults consume 2+ The amount should be less than 3.0 mg. Due to the wide application of copper, it has become an important metal pollutant. 2+ It is an important metal catalyst and intermediate in modern industry. Therefore, in addition to directly consuming Cu from food 2+ In addition, excessive Cu in the environment 2+ The discharge of industrial wastewater will also indirectly affect the Cu content in the human body. 2+ Therefore, a method with high selectivity and high sensitivity for the detection of Cu 2+ It is crucial to develop effective methods for early diagnosis of related diseases and environmental problems.

[0003] So far, a variety of analytical techniques such as atomic absorption spectroscopy, atomic emission spectroscopy, gas chromatography, mass spectrometry, and electrochemical methods have been developed and applied to the detection of Cu 2+ However, these traditional techniques have limitations such as high daily maintenance costs and cumbersome procedures. Compared with the above methods, fluorescent probes have attracted attention due to their simple operation, high sensitivity, rapid response, good selectivity, low toxicity and great potential for real-time bioimaging. In recent years, more and more Cu 2+ Fluorescent probes. Although these probes are 2+ However, most of the probes have shown excellent capabilities in detecting Cu in the environment. 2+ Therefore, a novel method with simple synthesis path, fast response speed, good selectivity, high sensitivity and high sensitivity for detecting Cu in the environment is constructed. 2+ New fluorescent probes are crucial. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a method for detecting Cu 2+ The fluorescent probe compound DM-2 and its preparation method and application method, DM-2 has the properties of a ratio probe, has a large Stokes shift, the probe DM-2 and Cu 2+ After the reaction, the solution color changes from colorless to yellow, and the fluorescence color changes from green to orange, thus achieving the Cu 2+ Visual inspection and analysis showed that the fluorescent probe DM-2 had simple synthesis steps, high yield and good stability.

[0005] The present invention provides a method for detecting Cu 2+ The fluorescent probe compound DM-2 has the chemical structural formula:

[0006] .

[0007] The present invention provides a method for detecting Cu 2+ The preparation method of the fluorescent probe compound DM-2 comprises the following steps:

[0008] Under Ar2 protection, 1-ethyl-2,3,3-trimethylindol iodonium and 6-formylnaphthalene-2-pyridinecarboxylate were mixed, and then an organic solvent was added to completely dissolve them. After heating, a catalyst was added and stirred under reflux for reaction. After completion, the reaction product was poured into water for quenching, filtered, washed, dried and column chromatographed in sequence to obtain the fluorescent probe DM-2.

[0009] According to the preparation method provided by the present invention, the organic solvent is acetic anhydride, the catalyst is sodium acetate, and the molar volume ratio of the 1-ethyl-2,3,3-trimethylindolyl iodonium, 6-formylnaphthalene-2-pyridinecarboxylate, acetic anhydride and sodium acetate is 1 mmol:1 mmol:2 mL:1.5 mmol.

[0010] According to the preparation method provided by the present invention, the temperature after heating is 80°C, the time of stirring and reflux reaction is 6 hours, and the temperature of stirring and reflux reaction is 80°C.

[0011] According to the preparation method provided by the present invention, the amount of water used for quenching is 8 mL, the washing detergent is a mixture of water and ethyl acetate in a volume ratio of 1:1, and the drying condition is drying at a temperature of 50°C for 0.5 h.

[0012] According to the preparation method provided by the present invention, the column chromatography conditions are: in a silica gel column, dichloromethane / methanol is used as the eluent, and the elution ratio is 10 / 1.

[0013] The present invention also provides a method for detecting Cu 2+ The application method of the fluorescent probe compound DM-2 comprises the following steps:

[0014] S1. Prepare standard curve: Use 400-430 nm as excitation wavelength to measure a series of different concentrations of Cu 2+ The fluorescence intensity value of the standard sample solution at the emission wavelength of 500-610 nm is recorded as F and the standard curve is drawn on the vertical axis;

[0015] S2. Detection of Cu in samples 2+ Concentration: Change the Cu 2+ The standard solution is replaced with the sample to be tested, and the probe DM-2 solution is added thereto. The fluorescence intensity of the sample to be tested when the emission wavelength is the same as that of S1 is detected and recorded as F'. The F' is substituted into the standard curve obtained by S1 to obtain the Cu content in the sample to be tested. 2+ concentration.

[0016] Compared with the prior art, the present invention has the following advantages:

[0017] The present invention provides a method for detecting Cu 2+ The fluorescent probe compound DM-2 and its preparation method and application method are described. The probe DM-2 emits weak green fluorescence in weakly alkaline HEPES buffer. 2+ After that, the fluorescence intensity at 600 nm increased rapidly and reached stability in 10 min. Its main advantages are three points: first, DM-2 has the properties of a ratiometric probe and has a large Stokes shift; second, the probe DM-2 is closely related to Cu 2+ After the reaction, the solution color changes from colorless to yellow, and the fluorescence color changes from green to orange, thus achieving the Cu 2+ Visual inspection and analysis; thirdly, the synthesis steps of the fluorescent probe DM-2 are simple, the yield is high, and the stability is good. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the technical solutions in the present invention or the prior art, a brief introduction will be given below to the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0019] Figure 1 is the mass spectrum of fluorescent probe DM-2;

[0020] Figure 2 This is the NMR spectrum of the fluorescent probe DM-2;

[0021] Figure 3The probe DM-2 reacts with Cu at room temperature. 2+ Absorption spectra (a) and fluorescence spectra (b) before and after the reaction;

[0022] Figure 4 The probe DM-2 and Cu 2+ Fluorescence intensity change graph of the reaction;

[0023] Figure 5 The probe DM-2 and Cu 2+ Spectrum (a) and kinetic diagram (b) of the change of fluorescence intensity over time after the reaction;

[0024] Figure 6 The fluorescence of probe DM-2 changes with Cu 2+ Concentration change diagram (a) and Cu 2+ Linear relationship between concentration and fluorescence intensity of probe DM-2 (b);

[0025] Figure 7 The fluorescence spectra (a) and fluorescence intensities (b) of probe DM-2 and different analytes;

[0026] Figure 8 The fluorescent probe DM-2 and Cu 2+ Response mechanism diagram of the reaction;

[0027] Figure 9 The probe DM-2 and Cu 2+ HRMS chart after reaction. DETAILED DESCRIPTION

[0028] Example 1

[0029] This embodiment provides a method for detecting Cu 2+ The preparation method of the fluorescent probe compound DM-2 comprises the following specific steps:

[0030] According to the literature (A hemicyanine-based selective and sensitive colorimetricandfluorescent turn-on probe for Cu 2+ Dongjian Zhu, Tetrahedron Letters, 2016, 57(48): 5326-5329.) Preparation of 1-ethyl-2,3,3-trimethylindolyl iodonium (Compound 1) and 6-formylnaphthalene-2-pyridinecarboxylate (Compound 2);

[0031] Under Ar2 protection, 1 mmol of compound 1 and 1 mmol of compound 2 were added to a round-bottom flask to obtain a mixture, and then 2 mL of acetic anhydride was added to completely dissolve the mixture. The temperature was raised to 80 °C, and 1.5 mmol of sodium acetate was added. The mixture was then stirred and refluxed at 80 °C for 6 h. The reaction progress was monitored by TLC. After the reaction was completed, the reaction product was poured into 8 mL of water to quench the reaction. After filtration, the product was washed with water and ethyl acetate in a volume ratio of water to ethyl acetate of 1:1. The crude product was dried at 50 °C for 0.5 h to obtain the crude product, which was then purified using a chromatography column to obtain the orange solid fluorescent probe DM-2.

[0032] The column chromatography conditions were as follows: using a silica gel column with dichloromethane / methanol as the eluent at an elution ratio of 10 / 1.

[0033]

[0034] Example 2

[0035] This example is the probe DM-2 prepared in Example 1 to Cu 2+ Feasibility study of the response.

[0036] By comparing the addition of Cu 2+ The changes in the UV-visible absorption spectrum and fluorescence spectrum of the probe DM-2 before and after were used to verify the detection of Cu by the probe DM-2. 2+ feasibility.

[0037] like Figure 3 As shown in a, the maximum absorption peak of probe DM-2 is at 425 nm. 2+ After that, the absorption peak of the probe DM-2 solution at 425 nm disappeared, and a new absorption band appeared at 450 nm, and the color of the solution changed from colorless to yellow.

[0038] like Figure 3 As shown in b, the maximum fluorescence emission peak of the probe DM-2 solution is at 543 nm. 2+ After addition of 4% CO, the fluorescence emission peak at 543 nm disappeared, while a new fluorescence emission peak appeared at 600 nm and the fluorescence intensity was significantly enhanced, and the fluorescence color of the solution changed from green to orange.

[0039] The above experimental results show that the probe DM-2 can 2+ Generate a response.

[0040] Example 3

[0041] This example is a probe DM-2 prepared in Example 1 and Cu 2+ Response studies under different pH conditions.

[0042] In order to study the effect of pH on the probe DM-2, HEPES buffer with different pH values ​​at a concentration of 10.0 mmol / L was added to the reaction solution at room temperature to adjust the pH of the system.

[0043] like Figure 4 As shown, no Cu was added 2+ When the probe DM-2 showed weak fluorescence in the reaction solution with different pH values, the addition of Cu 2+ After that, the fluorescence intensity of probe DM-2 at λ = 600 nm was enhanced to varying degrees at pH 6.8, 7.2, 7.4, 7.8 and 8.2. Especially at pH 7.8, the fluorescence intensity of probe DM-2 at λ = 600 nm changed most significantly. Therefore, when the pH of the system was 7.8, the probe DM-2 and Cu 2+ The response effect is best.

[0044] Example 4

[0045] This example is a study on the response time of the probe DM-2 prepared in Example 1.

[0046] The probe DM-2 was investigated for Cu 2+ The response time, such as Figure 5 As shown in a, a 10 μmol / L Cu 2+ After that, the fluorescence signal at 600 nm almost stopped increasing. It can be seen that the reaction was rapid within 10 minutes and the fluorescence intensity tended to be stable after 10 minutes, indicating that the reaction was basically completed. Figure 5 As shown in b.

[0047] Therefore, the probe DM-2 is sensitive to Cu 2+ The reaction time should be controlled within 10 min.

[0048] Example 5

[0049] This example is the quantitative detection of Cu by the probe DM-2 prepared in Example 1. 2+ research.

[0050] Under the optimal experimental conditions, the effect of probe DM-2 on different concentrations of Cu 2+ Spectral characteristics of the response.

[0051] like Figure 6 As shown in a, at 543 nm, the fluorescence intensity of probe DM-2 increases with the increase of Cu 2+ The fluorescence intensity of probe DM-2 decreased with the increase of Cu concentration from 0 to 1 μmol / L. At 600 nm, the fluorescence intensity of probe DM-2 increased with the increase of Cu concentration from 0 to 1 μmol / L. 2+The fluorescence intensity of the probe DM-2 at 600 nm was significantly higher than that of Cu 2+ There is a good linear relationship between the concentrations of β-catenin and β-catenin, with a linear range of 1 to 10 μmol / L. The linear regression equation is y = 70.13 x + 173.8, R 2 = 0.9907; where y is the relative fluorescence intensity of probe DM-2, x is the relative fluorescence intensity of Cu 2+ The concentration, such as Figure 6 As shown in b.

[0052] Then, a probe solution with a concentration of 10.0 μmol / L was prepared and added to the sample to be tested. The fluorescence intensity at 600 nm was measured with an excitation wavelength of 425 nm. Substituting it into the above curve equation, the Cu content in the sample to be tested was obtained. 2+ concentration.

[0053] The results showed that the probe DM-2 can realize the 2+ Quantitative detection of .

[0054] Example 6

[0055] This example is the specific detection of the probe DM-2 prepared in Example 1.

[0056] In order to study the specificity of DM-2 and considering its applicability in practical applications, e.g. Figure 7 As shown in a, different analytes 1 to 26 were measured for K + 、HSO3 - 、Na + 、Cr2O7 2- Cr 3+ 、Ni 2+ , Ca 2+ Mg 2+ 、CO3 2- 、NO3 - 、SO4 2- 、Al 3+ 、Fe 3 + 、Mn 2+ 、Zn 2+ 、Cl - 、HCO3 - NH4 + Br - 、CH3COO - 、SO3 - 、S2O3 2- , Hcy, Gsh, Cys and Cu 2+ The fluorescence intensity of the Cu 2+, Al concentration of 40 μmol / L 3+ 、Fe 3+ 、Mn 2+ and Zn 2+ , and other ions at a concentration of 1 mmol / L.

[0057] Cu 2+ Detection interference such as Figure 7 As shown in b, only Cu 2+ The fluorescence intensity of the probe DM-2 at λ = 600 nm increased significantly, while other analytes had almost no effect on it. This result indicates that the fluorescent probe DM-2 has high selectivity.

[0058] Example 7

[0059] This example is the probe DM-2 prepared in Example 1 to detect Cu in actual samples. 2+ Detection.

[0060] In order to further verify whether the probe DM-2 has the ability to detect Cu in real samples 2+ The standard addition method was used to determine the Cu content in three different liquid samples: beer, milk and water. 2+ Beer and milk were purchased from local supermarkets, and water samples were collected from the Han River.

[0061] The analysis results are shown in Table 1. When the same volume of different liquid samples was added to the probe DM-2, no obvious fluorescence change occurred. Subsequently, 2 μM, 4 μM and 6 μM of Cu were added to the probe under the above conditions. 2+ , and the recovery rates ranged from 98.0% to 104.5%.

[0062] Table 1 Cu in actual samples 2+ Test results (n=3)

[0063]

[0064] The results show that the probe DM-2 can accurately measure the Cu 2+ content.

[0065] Example 8

[0066] This embodiment provides a method for applying the probe DM-2 prepared in Example 1, comprising the following steps:

[0067] S1. Prepare standard curve: Use 400-430 nm as excitation wavelength to measure a series of different concentrations of Cu 2+ The fluorescence intensity value of the standard sample solution at the emission wavelength of 500-610 nm is recorded as F and the standard curve is drawn on the vertical axis;

[0068] S2. Detection of Cu in samples 2+ Concentration: Change the Cu 2+ The standard solution is replaced with the sample to be tested, and the probe DM-2 solution is added thereto. The fluorescence intensity of the sample to be tested when the emission wavelength is the same as that of S1 is detected and recorded as F'. The F' is substituted into the standard curve obtained by S1 to obtain the Cu content in the sample to be tested. 2+ concentration.

[0069] The probe DM-2 was verified by HRMS to be a Cu 2+ reaction mechanisms, such as Figure 9 As shown. 2+ Added to HEPES buffer (10 mmol L -1 , pH = 7.8), and after the reaction was completed, the substances before and after the reaction were detected by HRMS.

[0070] like Figure 1 The peak of m / z=447.2159 appeared in the mass spectrum data, corresponding to the probe DM-2[M] + Mass spectrum peak, Figure 9 A new peak with m / z=342.1580 appeared in the mass spectrum data, corresponding to DM-1[M] after the reaction. + Mass spectrometric peaks of the mixture.

[0071] From the mass spectra before and after the reaction, it can be inferred that the probe DM-2 and Cu 2+ After the reaction, picolinate acts as a specific response group, and the wavelength of the fluorophore blue-shifts after the reaction with picolinate, which is due to the closure of the intramolecular charge transfer (ICT) process between the two. 2+ When the reaction occurs, Cu 2+ Catalyzing the hydrolysis reaction of picolinate itself can restore the intramolecular charge transfer (ICT) process, thereby disengaging the recognition group, releasing the fluorophore from the probe, and enhancing the fluorescence. The solution color changes from colorless to orange, and the fluorescence color changes from green to orange.

[0072] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any simple modification, change and equivalent variation made to the above embodiment based on the essence of the invention technology shall still fall within the scope of protection of the technical solution of the present invention.

Claims

1. A method for detecting Cu 2+ The fluorescent probe compound DM-2 is characterized in that The chemical structural formula of the fluorescent probe compound DM-2 is: 。 2. A method for detecting Cu as claimed in claim 1 2+ The preparation method of the fluorescent probe compound DM-2 is characterized in that: The following steps are involved: exist Under Ar2 protection, 1-ethyl-2,3,3-trimethylindol iodonium and 6-formylnaphthalene-2-pyridinecarboxylate were mixed, and then an organic solvent was added to completely dissolve them. After heating, a catalyst was added and stirred under reflux for reaction. After completion, the reaction product was poured into water for quenching, filtered, washed, dried and subjected to column chromatography to obtain the fluorescent probe DM-2.

3. The preparation method according to claim 2, characterized in that The organic solvent is acetic anhydride, the catalyst is sodium acetate, and the molar volume ratio of the 1-ethyl-2,3,3-trimethylindolyl iodonium, 6-formylnaphthalene-2-pyridinecarboxylate, acetic anhydride and sodium acetate is 1 mmol:1 mmol:2 mL:1.5 mmol.

4. The preparation method according to claim 2, characterized in that The temperature after heating is 80°C, the time of stirring and reflux reaction is 6 hours, and the temperature of stirring and reflux reaction is 80°C.

5. The preparation method according to claim 2, characterized in that The amount of water used for quenching is 8 mL, the washing detergent is a mixture of water and ethyl acetate in a volume ratio of 1:1, and the drying condition is drying at a temperature of 50° C. for 0.5 h.

6. The preparation method according to claim 2, characterized in that The column chromatography conditions are as follows: using dichloromethane / methanol as eluent in a silica gel column at an elution ratio of 10 / 1.

7. A method for detecting Cu as claimed in claim 1 2+ The application method of the fluorescent probe compound DM-2 is characterized in that: The following steps are involved: S1. Prepare standard curve: Use 400-430 nm as excitation wavelength to measure a series of different concentrations of Cu 2+ The fluorescence intensity value of the standard sample solution at the emission wavelength of 500-610 nm is recorded as F and the standard curve is drawn on the vertical axis; S2. Detection of Cu in samples 2+ Concentration: Change the Cu 2+ The standard solution is replaced with the sample to be tested, and the probe DM-2 solution is added thereto. The fluorescence intensity of the sample to be tested when the emission wavelength is the same as that of S1 is detected and recorded as F'. The F' is substituted into the standard curve obtained by S1 to obtain the Cu content in the sample to be tested. 2+ concentration.

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