Fluorescent probe compound DM-2 for detecting Cu < 2 + > as well as preparation method and application method thereof

Abstract

The invention relates to the technical field of fluorescent probes, in particular to a fluorescent probe compound for detecting Cu < 2 + > as well as a preparation method and an application method thereof. The invention provides a fluorescent probe compound DM-2 for detecting Cu < 2 + > as well as a preparation method and an application method of the fluorescent probe compound DM-2, the probe DM-2 emits weak green fluorescence in a weakly alkaline HEPES buffer solution, and after Cu < 2 + > is added, the fluorescence intensity at 600 nm is rapidly increased and is stable within 10 minutes, and the fluorescent probe compound DM-2 has the main advantages that 1, the DM-2 has the properties of a ratio type probe, and the fluorescence intensity of the probe compound DM-2 is greatly improved; a large Stokes shift is achieved; 2, after the probe reacts with Cu < 2 + >, the color of a solution is changed from colorless to yellow, and the color of fluorescence is changed from green to orange, so that visual detection and analysis of Cu < 2 + > can be realized; and thirdly, the fluorescent probe is simple in synthesis step, high in yield and relatively good in stability.

Description

Technical Field

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

[0002] Copper is the third most abundant essential trace element in the human body (after iron and zinc), and plays a crucial role in various physiological processes. Cu 2+ is crucial for our physiological and pathological activities, including redox processes, enzyme-catalyzed reactions, and oxygen transport. Deviations from normal values of Cu 2+ can lead to various serious diseases, including myelopathy, 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. Therefore, there are strict regulations on the daily intake of Cu 2+ . For example, the National Research Council of the United States recommends that the daily intake of Cu 2+ by adults should be less than 3.0 mg. Due to the wide application of copper, it has become an important metal pollutant. Because Cu 2+ is an important metal catalyst and intermediate in modern industry. Therefore, in addition to directly ingesting Cu 2+ from food, excessive Cu 2+ in the environment can also indirectly affect the content of Cu 2+ in the human body due to the discharge of industrial wastewater. Therefore, it is crucial to establish an effective method for highly selective and sensitive detection of Cu 2+ for early diagnosis of related diseases and environmental problems.

[0003] So far, a variety of analytical techniques such as atomic absorption spectrometry, atomic emission spectrometry, 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 have been developed. Although these probes exhibit many excellent capabilities in Cu 2+ imaging, most of the probes still have disadvantages such as high detection limits and poor anti-interference ability in detecting Cu 2+ in the environment. Therefore, it is crucial to construct a new type of fluorescent probe with a simple synthetic route, fast response, good selectivity, high sensitivity, and capable of detecting Cu 2+ in the environment. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a fluorescent probe compound DM-2 for detecting Cu 2+ and its preparation method and application method in view of the deficiencies of the above-mentioned prior art. DM-2 has the properties of a ratiometric probe and a large Stokes shift. After the probe DM-2 reacts with Cu 2+ , the color of the solution 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. The synthesis steps of the fluorescent probe DM-2 are simple, the yield is high, and the stability is good.

[0005] The present invention provides a fluorescent probe compound DM-2 for detecting Cu 2+ , and the chemical structural formula of the fluorescent probe compound DM-2 is: .

[0006] The present invention provides a preparation method of the above-mentioned fluorescent probe compound DM-2 for detecting Cu 2+ , which includes the following steps: Under Ar 2 protection, 1-ethyl-2,3,3-trimethylindolium iodide and 6-formylnaphthalene-2-picolinate are mixed, then an organic solvent is added to completely dissolve them, after heating, a catalyst is added and the mixture is stirred and refluxed. After completion, the reaction product is poured into water for quenching, and after filtration, it is washed, dried and subjected to column chromatography to obtain the fluorescent probe DM-2.

[0007] 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 1-ethyl-2,3,3-trimethylindolium iodide, 6-formylnaphthalene-2-picolinate, acetic anhydride and sodium acetate is 1 mmol: 1 mmol: 2 mL: 1.5 mmol.

[0008] According to the preparation method provided by the present invention, the temperature after heating is 80 °C, the time of the stirring and reflux reaction is 6 h, and the temperature of the stirring and reflux reaction is 80 °C.

[0009] According to the preparation method provided by the present invention, the amount of water used for quenching by pouring into water is 8 mL, the detergent for washing is a mixed solution of water and ethyl acetate with a volume ratio of 1:1, and the drying condition is drying at a temperature of 50 °C for 0.5 h.

[0010] According to the preparation method provided by the present invention, the conditions for column chromatography are: in a silica gel column, dichloromethane / methanol is used as the eluent, and the elution ratio is 10 / 1.

[0011] The present invention also provides an application method of the above-mentioned fluorescent probe compound DM-2 for detecting Cu 2+ , which includes the following steps: S1. Prepare a standard curve: Using 400 - 430 nm as the excitation wavelength, measure the fluorescence intensity values of a series of standard sample solutions of Cu 2+ at the emission wavelength of 500 - 610 nm and plot a standard curve with the fluorescence intensity value F as the ordinate; S2. Detect the concentration of Cu 2+ in the sample: Replace the standard solution of Cu 2+ in S1 with the sample to be tested, add the probe DM-2 solution thereto, detect the fluorescence intensity at the same emission wavelength as that in S1 and record it as F', and substitute the F' into the standard curve obtained in S1 to further obtain the concentration of Cu 2+ in the sample to be tested.

[0012] The present invention has the following advantages compared with the prior art: The present invention provides a fluorescent probe compound DM-2 for detecting Cu 2+ , its preparation method and application method. The probe DM-2 emits weak green fluorescence in a weakly alkaline HEPES buffer solution. After adding Cu 2+ , the fluorescence intensity at 600 nm rapidly increases and reaches stability in 10 minutes. Its main advantages are threefold: First, DM-2 has the property of a ratio-type probe and has a large Stokes shift; second, after the probe DM-2 reacts with Cu 2+ , the color of the solution 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; third, 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

[0013] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the following drawings are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0014] Figure 1 is the mass spectrum of the fluorescent probe DM-2; Figure 2 is the nuclear magnetic resonance spectrum of the fluorescent probe DM-2; Figure 3 is the absorption spectrum (a) and fluorescence spectrum (b) of the probe DM-2 before and after reacting with Cu 2+ at room temperature; Figure 4 The fluorescence intensity change diagram of the reaction between the probe DM-2 and Cu at different pH values; 2+ ; Figure 5 The spectrogram (a) of the fluorescence intensity change with time and the kinetic diagram (b) after the reaction between the probe DM-2 and Cu; 2+ ; Figure 6 The change diagram (a) of the fluorescence of the probe DM-2 with the concentration of Cu and the linear relationship diagram (b) between the concentration of Cu and the fluorescence intensity of the probe DM-2; 2+ ; 2+ ; Figure 7 The fluorescence spectra (a) and fluorescence intensities (b) of the probe DM-2 with different analytes; Figure 8 The response mechanism diagram of the reaction between the fluorescent probe DM-2 and Cu; 2+ ; Figure 9 The HRMS diagram after the reaction between the probe DM-2 and Cu; 2+ ; Detailed implementation manners

[0015] Example 1

[0016] This example provides a preparation method of a fluorescent probe compound DM-2 for detecting Cu, and the specific steps are as follows: 2+ ; Prepare 1-ethyl-2,3,3-trimethylindolium iodide (Compound 1) and 6-formylnaphthalene-2-pyridinecarboxylate (Compound 2) according to the literature (A hemicyanine-based selective and sensitive colorimetric and fluorescent turn-on probe for Cu, Dongjian Zhu, Tetrahedron Letters, 2016, 57(48): 5326-5329.); 2+ ; Under Ar 2Under protection, 1 mmol of Compound 1 and 1 mmol of Compound 2 were added to a round-bottom flask to obtain a mixture. 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. After that, the mixture was 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, it was washed with water and ethyl acetate, and the volume ratio of water to ethyl acetate was 1:1. It was dried at 50 °C for 0.5 h to obtain the crude product, and then purified using a chromatography column to obtain the orange solid fluorescent probe DM-2; The conditions for column chromatography were as follows: in a silica gel column, dichloromethane / methanol was used as the eluent, and the elution ratio was 10 / 1.

[0017]

[0018] Example 2 This example was a feasibility study on the response of the probe DM-2 prepared in Example 1 to Cu 2+

[0019] By comparing the changes in the UV-visible absorption spectrum and fluorescence spectrum of the probe DM-2 before and after the addition of Cu 2+ to verify the feasibility of the probe DM-2 for detecting Cu 2+

[0020] As Figure 3 shown in a, the maximum absorption peak of the probe DM-2 was at 425 nm. After the addition of Cu 2+ , the absorption peak of the probe DM-2 solution at 425 nm disappeared, and at the same time, a new absorption band appeared at 450 nm, and the solution color changed from colorless to yellow.

[0021] As Figure 3 shown in b, the maximum fluorescence emission peak of the probe DM-2 solution was at 543 nm. After the addition of Cu 2+ , the fluorescence emission peak at 543 nm disappeared, and a new fluorescence emission peak appeared at 600 nm and the fluorescence intensity increased significantly, and the fluorescence color of the solution changed from green to orange.

[0022] The above experimental results showed that the probe DM-2 could respond to Cu 2+ .

[0023] Example 3 This example was a response study of the probe DM-2 prepared in Example 1 to Cu 2+ under different pH conditions.

[0024] To study the effect of pH on the probe DM-2, at room temperature, HEPES buffer solutions with different pH values and a concentration of 10.0 mmol / L were added to the reaction solution to adjust the pH of the system.

[0025] As Figure 4 shown, when Cu 2+ was not added, the fluorescence of the probe DM-2 was weak in the reaction solutions with different pH values. After adding Cu 2+ , when the pH was 6.8, 7.2, 7.4, 7.8, and 8.2, the fluorescence intensities of the probe DM-2 at λ = 600 nm were enhanced to varying degrees. Especially when the pH was 7.8, the change in the fluorescence intensity of the probe DM-2 at λ = 600 nm was the most obvious. Therefore, when the system pH was 7.8, the reaction effect of the probe DM-2 with Cu 2+ was the best.

[0026] Example 4 This example was a study on the response time of the probe DM-2 prepared in Example 1.

[0027] The response time of the probe DM-2 to Cu 2+ was investigated at room temperature. As Figure 5 shown in a, after adding Cu 2+ with a concentration of 10 μmol / L to the probe solution, the fluorescence signal at 600 nm hardly increased any more. It can be seen that the reaction was rapid within 10 min and the fluorescence intensity tended to be stable after 10 min, and the reaction basically ended, as Figure 5 shown in b.

[0028] Therefore, the reaction time of the probe DM-2 to Cu 2+ should be controlled within 10 min.

[0029] Example 5 This example was a study on the quantitative detection of Cu 2+ by the probe DM-2 prepared in Example 1.

[0030] Under the optimal experimental conditions, the spectral characteristics of the response of the probe DM-2 to different concentrations of Cu 2+ were measured.

[0031] As Figure 6 shown in a, at 543 nm, the fluorescence intensity of the probe DM-2 decreased with the increase in the concentration of Cu 2+ from 0 to 1 μmol / L, while at 600 nm, the fluorescence intensity of the probe DM-2 increased with the increase in the concentration of Cu 2+ from 1 to 10 μmol / L, and the fluorescence intensity of the probe DM-2 at 600 nm and Cu2+ There is a good linear relationship between the concentrations, and the linear range is 1 - 10 μmol / L. The linear regression equation is y = 70.13x + 173.8, with 2 R 2+ = 0.9907; where y is the relative fluorescence intensity of the probe DM-2, and x is the concentration of Cu Figure 6 , as shown in

[0032] Subsequently, a probe solution with a concentration of 10.0 μmol / L was prepared and added to the sample to be measured. Using 425 nm as the excitation wavelength, the fluorescence intensity at 600 nm was measured. Substituting it into the above curve equation, the concentration of Cu in the sample to be measured can be obtained. 2+ Concentration.

[0033] The results show that the probe DM-2 can achieve quantitative detection of Cu 2+ .

[0034] Example 6 This example is for the specific detection of the probe DM-2 prepared in Example 1.

[0035] To study the specificity of DM-2 and considering its applicability in practical applications, as shown in Figure 7 a, different analytes 1 - 26 were measured, namely K + , HSO 3 - , Na + , Cr 2 O 7 2- , Cr 3+ , Ni 2+ , Ca 2+ , Mg 2+ , CO 3 2- , NO 3 - , SO 4 2- , Al 3+ , Fe 3 + , Mn 2+ , Zn 2+ , Cl - , HCO 3 - , NH 4 + , Br - , CH 3 COO - , SO 3 - , S 2 O 32- , the fluorescence intensities of Hcy, Gsh, Cys, and Cu 2+ , where the concentration of Cu is 20 μmol / L 2+ , the concentration of Al is 40 μmol / L 3+ , Fe 3+ , Mn 2+ , and Zn 2+ , as well as other ions with a concentration of 1 mmol / L.

[0036] The interference to the detection of Cu 2+ is shown in Figure 7 b. Only Cu 2+ significantly increases the fluorescence intensity of the probe DM-2 at λ = 600 nm, while other analytes have little effect on it. This result indicates that the fluorescent probe DM-2 has high selectivity.

[0037] Example 7 This example is the detection of Cu 2+ in the actual sample by the probe DM-2 prepared in Example 1.

[0038] To further verify the practicability of the probe DM-2 for detecting Cu 2+ in real samples. The standard addition method was used to determine the content of Cu 2+ in three different liquid samples: beer, milk, and water sample. Beer and milk were purchased from a local supermarket, and the water sample was collected from the Hanjiang River.

[0039] The analysis results are shown in Table 1. When the same volume of different liquid samples was added to the probe DM-2, there was no obvious fluorescence change. Subsequently, 2 μM, 4 μM, and 6 μM of Cu 2+ at different concentrations were added under the above conditions, and the recovery rate ranged from 98.0% to 104.5%.

[0040] Table 1 Detection results of Cu 2+ in actual samples (n = 3)

[0041] The results show that the probe DM-2 can accurately measure the content of Cu 2+ in actual samples.

[0042] Example 8 This example provides an application method of the probe DM-2 prepared in Example 1, including the following steps: S1. Prepare a standard curve: Using 400 - 430 nm as the excitation wavelength, 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 denoted as F, and a standard curve is plotted with F as the ordinate; S2. Detection of Cu in the sample 2+ concentration: Replace the Cu 2+ standard solution in S1 with the sample to be tested, add the probe DM-2 solution thereto, detect the fluorescence intensity when the emission wavelength of the sample to be tested is the same as that in S1 and denote it as F', substitute the F' into the standard curve obtained in S1, and then obtain the concentration of Cu 2+ in the sample to be tested.

[0043] The reaction mechanism between the probe DM-2 and Cu 2+ was verified by HRMS, as Figure 9 shown. Excessive Cu 2+ was added to the HEPES buffer solution (10 mmol·L -1 , pH = 7.8) containing the probe DM-2. After the reaction was completed, the substances before and after the reaction were detected by HRMS respectively.

[0044] As Figure 1 shown in the mass spectrometry data, a peak at m / z = 447.2159 appeared, corresponding to the mass spectrometry peak of the probe DM-2 [M] + , Figure 9 and a new peak at m / z = 342.1580 appeared in the mass spectrometry data, corresponding to the mass spectrometry peak of the DM-1 [M] + mixture after the reaction.

[0045] It can be inferred from the mass spectrometry diagrams before and after the reaction that after the reaction between the probe DM-2 and Cu 2+ , as the specific response group, the pyridinecarboxylate ester causes a blue shift in the wavelength after the fluorophore reacts with it. This is because the intramolecular charge transfer (ICT) process between the two is turned off. When the probe DM-2 reacts with Cu 2+ , Cu 2+ catalyzes the hydrolysis reaction of the pyridinecarboxylate ester itself to restore the intramolecular charge transfer (ICT) process, thereby detaching the recognition group, and the probe releases the fluorophore with enhanced fluorescence. The solution color changes from colorless to orange, and the fluorescence color changes from green to orange.

[0046] The above are only the preferred embodiments of the present invention and do not impose any limitations on the present invention. Any simple modifications, changes, and equivalent variations made to the above embodiments based on the technical essence of the invention still fall within the protection scope 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 method for preparing the fluorescent probe compound DM-2 is characterized in that: The following steps are involved: exist Under the protection of Ar2, 1-ethyl-2,3,3-trimethylindolyl iodonium and 6-formylnaphthalene-2-pyridinecarboxylate are mixed, and then an organic solvent is added to completely dissolve them. After heating, a catalyst is added and stirred to reflux for reaction. After completion, the reaction product is poured into water for quenching, filtered, washed, dried and column chromatographed in sequence 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 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 reflux reaction is 6 hours, and the temperature of stirring 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: in a silica gel column, dichloromethane / methanol is used as the eluent, and the elution ratio is 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 range of 500-610 nm is recorded as F and the standard curve is drawn as the ordinate; S2. Detection of Cu in samples 2+ Concentration: 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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