Fluorescent molecular probe, its preparation method and application

Abstract

The present invention relates to the technical field of copper ion detection, and discloses a fluorescent molecular probe, a preparation method thereof and an application. The structural formula of the fluorescent molecular probe is as follows. The method includes: reacting 5-(4-(diphenylamino)phenyl)thiophene-2-carbaldehyde with salicylhydrazide to obtain the fluorescent molecular probe. The present invention also relates to the application of the fluorescent molecular probe in the sensing detection of copper ions. The synthesis of the fluorescent molecular probe provided by the present invention can be completed in only two steps or even one step, the raw materials are economically available, and the post-treatment process is relatively simple; the present invention realizes the quantitative sensing detection of copper ions by the different functions demonstrated by the probe through ultraviolet and fluorescence spectra, proving that it has good selectivity, strong reversibility, can be recycled multiple times, has strong anti-interference ability against other metal ions, and has a low detection limit. This fluorescent molecular probe can be widely used to detect toxic metal ions present in plant tissues and cells.

Description

Technical Field

[0001] The present invention relates to the technical field of copper ion detection, and in particular to a fluorescent molecular probe and a preparation method and application thereof. Background Art

[0002] In the human body, copper (II) is the third most abundant metal transition ion and plays a key role in many important physiological processes. Copper is associated with many diseases in the human body, including Menkes syndrome, Parkinson's disease, Alzheimer's disease, Wilson's disease, and coronary heart disease. On the other hand, with the widespread use of copper in industry and agriculture, excessive Cu 2+ It may be released into the environment, causing environmental pollution. Of course, excessive intake of copper in the environment will cause its long-term insoluble precipitation in the body, causing harm to the organism, leading to cirrhosis, sensory nerve disorders, movement disorders and other diseases. According to the World Health Organization, the amount of Cu allowed in beverages is 2+ The level is currently below 31.5 μM. Therefore, the presence of Cu in environmental or biological samples is 2+ On-site monitoring of copper levels has become a very important indicator in environmental monitoring. Therefore, the development of new methods for detecting copper compounds is of great research significance for protecting ecosystems and humans from the harm of copper ions.

[0003] Various analytical techniques such as atomic absorption spectroscopy (AAS), atomic fluorescence spectroscopy (AFS) and inductively coupled plasma mass spectrometry (ICP-MS) have been developed to detect copper ions (Cu 2+ ), most of which can provide satisfactory sensitivity. However, these methods are expensive and usually require complex and time-consuming operations, which limits their use for fast and convenient on-site real-time detection. In contrast, fluorescence sensing detection has the advantages of real-time monitoring and rapid response in terms of cost-effectiveness, simple operation, high specificity, and analytical visualization. In addition, fluorescence imaging combined with fluorescent probes has been proven to be one of the most promising techniques to visualize biologically relevant analytes in plant tissues or cells. In addition, fluorescent probes have large Stokes shifts, which help to reduce self-absorption and improve sensitivity. Therefore, for Cu 2+ Therefore, a new fluorescent chemical sensor with specific recognition performance is used to sense and detect Cu 2+ is desirable and still needs further exploration. Summary of the invention

[0004] The purpose of the present invention is to overcome the problems of complex synthesis steps, low selectivity and sensitivity of the copper ion detection probe in the prior art, and to provide a fluorescent molecular probe and a preparation method and application thereof.

[0005] To achieve the above object, a first aspect of the present invention provides a fluorescent molecular probe, wherein the structural formula of the fluorescent molecular probe is as follows:

[0006]

[0007] A second aspect of the present invention provides a preparation method of the fluorescent molecular probe described in the first aspect, wherein the method includes: reacting 5-(4-(diphenylamino)phenyl)thiophene-2-carbaldehyde with salicylhydrazide to obtain the fluorescent molecular probe.

[0008] A third aspect of the present invention provides the application of the fluorescent molecular probe described in the first aspect or the fluorescent molecular probe obtained by the preparation method described in the second aspect in the sensing detection of copper ions.

[0009] Through the above technical solutions, the beneficial technical effects achieved by the present invention are as follows:

[0010] 1) The synthesis of the fluorescent molecular probe provided by the present invention can be completed in only two steps or even one step. The raw materials are economically available and easy to obtain, and the post-treatment process is relatively simple;

[0011] 2) The present invention realizes the quantitative sensing detection of copper ions by the different functions shown by the probe through ultraviolet and fluorescence spectra, proving that it has good selectivity, strong reversibility, can be recycled multiple times, has strong anti-interference ability to other metal ions, and has a lower detection limit. For copper ions: the ultraviolet detection limit is 3.39×10 -7 M, and the fluorescence detection limit is 2.02×10 -7 M. This kind of fluorescent molecular probe can be widely used to detect the toxic metal ions existing in plant tissues and cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 It is the high-resolution mass spectrum of the fluorescent molecular probe J-1 synthesized in Example 1 of the present invention.

[0013] Figure 2 It is the nuclear magnetic resonance hydrogen spectrum 1 1H NMR spectrum of the fluorescent molecular probe J-1 synthesized in Example 1 of the present invention.

[0014] Figure 3 It is the nuclear magnetic resonance carbon spectrum 13 13C NMR spectrum of the fluorescent molecular probe J-1 synthesized in Example 1 of the present invention.

[0015] Figure 4 It is the normalized fluorescence spectrum and color change trend diagram of the probe J-1 at different pH values.

[0016] Figure 5Selectivity of probe J-1 for different metal ions in DMF / H2O (6:4, v / v, 0.01 M Hepes, pH = 7.4).

[0017] Figure 6 For probe J-1 + Cu 2+ Competition for different metal cations.

[0018] Figure 7 :(a) UV spectral response diagram of probe J-1 for different concentrations of Cu 2+ in DMF / H2O (6:4, v / v, 0.01 M Hepes, pH = 7.4); (b) Linear relationship between the UV absorption value at 405 nm and the concentration of Cu 2+ (2 - 7 μM).

[0019] Figure 8 :(a) Fluorescence spectral response diagram of probe J-1 for different concentrations of Cu 2+ in DMF / H2O (6:4, v / v, 0.01 M Hepes, pH = 7.4); (b) Linear relationship between the fluorescence intensity at 548 nm and the concentration of Cu 2+ (1 - 10 μM).

[0020] Figure 9 Appearance color change diagram of probe J-1 solution before and after adding copper ions under sunlight.

[0021] Figure 10 Reversible fluorescence spectrogram of probe J-1 for copper ion recognition. Detailed implementation mode

[0022] The endpoints and any values within the ranges disclosed herein are not limited to the exact ranges or values. These ranges or values should be understood to include values close to these ranges or values. For numerical ranges, between the endpoint values of each range, between the endpoint values of each range and individual point values, and between individual point values, they can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be regarded as specifically disclosed herein.

[0023] The first aspect of the present invention provides a fluorescent molecular probe, wherein the structural formula of the fluorescent molecular probe is as follows:

[0024]

[0025] The fluorescent molecular probe provided by the present invention is a Schiff base structure formed by condensation of 5-(4-(diphenylamino)phenyl)thiophene-2-carbaldehyde and salicylhydrazide (which can also be called a covalent conjugate of the two).

[0026] Compared with other fluorescence probes that only quantitatively detect metal ions using fluorescence spectra, the fluorescence molecular probe of the present invention also combines ultraviolet ratio quantitative detection with fluorescence spectrum quantitative detection to provide a variety of different data, increasing the accuracy and reliability of the detection of metal ions, namely copper ions. Moreover, the ultraviolet spectrum provides higher contrast and sensitivity. The most intuitive phenomenon is that the fluorescence molecular probe of the present invention has a chromogenic sensing function, not only causing a change in the color of the solution under sunlight, but also producing a change in fluorescence color under irradiation with a 365 nm ultraviolet lamp. This dual observation mode makes it a chromogenic sensing fluorescence probe suitable for different lighting conditions. Based on specific recognition, the fluorescence molecular probe of the present invention can be used as a specific indicator for the presence of copper ions in different test papers, and can provide real-time qualitative and quantitative information through fluorescence detection, accompanied by a color change, making it a highly specific indicator suitable for visual colorimetry detection. At the same time, the probe has highly reversible repeatability for the detection of copper ions and can also be used as a sensor for detecting Cu 2+ in living cells and can be repeatedly detected without damaging its performance. Therefore, the present invention provides a simple, rapid, highly specific, and highly sensitive fluorescence detection agent for copper ions, which has broad application prospects and is particularly suitable for the fields of cell detection and bioimaging.

[0027] The second aspect of the present invention provides a preparation method of the fluorescence molecular probe described in the first aspect, wherein the method includes: reacting 5-(4-(diphenylamino)phenyl)thiophene-2-carbaldehyde with salicylhydrazide to obtain the fluorescence molecular probe.

[0028] In the preparation process of the fluorescence molecular probe of the present invention, the reaction that occurs is:

[0029]

[0030] Among them, the 5-(4-(diphenylamino)phenyl)thiophene-2-carbaldehyde is obtained by the reaction of triphenylamine boronic acid and 5-bromothiophene-2-carbaldehyde, or can be commercially obtained.

[0031] In some embodiments of the present invention, the method includes: dissolving 5-(4-(diphenylamino)phenyl)thiophene-2-carbaldehyde in an organic solvent, then adding salicylhydrazide, and heating under reflux to react to obtain the fluorescence molecular probe.

[0032] In some embodiments of the present invention, the molar ratio of 5-(4-(diphenylamino)phenyl)thiophene-2-carbaldehyde to salicylhydrazide is 14:15.

[0033] In some embodiments of the present invention, the organic solvent is ultradry ethanol.

[0034] In some embodiments of the present invention, the reaction time is 4 - 6 h.

[0035] In some embodiments of the present invention, TLC is used to monitor the reaction.

[0036] In some embodiments of the present invention, after the reaction is complete, precipitation occurs upon cooling to room temperature. After filtration, washing, and drying, the fluorescent molecular probe is obtained.

[0037] The third aspect of the present invention provides the application of the fluorescent molecular probe described in the first aspect or the fluorescent molecular probe obtained by the preparation method described in the second aspect in the sensing detection of copper ions.

[0038] In some embodiments of the present invention, the fluorescent molecular probe is used for the sensing detection of copper ions in plant tissues and cells.

[0039] In some embodiments of the present invention, the fluorescent molecular probe is used for the fluorescence spectral detection and qualitative detection of copper ions under different pH conditions.

[0040] In some embodiments of the present invention, the sensing detection is ultraviolet light detection, fluorescence detection, visual qualitative detection, and reversible detection of copper ions.

[0041] In some embodiments of the present invention, the sensing detection is selective sensing detection and competitive sensing detection.

[0042] The present invention will be described in detail below through examples.

[0043] For those not specified in the following examples, they are carried out under conventional conditions or conditions recommended by the manufacturer. For reagents or instruments without indicating the manufacturer, they are all conventional products that can be obtained through commercial channels.

[0044] Example 1

[0045] (1) Synthesis of intermediate TPA - 1 (5 - (4 - (diphenylamino)phenyl)thiophene - 2 - carbaldehyde):

[0046]

[0047] Triphenylamine boronic acid (3.47 g, 12 mmol), 5-bromothiophene-2-carbaldehyde (1.91 g, 10 mmol), potassium carbonate (2.49 g, 18 mmol) and tetrakis(triphenylphosphine)palladium(0) (0.139 g, 0.12 mmol) were added to a 30 mL mixed solution of tetrahydrofuran / water (1 / 1, v / v). Under the protection of nitrogen, the mixture was refluxed at 70 °C until the starting material spots disappeared on TLC plate. The reaction was stopped, cooled to room temperature, and extracted three times with dichloromethane (30 mL × 3). The organic layer was taken out. The organic layer was further extracted three times with saturated sodium chloride solution (15 mL × 3), and the organic layer was separated, dried over anhydrous sodium sulfate, and the solvent was rotary evaporated to obtain a viscous black crude product. Finally, it was separated and purified by column chromatography to obtain 1.93 g of green solid with a yield of 54.4%.

[0048] (2) Synthesis of the target compound:

[0049]

[0050] Intermediate TPA-1 (100 mg, 0.28 mmol) was dissolved in 20 mL of ultradry ethanol, and then 0.30 mmol of another reactant salicylhydrazide was added. The mixture was refluxed for 6 h until the reaction was monitored to be complete by TLC. The reaction was stopped, and the reaction solution was cooled to room temperature. A large amount of precipitate was formed, filtered, and the filter cake was washed three times with 20 mL of cold ethanol to obtain the target compound J-1.

[0051] The target compound J-1 obtained in this example is a new compound that has not been reported. After purification, it was characterized by high-resolution mass spectrometry (HRMS), nuclear magnetic resonance spectroscopy (1H NMR, 13C NMR). The high-resolution mass spectrum of probe J-1 is as Figure 1 shown. A molecular ion peak with a mass-to-charge ratio of 490.1587 was measured. The theoretical mass-to-charge ratio of J-1 is 450.1589, and the relative error between the two is 4.4e -7 , within the allowable error range. The 1H nuclear magnetic resonance spectrum of probe J-1 1 1H NMR is as Figure 2 shown. 1H NMR (400 MHz, DMSO-d6) δ 8.62 (s, 1H), 7.87 (d, J = 7.7 Hz, 1H), 7.64 (d, J = 8.5 Hz, 2H), 7.50–7.41 (m, 4H), 7.35 (t, J = 7.8 Hz, 4H), 7.10 (dd, J = 13.2, 7.3 Hz, 7H), 6.97 (t, J = 11.1 Hz, 4H). 116.38. The 13C nuclear magnetic resonance spectrum of probe J-1 13 13C NMR is as Figure 3As shown, 13C NMR (101 MHz, DMSO-d6) δ 165.02, 159.52, 147.97, 147.17, 146.44, 144.16, 137.50, 134.26, 133.26, 130.17, 128.91, 127.27, 125.05, 124.17, 123.68, 123.00, 119.40, 117.76.

[0052] Application Example 1

[0053] Figure 4 It is the fluorescence spectral changes of the solution of probe J-1 (concentration of 1×10 -5 mol / L) in DMF / H2O (6:4, v / v, 0.01 M Hepes) at different pH values. It can be seen that the fluorescence intensity of probe J-1 is relatively strong and stable between pH = 4 - 8. Therefore, a buffer solution with pH = 7.4 is selected as the test system.

[0054] Application Example 2

[0055] Dissolve the copper ion fluorescent molecular probe J-1 synthesized in Example 1 in DMF to prepare a system of DMF / H2O (6:4, v / v, 0.01 M Hepes, pH = 7.4) with a concentration of 1×10 -5 mol / L, and detect its selectivity for different metal ions Al 3+ , K + , Fe 3+ , Fe 2 + , Na + , Cr 3+ , Ca 2+ , Zn 2+ , Mg 2+ , Mn 2+ , Ba 2+ , Hg 2+ , Pb 2+ , Cd 2+ , Co 2+ , Ni 2+ , Ag + and Cu 2+ .

[0056] Figure 5 It is the fluorescence selectivity spectrogram. As can be seen from Figure 5 , according to the ultraviolet maximum absorption peak of probe J-1, we use 410 nm as the excitation wavelength for fluorescence spectral testing. Probe J-1 itself has a fluorescence emission peak at 548 nm, corresponding to the solution emitting bright yellow fluorescence. After adding different metal ions, only Cu 2+Quenches the fluorescence of probe J-1, and there is almost no change when other metal ions are added. Therefore, probe J-1 can well distinguish Cu 2+ from other ions by fluorescence color.

[0057] Application Example 3

[0058] By conducting competitive experiments, we further studied and tested whether the coexistence with other metal cations would affect the selectivity for copper ions. The results Figure 6 are shown ( Figure 6 in the back is the fluorescence intensity when the probe is added with a certain metal ion alone, and in the front is the fluorescence intensity when other metal cations are added after adding copper ions).

[0059] Figure 6 Figure is the influence of different coexisting cations on the determination of copper ions. In Figure 6 the one marked "Cu 2+ " is the fluorescence intensity of the system when 1.0×10 -5 mol / L of copper ions exist alone, and the rest are the fluorescence intensities of the system when copper ions coexist with various metal ions at the same concentration. As can be seen from the figure, the presence of the above different coexisting cations does not significantly change the detection result of this probe molecule for copper ions, indicating that the fluorescent probe of the present invention can still maintain high selectivity for copper ions in the presence of other metal ions, verifying its reliability in complex systems.

[0060] According to fluorescence spectral analysis, the coexistence with other metal cations does not affect the recognition of copper ions by the probe.

[0061] Application Example 4

[0062] Different concentrations of copper ions are added to the solution, and the change in its ultraviolet absorption intensity is detected, as Figure 7 shown. Figure 7 Figure is the ultraviolet spectral response diagram of probe J-1 (concentration of 1×10 -5 mol / L) to different concentrations of Cu 2+ in DMF / H2O (6:4, v / v, 0.01M Hepes, pH = 7.4). In Figure 7 (a), the abscissa is the wavelength (nm), the ordinate is the ultraviolet absorption intensity, the excitation wavelength is 405nm, and the copper ion concentration ranges from 0 to 18 μM. In Figure 7 (b), when the concentration of Cu 2+ is in the range of 2 - 7 μM, the ultraviolet absorption value of the probe J-1 solution at 405nm shows a good linear relationship with the concentration of Cu 2+ , successfully realizing the quantitative detection of copper ions.

[0063] Figure 8 For the fluorescence spectral response of probe J-1 (concentration 1×10 -5 mol / L) to different concentrations of Cu 2+ in DMF / H2O (6:4, v / v, 0.01 M Hepes, pH = 7.4). In Figure 8 (a), the abscissa is wavelength (nm) and the ordinate is fluorescence intensity. The concentration of copper ions ranges from 0 to 18 μM. In Figure 8 (b), when the concentration of added Cu 2+ is 1 - 10 μM, there is a good linear relationship with the fluorescence intensity at 548 nm of the probe solution, and quantitative detection of copper ions is successfully achieved.

[0064] Application Example 5

[0065] Figure 9 For the color change of the solution of probe J-1 (concentration 1×10 -5 mol / L) in DMF / H2O (6:4, v / v, 0.01 M Hepes, pH = 7.4) after adding 5×10 -5 mol / L copper ions under irradiation of a 365 nm ultraviolet lamp (changing from yellow on the right to colorless on the left).

[0066] Figure 10 For the reversible change after adding an equal amount of copper ions to the solution of probe J-1 (concentration 1×10 -5 mol / L) in DMF / H2O (6:4, v / v, 0.01 M Hepes, pH = 7.4) and then adding 1×10 -5 mol / L Na2EDTA. The fluorescence intensity of the probe J-1 solution is strong. After adding copper ions of the same concentration (1×10 -5 mol / L) to the probe solution, the fluorescence is quenched. Subsequently, adding 1×10 -5 mol / L Na2EDTA solution to the mixture restores the fluorescence intensity. Then adding 1×10 -5 mol / L Cu 2+ again quenches the fluorescence, proving that the probe has good reversibility.

[0067] The present invention designs and synthesizes a fluorescence probe for specific recognition of Cu 2+ . Probe J-1 has obvious fluorescence emission in the absence of Cu 2+ , but has obvious fluorescence quenching at 548 nm after adding Cu 2+ . It has high selectivity and anti-interference ability, and further enables the [J-1 + Cu 2+ complex to effectively determine Cu 2+concentration. In addition, the detection limit of J-1 for Cu 2+ is also very low, and intracellular Cu 2+ was successfully observed through bioimaging experiments.

[0068] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited thereto. Within the technical concept scope of the present invention, various simple modifications can be made to the technical solutions of the present invention, including any other suitable combination of each technical feature. These simple modifications and combinations should also be regarded as the content disclosed by the present invention and fall within the protection scope of the present invention.

Claims

1. Application of the fluorescent molecular probe shown in the following formula in the preparation of reagents for copper ion sensing and detection in plant tissues and cells, in, The application uses the following test system: the probe concentration is 1×10 -5 mol / L, the solvent was DMF / H2O with a volume ratio of 6:4; the test system contained 0.01 M Hepes and pH = 7.

4.

2. The use according to claim 1, wherein: The sensing detection includes ultraviolet light detection, fluorescence detection, visual qualitative detection and copper ion reversible detection.

3. The use according to claim 1, wherein: The sensing assay is a selective sensing assay and a competitive sensing assay.

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