Dual-function fluorescent molecular probe, preparation method thereof, and application thereof in detecting heavy metal ions in Chinese herbal medicine

Abstract

The present invention relates to the technical field of copper ion and mercury ion detection, and discloses a bifunctional fluorescent molecular probe and a preparation method and application thereof. The bifunctional fluorescent molecular probe is obtained by a condensation reaction of 5-(4-(diphenylamino)phenyl)thiophene-2-carboxaldehyde and benzoyl hydrazide, and can be applied to the sensing and detection of copper ions and mercury ions in water environments, Chinese herbal medicines, living cells and living zebrafish, showing good selectivity, strong anti-interference performance to other metal ions, and low detection limit.

Description

Technical Field

[0001] The invention relates to the technical field of copper ion and mercury ion detection, and in particular to a dual-function fluorescent molecular probe and a preparation method thereof and application thereof in detecting heavy metal ions in Chinese herbal medicines. Background Art

[0002] Copper is an essential trace element in nature and participates in the basic life processes of humans and animals. It plays a key role in many enzyme catalysis and redox reactions and is necessary for health and normal function. Appropriate amounts of copper ions can effectively participate in the biosynthesis and cofactors of a variety of enzymes, help effectively inhibit the proliferation and growth of human bacteria, and enhance the body's immune function. Insufficient intake can lead to bone deformities, pigmentation, abnormal cholesterol metabolism, and serious neurological problems. Of course, excessive intake of copper in the environment can lead to its long-term insoluble precipitation in the body, causing harm to the organism, leading to cirrhosis, movement disorders, sensory nerve disorders and many other diseases.

[0003] In addition, mercury is one of the most toxic heavy metal elements in the environment. Its ions can strongly interact with proteins and various enzymes in organisms, stripping them of their activity, and ultimately leading to pathological changes or toxic manifestations in the body. Mercury ions are non-degradable, easy to migrate, and have high bioaccumulation potential. When they accumulate to a certain level in the human body, they will show toxic effects, thus endangering human health. Today, the World Health Organization (WHO) has released a list of the top ten chemicals or substances that pose a major risk to public health, including Hg. 2+ Therefore, from a development perspective, it is of great significance to establish a feasible, accurate, and effective method for trace detection of copper and mercury ions in biological systems and the pharmaceutical field.

[0004] At present, the detection of copper ions and mercury ions mainly relies on traditional technologies, such as inductively coupled plasma mass spectrometry (ICP-MS), flame atomic absorption spectroscopy (AAS) and anodic stripping voltammetry (ASV), which have the advantages of high sensitivity and reliability. However, the above technologies require expensive equipment, complex sample processing and time-consuming detection processes, resulting in low detection efficiency. In contrast, the research on fluorescent biosensors has made significant progress. Compared with traditional technologies, fluorescent probe technology has the advantages of simple operation, low cost, high specificity, analytical visualization, and non-interference, and has received widespread attention in the fields of food testing, environmental monitoring, and biological analysis. Fluorescent molecular probes also have greater sensitivity. Therefore, the development of fluorescent probes with specific recognition properties has become one of the current research hotspots. Summary of the invention

[0005] The purpose of the present invention is to overcome the problems of the prior art, such as complex synthesis process, poor selectivity, low sensitivity and inability to adapt to complex application environments, and to provide a bifunctional fluorescent molecular probe and a preparation method thereof and an application thereof in detecting heavy metal ions in Chinese herbal medicines.

[0006] In order to achieve the above object, the first aspect of the present invention provides a bifunctional fluorescent molecular probe for the detection of copper ions and mercury ions, wherein the bifunctional fluorescent molecular probe has the following structural formula:

[0007]

[0008] The second aspect of the present invention provides a method for preparing a bifunctional fluorescent molecular probe, wherein the preparation method comprises: allowing 5-(4-(diphenylamino)phenyl)thiophene-2-carboxaldehyde and benzoylhydrazide to undergo a condensation reaction to obtain a bifunctional fluorescent molecular probe for detecting copper ions and mercury ions.

[0009] The third aspect of the present invention provides a bifunctional fluorescent molecular probe obtained according to the preparation method described in the second aspect.

[0010] The fourth aspect of the present invention provides the use of the bifunctional fluorescent molecular probe described in the first aspect or the third aspect in the sensing and detection of copper ions and mercury ions.

[0011] A fifth aspect of the present invention provides a method for detecting copper ions or mercury ions using the bifunctional fluorescent molecular probe described in the first aspect or the third aspect, wherein the method comprises the following steps:

[0012] (1) dissolving the bifunctional fluorescent molecular probe in a mixture of N,N-dimethylformamide and Hepes buffer to obtain a fluorescent molecular probe solution;

[0013] (2) adding the sample to be tested to the fluorescent molecular probe solution obtained in step (1) to obtain a mixed solution;

[0014] (3) Qualitative analysis of whether the sample contains Cu based on the color change of the mixed solution under sunlight and ultraviolet light 2+ and Hg 2+ ; Determine the ultraviolet spectrum and fluorescence spectrum of the mixed solution, obtain the ultraviolet absorption and fluorescence intensity at the corresponding wavelength according to the test, and determine the Cu in the sample to be tested through the corresponding linear observation 2+ and Hg 2+ The content.

[0015] Through the above technical solution, the beneficial technical effects achieved by the present invention are as follows:

[0016] (1) The bifunctional fluorescent molecular probe provided by the present invention can be used for the specific identification of copper (II) ions or mercury ions in water environments, Chinese herbal medicines, living cells and living zebrafish, can display different functions through ultraviolet and fluorescence spectra, and can be used for quantitative and sensing detection of copper ions and mercury ions, showing good selectivity, strong anti-interference performance against other metal ions, and low detection limit.

[0017] (2) The bifunctional fluorescent molecular probe of the present invention can provide real-time qualitative and quantitative information through fluorescence spectroscopy and ultraviolet spectroscopy detection, and can undergo color changes, making it a highly specific indicator suitable for visual colorimetric detection.

[0018] (3) The raw materials for the synthesis of the bifunctional fluorescent molecular probe of the present invention are economical and readily available, the synthesis method is simple to operate, and the post-reaction treatment process is relatively simple. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a high-resolution mass spectrum of the fluorescent molecular probe J-2 synthesized in Example 1 of the present invention.

[0020] Figure 2 The nuclear magnetic resonance of the fluorescent molecular probe J-2 synthesized in Example 1 of the present invention is 1 H NMR spectra.

[0021] Figure 3 The nuclear magnetic resonance of the fluorescent molecular probe J-2 synthesized in Example 1 of the present invention is 13 C NMR spectrum.

[0022] Figure 4 J-2+Cu 2+ 、J-2+Hg 2+ , J-2+ fluorescence spectra of other metal cations.

[0023] Figure 5 J-2, J-2+Cu 2+ 、J-2+Hg 2+ Ultraviolet absorption spectrum of .

[0024] Figure 6 (A) Probe J-2 recognizes Cu 2+ Competitive detection diagram of different metal cations in DMF / water (6:4, v / v, 0.01 M Hepes, pH = 7.4) system, (B) is J-2+Cu 2+ Identification of Hg in DMF / water (6:4, v / v, 0.01 M Hepes, pH = 7.4) system 2+ Competition assay plots for different cations.

[0025] Figure 7(A)J-2+Cu 2+ UV titration curve, (B) is J-2+Hg 2+ UV titration curve.

[0026] Figure 8 (A) is J-2+Cu 2+ Fluorescence titration curve of J-2+Hg 2+ Fluorescence titration curve.

[0027] Fig. 9 Probe J-2, J-2+Cu 2+ and J-2+Hg 2+ The effect in daylight.

[0028] Fig.10 Probe J-2, J-2+Cu 2+ and J-2+Hg 2+ The effect diagram under 365nm ultraviolet light. DETAILED DESCRIPTION

[0029] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0030] The first aspect of the present invention provides a bifunctional fluorescent molecular probe for detecting copper ions and mercury ions, wherein the bifunctional fluorescent molecular probe has the following structural formula:

[0031]

[0032] The dual-function fluorescent molecular probe of the present invention can specifically identify Cu 2+ and Hg 2+ Probe J-2 has no Cu 2 + and Hg 2+ In the case of adding Cu 2+ and Hg 2+ After that, there is different degree of fluorescence quenching at 548nm. 2+ After addition, the fluorescence was almost completely quenched, and Hg 2+ After addition, the fluorescence is quenched by about half. It has high selectivity and anti-interference, which enables J-2 to effectively determine Cu with high sensitivity. 2+ and Hg 2+ In addition, J-2 has a 2+and Hg 2+ The detection limits were low.

[0033] The bifunctional fluorescent molecular probe of the present invention has the characteristics of simplicity, rapidity, high specificity and high sensitivity when used for the detection of copper ions and mercury ions, and therefore has broad application prospects, and is particularly suitable for the field of in vivo imaging.

[0034] The second aspect of the present invention provides a method for preparing a bifunctional fluorescent molecular probe, wherein the preparation method comprises: allowing 5-(4-(diphenylamino)phenyl)thiophene-2-carboxaldehyde and benzoylhydrazide to undergo a condensation reaction to obtain a bifunctional fluorescent molecular probe for detecting copper ions and mercury ions.

[0035] In some embodiments of the present invention, the bifunctional fluorescent molecular probe has the following structural formula:

[0036]

[0037] During the preparation of the bifunctional fluorescent molecular probe of the present invention, the reaction that occurs is:

[0038]

[0039] The 5-(4-(diphenylamino)phenyl)thiophene-2-carboxaldehyde is obtained by reacting triphenylamine boric acid and 5-bromothiophene-2-carboxaldehyde, and can also be obtained from a reagent company.

[0040] In some embodiments of the invention, the method comprises the following steps:

[0041] (1) preparing a solution of 5-(4-(diphenylamino)phenyl)thiophene-2-carbaldehyde;

[0042] (2) Adding benzoyl hydrazide to the solution of step (1) to carry out a condensation reaction to obtain the bifunctional fluorescent molecular probe.

[0043] In some embodiments of the present invention, the step (1) uses dry anhydrous ethanol as solvent.

[0044] In some embodiments of the present invention, in step (2), the molar ratio of 5-(4-(diphenylamino)phenyl)thiophene-2-carboxaldehyde to benzohydrazide is 14:15.

[0045] In some embodiments of the present invention, the conditions of the condensation reaction include: heating under reflux for 4-6 hours. After the reaction is complete, the reaction is cooled to room temperature to precipitate, and the precipitate is filtered, washed and dried to obtain the fluorescent molecular probe.

[0046] The third aspect of the present invention provides a bifunctional fluorescent molecular probe obtained according to the preparation method described in the second aspect.

[0047] The fourth aspect of the present invention provides the use of the bifunctional fluorescent molecular probe described in the first aspect or the third aspect in the sensing and detection of copper ions and mercury ions.

[0048] In some embodiments of the present invention, the objects of application include water environment, Chinese herbal medicine, living cells and living zebrafish.

[0049] The sensing detection is selective sensing detection and competitive sensing detection. The sensing detection is visual qualitative detection, ultraviolet light detection, and fluorescence detection. The sensing detection is quantitative detection under ultraviolet spectrum and fluorescence spectrum.

[0050] A fifth aspect of the present invention provides a method for detecting copper ions or mercury ions using the bifunctional fluorescent molecular probe described in the first aspect or the third aspect, wherein the method comprises the following steps:

[0051] (1) dissolving the bifunctional fluorescent molecular probe in a mixture of N,N-dimethylformamide and Hepes buffer to obtain a fluorescent molecular probe solution;

[0052] (2) adding the sample to be tested to the fluorescent molecular probe solution obtained in step (1) to obtain a mixed solution;

[0053] (3) Qualitative analysis of whether the sample contains Cu based on the color change of the mixed solution under sunlight and ultraviolet light 2+ and Hg 2+ ; Determine the ultraviolet spectrum and fluorescence spectrum of the mixed solution, obtain the ultraviolet absorption and fluorescence intensity at the corresponding wavelength according to the test, and determine the Cu in the sample to be tested through the corresponding linear observation 2+ and Hg 2+ The content.

[0054] In the present invention, when the quantitative detection of Cu 2+ When the UV spectrum is used for detection, the corresponding wavelength can be 405 nm, and the corresponding wavelength can be 542 nm for fluorescence spectrum detection; when the quantitative detection of Hg 2+ When the UV spectrum is detected, the corresponding wavelength can be 405 nm, and the corresponding wavelength can be 548 nm for fluorescence spectrum detection. The corresponding linear observation refers to the standard curve obtained by UV or fluorescence titration.

[0055] In some embodiments of the present invention, in the fluorescent molecular probe solution of step (1), the concentration of the fluorescent molecular probe is 10 μM.

[0056] In some embodiments of the present invention, the pH of the fluorescent molecular probe solution is 7.4.

[0057] In some embodiments of the present invention, the volume ratio of N,N-dimethylformamide to water is 6:4.

[0058] In some embodiments of the present invention, the concentration of Hepes buffer is 0.01M.

[0059] The present invention will be described in detail below through examples.

[0060] In the following examples and comparative examples, if no specific conditions are specified, the experiments were carried out under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used, if no manufacturer is specified, are all conventional products that can be obtained through commercial channels.

[0061] Example 1

[0062] This example is used to illustrate the preparation of the target compound J-2

[0063] 1) Synthesis of intermediate TPA-1 (5-(4-(diphenylamino)phenyl)thiophene-2-carboxaldehyde):

[0064]

[0065] Triphenylamine boric acid (3.47g, 12mmol), 5-bromothiophene-2-carboxaldehyde (1.91g, 10mmol), potassium carbonate (2.49g, 18mmol) and catalyst tetrakis(triphenylphosphine)palladium (0.139g, 0.12mmol) were added to a 30mL THF / water (1 / 1, v / v) mixed solution, and refluxed at 70°C under the protection of nitrogen until the TLC spot plate raw material point disappeared. After stopping the reaction, it was cooled to room temperature, extracted three times with dichloromethane (30mL×3 times), and the organic layer was taken out. The organic layer was extracted three times with a saturated sodium chloride solution (15mL×3 times), the organic layer was separated, anhydrous sodium sulfate was added to dry, and the solvent was spin-dried to obtain a viscous black crude product. Finally, it was separated and purified by column chromatography to obtain 1.93g of a green solid.

[0066] 2) Synthesis of target compound J-2:

[0067]

[0068] The intermediate TPA-1 (100 mg, 0.28 mmol) was dissolved in 20 mL of ultra-dry ethanol. Subsequently, 0.30 mmol of another reactant, benzoyl hydrazide, was added and heated under reflux for 6 hours until the reaction was complete as monitored by TLC, the reaction was stopped and cooled to room temperature, at which a large amount of precipitation was generated. Then a filtering operation was performed, and the filter cake was rinsed three times with 20 mL of cold ethanol to finally obtain the target compound J-2.

[0069] The target compound J-2 obtained in this example is a new compound that has not been reported before. After purification, high-resolution mass spectrometry (HRMS), nuclear magnetic resonance spectroscopy ( 1 H NMR, 13 C NMR) to characterize it. The high-resolution mass spectrum of probe J-2 is shown in Figure 1 As shown, the NMR of probe J-2 1 H NMR Figure 2 As shown, the NMR of probe J-2 13 C NMR Figure 3 shown. 1 H NMR (500MHz, DMSO-d6) δ (ppm) 11.83 (s, 1H), 8.62 (s, 1H), 7.90 (d, J = 7.4Hz, 2H), 7.64 (d, J = 8.3Hz, 2H), 7.61–7.57 (m, 1H) ,7.53(t,J=7.4Hz,2H),7.44(d,J=5.6Hz,2H),7.34(t,J=7.6Hz,4H),7.09(dd,J=15.8,7.7Hz,6H),6.98(d,J=8.4Hz,2H). 13 C NMR (126MHz, DMSO-d6) δ163.37,147.89,147.17,146.04,143.26,137.83,133.90,132. 80,132.20,130.17,128.96,128.04,127.37,127.21,125.01,124.14,123.64,123.05.

[0070] The fluorescent molecular probe J-2 synthesized in Example 1 was dissolved in DMF to prepare a 10 μM DMF / water (6:4, v / v, 0.01 M Hepes, pH=7.4) buffer system (hereinafter referred to as: probe J-2 solution) for the following tests.

[0071] Test Example 1

[0072] Different metal cations (Al 3+ , K + , Fe 3+ , Fe 2+ 、Na + Cr 3+ , Ca 2+ 、Zn 2+ Mg 2 + , Mn 2+ , Ba 2+ , Hg 2+ , Pb2+ 、Cd 2+ 、Co 2+ 、Ni 2+ 、Ag + and Cu 2+ ), fluorescence spectra such as Figure 4 As shown. Figure 4 It can be seen that the addition of Cu 2+ and Hg 2+ After that, at 548 nm, the fluorescence intensity decreased significantly (Cu 2+ The fluorescence of probe J-2 can be almost completely quenched. 2+ The fluorescence of probe J-2 can be quenched by about half), and the addition of other metal ions will not cause obvious changes in fluorescence intensity.

[0073] Figure 5 The probe J-2 solution and the addition of Cu 2+ and Hg 2+ The UV spectrum after adding Cu 2+ and Hg 2+ After that, the 405nm ultraviolet absorption peak decreased and red-shifted to 435nm and 425nm respectively.

[0074] The above results show that Cu 2+ and Hg 2+ Uniquely compared to other ions, probe J-2 can specifically recognize Cu 2+ and Hg 2+ .

[0075] Test Example 2

[0076] Detection of Cu using probe J-2 solution 2+ For different metal cations 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 + The fluorescence intensity of the probe J-2 solution and after adding different metal cations to it was measured, and the fluorescence intensity of the probe J-2 solution after adding other metal cations first and then adding an equal amount of Cu2+ The fluorescence intensity of the system is Figure 6 As shown in (A). It can be seen that probe J-2 has a strong affinity for Cu 2+ The recognition has good anti-interference ability, and the presence of other metal ions in the system will not affect its recognition effect.

[0077] Detection of Hg using probe J-2 solution 2+ For different metal cations Al 3+ , K + , Fe 3+ , Fe 2+ 、Na + Cr 3+ , Ca 2+ 、Zn 2 + Mg 2+ , Mn 2+ , Ba 2+ , Cu 2+ , Pb 2+ 、Cd 2+ 、Co 2+ 、Ni 2+ 、Ag + competitiveness, such as Figure 6 (B) As shown. It can be seen that in addition to Cu 2+ In addition, probe J-2 is sensitive to Hg 2+ It also has good anti-interference ability.

[0078] Test Example 3

[0079] Different concentrations of copper ions / mercury ions were added to solution J-2, and the changes in its UV spectrum were detected. Figure 7 shown.

[0080] Figure 7 (A) shows the change of the UV spectrum of the system and Cu 2+ The relationship between the concentration of copper ions and the wavelength of 405 nm is shown in Figure 2. With the increase of copper ion concentration, the UV intensity at a wavelength of 405 nm gradually weakens, and the change of UV intensity is obvious, which proves that probe J-2 can be used for Cu 2+ The quantitative detection is clear and easy to identify.

[0081] Figure 7 (B) shows the change of the UV spectrum of the system and Hg 2+ The relationship between the concentration of mercury ions and the UV intensity at a wavelength of 405 nm gradually decreases with the increase of mercury ion concentration. The change of UV intensity is obvious, which proves that probe J-2 can be used for Hg 2+ The quantitative detection is clear and easy to identify.

[0082] Different concentrations of copper ions / mercury ions were added to the J-2 solution, and the changes in its fluorescence spectrum were detected. Figure 8 shown.

[0083] Figure 8 (A) shows the fluorescence spectrum change of the system and Cu 2+ The relationship between the concentration of copper ions and the fluorescence spectrum at a wavelength of 542 nm was obvious with the increase of copper ion concentration, which proved that probe J-2 can be used for the detection of Cu 2+ The quantitative detection is clear and easy to identify.

[0084] like Figure 8 As shown in (B), the fluorescence spectrum changes with Hg 2+ The relationship between the concentration of mercury ions and the fluorescence spectrum at a wavelength of 548 nm was obvious when the mercury ion concentration increased, proving that probe J-2 can be used to detect Hg 2+ The quantitative detection is clear and easy to identify.

[0085] Test Example 4

[0086] Fig. 9 To add 1×10 -5 mol / L copper ion and 1×10 -5 mol / L mercury ions, and observe the color change of the solution under sunlight. It was found that the J-2 probe solution turned red after adding copper ions, and the color of the solution deepened after adding mercury ions.

[0087] Fig.10 To add 1×10 -5 mol / L copper ion and 1×10 -5 mol / L mercury ions, and the color change of the solution was observed under a 365nm ultraviolet lamp. It was found that the fluorescence of the J-2 probe solution was quenched when copper ions and mercury ions were added.

[0088] The above series of spectral experiments show that J-2 and Cu 2+ and Hg 2+ There is an interaction between them, which improves the J-2-Cu 2+ and J-2-Hg 2+ The J-2 probe has the potential to be developed into a bifunctional fluorescent biosensor capable of simultaneously detecting copper and mercury ions, which can be used for ratiometric and colorimetric detection of copper and mercury ions by the naked eye.

[0089] Test Example 5

[0090] Tap water, wolfberry extract and honeysuckle extract were selected to study the detection ability of probe J-2 in actual water samples and traditional Chinese medicine extracts.

[0091] The Chinese medicine extract was filtered to remove insoluble impurities, and then the filtered extract was mixed with probe J-2 to make a test solution to test its fluorescence spectrum. The fluorescence intensity was almost unchanged, indicating that Cu in the Chinese medicine extract 2+ / Hg 2+ The content was very small and could not reach the detection limit of J-2. Then tap water and Chinese medicine extract were used to prepare the concentration of 2.5×10 -5 mol / L、5×10 - 5 mol / L、10×10 -5 mol / L Cu 2+ / Hg 2+ Solution.

[0092] Table 1 shows the fluorescence molecular probe J-2 for the determination of Cu in tap water, wolfberry extract, and honeysuckle extract 2+ The experimental results.

[0093] Table 1

[0094]

[0095] Note: n is the number of test repetitions.

[0096] Table 2 shows the results of the fluorescent molecular probe J-2 for the determination of Hg in tap water, wolfberry extract, and honeysuckle extract. 2+ The experimental results.

[0097] Table 2

[0098]

[0099] As shown in Table 1, the recovery rate of spiked water samples was 94.0-102.8%, and the relative standard deviation was less than 10%; as shown in Table 2, the recovery rate of spiked water samples was 98.0-106.8%, and the relative standard deviation was less than 10%. Therefore, probe J-2 can be used for quantitative detection of Cu in tap water and traditional Chinese medicine extracts. 2+ / Hg 2+ content.

[0100] The present invention designs and synthesizes a dual-functional fluorescent molecular probe for specific recognition of copper (II) ions and mercury (II) ions. Probe J-2 has a strong fluorescence intensity in the absence of copper (II) ions, but has obvious fluorescence quenching after adding copper (II) ions, and has high selectivity and anti-interference. At the same time, probe J-2 has obvious fluorescence quenching after adding mercury (II) ions, and also shows high selectivity and anti-interference. In addition, probe J-2 has a low detection limit for copper (II) ions, and the ultraviolet detection limit is 3.39×10 -7 M, the fluorescence detection limit is 3.31×10 -7M; The detection limit of probe J-2 for mercury ions is low, and the UV detection limit is 3.31×10 -7 M, the fluorescence detection limit is 4.93×10 -7 M;

[0101] The preferred embodiments of the present invention are described in detail above, but the present invention is not limited thereto. Within the technical concept of the present invention, the technical solution of the present invention can be subjected to a variety of simple modifications, including the combination of various technical features in any other suitable manner, and these simple modifications and combinations should also be regarded as the contents disclosed by the present invention and belong to the protection scope of the present invention.

Claims

1. A bifunctional fluorescent molecular probe for the detection of copper ions and mercury ions, characterized in that: The bifunctional fluorescent molecular probe has the following structural formula:

2. A method for preparing a bifunctional fluorescent molecular probe, characterized in that: The preparation method comprises: making 5-(4-(diphenylamino)phenyl)thiophene-2-carboxaldehyde and benzoyl hydrazide undergo condensation reaction to obtain a bifunctional fluorescent molecular probe for detecting copper ions and mercury ions.

3. The preparation method according to claim 2, wherein The bifunctional fluorescent molecular probe has the following structural formula:

4. The preparation method according to claim 2 or 3, wherein The method comprises the following steps: (1) preparing a solution of 5-(4-(diphenylamino)phenyl)thiophene-2-carbaldehyde; (2) Adding benzoyl hydrazide to the solution of step (1) to carry out a condensation reaction to obtain the bifunctional fluorescent molecular probe.

5. The preparation method according to claim 4, wherein In the step (1), dry anhydrous ethanol is used as solvent.

6. The preparation method according to claim 4 or 5, wherein: In the step (2), the molar ratio of 5-(4-(diphenylamino)phenyl)thiophene-2-carboxaldehyde to benzoylhydrazide is 14:

15.

7. The preparation method according to claim 4 or 5, wherein: The conditions of the condensation reaction include: heating under reflux; and a reaction time of 4-6 hours.

8. Use of the bifunctional fluorescent molecular probe according to claim 1 or 7 in sensing and detecting copper ions and mercury ions.

9. The use according to claim 8, wherein: The objects of application include water environment, Chinese herbal medicine, living cells and living zebrafish.

10. A method for detecting copper ions or mercury ions using the bifunctional fluorescent molecular probe according to claim 1, characterized in that: The method comprises the following steps: (1) dissolving the bifunctional fluorescent molecular probe in a mixture of N,N-dimethylformamide and Hepes buffer to obtain a fluorescent molecular probe solution; (2) adding the sample to be tested to the fluorescent molecular probe solution obtained in step (1) to obtain a mixed solution; (3) Qualitative analysis of whether the sample contains Cu based on the color change of the mixed solution under sunlight and ultraviolet light 2+ and Hg 2+ ; Determine the ultraviolet spectrum and fluorescence spectrum of the mixed solution, obtain the ultraviolet absorption and fluorescence intensity at the corresponding wavelength according to the test, and determine the Cu in the sample to be tested through the corresponding linear observation 2+ and Hg 2+ The content.

11. The method according to claim 10, wherein: In the fluorescent molecular probe solution of step (1), the concentration of the fluorescent molecular probe is 10 μM.

12. The method according to claim 10, wherein: The pH of the fluorescent molecular probe solution is 7.

4.

13. The method according to claim 10, wherein: The volume ratio of N,N-dimethylformamide to water is 6:

4.

14. The method according to claim 10, wherein: The concentration of Hepes buffer was 0.01M.

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