Coumarin-based fluorescent probe for sequential detection as well as preparation method and application of coumarin-based fluorescent probe

Abstract

The invention provides a coumarin-based fluorescent probe for sequential detection as well as a preparation method and application thereof, and relates to the technical field of chemical analysis, a diphenylamine electron-donating group and a pyridine hydrazide coordination recognition unit are introduced into a coumarin fluorescent skeleton, and a molecular system with a D-pi-A conjugated structure is constructed, so that the coumarin-based fluorescent probe for sequential detection is obtained. According to the present invention, the copper ions are introduced into the probe, such that the probe and the copper ions can produce the coordination effect, and the fluorescence recovery is achieved through the competitive coordination in the presence of the biological mercaptan so as to form the on-off-re-on sequential fluorescence response mode, such that the two analytes can be sequentially detected in the single probe system, the defect that most fluorescent probes in the prior art can only detect a single target object is overcome.

Description

Technical Field

[0001] This invention relates to the field of chemical analysis technology, and in particular to a coumarin-based fluorescent probe for sequential detection, its preparation method, and its application. Background Technology

[0002] Copper ions are an important trace element in living systems, participating in various redox processes, enzyme-catalyzed reactions, and the regulation of intracellular homeostasis. Abnormal copper ion concentrations are closely related to oxidative stress, neurodegenerative diseases, and environmental pollution. Meanwhile, biothiols such as glutathione (GSH), cysteine ​​(Cys), and homocysteine ​​(Hcy), as important reducing small molecules in cells, not only participate in maintaining redox balance but also exhibit strong coordination interactions with copper ions, thereby affecting their chemical form and bioavailability.

[0003] Existing methods for copper ion detection mainly include atomic absorption spectrometry, inductively coupled plasma mass spectrometry, and electrochemical analysis. Although these methods offer high sensitivity, they typically require large-scale instruments, making them unsuitable for rapid on-site detection and real-time visualization analysis in biological systems. Fluorescent probe methods have attracted widespread attention due to their sensitivity, ease of operation, and high spatiotemporal resolution. However, most existing fluorescent probes target single analytes, making it difficult to achieve sequential recognition of copper ions and biothiols on the same molecular platform. Furthermore, existing systems generally suffer from small Stokes shifts, strong background interference, lack of naked-eye visualization capabilities, and difficulty in integrating with smart terminals.

[0004] Therefore, developing a fluorescent probe that can sequentially respond to copper ions and biothiols in a single molecular system, while also possessing large Stokes shift, naked-eye recognition, smartphone-assisted readout, and bioimaging capabilities, has significant application value. Summary of the Invention

[0005] This invention addresses the shortcomings of existing technologies by proposing a coumarin-based fluorescent probe capable of sequential detection.

[0006] To achieve the above objectives, the present invention employs the following technical solution: a coumarin-based fluorescent probe for sequential detection. This probe constructs a molecular system with a D–π–A conjugated structure by introducing a diphenylamine electron-donating group and a pyridylhydrazine coordination recognition unit into the coumarin fluorescent backbone. This enables the probe to coordinate with copper ions and achieve fluorescence recovery through competitive coordination in the presence of biothiols, thereby forming a sequential fluorescence response mode of "on-off-on-off".

[0007] When copper ions are added to the fluorescent probe, its coordination recognition unit complexes with the copper ions, causing a change in the molecular electronic structure and a significant decrease in the fluorescence signal. When biothiols are further added to the system, biothiols have a stronger coordination ability with copper ions and can compete with copper ions for binding, causing the probe-copper ion complex to dissociate and restore the original fluorescence signal of the probe, thereby realizing the sequential detection of copper ions and biothiols.

[0008] Furthermore, the probe causes a significant color change in the solution in the presence of copper ions, allowing for direct visual identification under natural light conditions. Simultaneously, by capturing solution images and extracting RGB color parameters via a smartphone, digital analysis of the detection signal can be achieved, thus establishing a dual-mode detection system combining fluorescence detection and visual recognition.

[0009] This fluorescent probe is mainly composed of the following structural units: (1) Coumarin fluorescent core, used to provide fluorescence emission center; (2) Diphenylamine electron-donating unit, used to construct electron-donating-π-conjugated-electron-accepting (D-π-A) structural system to enhance intramolecular charge transfer effect and improve fluorescence emission performance; (3) Pyridine hydrazide recognition unit, used as a coordination recognition site for copper ions, causes changes in molecular electronic structure by forming a complex with copper ions.

[0010] In the above structure, the coumarin core and the diphenylamine electron-donating unit are connected by a conjugated structure, forming a fluorescent system with obvious intramolecular charge transfer characteristics. The nitrogen atom in the pyridylhydrazine structure can coordinate with copper ions to form a stable probe-copper ion complex.

[0011] The coumarin-based fluorescent probe described in this invention can be used for the following purposes: (1) Sequential detection of copper ions and biothiols; (2) Visual detection of copper ions without the aid of the naked eye; (3) Portable detection based on smartphone RGB analysis; (4) Detection of copper ions in environmental water samples; (5) Fluorescence imaging of copper ions and biothiols in living cells; (6) Fluorescence imaging of copper ions and biothiols in zebrafish.

[0012] In the presence of copper ions, the fluorescence signal of the fluorescent probe is significantly reduced, accompanied by a change in solution color; in the presence of biothiols, the probe fluorescence is restored due to the competitive coordination of biothiols with copper ions, thereby achieving sequential detection.

[0013] Probe preparation method This invention also provides a method for preparing a coumarin-based fluorescent probe XDP, which includes the following steps: Step 1: Preparation of intermediate compound 3 Under nitrogen protection, 6-bromo-2,3-dihydro-1H-xanthocyanin-4-carboxaldehyde, 4,4-dimethoxydiphenylamine, palladium catalyst, ligand, and base were added to an organic solvent, and a coupling reaction was carried out under heating. After the reaction was completed, the mixture was cooled and filtered, and then purified by extraction, drying, and column chromatography to obtain intermediate compound 3.

[0014] Step 2: Preparation of the fluorescent probe XDP Intermediate compound 3 was condensed with 2-pyridinecarboxylhydrazine in an alcohol solvent under acidic catalytic conditions. After the reaction was completed, the solvent was removed, and the target fluorescent probe XDP was obtained by extraction and column chromatography purification.

[0015] The coumarin-based fluorescent probe XDP with a D–π–A conjugated structure can be obtained through the above steps.

[0016] 2. Detection methods for copper ions and biothiols The present invention also provides a method for detecting copper ions and biothiols using the above-mentioned fluorescent probe, which includes the following steps: Step 1: Prepare probe solution The fluorescent probe XDP is dissolved in a mixture of organic solvent and buffer solution to form a working solution of a certain concentration.

[0017] Step 2: Copper ion detection Add the copper ion solution to be tested to the probe working solution and mix thoroughly. Record the fluorescence change or observe the solution color change under excitation light. When copper ions are present in the system, the probe coordinates with the copper ions, resulting in a significant decrease in fluorescence signal and a change in solution color, thereby achieving the detection of copper ions.

[0018] Step 3: Biothiol Detection The biothiol solution to be tested is added to the probe-copper ion complex system formed above. Due to the strong coordination between biothiols and copper ions, biothiols can competitively bind with copper ions, thereby destroying the probe-copper ion complex, restoring the probe to a free state and generating a fluorescent signal, thus realizing the detection of biothiols.

[0019] Furthermore, the biothiols include: glutathione (GSH), cysteine ​​(Cys), and homocysteine ​​(Hcy).

[0020] Furthermore, during the detection process, the solution image can be captured and RGB color parameters extracted to achieve smartphone-based assisted detection, thereby enabling digital analysis of the detection results.

[0021] The above method can be used to achieve the sequential detection of copper ions and biothiols.

[0022] Compared with the prior art, the present invention has the following advantages: 1. Achieve sequential recognition of copper ions and biothiols.

[0023] The coumarin-based fluorescent probe provided by this invention can respond sequentially to copper ions and biothiols in the same molecular system. It achieves a "turn-on-off-turn-on" fluorescence change mode through a coordination-competition mechanism, thereby enabling the sequential detection of two types of analytes in a single probe system. This overcomes the limitation of most fluorescent probes in the prior art that can only detect a single target analyte.

[0024] 2. It has a large Stokes shift, which improves the resolution of the detection signal.

[0025] The fluorescent probe described in this invention has a large Stokes shift, which can effectively reduce the spectral overlap between excitation and emission light, thereby reducing background interference, improving the resolution and reliability of the detection signal, and making it more suitable for detection in complex environmental systems and biological systems.

[0026] 3. It has both fluorescence detection and naked-eye visualization detection functions.

[0027] The probe described in this invention not only produces a significant fluorescence change in the presence of copper ions, but also a change in solution color. Therefore, it can be directly identified by the naked eye under natural light conditions, thereby achieving rapid detection without the need for complex instruments.

[0028] 4. It can achieve smartphone-assisted detection.

[0029] This invention uses a smartphone to capture solution images and extract RGB color parameters, enabling digital analysis of color changes and achieving a portable detection method, thereby improving the practicality and application scope of the detection method.

[0030] 5. It has good selectivity and sensitivity.

[0031] The probe described in this invention exhibits significant responses to both copper ions and biothiols, while showing minimal interference from common metal ions and other amino acids, demonstrating good selectivity and high detection sensitivity.

[0032] 6. Applicable to complex environments and biological systems.

[0033] The probe described in this invention can be used not only for detection in buffer solution systems, but also for detection in environmental water samples and for fluorescence imaging in live cells and zebrafish, indicating that the probe can maintain a stable response in complex systems and has good application prospects.

[0034] In summary, the fluorescent probe provided by this invention is superior to existing technologies in terms of sequence recognition capability, detection mode, visual detection, and practical application, and has good scientific research value and application potential. Attached Figure Description

[0035] Figure 1 This is a flowchart illustrating the preparation of coumarin-based fluorescent probes.

[0036] Figure 2 Figure A in the figure is the absorbance curve of the probe XDP; Figure 2 Figure B in the figure shows the fluorescence intensity curve of the probe XDP.

[0037] Figure 3 Figure A in the figure shows the relationship between fluorescence intensity and pH value when the probe XDP detects copper ions.

[0038] Figure 3 Figure B in the figure shows the relationship between fluorescence intensity and pH value when the probe XDP detects biothiols.

[0039] Figure 4 Figure A in the figure shows the curve of the reaction between the probe XDP and copper ions over time.

[0040] Figure 4 Figure B in the image shows the XDP-Cu probe. 2+ A graph showing the reaction with biothiols over time.

[0041] Figure 5 Figure A in the figure shows the curve of the reaction between the probe XDP and copper ions as a function of temperature.

[0042] Figure 5 Figure B in the image shows the XDP-Cu probe. 2+ A graph showing the reaction of the product with biothiols as a function of temperature.

[0043] Figure 6 Figure A in the figure shows the curve of the fluorescence intensity of the probe XDP as a function of copper ion concentration.

[0044] Figure 6 Figure B in the figure is a linear graph of the fluorescence intensity of the probe XDP in the range of copper ion concentration from 1 to 80 μM.

[0045] Figure 6 Figure C in the graph shows the curve of the fluorescence intensity of the probe XDP as a function of GSH concentration.

[0046] Figure 6 Figure D in the graph is a linear graph of the fluorescence intensity of the probe XDP within the GSH concentration range.

[0047] Figure 6 Figure E in the figure is a curve showing the change in fluorescence intensity of the probe XDP as a function of Cys concentration.

[0048] Figure 6 The F-plot in the figure is a linear graph of the XDP probe fluorescence intensity within the Cys concentration range.

[0049] Figure 6 The G figure in the figure is a curve showing the change in fluorescence intensity of the probe XDP with Hcy concentration.

[0050] Figure 6 The H-plot in the figure is a linear graph of the XDP probe fluorescence intensity within the Hcy concentration range.

[0051] Figure 7 The graph shows the variation of parameter G / (R+B) with Cu²⁺ concentration.

[0052] Figure 8 Figure A in the diagram shows the fluorescence intensity of the probe XDP in response to different metal ions.

[0053] Figure 8 Figure B in the image shows the XDP-Cu probe. 2+ Fluorescence intensity diagrams for different amino acids.

[0054] Figure 9 Fluorescence micrographs of different analytes added.

[0055] Figure 10 Fluorescence micrographs of different analytes in zebrafish. Detailed Implementation

[0056] The following are specific embodiments of the present invention, which are described in conjunction with the accompanying drawings. However, the present invention is not limited to these embodiments. Example

[0057]

[0058] Based on such Figure 1The prepared route, as shown, involved adding 6-bromo-2,3-dihydro-1H-caryophyllene-4-carboxaldehyde (250.0 mg, 0.86 mmol), 4,4-dimethoxydiphenylamine (197.2 mg), Pd2(dba)3 (11.8 mg), DavePhos (5.0 mg), and cesium carbonate (700.5 mg) to 30 mL of 1,4-dioxane under nitrogen protection. The reaction was stirred at 95 °C for 18 h. After the reaction was complete, the mixture was cooled to room temperature and filtered to remove solid residue. The filtrate was washed with water and extracted with ethyl acetate. The combined organic phases were dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (PE / EA = 10:1) to give an orange solid, intermediate compound 3. 1 H NMR (600 MHz, CDCl3) δ 10.18 (s, 1H), 7.11–7.07 (m, 4H), 6.92 (d, J = 8.4 Hz, 1H), 6.89–6.85 (m, 4H), 6.60 (s, 1H), 6.59–6.54 (m, 2H), 3.81 (s, 6H), 2.54 (t, J = 5.5 Hz, 2H), 2.42 (t, J = 6.0Hz, 2H), 1.70 (q, J = 6.1 Hz, 2H). 13 C NMR (151 MHz, CDCl3) δ 187.43, 161.10,156.75, 153.22, 150.75, 139.52, 127.45, 127.12, 126.94, 125.52, 114.92,114.55, 113.78, 112.07, 104.71, 55.47, 29.94, 21.53, 20.53. Example

[0059]

[0060] Based on such Figure 1 The prepared route shown involves dissolving intermediate compound 3 (100.0 mg, 0.23 mmol) and 2-pyridinecarboxylhydrazide (31.5 mg) in anhydrous ethanol under nitrogen protection, with one drop of glacial acetic acid added as a catalyst. The reaction is carried out under reflux for 4 h. After the reaction is complete, the solvent is removed, the mixture is washed with water and extracted with ethyl acetate. The organic phase is dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product is purified by column chromatography to obtain a deep orange solid, namely the fluorescent probe XDP. 1H NMR (600 MHz, CDCl3) δ 10.77 (s, 1H), 8.57 (dt, J = 4.6, 1.3 Hz, 1H), 8.47(s, 1H), 8.29 (dt, J = 7.9, 1.1 Hz, 1H), 7.87 (td, J = 7.7, 1.7 Hz, 1H), 7.45(ddd, J = 7.6, 4.7, 1.2 Hz, 1H), 7.09 (d, J = 8.9 Hz, 4H), 6.88 (d, J = 8.9Hz, 4H), 6.81 (d, J = 8.3 Hz, 1H), 6.53 (d, J = 2.2 Hz, 1H), 6.49 (dd, J =8.3, 2.3 Hz, 1H), 6.29 (s, 1H), 3.82 (s, 6H), 2.70 (t, J = 6.0 Hz, 2H), 2.50(t, J = 6.1 Hz, 2H), 1.76–1.71 (m, 1H). 13 C NMR (151 MHz, CDCl3) δ 159.33,156.34, 153.49, 152.47, 149.82, 149.62, 147.92, 146.07, 140.08, 137.48,127.19, 126.98, 126.33, 126.28, 122.73, 122.50, 114.80, 114.64, 114.37,108.11, 105.36, 55.46, 29.98, 23.63, 20.77. Example

[0061] S1. Prepare probe stock solution: Accurately weigh 0.00279 g of the near-infrared fluorescent probe (XDP) prepared in Example 1, dissolve it in 5 mL of DMSO (analytical grade), mix thoroughly until completely dissolved, to obtain a concentration of 1×10⁻⁶. -3 The probe stock solution of M is stored in a 4°C refrigerator for later use.

[0062] S2. Detection of copper ions: Add appropriate amounts of probe stock solution and copper ion stock solution to a 5 mL EP tube, and bring the volume to 3 mL with Tris-HCl buffer (pH = 7.4) to achieve a final concentration of 10 μM for both XDP and Cu²⁺. A control experiment was also set up: an equal volume of Tris-HCl buffer (pH = 7.4) was used to replace the copper ion stock solution, maintaining the XDP concentration at 10 μM and the copper ion concentration at 0 μM. After thoroughly mixing the solutions, incubate them in a 37 ℃ water bath for 2 min. Then transfer the solutions to a 3 cm quartz cuvette, and measure the absorption spectrum using a UV-Vis spectrophotometer and the fluorescence spectrum using a fluorescence spectrometer with an excitation wavelength of 470 nm.

[0063] S3. Detection of biothiols: Add probe stock solution and copper ion stock solution to a 5 mL EP tube, and bring the volume to 2910 μL with Tris-HCl buffer (pH = 7.4). Then add 90 μL of biothiols stock solution to achieve a final concentration of 10 μM for the XDP–Cu²⁺ complex and 30 μM for the biothiols. Incubate the resulting solution in a 37 ℃ water bath for 15 min, then transfer it to a 3 cm quartz cuvette. Measure the absorption spectrum using a UV-Vis spectrophotometer and the fluorescence spectrum using a fluorescence spectrometer with an excitation wavelength of 470 nm.

[0064] like Figure 2 As shown in Figure A, the probe XDP exhibits a distinct absorption peak at 605 nm. This absorption peak significantly weakens upon the addition of copper ions; however, the absorption peak strengthens again upon further addition of biothiols (GSH, Hcy, or Cys) to the mixed solution of XDP and copper ions. A fluorescence emission spectrum is plotted with wavelength on the x-axis and fluorescence intensity on the y-axis, as shown in Figure A. Figure 2 As shown in Figure B, the XDP probe exhibits a strong fluorescence emission peak at 605 nm. Upon the addition of copper ions, the fluorescence intensity at 605 nm significantly decreases under 470 nm excitation due to the coordination interaction between XDP and Cu²⁺. When biothiols (GSH, Hcy, or Cys) are further added to the XDP–Cu²⁺ system, the XDP–Cu²⁺ complex dissociates due to the competitive coordination between the biothiols and Cu²⁺, and the probe fluorescence signal is recovered. The results indicate that the fluorescent probe XDP described in this invention can achieve sequential detection of copper ions and biothiols.

[0065] Experiment Example 4 The preparation of the probe stock solution, copper ion mother liquor, and probe copper ion system solution in this embodiment is the same as in Example 3.

[0066] Add probe stock solution and copper ion stock solution to Tris-HCl buffer (pH 2-12), and bring the volume to 3 mL to achieve a final concentration of 10 μM for both XDP and copper ions. Mix thoroughly. Use a control without added copper ions. Incubate at 37°C for 2 min, then transfer to a 3 cm quartz cuvette. Measure the fluorescence spectrum at 605 nm using a fluorescence spectrometer with an excitation wavelength of 470 nm. The control is denoted as F0. Plot pH on the x-axis and fluorescence intensity (F0-F) on the y-axis.

[0067] The results are as follows Figure 3 As shown in a, it can effectively respond to copper ions in the pH range of 6 to 7.4, and exhibits the strongest fluorescence signal at pH 7.4.

[0068] Add the probe copper ion system solution and biothiol stock solution to Tris-HCl buffer solution with pH = 2-12, respectively, and adjust the volume to 3 mL to make the probe XDP-Cu 2+ The final concentration of the active ingredient was 10 μM, and the final concentration of the biothiol was 30 μM. The mixture was thoroughly mixed. A control without added biothiol was used. The mixture was incubated in a 37℃ water bath for 15 min, then added to a 3 cm quartz cuvette. The fluorescence spectrum at 605 nm was measured using a fluorescence spectrometer with an excitation wavelength of 470 nm. The control was denoted as F0. The plot was calculated with pH on the x-axis and fluorescence intensity (F-F0) on the y-axis.

[0069] The results are as follows Figure 3 As shown in b, it can effectively respond to biothiols in the pH range of 6 to 7.4, and exhibits the strongest fluorescence signal at pH 7.4. Example

[0070] Effect of pH on the detection system: In this example, the preparation methods of the probe stock solution, copper ion stock solution, and probe-copper ion system solution are the same as in Example 3. The probe stock solution and copper ion stock solution were added to a Tris-HCl buffer solution with pH = 2–12, and the volume was adjusted to 3 mL to achieve a final XDP concentration of 10 μM and a Cu²⁺ concentration of 10 μM. A system without added copper ions was used as a control. After incubation at 37 °C for 2 min, the fluorescence intensity at 605 nm (λ_ex = 470 nm) was measured. The control fluorescence intensity was represented by F0, and a curve showing the relationship between (F−F0) and pH was plotted.

[0071] The results are as follows Figure 3As shown in Figure A, the system exhibited a good response to Cu²⁺ within the pH range of 6–7.4, with the most significant response at pH 7.4. Subsequently, the probe-copper ion system solution and biothiol stock solution were added to Tris-HCl buffer at pH = 2–12 to achieve a final concentration of 10 μM for XDP-Cu²⁺ and 30 μM for biothiol. Fluorescence intensity was measured after incubation at 37 °C for 15 min. The results are as follows. Figure 3 As shown in B, the system can effectively respond to biothiols in the pH range of 6 to 7.4, with the best response at pH 7.4. Example

[0072] Effect of reaction time on the detection system: The probe stock solution and copper ion stock solution were added to Tris-HCl buffer (pH=7.4) and the volume was adjusted to 3 mL to make the final concentration of XDP 10 μM and Cu²⁺ 10 μM. After incubation at 37 °C for 0–15 min, the fluorescence intensity at 605 nm (λ_ex = 470 nm) was measured.

[0073] The results are as follows Figure 4 As shown in Figure A, the fluorescence intensity reached its lowest point and tended to stabilize after approximately 2 minutes of reaction. Therefore, 2 minutes was selected as the optimal reaction time between the probe and copper ions. Subsequently, biothiol was added to the XDP–Cu²⁺ system to achieve a final concentration of 30 μM. After incubation at 37 °C for 0–30 minutes, the fluorescence intensity was measured. The results are as follows: Figure 4 As shown in Figure B, the fluorescence recovery of the system reaches its maximum and remains stable at approximately 15 min. Therefore, 15 min was selected as the optimal reaction time for detecting biothiols. Example

[0074] Effect of temperature on the detection system: The fluorescence intensity was measured after incubating the probe XDP and Cu²⁺ in Tris-HCl buffer (pH=7.4) for 2 min at 15–50 °C.

[0075] The results are as follows Figure 5 As shown in Figure A, the fluorescence quenching effect was most pronounced at 37 °C; therefore, 37 °C was selected as the optimal reaction temperature for detecting Cu²⁺. Subsequently, biothiols were added to the XDP–Cu²⁺ system within the same temperature range and incubated for 15 min before measuring the fluorescence intensity. The results are as follows: Figure 5 As shown in Figure B, the fluorescence recovery was most obvious at 37 °C, therefore 37 °C was selected as the optimal reaction temperature for detecting biothiols. Example

[0076] Probe response to the concentration of copper ions and biothiols Add probe stock solution and copper ion stock solution to a 5 mL EP tube, and bring the volume to 3 mL with Tris-HCl buffer (pH = 7.4) to achieve a final XDP concentration of 10 μM and a Cu²⁺ concentration range of 0–80 μM. After thoroughly mixing, incubate the solution in a 37°C water bath for 2 min, then transfer it to a 3 cm quartz cuvette and measure the fluorescence spectrum using a fluorescence spectrometer (λ_ex = 470 nm). Plot a response curve with Cu²⁺ concentration on the x-axis and fluorescence intensity at 605 nm on the y-axis.

[0077] The results are as follows Figure 6 As shown in Figure A, the fluorescence intensity of the system at 605 nm gradually decreases with increasing Cu²⁺ concentration. Figure 6 B indicates that there is a good linear relationship between fluorescence intensity and Cu²⁺ concentration in the range of 1–80 μM.

[0078] Subsequently, the probe-copper ion system solution and biothiol stock solution were added to a 5 mL EP tube, and the volume was adjusted to 3 mL with Tris-HCl buffer (pH = 7.4) to achieve a final concentration of 10 μM for the XDP-Cu²⁺ system and a biothiol concentration range of 0–30 μM. The solution was incubated in a 37 ℃ water bath for 15 min before fluorescence spectra were measured.

[0079] The results are as follows Figure 6 As shown in Figures C to 6H, the fluorescence intensity of the system at 605 nm gradually increases with the increase of the concentration of biothiols (GSH, Cys, Hcy), and shows a good linear relationship with the concentration of biothiols within a certain concentration range. Figures A, C, E, and G all show multiple curves in the vertical direction, and the concentration difference represented by adjacent curves in the vertical direction is the same. Taking Figure G as an example, the concentration difference between adjacent curves in the vertical direction is 3 μM. There are a total of 11 curves, and the top and bottom curves correspond to the two extreme values ​​of the selected concentration range.

[0080] In addition, photos of sample solutions of different concentrations were taken using a smartphone, and the images were analyzed using a colorimetric application to obtain RGB values. The results are as follows: Figure 7 As shown, the parameter G / (R+B) decreases linearly with increasing Cu²⁺ concentration, while it increases linearly with increasing GSH concentration, indicating that the system can achieve visualized quantitative analysis based on RGB signals. Example

[0081] Selectivity study of probe for Cu²⁺ and biothiols Add probe stock solution, copper ion stock solution, and solutions of different metal ion interfering substances to a 5 mL EP tube, and bring the volume to 3 mL with Tris-HCl buffer (pH = 7.4). Measure the fluorescence intensity at 605 nm (λ_ex = 470 nm) using a fluorescence spectrometer. Use a system without added copper ions as a blank control.

[0082] The results are as follows Figure 8 As shown in Figure A, except for Cu²⁺, other metal ions did not cause significant fluorescence changes. Even under conditions where multiple interfering substances coexisted, the fluorescence response of the probe to Cu²⁺ remained stable, indicating that the XDP probe has good selectivity and anti-interference ability for Cu²⁺. Subsequently, the probe-copper ion system solution, biothiol stock solution, and solutions of different amino acids were added to a 5 mL EP tube, and the volume was adjusted to 3 mL with Tris-HCl buffer (pH = 7.4) to make the final concentration of XDP-Cu²⁺ 10 μM, and the concentrations of biothiol and amino acids 30 μM. After incubation in a 37 ℃ constant temperature water bath for 15 min, the fluorescence spectrum was measured. The results are as follows. Figure 8 As shown in Figure B, except for GSH, Cys, and Hcy, the other amino acids did not cause significant fluorescence recovery, indicating that the XDP–Cu²⁺ system has good selectivity for biothiols. Example

[0083] Fluorescence imaging of probes in live cells HeLa cells were fed at a concentration of 1.5 × 10⁻⁶. 5 Cells were seeded at 15 mm confocal culture dishes and cultured at 37 ℃ and 5% CO2 for 24 h. Cells were divided into five groups: group a (XDP + NEM), group b (XDP + NEM + Cu²⁺), group c (XDP–Cu²⁺ + NEM), group d (XDP–Cu²⁺), and group e (XDP–Cu²⁺ + NEM + GSH). In group a, cells were first treated with 200 μM NEM for 30 min, then the NEM was carefully washed off the cell surface with buffer, followed by incubation with 10 μM XDP for 1 h. In group b, cells were treated with NEM and then incubated with XDP for 1 h, followed by incubation with 30 μM Cu²⁺. In group c, cells were treated with NEM and then incubated with the XDP–Cu²⁺ system for 1 h. In group d, cells were directly incubated with the XDP–Cu²⁺ system for 1 h. In group e, cells were treated with NEM and then incubated with the XDP–Cu²⁺ system for 1 h, followed by incubation with 50 μM GSH. After treatment, cells were washed with PBS and subjected to confocal fluorescence imaging. The excitation wavelength was 470 nm, and the emission wavelength was collected in the range of 600–700 nm. Results are as follows: Figure 9As shown, compared with group a, the fluorescence of cells in groups b and c with the addition of Cu²⁺ was significantly quenched; while in group e with the addition of GSH, the fluorescence signal was significantly restored, indicating that the probe can realize the sequential detection of Cu²⁺ and biothiols in living cells. Example

[0084] Fluorescence imaging of the probe in zebrafish Zebrafish fertilized eggs were placed in culture medium and incubated at 28 °C until three days of age. The zebrafish were then randomly divided into five groups: group a (XDP + NEM), group b (XDP + NEM + Cu²⁺), group c (XDP–Cu²⁺ + NEM), group d (XDP–Cu²⁺), and group e (XDP–Cu²⁺ + NEM + GSH). Each group was incubated using a treatment similar to that used in cell experiments, with final NEM concentrations of 50 μM, XDP concentrations of 10 μM, Cu²⁺ concentrations of 30 μM, and GSH concentrations of 50 μM. After incubation, the eggs were washed with culture medium and fixed with 4% paraformaldehyde.

[0085] Fluorescence imaging was then performed using confocal microscopy, with an excitation wavelength of 470 nm and an emission wavelength collection range of 600–700 nm. The results are as follows: Figure 10 As shown, a distinct "fluorescence quenching-fluorescence recovery" process was also observed in zebrafish, indicating that the probe can achieve sequential detection of Cu²⁺ and biothiols at the in vivo level.

[0086] The specific embodiments described herein are merely illustrative of the spirit of the invention. Those skilled in the art to which this invention pertains may make various modifications or additions to the described specific embodiments or use similar methods to substitute them, without departing from the spirit of the invention or exceeding the scope defined by the appended claims.

Claims

1. A coumarin-based fluorescent probe for sequential detection, characterized in that, The probe is constructed by introducing a diphenylamine electron-donating group and a pyridylhydrazine coordination recognition unit into the coumarin fluorescent backbone to create a molecular system with a D–π–A conjugated structure. This allows the probe to coordinate with copper ions and achieve fluorescence recovery through competitive coordination in the presence of biothiols, thus forming a sequential fluorescence response mode of "on-off-on-off".

2. A method for preparing the coumarin-based fluorescent probe as described in claim 1, characterized in that, Includes the following steps: Preparation of fluorescent probe XDP Intermediate compound 3 and 2-pyridinecarboxylhydrazine were dissolved in an alcohol solvent and condensed under acidic catalysis. After the reaction was completed, the solvent was removed, and the target fluorescent probe XDP was obtained by extraction and column chromatography purification.

3. An application of a coumarin-based fluorescent probe, characterized in that, The coumarin-based fluorescent probe described in claim 1 or 2 is used for the detection of copper ions, the detection of biothiols, and the sequential detection of copper ions and biothiols. Specifically, the fluorescent probe XDP is dissolved in a mixture of an organic solvent and a buffer solution to form a working probe solution of a certain concentration. A copper ion solution to be tested is added to the working probe solution and thoroughly mixed. Fluorescence changes are recorded or the color changes of the solution are observed under excitation light, thereby achieving the detection of copper ions. A biothiols solution to be tested is added to the copper ion complex system formed above. Due to the strong coordination between biothiols and copper ions, biothiols can competitively bind with copper ions, thereby destroying the probe-copper ion complex, restoring the probe to a free state, and generating a fluorescent signal, thus achieving the detection of biothiols. The sequential detection of copper ions and biothiols is achieved.

4. The application of the coumarin-based fluorescent probe according to claim 3, characterized in that, The biothiols include: glutathione (GSH), cysteine ​​(Cys), and homocysteine ​​(Hcy).

5. The application of the coumarin-based fluorescent probe according to claim 3, characterized in that, The fluorescent probe XDP is used for portable detection using smartphone RGB analysis as a tool.

6. The application of the coumarin-based fluorescent probe according to claim 3, characterized in that, The fluorescent probe XDP is used for the detection of copper ions in environmental water samples.

7. The application of the coumarin-based fluorescent probe according to claim 3, characterized in that, The fluorescent probe XDP is used for fluorescence imaging of copper ions and biothiols in live cells.

8. The application of the coumarin-based fluorescent probe according to claim 3, characterized in that, The fluorescent probe XDP was used for fluorescence imaging of copper ions and biothiols in zebrafish.

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