A copper nano fluorescent probe and a preparation method and application thereof

Abstract

The application provides a high-performance copper nano fluorescent probe, a rapid preparation method and application thereof, and belongs to the technical field of fluorescent probe preparation. The application provides a preparation method of a copper nano fluorescent probe, which comprises the following steps: mixing an aqueous solution of a divalent copper salt, an aqueous solution of an organic ligand, polyethylene glycol, water and an aqueous solution of a divalent calcium salt, stirring the obtained mixed solution at room temperature for 1-20 min, and obtaining the copper nano fluorescent probe by synergistically using reduction, coordination and hydrogen bonding; the organic ligand contains a mercapto group and a carboxyl group. The copper nano fluorescent probe provided by the application has the advantages of high fluorescence intensity, good stability and good batch repeatability; the copper nano fluorescent probe provided by the application can be used as a "mix-and-use" type fluorescent detection reagent and is used for fluorescence detection of uric acid and xanthine.

Description

Technical Field

[0001] This invention belongs to the field of fluorescent probe preparation technology, specifically relating to a copper nanoparticle fluorescent probe, its preparation method, and its application. Background Technology

[0002] Uric acid and xanthine are products of purine oxidation metabolism in the human body. When uric acid levels are too high, uric acid crystals may deposit in joints and other areas, leading to diseases such as gout. Xanthine can be metabolized into uric acid in the body and is an important indicator for assessing uric acid metabolism. Simultaneous detection of uric acid and xanthine can provide a more comprehensive reflection of the body's purine metabolism, offering more accurate information for the diagnosis and treatment of diseases such as gout, acute and chronic nephritis, and renal tuberculosis. Currently, the detection of uric acid and xanthine mainly relies on electrochemical methods, high-performance liquid chromatography, and mass spectrometry. Research on fluorescence analysis methods that can be applied simultaneously to the detection of uric acid and xanthine is limited.

[0003] Copper nanoparticle fluorescent probes possess advantages such as low preparation cost and good biocompatibility, making them a promising candidate to replace organic dyes, semiconductor quantum dots, and noble metal-based nanoparticles in fluorescence sensing and imaging. However, due to the highly reactive nature of copper, copper nanomaterials are prone to oxidative degradation during preparation and storage, resulting in problems such as low quantum yield, poor luminescence intensity, large batch-to-batch variability, and short shelf life. Currently, effective strategies for improving the luminescence performance and stability of copper nanoparticle fluorescent probes mainly focus on precise ligand design, matrix confinement, aggregation-induced emission, and optimization of preparation conditions (high temperature, inert gas protection, precise pH control, etc.). However, challenges remain, including high ligand synthesis costs, long purification times, high energy consumption, and the need to use toxic and harmful solvents and reagents. Summary of the Invention

[0004] In view of this, the purpose of this invention is to provide a copper nanoparticle fluorescent probe, its preparation method, and its application. The preparation method provided by this invention is green and rapid, requiring no toxic reagents, no stringent reaction conditions, and no separation or purification steps. The prepared copper fluorescent probe has the advantages of high fluorescence intensity, good stability, and good batch reproducibility, and can be used as a "ready-to-use" fluorescent detection reagent for clinical detection and home self-testing of disease markers such as uric acid and xanthine.

[0005] To achieve the above objectives, the present invention provides the following technical solution:

[0006] This invention provides a method for preparing copper nanofluorescent probes, comprising the following steps:

[0007] An aqueous solution of a divalent copper salt, an aqueous solution of an organic ligand, polyethylene glycol, water, and an aqueous solution of a divalent calcium salt are mixed. The resulting mixture is stirred at room temperature for 1–20 min to induce coordination and reduction reactions, thereby obtaining the copper nanofluorescent probe. The organic ligand contains thiol and carboxyl groups.

[0008] Preferably, the mixture includes:

[0009] Water and polyethylene glycol are mixed to obtain a water-polyethylene glycol mixed solvent;

[0010] An aqueous solution of a divalent copper salt and an aqueous solution of an organic ligand are mixed and then added to the water-polyethylene glycol mixed solvent. An aqueous solution of a divalent calcium salt is then added to the resulting mixed solution.

[0011] Preferably, the divalent copper salt includes one or more of copper chloride, copper nitrate, and copper sulfate; the organic ligand is one or more of a thiol-containing amino acid, a thiol-containing polypeptide, and acetylcysteine.

[0012] Preferably, the molar ratio of the divalent copper salt to the organic ligand is 1:(1-10).

[0013] Preferably, the mass ratio of polyethylene glycol to water in the resulting mixture is 9:2.

[0014] Preferably, the divalent calcium salt includes one or more of calcium chloride, calcium nitrate, and calcium sulfate.

[0015] Preferably, the molar concentration of divalent calcium salt in the obtained mixture is 0.05–10.0 mmol / L.

[0016] Preferably, the molar ratio of the divalent copper salt to the divalent calcium salt is (1-5):(1-200).

[0017] This invention provides a copper nanoparticle fluorescent probe prepared by the above-described method, comprising zero-valent copper and monovalent copper, an organic ligand, and Ca. 2+ Ions and water-polyethylene glycol medium; the zero-valent copper and monovalent copper are connected to the organic ligands via Cu-S coordination bonds, and the Ca 2+ Ions are linked to the organic ligands through electrostatic and coordination interactions.

[0018] This invention provides the application of the copper nanofluorescent probe described above in the detection of uric acid or xanthine for non-disease diagnosis and treatment purposes.

[0019] This invention provides a method for preparing copper nanoparticle fluorescent probes, comprising the following steps: mixing an aqueous solution of a divalent copper salt, an aqueous solution of an organic ligand, polyethylene glycol, water, and an aqueous solution of a divalent calcium salt; stirring the resulting mixture at room temperature for 1–20 min to induce coordination and reduction reactions, thereby obtaining the copper nanoparticle fluorescent probes. The preparation method provided by this invention is green and convenient. Copper nanoparticle fluorescent probes can be prepared at room temperature through simple reagent mixing in just 1–20 min, without the need for toxic reagents, heating, inert gas protection, magnetic stirring, pH adjustment, or separation and purification steps.

[0020] In this invention, a water-polyethylene glycol (PEG) mixture is used instead of pure water as the solvent. PEG is non-toxic, inexpensive, and readily biodegradable, making it a high-quality green solvent. Furthermore, PEG can significantly reduce the dissolved oxygen content in the solvent, thereby effectively inhibiting the oxidative deterioration of copper nanoparticle fluorescent probes during preparation and storage. Simultaneously, PEG can also react with Ca... 2+ Synergistically, through solvent hydrogen bonding, electrostatic interactions, and coordination, a confined fluorescence effect is generated, significantly increasing the radiative transition rate of the copper nanoprobe while decreasing its non-radiative transition rate, thereby effectively improving the fluorescence quantum yield and luminescence intensity of the copper nanoprobe. Polyethylene glycol can also enhance the dispersion stability of the copper nanoprobe through hydrogen bonding and van der Waals interactions, effectively preventing probe aggregation and precipitation. Therefore, the copper nanoprobe provided by this invention has the advantages of rapid preparation, environmental friendliness, good batch repeatability, high fluorescence intensity, and good stability; the copper nanoprobe provided by this invention can be used as a "ready-to-use" fluorescent detection reagent for the fluorescence detection of uric acid and xanthine. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 High-resolution transmission electron microscope image of the copper nanofluorescent probe prepared in Example 1;

[0023] Figure 2 XPS spectrum of the copper nanofluorescent probe prepared in Example 1;

[0024] Figure 3 The fluorescence emission spectrum of the copper nanoparticle fluorescent probe prepared in Example 1;

[0025] Figure 4The graph shows the fluorescence emission intensity of the copper nanofluorescent probe prepared in Example 1 as a function of pH.

[0026] Figure 5 The graph shows the change in fluorescence emission intensity of the copper nanofluorescent probe prepared in Example 1 as a function of ion concentration.

[0027] Figure 6 The graph shows the fluorescence emission intensity of the copper nanofluorescent probe prepared in Example 1 as a function of temperature.

[0028] Figure 7 The graph shows the change in fluorescence emission intensity over time for the copper nanofluorescent probe prepared in Example 1.

[0029] Figure 8 The figure shows the batch repeatability test results of the copper nanofluorescent probe prepared in Example 1;

[0030] Figure 9 The fluorescence emission spectrum of the copper nanoparticle fluorescent probe prepared in Example 2;

[0031] Figure 10 The fluorescence emission spectrum of the copper nanoparticle fluorescent probe prepared in Example 3;

[0032] Figure 11 A comparison of the fluorescence emission spectra of the copper nanofluorescent probes prepared in Comparative Example 1 and Example 1;

[0033] Figure 12 A comparison graph showing the change in fluorescence emission intensity over time for the copper nanofluorescent probes prepared in Comparative Example 1 and Example 1.

[0034] Figure 13 The fluorescence emission spectra of the copper nanoparticle fluorescent probe prepared in Example 1 for detecting uric acid at different concentrations are shown.

[0035] Figure 14 The fluorescence emission spectra of the copper nanoparticle fluorescent probe prepared in Example 1 for detecting xanthine at different concentrations are shown. Detailed Implementation

[0036] This invention provides a method for preparing copper nanofluorescent probes, comprising the following steps:

[0037] An aqueous solution of a divalent copper salt, an aqueous solution of an organic ligand, polyethylene glycol, water, and an aqueous solution of a divalent calcium salt are mixed. The resulting mixture is stirred at room temperature for 1–20 min to induce coordination and reduction reactions, thereby obtaining the copper nanofluorescent probe. The organic ligand contains thiol and carboxyl groups.

[0038] Unless otherwise specified, all materials and equipment used in this invention are commercially available.

[0039] In this invention, the mixing preferably includes the following steps:

[0040] Water and polyethylene glycol are mixed to obtain a water-polyethylene glycol mixed solvent;

[0041] An aqueous solution of a divalent copper salt and an aqueous solution of an organic ligand are mixed and then added to the water-polyethylene glycol mixed solvent. An aqueous solution of a divalent calcium salt is then added to the resulting mixed solution.

[0042] This invention mixes water and polyethylene glycol to obtain a water-polyethylene glycol mixed solvent.

[0043] In this invention, the polyethylene glycol preferably includes polyethylene glycol 200 and / or polyethylene glycol 400; the water is preferably deionized water; the mass ratio of water to polyethylene glycol is preferably (1-4):(6-9), and more preferably (1-3):(7-9).

[0044] After obtaining the water-polyethylene glycol mixed solvent, the present invention mixes the aqueous solution of divalent copper salt and the aqueous solution of organic ligand, and then adds them to the water-polyethylene glycol mixed solvent. Then, the aqueous solution of divalent calcium salt is added to the resulting mixed solution. The resulting mixture (i.e. the final mixed system) is stirred at room temperature for 1 to 20 minutes to allow coordination and reduction reactions to occur, thereby obtaining the copper nanofluorescent probe.

[0045] In this invention, the divalent copper salt preferably includes one or more of copper chloride, copper nitrate, and copper sulfate; the organic ligand is preferably one or more of a thiol-containing amino acid, a thiol-containing polypeptide, and acetylcysteine; the thiol-containing polypeptide preferably includes glutathione; the thiol-containing amino acid preferably includes cysteine; the molar ratio of the divalent copper salt to the organic ligand is preferably 1:(1-10), more preferably 1:(1-5), and even more preferably 1:5.

[0046] In this invention, the divalent calcium salt preferably includes one or more of calcium chloride, calcium nitrate, and calcium sulfate. In this invention, the molar ratio of the divalent copper salt to the divalent calcium salt is preferably (1-5):(1-200), more preferably (1-5):(1-150), and even more preferably 1:5.

[0047] In this invention, the molar concentration of divalent calcium salt in the obtained mixture (i.e., the final mixture system) is preferably 0.05 to 10.0 mmol / L, more preferably 1 to 5 mmol / L, and even more preferably 2 mmol / L; the mass ratio of polyethylene glycol to water in the obtained mixture (i.e., the final mixture system) is preferably 9:2.

[0048] In this invention, the resulting mixture is stirred at room temperature for 1 to 20 minutes, preferably 5 to 15 minutes, and more preferably 10 minutes.

[0049] During the stirring process at room temperature, the thiol group in the organic ligand undergoes a coordination reaction with divalent copper, while the organic ligand reduces the divalent copper salt to zero-valent copper and monovalent copper. At the same time, the divalent calcium salt assembles with the negatively charged carboxyl group on the surface of the organic ligand through electrostatic effects.

[0050] The preparation method provided by this invention is green and convenient. Copper nano fluorescent probes can be prepared in just 1 to 20 minutes at room temperature by simply mixing reagents. It does not require the use of toxic solvents and reagents, heating, inert gas protection, magnetic stirring device, pH adjustment of solution, or separation and purification steps.

[0051] This invention provides a copper nanoparticle fluorescent probe prepared by the method described above, comprising zero-valent copper and monovalent copper, an organic ligand, calcium ions, and a water-polyethylene glycol medium; the zero-valent copper and monovalent copper are connected to the organic ligand via Cu-S coordination bonds, and the Ca... 2+ Ions connect to the organic ligands through electrostatic and coordination interactions.

[0052] In this invention, a water-polyethylene glycol (PEG) mixture is used instead of pure water as the solvent. PEG is non-toxic, inexpensive, and readily biodegradable, making it a high-quality green solvent. Furthermore, PEG can significantly reduce the dissolved oxygen content in the solvent, thereby effectively inhibiting the oxidative deterioration of copper nanoparticle fluorescent probes during preparation and storage. Simultaneously, PEG can also react with Ca... 2+ Synergistically, through solvent hydrogen bonding, electrostatic interactions, and coordination, a confined fluorescence effect is generated, significantly increasing the radiative transition rate of the copper nanoprobe while decreasing its non-radiative transition rate, thereby effectively improving the fluorescence quantum yield and luminescence intensity of the copper nanoprobe. Polyethylene glycol can also enhance the dispersion stability of the copper nanoprobe through hydrogen bonding and van der Waals interactions, effectively preventing probe aggregation and precipitation. Therefore, the copper nanoprobe provided by this invention has the advantages of good batch repeatability, high fluorescence intensity, and good stability; the copper nanoprobe provided by this invention can be used as a "ready-to-use" fluorescent detection reagent for the fluorescence detection of uric acid and xanthine.

[0053] This invention provides the application of the copper nanofluorescent probe described above in the detection of uric acid or xanthine for non-disease diagnosis and treatment purposes.

[0054] The present invention does not have any special requirements for the detection method; any method well known in the art can be used.

[0055] To further illustrate the present invention, a copper nanofluorescent probe, its preparation method, and its application are described in detail below with reference to the accompanying drawings and embodiments. However, these descriptions should not be construed as limiting the scope of protection of the present invention.

[0056] Example 1

[0057] A method for preparing a copper nanoparticle fluorescent probe, comprising the following steps:

[0058] Polyethylene glycol 200 and deionized water were mixed at a mass ratio of 9:1 to obtain a water-polyethylene glycol 200 mixed solvent.

[0059] 100 μL of 40 mM copper nitrate aqueous solution and 500 μL of 40 mM glutathione aqueous solution were mixed at room temperature and then added to the water-polyethylene glycol 200 mixed solvent; then calcium chloride solution (500 μL, 40 mM) was added and stirred at room temperature for 10 min to obtain the copper nanofluorescent probe.

[0060] High-resolution transmission electron microscopy revealed that the size of the copper nanoprobe is ~7 nm (see...). Figure 1 X-ray photoelectron spectroscopy (XPS) showed that Cu in the copper nanoprobe had a 2p... 3 / 2 and 2p 1 / 2 The orbital binding energies are 931.9 eV and 951.7 eV, respectively. Figure 2 This indicates that divalent copper was reduced to zero-valent / monovalent copper during the synthesis process.

[0061] Fluorescence characterization showed that the copper nanoparticle fluorescent probe emitted red fluorescence under 365 nm UV excitation at a wavelength of 610 nm. The fluorescence emission spectrum is shown in the figure. Figure 3 .

[0062] Fluorescence quantum yield testing showed that the probe prepared in Example 1 had a quantum yield of 17.12%.

[0063] Monitoring different pH values, ion concentrations, temperatures, and storage times (see...) Figure 4 , 5 The fluorescence emission intensity of the copper nanoprobe was measured at (6, 7). The results showed that the fluorescence intensity did not decrease significantly, proving that the probe has good stability.

[0064] Following the method described in Example 1, ten batches of copper nanofluorescent probes were prepared. The fluorescence emission intensity of each batch of probes at 610 nm was measured under 365 nm excitation. Figure 8 As shown, the copper nanofluorescent probe prepared according to the method described in Example 1 has a batch-to-batch relative standard deviation of fluorescence intensity of 1.9%, indicating that the preparation method has excellent batch repeatability.

[0065] Example 2

[0066] A method for preparing a copper fluorescent probe, comprising the following steps:

[0067] Polyethylene glycol 400 and deionized water were mixed at a mass ratio of 8:2 to obtain a water-polyethylene glycol 400 mixed solvent.

[0068] 100 μL of 40 mM copper nitrate aqueous solution and 500 μL of 40 mM glutathione aqueous solution were mixed at room temperature and then added to the water-polyethylene glycol 400 mixed solvent; then calcium chloride solution (500 μL, 50 mM) was added and stirred at room temperature for 10 min to obtain the copper nanofluorescent probe.

[0069] Under 365nm ultraviolet light excitation, the fluorescence emission spectrum is shown below. Figure 9 .

[0070] Fluorescence quantum yield testing showed that the probe prepared in Example 2 had a quantum yield of 16.31%.

[0071] Example 3

[0072] A method for preparing a copper fluorescent probe, comprising the following steps:

[0073] Polyethylene glycol 200 and deionized water were mixed at a mass ratio of 8:2 to obtain a water-polyethylene glycol 200 mixed solvent.

[0074] 100 μL of 40 mM copper chloride aqueous solution and 500 μL of 40 mM glutathione aqueous solution were mixed at room temperature and then added to the water-polyethylene glycol 200 mixed solvent. Subsequently, 500 μL of calcium chloride solution (40 mM) was added, and the mixture was stirred at room temperature for 10 min to obtain the copper nanofluorescent probe.

[0075] Under 365nm ultraviolet light excitation, the fluorescence emission spectrum is shown below. Figure 10 .

[0076] Fluorescence quantum yield testing showed that the probe prepared in Example 2 had a quantum yield of 17.05%.

[0077] Comparative Examples 1-2

[0078] To demonstrate the important role of polyethylene glycol in the preparation of copper nanoprobes, this invention compares the copper nanoprobes prepared in Example 1 with those prepared without the addition of polyethylene glycol.

[0079] 100 μL of 40 mM copper nitrate aqueous solution and 500 μL of 40 mM glutathione aqueous solution were mixed at room temperature, and then deionized water was added. The mixture was stirred at room temperature for 10 min to obtain the copper nanofluorescent probe, denoted as probe 1. The fluorescence emission spectrum of the copper fluorescent probe at an excitation wavelength of 365 nm was monitored.

[0080] 100 μL of 40 mM copper nitrate aqueous solution and 500 μL of 40 mM glutathione aqueous solution were mixed at room temperature and then added to the deionized water. Subsequently, 500 μL of 40 mM calcium chloride solution was added, and the mixture was stirred at room temperature for 10 min to obtain the copper nanofluorescent probe, denoted as probe 2. Probe 2 precipitated during the preparation process, making it impossible to monitor its fluorescence spectrum.

[0081] The copper nanofluorescent probe prepared in Example 1 is designated as probe 3, and the fluorescence emission spectrum of the copper fluorescent probe at an excitation wavelength of 365 nm is monitored.

[0082] like Figure 11 As shown, compared to probe 1, probe 3 has an emission intensity that is nearly 60 times higher and a quantum yield that is increased from 0.02% to 17.12%.

[0083] Furthermore, probe 1, prepared without the addition of polyethylene glycol, exhibited a rapid decrease in fluorescence emission intensity over time, demonstrating poor stability. In the presence of polyethylene glycol, probe 3, prepared according to Example 1, remained stable at 4°C for at least two weeks. A comparison of the fluorescence emission intensity changes of probes 1 and 3 over time is shown below. Figure 12 .

[0084] Performance testing

[0085] Copper nanofluorescent probe for detecting uric acid

[0086] 50 μL of uricase (0.1 U / mL) was mixed with aqueous solutions of uric acid at different concentrations (final concentrations were 0.4 μM, 0.8 μM, 10 μM, 15 μM, 20 μM, 30 μM, 40 μM, 50 μM, 60 μM, 70 μM, 80 μM, 90 μM, 100 μM, 150 μM, 200 μM, 300 μM, and 400 μM, respectively). The mixture was incubated at 37°C for 30 min, then mixed with 1.0 mL of the copper nanoparticle fluorescent probe prepared in Example 1, shaken well, and allowed to stand at room temperature for 15 min. The fluorescence spectrum was then measured at an excitation wavelength of 365 nm. According to... Figure 13 It can be seen that as the concentration of uric acid increases, the fluorescence intensity gradually decreases, thus achieving sensitive detection of uric acid.

[0087] Detection of xanthine by copper nanoparticle fluorescent probe

[0088] 50 μL of xanthine oxidase (10 U / mL) was mixed with xanthine aqueous solutions of different concentrations (final concentrations were 0.5 μM, 5 μM, 10 μM, 15 μM, 20 μM, 30 μM, 40 μM, 50 μM, 60 μM, 80 μM, 100 μM, 140 μM, 180 μM, 200 μM, 300 μM, and 400 μM, respectively). The mixture was incubated at 37°C for 30 min, then mixed with 1.0 mL of the copper nanoparticle fluorescent probe prepared in Example 1, shaken well, and allowed to stand at room temperature for 15 min. The fluorescence spectrum was then measured at an excitation wavelength of 365 nm. According to... Figure 14 It can be seen that as the concentration of xanthine increases, the fluorescence intensity gradually decreases, thus achieving sensitive detection of xanthine.

[0089] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for preparing a copper nanoparticle fluorescent probe, characterized in that, Includes the following steps: An aqueous solution of divalent copper salt, an aqueous solution of organic ligand, polyethylene glycol, water, and an aqueous solution of divalent calcium salt were mixed. The resulting mixture was stirred at room temperature for 1–20 min to induce coordination and reduction reactions, thereby obtaining the copper nanofluorescent probe. The organic ligand was glutathione.

2. The preparation method according to claim 1, characterized in that, The mixture includes: Water and polyethylene glycol are mixed to obtain a water-polyethylene glycol mixed solvent; An aqueous solution of a divalent copper salt and an aqueous solution of an organic ligand are mixed and then added to the water-polyethylene glycol mixed solvent. An aqueous solution of a divalent calcium salt is then added to the resulting mixed solution.

3. The preparation method according to claim 1, characterized in that, The divalent copper salt is one or more of copper chloride, copper nitrate, and copper sulfate.

4. The preparation method according to claim 1, 2 or 3, characterized in that, The molar ratio of the divalent copper salt to the organic ligand is 1:(1-10).

5. The preparation method according to claim 1, characterized in that, The mass ratio of polyethylene glycol to water in the resulting mixture is 9:

2.

6. The preparation method according to claim 1, characterized in that, The divalent calcium salt is one or more of calcium chloride, calcium nitrate, and calcium sulfate.

7. The preparation method according to claim 1 or 6, characterized in that, The molar concentration of divalent calcium salt in the resulting mixture was 0.05–10.0 mmol / L.

8. The preparation method according to claim 1 or 2, characterized in that, The molar ratio of the divalent copper salt to the divalent calcium salt is (1-5):(1-200).

9. The copper nanofluorescent probe prepared by the preparation method according to any one of claims 1 to 8, characterized in that, Including zero-valent copper and monovalent copper, organic ligands and Ca 2+ Ions and water-polyethylene glycol medium; the zero-valent copper and monovalent copper are connected to the organic ligands via Cu-S coordination bonds, and the Ca 2+ Ions are linked to the organic ligands through electrostatic and coordination interactions.

10. The use of the copper nanofluorescent probe of claim 9 in the detection of uric acid or xanthine for non-disease diagnosis and treatment purposes.

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