Glutathione-Encapsulated Fluorescent Copper Nanoclusters GSH-CuNCs and Preparation Method and Application Thereof

Abstract

The present invention discloses a method for preparing stable copper nanoclusters (CuNCs) based on glutathione (GSH) as a ligand. The method includes: first, preparing an aqueous solution of copper chloride, then adding a glutathione solution to the above solution in a certain proportion, fully stirring for five minutes at room temperature to form a white turbid solution, dropping a certain amount of sodium hydroxide solution until the solution becomes clear, then vigorously stirring at room temperature, transferring the solution into a dialysis bag for further dialysis, and then filtering through a microporous membrane. The copper nanoclusters prepared by the present invention have high sensitivity, good selectivity, strong anti-interference ability, a low detection limit for uranyl ions (the detection limit is 6.7 nM), and are used for real-time detection in real water samples including lake water, seawater, and pure water to evaluate the applicability of the copper cluster probe in the environment, having practical application value.

Description

Technical Field

[0001] The present invention relates to a stable fluorescent copper nanocluster prepared based on glutathione as a ligand and its application, belonging to the technical field of fluorescent nanomaterials. Background Art

[0002] In recent years, with the increasing popularity of nuclear technology, actinide uranium and its compounds have become an important class of industrial materials, widely used in civil nuclear fuels, military nuclear industries and other fields. The toxicity of uranium compounds is mainly manifested as radioactive toxicity and heavy metal toxicity, which are mainly harmful to bones, the circulatory and urinary systems and can cause lung cancer. Therefore, uranium compound pollution caused by nuclear leakage and uranium mine tailings will cause serious social and environmental problems, thus becoming an important issue in the fields of environmental and energy sciences. Since uranium mainly exists in the environment in the form of uranyl ions (UO 2+ ), the development of highly sensitive and selective uranyl probes for simply and accurately monitoring trace uranyl in the environment, food and biology has attracted extensive attention in recent years.

[0003] Currently, commonly used methods such as inductively coupled plasma mass spectrometry, surface-enhanced Raman spectroscopy, reflection X-ray fluorescence and ultraviolet-visible spectroscopy, optical emission, cathodic stripping voltammetry, ion chromatography, etc. can detect uranium with high precision. For most of these traditional methods, expensive instruments, skilled operations and cumbersome sample preparations are required. Therefore, there is still a need for simple and convenient methods to detect uranyl ions. Fluorescent probe methods have been widely used by researchers as an effective method for detecting uranyl using fluorescent molecules such as quantum dots, small molecules and conjugated organic polymers. However, the development of highly selective, sensitive, simple and rapid fluorescent probes for detecting uranyl in environmental applications remains a challenge.

[0004] Metal nanoclusters (NCs) have attracted the interest of researchers due to their unique properties. As a fluorescent material, metal nanoclusters have advantages such as simple synthesis and low toxicity compared with quantum dots, organic molecules, etc., and are ideal candidates for bioimaging, catalysis and environmental detection. Among them, compared with gold and silver nanoclusters, copper nanoclusters (CuNCs) have rich sources and price advantages, and their development has received increasing attention. However, the preparation of CuNCs is more challenging due to its sensitivity to oxidation and difficulty in size control. So far, various stabilizers have been used to prepare nanoclusters, including proteins, DNA, peptides, etc. For example, Chinese Patent CN113322060A synthesizes nanoclusters based on glutathione S-transferase for the detection of chlortetracycline. Another example is Chinese Patent CN114563385A based on glutathione-protected gold-copper nanoclusters and Ce 3+Form an assembly for the detection of myricetin. However, these raw materials often contain precious metals, proteases, etc., which are expensive, have relatively cumbersome synthesis steps, and are inconvenient to store. Therefore, it is particularly important to further develop good nano copper clusters. Summary of the Invention

[0005] The purpose of the present invention is to provide a glutathione-encapsulated fluorescent copper nanocluster GSH-CuNCs and a preparation method thereof, and further apply the nanocluster GSH-CuNCs to the sensitive analysis and detection of uranyl ions UO 2+ to achieve the visual detection of the pollutant uranyl ions. Based on the good properties of reduced glutathione, the present invention avoids expensive raw materials and cumbersome synthesis steps, and successfully synthesizes glutathione-encapsulated copper nanoclusters, which have the advantages of good stability, strong antioxidant ability, high fluorescence signal, etc., and can quickly analyze and detect uranyl ions, thereby providing a basis for the direction of environmental and biological detection. To achieve the above purpose, the present invention adopts the following technical solutions:

[0006] The present invention provides a preparation method of a glutathione-encapsulated fluorescent copper nanocluster GSH-CuNCs, including the following steps:

[0007] First, prepare a copper salt solution, then add the glutathione solution thereto, stir and react at room temperature to form a white turbid solution, dropwise add an alkali solution until the solution becomes clear, then vigorously stir at room temperature for 1-3 hours, dialyze for 12-48 hours, and filter through a microporous membrane.

[0008] In the above preparation method, preferably, the molar ratio of copper ions to glutathione in the copper salt solution is 1:7-9.

[0009] In the above preparation method, preferably, the copper ion concentration in the copper salt solution is 8-12 mM, and the glutathione solution concentration is 20-30 mg / mL.

[0010] In the above preparation method, preferably, the copper salt is a soluble copper salt, including at least one of copper chloride, copper sulfate, and copper nitrate.

[0011] In the above preparation method, preferably, the alkali solution is a sodium hydroxide solution or a potassium hydroxide solution.

[0012] In the above preparation method, preferably, the dialysis uses a dialysis bag with a molecular weight cut-off of 500-2000 Da.

[0013] In the above preparation method, preferably, the diameter of the microporous membrane is 0.22-0.45 μm, more preferably 0.22 or 0.45 μm.

[0014] In the above preparation method, as a more preferred scheme, the following specific steps can be adopted:

[0015] First, prepare an 8 - 12 mM CuCl2 solution. Then, add 5 ml of GSH solution (20 - 30 mg / mL) to 5 ml of the CuCl2 solution and stir for 4 - 6 minutes to fully mix the solution, forming a white turbid solution. Then, add 300 - 500 μl of 1 mol / l sodium hydroxide dropwise and stir for 1 - 3 h until the solution becomes clear. Dialyze through a dialysis bag with a molecular weight cut-off of 500 - 2000 Da for 12 - 48 h. The filtration diameter of the microporous filtration membrane used for filtration is 0.22 - 0.45 μm to remove small molecules and unreacted reagents. Then, store the prepared copper nanocluster solution sealed in a refrigerator at 4°C.

[0016] In the above preparation method, as a most preferred scheme, the following specific steps can be adopted:

[0017] First, prepare a 10 mM CuCl2 solution. Then, add 5 ml of GSH solution (25 mg / mL) to 5 ml of the CuCl2 solution and stir for 5 minutes to fully mix the solution, forming a white turbid solution. Then, add 400 μl of NaOH solution (1 M) until the solution becomes clear. Then, stir the solution vigorously at room temperature for 2 hours. Then, transfer the solution to a dialysis bag (molecular weight cut-off (MWCO) = 1000 Da) for further dialysis for about 24 hours. Then, filter the solution through a microporous membrane (0.22 μm) to obtain the CuNCs solution. Store the GSH-CuNCs solution in a refrigerator at 4°C until further use.

[0018] Through the above preparation method, the present invention obtains a glutathione-encapsulated fluorescent copper nanocluster GSH-CuNCs. GSH-CuNCs exhibits orange-red fluorescence and a high quantum yield (QY = 55.1%). At the same time, it can be applied in the fluorescence detection of uranyl ions. Specifically, the application is to mix the copper nanocluster solution with ethanol and phosphate buffer to make a probe solution for detecting uranyl ions; more specifically, the application is to take 10 - 30 μl of the copper nanocluster solution, 1 - 3 mL of ethanol and phosphate buffer (v / v = 3:1, pH = 4.0 - 6.0) to prepare a probe solution for detecting uranyl ions; preferably, take 20 μl of the copper nanocluster solution, 2 mL of a mixed solution of ethanol and phosphate buffer (pH = 5.0) to prepare a probe solution for detecting uranyl ions, where the volume ratio of ethanol to phosphate buffer is 3:1. It is better that the ethanol concentration is above 80%, and absolute ethanol is the best. The pH of the buffer solution is 5, which can effectively eliminate the interference of the uranyl ion solution under alkaline conditions.

[0019] In the application of the present invention for detecting uranyl ions, by taking the fluorescent probe solution for detecting metal ions, successively adding uranyl ion standard solution to the probe solution, and respectively recording the changes in fluorescence intensity and absorbance, a standard change curve can be plotted.

[0020] In the application of the present invention for detecting uranyl ions, the method for selectively detecting metal cations is as follows: Under the same conditions, take the fluorescent probe solution for detecting metal ions, add metal ion standard solution to the probe solution, and respectively record the changes in fluorescence intensity and absorbance.

[0021] In the application of the present invention for detecting uranyl ions, the method for detecting the sensitivity of uranyl ions is: (a) Take 20 μl of the nanocopper cluster solution in a cuvette, dilute it to 2 ml with ethanol and phosphate buffer (v / v = 3:1), and then gradually add uranyl ion standard solution. By testing, a standard curve of fluorescence intensity versus uranyl ion concentration can be obtained respectively to calculate the sensitivity of the probe for detecting uranyl ions.

[0022] The principle of fluorescence detection of uranyl ions in the present invention is based on the "turn-off" strategy of the probe. Specifically, it is manifested in the electrostatic interaction between uranyl ions and the synthesized nanocopper clusters, which causes the fluorescence quenching of the probe. Due to the electrostatic interaction, it is easier for electrons to transfer between UO 2+ and GSH-CuNCs, thus realizing highly accurate and real-time visual detection of uranyl ions.

[0023] The present invention prepares nanocopper clusters based on glutathione-encapsulated copper chloride, and develops a probe with orange-red fluorescence. Its preparation method is simple and the cost is low. The detection application of the present invention is based on the electron transfer caused by the electrostatic interaction between UO 2+ and GSH-CuNCs, resulting in fluorescence quenching, so it can be used for sensitive analysis and detection of UO 2+ , and the detection limit is as low as 6.7 nM. At the same time, UO in different real water samples 2+ was analyzed. The results show that GSH-CuNCs has a good basis for detecting real environmental water samples, providing an important basis for the environmental and biological directions. The nanocopper clusters prepared by the present invention have high sensitivity, good selectivity, strong anti-interference ability, a low detection limit for uranyl ions (the detection limit is 6.7 nM), and are used for real-time detection in real water samples including lake water, seawater, and pure water to evaluate the applicability of the copper cluster probe in the environment, having practical application value. Description of the Drawings

[0024] Figure 1 It is the transmission electron microscope image of the nanocopper clusters prepared by the present invention;

[0025] Figure 2UV-Vis absorption spectrum and fluorescence spectrum of the nano copper clusters prepared in the present invention;

[0026] Figure 3 X-ray energy spectrum of the nano copper clusters prepared in the present invention;

[0027] Figure 4 X-ray diffraction pattern of the nano copper clusters prepared in the present invention;

[0028] Figure 5 Linear change curve of fluorescence response of the probe for detecting metal ions prepared by mixing the nano copper cluster solution prepared in the present invention with ethanol and phosphate buffer solution to uranyl ions at room temperature;

[0029] Figure 6 Linear relationship diagram of the fluorescence intensity ratio (F0 - F) / F0 of the probe for detecting metal ions prepared by mixing the nano copper cluster solution prepared in the present invention with ethanol and phosphate buffer solution to the concentration of uranyl ions at room temperature;

[0030] Figure 7 Selective fluorescence detection diagram of the probe for detecting metal ions prepared by mixing the nano copper cluster solution prepared in the present invention with ethanol and phosphate buffer solution to different metal ions;

[0031] Figure 8 Interference fluorescence detection diagram of the probe for detecting metal ions prepared by mixing the nano copper cluster solution prepared in the present invention with ethanol and phosphate buffer solution to different metal ions;

[0032] Figure 9 Real-time change diagram of the color of the probe for detecting metal ions prepared by mixing the nano copper cluster solution prepared in the present invention with ethanol and phosphate buffer solution with the addition of uranyl ions. Detailed implementation mode

[0033] The following embodiments are further explanations of the content of the present invention to illustrate the technical content of the present invention. However, the substantial content of the present invention is not limited to the following embodiments. Those of ordinary skill in the art can and should know that any simple change or substitution based on the substantial spirit of the present invention should belong to the protection scope required by the present invention.

[0034] Example 1

[0035] 1. Preparation of nano copper clusters GSH-CuNCs

[0036] First, a 10 mM CuCl₂ solution was prepared. Then, 5 ml of GSH solution (25 mg / mL) was added to the 5 ml CuCl₂ solution and stirred for 5 minutes to allow the solution to mix thoroughly, forming a white, turbid solution. Then, 400 μl of 1 M NaOH solution was added dropwise until the solution became clear. The solution was then stirred vigorously at room temperature for 2 hours. The solution was then transferred to a dialysis bag (molecular weight cutoff (MWCO) = 1000 Da) and further dialyzed for approximately 24 hours. The solution was then filtered through a microporous filter membrane (0.22 μm) to obtain a CuNCs solution. The GSH-CuNCs solution was stored in a refrigerator at 4°C until further use.

[0037] 2. Preparation of Fluorescent Probes

[0038] 20 μl of the nano-copper cluster solution was fully mixed with 2 ml of anhydrous ethanol and phosphate buffer (v / v=3:1, pH=5.0) and added to a cuvette to prepare a probe solution.

[0039] 3. Fluorescence response of the probe to uranyl ions

[0040] To demonstrate the role of the probe in uranyl ion fluorescence detection, 0-22 μM uranyl ion standard solutions were added to the probe solution, and the changes in fluorescence intensity were recorded. As the uranyl ion concentration increased, the fluorescence intensity gradually decreased.

[0041] Figure 1 This is a transmission electron microscope image of the nano-copper cluster prepared by the present invention, and the lattice spacing of the nano-copper cluster is 0.2-0.3nm; Figure 2 The UV-visible absorption spectrum and fluorescence spectrum of the nano-copper cluster prepared by the present invention are shown in FIG. Figure 3 This is the X-ray energy spectrum of the nano-copper cluster prepared by the present invention. Figure 4 This is the X-ray diffraction pattern of the nano-copper cluster prepared in the present invention.

[0042] 4. Selectivity of fluorescent probes for metal cations

[0043] In order to evaluate the selectivity of the probe for detecting uranyl ions, Al 3+ ,K + ,Ca 2+ ,Cd 2+ ,Cr 3+ ,Cu 2+ ,Fe 3+ ,Mg 2+ ,Mn 2+ ,Na + ,Ni 2+ ,Pb 2+ ,Zn 2+ ,Hg 2+ ,UO2+ Fluorescence selectivity experiments were conducted on a series of metal cations, and the results showed that the probe had good selectivity for uranyl ions. Figure 5 The fluorescence response linear change curve of the probe for detecting metal ions prepared by mixing the nano copper cluster solution prepared in this invention with ethanol and phosphate buffer at room temperature, Figure 6 The linear relationship diagram of the fluorescence intensity ratio (F0 - F) / F0 of the probe for detecting metal ions prepared by mixing the nano copper cluster solution prepared in this invention with ethanol and phosphate buffer with the concentration of uranyl ions at room temperature, Figure 7 The selective fluorescence detection diagram of the probe for detecting metal ions prepared by mixing the nano copper cluster solution prepared in this invention with ethanol and phosphate buffer for different metal ions, Figure 8 The interference fluorescence detection diagram of the probe for different metal ions prepared by mixing the nano copper cluster solution prepared in this invention with ethanol and phosphate buffer (detecting by mixing other metal ions with uranyl ions respectively).

[0044] Example 2

[0045] Detection of uranyl ions in actual water samples

[0046] 10 μl of the probe solution in Example 1 was fully mixed with 2 ml of different water samples (lake water, seawater, pure water) and added to a cuvette, and then standard solutions of uranyl ions with different concentrations were added. Finally, the recovery rates of uranyl ions detected by the fluorescence mode were 93.5% - 103.6% respectively, and the results showed that it could be used for the real-time detection of uranyl ions in environmental samples. Figure 9 The real-time change diagram of the color of the probe prepared by mixing the nano copper cluster solution prepared in this invention with absolute ethanol and phosphate buffer with the addition of uranyl ions. From left to right in the figure, the concentrations of uranyl ions added were 0, 2, 4, 8, 12, 16, 20, 22 μM in sequence. It can be seen that with the increase of the concentration of uranyl ions added, the orange-red fluorescence brightness of the probe gradually faded until it completely disappeared, indicating that fluorescence quenching occurred after the combination of uranyl ions and the probe.

[0047] Example 3

[0048] The preparation method of the nano copper cluster solution is similar to that in Example 1, except that the molar ratio of copper chloride to glutathione is 1:7.

[0049] Example 4

[0050] The preparation method of the nano copper cluster solution is similar to that in Example 1, except that the molar ratio of copper chloride to glutathione is 1:8.

[0051] Example 5

[0052] The preparation method of the copper nanocluster solution is similar to that of Example 1, except that the molar ratio of copper chloride to glutathione is 1:9.

[0053] Example 6

[0054] The preparation method of the copper nanocluster solution is similar to that of Example 1, except that the mixing and stirring time of copper chloride and glutathione is 4 min, then 300 μl of sodium hydroxide with a concentration of 1 mol / l is added dropwise, and the stirring time is 1 h.

[0055] Example 7

[0056] The preparation method of the copper nanocluster solution is similar to that of Example 1, except that the mixing and stirring time of copper chloride and glutathione is 6 min, then 500 μl of sodium hydroxide with a concentration of 1 mol / l is added dropwise, and the stirring time is 3 h.

[0057] Example 8

[0058] The preparation method of the copper nanocluster solution is similar to that of Example 1, except that the mixing and stirring time of copper chloride and glutathione is 7 min, then 600 μl of sodium hydroxide with a concentration of 1 mol / l is added dropwise, and the stirring time is 4 h.

[0059] Example 9

[0060] The preparation method of the copper nanocluster solution is similar to that of Example 1, except that dialysis is carried out through a dialysis bag with a molecular weight cut-off of 500 Da for 12 h, and the filtration membrane is 0.22 μm.

[0061] Example 10

[0062] The preparation method of the copper nanocluster solution is similar to that of Example 1, except that dialysis is carried out through a dialysis bag with a molecular weight cut-off of 1500 Da for 24 h, and the filtration membrane is 0.25 μm.

[0063] Example 11

[0064] The preparation method of the copper nanocluster solution is similar to that of Example 1, except that dialysis is carried out through a dialysis bag with a molecular weight cut-off of 2000 Da for 48 h, and the filtration membrane is 0.45 μm.

[0065] Example 12

[0066] The preparation method of the fluorescent probe is similar to that of Example 1, except that 10 μl of the copper nanocluster solution is fully mixed with 1.5 ml of ethanol and phosphate buffer solution and added to a cuvette to prepare a probe solution.

[0067] Example 13

[0068] The preparation method of the fluorescent probe is similar to that of Example 1, except that 15 μl of the nano copper cluster solution is fully mixed with 2.5 ml of ethanol and phosphate buffer solution and added to a cuvette to prepare a probe solution.

[0069] It should be noted that the above technical content of the present invention is only an explanation and clarification to enable those skilled in the art to understand the technical essence of the present invention. Therefore, the above technical content is not used to limit the scope of the substantive protection of the present invention. The scope of the substantive protection of the present invention shall be subject to what is described in the claims. Those skilled in the art should know that any modifications, equivalent replacements, and improvements made based on the substantive spirit of the present invention shall fall within the scope of the substantive protection of the present invention.

Claims

1. Application of glutathione-encapsulated fluorescent copper nanoclusters GSH-CuNCs in fluorescence detection of uranyl ions, wherein, The preparation of the glutathione-encapsulated fluorescent copper nanoclusters GSH-CuNCs comprises the following steps: First, a copper salt solution is prepared, and then the glutathione solution is added thereto. A white turbid solution is formed by stirring the reaction at room temperature. An alkali solution is added dropwise until the solution becomes clear, and then the mixture is vigorously stirred at room temperature for 1-3 hours, dialyzed for 12-48 hours, and filtered through a microporous membrane to obtain a GSH-CuNCs solution. The GSH-CuNCs solution is mixed with ethanol and phosphate buffer solution to prepare a probe solution for detecting uranyl ions.

2. The application according to claim 1, characterized in that The molar ratio of copper ions to glutathione in the copper salt solution is 1:7-9.

3. The application according to claim 1, wherein The concentration of copper ions in the copper salt solution is 8-12 mM, and the concentration of the glutathione solution is 20-30 mg / mL.

4. The application according to claim 1, characterized in that, The copper salt is a soluble copper salt, including at least one of copper chloride, copper sulfate, and copper nitrate.

5. The application according to claim 1, characterized in that, The alkali solution is sodium hydroxide solution or potassium hydroxide solution.

6. The application according to claim 1, wherein The dialysis is carried out using a dialysis bag with a molecular weight cut-off of 500-2000 Da.

7. The application according to claim 1, characterized in that, The diameter of the microporous membrane is 0.22-0.45 μm.

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