Preparation method and application of red fluorescent copper nano-cluster

Abstract

The invention discloses a preparation method and application of a red fluorescent copper nano-cluster. According to the preparation method, a red fluorescent copper nano-cluster aqueous solution is prepared at room temperature by using polyvinylpyrrolidone as a protective agent, 3-mercaptophenylboronic acid as a stabilizer and a reducing agent and water-soluble copper salt as a copper source. The preparation method has the advantages of being simple in preparation process, low in instrument requirement, short in treatment time, low in cost and the like, the fluorescence quantum yield of the prepared red fluorescent copper nano-cluster is 4.9%, and the prepared red fluorescent copper nano-cluster has the advantages of being good in water solubility, good in stability, high in photobleaching resistance, high in salt resistance, long in fluorescence service life and the like, has high sensitivity and selectivity to Congo red, and is suitable for industrial production. The method can be applied to detection of fluorescence quenching type Congo red.

Description

Technical Field

[0001] The invention belongs to the technical field of metal nanocluster preparation, in particular to a preparation method and application of red fluorescent copper nanocluster. Background Art

[0002] Congo red is a dangerous azo dye derived from benzidine, which is highly toxic to the environment and humans. Due to the use of pigments and dyes in industries such as dyes, plastics, papermaking, leather, textiles, cosmetics, food and pharmaceuticals, a large amount of wastewater containing Congo red is generated. Congo red is not easily degraded by microorganisms and is highly stable in the environment. Worse still, Congo red is converted into benzidine, a carcinogen and mutagen, after entering the human body. In addition, Congo red has many other negative effects on human health, including diarrhea, vomiting, nausea and allergic problems.

[0003] At present, a variety of methods for detecting Congo red have been developed, including high performance liquid chromatography-ultraviolet detection, ultraviolet-visible spectrophotometry, electrochemical method and fluorescence detection. Among them, fluorescence detection is more attractive due to its advantages of high selectivity, high sensitivity, simple operation and low instrument cost. So far, several fluorescence methods based on carbon quantum dots have been developed to detect Congo red. For example, Durrani et al. used willow leaf purslane and m-phenylenediamine as raw materials, and obtained fluorescent carbon quantum dots by hydrothermal method at 180 ° C for 12 hours, and applied them to the detection of Congo red (Analytica ChimicaActa, 2022, 1202, 339672). Liu et al. used p-phenylenediamine and citric acid as precursors, and obtained fluorescent carbon quantum dots by hydrothermal method at 200 ° C for 8 hours, and used them for the detection of Congo red (Microchemical Journal, 2021, 168, 106420). Zulfajri et al. used rambutan seeds and L-aspartic acid as precursors, and obtained nitrogen-doped fluorescent carbon quantum dots by hydrothermal method at 200 ° C for 20 hours, and used them for the detection of Congo red (RSC Adv., 2023, 13, 6422). However, these carbon quantum dots usually require complex synthesis steps, high reaction temperatures and long reaction times. Therefore, it is still very important to develop a simple and practical method for the fluorescence sensing of Congo red.

[0004] In recent years, fluorescent probes based on copper nanoclusters have attracted widespread attention due to their unique advantages such as high catalytic activity, high cost-effectiveness, high conductivity and low cost. In recent years, a series of fluorescent copper nanoclusters (Cu NCs) have been successfully synthesized using different ligands and applied in many fields. However, most synthesis methods are characterized by long synthesis time, high reaction temperature, or need to be prepared under ultrasonic conditions. Therefore, it is very important to develop a simple and easy-to-operate method for synthesizing copper nanoclusters. And so far, no copper nanocluster materials have been found to be used for the detection of Congo red. Summary of the invention

[0005] The object of the present invention is to overcome the deficiencies of the prior art and provide a method for preparing and applying red fluorescent copper nanoclusters. The present invention uses polyvinyl pyrrolidone as a protective agent, 3-mercaptophenylboronic acid as a stabilizer and a reducing agent, and a water-soluble copper salt as a copper source to prepare a red fluorescent copper nanocluster solution with strong fluorescence characteristics at room temperature. The method has a short preparation time and a simple preparation process, and the prepared copper nanoclusters have the advantages of good water solubility, good stability, strong resistance to photobleaching, strong salt resistance and long fluorescence lifetime. The fluorescence of the red fluorescent copper nanocluster can be quenched by Congo red, and a new method for determining Congo red is established accordingly. The method is simple to operate and can detect Congo red quickly and accurately. The present invention uses red fluorescent copper nanoclusters as probes to detect Congo red based on fluorescence spectrophotometry, which provides a new idea for the detection of Congo red.

[0006] In order to achieve the above purpose, the technical solutions adopted are as follows:

[0007] The invention discloses a red fluorescent copper nanocluster, which is prepared at room temperature by using polyvinyl pyrrolidone as a protective agent, a copper salt solution as a copper source, and 3-mercaptophenylboronic acid as a stabilizer and a reducing agent.

[0008] A method for preparing red fluorescent copper nanoclusters comprises the following steps: mixing 2000 μL of a polyvinyl pyrrolidone solution with a mass fraction of 2% to 20% and 1000 μL of a 10 mmol / L copper salt aqueous solution, adding 1000 μL of a 3-mercaptophenylboric acid solution with a concentration of 10 to 50 mmol / L to the mixed solution, mixing evenly, reacting at room temperature for 5 to 30 minutes, and obtaining a red fluorescent copper nanocluster solution. An ultrafiltration centrifuge tube with a molecular weight cutoff of 10KD is used for ultrafiltration centrifugation for 20 minutes to remove excess small molecules in the solution. The purified red fluorescent copper nanoclusters are redispersed in 4 mL of a phosphate buffer solution with a pH of 7.0 and a concentration of 20 mmol / L, and stored in a refrigerator at 4°C.

[0009] Furthermore, the mass fraction of polyvinyl pyrrolidone is 2% to 20%, and preferably, the mass fraction of polyvinyl pyrrolidone is 14%.

[0010] Furthermore, the copper salt aqueous solution includes copper nitrate, copper chloride or copper sulfate; preferably, the copper salt aqueous solution is copper chloride.

[0011] Furthermore, the concentration of 3-mercaptophenylboric acid is 10 to 50 mmol / L, and preferably, the concentration of 3-mercaptophenylboric acid is 30 mmol / L.

[0012] Furthermore, the reaction time at room temperature is 5 to 30 minutes, preferably, the reaction time at room temperature is 15 minutes.

[0013] Application of red fluorescent copper nanoclusters in the detection of Congo red.

[0014] Furthermore, the detection method of Congo red includes the following steps: taking 100 μL of red fluorescent copper nanocluster aqueous solution and 1 mL of phosphate buffer solution with a pH of 7.0 and a concentration of 40 mmol / L, adding them into a fluorescence cuvette, adding Congo red standard solution of different concentrations, fixing the volume to 2 mL, taking 335 nm as the excitation wavelength, measuring the fluorescence intensity of the solution, and detecting Congo red by quenching the fluorescence of copper nanoclusters by Congo red.

[0015] The final concentration of the Congo red solution in the red fluorescent copper nanoclusters is 0 to 180 μM.

[0016] In the detection method, the concentration of Congo red and the fluorescence quenching ratio have a good linear relationship. The fluorescence quenching ratio refers to F0 / F, where F0 represents the fluorescence intensity of the red fluorescent copper nanocluster solution at a wavelength of 335nm when Congo red is not added; F represents the fluorescence intensity of the red fluorescent copper nanocluster solution at a wavelength of 335nm after Congo red is added. When the concentration of Congo red is in the range of 0 to 100 μM, its linear correlation coefficient is 0.995, and when the concentration of Congo red is in the range of 100 to 180 μM, its linear correlation coefficient is 0.991. According to this linear relationship between them, the content of Congo red in the solution can be judged by detecting the quenching ratio of the fluorescence intensity of the red fluorescent copper nanocluster by the solution.

[0017] Compared with the prior art, the advantages of the present invention are:

[0018] (1) The present invention adopts a one-step method to synthesize red fluorescent copper nanoclusters at room temperature, which has the advantages of simple preparation process, low instrument requirements, short processing time and low cost.

[0019] (2) The prepared red fluorescent copper nanoclusters have a fluorescence quantum yield of 4.9% and have the advantages of good water solubility, good stability, strong resistance to photobleaching, strong salt resistance, and long fluorescence lifetime.

[0020] (3) Congo red has a fluorescence quenching effect on the prepared red fluorescent copper nanoclusters, and the prepared red fluorescent copper nanoclusters can be used for the detection of Congo red. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The drawings constituting a part of this application are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0022] Figure 1 TEM image of the red fluorescent copper nanoclusters in Example 1;

[0023] Figure 2 is the infrared spectrum of the red fluorescent copper nanoclusters in Example 1;

[0024] Figure 3 is the UV-visible absorption spectrum of the red fluorescent copper nanoclusters in Example 2;

[0025] Figure 4 It is the fluorescence excitation-emission spectrum of the red fluorescent copper nanoclusters in Example 2; Inset: I is a picture of the copper nanoclusters under sunlight, Ⅱ is a picture of the copper nanoclusters under 365nm ultraviolet light.

[0026] Figure 5 This is the time-resolved fluorescence decay spectrum of the red fluorescent copper nanoclusters in Example 2;

[0027] Figure 6 a is the stability of the red fluorescent copper nanoclusters over time in Example 2; Figure 6 b is the photostability of the red fluorescent copper nanoclusters in Example 2; Figure 6 c is the effect of different sodium chloride concentrations on the fluorescence intensity of red fluorescent copper nanoclusters in Example 2;

[0028] Figure 7 The fluorescence spectra after adding different concentrations of Congo red to the red fluorescent copper nanocluster solution in Example 2;

[0029] Figure 8 It is the linear relationship diagram between Congo red concentration and fluorescence quenching ratio F0 / F;

[0030] Fig. 9 The selectivity experimental diagram of the red fluorescent copper nanocluster solution for Congo red in Example 2; 1 is a red fluorescent copper nanocluster solution, 2-13 are Ba 2+ , K + 、Na + 、Cd 2+ , Mn 2+ , Ca 2+ 、SO4 2- , Cl- Br - , methylene orange, malachite green, and Congo red red fluorescent copper nanocluster solutions. DETAILED DESCRIPTION

[0031] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present application can be combined with each other. The present invention will be described in detail below with reference to the accompanying drawings and in combination with the embodiments.

[0032] In order to enable those skilled in the art to better understand the solution of the present application, the technical solution in the embodiments of the present application will be clearly and completely described below in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present application.

[0033] Example 1

[0034] A method for preparing red fluorescent copper nanoclusters comprises the following steps: mixing 2000 μL of a polyvinyl pyrrolidone solution with a mass fraction of 14% and 1000 μL of a 10 mmol / L copper chloride aqueous solution, adding 1000 μL of 30 mmol / L 3-mercaptophenylboronic acid to the solution, reacting at room temperature for 15 minutes, and obtaining a red fluorescent copper nanocluster solution. An ultrafiltration centrifuge tube with a molecular weight cutoff of 10 KD is used for ultrafiltration centrifugation for 20 minutes to remove excess small molecules in the solution. The purified red fluorescent copper nanoclusters are redispersed in 4 mL of aqueous solution.

[0035] The obtained red fluorescent copper nanocluster solution was subjected to transmission electron microscopy test, and its TEM image is as follows Figure 1 As shown in the figure, it can be seen that the particle size of the red fluorescent copper nanoclusters prepared by the method of the present invention is about 2.9nm, which meets the size requirements of nanomaterials. Figure 2 shown.

[0036] Example 2

[0037] A method for preparing red fluorescent copper nanoclusters comprises the following steps: mixing 2000 μL of a polyvinyl pyrrolidone solution with a mass fraction of 14% and 1000 μL of a 10 mmol / L copper chloride aqueous solution, adding 1000 μL of 30 mmol / L 3-mercaptophenylboronic acid to the solution, reacting at room temperature for 15 minutes, and obtaining a red fluorescent copper nanocluster solution. An ultrafiltration centrifuge tube with a molecular weight cutoff of 10 KD is used for ultrafiltration centrifugation for 20 minutes to remove excess small molecules in the solution. The purified red fluorescent copper nanoclusters are redispersed in 4 mL of a phosphate buffer solution with a pH of 7.0 and a concentration of 20 mmol / L, and stored in a refrigerator at 4°C.

[0038] 100 μL of red fluorescent copper nanocluster aqueous solution and 1.9 mL of 20 mmol / L phosphate buffer solution at pH = 7.0 were added to a UV cuvette and a fluorescence cuvette to measure their UV-visible absorption spectrum, fluorescence excitation and emission spectrum, and time-resolved fluorescence decay spectrum. The UV-visible absorption spectrum of red fluorescent copper nanocluster is shown in Figure 3 As shown. Figure 3 As can be seen from the figure, the red fluorescent copper nanoclusters have strong absorption in the ultraviolet region, but no obvious absorption in the visible region, indicating that the synthesized copper nanoclusters are not large copper nanoparticles. Under 365nm ultraviolet light, they show red fluorescence; the fluorescence spectrum of the red fluorescent copper nanoclusters is shown in Figure 4 As shown. Figure 4 As can be seen from the figure, the maximum excitation and emission peaks of the red fluorescent copper nanoclusters are 335nm and 635nm, respectively. The time-resolved fluorescence decay spectrum of the red fluorescent copper nanoclusters is shown in Figure 5 As shown, the fluorescence lifetime of the red fluorescent copper nanoclusters is 837ns. The results show that the red fluorescent copper nanoclusters synthesized by the present invention have a long fluorescence lifetime.

[0039] The effect of storage time on the fluorescence intensity of the prepared red fluorescent copper nanocluster solution was investigated. Figure 6 The red fluorescent copper nanocluster solution still maintains good luminescence performance after being stored at 4° C. for 5 months, indicating that the red fluorescent copper nanocluster synthesized in the present invention has good storage stability.

[0040] The prepared red fluorescent copper nanocluster solution was continuously irradiated with a 150W xenon lamp to measure its photostability. Figure 6 As shown in b. The experimental results show that the red fluorescent copper nanoclusters synthesized by the present invention have good light stability.

[0041] The prepared red fluorescent copper nanocluster solution was used to investigate the effect of different sodium chloride solutions on its fluorescence intensity. Figure 6c shows that the fluorescence intensity of the red fluorescent copper nanoclusters does not substantially change with the change of the sodium chloride concentration, indicating that the red fluorescent copper nanoclusters synthesized in the present invention have good salt resistance.

[0042] Example 3

[0043] A method for preparing red fluorescent copper nanoclusters comprises the following steps: mixing 2000 μL of a 10% polyvinyl pyrrolidone solution and 1000 μL of a 10 mmol / L copper chloride aqueous solution, adding 1000 μL of 20 mmol / L 3-mercaptophenylboronic acid to the solution, reacting at room temperature for 10 min, and obtaining a red fluorescent copper nanocluster solution. An ultrafiltration centrifuge tube with a molecular weight cutoff of 10 KD is used for ultrafiltration centrifugation for 30 min to remove excess small molecules in the solution. The purified copper nanoclusters are redispersed in 4 mL of a phosphate buffer solution with a pH of 7.0 and a concentration of 20 mmol / L, and stored in a refrigerator at 4°C.

[0044] Example 4

[0045] A method for preparing red fluorescent copper nanoclusters comprises the following steps: mixing 2000 μL of a polyvinyl pyrrolidone solution with a mass fraction of 18% and 1000 μL of a 10 mmol / L copper chloride aqueous solution, adding 1000 μL of 3-mercaptophenylboronic acid with a concentration of 40 mmol / L to the solution, reacting at room temperature for 20 minutes, and obtaining a red fluorescent copper nanocluster aqueous solution. An ultrafiltration centrifuge tube with a molecular weight cutoff of 10 KD is used for ultrafiltration centrifugation for 15 minutes to remove excess small molecules in the solution. The purified red fluorescent copper nanoclusters are redispersed in 4 mL of a phosphate buffer solution with a pH of 7.0 and a concentration of 20 mmol / L, and stored in a refrigerator at 4°C.

[0046] Example 5: Application of red fluorescent copper nanoclusters in Congo red detection

[0047] 100 μL of the red fluorescent copper nanocluster aqueous solution prepared in Example 2 and 1 mL of a phosphate buffer solution with a pH of 7.0 and a concentration of 40 mmol / L were added to a 2 mL centrifuge tube, and different concentrations of Congo red standard solutions were added to make the volume 2 mL. The fluorescence spectrum was measured with an excitation wavelength of 335 nm. Figure 7 As shown in the figure, as the concentration of Congo red standard solution increases, the fluorescence of the red fluorescent copper nanoclusters gradually decreases. With the concentration of Congo red as the horizontal axis and F0 / F as the vertical axis, the working curve of Congo red is obtained, as shown in Figure 8 In the range of 1-100 μM, the linear equation is: F0 / F=0.0148C+0.967 (linear coefficient R 2=0.995, the unit of C is μM); in the range of 100-180 μM, the linear equation is: F0 / F=0.0253C-0.014 (linear coefficient R 2 =0.991, the unit of C is μM).

[0048] Example 6: Selective experiment of red fluorescent copper nanoclusters for Congo red detection

[0049] 100 μL of the red fluorescent copper nanocluster solution synthesized in Example 2 and 1000 μL of a phosphate buffer solution with a pH of 7.0 and a concentration of 40 mmol / L were mixed, and Congo red and a common coexisting substance (such as Ba) at an appropriate concentration were added respectively. 2+ , K + , Na + , Cd 2+ , Mn 2+ , Ca 2+ , SO4 2- , Cl - ,Br - , methylene orange and malachite green), the volume was adjusted to 2 mL, and fluorescence measurements were performed respectively. A bar graph was drawn with F0 / F as the ordinate and Congo red and different coexisting substances as the abscissa. Fig. 9 The results showed that the detection of Congo red by red fluorescent copper nanoclusters was not interfered by these coexisting substances.

[0050] Example 7: Determination of Congo Red in Tap Water and Drinking Water by Red Fluorescent Copper Nanoclusters

[0051] The content of Congo red in drinking water and tap water was determined by the standard addition method, and the specific steps were as follows: 100 μL of the red fluorescent copper nanocluster solution synthesized in Example 2, 1000 μL of a phosphate buffer solution with a pH of 7.0 and a concentration of 40 mmol / L, and 500 μL of a water sample were mixed, and 400 μL of a standard solution of Congo red of different concentrations was added, and the final concentration of Congo red in the water sample was 20 μM, 50 μM, and 100 μM. The reaction was carried out at room temperature for 1 min, and the mixture was transferred to a fluorescence cuvette, and fluorescence was measured respectively, F0 / F was calculated, the sample concentration C was calculated according to the linear equation, and the recovery rate was calculated, and the results are shown in Table 1.

[0052] Table 1. Congo red determination results in drinking water and tap water

[0053]

[0054] As can be seen from Table 1, the spiked recovery of red fluorescent copper nanoclusters for detecting Congo red is between 91.5% and 105.2%, so this method can be used for the quantitative detection of Congo red.

[0055] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement or combination made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for preparing red fluorescent copper nanoclusters, characterized in that: include: Polyvinyl pyrrolidone, 3-mercaptophenylboric acid and a copper salt aqueous solution are mixed, the mixed solution is stirred evenly and then placed at room temperature for a period of time to obtain a red fluorescent copper nanocluster material.

2. The method for preparing red fluorescent copper nanoclusters according to claim 1, characterized in that: Specifically, 2000 μL of a 2% to 20% polyvinyl pyrrolidone solution and 1000 μL of a 10 mmol / L copper salt aqueous solution were mixed, and then 1000 μL of a 10 to 50 mmol / L 3-mercaptophenylboric acid solution was added to the mixed solution, mixed evenly, and reacted at room temperature for 5 to 30 minutes to obtain a red fluorescent copper nanocluster solution. An ultrafiltration centrifuge tube with a molecular weight cutoff of 10KD was used for ultrafiltration centrifugation for 20 minutes to remove excess small molecules in the solution. The purified red fluorescent copper nanocluster was redispersed in 4 mL of a phosphate buffer solution with a pH of 7.0 and a concentration of 20 mmol / L, and stored in a 4°C refrigerator.

3. The method for preparing red fluorescent copper nanoclusters according to claim 2, characterized in that: The mass fraction of polyvinyl pyrrolidone is 14%.

4. The method for preparing red fluorescent copper nanoclusters according to claim 2, characterized in that: The copper salt aqueous solution is copper nitrate, copper chloride or copper sulfate.

5. The method for preparing red fluorescent copper nanoclusters according to claim 4, characterized in that: The aqueous solution of copper salt is copper chloride.

6. The method for preparing red fluorescent copper nanoclusters according to claim 2, characterized in that: The concentration of 3-mercaptophenylboronic acid is 30 mmol / L.

7. The method for preparing red fluorescent copper nanoclusters according to claim 2, characterized in that: The reaction time of the mixed solution at room temperature was 15 min.

8. An application of red fluorescent copper nanoclusters, characterized in that: The method comprises the following steps: taking 100 μL of the red fluorescent copper nanocluster solution described in claim 1 and 1 mL of a phosphate buffer solution with a pH value of 7.0 and a concentration of 40 mmol / L, adding them into a fluorescent cuvette, adding Congo red standard solutions of different concentrations, fixing the volume to 2 mL, taking 335 nm as an excitation wavelength, measuring the fluorescence intensity of the solution, and detecting Congo red by quenching the fluorescence of the copper nanocluster solution with Congo red.

9. The use of a red fluorescent copper nanoclusters according to claim 8, characterized in that: The final concentration of the Congo red in the red fluorescent copper nanocluster solution is 0-180 μM.

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