Application of ascorbic acid-stabilized copper nano-cluster in p-nitrophenol detection
By using ascorbic acid-stabilized copper nanoclusters as fluorescent probes, the problem of efficient detection of p-nitrophenol was solved using fluorescence sensing methods, achieving a simple, sensitive, and highly selective detection effect, which is applicable to food and pharmaceuticals.
Patent Information
- Application Number
- CN202510883113.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-06-28
- Publication Date
- 2025-10-31
AI Technical Summary
Existing technologies are difficult to detect p-nitrophenol efficiently, conveniently, rapidly, and selectively. Furthermore, copper nanoclusters are easily oxidized and have low quantum yields, which limits their application in pollutant detection.
Ascorbic acid-stabilized copper nanoclusters were used as fluorescent probes to detect p-nitrophenol using fluorescence sensing methods, and quantitative analysis was performed using changes in fluorescence intensity.
It enables a simple, sensitive, and highly selective detection of p-nitrophenol, with a low detection limit and a wide linear detection range, making it suitable for the detection of food and pharmaceuticals.
Smart Images
Figure CN120870069A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the application of ascorbic acid-stabilized copper nanoclusters in the detection of p-nitrophenol. It belongs to the field of biodetection technology. Background Technology
[0002] p-Nitrophenol (p-NP) is a persistent environmental pollutant that poses a serious threat to human health and ecosystems due to its low biodegradability and high toxicity. p-NP is widely used in agriculture and industry, including in the manufacture of pharmaceuticals, dyes, fungicides, and pesticides. Its high water solubility and low biodegradability make the removal of p-NP from contaminated water resources and crops extremely challenging. The U.S. Environmental Protection Agency has set the maximum permissible concentration of p-nitrophenol in drinking water at 60 μg / L, highlighting the importance of developing efficient, simple, rapid, highly selective, and highly sensitive p-NP detection strategies.
[0003] Metal nanoclusters (MNCs) are a novel class of fluorescent nanomaterials renowned for their unique physicochemical properties. These are extremely small metal-organic aggregates exhibiting molecular-like characteristics. MNCs offer numerous advantages, such as short preparation time, compact size, high stability, and ease of modification. Furthermore, when combined with other functional materials, they exhibit unique properties, making them promising candidate materials for pollutant detection, biosensing, and bioimaging—fields that have attracted widespread attention. MNCs enable rapid and sensitive detection of pollutants that tend to accumulate, resist degradation, and pose serious threats to human health and the ecological environment.
[0004] Metal nanoclusters (MNCs), as a novel class of fluorescent nanomaterials, are renowned for their unique physicochemical properties and molecular-like characteristics. MNCs offer advantages such as short preparation time, compact size, high stability, and ease of modification, making them particularly suitable for pollutant detection, biosensing, and bioimaging. Compared to precious metals like gold and silver, copper, due to its abundant resources, cost-effectiveness, and excellent physicochemical and optical properties, shows great potential in sensor development. However, copper nanoclusters are easily oxidized and have low quantum yields, thus requiring further research to improve their stability. Summary of the Invention
[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide the application of ascorbic acid-stabilized copper nanoclusters in the detection of p-nitrophenol.
[0006] To achieve the above objectives, the present invention adopts the following technical solution:
[0007] 1. Application of ascorbic acid-stabilized copper nanoclusters AA-CuNCs in the detection of p-nitrophenol.
[0008] Preferably, ascorbic acid-stabilized copper nanoclusters are used as fluorescent probes to detect p-nitrophenol.
[0009] Preferably, the ascorbic acid-stabilized copper nanoclusters are prepared by the following method: first, copper nitrate aqueous solution is added to deionized water, then ascorbic acid aqueous solution is added dropwise under vigorous stirring, stirred at room temperature, heated and stirred in the dark, and the supernatant is collected by centrifugation.
[0010] More preferably, the volume ratio of deionized water, copper nitrate aqueous solution, and ascorbic acid aqueous solution is 1.4:0.3:0.3, and the concentration of both copper nitrate aqueous solution and ascorbic acid aqueous solution is 0.1 mol / L.
[0011] Further preferred, the room temperature stirring time is 20 minutes; the heating and stirring conditions are: heating and stirring at 70°C for 8 hours.
[0012] Preferably, the supernatant is stored at 4°C for later use.
[0013] 2. A fluorescence sensing method for detecting p-nitrophenol, the specific steps of which are as follows:
[0014] (1) Add 20 μL of p-nitrophenol at different concentrations (0-400 μM) in order from low to high to a 100 μL reaction system containing 10 μL of ascorbic acid-stabilized copper nanoclusters and 20 μL of PB (pH=8.0) buffer solution. Mix the solutions evenly, record the fluorescence emission spectrum, and plot the fluorescence spectrum curve.
[0015] (2) Add 20 μL of a solution containing p-nitrophenol to a 100 μL reaction system containing 10 μL of ascorbic acid-stabilized copper nanoclusters and 20 μL of PB (pH=8.0) buffer solution. Mix the solution evenly, record the fluorescence emission spectrum, and calculate the p-nitrophenol content in the solution through the fluorescence spectrum curve.
[0016] Preferably, the excitation wavelength is 370 nm.
[0017] Preferably, the pH of the phosphate buffer is 8.0.
[0018] The beneficial effects of this invention are:
[0019] This invention provides the application of ascorbic acid-stabilized copper nanoclusters in the detection of p-nitrophenol. Ascorbic acid-stabilized copper nanoclusters are used as fluorescent probes to detect p-nitrophenol through fluorescence sensing.
[0020] Due to the quenching effect of p-nitrophenol, the fluorescence intensity of this system is significantly reduced in the presence of nitrophenol. By comparing the fluorescence intensity of copper nanoclusters and the quenching system, the concentration of nitrophenol can be quantitatively detected. This method is simple, sensitive, and highly selective, requires no complex fluorescent dye labeling, and shows significant potential for nitrophenol detection, with promising applications in food and pharmaceuticals. Details are as follows:
[0021] (1) The synthesized copper nanoclusters have stable optical properties, the synthesis method is simple, fast, and low in cost, and the synthesized materials have good fluorescence properties.
[0022] (2) Using synthesized copper nanoclusters with unique optical properties as fluorescent probes, the content of nitrophenol is detected with high specificity. The operation is simple and fast, and the specific detection of nitrophenol can be directly achieved.
[0023] Copper nanoclusters, as a novel type of functional nanoparticle, possess broad application prospects in the field of biosensing due to their simple synthesis, good photostability, and excellent dispersibility in aqueous solutions. This invention utilizes ascorbic acid (AA) as a stabilizer and reducing agent to synthesize a water-soluble, monodisperse, and highly fluorescent copper nanocluster probe. The probe was characterized by fluorescence spectroscopy and UV-Vis absorption spectroscopy.
[0024] Fluorescent metal nanoparticle analysis methods have advantages such as simple operation, high speed, high sensitivity, and good selectivity, which have attracted widespread research interest from researchers.
[0025] (3) The fluorescence intensity of copper nanoclusters decreases after nitrophenol is added to the copper nanocluster system. This method can achieve quantitative detection of p-NP with a wide detection linear range and low detection limit.
[0026] (4) The present invention can conveniently detect the content of nitrophenol in drug samples.
[0027] (5) Comparing the detection limits of different fluorescent nanomaterials in different detection ranges, it can be seen that the detection limit used in this work is lower and the sensitivity is higher than that of many reported methods based on fluorescent nanomaterials. (Table 1)
[0028] Table 1 Comparison of p-NP detection using different probes
[0029] Attached Figure Description
[0030] Figure 1 This is a schematic diagram of a p-NP detection fluorescence sensing strategy based on AA-CuNCs.
[0031] Figure 2(a) TEM image of AA-CuNCs. (b) Fluorescence excitation (1) and emission (2) spectra of AA-CuNCs. (c) Relationship between pH and spectral intensity of AA-CuNCs. (d) Fluorescence emission spectra of AA-CuNCs at different excitation wavelengths.
[0032] Figure 3 These are the fluorescence emission spectra of AA-CuNCs(1) and AA-CuNCs+200μMp-NP(2).
[0033] Figure 4 The effects of (a) pH on the fluorescence intensity of AA-CuNCs in the presence of 200 μM p-nitrophenol (p-NP) and (b) incubation time on the fluorescence intensity of AA-CuNCs in the presence of 200 μM p-nitrophenol (p-NP).
[0034] Figure 5 The results are: (a) fluorescence emission spectra of AA-CuNCs in the presence of different concentrations of p-nitrophenol (p-NP, 0 μM, 0.1 μM, 0.5 μM, 1 μM, 2 μM, 5 μM, 10 μM, 20 μM, 50 μM, 100 μM, 150 μM, 200 μM, 250 μM, 300 μM, 400 μM); (b) quantitative relationship between fluorescence intensity at 440 nm and p-NP concentration; and (c) calibration curve of fluorescence response ratio (F0 / F) versus p-NP concentration.
[0035] Figure 6 It is the selectivity of AA-CuNCs-based sensors for 200 μM structural analogs and a variety of other compounds. Detailed Implementation
[0036] The present invention will be further described below with reference to the accompanying drawings and embodiments. It should be noted that the following description is only for explaining the present invention and does not limit its content.
[0037] All reagents used in this invention are of analytical grade, and the reagents and their manufacturers are as follows: ascorbic acid (AA, >99%), copper nitrate trihydrate (Cu(NO3)2·3H2O) and p-nitrophenol (p-NP) were all purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.
[0038] Example 1
[0039] like Figure 1 As shown, the preparation of ascorbic acid-stabilized copper nanoclusters was carried out according to the following steps:
[0040] (1) Preparation of 0.1 mol / L copper nitrate solution: Dissolve 0.1876 g of copper nitrate in ultrapure water, and then make up to 10 mL with ultrapure water for later use.
[0041] (2) Preparation of 0.1 mmol / L oxalic acid solution: Dissolve 0.1761 g of oxalic acid in ultrapure water, then make up to 10 mL with ultrapure water and store in the dark for later use.
[0042] (3) Preparation of copper nanoclusters: Fluorescent copper nanoclusters were synthesized using ascorbic acid (AA) as a reducing agent and stabilizer. First, 0.3 mL of 0.1 M copper nitrate aqueous solution was added to 1.4 mL of deionized water. Then, 0.3 mL of 0.1 M AA was added dropwise under vigorous stirring. The mixture was stirred at room temperature for 20 min. After that, the thoroughly mixed solution was continuously stirred in a dark environment at 70 °C for 8 h. After the solution cooled to room temperature, it was centrifuged at 12000 r / min for 10 min to obtain pale yellow fluorescent copper nanoclusters. The supernatant of the purified copper nanoclusters was stored at 4 °C for later use.
[0043] Example 2 ( Figure 2 )
[0044] 1. Preparation of copper nanoclusters stabilized with ascorbic acid is described in Example 1;
[0045] 2. Characterization and analysis of the properties of ascorbic acid-stabilized copper nanoclusters, characterized by the following steps:
[0046] (1) Transmission electron microscopy (TEM) characterization of ascorbic acid-stabilized copper nanoclusters. The ascorbic acid-stabilized copper nanoclusters exhibited an approximately spherical shape with an average particle size of about 3–5 nm. Figure 2 a)
[0047] (2) 100 μL of ascorbic acid-stabilized copper nanoclusters solution was taken and its fluorescence intensity was measured using a fluorescence spectrophotometer. The emission spectrum was scanned under excitation wavelength of 370 nm. The probe showed strong emission at 440 nm. Figure 2 (b)
[0048] (3) Add 10 μL of ascorbic acid-stabilized copper nanoclusters solution, 20 μL of PB buffer solution with different pH values (pH = 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0), and 70 μL of deionized water to a centrifuge tube. After mixing thoroughly, measure the fluorescence intensity using a fluorescence spectrophotometer. The fluorescence intensity of the fluorescent copper nanoclusters is stronger when pH = 6.0–9.0 and in the absence of the target analyte p-NP. Figure 2 c)
[0049] (4) Take 100 μL of ascorbic acid-stabilized copper nanoclusters solution and measure its fluorescence intensity at different excitation wavelengths (350 nm, 360 nm, 370 nm, 380 nm, 390 nm) using a fluorescence spectrophotometer. It was found that the ascorbic acid-stabilized copper nanoclusters had the strongest fluorescence intensity at an excitation wavelength of 370 nm. Figure 2 (d)
[0050] Example 3
[0051] 1. Preparation of copper nanoclusters stabilized with ascorbic acid is described in Example 1;
[0052] 2. Feasibility analysis of using ascorbic acid-stabilized copper nanoclusters as fluorescent probes for detecting p-nitrophenol in solution, characterized by the following steps:
[0053] (1) Preparation of p-nitrophenol stock solution: Dissolve 0.0014g of nitrophenol in ultrapure water, and then make up to 10mL with ultrapure water to prepare a 1mmol / L p-nitrophenol stock solution for later use.
[0054] (4) Add 10 μL of ascorbic acid-stabilized copper nanoclusters solution, 20 μL of 1 μmol / L p-nitrophenol solution, 20 μL of LPB buffer (pH = 8.0), and 50 μL of deionized water to a centrifuge tube. After mixing thoroughly, measure the fluorescence intensity using a fluorescence spectrophotometer. The fluorescence intensity is significantly reduced at this point. The decrease in fluorescence emission intensity can be used to demonstrate the feasibility of using ascorbic acid-stabilized copper nanoclusters as a fluorescent probe to detect p-nitrophenol. Figure 3 )
[0055] Example 4
[0056] 1. Preparation of copper nanoclusters stabilized with ascorbic acid is described in Example 1;
[0057] 2. Optimization of the pH value of the buffer solution in the reaction system of p-nitrophenol using ascorbic acid-stabilized copper nanoclusters as a fluorescent probe, characterized by the following steps:
[0058] 20 μL of PB buffer solution with different pH values (pH 6.0–9.0), 10 μL of ascorbic acid-stabilized copper nanoclusters, and 20 μL of 1 μmol / L p-nitrophenol solution were added to a 100 μL reaction system and mixed thoroughly. The fluorescence intensity was measured using a fluorescence spectrophotometer. The results showed that the fluorescence intensity of the ascorbic acid-stabilized copper nanoclusters in the presence of p-nitrophenol decreased slowly with increasing pH. A significant fluorescence quenching phenomenon was observed at pH 8.0, reaching a stable peak value. Therefore, pH 8.0 was selected as the fluorescence detection condition for p-nitrophenol. Figure 4 a)
[0059] 2. Optimization of the incubation time for p-nitrophenol in a reaction system using ascorbic acid-stabilized copper nanoclusters as a fluorescent probe, characterized by the following steps:
[0060] 10 μL of ascorbic acid-stabilized copper nanoclusters, 20 μL of LPB buffer (pH 8.0), and 20 μL of 1 μmol / L p-nitrophenol solution were added to a centrifuge tube and mixed thoroughly. The reaction was carried out at room temperature for 0, 5, 10, 15, 20, 25, and 30 min, and the fluorescence intensity was measured using a fluorescence spectrophotometer. The experimental results show that the fluorescence of the ascorbic acid-stabilized copper nanoclusters was rapidly quenched by p-nitrophenol, and no significant change was observed at different incubation times. Therefore, it can be inferred that the effect of incubation time on the fluorescence intensity of the ascorbic acid-stabilized copper nanoclusters is negligible. Figure 4 (b)
[0061] Example 5
[0062] 1. Preparation of copper nanoclusters stabilized with ascorbic acid is described in Example 1;
[0063] 2. An analysis of the content of p-nitrophenol in solution using ascorbic acid-stabilized copper nanoclusters as a fluorescent probe, characterized by the following steps:
[0064] 20 μL of p-nitrophenol at different concentrations (0–400 μM) were added, in ascending order, to 100 μL of a reaction system containing 10 μL of ascorbic acid-stabilized copper nanoclusters and 20 μL of PB (pH = 8.0) buffer solution. The solutions were mixed thoroughly, and fluorescence emission spectra were recorded. The fluorescence intensity was then measured using a fluorescence spectrophotometer. The experimental results showed that (…). Figure 5 Within the range of 0.1 μM to 250 μM, the fluorescence response (F0 / F) exhibited a good linear relationship with the concentration of p-nitrophenol, with the linear equation being F0 / F = 0.0071C + 1.0129, R0. 2 =0.9929, detection limit is 30 nM.
[0065] Example 6
[0066] 1. Preparation of copper nanoclusters stabilized with ascorbic acid is described in Example 1;
[0067] 2. The preparation of the p-nitrophenol solution is as described in Example 3;
[0068] 3. A specific analysis of p-nitrophenol in solution using ascorbic acid-stabilized copper nanoclusters as fluorescent probes, characterized by the following steps:
[0069] (1) p-NP (p-nitrophenol), o-NP (o-nitrophenol), m-NP (m-nitrophenol), MB (toluene), TNT (2,4,6-trinitrotoluene), Na + Co 2+ F - Cl - Solution preparation:
[0070] (2) Add 20 μL of o-NP (o-nitrophenol) solution for specific analysis, 10 μL of fluorescent copper nanoclusters, 20 μL of LPB buffer (pH = 8.0), and 50 μL of deionized water to a centrifuge tube and mix thoroughly. Measure the fluorescence intensity using a fluorescence spectrophotometer. Except for p-nitrophenol, other interfering substances did not cause a significant fluorescence response (F0 / F). More importantly, it can selectively distinguish p-nitrophenol and its coexisting isomers (o-nitrophenol and m-nitrophenol). The results show that this method has good selectivity for p-nitrophenol. Figure 6 )
[0071] While the specific embodiments of the present invention have been described above in conjunction with the accompanying drawings, this is not intended to limit the scope of protection of the present invention. Based on the technical solutions of the present invention, various modifications or variations that can be made by those skilled in the art without creative effort are still within the scope of protection of the present invention.
Claims
1. Application of ascorbic acid-stabilized copper nanoclusters AA-CuNCs in the detection of p-nitrophenol.
2. The application according to claim 1, characterized in that, Ascorbic acid-stabilized copper nanoclusters were used as fluorescent probes to detect p-nitrophenol.
3. The application according to claim 1, characterized in that, The ascorbic acid-stabilized copper nanoclusters were prepared by the following method: copper nitrate aqueous solution was added to deionized water, and then ascorbic acid aqueous solution was added dropwise under vigorous stirring. The mixture was stirred at room temperature, heated and stirred in the dark, and the supernatant was collected by centrifugation.
4. The application according to claim 3, characterized in that, The volume ratio of deionized water, copper nitrate aqueous solution, and ascorbic acid aqueous solution is 1.4:0.3:0.3, and the concentration of copper nitrate aqueous solution and ascorbic acid aqueous solution is 0.1 mol / L.
5. The application according to claim 3, characterized in that, The stirring time at room temperature is 20 minutes; the heating and stirring conditions are: heating and stirring at 70℃ for 8 hours.
6. The application according to claim 3, characterized in that, The supernatant is stored at 4°C for later use.
7. A fluorescence sensing method for detecting p-nitrophenol, characterized in that, The specific steps are as follows: (1) Add 20 μL of p-nitrophenol at different concentrations (0-400 μM) in order from low to high to a 100 μL reaction system containing 10 μL of ascorbic acid-stabilized copper nanoclusters and 20 μL of PB buffer solution, mix the solutions evenly, record the fluorescence emission spectrum, and plot the fluorescence spectrum curve. (2) Add 20 μL of a solution containing p-nitrophenol to a 100 μL reaction system containing 10 μL of ascorbic acid-stabilized copper nanoclusters and 20 μL of PB buffer solution. Mix the solution evenly, record the fluorescence emission spectrum, and calculate the p-nitrophenol content in the solution through the fluorescence spectrum curve.