Ratio-fluorescence system based on o-phenylenediamine and sodium ascorbate and detection of copper ions in water by ratiofluorescence system
By constructing a ratiometric fluorescence system of o-phenylenediamine and sodium ascorbate, and utilizing the intensity ratio of the fluorescence emission peak generated by Cu2+ to detect copper ions, the problem of complex detection in existing technologies is solved, and a simple and sensitive detection of copper ions is achieved.
Patent Information
- Application Number
- CN202410696318.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-05-31
- Publication Date
- 2025-12-02
AI Technical Summary
Existing ratio fluorescence systems for detecting copper ions are complex, require nanomaterials, and lack simple and rapid detection methods.
A ratiometric fluorescence system based on o-phenylenediamine and sodium ascorbate was constructed. Cu2+ reacts with o-phenylenediamine and sodium ascorbate to generate quinoxaline and 2,3-diaminophenazine. The ratio of the fluorescence emission peak intensities of the two products at different wavelengths was used to detect copper ions.
It achieves sensitive and reliable detection of copper ions, with accurate detection results, simple system construction, low cost, and no dependence on nanomaterials.
Smart Images
Figure HDA0004869851880000011 
Figure HDA0004869851880000012 
Figure HDA0004869851880000021
Abstract
Description
Technical Field
[0001] This invention relates to the field of detection technology, and in particular to a method for detecting copper ions. Background Technology
[0002] Copper is an essential trace element for living organisms such as humans and animals. However, excessive copper can lead to physiological dysfunction in humans and animals, inducing various diseases. 2+ Its widespread industrial applications have caused serious environmental pollution to water and soil systems. Due to its aquatic use and high environmental stability, these water and soil systems can enter the human body through the food chain. When copper intake exceeds the normal levels required by the human body, it can damage human organs.
[0003] Currently, there are various analytical methods for detecting copper ions, including colorimetry, atomic absorption spectrophotometry, fluorescence methods, ultraviolet-visible spectrophotometry, inductively coupled plasma atomic emission spectrometry (ICP-AES), and ICP-AES mass spectrometry. Among these, fluorescence methods are commonly used for the detection of trace and ultra-trace copper ions due to their high sensitivity. Compared to single-emission fluorescence systems, ratiometric fluorescence systems, with their two emission peaks of good resolution, can effectively eliminate background interference through their fluorescence intensity ratio, thus attracting widespread research and attention. However, most ratiometric fluorescence systems used for copper ion detection rely on nanomaterials such as carbon dots and quantum dots, making the experimental process relatively complex. Therefore, it is necessary to seek a simple ratiometric fluorescence system for rapid and sensitive detection of copper ions. Summary of the Invention
[0004] To address the shortcomings of existing technologies, the purpose of this invention is to construct a ratiometric fluorescence system based on o-phenylenediamine and sodium ascorbate, aiming to achieve simple and rapid detection of copper ions.
[0005] To achieve the above objectives, the present invention adopts the following technical solution, which includes the following steps:
[0006] S1: Prepare o-phenylenediamine solution, sodium ascorbate solution, and copper ion solution;
[0007] S2: Construct a ratiometric fluorescence system based on o-phenylenediamine and sodium ascorbate. After the system has reacted for 15 minutes, add the Cu to be tested. 2+ The solution reacts;
[0008] S3: After the reaction is complete, the fluorescence emission intensity of the reaction system at 435 nm and 560 nm is detected respectively.
[0009] The reaction principle is Cu 2+ Sodium ascorbate is oxidized to produce dehydroascorbic acid, which then reacts with o-phenylenediamine to form quinoxaline; additionally, Cu... 2+It can undergo a redox reaction with o-phenylenediamine to produce 2,3-diaminophenazine. The two products generated by the above reactions, quinoxaline and 2,3-diaminophenazine, exhibit fluorescence emission peaks at 435 nm and 560 nm, respectively, and can be used to construct ratiometric fluorescence systems. With Cu... 2+ With increasing concentration, the fluorescence emission peak intensity (F) of quinoxaline at 435 nm increased. 435 The fluorescence emission peak intensity of 2,3-diaminophenazine at 560 nm remained almost unchanged, while the fluorescence emission peak intensity (F) of 2,3-diaminophenazine remained almost unchanged. 560 As the fluorescence intensity gradually increases, the ratio of the two fluorescence emission peak intensities (F) is calculated. 560 / F 435 The obtained fluorescence intensity ratio is compared with Cu. 2+ The concentration of copper ions showed a positive correlation, enabling sensitive and reliable detection of copper ions.
[0010] In the above steps:
[0011] The order of adding the solutions is as follows: first add sodium ascorbate solution to the o-phenylenediamine solution, then add copper ion solution.
[0012] The concentration of the o-phenylenediamine solution can be 1.67, 1.78, 1.89, 2, 2.11, 2.22, or 2.33 (unit: mmol / L).
[0013] The concentration of the sodium ascorbate solution can be 14, 16, 18, 20, 22, 24, or 26 (unit: μmol / L).
[0014] The experimental system is described by detecting the fluorescence intensity of reaction systems containing different concentrations of copper ions and plotting a standard curve.
[0015] According to some optimized embodiments of the present invention, the ratiometric fluorescence system exhibits the highest fluorescence intensity when the reaction time is 30 minutes, the optimal concentration of o-phenylenediamine is 2 mmol / L, the optimal concentration of sodium ascorbate is 20 μmol / L, and the pH is 7.
[0016] This invention provides the fluorescence intensity ratio (F) of 2,3-diaminophenazine and quinoxaline. 560 / F 435 The correlation between the increase of copper ion concentration and the increase of copper ion concentration was investigated, and a standard curve was plotted.
[0017] The present invention further provides the results of a selectivity test, which shows that the reaction system has good anti-interference properties; it also provides the results of a recovery test, in which the recovery rate of copper ions is between 92.3% and 103.5%.
[0018] The present invention has the following advantages:
[0019] This invention proposes a ratiometric fluorescence system based on o-phenylenediamine and sodium ascorbate for the detection of copper ions in water. It utilizes the fluorescence intensity ratio of two products generated from the reaction of several substances in the system as the analytical signal, resulting in more accurate detection. Furthermore, the system is simple to construct, does not require the synthesis of nanomaterials, and has low cost. Attached Figure Description
[0020] Figure 1 The reaction equation involved in Example 1 is shown below.
[0021] Figure 2 This illustrates the effect of reaction time on the fluorescence emission intensity at 560 nm in Example 2.
[0022] Figure 3 This is a graph showing the relationship between the concentration of o-phenylenediamine and the fluorescence intensity ratio in Example 2.
[0023] Figure 4 This is a graph showing the relationship between sodium ascorbate concentration and fluorescence intensity ratio in Example 2.
[0024] Figure 5 This is a graph showing the relationship between pH and fluorescence intensity at 560 nm in Example 2.
[0025] Figure 6 This is a superimposed fluorescence spectrum of the reaction system at 560 nm when different concentrations of copper ions were added in Example 3.
[0026] Figure 7 This is a superimposed fluorescence spectrum of the reaction system at 435 nm when different concentrations of copper ions were added in Example 3.
[0027] Figure 8 This is the standard curve graph in Example 3.
[0028] Figure 9 This is a graph showing the relationship between the concentration of interfering ions and the fluorescence intensity of the reaction system in Example 4. Detailed Implementation
[0029] The following examples illustrate the present invention.
[0030] Example 1: Establishment of a ratiometric fluorescence system based on o-phenylenediamine and sodium ascorbate and its detection of copper ions in water.
[0031] This invention constructs a ratiometric fluorescence system based on o-phenylenediamine and sodium ascorbate and its detection of copper ions in water.
[0032] See attached document Figure 1 Because Cu 2+ It possesses oxidizing properties and can react with sodium ascorbate to form dehydroascorbic acid, which then reacts with o-phenylenediamine to form quinoxaline; additionally, Cu...2+ It can undergo a redox reaction with o-phenylenediamine to produce 2,3-diaminophenazine. The two products generated by the above reactions, quinoxaline and 2,3-diaminophenazine, exhibit fluorescence emission peaks at 435 nm and 560 nm, respectively, and can be used to construct ratiometric fluorescence systems. With Cu... 2+ With increasing concentration, the fluorescence emission peak intensity (F) of quinoxaline at 435 nm increased. 435 The fluorescence emission peak intensity of 2,3-diaminophenazine at 560 nm remained almost unchanged, while the fluorescence emission peak intensity (F) of 2,3-diaminophenazine remained almost unchanged. 560 As the fluorescence intensity gradually increases, the ratio of the two fluorescence emission peak intensities (F) is calculated. 560 / F 435 The obtained fluorescence intensity ratio is compared with Cu. 2+ The concentration of copper ions showed a positive correlation, enabling sensitive and reliable detection of copper ions.
[0033] Example 2: Optimization of Reaction Conditions
[0034] 1. Optimization of reaction time
[0035] To obtain the optimal reaction time, the concentration of o-phenylenediamine was set to 2 mmol / L and the concentration of ascorbic acid was set to 20 μmol / L. First, 900 μL of o-phenylenediamine (2 mmol / L) and 50 μL of sodium ascorbate (20 μmol / L) were added to a 15 mL centrifuge tube and mixed thoroughly. The mixture was then reacted at room temperature for 15 minutes. Next, a certain amount of copper ions was added to the system, followed by a certain amount of buffer solution to bring the volume to a suitable level. After mixing thoroughly, 300 μL of the reaction solution was transferred to a quartz microcuvette. The sample was excited using an excitation wavelength of 418 nm to obtain a fluorescence emission spectrum, and a graph showing the relationship between fluorescence emission intensity and reaction time was plotted.
[0036] See attached document Figure 2 As time increases, the fluorescence intensity gradually increases, then tends to level off after a certain period. When the fluorescence intensity reaches 90% of its maximum value, the reaction is considered essentially complete. This point in time is the optimal reaction time. Therefore, the optimal reaction time is set at 30 minutes.
[0037] 2. Optimization of o-phenylenediamine concentration
[0038] In the o-phenylenediamine concentration optimization experiment, the sodium ascorbate concentration was set at 20 μmol / L. Next, reaction system solutions with different concentrations of o-phenylenediamine were prepared. First, a certain volume of o-phenylenediamine solution was transferred to a 15 mL centrifuge tube and mixed thoroughly with 50 μL of sodium ascorbate (20 μmol / L). The mixture was then reacted at room temperature for 15 minutes. Afterward, 200 μL of copper ion solution was added to the system, followed by a certain amount of buffer solution to bring the volume to a suitable level. After mixing thoroughly, 300 μL of the reaction system solution was transferred to a quartz microcuvette. The fluorescence intensity spectra of the reaction system were measured using 348 nm and 418 nm as excitation wavelengths, respectively. By analyzing the obtained fluorescence intensity ratios, the optimal o-phenylenediamine concentration was determined.
[0039] See attached document Figure 3 By constructing fluorescence intensity ratio (F 560 / F 435 The relationship between fluorescence intensity and o-phenylenediamine concentration, and the fluorescence intensity ratio (F) 560 / F 435 When the concentration of o-phenylenediamine reaches its maximum, the concentration in the reaction system is 2 mmol / L. Therefore, the optimal concentration of o-phenylenediamine is 2 mmol / L.
[0040] 3. Optimization of sodium ascorbate concentration
[0041] The reaction time was set to 30 minutes, and the concentration of o-phenylenediamine was set at 2 mmol / L. First, 900 μL of o-phenylenediamine solution was transferred to a 15 mL centrifuge tube and mixed thoroughly. The mixture was then allowed to react at room temperature for 15 minutes. Next, 200 μL of copper ion solution was added to the system, followed by a buffer solution to bring the volume to a suitable level. After mixing thoroughly, the mixture was allowed to react at room temperature for 30 minutes. Then, 300 μL of the reaction solution was transferred to a quartz microcuvette. The fluorescence intensity spectra of the reaction system were measured using excitation wavelengths of 348 nm and 418 nm, respectively. By analyzing the obtained fluorescence intensity ratios, the optimal sodium ascorbate concentration was determined.
[0042] See attached document Figure 4 By constructing fluorescence intensity ratio (F 560 / F 435 The relationship between fluorescence intensity and sodium ascorbate concentration, and the fluorescence intensity ratio (F) 560 / F 435 When the maximum value is reached, the concentration of sodium ascorbate in the reaction system is 20 μmol / L. Therefore, the optimal concentration of sodium ascorbate is 20 μmol / L.
[0043] 4. pH optimization
[0044] The reaction time was set to 30 minutes, the o-phenylenediamine concentration to 2 mmol / L, and the ascorbic acid concentration to 20 μmol / L. First, 900 μL of o-phenylenediamine solution and 50 μL of sodium ascorbate were added to a 15 mL centrifuge tube and mixed thoroughly. The mixture was then allowed to react at room temperature for 15 minutes. Next, 200 μL of copper ion solution was added to the system, and the volume was adjusted to an appropriate level using buffer solutions of different pH values. After mixing thoroughly, the mixture was allowed to react at room temperature for 30 minutes. Then, 300 μL of the reaction solution was transferred to a quartz microcuvette. The fluorescence intensity spectrum of the reaction system at 560 nm was measured using 418 nm as the excitation wavelength. The optimal pH was determined by analyzing the obtained fluorescence intensity.
[0045] See attached document Figure 5 As the pH increases, the fluorescence intensity F565 of the reaction system at 560 nm gradually increases. The fluorescence intensity reaches its maximum at pH 7.0. Therefore, the optimal pH for the reaction system is 7.0.
[0046] Example 3: Standard Curve and Linear Range
[0047] First, 900 μL of 2 mmol / L o-phenylenediamine solution and 50 μL of 20 μmol / L sodium ascorbate were added to a 15 mL centrifuge tube and mixed thoroughly. The mixture was then allowed to react at room temperature for 15 minutes. Next, copper ion solutions of different concentrations were added to the system, followed by a buffer solution (pH 7.0) to bring the volume to a suitable level. The mixture was shaken on a high-speed mixer for 30 seconds and then allowed to stand at room temperature for 30 minutes. Finally, 300 μL of the reaction solution was transferred to a quartz microcuvette, and the fluorescence intensity spectra of the reaction system at 418 nm and 560 nm were measured using 348 nm and 418 nm as excitation wavelengths, respectively. The fluorescence intensity ratios were analyzed, and the results were expressed as F... 560 / F 435 The Y-axis data is used, and the copper ion concentration is used as the X-axis data. An F-axis is constructed. 560 / F 435 A standard curve can be obtained by plotting a scatter plot of copper ion concentration versus copper ion concentration and performing linear fitting.
[0048] After adding copper ion solutions of different concentrations (5-35 μmol / L), the fluorescence intensity at 418 nm and 560 nm was recorded, and the fluorescence intensity ratio F was obtained. 560 / F 435 See attached document. Figure 6 and attached Figure 7 As the copper ion concentration increased, the fluorescence intensity of 2,3-diaminophenazine at 560 nm gradually increased, while the fluorescence intensity of quinoxaline at 435 nm remained almost unchanged, and the fluorescence intensity ratio of the two products, F...560 / F 435 It exhibits a linear correlation with the concentration of copper ions, thereby enabling sensitive detection of copper ions.
[0049] See attached document Figure 8 Fluorescence intensity ratio F 560 / F 435 A linear relationship was observed with copper ion concentration in the range of 5–35 μmol / L. The calibration equation for this linear relationship is: y = 0.11427x + 0.85757(R²). 2 =0.98774). Where y represents the fluorescence intensity ratio and x represents the copper ion concentration. Based on a signal-to-noise ratio of 3, the detection limit for copper ions is calculated to be 1.6 μmol / L.
[0050] Example 4: Selective Test
[0051] To verify the selectivity of this invention for copper ion detection, Ca was added to the reaction system under optimal conditions. 2+ Pb 2+ Na + Co 2+ K + Zn 2+ Fe 2+ Mg 2+ Mn 2+ Plasma was used to detect the fluorescence intensity of the system at 560 nm. Then, the fluorescence intensity difference ΔF of the reaction system under different interfering substances was calculated. ΔF is the difference between the fluorescence intensity F of the reaction system at 560 nm and the fluorescence intensity F0 of the blank solution, i.e., ΔF = F - F0.
[0052] The concentration of interfering ions was 10 times that of copper ions. (See attached document.) Figure 9 , except Fe 2+ Besides, other metal ions had little effect on the fluorescence intensity of the system, indicating that they would not interfere with the detection of copper ions. Fe... 2+ This can interfere with the detection of copper ions. This is because Fe 2+ It has weak oxidizing properties and slightly inhibits the reaction of copper ions with o-phenylenediamine and sodium ascorbate, resulting in a reduction in the amount of 2,3-diaminophenazine and quinoxaline generated, which may cause some interference to the detection signal.
[0053] Example 5 Recovery Test
[0054] To demonstrate the applicability of this invention, copper ions of different concentrations were added to tap water as actual complex samples to prepare ratiometric fluorescence systems, with copper ion concentrations of 10 μmol / L, 20 μmol / L, and 30 μmol / L in the system solutions. After thorough mixing and reaction for 30 minutes, 300 μL of the reaction system solution was transferred to a quartz microcuvette. Using 348 nm and 418 nm as excitation wavelengths, the fluorescence intensity spectra of the reaction system at 435 nm and 560 nm were measured, and the corresponding fluorescence intensity ratio F was calculated. 560 / F 435 By substituting the calculated fluorescence intensity ratio into the linear calibration equation obtained in Example 3, the concentration of copper ions can be calculated, and then the recovery rate can be calculated to determine the accuracy of the method.
[0055] The results showed that the sample recovery rate was 92.3%–103.5%, and the relative standard deviation (RSD) was 4.7%–6.5%. The good recovery results demonstrate that this method is suitable for the detection of copper ions in real water samples.
[0056] Although the description of this disclosure has been quite detailed and particularly of several described embodiments, it is not intended to limit itself to any of these details or embodiments or any particular embodiments, thereby effectively covering the intended scope of this disclosure. Furthermore, the disclosure has been described above with respect to embodiments foreseeable by the inventors for the purpose of providing a useful description, and non-substantial modifications to this disclosure that have not yet been foreseen may still represent equivalent modifications.
Claims
1. A ratiometric fluorescence system based on o-phenylenediamine and sodium ascorbate and its detection of copper ions in water, characterized in that, It includes the following steps: S1: Prepare o-phenylenediamine solution, sodium ascorbate solution, and copper ion solution; S2: Construct a ratiometric fluorescence system based on o-phenylenediamine and sodium ascorbate. After the system has reacted for 15 minutes, add the Cu to be tested. 2+ The solution reacts; S3: After the reaction is complete, the fluorescence emission intensity of the reaction system at 435 nm and 560 nm is detected respectively.
2. The ratiometric fluorescence system based on o-phenylenediamine and sodium ascorbate according to claim 1 and its detection of copper ions in water, characterized in that, The optimal concentration of o-phenylenediamine in step S1 is 2 mmol / L.
3. The ratiometric fluorescence system based on o-phenylenediamine and sodium ascorbate according to claim 1 and its detection of copper ions in water, characterized in that, The optimal concentration of sodium ascorbate in step S1 is 20 μmol / L.
4. The ratiometric fluorescence system based on o-phenylenediamine and sodium ascorbate according to claim 1 and its detection of copper ions in water, characterized in that, The optimal pH for the ratiometric fluorescence system is 7.
5. The ratiometric fluorescence system based on o-phenylenediamine and sodium ascorbate according to claim 1 and its detection of copper ions in water, characterized in that, The reaction time required for the reaction in step S3 to be complete is 30 minutes.
6. A ratiometric fluorescence system based on o-phenylenediamine and sodium ascorbate and its detection of copper ions in water, characterized in that, The standard curve for detecting copper ion concentration is plotted based on the fluorescence intensity ratio of the ratio fluorescence system.
7. The method for plotting the standard curve as described in claim 6, characterized in that, The fluorescence intensity was determined using a fluorescence spectrophotometer, measuring the fluorescence emission intensities of the two products of the ratiometric fluorescence system, namely quinoxaline and 2,3-diaminophenazine, at 435 nm and 560 nm, respectively.
8. The method for plotting the standard curve as described in claim 4, characterized in that, As the concentration of copper ions increases, the ratio of fluorescence intensity (Fi) of the ratiometric fluorescence system at 435 nm and 560 nm decreases. 560 / F 435 The concentration of copper ions showed a linear relationship with the concentration of copper ions in the range of 5-35 μmol / L.
9. The ratiometric fluorescence system based on o-phenylenediamine and sodium ascorbate as described in claim 1 and its detection of copper ions in water, characterized in that, The method has good selectivity.
10. The ratiometric fluorescence system based on o-phenylenediamine and sodium ascorbate as described in claim 1 and its detection of copper ions in water, characterized in that... The method exhibits good recovery rates in tap water sample testing.