Method for accurately detecting urea in water by using fluorescent probe technology
The fluorescent probe technology reacts with urea to detect urea in water, which solves the problems of unstable detection, high cost, complex operation and environmental pollution in traditional methods, and achieves high sensitivity, low cost, fast and environmentally friendly urea detection in water.
Patent Information
- Application Number
- CN202510401105.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-01
- Publication Date
- 2025-07-18
AI Technical Summary
Traditional water urea testing methods have problems such as unstable test results, high cost, complex operation and unenvironmental protection, and it is difficult to meet the fast, accurate, low-cost and environmentally friendly testing needs.
The fluorescent probe technology is used to react specifically with urea, and the urea content in water is accurately measured by detecting changes in fluorescence intensity, a fluorescent probe solution of a specific concentration is configured, and a simple equipment is used for detection, a standard curve is drawn and the urea concentration is calculated.
It achieves high sensitivity and accuracy, reduces detection errors, simplifies operational processes, reduces costs, and reduces environmental pollution. It is suitable for batch rapid testing and provides reliable water quality assessment data.
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of water quality detection, and specifically to a precise detection method for urea in water using fluorescence probe technology. Background Art
[0002] In the field of water quality detection, urea, as a common nitrogen-containing pollutant, its accurate determination is crucial for evaluating the degree of water body pollution, ensuring the safety of the water ecological environment, and the quality of drinking water. Traditional methods for detecting urea in water have many limitations. For example, although the enzyme colorimetric method has a certain degree of accuracy, the activity of the enzyme is easily affected by environmental factors such as temperature and pH value, resulting in poor stability of the detection results. In addition, the enzyme reagents are expensive, and the detection cost is relatively high. The diacetyl monoxime method requires the use of a large amount of toxic and harmful chemical reagents, such as concentrated sulfuric acid and thiosemicarbazide, which not only pose a potential threat to the health of operators but also cause environmental pollution. At the same time, this method has a cumbersome operation and a long detection process. In addition, ion chromatography equipment is expensive, with high maintenance costs, and requires high professional technical requirements for the detection personnel, which limits its application in grass-roots laboratories and on-site detections. These traditional methods are difficult to meet the requirements of rapid, accurate, low-cost, and environmentally friendly detection of urea in water, and there is an urgent need for a more advanced and efficient detection technology to solve these problems. Summary of the Invention
[0003] (I) Technical Problems to be Solved Aiming at the deficiencies of the existing technology, the present invention provides a precise detection method for urea in water using fluorescence probe technology, which has the advantages of high sensitivity and accuracy, and solves the problem that traditional methods are difficult to meet the requirements of rapid, accurate, low-cost, and environmentally friendly detection of urea in water.
[0004] (II) Technical Solutions To achieve the above-mentioned purposes of high sensitivity and accuracy, the present invention provides the following technical solutions: A precise detection method for urea in water using fluorescence probe technology, including the following detection steps: The first step: Prepare materials and instruments. Fluorescent probe solution: Select a fluorescent probe with high selectivity and sensitivity to urea, and configure a probe solution with a concentration of 1×10 -5 mol / L. The solvent is a phosphate buffer solution (PBS) with a buffer capacity to maintain the pH value at 7.4, ensuring the stability and activity of the probe during the detection process; Urea standard solution: Accurately weigh a certain amount of analytical grade urea, dissolve it in ultrapure water, and configure a series of standard solutions with different concentration gradients, such as 0.1 μmol / L, 0.5 μmol / L, 1 μmol / L, 5 μmol / L, 10 μmol / L, 50 μmol / L, and 100 μmol / L, for making a standard curve; Fluorescence Spectrophotometer: It has the functions of exciting wavelength and emission wavelength scanning, with a wavelength accuracy of ±1 nm, and can accurately measure fluorescence intensity; Cuvette: A quartz cuvette with a 1-cm optical path, used to hold the test solution to reduce errors caused by light absorption and scattering; Pipette and its matching tips: With measuring ranges of 10 μL, 100 μL, and 1000 μL respectively, used to accurately transfer various solutions; Ultra-pure Water Machine: It prepares ultra-pure water with a resistivity greater than 18 MΩ·cm, used for preparing solutions and cleaning experimental instruments; Standard Curve Plotting: Second step: Respectively transfer 1 mL of urea standard solutions with different concentrations into a series of clean cuvettes; Third step: Add 100 μL of the fluorescence probe solution to each cuvette, shake gently to make the probe fully react with urea; Fourth step: On the fluorescence spectrophotometer, set the excitation wavelength to 488 nm (adjusted according to the optimal excitation wavelength of the selected fluorescence probe), set the emission wavelength scanning range to 500 - 600 nm, measure the fluorescence emission intensity of each solution, and record the fluorescence intensity value at the emission wavelength of 520 nm (the characteristic emission wavelength after the probe binds to urea); Fifth step: Using the urea standard solution concentration as the abscissa and the corresponding fluorescence intensity value as the ordinate, plot the standard curve; Water Sample Detection: Sixth step: Collect representative water samples. If the water sample is turbid or has suspended matter, it needs to be filtered through a 0.45-μm filter membrane first to remove impurities and avoid interfering with the detection results; Seventh step: Take 1 mL of the filtered water sample into a clean cuvette, add 100 μL of the fluorescence probe solution, and shake well; Eighth step: According to the instrument parameters when plotting the standard curve, measure the fluorescence emission intensity of the water sample on the fluorescence spectrophotometer, and record the fluorescence intensity value at 520 nm; Result Calculation: Ninth step: Substitute the measured fluorescence intensity value of the water sample into the standard curve equation to calculate the concentration of urea in the water; Tenth step: To ensure the accuracy of the detection results, each water sample needs to be measured in parallel 3 times, take the average value as the final detection result, and calculate the relative standard deviation (RSD).
[0005] Preferably, in the fifth step, the equation of the standard curve y = 100x + 50 (y is the fluorescence intensity, x is the urea concentration, this equation is only an example, and it actually needs to be fitted according to experimental data) is obtained through linear regression analysis, and the linear correlation coefficient R² of the curve is determined. It should be ensured that R² is greater than 0.995 to ensure the reliability of the standard curve.
[0006] Preferably, in the seventh step, react for 10 minutes (the optimal reaction time determined according to the probe reaction kinetics) to allow the probe to fully bind to urea in the water sample.
[0007] Preferably, in the ninth step, if the measured fluorescence intensity value of the water sample is 150, according to the standard curve equation y = 100x + 50, the urea concentration x = (150 - 50) / 100 = 1 μmol / L can be calculated.
[0008] Preferably, in the tenth step, the RSD is less than 5%. If the RSD is greater than 5%, the detection needs to be performed again.
[0009] (III) Beneficial effects Compared with the prior art, the present invention provides an accurate detection method for urea in water using a fluorescence probe technology, having the following beneficial effects: 1. The accurate detection method for urea in water using a fluorescence probe technology realizes high sensitivity and accuracy: The fluorescence probe technology can accurately capture the subtle changes in fluorescence intensity, enabling high-precision detection of low-concentration urea. By setting specific excitation and emission wavelengths and strictly controlling the standard curve (linear correlation coefficient R² greater than 0.995), the urea content in water can be accurately determined, effectively reducing the detection error, significantly improving the detection accuracy compared with traditional methods, and providing more reliable data support for water quality assessment.
[0010] 2. The accurate detection method for urea in water using a fluorescence probe technology is simple and fast to operate: The operation process of this method is relatively simple. It only needs to transfer solutions, mix and react according to the steps and then measure the fluorescence intensity, without complex pretreatment processes. From the standard curve drawing to the water sample detection, the whole process takes a short time. Especially in the water sample detection link, the result can be obtained in a short time from sampling, greatly improving the detection efficiency and being suitable for the rapid detection of batch water samples.
[0011] 3. The accurate detection method for urea in water using a fluorescence probe technology has significant cost-effectiveness: Compared with the enzyme colorimetric method that uses expensive enzyme reagents and the ion chromatography method that relies on high-cost equipment, the preparation of the fluorescence probe solution required by the fluorescence probe technology is relatively simple and the cost is low. Moreover, the instrument equipment (fluorescence spectrophotometer) is common in general laboratories, without the need to purchase expensive special equipment additionally, reducing the detection cost and being conducive to popularization and application in a wider range of scenarios.
[0012] 4. The precise detection method of urea in water using fluorescence probe technology is environmentally friendly: This method avoids the use of a large amount of toxic and harmful chemical reagents, reduces environmental pollution and harm to the health of operators. The entire detection process is green and environmentally friendly, meeting the current development needs of environmentally friendly detection technologies. Strong stability: A phosphate buffer solution (PBS) with a pH value maintained at 7.4 is selected as the solvent for the fluorescence probe, ensuring the stability and activity of the probe during the detection process, reducing the interference of environmental factors on the detection results, and making the detection results have better stability and repeatability. Specific implementation method
[0013] Next, in combination with the embodiments of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.
[0014] Principle overview: This method is based on the specific reaction between the fluorescence probe and urea, which causes a change in fluorescence intensity. By detecting the change amount of fluorescence intensity, the content of urea in water can be accurately determined. After a specific fluorescence probe molecule binds to a urea molecule, its fluorescence emission spectrum will produce a quantifiable change, and this change has a linear relationship with the urea concentration, thereby realizing the quantitative analysis of urea in water: The present invention provides a technical solution, specifically, a precise detection method of urea in water using fluorescence probe technology, including the following detection steps: First step: Prepare materials and instruments, fluorescence probe solution: Select a fluorescence probe with high selectivity and sensitivity to urea, and configure a probe solution with a concentration of 1×10 -5 mol / L, and the solvent is a phosphate buffer solution (PBS) with a buffering capacity and a pH value maintained at 7.4 to ensure the stability and activity of the probe during the detection process; Urea standard solution: Accurately weigh a certain amount of analytical reagent grade urea, dissolve it in ultrapure water, and configure a series of standard solutions with different concentration gradients, such as 0.1 μmol / L, 0.5 μmol / L, 1 μmol / L, 5 μmol / L, 10 μmol / L, 50 μmol / L, and 100 μmol / L, for making a standard curve; Fluorescence spectrophotometer: It has functions of excitation wavelength and emission wavelength scanning, with a wavelength accuracy of ±1 nm, and can accurately measure fluorescence intensity; Cuvette: A quartz cuvette with a 1 cm optical path is used to hold the detection solution to reduce errors caused by light absorption and scattering; Pipettes and matching tips: with measuring ranges of 10 μL, 100 μL, and 1000 μL respectively, used for accurately pipetting various solutions; Ultra-pure water machine: used to prepare ultra-pure water with a resistivity greater than 18 MΩ·cm for preparing solutions and cleaning experimental instruments; Standard curve plotting: Second step: Pipette 1 mL of urea standard solutions with different concentrations into a series of clean cuvettes respectively; Third step: Add 100 μL of the fluorescent probe solution to each cuvette and gently shake well to allow the probe to fully react with urea; Fourth step: On the fluorescence spectrophotometer, set the excitation wavelength to 488 nm (adjusted according to the optimal excitation wavelength of the selected fluorescent probe), and the emission wavelength scanning range to 500 - 600 nm. Measure the fluorescence emission intensity of each solution and record the fluorescence intensity value at the emission wavelength of 520 nm (the characteristic emission wavelength after the probe binds to urea); Fifth step: Plot the standard curve with the urea standard solution concentration as the abscissa and the corresponding fluorescence intensity value as the ordinate; Among them, the equation of the standard curve y = 100x + 50 (where y is the fluorescence intensity and x is the urea concentration. This equation is only an example, and it actually needs to be fitted according to experimental data) is obtained through linear regression analysis. Determine the linear correlation coefficient R² of the curve, and ensure that R² is greater than 0.995 to ensure the reliability of the standard curve; Water sample detection: Sixth step: Collect representative water samples. If the water sample is turbid or has suspended matter, it needs to be filtered through a 0.45 μm filter membrane first to remove impurities and avoid interfering with the detection results; Seventh step: Take 1 mL of the filtered water sample into a clean cuvette, add 100 μL of the fluorescent probe solution, and shake well; Among them, react for 10 minutes (the optimal reaction time determined according to the probe reaction kinetics) to allow the probe to fully bind to the urea in the water sample; Eighth step: Measure the fluorescence emission intensity of the water sample on the fluorescence spectrophotometer according to the instrument parameters when plotting the standard curve, and record the fluorescence intensity value at 520 nm; Result calculation: Ninth step: Substitute the measured fluorescence intensity value of the water sample into the standard curve equation to calculate the urea concentration in the water; Among them, if the measured fluorescence intensity value of the water sample is 150, according to the standard curve equation y = 100x + 50, the urea concentration x = (150 - 50) / 100 = 1 μmol / L can be calculated; Step 10: To ensure the accuracy of the detection results, each water sample needs to be measured in parallel 3 times, and the average value is taken as the final detection result, and the relative standard deviation (RSD) is calculated; Among them, RSD is less than 5%. If RSD is greater than 5%, the detection needs to be carried out again.
[0015] Furthermore, this detection method realizes high sensitivity and accuracy: by using the fluorescence probe technology, the subtle changes in fluorescence intensity can be accurately captured, and high-precision detection of low-concentration urea can also be achieved. By setting specific excitation and emission wavelengths, and strictly controlling the standard curve (linear correlation coefficient R² is greater than 0.995), the urea content in water can be accurately measured, effectively reducing the detection error, significantly improving the detection accuracy compared with traditional methods, and providing more reliable data support for water quality assessment.
[0016] Furthermore, this detection method is simple and fast to operate: the operation process of this method is relatively simple. Only need to transfer the solution step by step, mix and react, and then measure the fluorescence intensity, without complex pretreatment processes. From the drawing of the standard curve to the detection of water samples, the whole process takes a short time. Especially in the water sample detection link, the result can be obtained in a short time from sampling, greatly improving the detection efficiency and being suitable for the rapid detection of batch water samples.
[0017] Furthermore, this detection method has significant cost-effectiveness: compared with the enzyme colorimetric method that uses expensive enzyme reagents and the ion chromatography method that relies on high-cost equipment, the preparation of the fluorescence probe solution required by the fluorescence probe technology is relatively simple and the cost is low, and the instrument equipment (fluorescence spectrophotometer) is common in general laboratories, without the need to purchase expensive special equipment additionally, reducing the detection cost and being conducive to popularization and application in a wider range of scenarios.
[0018] Furthermore, this detection method is environmentally friendly: this method avoids the use of a large number of toxic and harmful chemical reagents, reduces environmental pollution and harm to the health of operators, and the whole detection process is green and environmentally friendly, meeting the current development needs of environmentally friendly detection technologies; Strong stability: Select a phosphate buffer solution (PBS) with a pH value maintained at 7.4 as the solvent for the fluorescence probe, ensuring the stability and activity of the probe during the detection process, reducing the interference of environmental factors on the detection results, and making the detection results have better stability and repeatability.
[0019] Although the embodiments of the present invention have been shown and described, for those of ordinary skill in the art, it can be understood that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and spirit of the present invention. The scope of the present invention is defined by the appended claims and their equivalents.
Claims
1. An accurate detection method for urea in water using fluorescence probe technology, characterized in that: It includes the following detection steps: Step 1: Prepare materials and instruments. Fluorescent probe solution: Select a fluorescent probe with high selectivity and sensitivity to urea, and prepare a probe solution with a concentration of 1×10 -5 mol / L. The solvent is phosphate buffer solution (PBS) with a buffer capacity to maintain a pH value of 7.4 to ensure the stability and activity of the probe during the detection process; Urea standard solution: Weigh a certain amount of analytical reagent grade urea accurately and dissolve it in ultrapure water to prepare a series of standard solutions with different concentration gradients, such as 0.1 μmol / L, 0.5 μmol / L, 1 μmol / L, 5 μmol / L, 10 μmol / L, 50 μmol / L, and 100 μmol / L, for making the standard curve; Fluorescence spectrophotometer: It has the functions of excitation wavelength and emission wavelength scanning, with a wavelength accuracy of ±1 nm, and can accurately measure the fluorescence intensity; Cuvette: A quartz cuvette with a 1-cm optical path length, used to hold the detection solution to reduce the errors caused by light absorption and scattering; Pipette and matching tips: With measuring ranges of 10 μL, 100 μL, and 1000 μL respectively, used to accurately transfer various solutions; Ultrapure water machine: Prepare ultrapure water with a resistivity greater than 18 MΩ·cm, used for preparing solutions and cleaning experimental instruments; Drawing of the standard curve: The second step: Transfer 1 mL of urea standard solutions with different concentrations to a series of clean cuvettes respectively; The third step: Add 100 μL of the fluorescence probe solution to each cuvette, shake gently to make the probe react fully with urea; The fourth step: On the fluorescence spectrophotometer, set the excitation wavelength to 488 nm (adjusted according to the optimal excitation wavelength of the selected fluorescence probe), and the emission wavelength scanning range to 500 - 600 nm. Measure the fluorescence emission intensity of each solution, and record the fluorescence intensity value at the emission wavelength of 520 nm (the characteristic emission wavelength after the probe binds to urea); The fifth step: Take the concentration of the urea standard solution as the abscissa and the corresponding fluorescence intensity value as the ordinate to draw the standard curve; Detection of water samples: The sixth step: Collect representative water samples. If the water samples are turbid or have suspended substances, they need to be filtered through a 0.45-μm filter membrane first to remove impurities and avoid interfering with the detection results; The seventh step: Take 1 mL of the filtered water sample into a clean cuvette, add 100 μL of the fluorescence probe solution, and shake well; The eighth step: According to the instrument parameters when drawing the standard curve, measure the fluorescence emission intensity of the water sample on the fluorescence spectrophotometer, and record the fluorescence intensity value at 520 nm; Result calculation: The ninth step: Substitute the measured fluorescence intensity value of the water sample into the standard curve equation to calculate the concentration of urea in the water; The tenth step: To ensure the accuracy of the detection results, each water sample needs to be measured in parallel 3 times, and the average value is taken as the final detection result, and the relative standard deviation (RSD) is calculated.
2. The precise detection method of urea in water using the fluorescence probe technique according to claim 1, characterized in that: In the fifth step, the equation of the standard curve y = 100x + 50 (y is the fluorescence intensity, x is the urea concentration, this equation is only an example, and it actually needs to be fitted according to the experimental data) is obtained through linear regression analysis, and the linear correlation coefficient R² of the curve is determined. It should be ensured that R² is greater than 0.995 to ensure the reliability of the standard curve.
3. The precise detection method of urea in water using the fluorescence probe technology according to claim 1, characterized in that: In the seventh step, react for 10 minutes (the optimal reaction time determined according to the probe reaction kinetics) to make the probe bind fully with the urea in the water sample.
4. The precise detection method of urea in water using a fluorescence probe technique according to claim 1, characterized in that: In the ninth step, if the measured fluorescence intensity value of the water sample is 150, according to the standard curve equation y = 100x + 50, the urea concentration x = (150 - 50) / 100 = 1 μmol / L can be calculated.
5. The precise detection method for urea in water using the fluorescence probe technology according to claim 1, characterized in that: In the tenth step, the RSD is less than 5%. If the RSD is greater than 5%, re-detection is required.