Method for determining activity of trace aryl sulfatase and activity of beta-glucuronidase in water body
By adding a specific substrate to the water sample and concentrating the product by solid-phase extraction method, the amount of nitrophenol was determined, and the problems of low sensitivity and high background interference in the prior art were solved, and high sensitivity enzyme activity detection was achieved.
Patent Information
- Application Number
- CN202510338908.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-21
- Publication Date
- 2025-06-24
AI Technical Summary
The prior art has low sensitivity and high background interference when determining the activity of trace aromatic sulfate and β-glucuronidase in water bodies, so it is impossible to effectively detect the enzyme activity in samples such as urine, environmental water bodies, etc.
The product p-nitrophenol potassium sulfate or p-nitrophenol-β-glucuronidin sodium salt was added to the water sample, and the product p-nitrophenol was concentrated by solid phase extraction. The amount of p-nitrophenol was then determined by spectrophotometer to calculate the enzyme activity. This method inhibits β-glucuronidase by using 28-32% hydrogen peroxide and a solution with pH = 2 to inhibit β-glucuronidase, reducing background interference.
It significantly improves the sensitivity to detect the activities of aromatic sulfate esterase and β-glucuronidase in water samples, reduces background interference, and can accurately determine the activity of trace enzymes in the samples, with a wide range of medical and environmental application prospects.
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Figure CN120193048A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of determination of enzyme activity, and particularly relates to a method for determining the activities of trace arylsulfatase and β-glucuronidase in water bodies. Background Art
[0002] Arylsulfatase and β-glucuronidase are widely present in animals, humans and the environment. In the human body, the functions of these two enzymes are to participate in the metabolism of organisms. Abnormalities in the activities of the two enzymes can lead to the occurrence of various diseases. In medicine, the activities of these two enzymes in the human body are often measured to diagnose diseases such as breast cancer, rectal cancer, pancreatic cancer, etc. In the environment, these two enzymes will decompose the conjugates of human metabolites such as estrogens, triclosan and various drugs discharged into the environment by the human body, and the levels of the activities of these two enzymes will affect the degradation rates of these pollutants. Therefore, detecting the activities of trace (less than 0.1 U / mL) arylsulfatase and β-glucuronidase is of great significance both in the environmental and medical fields. However, the current traditional methods have disadvantages such as low sensitivity and large background interference when measuring the activities of the two enzymes. Moreover, the activities of the above two enzymes in samples such as urine and environmental water bodies are extremely low, and the traditional methods cannot be used for determination. Therefore, there is an urgent need for a method for determining the activities of trace arylsulfatase and β-glucuronidase. Summary of the Invention
[0003] In order to overcome the above-mentioned disadvantages and deficiencies of the prior art, the purpose of the present invention is to provide a method for determining the activity of trace arylsulfatase in water bodies, which significantly improves the sensitivity of detecting the activity of arylsulfatase in water body samples.
[0004] Another purpose of the present invention is to provide a method for determining the activity of trace β-glucuronidase in water bodies, which significantly improves the sensitivity of detecting the activity of β-glucuronidase in water body samples.
[0005] The purpose of the present invention is achieved by the following technical solutions:
[0006] The present invention provides a method for determining the activity of trace arylsulfatase in water bodies, comprising the following steps:
[0007] (1) Add potassium p-nitrophenyl sulfate to the water body sample, seal the reaction container, react at 37 °C for 1 to 4 hours, and add hydrogen peroxide with a mass concentration of 28 to 32% to terminate the reaction;
[0008] (2) Concentrate p-nitrophenol in the solution obtained in step (1) by solid-phase extraction, measure the amount of p-nitrophenol in the concentrated p-nitrophenol solution with a spectrophotometer, and then calculate the activity of arylsulfatase.
[0009] Preferably, the p-nitrophenol in the solution obtained in step (1) is concentrated by solid-phase extraction, and the concentrated p-nitrophenol solution is measured by a spectrophotometer, specifically as follows:
[0010] The solution obtained in step (1) is filtered to remove impurities, and the filtered solution is passed through a solid-phase extraction column. After the solid-phase extraction is completed, the solid-phase extraction column is rinsed. After the rinsing is completed, the target substance in the solid-phase extraction column is eluted with a methanol solution, and the eluate is collected in the dark. Then, the amount of p-nitrophenol in the eluate is measured by a spectrophotometer;
[0011] Among them, the volume of the eluate is 4 - 10 mL.
[0012] Preferably, the concentration of hydrogen peroxide in the solution obtained in step (1) is greater than 10 mM.
[0013] Preferably, adding potassium p-nitrophenol sulfate to the water sample in step (1) is specifically as follows:
[0014] The water sample is preheated in a water bath at 37°C for 8 - 12 minutes, and then potassium p-nitrophenol sulfate with a concentration of 4 - 5 mM is added to the water sample; among them, the volume ratio of potassium p-nitrophenol sulfate to the volume of the water sample is 1:(500 - 1000).
[0015] Preferably, concentrating the p-nitrophenol in the solution obtained in step (1) by solid-phase extraction in step (2) is specifically as follows:
[0016] The solution obtained in step (1) is filtered, and the filtered solution passes through the solid-phase extraction column at a speed of 15 - 25 mL / min;
[0017] After the solid-phase extraction is completed, the solid-phase extraction column is rinsed successively with an aqueous solution with pH = 2 and a methanol / aqueous solution at a speed of 0.8 - 1.2 mL / min, where the volume ratio of methanol to water in the methanol / aqueous solution is 6:4;
[0018] After the rinsing is completed, the target substance in the solid-phase extraction column is eluted with a methanol solution at a speed of 0.8 - 1.2 mL / min, and the eluate is collected in the dark.
[0019] Preferably, when the water sample is river water, the volume of the water sample is 0.5 - 1 L; when the water sample is sewage, the volume of the water sample is 0.1 - 1 L; when the water sample is urine, the volume of the water sample is 10 - 50 mL.
[0020] Preferably, in step (2), the solution obtained in step (1) is filtered to remove impurities, specifically: the solution is further passed through a glass fiber filter membrane with a pore size of 0.22 or 0.45 μm to remove impurities.
[0021] The present invention provides a method for determining the activity of trace β-glucuronidase in water, comprising the following steps:
[0022] (1) Add sodium salt of p-nitrophenyl-β-glucuronide to the water sample, seal the reaction vessel, react at 37 °C for 1 to 4 hours, and adjust the pH of the solution to 2 to terminate the reaction;
[0023] (2) Concentrate p-nitrophenol in the solution obtained in step (1) by solid-phase extraction, measure the amount of p-nitrophenol in the concentrated p-nitrophenol solution with a spectrophotometer, and then calculate the activity of arylsulfatase.
[0024] Preferably, in step (2), the method of concentrating p-nitrophenol in the solution obtained in step (1) by solid-phase extraction and measuring the amount of p-nitrophenol in the concentrated p-nitrophenol solution with a spectrophotometer is specifically as follows:
[0025] Filter the solution obtained in step (1) to remove impurities, pass the filtered solution through a solid-phase extraction column, wash the solid-phase extraction column after the solid-phase extraction is completed, after the washing is completed, elute the target substance in the solid-phase extraction column with a methanol solution, collect the eluate in the dark, and then measure the amount of p-nitrophenol in the eluate with a spectrophotometer;
[0026] Among them, the volume of the eluate is 4 to 10 mL.
[0027] Preferably, the volume concentration of hydrochloric acid in step (1) is 25 to 50%.
[0028] Preferably, in step (1), the addition of potassium p-nitrophenyl sulfate to the water sample is specifically as follows:
[0029] Preheat the water sample in a water bath at 37 °C for 8 to 12 minutes, and then add potassium p-nitrophenyl sulfate with a concentration of 4 to 5 mM to the water sample; the ratio of the volume of potassium p-nitrophenyl sulfate to the volume of the water sample is 1:(500 to 1000).
[0030] Preferably, in step (2), the method of concentrating p-nitrophenol in the solution obtained in step (1) by solid-phase extraction is specifically as follows:
[0031] Filter the solution obtained in step (1), and pass the filtered solution through the solid-phase extraction column at a speed of 15 to 25 mL / min;
[0032] After solid-phase extraction, the solid-phase extraction column was sequentially rinsed with an aqueous solution with a pH of 2 and a methanol / water solution at a rate of 0.8 - 1.2 mL / min, where the volume ratio of methanol to water in the methanol / water solution was 6:4;
[0033] After rinsing, the target substance in the solid-phase extraction column was eluted with a methanol solution at a rate of 0.8 - 1.2 mL / min, and the eluate was collected in the dark.
[0034] Preferably, when the water sample is river water, the volume of the water sample is 0.5 - 1 L; when the water sample is sewage, the volume of the water sample is 0.1 - 1 L; when the water sample is urine, the volume of the water sample is 10 - 50 mL.
[0035] Preferably, in step (2), the solution obtained in step (1) was filtered to remove impurities, specifically: the solution was further passed through a glass fiber filter membrane with a pore size of 0.22 or 0.45 μm to remove impurities.
[0036] Compared with the prior art, the present invention has the following advantages and beneficial effects:
[0037] In the present invention, potassium 4-nitrophenyl sulfate, an arylsulfatase substrate, is added to the water sample or sodium 4-nitrophenyl-β-D-glucuronide, a β-glucuronidase substrate, is added to the water sample. Both of these enzymes can produce the product 4-nitrophenol. Since the amount of the product is extremely low, it cannot be directly measured. Therefore, the product 4-nitrophenol is concentrated using a solid-phase extraction column, and then the amount of 4-nitrophenol is measured with a spectrophotometer to measure the enzyme activity. To prevent the continuous production of the product during the concentration of the water sample from affecting the accuracy of the measurement results, 28 - 32% hydrogen peroxide is used in the present invention to effectively inhibit arylsulfatase, and a solution with a pH of 2 is used to effectively inhibit β-glucuronidase. The present invention has a low detection limit and little background interference, and can accurately measure the activities of the two enzymes in the sample when the traditional methods for measuring the activities of the two enzymes are ineffective, and has a wide application prospect in the medical and environmental fields. BRIEF DESCRIPTION OF THE DRAWINGS
[0038] Figure 1 It is the standard curve of 4-nitrophenol drawn in the examples of the present invention. DETAILED DESCRIPTION OF THE INVENTION
[0039] The following is a further detailed description of the present invention in conjunction with the examples, but the implementation manners of the present invention are not limited thereto.
[0040] For the examples of the present invention, p-nitrophenol (purity > 98%) was purchased from Sigma (Shanghai) Company; p-nitrophenyl potassium sulfate (purity > 98%) was purchased from Sigma (Shanghai) Company; p-nitrophenyl-β-D-glucuronide sodium salt (purity > 98%) was purchased from Sigma (Shanghai) Company. The solid phase extraction column used was an Oasis HLB column (200 mg, 6 cc, 30 μm pore size) purchased from Waters Company.
[0041] In the following examples, the standard curve was drawn by the following method:
[0042] Take 7 10-mL test tubes, and add 0, 40, 80, 120, 160, 200, 240 μL of p-nitrophenol solution with a concentration of 100 μg / mL respectively, and then make up to 4 mL with methanol solution. At this time, the concentrations of p-nitrophenol in each test tube are 0, 1, 2, 3, 4, 5, 6 μg / mL respectively. Then add 1 mL of 0.5 M NaOH solution to each test tube, shake well, and measure the absorbance at 400 nm with a spectrophotometer to draw the p-nitrophenol standard curve.
[0043] Example 1
[0044] Determination of arylsulfatase activity in river water:
[0045] Add 1 L of river water sample to a 1-L conical flask, preheat it in a 37°C water bath for 10 minutes, then add 1 mL of p-nitrophenyl potassium sulfate with a concentration of 5 mM, and shake well. Seal it with a sealing film to prevent liquid evaporation and react at 37°C for 4 hours. After the reaction, add 1 mL of 30% hydrogen peroxide solution to terminate the reaction. Then pass the solution through a 0.22-μm glass fiber filter membrane to remove impurities. Pass the filtered solution through a solid phase extraction device and flow through the solid phase extraction column at a speed of 20 mL / min. The solid phase extraction column was pre-activated with 10 mL of methanol and 10 mL of ultrapure water. After solid phase extraction, wash the solid phase extraction column with 10 mL of aqueous solution with pH = 2 and methanol / water (6 / 4) solution at a speed of 1 mL / min respectively. After washing, elute the target substance in the solid phase extraction column with 4 mL of methanol solution at a speed of 1 mL / min, and collect the eluate with a 10-mL brown glass tube. Then add 1 mL of 0.5 M NaOH solution to the glass tube, shake well, and measure the absorbance at 400 nm wavelength with a spectrophotometer.
[0046] Example 2
[0047] Determination of β-glucuronidase activity in river water:
[0048] Add 1 L of river water sample into a 1-L conical flask, preheat it in a water bath at 37 °C for 10 minutes, then add 1 mL of sodium 4-nitrophenyl-β-D-glucuronide with a concentration of 5 mM, and shake well. Seal it with a sealing film to prevent liquid evaporation and react at 37 °C for 4 hours. After the reaction, adjust the pH of the solution to 2 with 50% hydrochloric acid to terminate the reaction. Then pass the solution through a 0.22-μm glass fiber filter membrane to remove impurities. Pass the filtered solution through a solid-phase extraction device and flow through the solid-phase extraction column at a speed of 20 mL / min. The solid-phase extraction column has been pre-activated with 10 mL of methanol and 10 mL of ultrapure water. After the solid-phase extraction, rinse the solid-phase extraction column with 10 mL of aqueous solution with pH = 2 and methanol / water (6 / 4) solution at a speed of 1 mL / min respectively. After the rinsing, elute the target substance in the solid-phase extraction column with 4 mL of methanol solution at a speed of 1 mL / min, and collect the eluate with a 10-mL brown glass tube. Then add 1 mL of 0.5 M NaOH solution into the glass tube, shake well and measure the absorbance with a spectrophotometer at a wavelength of 400 nm.
[0049] Example 3
[0050] Determination of arylsulfatase activity in sewage:
[0051] Add 500 mL of sewage sample into a 1-L conical flask, preheat it in a water bath at 37 °C for 10 minutes, then add 1 mL of potassium 4-nitrophenyl sulfate with a concentration of 5 mM, and shake well. Seal it with a sealing film to prevent liquid evaporation and react at 37 °C for 4 hours. After the reaction, add 1 mL of 30% hydrogen peroxide solution to terminate the reaction. Then pass the solution through a 0.22-μm glass fiber filter membrane to remove impurities. Pass the filtered solution through a solid-phase extraction device and flow through the solid-phase extraction column at a speed of 20 mL / min. The solid-phase extraction column has been pre-activated with 10 mL of methanol and 10 mL of ultrapure water. After the solid-phase extraction, rinse the solid-phase extraction column with 10 mL of aqueous solution with pH = 2 and methanol / water (6 / 4) solution at a speed of 1 mL / min respectively. After the rinsing, elute the target substance in the solid-phase extraction column with 4 mL of methanol solution at a speed of 1 mL / min, and collect the eluate with a 10-mL brown glass tube. Then add 1 mL of 0.5 M NaOH solution into the glass tube, shake well and measure the absorbance with a spectrophotometer at a wavelength of 400 nm.
[0052] Example 4
[0053] Determination of β-glucuronidase activity in sewage:
[0054] Add 500 mL of sewage sample into a 1 L conical flask, preheat it in a water bath at 37 °C for 10 minutes, then add 1 mL of sodium nitrophenyl-β-D-glucuronide with a concentration of 5 mM, and shake well. Seal it with a sealing film to prevent liquid evaporation, and react at 37 °C for 4 hours. After the reaction, adjust the pH of the solution to 2 with 50% hydrochloric acid to terminate the reaction. Then pass the solution through a 0.22 μm glass fiber filter membrane to remove impurities. Pass the filtered solution through a solid-phase extraction device and flow through the solid-phase extraction column at a speed of 20 mL / min. The solid-phase extraction column has been pre-activated with 10 mL of methanol and 10 mL of ultrapure water. After solid-phase extraction, rinse the solid-phase extraction column with 10 mL of aqueous solution with pH = 2 and methanol / water (6 / 4) solution at a speed of 1 mL / min respectively. After rinsing, elute the target substance in the solid-phase extraction column with 4 mL of methanol solution at a speed of 1 mL / min, and collect the eluate with a 10 mL brown glass tube. Then add 1 mL of 0.5 M NaOH solution to the glass tube, shake well, and measure the absorbance with a spectrophotometer at a wavelength of 400 nm.
[0055] Example 5
[0056] Determination of arylsulfatase activity in urine:
[0057] Add 50 mL of urine sample into a 100 mL conical flask, preheat it in a water bath at 37 °C for 10 minutes, then add 1 mL of potassium p-nitrophenyl sulfate with a concentration of 5 mM, and shake well. Seal it with a sealing film to prevent liquid evaporation, and react at 37 °C for 4 hours. After the reaction, add 1 mL of 30% hydrogen peroxide solution to terminate the reaction. Then pass the solution through a 0.22 μm glass fiber filter membrane to remove impurities. Pass the filtered solution through a solid-phase extraction device and flow through the solid-phase extraction column at a speed of 20 mL / min. The solid-phase extraction column has been pre-activated with 10 mL of methanol and 10 mL of ultrapure water. After solid-phase extraction, rinse the solid-phase extraction column with 10 mL of aqueous solution with pH = 2 and methanol / water (6 / 4) solution at a speed of 1 mL / min respectively. After rinsing, elute the target substance in the solid-phase extraction column with 4 mL of methanol solution at a speed of 1 mL / min, and collect the eluate with a 10 mL brown glass tube. Then add 1 mL of 0.5 M NaOH solution to the glass tube, shake well, and measure the absorbance with a spectrophotometer at a wavelength of 400 nm.
[0058] Example 6
[0059] Determination of β-glucuronidase activity in urine:
[0060] Add 50 mL of urine sample into a 100 mL conical flask, preheat it in a water bath at 37 °C for 10 minutes, then add 1 mL of sodium 4-nitrophenyl-β-D-glucuronide with a concentration of 5 mM, and shake well. Seal it with a sealing film to prevent liquid evaporation and react at 37 °C for 4 hours. After the reaction, adjust the pH of the solution to 2 with 50% hydrochloric acid to terminate the reaction. Then pass the solution through a 0.22 μm glass fiber filter membrane to remove impurities. Pass the filtered solution through a solid-phase extraction device and flow through the solid-phase extraction column at a speed of 20 mL / min. The solid-phase extraction column has been pre-activated with 10 mL of methanol and 10 mL of ultrapure water. After solid-phase extraction, wash the solid-phase extraction column with 10 mL of aqueous solution with pH = 2 and methanol / water (6 / 4) solution at a speed of 1 mL / min respectively. After washing, elute the target substance in the solid-phase extraction column with 4 mL of methanol solution at a speed of 1 mL / min, and collect the eluate with a 10 mL brown glass tube. Then add 1 mL of 0.5 M NaOH solution to the glass tube, shake well and measure the absorbance with a spectrophotometer at a wavelength of 400 nm.
[0061] Control example
[0062] Take six test tubes, three with a specification of 10 mL and three with a specification of 20 mL. Use the traditional method to measure the activities of arylsulfatase (refer to Tabatabai and Bremner, Soil Science Society of America Journal, 1970, 34: 225-229) and β-glucuronidase (refer to Jung et al., The role of intestinal microflora in anti-inflammatory effect of baicalin in mice, Biomol Ther (Seoul), 20(1)(2012), pp. 36-42) in river water, sewage and urine respectively.
[0063] Perform the following operations on these six test tubes:
[0064] Test tube 1:
[0065] Add 4 mL of 0.5 M acetic acid-sodium acetate buffer (pH 5.8) into test tube 1, then add 1 mL of sodium 4-nitrophenyl sulfate solution with a concentration of 5 mM, and then add 1 mL of river water to be tested. After shaking well, place the test tube in a water bath at 37 °C and react for 1 hour, then add 4 mL of 0.5 mol / L sodium hydroxide to terminate the reaction, and then measure the absorbance with a spectrophotometer at a wavelength of 400 nm. This is the method for measuring arylsulfatase in river water by the conventional method.
[0066] Test tube 2:
[0067] Add 0.8 mL of 0.1 M disodium hydrogen phosphate - sodium dihydrogen phosphate solution (pH 7.0) to test tube 2, then add 0.2 mL of a 5 mM solution of sodium salt of p-nitrophenyl-β-glucuronide, and then add 0.2 mL of the river water to be tested. After shaking well, place the test tube in a water bath at 37 °C and react for 1 hour. Then add 0.8 mL of 0.5 mol / L sodium hydroxide to terminate the reaction, and then measure the absorbance with a spectrophotometer at a wavelength of 400 nm. This is the conventional method for measuring β-glucuronidase in river water.
[0068] Test tube 3:
[0069] Add 4 mL of 0.5 M acetic acid - sodium acetate buffer solution (pH 5.8) to test tube 3, then add 1 mL of a 5 mM solution of sodium p-nitrophenyl sulfate, and then add 1 mL of the sewage to be tested. After shaking well, place the test tube in a water bath at 37 °C and react for 1 hour. Then add 4 mL of 0.5 mol / L sodium hydroxide to terminate the reaction, and then measure the absorbance with a spectrophotometer at a wavelength of 400 nm. This is the conventional method for measuring arylsulfatase in sewage.
[0070] Test tube 4:
[0071] Add 0.8 mL of 0.1 M disodium hydrogen phosphate - sodium dihydrogen phosphate solution (pH 7.0) to test tube 4, then add 0.2 mL of a 5 mM solution of sodium salt of p-nitrophenyl-β-glucuronide, and then add 0.2 mL of the sewage to be tested. After shaking well, place the test tube in a water bath at 37 °C and react for 1 hour. Then add 0.8 mL of 0.5 mol / L sodium hydroxide to terminate the reaction, and then measure the absorbance with a spectrophotometer at a wavelength of 400 nm. This is the conventional method for measuring β-glucuronidase in sewage.
[0072] Test tube 5:
[0073] Add 4 mL of 0.5 M acetic acid - sodium acetate buffer solution (pH 5.8) to test tube 5, then add 1 mL of a 5 mM solution of sodium p-nitrophenyl sulfate, and then add 1 mL of the urine to be tested. After shaking well, place the test tube in a water bath at 37 °C and react for 1 hour. Then add 4 mL of 0.5 mol / L sodium hydroxide to terminate the reaction, and then measure the absorbance with a spectrophotometer at a wavelength of 400 nm. This is the conventional method for measuring arylsulfatase in urine.
[0074] Test tube 6:
[0075] Add 0.8 mL of 0.1 M disodium hydrogen phosphate - sodium dihydrogen phosphate solution (pH 7.0) to test tube 6, then add 0.2 mL of 5 mM sodium salt solution of p-nitrophenyl-β-glucuronide, and then add 0.2 mL of the urine sample to be tested. After shaking well, place the test tube in a water bath at 37 °C and react for 1 hour. Then add 0.8 mL of 0.5 mol / L sodium hydroxide to terminate the reaction, and then measure the absorbance with a spectrophotometer at a wavelength of 400 nm. This is the conventional method for measuring β-glucuronidase in urine.
[0076] The test results of Examples 1 - 6 and the comparative examples are shown in Table 1.
[0077] Table 1 Summary of Test Results
[0078]
[0079] *U is defined as the production of 1 μg of p-nitrophenol per hour;
[0080] *ND indicates that no enzyme activity was detected
[0081] As can be seen from Table 1, the present invention can accurately measure the enzyme activities of trace arylsulfatase and β-glucuronidase in river water, sewage, and urine samples. The measurement results show that in river water, the enzyme activities of these two enzymes are the lowest, while in urine, the enzyme activities of these two enzymes are the highest. However, the traditional methods cannot detect the enzyme activities of arylsulfatase and β-glucuronidase in river water, sewage, and urine samples.
[0082] The accuracy of the present invention is mainly verified by measuring the recovery rate of p-nitrophenol in water samples.
[0083] Recovery rate of p-nitrophenol in river water:
[0084] Take 12 conical flasks, and add 1 L of river water samples to each. Then divide these 12 conical flasks into four groups. Add 80 μL of p-nitrophenol solution with a concentration of 100 μg / mL to the first group, 120 μL of p-nitrophenol solution with a concentration of 100 μg / mL to the second group, 240 μL of p-nitrophenol solution with a concentration of 100 μg / mL to the third group, and the fourth group without adding p-nitrophenol solution serves as the blank control group. Then mix the solutions and remove impurities through a 0.22-μm glass fiber filter membrane. Pass the filtered solution through a solid-phase extraction device and let it flow through the solid-phase extraction column at a speed of 20 mL / min. The solid-phase extraction column has been pre-activated with 10 mL of methanol and 10 mL of ultrapure water. After the solid-phase extraction is completed, wash the solid-phase extraction column with 10 mL of aqueous solution with pH = 2 and methanol / water (6 / 4) solution at a speed of 1 mL / min respectively. After the washing is completed, elute the target substance in the solid-phase extraction column with 4 mL of methanol solution at a speed of 1 mL / min, and collect the eluate with a 10-mL brown glass tube. Then add 1 mL of 0.5 M NaOH solution to the glass tube, shake well, and measure the absorbance with a spectrophotometer at a wavelength of 400 nm.
[0085] Recovery rate of p-nitrophenol in sewage:
[0086] Take 12 conical flasks, and add 500 mL of sewage samples to each. Then divide these 12 conical flasks into four groups. Add 80 μL of p-nitrophenol solution with a concentration of 100 μg / mL to the first group, 120 μL of p-nitrophenol solution with a concentration of 100 μg / mL to the second group, 240 μL of p-nitrophenol solution with a concentration of 100 μg / mL to the third group, and the fourth group without adding p-nitrophenol solution serves as the blank control group. Then mix the solutions and remove impurities through a 0.22-μm glass fiber filter membrane. Pass the filtered solution through a solid-phase extraction device and let it flow through the solid-phase extraction column at a speed of 20 mL / min. The solid-phase extraction column has been pre-activated with 10 mL of methanol and 10 mL of ultrapure water. After the solid-phase extraction is completed, wash the solid-phase extraction column with 10 mL of aqueous solution with pH = 2 and methanol / water (6 / 4) solution at a speed of 1 mL / min respectively. After the washing is completed, elute the target substance in the solid-phase extraction column with 4 mL of methanol solution at a speed of 1 mL / min, and collect the eluate with a 10-mL brown glass tube. Then add 1 mL of 0.5 M NaOH solution to the glass tube, shake well, and measure the absorbance with a spectrophotometer at a wavelength of 400 nm.
[0087] Recovery rate of p-nitrophenol in urine:
[0088] Take 12 conical flasks, and add 50 mL of urine samples to each. Then divide these 12 conical flasks into four groups. Add 80 μL of p-nitrophenol solution with a concentration of 100 μg / mL to the first group, 120 μL of p-nitrophenol solution with a concentration of 100 μg / mL to the second group, 240 μL of p-nitrophenol solution with a concentration of 100 μg / mL to the third group, and the fourth group without adding p-nitrophenol solution as a blank control group. Then mix the solutions and remove impurities through a 0.22 μm glass fiber filter membrane. Pass the filtered solution through a solid-phase extraction device and flow through the solid-phase extraction column at a speed of 20 mL / min. The solid-phase extraction column has been pre-activated with 10 mL of methanol and 10 mL of ultrapure water. After the solid-phase extraction is completed, wash the solid-phase extraction column with 10 mL of aqueous solution with pH = 2 and methanol / water (6 / 4) solution at a speed of 1 mL / min respectively. After the washing is completed, elute the target substance in the solid-phase extraction column with 4 mL of methanol solution at a speed of 1 mL / min, and collect the eluate with a 10 mL brown glass tube. Then add 1 mL of 0.5 M NaOH solution to the glass tube, shake well, and measure the absorbance with a spectrophotometer at a wavelength of 400 nm.
[0089] The recovery rate of p-nitrophenol is calculated according to the following formula:
[0090]
[0091] Where: Ci represents the measured concentration of p-nitrophenol in the spiked sample (μg / L); C0 represents the background concentration of p-nitrophenol in the unspiked sample (μg / L); C S represents the spiked concentration (μg / L).
[0092] The recovery rates of the sewage, river water and urine samples used in the examples and comparative examples are shown in Table 2. The recovery rates of p-nitrophenol in different matrices are between 87.6% - 95.1%, indicating that this method has good accuracy.
[0093] Table 2 Recovery rates of p-nitrophenol in sewage, river water and urine (RSD%, n = 3)
[0094]
[0095] *RSD represents the standard deviation of three repeated samples
[0096] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the described embodiments. Any other changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.
Claims
1. A method for determining trace amounts of aromatic sulfatase activity in water, characterized in that: The following steps are involved: (1) adding p-nitrophenol potassium sulfate to a water sample, sealing the reaction container, reacting at 37° C. for 1 to 4 hours, and then adding hydrogen peroxide with a mass concentration of 28 to 32% to terminate the reaction; (2) Concentrating the p-nitrophenol in the solution obtained in step (1) by solid phase extraction, measuring the amount of p-nitrophenol in the concentrated p-nitrophenol solution by a spectrophotometer, and then calculating the aromatic sulfatase activity.
2. The method for determining trace aromatic sulfatase activity in water according to claim 1, characterized in that: Step (2) is to concentrate the p-nitrophenol in the solution obtained in step (1) by solid phase extraction, and measure the amount of p-nitrophenol in the concentrated p-nitrophenol solution by a spectrophotometer, specifically: The solution obtained in step (1) is filtered to remove impurities, and the filtered solution is passed through a solid phase extraction column. After the solid phase extraction is completed, the solid phase extraction column is rinsed. After the elution is completed, the target substance in the solid phase extraction column is eluted with a methanol solution, the eluate is collected in a dark place, and the amount of p-nitrophenol in the eluate is determined by a spectrophotometer; The volume of the eluent is 4 to 10 mL.
3. The method for determining trace aromatic sulfatase activity in water according to claim 1, characterized in that: The concentration of hydrogen peroxide in the solution obtained in step (1) is greater than 10 mM.
4. The method for determining trace aromatic sulfatase activity in water according to claim 1, characterized in that: The step (1) of adding potassium p-nitrophenol sulfate to the water sample is specifically: Preheat the water sample in a 37° C. water bath for 8 to 12 minutes, and then add p-nitrophenol potassium sulfate with a concentration of 4 to 5 mM to the water sample; wherein the ratio of the volume of p-nitrophenol potassium sulfate to the volume of the water sample is 1:(500 to 1000).
5. The method for determining trace aromatic sulfatase activity in water according to claim 1, characterized in that: Step (2) wherein the p-nitrophenol in the solution obtained in step (1) is concentrated by solid phase extraction, specifically: Filtering the solution obtained in step (1), and passing the filtered solution through a solid phase extraction column at a rate of 15 to 25 mL / min; After the solid phase extraction is completed, the solid phase extraction column is eluted with a pH = 2 aqueous solution and a methanol / water solution at a rate of 0.8 to 1.2 mL / min, wherein the volume ratio of methanol to water in the methanol / water solution is 6:4; After the elution is completed, the target substance in the solid phase extraction column is eluted with a methanol solution at a rate of 0.8 to 1.2 mL / min, and the eluate is collected in a dark place.
6. A method for determining trace β-glucuronidase activity in water, characterized in that: The following steps are involved: (1) adding p-nitrophenol-β-glucuronide sodium salt to a water sample, sealing the reaction container, reacting at 37° C. for 1 to 4 hours, and adjusting the solution pH to 2 to terminate the reaction; (2) Concentrating the p-nitrophenol in the solution obtained in step (1) by solid phase extraction, measuring the amount of p-nitrophenol in the concentrated p-nitrophenol solution by a spectrophotometer, and then calculating the aromatic sulfatase activity.
7. The method for determining trace β-glucuronidase activity in water according to claim 6, characterized in that: Step (2) is to concentrate the p-nitrophenol in the solution obtained in step (1) by solid phase extraction, and measure the amount of p-nitrophenol in the concentrated p-nitrophenol solution by a spectrophotometer, specifically: The solution obtained in step (1) is filtered to remove impurities, and the filtered solution is passed through a solid phase extraction column. After the solid phase extraction is completed, the solid phase extraction column is rinsed. After the elution is completed, the target substance in the solid phase extraction column is eluted with a methanol solution, the eluate is collected in a dark place, and the amount of p-nitrophenol in the eluate is determined by a spectrophotometer; The volume of the eluent is 4 to 10 mL.
8. The method for determining trace β-glucuronidase activity in water according to claim 6, characterized in that: The volume concentration of hydrochloric acid in step (1) is 25-50%.
9. The method for determining trace β-glucuronidase activity in water according to claim 6, characterized in that: The step (1) of adding potassium p-nitrophenol sulfate to the water sample is specifically: Preheat the water sample in a 37° C. water bath for 8 to 12 minutes, and then add p-nitrophenol potassium sulfate with a concentration of 4 to 5 mM to the water sample; wherein the ratio of the volume of p-nitrophenol potassium sulfate to the volume of the water sample is 1:(500 to 1000).
10. The method for determining trace β-glucuronidase activity in water according to claim 6, characterized in that: Step (2) wherein the p-nitrophenol in the solution obtained in step (1) is concentrated by solid phase extraction, specifically: Filtering the solution obtained in step (1), and passing the filtered solution through a solid phase extraction column at a rate of 15 to 25 mL / min; After the solid phase extraction is completed, the solid phase extraction column is eluted with a pH = 2 aqueous solution and a methanol / water solution at a rate of 0.8 to 1.2 mL / min, wherein the volume ratio of methanol to water in the methanol / water solution is 6:4; After the elution is completed, the target substance in the solid phase extraction column is eluted with a methanol solution at a rate of 0.8 to 1.2 mL / min, and the eluate is collected in a dark place.