CN110632052A - Fluorescence spectrum detection method for activity of soil arylsulfatase - Google Patents
Fluorescence spectrum detection method for activity of soil arylsulfatase Download PDFInfo
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Abstract
The invention discloses a fluorescence spectrum detection method for activity of soil arylsulfatase, and relates to the field of methods for detecting the activity of the soil enzyme. The method comprises the following steps: firstly, preparing a reagent: comprises an arylsulfatase fluorogenic substrate, an acetate buffer solution, a sodium hydroxide terminator, an MU standard substance and a sodium azide solution; II, a test process: the method comprises the steps of (1) preparing a microporous plate; (2) preparing a soil sample; (3) and (3) detection process: respectively transferring the soil suspension into all columns of the sample group by using an 8-channel liquid transfer gun, and then placing the microplate in an incubator with a standard measurement temperature of 30 ℃ for culturing for 3 hours; the microplate was removed, and 50 μ L of 0.5M NaOH was added to all wells to terminate the assay; setting parameters of an enzyme-labeling instrument, then placing a microporous plate into a feed inlet, and reading a measured value of a fluorescent plate of the enzyme-labeling instrument; and finally, calculating. Compared with the prior determination method, the method has the advantages of simple operation, stable and reliable analysis result and good reproducibility; the dosage of the medicine is small.
Description
Technical Field
The invention relates to the field of a method for measuring enzyme activity in soil, in particular to a fluorescence spectrum detection method for soil arylsulfatase activity.
Background
The soil arylsulfatase is a key enzyme in the soil thionin circulation process, can hydrolyze organic sulfur into sulfate, has strong and weak activity on the sulfur metabolism in soil and the activity of microorganisms, and has an indication effect on the soil fertility condition. The activity of the arylsulfatase in the soil is influenced by the physicochemical properties of the soil, such as soil moisture, temperature, texture, farmland farming measures, crop planting and artificial management measures, and therefore, the detection of the arylsulfatase is of great significance. So far, the determination of soil aryl sulfatase is mostly ultraviolet spectrophotometry, the activity of aryl sulfatase is estimated by using the content of released p-nitrophenol measured by 410nm colorimetric determination, but the decomposition of organic matters in the experimental culture process can generate brown substances, which interfere the contrast color and influence the colorimetric result. Therefore, the existing method for measuring the soil arylsulfatase needs to be improved, so that the sample consumption is less, and the method is quicker, more accurate and more economical.
Disclosure of Invention
The invention provides an improved fluorescence spectrum detection method for soil arylsulfatase activity, aiming at solving the problems of large sample consumption and low test efficiency of the existing method for detecting soil arylsulfatase.
The invention is realized by the following technical scheme: a fluorescence spectrum detection method for soil arylsulfatase activity comprises the following steps:
firstly, preparing a reagent:
preparation of arylsulfatase fluorogenic substrate: the fluorescent substrate of aryl sulfatase, namely 4-methyl umbelliferyl sulfate potassium salt (molecular formula is C)10H7KO6S, molecular weight 294.3) 14.72mg were diluted to 250 mL with sterile deionized water and stored in 50 mL plastic tubes for freezing if not used within 7 days;
acetate buffer solution: 4.1 g of Sodium acetate (Sodium acetate, C)2H3O2Na, FW 82.03) in 200 mL deionized water; dropwise adding acetic acid to adjust the pH value to 5.0; diluting to 250 mL with deionized water, and storing at 4 ℃ for later use, wherein the storage period is at most two weeks;
③ sodium hydroxide terminator: dissolving 20 g of NaOH in 500 mL of deionized water, cooling, and diluting to 1L in a volumetric flask;
(iv) MU standard: 19.82 mg of Methylumbelliferone (sodium salt, C)10H7O3Na, molecular weight 198.16) was dosed in a 100 mL volumetric flask with deionized water, the solution contained 1 nmol MU mL of standard-1(ii) a MU standard solution was frozen in 1 mL aliquots; on the day of analysis, melting the mixture, sucking 0.5mL of the mixture, diluting the mixture with deionized water, and fixing the volume to a 50 mL volumetric flask;
sodium azide solution: 0.65 g of NaN3Dissolving in 10L deionized water;
II, testing:
(1) preparing a microporous plate: adopting a 96-hole microporous plate;
firstly, each 8 holes correspond to one row, a control group and a sample group respectively comprise one row of blanks, one row of MU standard products and one row of arylsulfatase fluorogenic substrates, and 50 MU L of buffer solution is added into each row of holes by using an 8-channel pipette gun;
sample group part: blank corresponding column, and adding 100 muL NaN into each hole3A solution; adding 100 MU L of MU standard substance into each hole in a corresponding row of MU standard substances; correspondingly arranging the aryl sulfatase fluorogenic substrates, and adding 100 mu L of the aryl sulfatase fluorogenic substrates into each hole;
③ part of the control group: blank corresponding column, adding 150 muL NaN into each hole3Solution, corresponding row of MU standard substance, adding 50 muL NaN into each hole3Solution and 100 muL MU standard solution; correspondingly arranging the substrates of the arylsulfatase fluorogenic substrate, and adding 50 mu L NaN into each hole3Solution and 100 μ L arylsulfatase fluorogenic substrate;
(2) preparing a soil sample:
weighing fresh soil sample with mass of 2.00 +/-0.05 g into a 1L plastic beaker, and adding 200 mL of NaN3A solution;
secondly, adding a stirring rod, and stirring for 15 ~ 20 minutes by using a magnetic stirring plate, so as to ensure that the soil is uniformly and well dispersed before measurement;
(3) a detection step:
firstly, respectively sucking and transferring 50 muL of soil suspension into all columns of a sample group by using an 8-channel liquid transfer gun, and then putting a microporous plate into an incubator with a standard measurement temperature of 30 ℃ for culturing for 3 hours;
taking out the microporous plate, and adding 50 muL NaOH into all the holes to terminate the measurement;
setting parameters of the microplate reader, then placing the microporous plate into the feed inlet, and reading the measured value of the fluorescent plate of the microplate reader;
fourthly, after the microporous plate is used, firstly, the microporous plate is washed under a tap, and then, the microporous plate is cleaned by ultrasonic waves in deionized water and dried;
calculating the average fluorescence of each row of holes, and then calculating the activity of the soil arylsulfatase according to the following mode, wherein the final unit is nmol MU g-1Soil min-1：
i) Fluorescence from the arylsulfatase fluorogenic substrate (nmol MU) was calculated for the control group: standard: (control-blank)/(standard-blank);
ii) calculating the fluorescence (nmol MU) derived from the arylsulfatase fluorogenic substrate for the sample set: standard (assay-blank)/(standard-blank);
iii) multiplication by dilution factor: total solution volume (mL)/volume of soil suspension used in the analysis (mL)/weight of dry soil (g);
iv) dividing by the analysis time (min) to obtain the activity value.
The invention provides a fluorescence spectrum detection method for soil arylsulfatase activity, which has the following determination principle: methyl umbelliferone can be excited at 360nm wavelength and fluorescence can be detected at 460nm, the fluorescence property disappears when it is combined with some substances, methyl umbelliferone is released by hydrolysis of aryl sulfatase, and enzyme activity is characterized by fluorescence quantity.
Compared with the prior art, the invention has the following beneficial effects: compared with the prior analysis method, the fluorescence spectrum detection method for the activity of the soil aryl sulfatase provided by the invention has the advantages that the culture time in the determination process is short, the terminator is directly added after the culture is finished and is determined by using the microplate reader, and the fluorescence data on the microplate can simultaneously obtain parallel data within a few seconds, so that the determination of a large number of samples can be carried out, and the working efficiency is improved; the operation is simple, the analysis result is stable and reliable, and the reproducibility is good; the dosage of the medicine is small.
Detailed Description
The present invention is further illustrated by the following specific examples.
A fluorescence spectrum detection method for soil arylsulfatase activity comprises the following steps:
firstly, preparing a reagent:
preparation of arylsulfatase fluorogenic substrate: diluting 14.72mg of a fluorescent substrate 4-methylumbelliferyl sulfate potassium salt of aryl sulfatase to 250 mL by using sterile deionized water, and storing in a 50 mL plastic tube for freezing if the potassium salt is not used within 7 days;
acetate buffer solution: 4.1 g of Sodium acetate (Sodium acetate, C)2H3O2Na, FW 82.03) in 200 mL deionized water; dropwise adding acetic acid to adjust the pH value to 5.0; diluting to 250 mL with deionized water, and storing at 4 ℃ for later use, wherein the storage period is at most two weeks;
③ sodium hydroxide terminator: dissolving 20 g of NaOH in 500 mL of deionized water, cooling, and diluting to 1L in a volumetric flask;
(iv) MU standard: 19.82 mg of Methylumbelliferone (sodium salt, C)10H7O3Na, molecular weight 198.16) was dosed in a 100 mL volumetric flask with deionized water, the solution contained 1 nmol MU mL of standard-1(ii) a MU standard solution was frozen in 1 mL aliquots; on the day of analysis, melting the mixture, sucking 0.5mL of the mixture, diluting the mixture with deionized water, and fixing the volume to a 50 mL volumetric flask;
sodium azide solution: 0.65 g of NaN3Dissolving in 10L deionized water;
II, testing:
(1) preparing a microporous plate: adopting a 96-hole microporous plate;
firstly, each 8 holes correspond to one row, a control group and a sample group respectively comprise one row of blanks, one row of MU standard products and one row of arylsulfatase fluorogenic substrates, and 50 MU L of buffer solution is added into each row of holes by using an 8-channel pipette gun;
sample group part: as shown on the right side of Table 1, the blank corresponds to a column, and 100. mu.L of NaN was added to each well3A solution; adding 100 MU L of MU standard substance into each hole in a corresponding row of MU standard substances; correspondingly arranging the aryl sulfatase fluorogenic substrates, and adding 100 mu L of the aryl sulfatase fluorogenic substrates into each hole;
③ part of the control group: as shown in the left side of Table 1, the blank corresponds to a column, and 150. mu.L of NaN was added to each well3Solution, corresponding row of MU standard substance, adding 50 muL NaN into each hole3Solution and 100 muL MU standard solution; correspondingly arranging the substrates of the arylsulfatase fluorogenic substrate, and adding 50 mu L NaN into each hole3Solution and 100 μ L arylsulfatase fluorogenic substrate;
(2) preparing a soil sample:
weighing fresh soil sample with mass of 2.00 +/-0.05 g into a 1L plastic beaker, and adding 200 mL of NaN3A solution;
secondly, adding a stirring rod, and stirring for 15 ~ 20 minutes by using a magnetic stirring plate, so as to ensure that the soil is uniformly and well dispersed before measurement;
(3) a detection step:
firstly, respectively sucking and transferring 50 muL of soil suspension into all columns of a sample group by using an 8-channel liquid transfer gun, and then putting a microporous plate into an incubator with a standard measurement temperature of 30 ℃ for culturing for 3 hours;
taking out the microporous plate, and adding 50 muL NaOH into all the holes to terminate the measurement;
setting parameters of the microplate reader, then placing the microporous plate into the feed inlet, and reading the measured value of the fluorescent plate of the microplate reader; the parameters of the microplate reader are set as follows: setting general parameters: position delay 0.2s, kinetic window number 1, cycle number 1, measurement start time 0.0s, scintillation number per well and cycle 10, cycle 1 min; filter and integration parameter settings: fluorescence intensity Check, excitation filter 360nm, emission filter 460nm, obtaining value 1500, scanning well None;
fourthly, after the microporous plate is used, firstly, the microporous plate is washed under a tap, and then, the microporous plate is cleaned by ultrasonic waves in deionized water and dried;
calculating the average fluorescence of each row of holes, and then calculating the activity of the soil arylsulfatase according to the following mode, wherein the final unit is nmol MU g-1Soil min-1：
i) Fluorescence from the arylsulfatase fluorogenic substrate (nmol MU) was calculated for the control group: standard: (control-blank)/(standard-blank);
ii) calculating the fluorescence (nmol MU) derived from the arylsulfatase fluorogenic substrate for the sample set: standard (assay-blank)/(standard-blank);
iii) multiplication by dilution factor: total solution volume (mL)/volume of soil suspension used in the analysis (mL)/weight of dry soil (g);
iv) dividing by the analysis time (min) to obtain the activity value.
Example 1
The embodiment is a pot experiment, the soil adopted is brown soil developed on loess matrix, and the black Feng No. 1 tartary buckwheat is planted on the soil and is provided by the alpine crop research institute of the academy of agricultural sciences of Shanxi province. Three different treatments were set: no. 1 is nitrogen treatment (1.6 g 10 kg)-1) No. 2 is low-nitrogen treatment (0.8 g 10 kg)-1) No. 3 is a control (no nitrogen fertilizer is applied), the nitrogen fertilizer is urea (the nitrogen content is 46.4%), and the phosphate fertilizer (P)2O5，150mg·kg-1) And potash fertilizer (K)2O，60mg·kg-1) All are applied as base fertilizers. The method for detecting the activity of the arylsulfatase of the soil after three different treatments comprises the following steps:
firstly, preparing a soil sample:
weighing fresh soil sample with mass of 2.00 +/-0.05 g into a 1L plastic beaker, and adding 200 mL of NaN3A solution;
secondly, adding a stirring rod, selecting larger stirring rod, and violently stirring for 15 ~ 20 minutes by using a magnetic stirring plate, so as to ensure that the soil is well dispersed before measurement;
II, detection step:
respectively sucking and transferring 50 muL of soil suspension into all measurement holes of a sample group by using an 8-channel pipette gun (the vacant positions refer to table 1), cutting off the tail end from a standard pipette suction head to prevent blockage (cutting off the tail end by 2 mm), and then placing a microporous plate in an incubator with a standard measurement temperature of 30 ℃ for culturing for 3 hours;
taking out the microporous plate, and adding 50 muL NaOH into all the holes to terminate the measurement;
setting parameters of the microplate reader, then placing the microporous plate into the feed inlet, and reading the measured value of the fluorescent plate of the microplate reader; microplate reader parameter settings are shown in table 2: setting general parameters: position delay 0.2s, kinetic window number 1, cycle number 1, measurement start time 0.0s, scintillation number per well and cycle 10, cycle 1 min; filter and integration parameter settings: fluorescence intensity Check, excitation filter 360nm, emission filter 460nm, obtaining value 1500, scanning well None;
fourthly, after the microporous plate is used, firstly, the microporous plate is washed under a tap, and then, the microporous plate is cleaned by ultrasonic waves in deionized water and dried;
calculating the average fluorescence of each row of holes, and then calculating the activity of the soil arylsulfatase according to the following mode, wherein the final unit is nmol MU g-1Soil min-1：
i) Fluorescence from the arylsulfatase fluorogenic substrate (nmol MU) was calculated for the control group: standard: (control-blank)/(standard-blank);
ii) calculating the fluorescence (nmol MU) derived from the arylsulfatase fluorogenic substrate for the sample set: standard (assay-blank)/(standard-blank);
iii) multiplication by dilution factor: total solution volume (mL)/volume of soil suspension used in the analysis (mL)/weight of dry soil (g);
iv) dividing by the analysis time (min) to obtain the activity value.
Table 1 control and sample microplate reagents and sample amounts
TABLE 2 microplate reader parameters
The test results are as follows:
as can be seen from the data in the table, the smaller standard deviation shows that the analysis method has smaller deviation between the results, higher accuracy and good reproducibility.
Example 2
The test position is in an improved variety field (32 '35' 5 'N, 119' 42 '0' E) of small-town Mallingua villages in Jiangsu province, China, and the soil type is lower sand ginger soil. In this example, the rice-wheat rotation FAOE (Free-Air ozone enrichment) system platform was used to set 3 treatments for rhizosphere soil arylsulfatase activity at elevated ozone concentrations: no. 1 is Yannong 19 (Y19), No. 2 is Yanmai 16 (Y16), and No. 3 is Yanmai 15 (Y15). The specific steps are the same as those in embodiment 1.
The test results are as follows:
the data in the table show the stability of the analysis results and the higher precision.
The scope of the invention is not limited to the above embodiments, and various modifications and changes may be made by those skilled in the art, and any modifications, improvements and equivalents within the spirit and principle of the invention should be included in the scope of the invention.
Claims (3)
1. A fluorescence spectrum detection method for soil arylsulfatase activity is characterized in that: the method comprises the following steps:
firstly, preparing a reagent:
preparation of arylsulfatase fluorogenic substrate: diluting 14.72mg of a fluorescent substrate 4-methylumbelliferyl sulfate potassium salt of aryl sulfatase to 250 mL by using sterile deionized water, and storing in a 50 mL plastic tube for freezing if the potassium salt is not used within 7 days;
acetate buffer solution: 4.1 g of sodium acetate is dissolved in 200 mL of deionized water; dropwise adding acetic acid to adjust the pH value to 5.0; diluting to 250 mL with deionized water, and storing at 4 ℃ for later use, wherein the storage period is at most two weeks;
③ sodium hydroxide terminator: dissolving 20 g of NaOH in 500 mL of deionized water, cooling, and diluting to 1L in a volumetric flask;
(iv) MU standard: 19.82 mg of methyl umbelliferone was dosed into a 100 mL volumetric flask with deionized water, the solution containing 1 nmol of the standard MU mL-1(ii) a MU standard solution was frozen in 1 mL aliquots; on the day of analysis, melting the mixture, sucking 0.5mL of the mixture, diluting the mixture with deionized water, and fixing the volume to a 50 mL volumetric flask;
sodium azide solution: 0.65 g of NaN3Dissolving in 10L deionized water;
II, testing:
(1) preparing a microporous plate: adopting a 96-hole microporous plate;
firstly, each 8 holes correspond to one row, a control group and a sample group respectively comprise one row of blanks, one row of MU standard products and one row of arylsulfatase fluorogenic substrates, and 50 MU L of buffer solution is added into each row of holes by using an 8-channel pipette gun;
sample group part: blank corresponding column, and adding 100 muL NaN into each hole3A solution; adding 100 MU L of MU standard substance into each hole in a corresponding row of MU standard substances; correspondingly arranging the aryl sulfatase fluorogenic substrates, and adding 100 mu L of the aryl sulfatase fluorogenic substrates into each hole;
③ part of the control group: blank corresponding column, adding 150 muL NaN into each hole3Solution, corresponding row of MU standard substance, adding 50 muL NaN into each hole3Solution and 100 muL MU standard solution; correspondingly arranging the substrates of the arylsulfatase fluorogenic substrate, and adding 50 mu L NaN into each hole3Solution and 100 μ L arylsulfatase fluorogenic substrate;
(2) preparing a soil sample:
weighing fresh soil sample with mass of 2.00 +/-0.05 g into a 1L plastic beaker, and adding 200 mL of NaN3A solution;
secondly, adding a stirring rod, and stirring for 15 ~ 20 minutes by using a magnetic stirring plate, so as to ensure that the soil is uniformly and well dispersed before measurement;
(3) a detection step:
firstly, respectively sucking and transferring 50 muL of soil suspension into all columns of a sample group by using an 8-channel liquid transfer gun, and then putting a microporous plate into an incubator with a standard measurement temperature of 30 ℃ for culturing for 3 hours;
taking out the microporous plate, and adding 50 muL NaOH into all the holes to terminate the measurement;
setting parameters of the microplate reader, then placing the microporous plate into the feed inlet, and reading the measured value of the fluorescent plate of the microplate reader;
fourthly, after the microporous plate is used, firstly, the microporous plate is washed under a tap, and then, the microporous plate is cleaned by ultrasonic waves in deionized water and dried;
calculating the average fluorescence of each row of holes, and then calculating the activity of the soil arylsulfatase according to the following mode, wherein the final unit is nmol MU g-1Soil min-1：
i) Fluorescence from the arylsulfatase fluorogenic substrate (nmol MU) was calculated for the control group: standard: (control-blank)/(standard-blank);
ii) calculating the fluorescence (nmol MU) derived from the arylsulfatase fluorogenic substrate for the sample set: standard (assay-blank)/(standard-blank);
iii) multiplication by dilution factor: total solution volume (mL)/volume of soil suspension used in the analysis (mL)/weight of dry soil (g);
iv) dividing by the analysis time (min) to obtain the activity value.
2. The fluorescence spectroscopic detection method of soil arylsulfatase activity according to claim 1, wherein: in arylsulfatase fluorogenic substrate preparation, 14.72mg of the arylsulfatase fluorogenic substrate 4-methylumbelliferyl sulfate potassium salt was dissolved in deionized water in an ultrasonic bath for 3-5 seconds to aid dissolution.
3. The fluorescence spectroscopic detection method of soil arylsulfatase activity according to claim 1, wherein: the parameters of the microplate reader are set as follows:
setting general parameters: position delay 0.2s, kinetic window number 1, cycle number 1, measurement start time 0.0s, scintillation number per well and cycle 10, cycle 1 min;
filter and integration parameter settings: fluorescence intensity Check, excitation filter 360nm, emission filter 460nm, yield 1500, scan well None.
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