CN114624205A - Detection method of alkaline phosphatase - Google Patents
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- CN114624205A CN114624205A CN202210189861.3A CN202210189861A CN114624205A CN 114624205 A CN114624205 A CN 114624205A CN 202210189861 A CN202210189861 A CN 202210189861A CN 114624205 A CN114624205 A CN 114624205A
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- 102000002260 Alkaline Phosphatase Human genes 0.000 title claims abstract description 61
- 108020004774 Alkaline Phosphatase Proteins 0.000 title claims abstract description 61
- 238000001514 detection method Methods 0.000 title claims abstract description 16
- 239000000243 solution Substances 0.000 claims abstract description 66
- 239000002689 soil Substances 0.000 claims abstract description 55
- -1 sodium fatty alcohol Chemical class 0.000 claims abstract description 36
- 239000007864 aqueous solution Substances 0.000 claims abstract description 26
- BDHFUVZGWQCTTF-UHFFFAOYSA-M sulfonate Chemical compound [O-]S(=O)=O BDHFUVZGWQCTTF-UHFFFAOYSA-M 0.000 claims abstract description 24
- 229910000365 copper sulfate Inorganic materials 0.000 claims abstract description 23
- ARUVKPQLZAKDPS-UHFFFAOYSA-L copper(II) sulfate Chemical compound [Cu+2].[O-][S+2]([O-])([O-])[O-] ARUVKPQLZAKDPS-UHFFFAOYSA-L 0.000 claims abstract description 23
- VHRDVBFFZCBVAL-UHFFFAOYSA-N (2-aminophenyl) dihydrogen phosphate Chemical compound NC1=CC=CC=C1OP(O)(O)=O VHRDVBFFZCBVAL-UHFFFAOYSA-N 0.000 claims abstract description 18
- 229920003171 Poly (ethylene oxide) Polymers 0.000 claims abstract description 12
- 239000011734 sodium Substances 0.000 claims abstract description 12
- 229910052708 sodium Inorganic materials 0.000 claims abstract description 12
- 239000012085 test solution Substances 0.000 claims abstract description 10
- MCHZKGNHFPNZDP-UHFFFAOYSA-N 2-aminoethane-1,1,1-triol;hydrochloride Chemical compound Cl.NCC(O)(O)O MCHZKGNHFPNZDP-UHFFFAOYSA-N 0.000 claims abstract description 3
- 238000000034 method Methods 0.000 claims description 23
- 239000006228 supernatant Substances 0.000 claims description 22
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 16
- 238000002360 preparation method Methods 0.000 claims description 12
- 238000000870 ultraviolet spectroscopy Methods 0.000 claims description 7
- ZYPZVOKVDNSKLP-UHFFFAOYSA-N tris(4-aminophenyl) phosphate Chemical compound C1=CC(N)=CC=C1OP(=O)(OC=1C=CC(N)=CC=1)OC1=CC=C(N)C=C1 ZYPZVOKVDNSKLP-UHFFFAOYSA-N 0.000 claims description 3
- 239000000523 sample Substances 0.000 description 29
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 24
- 230000000052 comparative effect Effects 0.000 description 20
- 150000002191 fatty alcohols Chemical class 0.000 description 12
- 239000012488 sample solution Substances 0.000 description 9
- 239000004480 active ingredient Substances 0.000 description 7
- 238000012258 culturing Methods 0.000 description 6
- 102000004190 Enzymes Human genes 0.000 description 5
- 108090000790 Enzymes Proteins 0.000 description 5
- 238000012360 testing method Methods 0.000 description 5
- HVBSAKJJOYLTQU-UHFFFAOYSA-N 4-aminobenzenesulfonic acid Chemical compound NC1=CC=C(S(O)(=O)=O)C=C1 HVBSAKJJOYLTQU-UHFFFAOYSA-N 0.000 description 4
- FXZCCINCKDATPA-UHFFFAOYSA-N (4-aminophenyl) dihydrogen phosphate Chemical compound NC1=CC=C(OP(O)(O)=O)C=C1 FXZCCINCKDATPA-UHFFFAOYSA-N 0.000 description 3
- 239000008351 acetate buffer Substances 0.000 description 3
- 238000011534 incubation Methods 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 230000000694 effects Effects 0.000 description 2
- 239000003864 humus Substances 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 230000004044 response Effects 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- 239000012086 standard solution Substances 0.000 description 2
- 229950000244 sulfanilic acid Drugs 0.000 description 2
- 238000001069 Raman spectroscopy Methods 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000003851 biochemical process Effects 0.000 description 1
- 238000005842 biochemical reaction Methods 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 238000004737 colorimetric analysis Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000005518 electrochemistry Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 230000035558 fertility Effects 0.000 description 1
- 238000001917 fluorescence detection Methods 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
- G01N21/33—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using ultraviolet light
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- Physics & Mathematics (AREA)
- Spectroscopy & Molecular Physics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention provides a detection method of alkaline phosphatase, which comprises the steps of incubating soil containing the alkaline phosphatase, an aminophenylphosphoric acid aqueous solution and sodium fatty alcohol polyoxyethylene sulfonate in a constant temperature box for 3-5h, adding a copper sulfate solution for mixing, taking supernate to react with a trihydroxymethyl aminomethane hydrochloride solution to prepare an alkaline phosphatase test solution, detecting the alkaline phosphatase test solution at the wavelength of 500-550nm by adopting an ultraviolet spectrophotometer, and showing that the accuracy is 99% and the detection limit is 0.01mg/g through experimental results.
Description
Technical Field
The invention relates to the field of alkaline phosphatase detection, and particularly relates to a detection method of alkaline phosphatase.
Background
Soil acts as a "life-like body" in that almost all biochemical reactions are driven by enzymes. The soil enzyme activity can be used as important indexes of soil biological activity and productivity such as soil fertility, soil quality, soil health and the like. Soil enzymes promote the formation of soil humus, and the conversion of organic substances and organic residues entering the soil into soil humus is a very complex biochemical process which cannot be catalyzed by various enzymes.
The invention relates to a method for detecting alkaline phosphatase in soil, and various methods for detecting alkaline phosphatase activity have been developed through different analysis technologies such as chromatography, colorimetry, chemiluminescence, electrochemistry, fluorescence, surface enhanced resonance Raman scattering and the like. Among them, fluorescence detection techniques have received much attention due to their advantages such as simplicity, sensitivity, and real-time property. The content of alkaline phosphatase in soil is detected, so that the detection result is inaccurate due to the influence of soil color, other elements contained in the soil and soil enzymes, and therefore, the invention provides the method for detecting the alkaline phosphatase with high accuracy by removing background interference.
Disclosure of Invention
Accordingly, the present invention provides a method for detecting alkaline phosphatase, which solves the above problems.
The technical scheme of the invention is realized as follows:
a method for detecting alkaline phosphatase, comprising the steps of:
(1) solution preparation: adding sample soil into aqueous solution of aminophenylphosphoric acid, oscillating for 50-70s, adding sodium fatty alcohol polyoxyethylene sulfonate, mixing, standing for 8-10min, placing in a constant temperature incubator at 36-38 deg.C for 3-5h, adding copper sulfate solution, centrifuging, and collecting supernatant; adding tris (hydroxymethyl) aminomethane hydrochloride solution into the supernatant, and shaking for 1-3min to obtain alkaline phosphatase sample solution;
(2) sample detection: and (3) detecting the alkaline phosphatase test solution prepared in the step (1) by using an ultraviolet-visible spectrophotometer.
Further, in the step (1), the mass ratio of the sample soil, the aqueous solution of p-aminophenylphosphate and the sodium fatty alcohol polyoxyethylene sulfonate is 1:5-7: 0.5-0.8.
Further, in the step (1), the concentration of the aqueous solution of p-aminophenylphosphoric acid is 8-10 mmol/L.
Further, in the step (1), the concentration of the copper sulfate solution is 7-10 mmol/L.
Further, in the step (1), the mass ratio of the copper sulfate solution to the soil sample is 2-3: 1.
Further, in the step (1), the concentration of the tris hydrochloride solution is 200-250mmol/L, and the pH value is adjusted to 7.5-8.5.
Further, in the step (1), the volume ratio of the tris hydrochloride solution to the supernatant is 1-3: 1.
Further, in the step (2), the detection wavelength of the ultraviolet-visible spectrophotometer is 500-550 nm.
Further, the detection wavelength of the ultraviolet-visible spectrophotometer is 530 nm.
Compared with the prior art, the invention has the beneficial effects that:
the method for detecting the alkaline phosphatase can accurately detect the content of the alkaline phosphatase in the soil, and has the advantages of high sensitivity and accuracy, strong anti-interference capability and the like. The method has the advantages that the content of the alkaline phosphatase enzymolysis active ingredients can be improved by taking the aminophenylphosphoric acid aqueous solution and the sodium fatty alcohol polyoxyethylene sulfonate as substrates, the active ingredients are combined into a test sample solution system, the incubated active ingredients react with a copper sulfate solution and a trihydroxymethyl aminomethane hydrochloride solution, the signal of the active ingredients in the alkaline phosphatase can be improved, the alkaline phosphatase test method has the accuracy rate of 99 percent, and the detection limit is 0.01 mg/g.
Detailed Description
In order to better understand the technical content of the invention, specific examples are provided below to further illustrate the invention.
The experimental methods used in the examples of the present invention are all conventional methods unless otherwise specified.
The materials, reagents and the like used in the examples of the present invention can be obtained commercially without specific description.
EXAMPLE 1 method for detecting alkaline phosphatase
(1) Solution preparation: adding sample soil into an aminophenylphosphoric acid aqueous solution with the concentration of 8mmol/L, oscillating for 50s, adding fatty alcohol polyoxyethylene sodium sulfonate, mixing, placing the sample soil, the aminophenylphosphoric acid aqueous solution and the fatty alcohol polyoxyethylene sodium sulfonate in a mass ratio of 1:5:0.5 for standing for 8min, placing the mixture into a constant-temperature incubator at 37 ℃ for incubation for 3h, adding a copper sulfate solution with the concentration of 7mmol/L, shaking for 5min, centrifuging for 2min at 1100r/min, and taking supernatant; adding 200mmol/L Tris-HCl solution into the supernatant, adjusting pH to 7.5 with 1mol/L NaOH solution, and shaking for 1-3min to obtain alkaline phosphatase sample solution.
EXAMPLE 2 method for detecting alkaline phosphatase
(1) Solution preparation: adding sample soil into an aminophenylphosphoric acid aqueous solution with the concentration of 9mmol/L, oscillating for 60s, adding fatty alcohol polyoxyethylene sodium sulfonate, mixing, wherein the mass ratio of the sample soil, the aminophenylphosphoric acid aqueous solution and the fatty alcohol polyoxyethylene sodium sulfonate is 1:6:0.65, standing for 9min, putting into a constant-temperature incubator at 37 ℃ for culturing for 4, adding a copper sulfate solution with the concentration of 8mmol/L, the mass ratio of the copper sulfate solution to the soil sample is 2.5:1, shaking for 5min, centrifuging for 2min at 1100r/min, and taking supernatant; adding 230mmol/L Tris-HCl solution into the supernatant, adjusting pH to 8.0 with 1mol/L NaOH solution, and shaking for 2min to obtain alkaline phosphatase sample solution.
EXAMPLE 3 method for detecting alkaline phosphatase
(1) Solution preparation: adding sample soil into a 10mmol/L aqueous solution of sulfanilic acid, oscillating for 70s, adding sodium fatty alcohol polyoxyethylene sulfonate, mixing, wherein the mass ratio of the sample soil, the aqueous solution of sulfanilic acid and the sodium fatty alcohol polyoxyethylene sulfonate is 1:7:0.8, standing for 10min, putting into a constant-temperature incubator for incubation for 5h, adding a 10mmol/L copper sulfate solution, the mass ratio of the copper sulfate solution to the soil sample is 3:1, shaking for 5min, centrifuging for 2min at 1100r/min, and taking supernatant; adding 250mmol/L Tris-HCl solution into the supernatant, adjusting pH to 8.5 with 1mol/L NaOH solution, and shaking for 1-3min to obtain alkaline phosphatase sample solution.
Comparative example 1
On the basis of the embodiment 2, the preparation of the solution does not use sodium fatty alcohol polyoxyethylene sulfonate, and specifically comprises the following steps: adding sample soil into an aminophenylphosphoric acid aqueous solution with the concentration of 9mmol/L, oscillating for 60s, wherein the mass ratio of the sample soil to the aminophenylphosphoric acid aqueous solution is 1:6, standing for 9min, putting the mixture into a constant-temperature incubator at 37 ℃ for culturing for 4, adding a copper sulfate solution with the concentration of 8mmol/L, wherein the mass ratio of the copper sulfate solution to the soil sample is 2.5:1, shaking for 5min, centrifuging for 2min at 1100r/min, and taking supernatant; adding 230mmol/L Tris-HCl solution into the supernatant, adjusting pH to 8.0 with 1mol/L NaOH solution, and shaking for 2min to obtain alkaline phosphatase sample solution.
Comparative example 2
On the basis of example 2, the concentration of the tris hydrochloride solution was changed during the solution preparation, specifically: adding sample soil into an aminophenylphosphoric acid aqueous solution with the concentration of 9mmol/L, oscillating for 60s, adding fatty alcohol polyoxyethylene sodium sulfonate, mixing, wherein the mass ratio of the sample soil, the aminophenylphosphoric acid aqueous solution and the fatty alcohol polyoxyethylene sodium sulfonate is 1:6:0.65, standing for 9min, putting into a constant-temperature incubator at 37 ℃ for culturing for 4, adding a copper sulfate solution with the concentration of 8mmol/L, the mass ratio of the copper sulfate solution to the soil sample is 2.5:1, shaking for 5min, centrifuging for 2min at 1100r/min, and taking supernatant; adding 100mmol/L Tris-HCl solution into the supernatant, adjusting pH to 8.0 with 1mol/L NaOH solution, and shaking for 2min to obtain alkaline phosphatase sample solution.
Comparative example 3
On the basis of example 2, an acetate buffer solution is used in the solution preparation instead of a tris hydrochloride solution, specifically: adding sample soil into an aminophenylphosphoric acid aqueous solution with the concentration of 9mmol/L, oscillating for 60s, adding fatty alcohol polyoxyethylene sodium sulfonate, mixing, wherein the mass ratio of the sample soil, the aminophenylphosphoric acid aqueous solution and the fatty alcohol polyoxyethylene sodium sulfonate is 1:6:0.65, standing for 9min, putting into a constant-temperature incubator at 37 ℃ for culturing for 4, adding a copper sulfate solution with the concentration of 8mmol/L, the mass ratio of the copper sulfate solution to the soil sample is 2.5:1, shaking for 5min, centrifuging for 2min at 1100r/min, and taking supernatant; adding 230mmol/L acetate buffer solution into the supernatant, adjusting pH to 8.0 with 1mol/L sodium hydroxide solution, and shaking for 2min to obtain alkaline phosphatase test solution.
Comparative example 4
On the basis of the embodiment 2, the mass ratio of the sample soil, the aqueous solution of aminophenylphosphoric acid and the sodium fatty alcohol polyoxyethylene sulfonate is changed in the solution preparation, and the method specifically comprises the following steps: adding sample soil into an aminophenylphosphoric acid aqueous solution with the concentration of 9mmol/L, oscillating for 60s, adding fatty alcohol polyoxyethylene sodium sulfonate, mixing, placing the sample soil, the aminophenylphosphoric acid aqueous solution and the fatty alcohol polyoxyethylene sodium sulfonate in a mass ratio of 1:3:1, standing for 9min, placing the mixture into a constant-temperature incubator at 37 ℃ for culturing 4, adding a copper sulfate solution with the concentration of 8mmol/L, adding the copper sulfate solution and the soil sample in a mass ratio of 2.5:1, shaking for 5min, centrifuging for 2min at 1100r/min, and taking supernatant; adding 230mmol/L Tris-HCl solution into the supernatant, adjusting pH to 8.0 with 1mol/L NaOH solution, and shaking for 2min to obtain alkaline phosphatase sample solution.
Test example 1
Standard curve 1: respectively adding 0, 0.1, 0.2, 0.5, 1, 2, 3, 4 and 5mg/L alkaline phosphatase standard solution into a 9mmol/L aqueous solution of p-aminophenylphosphoric acid, oscillating for 60s, adding fatty alcohol polyoxyethylene sodium sulfonate, mixing, wherein the mass ratio of the sample soil, the aqueous solution of p-aminophenylphosphoric acid and the fatty alcohol polyoxyethylene sodium sulfonate is 1:6:0.65, standing for 9min, placing into a 37 ℃ constant-temperature incubator for culturing for 4, adding a copper sulfate solution with the concentration of 8mmol/L, the mass ratio of the copper sulfate solution to the alkaline phosphatase standard solution is 2.5:1, shaking for 5min, centrifuging for 2min at 1100r/min, and taking supernatant; adding 230mmol/L Tris-HCl solution into the supernatant, adjusting pH to 8.0 with 1mol/L NaOH solution, and shaking for 2min to obtain alkaline phosphatase sample solution.
And detecting by using an ultraviolet-visible spectrophotometer at the wavelength of 530 nm.
Drawing a standard curve equation by taking the x axis as concentration and the y axis as absorbance, wherein the y is 0.0243x +0.0012, and R2＝0.9998。
In the experimental process, the soil without alkaline phosphatase is used to prepare the sample soil containing 1.5mg/g of alkaline phosphatase in the soil. Alkaline phosphatase test solutions were prepared according to the solution preparation methods of examples 1 to 3 and comparative examples 1 to 4, and the solutions were measured at a wavelength of 530nm using an ultraviolet-visible spectrophotometer, and two parallel tests were performed. The accuracy is calculated, where (%) -
| Name (R) | Accuracy (%) |
| Example 1 | 99.51 |
| Example 2 | 99.89 |
| Example 3 | 99.63 |
| Comparative example 1 | 93.85 |
| Comparative example 2 | 96.17 |
| Comparative example 3 | 94.11 |
| Comparative example 4 | 92.88 |
Experimental results show that the detection method provided by the invention has high accuracy, and can shield the interference of soil color and metal elements on alkaline phosphatase detection. In the comparative example 1, no sodium fatty alcohol polyoxyethylene sulfonate is used in the incubation of alkaline phosphatase, so that the content of the enzymolysis active ingredients is reduced in the test solution; comparative example 2 changing the concentration of tris hydrochloride solution resulted in a decrease in response signal; comparative example 3 acetate buffer was used instead of tris hydrochloride solution, resulting in poor combination of the enzymatically active ingredient with the test solution system and reduced response signal; comparative example 4 the mass ratio of the sample soil, the aqueous solution of aminophenylphosphoric acid and sodium fatty alcohol polyoxyethylene sulfonate was changed, resulting in a decrease in the content of the alkaline phosphatase enzymatic active ingredient.
Test example 2
In the experimental process, the soil without alkaline phosphatase was used to prepare the sample soil containing 0.05 mg/g alkaline phosphatase and 0.01mg/g alkaline phosphatase in the soil. Alkaline phosphatase test solutions were prepared according to the solution preparation methods of examples 1 to 3 and comparative examples 1 to 4, and the solutions were measured at a wavelength of 530nm using an ultraviolet-visible spectrophotometer, and two parallel tests were performed. The accuracy is calculated, where (%) -
| Concentration (mg/g) | 0.05 | 0.01 |
| Item | Accuracy (%) | Accuracy (%) |
| Example 1 | 99.12 | 98.84 |
| Example 2 | 99.68 | 99.53 |
| Example 3 | 99.10 | 98.64 |
| Comparative example 1 | Not detected out | Not detected out |
| Comparative example 2 | 60.47 | Not detected out |
| Comparative example 3 | 53.75 | Not detected out |
| Comparative example 4 | 44.31 | Not detected out |
The experimental result shows that the detection method of the alkaline phosphatase can enhance the signal of the alkaline phosphatase, the detection limit is 0.01mg/g, the comparative examples 1 to 4 change the preparation method of the solution, the signal of the alkaline phosphatase is reduced, and when the content of the alkaline phosphatase in the soil is 0.01mg/g, the comparative examples 1 to 4 can not detect the content of the alkaline phosphatase in the soil.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Claims (9)
1. A method for detecting alkaline phosphatase, comprising the steps of:
(1) solution preparation: adding sample soil into aqueous solution of aminophenylphosphoric acid, oscillating for 50-70s, adding sodium fatty alcohol polyoxyethylene sulfonate, mixing, standing for 8-10min, placing in a constant temperature incubator at 36-38 deg.C for 3-5h, adding copper sulfate solution, centrifuging, and collecting supernatant; adding a trihydroxymethyl aminomethane hydrochloride solution into the supernatant, and shaking for 1-3min to obtain an alkaline phosphatase test solution;
(2) sample detection: and (3) detecting the alkaline phosphatase test solution prepared in the step (1) by using an ultraviolet-visible spectrophotometer.
2. The method for detecting alkaline phosphatase according to claim 1, wherein in the step (1), the mass ratio of the sample soil, the aqueous solution of p-aminophenylphosphate and the sodium fatty alcohol polyoxyethylene sulfonate is 1:5 to 7:0.5 to 0.8.
3. The method for detecting alkaline phosphatase according to claim 1 or 2, wherein the concentration of the aqueous solution of p-aminophenylphosphate in the step (1) is 8 to 10 mmol/L.
4. The method for detecting alkaline phosphatase according to claim 1, wherein the copper sulfate solution has a concentration of 7 to 10mmol/L in step (1).
5. The method for detecting alkaline phosphatase according to claim 1, wherein the mass ratio of the copper sulfate solution to the soil sample in step (1) is 2-3: 1.
6. The method for detecting alkaline phosphatase as claimed in claim 1, wherein in the step (1), the concentration of the tris hydrochloride solution is 200-250mmol/L, and the pH value is adjusted to 7.5-8.5.
7. The method for detecting alkaline phosphatase according to claim 1, wherein in the step (1), the volume ratio of the tris hydrochloride solution to the supernatant is 1-3: 1.
8. The method for detecting alkaline phosphatase according to claim 1, wherein in the step (2), the UV-Vis spectrophotometer detects the wavelength at 500-550 nm.
9. The method for detecting alkaline phosphatase according to claim 8, wherein the UV-VIS spectrophotometer detects the alkaline phosphatase at a wavelength of 530 nm.
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