土壤磷酸酶是一类催化土壤有机磷矿化的酶，其活性的高低直接影响着土壤中有机磷的分解转化及其生物有效性，是评价土壤磷素生物转化方向与强度的指标。根据其最适pH 土壤磷酸酶通常分为酸性、中性和碱性三种类型。
本试剂盒主要检测的是土壤中碱性磷酸酶活性。其原理为土壤中的碱性磷酸酶（S-AKP/ALP）能够在碱性环境中催化磷酸苯二钠水解生成苯酚和磷酸氢二钠，通过测定酚的生成量即可表征土壤碱性磷酸酶的活性。
S1515811 | 组件 | 物料外观 | 48T | 96 T | 储存条件 | 单次测试使用量 |
S1515811A | 检测缓冲液 | 无色液体 | 10 mL | 20 mL | 2-8 ℃. | 200 µL |
S1515811B | 显色底物 | 无色液体 | 10 mL | 20 mL | 2-8 ℃.避光. | 200 µL |
S1515811C | 显色液 A | 浅黄液体 | 6.0 mL | 12 mL | 2-8 ℃.避光. | 120 µL |
S1515811D | 显色液 B | 浅黄液体 | 25 mL | 50 mL | 2-8 ℃.避光. | 480 µL |
S1515811E | 标准品(10 mM) | 无色液体 | 1.0 mL | 1.5 mL | 2-8 ℃.避光. | 按需 |
注意：显色液B和标准品有一定刺激性或毒性，请规范穿戴实验服和手套，避免接触到皮肤。
使用方法
1. 自备试剂耗材
(1) 96 孔板，1.5 mL离心管以及30-50 目筛。
(2) 37℃恒温培养箱或者水浴锅，酶标仪或可见分光光度计（能测 510 nm 处的吸光度）。
(3) 甲苯，去离子水。
2. 样品检测
建议正式实验前，选取2个样本做预测定，了解实验样品情况，熟悉流程，避免样本和试剂浪费。
(1) 样品制备：取新鲜土样自然风干或 37℃烘箱风干，研磨后过30-50目筛备用。
(2) 标曲稀释：取100 μL标准品(10 mM) + 400 μL去离子水，配制成2 mM的标准品工作液，然后将标准品工作液用去离子水分别稀释成0.2 mM，0.4 mM，0.8 mM，1.2 mM，1.6 mM和2.0 mM。
(3) 显色工作液液配制：按照显色液A:显色液B=1:4比例混匀配制，以配制600 µL 显色工作液为例，取120 µL 显色液A和480 µL 显色液B混匀即可。
(4) 上机检测：在1.5 mL离心管中按照下表，依次加入各试剂
试剂名称 | 测定管 | 对照管 | 空白管 | 标准管 | 土样 | 0.1 g | 0.1 g | - | - | 甲苯 | 50 μL | 50 μL | 50 μL | - | 显色底物 | 200 μL | - | 200 μL | 200 μL | 去离子水 | - | 200 μL | - | - | 标准品 | - | - | - | 50 μL | 检测缓冲液 | 200 μL | 200 μL | 200 μL | 200 μL |
振荡混匀后于37 ℃孵育1 h。 显色工作液 | 600 μL | 600 μL | 600 μL | 600 μL |
混匀后，静置2 min，12000 rpm离心10 min，吸取200 uL上清至96孔板中，读取 A₅₁₀ 。ΔA=A测定 - A对照；Δ标=A标准 - A空白 |
3. 结果计算
(1) 标准曲线：y=0.008*X + 0.0021(仅供参考)；x是酚的摩尔质量(nmol)，y 是△A。
(2) 酶活性定义：每克土壤每小时水解底物（磷酸苯二钠）产生 1nmol产物（酚）为一
个酶活单位，计为U。
U=[(△A-0.0021)÷0.008]÷W÷T×D
W-土壤样品质量，g。
T-反应时间，1 h。
D-稀释倍数，未稀释即为1。
注意事项
1. 显色工作液需现用现配，需避光放置。
2. 显色液B和标准品有一定刺激性或者毒性，在操作中，请规范穿戴实验服和手套，避免接触到皮肤。
3. 本产品仅供科学研究使用，不适用于临床诊断。
Soil phosphatases are a group of enzymes that catalyze the mineralization of soil organic phosphorus. Their activity levels directly govern the decomposition, transformation and bioavailability of organic phosphorus in soil, serving as an indicator for evaluating the direction and intensity of biological phosphorus transformation in soil. Based on their optimum pH values, soil phosphatases are generally categorized into three types: acid phosphatase, neutral phosphatase and alkaline phosphatase.
This assay kit is designed primarily for detecting alkaline phosphatase activity in soil. The detection principle is as follows: soil alkaline phosphatase (S-AKP/ALP) catalyzes the hydrolysis of disodium phenyl phosphate under alkaline conditions to produce phenol and disodium hydrogen phosphate. The activity of soil alkaline phosphatase can be quantified by measuring the yield of phenol generated in the reaction system.
S1515811 | Components | Appearance | 48T | 96T | Storage | Usage volume per test |
S1515811A | Assay Buffer | Colorless liquid | 10 mL | 20 mL | 2-8 ℃. | 200 µL |
S1515811B | Chromogenic substrate | Colorless liquid | 10 mL | 20 mL | 2-8 ℃.Store in the dark. | 200 µL |
S1515811C | Chromogenic Solution A | Light yellow liquid | 6.0 mL | 12 mL | 2-8 ℃.Store in the dark. | 120 µL |
S1515811D | Chromogenic Solution B | Light yellow liquid | 25 mL | 50 mL | 2-8 ℃.Store in the dark. | 480 µL |
S1515811E | Standard (10 mM) | Colorless liquid | 1.0 mL | 1.5 mL | 2-8 ℃.Store in the dark. | As need |
Note: Chromogenic Solution B and standard solution are irritant and toxic. Wear lab coat and gloves properly during operation to avoid skin contact.
Usage Method
1. Self-supplied Reagents and Consumables
(1) 96-well plates, 1.5 mL centrifuge tubes, and 30–50 mesh sieves.
(2) Incubator or water bath set at 37°C; microplate reader or visible spectrophotometer (capable of measuring absorbance at 510 nm).
(3) Toluene, deionized water.
2. Sample Assay
It is recommended to perform a pre-test with 2 samples prior to the formal experiment to characterize sample properties and get familiar with the operating procedure, so as to avoid waste of samples and reagents.
(1) Sample Preparation: Air-dry fresh soil samples naturally or dry them in an oven at 37 °C. Grind the dried soil and pass it through a 30–50 mesh sieve for later use.
(2) Standard Curve Dilution: Pipette 100 μL of standard stock solution (10 mM) and mix with 400 μL deionized water to prepare 2 mM standard working solution. Subsequently, dilute the standard working solution with deionized water to obtain gradient concentrations of 0.2 mM, 0.4 mM, 0.8 mM, 1.2 mM, 1.6 mM and 2.0 mM respectively.
(3) Preparation of Chromogenic Working Solution: Mix Chromogenic Solution A and Chromogenic Solution B at a volume ratio of 1:4. For example, to prepare 600 μL chromogenic working solution, combine 120 μL Chromogenic Solution A with 480 μL Chromogenic Solution B and mix thoroughly.
(4) Microplate Assay Setup: Add each reagent sequentially into 1.5 mL centrifuge tubes according to the table below.
Reagent Name | Sample Tube | Control Tube | Blank Tube | Standard Tube | Soil sample | 0.1 g | 0.1 g | - | - | Toluene | 50 μL | 50 μL | 50 μL | - | Chromogenic substrate | 200 μL | - | 200 μL | 200 μL | Deionized Water | - | 200 μL | - | - | Standard | - | - | - | 50 μL | Assay Buffer | 200 μL | 200 μL | 200 μL | 200 μL |
Mix thoroughly by shaking, then incubate at 37 °C for 1 h.
Chromogenic Working Solution | 600 μL | 600 μL | 600 μL | 600 μL |
Mix well and stand for 2 min. Centrifuge at 12000 rpm for 10 min. Transfer 200 μL of supernatant to a 96-well plate and record absorbance at 510 nm ( A₅₁₀ )。ΔA = Aₛₐₘₚₗₑ− AControl; ΔStandard = Aₛₜₐₙdₐᵣd-Abₗₐₙₖ. |
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3. Result Calculation
(1) Standard curve: (y = 0.008*X + 0.0021) (for reference only); x refers to the molar amount of phenol (nmol), and y represents ΔA.
(2) Definition of Enzyme Activity: One unit of enzyme activity (U) is defined as the amount of alkaline phosphatase that hydrolyzes the substrate (disodium phenyl phosphate) to produce 1 nmol of the product (phenol) per gram of soil per hour.
U = [ ΔA - 0.0021) \0.008] \W \T×D
W: Mass of soil sample, g
T: Reaction time, 1 h
D: Dilution factor; set as 1 if no dilution is performed
Precautions
1. The chromogenic working solution shall be freshly prepared before use and stored away from light.
2. Chromogenic Solution B and standard solution are irritant and toxic. Wear lab coat and gloves properly during operation to avoid skin contact.
3. This product is for research use only and not for clinical diagnostic applications.
ZJ26F0838236
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