BioReagent 级 ，适用于对基线干扰要求严格的色谱和分析工作流程。
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在色谱分析、有机合成和交叉偶联反应领域已被 0 篇同行评审文献引用。
土壤铵态氮是土壤速效氮的重要组成部分，氮素作为植物生长发育所必须的基本营养元素，在植物生长发育和形态建成中起着重要作用，铵态氮可以被植物直接吸收利用，其含量对农业生产具有极其重要的影响，可作为判断土壤肥力和氮素营养水平的重要指标。
检测原理：土壤中的铵态氮在强碱性介质中与次氯酸盐和苯酚作用，生成水溶性染料靛酚蓝，在 625 nm 处有特征吸收峰，吸光值与铵态氮含量成正比。
适用样本：土壤样本
产品组分表
| S1506761 | Component | 96T | Storage |
| S1506761A | Extraction Buffer | 70 mL×2 | 2-8℃ |
| S1506761B | Reagent Ⅰ | 2 EA | 2-8℃. Store in the dark. |
| S1506761C | Reagent Ⅱ | 11.2 mL | 2-8℃. Store in the dark. |
| S1506761D | Reagent Ⅲ | 2.8 mL | 2-8℃ |
| S1506761E | Standard | 1 mL | 2-8℃ |
注意：正式检测前，建议选择2-3个预期差异较大的样本进行预实验。
自备仪器和试剂
1、酶标仪或可见分光光度计（能测 625 nm 处的吸光度）
2、96 孔板或微量玻璃比色皿、可调节式移液枪及枪头
3、恒温摇床、台式离心机
4、去离子水
实验流程
1、试剂准备
| 试剂名称 | 试剂准备 | 注意事项 |
| Extraction Buffer | 即用型；使用前平衡到室温 | 4℃保存 |
| Working Reagent Ⅰ | 临用前配制，根据用量每支加入 5.6 mL 去离子水充分溶解后使用 | 用不完的试剂4℃避光保存可稳定2周；Reagent Ⅰ有毒且有刺激性气味，建议在通风橱进行实验 |
| Reagent Ⅱ | 即用型；使用前平衡到室温 | 4℃保存 |
| Reagent Ⅲ | 即用型；使用前平衡到室温 | 4℃保存 |
| Standard | 即用型；使用前平衡到室温 | 4℃保存 |
标准品制备：使用 500 μmol/mL 标准品，按照下表所示，进一步稀释标准品：
| 序号 | Standard 体积 | Extraction Buffer 体积（µL） | 浓度（μmol/mL） |
| Std.1 | 5µL 500 μmol/mL | 995 | 2.5 |
| Std.2 | 100µL of Std.1 (2.5 μmol/mL) | 100 | 1.25 |
| Std.3 | 100µL of Std.2 (1.25 μmol/mL) | 100 | 0.625 |
| Std.4 | 100µL of Std.3 (0.625 μmol/mL) | 100 | 0.313 |
| Std.5 | 100µL of Std.4 (0.313 μmol/mL) | 100 | 0.156 |
| Std. | 100µL of Std.5 (0.156 μmol/mL) | 100 | 0.078 |
| Std. | 100µL of Std.6 (0.078 μmol/mL) | 100 | 0.039 |
| Blank | 0 | 100 | 0 |
注意：每次实验都要做一次标准品检测，制作标曲；稀释后的标准品溶液不稳定，必须在 4 小时内使用。
2、样本制备
注意：土壤经风干或者烘干很容易引起铵态氮含量的变化，所以建议采用新鲜土壤或者同样处理的样品进行测定。如果不立即进行实验，样本可在-80℃保存数周。测定时，应控制解冻的温度和时间。室温环境下解冻时，需在 4 h 内完成样品解冻。
按照土壤质量（g）：Extraction Buffer 体积（mL）为 1：5-10 的比例（建议称取约0.1g，加入1mL Extraction Buffer）加入Extraction Buffer，37℃振荡提取1h，然后25℃，10,000g离心10min，取上清待测。
3、实验步骤
3.1 酶标仪或可见分光光度计预热30min，调节波长到625nm。可见分光光度计用去离子水调零。
3.2 操作表（下述操作在 96 孔板或微量玻璃比色皿中进行）：
| 试剂（μL） | 空白孔 | 标准孔 | 测定孔 |
| 样本上清 | 0 | 0 | 20 |
| Extraction Buffer | 20 | 0 | 0 |
| Standards | 0 | 20 | 0 |
| Working Reagent Ⅰ | 80 | 80 | 80 |
| Reagent Ⅱ | 80 | 80 | 80 |
充分混匀，25℃静置1h。
| Reagent Ⅲ | 20 | 20 | 20 |
3.3 充分混匀后立即测定625nm处的吸光值，分别记为A空白、A标准、A测定，计算ΔA测定=A测定-A空白、ΔA标准=A标准-A空白。
注意：空白孔和标准孔只需测定1次。实验之前建议选择2-3个预期差异大的样本做预实验。如果ΔA测定 小于0.02， 可适当加大样本量。如果ΔA测定大于2.5 μmol/mL的ΔA标准，样本可用Extraction Buffer进一步稀释，计算结果乘以稀释倍数，或减少提取用样本量。
4、结果计算
4.1 标准曲线的绘制
以标准溶液浓度为x轴，ΔA标准为y轴，绘制标准曲线，得到标准方程 y=kx+b，将ΔA测定带入方程得到x（μmol/mL）。
4.2 铵态氮含量的计算
NH₄⁺-N含量（μg/g 土样）=x×V样总×18÷W=180×x
参数说明：
V样总：加入提取液体积，1mL；
W：样本质量，0.1g；
18： NH₄⁺摩尔质量，μg/μmol。
5、结果展示
图1：本试剂盒土壤中铵态氮的含量
注意事项
1、如果要比较不同样本间的铵态氮含量，需保持相同的采样深度，将土样烘干，按干重计算后再进行比较。
2、操作时需做好防护措施。
3、本产品仅供科学研究使用，不适用于临床诊断。为了您的安全和健康，请穿实验服并戴一次性手套操作。
Soil ammonium nitrogen (NH₄⁺-N) is a crucial component of soil available nitrogen. As an essential fundamental nutrient for plant growth and development, nitrogen plays a vital role in plant morphogenesis and development. Ammonium nitrogen can be directly absorbed and utilized by plants. Its content significantly impacts agricultural production and serves as an important indicator for assessing soil fertility and nitrogen nutrition levels.
Detection Principle
Ammonium nitrogen in the soil reacts with hypochlorite and phenol in a strongly alkaline medium to form the water-soluble dye indophenol blue, which has a characteristic absorption peak at 625 nm. The absorbance value is proportional to the ammonium nitrogen content.
Applicable Sample: Soil samples
| S1506761 | Component | 96T | Storage |
| S1506761A | Extraction Buffer | 70 mL×2 | 2-8℃ |
| S1506761B | Reagent Ⅰ | 2 EA | 2-8℃. Store in the dark. |
| S1506761C | Reagent Ⅱ | 11.2 mL | 2-8℃. Store in the dark. |
| S1506761D | Reagent Ⅲ | 2.8 mL | 2-8℃ |
| S1506761E | Standard | 1 mL | 2-8℃ |
Note: Before formal testing, it is recommended to perform a preliminary assay using 2-3 samples expected to have significant differences.
Required Instruments and Reagents
Microplate reader or visible spectrophotometer (capable of measuring absorbance at 625 nm)
96-well plate or micro glass cuvettes, adjustable pipettes, and tips
Constant temperature shaker, benchtop centrifuge
Deionized water
Experimental Procedure
1. Reagent Preparation
| Reagent Name | Preparation | Notes |
| Extraction Buffer | Ready-to-use; equilibrate to room temperature before use. | Store at 4°C. |
| Working Reagent Ⅰ | Prepare before use. Dissolve each vial in 5.6 mL deionized water according to the required amount. | Unused reagent can be stored at 4°C protected from light for up to 2 weeks. Reagent I is toxic and has an irritating odor. Perform subsequent steps in a fume hood. |
| Reagent Ⅱ | Ready-to-use; equilibrate to room temperature before use. | Store at 4°C. |
| Reagent Ⅲ | Ready-to-use; equilibrate to room temperature before use. | Store at 4°C. |
| Standard | Ready-to-use; equilibrate to room temperature before use. | Store at 4°C. |
Standard Preparation: Using the 500 μmol/mL standard, prepare a dilution series as shown in the table below:
| Tube | Standard Volume Added (to previous tube) | Extraction Buffer Volume (µL) | Final Concentration (μmol/mL) |
| Std.1 | 5 µL of 500 μmol/mL Std. | 995 | 2.5 |
| Std.2 | 100µL of Std.1 (2.5 μmol/mL) | 100 | 1.25 |
| Std.3 | 100µL of Std.2 (1.25 μmol/mL) | 100 | 0.625 |
| Std.4 | 100µL of Std.3 (0.625 μmol/mL) | 100 | 0.313 |
| Std.5 | 100µL of Std.4 (0.313 μmol/mL) | 100 | 0.156 |
| Std. | 100µL of Std.5 (0.156 μmol/mL) | 100 | 0.078 |
| Std. | 100µL of Std.6 (0.078 μmol/mL) | 100 | 0.039 |
| Blank | 0 | 100 | 0 |
Note: A standard curve must be generated with each experiment. Diluted standard solutions are unstable and must be used within 4 hours.
2. Sample Preparation
Note: Air-drying or oven-drying soil can easily alter ammonium nitrogen content. Therefore, it is recommended to use fresh soil or samples processed identically. If not used immediately, samples can be stored at -80°C for several weeks. Control thawing temperature and time during assay. If thawing at room temperature, complete sample thawing within 4 hours.
Add Extraction Buffer to the soil sample at a ratio of 1:5-10 (soil mass (g): Extraction Buffer volume (mL)) (recommended: weigh approximately 0.1 g soil, add 1 mL Extraction Buffer). Shake at 37°C for 1 hour for extraction. Then centrifuge at 25°C, 10,000 × g for 10 minutes. Collect the supernatant for assay.
3. Assay Steps
3.1 Preheat the microplate reader or visible spectrophotometer for 30 minutes and set the wavelength to 625 nm. Zero the visible spectrophotometer with deionized water.
3.2 Operation Table (perform the following steps in a 96-well plate or micro glass cuvette):
| Reagent (μL) | Blank Well | Standard Well | Test Well |
| Sample Supernatant | 0 | 0 | 20 |
| Extraction Buffer | 20 | 0 | 0 |
| Standards | 0 | 20 | 0 |
| Working Reagent Ⅰ | 80 | 80 | 80 |
| Reagent Ⅱ | 80 | 80 | 80 |
Mix thoroughly and incubate at 25°C for 1 hour.
| Reagent Ⅲ | 20 | 20 | 20 |
3.3 Mix thoroughly and immediately measure the absorbance at 625 nm. Record the values as Ablank, Astandard, and Atest. Calculate ΔAtest = Atest - Ablank and ΔAstandard = Astandard - Ablank.
Note: The blank and standard wells only need to be measured once. Preliminary tests with 2-3 samples expected to have significant differences are recommended before the formal experiment. If ΔAtest is less than 0.02, consider increasing the sample amount appropriately. If ΔAtest is greater than the ΔAstandard of the 2.5 μmol/mL standard, the sample supernatant can be further diluted with Extraction Buffer (multiply the result by the dilution factor) or the sample amount used for extraction can be reduced.
4. Calculation of Results
Note: We provide two formulas, including the detailed derivation formula and the simplified formula. They are exactly equivalent. The simplified formula (in bold) is recommended as the final calculation formula.
4.1 Plotting the Standard Curve
Plot the standard solution concentration (x-axis) against ΔAstandard (y-axis) to draw the standard curve. Obtain the standard equation y = kx + b. Substitute ΔAtest into the equation to calculate x (μmol/mL).
4.2 Calculation of Ammonium Nitrogen Content NH₄⁺-N content (μg/g soil) = x × Vsample total × 18 ÷ W = 180 × x
Parameter Description:
Vsample total: Volume of extraction buffer added, 1 mL;
W: Sample mass, 0.1 g;
18: Molar mass of NH₄⁺, μg/μmol.
Precautions
When comparing ammonium nitrogen content between different samples, ensure the same sampling depth. Calculate based on dry weight after oven-drying the soil samples before comparison.
Take appropriate protective measures during operation.
This product is for research use only. Not for use in diagnostic procedures. For your safety and health, please wear lab coats and disposable gloves during operation.
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| 批号(Lot Number) | 证书类型 | 货号 |
|---|---|---|
 ZJ26F0131479 |
分析证书 | S1506761 |
危险品化学品经营许可证（带存储）