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硝态氮是植物最主要的氮源，植物体内硝态氮含量反映了土壤中硝态氮的供应情况，可作为土壤氮肥的指标，测定植物体内的硝态氮含量,不仅能够反映出植物的氮素营养情况，而且对鉴定蔬菜和以植物为原料的加工制品的品质也有重要的意义。
检测原理：在浓酸条件下，NO₃⁻与水杨酸反应生成硝基水杨酸，后者在碱性条件下呈黄色，其颜色深浅与氮含量在一定范围内呈正比，以分光光度计或酶标仪测定410nm处吸光度，根据NO₃⁻的标准曲线即可计算出样品的硝态氮含量。
适用样本：植物组织
自备仪器和试剂
操作步骤
1、样品准备
1.1 植物样品
取植物组织1g，清洗干净，擦干，剪碎成1~2mm的碎片，加入10ml蒸馏水，沸水浴中提取30min，取出后用自来水冷却至室温，中速滤纸过滤，补蒸馏水至10ml，即为硝态氮提取液。
1.2 血浆、血清和尿液样品
血浆、血清按照常规方法制备后可以直接用于本试剂盒的测定，用于硝态氮的检测。
1.3 高活性样品
如果样品中含有较高浓度的硝态氮，可以使用硝态氮裂解液或蒸馏水等进行恰当的稀释后再行测定。
2、配制水杨酸工作液
按水杨酸：浓硫酸=1g：20ml的比例，把水杨酸溶解于浓硫酸，4℃避光保存，1周有效。
注意：浓硫酸为强酸，具有强腐蚀性，请小心操作。
3、配制NO₃⁻ Assay Buffer (1×)
用蒸馏水将NO₃⁻ Assay Buffer (2×) 稀释一倍即可。
4、配制系列NO₃⁻标准溶液
取NO₃⁻ 标准 (200μg/mL) 按下表加蒸馏水稀释：
加入物（mL） | 1 | 2 | 3 | 4 | 5 | 6 | 7 |
NO₃⁻ 标准 (200μg/mL) | 0.05 | 0.1 | 0.2 | 0.3 | 0.4 | 0.5 | 0.6 |
蒸馏水 | 0.95 | 0.9 | 0.8 | 0.7 | 0.6 | 0.5 | 0.4 |
NO₃⁻ 浓度（μg/mL） | 10 | 20 | 40 | 60 | 80 | 100 | 120 |
5、NO₃⁻ 加样
按照下表设置空白管、标准管、测定管，溶液应按照顺序依次加入，并注意避免产生气泡。如果样品中的硝态氮浓度过高，可以减少样品用量或适当稀释后再进行测定，样品的检测最好能设置平行管。
加入物（mL） | 空白管 | 标准管 | 测定管 |
蒸馏水 | 0.02 | — | — |
系列NO₃⁻ 标准（1~7号管） | — | 0.02 | — |
待测样品 | — | — | 0.02 |
水杨酸工作液 | 0.08 | 0.08 | 0.08 |
混匀，室温放置20min，慢慢加入NO₃⁻ Assay Buffer (1×)。
NO₃⁻ Assay Buffer (1×) | 1.9 | 1.9 | 1.9 |
6、NO₃⁻ 测定
冷却至室温，以空白管调零，分光光度计（1cm光径比色杯）或酶标仪测定标准管、测定管410nm处吸光度（记为A标准、A测定）。
7、结果计算
以系列NO₃⁻ 标准（1~7号管）浓度 (μg/ml) 为横坐标，对应吸光度为纵坐标，制作标准曲线，根据测定管的吸光度参考如下公式计算出样品的NO₃⁻含量：
7.1 植物组织样品硝态氮 (μg/g)=C×V/m
参数说明：
C：从标准曲线上查得的NO₃⁻浓度 (μg/ml)
V：硝态氮提取液的总体积 (ml)
m：植物样品重量 (g)
7.2 血清、尿液等样品硝态氮 (μg/ml)=C×N
参数说明：
C：从标准曲线上查得的NO₃⁻浓度 (μg/ml)
N：稀释倍数
注意事项
1、实验材料应尽量新鲜，如取材后不立即使用，应存于4℃。
2、需自备浓硫酸。浓硫酸为强酸，具有强腐蚀性和氧化性，请小心操作。
3、如果没有分光光度计，也可以使用普通的酶标仪测定。
4、所测样品的浓度过高，应用蒸馏水稀释样品后重新测定。
5、为了您的安全和健康，请穿实验服并戴一次性手套操作。
6、试剂开封后请尽快使用，以防影响后续实验效果。
附录
参考标准曲线范围：
在室温条件下通过酶标仪405nm测定NO₃⁻标准在10、20、40、60、80、100、120μg/mL时的吸光度，据此作出其参考标准曲线如下：
注意：由于检测仪器和操作手法等条件的不同，参考值范围会有波动，该值仅供参考。
Nitrate nitrogen is the primary nitrogen source for plants. The nitrate nitrogen content in plant tissues reflects the supply of nitrate nitrogen in the soil and can serve as an indicator of soil nitrogen fertility. Measuring the nitrate nitrogen content in plants not only reveals the nitrogen nutrition status of the plants but is also significant for assessing the quality of vegetables and processed products derived from plants.
Detection Principle
Under concentrated acidic conditions, NO₃⁻ reacts with salicylic acid to form nitrosalicylic acid, which turns yellow under alkaline conditions. The intensity of the yellow color is proportional to the nitrogen content within a certain range. The absorbance at 410 nm is measured using a spectrophotometer or microplate reader, and the nitrate nitrogen content of the sample is calculated based on a standard curve for NO₃⁻.
Applicable Samples: Plant tissues
Reagents, consumables and Equipments not provided
Operating Steps
1. Sample Preparation
1.1 Plant Samples
Take 1 g of plant tissue, clean thoroughly, dry, and cut into 1–2 mm fragments. Add 10 ml of distilled water and extract in a boiling water bath for 30 minutes. After removal, cool to room temperature with tap water, filter through medium-speed filter paper, and bring the volume to 10 ml with distilled water. This is the nitrate nitrogen extraction solution.
1.2 Plasma, Serum, and Urine Samples
Plasma and serum, prepared by conventional methods, can be directly used for detection with this kit for nitrate nitrogen measurement.
1.3 High-Activity Samples
If the sample contains a high concentration of nitrate nitrogen, appropriate dilution with nitrate nitrogen lysis buffer or distilled water can be performed before measurement.
2. Preparation of Salicylic Acid Working Solution
Dissolve salicylic acid in concentrated sulfuric acid at a ratio of 1 g salicylic acid to 20 ml concentrated sulfuric acid. Store at 4°C protected from light. The solution is stable for one week.
Note: Concentrated sulfuric acid is a strong acid with high corrosivity. Handle with care.
3. Preparation of NO₃⁻ Assay Buffer (1×)
Dilute the NO₃⁻ Assay Buffer (2×) with distilled water to achieve a 1× concentration.
4. Preparation of NO₃⁻ Standard Solutions Series
Take the NO₃⁻ Standard (200 μg/mL) and dilute with distilled water according to the table below:
Tube Number | 1 | 2 | 3 | 4 | 5 | 6 | 7 |
NO₃⁻ Standard (200 μg/mL) (mL) | 0.05 | 0.1 | 0.2 | 0.3 | 0.4 | 0.5 | 0.6 |
Distilled Water (mL) | 0.95 | 0.9 | 0.8 | 0.7 | 0.6 | 0.5 | 0.4 |
NO₃⁻ Concentration (μg/mL) | 10 | 20 | 40 | 60 | 80 | 100 | 120 |
5. NO₃⁻ Sample Addition
Set up blank, standard, and test tubes according to the table below. Add solutions in the specified order, taking care to avoid bubbles. If the nitrate nitrogen concentration in the sample is too high, reduce the sample volume or dilute appropriately before measurement. It is recommended to set up duplicate tubes for sample detection.
Substance Added (mL) | Blank Tube | Standard Tube | Test Tube |
Distilled Water | 0.02 | — | — |
NO₃⁻ Standard Series (Tubes 1–7) | — | 0.02 | — |
Sample | — | — | 0.02 |
Salicylic Acid Working Solution | 0.08 | 0.08 | 0.08 |
Mix well, let stand at room temperature for 20 min, then slowly add NO₃⁻ Assay Buffer (1×).
NO₃⁻ Assay Buffer (1×) | 1.9 | 1.9 | 1.9 |
6. NO₃⁻ Measurement
Cool to room temperature. Zero the spectrophotometer or microplate reader with the blank tube, and measure the absorbance of the standard and test tubes at 410 nm (recorded as A<sub>Standard</sub> and A<sub>Test</sub>) using a 1 cm light path cuvette.
7. Result Calculation
Using the NO₃⁻ Standard Series (Tubes 1–7) concentrations (μg/mL) as the x-axis and the corresponding absorbance values as the y-axis, plot a standard curve. Calculate the NO₃⁻ content of the sample based on the absorbance of the test tube and the following formulas:
7.1 For plant tissue samples:
Nitrate nitrogen (μg/g) = C × V / m
Parameter Explanation:
C: NO₃⁻ concentration obtained from the standard curve (μg/mL)
V: Total volume of the nitrate nitrogen extraction solution (mL)
m: Weight of the plant sample (g)
7.2 For serum, urine, and other liquid samples:
Nitrate nitrogen (μg/mL) = C × N
Parameter Explanation:
C: NO₃⁻ concentration obtained from the standard curve (μg/mL)
N: Dilution factor
Precautions
Experimental materials should be as fresh as possible. If not used immediately after collection, store at 4°C.
Concentrated sulfuric acid must be prepared by the user. Concentrated sulfuric acid is a strong acid with high corrosivity and oxidizing properties. Handle with care.
If a spectrophotometer is unavailable, a standard microplate reader can also be used for measurement.
If the concentration of the measured sample is too high, dilute the sample with distilled water and repeat the measurement.
For your safety and health, please wear a lab coat and disposable gloves during operation.
Please use the reagent as soon as possible after opening to avoid affecting subsequent experimental results.
Appendix
Reference Standard Curve Range:
The absorbance of NO₃⁻ standards at concentrations of 10, 20, 40, 60, 80, 100, and 120 μg/mL was measured at 405 nm using a microplate reader at room temperature. Based on this, the reference standard curve is as follows:
Note: Due to variations in detection instruments, operational techniques, and other conditions, the reference value range may fluctuate. These values are for reference only.
| N1509264 | Component | 50T | 100T | Storage |
| N1509264A | NO₃⁻ 标准 (200μg/mL) | 0.5 mL | 1 mL | 2-8℃. |
| N1509264B | 水杨酸 | 1.5 g | 3 g | RT. |
| N1509264C | NO₃⁻ Assay Buffer (2×) | 50 mL | 100 mL | RT. |
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| 批号(Lot Number) | 证书类型 | 货号 |
|---|---|---|
 ZJ26F0232069 |
分析证书 | N1509264 |
| ZJ26F0232070 |
分析证书 | N1509264 |
| ZJ26F0232071 |
分析证书 | N1509264 |
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