超氧阴离子自由基作为生物体代谢过程中产生的一种自由基，可攻击生物大分子，如脂质、蛋白质、核酸和聚不饱和脂肪酸等，使其交联或者断裂，引起细胞结构和功能的破坏，与机体衰老和病变有很密切的关系，清除超氧阴离子自由基的研究已经得到了广泛的关注。生物体内的分子氧可以经过单电子还原转变为超氧阴离子自由基（O₂⁻），O₂⁻既可以直接作用于蛋白质和核酸等大分子，也可以衍生为羟自由基、单线态氧、过氧化氢及脂质过氧物自由基等对细胞结构和功能具有破坏作用的活性氧。
检测原理：在酸性条件下，2份O₂⁻与羟胺反应生成1份NO₂⁻，NO₂⁻在对氨基苯磺酸和萘胺的作用下反应生成粉红色的偶氮物质，以分光光度计测定530nm处吸光度，在一定范围内颜色深浅与O₂⁻成正比，根据A530及NO₂⁻和O₂⁻的相关反应中物质的量的关系，可算出样品中O₂⁻的浓度，主要用于测定植物组织中的超氧阴离子自由基含量或超氧阴离子的产生速率。
自备仪器和试剂
- 分光光度计（能测530nm处的吸光度）
- 研钵或匀浆器、低温离心机、恒温箱或水浴锅
- 离心管或试管、比色杯
- 蒸馏水等
操作步骤
1、样品准备
1.1 植物样品
取正常或逆境下的新鲜植物组织，清洗干净，擦干，切碎，迅速称取1~1.5g，加入2ml预冷的O₂⁻ Lysis Buffer后冰浴条件下匀浆或研磨，4℃ 10000g离心10min，上清液即为超氧阴离子自由基提取液，4℃保存备用。
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该试剂盒的测定，4℃保存，用于超氧阴离子自由基的检测。
1.3 高活性样品
如果样品中含有较高浓度的超氧阴离子自由基，可以使用O₂⁻ Lysis Buffer进行恰当的稀释。
2、配制系列NO₂⁻标准溶液：取出NO₂⁻标准 (1mM) 恢复至室温后，以NO₂⁻标准 (1mM) 按下表继续稀释：
加入物 (ml) | Std.1 | Std.2 | Std.3 | Std.4 | Std.5 | Std.6 | NO₂⁻标准 (1mM) | 0.01 | 0.02 | 0.03 | 0.04 | 0.05 | 0.06 | 蒸馏水 | 0.99 | 0.98 | 0.97 | 0.96 | 0.95 | 0.94 | NO₂⁻含量 (μM) | 10 | 20 | 30 | 40 | 50 | 60 |
|
3、O₂⁻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；如果样品中的超氧阴离子自由基浓度过高，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
| 1
| —
| —
|
系列NO₂⁻标准(1~6号)
| —
| 1
| —
|
待测样品
| —
| —
| 0.25
|
O₂⁻ Lysis Buffer
| —
| —
| 0.25
|
羟胺溶液
| —
| —
| 0.5 |
混匀，25℃水浴孵育20min。
氨基苯磺酸显色液
| 0.5
| 0.5
| 0.5
|
萘胺显色液
| 0.5
| 0.5
| 0.5
|
混匀，30℃水浴孵育30min。
4、O₂⁻测定
以空白调零，比色杯光径1cm，分光光度计测定标准管、测定管530nm处吸光度（即为A标准、A测定）。
5、结果计算
以系列NO₂⁻标准（1～6号）含量 (μM) 为横坐标，以对应的吸光度为纵坐标，制作标准曲线，根据测定管的吸光度进而计算NO₂⁻含量。根据如下公式计算具体样品中超氧阴离子自由基（O₂⁻）的含量：
植物组织样品O₂⁻ (μM/g)=2×n×VT/(W×VS)
血清、尿液等样品O₂⁻ (μM/ml)=2×n×N/VS
超氧阴离子的产生速率的定义：每分钟每克鲜重组织产生的超氧阴离子的物质的量，计算公式如下：
超氧阴离子产生速率【μmol/(min*g)】=2×n×VT/(W×t×VS)
测得样品中蛋白质含量后，可用下面公式表示：
超氧阴离子产生速率【μmol/(min*mg)】=2×n×VT/(m×t×VS)
参数说明：
2：NO₂⁻与O₂⁻间的化学计量数
n：从标准曲线上查得的NO₂⁻含量(μM)
VT：超氧阴离子自由基提取液总体积(ml)
N：样品稀释倍数
W：样品鲜重(g)
m：样品中蛋白质含量(mg)
t：样品与羟胺的反应时间(min)=20
VS：测定时加入提取液体积(ml)
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、如果样品中含有较多的叶绿素，会干扰测定结果，可在加入羟胺溶液25℃水浴孵育20min后用等体积乙醚或三氯甲烷萃取叶绿素，再行显色反应。
3、如果没有分光光度计，也可以使用普通的酶标仪测定，但应考虑酶标仪的最大检测体积。
4、所测样本的浓度过高，应用O₂⁻ Lysis Buffer稀释样品后重新测定。
5、氨基苯磺酸显色液和萘胺显色液有强烈的刺激性，尽量在通风条件好的地方操作，用后需拧紧瓶盖，避免挥发。
6、试剂开封后请尽快使用，以防影响后续实验效果。
7、该试剂盒仅用于科研领域，不适用于临床诊断或其他用途。
The superoxide anion radical, a type of free radical produced during organism metabolism, can attack biological macromolecules such as lipids, proteins, nucleic acids, and polyunsaturated fatty acids, causing cross-linking or fragmentation, and damaging cellular structure and function. It is closely related to aging and pathological conditions, and research into scavenging superoxide anion radicals has garnered widespread attention. Molecular oxygen in organisms can undergo single‑electron reduction to form the superoxide anion radical (O₂⁻). O₂⁻ can directly act on macromolecules like proteins and nucleic acids, and can also generate reactive oxygen species such as hydroxyl radicals, singlet oxygen, hydrogen peroxide, and lipid peroxide radicals, which are damaging to cellular structure and function.
Detection Principle
Under acidic conditions, two molecules of O₂⁻ react with hydroxylamine to produce one molecule of NO₂⁻. NO₂⁻ then reacts with sulfanilic acid and naphthylamine to form a pink azo compound. The absorbance at 530 nm is measured using a spectrophotometer. Within a certain range, the color intensity is proportional to the O₂⁻ concentration. Based on the A<sub>530</sub> value and the stoichiometric relationship between NO₂⁻ and O₂⁻ in the relevant reactions, the concentration of O₂⁻ in the sample can be calculated. This method is primarily used to determine the content or production rate of superoxide anion radicals in plant tissues.
S1508296
| Component
| 50T | Storage
|
S1508296A
| NO₂⁻ Standard (1 mM)
| 1 mL | 2-8℃. Store in the dark.
|
S1508296B
| O₂⁻ Lysis Buffer | 125 mL | 2-8℃. |
S1508296C
| Hydroxylamine Solution
| 30 mL | 2-8℃. Store in the dark. |
S1508296D
| Sulfanilic Acid Color Development Solution
| 30 mL
| 2-8℃. Store in the dark.
|
S1508296E
| Naphthylamine Color Development Solution
| 30 mL
| 2-8℃. Store in the dark. |
Reagents, consumables and Equipments not provided
- Spectrophotometer (capable of measuring absorbance at 530 nm)
- Mortar or homogenizer, Low‑temperature centrifuge, Incubator or water bath
- Centrifuge tubes or test tubes, Cuvette
- Distilled water, etc.
Procedure
1. Sample Preparation
1.1 Plant Samples
Take fresh plant tissue (normal or under stress), wash clean, dry, cut into small pieces, and quickly weigh 1–1.5 g. Add 2 mL of pre‑chilled O₂⁻ Lysis Buffer and homogenize or grind on ice. Centrifuge at 4°C, 10,000 g for 10 minutes. The supernatant is the superoxide anion radical extract. Store at 4°C for use.
1.2 Plasma, Serum, and Urine Samples
Plasma and serum prepared by standard methods can be directly used for assay with this kit. Store at 4°C for detection of superoxide anion radicals.
1.3 High‑Activity Samples
If the sample contains a high concentration of superoxide anion radicals, dilute appropriately with O₂⁻ Lysis Buffer.
2. Preparation of Series NO₂⁻ Standard Solutions
Bring the NO₂⁻ Standard (1 mM) to room temperature, then continue dilution according to the table below:
Additive (mL) | Std.1 | Std.2 | Std.3 | Std.4 | Std.5 | Std.6 | NO₂⁻ Standard (1 mM) | 0.01 | 0.02 | 0.03 | 0.04 | 0.05 | 0.06 | Distilled Water | 0.99 | 0.98 | 0.97 | 0.96 | 0.95 | 0.94 | NO₂⁻ Content (μM) | 10 | 20 | 30 | 40 | 50 | 60 |
|
3. O₂⁻ Sample Loading
Set up blank, standard, and test tubes according to the table below. Add solutions in the specified order, avoiding bubble formation. If the superoxide anion radical concentration in the sample is too high, reduce the sample volume or dilute appropriately before measurement. It is best to set up replicate tubes for each sample.
Additive (mL)
| Blank Tube
| Standard Tube
| Test Tube
|
Distilled Water
| 1
| —
| —
|
Series NO₂⁻ Standards (No. 1–6)
| —
| 1
| —
|
Test Sample
| —
| —
| 0.25
|
O₂⁻ Lysis Buffer
| —
| —
| 0.25
|
Hydroxylamine Solution
| —
| —
| 0.5 |
Mix well and incubate in a 25°C water bath for 20 min.
Sulfanilic Acid Color Development Solution
| 0.5
| 0.5
| 0.5
|
Naphthylamine Color Development Solution
| 0.5
| 0.5
| 0.5
|
Mix well and incubate in a 30°C water bath for 30 min.
4. O₂⁻ Assay
Zero the instrument with the blank. Using a cuvette with a 1 cm light path, measure the absorbance at 530 nm for the standard and test tubes using a spectrophotometer (recorded as A<sub>Standard</sub> and A<sub>Test</sub>).
5. Calculation of Results
Plot a standard curve using the series NO₂⁻ standard contents (μM) from tubes 1–6 as the x‑axis and the corresponding absorbance as the y‑axis. Calculate the NO₂⁻ content based on the absorbance of the test tube. Calculate the superoxide anion radical (O₂⁻) content in the specific sample using the following formulas:
For plant tissue samples:
O₂⁻ (μM/g) = 2 × n × VT / (W × VS)
For serum, urine, and other liquid samples:
O₂⁻ (μM/mL) = 2 × n × N / VS
Definition of Superoxide Anion Production Rate: The amount of superoxide anion produced per minute per gram of fresh tissue weight. Calculation formula:
Superoxide Anion Production Rate [μmol/(min·g)] = 2 × n × VT / (W × t × VS)
After measuring the protein content in the sample, the following formula can be used:
Superoxide Anion Production Rate [μmol/(min·mg)] = 2 × n × VT / (m × t × VS)
Parameter Description:
2: Stoichiometric coefficient between NO₂⁻ and O₂⁻
n: NO₂⁻ content obtained from the standard curve (μM)
V<sub>T</sub>: Total volume of superoxide anion radical extract (mL)
N: Sample dilution factor
W: Sample fresh weight (g)
m: Protein content in the sample (mg)
t: Reaction time between sample and hydroxylamine (min) = 20
V<sub>S</sub>: Volume of extract added during measurement (mL)
Notes
Experimental materials should be as fresh as possible. If not used immediately after collection, store at 4°C.
If the sample contains a high amount of chlorophyll, which may interfere with the assay, extract chlorophyll with an equal volume of ether or chloroform after incubation with hydroxylamine solution at 25°C for 20 minutes, then proceed with the color development reaction.
If a spectrophotometer is not available, a standard microplate reader can be used, but the maximum detection volume of the microplate reader should be considered.
If the measured sample concentration is too high, dilute the sample with O₂⁻ Lysis Buffer and repeat the assay.
Sulfanilic Acid Color Development Solution and Naphthylamine Color Development Solution are highly irritating. Operate in a well‑ventilated area and tighten bottle caps after use to avoid evaporation.
Use reagents as soon as possible after opening to avoid affecting subsequent experimental results.
This kit is intended for research use only and is not suitable for clinical diagnosis or other purposes.
ZJ26F0939190
危险品化学品经营许可证（带存储）