过氧化氢（H₂O₂）是生物体内最常见的活性氧分子，是一种活性氧代谢的副产物，主要由SOD和XOD 等催化产生，由 CAT 和 POD 等催化降解。过氧化氢不仅是重要的活性氧之一，也是活性氧相互转化的枢纽。一方面，H₂O₂可以直接或间接地氧化细胞内核酸，蛋白质等生物大分子，使细胞膜遭受损害，从而加速细胞的衰老和解体；另一方面，H₂O₂也是许多氧化应急反应中的关键调节因子。H₂O₂可以激活NF-κB 等因子，这些过氧化氢相关的信号途径和哮喘、炎症性关节炎、动脉硬化以及神经退行性疾病等许多疾病相关。H₂O₂也和细胞凋亡、细胞增殖等密切相关。
检测原理：H₂O₂与硫酸钛反应生成的过氧化物-钛复合物黄色沉淀，溶解于强酸中，其黄色深浅与H₂O₂浓度在一定范围内呈线性关系，通过比色法测定412nm处吸光度，主要用于检测植物组织、血清、血浆等样品中H₂O₂含量。该试剂盒仅用于科研领域，不适用于临床诊断或其他用途。
组分表：
H1505496
| Component
| 50T | Storage
|
H1505496A
| H₂O₂基液
| 1 mL | 2-8℃. Store in the dark.
|
H1505496B
| 碱性基液 | 10.5 mL | RT. |
H1505496C
| 硫酸钛 | 0.3 g | RT.
|
H1505496D
| 酸性基液 | 100 mL | RT. |
自备仪器和试剂
1、蒸馏水、丙酮
2、匀浆器或研钵、低温离心机、分光光度计、比色杯
实验流程
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5g，加入5ml预冷的丙酮，在冰浴条件下迅速匀浆或研磨，4℃ 12000g离心20min，收集上清液，测量提取液总体积，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，用于过氧化氢的测定。
1.3 高活性样品
如果样品中含有较高浓度的过氧化氢，可以使用丙酮进行恰当的稀释。
2、10mM H₂O₂标准溶液的配制
由于H₂O₂不是非常稳定，使用前需自行测定H₂O₂的实际浓度，本试剂盒提供的H₂O₂基液的H₂O₂浓度约为1M，用蒸馏水稀释100倍，使H₂O₂浓度约为10mM，蒸馏水调零，分光光度计测定A₂₄₀，根据公式H₂O₂浓度(mM)=22.94×A₂₄₀计算出H₂O₂基液中H₂O₂的实际浓度，再用丙酮稀释H₂O₂基液配制10mM H₂O₂-丙酮标准溶液（一般情况下新配制的10mM H₂O₂基液A₂₄₀为0.4~0.45，3个月以后A₂₄₀为0.35~0.42）。
按下表依次稀释（常用浓度0.3-3mM，即1~5号）：
| 序号 | H₂O₂-丙酮标准 (10mM) (ml)
| 预冷丙酮 (ml)
| H₂O₂浓度 (mM) |
| 1 | 0.03 | 0.97 | 0.3 |
| 2 | 0.05 | 0.95 | 0.5 |
| 3 | 0.08 | 0.92 | 0.8 |
| 4 | 0.1 | 0.9 | 1 |
| 5 | 0.3 | 0.7 | 3 |
| 6 | 0.5 | 0.5 | 5 |
| 7 | 0.8 | 0.2 | 8 |
3、硫酸钛溶液的配制
0.3g硫酸钛加入6ml蒸馏水中，即为5%硫酸钛溶液，4℃保存。
4、H₂O₂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H₂O₂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
|
预冷丙酮
| 1 | —
| —
|
系列H₂O₂-丙酮溶液（1~7号）
| —
| 1
| —
|
待测样品
| —
| —
| 1
|
碱性基液
| 0.2 | 0.2
| 0.2
|
硫酸钛溶液
| 0.1 | 0.1
| 0.1
|
注：加入碱性基液、硫酸钛溶液时，应直接加至溶液中，不要粘到管壁。
5、H₂O₂测定
混匀，室温放置5min，12000g离心15min，弃上清液，留取沉淀，如有必要可加入预冷丙酮反复洗涤沉淀物，向各管的沉淀中加入2ml酸性基液，摇动，使沉淀完全溶解；比色杯光径1cm，空白管调零，分光光度计测定412nm处各标准管、测定管的吸光度，如果没有分光光度计，亦可用酶标仪检测。
6、结果计算
6.1 标准曲线的制作
以系列H₂O₂-丙酮标准（0.3、0.5、0.8、1、3、5、8 mM）为横坐标，以对应的吸光度为纵坐标，绘制标准曲线，求得回归方程；
6.2 样本H₂O₂浓度计算
以测定管吸光度代入回归方程求得待测样品中H₂O₂的浓度（C₀）。
组织样品H₂O₂ (mmol/g)=(C₀×VT×N)/m
液体样品H₂O₂ (mmol/L)=C₀×N
参数说明
C₀：根据待测样品的吸光度在标准曲线求得H₂O₂浓度 (mM)；
VT：待测样品的总体积 (L)；
m：样品质量 (g)；
N：样本稀释倍数。
结果展示
标准曲线制作：在室温条件下按说明书操作，对系列标准进行吸光度的测定，其吸光度及标准曲线如下（仅供参考），我们采用H₂O₂标准（0.1、0.3、0.5、0.8、1、3、5、8、10mM）绘制标准曲线（标准品浓度过高或过低都有可能影响标准曲线的准确性）：
H₂O₂标准 (mM)
| 0.1 | 0.3 | 0.5 | 0.8 | 1 | 3 | 5 | 8 | 10 |
| 吸光度 | 0.009 | 0.065 | 0.141 | 0.216 | 0.264 | 0.812 | 1.262 | 2.010 | 2.355 |
注意：H₂O₂浓度低于0.3mM基本无色，0.3~1mM为黄色，3~10mM为橙黄色，效果参考如下。
_%E5%89%AF%E6%9C%AC.png)
注意事项
1、该试剂盒亦可用酶标仪进行检测，但检测的样本数相应增加。
2、加入碱性基液、硫酸钛溶液时，应直接加入至溶液中，不要粘到管壁。
3、过氧化物-钛复合物黄色沉淀溶解于酸性基液时需要一段时间，需完全溶解，否则有可能影响测定结果。
4、H₂O₂基液和碱性基液应严格密闭保存，避免挥发，否则效率会下降。
5、H₂O₂基液和酸性基液有一定腐蚀性，请小心操作。
6、硫酸钛溶解于水后应尽早使用，如暂时不用，可短期放置4℃冰箱保存；亦可用分析天平称取一定量的粉剂，配置5%的浓度即可。
7、为了您的安全和健康，请穿实验服并戴一次性手套操作。
8、试剂开封后请尽快使用，以防影响后续实验效果。
特别说明：
整套试剂盒未拆封时于2-8°C 避光保存，自生产之日起有效期 1 年；拆封后各组分稳定性不同，部分组分可转至室温（有效期仍为 1 年），其余需继续冷藏。请严格按照组分表标注温度分类存放，保障试剂性能。
Hydrogen peroxide (H₂O₂) is the most common reactive oxygen species (ROS) molecule in living organisms. It is a byproduct of active oxygen metabolism, primarily generated by enzymes like SOD and XOD, and degraded by enzymes such as CAT and POD. H₂O₂ is not only a significant ROS but also a hub for the interconversion of various reactive oxygen species. On one hand, H₂O₂ can directly or indirectly oxidize biological macromolecules like nucleic acids and proteins within cells, damaging cell membranes and thereby accelerating cellular aging and disintegration. On the other hand, H₂O₂ is a key regulatory factor in many oxidative stress responses. It can activate factors like NF-κB, and these H₂O₂-related signaling pathways are associated with numerous diseases such as asthma, inflammatory arthritis, arteriosclerosis, and neurodegenerative disorders. H₂O₂ is also closely linked to processes like cell apoptosis and proliferation.
Assay Principle
H₂O₂ reacts with titanium sulfate to form a yellow peroxide-titanium complex precipitate. This precipitate dissolves in strong acid, and the intensity of the yellow color is linearly proportional to the H₂O₂ concentration within a certain range. The absorbance at 412 nm is measured using a colorimetric method. This kit is primarily used to detect H₂O₂ content in samples such as plant tissues, serum, and plasma. It is intended for research use only and not suitable for clinical diagnosis or other purposes.
H1505496
| Component
| 50T | Storage
|
H1505496A
| H₂O₂ Stock Solution
| 1 mL | 2-8℃. Store in the dark.
|
H1505496B
| Alkaline Matrix Solution
| 10.5 mL | RT. |
H1505496C
| Titanium Sulfate
| 0.3 g | RT.
|
H1505496D
| Acidic Matrix Solution
| 100 mL | RT. |
Required Materials Not Provided
1. Distilled water, Acetone
2. Homogenizer or mortar and pestle, Refrigerated centrifuge, Spectrophotometer, Cuvettes
Experimental Procedure
1. Sample Preparation
1.1 Plant Samples
Take fresh plant tissue (under normal or stress conditions), wash clean, dry, and mince.
Quickly weigh 5 g, add 5 mL of pre-chilled acetone.
Homogenize or grind rapidly in an ice bath.
Centrifuge at 12,000 g for 20 min at 4°C.
Collect the supernatant, measure the total volume of the extract, and store at 4°C for later use.
1.2 Plasma, Serum, and Urine Samples
1.3 High-Activity Samples
2. Preparation of 10 mM H₂O₂ Standard Solution
Since H₂O₂ is not very stable, its actual concentration needs to be determined before use.
The H₂O₂ Stock Solution provided has an H₂O₂ concentration of approximately 1 M. Dilute it 100-fold with distilled water to achieve ~10 mM.
Zero the spectrophotometer with distilled water and measure the absorbance at 240 nm (A₂₄₀).
Calculate the actual H₂O₂ concentration using the formula: H₂O₂ concentration (mM) = A₂₄₀ × 22.94.
Then, dilute the H₂O₂ Stock Solution with acetone to prepare a 10 mM H₂O₂-acetone standard solution.
Prepare serial dilutions according to the table below (common range 0.3-3 mM, i.e., Tubes 1-5):
| Tube | 10 mM H₂O₂-Acetone Standard (mL)
| Pre-chilled Acetone (mL)
| Final H₂O₂ Concentration (mM)
|
| 1 | 0.03 | 0.97 | 0.3 |
| 2 | 0.05 | 0.95 | 0.5 |
| 3 | 0.08 | 0.92 | 0.8 |
| 4 | 0.1 | 0.9 | 1 |
| 5 | 0.3 | 0.7 | 3 |
| 6 | 0.5 | 0.5 | 5 |
| 7 | 0.8 | 0.2 | 8 |
3. Preparation of Titanium Sulfate Solution
4. H₂O₂ Assay Setup
Set up Blank, Standard, and Test tubes as per the table below.
Add reagents in the specified order, avoiding bubbles.
If the sample H₂O₂ concentration is too high, reduce the sample volume or dilute appropriately before assay. Duplicate tubes are recommended.
Reagent (mL)
| Blank Tube
| Standard Tube
| Test Tube
|
Pre-chilled Acetone
| 1.0 | —
| —
|
Serial H₂O₂-Acetone Standards
| —
| 1.0
| —
|
Sample Extract
| —
| —
| 1.0
|
Alkaline Matrix Solution
| 0.2 | 0.2
| 0.2
|
Titanium Sulfate Solution
| 0.1 | 0.1
| 0.1
|
Note: When adding Alkaline Matrix Solution and Titanium Sulfate Solution, add them directly into the liquid, avoiding the tube walls.
5. H₂O₂ Measurement
- Mix well and incubate at room temperature for 5 minutes.
- Centrifuge at 12,000 g for 15 minutes.
- Discard the supernatant, retaining the precipitate.
- If necessary, wash the precipitate repeatedly with pre-chilled acetone.
- Add 2 mL of Acidic Matrix Solution to the precipitate in each tube.
- Shake to dissolve the precipitate completely.
- Using a 1 cm light path cuvette, zero the spectrophotometer with the blank solution.
- Measure the absorbance of standard and test tubes at 412 nm.
- Note: A microplate reader can also be used if a spectrophotometer is unavailable.
6. Calculation of Results
6.1 Standard Curve Plotting
Plot the standard curve with H₂O₂-acetone standard concentrations (0.3, 0.5, 0.8, 1, 3, 5, 8 mM) on the X-axis and corresponding absorbances on the Y-axis. Determine the regression equation.
6.2 Sample H₂O₂ Concentration Calculation
Substitute the test sample absorbance into the regression equation to find its H₂O₂ concentration (C₀).
For tissue samples: H₂O₂ (mmol/g weight) = (C₀ × VT × N) / m
For liquid samples: H₂O₂ (mmol/L) = C₀ × N
Parameter Definitions:
C₀: H₂O₂ concentration (mM) obtained from the standard curve based on the sample's absorbance.
VT: Total volume of the test sample extract (L).
m: Sample mass (g).
N: Sample dilution factor.
Results Presentation
Standard Curve Example: When measured according to the instructions at room temperature, the absorbance values for the serial standards and a typical standard curve are as follows (for reference only). A standard curve was plotted using H₂O₂ standards (0.1, 0.3, 0.5, 0.8, 1, 3, 5, 8, 10 mM). Note: Standard concentrations that are too high or too low may affect the accuracy of the standard curve.
H₂O₂ Standard (mM)
| 0.1 | 0.3 | 0.5 | 0.8 | 1 | 3 | 5 | 8 | 10 |
| Absorbance | 0.009 | 0.065 | 0.141 | 0.216 | 0.264 | 0.812 | 1.262 | 2.010 | 2.355 |
Note: H₂O₂ concentrations below 0.3 mM are basically colorless, 0.3~1 mM appear yellow, and 3~10 mM appear orange-yellow. Refer to the image below for expected results.
Precautions
1. This kit can also be used with a microplate reader, allowing for a corresponding increase in the number of samples processed.
2. When adding Alkaline Matrix Solution and Titanium Sulfate Solution, add them directly into the solution, avoiding the tube walls.
3. Dissolving the yellow peroxide-titanium complex precipitate in the Acidic Matrix Solution takes time. Ensure complete dissolution to prevent affecting the results.
4. The H₂O₂ Stock Solution and Alkaline Matrix Solution should be tightly sealed to prevent evaporation, which could reduce efficiency.
5. The H₂O₂ Stock Solution and Acidic Matrix Solution are corrosive. Handle with care.
6. The Titanium Sulfate solution should be used promptly after preparation. If not used immediately, it can be stored short-term at 4°C. Alternatively, prepare a 5% solution by weighing the appropriate amount of powder as needed.
7. For your safety and health, wear lab coats and disposable gloves during operation.
8. Use reagents promptly after opening to avoid affecting subsequent experimental results.
ZJ26F1039895
危险品化学品经营许可证（带存储）