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。
羟基自由基·OH是氢氧根离子（OH⁻）的中性形式，具有较强的氧化能力。羟自由基作用于体内蛋白质、核酸、脂类等生物分子，造成细胞结构和功能受损，进而导致体内代谢紊乱，引起疾病。羟自由基清除能力是抗氧化能力的重要指标之一，在抗氧化类保健品和药品研究中得到广泛应用。
检测原理：H₂O₂/Fe²⁺ 通过Fenton反应产生羟自由基，水杨酸能有效捕捉产生的羟自由基并与其反应生成紫色的2,3-二羟基苯甲酸。加入具有清除羟自由基的物质后，抑制紫色产物的形成。因此显色越深，说明羟自由基含量越高，反之含量越低。测定其在520nm处吸光值的变化即可计算出样本清除羟自由基的能力。
适用样本：动植物组织、血清（浆）、细胞、细菌、细胞上清、果汁、蜂蜜、尿液等样本
| P1501782 | Component | 48 T | 96 T | Storage |
| P1501782A | Ferrous Salt | 10 mL | 20 mL | 2-8℃. Store in the dark. |
| P1501782B | H₂O₂ | 5 mL | 10 mL | 2-8℃. Store in the dark. |
| P1501782C | Salicylic Acid | 10 mL | 20 mL | 2-8℃. Store in the dark. |
请在实验前检查各组分的量。
各组分在规格基础上额外提供10%的量用于进行标准曲线制作或预实验。
自备仪器和试剂
| 自备资源类型 | 资源名称 | 备注 |
| 仪器 | 酶标仪 | 能测520nm处的吸光度 |
| 耗材 | 96孔酶标板 | 普通酶标板 |
| 试剂 | PBS (pH7.4) | 用于清洗样本 |
| 其他 | 匀浆器（组织样本）、恒温箱、冰盒、低温离心机、可调节式移液枪及枪头 | 检测量较大时合理使用多通道移液器有助于提高实验效率 |
实验流程
1、试剂准备
| 试剂名称 | 试剂准备 | 注意事项 |
| Ferrous Salt | 即用型；使用前平衡到室温 | 4℃避光保存。具有腐蚀性，建议使用时做好个人防护。 |
| H₂O₂ | 即用型；使用前平衡到室温 | 4℃避光保存 |
| Salicylic Acid | 即用型；使用前平衡到室温 | 4℃避光保存。对皮肤和黏膜具有刺激性，建议使用时做好个人防护。 |
2、样本准备
注意：推荐使用新鲜样本，如果不立即进行实验，样本可在-80℃保存1个月。为了比较不同样本羟自由基清除能力，对于同一批样本必须稀释倍数相同，提取物或者药物配制成同样浓度。
2.1 动物组织样本
称取约0.1g样本，加入1mL去离子水，冰浴匀浆，10000g，4°C离心10min，取上清液置冰上待测。
2.2 植物组织样本
称取约0.1g样本，加入1mL去离子水捣碎，冰浴超声破碎5min（功率20%或200W，超声3s，间隔7s，重复30次），10000g，4°C离心10min，取上清液置冰上待测。
2.3 细胞（菌）
收集5×10⁶个细胞或细菌到离心管中，用预冷的PBS清洗细胞或细菌，离心后弃上清，加入1mL去离子水，冰浴超声破碎5min（功率20%或200W，超声3s，间隔7s，重复30次），然后10000g，4°C离心10min，取上清液置冰上待测。
2.4 血清（浆）等蛋白含量高或浑浊的液体
取0.1mL样本，加入1mL去离子水混匀，10000g，4°C离心10min，取上清液置冰上待测。
2.5 蜂蜜和尿液等蛋白含量低且清澈的液体
直接检测。
2.6 提取物或者药物
可配制成一定浓度，如0.5 mg/mL进行测定。
3、实验步骤
3.1 酶标仪准备：预热30min以上，调节波长到520nm。
3.2 检测体系配制：在96孔板中按照如下方式操作。空白管和标准管只需做1-2次。每个测定孔需要设置一个对照孔。
| 试剂 | 空白孔 (μL) | 标准孔 (μL) | 测定孔 (μL) | 对照孔 (μL) |
| Ferrous Salt | 40 | 40 | 40 | 40 |
| H₂O₂ | 0 | 40 | 40 | 0 |
| 去离子水 | 120 | 80 | 40 | 80 |
| Salicylic Acid | 40 | 40 | 40 | 40 |
| 样本 | 0 | 0 | 40 | 40 |
3.3 吸光度检测：混匀，37°C反应20min，在520nm处读取吸光度值，分别记为A 空白 、A 标准 、A 测定 和A 对照 。
4、结果计算
以下为您提供的推导过程计算公式和简洁计算公式，两者完全相等。
4.1 数据处理
计算ΔA 测定 =A 测定 -A 对照 ，ΔA 标准 =A 标准 -A 空白 。
4.2 样本羟自由基清除率计算
羟自由基清除率D%=(ΔA 标准 -ΔA 测定 )÷ΔA 标准 ×100%
5、结果展示
示例：取0.1g油桃果肉，按照测定步骤操作，用96孔板进行检测。测得ΔA 标准 =A 标准 -A 空白 =1.020-0.051=0.969，ΔA 测定 =A 测定 -A 对照 =0.465-0.052=0.413，计算羟自由基清除率D%=(ΔA 标准 -ΔA 测定 )÷ΔA 标准 ×100%=(0.969-0.413)÷0.969×100%=57.38%
注意事项
1、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。
2、对于组织样本及细胞样本等，可通过测定蛋白浓度来进行样本间结果均一化。推荐使用阿拉丁B665595 BCA蛋白定量试剂盒或R1491648 即用型BCA蛋白定量试剂盒。
3、本试剂盒兼容分光光度计检测，请按照分光光度计检测要求按比例调节检测试剂配制量。
4、生化检测试剂普遍具有刺激性、生物毒性等，为了您的安全和健康，请在实验全程做好生物安全防护，穿戴实验服、口罩、手套、头套等安全护具，在通风橱、生物安全柜中进行实验。
5、本产品仅供科学研究使用，不适用于临床诊断。
A：如果样本ΔA 测定 ＜0.02，可适当增加样本量，再重新进行测定。如果样本ΔA 测定 ＞ΔA 标准 ，样本可用去离子水进一步稀释或减少提取用样本量，再重新进行测定。
The hydroxyl radical (·OH) is the neutral form of the hydroxide ion (OH⁻) and possesses strong oxidizing capacity. Hydroxyl radicals act on biological molecules such as proteins, nucleic acids, and lipids within the body, damaging cellular structure and function, which can lead to metabolic disorders and disease. The hydroxyl radical scavenging capacity is a key indicator of antioxidant ability and is widely used in research on antioxidant health products and pharmaceuticals.
Detection Principle: H₂O₂/Fe²⁺ generates hydroxyl radicals via the Fenton reaction. Salicylic acid effectively captures these generated hydroxyl radicals and reacts with them to produce a purple compound, 2,3-dihydroxybenzoic acid. When a substance capable of scavenging hydroxyl radicals is added, it inhibits the formation of this purple product. Therefore, a darker color indicates a higher hydroxyl radical content, and vice versa. The change in absorbance at 520 nm is measured to calculate the sample's hydroxyl radical scavenging capacity.
Applicable Samples: Animal and plant tissues, serum (plasma), cells, bacteria, cell culture supernatants, fruit juice, honey, urine, and other samples.
| P1501782 | Component | 48 T | 96 T | Storage |
| P1501782A | Ferrous Salt | 10 mL | 20 mL | 2-8℃. Store in the dark. |
| P1501782B | H₂O₂ | 5 mL | 10 mL | 2-8℃. Store in the dark. |
| P1501782C | Salicylic Acid | 10 mL | 20 mL | 2-8℃. Store in the dark. |
Please check the quantities of all components before starting the experiment.
An additional 10% of each component is provided beyond the specified volumes for standard curve preparation or preliminary experiments.
User-Prepared Instruments and Reagents
| Type | Name | Notes |
| Instrument | Microplate Reader | Capable of measuring absorbance at 520 nm. |
| Consumables | 96-well Microplate | Standard microplate. |
| Reagents | PBS (pH7.4) | For washing samples. |
| Others | Homogenizer (for tissue samples), Incubator, Ice Box, Refrigerated Centrifuge, Adjustable Micropipettes and Tips | Using a multi-channel pipette can improve efficiency for large-scale assays. |
Experimental Procedure
1. Reagent Preparation
| Reagent Name | Reagent Preparation | Notes |
| Ferrous Salt | Ready-to-use; Equilibrate to room temperature before use. | Store at 4°C protected from light. Corrosive. Use appropriate personal protective equipment. |
| H₂O₂ | Ready-to-use; Equilibrate to room temperature before use. | Store at 4°C protected from light. |
| Salicylic Acid | Ready-to-use; Equilibrate to room temperature before use. | Store at 4°C protected from light. Irritating to skin and mucous membranes. Use appropriate personal protective equipment. |
2. Sample Preparation
Note: Fresh samples are recommended. If not used immediately, samples can be stored at -80°C for one month. To compare the hydroxyl radical scavenging capacity of different samples, the dilution factor must be the same for the same batch of samples, and extracts or drugs should be prepared at the same concentration.
2.1 Animal Tissue Samples
Weigh approximately 0.1 g of tissue, add 1 mL of deionized water, and homogenize in an ice bath. Centrifuge at 10,000 g, 4°C for 10 minutes. Collect the supernatant and keep it on ice for assay.
2.2 Plant Tissue Samples
Weigh approximately 0.1 g of tissue, add 1 mL of deionized water and grind. Sonicate in an ice bath for 5 minutes (power 20% or 200W, pulse 3s on, 7s off, repeat 30 times). Centrifuge at 10,000 g, 4°C for 10 minutes. Collect the supernatant and keep it on ice for assay.
2.3 Cells or Bacteria
Collect 5×10⁶ cells or bacteria into a centrifuge tube. Wash with pre-cooled PBS, centrifuge, and discard the supernatant. Add 1 mL of deionized water. Sonicate in an ice bath for 5 minutes (power 20% or 200W, pulse 3s on, 7s off, repeat 30 times). Centrifuge at 10,000 g, 4°C for 10 minutes. Collect the supernatant and keep it on ice for assay.
2.4 Serum (Plasma) and Other Protein-Rich or Turbid Liquids
Take 0.1 mL of sample, add 1 mL of deionized water and mix well. Centrifuge at 10,000 g, 4°C for 10 minutes. Collect the supernatant and keep it on ice for assay.
2.5 Honey, Urine, and Other Clear Liquids with Low Protein Content
Assay directly.
2.6 Extracts or Drugs
Can be prepared to a specific concentration, e.g., 0.5 mg/mL.
3. Assay Steps
3.1 Microplate Reader Preparation: Preheat for at least 30 minutes. Set the wavelength to 520 nm.
3.2 Assay System Setup: Perform the following operations in a 96-well plate. The Blank and Standard wells only need to be set up 1-2 times. Each test well requires a corresponding control well.
| Reagent | Blank Well (μL) | Standard Well (μL) | Test Well (μL) | Control Well (μL) |
| Ferrous Salt | 40 | 40 | 40 | 40 |
| H₂O₂ | 0 | 40 | 40 | 0 |
| Deionized Water | 120 | 80 | 40 | 80 |
| Salicylic Acid | 40 | 40 | 40 | 40 |
| Sample | 0 | 0 | 40 | 40 |
3.3 Absorbance Measurement: Mix well, incubate at 37°C for 20 minutes. Read the absorbance at 520 nm, recorded as A blank , A standard , A test , and A control respectively.
4. Calculation of Results
Both the derived formula and the simplified formula provided below are equivalent.
4.1 Data Processing
Calculate ΔA test = A test - A control
Calculate ΔA standard = A standard - A blank
4.2 Calculation of Hydroxyl Radical Scavenging Rate
Hydroxyl Radical Scavenging Rate D% = (ΔA standard - ΔA test ) / ΔA standard × 100%
5. Representative Results
Example: 0.1 g of nectarine pulp was taken and assayed according to the procedure using a 96-well plate.
Measured: ΔA standard = A standard - A blank = 1.020 - 0.051 = 0.969
ΔA test = A test - A control = 0.465 - 0.052 = 0.413
Calculated Hydroxyl Radical Scavenging Rate D% = (0.969 - 0.413) / 0.969 × 100% = 57.38%
Precautions
1. Before formal testing, it is recommended to perform a preliminary test with 2-3 samples expected to have significant differences.
2. For tissue samples, cell samples, etc., results can be normalized between samples by measuring protein concentration. Aladdin's BCA Protein Quantification Kit (B665595) or Ready-to-Use BCA Protein Quantification Kit (R1491648) is recommended.
3. This kit is compatible with spectrophotometer detection. Adjust the reagent preparation volumes proportionally according to the spectrophotometer's requirements.
4. Biochemical reagents are generally irritating and potentially biologically toxic. For your safety and health, implement appropriate biosafety precautions throughout the experiment, including wearing lab coats, masks, gloves, and head covers. Perform experiments in a fume hood or biosafety cabinet.
5. This product is for research use only. Not for use in clinical diagnosis.
A: If ΔA test < 0.02, appropriately increase the sample volume and re-run the assay. If ΔA test > ΔA standard , further dilute the sample with deionized water or reduce the amount of sample used for extraction, and re-run the assay.
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| 批号(Lot Number) | 证书类型 | 货号 |
|---|---|---|
 ZJ26F0535751 |
分析证书 | P1501782 |
| ZJ25F1129292 |
分析证书 | P1501782 |
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