计算溶液所需的质量、体积或浓度。
DPPH自由基清除率检测试剂盒 (微量法)
有货
为什么选择此级别
BioReagent,标曲法 级 ，适用于对基线干扰要求严格的色谱和分析工作流程。
储存与运输
2-8°C储存,避光。低温运输 。请查阅批次 COA 获取详细规格。
质量文档
SDS、COA、产品数据表及规格说明书均可下载。可通过批号查询获取批次 COA。
文献证明
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。
概述
在生命活动的代谢过程中不断产生各种自由基，而自由基的积累会使机体产生氧化损伤而引起衰老、肿瘤、中风、心肌炎和糖尿病等慢性疾病，在众多自由基中2，2-二苯基-1-苦基苯肼 (DPPH)是一种较稳定的自由基，当有自由基清除剂存在时颜色由紫色向黄色转变，吸光度随之变小。
DPPH自由基清除率检测试剂盒 (微量法) 又称氮自由基清除能力检测试剂盒或氮自由基清除率检测试剂盒，其检测原理是DPPH自由基是一种较稳定的含氮自由基，含一个单电子，溶于乙醇后溶液呈紫色，在517nm下有强吸收，如果有其他物质提供一个电子与此单电子配对，溶液会褪色，褪色程度与接受电子的量呈正比，通过吸光度下降的程度来反应样品的氮自由基清除能力，主要用于检测血清、植物组织、抗氧化类食品、保健品及药品等样本。该试剂盒仅用于科研领域，不适用于临床诊断或其他用途。
组分与储存：
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自备材料：
1. 植物组织 (芹菜、玉米等叶片)、血液、蒸馏水、无水乙醇、氢氧化钠、盐酸等实验材料
2. 研钵或匀浆器、粉碎机或超声破碎机、离心机、离心管、恒温水浴锅、恒温干燥箱电子天平、30-50目筛、酶标仪、96孔板
操作步骤（仅供参考）：
1. 准备样品：
① 植物样品：取新鲜植物样本，清洗干净，研钵研碎 (粉碎)，干燥箱烘干后过筛，称取0.05g样品，加入0.8ml氮自由基提取液，40℃水浴浸提30-60min，10000rpm 离心10min，上清液待用，4℃保存备用 (亦可参考相关资料提取方法提取)。
② 血浆、血清和尿液样品：血浆 (用肝素或枸橼酸钠抗凝，不宜使用 EDTA抗凝) 4℃ 5000rpm 离心10min，取上清液待用；血清或尿液样品可直接测定。
③ 细胞样品：按每5x10⁶个细胞加入0.8ml氮自由基提取液，超声破碎 (功率200W，超声3s，间隔10s，重复30次)，10000rpm 离心10min，取上清液待用。
④ 果汁、葡萄酒等样品：按样品：氮自由基提取液=1:9 的比例震荡混匀，10000rpm离心10min，上清液待用，4℃保存备用。
⑤ 高活性样品：如果样品中有效浓度较高，可用提取液进行恰当的稀释。
2. 配制维生素C溶液：将一支10mg 维生素C 充分溶解于1ml氮自由基提取液中，即成10mg/ml 维生素C 溶液；可分成 0.1ml的小份，-20°℃保存。
3. 配制 VC 标准工作液：如需测线性关系，建议用氮自由基提取液将10mg/ml 维生素C 溶液稀释至50、40、30、20、10、5μg/ml 的 VC 标准工作液；如需清除率大于90%的阳性对照，建议选用大于50μg/ml的 VC 标准工作液。
4. 酶标仪开机预热30min以上，调节波长517nm (500-530nm亦可)，无水乙醇调零。
5. DPPH加样：按照下表设置空白管、样本测定管、样本对照管、阳性对照管，溶液应按照顺序依次加入，混匀，室温避光静置30min。
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6. OD值测定：将各管溶液室温 12000rpm 离心5min，取上清300μl加至96孔板中，用酶标仪检测各管吸光度值，依次记为A₀、A₁、A₂、A₃。
计算：
阳性对照 ·DPPH清除率(%)=(A₀-A₃)/A₀×100%
待测样本 ·DPPH清除率(%)=[A₀-(A₁-A₂)]/A₀×100%
备注：
A₀=空白管的吸光度值
A₁=样本测定管的吸光度值
A₂=样本对照管的吸光度值
A₃=阳性对照管的吸光度值
注意事项：
1. 正式测定之前建议选择2-3个预期差异较大的样本做预实验。
2. 实验材料应尽量新鲜，当天提取当天测定。
3. 不同样本清除DPPH自由基的能力差异很大。如样本 DPPH清除率大于90%，应用提取液稀释；如样本DPPH 清除率小于5%，应提高样本用量以提高有效成分浓度。
4. 如样品活性较低时，可通过提高上清液加入量或减少DPPH溶液加入量来增加检测的灵敏度。
5. 不同批次生产的DPPH 溶液初始吸光度值可能有差异，根据实际情况，DPPH溶液可用无水乙醇稀释1-2倍后使用。(参见附录2)
6. 样品中不宜添加 Triton、DTT 等可能影响氧化还原反应的成分。
7. 本方法受反应时间、DPPH初浓度、样品性质的影响，还受到光照、氧气、pH值、有机离子、无机离子等外界因素的影响。
8. 如果样品提取液 pH 值偏碱性，将可能导致样本测定管吸光度大于空白管吸光度，使清除率为负值，可用氢氧化钠等调整 pH至中性。
9. 为了您的安全和健康，请穿实验服并戴一次性手套操作。
10. 试剂开封后请尽快使用，以防影响后续实验效果。
附录1：
标准曲线制作：在室温条件下按说明书操作，对系列 VC 标准工作液 (60、50、40、30、20、10μg/ml)进行吸光度的测定，其数值及标准曲线如下 (仅供参考)：
| 初始吸光度值 | 调零吸光度值 | DPPH清除率 | |
| 无水乙醇 | 0.039 | / | / |
| 空白管 | 0.641 | 0.602 | 0 |
| VC 标准 10μg/ml | 0.557 | 0.518 | 13.95348837 |
| VC 标准 20μg/ml | 0.451 | 0.376 | 37.54152824 |
| VC 标准 30μg/ml | 0.328 | 0.289 | 51.99335548 |
| VC 标准 40μg/ml | 0.210 | 0.171 | 71.59468439 |
| VC 标准 50μg/ml | 0.092 | 0.053 | 91.19601329 |
| VC 标准 60μg/ml | 0.075 | 0.036 | 94.01993355 |
由上图可知： VC 标准工作液在0~50μg/mI时线性关系良好，DPPH清除率与 VC 浓度呈正相关，当 VC 浓度大于50μg/mI时，DPPH清除率接近最大值，数据有偏差。
附录2：
用无水乙醇将 DPPH溶液分别稀释1倍和2倍后， VC 标准分别在0~28μg/ml和0~20μg/mI时线性关系良好，DPPH清除率与 VC 浓度呈正相关，数据参考如下：
| 无水乙醇+DPPH溶液(1+1) | 无水乙醇+DPPH溶液(2+1) | |||
| 调零吸光度值 | DPPH 清除率 | 调零吸光度值 | DPPH 清除率 | |
| 空白管 | 0.304 | 0 | 0.209 | 0 |
| VC 标准 1μg/ml | 0.274 | 9.868421053 | 0.204 | 2.392344498 |
| VC 标准 4μg/ml | 0.256 | 15.78947368 | 0.171 | 18.18181818 |
| VC 标准 6μg/ml | 0.237 | 22.03947368 | 0.143 | 31.57894737 |
| VC 标准 8μg/ml | 0.219 | 27.96052632 | 0.127 | 39.23444976 |
| VC 标准 10μg/ml | 0.190 | 37.5 | 0.097 | 53.58851675 |
| VC 标准 15μg/ml | 0.157 | 48.35526316 | 0.053 | 74.64114833 |
| VC 标准 20μg/ml | 0.079 | 74.01315789 | 0.012 | 94.25837321 |
| VC 标准 30μg/ml | 0.018 | 94.07894737 | 0.010 | 95.215311 |
Various free radicals are continuously generated during metabolic processes of life activities. Accumulation of free radicals causes oxidative damage to the organism, leading to chronic diseases such as aging, tumors, stroke, myocarditis and diabetes. Among numerous free radicals, 2,2-diphenyl-1-picrylhydrazyl (DPPH) is a relatively stable free radical. In the presence of free radical scavengers, its color changes from purple to yellow accompanied by a decrease in absorbance.
The DPPH Free Radical Scavenging Capacity Assay Kit (Microplate Method), also known as Nitrogen Radical Scavenging Capacity Assay Kit or Nitrogen Radical Scavenging Rate Assay Kit, is based on the following principle: DPPH free radical is a stable nitrogen-containing free radical with an unpaired single electron. Its ethanol solution displays purple color and exhibits strong absorbance at 517 nm. When other substances donate an electron to pair with this single electron, the solution fades. The degree of discoloration is proportional to the amount of electrons accepted. The nitrogen radical scavenging capacity of samples can be reflected by the reduction of absorbance. This kit is mainly applied to samples including serum, plant tissues, antioxidant foods, health supplements and pharmaceuticals. This kit is for research use only and is not intended for clinical diagnosis or any other applications.
Contents & Storage:
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Materials to be prepared:
1. Experimental materials: plant tissues (celery, corn leaves, etc.), blood, distilled water, anhydrous ethanol, sodium hydroxide, hydrochloric acid, etc.
2. Mortar or homogenizer, grinder or ultrasonicator, centrifuge, centrifuge tubes, thermostatic water bath, constant-temperature drying oven, electronic balance, 30–50 mesh sieve, microplate reader, 96-well plate
Operating Procedures (For Reference Only):
1. Sample Preparation
① Plant samples: Take fresh plant samples, clean thoroughly, grind in a mortar. Dry the ground material in a drying oven and sieve. Weigh 0.05 g sample, add 0.8 mL nitrogen radical extract, extract in a 40 °C water bath for 30–60 min, then centrifuge at 10,000 rpm for 10 min. Retain the supernatant and store at 4 °C for later use (alternative extraction protocols from published references are also acceptable).
② Plasma, serum and urine samples: Plasma samples should be anticoagulated with heparin or sodium citrate (EDTA anticoagulant is not recommended). Centrifuge plasma at 5,000 rpm for 10 min at 4 °C and collect supernatant. Serum or urine samples can be tested directly.
③ Cell samples: Add 0.8 mL nitrogen radical extract per 5×10⁶ cells. Lyse cells by sonication (power: 200 W, sonicate for 3 s, interval 10 s, repeat 30 times). Centrifuge at 10,000 rpm for 10 min, collect supernatant for subsequent assay.
④ Fruit juice, wine and similar samples: Mix sample and nitrogen radical extract at a volume ratio of 1:9 with shaking. Centrifuge at 10,000 rpm for 10 min. Collect supernatant and store at 4 °C.
⑤ Highly active samples: If the effective concentration of the sample is high, appropriately dilute the sample with nitrogen radical extract.
2. Preparation of Vitamin C Stock Solution
Fully dissolve one vial of 10 mg Vitamin C in 1 mL nitrogen radical extract to prepare 10 mg/mL Vitamin C solution. Aliquot into 0.1 mL portions and store at −20 °C.
3. Preparation of VC Standard Working Solution
If linear relationship measurement is required, dilute the 10 mg/mL Vitamin C stock solution with nitrogen radical extract to prepare VC standard working solutions at concentrations of 50, 40, 30, 20, 10 and 5 μg/mL. For positive control with DPPH scavenging rate over 90%, standard working solution above 50 μg/mL is recommended.
4. Turn on the microplate reader and preheat for no less than 30 min. Set detection wavelength to 517 nm (500–530 nm is also acceptable). Perform zero calibration using anhydrous ethanol.
5. DPPH Reagent Addition
Set blank tube, sample detection tube, sample control tube and positive control tube (standard tube) according to the table below. Add reagents sequentially, mix thoroughly, incubate at room temperature in the dark for 30 min.
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6. OD Measurement
Centrifuge all tube solutions at 12,000 rpm for 5 min at room temperature. Transfer 300 μL supernatant into a 96-well plate. Measure absorbance values with a microplate reader, recorded sequentially as A₀, A₁, A₂, A₃.
Calculations:
Positive control ·DPPH Scavenging Rate (%)=(A₀-A₃)/A₀×100%
Test sample ·DPPH Scavenging Rate (%) =[A₀-(A₁-A₂)]/A₀×100%
Notes:
A₀ = Absorbance value of blank tube
A₁ = Absorbance value of sample detection tube
A₂ = Absorbance value of sample control tube
A₃ = Absorbance value of positive control tube
Precautions:
1. It is recommended to conduct a preliminary experiment with 2–3 samples with expected significant differences before formal detection.
2. All experimental materials should be kept fresh. Perform extraction and determination on the same day.
3. The DPPH radical scavenging capacity varies greatly among different samples. If the DPPH scavenging rate exceeds 90%, dilute the sample with extraction solution; if the scavenging rate is lower than 5%, increase the sample dosage to raise the concentration of active ingredients.
4. For samples with low activity, the detection sensitivity can be improved by increasing the addition amount of supernatant or reducing the dosage of DPPH solution.
5. The initial absorbance of DPPH solution may vary between different batches. If necessary, DPPH solution can be diluted 1–2 times with absolute ethanol before use (see Appendix 2).
6. Avoid adding substances that interfere with redox reactions, such as Triton and DTT, to samples.
7. This method is affected by reaction time, initial DPPH concentration and sample properties, as well as external factors including light, oxygen, pH value, organic ions and inorganic ions.
8. Alkaline sample extract may result in a higher absorbance of the sample assay tube than the blank tube, leading to a negative scavenging rate. Adjust the pH to neutral with sodium hydroxide or other neutralizers.
9. Wear laboratory coats and disposable gloves during operation for personal safety and health protection.
10. Use reagents as soon as possible after opening to avoid affecting subsequent experimental results.
Appendix 1:
Standard Curve Preparation: Operate in accordance with the instructions at room temperature. Determine the absorbance of a series of VC standard working solutions (60, 50, 40, 30, 20, 10 μg/mL). The measured data and standard curve are shown below (for reference only).
| Initial absorbance value | Zero-set absorbance value | DPPH scavenging rate | |
| Absolute ethanol | 0.039 | / | / |
| Blank Tube | 0.641 | 0.602 | 0 |
| VC Standard 10μg/ml | 0.557 | 0.518 | 13.95348837 |
| VC Standard 20μg/ml | 0.451 | 0.376 | 37.54152824 |
| VC Standard 30μg/ml | 0.328 | 0.289 | 51.99335548 |
| VC Standard 40μg/ml | 0.210 | 0.171 | 71.59468439 |
| VC Standard 50μg/ml | 0.092 | 0.053 | 91.19601329 |
| VC Standard 60μg/ml | 0.075 | 0.036 | 94.01993355 |
As shown in the figure above, the VC standard working solution presents a good linear relationship within the concentration range of 0–50 μg/mL, and the DPPH scavenging rate is positively correlated with VC concentration. When the VC concentration exceeds 50 μg/mL, the DPPH scavenging rate approaches the maximum value, resulting in data deviation.
Appendix 2:
After the DPPH solution is diluted 1-fold and 2-fold with absolute ethanol respectively, the linearity remains favorable for st VC andards at 0–28 μg/mL and 0–20 μg/mL correspondingly. The DPPH scavenging rate is positively correlated with VC concentration. Relevant reference data are listed below.
| Absolute Ethanol + DPPH Solution (1+1) | Absolute Ethanol + DPPH Solution (2+1) | |||
| Zero-set absorbance value | DPPH scavenging rate | Zero-set absorbance value | DPPH scavenging rate | |
| Blank Tube | 0.304 | 0 | 0.209 | 0 |
| VC Standard 1μg/ml | 0.274 | 9.868421053 | 0.204 | 2.392344498 |
| VC Standard 4μg/ml | 0.256 | 15.78947368 | 0.171 | 18.18181818 |
| VC Standard 6μg/ml | 0.237 | 22.03947368 | 0.143 | 31.57894737 |
| VC Standard 8μg/ml | 0.219 | 27.96052632 | 0.127 | 39.23444976 |
| VC Standard 10μg/ml | 0.190 | 37.5 | 0.097 | 53.58851675 |
| VC Standard 15μg/ml | 0.157 | 48.35526316 | 0.053 | 74.64114833 |
| VC Standard 20μg/ml | 0.079 | 74.01315789 | 0.012 | 94.25837321 |
| VC Standard 30μg/ml | 0.018 | 94.07894737 | 0.010 | 95.215311 |
规格
别名
氮自由基清除能力测试盒 | DPPH自由基清除能力检测试剂盒 | 氮自由基（DPPH）清除能力测试盒
英文别名
DPPH Free Radical Scavenging Capacity Assay Kit | DPPH Free Radical Scavenging Capacity Assay Kit, Colorimetric Method
规格或纯度
BioReagent, 标曲法
英文名称
DPPH Free Radical Scavenging Assay Kit (Micro Method)
应用
细胞代谢
样品类型
保健品, 抗氧化类食品, 植物组织, 药品, 血清
检测方法
分光光度法
定量&半定量
定量
检测仪器
酶标仪
检测波长
517nm
敏感性
Light-sensitive
储存与运输
储存条件
2-8°C储存,避光
运输条件
低温运输
稳定性与储存
Store at 2-8℃ long term (6 months). Store in the dark.
组分与储存
| 组分 | 名称 | 物理外观 | 100T | 储存 |
| D1522019A | 氮自由基提取液 | 液体 | 100 mL | 2-8℃. |
| D1522019B | DPPH溶液 | 液体 | 25 mL | 2-8℃，避光 |
| D1522019C | 维生素C | 粉末 | 10 mg | 2-8℃，避光 |
技术文档
高级数据
质检证书（CoA,COO,BSE/TSE 和分析图谱)
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输入批号以搜索分析图谱:
通过匹配包装上的批号来查找并下载产品的 COA，每批产品都进行了严格的验证，您可放心使用！
| 批号(Lot Number) | 证书类型 | 货号 |
|---|---|---|
 ZJ26F0939288 |
分析证书 | D1522019 |
技术文档和文章
溶液计算器
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