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。
总酚是一类具有抗氧化和清除自由基功能的活性物质。本试剂盒采用福林酚法测定总酚含量，在碱性条件下，酚类物质将钨钼酸还原，产生蓝色化合物，在 760 nm 处有特征吸收峰，在 760 nm 处的读取吸光值，进而计算总酚含量。实验器材：
研钵（匀浆机）、冰盒（制冰机）、台式离心机、可调式移液枪、水浴锅（烘箱、培养箱、金属浴）、1 mL 比色皿、离心管、分光光度计、60% 乙醇、蒸馏水（去离子水、超纯水均可）。
组分与储存
| 组分货号 | 组分名称 | 物理外观 | 48T | 储存 |
| T1527571A | 试剂一 | 液体 | 12mL | 2-8℃.避光保存 |
| T1527571B | 试剂二 | 液体 | 12mL | 2-8℃. |
| T1527571C | 标准品 | 粉末 | 1EA | 2-8℃. |
试剂注意事项：
标准品：
1. 若重新做标曲，则用到该试剂；
2. 按照说明书中标曲制作步骤进行配制；
3. 溶解后的标品一周内用完。
指标测定：
建议先选取 1-3 个差异大的样本（例如不同类型或分组）进行预实验，熟悉操作流程，根据预实验结果确定或调整样本浓度，以防造成样本或试剂不必要的浪费！
1、样本提取：
① 组织样本：
称取约 0.1 g 新鲜样本（若水分充足，可增加样本取样质量）；或者称取约 0.03 g 烘干样本（将样本在 105℃ 下杀青 3 min，然后 60℃ 烘干至恒重，粉碎，过 40-60 目筛，得到烘干样本），加入 1.5 mL 的 60% 乙醇（若鲜样需研磨均质），60℃ 振荡提取 2 h（若蒸发用 60% 乙醇定容至 1.5 mL），25℃×12000 rpm 离心 10 min，取上清待测。
注：若样本量较少，可同比例缩减样本量，如取 0.02 g 干样，加入 1 mL 60% 乙醇，60℃ 振荡提取 2 h。25℃×12000 rpm 离心 10 min，取上清，用 60% 乙醇定容至 1 mL 待测。
② 液体样本：直接检测；若浑浊，离心后取上清检测。
③ 细菌/培养细胞：
先收集细菌或细胞到离心管内，离心后弃上清；取约 500 万细菌或细胞至 EP 管中，加入 1 mL 的 60% 乙醇，60℃ 振荡提取 2 h（若蒸发用 60% 乙醇定容至 1 mL），25℃×12000 rpm，离心 10 min，取上清待测。
注：若增加样本量，可按照细菌或细胞数量（10⁴ 个）：提取液体积（mL）为 500:1 比例进行提取。
2、检测步骤：
① 可见分光光度计预热 30 min（等待仪器过自检程序亦可），调节波长至 760 nm，蒸馏水调零。
② 对于总酚含量较高的样本如茶叶，一般需用蒸馏水稀释后再检测如稀释 50 倍，也可先选取 2 个样本做预测定，找出适合本次检测样本的稀释倍数 D。
③ 在 EP 管中依次加入：
1.加入试剂一后混匀，25℃室温件下，暗处静置 3 min。 2.加入蒸馏水后混匀，25℃室温静置 30 min，全部液体转移至 1mL 玻璃比色皿中，测定 760nm 吸光值 A，ΔA = A测定-A对照。 |
注：1. 吸光值大于 1，样品适当稀释再测定，计算公式里乘以稀释倍数 D。2. 若 ΔA 在零附近，可增加样本取样质量 W，或加大样本上样量 V₁（如增至 50 μL，则蒸馏水相应减少，保持总体积不变），则改变后 W 和 V₁ 需代入计算公式重新计算。3. 若待检测样本有强背景色（如蓝色），需做一个样本自身对照：即试剂一用 200 μL 蒸馏水替换，其他步骤同测定管，ΔA = A测定-A对照。
结果计算：
1、标准曲线：y = 0.1709x - 0.0024，x 是标准品质量（μg），y 是 ΔA。
2、总酚（TP）含量（mg/g 干重）= (ΔA + 0.0024)÷0.1709×10⁻³÷(V₁÷V×W)×D= 0.1463×(ΔA + 0.0024)×V÷W×D
3、总酚（TP）含量（mg/mg prot）= (ΔA + 0.0024)÷0.1709×10⁻³÷(V₁÷V×Cpr)×D= 0.1463×(ΔA + 0.0024)×V÷Cpr×D
4、总酚（TP）含量（μg/mL）= (ΔA + 0.0024)÷0.1709÷V₁×D= 146.28×(ΔA + 0.0024)×D
5、总酚（TP）含量（mg/10⁴cell）= (ΔA + 0.0024)÷0.1709×10⁻³÷(V₁÷V×500)×D= 0.1463×(ΔA + 0.0024)×V÷500×D
参数说明：
V：加入提取液体积；
V₁：反应中样品体积，0.04 mL
D：稀释倍数，未稀释即为 1；
W：样品质量，g；
500：细菌或细胞总数，万；
Cpr：上清液蛋白浓度，mg/mL。
附：标准曲线制作过程：
1.标曲为非必做实验，用户可根据实验需求制作标曲，亦可直接采用说明书计算公式进行结果计算。
2.制备标准品母液（10 mg/mL）：向标准品 EP 管里面加入 1 mL 提取液（60% 乙醇），超声完全溶解；
3.将母液用 60% 乙醇稀释成六个浓度梯度的标准品，例如：0, 0.02, 0.04, 0.06, 0.08, 0.1 mg/mL。也可根据实际样本调整标准品浓度。
4.标品稀释参照表如下：
吸取标准品母液 50uL，加入 4.95 mL60%乙醇，混匀得到 0.1 mg/mL 的标品稀释液待用。
各标准管混匀待用。 |
5. 依据测定管的加样表操作，根据结果，以各浓度吸光值减去 0 浓度吸光值，过0点制作标准曲线。
1. 加入试剂一后混匀，25℃室温件下，暗处静置 3 min。 2.加入蒸馏水后混匀，25℃室温静置 30 min，全部液体转移至 1mL 玻璃 比色皿中，测定 760nm 吸光值 A，ΔA= A标准-A0浓度 。 |
Total phenols are a class of bioactive substances with antioxidant and free‑radical‑scavenging capacities. This kit determines total phenolic content via the Folin‑Ciocalteu method. Under alkaline conditions, phenolic compounds reduce phosphotungstic‑phosphomolybdic acid to produce blue‑colored complexes, which exhibit a characteristic absorption peak at 760 nm. The absorbance is measured at 760 nm to calculate total phenolic content.
Contents & Storage
| Component No. | Component Name | Appearance | 48T | Storage |
| T1527571A | Reagent Ⅰ | Liquid | 12mL | 2-8℃.Store in the dark. |
| T1527571B | Reagent Ⅱ | Liquid | 12mL | 2-8℃. |
| T1527571C | Standard | Powder | 1EA | 2-8℃. |
Experimental Equipment
Mortar (homogenizer), ice box (ice maker), table‑top centrifuge, adjustable pipettes, water bath (oven, incubator, metal bath), 1 mL cuvettes, centrifuge tubes, spectrophotometer, 60% ethanol, distilled water (deionized water or ultrapure water is acceptable).
Reagent Precautions
Standard:
1. This reagent is required if you prepare a new standard curve.
2. Prepare the standard according to the standard‑curve preparation steps in the manual.
3. Dissolved standard should be used within one week.
Index Determination
It is recommended to perform a preliminary experiment using 1‑3 samples with large differences (e.g., different types or groups) to familiarize yourself with the procedure. Adjust sample concentration based on preliminary results to avoid unnecessary waste of samples and reagents.
1. Sample Extraction
① Tissue samples:
Weigh approximately 0.1 g fresh sample (increase sample weight for high‑moisture tissues); or weigh about 0.03 g dried sample (sample is blanched at 105 °C for 3 min, then dried at 60 °C to constant weight, ground and sieved through 40‑60 mesh to obtain dried powder). Add 1.5 mL of 60% ethanol (fresh samples require grinding and homogenization). Extract with shaking at 60 °C for 2 h. If evaporation occurs, replenish to 1.5 mL with 60% ethanol. Centrifuge at 12 000 rpm for 10 min at 25 °C, collect supernatant for assay.
Note: For limited sample quantity, scale down proportionally. For example: weigh 0.02 g dried sample, add 1 mL 60% ethanol, extract with shaking at 60 °C for 2 h. If evaporation occurs, replenish to 1 mL with 60% ethanol. Centrifuge at 12 000 rpm for 10 min at 25 °C, collect supernatant and make up to 1 mL with 60% ethanol for assay.
② Liquid samples: Assay directly. Centrifuge and collect supernatant if the sample is turbid.
③ Bacteria / cultured cells:
Collect bacteria or cells into centrifuge tubes, centrifuge and discard supernatant. Transfer approximately 5 million bacteria or cells to an EP tube. Add 1 mL of 60% ethanol, extract with shaking at 60 °C for 2 h. If evaporation occurs, replenish to 1 mL with 60% ethanol. Centrifuge at 12 000 rpm for 10 min at 25 °C, collect supernatant for assay.
Note: For larger sample amounts, follow the ratio: 500 × 10⁴ cells : 1 mL extraction solution.
2. Assay Procedure
① Pre‑warm the visible‑light spectrophotometer for 30 min (or wait for instrument self‑check). Set wavelength to 760 nm and zero the instrument with distilled water.
② For samples with high total phenolic content (e.g., tea), dilute with distilled water (e.g., 50‑fold dilution) before measurement. Alternatively, test two samples first to determine the appropriate dilution factor D for your samples.
③ Add reagents sequentially into EP tubes as follows:
1. Mix well after adding Reagent Ⅰ, and incubate in the dark at 25 °C for 3 min. 2. Mix well after adding distilled water, incubate at 25 °C for 30 min. Transfer the whole solution into a 1 mL glass cuvette and measure absorbance A at 760 nmΔA = Aassay − Ablank. |
Notes:
1. If absorbance exceeds 1, dilute the sample and multiply the result by dilution factor D in the calculation formula.
2. If ΔA is close to zero, increase sample weight W or sample loading volume V₁ (e.g., increase to 50 μL; reduce distilled water accordingly to keep total volume unchanged). Use the revised W and V₁for calculation.
3. For samples with strong intrinsic background color (e.g., blue), prepare a sample blank control: replace Reagent 1 with 200 μL distilled water, keep other steps identical to the test tube. ΔA = Atest − Ablank.
Result Calculation:
Standard curve: y = 0.1709x - 0.0024, x is the standard mass (μg), y is ΔA.
Total Phenols (TP) content (mg/g dry weight) = (ΔA + 0.0024)÷0.1709×10⁻³÷(V₁÷V×W)×D= 0.1463×(ΔA + 0.0024)×V÷W×D
Total Phenols (TP) content (mg/mg prot) = (ΔA + 0.0024)÷0.1709×10⁻³÷(V₁÷V×Cpr)×D
= 0.1463×(ΔA + 0.0024)×V÷Cpr×D
Total Phenols (TP) content (μg/mL) = (ΔA + 0.0024)÷0.1709÷V₁×D
= 146.28×(ΔA + 0.0024)×D
Total Phenols (TP) content (mg/10⁴cell) = (ΔA + 0.0024)÷0.1709×10⁻³÷(V₁÷V×500)×D= 0.1463×(ΔA + 0.0024)×V÷500×D
Parameter Description：
V: Volume of extraction liquid added;
V₁: Sample volume in the reaction, 0.04 mL;
D: Dilution factor, 1 if undiluted;
W: Sample mass, g;
500; Total number of bacteria or cells, in ten thousand;
Cpr: Protein concentration of the supernatant, mg/mL.
Appendix: Standard Curve Preparation Procedure:
1.The standard curve is not a mandatory experiment. Users can prepare the standard curve according to experimental needs, or directly use the calculation formula in the instructions for result calculation.
2.Prepare the standard stock solution (10 mg/mL): Add 1 mL of extraction liquid (60% ethanol) to the standard EP tube, and ultrasonicate to completely dissolve;
3.Dilute the stock solution with 60% ethanol into six concentration gradient standards, for example: 0, 0.02, 0.04, 0.06, 0.08, 0.1 mg/mL. The standard concentrations can also be adjusted according to actual samples.
4.The standard dilution reference table is as follows:
Pipette 50 μL of standard stock solution and add it to 4.95 mL of 60% ethanol. Mix well to prepare the standard working solution at 0.1 mg/mL for later use.
Mix each standard tube well and set aside for use. |
5.Operate according to the loading table of the determination tube. According to the results, subtract the absorbance value of the 0 concentration from the absorbance value of each concentration, and prepare the standard curve through the 0 point.
2. Mix well after adding distilled water, incubate at 25 °C for 30 min. Transfer the entire solution into a 1 mL glass cuvette and measure absorbance A at 760 nm.ΔA =Astandard − A0concentration. |
| 组分货号 | 组分名称 | 物理外观 | 48T | 储存 |
| T1527571A | 试剂一 | 液体 | 12mL | 2-8℃. 避光保存 |
| T1527571B | 试剂二 | 液体 | 12mL | 2-8℃. |
| T1527571C | 标准品 | 粉末 | 1EA | 2-8℃. |
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 |
|---|---|---|
 ZJ26F1040119 |
分析证书 | T1527571 |
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