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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。
植物根系是活跃的吸收器官和合成器官，根的生长情况和代谢水平即根系活力将直接影响植物地上部分的生长和营养状况以及最终产量，是植物生长的重要生理指标之一，TTC (2，3，5-氯化三苯基四氮唑) 是一种氧化还原物质，是标准氧化电位为80mV的氧化还原色素，溶解于水为无色，但还原后即生成红色而不溶于水的三苯基甲腙(TTF)，TTF比较稳定，不会被空气中的氧自动氧化，所以TTC常被用作酶试验的氢受体。
检测原理：当测定植物根系活力时，以TTC为底物，保温1~4小时，根系中脱氢酶能够还原TTC并生成不溶于水的红色TTF，再用有机溶剂（乙酸乙酯或丙酮等）将其从根中提取出来，用分光光度计或酶标仪检测485nm处吸光度，进而计算出TTC的还原量，以该还原量表示脱氢酶活性，并作为植物根系活力的指标，该试剂盒主要用于定量测定植物的根系活力或脱氢酶活性。该试剂盒仅用于科研领域，不适用于临床诊断或其他用途。
| P1505878 | Component | 50T | Storage |
| P1505878A | TTC | 1 g | RT. Store in the dark. |
| P1505878B | TTC Assay Buffer | 250 mL | RT |
| P1505878C | TTC终止液 | 100 mL | RT |
| P1505878D | Na₂S₂O₄粉末 | 0.5 g | RT |
自备仪器和试剂
1、蒸馏水、乙酸乙酯或丙酮
2、精密天平、离心管、容量瓶、滤纸、水浴锅、研钵或匀浆器、分光光度计、比色杯
实验流程
1、配制TTC溶液 (0.4%)
称取0.4g TTC完全溶解于100mL蒸馏水即成，配制成溶液后应4℃避光保存，若变红应弃用。
2、配制TTC Assay Buffer工作液
将TTC溶液 (0.4%) 与TTC Assay Buffer等比例混合即成，建议现用现配，4℃避光保存，若变红应弃用。
3、配制TTF标准工作液
取0.25mL TTC溶液 (0.4%)置于10mL离心管或容量瓶，加入少许Na₂S₂O₄粉末（一般30mg左右），混匀，产生红色的不溶于水的红色颗粒物TTF，加10mL乙酸乙酯，充分混合溶解颗粒，静置分层（下层0.25ml为无色水层，不可用），上层红色溶液中TTF浓度为100μg/mL。
按下表分别移取上层红色溶液0.25、0.5、1、1.5、2mL置于离心管或容量瓶中，用乙酸乙酯补加至10mL，即获得TTF系列标准管。
| 试剂 (mL) | Std.0 | Std.1 | Std.2 | Std.3 | Std.4 | Std.5 |
| TTF标准工作液 (100μg/ml) | 0 | 0.25 | 0.5 | 1 | 1.5 | 2 |
乙酸乙酯 | 10 | 9.75 | 9.5 | 9 | 8.5 | 8 |
相当于TTF含量 (μg) | 0 | 25 | 50 | 100 | 150 | 200 |
4、准备样品
取0.2~0.5g植物须根系，洗净，用滤纸吸干，完全浸没于TTC Assay buffer工作液中，37℃避光孵育1~3h，加入2mL TTC终止液终止反应；同时做一空白对照实验，即根系先完全浸没于2mL TTC终止液以杀死根样，再加入10mL TTC Assay Buffer工作液，37℃避光孵育1~3h。
5、提取
分别将上述实验组和空白对照组的根系取出，滤纸吸干水分，放入研钵或匀浆器中，加入3~4mL乙酸乙酯，充分研磨或匀浆，以提取出TTF，把红色提取液 (TTF) 转移至离心管，并用少量乙酸乙酯把残渣洗涤2~3次，再将洗涤液一并转移至离心管，最后补加乙酸乙酯至10mL，摇匀。
6、测定
比色杯光径1.0cm，系列标准管以“Std.0”号管调零，测定Std.1~Std.5号管485nm的吸光度值；样品管以空白对照组样品提取液做参比，调零，以分光光度计或酶标仪测定实验组样品提取液在485nm的吸光度值。
7、计算
以系列标准溶液TTF含量（25、50、100、150、200μg）为横坐标，以对应吸光度为纵坐标，绘制TTF标准曲线，根据回归方程计算待测样品的TTF含量或TTC还原量 (μg)，即为根系活力或脱氢酶活性。
根系 (脱氢酶) 活力 [mg/(g·h)]=m/(1000×W×t)
参数说明：
m：根据标准曲线查得的样品提取液TTF含量 (μg)= TTC还原量 (μg)
W：植物须根系的重量，g
t：孵育时间，h
8、标准曲线参考
参考标准曲线范围：测定空白对照，通过分光光度计测定其吸光度多在0.03-0.07之间；测定TTF标准在5-500μg时吸光度，据此作出其标准曲线：
注：由于检测仪器和操作手法等条件的不同，参考值范围会有波动，该值仅供参考，对于要求精确计算植物根系含量的，可以采用标准曲线多点重复测定；根据测定经验显示，TTF含量在5μg以下，500μg以上，标准曲线会有偏差。
注意事项
1、配制好的TTC溶液 (0.4%) 和TTC Assay buffer工作液应避光保存，若变红应弃用。
2、根系种类不同，取样量可有不同。
3、根系应吸干水分但不能挤压伤及细胞，才能测定准确。
4、提取用的研钵或匀浆器、试管、比色皿均应保持干燥，没有水分。
5、由于本实验采用有机溶剂(乙酸乙酯或丙酮)提取TTF，因此不能用一次性塑料比色皿或者酶标条进行检测；建议用石英比色皿或玻璃比色皿检测，检测完成后用乙醇清洗并用水冲洗干燥即可。
6、如果没有分光光度计，也可以使用酶标仪测定，但应注意酶标板每孔检测体积。
7、试剂开封后请尽快使用，以防影响后续实验效果。
8、为了您的安全和健康，请穿实验服并戴一次性手套操作。
Plant root systems are active organs for absorption and synthesis. The growth status and metabolic level of roots, known as root system activity, directly affect the growth, nutritional status, and final yield of the aerial parts of plants, making it one of the important physiological indicators of plant growth. TTC (2,3,5-Triphenyltetrazolium Chloride) is a redox substance, a redox pigment with a standard oxidation potential of 80 mV. It is colorless when dissolved in water but turns red upon reduction, generating water-insoluble Triphenylformazan (TTF). TTF is relatively stable and is not auto-oxidized by air. Therefore, TTC is often used as a hydrogen acceptor in enzyme assays.
Assay Principle
When determining plant root system activity, TTC is used as the substrate and incubated for 1-4 hours. Dehydrogenases in the root system can reduce TTC, generating insoluble red TTF. TTF is then extracted from the roots using an organic solvent (such as ethyl acetate or acetone). The absorbance at 485 nm is measured using a spectrophotometer or microplate reader, and the amount of TTC reduced is calculated. This reduction amount represents dehydrogenase activity and serves as an indicator of plant root system vitality. This kit is primarily used for the quantitative determination of plant root system activity or dehydrogenase activity. This product is for research use only and is not suitable for clinical diagnosis or other purposes.
| P1505878 | Component | 50T | Storage |
| P1505878A | TTC | 1 g | RT. Store in the dark. |
| P1505878B | TTC Assay Buffer | 250 mL | RT |
| P1505878C | TTC Stop Solution | 100 mL | RT |
| P1505878D | Na₂S₂O₄ Powder | 0.5 g | RT |
Required Materials Not Provided
1. Distilled water, Ethyl acetate or Acetone
2. Precision balance, Centrifuge tubes, Volumetric flasks, Filter paper, Water bath, Mortar and pestle or homogenizer, Spectrophotometer, Cuvettes
Experimental Procedure
1. Preparation of TTC Solution (0.4%)
Weigh 0.4 g of TTC and dissolve completely in 100 mL of distilled water.
Store the prepared solution at 4°C protected from light. Discard if it turns red.
2. Preparation of TTC Assay Buffer Working Solution
Mix the TTC Solution (0.4%) with TTC Assay Buffer in equal proportions.
Prepare fresh before use. Store at 4°C protected from light. Discard if it turns red.
3. Preparation of TTF Standard Working Solution
Pipette 0.25 mL of TTC Solution (0.4%) into a 10 mL centrifuge tube or volumetric flask.
Add a small amount of Na₂S₂O₄ powder (approx. 30 mg) and mix. Red, water-insoluble TTF particles will form.
Add 10 mL of ethyl acetate, mix thoroughly to dissolve the particles, and let stand for phase separation (the lower 0.25 mL colorless aqueous layer is discarded).
The TTF concentration in the upper red solution is 100 μg/mL.
Prepare serial standard tubes according to the table below by pipetting the upper red solution and diluting with ethyl acetate to 10 mL.
| Reagent (mL) | Std.0 | Std.1 | Std.2 | Std.3 | Std.4 | Std.5 |
| TTF Std. Working Sol. (100 μg/mL) | 0 | 0.25 | 0.5 | 1 | 1.5 | 2 |
Ethyl Acetate | 10 | 9.75 | 9.5 | 9 | 8.5 | 8 |
Equivalent TTF (μg) | 0 | 25 | 50 | 100 | 150 | 200 |
4. Sample Preparation
Take 0.2~0.5 g of plant fibrous roots, wash, blot dry with filter paper.
Completely immerse the roots in the TTC Assay Buffer Working Solution.
Incubate at 37°C protected from light for 1~3 hours.
Add 2 mL of TTC Stop Solution to terminate the reaction.
Prepare a Blank Control: Immerse roots first in 2 mL TTC Stop Solution to kill the root sample, then add 10 mL TTC Assay Buffer Working Solution. Incubate at 37°C protected from light for 1~3 hours.
5. Extraction
Remove the roots from both the test and blank control groups, blot dry with filter paper.
Place the roots in a mortar or homogenizer, add 3~4 mL of ethyl acetate.
Grind or homogenize thoroughly to extract TTF.
Transfer the red extract (TTF) to a centrifuge tube.
Rinse the residue 2-3 times with a small amount of ethyl acetate and combine the washes into the centrifuge tube.
Adjust the final volume to 10 mL with ethyl acetate and mix well.
6. Measurement
Using a 1.0 cm light path cuvette:
Zero the spectrophotometer using the "Std.0" tube.
Measure the absorbance of Std.1 to Std.5 tubes at 485 nm.
Zero the instrument using the blank control group extract.
Measure the absorbance of the test sample extract at 485 nm using a spectrophotometer or microplate reader.
7. Calculation
Plot the standard curve with the TTF content of the standard solutions (25, 50, 100, 150, 200 μg) on the X-axis and the corresponding absorbance on the Y-axis.
Use the regression equation to calculate the TTF content or TTC reduction amount (μg) in the test sample, which represents the root system activity or dehydrogenase activity.
Root System (Dehydrogenase) Activity [mg/(g·h)] = m / (1000 × W × t)
Parameter Definitions:
m: TTF content (μg) of the sample extract obtained from the standard curve = TTC reduction amount (μg)
W: Weight of the plant fibrous roots (g)
t: Incubation time (h)
8. Standard Curve Reference
Typical Blank Control Absorbance: 0.03 - 0.07
A standard curve can be constructed by measuring the absorbance of TTF standards in the range of 5-500 μg.
*Note: Reference ranges may vary due to differences in instruments and operational techniques. For precise quantification, use a multi-point standard curve with replicate measurements. Empirical data suggests deviations in the standard curve at TTF contents below 5 μg and above 500 μg.*
Precautions
Prepared TTC Solution (0.4%) and TTC Assay Buffer Working Solution should be stored protected from light. Discard if they turn red.
The sample amount may vary depending on the root type.
Roots should be blotted dry but not squeezed to damage cells for accurate measurement.
The mortar/homogenizer, tubes, and cuvettes used for extraction should be kept dry, free of moisture.
Since organic solvents (ethyl acetate or acetone) are used to extract TTF, disposable plastic cuvettes or microplate strips cannot be used. Use quartz or glass cuvettes. Clean them with ethanol after use, followed by water rinsing and drying.
If a spectrophotometer is unavailable, a microplate reader can be used, but note the detection volume per well.
Use reagents promptly after opening to avoid affecting subsequent experimental results.
For your safety and health, wear lab coats and disposable gloves during operation.
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| 批号(Lot Number) | 证书类型 | 货号 |
|---|---|---|
 ZJ26F0131478 |
分析证书 | P1505878 |
危险品化学品经营许可证（带存储）