经改良和优化的邻甲苯胺试剂(o-Toluidine Reagent)与葡萄糖发生缩合反应，生成最大吸收波长为630nm的蓝绿色Schiff 碱，并且630nm处的吸光度值与样品中的葡萄糖浓度成正比。在样本体积为25 μL时能检测样本中低至4 mg/dL的葡萄糖，在4-300 mg/dL检测范围内具有良好的线性关系。
产品组分
G1522839 | 组分名称 | 物理外观 | 200 T | 1000 T | 储存 | 单次测试使用量 |
G1522839A | 邻甲苯胺试剂 | 液体 | 100mL | 500 mL | -20℃.避光 | 0.5 mL |
G1522839B | 葡萄糖标准品 (300 mg/dL) | 液体 | 10mL | 50 mL | -20℃.避光 | 按需 |
使用方法
1. 样本处理
(1) 动物组织：称取0.1 g样本，加入1 mL的1×PBS，冰浴匀浆，12,000 g，4℃离心5 min，取上清液，置冰上待测。
(2) 植物组织：称取0.1 g样本，加入1 mL的1×PBS捣碎，冰浴超声波破碎 5 min（功率 20％或200 W，超声3 s，间隔7 s，重复30 次），12,000 g，4℃离心5 min，取上清液，置冰上待测。
(3) 细胞或细菌：收集500万细胞或细菌到离心管内，用冷1×PBS清洗细胞，离心后弃上清，加入1 mL的1×PBS，冰浴超声波破碎细胞或细菌5 min（功率20%或200 W，超声3 s，间隔7 s，重复30 次），然后12,000 g，4℃离心 5 min，取上清液，置冰上待测。
(4) 血浆，血清和尿液（和其他生物液体）：直接检测。
2. 标准曲线设置
按下表所示，用去离子水将葡萄糖标准品 (300 mg/dL)稀释为300、200、100、50、20、10、4 mg/dL的标准溶液。每次实验，请使用新配制的标准品, 配制好的标准品需要在4 h 之内使用。
序号 | 葡萄糖标准品 (300 mg/dL)体积 (μL) | 去离子水(μL) | 标准品浓度(mg/dL) |
Std.1 | 150 | 0 | 300 |
Std.2 | 100 | 50 | 200 |
Std.3 | 50 | 100 | 100 |
Std.4 | 25 | 125 | 50 |
Std.5 | 10 | 140 | 20 |
Std.6 | 5 | 145 | 10 |
Std.7 | 2 | 148 | 4 |
3. 检测步骤
（1）酶标仪或可见光分光光度计预热30 min 以上，调节波长到630 nm，可见光分光光度计用去离子水调零。
（2）按照下列操作表进行实验（因涉及到加热步骤，下述操作在EP管中进行）：
试剂 | 空白管 (μL) | 标准管 (μL) | 测定管 (μL) |
样本 | 0 | 0 | 25 |
标准品 | 0 | 25 | 0 |
去离子水 | 25 | 0 | 0 |
邻甲苯胺试剂 | 500 | 500 | 500 |
（3）沸水浴中加热8 min，冷水浴（4℃）中冷却4 min。取200 µL到96孔板或微量玻璃玻璃比色皿，检测630 nm 处吸光值。
4. 结果计算
空白孔、标准孔和测定孔的OD值分别记为A空白 ， A标准 和 A测定 。计算ΔA标准 = A标准 - A空白 ，ΔA测定= A测定- A空白。以葡萄糖标准品的浓度（mg/dL）为横坐标， ΔA标准 为纵坐标建立标准曲线。根据标准曲线计算出样品中的葡萄糖浓度。对于细胞或组织样品，通常需测定蛋白浓度计算出单位蛋白量样品中的葡萄糖含量，或根据组织重量计算出单位重量组织中的葡萄糖含量，并将葡萄糖浓度表述为µg/mg蛋白或µg/mg组织。1 mg/dL葡萄糖的浓度相当于55.5 µM，0.001%或10 ppm。
5. 结果展示
从反应后的标准品管中取200 µL反应液到96孔板中，酶标仪检测630 nm 处吸光值。
以萄糖标准品的浓度（mg/dL）为横坐标，A标准为纵坐标建立标准曲线，结果如图所(仅供参考)：
注意事项
1. 邻甲苯胺试剂为淡黄色，长期放置可能会产生棕色物质，该棕色物质会干扰比色检测。若使前出现很明显的棕色物质，本试剂盒不宜继续使用。
2. 邻甲苯胺试剂呈酸性，有一定的腐蚀性，需注意防护。
3. 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。
The modified and optimized o-toluidine reagent undergoes a condensation reaction with glucose to form a blue-green Schiff base with a maximum absorption wavelength of 630 nm, and the absorbance at 630 nm is directly proportional to the glucose concentration in the sample. With a sample volume of 25 μL, it can detect glucose as low as 4 mg/dL in samples, and exhibits a good linear relationship within the detection range of 4-300 mg/dL.
Components
G1522839 | Component Name | Appearance | 200 T | 1000 T | Storage | Quantity Per Test |
G1522839A | o-Toluidine Reagent | Liquid | 100 mL | 500 mL | -20°C. Store in the dark | 0.5 mL |
G1522839B | Glucose Standard (300 mg/dL) | Liquid | 10 mL | 50 mL | -20°C. Store in the dark | As needed |
Usage Protocol
1. Sample Preparation
1) Animal tissue: Weigh 0.1 g of sample, add 1 mL of 1× PBS, homogenize on ice, centrifuge at 12,000 g for 5 min at 4°C, collect the supernatant, and keep on ice for testing.
2) Plant tissue: Weigh 0.1 g of sample, add 1 mL of 1× PBS, mash, and disrupt by ultrasonication on ice for 5 min (power 20% or 200 W, sonicate for 3 s, pause for 7 s, repeat 30 times). Centrifuge at 12,000 g for 5 min at 4°C, collect the supernatant, and keep on ice for testing.
3) Cells or bacteria: Collect 5 × 10⁶ cells or bacteria into a centrifuge tube, wash the cells with cold 1× PBS, centrifuge and discard the supernatant, add 1 mL of 1× PBS, and disrupt the cells or bacteria by ultrasonication on ice for 5 min (power 20% or 200 W, sonicate for 3 s, pause for 7 s, repeat 30 times). Then centrifuge at 12,000 g for 5 min at 4°C, collect the supernatant, and keep on ice for testing.
4) Plasma, serum, and urine (and other biological fluids): Measure directly.
2. Standard Curve Setup
As shown in the table below, dilute the Glucose Standard (300 mg/dL) with deionized water to prepare standard solutions of 300, 200, 100, 50, 20, 10, and 4 mg/dL. For each experiment, use freshly prepared standards. The prepared standards must be used within 4 h.
No. | Glucose Standard (300 mg/dL) Volume (μL) | Deionized Water (μL) | Standard Concentration (mg/dL) |
Std.1 | 150 | 0 | 300 |
Std.2 | 100 | 50 | 200 |
Std.3 | 50 | 100 | 100 |
Std.4 | 25 | 125 | 50 |
Std.5 | 10 | 140 | 20 |
Std.6 | 5 | 145 | 10 |
Std.7 | 2 | 148 | 4 |
3. Assay Procedures
1) Preheat the microplate reader or visible light spectrophotometer for more than 30 min, adjust the wavelength to 630 nm, and zero the visible light spectrophotometer with deionized water.
2) Perform the experiment according to the following operation table (because a heating step is involved, the following operations are performed in EP tubes):
Reagent | Blank tube (μL) | Standard tube (μL) | Sample well (μL) |
Sample | 0 | 0 | 25 |
Standard | 0 | 25 | 0 |
Deionized water | 25 | 0 | 0 |
o‑Toluidine Reagent | 500 | 500 | 500 |
3) Heat in a boiling water bath for 8 min, cool in a cold water bath (4°C) for 4 min. Transfer 200 µL to a 96-well plate or a micro glass cuvette, and measure the absorbance at 630 nm.
4. Result Calculation
The OD values of the blank well, standard well, and sample well are recorded as Abₗₐₙₖ, Aₛₜₐₙdₐᵣd, and Aₛₐₘₚₗₑ, respectively. Calculate ΔAₛₜₐₙdₐᵣd = Aₛₜₐₙdₐᵣd - Abₗₐₙₖ, and ΔAₛₐₘₚₗₑ = Aₛₐₘₚₗₑ - Abₗₐₙₖ Plot a standard curve with the glucose standard concentration (mg/dL) as the x-axis and ΔAₛₜₐₙdₐᵣd as the y-axis. Calculate the glucose concentration in the sample according to the standard curve.
For cell or tissue samples, it is usually necessary to determine the protein concentration to calculate the glucose content per unit protein amount, or calculate the glucose content per unit weight based on tissue weight, and express the glucose concentration as µg/mg protein or µg/mg tissue. A glucose concentration of 1 mg/dL is equivalent to 55.5 µM, 0.001%, or 10 ppm.
5. Results Presentation
From the reacted standard tubes, take 200 µL of reaction solution into a 96-well plate, and measure the absorbance at 630 nm using a microplate reader.
Plot a standard curve with the glucose standard concentration (mg/dL) as the x-axis and Aₛₜₐₙdₐᵣd as the y-axis. The results are shown in the figure (for reference only):
Precautions
1. The o-Toluidine Reagent is light yellow. Prolonged storage may produce a brown substance, which can interfere with colorimetric detection. If a very obvious brown substance appears before use, this kit should not be used further.
2. The o-Toluidine Reagent is acidic and corrosive to a certain extent; protective measures should be taken.
3. Biochemical assay reagents generally have irritancy, biological toxicity, etc. For your safety and health, please take proper biological safety precautions throughout the experiment.
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