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。
背景介绍：
类胡萝卜素(Carotenoids)是一类重要天然色素的总称，广泛存在于动物、高等植物、真菌及藻类的黄色、橙红色或红色组织中。类胡萝卜素是体内维生素 A 的前体，同时具备抗氧化、免疫调节、抗肿瘤、降低心血管疾病风险等生理活性，也可用作着色剂。检测原理：植物中的类胡萝卜素定位于有色体内，例如黄叶、黄色花朵、黄/红色果实、黄色块根等组织。样本经溶剂萃取分离提取类胡萝卜素，其在 440 ±10 nm 处具有特征吸收峰，可于该波长测定其含量。多数高等植物与藻类的叶绿体中同样含有类胡萝卜素；类胡萝卜素主要吸收蓝紫光，而叶绿素 a 与叶绿素 b 可同时吸收红光与蓝紫光。因此对于含叶绿体的组织，为消除叶绿素 a、叶绿素 b 对类胡萝卜素测定的干扰，需通过经验公式先计算叶绿素 a、叶绿素 b 含量，再推算类胡萝卜素含量；对于不含叶绿素的组织，可直接利用类胡萝卜素的经验消光系数进行计算。
该试剂盒仅用于科研领域，不适用于临床诊断或其他用途。
注意事项：
1. 为避免色素见光分解，操作全程尽量避光，研磨或匀浆操作应尽量缩短时间。
2. 若使用聚苯乙烯材质 96 孔板，加样后请于 5 分钟内完成测定。
3. 若不确定组织中是否存在叶绿素干扰，可取样本浸提液，用分光光度计在 400-700 nm 波长范围扫描；如在 640-670 nm 出现吸收峰，表明样本含有叶绿素。
4. 若测得吸光度大于 1.0，建议使用提取液对浸提液适当稀释后再测定，计算时按实际稀释倍数调整公式中的 F；若测得吸光度小于 0.05，可在提取时适当减少提取液用量，再进行测定，计算时按提取后实际总体积调整公式中的 V样总。
5. 提取液易挥发，且具有刺激性气味，操作时应采取适当防护。
| 组分货号 | 组分名称 | 物理外观 | 48T | 96T | 储存 | 单次测试使用量 |
| - | 提取液(自备) | - | - | - | 2-8℃, 避光. | 按需 |
| C1515815A | 试剂 一 | 白色粉末 | 3 g | 6 g | 2-8℃. | 10 mg |
使用说明：
1. 自备仪器、试剂与耗材
(1) 所需仪器：酶标仪或可见分光光度计(能测440 / 470 / 646 / 663 nm处的吸光度)。
(2) 所需试剂：丙酮。
(3) 其它：96孔板(非聚苯乙烯材质)或微量玻璃比色皿、可调节式移液枪及枪头、10 mL离心管/试管、匀浆器、去离子水、分析天平。
注：丙酮会溶解聚苯乙烯(PS)微孔板，请勿使用普通聚苯乙烯 96 孔板。
2. 试剂准备
(1) 提取液：自备 80 % 丙酮，将丙酮：去离子水(V:V) = 4:1混合待用, 4 ℃ 避光保存1周。
(2) 试剂一：即用型；使用前，平衡到室温；4 ℃保存。
3. 样本制备
(1) 新鲜植物叶片(去掉中脉)或其他组织用去离子水洗干净，然后吸干表面水分，称取约 0.1 g，剪碎放入研钵或匀浆器中。
(2) 加入1 mL去离子水，少量试剂 一(约10 mg)，在黑暗或弱光条件下充分研磨，转入10 mL离心管或试管中。
(3) 使用提取液冲洗研钵，将所有冲洗液转入15 mL试管/离心管中，提取液定容至10 mL，置于黑暗条件下或锡箔纸避光浸提3 h (期间颠倒混合2-3次)，观察底部组织残渣接近于白色则提取完全，若组织残渣未完全变白，继续浸提至组织残渣颜色接近白色即为浸提液。
注：推荐使用新鲜样本，如果不立即进行实验，样本可在 -80 ℃ 保存2周。测定时，应控制解冻的温度和时间。室温环境下解冻 时，需在 4 h 内完成样品解冻。
4. 实验步骤
(1) 黄色或其他非绿色组织(无叶绿素)类胡萝卜素含量测定步骤:
a) 酶标仪或可见分光光度计预热 30 分钟，设置波长为 440 nm。可见分光光度计使用提取液调零；
b) 采用微量玻璃比色皿测定时，无需设置空白管；采用 96 孔板(非聚苯乙烯材质)测定时，空白孔仅需检测 1-2 次作为空白基准。
试剂 | 测定孔 (μL) | 空白孔 (μL) |
样本上清 | 200 | 0 |
提取液 | 0 | 200 |
用分光光度计检测：使用微量玻璃比色皿快速测定 440 nm 处测定孔吸光度，记为 A₄₄₀。
用酶标仪检测：于96孔板(非聚苯乙烯材质)快速测定 440 nm 处吸光值，分别记为A440 测定 、A440 空白，ΔA ₄₄₀= A440 测定 - A440 空白
(2) 新鲜植物叶片或其他绿色组织(含叶绿素)类胡萝卜素含量测定步骤：
a) 酶标仪或可见光分光光度计预热30 分钟，调节多波长至 470 nm、646 nm 和 663 nm。可见分光光度计采用提取液调零。
b) 样本检测: 采用微量玻璃比色皿测定时，无需测空白管; 用96孔板(非聚苯乙烯材质)测定时，空白孔仅需检测1-2次作为空白基准)。
试剂 | 测定孔 (μL) | 空白孔 (μL) |
样本上清 | 200 | 0 |
提取液 | 0 | 200 |
注 1: 若上层浸提液含有残渣，可取 1 mL 上层浸提液转移至 1.5 mL 棕色 EP 管，常温 4000 rpm 离心 5 分钟，取上清用于检测；
注 2: 若使用聚苯乙烯材质 96 孔板，请在加样后 5 分钟 内完成测定。若吸光度 A＜0.1，可适当增加样本用量；若 A > 1.0，用提取液稀释样本后重新测定，计算时乘以最终稀释倍数。
5. 结果计算
(1) 黄色或其他非绿色组织(无叶绿素)类胡萝卜素含量的计算公式：
a) 使用96孔板(非聚苯乙烯材质)测定的计算公式如下:
类胡萝卜素含量(mg/g 质量)= A440 ÷ (ε × d) × Vtotal × 1,000 ÷ W × F = 0.08 × A440 × F ÷ W V 样总：提取液总体积, 0.01 L；
1,000：单位换算系数, 1 g=1,000 mg；
ε：类胡萝卜素经验消光系数, 250 L/g/cm；
d：96 孔板 (非聚苯乙烯材质)光径, 0.5 cm；
F：稀释倍数；
W：样本质量, g。
b) 使用微量玻璃比色皿测定的计算公式:
将上述计算公式中光径d:0.5 cm调整为d:1 cm进行计算即可。
(2) 新鲜植物叶片或其他绿色组织(含叶绿素)类胡萝卜素含量的计算公式：
a) 使用96孔板(非聚苯乙烯材质)测定的计算公式如下:
Ca(mg/L)= (12.21 × ΔA663 - 2.81 × ΔA646) ÷ 0.5 = 24.42 × ΔA663 - 5.62 × ΔA646。
C b(mg/L) = (20.13 × ΔA646 - 5.03 × ΔA663) ÷ 0.5 = 40.26 × ΔA646 - 10.06 × ΔA663。
类胡萝卜素浓度： Cc(mg/L) = (1,000 × ΔA470 ÷ 0.5 - 3.27 × Ca - 104 × Cb ) ÷ 229 = 8.734 × ΔA470 - 0.014 × Ca - 0.454 × Cb。
类胡萝卜素含量 (mg/g 质量)=Cc ×Vtotal×F÷W=0.01×C c ×F÷W。
V样总 : 提取液总体积, 0.01 L；
F：稀释倍数；
W：样本质量, g。
b) 使用微量玻璃比色皿测定的计算公式:
将上述计算公式中光径 d: 0.5 cm调整为 d: 1 cm进行计算即可。
Background Introduction
Carotenoids are a general term for an important class of natural pigments, widely present in the yellow, orange-red or red tissues of animals, higher plants, fungi and algae. As precursors of vitamin A in vivo, carotenoids possess physiological activities including antioxidant, immunomodulatory and anti-tumor effects, and can reduce the risk of cardiovascular diseases. They can also be used as colorants.
Assay Principle: In plants, carotenoids are localized in chromoplasts, such as yellow leaves, yellow flowers, yellow and red fruits, and yellow tuberous roots. Carotenoids are isolated and extracted from samples via solvent extraction. They have a characteristic absorption peak at 440±10 nm, and their content can be determined at this wavelength.
Carotenoids are also found in the chloroplasts of most higher plants and algae. Carotenoids mainly absorb blue-violet light, whereas chlorophyll a and chlorophyll b absorb both red light and blue-violet light. Therefore, for chlorophyll-containing tissues, to eliminate the interference of chlorophyll a and chlorophyll b on carotenoid quantification, the concentrations of chlorophyll a and chlorophyll b are calculated first using empirical formulas, and then the carotenoid content is derived. For chlorophyll-free tissues, the empirical extinction coefficient of carotenoids can be applied directly for calculation.
This kit is only for scientific research purposes and shall not be used for clinical diagnosis or other applications.
Precautions
1. To prevent photodegradation of pigments, perform all operations away from light as much as possible, and minimize the duration of grinding or homogenization.
2. If polystyrene 96-well plates are used, complete the measurement within 5 min after sample loading.
3. If the interference from chlorophyll in the tissue is unknown, scan the sample extract with a spectrophotometer over the wavelength range of 400-700 nm. An absorption peak between 640-670 nm indicates the presence of chlorophyll in the sample.
4. If the measured absorbance exceeds 1.0, dilute the extract appropriately with extraction solution before measurement, and adjust parameter F in the calculation formula according to the actual dilution factor. If the measured absorbance is less than 0.05, reduce the volume of extraction solution during extraction prior to measurement, and adjust parameter Vtotal in the formula according to the actual total volume after extraction.
5. The extraction solution is volatile and has a pungent odor. Take appropriate protective measures during operation.
| Component No. | Component Name | Appearance | 48T | 96T | Storage | Quantity Per Test |
| - | Extraction Buffer (user-supplied) | - | - | - | 2-8℃, 避光. | As needed |
| C1515815A | Reagent I | White powder | 3 g | 6 g | 2-8℃. | 10 mg |
Instructions for Use
1. User-supplied Instruments, Reagents and Consumables
(1) Instruments required: Microplate reader or visible spectrophotometer (capable of measuring absorbance at 440 / 470 / 646 / 663 nm).
(2) Reagents required: Acetone
(3) Others: 96-well plate (non-polystyrene) or micro glass cuvettes, adjustable pipettors and pipette tips, 10 mL centrifuge tubes/test tubes, homogenizer, deionized water, analytical balance.
Note: Acetone dissolves polystyrene (PS) microplates. Do not use ordinary polystyrene 96-well plates.
2. Reagent Composition and Storage
(1) Extraction solution: Prepare 80% acetone by mixing acetone and deionized water at v/v = 4:1 for immediate use. Store at 4 °C away from light, valid for 1 week.
(2) Reagent I: Ready-to-use. Equilibrate to room temperature before use. Store at 4 °C.
3. Sample Pretreatment
(1) Rinse fresh plant leaves (remove midribs) or other tissues with deionized water and blot surface moisture. Weigh approximately 0.1 g sample, cut into pieces and place in a mortar or homogenizer.
(2) Add 1 mL deionized water and a small amount of Reagent I (approx. 10 mg). Fully grind under dark or low-light conditions, then transfer the homogenate into a 10 mL centrifuge tube or test tube.
(3) Rinse the mortar with extraction solution, combine all rinsates and transfer to a 15 mL centrifuge tube/test tube. Make up the volume to 10 mL with extraction solution. Wrap with aluminum foil and extract in darkness for 3 h (invert and mix 2-3 times during incubation). Extraction is complete when the residual tissue pellet at the bottom turns nearly white. If not, continue extraction until the residue is nearly white; the resulting solution is the sample extract.
Note: Fresh samples are recommended. If the assay cannot be performed immediately, samples can be stored at -80 °C for up to 2 weeks. Control thawing temperature and duration during measurement. For samples thawed at room temperature, extraction and detection must be completed within 4 h after thawing.
4. Experimental Procedures
(1) Procedure for determination of carotenoid content in yellow or other non-green tissues (chlorophyll-free):
a) Preheat the microplate reader or visible spectrophotometer for 30 min and set the wavelength to 440 nm. Zero the visible spectrophotometer with extraction solution.
b) Sample detection: No blank tube is required when using micro glass cuvettes. For 96-well plate (non-polystyrene) detection, measure the blank well only 1-2 times for blank baseline correction.
Reagent | Test Well (μL) | Blank Well (μL) |
Sample supernatant | 200 | 0 |
Extraction solution | 0 | 200 |
For spectrophotometer measurement: Quickly measure the absorbance of the assay well at 440 nm using a micro glass cuvette, and record the value as A₄₄₀.
For microplate reader measurement: Quickly measure the absorbance at 440 nm using a 96-well plate (non-polystyrene). Record values as A440 assay and A440 blank, A₄₄₀ = A440 assay- A440 blank.
(2) Procedure for determination of carotenoid content in fresh plant leaves or other green tissues (chlorophyll-containing):
a) Preheat the microplate reader or visible spectrophotometer for 30 min, and set multiple wavelengths to 470 nm, 646 nm and 663 nm. Zero the visible spectrophotometer with extraction solution.
b) Sample detection: No blank tube is required when using micro glass cuvettes. For 96-well plate (non-polystyrene) detection, measure the blank well only 1-2 times for blank baseline correction.
Reagent | Test Well (μL) | Blank Well (μL) |
Sample supernatant | 200 | 0 |
Extraction solution | 0 | 200 |
Note 1: If the upper extract contains residues, transfer 1 mL of the upper extract into a 1.5 mL brown EP tube, centrifuge at 4000 rpm for 5 min at ambient temperature, and collect the supernatant for detection.
Note 2: If polystyrene 96-well plates are used, complete the measurement within 5 min after sample loading. If absorbance A＜0.1, appropriately increase the sample amount. If A＞1.0, dilute the sample with extraction solution before re-testing, and multiply by the final dilution factor during calculation.
5. Result Calculation
(1) Calculation formula for carotenoid content in yellow or other non-green tissues (chlorophyll-free):
a) Measurement with 96-well plate (non-polystyrene):
Carotenoid content (mg/g fresh weight) = A440 ÷ (ε × d) × Vtotal × 1,000 ÷ W × F = 0.08 × A440 × F ÷ W.
Vtotal: Total volume of extraction solution, 0.01 L;
1000: Unit conversion factor, 1 g = 1000 mg;
ε: Empirical extinction coefficient of carotenoids, 250 L/g/cm;
d: Optical path of 96-well plate (non-polystyrene), 0.5 cm;
F: Dilution factor;
W: Sample weight, g.
b) Measurement with micro glass cuvette:
Use the formula above and change the optical path d from 0.5 cm to 1 cm for calculation.
(2) Calculation formula for carotenoid content in fresh plant leaves or other green tissues (chlorophyll-containing):
a) Measurement with 96-well plate (non-polystyrene):
Carotenoid concentration:
Carotenoid content (mg/g fresh weight)=
Ca(mg/L)= (12.21 × ΔA663 - 2.81 × ΔA646) ÷ 0.5 = 24.42 × ΔA663 - 5.62 × ΔA646.
C b(mg/L) = (20.13 × ΔA646 - 5.03 × ΔA663) ÷ 0.5 = 40.26 × ΔA646 - 10.06 × ΔA663.
Carotenoid concentration: Cc(mg/L) = (1,000 × ΔA470 ÷ 0.5 - 3.27 × Ca - 104 × Cb ÷ 229 = 8.734 × ΔA470 - 0.014 × Ca - 0.454 × Cb .
Carotenoid content (mg/g fresh weight) = Cc ×Vtotal×F÷W=0.01×C c×F÷W.
Vtotal: Total volume of extraction solution, 0.01 L;
F: Dilution factor;
W: Sample weight, g.
b) Measurement with micro glass cuvette:
Use the formula above and change the optical path d from 0.5 cm to 1 cm for calculation.
| 组分货号 | 组分名称 | 物理外观 | 48T | 96T | 储存 | 单次测试使用量 |
| - | 提取液(自备) | - | - | - | 2-8℃, 避光. | 按需 |
| C1515815A | 试剂 一 | 白色粉末 | 3 g | 6 g | 2-8℃. | 10 mg |
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