Fluorescein diacetate (FDA), with the molecular formula C24H16O7, CAS number 596-09-8, molecular weight 416.380, usually appears as a light yellow powder with good crystallinity. The solubility in acetone is relatively high, reaching up to 25mg/mL. However, there is limited information about its solubility in other solvents, but it is generally believed that its solubility is affected by solvent polarity and temperature. It is a lipophilic compound that can penetrate the cell membrane in living cells and be hydrolyzed as an esterase substrate to produce fluorescent fluorescein. This characteristic makes it widely applicable in fields such as cell viability detection, microbial detection, and enzyme activity evaluation. It does not emit light itself, but the fluorescein produced by hydrolysis in living cells can emit green fluorescence. Its excitation and emission wavelengths are 488 nm and 530 nm, respectively, making it a commonly used fluorescent probe in flow cytometry and other biological detection instruments.
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Chemical Formula |
C24H16O7 |
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Exact Mass |
416 |
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Molecular Weight |
416 |
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m/z |
416 (100.0%), 417 (26.0%), 418 (2.7%), 418 (1.4%) |
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Elemental Analysis |
C, 69.23; H, 3.87; O, 26.90 |
Fluorescein Diacetate (FDA) is a chemical substance widely used in biology, medicine, environmental monitoring, and industrial fields.
Biology And Medical Research
1. Cell viability and activity detection
(1) Flow cytometry analysis: As an esterase substrate, it can enter live cells through the cell membrane and be hydrolyzed into fluorescein by esterases inside the cells, emitting green fluorescence. Therefore, it is often used in flow cytometry to detect cell viability and evaluate the survival status of cells by measuring fluorescence intensity.
(2) Cytotoxicity assessment: In cytotoxicity studies, it can be used to evaluate the effects of drugs, chemicals, etc. on cell activity. By comparing the changes in fluorescence intensity of cells before and after treatment, it can be determined whether these substances have toxic effects on cells.
2. Microbial detection and identification
(1) Microbial activity detection: can be used to detect microbial activity in the environment, such as total microbial activity in soil. By measuring the fluorescence intensity produced by the hydrolysis of FDA by microorganisms, the activity level of microorganisms can be evaluated.
(2) Bacterial and virus detection: In biological and medical research, it can also be used to detect the presence of bacteria and viruses. Through specific staining or labeling techniques, rapid identification and counting of bacteria and viruses can be achieved.
3. Biomarkers and enzyme activity detection
(1) Enzyme activity evaluation: can be used as a substrate for various enzymes to evaluate their activity. For example, it can serve as a fluorescent substrate for human glutathione S-transferase Pi (hGTP1-1), and its activity can be evaluated by measuring the fluorescence intensity.
(2) Biomarker detection: In disease diagnosis, it can be used to detect the presence and level of certain biomarkers, such as markers for cancer cells. Early diagnosis and treatment monitoring of diseases can be achieved through specific detection methods.
Environmental Monitoring
1. Water quality monitoring
(1) Detecting microorganisms in water: can be used to detect microorganisms in water, such as live Giardia cysts. By measuring the fluorescence intensity produced by the hydrolysis of FDA by microorganisms in water samples, the microbial contamination of water quality can be evaluated.
(2) Algae vitality assessment: In environmental monitoring, it can also be used to evaluate the vitality status of algae in water bodies.
By measuring the uptake and conversion ability of algal cells towards FDA, the growth status and ecological environment quality of algae can be determined.
2. Soil monitoring
Soil microbial activity: As mentioned earlier, it can be used to measure the total microbial activity in soil. This is of great significance for assessing the health status and fertility level of soil ecosystems.
Industrial Applications
1. LED equipment
Fluorescent material: It can be used as a fluorescent material in LED devices to convert and adjust light color by absorbing the light emitted by the LED and emitting different colors of fluorescence.
2. Oil marking
Fluorescent labeling agent: In the petroleum industry, it can be used as a fluorescent labeling agent. By adding it to petroleum products to give them fluorescent properties, it facilitates identification and localization in leak detection, tracking, and recovery processes.
Other Applications
1. Temperature sensing in medical research
Temperature sensitive probe: Fluorescence properties are temperature sensitive, therefore it can be used as a temperature sensitive probe in medical research. By measuring changes in fluorescence intensity, real-time monitoring of temperature changes in the in vivo or in vitro environment can be achieved.
2. Treatment of ischemic diseases
Treating ischemia: Studies have shown that it can be used to treat ischemic diseases in certain situations. Through its specific biochemical mechanism, it can promote the recovery and repair of ischemic tissues.
The synthesis of fluorescein diacetate (FDA) is a complex but important chemical process, and its products have wide applications in biology, medicine, environmental monitoring, and other fields.
The synthesis of FDA is usually based on fluorescein or its precursor compounds, which introduce acetic acid groups through esterification reactions. Esterification reaction is a commonly used organic synthesis reaction, which usually involves the reaction of carboxylic acid and alcohol in the presence of an acid catalyst to produce esters. In synthesis, fluorescein or its precursor (such as diacetylfluorescein) reacts with acetic anhydride to produce the substance.
1. Raw material preparation
Fluorescein:
As a starting material, fluorescein can be synthesized or purchased through various methods.
Acetic anhydride:
As an acylating agent in esterification reactions, purity and stability need to be ensured.
Catalysts:
Such as pyridine and sulfuric acid, are used to promote esterification reactions.
Solvent:
Such as anhydrous ethanol, DMSO, etc., used to dissolve reactants and products, promote the uniform progress of the reaction.
2. Esterification reaction
(1) Reaction conditions:
Usually carried out at a certain temperature (such as 90-120 ° C) and pressure to ensure thorough mixing and reaction of reactants.
(2) Operation steps:
Dissolve fluorescein in an appropriate amount of solvent, add acetic anhydride and catalyst, stir evenly, and then heat and reflux for a period of time (such as several hours). Continuous monitoring of the reaction progress is required during the reaction process to ensure complete reaction.
(3) Post processing:
After the reaction is complete, the crude product is obtained through steps such as filtration, washing, and drying. Then, purification was carried out using methods such as recrystallization and column chromatography to obtain high-purity FDA.
3. Specific synthesis examples
(1) Preparation of fluorescein:
Firstly, fluorescein is prepared through a series of chemical reactions. This process may include steps such as melting, adding zinc chloride, reaction, solidification, cooling, acid treatment, washing, drying, etc.
(2) Esterification reaction:
Take a certain amount of fluorescein (such as 6.3 g, about 0.02 mol), add an appropriate amount of acetic anhydride (such as 16 ml, about 5 times the theoretical amount) and catalyst (such as pyridine), heat to a certain temperature (such as 90 ° C) in an oil bath, stir and mix evenly. Then gradually raise the temperature to a higher temperature (such as 120 ° C) and react for a period of time (such as 7 hours).
(3) Post processing:
After the reaction is complete, cool naturally to room temperature and filter to obtain the crude product. Wash and dry the crude product to obtain the finished product.
Safe operation:
During the synthesis process, toxic and harmful chemicals such as acetic anhydride and pyridine are involved. It is necessary to operate in a fume hood and wear appropriate protective equipment (such as protective goggles, lab coats, gloves, etc.).
Reaction condition control:
Esterification reaction is sensitive to temperature, pressure, reaction time and other conditions, and strict control is required to ensure smooth reaction and product quality.
Purification steps:
The purification steps during post-processing are crucial for obtaining high-purity products. It is necessary to choose a suitable purification method according to the actual situation and conduct sufficient washing and drying treatment.
In order to improve synthesis efficiency and product purity, the synthesis method can be optimized. For example:
(1) Improving catalysts:
Choosing appropriate catalysts can increase the rate and yield of esterification reactions.
(2) Optimizing reaction conditions:
By adjusting reaction temperature, pressure, time, and other conditions, the purity and yield of the product can be further improved.
(3) Improving purification methods:
Adopting more efficient purification methods (such as chromatographic separation, membrane separation, etc.) can improve the purity and recovery rate of the Fluorescein Diacetate.
What are the side effects of this substance?
Cell viability testing:
FDA is widely used for cell viability testing because it can enter live cells and be broken down inside the cells to produce fluorescence, allowing for a visual assessment of the cell's viability status.
Low toxicity:
At normal usage concentrations, FDA has low toxicity to cells and usually does not have significant negative effects on cells.
Cell damage:
If the concentration of FDA is too high or the processing time is too long, it may cause certain damage to cells. This may be due to the impact of FDA or its decomposition products on the permeability of cell membranes or internal metabolism of cells.
Allergic reactions:
For certain individuals, FDA may cause allergic reactions. This reaction is usually related to an individual's constitution and immune system status, but the probability of occurrence is relatively low.
Environmental pollution:
The FDA needs to handle it properly after use to avoid potential pollution to the environment and ecosystem.
Although the FDA itself is not a highly hazardous chemical substance, improper handling of any chemical substance can have a negative impact on the environment.
Concentration control:
When using FDA for cell viability testing, the concentration and processing time should be strictly controlled to avoid unnecessary damage to cells.
Personal protection:
Operators should wear appropriate personal protective equipment such as gloves, masks, and goggles when using FDA to prevent direct contact with skin, eyes, or respiratory tract.
Waste disposal:
After use, FDA solutions should be properly disposed of in accordance with relevant regulations to ensure that they do not cause pollution to the environment.
FAQ
What is fluorescein diacetate?
Fluorescein diacetate (FDA) is defined as a fatty acid ester that becomes fluorescent after enzymatic hydrolysis by acetyltransferases in live mycobacterial cells, allowing for the identification of viable bacilli in viability assays.
What is fluorescein used for?
Fluorescein injection is used to help certain parts of the eye (eg, retina, iris) become more visible during eye medical procedures. This medicine is to be given only by or under the direct supervision of your doctor. This product is available in the following dosage forms: Solution.
What is the fluorescein diacetate analysis?
Fluorescein diacetate (FDA) is used to determine microbial activity. The determination of this activity can be carried out within the frame of various study objects using soil, litter, peat, etc. This method is based on a hydrolysis reaction of converting fluorescein diacetate (FDA) to fluorescein
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