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Fluorescein isothiocyanate orange red, isothiocyanate fluorescent yellow, isothiocyanate fluorescent red, isothiocyanate fluorescein isomer I, 5-isothiocyanate fluorescein. Yellow powder. It is hygroscopic. It can bind with various antibody proteins, and the antibody after binding does not lose the specificity of binding with certain antigen. It still has strong green fluorescence in alkaline solution.
After adding acid, it precipitates, and the fluorescence disappears. It is slightly soluble in acetone, ether and petroleum ether. It is a biochemical reagent as well as a medical diagnostic drug, which can quickly diagnose diseases caused by bacteria, viruses, parasites, etc. In addition, it can bind with various antibody proteins, and the antibody after binding does not lose the specificity of binding with certain antigen, and there is still strong green fluorescence in alkaline solution. After adding acid, it precipitates, and the fluorescence disappears, and it is slightly soluble in acetone, ether and petroleum ether.
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Chemical Formula |
C21H11NO5S |
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Exact Mass |
389 |
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Molecular Weight |
389 |
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m/z |
389 (100.0%), 390 (22.7%), 391 (4.5%), 391 (2.5%), 391 (1.0%), 392 (1.0%) |
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Elemental Analysis |
C, 64.78; H, 2.85; N, 3.60; O, 20.54; S, 8.23 |
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Study on the properties of fluorescein isothiocyanate labeled insulin for aminated gelatin and its application in nasal mucosa administration:
Under the condition of no external emulsifier, the aqueous solution of aminated gelatin was dispersed in olive oil, and a new type of positively charged gel microspheres, aminated gelatin microspheres, was prepared through the processes of emulsification, gelling, dehydration and cross-linking. The average particle size of microspheres is 40-50 μ m.
The size is evenly distributed. The dried microspheres can absorb about 20 times of their own weight of water to form hydrogels. Aminoated gelatin microspheres have higher amino content than gelatin microspheres. They are positively charged in distilled water and phosphate buffer solution, and the Zata potential in distilled water is 41.5 mV.
With the increase of glutaraldehyde concentration or the extension of crosslinking reaction time, the amino content of microspheres decreased. However, regardless of the crosslinking reaction conditions, the aminated gelatin microspheres prepared by this method have higher primary amino content than the gelatin microspheres. Aminoated gelatin microspheres can be degraded rapidly (<1h) in the presence of trypsin, but slowly (>20h) in pepsin and acid-base medium.
The interaction between aminated gelatin microspheres and mucin is stronger than that between gelatin microspheres. The high positive charge density of aminated gelatin microspheres is the main reason for its strong interaction with mucin. The bioadhesive properties of aminated gelatin microspheres were studied by fluorescence labeling method and in vitro enzyme degradation technology, using the isolated rat gastric perfusion washing model and in vivo experiment in rats.
The results showed that under the same experimental conditions, the number of aminated gelatin microspheres retained in the isolated rat stomach was more than that of gelatin microspheres, and the type of irrigation solution (artificial gastric juice or PH7 human phosphate buffer) had no significant effect on the gastric retention of microspheres, which indicated that the electrostatic interaction between the microspheres and gastric mucus was not the decisive factor for the gastric retention of microspheres in this model.
However, in the experiment, it was found that the amount of aminated gelatin microspheres retained in the isolated rat stomach decreased with the increase of the concentration of the crosslinking agent glutaraldehyde when preparing the microspheres, which indicated that the gastric mucosal adhesion of the aminated gelatin microspheres might be affected by the amino content on the surface of the microspheres or the degree of crosslinking.
The experimental results in vivo in rats showed that amino gelatin microspheres had better mucosal adhesion than gelatin microspheres. The effects of aminated gelatin on the nasal absorption of glucan and insulin were investigated in healthy rats. The results showed that 0.2% (WN) amino gelatin could significantly promote the absorption of fluorescein isothiocyanat glucan from rat nasal mucosa with a bioavailability of 23.8%.
After intranasal administration of 10 IU insulin phosphate buffer containing 0 2% (w/v) amino gelatin, the blood glucose level was significantly lower than that of insulin phosphate buffer.
Fluorescein isothiocyanate (FITC), as a classic fluorescent labeling reagent, has become an indispensable tool in the fields of biology, medicine, and materials science due to its high quantum yield, excellent photostability, and good biocompatibility.
The isothiocyanate group (- N=C=S) of FITC can undergo covalent reactions with active groups such as amino and thiol groups in biomolecules, forming stable fluorescent labeling complexes. This characteristic makes it the preferred reagent for protein, nucleic acid, and peptide labeling.
Protein labeling and functional research
FITC achieves specific labeling by binding to lysine residues of proteins such as antibodies and lectins. For example, in immunofluorescence experiments, FITC labeled antibodies can bind to cell surface antigens and quantitatively analyze antigen expression levels through fluorescence microscopy or flow cytometry. The experimental data shows that the fluorescence intensity of FITC IgG conjugate is linearly related to the antigen concentration, and the detection sensitivity can reach the Danak level. In addition, FITC labeled proteins can also be used for protein interaction studies, such as analyzing protein conformational changes through fluorescence resonance energy transfer (FRET) technology.
Nucleic acid labeling and molecular hybridization
FITC can be chemically modified to label the 5 'or 3' end of nucleic acids for fluorescence in situ hybridization (FISH) technology. In cancer research, FITC labeled probes can specifically bind to chromosomal abnormal regions to assist in the diagnosis of hematological diseases such as leukemia and lymphoma. For example, FITC probes targeting BCR-ABL fusion genes can display yellow fluorescent signals in interphase cell nuclei with an accuracy rate of over 95%.
Peptide labeling and drug delivery
FITC labeled peptides play an important role in drug delivery systems. Taking M2PEP-FITC as an example, this molecule is composed of a targeting peptide M2PEP and a FITC fluorescent group, which can specifically recognize tumor cell surface receptors and achieve targeted drug delivery. Experiments have shown that the accumulation of M2PEP-FITC labeled liposomes in tumor tissues is 3.2 times that of free drugs, significantly improving therapeutic efficacy and reducing side effects.
The fluorescence properties of FITC make it the "gold standard" in the field of in vitro diagnostics, widely used in techniques such as immunochromatography, flow cytometry, and ELISA.
Rapid detection of pathogens
FITC labeled antibodies can be used for rapid diagnosis of bacterial, viral, and parasitic infections.
For example, in malaria diagnosis, FITC labeled anti malaria antibodies bind to the patient's blood smear, and the green fluorescence signal of the parasite is observed through fluorescence microscopy. The detection time is shortened from 2 hours in traditional methods to 15 minutes, and the sensitivity reaches 98%. In addition, FITC labeled nucleic acid probes can also be used to detect respiratory virus nucleic acids, such as the ORF1ab gene of SARS-CoV-2, with a specificity of 99.2%.
Tumor markers
In flow cytometry, fuorescein Isothiocyanate labeled anti-CD45 antibodies can distinguish leukemia cells from normal blood cells. The data shows that the positive rate of FITC labeled CD34 antibody in the diagnosis of acute myeloid leukemia (AML) is 85%, which is 20% higher than traditional morphological examination. In terms of early screening of tumors, FITC labeled circulating tumor cells (CTC) detection technology can capture tumor cells in extremely low concentrations in peripheral blood, providing a new method for early diagnosis of solid tumors such as breast cancer and lung cancer.
Diagnosis of autoimmune diseases
In ELISA technology, FITC labeled autoantibodies (such as anti nuclear antibodies and anti double stranded DNA antibodies) can be used for the diagnosis of diseases such as systemic lupus erythematosus (SLE). Experiments have shown that FITC-ELISA has a sensitivity of 92% and a specificity of 95% for detecting anti Sm antibodies, which is significantly better than traditional immunofluorescence methods.
The cell permeability and low toxicity of FITC make it an ideal tool for cell imaging, widely used in organelle localization, cell division observation, and apoptosis research.
Cell organelle labeling and dynamic observation
FITC labeled mitochondrial probes (such as MitoTracker Green) can specifically bind to mitochondrial membrane potential and monitor mitochondrial morphological changes in real-time.
In neuronal research, FITC labeled microtubule protein antibodies can display the dynamic extension of axon growth cones, revealing the mechanism of neural development. In addition, FITC labeled lysosome probes (LysoTracker Green) can also be used to study autophagy processes, quantifying the number of autophagosomes through changes in fluorescence intensity.
Cell cycle and apoptosis analysis
In flow cytometry, FITC labeled propidium iodide (PI) can bind to DNA and distinguish different stages of the cell cycle (G0/G1, S, G2/M) by fluorescence intensity. Experimental data shows that the sensitivity of FITC-PI double staining method for detecting cell apoptosis is 98%, which is 30% higher than the traditional trypan blue staining method. In addition, FITC labeled Annexin V probes can specifically bind to phosphatidylserine exposed on the surface of apoptotic cells, achieving precise identification of early apoptotic cells.
Research on Cell Migration and Invasion
In Transwell experiments, FITC labeled cells can monitor the migration process in real-time. For example, in the study of tumor metastasis, the migration rate of FITC labeled breast cancer cells (MCF-7) through the matrix glue is positively correlated with the invasion ability, providing quantitative indicators for the screening of anti-tumor drugs.
FITC plays multiple roles in drug development, including target validation, drug delivery system evaluation, and pharmacokinetic studies.
Target validation and mechanism of action research
FITC labeled small molecule inhibitors can be used to validate drug targets.
For example, FITC labeled EGFR inhibitors (such as gefitinib) can demonstrate the binding of drugs to EGFR receptors on the surface of tumor cells, quantifying the binding affinity (Kd value) through fluorescence intensity. In addition, FITC labeled siRNA can also be used to study gene silencing efficiency, providing a basis for RNA interference therapy.
Drug delivery system evaluation
FITC labeled nanoparticles (such as liposomes and polymer microspheres) can monitor the distribution and release behavior of drugs in vivo in real-time.
Experiments have shown that the accumulation of FITC labeled PEGylated liposomes in tumor tissues is 2.8 times that of free drugs, and the fluorescence signal can last for 24 hours, providing visual data for optimizing drug delivery systems.
Pharmacokinetic and Toxicological Studies
FITC labeled drug molecules can be quantitatively analyzed for their absorption, distribution, metabolism, and excretion (ADME) processes in vivo through fluorescence imaging technology. For example, the fluorescence intensity of FITC labeled antibiotics (such as vancomycin) in the kidneys is linearly related to drug concentration, providing a basis for optimizing drug dosage. In addition, FITC labeled cytotoxicity assays can also evaluate the damage of drugs to normal cells and guide safety evaluations.
The fluorescence response characteristics of FITC make it uniquely applicable in the field of materials science, including the development of pH sensors, temperature sensors, and metal ion probes.
PH responsive fluorescence sensor
The fluorescence intensity of fluorescein Isothiocyanate is significantly affected by the pH value of the solution.
In weakly acidic environments (pH 5.0-6.5), protonation of FITC groups leads to increased fluorescence intensity; Under alkaline conditions (pH>8.0), fluorescence is quenched. Based on this characteristic, FITC modified nanoparticles can be used to monitor intracellular acid-base fluctuations. For example, the fluorescence intensity of FITC Biliribin complex in tumor microenvironment is 2.3 times that of normal tissue, providing a new method for tumor diagnosis.
Temperature responsive fluorescent probe
The fluorescence lifetime of FITC changes with temperature. Under low temperature conditions (4 ℃), the fluorescence lifetime of FITC labeled polymer microspheres is extended to 5.2 nanoseconds, which is 1.8 times higher than room temperature (25 ℃). This characteristic makes it an ideal tool for studying phase transition processes and cell cryopreservation.
Metal ion detection probe
FITC significantly reduces fluorescence intensity when combined with metal ions such as Cu ² ⁺ and Hg ² ⁺, making it suitable for environmental monitoring. For example, FITC modified silica gel nanoparticles have a detection limit of 0.1 nM for Hg ² ⁺ in water, which is 100 times higher than traditional atomic absorption spectroscopy.
The application of FITC in the agricultural field is gradually expanding, including plant virus detection, animal disease diagnosis, and variety purity identification.
Rapid detection of plant viruses
FITC labeled virus antibodies can be used to detect tobacco mosaic virus (TMV), cucumber mosaic virus (CMV), and other viruses. Experiments have shown that the sensitivity of FITC-ELISA for detecting TMV is 0.1 ng/mL, which is 10 times higher than traditional serological methods.
In addition, FITC labeled nucleic acid probes can also be used to detect exogenous genes in genetically modified crops, such as insect resistant Bt genes.
Diagnosis of animal diseases
FITC labeled antibodies can be used to detect swine fever virus (CSFV), avian influenza virus (AIV), and other viruses.
In the diagnosis of swine fever, FITC labeled anti CSFV antibodies bind to tissue sections and observe virus distribution through fluorescence microscopy, reducing the detection time from traditional methods of 3 days to 6 hours. In addition, FITC labeled flow cytometry can also be used to detect pathogen specific T cells in animal blood and evaluate immune response intensity.
Variety purity identification
FITC labeled DNA probes can be used to identify the purity of crop varieties. For example, the FITC probe targeting the rice blast disease resistance gene Pi ta can accurately distinguish between homozygotes and heterozygotes in hybrid rice seeds, with a purity identification accuracy of 99.5%. In addition, FITC labeled SSR molecular markers can also be used for genetic diversity analysis of livestock and poultry breeds, providing scientific basis for breeding.
Synthesis of FITC: 4-nitrophthalic anhydride (see C8H3NO5, [5466-84-2]) reacts with resorcinol at 195-200 ° C for 10h, cyclizes into a mixture of 3 'nitrofluorescein and 4' - nitrofluorescein, acetylates with acetic anhydride, hydrolyzes with saturated sodium hydroxide ethanol solution, 4 "nitrofluorescein is obtained. Then 4 '- nitrofluorescein is dissolved in ammonia water, reacted with nickel Rexhlet and hydrazine hydrate at room temperature for 2h, and reduced to 4' - amino fluorescein. The most radical is acylated and eliminated to obtain fluorescein isothiocyanate.
FITC pure product is yellow or orange crystalline powder, which is easily soluble in water and alcohol solvent. There are two isomers, among which isomer type I is better in efficiency, stability and protein binding. The molecular weight of FITC is 389.4, the maximum absorption wavelength is 490~495 nm, and the maximum emission wavelength is 520~530 nm, showing bright yellow green fluorescence. FITC can be stored for many years in cold, dark and dry places, and is the most widely used fluorescein. Its main advantage is that the human eye is more sensitive to yellow green, and the green fluorescence in the section specimen is usually less than red.
Frequently Asked Questions
Q: Why does FITC sometimes show decreased fluorescence after conjugation to proteins at high pH?
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A: At pH >9.0, FITC can undergo side reactions with amines to form substituted thiourea derivatives that are weakly fluorescent or non-fluorescent. In addition, hydrolysis of the isothiocyanate group to amine occurs competitively, reducing labeling efficiency and final brightness.
Q: Does FITC exhibit pH-dependent fluorescence lifetime changes, and what is the practical consequence?
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A: Yes. The fluorescence lifetime of FITC shortens significantly below pH 6 due to protonation of the xanthene ring. This makes it usable as a ratiometric or lifetime-based pH probe in microenvironments, but also causes unreliable signal intensity in live-cell endosomes or acidic organelles.
Q: Why is FITC more prone to photobleaching in degassed solutions than in air-saturated buffers?
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A: Normally, oxygen can quench triplet-state intermediates and reduce radical formation. Under degassed conditions, triplet accumulation increases, promoting irreversible photobleaching via covalent photodamage to the aromatic ring system.
Q: Can FITC conjugates undergo unexpected thiol-mediated side reactions during purification?
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A: Yes. Residual isothiocyanate groups can react with thiols (DTT, β-mercaptoethanol, or cysteine residues) to form thiocarbamates, which can cause protein crosslinking, aggregation, or loss of fluorescence during storage.
Q: Why does FITC show unusual polarity-dependent spectral shifts compared to other fluoresceins?
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A: The isothiocyanate group is strongly electron-withdrawing and polarizable. In low-polarity solvents, intramolecular charge transfer (ICT) is enhanced, leading to a red shift in emission and reduced quantum yield, which is rarely observed in unmodified fluorescein.
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