Chemistry:Fluorescein

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Short description: Synthetic organic compound used as dye and fluorescent tracer
Fluorescein
Skeletal formula
Ball-and-stick model
Sample of dark red powder
Names
Pronunciation /flʊəˈrɛsi.ɪn, flʊəˈrɛsiːn/
IUPAC name
3′,6′-dihydroxyspiro[isobenzofuran-1(3H),9′-[9H]xanthen]-3-one
Other names
Fluorescein, resorcinolphthalein, C.I. 45350, solvent yellow 94, D & C yellow no. 7, angiofluor, Japan yellow 201, soap yellow
Identifiers
3D model (JSmol)
ChEBI
ChEMBL
ChemSpider
DrugBank
EC Number
  • 219-031-8
KEGG
MeSH Fluorescein
UNII
Properties
C20H12O5
Molar mass 332.311 g·mol−1
Melting point 314 to 316 °C (597 to 601 °F; 587 to 589 K)
Slightly
Pharmacology
1=ATC code }} S01JA01 (WHO)
Hazards
GHS pictograms GHS07: Harmful
GHS Signal word Warning
H319
P305, P351, P338
Except where otherwise noted, data are given for materials in their standard state (at 25 °C [77 °F], 100 kPa).
☒N verify (what is ☑Y☒N ?)
Infobox references

Fluorescein is an organic compound and dye based on the xanthene tricyclic structural motif, formally belonging to triarylmethine dyes family. It is available as a dark orange/red powder slightly soluble in water and alcohol. It is widely used as a fluorescent tracer for many applications.[1]

The color of its aqueous solutions is green by reflection and orange by transmission (its spectral properties are dependent on pH of the solution),[2] as can be noticed in bubble levels, for example, in which fluorescein is added as a colorant to the alcohol filling the tube in order to increase the visibility of the air bubble contained within. More concentrated solutions of fluorescein can even appear red (because under these conditions nearly all incident emission is re-absorbed by the solution).

It is on the World Health Organization's List of Essential Medicines.[3]

Uses

Main page: Medicine:Fluorescein (medical use)

Fluorescein sodium, the sodium salt of fluorescein, is used extensively as a diagnostic tool in the field of ophthalmology and optometry, where topical fluorescein is used in the diagnosis of corneal abrasions, corneal ulcers and herpetic corneal infections. It is also used in rigid gas permeable contact lens fitting to evaluate the tear layer under the lens. It is available as sterile single-use sachets containing lint-free paper applicators soaked in fluorescein sodium solution.[4]

The thyroxine ester of fluorescein is used to quantify the thyroxine concentration in blood.[1]

Fluorescein is also known as a color additive (D&C Yellow no. 7). The disodium salt form of fluorescein is known as uranine or D&C Yellow no. 8.

Fluorescein is a precursor to the red dye eosin Y by bromination.[1]

Safety

Oral and intravenous use of fluorescein can cause adverse reactions, including nausea, vomiting, hives, acute hypotension, anaphylaxis and related anaphylactoid reaction,[5][6] causing cardiac arrest[7] and sudden death due to anaphylactic shock.[8][9]

Intravenous use has the most reported adverse reactions, including sudden death, but this may reflect greater use rather than greater risk. Both oral and topical uses have been reported to cause anaphylaxis,[10][11] including one case of anaphylaxis with cardiac arrest (resuscitated) following topical use in an eye drop.[7] Reported rates of adverse reactions vary from 1% to 6%.[12][13][14][15] The higher rates may reflect study populations that include a higher percentage of persons with prior adverse reactions. The risk of an adverse reaction is 25 times higher if the person has had a prior adverse reaction.[14] The risk can be reduced with prior (prophylactic) use of antihistamines[16] and prompt emergency management of any ensuing anaphylaxis.[17] A simple prick test may help to identify persons at greatest risk of adverse reaction.[15]

Chemistry

Fluorescein under UV illumination
Fluorescence excitation and emission spectra of fluorescein

The fluorescence of this molecule is very intense; peak excitation occurs at 495 nm and peak emission at 520 nm. Values for the deprotonated form in basic solution.

Fluorescein has a pKa of 6.4,[18] and its ionization equilibrium leads to pH-dependent absorption and emission over the range of 5 to 9. Also, the fluorescence lifetimes of the protonated and deprotonated forms of fluorescein are approximately 3 and 4 ns, which allows for pH determination from nonintensity based measurements. The lifetimes can be recovered using time-correlated single photon counting or phase-modulation fluorimetry. Upon exhaustive irradiation with visible light fluorescein decomposes to release phthalic and formic acids and carbon monoxide, effectively acting as a photoCORM.[19]

Fluorescein has an isosbestic point (equal absorption for all pH values) at 460 nm.

Derivatives

Fluorescein isothiocyanate and 6-FAM phosphoramidite

Many derivatives of fluorescein are known. Examples are:

In oligonucleotide synthesis, several phosphoramidite reagents containing protected fluorescein, e.g. 6-FAM phosphoramidite 2,[20] are used for the preparation of fluorescein-labeled oligonucleotides.

The extent to which fluorescein dilaurate is broken down to yield lauric acid can be detected as a measure of pancreatic esterase activity.

Synthesis

Approximately 250 tons/y were produced in the year 2000. The method involves the fusion of phthalic anhydride and resorcinol,[1] similar to the route described by Adolf von Baeyer in 1871.[21] In some cases, acids such as zinc chloride and methanesulfonic acid are employed to accelerate the Friedel-Crafts reaction.[22][23]

Preparation of Fluorescein.svg

Research

Fluorescein is a fluorophore commonly used in microscopy, in a type of dye laser as the gain medium, in forensics and serology to detect latent blood stains, and in dye tracing. Fluorescein has an absorption maximum at 494 nm and emission maximum of 512 nm (in water). The major derivatives are fluorescein isothiocyanate (FITC) and, in oligonucleotide synthesis, 6-FAM phosphoramidite.

Biosciences

In cellular biology, the isothiocyanate derivative of fluorescein is often used to label and track cells in fluorescence microscopy applications (for example, flow cytometry). Additional biologically active molecules (such as antibodies) may also be attached to fluorescein, allowing biologists to target the fluorophore to specific proteins or structures within cells. This application is common in yeast display.

Fluorescein can also be conjugated to nucleoside triphosphates and incorporated into a probe enzymatically for in situ hybridisation. The use of fluorescein amidite, shown below right, allows one to synthesize labeled oligonucleotides for the same purpose. Yet another technique termed molecular beacons makes use of synthetic fluorescein-labeled oligonucleotides. Fluorescein-labelled probes can be imaged using FISH, or targeted by antibodies using immunohistochemistry. The latter is a common alternative to digoxigenin, and the two are used together for labelling two genes in one sample.[24]

Fluorescein drops being instilled for an eye examination

Intravenous or oral fluorescein is used in fluorescein angiography in research and to diagnose and categorize vascular disorders including retinal disease, macular degeneration, diabetic retinopathy, inflammatory intraocular conditions, and intraocular tumors. It is also being used increasingly during surgery for brain and spine tumors.[25]

Diluted fluorescein dye has been used to localise multiple muscular ventricular septal defects during open heart surgery and confirm the presence of any residual defects.[26]

The Gemini 4 spacecraft releases dye into the water, to aid location after splashdown, June 1965.

Earth sciences

Fluorescein is used as a rather conservative flow tracer in hydrological tracer tests to help in understanding of water flow of both surface waters and groundwater. The dye can also be added to rainwater in environmental testing simulations to aid in locating and analyzing any water leaks, and in Australia and New Zealand as a methylated spirit dye.

As fluorescein solution changes its color depending on concentration,[27] it has been used as a tracer in evaporation experiments.

One of its more recognizable uses was in the Chicago River, where fluorescein was the first substance used to dye the river green on St. Patrick's Day in 1962. In 1966, environmentalists forced a change to a vegetable-based dye to protect local wildlife.[28]

Fluorescein dye solutions, typically 15% active, are commonly used as an aid to leak detection during hydrostatic testing of subsea oil and gas pipelines and other subsea infrastructure. Leaks can be detected by divers or ROVs carrying an ultraviolet light.

Plant science

Fluorescein has often been used to track water movement in groundwater to study water flow and observe areas of contamination or obstruction in these systems. The fluorescence that is created by the dye makes problem areas more visible and easily identified. A similar concept can be applied to plants because the dye can make problems in plant vasculature more visible. In plant science, fluorescein, and other fluorescent dyes, have been used to monitor and study plant vasculature, particularly the xylem, which is the main water transportation pathway in plants. This is because fluorescein is xylem-mobile and unable to cross plasma membranes, making it particularly useful in tracking water movement through the xylem.[29] Fluorescein can be introduced to a plant's veins through the roots or a cut stem. The dye is able to be taken up into the plant the same way as water and moves from the roots to the top of the plant due to a transpirational pull.[30] The fluorescein that has been taken up into the plant can be visualized under a fluorescent microscope.

See also

References

  1. ↑ 1.0 1.1 1.2 1.3 Gessner, Thomas; Mayer, Udo (2000). "Ullmann's Encyclopedia of Industrial Chemistry". Ullmann's Encyclopedia of Industrial Chemistry. Weinheim: Wiley-VCH. doi:10.1002/14356007.a27_179. 
  2. ↑ Sjöback, Robert; Nygren, Jan; Kubista, Mikael (1995-06-01). "Absorption and fluorescence properties of fluorescein" (in en). Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 51 (6): L7–L21. doi:10.1016/0584-8539(95)01421-P. ISSN 1386-1425. Bibcode: 1995AcSpA..51L...7S. https://dx.doi.org/10.1016/0584-8539%2895%2901421-P. 
  3. ↑ World Health Organization model list of essential medicines: 21st list 2019. Geneva: World Health Organization. 2019. WHO/MVP/EMP/IAU/2019.06. License: CC BY-NC-SA 3.0 IGO. 
  4. ↑ "New Drugs". Can Med Assoc J 80 (12): 997–998. 1959. PMID 20325960. 
  5. ↑ "The diagnosis and management of anaphylaxis. Joint Task Force on Practice Parameters, American Academy of Allergy, Asthma and Immunology, American College of Allergy, Asthma and Immunology, and the Joint Council of Allergy, Asthma and Immunology". The Journal of Allergy and Clinical Immunology 101 (6 Pt 2): S465–528. 1998. doi:10.1016/S0091-6749(18)30566-9. PMID 9673591. http://www.allergy123.com/library/documents/anaphylaxis.pdf. 
  6. ↑ The diagnosis and management of anaphylaxis: an updated practice parameter. National Guideline Clearinghouse.
  7. ↑ 7.0 7.1 El Harrar, N; Idali, B; Moutaouakkil, S; El Belhadji, M; Zaghloul, K; Amraoui, A; Benaguida, M (1996). "Anaphylactic shock caused by application of fluorescein on the ocular conjunctiva". Presse Médicale 25 (32): 1546–7. PMID 8952662. 
  8. ↑ "Fatal anaphylactic shock during a fluorescein angiography". Forensic Sci. Int. 100 (1–2): 137–42. 1999. doi:10.1016/S0379-0738(98)00205-9. PMID 10356782. 
  9. ↑ "An autopsy case of fatal anaphylactic shock following fluorescein angiography: a case report". Med Sci Law 44 (3): 264–5. 2004. doi:10.1258/rsmmsl.44.3.264. PMID 15296251. 
  10. ↑ "Anaphylaxis following oral fluorescein angiography". Am. J. Ophthalmol. 106 (6): 745–6. 1988. doi:10.1016/0002-9394(88)90716-7. PMID 3195657. 
  11. ↑ "Severe anaphylactic reaction to orally administered fluorescein". Am. J. Ophthalmol. 112 (1): 94. 1991. doi:10.1016/s0002-9394(14)76222-1. PMID 1882930. 
  12. ↑ "Fluorescein angiography and adverse drug reactions revisited: the Lions Eye experience". Clin. Experiment. Ophthalmol. 34 (1): 33–8. 2006. doi:10.1111/j.1442-9071.2006.01136.x. PMID 16451256. 
  13. ↑ "Adverse reactions during retinal fluorescein angiography". J Am Optom Assoc 65 (7): 465–71. 1994. PMID 7930354. 
  14. ↑ 14.0 14.1 "Frequency of adverse systemic reactions after fluorescein angiography. Results of a prospective study". Ophthalmology 98 (7): 1139–42. 1991. doi:10.1016/s0161-6420(91)32165-1. PMID 1891225. 
  15. ↑ 15.0 15.1 "[Usefulness of the prick test for anaphylactoid reaction in intravenous fluorescein administration]" (in ja). Nippon Ganka Gakkai Zasshi 100 (4): 313–7. 1996. PMID 8644545. 
  16. ↑ "Antihistamines as prophylaxis against side reactions to intravenous fluorescein". Trans Am Ophthalmol Soc 78: 190–205. 1980. PMID 7257056. 
  17. ↑ "Management of anaphylactic shock during intravenous fluorescein angiography at an outpatient clinic". J Chin Med Assoc 70 (8): 348–9. 2007. doi:10.1016/S1726-4901(08)70017-0. PMID 17698436. 
  18. ↑ Sjöback, Robert; Nygren, Jan; Kubista, Mikael (1995-06-01). "Absorption and fluorescence properties of fluorescein" (in en). Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 51 (6): L7–L21. doi:10.1016/0584-8539(95)01421-P. ISSN 1386-1425. Bibcode: 1995AcSpA..51L...7S. https://dx.doi.org/10.1016/0584-8539%2895%2901421-P. 
  19. ↑ Martínek, Marek; Ludvíková, Lucie; Šranková, Mária; Navrátil, Rafael; Muchová, Lucie; Huzlík, Jiří; Vítek, Libor; Klán, Petr et al. (2022-11-03). "Common xanthene fluorescent dyes are visible-light activatable CO-releasing molecules" (in en). Organic & Biomolecular Chemistry 21 (1): 93–97. doi:10.1039/D2OB01823C. ISSN 1477-0539. PMID 36326159. https://pubs.rsc.org/en/content/articlelanding/2022/ob/d2ob01823c. 
  20. ↑ Brush, C. K. "Fluorescein Labelled Phosphoramidites". U.S. Patent 5,583,236. Priority date July 19, 1991.
  21. ↑ Baeyer, Adolf (1871) "Uber ein neue Klasse von Farbstoffen" (On a new class of dyes), Berichte der Deutschen chemischen Gesellschaft zu Berlin, 4 : 555-558 ; see p. 558.
  22. ↑ Sun, W. C.; Gee, K. R.; Klaubert, D. H.; Haugland, R. P. (1997). "Synthesis of Fluorinated Fluoresceins". The Journal of Organic Chemistry 62 (19): 6469–6475. doi:10.1021/jo9706178. 
  23. ↑ Burgess, Kevin; Ueno, Yuichiro; Jiao, Guan-Sheng (2004). "Preparation of 5- and 6-Carboxyfluorescein". Synthesis 2004 (15): 2591–2593. doi:10.1055/s-2004-829194. 
  24. ↑ Noga E. J., Udomkusonsri, P. (2002). "Fluorescein: A Rapid, Sensitive, Nonlethal Method for Detecting Skin Ulceration in Fish". Vet Pathol 39 (6): 726–731(6). doi:10.1354/vp.39-6-726. PMID 12450204. http://www.vetpathology.org/cgi/reprint/39/6/726.pdf. Retrieved 2007-07-16. 
  25. ↑ Cardali, Salvatore Massimiliano; Ricciardo, Giuseppe; Garufi, Giada; Raffa, Giovanni; Messineo, Francesco; Scalia, Gianluca; Conti, Alfredo; Germanò, Antonino (2022). "Fluorescein-guided surgery for intradural spinal tumors: A single-center experience" (in en). Brain and Spine 2: 100908. doi:10.1016/j.bas.2022.100908. PMID 36248155. PMC 9560644. https://linkinghub.elsevier.com/retrieve/pii/S2772529422000492. 
  26. ↑ Mathew, Thomas (2014). "Use of Fluorescein Dye to Identify Residual Defects". Ann Thorac Surg 97 (1): e27-8. doi:10.1016/j.athoracsur.2013.10.059. ISSN 0003-4975. PMID 24384220. 
  27. ↑ Käss, W. Tracing Technique in Geohydrology. Rotterdam: Balkema. 
  28. ↑ The Story Behind Dyeing the River Green. Greenchicagoriver.com. Retrieved on 2014-08-28.
  29. ↑ Salih, Anya; Tjoelker, Mark G.; Renard, Justine; Pfautsch, Sebastian (2015-03-01). "Phloem as Capacitor: Radial Transfer of Water into Xylem of Tree Stems Occurs via Symplastic Transport in Ray Parenchyma". Plant Physiology 167 (3): 963–971. doi:10.1104/pp.114.254581. ISSN 0032-0889. PMID 25588734. PMC 4348778. http://www.plantphysiol.org/content/167/3/963. 
  30. ↑ "Fluorescein Transport Assay to Assess Bulk Flow of Molecules Through the Hypocotyl in Arabidopsis thaliana". Bio-Protocol 8 (7): e2791. July 2017. doi:10.21769/bioprotoc.2791. PMID 34286014. 

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